Environmental Fate, Aquatic Toxicology and Risk Assessment

of Polymeric Quaternary Ammonium Salts from Cosmetic

Uses

Author
Cumming, Janet L

Published
2008

Thesis Type
Thesis (PhD Doctorate)

School
School of Environment

DOI
https://doi.org/10.25904/1912/2683

Copyright Statement
The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from
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Environmental Fate, Aquatic Toxicology and Risk Assessment of
Polymeric Quaternary Ammonium Salts from Cosmetic Uses.

Janet L. Cumming
B.Sc. (Hons)

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

Griffith School of Environment

Science, Environment, Engineering and Technology Group
Griffith University

February 2008
 Removal Notice

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Figure 3.3 (p. 61) has been removed from the digital version of this thesis for
copyright reasons.

Figures 3.4 & 3.5 (p. 62) have been removed from the digital version of this thesis for
copyright reasons.
 Synopsis
The consumption of household and personal care products in Australia is similar to
that of more highly regulated agricultural and veterinary chemicals. One class of
chemical used in cosmetic applications, polymeric quaternary ammonium salts
(polyquaterniums), is thought to have adverse effects on aquatic organisms. These
polymers belong to a larger class of polymers, cationic polyelectrolytes, that are
widely used in industry, largely for water treatment, and that have been extensively
studied and regulated. The cosmetic polyquaterniums, however, have not been subject
to the same scrutiny, even though differences in, or expectations of, their behaviour
are known to exist.

The aim of this study was to examine the fate and toxicity of some cosmetic
polyquaterniums, and particularly to examine the impact of the presence of the
anionic surfactant, sodium dodecyl sulphate, that is often complexed with the
polyquaterniums in cosmetic formulations on fate and toxicity. The polyquaterniums
studied consisted of six samples of Polyquaternium-10 of provided by Amerchol (The
Dow Chemical Company, Midland, MI U.S.A.), five samples of three
polyquaterniums (Polyquaternium-11, Polyquaternium-28, Polyquaternium-55)
provided by International Specialty Products (ISP, Wayne, New Jersey, USA), and
polydimethyldiallyl ammonium chloride (poly(DADMAC), Polyquaternium-6),
widely used in water treatment but less commonly in cosmetic applications purchased
from Sigma-Aldrich (Castle Hill, NSW, Australia). The four-step risk assessment
paradigm (hazard identification, exposure assessment, hazard assessment, risk
characterisation) provided the framework for this study.

Metachromatic polyelectrolyte titration was used to analyse polyquaterniums in

aqueous solution. Although the method is generally not viable in the presence of other
ions due to interference, it was found to be viable in the presence of the anionic
surfactant sodium dodecyl sulphate. Further, the method was found to work with the
supernatant following a sorption experiment involving humic acid. It was not possible
to titrate solutions following exposure to bentonite, or in solutions prepared for
toxicity tests. Metachromatic Colloid Titration was found to be useful in determining
the charge density of the polyquaterniums, and in measuring the concentration of
polyquaterniums of known charge density.

ii
To establish the extent of exposure of vulnerable aquatic organisms to
polyquaterniums released from cosmetic usage, it is necessary to estimate the
concentration of polyquaterniums in the aquatic environment. The volume usage of
polyquaterniums was estimated from available published data and standard emission
scenarios used in the risk assessment of new and existing chemicals. The partitioning
of the polyquaternium from the aqueous to the biosolid phase from wastewater
treatment plants was estimated from the determination of the partition coefficient
between water and humic acid. The latter was assumed to be a suitable surrogate for
the biosolids. The fate of polyquaterniums in wastewater treatment plants was
modelled using a fugacity approach based on a typical wastewater treatment plant.
The import/manufacture volume of polyquaterniums for cosmetic uses was estimated
to be between 20 and 60 tonnes per annum. The partition coefficient for
polyquaterniums between the aqueous phase and humic acid was lower than expected,
generally between 100 and 1000 for the polyquaterniums in this study. Fugacity
modelling results suggested that the partitioning of polyquaterniums to the solid phase
in wastewater treatment may be less than the default values normally assumed in
regulatory risk assessment. Therefore, the estimate of the predicted environmental
concentration (PEC) of polyquaterniums in Australian waters is between 0.7 µg/L and
40 µg/L depending on the assumptions and methodology used.

Effects assessment, or hazard assessment, is concerned with determining the capacity

of the cosmetic polyquaterniums to cause harm to aquatic organisms in the
environment. In this study, the aim was to determine if the hazard of the
polyquaternium from cosmetic usage is the same as that of the better studied water
treatment polymers; and if the complexing of the polyquaternium with the anionic
surfactant makes any difference to the toxicity. One species from each of three trophic
levels, viz fish, crustacean and algae were selected. Using assessment factors
developed for the risk assessment of new chemicals, the environmental concentration
likely to be hazardous to the most sensitive species was estimated. The
polyquaterniums studied were found to be just as hazardous to the most sensitive
species for a typical cosmetic polyquaternium when complexed with the anionic
surfactant. The lowest concentration at which a toxic effect occurred was for 50%
growth inhibition for algae, 0.3 mg/L for the most toxic polyquaternium. With
assessment factors, and using the concentration at which cosmetic polyquaterniums

iii
were likely to be hazardous to aquatic organisms, the predicted no effect
concentration (PNEC) was estimated to be between 0.3 µg/L and 1.2 µg/L.

The risk characterisation process combines the information obtained from the effects
and exposure assessments to evaluate the nature of the potential risk. Commonly, the
level of risk is estimated based on the PEC/PNEC ratio. In this study, using point
estimates and probabilistic methods (Monte Carlo Simulation), the risk of
polyquaterniums from cosmetic uses was estimated. Based on the behaviour and
toxicity determined in this study, there may be some risk to aquatic organisms from
individual polyquaterniums at even low import volumes. As a class of compounds,
polyquaterniums from cosmetic uses may present a significant risk to environmental
waters in Australia. Sensitivity analysis showed that the prediction of risk was most
sensitive to those parameters for which the least amount of data was available, such as
the import volume and dilution to receiving waters.

A recently developed method of estimating potential risk based on the concept of an

Environmental Threshold of No Concern (ETNC), was applied to the use of cosmetic
polyquaterniums in Australia. Using the fugacity model approach, the usage volume
at which the environmental concentration would exceed the critical threshold was
estimated. The volume was found to be significantly lower than the estimated usage
determined by either of the methods employed in estimating the current usage
volume.

While some problems remain in identifying the risk from polyquaterniums to the
Australian environment, particularly those associated with the difficulties of
quantifying polymers in environmental samples, this thesis has made substantial
progress in the risk assessment. Particularly, it has been shown that the use of default
assumptions that are largely unsubstantiated, and the sensitivity of the methodology to
information that is often unavailable, may result in an estimation of risk that may not
be able to protect vulnerable environments.

iv
 Acknowledgements

This work was supported by the Society of Environmental Toxicology and Chemistry
(SETAC)/Procter & Gamble Company Global Fellowship for Doctoral Research in
Environmental Science, sponsored by the Procter & Gamble Company.

I would like to thank my supervisors, Dr Darryl Hawker, Dr Heather Chapman and Dr

Kerry Nugent for their guidance, support and encouragement through all aspects of
my candidature.

Thank you to the technical and support staff at the Griffith School of Environment,
particularly Scott Byrnes and Jane Gifkin for their assistance and support in the
laboratory.

Thank you to my research assistant, Melanie Crook, for many dedicated hours in the
field and the laboratory; Alan and Joyce Hodder for access to their property for
collecting Gambusia; and the undergrad students who helped sort the blighters.

I would like to acknowledge the CRC for Water Quality and Treatment and the Centre
for Environmental Systems Research for financial support and encouragement. Also
important were Jenny Stauber and her team at CSIRO for their endeavours in teaching
me algal toxicity testing.

I am especially grateful to SETAC and Procter & Gamble, whose support made a
significant contribution to this research, which might have been barely possible
otherwise.

Thank you to Fiona de Mestre for the final editing.

I would like to thank my fellow candidates at Griffith School of Environment; the

friendship and encouragement of peers is invaluable.

Finally, a special thank you to my family – my sister Alison, my daughters Jeanne and
Heather, their father Fred, and my niece Darcy; with their love and support, anything
is possible.

v
 Dedicated to

Alyssa Lenore Hannaford

(8/7/1989 to 16/9/2003)

and

Michael Thomas Wakeham

(8/10/1989 to 9/4/2007)

vi
 Declaration of Originality

The experimentation, analysis, presentation and interpretation of results presented in

this thesis represent my original work and have not previously been submitted for a
degree or diploma in any university. To the best of my knowledge and belief, the
thesis contains no material previously published or written by another person except
where due reference is made in the thesis itself.

Janet L. Cumming

Publications arising from this work

Cumming, J.L. 2007 ‘Polyelectrolytes’. in Chemicals of Concern in Wastewater
Treatment Plant Effluent. CRC for Water Quality and Treatment Occasional Paper
No 8. Cooperative Research Centre for Water Quality and Treatment, Adelaide.
Cumming, J.L, Hawker D.W., Nugent, K.W. and Chapman, H.F. 2008 Ecotoxicities of
Polyquaterniums and their associated polyelectrolyte surfactant aggregates (PSA) to
Gambusia holbrooki. Journal of Environmental Science and Health, Part A
Toxic/Hazardous Substances and Environmental Engineering, Volume 43 Issue 2, 113
Cumming, J.L, Hawker D.W., Nugent, K.W. and Chapman, H.F. 2006 Toxicity of
cosmetic polyquaterniums to Gambusia holbrooki. Oral Presentation, SETAC Asia
Pacific, Beijing.
Janet Cumming, 2006 Environmental Fate and Aquatic Toxicology of Polymeric
Quaternary Ammonium Salts. Oral Presentation, CRCWQT Postgraduate Students
Conference, Melbourne, July 2006.
Janet Cumming, Darryl Hawker, Heather Chapman. 2005 Mitigation of Toxicity of
Quaternary Polyelectrolytes – Fact or Fiction? Oral Presentation, Australian Society
for Ecotoxicology Conference, Melbourne.
Cumming, J.L. Hawker D.W and Chapman, H.F 2005 Polyquaternium Ammonium
Salts: Using data obtained in the study of water treatment polyelectrolytes in the risk
assessment of cosmetic polymers. Presentation and Paper (peer reviewed) Australian
Water Association Contaminants of Concern Conference, Canberra.

vii
 Table of Contents
Synopsis .....................................................................................................................ii
Acknowledgements....................................................................................................v
Declaration of Originality ........................................................................................vii
Publications arising from this work .........................................................................vii
Table of Contents......................................................................................................xi
List of Tables ..........................................................................................................xiv
List of Figures ...................................................................................................... xviii
Abbreviations..........................................................................................................xix
Definitions..............................................................................................................xxii
1. Introduction............................................................................................................1
1.1. Non-cosmetic Uses of Polyquaterniums ........................................................3
1.2. Regulation of Chemicals in Australia ............................................................7
1.3. The Risk Assessment Framework................................................................11
1.3.1. Hazard Identification ...........................................................................12
1.3.2. Effects Assessment ..............................................................................13
1.3.3. Exposure Assessment...........................................................................13
1.3.4. Risk Characterisation ...........................................................................13
1.3.5. Assessment Reports .............................................................................14
1.3.6. Data Requirements...............................................................................14
1.3.7. Globally Harmonised Labelling...........................................................15
1.3.8. GHS and the Aquatic Environment .....................................................16
1.4. Conclusion ...................................................................................................17
1.4.1. Aims.....................................................................................................18
1.4.2. Structure...............................................................................................18
2. Literature Review.................................................................................................19
2.1. Structure of Polyquaterniums ......................................................................19
2.1.1. Quaternary Ammonium Salts ..............................................................19
2.1.2. Polymers ..............................................................................................19
2.1.3. Polyelectrolytes....................................................................................20
2.1.4. Common Features of Polyquaterniums................................................20
2.1.5. Variation Between Polyquaterniums ...................................................21
2.1.6. Variation Within Polyelectrolytes........................................................23
2.2. Nomenclature...............................................................................................24
2.3. Relevant Polyquaternium Physical-Chemical Properties ............................24
2.3.1. Molecular Weight Distribution ............................................................25
2.3.2. Charge Density.....................................................................................27
2.3.3. Aqueous Solubility...............................................................................29
2.3.4. Biodegradation.....................................................................................31
2.3.5. Chemical/physical Degradation ...........................................................32
2.4. Exposure Assessment (Environmental Fate) ...............................................33
2.4.1. Sorption-desorption..............................................................................33
2.4.2. Sorption of Polymers ...........................................................................33
2.4.3. Sorption of Polyelectrolytes.................................................................35
2.5. Effects Assessment (Aquatic Toxicology)...................................................40
2.5.1. Toxicity of Surfactants.........................................................................40
2.5.2. Toxicity of Polymers............................................................................41
2.5.3. Toxicity of Polyelectrolytes – General Considerations .......................41
2.5.4. Meta-analysis .......................................................................................46
2.5.5. Mechanism...........................................................................................51

viii
 2.6. Conclusion ...................................................................................................53
3. Analysis of Polyquaterniums ...............................................................................55
3.1. Introduction..................................................................................................55
3.1.1. Metachromasy......................................................................................56
3.1.2. Colloid Titration...................................................................................56
3.2. Metachromatic Polyelectrolyte Titration .....................................................57
3.2.1. The Titrant – Choice of Chromotropic Polyanion and Cationic
Standard ...............................................................................................58
3.2.2. The Indicator – Choice of Metachromatic Dye ...................................58
3.2.3. Determination of The Visual Endpoint................................................59
3.2.4. Determination of the Endpoint Using Spectrophotometry ..................59
3.2.5. Calculations – Determining Charge Density and/or Concentration ....62
3.2.6. Method Validation ...............................................................................63
3.2.7. Problems and Limitations ....................................................................63
3.2.8. Aims and Objectives ............................................................................65
3.3. Analytical Methods......................................................................................65
3.3.1. Materials ..............................................................................................65
3.3.2. Standardisation of PVSK .....................................................................68
3.3.3. Titration of Polyquaternium Solutions (Visual Endpoint)...................68
3.3.4. Charge Density Determination of Polyquaterniums (Preparation of the
Standard Curve) ...................................................................................69
3.3.5. Titration (Spectrophotometric Endpoint).............................................70
3.4. Results..........................................................................................................71
3.4.1. PVSK ...................................................................................................71
3.4.2. Analysis of Results for Equivalent Weight..........................................72
3.4.3. Titration in the Presence of SDS..........................................................74
3.4.4. Titration (Spectrophotometric Endpoint).............................................74
3.5. Discussion ....................................................................................................76
3.6. Conclusion ...................................................................................................79
4. Chemistry and Fate – Exposure Assessment of Polyquaterniums.......................81
4.1. Introduction..................................................................................................81
4.2. Use patterns and release data .......................................................................82
4.2.1. Emission Scenarios ..............................................................................86
4.3. Environmental Fate......................................................................................87
4.3.1. Partitioning...........................................................................................87
4.3.2. Methods................................................................................................88
4.3.3. Results..................................................................................................90
4.3.4. Discussion ............................................................................................93
4.4. Fate Modelling .............................................................................................96
4.4.1. Partitioning Models..............................................................................96
4.4.2. Model Parameters ................................................................................98
4.4.3. Predicting Extent of Removal of Polyquaternium. ............................100
4.4.4. Results................................................................................................106
4.4.5. Discussion ..........................................................................................107
4.5. Predicted Environmental Concentration ....................................................108
4.6. Conclusion .................................................................................................112
5. Aquatic Toxicology – Effects Assessment of Polyquaterniums........................113
5.1. Introduction................................................................................................113
5.2. Methods......................................................................................................115
5.2.1. Experimental Design..........................................................................115

ix
 5.2.2. Test Solutions.....................................................................................116
5.2.3. Fish.....................................................................................................116
5.2.4. Brine Shrimp......................................................................................120
5.2.5. Algae ..................................................................................................121
5.3. Results, Observations and Data Analysis ..................................................122
5.3.1. Fish.....................................................................................................122
5.3.2. Brine Shrimp......................................................................................127
5.3.3. Algae ..................................................................................................128
5.4. Discussion ..................................................................................................129
5.4.1. Charge Density...................................................................................133
5.4.2. Polymer-surfactant Complex .............................................................136
5.4.3. Humic Acid........................................................................................137
5.5. PNEC .........................................................................................................139
5.6. Conclusions................................................................................................141
6. Risk Characterisation .........................................................................................142
6.1. Introduction................................................................................................142
6.1.1. Monte Carlo Simulation.....................................................................145
6.2. The Environmental Threshold of No Concern Method .............................146
6.2.1. Method ...............................................................................................147
6.2.2. Results................................................................................................152
6.3. The PEC/PNEC Ratio (The Quotient Method)..........................................154
6.3.1. Method ...............................................................................................155
6.3.2. Results................................................................................................156
6.4. Probabilistic Risk Characterisation............................................................158
6.4.1. Method ...............................................................................................158
6.4.2. Results................................................................................................162
6.5. Discussion ..................................................................................................165
6.5.1. Is This Risk? ......................................................................................165
6.5.2. Uncertainty in PNEC and PEC ..........................................................169
6.5.3. Conservatism and Precaution.............................................................173
6.5.4. Science, Policy, Assessment and Management .................................175
6.5.5. Characterising the Risk to the Aquatic Environment from Cosmetic
Polyquaternium Use in Australia .......................................................178
6.6. Conclusion .................................................................................................181
7. Overall Conclusions and Future Research.........................................................183
7.1. Conclusions................................................................................................183
7.1.1. Analysis..............................................................................................183
7.1.2. Environmental Fate............................................................................183
7.1.3. Toxicology .........................................................................................184
7.1.4. Risk Characterisation .........................................................................185
7.2. Implications and Future Research..............................................................185
7.3. Concluding Comments...............................................................................188
List of References ......................................................................................................189
Appendix 1. Database of cosmetic polyquaterniums..................................................i
Polyquaternium-1....................................................................................................i
Polyquaternium-2...................................................................................................ii
Polyquaternium-4................................................................................................. iii
Polyquaternium-5..................................................................................................iv
Polyquaternium-6...................................................................................................v
Polyquaternium-7..................................................................................................vi

x
 Polyquaternium-8.................................................................................................vii
Polyquaternium-9............................................................................................... viii
Polyquaternium-10................................................................................................ix
Polyquaternium-11.................................................................................................x
Polyquaternium-12................................................................................................xi
Polyquaternium-13...............................................................................................xii
Polyquaternium-14............................................................................................. xiii
Polyquaternium-15/polyquaternium-32 ..............................................................xiv
Polyquaternium-16...............................................................................................xv
Polyquaternium-17..............................................................................................xvi
Polyquaternium-18.............................................................................................xvii
Polyquaternium-19........................................................................................... xviii
Polyquaternium-20........................................................................................... xviii
Polyquaternium-22..............................................................................................xix
Polyquaternium-24...............................................................................................xx
Polyquaternium-26...............................................................................................xx
Polyquaternium-27..............................................................................................xxi
Polyquaternium-28.............................................................................................xxii
Polyquaternium-29........................................................................................... xxiii
Polyquaternium-30........................................................................................... xxiii
Polyquaternium-31............................................................................................xxiv
Polyquaternium-34.............................................................................................xxv
Polyquaternium-35............................................................................................xxvi
Polyquaternium-37...........................................................................................xxvii
Polyquaternium-39......................................................................................... xxviii
Polyquaternium-42............................................................................................xxix
Polyquaternium-43............................................................................................xxix
Polyquaternium-44.............................................................................................xxx
Polyquaternium-46............................................................................................xxxi
Polyquaternium-47...........................................................................................xxxii
Polyquaternium-51......................................................................................... xxxiii
Polyquaternium-55..........................................................................................xxxiv
Dimethylamine-epichlorohydrin copolymer....................................................xxxv
Polymethacrylamidopropyltrimonium chloride..............................................xxxvi
Guar hydroxypropyltrimonium chloride........................................................xxxvii
Other polyquaterniums................................................................................. xxxviii
Appendix 2. Published toxicity values for Cationic Polyelectrolytes......................xli
Appendix 3 Partitioning Models......................................................................... xlviii
a. Percent Removal Model.................................................................................. xlviii
b. Input Flux Model. ..............................................................................................xlix

xi
 List of Tables
Table 1.1 Comparison of the regulatory schemes for industrial chemicals and
therapeutics in Australia, USA and the European Union. .............................5
Table 1.2 Key elements of the regulatory and management structure of chemicals in
Australia (adapted from DEH 1998)..............................................................8
Table 1.3 Summary of assessment reports for polyquaterniums assessed by NICNAS
as new chemicals..........................................................................................10
Table 1.4 Hazard classifications for aquatic toxicity under Globally Harmonised
System for Classification and Labelling of Chemicals (GHS) (OECD 2001).
.....................................................................................................................17
Table 2.1 In Amerchol’s range of Polyquaternium-10, UCareTM, the polymer is
produced in a range of molecular weight (as indicated by viscosity) and
charge density combinations (as % amine-nitrogen) (Amerchol, 2005) .....23
Table 2.2 The number of high and low charge density polycations by Globally
Harmonised System for Classification and Labelling of Chemicals (GHS)
classification based on the OPPT data in Boethling and Nabholz (1997). ..49
Table 2.3 Histological observations of fish gill tissue exposed to cationic
polyelectrolytes (Biesinger and Stokes 1986)..............................................52
Table 3.1 The cosmetic polyquaterniums used in this study, supplied by Amerchol
and ISP. The water treatment polyquaternium polyDADMAC, which is also
sometimes occurs in cosmetic formulations, was also used. Both
International Nomenclature of Cosmetic Ingredients (INCI) and trade names
are given.......................................................................................................66
Table 3.2 The gram equivalence of the polyquaternium as determined by visual
titration of three solutions and a blank with PVSK and o-toluidine blue. ...73
Table 3.3 Equivalence of some polyquaterniums determined from the spectroscopic
titration and using the Matlab® method to determine the tipping point......76
Table 4.1 Confidentiality status and import volume details from NICNAS FPR for
assessed polyquaterniums. ...........................................................................83
Table 4.2 An extract from High Volume Industrial Chemical List maintained by
NICNAS to record the volumes of industrial chemicals manufactured in or
imported into Australia in quantities > 1000 tonnes pa (NICNAS 2002b). 84
Table 4.3 Some categories for which data is collected on imported products by ABS.
.....................................................................................................................85
Table 4.4 Estimation of volume of polyquaterniums imported or manufactured based
on estimates given in NICNAS New Chemical Notifications and the number
of polyquaterniums already listed on AICS.................................................86
Table 4.5. Estimation of volume of polyquaterniums based on the EC Guidance
document, Emission scenario for personal/domestic chemicals (ECB 2003).
.....................................................................................................................87
Table 4.6 Results of statistical analysis of humic acid particle size for the separated
supernatant and solid fraction showing that the difference was significant at
α = 0.05 given unequal variance. .................................................................92
Table 4.7 Partition coefficient KD determined for polyquaterniums and PSCs and one
cationic surfactant (cetyl pyridinium chloride)............................................92
Table 4.8 Results of a paired t-test of the difference between KD for the
polyquaternium and its PSC. .......................................................................93
Table 4.9 Results of a regression model of KD against charge density. The model is
significant if the high charge density poly(DADMAC) is included, but is not
significant for the cosmetic polymers alone. ...............................................94

xii
Table 4.10 Results of model calculation for proportion of influent polyquaternium
removed as a function of various values of KD and solids removal (PST
only and with WAS taken into account). .................................................107
Table 4.11 Determination of PEC for environmental risk assessment using
NICNAS/DEW method. ..........................................................................109
Table 4.12 Effluent discharge polyquaternium concentrations (µg/L) for a range of
import volumes and WWTP removal rates..............................................109
Table 4.13 PECs of polyquaterniums for different import volumes and environmental
dilution ratios (µg/L). The effluent concentration would also represent the
case where there was zero dilution. .........................................................111
Table 5.1 Water parameters before and after exposure of fish to the polyquaternium of
PSC for an acceptable test with G. holbrooki. ...........................................118
Table 5.2 Concentrations of polyquaterniums used in fish toxicity tests. .................119
Table 5.3 Concentration of polyquaternium as polymer-surfactant complex used in the
toxicity tests. ..............................................................................................119
Table 5.4 Concentrations of polyquaterniums, SDS and SDS as polyquaternium-
surfactant complexes used in brine shrimp tests........................................121
Table 5.5 Conditions for the culture and testing of algae. .........................................121
Table 5.6 Test concentrations of chemicals used in algal toxicity tests ....................122
Table 5.7 Result of probit analysis of data from 96 hour fish tests using G. holbrooki
with polyquaterniums and the monoquaternium cetyl pyridinium chloride
without surfactant. Where more than one test has been performed, the repeat
results are indicated by the letters in parenthesis.......................................125
Table 5.8 Result of probit analysis of data from 96 hour fish tests using G. holbrooki
with PSC and the monoquaternium cetylpyridinium chloride and SDS. ..126
Table 5.9 Results of paired t-test for fish toxicity studies. ........................................126
Table 5.10 Results of analysis of data from algal growth inhibition test for
polyquaterniums and for the PSC for UCareTM JR125 only....................128
Table 5.11 Results of statistical test for difference in algal toxicity between the
samples of UCare JR125, JR400 and JR30M (Polyquaternium-10) and
Gafquat® 440 and 734 (Polyquaternium-11). .........................................129
Table 5.12 Results of Dunnett's test for difference between test concentrations and
controls in algal growth inhibition test. ...................................................129
Table 5.13 Fish Acute Toxicity Syndromes (FATS) as described by McKim et al.
(1987) .......................................................................................................130
Table 5.14 Recommended assessment factors to be applied in determining the PNEC
are dependent on the confidence that can be attributed to the available data,
which is dependent on the amount and types of toxicity data available. .140
Table 6.1 Definitions of risk characterisation published in various guidelines and
articles since the introduction of the four-step paradigm in 1983. ............143
Table 6.2 Definitions of risk published in various guidelines and articles since the
introduction of the four-step paradigm in 1983. ........................................144
Table 6.3 Estimates of input volume and product usage for various ETNCaq for
polyquaterniums, calculated for KD values of 400 (UCareTM JR125) and
1000 (Gafquat® 755) and 10000. ..............................................................153
Table 6.4 Analysis of the PEC/PNEC estimates in NICNAS assessments. Note that
different assumptions regarding population size and water use volumes may
apply in these assessments. ........................................................................156

xiii
Table 6.5 Estimated PEC/PNEC for a polyquaternium at the modelled WWTP
fraction removed (21-73%) from Chapter 4 and the default assumption of
90% removal via sludge.............................................................................157
Table 6.6 Estimated PEC/PNEC for all polyquaternium use in Australia, assuming
additive toxicities, at the modelled WWTP removal rates (21-73%) from
Chapter 4 and the default assumption of 90% removal via sludge............158
Table 6.7 Probability density functions of variables used in all Monte Carlo
Simulation of the PEC calculation.............................................................160
Table 6.8 Probability density functions of import volumes used in Monte Carlo
Simulation for three simulations of the PEC calculation...........................162
Table 6.9 Ecotoxicological assessment criteria for pesticides used by USEPA to
estimate the hazard potential of pesticides to non-target aquatic organisms
(Bascietto 1990). ........................................................................................167
Table 6.10 Factors contributing to uncertainty in the estimation of the PEC of
polyquaterniums in Australia. ..................................................................172
Table 6.11 Factors contributing to uncertainty in the estimation of the PNEC of
polyquaterniums in Australia. ..................................................................173

xiv
 List of Figures
Figure 2.1 The structure of the ammonium cation, showing the degree of substitution
of hydrogen with organic groups on the nitrogen. .....................................19
Figure 2.2 The structure of Polyquaternium-10 (quaternised hydroxyethyl cellulose),
which is a bio-polymer, based on cellulose. Polymers such as
Polyquaternium-10 are often referred to as ‘natural’ polymers.................22
Figure 2.3 The structure of the polymerisable functional groups in monomers used in
the synthesis of some polyquaterniums. Polymers synthesised from these
monomers are known as ‘synthetic’ polymers...........................................22
Figure 2.4 Number Average Molecular Weight (NAMW) is the total weight of all the
polymer molecules in a sample, divided by the total number of polymer
molecules in a sample. It gives an indication of the average size of the
polymer chain, but not of the range of sizes of the chains (polydispersity)
of the polymer. ...........................................................................................25
Figure 2.5 Representation of the adsorption of a polymer onto a surface, showing the
formation of loops, tails and trains (Obey and Griffiths 1999)..................34
Figure 2.6 Scatter plot of charge density (as % amine-Nitrogen) against log molecular
weight for all cationic polymers in the data submitted with PMNs to OPPT
(Boethling and Nabholz 1997)...................................................................48
Figure 2.7 Median LC50 (mg/L) of water treatment cationic polyelectrolytes by
chemical class based on the data in Lyons and Vaconcellos (1997). The
median daphnid LC50 for Mannich polymers (48.95 mg/L) is not shown
due to scale.................................................................................................50
Figure 2.8 Median LC50 (mg/L) of water treatment cationic polyelectrolytes by use as
coagulants or flocculants based on the data in Lyons and Vaconcellos
(1997).........................................................................................................50
Figure 3.1 Structure of the metachromatic dye o-toluidine blue, a commonly used
indicator in metachromatic polyelectrolyte titration..................................59
Figure 3.2 A sample plot of a spectroscopic titration of a polyquaternium (UCareTM
JR125, 6.5 mg/L) with PVSK and o-toluidine blue at wavelength 630 nm
showing breakpoint, inflection point, and colour changes. From the
beginning of the titration to the break point, the added PVSK reacts with
the polyquaternium, from the break point to the final colour change, the
PVSK reacts with the indicator o-toluidine blue and from the final colour
change, and no reactions are taking place..................................................60
Figure 3.3 The method of endpoint determination used by Mikkelsen (2003). The
absorbance is plotted against time in a controlled automatic titration where
the concentration of the PVSK in the reaction chamber is directly
proportional to the titration time. The endpoint is determined as the
intersection of two straight lines corresponding to the first and second
stages of the titration reactions. .................................................................61
Figure 3.4 Determination of endpoint using the inflection point (Hutter et al. 1991)
where the endpoint is determined as the ‘point lying midway between lines
drawn tangent to the baselines’, that is, the inflection point......................62
Figure 3.5 Determination of endpoint from relative absorbance at 550 and 635 nm
(Horn and Heuck 1983) with the endpoint determined to be the inflection
point of the metachromatic shift. ...............................................................62
Figure 3.6 The visual titration of a polyquaternium with PVSK and o-toluidine blue,
showing the initial blue colour of the solution (left) and the pink colour at
the endpoint (right). ...................................................................................69

xv
Figure 3.7 Matlab® plot of the titration of a polyquaternium with PVSK and o-
toluidine blue, showing the intersection of the fitted curves of the straight
line and four-parameter logistic model, indicating the different stages of
the titration, and showing the break point (c) and inflection point (U). .71
Figure 3.8 Example of a plot of the results of titrations of three concentrations of
UCareTM JR125 with PVSK and o-toluidine blue used to determine the
charge density of the polyquaternium from the slope of the line of best fit.
....................................................................................................................73
Figure 3.9 Comparison of the titration of two polyquaternium samples, Styleze® W-
20 (Polyquaternium-55) and UCareTM JR125 (Polyquaternium-10), (a)
alone and in the presence of sodium dodecylsulphate at (b) 1:1
stoichiometry and in excess (c) (1:4 stoichiometry). .................................74
Figure 3.10 Spectrophotometric titration of a solution containing UCareTM JR125
(Polyquaternium-10) (6.5 mg/L) at 630 nm (■, recording the loss of blue
colour) and 512 nm (▲, recording the emergence of the pink colour) and
the titration of a blank sample at 630 nm (¡)..........................................75
Figure 3.11 A plot of the spectrophotometric titration of a blank solution and three
concentrations of Conditioneze® NT-20 with PVSK and o-toluidine blue
at 630 nm..................................................................................................75
Figure 4.1 Plot of particle size analysis of humic acid samples after separation by
centrifugation of coloured fraction (top) from the fraction used for the
partition experiment (bottom) showing the different size distribution of the
two samples................................................................................................91
Figure 4.2 Plot of Equation 4.2 as used in determining KD, in this case for
Conditioneze® W-20 (Polyquaternium-55)...............................................92
Figure 4.3 Plot of KD against charge density for polyquaterniums on which the
regression analysis is based. The data is also presented in tabular form in
Table 4.7. ...................................................................................................94
Figure 4.4 Conceptual model of the ‘box’ structure of the Oxley WWTP in SE
Queensland, showing the possible chemical fates, volatilization,
biotransformation and sedimentation in the three stages of the treatment
process. The numbered arrows represent the fluxes in Equations 4.11-4.13.
....................................................................................................................99
Figure 4.5 Diagram of water balance for Oxley WWTP, assuming 65% solids removal
in primary settling tank (PST)..................................................................100
Figure 4.6 Diagram of solids balance for Oxley WWTP, assuming 65% solids
removal in primary settling tank (PST). ..................................................100
Figure 4.7 Plot of 1/KD vs 1/p for values of p up to the total solids removal for the
WWTP showing linear relationship between these parameters...............104
Figure 4.8. Overall fraction of polyquaternium removed as a function of the partition
coefficient KD. Plot of removal fraction for varying solids removal rates.
..................................................................................................................107
Figure 5.1 The small dam on a private property in the Beenleigh area in SE
Queensland where the Gambusia used in the toxicity testing were caught.
The dam collected runoff from an area that is largely rural (hobby farms).
..................................................................................................................117
Figure 5.2 Gambusia in the polypropylene containers set up for a range-finding test.
The three tests in the foreground contain humic acid. .............................118

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Figure 5.3 Typical plots of concentration vs. mortality for two polyquaterniums,
poly(DADMAC) (a) and Conditioneze® W-20 (Polyquaternium-55) (b)
demonstrating the steepness of the curve in this all-or-nothing toxicity. 124
Figure 5.4 Toxicity to Artemia (% mortality) for SDS complexed with UCareTM
JR125. The non-standard toxicity curves show a reduced mortality at
higher concentrations of the complex. .....................................................127
Figure 5.5 Toxicity (% mortality) for SDS complexed with Gafquat® 734. The non-
standard toxicity curves show a reduced mortality at higher concentrations
of the complex..........................................................................................128
Figure 5.6 Plot of EC50 for fish in mass units (mg/L) against charge density (eq/g)
for eight cosmetic polyquaterniums (Gafquat® 440, 734, and HS100;
UCareTM JR125, JR30M, JR400; Styleze® W-20) and polyDADMAC.
..................................................................................................................134
Figure 5.7 Plot of EC50 for fish in equivalence units (eq/L) against charge density
(eq/g) for eight cosmetic polyquaterniums (Gafquat® 440, 734, and
HS100; UCareTM JR125, JR30M, JR400; Styleze® W-20) and
polyDADMAC.........................................................................................135
Figure 6.1 Representation of the mass balance in the Final Settling Tank of the
WWTP. ....................................................................................................149
Figure 6.2 Representation of the mass balance in the bioreactor of the WWTP. ......150
Figure 6.3 Representation of the mass balance in the Primary settling tank of the
WWTP. ....................................................................................................151
Figure 6.4 Probability density function for the input variable partition coefficient KD
used in the Monte Carlo Simulation of the ETNCaq model. ...................154
Figure 6.5 Forecast Probability Density Function (left) and sensitivity analysis (right)
for the influent flux of polyquaternium, from the Monte Carlo simulation
of the ETNCaq model. .............................................................................154
Figure 6.6 Probability density functions for variables common to all simulations of
probabilistic risk assessment (a) proportion of polyquaternium released to
sewer; (b) water use per person; (c) proportion of polyquaternium removed
in WWTP; and (d) dilution to receiving waters.......................................160
Figure 6.7 Probability density functions for input volumes for the three simulations of
the PEC (a) import volume < 1000 kg; (b) import volume < 16 tonnes; and
(c) estimated total import volume for all cosmetic polyquaterniums. .....161
Figure 6.8 Fish probability distribution function from data (a), and assumed for the
Monte Carlo Simulation (b) .....................................................................162
Figure 6.9 Probability Density Forecast (left) and sensitivity analysis (right) from a
Monte Carlo Simulation of the PEC for a polyquaternium at an import
volume of < 1tonne. .................................................................................163
Figure 6.10 Probability Density Function for the PEC input into PEC/PNEC Monte
Carlo Simulation for import volume < 1 tonne, a log normal distribution
with mean = 0.02; stddv = 0.08..............................................................163
Figure 6.11 Probability density forecast from a Monte Carlo Simulation for the
PEC/PNEC for a polyquaternium import volume of < 1 tonne .............163
Figure 6.12 Probability density forecast and sensitivity analysis from the Monte Carlo
Simulation of PEC for a polyquaternium at an import volume of 16
tonnes. ....................................................................................................164
Figure 6.13 Probability Density Function for PEC input into Monte Carlo Simulation
of the PEC/PNEC for import volume of < 16 tonnes, a log normal
distribution with mean = 0.34; stddv = 1.13. .........................................164

xvii
Figure 6.14 Probability density forecast from a Monte Carlo Simulation of
PEC/PNEC for polyquaternium at an import volume of < 16 tonnes....164
Figure 6.15 Probability density forecast (right) and sensitivity analysis (left) from a
Monte Carlo Simulation of the PEC for an estimated total cosmetic
polyquaternium import volume..............................................................165
Figure 6.16 Probability Density Function of PEC for input into PEC/PNEC Monte
Carlo Simulation for import volume for all polyquaterniums, a log normal
distribution with mean = 1.51; stddv = 1.17. .........................................165
Figure 6.17 Probability density forecast from a Monte Carlo Simulation of the
PEC/PNEC for estimated total cosmetic polyquaterniums import volume.
................................................................................................................165
Figure 6.18 Decision scheme for aquatic risk characterisation of new chemicals (ECB
2003). The fourth possibility, no further assessment if PEC/PNEC is < 1
is not shown on the diagram. .................................................................168

xviii
 Abbreviations
ABS Australian Bureau of Statistics
AETAC acroyloxy ethyl trimethyl ammonium chloride (N,N-
Dimethylaminoethyl Acrylate Methyl Chloride)
AICS Australian Inventory of Chemical Substances
APVMA Australian Pesticides & Veterinary Medicines Authority
ARTG Australian Register of Therapeutic Goods
ASCC Australian Safety and Compensation Council
CFPA Cationic Flocculant Producers Association
CMC Critical Micelle Concentration
CofA Commonwealth of Australia
CSTEE Committee for Toxicology, Ecotoxicology and the Environment
CTE Central Tendency Exposure
CTFA Cosmetic Toiletry and Fragrance Association
DADMAC Diallyldimethylammonium chloride
DEH Department of Environment and Heritage (previous name of
Department of Environment and Water)
DEW Department of Environment and Water
DOC Dissolved organic carbon
EC50 Median Effective Concentration
ECB European Chemical Bureau
EEC Estimated Environmental Concentration
EINECS European Inventory of Existing Commercial Chemical Substances
ELINCS European List of Notified Chemical Substances
EPA Environmental Protection Agency
EPAR European Public Assessment Reports
EPI/DMA dimethylamine-epichlorohydrin polymer
eq equivalence
EQWT Equivalent Weight
ErC50 Median Effective Concentration (growth inhibition)
ETNC Environmental Threshold of No Concern
FATS Fish Acute Toxicity Syndrome
FDA Food and Drug Administration (USA)
FFDCA Federal Food, Drug and Cosmetic Act (USA)
FPR Full Public Report
GHS Globally Harmonised System for Classification and Labelling of
Chemicals
HVICL High Volume Industrial Chemical List
ICNA Industrial Chemicals (Notification and Assessment) Act
INCI International Nomenclature of Cosmetic Ingredients
IOMC Inter Organisational Program for Sound Management of Chemicals
LC50 Median Lethal Concentration
LOAEL Lowest Observed Adverse Effect Level
LOEC Lowest Observed Effect Concentration
LRCC Low Regulatory Concern Chemical
MAPTAC methacrylamido propyl trimethyl ammonium chloride (3-
Trimethylammonium propyl methacrylamide chloride)
METAC methacroyloxy ethyl trimethyl ammonium chloride
MF Melamine-formaldehyde

xix
Mn Number Average Molecular Weight
MOA Mode of Action
MOU Memorandum of Understanding
MSDS Material Safety Data Sheet
N Normality
NAMW Number Average Molecular Weight
NChEM National Framework for Chemicals Environmental Management
NICNAS National Industrial Chemical Notification and Assessment Scheme
NLM National Library of Medicine
NOAEL No Observed Adverse Effect Level
NOEC No Observed Effect Concentration
NOEL No Observed Effect Level
NOHSC National Occupational Health and Safety Commission
NRC National Research Council
OCDD octochlorodibenzodioxin
OECD Organisation for Economic Cooperation and Development
OHS Occupational Health and Safety
OPPT Office of Pollution Prevention and Toxics (USA)
PAA Polyacrylic acid
PDAM poly(N,N,-dimethylaminoethyl methacrylate)
pdf Probability Distribution Function
PEC Predicted Environmental Concentration
PI Polydispersity Index
PLC Polymer of Low Concern
PMN Premanufacture Notice
PNEC Predicted No Effect Concentration
POP Persistent Organic Pollutant
PRA Probabilistic Risk Assessment
PSC Polyelectrolyte Surfactant Complex
PST Primary Settling Tank
PVSK Poly(vinyl sulphate), potassium salt
QSAR Quantitative Structure-Activity Relationships
REACH Registration, Evaluation and Authorisation of Chemicals
RME Reasonable Maximum Exposure
SAR Structure Activity Relationship
SDS Sodium dodecyl sulphate
SOCMA Specialty Organic Chemicals Manufacturers Association
SUSDP Standard for Uniform Scheduling of Drugs and Poisons
TGA Therapeutic Goods Administration
TL50 Median Tolerance Level
TOC Total Organic Carbon
TSCA Toxic Substances Control Act
TTC Threshold of Toxicological Concern
UNCED United Nations Conference on Environment and Development
USC United States Code
USEPA United States Environment Protection Agency
WAMW Weight Average Molecular Weight
WAS Waste Activated Sludge
WWTP Wastewater Treatment Plant

xx
 Definitions
Amphoteric Surfactant species that can be either cationic or anionic
surfactants depending on the pH of the solution, including also those which
are zwitterionic (possessing permanent charges of each type)
(Myers 1999).
Anionic Surfactants that carry a negative charge on the active portion of
surfactants the molecule (Myers 1999).
Antistatic agents Substances which are added to cosmetic products to reduce
static electricity by neutralising electrical charge on a surface
(Europa 1996).
Assessment factors Assessment factors were developed for the process of reviewing
premanufacture notices and are applied to acute toxicity values,
and take into account the uncertainties due to such variables as
test species’ sensitivities to acute and chronic exposures,
laboratory test conditions, and age-group susceptibility
(Bascietto 1990).
Bioconcentration An initial measure of the potential for accumulation of chemical
residues in the food chain (Jop 1997).
Biodegradability A measure of the ability of a chemical to be degraded to simpler
molecular fragments by the action of biological processes,
especially by the bacterial processes present in wastewater
treatment plants, the soil, and general surface water systems
(Myers 1999).
Biodegradation The removal or destruction of chemical compounds through the
biological action of living organisms (Myers 1999).
Biopolymers Polymers directly produced by living or once-living cells or
cellular components, or synthetic equivalents of such polymers,
or derivatives or modifications of such polymers in which the
original polymer remains substantially intact (CofA 1989)
Cationic Surfactants carrying a positive charge on the active portion of
surfactants the molecule (Myers 1999).
Cellulosic polymer A polymer having a backbone composed of cellulose.
Central Tendency A risk descriptor representing the average or typical individual
Exposure in a population, usually considered to be the mean or median of
the distribution.
Charge Density Proportional weight of cationic (e.g. quaternary ammonium) or
anionic (e.g. carboxylate) fragments in the polymer chain (Doi
1997).
Clarification The removal of small amounts of fine (2-100 µm) particulates
from liquids (Parke 2003).
Coacervate Complex phase formed by a polyelectrolyte in the presence of
an oppositely charged surfactant (Gruber 1999).
Coagulation The neutralisation of the charges on colloidal matter (Kemmer
1987).

xxi
Coalescence The irreversible union of two or more drops (emulsion) or
particles (dispersions) to produce a larger unit of lower
interfacial area (Myers 1999).
Colloid A system consisting of one substance, the dispersed phase (gas,
liquid or solid), finely divided and distributed evenly throughout
a second substance, the dispersion medium of continuous phase
(gas, liquid or solid) (Myers 1999).
Copolymer A polymer synthesised from two or more distinct monomers.
Counter ion The (generally) non-surface active portion of an ionic surfactant
species necessary for maintaining electrical neutrality (Myers
1999).
de minimis A level of risk to small to be concerned about. From de minimis
non curat lex, the law is not concerned with insignificant
matters.
Desorption The reverse process of sorption (Doi 1997).
dilution deposition The deposition of the conditioning agent on the skin or hair
during the rinsing (Gruber 1999).
Dispersion The distribution of finely divided solid particles in a liquid phase
to produce a system of very high solid/liquid interfacial area
(Myers 1999).
Dissociation Measure of the degree of ionisation of a polymer, which varies
Constant(s) with the pH of the solution (Doi 1997).
EC50 ‘The concentration that immobilises, inhibits growth or causes
other sub-lethal effects in 50% of test organisms . . . Used as and
effect endpoint in tests with fish, invertebrates and algae.’ (Jop
1997).
Ecological risk The component of Environmental risk assessment which is
assessment concerned with the effects of chemicals on non-human
populations, communities and ecosystems (Maltby 2006).
Ecotoxicology ‘… the science of assessing the effects of toxic substances on
ecosystems, with the goal of protecting entire ecosystems and
not merely isolated components.’ (Jop 1997).
Environmental The analysis of information on the environmental fate and
Risk Assessment behaviour of chemicals in the environment (Maltby 2006)
Emollients Substances which are added to cosmetic products to soften and
smoothen the skin (Europa 1996).
Emulsifying Surfactants or other materials added in small quantities to a
agents mixture of two immiscible liquids for the purpose of aiding in
the formation and stabilisation of an emulsion.
Emulsion A colloidal suspension of one liquid in another (Myers 1999).

xxii
Fatty acids A general term for the groups of saturated and unsaturated
monobasic aliphatic carboxylic acids with hydrocarbon chains
of 6-20 carbons, the name deriving from the original source of
such materials, namely animal and vegetable fats and oils
(Myers 1999).
Film Formers Substances which are added to cosmetic products to produce,
upon application, a continuous film on skin, hair or nails
(Europa 1996).
Flocculation The process of agglomerating coagulated particles into settleable
flocs, usually of a gelatinous nature (Kemmer 1987).
gram-equivalents The molar mass of a substance divided by the number of charges
of the same sign carried by the ions released by a molecule of
that substance in an aqueous solution is the gram-equivalent of
the substance (Dregrémont 1991).
Grandfathered Chemicals added to AICS at the time it was established, and are
chemicals exempt from the provisions of ICNA.
Head group A term referring to the portion of a surfactant molecule that
(surfactant) imparts solubility to the molecule. Generally used in the context
of water solubility (Myers 1999).
homopolymer A polymer synthesised from one monomer only.
Hydrolysis Reaction of a polymer (RX) with water (HOH), with the
resultant net exchange of a group (X) from the polymer for the
OH group from water at the reaction centre as shown: RX +
HOH ---> ROH + HX (Doi 1997).
Hydrophilic A descriptive term indicating the tendency on the part of a
(‘water loving’) species to interact strongly with water (Myers 1999).
Hydrophobic The opposite of hydrophilic, having little energetically
(‘water hating’) favourable interaction with water (Myers 1999).
Interface The boundary between two immiscible phases. The phases may
be solids, liquids or vapours, although there cannot be an
interface between two vapour phases (Myers 1999).
Isoelectric point The pH value of the dispersion medium of a colloidal
suspension at which the colloidal particles do not move in an
electric field (McGraw-Hill 2003).
LC50 The median effective concentration that is lethal to 50% of a test
population (Jop 1997).
LOEC Lowest Observed Effect Concentration. The lowest
concentration that has a statistically significant adverse effect on
the test organisms compared to control organisms (Jop 1997).
Macromolecule Very large molecules, including natural and synthetic polymers,
proteins, and biomolecules such as nucleic acids, proteins and
carbohydrates.
Median Tolerance The concentration at which 50% of test animals were able to
Level survive for a specified period of exposure.

xxiii
Metachromasy Metachromasy is the hypsochromic (shift in absorption to
shorter wavelength) and hypochromic (decrease in intensity of
colour) exhibited by certain basic aniline dyes in the presence of
water and under the following conditions: Increase in dye
concentration; temperature decrease; salting out; interaction with
certain substances whose metachromatic influence may be due
to serially arranged proximate anionic sites (Bergeron and
Singer. 1958).
Micelles Aggregated units composed of a number of molecules of a
surface active material, formed as a result of the
thermodynamics of the interactions between the solvent (usually
water) and the lyophobic (or hydrophobic) portions of the
molecule (Myers 1999).
monomer A molecule which is capable of combining with like or unlike
molecules to form a polymer; the repeating structure within a
polymer (McGraw-Hill 2003).
Monte Carlo A technique for characterising the uncertainty and variability in
Simulation risk estimates by repeatedly sampling the probability
distributions of the risk equation inputs and using these inputs to
calculate a range of risk values (USEPA 2001).
natural polymer A polymer having a polymer backbone of a natural material
such as cellulose, guar, or chitin.
NOAEC No Observed Adverse Effect Concentration. An endpoint used
in partial or full life-cycle tests for chronic toxicity that is the
highest concentration with no adverse effects when compared to
control animals.
NOEC No Observed Effect Concentration. An endpoint use in partial or
full life-cycle tests for chronic toxicity that is the highest
concentration that has no statistically significant effect on the
test organisms compared to the control organisms (Jop 1997).
Non-ionic Surfactants that carry no electrical charge, their water solubility
surfactants being derived from the presence of polar functionalities capable
of significant hydrogen bonding interaction with water, e.g.
polyoxyethylenes and polyglycidols (Myers 1999).
Number average The total weight of all the molecules in a polymer sample
molecular weight divided by the total number of moles present (Doi 1997).
Octanol/water Ratio of the concentration of any single molecular species in two
partition phases, n-octanol and water, when the phases are in equilibrium
coefficient with one another and the substance is in dilute solution in both
phases (Doi 1997).
oligomer A very low molecular weight polymer, usually with a degree of
polymerisation of 10 or less (Winnik 1999).
orthochromasy The absence of colour change when an aniline dye is in solution
or bound to a matrix (Bergeron and Singer 1958).

xxiv
Parameter A value that characterises the distribution of a random variable.
Parameters commonly characterise the location, scale, shape, or
bounds of the distribution. For example, a truncated normal
probability distribution may be defined by four parameters:
arithmetic mean [location], standard deviation [scale], and
minimum and maximum. It is important to distinguish between a
variable (e.g., ingestion rate) and a parameter (e.g. arithmetic
mean ingestion rate) (USEPA 2001).
Personal Care Cosmetics and toiletries that are substances or preparations used
Products externally on the body (including the oral cavity) for the purpose
of cleansing, perfuming, protection, or changing appearance
(CofA 1989).
Point estimate In statistical theory, a quantity calculated from values in a
sample to estimate a fixed but unknown population parameter.
Point estimates typically represent a central tendency or upper
bound estimate of variability (USEPA 2001).
Point Estimate A risk assessment in which a point estimate of risk is calculated
Risk Assessment from a set of point estimates for exposure and toxicity. Such
point estimates of risk can reflect the RME, or bounding risk
estimate depending on the choice of inputs
Polydispersity The breadth of the distribution of molecular weights in a
Index polymer (Mw/Mn) (Doi 1997).
polymer A chain of organic molecules produced by the joining of
primary units called monomers (Kemmer 1987).
Probabilistic Risk A risk assessment that yields a probability distribution for risk,
Assessment generally by assigning a probability distribution to represent
variability or uncertainty in one or more inputs to the risk
equation.
Probability A function representing the probability distribution of a
Density Function continuous random variable. The density at a point refers to the
probability that the variable will have a value in a narrow range
about that point.
Reasonable The highest exposure that is reasonably expected to occur at a
Maximum site (USEPA, 1989a). The intent of the RME is to estimate a
Exposure (RME) conservative exposure case (that is, well above the average case)
that is still within the range of possible exposures.
Risk Quotient The ratio of the Predicted Environmental Concentration and the
Predicted No Effect Concentration, sometimes also called
Hazard Quotient.
Safety Factor A safety factor is generally a margin of safety applied to a No
Observed effect Concentration to produce a value below which
exposures are assumed to be safe (Bascietto 1990).
Sorption The adhesion of molecules to surfaces of solid bodies with
which they are in contact (Doi 1997).

xxv
Substantivity The affinity of a compound for a given substrate. In cosmetics,
the affinity of the conditioning agent for skin or hair.
Surface active The descriptive generic term for materials that preferentially
agent adsorb at interfaces as a result of the presence of both lyophobic
and lyophilic structural units, the adsorption generally resulting
in the alteration of the surface or interfacial properties of the
system (Myers 1999).
Surface tension The property of a liquid evidenced by the apparent presence of
thin elastic membrane along the interface between the liquid and
vapour phase, resulting in the contraction of the interface and
the reduction of the total interfacial area. Thermodynamically,
the surface excess free energy per unit area of interface resulting
from an imbalance in the cohesion forces acting on liquid
molecules at the surface (Myers 1999).
Surfactant tail In surfactant science, usually used in reference to the
hydrophobic portion of the surfactant molecule (Myers 1999).
Surfactants Contraction for ‘surface active agents’. Substances which are
added to cosmetic products to lower the surface tension as well
as to aid the even distribution of the cosmetic product, when
used (CofA 1989).
synthetic polymer A synthetic polymer that is not a natural polymer, i.e. does not
have a backbone composed of a natural material.
Vapour pressure The force per unit area exerted by a gas in equilibrium with its
liquid or solid phase at a specific temperature. It can be thought
of as the solubility of a substance in air and is dependent on the
nature of the compound and the temperature (Doi 1997).
Water solubility The maximum amount of a polymer in solution and at
equilibrium with excess compound in the water at specific
environmental conditions (that is temperature, atmospheric
pressure and pH) (Doi 1997).
Weight average The mean of the weight distribution of molecular weights (Doi
molecular weight 1997).
Zeta potential The difference in voltage between the surface of a diffuse layer
surrounding a colloid particle and the bulk liquid beyond
(Kemmer 1987).

xxvi
1. Introduction
The environmental impact of pharmaceuticals and personal care products (PPCPs)
has, until recently, been a neglected area of research (Daughton and Ternes 1999).
Pharmaceuticals, including over-the-counter and prescription medications, and
complementary medicines, are chemicals designed to stimulate and inhibit
physiological responses in humans (Breton and Boxall 2003). Personal care products
(PCPs) (cosmetics and toiletries) are substances or preparations used externally on the
body (including the oral cavity) for the purpose of cleansing, perfuming, protection, or
changing appearance (CofA 1989; USC 2004). The consumption of these chemicals,
according to Daughton and Ternes (1999), may be on a par with the consumption of
the more highly regulated agricultural and veterinary chemicals.

In Australian in 1996-97, A$1,200 million was spent on non-aerosol cleaning

products, and A$906 million on personal hygiene products. In the same period, A$116
million worth of insecticides was consumed, part of a total agricultural and veterinary
expenditure on chemicals of A$1,662 million (DEH 1998). These figures include
veterinary medicines, but do not include pharmaceuticals for human consumption. It
is clear, therefore, that the consumption of household and PCPs is greater, in
monetary terms, than that of agricultural and veterinary chemicals in Australia.

More recently, effort has been focused on assessing the impacts of pharmaceuticals on
aquatic environments. Because their purpose is to stimulate and inhibit physiological
responses in humans (or animals), pharmaceuticals may have unforseen adverse
effects on non-target species when released into the environment (Breton and Boxall
2003). For chemical constituents in PCPs which do not have intended physiological
effects, however, even less is known about their effects on non-target species
(Daughton and Ternes 1999). One such class of chemicals that has ‘desirable’ effects
when used in cosmetic applications, but may have adverse effects on aquatic
organisms, is the polymeric quaternary ammonium salts.

Polymeric quaternary ammonium salts (polyquaterniums) are a class of polymer with

a wide variety of uses. These uses fall into two categories; commercial/industrial
flocculation and clarification, and use in cosmetics. In cosmetics their application is
generally described as ‘film formers and antistatic agents’ (Europa 1996). In the
language of the cosmetic industry, polyquaterniums are ‘substantive’ to skin and hair,

1
that is, they will sorb to proteinaceous surfaces. This is a very desirable property
providing they also impart cosmetic effects in terms of ‘feel’. This characteristic of
conditioning is subjectively reported by test subjects, and comprises one of four
measures used by cosmetic companies in assessing conditioning of hair. The others
are wet combability, dry combability and curl retention.

Because skin and hair are negatively charged at their surface, ‘conditioning’ has
become synonymous with cationic adsorption agents. Cleaning formulations, such as
shampoos and body scrubs, normally employ anionic (sometimes non-ionic)
surfactants. Without a conditioning agent, these formulations can cause fibre friction,
dryness and the ‘fly-away’ effect of electrostatic charge, conditions generally
regarded as sub-optimal by the cosmetic industry (Goddard 1999).

To overcome these apparent negative effects of anionic cleansing agents, cationic

agents, such as surfactants, but particularly polyelectrolytes, are used in the cosmetic
industry as hair and skin conditioners. The high adsorption efficiency of cationic
polyelectrolytes and the inherent properties of their adsorbed layers make them
particularly suitable as conditioning agents. Cationic polyelectrolytes have a further
advantage in that they can function as conditioners in formulations also containing the
anionic surfactants that would neutralise, and be neutralised by, a cationic surfactant
(Goddard 1999).

The combining of cationic polyelectrolytes and anionic surfactants in formulations is

one of two technologies that led to the development of the 2-in-1 shampoo
conditioner. This was developed and commercialised in the late 1960s and early
1970s, and is still employed. The alternative method involves a micro-suspension of
silicone, and essentially works in the same manner with the anionic surfactant (Burke
2005). Both involve the deposition of the conditioning agent, polyelectrolyte or
silicone, on the hair as the surfactant is rinsed away, a process known as ‘dilution
deposition’ (Gruber 1999).

At the present time all the major manufacturers produce ‘2-in-1’ formulations, and
over 20% of the shampoos sold are of this type (Gray 2003). Paradoxically, the advent
of the 2-in-1 conditioning shampoo has led to an increase in the number of people
using separate step conditioners (Burke 2005). Most shampoos now available have
some conditioning function, and cationic polyelectrolytes are found in a wide variety

2
of cosmetic products with or without anionic surfactants present – for example,
cleansing agents such as shampoos, liquid hand soaps, body scrubs and facial
cleansers, conditioning agents including hair conditioners, moisturisers and hand and
body crèmes, hair styling agents like gels, hairsprays and mousses, and even hair dyes
(NLM 2004).

Polyquaterniums have no known systemic toxicity to mammals (Hamilton et al.

1997), though some may be minor skin irritants. Although of concern in occupational
situations, irritation is unlikely to occur at cosmetic concentrations. No harmful
effects to human health are expected from the environmental release of these
polymers. Polyquaterniums, however, like other quaternary ammonium species, are
known to exhibit toxicity to aquatic organisms.

1.1. Non-cosmetic Uses of Polyquaterniums

Cationic polymers are also used extensively in industry, for processes including oil-
water separation, corrosion control, flocculation of iron ore slimes, improvement of
lime precipitation processes, clarification of titanium sulphate liquor, removal of
heavy metals, killing viruses, industrial wastewater treatment (Wang and Kao 1978).
They are particularly useful in water treatment applications because colloidal particles
in natural waters and wastewater are generally negatively charged. In sewage
treatment, they are used for flocculation, clarification and dewatering in the treatment
of effluent, often in conjunction with more conventional flocculants such as alum or
ferric chloride.

Polyelectrolytes used in these industrial applications are designed to sorb to colloidal

matter to produce a neutral precipitate. The sorption is thus expected to be
irreversible, and the polyelectrolyte removed from the water column in the process.
The dilution deposition of cosmetic polyquaterniums, on the other hand, is intended to
be reversible, and the polyquaternium is removed from the hair or skin with
subsequent washing.

Industrial applications have, or should have, zero release to waterways.

Environmental controls on these industries usually specify disposal of solid waste to
landfill or incineration, with either no release of water, or release of water only after
stringent treatment. In water treatment applications, the dosing with the polymer is
usually controlled, as overdosing can result in resuspension of the floc. Cosmetic uses,

3
in contrast, generally result in 100% release of the cosmetic ingredients (less any,
usually small fraction, that may be adsorbed through or degraded on human skin) via
wastewater treatment plants (WWTPs).

The fate of polymers used in water treatment has been fairly extensively studied. The
research has largely been driven by the requirements of the US Office of Pollution
Prevention and Toxics (OPPT), which administers the Toxic Substances Control Act
(TSCA) (USC 1976). Cosmetic ingredients in the US are regulated by the US Food
and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act
(FFDCA) (USC 2004), which has no provision for environmental risk assessment. As
a result of the differences between the regulatory schemes (Table 1.1), the
environmental fate of cosmetic polymers has not been as extensively studied.

4
Table 1.1 Comparison of the regulatory schemes for industrial chemicals and therapeutics in Australia, USA and the European Union.
Industrial Chemicals Agency Legislation Inventory Scope
Australia National Industrial Industrial Chemicals (Notification Australian Inventory Excludes articles, radioactive
Chemical Notification and and Assessment) Act 1989 (ICNA) of Chemical chemicals, medicines,
Assessment Scheme Substances (AICS) pesticides, veterinary
(NICNAS) chemicals, food or food
additives
USA Office of Pollution Toxic Substances Control Act Chemical Substances Excludes tobacco and certain
Prevention and Toxics 1976 (TSCA) Inventory (TSCA tobacco products, nuclear
(OPPT), Environment Inventory) materials, munitions, foods,
Protection Agency (EPA) food additives, drugs,
cosmetics, and substances
used solely as pesticides
Europe European Chemical Various EC regulations including European Inventory Exemption categories
Bureau 1967L0548EC, 1993R0793EC, of Existing include consumer products
1994R1488EC, 2006R1907EC Commercial pertaining to pharmaceutics,
Chemical Substances cosmetics and foodstuffs.
(EINECS), and The Directive is not
European List of applicable to pesticides,
Notified Chemical radioactive materials, wastes,
Substances and substances used in
(ELINCS) scientific research
Therapeutics Agency Legislation Inventory
Australia Therapeutic Goods Therapeutic Goods Act 1989 Australian Register Includes pharmaceuticals,
Administration of Therapeutic Goods over-the-counter
(ARTG) preparations and
complementary medicines
USA Federal Food and Drug Federal Food, Drug and Cosmetics none
Administration (FDA) Act (1938) (FFDA)

5
Europe European Agency for the Various Directives including On-line listing of
Evaluation of Medicines 67/548/EEC and 79/831/EEC) European Public
Assessment Reports
(EPAR)
Directorate General Cosmetics Directive 76/768/EEC
Enterprise and Industry
International
Nomenclature of
Cosmetic Ingredients
(INCI)

6
 1.2. Regulation of Chemicals in Australia
In Australia, there are four agencies responsible for the regulation of chemicals. The
responsibilities of each agency are outlined in Table 1.2. The regulation of industrial
chemicals, including cosmetics, is the responsibility of the National Industrial
Chemical Notification and Assessment Scheme (NICNAS), a statutory scheme which
is part of the Therapeutic Goods Administration (TGA) in the portfolio of the Minister
for Health and Ageing. NICNAS undertakes risk assessment in the areas of
Occupational Health and Safety (OHS), public health and environment. The
environmental assessment, generally carried out by the Department of Environment
and Water Resources (DEW), deals only with the non-human environment. Human
health impacts resulting from environmental exposure form part of the public health
assessment. NICNAS maintains an inventory of industrial chemicals approved for use
in Australia, called the Australian Inventory of Chemical Substances (AICS). The
inventory is used to distinguish between new and existing chemicals. A new chemical
is any chemical that is not on the AICS. Companies wishing to import or manufacture
any new chemical (known as ‘notifiers’ in the Industrial Chemicals (Notification and
Assessment) Act (ICNA)) must apply to NICNAS for a certificate or permit before
importing the chemical. Depending on the type of chemical and/or the volume to be
introduced, an assessment process is conducted and an assessment report published.
Existing chemicals include those that were in use in Australia between 1 January 1977
and 28 February 1990, and therefore placed on the inventory when it was established.
These chemicals are sometimes referred to as ‘grandfathered’ chemicals. In addition,
new chemicals are added to AICS after assessment. There are currently over 38,000
chemicals on AICS. All grandfathered chemicals, chemicals which have been
imported under certificate for five years, and chemicals where the notifier requests
early AICS listing comprise AICS. A small proportion of AICS chemicals are held on
a confidential section. Chemicals are introduced under an assessment certificate for
the first five years, unless the notifier requests early AICS listing. These chemicals do
not form part of AICS and are not able to be imported by other companies without
separate notification.

7
Table 1.2 Key elements of the regulatory and management structure of chemicals in Australia (adapted from DEH 1998).
Element Industrial Chemicals Agricultural and Veterinary Pharmaceuticals Food Additive
Products
Scheme responsible for National Industrial Chemicals Australian Pesticides & Therapeutic Goods Food Standards Australia
assessment Notification and Assessment Veterinary Medicines Administration (TGA) New Zealand (FSANZ)
Scheme (NICNAS) Authority (APVMA)
Ministry Health and Ageing Agriculture, Fisheries and Health and Ageing Health and Ageing
Forestry
Assessment and/or All new chemicals are assessed All new products and new All new therapeutic goods Stipulates food standards to
registration before use. Existing chemicals uses of products must be must be registered on the protect public health.
are reviewed on a priority basis. assessed and registered Australian Register of
before use. Existing Therapeutic Goods.
chemicals and products may
be reviewed.
Scope and definition Assessment of a chemical entity Registration of products Assessment and registration Control of contaminants and
(not product) – any chemical includes: an agricultural of therapeutic goods food additives that are
that has an industrial use may product used to stupefy, added to food to assist in
be included i.e., dyes, solvents, repel, inhibit the feeding of food processing or to
adhesives, plastics, laboratory or prevent, pests on plants achieve a technological
chemicals, paints, cleaning or other things; destroy a purpose in the food, for
products, cosmetics and plant or modify physiology; example, colouring or
toiletries. Excludes articles, or attract a pest to destroy it. flavouring.
radioactive chemicals or Veterinary product includes
chemicals solely in other a substance for preventing,
schemes. diagnosing curing or
alleviating disease in
animals. Excludes fertilisers
Controls of use If not on AICS (or has an If not registered may not be If not registered, may not be Food Standards Code
assessment certificate or permit used; registration may used. Licensing of (A14); control permissible
issued) may not be used specify how used; Manufacturer; Standard for additives, preservatives and
commercially; may be removed registration can be Uniform Scheduling of Drugs colours; application of
from AICS. Application of cancelled; use controlled by and Poisons (SUSDP); Maximum Residue Limits.

8
 assessment report State and Territory by controls use with Poison
recommendations by legislation application of registration Schedule Classification
at State and Territory level advice. (Labels must be
through adoption of NOHSC complied with)
National Model Regulations for
Hazardous Workplaces (OHS
only)
Supporting legislation Industrial Chemicals Agricultural & Veterinary Therapeutic Goods Act 1989, Food, Stock and Medicines
(Notification and Assessment) Chemicals; (Code) Act 1994 Poisons Act (various). Acts.
Act 1989, Various state and Administration Act
legislation on OHS and poisons 1994, State and Territory
complementary legislation
control of use legislation
including Pesticides,
Poisons & Food Acts.
Advisory products – Chemical Assessment Reports, Product Assessment SUSDP – Poison Schedule Labelling Maximum
including labels, assessment (NICNAS) Exposure Standards, Labelling Maximum Classification Residue Limits SUSDP –
reports, safe use advice Labelling, Material Safety Data Residue Limits, SUSDP – Poison Schedule
Sheets (ASCC) SUSDP Poison Schedule Classification
Classification
Assessment Assessed for risks to Assessed for risk to Assessed for quality, safety Assessed for risk to public
occupational health and safety, human health, occupational (consumer) and efficacy. No health (consumer) only. No
public health, and environment. health and safety, environmental risk environmental risk
environment and trade; and assessment. assessment.
efficacy.

9
There are at least 18 cationic polyquaterniums on AICS, and NICNAS has published
risk assessments of five cosmetic polyquaterniums under the new chemicals
notification and assessment arrangements (Table 1.3). Three of the polyquaterniums
assessed have since been added to the public section of AICS.

Table 1.3 Summary of assessment reports for polyquaterniums assessed by NICNAS as new
chemicals.
NICNAS Publication Polymer Trade Name Notifier
Report Date Identity
NA/475 Sept 1997 Polyquaternium- Polyquaternium- L'Oreal (NICNAS
34 34 Paris 1997a)
NA/533 Nov 1997 Polyquaternium- Luviquat Hold BASF (NICNAS
46 Australia 1997b)
Ltd
NA/89 Nov 1999 Polyquaternium- Gafquat® HS- ISP (NICNAS
28 100 (Australasia) 1999)
Pty Ltd
NA/961 Dec 2001 Polyquaternium- Luviquat Care BASF (NICNAS
44 Australia 2001)
Pty Ltd and
Johnson and
Johnson
Pacific Pty
Ltd
NA/896 Jan 2002 Polyquaternium- Merquat 2001 Nalco (NICNAS
47 Australia 2002a)
Pty Ltd

The environmental risk assessment in each case involved the calculation of a

predicted environmental concentration (PEC) for the polyquaternium when released
into sewers. Although the figures have been modified over the years, for example with
changing population and water use patterns, the most recent calculation, for Merquat
2001, is typical:

Amount of polymer entering sewer 16 tonnes

Population of Australia 19 million
Amount of water used per person per day 150 Litres
Estimated PEC in sewage 15.4 μg L-1
Estimated PEC in receiving waters 1.54 μg L-1
(NICNAS 2002a)
The estimated PEC in receiving waters is based on an assumption of a 1:10 dilution
without any partitioning of the polyquaternium to sewage solids, a worst case
scenario. However, it is noted in the reports that partitioning to solids is likely to
occur. Where toxicological data is provided, the Full Public Report (FPR) also

10
includes a Predicted No Effect Concentration (PNEC) based on the toxicity of the
polymer to the most sensitive aquatic species. The environmental risk is then
determined as the ratio PNEC/PEC, sometimes called the Risk Quotient or Hazard
Quotient (Q). Only Merquat 2001, with its high import volume, had a PNEC/PEC
value of less than one, indicating a possible environmental risk. However, according
to the report, the partitioning of the polyquaternium to sewage sludge was expected to
fully mitigate the risk.

The expectation of partitioning to sludge is based on references including Nabholz et

al. (1993), and Boethling and Nabholz (1997), which rely on data submitted with
Premanufacture Notices (PMN) under TSCA for their models and assessment of
polyelectrolytes in the environment. As cosmetic polymers in the United States are
not included under the TSCA, but are regulated under the Federal Food, Drug and
Cosmetic Agency, the TSCA data is based upon only water treatment and other
industrial polyelectrolytes, but not cosmetic polymers.

1.3. The Risk Assessment Framework

Regulatory risk assessment of chemicals in most jurisdictions follows the framework
developed by the United States Environment Protection Agency (USEPA) in the early
1980s. This process, known as the four-step paradigm, was first outlined by the
National Research Council (NRC) in a 1983 publication known as ‘the red book’,
(NRC 1994). The four steps of the process are:

1. Hazard Identification

2. Dose-Response Assessment

3. Exposure Assessment

4. Risk Characterisation.

Although there are many published versions of the four-step framework for risk
assessment, most are focused on human health impacts, either from direct exposure
(i.e. from food and water consumption) or indirect exposure from the environment
(i.e. air toxics, recreational water use etc). There are fewer examples of guidelines for
risk assessment of chemicals as they affect the health of the environment, or non-
human species. From the basic framework, many variations of the method, and of
terminology, have developed. Much of this variation results from the purpose of the
risk assessment, for example whether the risk assessment is remedial (for

11
contaminated sites) or predictive (for new chemicals) and whether the assessment is
conducted for the purpose of protecting human health, endangered species or
ecosystems.

This study concerns the evaluation of risk from polyquaterniums, both new and
existing, in Australia, and therefore follows the framework of regulatory chemical risk
assessment. There is less variation in the assessment methodology and terminology
across jurisdictions, largely because of the cooperative arrangements that have been
developed, such as the Canada-Australia Bilateral cooperative arrangements and the
Organisation for Economic Cooperation and Development (OECD) New Chemicals
Taskforce on mutual acceptance of assessments (NICNAS 2007). Where alternative
methods and terminology are available, this study will reflect those used in NICNAS
assessment reports.

1.3.1. Hazard Identification

The first step in risk assessment is to determine if the chemical is or can be causally
linked to an adverse outcome (NRC 1994) that is, does the chemical have a known
hazard, belong to a class of compounds known to be toxic, or does the chemical have
characteristics that suggest it may be toxic. For existing chemicals, the hazard may be
identified from observed environmental effects in the field. In Australia, the chemical
can be nominated as a candidate for assessment by any person, group or organisation,
but the criteria used by NICNAS to select nominated chemicals for assessment have
not been published and are currently under review (NICNAS 2006). For new
chemicals, identification of a specific hazard is not required, although manufacturers
and importers, are required to have checked the chemical against criteria specified
under various guidelines, for example Approved Criteria for Classifying Hazardous
Substances (NOHSC 1999), and prepared appropriate hazard labelling and safety data
sheets (NICNAS 2004b). The nature of the risk assessment process is determined by
the volume of the chemical to be introduced (< or ≥ 1000 kg) and/or its classification
as a Polymer of Low Concern (PLC). Recent review and amendments of ICNA have
resulted in a new classification of Low Regulatory Concern Chemicals (LRCC),
which will enable manufacturers and importers (known as ‘notifiers’ in ICNA) to
undertake audited self-assessment of chemicals and polymers considered to be of low
hazard or low risk potential.

12
 1.3.2. Effects Assessment
The second step of the risk assessment, dose-response assessment in the red book
(NRC 1983), is generally called effects assessment in NICNAS assessment reports.
Using available toxicity data, the relationship between the exposure and the outcome
in an exposed population is predicted, generally from the No Observed Effect Level
(NOEL), or the No Observed Adverse Effect Level (NOAEL) in a laboratory study.
Safety margins can be determined from the ratio of the NOAEL and the Lowest
Observed Adverse Effect (LOAEL) (NRC 1994), or from the ratio of chronic to acute
studies (Giolando et al. 1995; Jop 1997). Other factors that may be important in
hazard assessment include the existence of a threshold level (i.e. minimum
concentration below which no effects are expected), the level of uncertainty,
reversibility of effect, interaction between species, host characteristics, reproductive
status, and population characteristics such as mobility.

1.3.3. Exposure Assessment

The extent of the exposure of a vulnerable organism to the chemical before or after
application of regulatory controls requires determination of the fate of the chemical
and exposure pathways. Factors that need to be considered include frequency and
duration of exposure, rates of uptake or contact, and rates of absorption (NRC 1994).
Methods and approaches to exposure are medium-specific (air, water, soil), and unless
measured environmental data are available, require transport and fate models (NRC
1994). The models, in turn, rely on specified physicochemical characteristics of the
chemical, such as solubility, vapour pressure, and partition coefficients. Other
important factors in assessing exposure include release patterns, cumulative versus
non-cumulative exposure, persistence, failure of exposure controls, quality of data and
quality of models (EnHealth 2002).

1.3.4. Risk Characterisation

The final stage in the risk assessment integrates the information from effects and
exposure assessment (EnHealth 2002) to provide a description of the nature and
magnitude of the risk, including uncertainty. The outcome of the risk assessment can
be qualitative or quantitative. Qualitative risk assessments provide a descriptive
indication of risk (e.g. high, medium, low), usually determined against a pre-existing
set of criteria or guidelines developed for that purpose (NRC 1994). A quantitative
risk assessment, on the other hand, attempts to put a numeric value to the risk, for
example lifetime and population risks that have been determined for some

13
carcinogens. The use of numerical methods in the risk assessment does not, however,
indicate that the outcome will be a quantitative risk assessment, that is a measure of
the risk. For example, the hazard index used for non-carcinogens in humans is a
benchmark used in the estimation of risk, but is not a quantitative measure of risk
(NRC 1994). While weighing the risks against other societal costs and benefits is
often considered an important part of risk assessment, this is not a part of the risk
assessment of industrial chemicals in Australia. Each chemical is assessed on the basis
of its hazard and exposure only. Positive benefits, including the replacement of a more
hazardous chemical, are not considered. The determination of labelling classifications
and recommendations for control are part of the NICNAS risk assessment process. It
should be noted, however, that labelling classifications used in Australia (and
globally) are hazard based, not risk based.

1.3.5. Assessment Reports

Unlike in the USA and Canada where the assessment reports remain confidential,
reports of new and existing chemicals assessed by NICNAS are published and freely
available. Although draft guidelines on the assessment process as it is applied have
only recently been published by DEW (Lee-Steere 2007), it can be ascertained from
the published reports that the four-step risk assessment process is used. An assessment
report contains the information on which the assessment is based, under the headings
Process and Release Information, Physical and Chemical Properties and Toxicological
Investigations. The following section of the report, Risk Assessment, is divided into
OHS, public health, and environment, and each of these is further divided into
exposure assessment, effects assessment and risk characterisation. The final section
contains the risk assessment for each category, plus the recommendations for hazard
classification, labelling, and the Material Safety Data Sheet (MSDS). In the FPR,
which is available on the NICNAS website, the data that the notifier has requested be
kept confidential is excluded. If no request is made to keep data confidential, the
Assessment Report and the FPR will be identical.

1.3.6. Data Requirements

In chemical assessment, the data that needs to be made available for the risk
assessment is usually determined by the legislation and/or regulations, and may
depend on the type of chemical or polymer and the volume to be manufactured or
imported. The data requirements for new chemical assessment under ICNA are

14
detailed in the Handbook for Notifiers (NICNAS 2004b). The requirements for
existing chemicals are set on a case by case basis by NICNAS. There is provision for
the regulator to request for more data if there is a valid reason e.g. suspected
Persistent Organic Pollutant (POP). In Australia, the ICNA provides that a notifier
must supply all data available to them regardless of whether it is specifically required
by the Act (NICNAS 2004b).

1.3.7. Globally Harmonised Labelling

Although the regulatory assessment of chemicals is risk based, the labelling of
chemicals and the communication of chemical safety information is generally hazard
based. The need for adequate labelling of chemicals and dissemination of chemical
safety information was raised as part of United Nations Conference on Environment
and Development (UNCED) (1992) Agenda 21 with the aim that a ‘globally
harmonised hazard classification and compatible labelling system, including material
safety data sheets and easily understandable symbols, should be available, if feasible,
by the year 2000’. Oversight of the program to develop the Globally Harmonised
System for Classification and Labelling of Chemicals (GHS) eventually became the
responsibility of Inter Organisational Program for the Sound Management of
Chemicals (IOMC). According to the terms of reference, the goals of the program
were to:

a) enhance the protection of people and the environment by providing an

internationally comprehensible system for hazard communication;

b) provide a recognised framework for those countries without an existing

system;

c) reduce the need for testing and evaluation of chemicals;

d) facilitate international trade in chemicals whose hazards have been properly

assessed and identified on an international basis (OECD 2001).

The classification of chemicals in accordance with GHS became part of the NICNAS
assessment process, in preparation for the Australian Government implementation of
the classification and labelling system (NICNAS 2005). Previously, industrial
chemicals were labelled for occupational health and safety concerns according to
guidelines published by the National Occupational Health and Safety Commission
(NOHSC), for public safety according to the Poisons Schedule, and according to the

15
requirements of the Dangerous Goods Act for transport and storage purposes, but
there was no system for the classification and labelling of environmentally hazardous
chemicals.

1.3.8. GHS and the Aquatic Environment

The classification of hazards to aquatic environments under GHS is based on the
impacts of the chemical on aquatic organism and the ecosystems which they inhabit,
and not in terms of public health impacts (OECD 2001). The basic data elements used
in the classification of environmental hazard under the GHS are:

e) acute aquatic toxicity;

f) potential for or actual bioaccumulation;

g) degradation (biotic or abiotic) for organic chemicals;

h) chronic aquatic toxicity.

The hazard classifications for the aquatic environment under the GHS are given in
Table 1.4.

16
Table 1.4 Hazard classifications for aquatic toxicity under Globally Harmonised System for
Classification and Labelling of Chemicals (GHS) (OECD 2001).
Category: Acute I
Acute toxicity:
96 hr Median Lethal Concentration (LC50) (for fish) ≤ 1 mg/L and/or
48 hr Median Effective Concentration (EC50) (for crustacea) ≤ 1 mg/L and/or
72 or 96 hr Median Effective Concentration – growth inhibition ( ErC50)
(for algae or other aquatic plants) ≤ 1 mg/L.
Category: Acute I may be subdivided for some regulatory systems to include a lower band
at L(E)C50 ≤ 0.1 mg/L.
Category: Acute II
Acute toxicity:
96 hr LC50 (for fish) > 1 - ≤ 10 mg/L and/or
48 hr EC50 (for crustacea) > 1 - ≤ 10 mg/L and/or
72 or 96 hr ErC50 (for algae or other aquatic plants) > 1 - ≤ 10 mg/L.
Category: Acute III
Acute toxicity:
96 hr LC50 (for fish) > 10 - ≤ 100 mg/L and/or
48 hr EC50 (for crustacea) > 10 - ≤ 100 mg/L and/or
72 or 96 hr ErC50 (for algae or other aquatic plants) > 10 - ≤ 100 mg/L.
Some regulatory systems may extend this range beyond an L(E)C50 of 100 mg/L through
the introduction of another category.
Category: Chronic I
Acute toxicity:
96 hr LC50 (for fish) ≤ 1 mg/L and/or
48 hr EC50 (for crustacea) ≤ 1 mg/L and/or
72 or 96 hr ErC50 (for algae or other aquatic plants) ≤ 1 mg/L
and the substance is not rapidly degradable and/or the log KOW ≥ 4 (unless the
experimentally determined BCF < 500).
Category: Chronic II
Acute toxicity
96 hr LC50 (for fish) > 1 to ≤ 10 mg/L and/or
48 hr EC50 (for crustacea) > 1 to ≤ 10 mg/L and/or
72 or 96 hr ErC50 (for algae or other aquatic plants) > 1 to ≤ 10 mg/L
and the substance is not rapidly degradable and/or the log KOW ≥ 4 (unless the
experimentally determined BCF < 500), unless the chronic toxicity NOECs are > 1 mg/L.
Category: Chronic III
Acute toxicity:
96 hr LC50 (for fish) > 10 to ≤ 100 mg/L and/or
48 hr EC50 (for crustacea) > 10 to ≤ 100 mg/L and/or
72 or 96 hr ErC50 (for algae or other aquatic plants) > 10 to ≤ 100 mg/L
and the substance is not rapidly degradable and/or the log KOW ≥ 4 (unless the
experimentally determined BCF < 500) unless the chronic toxicity NOECs are > 1 mg/L.

1.4. Conclusion
Polyquaterniums are in use in Australia in cosmetic preparations both as
grandfathered chemicals and as newly introduced polymers under certificates issued
by NICNAS. In the latter case, the polyquaterniums have undergone assessment for
OHS, public health and environmental risks. There has been no assessment of the
polyquaterniums in use prior to the introduction of ICNA, and no assessment of

17
polyquaterniums as a chemical class. Further, the risk assessment of the new polymers
does not take into account other ingredients which occur in cosmetic preparations
with polyquaterniums and in particular, the interactions that occur between the
polyquaterniums and anionic surfactants. Little is known about the fate of cosmetic
polyquaterniums. All are released to WWTP where they are assumed to sorb to
sludge. Due to their toxicity to aquatic biota, the release of polyquaterniums into
receiving waters with WWTP effluent could be a potential risk to the environment.

1.4.1. Aims
It is the aim of this research to provide reliable data to facilitate the risk assessment
for polyquaterniums used in cosmetics applications. Specifically, this research aims to

a) determine the sorption behaviour of the polyquaternium and the

polyquaternium/surfactant complex (PSC) on environmental surfaces (e.g.
dissolved organic carbon (DOC), humic acid);

b) determine the toxicity of the polyquaternium and the PSC to algae, aquatic
invertebrates and fish;

c) investigate the mitigating effects of humic acids and electrolytes on the

toxicity of polyquaterniums and PSC that are found to be toxic;

d) investigate models and/or predictive tests that may assist in determining the
environmental risk of polyquaterniums of different chemistries, molecular
weights and charge densities.

1.4.2. Structure
The four-step risk assessment paradigm described above has been adopted for this
study. In addition to providing a framework for the research on environmental fate
and aquatic toxicology of the polyquaterniums and PSCs, this will facilitate a critique
of the method as it is used for the assessment of new and existing chemicals, and a
review the alternative methods that may be available.

18
2. Literature Review
2.1. Structure of Polyquaterniums
2.1.1. Quaternary Ammonium Salts
Ammonium salts are inorganic compounds containing the ammonium cation [NH4]+,
and an anionic counterion such as a halide (Cl-, Br-, I-) or a sulphate (SO42-).
Substituted ammonium salts are classified by the degree of substitution of the nitrogen
atom, that is, the number of hydrogen atoms replaced by an organic group as
illustrated in Figure 2.1.

H
H
N H
N H R
R R'
H
Primary Secondary

H R'

N R'' N R"
R R
R' R'''
Tertiary Quaternary

Where R, R', R'' and R''' are a methyl or larger organic goup,
or a polymer chain.

Figure 2.1 The structure of the ammonium cation, showing the degree of substitution of hydrogen
with organic groups on the nitrogen.
2.1.2. Polymers
Polymers are often defined as large molecules made up of repeating monomers, while
monomers are defined as molecules that join together to make a polymer. The term
polymer comes from the Greek polys ‘many’, and meros ‘parts’. The term was coined
by Jons Jakob Berzelius to denote molecular substances of high molecular mass
formed by the polymerisation (joining together) of monomers, molecules of low
molecular mass (Brown et al. 2006). Further, the term macromolecule is often used
synonymously with polymer. While polymers are macromolecules, there is an
important difference between a polymer and other macromolecules – a polymer has
variable molecular weight. The definition of a polymer according to the Industrial
Chemicals (Notification and Assessment) Act (ICNA) (CofA 1989) is:

Polymer means a chemical:

19
 a) consisting of molecules that are:

i) characterised by the sequence of one or more types of monomer units;

and
ii) distributed over a range of molecular weights whose differences in the
molecular weight are primarily attributable to differences in the
number of monomer units; and
b) comprising a simple weight majority of molecules containing at least 3
monomer units which are covalently bound to at least one other monomer unit
or other reactant; and

c) comprising less than a simple weight majority of molecules of the same

molecular weight.

A polymeric quaternary ammonium salt (polyquaternium) is a polymer based on a

monomer that is a quaternary ammonium salt. The polymer may be a homopolymer,
consisting of only the one quaternary ammonium monomer. Alternatively it may be a
copolymer, consisting of one or more monomers that are quaternary ammonium salts
and one or more monomers which are not. These other monomers are often called
‘spacer’ units, and are used to control the amount of charge on the polymer (charge
density).

2.1.3. Polyelectrolytes
Ammonium compounds, including polyquaterniums, are ionic compounds. Ionic
compounds that undergo complete or partial dissociation into ions in solution are
called electrolytes. Ammonium compounds are cationic electrolytes, and
polyquaterniums are cationic polyelectrolytes (or polycations). Cationic, anionic and
related classes of non-ionic polymers have many similar uses in industry, water
treatment and cosmetics, and are often studied as a group. In the discussion that
follows, the terms polyelectrolytes, cationic polyelectrolytes and polyquaterniums will
be used. The terms are not used interchangeably, but occur where the characteristic
being discussed can be said to belong to all polyelectrolytes, only cationic
polyelectrolytes, or exclusively to polyquaterniums.

2.1.4. Common Features of Polyquaterniums

A polyquaternium is a polymer according to the criteria outlined in Section 2.1.2, that
contains quaternary substituted ammonium functional groups (Figure 2.1). A

20
polyquaternium is a distinct entity when it is composed of a unique combination of
monomers and other reactive components, provided that each component represents at
least 2% by weight of the polymer. A polymer made of, for instance, 2-Propen-1-
aminium, N,N-dimethyl-N-2-propenyl-, chloride (diallyldimethylammonium chloride,
DADMAC) and propanoic acid is regarded as the same polymer if it has 80%
DADMAC and 20% propanoic acid, or any other ratio of these monomers. It is also
regarded as the same polymer regardless of whether the chain length is 1000 or
1,000,000. It is also the same polymer if it has ≤ 2% by weight of another monomer as
monomers and reactants present at this level are not considered part of the polymer
identity (USEPA 1997b). It is not the same polymer if it has > 2% of another
monomer, or if it has a bromide rather than a chloride counter ion, though in the latter
case it could be considered an analogue (NICNAS 2004b).

2.1.5. Variation Between Polyquaterniums

There are a large number of monomers from which polyquaterniums can be
synthesised. Polymers are often classified according to the monomer that provides the
reactive functional group in the polymerisation process, and thus the backbone
structure of the polymer. One of the ways in which polyquaterniums can be grouped
according to backbone structure, which is determined by the choice of monomer, is
into ‘natural’ or ‘synthetic’ polymers.

Natural polymers are also called biopolymers and are defined as polymers directly
produced by living or once-living cells or cellular components, or synthetic
equivalents of such polymers, or derivatives or modifications of such polymers in
which the original polymer remains substantially intact (CofA 1989). Bio-
polyquaterniums are most commonly derivatives of cellulose, but chitin and alginate
derivatives also exist. Polyquaternium-10 (Appendix 1, ix), for example, is produced
by the quaternary ammonium functionalisation of the cellulosic derivative,
hydroxyethyl cellulose (Figure 2.2).

21
 HO
HO HO
HO O O
n
O
O
O O
O O
O O
O O
* O O
O
O
OH
O
HO
HO
O

+
N (CH3)3

Figure 2.2 The structure of Polyquaternium-10 (quaternised hydroxyethyl cellulose), which is a

bio-polymer, based on cellulose. Polymers such as Polyquaternium-10 are often referred to as
‘natural’ polymers.

A synthetic polymer is any polymer that is not a biopolymer. Synthetic polymers can
be further classified by the polymerisable functional group(s) of their monomers, for
example as polyvinylic, polyacrylic, and polyamide (Figure 2.3). It is not necessary
that the backbone of the polymer be predominately carbon. Quaternised dimethicones
(polymers with silicone backbones) also exist.

The location of the ammonium functional group is also important in polyelectrolyte

structure. It can be located in the backbone of the polymer, on a pendant group
attached to the backbone, or on a side chain. The location of the group is often a
function of its location on the monomer, but can also be determined by polymerisation
processes such as cross-linking, where the charged group participates in the
polymerisation process.

R' R" R
R allylic
R"' C(O)R vinylic
acrylic
O
O R N R'
amide
R H
epoxide

Figure 2.3 The structure of the polymerisable functional groups in monomers used in the
synthesis of some polyquaterniums. Polymers synthesised from these monomers are known as
‘synthetic’ polymers.

22
Polyionenes are copolymers that have linkages through cationic and anionic charged
groups, and consequently are cross-linked. The anti-microbial Polyquaternium-1, used
in contact lens solutions, is an example of a polyionene (Appendix 1, i).

2.1.6. Variation Within Polyelectrolytes

In addition to the structural variations between polyquaterniums, there is considerable
scope for variation within a given polymer. These variations are brought about by
varying conditions in the polymerisation process. The molecular weight of the
polymer and the percentage of low molecular weight species can be varied depending
on the reaction conditions. Polyquaternium-10, for example, is produced by the Dow
Chemical Company subsidiary Amerchol in a variety of molecular weights in the
UCareTM range (Table 2.1). The number of functional groups in the polymer (except
for homopolymers of monomers with charged groups) can be varied by changing the
ratios of monomers in the reaction. Again, Amerchol’s UCareTM range of
Polyquaternium-10, is produced with a variety of charge densities (Table 2.1).

Table 2.1. In Amerchol’s range of Polyquaternium-10, UCareTM, the polymer is produced in a

range of molecular weight (as indicated by viscosity) and charge density combinations (as %
amine-nitrogen) (Amerchol, 2005)
Trade Name Charge Density Molecular Weight
UCareTM JR400 High Low
UCareTM JR30M High High
UCareTM JR125 High Low
UCareTM LR400 Low Low
UCareTM LR30M Low High
UCareTM LK Low Low

For polymers that are quaternised during or after the manufacture of the polymer, the
degree of quaternisation can be varied, allowing the polymer to be produced with a
mixture of quaternary, tertiary and secondary ammonium groups. Polyquaternium-11
is an example of a polyquaternium produced from a tertiary amine monomer (2-
Propenoic acid, 2-methyl-, 2-(dimethylamino)ethyl ester and quaternised following
polymerisation with diethyl sulphate (Appendix 1, x).

Another important variation in polyelectrolytes is hydrophobic substitution, i.e. the

addition of short or long hydrocarbon side chains to the polymer backbone. The
addition of the chains is usually not enough to make the polymer insoluble, but does
alter other important aspects of solution behaviour. Polyquaternium-24 (Appendix 1,
xx), for example, is a hydrophobically modified version of Polyquaternium-10, and is

23
also produced by Amerchol under the trade name Quatrisoft®. It has an 11-carbon
chain attached to the ammonium functional group.

2.2. Nomenclature
Most polymers are named in terms of the monomers from which they are
manufactured. In the nomenclature of the Chemical Abstract Service of the American
Chemical Society, the basic form of the monomer-based name of a polymer is
‘Reactant A, polymer with reactant B and reactant C’. For example, the formal name
for Polyquaternium-13 (Appendix 1, xii) is 2-Propenoic acid, 2-methyl-, 2-
(diethylamino)ethyl ester, polymer with ethyl 2-methyl-2-propenoate and (9Z)-9-
octadecenyl 2-methyl-2-propenoate, compound with dimethyl sulphate (9CI).
Polymers with only one monomer generally have the term ‘homopolymer’ added to
the name, for example, Polyquaternium-6 (Appendix 1, v) is 2-Propen-1-aminium,
N,N-dimethyl-N-2-propenyl-, chloride, homopolymer (9CI). Although this
nomenclature system is required for regulatory purposes, common names for
monomers are still used in naming polymers. Polyquaternium-6 is most often referred
to in both product catalogues and scientific literature as poly(diallyldimethyl
ammonium chloride) or poly(DADMAC).

Polymeric quaternary ammonium salts are recognised by Cosmetic Toiletry and

Fragrance Association US (CTFA) under the label ‘Polyquaternium-X’, where X is
sequentially chosen. This naming system is widely accepted by cosmetic
manufacturers and was adopted by the European Union (96/335/EC) as the common
nomenclature for cosmetic ingredients. It appears on the International Nomenclature
for Cosmetic Ingredients (INCI) and is the name under which these polymers are
listed on product labels in Australia.

2.3. Relevant Polyquaternium Physical-Chemical

Properties
Properties that are important in determining the fate of polymers such as
polyquaterniums in the environment include water solubility, molecular weight
distribution, acid dissociation constant(s), degradation half-lives, and sorption-
desorption data. Polymers tend to have low volatility in air and are not generally
absorbed through biological membranes to bioaccumulate in tissues (Doi 1997).
Therefore, the octanol-water partition coefficients and vapour pressures are generally
not considered important for predicting the environmental fate of polymers (Doi

24
1997). The risk assessment of polymers under the Toxic Substances Control Act
(TSCA) focuses on the monomer content of the polymer, molecular weight
distribution, equivalent weight of any reactive functional groups and/or cationic
charge density, properties such as physical form, particle size distribution,
swellability, aqueous solubility and water disposability (Boethling and Nabholz
1997). In the USA, the Office of Pollution Prevention and Toxics (OPPT) has
identified a group of polymers that are believed to pose low or no risk to human
health or environment and exempted them from the notification process (USEPA
1997b). In Australia, the separate notification category, PLC, with a reduced data
requirement, was introduced for these polymers (CofA 1989). Exempt polymers in the
USA, or PLCs in Australia, have to meet particular requirements with regard to
molecular weight and elemental composition. Polymers are excluded from the
category if they contain reactive functional groups, are biodegradable or unstable, are
water absorbing or contain monomers that are not on the respective inventory.
Importantly, polymers that are cationic, potentially cationic, or reasonably anticipated
to be cationic are excluded from the exemption (USEPA, 1997b).

2.3.1. Molecular Weight Distribution

Polymers contain mixtures of different size molecules, and individual polymers can
be made in a range of molecular weights. In the case of the Amerchol’s
Polyquaternium-10 range with the trade name UCareTM, the name may give some
indication of the molecular weight (e.g. JR400 ≈ 4 x 105 amu, LR30M ≈ 3 x 107 amu).
However, the classification of polymers as high, medium or low molecular weight is
somewhat arbitrary, and makes comparison between polymers difficult if the actual
molecular weights are not known (Figure 2.4).

NAMW
1 3 10 102 103 104 105 106 107

Monomer
Oligomer
Low Molecular Weight Polymer
Medium Molecular Weight Polymer
High Molecular Weight Polymer

Figure 2.4 Number Average Molecular Weight (NAMW) is the total weight of all the polymer
molecules in a sample, divided by the total number of polymer molecules in a sample. It gives an
indication of the average size of the polymer chain, but not of the range of sizes of the chains
(polydispersity) of the polymer.

25
There are several ways of measuring molecular weight. The molecular weight can be
determined from measurements of the colligative properties of the polymer, such as
osmotic pressure or vapour pressure lowering. When determined in this manner, it is
known as the number-average molecular weight (NAMW or Mn) of a polymer and is
defined as the total weight of all the molecules in a polymer sample divided by the
number of molecules present (Equation 2.1). The NAMW is sensitive to small weight
fractions of low molecular weight molecules and insensitive to small molecular
weight fractions of high molecular weight molecules (Stille 1962).

∑ (M N ) i i
Equation 2.1
Mn = i

∑N i
i

Where Ni is the number of molecules at a given molecular weight

and Mi is the molecular weight of the polymer fraction

Molecular weight can also be determined by light scattering, and the result is known
as the weight-average molecular weight (WAMW or Mw). The WAMW is calculated
from the total weight of all molecules, without consideration of the number of
molecules at each individual weight (Equation 2.2). Therefore this method can be
biased by a small percentage of large molecules, and give a false impression of the
majority of molecules in the sample (USEPA 1997b).
∞

∑N M i
2
i

Mw = i =1
∞ Equation 2.2
∑N M
i =1
i i

Because of the inherent bias in both methods, WAMW is always greater than
NAMW. For example, a polymer in which half the molecules are 30,000 amu and half
60,000 amu has NAMW of 45,000 amu, and WAMW of 50,000. Because molecules
of 60,000 amu scatter twice as much light as those of 30,000 amu, one third of the
contribution to light scattering comes from small molecules, and two thirds from the
large molecules (Stille 1962).

The NAMW can also be determined by gel permeation chromatography (OECD

1996). According to this method, the NAMW is calculated from the level of the
detector signal from the baseline for the retention volume. Assuming that the signal

26
peak is proportional to N, these equations are also given for NAMW (Equation 2.3)
and WAMW (Equation 2.4) in the Polymer Exemption Guidance Manual (USEPA
1997b).

∑N i
Equation 2.3
Mn = i
Ni
∑M
i

∑ (M N ) i i
MW = i

∑N i
i
Equation 2.4

However, for the example of the polymer given above, NAMW by this method would
be 40,000 amu and WAMW of 45,000. It is clear therefore, that it is not only
necessary to know whether the molecular weight stated is NAMW or WAMW, but
also which definition and method has been applied in its determination.

The polydispersity of a polymer is the distribution of molecular weights in the sample.

The Polydispersity Index (PI) is the ratio of the weight average molecular weight to
number average molecular weight. As the PI approaches 1, the length of polymer
chains in the sample becomes more uniform. An important characteristic of the
molecular weight distribution for environmental risk assessment is the percentage of
low molecular weight chains in the sample. To be classed as a PLC, a polymer sample
must have < 25% molecules with Mn < 1000; and < 10% with < 500 (NICNAS
2004b). As stated previously, cationic polyelectrolytes can not usually be classed as
PLCs, however, the proportion of low molecular weight species is still considered
important in predicting the toxicity of the polymer.

2.3.2. Charge Density

The charge density of a polymer is the proportional weight of cationic or anionic
fragments in the polymer chain (Doi 1997). The charge density of a polyquaternium
therefore is a measure of the quaternary functionality of the polymer. Polymer
characteristics such as water solubility, sorption behaviour and aquatic toxicity are
dependent to some extent on the charge density of the polymer.

27
There are several methods of measuring charge density.

Charge percent: In this case charge does not refer to the cationic charge, but to the
proportion of the monomer in the polymer mix, and can be applied to any monomer in
a polymer. By this method, a homopolymer, for example, has a charge density of
100% (USEPA 1997b).

Percent amine-nitrogen (% a-N): A measure commonly used by OPPT for cationic

polyelectrolytes is the percentage of amine-nitrogen present in the polymer, as 99% of
cationic polymers have their charge based on nitrogen. It is the percentage of cationic
nitrogen in the polymer (Boethling and Nabholz 1997). This is simple for
homopolymers, but requires information on the charge percentages of the monomers
in the polymer for copolymers (Equation 2.5).

14
%a − N = ∗ 100 Equation 2.5
monomer mw

Equivalent weight (EQWT): The equivalent weight can be calculated for any reactive
functional group in a polymer, and is used by OPPT and the National Industrial
Chemicals Notification and Assessment Scheme (NICNAS) in polymer risk
assessment. Methods of calculating the EQWT are given in the guidelines for polymer
exemption (USEPA 1997b). For polyquaterniums, EQWT can be calculated from
% a-N (Equation 2.6) (Boethling and Nabholz 1997).

1400
EQWT = Equation 2.6
%a − N

Number of cations per 1000 molecular weight (#C/K): Although not a commonly
used measure of charge density, #C/K can be calculated simply from either % a-N
(Equation 2.7) or EQWT (Equation 2.8) according to Boethling and Nabholz (1997).

# C / K = % a − N ∗ 0.714286 Equation 2.7

1000
#C / K = Equation 2.8
EQWT

Gram-equivalents: The molar mass of a substance divided by the number of charges

of the same sign carried by the ions released by a molecule of that substance in an
aqueous solution is the gram-equivalent of the substance. For simple molecules, the
gram-equivalent is the weight of one mole of the substance divided by its valency. For

28
polymers, the gram-equivalent is usually determined by titration of a polymer
solution. The concentration of the polymer (or any molecule) in terms of gram-
equivalents is the normality (N) of the solution (Degrémont 1991). For polymers,
charge density is determined by electrolyte titration of solutions of known
concentration (Dentel 1989).

2.3.3. Aqueous Solubility

The low water solubility of the majority of nonionic polymers is due to the
hydrophobicity of the polymer chains. The polyquaternium conditioning polymers
used in cosmetics are, however, highly water soluble (Gruber 1999). The solubility is
largely the result of the charged centre of the amine functional group. In an aqueous
solution, repulsive forces between the cationic amines keep the polymers apart and
dispersed through the solution. Intra-chain repulsion between the charge centres
forces the polyelectrolytes to adopt a stretched conformation (Claesson et al. 2000). In
this conformation, the polymer is often described as resembling a string, but is
perhaps a very loosely coiled globular structure, similar to globular proteins, with the
charged centres facing into the water. This conformation occurs because, like
surfactants, cationic polyelectrolytes consist of two parts with different solubility
characteristics; one part that is soluble in water (a hydrophilic group) and one part that
is not water-soluble (hydrophobic). The hydrophilic group is the charged functional
group, the cationic amine in polyquaterniums. The hydrophobic group section is the
‘tail’ of the surfactant or the polymer backbone for polyelectrolytes. In the coiled
conformation, the hydrophobic domains of the polymer chain, like surfactant tails, are
contained within a micelle-like structure (Fundin et al. 1996).

Although polyquaterniums are surface active, they are only slightly so (Manuszak
Guerrini et al. 1998). When the concentration of surfactants approaches the solubility
limit, the molecules do not precipitate, but form micelles, tiny aggregates of 50 to 100
molecules. The concentration at which micelles begin to form is called the critical
micelle concentration (CMC), and can be detected by subtle changes in surface
tension or light scattering (Connell 1997). Above the CMC, the surface tension
remains fairly constant (Manuszak Guerrini et al. 1998). In a study using Polymer
JR400, Regismond et al. (1999c) reported a slight lowering of surface tension at
concentrations between 0.05 and 0.5% w/w. The amount of reduction in surface

29
tension ranged from 0.1 to 7.4 mJ/m2. However, at the lower concentration of 0.01 %
w/w, the polymer appeared to increase surface tension slightly (0.3 to 0.4 mJ/m2).

When an anionic surfactant is added to a solution with a cationic polyelectrolyte,

several types of behaviour may be seen. The phase diagram shows three distinct
zones, the clear zone, the precipitation zone and the resolubilisation zone. The
transition between stages is marked by changes in the relative concentrations of the
polyelectrolyte and the surfactant. The clear zone occurs where there is an excess of
polyelectrolyte, and the resolubilisation with an excess of surfactant. The precipitation
zone occurs when the stoichiometry of the surfactant and polymer is approximately
1:1 (Leung, et al. 1985).

When the polymer is in excess, the electrostatic salt formation between the charged
head of the surfactant and the charged group on the polymer is followed by the
hydrophobic interaction of the surfactant tails and the hydrophobic segments of the
polyelectrolytes. The interaction begins at low concentrations, below the critical
micelle concentration (CMC) of the surfactant. The concentration at which
complexation begins is called the critical aggregation concentrations (CAC)
(Manuszak-Guerrini et al. 1997). The binding process is cooperative, as the binding of
the polycation-surfactant is more favourable than the aggregation of the surfactant
(Goddard 1999). As the charge is neutralised, hydrophobic interactions between the
surfactant tails and uncharged segments of the polymer lead to further contraction of
the complex. Neutralisation results in reduced water solubility, and the hydrodynamic
radius of the polyelectrolyte-surfactant complex is significantly smaller than that of
the free polymer (Fundin et al. 1996). As the surfactant molecules bind to the charged
centres of the polymer, a change in the conformation of the polymer occurs. The
reduction of the intramolecular repulsion allows the polymer to ‘curl up’ (Leung et al.
1985). As the polyelectrolyte-surfactant complex is formed, there is a synergistic
lowering of surface tension (Regismond et al. 1998).

In a solution with a large excess of surfactant, the complex formed consists of

polyelectrolyte cross-linked or woven through surfactant micelles (Fundin et al. 1996,
Leung et al. 1985). The structure of the surfactant micelle is similar to the structure of
micelles without the polyelectrolyte present (Ananthapadmanabhan et al. 1985). The
binding results from hydrophobic interactions between the neutral complex and the
hydrophobic tails of the surfactant and produces a negatively charged complex. The

30
new complex is larger than the complexes in the previous two zones (Fundin et al.
1996), and is soluble due to the negative charge on the surface. In cosmetic
formulations, the polymer and the surfactant are dissolved in separate components of
the formula and then mixed together. Thus the binding occurs between the formed
surfactant micelle and the polyelectrolyte and results in a viscous solution with a
highly ordered structure (Goddard and Hannan 1976). If a system with excess
surfactant is diluted below the CAC for the polymer/surfactant system, the micelle
structure of the surfactant will break down (Gruber 1999).

2.3.4. Biodegradation
Degradation is an important component of the fate of cationic surfactants, but
generally, polymers are considered to be essentially nonbiodegradable. Even modified
natural polymers such as carboxymethylcellulose with an ‘appreciable’ degree of
substitution, regardless of the type of substituent, are not biodegradable. As
environmental fate assessment is commonly limited to the potential for sorption or
precipitation under various conditions, there are few studies on the biodegradability of
commercially available polymers (Boethling and Nabholz 1997).

Some studies have shown that anionic and non-ionic polymers may undergo some
degradation, but that cationic polymers are more resistant. For example, the secondary
nitrogen in anionic and non-ionic polyacrylamides was found to provide a nitrogen
source to bacteria isolated from soil, however cationic polyacrylamide was toxic to
Psuedomonas and strongly inhibited the growth of Desulfovibrio (Grula et al. 1994).
The anionic and non-ionic polyacrylamides did not provide a source of carbon, and
there was no evidence of breakdown of the polymer chain. The ability of soil bacteria
to use anionic polyacrylamide as a nitrogen source, but not as a carbon source, was
confirmed in studies by Kay-Shoemake et al. (1998). Complete removal of pendant
nitrogen from polyacrylamide would leave a residual chain similar to polyacrylic acid
(PAA), which has been shown to be incompletely mineralised by activated sludge
microorganisms even at low molecular weights. Both monomers and dimers of PAA
were completely mineralised, and low molecular weight oligomers of PAA (molecular
weights 500 and 700) were extensively but not completely degraded (Larson et al.
1997).

Cationic poly(acryloyloxyethyltrimethylammonium chloride) (AETAC) copolymer,

Percol (or Zetag) 787 (Polyquaternium-15, Appendix 1, xiv) was found by Chang et

31
al. (2001) to degrade aerobically leaving ammonia, trimethylamine, which is resistant
to further degradation, and a non-fragmented backbone of either polyacrylamide or
PAA. Anaerobically, the pendant ammonium group was also hydrolysed, and
completely degraded following removal. Again there was no evidence of breakdown
of the polymer chain. It should be noted that this test period was considerably longer
than retention times in Wastewater Treatment Plants (WWTPs). Anaerobic
degradation, as measured by increased gas production, was incomplete at the end of
the test period (840 hours). The incubation period prior to this increase in gas
production was 6 days. The authors suggest that ‘hydrolytic release of pendant group
may occur with many common flocculant polymers possessing an ester in the position
immediately adjacent to the main alkyl chain’ (Chang et al. 2001). That ester linkages
or other labile groups in the main chain of natural or synthetic backbone of polymers
may be biodegradable under favourable conditions has also been suggested (Boethling
and Nabholz 1997). Although ester linkages are common in the pendant groups of
natural polyquaterniums, they are much rarer in the synthetic polymer chains.
Quaternary polyesters are not represented in either the cosmetic or flocculant groups
of polyquaterniums.

2.3.5. Chemical/physical Degradation

In addition to biodegradation, polyelectrolytes may undergo degradation due to
mechanical wearing of the polymer chains, or by reaction with other chemical species
in the water. As cationic polyelectrolytes are often used in water treatment, of
particular interest is the effect of other water treatment chemicals, such as ozone and
chlorine. The possibility of chemical degradation in effluent is also relevant to
cationic polyelectrolytes used in cosmetics, which are disposed of in sewage.

Ozonation reduced the average molecular weight of several polymers to oligomer size
(250-560 amu) in 2 to 4 hours, but without significant improvement in the
biodegradation of the fragments (Suzuki et al. 1978). Polyvinyl alcohol and
polyvinylpyrrolidone showed no improvement in biodegradability for fragments with
molecular weight > 100. There was no improvement in the degradation of
polyacrylamide even for fragments with molecular weight < 100. Chlorination
reduced the average molecular weight of anionic polyacrylamide from 107 to
approximately 104 in three hours reducing the ability of the polymers to form flocs
(Aizawa et al. 1991).

32
 2.4. Exposure Assessment (Environmental Fate)
A major problem in the study of environmental fate of polyquaterniums has been the
absence of any method for identifying and quantifying polyelectrolytes in
environmental samples. Although around 6.5 million tons of polyelectrolytes are used
each year in the USA alone in industry and wastewater treatment (Chang et al. 2002),
there is no satisfactory way of quantifying any flocculant residual in the treated water
(Bennett et al. 2000). Although several techniques have been developed for analysing
polyelectrolytes in relatively clean water (Wickramanayake et al. 1987), no
established method is satisfactory in complex mixtures such as wastewater, biosolids
and environmental samples (Chang et al. 2002). The inability to monitor water
concentrations at levels of regulatory concern has been identified as one of the
problems facing the cationic flocculants industry.

2.4.1. Sorption-desorption
The partitioning to solids in the waste stream is generally considered the most
significant fate pathway for industrial and cosmetic cationic polyelectrolytes
(Boethling and Nabholz 1997). However, there have been few studies on sorption or
precipitation of cationic polyelectrolytes in the waste stream or in the environment.
Consequently, risk assessments of cationic polyelectrolytes are generally based on
default values for polymers. According to Boethling and Nabholz (1997) ‘non-ionic,
cationic and amphoteric polymers with molecular weight >1000 are assumed to
partition mainly to the solids phase and to be 90% removed relative to the total
influent concentration’. The 90% figure was selected because it represents a typical
level of solids removal in WWTPs (Boethling and Nabholz 1997).

The sorption of low molecular weight organic substances to soils and sediments is
dependent on the aqueous solubility of the substance and is normally proportional to
the organic carbon content of the sorbent. Generally, sorption increases with
decreasing solubility of the substance and with the increasing carbon content of the
sorbent (Podoll and Irwin 1988). Typically, the relationship between the mass of the
chemical adsorbed and the concentration remaining in solution at a given temperature,
the sorption isotherm, is linear or approximately so (Burchill et al. 1981).

2.4.2. Sorption of Polymers

For polymers, molecular weight and chemical structure dominate sorption behaviour.
For polydisperse polymers i.e. polymer samples with a wide range of sizes, small

33
polymers chains are adsorbed preferentially, but are also easier to desorb, and are
eventually replaced by higher molecular weight chains. Equilibrium sorption of
polydisperse polymers may take days or months to achieve, as a balance is established
between affinity for solvent and configurational restrictions on sorption. The shape or
configuration adopted by the polymer in the solution is important to sorption. It is
possible for polymers to adopt shapes that are stretched or compact, looped or coiled.
Consequently, the adsorbed polymer may adopt a conformation on the surface that is
either completely flat, or extending away from the surface (Jaycock and Parfitt 1981).
In particular, the polymer may be expected to adopt a formation consisting of
segments close to the surface (trains), segments extending into the solution but
attached to a train at each end (loops) and sections extending into the solution
attached to a train at only one end (tails) (Figure 2.5). For relatively high molecular
weight polymers, the adsorbed layer is generally between 3 and 30 nm thick (Myers
1999). The configuration adopted by the polymer at the surface will result from a
balance of solution characteristics, plus the net energy change on sorption, the
decrease in entropy of the chain that accompanies sorption and the gain in entropy due
to freeing of solvent molecules (Myers 1999). The sorption of some polymers
increases with temperature, indicating that the sorption process is entropically rather
than enthalpically controlled. According to Jaycock and Parfitt (1981), this suggests
release of solvent from the surface on sorption of the polymer.

Figure removed, please consult

print copy of the thesis held in
Griffith University Library

Figure 2.5 Representation of the adsorption of a polymer onto a surface, showing the formation
of loops, tails and trains (Obey and Griffiths 1999).

While sorption with polymers may take a relatively long time to reach equilibrium, it
is effectively irreversible, except for low molecular weight fractions (Myers 1999).
The sorption isotherm is usually of a Langmuir type (Jaycock and Parfitt 1981) or
high affinity (Myers 1999). High affinity isotherms result when the entire polymer is

34
adsorbed below some threshold concentration. The shape of the isotherm suggests the
sorption process occurs in three stages,

1) rapid sorption of polymer chains reaching the surface as indicated by change

in polymer concentration in solution;

2) polymer chains reaching the surface can only be adsorbed by reconfiguration

of the polymer already adsorbed, either by desorbing low molecular weight
species, or increasing the amount of polymer in loops and tails as indicated by
change in the rate of reduction of the solutions concentration of polymer;

3) little or no change in polymer concentration in solution indicating

accommodation of additional chains has reached a maximum.

In the latter stage of the sorption process, the thickness of the adsorbed polymer layer
is increased, due to the increased amount of polymer extending into the solution in the
form of loops and tails (Myers 1999).

As noted above, the sorption of polymers is generally considered to be irreversible

(Podoll and Irwin 1988; Myers 1999). Although a polymer segment might be
adsorbed reversibly, for the polymer chain to desorb, all segments must desorb
simultaneously. The simultaneous desorption of all segments is considered
statistically unlikely. The problem with this model of desorption is that it implies
several assumptions that are not normally stated when the model is invoked, for
example

a) the desorbing of polymer segments is random;

b) no agent can initiate or facilitate desorption;

c) the desorption of one segment has no effect on the probability of the

desorption of any other segment.

2.4.3. Sorption of Polyelectrolytes

Consideration of the sorption of polyelectrolytes must take into account any charge on
the surface of the sorbent. It is expected that surfaces with the same charge as the
polyelectrolyte would repel the charged centre on the polymer chain resulting in
reduced sorption at the surface, while sorption of polyelectrolytes to surfaces with
opposite charge would be facilitated by the attraction between the charge centres. As
flocculants and coagulants, cationic polyelectrolytes are intended to bind irreversibly

35
to anionic biosolids and separate into the solid phase during effluent treatment. In the
case of cationic polyelectrolytes, the binding is expected to be coulombic (Podoll et
al. 1987).

A major study of the adsorption behaviour of the low molecular weight fraction of
cationic and non-ionic polyelectrolytes was undertaken to assist the OPPT in
assessing the fate of water treatment polymers. These low molecular weight fractions,
or oligomers, are larger than most organic compounds, but smaller than the
commercially used polymers. They are not commercially significant, but are by-
products of the polymer synthesis process and released with the polymer on use
(Podoll et al. 1987).

This study of the sorption/desorption behaviour of oligomers of non-ionic and cationic

water treatment polymers on sediments compared non-ionic poly(ethylene glycol)
(PEG) with molecular weight from 194 (4 repeat units) to 3400 (≈ 77 units);
poly(ethylenimine) (PEI), a branched cationic polymer with primary, secondary and
tertiary substituted amino groups and molecular weight 600, 1200 and 1800 (n = 14-
42) Podoll et al. (1987); and tertiary cationic polymer poly(N,N,-dimethylaminoethyl
methacrylate) (PDAM) molecular weight of 920, 1598 and 4115 (Podoll and Irwin
1988). The sediments had varying compositions of clay, sand and silt, with organic
carbon contents varying from 0.48 – 2.33%, cation exchange capacities (CEC) of
13.5 – 33 mequiv/100 g and pH from 4.3 – 8.25.

All polymers showed strong, site-specific sorption to all sediments. While the sorption
of non-ionic PEG correlated with the clay content of the sediment, and was also
influenced by the pH of the sediment and the solution, the amount of cationic PEI and
PDAM adsorbed correlated with the CEC of the sediment and was independent of pH.
The coulombic interaction between the tertiary nitrogens of PDAM and the cation
exchange sites was found to be weaker than the coulombic interaction of PEI at these
sites.

The sorption isotherms were non-linear for both the non-ionic and the cationic
polymers. A Langmuir isotherm fitted well with cationic PEI and PDAM but less well
with non-ionic PEG. For a given molecular weight and sediment, the extent of
sorption for PEI was greater than for PEG. Sorption increased with molecular weight,
more noticeably for PEG than PEI. The plateau sorption values increased with

36
increasing molecular weight for PEI, but were independent of molecular weight for
PDAM. The number of nitrogens adsorbed per cation exchange site for PEI ranged
from about two to three and increased with increasing molecular weight, indicating
that PEI adsorbs with a significant number of the amino groups in the loops and tails.
PDAM, on the other hand, adsorbed to most soils with a ratio of amino groups to
cation exchange sites close to 1:1, indicating that it probably adsorbs in a train
conformation with almost all nitrogens attached to the sediment. These results suggest
that some flexibility is required to allow all cationic sites on the polymer to interact
with the array of anionic sites on the substrate. For example, if the separation of
anionic sites is greater than the distance between amines in PEI, then not all amines
can coulombically bond. Even if the separation is a little less, it is still difficult to
achieve an arrangement where all amines are associated with anionic sites, because
equilibration would require large amounts of desorption. With a more flexible link,
sectors can loop together and entangle more. The authors state the molecular weight
effect for PEI is mitigated by repulsive force of charged groups in the loops and tails
of the sorbed polymer.

It is possible that the non-ionic binding mechanisms (van der Waals) operating
between PEG and the clays are more sensitive to molecular weight than the charge
density dependent coulombic binding of the cationic PEI. Podoll et al. (1987) also
note that the increase in absorption with increase in molecular weight is greatest in the
lower molecular weight range, and that the rate of increase was expected to diminish
as molecular weight increased above 3400 amu. Sorption isotherms were constructed
and sorption coefficients (slope of the linear section of the isotherm) and the sorption
capacities (plateau value of the isotherm) were calculated for a range of sediments.
Sorption capacities of the sediments ranged from 17.6 mg/g to 142 mg/g and partition
coefficients from 880 to 480,000 mL/g (Podoll et al. 1987; Podoll and Irwin 1988).

Desorption was reported as being < 15% for PDAM at all concentrations, while
desorption for PEI increased with increasing solution concentration, from < 15% to as
high as 50% in the plateau region of the isotherm. However, the actual desorption data
is not published. Looking at desorption for both polymers, it was not below 10% for
any solution concentration tested, and approached 50% as the concentration
approached or exceeded 1000 ppm.

37
When added to humic acid, the water treatment algicide and cationic polymer
poly[oxy-1,2-ethanediyl(dimethyliminio)-1,2-ethanediyl(dimethyliminio)-1,2-
ethanediyl dichloride] (Busan 77, Polyquaternium-42) (Appendix 1, xxix), was found
to form insoluble precipitates with high molecular weight humic acids (Matthews et
al. 1995). The Polyquaternium-42 studied had an average molecular weight of 3900
amu, and molecular weight range from 600 to 5000 amu. However, soluble complexes
were formed with low molecular weight humic acid, with the maximum binding
capacity indicating 1:1 molar binding at saturation. The binding was reversible, and
gave a reasonable fit to a Langmuir isotherm. Between 10% and 20% of the polymer
dose was found to be bound in soluble complexes to naturally occurring humic acid
under expected levels of polymer contamination in aquatic conditions (Matthews et
al. 1995).

While the study of the sorption of polyquaterniums to environmental solids has not
received much attention, the sorptive behaviour in cosmetic applications has received
extensive attention. In addition to studies of the sorption of polyelectrolytes to hair,
various studies have examined the sorption of polyelectrolytes to mica (Fielden et al.
1998; Rojas et al. 2002), glass (Poptoshev and Claesson 2002), cellulose (Rojas et al.
2000) and silica (Samoshina et al. 2005).

Like environmental solids such as humic acid, human hair has a negatively charged
surface. The isolectric point of hair (the pH above which it has a net negative charge)
is 3.67, and its zeta potential is normally about -30 mV. A zeta potential of -25 mV is
needed for molecules to overcome van der Waals attraction. Van der Waals bonding
increases with increasing molecular surface area, and for polymers can approach the
strength of primary valence bonds (Robbins 1994). Studies with monofunctional
quaternary ammonium surfactants show substantivity to hair is only achieved when
there are 8 to 10 carbons in the hydrocarbon tail of the surfactant, indicating that van
der Waals forces in addition to ionic binding contribute to the sorption of quaternary
cationics to hair.

Many studies of the sorption of polyquaterniums to hair have been of the sorption of
the polymer alone to hair. Without the surfactant, the polymer sorbs strongly to hair,
with an excess of positive charges in the loops and tails of the sorbed polymer. The
zeta potential of the hair sample may be increased to 25-30 mV and remains positive
through continuous rinsing with water (Amerchol 2002). The amount of polymer

38
needed to saturate normal hair is 2 mg/g for
poly(methacrylamidopropyltrimethylammonium) chloride (poly(MAPTAC))
(Appendix 1, xxxvi) and 25 mg/g for UCare JR (Polyquaternium-10) (Jachowicz, et
al. 1985).

In the presence of a surfactant, however, only a fraction of the polymer may be

deposited on the hair (André et al. 1999), and the uptake of Polymer JR in the
presence of surfactants used in shampoos may be as low as 1 µg/mg (Goddard et al.
1975). The zeta potential of the hair is increased, but not to positive values, and
according to Hössel et al. (2000), this indicates the deposition of a charge neutral
polymer-surfactant complex. However, it is also possible that the lower deposition
may be responsible for the smaller change in zeta potential.

The addition of a surfactant to a preadsorbed polyquaternium layer has been studied

using mica or gold, as well as hair, as the substrate. For example, in a study of AM-
MAPTAC on gold Plunkett et al.(2002) reported swelling of the adsorbed polymer
when the surfactant, SDS, concentration was 20% of the CMC, with desorption of the
polymer occurring when the concentration was between 60% and 200% of the CMC.
Rojas et al. (2001) reported that low charge density AM-MAPTAC began to desorb
from mica at 0.1% of the CMC of SDS, and desorption increased as the surfactant
concentration was increased. Low charge density polyquaterniums adopt a more
extended conformation on the surface (more polymer in loops and tails), allowing the
association of the polyquaternium and the surfactant on the surface, thus incorporating
negative charges within the previously cationic layer of the double layer (Rojas et al.
2000). Regismond et al. (1999a) demonstrated the desorption of Polymer JR from hair
with SDS using fluorescence microscopy. Hair was treated with the fluorescently
labelled polymer JR 400 for periods ranging from 30 minutes to 12 hours. With SDS
the polyquaternium was completely desorbed from the hair samples treated for the
shorter times. Jachowicz et al. (1985) reported limited desorption of polyMAPTAC
and polyDADMAC with SDS as the polymer-surfactant complex remained bound to
the surface of the hair, however, the sorbed polycations formed complexes with the
anionic polymers PSS and PAA, and were subsequently desorbed from the hair.
Faucher et al. (1977) reported that the deposition of Polymer JR was hindered by the
presence of other electrolytes such as metal salts, with effectiveness being dependant

39
on valency. These metal salts were also able to desorb up to 50% of polymer JR,
however, the effect of valency was only apparent at low concentrations (< 0.01 M).

The studies outlined above would seem to indicate that in the presence of the
oppositely charged surfactant, sorption of the polymer may be reduced, and
desorption increased. Although humic acid has a similar isoelectric point to hair, the
behaviour of the polyquaternium/surfactant complex at infinite dilution is not known.

2.5. Effects Assessment (Aquatic Toxicology)

Some of the earliest published fish toxicity data on a cationic polyelectrolyte were
reported by Tooby et al. (1975). The toxicity to Rasbora heteromorpha Duncker of
Busan 77, a Polyquaternium-42 used as a microbicide, was included in a study of the
aquatic toxicity of pesticides. The 24 hour 10th percentile lethal concentration (LC10)
and the median lethal concentration (LC50) were reported as 0.47 and 0.66 mg/L,
respectively; the 48 hour LC10 and LC50 as 0.32 and 0.39 mg/L; and the 96 hour LC50
as 0.17 mg/L. Importantly, this study established that the toxicity of cationic
polyelectrolytes may be similar to that of cationic surfactants (Madsen et al. 2001).

2.5.1. Toxicity of Surfactants

As the toxicity of cationic surfactants and cationic polyelectrolytes appear to be
similar, it is useful to look briefly at the toxicity of cationic surfactants. In an
environmental and health assessment of household detergents for the Danish
Environmental Protection Agency, Madsen et al. (2001) reported that cationic
surfactants were very toxic to fish, invertebrates and algae. The LC50 values were
generally ≤ 1.0 mg/L and rarely more than ≥ 10.0 mg/L. Lewis and Suprenant (1983)
studied literature values of LC50 of cationic, anionic and non-ionic surfactants to
aquatic invertebrates. They found that the sensitivity of different species to the same
surfactant, cationic cetyl trimethyl ammonium chloride (CTAC), varied by a factor of
2300, while the sensitivity of the same species (Daphnia magna) to three surfactants
varied by a factor of up to 220. D. magna was the most sensitive species of those
considered to surfactants. The authors also tested three surfactants, CTAC, an anionic
linear alkylbenzene sulphonate and nonionic C14-15 alkyl ethoxylate to six species of
aquatic invertebrates. Clearly, the possession of cationic centres is an important factor
in the toxicity of surfactants, but variability between vulnerable organisms and
between chemical analogues appears to be high.

40
 2.5.2. Toxicity of Polymers
The toxicity of polymers is generally limited because their high molecular weight and
large steric size limit their ability to cross biological membranes, though chemical
reactivity of functional groups can influence ecotoxicology and fate (Hamilton et al.
1997). According to Jop (1997), with polymer dispersions, polymer toxicity may
result from mechanisms such as inhibition of light penetration or by clogging gills at
high concentrations.

2.5.3. Toxicity of Polyelectrolytes – General Considerations

Assessment of the published studies on the toxicity of cationic polyelectrolytes,
mostly for water treatment polymers, can be difficult. The polymers are often
identified only by their trade names, if at all, and the chemical identity or class may be
difficult to establish. Further, molecular weight and charge density of the
polyelectrolytes are not always stated, and it may not be possible to determine if the
charge centre is tertiary or quaternary substituted. When molecular weight and charge
density are given, different methods of reporting them can still make comparisons
difficult. A study by Biesinger et al. (1976), typifies the problems in identifying the
studied polymers. The 5 cationic, one anionic and one non-ionic polyelectrolytes in
the study are identified only by their trade names, Superfloc 330, Calgon M-500,
Gendriv 162, Magnifloc 570C and Magnifloc 521C (all cationic), Dow AP-30
(anionic) and Magnifloc 905N (nonioinc). While it is possible to find Chemical
Abstracts Service (CAS) listings for most of these polymers, details of their structures
are not available. Superfloc is a trade name of Cytec, who make a range of polyamine
and poly(DADMAC) water treatment polymers, however, Superfloc 330 is no longer
listed in the Cytec catalogue. Magnifloc is the trade name for a range of food grade
water treatment polymers also made by Cytec. The Magnifloc range includes both
Polyquaternium-6 (poly(DADMAC)) and Polyquaternium-33 (Appendix 1, xxiv), but
Magnifloc 521C and Magnifloc 570C are not listed as trade names in the CAS listings
for these polymers. Gendriv 162 is a trade name for guar gum, which is not cationic as
stated by the authors. Cationic guar gum (Appendix 1, xxxvii) has a different CAS
number (65497-29-2), and the trade name Gendriv is not listed in its chemical
abstracts listing. However, guar gum, hydroxypropyl guar gum and cationic guar gum
have many similar trade names. In many cases the trade name may still be in use, but
the specific polymer referred to may no longer be in the manufacturer’s catalogue.

41
No further details were given of the structures of the seven polyelectrolytes employed
by Biesinger et al. (1976). As all are flocculants, it is probable that they are high
molecular weight, high charge density polymers. The authors reported the toxicity
values as median tolerance limit, TL50, defined as the concentration at which 50% of
the test animals were able to survive for a specified period of exposure. The exposure
time was 48 hours for Daphnia and 96 hours for fish. The results for cationic
polyelectrolytes were generally less than 10 mg/L, and less than < 1 mg/L for some
species, and only Gendriv, which has a non-synthetic backbone (guar gum) had
relatively low toxicity, with TL50 > 100 mg/L. The authors concluded that ‘some
cationic polyelectrolytes tested were particularly toxic at certain concentrations that
might easily be released into the environment and cause serious problems for aquatic
life’ (Biesinger et al. 1976). They found that the 21 day TL50 for Daphnia was higher
by an order of magnitude than 48 hour TL50, which they attributed to the addition of
food during the test, and adsorption of the test material to the food particles (Biesinger
et al. 1976).

A number of studies have shown that the presence of suspended solids mitigated the
toxicity of cationic polyelectrolytes (Devore and Lyons 1986). For example, four
cationic polylectrolytes, two epichlorohydrin-amine condensates (molecular weight
20,000 and 400,000 amu), an acrylamide-vinyl quaternary amine copolymer
(molecular weight 3,000,000 amu) and poly(DADMAC), with LC50 values to
minnows and Daphnia < 1 mg/L, were tested in synthetic river water containing fixed
amounts kaolinite clay, humic acid and dissolved salts prepared in the laboratory. No
toxicity was evident until the polymer dose exceeded the dose required for
flocculation, around 10 mg/L. The lack of toxicity at concentrations below the
flocculant dose is therefore assumed to result from the lack of free polymer molecules
in the solution. The resuspension of the floc due to overdosing coincided with the
reappearance of free polymer molecules in the supernatant and observed toxic effects
in the test organisms. The mitigating effect of suspended solids has become a major
theme in toxicity studies of cationic polyelectrolytes.

Biesinger and Stokes. (1986) looked at detoxifying effects of anionic polymers and
various clays on cationic polyelectrolytes. Anionic polyelectrolytes were able to
negate the toxic effects of the cationic polyelectrolyte when present in about equal
quantities. The authors report the amounts of clay needed to detoxify a cationic

42
polymer at five times the toxic dose were 80 mg/L for red clay, 160 mg/L for
montmorillonite and 320 mg/L for kaolinite. These concentrations are a useful
indication of the relative adsorptive ability of the various clays. However, it appears
that only one polymer was tested in this manner (‘Polymer O’, Median Effective
Concentration (EC50) 96 hour fish 1.05 mg/l). Of the fifteen polyelectrolytes in this
study, most had effective concentration in tests with daphnids of < 1 mg/L and with
minnows < 10 mg/L, although some had effective concentrations > 100 mg/L for one
or both species. Eight polyelectrolytes were also tested with midges, and 13 with
gammarids. These organisms were less sensitive to the cationic polyelectrolytes than
fish and Daphnia. According to the authors, the ‘large differences in sensitivity
between these species suggest different modes of toxic action’, while the differences
in toxicity between various polymers ‘is probably accounted for by their chemical
structure’ (Biesinger and Stokes 1986). However, no details of the chemical structures
of the polymers in their study are given. Elucidating the structural characteristics that
influence the toxicity is the other major theme in toxicity studies of cationic
polyelectrolytes.

The mitigating effect of humic acid on the toxicity of polyquaterniums was an issue
for industrial manufacturers because of the United States Environment Protection
Agency’s (USEPA) data requirements pre-manufacture notice (PMN) under TSCA.
When acute toxicity studies submitted with a PMN for a chemical had LC50 values
< 1.0 mg/L, additional supporting data including chronic life-cycle studies on two to
three species were required (Cary et al. 1987; Cary et al. 1989). It was suggested that
results of acute toxicity tests ‘may be too stringent for estimating the effects of
cationic polyelectrolytes in receiving waters’ (Cary et al. 1987). This claim was
supported by the study of four quaternary polyelectrolytes of varying chemistries,
molecular weight, charge density and percent quaternisation, all with LC50 values to
three species of fish and Daphnia of < 1 mg/L. The toxicity of the polyelectrolytes
was tested in the presence of suspended solids (bentonite, illite, kaolin and pure silica)
at 50 mg/L, and dissolved organic carbon (DOC) (humic acid, tannic acid, fulvic acid,
lignin, and lignosite) at 10 mg/L. The presence of suspended solids or DOC reduced
the toxicity of the cationic polyelectrolytes regardless of molecular weight or charge
density. The reduction in LC50 was generally one to two orders of magnitude, though
illite, kaolin and silica were less effective (Cary et al. 1987).

43
To address chemical industry concerns regarding the costs of providing the extra data
with PMNs, the Cationic Flocculant Producer Association (CFPA) was formed as a
subgroup of Specialty Organic Chemical Manufacturers Association (SOCMA) (Cary
et al. 1989). The CFPA consisted of American Cyanimid Company, Betz Laboratories
Inc, Calgon Corporation, CPS Chemical Company Inc, Hercules Incorporated, Nalco
Chemical Company and Petrolite Corporation. The purpose of the group was to lobby
USEPA to treat cationic polyelectrolytes as a class of chemicals, rather than treating
each new PMN individually. It was felt that this would lead to lower data
requirements, and hence lower notification costs. Data was collected from members
on 386 toxicity studies of cationic polyelectrolytes. The data included chemical
classification, molecular weight, charge density, test species, toxicity values, water
hardness, suspended solids and type of Total Organic Carbon (TOC) and Suspended
Solids (SS). Sixty percent of the cationic polyelectrolytes had toxicity values between
0.1 and 1.0 mg/L, and only 8% had toxicity values > 10 mg/L. Nine classes of water
soluble cationic polyelectrolytes were identified, though data was collected on only
six of them. No correlation was reported between toxicity and molecular weight,
charge density or water hardness (Cary et al. 1989). The data have not been published.

To further support the case for mitigation, and therefore reduced data requirements for
PMNs, four cationic polyelectrolytes were tested in reconstituted laboratory water
with humic acid, fulvic acid, tannic acid lignin, and lignosite at concentrations of 5,
10, 20 mg/L were used for four compounds (quaternised polyethanolamine, molecular
weight 25,000 amu; poly(dimethylvinyl-pyridinium) chloride, molecular weight
≈ 1,200,000 amu; dimethylamine-epichlorohydrin copolymer, molecular weight
100,000 amu), and 5, 10, 20 mg/L for high charge density polymers
(epichlorohydrin/amine polymer, molecular weight 2-3000 amu). Again, a reduction
in the toxicity of around 2 orders of magnitude was reported. Further, a direct linear
relationship between LC50 and DOC was reported from regression analysis (Cary et
al. 1989). As a result of the efforts by CFPA, test procedures incorporating the
mitigating effect of humic acid were incorporated into the USEPA’s procedure for
environmental impact assessment (Cary et al. 1989). Humic acid was chosen as the
most representative DOC because of its median response and ‘ubiquitous’ nature
(Cary et al. 1989).

44
Having established cationic polyelectrolytes as a distinct class of polymers, further
studies on the toxicity of cationic polyelectrolytes have focused on the relative
toxicity of the polymers based on structural features such as molecular weight and
charge density, that is, within-class variation in toxicity. The results have been
somewhat contradictory. In a study to evaluate the effect of chemistry, charge density
and molecular weight on acute and chronic toxicity (Goodrich et al. 1991) found
useful comparisons were made difficult by variability in replicate data and the small
data sample. The polymers selected for this study included three
epichlorohydrin/dimethylamine polymers of varying molecular weight (up to 250,000
amu), and two acrylamide polymers (acrylamide/2-(N,N,N)-trimethyl ammonium
ethylacrylate chloride) with different charge densities. The polyelectrolytes were all
relatively toxic, with only the low charge density acrylamide polymer having a LC50
> 1.0 mg/L (≈ 1.7 mg/L). The authors did report a tendency of increasing toxicity with
increasing molecular weight. This was most noticeable in the chronic studies, due to
the replicate variability in the acute studies. What is important, however, is the low
acute to chronic LC50 ratios, with most mortality in the long-term test occurring in the
first seven days. The mitigating effects of humic acid were again reported with a 7 to
16-fold decrease in toxicity with as little as 5 mg/L humic acid. Although this study
included chronic studies, the mitigating effect of humic acid was not established in the
chronic tests.

A much larger study of 34 water treatment polymers, including 24 cationic

polyelectrolytes, appears to have been more successful in finding factors controlling
toxicity (Hall and Mirenda 1991). The cationic polyelectrolytes included in the study
were poly(methacryloyloxy ethyltrimethyl ammonium chloride) (METAC), AETAC,
dimethylamine-epichlorohydrin polymer EPI/DMA, poly(DADMAC), Mannich
amine (cationic tertiary amines), and one melamine-formaldehyde (MF) copolymer.
While the METAC, AETAC and DADMAC polymers are expected to be quaternary,
the degree of substitution of the EPI/DMA and MF polymers is not specified. Two
species, Daphnia pulex and Pimephales promelas (fathead minnow) were used in the
tests. There was an apparent increase in toxicity with increasing charge density
evident in the fish data for the METAC and AETAC polymers. The LC50 for fish for
the melamine-formaldehyde copolymer was > 170 mg/L, and this compound was also
only moderately toxic to Daphnia (12.31 mg/L). The Mannich amine polymers were

45
significantly less toxic to Daphnia, (LC50 42-70 mg/L) than to fish (1.1-3.3 mg/L).
The authors suggest that polymer chemistry is the controlling factor in toxicity of the
cationic polyelectrolytes to Daphnia, while toxicity to the minnow appeared to be
related to charge density. However, analysis of this data by Cumming et al. (2005)
using Pearson Correlation Coefficient (SAS 2002) shows that while fish LC50 is
correlated with charge density for AETAC polymers (0.83682, p = 0.0049), it was
less correlated for METAC polymers (0.8077, p = 0.0982) and not at all for all the
cationic polyelectrolytes (0.08799, p = 0.6827). There are two possible explanations
for this outcome. Either chemistry is also important in toxicity of cationic
polyelectrolytes to fish, or toxicity becomes asymptotic relative to charge density
above a certain value.

Further toxicity data for water treatment polymers were reported Beim and Beim.
(1994) and Fort and Stover (1995). In one study higher bioactivity with increasing
charge density was reported for three polymers, but no correlation with molecular
weight. Species including saprophytic bacteria, planaria and gammaridae were found
to be less susceptible than fish and Daphnia (Beim and Beim. 1994). Although LC50
values for algae were not published, they were found to be less sensitive than
Daphnia, but more sensitive than fish. In a comparison of the toxicity of four water
treatment polymers (including 3 polyquaterniums) and two inorganic flocculants,
ferric chloride and aluminium sulphate, to Daphnia, the polymers were found to be
significantly more toxic than the inorganic flocculants (Fort and Stover 1995).

A summary of the published toxicity values for cationic polyelectrolytes is given in

Appendix 2.

2.5.4. Meta-analysis
Meta-analysis is a technique of combining independent but related published studies
using statistical methods to synthesise results with the aim of evaluating results in
ways that may not have been possible in the original studies. Published data can also
be re-analysed using statistical techniques that may not have been available or
employed at the time the data as published. Review studies of cationic
polyelectrolytes have been published and provide suitable data for examination of the
role of polymer characteristics in toxicity. Boethling and Nabholz (1997) published
toxicity data for about 50 cationic polyelectrolytes that had been submitted with

46
PMNs to the OPPT under TSCA. Based on their interpretation of this data, the authors
suggested the following:

a) aquatic toxicity is strongly influenced by cationic charge density and type of

polymer backbone;

b) aquatic toxicity increases ‘exponentially’ with higher charge density until

toxicity becomes ‘asymptotic’;

c) aquatic toxicity is not influenced by pH dependence (degree of substitution),

position of cations (backbone or pendant) or molecular weight;

d) effect of molecular weight may be greatest with relatively high surface to

volume ratios of organisms: algae > daphnids > fish (Boethling and Nabholz
1997).

Establishing any relationships between polymer characteristics and toxicity based on

this published data is problematic. The data set is not balanced in terms of treatments
(polymer characteristics) and the data is neither independent nor normally distributed.
It should be possible, however, to determine if the conclusions that have been drawn
from the data are reasonable.

Importantly, it can be shown that there are significant correlations between the
polymer characteristics themselves. For example, in the OPPT data (Boethling and
Nabholz 1997), cationic polylectrolytes with natural (cellulosic) backbones tend to
have medium to high molecular weight and low charge density, while silicone based
polycations have low molecular weights and low charge densities. Synthetic carbon
polymers have an approximately equal distribution of molecular weights and charge
densities. Specifically, using correlation analysis (SAS 2002) it can be shown that
there is a small, but significant, negative correlation (-0.43811, p = 0.0008) between
charge density and molecular weight in the OPPT data. As can be seen from the
simple scatter plot of the two variables in Figure 2.6, the high variability in molecular
weight at low charge density narrows considerably as charge density increases.

47
 8

7
log(Molecular Weight)

2
0 5 10 15 20 25
Charge Density (%a-N)

Figure 2.6 Scatter plot of charge density (as % amine-Nitrogen) against log molecular weight for
all cationic polymers in the data submitted with PMNs to OPPT (Boethling and Nabholz 1997).

Correlations in the toxicity of the cationic polyelectrolytes also exist between species.
Fish toxicity is strongly correlated with both algal (R = 0.89134, p < 0.0001) and
daphnid toxicity (0.77621 p < 0.0001), but there is no correlation between toxicity to
algae and to Daphnia (-0.07406 p = 0.7548). Regression analysis shows that the
relationships between fish toxicity and algal toxicity, (algal toxicity = 0.97954*fish
toxicity + 5.85969; R2 = 0.7945, p < 0.0001) and fish toxicity and daphnid toxicity
(Daphnia toxicity = 0.64458*fish toxicity + 24.92703; R2 = 0.6358, p < 0.0001) are
significant.

There is a small, negative (-0.32483) but significant (p = 0.0214) correlation between

fish toxicity and charge density in the re-analysis of the OPPT data. There is no
significant correlation between charge density and either daphnid toxicity or algal
toxicity. Boethling and Nabholz (1997) presented Structure Activity Relationships
(SAR) for predicting toxicity of cationic polyelectrolytes with natural or synthetic
carbon backbones, in which the polymers are sorted into groups with charge density
≥ 3.5% a-N and < 3.5% a-N. Grouped in this manner, charge density group was

48
significant for all species in a nonparametric rank sums (Kruskal-Wallis) test. The
significant correlations in these data sets appear to be the result of a small number of
very low charge density polymers with very low toxicities. Importantly, this data
shows that high charge density polycations are almost always very toxic, but low
charge density polycations are still frequently toxic (Table 2.2). There is a significant
correlation between log molecular weight and acute and chronic algal toxicity
(0.61299, p = 0.0089, n = 17; and 0.61168, p = 0.0118, n = 16 respectively) but not
fish or daphnid toxicity in the TSCA data.

Table 2.2 The number of high and low charge density polycations by Globally Harmonised
System for Classification and Labelling of Chemicals (GHS) classification based on the OPPT
data in Boethling and Nabholz (1997).
>100 mg/L Harmful Toxic Very toxic Total
Fish:
High charge density 1 0 2 21 24
Low charge density 5 5 6 10 26
Daphnia:
High charge density 0 1 4 10 15
Low charge density 6 6 1 6 19
Algae:
High charge density 1 0 0 12 13
Low charge density 2 0 3 4 9

Lyons and Vaconcellos (1997) reviewed toxicity data for 61 polycations, drawn from
unpublished studies by Betz Laboratories, and published papers including Hall and
Mirenda (1991), Goodrich et al. (1991) and Cary et al. (1987). In a meta-analysis of
this data (SAS 2002), the relationship between charge density (ionicity) and molecular
weight was found to be significant in a Kruskal-Wallis test (p < 0.0001), and in a
generalised linear model (in which molecular weight is given as a class variable only)
molecular weight was found to account for about 48% of the variation in percent
ionicity. There was no correlation between fish toxicity and daphnid toxicity. There
was a correlation between percent ionicity and daphnid toxicity (r = 0.30772, p = 0.
0265), but not fish toxicity. As the correlation between percent ionicity and daphnid
toxicity is positive, LC50 is increasing, and therefore toxicity decreasing, as charge
density increases. The statistical significance in this data set, in contrast to the OPPT
data, appears to result from a small number of relatively very toxic, high charge
density polymers. Analysis by non-parametric rank (Kruskal-Wallis) indicates a
significant relationship between molecular weight and fish toxicity, but not daphnid
toxicity. Details of the chemistry were not given for the OPPT data, but the

49
polyelectrolytes in Lyons and Vaconcellos (1997) are grouped by chemistry. The
mean LC50 values for the chemistry groups from the data are given in Figure 2.7. Only
those groups with n ≥ 5 are shown. The particular use of each polyelectrolyte
(flocculant or coagulant) is also provided in this data, and appears to have a
significant effect on toxicity (Figure 2.8).

4.5
4
3.5
Median LC50

3
2.5 Daphnia
2 Fish
1.5
1
0.5
0
METAC AETAC EPI/DMA DADMAC Mannich
Chemistry

Figure 2.7 Median LC50 (mg/L) of water treatment cationic polyelectrolytes by chemical class
based on the data in Lyons and Vaconcellos (1997). The median daphnid LC50 for Mannich
polymers (48.95 mg/L) is not shown due to scale.

16

14

12
Median LC50

10
dapnia
8
fish
6

4
2

0
coagulants flocculants

Figure 2.8 Median LC50 (mg/L) of water treatment cationic polyelectrolytes by use as coagulants
or flocculants based on the data in Lyons and Vaconcellos (1997).

50
In summary, from published data, toxicity seems to be a complex function of the
polymer architecture, and only influenced to a lesser extent by individual polymer
characteristics such as charge density, molecular weight or type of polymer backbone.
The strong relationship between use and toxicity suggests that mode of action in water
treatment and mode of action in toxicity might be related. However, the interpretation
of the data by Boethling et al (1997), as outlined at the beginning of this section, that
aquatic toxicity is strongly influenced by cationic charge density and type of polymer
backbone appears to be unsupported even by the data published in their own studies,
and generally unsupported by other studies (Cary et al. 1987; Hall and Hall 1989).
This interpretation does not seem to provide an adequate basis for the risk assessment
of the aquatic toxicity of water treatment polymers, and perhaps even less so for
cosmetic polycations.

2.5.5. Mechanism
The mechanism of toxicity of surfactants results from binding at the cell surface,
causing membrane disruption and protein denaturation which leads to necrosis of the
exposed tissue (Juergensen et al. 2000). Cationic surfactants used in disinfection
disrupt the organisational structure of the bacterial cell membranes, altering
membrane permeability and causing cell leakage and lysis (Pelczar et al. 1993).
However, the mode of action of cationic polyelectrolytes to fish has been suggested to
result from sorption of the polymer to the gills resulting in suffocation (Brocksen
1971), although possible interference in ion exchange mechanisms has also been
suggested (Goodrich et al. 1991). Biesinger and Stokes (1986) examined the gills of
exposed minnows microscopically. The gills of the controls showed a regular filament
structure with red blood cells in the lamellar capillaries and a thin layer of respiratory
epithelium covering the lamellae. A summary of the observations of the exposed fish
is given below in Table 2.3. According to the authors, death results from suffocation
though toxic action by oligomers could also occur.

51
Table 2.3 Histological observations of fish gill tissue exposed to cationic polyelectrolytes
(Biesinger and Stokes 1986).
Concentration Time Observations
0.5 mg/L 24 hours lamellar epithelium thickened with a fuzzy
appearance, either from mucous or the polyelectrolyte
96 hours lamellar wall still visible along the gill filament, cells
accumulating in the interlamellar spaces, tips of the
filaments covered by infiltrating cells
1.0 mg/L 24 hours marked increase in interlamellar mass, white blood
cells, mucous cells and chloride cells present
48 hours cell masses extended to the tips of lamellae in some
areas. Lamellae still free of cell infiltration were
thickened and bent.
2.0 mg/L 24 hours cellular infiltration, particularly at the tips of
filaments, which were full of cells and covered with a
layer of epithelium, branching filament structure still
visible, but capillary spaces and red blood cells not
apparent, mucous cells evident near filament tips.
3.0 mg/L 24 hours surviving fish had very little lamellar structure, entire
filament was a mass of cells surrounded by a layer of
epithelium, areas within the filament had no definable
structure left, cell fragments and debris present and
accumulated on the surfaces and between the gill
filaments.

Examination of the tissue of rainbow trout following exposure to 14C labelled cationic
polyelectrolytes found significantly higher concentrations in the gills than in skin,
muscle or viscera (Muir et al. 1997). The fish were exposed at a concentration high
enough to elicit mortality under longer exposure conditions but not cause death in the
exposure period used in the study. The concentration of 14C in the gill was 10 times
higher than in other tissues mentioned above for an epichlorohydrin-dimethylamine
polymer, and 50 times higher for a cationic polyacrylamide and a cationic
polyacrylamide ester polymer. Fish exposed to high concentrations of the
epichlorohydrin-dimethylamine polymer until loss of equilibrium occurred (40 to 45
minutes) were found to have significantly decreased blood pH (from 7.1 to 6.6),
decreased sodium and chloride concentrations, and elevated concentrations of
potassium ions and total ammonia. Decreased levels of sodium and chloride and
increased ammonia concentration indicate a disruption of the ionic regulatory function
of the gill, which combined with impaired respiration, may cause lethality. Elevated
potassium concentration is thought to result from haemolysis caused by the reduction
in blood pH (Muir et al. 1997).

52
The depuration of the polymer, as loss of 14C from the gill, in water following short
one hour exposures then depuration for 6 hours was rapid for both cationic
polyacrylamide (half life 2.8 hours) and cationic polyacrylamide ester (2.6 hours). For
epichlorohydrin-dimethylamine, removal was somewhat slower, with a half life of 5.7
hours. The presence of humic acid in the water during depuration did not significantly
alter the depuration time for the polyacrylamides (3.4 and 3.5 hours respectively), but
removal of epichlorohydrin-dimethylamine was more rapid (half-life 1.2 hours).
There was no accumulation in tissues following three repeat exposures of three hours
followed by 24 hour depuration (Muir et al. 1997).

While it is feasible to extrapolate the mode of action to all organisms that have a gill
structure like fish, cationic polyelectrolytes are also expected to sorb strongly to other
biological membranes that are anionic, such as some bacterial surfaces (Boethling and
Nabholz 1997). However, it has been suggested that other factors may be important
for some smaller organisms. Cary et al. (1987) observed the mechanism of toxicity of
cationic polyelectrolytes to daphnids and reported that Daphnia acted as sites of
flocculation and were physically clumped together or entrapped within the floc.
Consequently, the toxicity did not follow a typical dose-response curve. Survival rates
increase above the optimum treatment dose due to the polymers’ tendency to
resuspend the floc. Mortality due to physical entrapment or clumping of daphnids was
also noted by Hall and Mirenda(1991), who attributed wide confidence intervals
around the LC50 values in this study to this phenomenon.

2.6. Summary
The above review shows that unlike compounds used in applications such as water
treatment, polyquaterniums used in cosmetic applications may not be released to
wastewater as a charged polycation. Cosmetic polyquaterniums are formulated in an
excess of anionic surfactant, deposited onto hair or skin as a polymer-surfactant
complex, and are desorbed from the hair or skin in subsequent washing with an
anionic surfactant. The chemical species or form released, therefore, is most likely to
be a charge-neutral polyquaternium/surfactant complex. Unlike water treatment
polycations, this complex is film-forming rather than floc forming and may have very
different environmental characteristics than the charged polycation. In particular, rates
of sorption and desorption may be significantly different for the polymer-surfactant
complex. While anionic species, particularly anionic polymers, have been shown to

53
mitigate the toxicity of some industrial polycations, the toxicity of the
polyquaternium/surfactant complex to aquatic organisms is not known.

54
3. Analysis of Polyquaterniums
3.1. Introduction
Many techniques have been developed for analysing polyelectrolytes in relatively
clean water. These techniques include turbidimetry/nephelometry, spectrofluorometry,
spectrophotometry, viscometry, colloid titration, luminescence titration, gel
permeation chromatography, bromine oxidation of primary amides, and
radioimmunoassay (Wickramanayake et al. 1987). Some of these clean water
analytical techniques are limited by their specificity to certain types of
polyelectrolytes, for example, the bromine oxidation for polyelectrolytes containing
primary amide functional groups, and radioimmunoassay for polyacrylamides. More
recent developments, such as the use of NMR (Chang et al. 2002) are also limited, so
far, to polyelectrolytes with trimethyl quaternary ammonium pendant functional
groups.

Fluorescent tagging, the attaching of a fluorescent chromophore to a polyelectrolyte,

has also been used in some studies of cationic polyelectrolytes. Fluorescently tagged
polyelectrolytes can be detected qualitatively by fluorescent microscopy (Regismond
et al. 1999b) and quantitatively by luminescence spectroscopy (Bennett et al. 2000).
The method has been used to study the sorption of cationic polyelectrolytes on hair
(Regismond et al. 1999b), polymer-surfactant interactions (Ananthapadmanabhan et
al. 1985; Winnik and Regismond 1996; Morishima et al. 1999) and as an indicator in
polyelectrolyte titration (Tanaka and Sakomoto 1993).

Fluorescently tagged poly(DADMAC) has also been suggested for possible use in the
determining of residual polymer after flocculant dosing in water treatment (Bennett et
al. 2000). Unlike the studies of sorption of polyquaterniums on hair using
Polyquaternium-10, which needs no modification prior to the addition of the
fluorescent tag, poly(DADMAC) has to be polymerised with an amine-functional
monomer at 1-2% charge in order to be tagged. This does not, however, amount to a
substantial change to the polymer, and does not change its legal identity. Of two
fluorescent tags trialled in the study by Bennett et al. (2000), one was found to be
quenched by the presence of organic carbon in the water. Many fluorescent indicators
are also quenched by the presence of surfactants, hence their use in the study of
polymer-surfactant interactions, and this may limit the usefulness of the method in
determining residuals following wastewater treatment.

55
 3.1.1. Metachromasy
The most widely used method of analysis of polyelectrolytes in the literature is colloid
titration, which is based on a phenomenon known as metachromasy. This method
arose from the observation of the behaviour of certain aniline dyes used in the staining
of histological samples (Bergeron and Singer. 1958). A history of the development of
the method in histology is given in Bergeron and Singer. (1958), who proposed the
following definition:

Metachromasy is the hypsochromic (shift in absorption to shorter

wavelength) and hypochromic (decrease in intensity of colour) change in
colour exhibited by certain basic aniline dyes in the presence of water and
under the following conditions:

a) increase in dye concentration;

b) temperature decrease;

c) salting out;

d) interaction with certain substrates whose metachromatic influence may

be due to serially arranged proximate anionic sites.

Metachromasy has been adapted for the measurement of concentration and charge
density of polyelectrolytes when used in conjunction with analytical techniques such
as colloid titration.

3.1.2. Colloid Titration

Colloid titration is the name given to the quantitative volumetric analysis of
polyelectrolytes in solution (Terayama 1952). The titration is possible because ionic
reactions between oppositely charged polyelectrolytes in dilute solutions are
stoichiometric by charge and very rapid (Terayama 1952). The endpoint of colloid
titration has been determined by changes in turbidity (Hanasaki et al. 1985),
streaming current detection (Kam et al. 1999), ion-selective electrodes (Séquaris and
Kalabokas et al. 1993), conductometric methods (Ghimici and Dragan 2002), and by
colour change of a metachromatic dye (Wang and Shuster 1975). The method has also
been used for charge determination of proteins (Horn and Heuck 1983) and cell
surfaces (Watanabe and Takesue 1976; Van Damme et al. 1994), as well as for
determining polyelectrolyte concentration in water (Wang and Shuster 1975; Parazak
et al. 1987; Majam and Thompson 2006). In the latter capacity it has also been used

56
for sorption studies. In these, the sorbed concentration was determined by difference
between the starting and equilibrium concentrations {Hutter, 1991 #100}. However, it
has limitations in some water industry applications due to interference by organics
(Hanasaki et al. 1985) and inorganic ions (Sjöedin and Öedberg 1996).

Cationic metachromatic dyes, for example o-toluidine blue, are useful indicators for
colloid titration as they do not interact with cationic polyelectrolytes due to
electrostatic repulsion, but produce a distinct metachromatic colour shift from blue to
red-violet on binding with anionic polyelectrolytes. The method, therefore, allows for
the direct titration of cationic polyelectrolytes, while anionic polyelectrolytes need to
be back-titrated with a cationic standard (Ueno and Kina 1985). In colloid titration
using a metachromatic dye as an endpoint indicator, the reaction between the cationic
analyte and anionic titrant is favoured over the reaction between the titrant and the
indicator, so the coupled reactions occur consecutively (Horn and Heuck 1983). Once
the cationic polyelectrolyte is reacted, the chromotropic polyanionic titrant begins to
combine with the dye, resulting in a colour change that is clear and instant according
to Terayama (1952).

3.2. Metachromatic Polyelectrolyte Titration

While colloid titration with metachromatic dyes has been in use for nearly fifty years,
there are still many unresolved issues in the method. The determination of the
concentration of a polyelectrolyte in water by titration relies on the measurement of
the amount of cationic or anionic functional groups present in the solution, and thus
determines the concentration of the solution in terms of equivalents per litre, i.e. the
normality of the solution. The method has been applied to charge determination not
only of water treatment polyelectrolytes (Dentel 1989; Kam et al. 1999), but also to a
variety of bio-molecules such as bovine pancreatic chymotrypsin A, bovine
ribonuclease A, porcine pepsin, equine heart muscle cytochrome c. (Horn and Heuck
1983), bone cartilage (Van Damme et al. 1992), heparin (Katayama et al. 1978),
together with humic acid and the extracellular polymers of activated sludge
(Mikkelsen 2003). Other colloids whose normality may be determined by colloid
titration include cellulose sulphate, plant mucilages (gum Arabic, gum tragacanth,
agar agar), lignin, and clay, all of which are anionic (Terayama 1952; Ueno and Kina
1985). While there are fewer positively charged colloids, derivatives of chitosan and

57
some proteins, such as clupein and salmine (Terayama 1952) have been measured
with polyelectrolyte titration.

Subsequently, in this work the term Metachromatic Polyelectrolyte Titration will be

used to refer to the analysis of polyelectrolytes using a metachromatic indictor, in
preference to the somewhat misleading term of Colloid Titration (Tanaka and
Sakomoto 1993).

3.2.1. The Titrant – Choice of Chromotropic Polyanion and

Cationic Standard
The most commonly used chromotropic polyanion in the literature is the potassium
salt of poly(vinylsulphate) (PVSK, sometimes also abbreviated as PVS-K or KPVS).
Dextran sulphate has also been used (Van Damme et al. 1992). In some cases, PVSK
is used as supplied (Horn and Heuck 1983). However, as commercial PVSK may be
rather impure and can leave some solid residue when dissolved in water (Kam et al.
1999), filtration of the gelatinous residue is recommended (Dentel 1989). If the PVSK
has not been filtered, the equivalence of the PVSK solution can be estimated from the
mass and monomer molecular weight (Horn and Heuck 1983; Ueno and Kina 1985),
however, PVSK does not serve as a good primary standard and standardising the
solution is often recommended. Standardisation is usually performed by titration with
a cationic surfactant such as with Zephiramin (tetradecyltrimethylbenzylammonium
chloride) (Katayama et al. 1978), cetyltrimethylammonium bromide (CTAB) (Kam et
al. 1999), or cetylpyridinium chloride (Ueno and Kina 1985). These cationic
surfactants give reproducible solution concentrations and have a defined molecular
weight, and are therefore used as primary standards.

For the determination of anionic polyelectrolytes by back-titration, the most

commonly used cation is polyDADMAC, (as cat-floc) (Katayama et al. 1978; Horn
and Heuck 1983; Van Damme et al. 1992; Mikkelsen 2003). Protamine sulphate
(Watanabe and Takesue 1976) and 1,5-dimethyl-1,5-diazaundecamethylene
polymethobromine (DDPM) (Wang and Shuster 1975) have also been used.

3.2.2. The Indicator – Choice of Metachromatic Dye

The most commonly used indicator in metachromatic polyelectrolyte titration is o-
toluidine blue (Figure 3.1), at a concentration of between 0.01 and 0.1% w/v. Other
metachromatic dyes include brilliant cresyl blue and methylene blue (Terayama
1952). Orthochromatic dyes, which do not change colour, but nevertheless undergo a

58
change in intensity of colour, can also be used in polyelectrolyte titration. Although
not suitable for visual titration, the change in intensity can be measured
spectroscopically. An example of an orthochromatic dye which can be used in this
manner is crystal violet (Masadome 2003). Anionic dyes have also been used to
directly measure concentrations of cationic polymers, for example, use of trypan blue
to measure the concentration of Polyquaternium-1 (Polyquad®) in a solution of
contact lens cleaner by Stevens and Eckardt, (1987).

H3C N Cl

+
H2N S N CH3
CH3
Figure 3.1 Structure of the metachromatic dye o-toluidine blue, a commonly used indicator in
metachromatic polyelectrolyte titration.

3.2.3. Determination of The Visual Endpoint

The colour change of the metachromatic dye on binding with the chromotropic
polyanion is usually described as a change from blue to red-violet (Watanabe and
Takesue 1976; Katayama et al. 1978; Ueno and Kina 1985; Kam et al. 1999),
however, it has also been described as a change to purple (Dentel 1989; Van Damme
et al. 1992), reddish-purple (Terayama 1952), bluish purple (Wang and Shuster 1975)
and pink (Sjöedin and Öedberg 1996). The colour change is generally considered
distinct (Wang and Shuster 1975; Kam et al. 1999), however the solution is
sometimes described as becoming suddenly colourless prior to the colour change
(Ueno and Kina 1985). This transition phase is usually attributed to the flocculation of
the neutral colloid (Wang and Shuster 1975; Ueno and Kina 1985). According to
Terayama (1952), in cases where the metachromatic change is uncertain, this
precipitation can be used to determine the endpoint. The endpoint is said to occur
when the solution remains red-violet for a few seconds (Ueno and Kina 1985), or
when the colour persists on the further addition of the titrant (Dentel 1989).

3.2.4. Determination of the Endpoint Using Spectrophotometry

The maximum absorption peak of the unbound o-toluidine blue has been determined
as 635 nm (Horn and Heuck 1983; Kam et al. 1999) or 620 nm {Hutter, 1991 #100},
(Mikkelsen 2003). The red-violet bound form peak is around 530 nm (Kam et al.
1999) or 550 nm (Horn and Heuck 1983). During the initial stage of the titration,

59
when the titrant is binding with the free polycations, little or no change in absorption
occurs. A break point occurs when the titrant begins to react with the dye, and again
when the dye is completely reacted. Consequently there are three potential end-points
on the titration curve, the upper and lower break points, and the inflection point based
on the logistic dose response equation (Kam et al. 1999). The lower break point
approximately corresponds to the final colour change in the visual titration (Figure
3.2).

0.3

0.25
Break Point
0.2
Absorbance

Inflection point
0.15
Clear and colourless
0.1 Pink

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Volume PVSK (mL)

Figure 3.2 A sample plot of a spectroscopic titration of a polyquaternium (UCareTM JR125, 6.5
mg/L) with PVSK and o-toluidine blue at wavelength 630 nm showing breakpoint, inflection
point, and colour changes. From the beginning of the titration to the break point, the added
PVSK reacts with the polyquaternium, from the break point to the final colour change, the
PVSK reacts with the indicator o-toluidine blue and from the final colour change, and no
reactions are taking place.

While it is possible to follow the titration at the wavelength of the absorption peak of
either the unbound initial colour or the bound dye species’ emerging colour, the
former is generally preferred as it is more distinct. In an example of the use of the first
break point in Figure 3.3, from Mikkelsen (2003), the absorption at 620 nm is plotted
against time in a controlled delivery titration. The intersection of two straight lines on
the first and second segments of the titration curve is taken as the endpoint, which
corresponds to the point at which the PVSK begins to react with the o-toluidine blue
as all the cationic polymer is used up.

60
 Figure removed, please consult
print copy of the thesis held in
Griffith University Library

Figure 3.3 The method of endpoint determination used by Mikkelsen (2003). The absorbance is
plotted against time in a controlled automatic titration where the concentration of the PVSK in
the reaction chamber is directly proportional to the titration time. The endpoint is determined as
the intersection of two straight lines corresponding to the first and second stages of the titration
reactions.

In an example of the inflection point method, Figure 3.4, three straight lines have been
drawn on each of the three segments of the titration curve, and the endpoint is taken as
the midpoint on the second line between the intersection with the first and second
points {Hutter, 1991 #100}. Horn and Heuck (1983) followed the titration at both 550
and 635 nm with a double beam spectrophotometer, and plotted the relative
absorbance of the two wavelengths (Figure 3.5). The inflection point in this plot
corresponds to the point where rate of change between the absorbance at the two
wavelengths is greatest. According to Kam et al. (1999), deduction of a blank is not
required, however, both Hutter et al. (1991) and Mikkelsen et al. (2003) deducted
blanks in their calculations.

61
 Figure removed, please consult
print copy of the thesis held in
Griffith University Library

Figure 3.4 Determination of endpoint using the inflection point (Hutter et al. 1991) where the
endpoint is determined as the ‘point lying midway between lines drawn tangent to the baselines’,
that is, the inflection point.

Figure removed, please consult

print copy of the thesis held in
Griffith University Library

Figure 3.5 Determination of endpoint from relative absorbance at 550 and 635 nm (Horn and
Heuck 1983) with the endpoint determined to be the inflection point of the metachromatic shift.

3.2.5. Calculations – Determining Charge Density and/or

Concentration
The calculation of the concentration of the unknown analyte is based on the known
equivalence of titrant, according to Equation 3.1 (Wang and Shuster 1975).

62
 N PVSKVPVSK
Na = Equation 3.1
Va
where Na is the normality of the cationic analyte in eq/L
NPVSK is the normality of the titrant in eq/L
VPVSK is the volume of the titrant solution to endpoint
and Va is the volume of the analyte solution titrated.

The charge density of the cationic polymer/colloid can be determined from the
nominal equivalence of the PVSK (6.17 meq/g) if the solution has not been
standardised (Horn and Heuck 1983). This assumes that the PVSK is fully dissociated
and completely dissolved. Alternatively, the equivalence of the PVSK can be
determined by standardisation on titration with a cationic surfactant as mentioned
above (Dentel 1989).

3.2.6. Method Validation

The results of charge density determination using metachromatic polyelectrolyte
titration have been found to be within the range of charge density quoted by the
polymer manufacturer (Kam et al. 1999), and consistent with the results from other
methods such as the Zeisel method of hydroxyethyl molar substitution and Kjeldahl
determination of nitrogen content (Hutter et al. 1991), streaming current detection
(Kam et al. 1999), turbidimetric and electrochemical titration (Koetz et al. 1996) and
1
H NMR determination of quaternary methyl substituted ammonium functional
groups (Chang et al. 2002). A detection limit of 10-5 eq/g was reported by Mikkelsen
(2003).

In addition, the surface charge determined for proteins using this method has been
shown to have good agreement with the literature values measured by alternative
methods (Van Damme et al. 1992). For the determination of concentration of
polyelectrolyte solutions, metachromatic polyelectrolyte titration has been found to be
suitable for concentration ranges of 10-3–10-4 N (Terayama 1952), and 5 x 10-3 –
2 x 10-4 N (Wang and Shuster 1975). A linear relationship between concentration of
the titrant and amount of polyquaternium was established for concentrations of
polymer between 0.05 and 0.20% w/v, provided the amount of polyquaternium in the
aliquot did not exceed 400 µg (Hutter et al. 1991).

3.2.7. Problems and Limitations

For polyquaterniums a constant charge density should be observed across a wide
range of solution pH values (Katayama et al. 1978; Horn and Heuck 1983; Dentel

63
1989). If the polyelectrolyte is not quaternised, the charge density will vary with
solution pH, with ionicity inversely proportional to pH. It is recommended that the
titration be performed in solutions adjusted to pH 3, 5, 7 and 9 with NaOH or HCl
(Dentel 1989). Likewise, for the determination of charge density of anionic polymers
by back-titration, pH should be monitored and adjusted throughout the titration, as the
charge density of anionic polyelectrolytes is directly related to solution pH, with
maximum ionicity occurring at high pH (Dentel 1989).

The presence of other electrolytes in the solution can interfere with metachromatic
polyelectrolyte titration as they inhibit the complexation of o-toluidine blue and
PVSK, making the determination of the endpoint difficult (Sjöedin and Öedberg
1996). While the titration is not affected by non-electrolytes such as sucrose, glucose
or glycerol at concentrations up to 30%, it is not possible in the presence of > 0.3%
NaCl (Katayama et al. 1978). According to Ueno and Kina (1985), uncharged organic
molecules do not interfere up to concentrations of 10%; NaCl does not interfere up to
0.1%; but electrolytes involving ions of higher valency such as CaCl2 can seriously
interfere and their concentration should be kept below 0.005%. It has been suggested
that the critical electrolyte concentration above which titration is impossible is
roughly proportional to 10z where z is the valency of the cation (Sjöedin and Öedberg
1996). The presence of electrolytes may be overcome by increasing the concentration
of o-toluidine blue; however if the concentration of the indicator exceeds 20 µM,
insoluble precipitates can be formed between o-toluidine blue and PVSK (Sjöedin and
Öedberg 1996).

Steric effects of macromolecules may also interfere with the stoichiometric reaction of
polyelectrolytes in solution (Terayama 1952). It has been suggested that chain
flexibility favours 1:1 stoichiometry (Kam et al. 1999), however Koetz et al. (1996)
found variation from 1:1 stoichiometry with increasing spacer length between charge
centres. Steric hindrance resulting from increasing alkyl chain length surrounding the
ammonium group of polyquaterniums (for example, from methyl to ethyl groups) also
resulted in deviations from 1:1 stoichiometry (Koetz et al. 1996). Increasing the
spacer length between charges, and/or increasing the length of the alkyl chain attached
to the charged centre, can result in increased hydrophobicity of the polyelectrolyte
(Koetz et al. 1996). When 1:1 stoichiometry cannot be established, the possibility that
there are untitratable cationic sites exists, and accordingly the measured equivalence

64
of the polyelectrolyte should be considered a measure of the ‘free’, ‘dissolved’ or net
charge of the polyelectrolyte (Wang and Shuster 1975).

3.2.8. Aims and Objectives

From among the various methods published for cationic polyelectrolytes, to determine
a procedure suitable to the polyquaterniums being used in this study. In this study, it
would be necessary to determine the charge density of the polymer, thereby allowing
unknown concentrations of the polyquaterniums to be determined by measurement of
the charge in the solution, that is, the normality of the solution. To adequately address
the behaviour of the polymer-surfactant complex, it would be necessary for the
analytical method to work in the presence of an anionic surfactant. Further, it would
be useful to determine if the method could be made to work in the presence of an
organic contaminant such as humic acid.

The aims of this section are:

• To determine the appropriate parameters for the analysis of cosmetic

polyquaterniums by metachromatic polyelectrolyte titration;

• To determine the appropriate method of endpoint determination to facilitate

the determination of the polyquaternium concentration of a solution by
metachromatic colloid titration;

• To determine if metachromatic colloid titration could be carried out in the

presence of anionic surfactants and humic acid.

3.3. Analytical Methods

3.3.1. Materials
Water: All water used in the preparation of solutions, in titrations and for final rinsing
of glassware was Milli-Q™ Ultrapure water, with resistivity 18 MΩ·cm (0.06 µS).

Glassware: To prevent sorption of the polyquaterniums to the surface of storage and

reaction vessels, glassware was treated with Coatasil Glass Treatment containing 1,1-
dichloro-1-fluoroethane 98% w/w and dimethyldichlorosilane 2 % w/w. The
glassware was first acid washed, air dried, rinsed five times with demineralised water
and five times with Milli-Q™ water and air dried. It was then coated with the
silanising solution and allowed to dry for 24 hours. It was rinsed in demineralised
water until foaming ceased (20+ times), rinsed again with Milli-Q™ water, air dried

65
and acid washed again. Silanised glassware was acid washed between each use, and
retreated after six months.

Polyquaterniums: Samples of six variations of Polyquaternium-10 of varying

molecular weight and charge density were provided by Amerchol (The Dow Chemical
Company, Midland, MI USA), and five samples of three polyquaterniums
(Polyquaternium-11, Polyquaternium-28, Polyquaternium-55) were provided by
International Specialty Products (ISP, Wayne, New Jersey, USA) (Table 3.1).
Amerchol’s UCareTM polyquaterniums (JR125, JR 400, JR 30M, LR400, LR30M and
LK) were supplied in powder form as 90% active substance. The ISP polymers
Conditioneze® NT-20 (Polyquaternium-28) and Gafquat® HS100 were supplied as
viscous solutions in water as 19-21% active substance by weight. Gafquat® 734 and
Gafquat® 440 were supplied as viscous solutions in 48-52% and 68-72% ethanol
respectively. Polydimethyldiallyl ammonium chloride (poly(DADMAC),
Polyquaternium-6) was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).
All the polyquaterniums, except poly(DADMAC) were commercial grade, as reagent
grade versions of these polyquaterniums were not available for purchase.

Table 3.1 The cosmetic polyquaterniums used in this study, supplied by Amerchol and ISP. The
water treatment polyquaternium polyDADMAC, which is also sometimes occurs in cosmetic
formulations, was also used. Both International Nomenclature of Cosmetic Ingredients (INCI)
and trade names are given.
Supplier INCI Name Trade Name(s) Abbreviation used.
Amerchol Polyquaternium-10 UCareTM JR125 JR125
UCareTM JR400 JR400
UCareTM JR30M JR30M
UCareTM LR400 LR400
UCareTM LR30M LR30M
UCareTM LK LK
ISP Polyquaternium-11 Gafquat® 734 G734
Gafquat® 440 G440
Polyquaternium-28 Gafquat® HS-100 HS100
Conditioneze NT-20 NT20
Polyquaternium-55 Styleze W-20 W20

Stock solutions were prepared in 100 mL silanised volumetric flasks at concentrations

of 10 g/L active substance. The polyquaternium as supplied was weighed directly into
the volumetric flask and approximately 90 mL Milli-Q™ water added. It was placed
on a magnetic stirring plate for at least 24 hours. The solution was then slowly made
up to the final volume, with repeated shaking by hand, until the volume remained
consistent, and was allowed to settle before use. The solutions were stored in 100 mL

66
silanised Schott bottles. Working solutions (1:10 dilution) were prepared, again in
silanised 100 mL volumetric flasks. The working solutions were stored in 100 mL
Nalgene® bottles. Subsequent dilutions were prepared in silanised volumetric flasks
as required. Wherever possible, transfers and measurements were made with plastic
tip automatic pipettes. Where the use of glass volumetric or graduated pipettes was
unavoidable, the first uptake of the solution was discarded.

PVSK: Solutions of PVSK were prepared by adding approximately 0.5 g of PVSK to

one litre of Milli-Q™ water and stirring on a magnetic plate for at least 24 hours, but
generally significantly longer. The solution was then filtered under vacuum using
Whatman 41 filters, which were changed approximately every 100 mL. A significant
difference was found in the solubility of two successive batches of PVSK. Solutions
made from the first batch were slow to filter, with significant amounts of gelatinous
material removed during filtering. The measured normality of these solutions,
determined by titration with cetylpyridinium chloride, was generally between 8% and
10% of the theoretical normality. The second batch of PVSK purchased filtered
quickly, with no observable material removed during filtering. The measured
normality of the solution was over 50% of the theoretical normality.

Cetylpyridinium chloride: Approximately 0.07 g cetylpyridinium chloride was

weighed and the actual mass recorded, then added to 1 L Milli-Q™ water in a
silanised volumetric flask and stirred with a magnetic stirrer for approximately 3
hours, or until there were no visible solids in the solution. A fresh solution was made
for each equilibration of PVSK and kept for no more than 2 days.

o-toluidine blue: The indicator was prepared by adding approximately 0.1 g of the
solid into a 100 mL volumetric flask, with 100 mL Milli-Q™ water and stirred with a
magnetic stirrer for at least 1 hour, ensuring that no solids were observed when the
flask was inverted.

Sodium dodecyl sulphate: Sodium dodecyl sulphate was prepared by the addition of a
pre-weighed amount to Milli-Q™ water in a 1 L volumetric flask and stirring for 2–3
hours. Generally, the solution was prepared to match the normality of the
polyquaternium solution with which it was being used.

67
 3.3.2. Standardisation of PVSK
The normality of the PVSK solutions was determined by titration with
cetylpyridinium chloride. Cetylpyridinium chloride solution (10 mL) and o-toluidine
blue solution (0.5 mL) were placed in a silanised conical flask on a magnetic stirring
plate with approximately 20 mL of Milli-Q™ water. PVSK solution was added very
slowly drop-wise from a graduated 10 mL burette until a colour change from blue to
pink was observed (Figure 3.6). The solution was allowed to stand for several minutes
to ensure a return to blue did not occur. Instability of the colour change can occur if
the titration is conducted too quickly. Generally, three titrations were performed;
however, an additional three titrations were performed if the variation between the
titrant volumes was greater than 0.2 mL. A blank titration of the indicator was also
performed, and the blank titration deducted from the cetylpyridinium chloride titre.

3.3.3. Titration of Polyquaternium Solutions (Visual Endpoint)

Between 5 and 15 mL of the polyquaternium solution was placed in a silanised
conical flask with 0.5 mL o-toluidine blue solution and the volume made up to
approximately 30 mL with Milli-Q™ water. PVSK solution was added very slowly
drop-wise from a graduated 10 mL burette until a colour change from blue to pink
was observed, as for the standardisation of PVSK. The solution was allowed to stand
for several minutes to ensure a return to blue did not occur. A blank titration of the
indicator was also performed, and the blank titre deducted from the polyquaternium
titre.

68
Figure 3.6 The visual titration of a polyquaternium with PVSK and o-toluidine blue, showing the
initial blue colour of the solution (left) and the pink colour at the endpoint (right).

3.3.4. Charge Density Determination of Polyquaterniums

(Preparation of the Standard Curve)
The charge density of the polyquaterniums was determined by visual titration with
PVSK using o-toluidine blue as an indicator. The method of titration is the same as
that used for the standardisation of PVSK. Three dilutions of the 1 g/L working
solution (30, 65 and 100 mg/L) and a blank were prepared and titrated with a
standardised PVSK solution. The normality of each solution was determined from the
normality of the PVSK using Equation 3.2.

(VPVSK − Vblank ) × N PVSK Equation 3.2

N Pq =
VPq
Where:
NPq is the normality of the polyquaternium solution in eq/L.
VPq is the volume of the polyquaternium solution in mL
NPVSK is the normality of the PVSK solution eq/L
VPVSK is the volume of the PVSK solution added in mL.

The equivalence of the three solutions was plotted against the concentration in g/L of
the polyquaternium solution and the line of best fit determined. The slope of the line
is the equivalent weight of the polyquaternium. The concentration of unknown
polyquaternium solutions can then be determined from the equivalent weight using
Equation 3.3.

69
 Equation 3.3
CPq = N × EqW

Where:
N is the normality of the solution in eq/L
CPq is the concentration in g/L
EqW is the equivalent weight of the polyquaternium in g/eq

3.3.5. Titration (Spectrophotometric Endpoint)

For the spectrophotometric titration, solution concentrations of 3, 6.5 and 10 mg/L
were prepared. The solution (2.5–3.0 mL) was added to a 1 mm plastic cuvette with
40 μL of o-toluidine blue. The solution was placed in the spectrophotometer and
titrated with the PVSK solution in situ using a Gilson repeater micropipette. The
PVSK (N ≈ 10-4 eq/L) was added in aliquots of 5 µL and the solution mixed with a
plastic transfer pipette. A reading was taken after each addition.

In this work, the endpoint of the spectrophotometric titration was established by

plotting the absorbance of the solution against the titrant volume. A Matlab® program
was written (Matthews 2006) to fit curves to the two sections of the titration curve,
representing the two stages of the titration process: 1) the binding of the PVSK titrant
to the polyquaternium analyte, and 2) the binding of the metachromatic dye, o-
toluidine blue to the excess PVSK. The former was fitted as a straight line (Equation
3.4) and the latter as a four-parameter logistic model (Equation 3.5). The endpoint
was the point where the two curves intersect, representing the point at which the
polyquaternium is fully bound to the PVSK titrant (Figure 3.7). The equivalence of
the polymer is determined in the same manner as for the visual titration, i.e. from the
volume of PVSK at the endpoint, according to Equation 3.2.

y ( x) = mx + c
Equation 3.4

Where:
y(x) = absorbance
x = volume of PVSK (ml)
c = initial absorbance
φ2 − φ1
y ( x) = φ1 +
⎡ (φ − x ) ⎤ Equation 3.5
1+ exp ⎢ 3 ⎥
⎣ φ4 ⎦

Where:
φ1 = the horizontal asymptote as x → ∞

70
 φ2 = the horizontal asymptote as x → −∞
φ3 = the value of x at the inflection point. (At this value of x the response is
midway between the asymptotes)
φ4 = a scale parameter on the x axis.
(Pinheiro and Bates
2000)

0.35
experimental data
fitted curve
0.3

0.25
Absorbance

0.2

0.15

0.1

0.05

0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
PVSK (ml)

Figure 3.7 Matlab® plot of the titration of a polyquaternium with PVSK and o-toluidine blue,
showing the intersection of the fitted curves of the straight line and four-parameter logistic
model, indicating the different stages of the titration, and showing the break point (c) and
inflection point (U).

3.4. Results
3.4.1. PVSK
Solutions of PVSK (Sigma-Aldrich), prepared by dissolving 0.4–0.5 g of the dry
powder in 1 L Milli-Q™ water, have a nominal normality of 2.46–3.85 meq/L.
However solutions prepared from the first batch of PVSK were found to have
normality of between 0.186 and 0.786 meq/L. This was due to the large amount of
gelatinous material removed from the solution during filtration. Solutions prepared
from the second batch of PVSK filtered quickly and no gelatinous material was
observed on the filters, indicating that this batch of PVSK had dissolved more
thoroughly. The normality of these solutions when standardised was found to be

71
between 0.59 and 1.78 meq/L. These solutions were diluted and recalibrated before
use. Solutions of PVSK were found to be very stable. Generally, solutions were used
within one month, with re-calibration indicated if the blank titration varied more than
10% from the initial blank titration. Only one solution was used for more than one
month, and was recalibrated twice. The normality of the solution was found to be
stable over that time.

3.4.2. Analysis of Results for Equivalent Weight

Generally, new solutions of polyquaterniums were made for each new experiment.
Where possible, as many experiments as possible were conducted using the same
solution. As can be seen from Table 3.2, subsequent batches of the same
polyquaternium did not always have the same equivalence, although the results were
reasonably consistent given the difficulties in preparing dilutions from highly viscous
solutions. An example of a plot used to determine the equivalent weight of a
polyquaternium sample is shown in Figure 3.8. Analysis of the results using a
generalised linear model with Tukey test for multiple comparisons which included all
samples was significant (F = 20.70, Pr > F <0.0001), with poly(DADMAC) being
significantly different to all the personal care polyquaterniums. Removing
poly(DADMAC), and looking only at the personal care polyquaterniums, the Tukey
test showed a significant difference between Styleze® W-20 and the low charge
density UCareTM LR/LK range, Gafquat® HS100 and Conditioneze® NT-20, and
significant differences also existed between UCareTM LK and the JR polymers, and
between JR30M and LR40M (F = 7.81, Pr > F = 0.0016).

72
Table 3.2 The gram equivalence of the polyquaternium as determined by visual titration of three
solutions and a blank with PVSK and o-toluidine blue.
Polymer Equivalent R^2
Weight (eq/g)
Gafquat® 440 Polyquaternium-11 0.0009 0.9587
Gafquat® 734 0.001 0.9982
Gafquat® 734 Polyquaternium-11 0.0009 0.9985
Gafquat® HS100 Polyquaternium-28 0.0009 0.8810
Gafquat® HS100 0.0007 0.9845
Gafquat® HS100 0.0007 0.9875
UCareTM JR125 Polyquaternium-10 0.0009 0.9982
UCareTM JR125 0.0009 0.9982
UCareTM JR125 0.0011 0.9985
UCareTM JR30M Polyquaternium-10 0.0010 0.9974
UCareTM JR30M 0.0014 0.9987
UCareTM JR400 Polyquaternium-10 0.0012 0.9391
UCareTM LK Polyquaternium-10 0.0003 0.9810
UCareTM LR30M Polyquaternium-10 0.0004 0.9976
UCareTM LR400 Polyquaternium-10 0.0007 0.9903
UCareTM LR400 0.0006 0.9941
Conditioneze® NT-20 Polyquaternium-28 0.0007 0.9716
Conditioneze® NT-20 0.0006 0.9915
poly(DADMAC) Polyquaternium-6 0.0048 0.9868
poly(DADMAC) 0.0050 0.9803
poly(DADMAC) 0.0070 0.9943
Styleze® W-20 Polyquaternium-55 0.0011 0.9983
Styleze® W-20 0.0012 0.9986
Styleze® W-20 0.0014 0.9967

1.E-04
9.E-05 y = 0.0009x
8.E-05 R2 = 0.9982
Volume PVSK (N)

7.E-05
6.E-05
5.E-05
4.E-05
3.E-05
2.E-05
1.E-05
0.E+00
0 0.02 0.04 0.06 0.08 0.1 0.12
[JR125] (g/L)

Figure 3.8 Example of a plot of the results of titrations of three concentrations of UCareTM JR125
with PVSK and o-toluidine blue used to determine the charge density of the polyquaternium
from the slope of the line of best fit.

73
 3.4.3. Titration in the Presence of SDS
The equivalence of the polymer solutions was not altered by the presence of the
anionic surfactant SDS. Titrations were possible with the polyquaternium:surfactant
ratio of approximately 1:1 and in a large excess of surfactant (1:4). Examples of these
results are given in Figure 3.9. However, it was observed that the colour change was
not as stable when the surfactant was present, with the colour returning to blue after a
period of several minutes.

1.4
(a) (c)
(b)
1.2

1 (a) (b) (c)

0.8
meq/L

0.6

0.4

0.2

0
W20 JR125

Figure 3.9 Comparison of the titration of two polyquaternium samples, Styleze® W-20
(Polyquaternium-55) and UCareTM JR125 (Polyquaternium-10), (a) alone and in the presence of
sodium dodecylsulphate at (b) 1:1 stoichiometry and in excess (c) (1:4 stoichiometry).

3.4.4. Titration (Spectrophotometric Endpoint)

By following the titration at the higher wavelength of 630 nm, which records the loss
of blue colour, and the lower wavelength of 512 nm, which records the emergence of
the red/violet colour, it was found that a clearer endpoint was produced when the
titration of the higher wavelength was plotted (Figure 3.10).

74
 0.35

0.3 630nm

0.25
Absorbance

Blank
0.2

0.15

0.1 512nm

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Volume PVSK (ml)

Figure 3.10 Spectrophotometric titration of a solution containing UCareTM JR125

(Polyquaternium-10) (6.5 mg/L) at 630 nm (■, recording the loss of blue colour) and 512 nm (▲,
recording the emergence of the pink colour) and the titration of a blank sample at 630 nm (¡).

The data from the titration of each concentration, a graphical example of which is
given in Figure 3.11, was analysed in the Matlab® program to determine the
endpoint, as described in Section 3.3.4. Using this method, it was not possible to
determine the charge density of the UCareTM LR and LK (Polyquaternium-10), as no
clear endpoint was discernable; however, the method was successful for all other
polyquaterniums (Table 3.3)

0.3

0.25

0.2
Absorbance

Blank
0.15 3 mg/L
6.5 mg/L
0.1
10 mg/L
0.05

0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
Volume PVSK (ml)

Figure 3.11 A plot of the spectrophotometric titration of a blank solution and three
concentrations of Conditioneze® NT-20 with PVSK and o-toluidine blue at 630 nm.

75
Table 3.3 Equivalence of some polyquaterniums determined from the spectroscopic titration and
using the Matlab® method to determine the tipping point.
Polyquaternium Equivalent weight (meq/g) R2
Conditioneze® NT-20 0.6 0.9988
UCareTM JR400 0.1 0.9999
UCareTM JR125 1.0 0.9937
UCareTM JR30M 1.1 0.974
UCareTM HS-100 0.8 0.9355
Gafquat® 734 1.2 0.9598

3.5. Discussion
Repeat titrations of the same solutions gave consistent results, however some
variations occurred between different solutions of the same polyquaternium made to
the same concentration. These differences were probably due to the difficulties in
measuring and mixing the polyquaternium solutions, which were often highly viscous.
During the method development stage of the project, solutions were kept for several
months, with no alteration to titrated normality of solutions. A cotton-like floc was
observed in some of the UCareTM cellulosic polyquaterniums. A similar precipitate
has been described by Hattori et al. (1997) in solutions containing an ion complex of
poly(vinyl sulphate) and poly(vinylamine) during potentiometric titration. However,
the precipitate observed in the UCareTM cellulosic polyquaterniums in this work
occurred in standard solutions of the polyquaternium but only at relatively high
concentrations (10 mg/L active substance).

The use of a non-silanised flask during visual titration resulted in a 10% reduction in
the volume of PVSK required to complete the titration. In the spectrophotometric
titrations, the use of a glass pipette for stirring was found to have a significant effect
on the outcome of the titration. Although the phenomenon of cationic polyelectrolyte
sorption to glass has been well studied (Goddard and Chandar 1989; Poptoshev and
Claesson 2002), it does not seem to have been investigated as a possible cause of the
poor success rate in the titration of low charge density polyelectrolytes, or low
concentrations of polyelectrolyte. In addition to problems with the loss of
polyquaterniums to glass surfaces, difficulties were encountered with the adsorption
of the metachromatic indicator to, and subsequent staining of, glass surfaces. For this
reason alone, plastic cuvettes and transfer pipettes were preferred, as stained
glassware needed regular cleaning in chromic acid.

76
It has been recommended that the titration of polyelectrolytes be timed (Dentel 1989).
If the titration is conducted too rapidly, a colour change may result that is not stable,
with the return to the blue colour indicating that the polyelectrolyte was not fully
bound to the PVSK. Some binding of the PVSK to the indicator may occur before the
titre is fully reacted, with equilibrium being achieved only after several seconds.
Although this colour reversal was observed during some titrations, it was not found
necessary to time the titrations in this study. However, the titrations were conducted at
the slowest possible addition rate, and the solution allowed to stand for a few minutes
after completion to ensure that equilibrium had been reached.

The deduction of the blank titre, though not strictly necessary, nevertheless provided
specific advantages in terms of the calculation of the solution normality and for
quality control during the titration. In the latter case, the blank titration provided an
indication of the stability of both dye and titrant. Variations in the amount of titrant
used in the blank can indicate changes in titrant and/or indicator that may affect the
outcome of the titrations. Generally, variations of more than 10% in the blank titration
were taken to indicate unacceptable changes in the titration conditions. Further, the
blank titration provided a check on the acceptability of the lowest titrated
concentration. Generally, the titrant volume at end point of 2.5 times the blank
titration was considered desirable for the lowest concentration. If the titrant volume
was less than 2.5 times the blank titrant volume, the volume of the solution being
titrated was increased.

No precipitation was observed in any titrations. Solutions were always blue and clear,
never blue and turbid. The turbidity resulting from sudden precipitation just prior to
the endpoint has been suggested as a method of aiding in endpoint detection
(Terayama 1952), however, it has also been suggested that the disappearance of
colour just prior to the endpoint is also a result of ‘coagulation of flocculant
precipitates’ (Ueno and Kina 1985). By monitoring the colour during titration with the
spectrophotometer, it was observed that the solution became clear consistently at
approximately the same absorption, regardless of other conditions, such as
concentration of the titre, concentration of the titrant, volume of solution. The clear
stage before colour transition was also observed in blank titrations. Only in high
concentrations of the indicator, when initial absorbance was greater than about > 0.5
was no clear stage observed in the titrations. It is suggested, therefore, that the clear

77
stage observed prior to colour change is the result of the hypochromic shift that occurs
on the binding of PVSK to o-toluidine blue.

The endpoint in all visual titrations in this study is most suitably described as pink,
and followed a stage in which the solution changed from blue to clear. Solutions at all
stages were clear rather than turbid. Of the two breakpoints in the spectroscopic
titration, which coincide with changes in the reaction processes, the first breakpoint
was the more distinct and was used in this study for the determination of the
spectroscopic endpoint. There was good agreement between the endpoint determined
by visual titration with the blank subtracted, and the first break point in the
spectroscopic titration. Both these points represent the stage in the titration where the
polyquaternium and the PVSK are fully reacted, and the PVSK begins to bind with
the indicator, o-toluidine blue. Unlike more conventional acid-base titrations, where
the titration curve is very steep and there is no intermediate stage in the titration, the
inflection point on the polyelectrolyte does not correspond to any significant chemical
process in the titration reaction and is therefore not particularly useful as an endpoint
when there is a better one available.

As previously mentioned, the equivalence of the polyquaternium in this study was

determined from the normality of the titre solutions according to Equation 3.3. An
alternative method, where the amount of polymer is plotted against the volume of the
titrant, and the equivalence determined from the slope of the regression line was used
by Hutter et al. (1991). In addition to being mathematically more complex, this
method allows less flexibility in the titration procedure, as it requires that the volume
of the titre be identical for all concentrations in a series. In the method used in this
study, the volume of the titre could be varied to ensure the lowest concentration was
significantly differentiated from the blank, and the maximum concentration titratable
within the volume of the burette. Consequently, a wider range of concentrations could
be titrated. While it is not strictly necessary to have a wide range of concentrations for
the determination of charge density, such variation can and does occur in the
applications where concentration is being determined for difference studies.

The method of direct titration using trypan-blue (Stevens and Eckardt 1987) was
found to be non-linear and variable at the concentration range required for this study.
Polyquaternium-1, the polyelectrolyte used in the study by Stevens and Eckardt
(1987) is a very high charge density polyelectrolyte, with two charge centres on each

78
monomer, which may account for its titratability at the low concentrations (0.0005 to
0.0015% m/V).

In order to investigate the behaviour of the polyquaternium in the presence of the

surfactant, it was necessary to be able to determine if the metachromatic
polyelectrolyte titration could be performed in the presence of the surfactant. The
presence of SDS, either at 1:1 stoichiometry or in excess, had no effect on the titration
of the polyquaternium with PVSK. The binding of the polyquaternium with PVSK,
and of PVSK with o-toluidine blue, were therefore found to be preferred to
association with the surfactant. However, the colour change in the presence of the
surfactant was not as stable as the colour change in the standard titration (though it
was sufficiently stable for the determination of the endpoint), indicating that some
desorption/dissociation of the polyquaternium and PVSK in the presence of the
surfactant could occur over time. It was possible, therefore, using this method, to
determine the concentration of polyelectrolyte in solution when SDS was present, but
not the extent to which it was complexed with the surfactant.

It was not possible to conduct the titration in solutions of either polyquaterniums that
had been exposed to bentonite clay, or polyquaterniums prepared in a solution
containing tap water, such as the medium used in the fish toxicity tests in Chapter 5.
In each case, no colour change occurred, indicating interference in the reaction
between the PVSK and o-toluidine blue.

3.6. Conclusion
Metachromatic polyelectrolyte titration was found to be a reliable method for
determining the charge density of polyquaterniums, and for subsequent determination
of concentration of unknown solutions of polyquaterniums. The most suitable
endpoint in visual titration was found to be the point at which colour change is
complete. With the deduction of a blank titration, this point was consistent with the
first breakpoint in the spectrophotometric titration. In this study, metachromatic
polyelectrolyte titration was effective for concentrations as low as 10-4 N with the
visual endpoint, and 10-5 N with the spectrophotometric endpoint.

The most significant disadvantages of the method are its inability to distinguish
between polyelectrolytes, and the interference of other ions. The method is limited to
solutions in simple matrices, and therefore is not suitable for the determination of

79
polyelectrolytes in environmental samples. While it may be possible to achieve
further improvements in the concentration range for metachromatic polyelectrolyte
titration using automated titration techniques and improved mathematical models for
endpoint detection, the development of methods able to overcome the problems of
electrolyte interference, and to distinguish specific cationic polyelectrolytes, would be
more useful in environmental studies of polyquaterniums. However, metachromatic
polyelectrolyte titration is a useful method for laboratory analysis of polyelectrolytes.

80
4. Chemistry and Fate – Exposure Assessment of
Polyquaterniums
4.1. Introduction
In the previous chapter, a method of measuring the concentration of polyquaterniums,
at least in the laboratory, was described. This method can be used to investigate some
aspects of the behaviour of polyquaterniums that may influence their fate in the
environment. As outlined in Chapter 1, the exposure assessment step of the four-step
risk assessment paradigm uses various measurements and/or models to estimate the
contact that may occur between a chemical and a vulnerable organism in the
environment.

Exposure assessment examines the contact between an organism and a chemical in

terms of the intensity, frequency and duration of the contact (USEPA 1998). The site
of interaction between the organism and the chemical is somewhat controversial, and
can be taken to mean either at the outer visible boundary, the visible exterior of the
organism (the skin and openings into the body, such as mouth, nostrils) or the so-
called exchange boundaries where absorption takes place (skin, lung, gastrointestinal
tract) (USEPA 1992). Exposure assessment can also evaluate the rate at which a
chemical crosses the boundary. In the former case, the exposure can be expressed as a
concentration of the chemical in the exposure medium, such as air, or water. In the
latter case, the exposure can only be expressed in terms of dose, and must take into
account the rates of uptake and absorption (USEPA 1992). Generally, the exposure
assessment should be expressed in units that allow it to be combined with the hazard
assessment. If the hazard assessment is expressed in terms of dose or internal
concentration, then the exposure assessment should also be expressed in terms of dose
or internal concentration. Fortunately, aquatic toxicity is usually expressed in terms of
concentration of the chemical in the aquatic environment, and the exposure can thus
be expressed as the aqueous concentration of the chemical in receiving waters.

Where an environmental risk assessment is undertaken in response to an existing

exposure, the extent of the exposure can be directly measured. However, in some
cases, such as the assessment of a new chemical or where analytical methods are not
available, the exposure must be estimated by means of models. Where the exposure is
to be expressed in terms of concentration in the exposure medium, the exposure can
be estimated with fate and transport models based on use and release data and the

81
physico-chemical properties of the chemical. This is the approach adopted by
National Industrial Chemicals Notification and Assessment Scheme/Department of
Environment and Water Resources (NICNAS/DEW) in the assessment of new
chemicals, as can be seen from the published reports (NICNAS 2004b). This
assessment takes into account relevant properties of the chemical that may determine
fate (volatility, aqueous solubility, etc), use patterns and potential release estimates to
predict an environmental concentration. These estimates also take into account basic
(human) population data and the water use patterns of the population.

4.2. Use patterns and release data

An important first step in modelling the predicted environmental concentration is to
determine the amount of the chemical being used. However, unless required by
regulation to publish such data, many companies regard volume data as commercially
confidential information. In new chemical notifications to NICNAS, use and
manufacture/import volume is a requirement for all notifications (a Part B data
requirement), however, the guidance notes for notifiers states

‘In some cases, for example, “Maximum Introduction Volume of Notified

Chemical (100%) Over Next 5 Years”, the exact details can be claimed as
confidential; however, a generic or sanitised description is required for the
public report’ (NICNAS undated).

Of the five published assessment reports of polyquaterniums (Table 4.1), only one
claimed confidentiality as evidenced by this standard statement in the Full Public
report (FPR)

The chemical name, CAS number, molecular and structural formulae,

molecular weight, spectral data, details of the polymer composition and
details of exact import volume and uses have been exempted from
publication in the Full Public Report and the Summary Report. (NICNAS
2002a).

The stated import volume in this notification is < 16 tonnes. It may be considerably
less than 16 tonnes, but not less than one tonne, and certainly cannot be more than 16
tonnes. Three of the notifications are ‘limited’ notifications, implying that the import
volume is less than one tonne pa (it cannot exceed one tonne pa without further
notification). The remaining report is a standard notification (volume > 1000 kg), no

82
confidentiality is claimed, and the volume is stated as “up to” 5 tonnes. As the stated
volume is a legal limit imposed by the certificate conditions, the import volume of this
polyquaternium cannot exceed that amount. There are a further 18 polyquaterniums
on the Australian Inventory of Chemical Substances AICS for which no assessment
reports are available, so no import data, ‘generic’ or otherwise, is available on these.

Table 4.1 Confidentiality status and import volume details from NICNAS FPR for assessed
polyquaterniums.
Notification Product Notifier Confidentiality Maximum
Volume
(tonnes pa)
NA89 Polyquaternium- ISP Corp No 5
28 (Gafquat®
HS100)
NA475 Polyquaternium- L’Oreal Paris No <1
34
NA533 Polyquaternium- BASF No <1
46 Australia Ltd
NA896 Polyquaternium- Nalco Yes < 16
47 (Merquat Australia Pty
2001) Ltd
NA961 Polyquaternium- Johnson and No <1
44 (Luviquat Johnson
Care) Pacific Pty
Ltd

With respect to existing chemicals, NICNAS is able to acquire import data on

chemicals as part of the evaluation of a potential Priority Existing Chemical
declaration under Section 48 of the Industrial Chemicals (Notification and
Assessment) Act (ICNA). When deciding whether to make a recommendation for the
declaration of a chemical as a Priority Existing Chemicals, the Director may require
additional information. This may include:

a) specified information about a particular chemical, such as use and health

and/or environmental effects;

b) the quantities of the chemical used for a specified purpose in a specified

period, for example, use in cosmetics over the past three years;

c) the quantities of the chemical manufactured or imported in a specified

period (CofA 1989).

Following declaration, information required to be submitted by companies importing

and/or manufacturing, or intending to manufacture or import the chemical, either as is

83
or part of a mixture, is much the same as for a new chemical, and includes use,
volume and release data. However, polyquaterniums are not currently listed as
candidate chemicals for Priority Existing Chemical assessment.

If a chemical is imported or manufactured in a significantly high volume, companies

may be required to report the volume of their manufacture or import to NICNAS for
inclusion on the High Volume Industrial Chemicals List (HVICL). Companies are
required to report volumes greater than 20 tonnes pa (under new guidelines) or 100
tonnes pa (under the previous guidelines), or 100 tonnes pa as part of a mixture. The
chemicals are listed by name, CAS number, industrial category and use (the latter two
selected from a list), and reported in threshold categories 1,000 to 9,999 tonnes pa,
10,000 to 999,999 tonnes pa and > 1,000,000 tonne pa. The current list has only
chemicals imported in volumes greater than 100 tonnes pa (per importer), however
future lists will include chemicals imported at the lower volume of 20 tonnes pa (per
importer). An extract from the list, showing the type of data available is given in
Table 4.2.

Table 4.2 An extract from High Volume Industrial Chemical List maintained by NICNAS to
record the volumes of industrial chemicals manufactured in or imported into Australia in
quantities > 1000 tonnes pa (NICNAS 2002b).
Chemical/Chemical CAS No Use Category Industry Category
Group Name

Threshold Range : Between 1,000 and 9,999 Tonnes/Year

1,2,3- 77-92-9 Cleaning/washing Chemical industry:

Propanetricarboxylic agents and basic chemicals
acid, 2-hydroxy- additives (supply)
Cosmetics Domestic/Cleaners
Others Leather processing
pH-regulating industry
agents Textile processing
Tanning agents industry

1,2,3-Propanetriol 56-81-5 Cleaning/washing Chemical industry:

agents and basic chemicals
additives (supply)
Complexing agents Chemical industry:
Construction chemicals used in
materials additives synthesis
Cosmetics Domestic/Cleaners
Explosives Mineral oil and fuel
Others industry

84
 Softeners Plastics industry
Solvents

No polyquaterniums, either for personal care use or water treatment use, currently
appear on the list. As the list has no provision for classes of compounds, only any
individual polyquaterniums imported at greater than 20 tonnes pa (per importer) will
be on future lists.

The Australian Bureau of Statistics (ABS) collects some data on some products,
including chemicals, imported into, but not manufactured in, Australia. The relevant
categories that may include some polyquaterniums are given in Table 4.3. However,
as the import quantity is reported as a dollar value for the first four categories, there is
no way of determining the polyquaternium volume from this data (ABS 2006).

Table 4.3 Some categories for which data is collected on imported products by ABS.
Code Description
3305100024 Shampoos
3305200025 Preparations for permanent waving or straightening of hair
3305300026 Hair lacquers
3305900027 Preparations for use on the hair (excl. shampoos,
preparations for permanent waving or straightening,
lacquers)
3307100008 Shaving preparations
3307100009 Pre-shave or after-shave preparations
3402120059 Cationic organic surface-active agents, whether or not put
up for retail sale
3402200017 Liquid form preparations (incl. surface-active, washing and
auxiliary washing and cleaning), put up for retail sale (excl.
organic surface-active agents)
3402200018 Other preparations (incl. surface-active, washing and
auxiliary washing and cleaning), put up for retail sale (excl.
those in liquid form and organic surface-active agents)

Although the NICNAS data is somewhat incomplete, it provides the only basis for
estimation of the volume of polyquaternium use in Australia. An estimate of the total
volume imported can be made by assuming that the volumes given for the five
polyquaterniums for which assessment reports are available are representative of the
import volumes for all the polyquaterniums on AICS. The estimate, as shown in Table
4.4, is based on the assumption that the import volume for Polyquaternium-10 is

85
somewhat higher than the estimated import volume for Polyquaternium-28, and that
the remaining polyquaterniums have import volumes of ≤ 1 tonne. Although some of
these 17 polyquaterniums may not currently be in use, or not used in cosmetic
applications, some, such as Polyquaternium-11, may have import volumes
approaching that of Polyquaternium-10. It is conservatively estimated, based on the
declared import volumes for polyquaterniums for which certificates have been issued,
that the amount of polyquaterniums manufactured in or imported into Australia is
between 30 and 60 tonnes pa.

Table 4.4 Estimation of volume of polyquaterniums imported or manufactured based on

estimates given in NICNAS New Chemical Notifications and the number of polyquaterniums
already listed on AICS.
Data source Basis of estimate Maximum Conservative
(tonnes pa) (tonnes pa)
limited 3 @ < 1 tonne pa 3 1.5
notifications
standard PQ-28 up to 5 tonnes 5 2.5
notifications
PQ-47 < 16 tonnes 16 8
on AICS PQ-10, assuming highest market 20 10
share
all others (17 ≡ Limited 17 8.5
notifications)
TOTAL 61 30.5

4.2.1. Emission Scenarios

An alternative method of estimating release of chemicals is to use an emission
scenario based on the use of the chemical. In the case of personal care chemicals, this
would require an estimate of the amount of the product containing the chemical used
on a per capita basis. The EC Guidance document, ‘Emission scenario for
personal/domestic chemicals’ gives such an emission scenario for the prediction of
post-consumer release to sewer in ‘assessment of the environmental release of soaps,
fabric washing, dish cleaning and surface cleaning substances’ (ECB 2003), where the
consumption of shampoo is estimated at 2.3 g/capita.day. The release of ingredients in
these products can be determined based on their percentage in the product.

To estimate the amount of polyquaternium used and released, based on this default
shampoo use, it is necessary to make assumptions about the market share of PCPs
containing polyquaterniums. By assuming that consumers use either a shampoo
containing a polyquaternium, or an equivalent product (conditioner or styling agent)

86
containing a polyquaternium, several scenarios, outlined in Table 4.5, can be devised.
It should be noted that polyquaterniums appear in a range of products other than
shampoos and conditioners, such as body scrubs, shaving preparations etc. Although
some people will be using none of these products, others will use several of them.

Table 4.5. Estimation of volume of polyquaterniums based on the EC Guidance document,

Emission scenario for personal/domestic chemicals (ECB 2003).
Emission scenario Use % in Population Total Use Total
per product (g) Use
day (tonnes
(g) pa)
Scenario 1. Low 2.3 0.1 2.00E+07 16790000 16.8
concentration in product,
full population coverage
Scenario 2. Low 2.3 0.1 1.00E+07 8395000 8.4
concentration in product,
50% population coverage
Scenario 3. Medium 2.3 0.25 2.00E+07 41975000 42.0
concentration in product,
full population coverage
Scenario 4. Medium 2.3 0.25 1.00E+07 20987500 21.0
concentration in product,
50% population coverage
Scenario 5. High 2.3 0.5 2.00E+07 83950000 84.0
concentration in product,
full population coverage
Scenario 6. High 2.3 0.5 1.00E+07 41975000 42.0
concentration in product,
half population coverage

Given that the lower estimates from this method are not supported by the import
certificate coverage above, the medium to high estimates would seem to be more
realistic. There is a convergence in use in Australia data between the two methods of
estimation at between 30 and 60 tonnes pa.

4.3. Environmental Fate

4.3.1. Partitioning
A partition coefficient (KD) is the ratio of the activity of a substance in two phases and
provides a clear indication of how a substance will migrate from one environmental
phase to another (Samiullah 1990). At low concentrations, concentrations (expressed
as a mole fraction) are equal to activity. Sorption to solids is probably the most
important fate for cationic polyelectrolytes, and therefore the partitioning of these
polymers between aqueous and solid phases, for example, wastewater influent and

87
sludges, or wastewater effluent and stream suspended solids is fundamental to
modelling the environmental concentrations of polyquaterniums (Equation 4.1).

CS Equation 4.1
KD =
CW

where CS is the concentration of the substance on the sorbent

CW is the concentration of the substance in the aqueous phase

As mentioned in Section 2.5.3, humic acid has been selected by United States
Environmental Protection Agency (USEPA) as representative of dissolved organic
carbon (DOC) sources in natural waters for the purposes of determining the mitigating
effect of dissolved solids on the toxicity of cationic polyelectrolytes (Nabholz 1991).
Humic acid accounts for between 60 and 80% of DOM in surface waters (Matthews et
al. 1995), and between 40 and 60% of wastewater solids (Narkis and Rebhun 1983).
Other components of wastewater solids include fatty acids 8.3%; anionic detergents
13.9%; carbohydrates 11.5%; proteins 22.4%; tannins 1.7% (Narkis and Rebhun
1983). For these reasons, humic acid has been selected in this study to represent the
sorbent in wastewater sludges and surface receiving waters. Humic acid is a high
molecular weight anionic polyhydroxycarboxylate, comprised of polyaromatic and
aliphatic subunits. The degree of ionisation of this anionic polyelectrolyte is governed
by the amount of ionised phenolic and carboxylic groups of the humic core which is
pH dependent (Schmitt-Kopplin et al. 1998). Humic acid and wastewater sludges are
characterised by relatively high negative zeta potential (≈ -25 mV) and low isoelectric
point (Kerr et al. 2000; Mikkelsen 2003).

The aim of the following experimental section, therefore, is to determine the partition
coefficients of the polyquaterniums to humic acid. Polyquaterniums in solution will
be exposed to humic acid, and the uptake of polyquaternium from solution to the solid
phase will be determined by difference, using the metachromatic colloid titration
method described in the previous chapter. In later sections of this chapter, the partition
coefficients will be used in a fugacity model to estimate the partitioning of
polyquaterniums to the solid phase in Wastewater Treatment Plants (WWTPs).

4.3.2. Methods
Materials: All chemicals used were as described previously in Section 3.3.1.

88
Preparation of Humic Acid: As commercial humic acid produces a coloured solution
in water, metachromatic colloid titration cannot be used to analyse polyquaternium
concentrations without prior treatment. To separate out the water-colouring material,
the humic acid (Fluka, technical grade) was repeatedly washed in Milli-Q™ water.
When the water appeared clear following centrifugal separation of the water and
solids, the solid residual humic material was collected, filtered and dried, and stored
in an air-tight stoppered glass bottle for use in the partition experiments. The collected
supernatant with the dissolved coloured fraction of the humic acid from the repeated
washings (which was used in the toxicity experiments) and the filtered, dried humic
acid were analysed for particle size and total organic carbon content. Particle size
analysis was performed on a Multisizer™ 3 Coulter Counter.

Preparation of Solutions: A polyquaternium solution at each required concentration

was prepared in Milli-Q™ water. Polyquaternium-surfactant solutions were prepared
by mixing equal volumes of the polyquaternium solution with an SDS solution of the
same equivalence. The resulting solution was gently mixed on a rotary mixer prior to
addition to the exposure container.

Polyquaternium Sorption to Humic Acid: The humic acid (0.02 ± 0.002 g) was
weighed directly into 50 ml centrifuge tubes, and solutions of either polyquaternium,
or polyquaternium and SDS at 1:1 stoichiometry (50 mL) were added. The tubes were
placed on a mixer for 24 hours, centrifuged, and the supernatant analysed for
polyquaternium concentration. Initial polyquaternium concentration of the solutions
ranged from 20 to 160 mg/L. Four concentrations from within this range were
selected, with higher concentrations of polyquaternium being used for lower charge
density polyquaterniums to ensure an analysable supernatant. Blank and control
samples containing 50 mg/L polyquaternium or polyquaternium-surfactant only,
humic acid only, Milli-Q™ water only, and where appropriate, surfactant only, were
also prepared and treated in the same manner as the exposed samples. The amount of
polyquaternium sorbed was calculated from the difference between the initial
concentration and the final concentration following exposure and separation by
centrifugation. The partition coefficient was found from the plot of Equation 4.2
(Vowles and Hawker 1992).

89
 mHA
Ci − C f = K D × C f × Equation 4.2
mw

where Ci is the initial concentration in water

Cf is the concentration in water after exposure to humic acid
mHA is the mass of humic acid
and mw is the mass of water

A selection of humic acid samples exposed to polyquaternium in the sorption

experiment were also analysed for particle size.

4.3.3. Results
The total organic carbon TOC level of the humic acid was 54% for the humic acid as
purchased, and 51% for the coloured humic acid fraction removed by centrifugation.
The separation of the humic acid into the coloured and non-coloured fractions
effectively divided the humic acid by particle size. The humic acid in the supernatant
had a mean diameter of 10.6 μm, while the mean diameter of the insoluble fraction
was > 70 μm (Figure 4.1).

90
Figure 4.1 Plot of particle size analysis of humic acid samples after separation by centrifugation
of coloured fraction (top) from the fraction used for the partition experiment (bottom) showing
the different size distribution of the two samples.

The difference was significant in a Satterthwaite t-test for group means with unequal
variances (SAS 2002). There was no significant difference at α = 0.05in the particle
size of the control and exposed humic acid following the sorption experiment,
indicating that the adsorption of the polyquaternium did not result in coagulation of
the humic acid.(Table 4.6).

91
Table 4.6 Results of statistical analysis of humic acid particle size for the separated supernatant
and solid fraction showing that the difference was significant at α = 0.05 given unequal variance.
Variable Method Variances DF t Value Pr > |t|
Size Pooled Equal 2 34.34 0.0008
Size Satterthwaite Unequal 1.37 34.34 0.0056
Test for Equality of Variances
Variable Method Num DF Den DF F Value Pr > F
Size Folded F 1 1.37 5.22 0.5252

The calculated KD values are given in Table 4.7, and a sample of the plot of Equation
4.2 as used in determining KD, in this case for Conditioneze W-20 is shown in Figure
4.2.

16
y = 500.18x
14
R2 = 0.9093
12

10
Ci - Cf

8
6

0
0 0.005 0.01 0.015 0.02 0.025 0.03

Cf*Ms/Mw
Figure 4.2 Plot of Equation 4.2 as used in determining KD, in this case for Conditioneze® W-20
(Polyquaternium-55).

Table 4.7 Partition coefficient KD determined for polyquaterniums and PSCs and one cationic
surfactant (cetyl pyridinium chloride).
Polyquaternium KD KD with SDS
Gafquat® 440 1038 1226
Gafquat® 734 986 1436
Gafquat® HS100 186 80
UCareTM JR125 359 795
UCareTM JR30M 634 1484
UCareTM JR400 440 382
Conditioneze® NT-20 309 168
poly(DADMAC) 2238 3329
Styleze® W-20 500 400
Cetyl pyridinium chloride 55,000 -

In a Paired T-test (SAS 2002), there was no significant difference between the KD
values for the polyquaternium or the polyquaternium-surfactant complex (PSC) at α =
0.05 (Table 4.8).

92
Table 4.8 Results of a paired t-test of the difference between KD for the polyquaternium and its
PSC.
N Lower CL Mean Upper CL Std Std DF t PR > |t|
Mean Mean Dev Err
KD (Pq) – 9 -636.6 - 57.759 451.69 150.56 8 - 0.09808
(PSC) 289.4 1.92

4.3.4. Discussion
The concentration of humic acid in the tests in this study was around 400 mg/L,
significantly higher than the suspended solids concentration in local WWTPs of
approximately 10 mg/L. An equivalent dose (in terms of charge) for the
polyquaterniums in this study is between 53 and 55 mg/L for UCareTM JR12, JR400,
JR30M, Gafquat® 734, 440, Styleze® W-20; 70 to 80 mg/L for Conditioneze® NT-
20 and Gafquat® HS100; and 80 to 160 mg/L for UCareTM LR/LK polyquaterniums.
According to Bennett et al. (2000), the optimum flocculant dose for 10 mg/L Aldrich
sodium humate was 12-14 mg/L poly(DADMAC). The concentration of
poly(DADMAC) needed to floc the concentration used in this work would be around
520 mg/L, and the concentration of the cosmetic polyquaterniums in excess of 1400
mg/L. Tests in this study were conducted with concentrations in the range of 10 to 80
mg/L for UCareTM JR125, JR400, JR30M, Gafquat® 734, 440, Styleze® W-20, 10 to
90 mg/L for Conditioneze® NT-20 and 30 to 130 mg/L for Gafquat® HS100. All test
concentrations, therefore, are expected to be well below the flocculant dose, and the
measured concentration of polyquaternium following exposure to humic acid cannot
be attributed to saturation of the humic acid.

As expected from its wide use as a flocculant, poly(DADMAC), with highest charge
density, has the largest partition coefficient. Regression analysis of all polyquaternium
partition coefficients with charge density is significant (SAS 2002), but all
significance comes from the poly(DADMAC) result, and regression analysis of the
non-flocculant polyquaterniums without poly(DADMAC) is not significant (Table
4.9).

93
 2500

2000 poly(DADMAC)

1500

1000

500

0
0 0.001 0.002 0.003 0.004 0.005 0.006

Figure 4.3 Plot of KD against charge density for polyquaterniums on which the regression analysis
is based. The data is also presented in tabular form in Table 4.7.

Table 4.9 Results of a regression model of KD against charge density. The model is significant if
the high charge density poly(DADMAC) is included, but is not significant for the cosmetic
polymers alone.
Regression F Pr > F R2
All polyquaterniums 25.63 0.0015 0.7854
Without poly(DADMAC) 0.08 0.7832 0.0136

The sorption coefficients overall are perhaps lower than expected. For example,
Busan 77 (Polyquaternium-42), a high charge density bactericide homopolymer with
two quaternary ammonium charged centres in each monomeric unit and a nominal
equivalence/gram of 0.00775, has been found to have a log KD between 4.3 and 4.7
(≈ 20,000 – 50,000) when tested with acid-precipitable fraction humic acid (Matthews
et al. 1995). Similarly, when cetylpyridinium chloride was tested in this study using
the same method, the sorption coefficient was found to be 55,000 L/kg. The results
were similar, however, to the results obtained by Podoll and Irwin (1988) for the
tertiary cationic poly(N,N,-dimethylaminoethyl methacrylate) (PDAM) oligomers on
sediments (880 – 4,100 mL/g). One possible reason for an apparently low partition
coefficient could be the lower surface area to mass ratio due to the larger particles of
the humic acid used in the sorption experiment. Roughness of the surface in terms of
pores not accessible to the polyelectrolyte of the large particles may also be a factor in
lower adsorption, as has been suggested for the binding of flocculant polymers to
wastewater sludge (Mikkelsen 2003). Due to this roughness, neutralisation of the
‘polymer accessible surface charges’ would occur at a polymer dose that was below
the zeta potential for full neutralisation.

94
It has been suggested that the calculation of a partition coefficient for highly soluble
substances such as polyquaterniums may not be appropriate, as this model of
environmental behaviour assumes that the binding of the substance is not saturable
(Matthews et al. 1995). Poorly soluble substances form micelle-like structures with
humic acid and the binding capacity is unlimited. There is evidence to suggest,
however, that binding of the polyquaternium to humic acid or suspended solids
continues beyond the flocculant dose, that is, charge neutralisation, to form a stable
dispersion of opposite charge. This phenomenon is seen in the re-suspension of flocs
following over dosing with flocculants in water treatment applications (Narkis and
Rebhun 1983).

Nevertheless, in general, care should be taken in extrapolating from the conditions

pertaining to an experimental sorbent-water partition coefficient derived for highly
soluble substances. The polyquaternium-surfactant complex, however, is insoluble,
and thus this constraint to the use of partition coefficient would not apply.

The observation that binding of the polyquaternium with humic acid is not
significantly affected by the presence of the anionic surfactant is not surprising; as it
is this aspect of the behaviour of these polyelectrolytes that makes them suitable for
use many personal care applications. The zeta potential and isoelectric point of hair (-
30 mV, 3.6) are roughly the same as those of humic acid. In studies of the behaviour
of a polyquaternium acrylamide-methacrylamido propyl trimethyl ammonium
chloride (AM-MAPTAC) and the anionic surfactant SDS at a negatively charged
surface (mica), sorption of a high charge density polymer (10%) was found to be
enhanced at SDS concentrations above one tenth of critical micelle concentration
(CMC) (8 x 10-4 M), and at a maximum at charge neutralisation (Rojas et al. 2004).
Low charge density AM-MAPTAC (1%) began to desorb when surfactant
concentration was one tenth of CMC (Rojas et al. 2001). In terms of the sorption
studies here, the concentration of SDS at charge neutralisation of poly(DADMAC)
was > 0.5 CMC, while for the personal care polymers it was between 0.07 and 0.15
CMC. Therefore, enhanced sorption of the poly(DADMAC) could be expected, and
was observed. The lower partition coefficients observed for the low charge density
polyquaterniums, Conditioneze® NT-20 and Gafquat® HS100, in the presence of the
surfactant could result from desorption.

95
 4.4. Fate Modelling
Fate modelling of chemical species in an aquatic environment is often thought of in
terms of four pathways – adsorption, biodegradation, chemical degradation and
volatilisation. Two of these, adsorption and volatilisation are transfers of the chemical
of interest from the dissolved phase to another phase. In wastewater treatment, this
generally means transfer to air, sludge, or suspended solids. The remaining two
pathways, chemical degradation and biodegradation involve the transformation of the
chemical of interest into a different chemical, or even a suite of different chemicals.
These pathways are often referred to as ‘removal’ pathways, but from a risk
assessment perspective only complete mineralisation can be regarded as complete
removal.

In the context of chemicals such as polyquaterniums, ‘transfer’ from the water column
to the sludge in the wastewater treatment process has traditionally been regarded as a
‘removal’ process in chemical risk assessment in Australia, for example in the FPR
for Luviquat Care (Polyquaternium-44) (NICNAS 2001). Traditional disposal
methods of wastewater sludges by landfill or incineration were thought to result in
immobilisation of the chemical in the former, and transformation to oxides of carbon
and nitrogen in the latter. However, recent developments in the re-use of sludge in
land applications could result in further mobility of chemicals, and their potential
return to the aquatic environment via leaching and surface water movements.

In the risk assessment of chemicals that are transformed by either biodegradation or

chemical degradation (hydrolysis, photolysis), it must be established in the risk
assessment process that such transformation products are less hazardous than the
original chemical. In the case of polymers, the ability to degrade is not generally
regarded as a favourable outcome, due to the complexity of possible degradation
products, their higher potential for mobility and systemic absorption compared with
the high MW polymer. It is for this reason that biopolymers and other biodegradable
polymers are not considered to be PLCs.

4.4.1. Partitioning Models

The environmental fate of chemicals may as a first estimate be considered by
determining the distribution of chemicals between phases or compartments in the
environment based on the thermodynamic principle that the chemical will tend to
reach an equilibrium state between phases or compartments. The operation of such

96
models is based on the estimation of partition coefficients between the various phases;
air-water (Equation 4.3), soil-water (Equation 4.4), and biota-water (Equation 4.5).

Henry’s Law Constant Equation 4.3

Ca
H=
CW
Soil sorption coefficient Equation 4.4
Cs
KP =
CW
Bioconcentration Factor Equation 4.5
Cf
BF =
CW
where C is the concentration in water (w), soil (s), biota (f) and air (a)

Where there is no empirical partition coefficient, mathematical methods have been

devised to estimate these parameters from more accessible physical-chemical
characteristics such as vapour pressure, molecular weight, aqueous solubility, and
octanol/water partition coefficient (Samiullah 1990).

Partitioning, the tendency towards equilibrium, is the maximising of entropy in the

system, and can therefore be regarded as ‘equating the chemical potential of the
substance in each phase’ (Samiullah 1990). Equilibrium is defined as the point where
the chemical potential or ‘escaping tendency’ of a chemical in two phases is equal.
This can be compared conceptually to thermal equilibrium where the temperature of
two bodies is the same. The equivalent of temperature in thermal equilibrium for
chemical partitioning is fugacity. Chemical potential has units of energy per mole and
is conceptually difficult. Fugacity, with units of pressure, is a more convenient
descriptor of phase equilibrium (Samiullah 1990). Fugacity is related to concentration
using a fugacity capacity constant Z (mol m-3 pa) (Clark et al. 1995), which is specific
to the compound, the phase in which the compound is found, and the temperature
(Equation 4.6).

97
C = Zf Equation 4.6

Where C is concentration (mol m-3)

Z is fugacity capacity constant (mol m-3 Pa-1)
f is fugacity

4.4.2. Model Parameters

The modelling of the environmental fate of chemicals requires knowledge of the
characteristics of the chemical that contribute to its partitioning behaviour. These
characteristics include aqueous solubility, vapour pressure, and various partition
coefficients (KOW and KD, Henry’s Law Constant). These characteristics form an
important part of the data requirements in the assessment of chemicals and are
required by NICNAS in the notification of all new chemicals (NICNAS 2004b).

As previously demonstrated for Merquat 2001 in Section 1.2, the imported volume of
the cosmetic polyquaternium is assumed to be released to sewers after use, so that the
study of the environmental fate of polyquaterniums is largely a study of the progress
of the polyquaternium in the wastewater treatment process. Generally, it is assumed
that the adsorption of the polyquaternium to sewage solids is the most important
pathway of removal in wastewater treatment, with degradation processes being of
significantly less importance (Boethling and Nabholz 1997). Omitting transformation
processes, the WWTP can be viewed as a system with phases in which the
partitioning of chemicals between phases occurs as illustrated in Figure 4.4. The
phases in this case are the water, air and the solids (sludge and suspended). The input
parameters relevant to the system include influent concentration of chemical, influent
suspended solids concentration, flow rates, percent removal of suspended solids. It is,
in essence, a mass balance approach which estimates how the chemical is distributed
from the water column to air or solids as the solids and water are separated and treated
through the system.

98
 Volatilization

1 Primary 3 4 Final 6
Settling Bioreactor Settling
Tank Tank

2
5

Sedimentation
Biotransformation

Figure 4.4 Conceptual model of the ‘box’ structure of the Oxley WWTP in SE Queensland,
showing the possible chemical fates, volatilization, biotransformation and sedimentation in the
three stages of the treatment process. The numbered arrows represent the fluxes in Equations
4.11-4.13.

The application of the fugacity/partition models to WWTP is therefore specific to

particular plants, as the mass balance of each plant will be different. For the purposes
of this study, the model has been applied to Oxley WWTP, a conventional activated
sludge WWTP with a mix of industrial and domestic influent of 240,000 people
equivalents, and typical in configuration of those found in South East Queensland
(Tan et al. 2007). The mass balance of water and solids in the Oxley WWTP is given
in Figure 4.5 and Figure 4.6 respectively (Tan et al. 2007).

99
 Water Balance

583 m3 h-1 580 m3 h-1 1163 m3 h-1 571 m3 h-1

PST Bioreactor FST

12.5 m3 h-1
583 m3 h-1
2.6 m3 h-1
This assumes
the 1º is 4% Returned Waste Activated
solids Activated Sludge Sludge

Figure 4.5 Diagram of water balance for Oxley WWTP, assuming 65% solids removal in primary
settling tank (PST).

Solids Balance

56,700 h-1 2,330,400 g h-1 5710 m3 h-1

PST Bioreactor FST
10 g m3 solids
& 572 m3 h-1

105,300 g h-1
2,273,300 g h-1 48750 g h-1
i.e. 65%
removal From 3.9 g l-1 & 12.5 m3 h-1
Returned
Activated Sludge Waste Activated
Sludge
Figure 4.6 Diagram of solids balance for Oxley WWTP, assuming 65% solids removal in primary
settling tank (PST).
4.4.3. Predicting Extent of Removal of Polyquaternium.
For the model, the WWTP is considered as a series of interlinked ‘boxes’,
representing the sequence of treatments in the plant. A steady state is assumed to exist
within each ‘box’, i.e. inputs = outputs. Transport process parameters, D (mol/h Pa),
are used to determine the importance of the fates of the chemical in each ‘box’. Three
types of fate processes exist within the process, each represented by their own D value
(Equation 4.7 – Equation 4.9). The steady state situation in the context of D values is
given in Equation 4.10.

100
Advection (transport in liquid)
Equation 4.7
D = Q×Z
Volatilization
Equation 4.8
D = KV × A × Z
Biotransformation
Equation 4.9
D = k ×V × Z
where Q is the volumetric flow rate of the phase (m3 h-1)
k is the first order biotransformation rate constant (h-1)
V is the phase volume (m3)
A is the air/water interfacial area
KV is the overall mass transfer coefficient
Z is the fugacity capacity constant (mol m-3 Pa-1)

Input flux = f × outputs = f (ΣD) Equation 4.10

where f is the fugacity of the compound of interest

For the modelling of polyquaterniums, sorption is expected to be a significantly more

important fate than either volatilisation or biotransformation. Test reports provided
with the notification of Luviquat Care (Polyquaternium-44) indicate that the polymer
is not readily biodegradable (NICNAS 2001). In addition, due the relatively high
water solubility of polyquaterniums and the expected low vapour pressure, the
Henry’s Law Constant is also expected to be very low.

As a preliminary simplification, the fugacity model can be expressed in terms of

fluxes only (Figure 4.4), ignoring Waste Activated Sludge;

fD1 = fD2 + fD3 Equation 4.11

for the Bioreactor;

fD3 = fD5 + fD4 Equation 4.12

and for the Final Settling Tank (FST).

fD4 = fD5 + fD6 Equation 4.13

Solving simultaneously

fD1 = fD2 + fD6 Equation 4.14

Since removal is the difference between input and output fluxes, expressing removal
as a percentage or fraction of input flux

101
 ( fD1 − fD6 ) Equation 4.15
% removal = × 100
fD1
fD2 × 100
=
fD1
fD2 × 100
=
fD2 + fD3

and fraction removal

fD2
p= Equation 4.16
fD2 + fD3

Within an individual flux, there will be dissolved and sorbed fractions of

polyquaterniums. For example, with the influent

fD1 (mol hr −1 ) = f (Q1W ZW + Q1B Z B ) Equation 4.17

or

fD1 (mol hr −1 ) = f (Q1W + Q1B K D ) ZW Equation 4.18

where Q1W and Q1B are the flow rates of water (L/h) and solids (kg/h) respectively,
ZW (mol/Pa) and ZB (mol/Pa kg) dissolved and sorbed polyquaternium
fugacity capacity constant
and KD the solids/water partition coefficient (L/kg)

Therefore, the above equations for the removal of polyquaterniums may be expressed
as functions of flow rates, fugacity capacity constants and KD.

By simplifying the model process to exclude biotransformation and volatilization, it is

possible to predict the extent of removal as a function of the sorption coefficient, KD,
and separately, as a function of biosolids removal (for example primary sludge).
Generally, the formula for percent removal of a polyquaternium in the WWTP is the
amount of polyquaternium removed with the solids in the PST as a percentage of the
total amount entering the PST (Equation 4.19), which can also be expressed as a
fraction (Equation 4.20).

⎛ Q2W + Q2 B K D ⎞ Equation 4.19

% removal = ⎜⎜ ⎟⎟ × 100
⎝ Q2W + Q2 B K D + Q3W + Q3 B K D ⎠

And

102
 ⎛ Q2W + Q2 B K D ⎞ Equation 4.20
p = ⎜⎜ ⎟⎟
⎝ Q2W + Q2 B K D + Q3W + Q3 B K D ⎠
where p is the fraction of polyquaternium removed

This equation can be rearranged to express the relationship between p and the
partition coefficient KD (Equation 4.21).

p(Q2W + Q3W ) − Q2W Equation 4.21

KD =
Q2 B − p(Q2 B + Q3 B )

In the situation under consideration, the flow of water removed with the solids is
significantly less than the flow rate of water to the bioreactor, that is Q2W << Q3W, and
for most WWTPs, it can be expected that Q2W << p(Q2w+Q3W). Taking this
assumption into account, the relationship can be simplified (Equation 4.22), and
rearranged to Equation 4.23 and Equation 4.24, which describes the linear relationship
between 1/KD and 1/p.

p(Q2W + Q3W )
KD ≈ Equation 4.22
Q2 B − p(Q2 B + Q3 B )

and therefore

1 Q − p(Q2 B + Q3 B )
= 2B Equation 4.23
KD p(Q2W + Q3W )

1
=
Q2 B (Q + Q3B )
− 2B Equation 4.24
K D p(Q2W + Q3W ) (Q2W + Q3W )

If Q2W << Q3W, denominator term may also be approximated as ≈ Q3W.

1 Q Q + Q3 B
= 2B − 2B Equation 4.25
K D pQ3W Q3W

Employing the actual fluxes from Oxley WWTP mass balance data with this equation,
the plot of the inverse of KD and p for values of p up to the maximum solids removal
(65%) for this WWTP (Figure 4.7), demonstrates that as KD becomes larger, the
proportion removed in the primary sludge becomes larger. In fact, as KD → ∞, and
1/KD → 0, 1/p approaches 1/0.65. That is, if KD → ∞ and the polyquaternium were all
sorbed, the maximum proportion of the polyquaternium that could be removed is 0.65
or 65%.

103
 0.0035

0.003

0.0025

0.002
1/KD

0.0015

0.001

0.0005

0
0 5 10 15 20 25
-0.0005
1/p

Figure 4.7 Plot of 1/KD vs 1/p for values of p up to the total solids removal for the WWTP showing
linear relationship between these parameters.

To increase the accuracy of the model, polyquaternium removal by sorption to WAS

can be considered as well (Equation 4.26).

% removal =
(D2 + DWAS ) f × 100 Equation 4.26
(D2 + D3 ) f
As before, KD can be related to p as

p(Q2W + Q3W ) − Q2W − QWASW

KD = Equation 4.27
Q2 B + QWASB − p(Q2 B + Q3 B )

and if Q2W and QWASW << p(Q2W + Q3W)

p (Q2W + Q3W )
KD ≈ Equation 4.28
Q2 B + QWASB − p(Q2 B + Q3 B )

and

1 Q + QWASB
= 2B
(Q + Q3B )
− 2B Equation 4.29
K D p(Q2W + Q3W ) (Q2W + Q3W )

For Oxley WWTP, there is typically 65% solids removal (s) in the PST, and the
primary sludge is 4% solids by mass. That is

Q2 B = 0.65 Influent biosolids flux Equation 4.30

and

104
Q2 B = 0.65(Q2 B + Q3 B ) Equation 4.31

Given that the solids level in the primary sludge is 4%, the biosolids and water flow
rate from the PST in the primary sludge can be expressed as a proportion of the total
flow rate

Q2 B = 0.04(Q2 B + Q2W ) Equation 4.32

Q2W = 0.96(Q2 B + Q2W ) Equation 4.33

From Equation 4.20, the relationship between p and KD can be expressed taking into
account the proportion of solids removal (Equation 4.34).

0.96 × s × (Q2 B + Q3 B )
+ s(Q2 B + Q3 B )K D
p= 0 . 04 Equation 4.34
Q2W + Q3W + (Q2 B + Q3 B )K D
Where: s is the proportion of solids removed
Thus for a polyquaternium with KD = 400, when s = 0.65, using the flux values from
Oxley WWTP, the proportion of polyquaternium removed is about 7%. As s → 1, the
equation can be simplified (Equation 4.35) and it can be shown that if KD is 400, and
all biosolids removed as primary sludge, only maximum of 10.6% of the
polyquaternium can be expected to be removed.

⎛ 0.96 ⎞
⎜ + K D ⎟(Q2 B + Q3 B )
0.04
p= ⎝ ⎠ Equation 4.35
Q2W + Q3W + (Q2 B + Q3 B )K D

Where: Q2B = 105 kg/h

Q3B = 56.7 kg/h
Q2W = 2.6x103 L/h
Q3W = 580x103 L/h
p→
(24 + 400)161.7
582.6 × 103 + 161.7 × 400
6.86 × 104
= = 0.106
6.47 × 10

To take into account WAS, Equation 4.34 can be expanded to include sorption to
WAS (Equation 4.36).

105
 ⎛ 0.96 × s × (Q3 B + Q2 B ) ⎞
⎜ + s (Q2 B + Q3 B )K D + QWASW + QWASB K D ⎟
p =⎜ 0.04 ⎟ Equation 4.36
⎜ Q2W + Q3W + (Q2 B + Q3 B ) K D ⎟
⎜ ⎟
⎝ ⎠
2.52 × 10 + 4.2 × 10 + 12.5 × 10 + 48.7 × 400
3 4 3
p=
582.6 × 103 + 161.7 × 400

7.65 × 104
= = 0.118
6.47 × 105

Again, for a polyquaternium with KD = 400, when s = 0.65, using the flux values from
Oxley WWTP, the proportion of polyquaternium removed increases from 7% to
11.8%. As s → 1, only a maximum of 15.6% of the polyquaternium can be expected
to be removed.

6.86 × 104 + 12.5 × 103 + 48.7 × 100

p→
6.47 × 105
101 × 105
= = 0.156
6.47 × 105

4.4.4. Results
In an Excel spreadsheet (Appendix 3a), the model was run using Oxley WWTP flux
parameters for values of s = 0.65, the removal fraction for Oxley; s = 0.9, the typical
level of solids removal generally assumed for risk assessment of polymers (Boethling
and Nabholz 1997); and s = 1, representing the theoretical maximum removal
fraction. Varying values of KD were also used in the models. KD values of 400 and
1000 represent typical values determined in this study Table 4.7. Values an order of
magnitude higher, 104, enable an estimation of the consequences of an
underestimation of the true partition coefficient. Higher values, up to 107, enable the
theoretical maximum removal fraction to be determined. The removal for both
versions of the model, PST only and WAS included, were determined (Table 4.10).
The relationship between the overall fraction of the polyquaternium removed and KD
is illustrated in Figure 4.8.

106
Table 4.10 Results of model calculation for proportion of influent polyquaternium removed as a
function of various values of KD and solids removal (PST only and with WAS taken into account).
s (PST only) s (WAS included)
KD 0.65 0.9 1 0.65 0.9 1
4 x 102 0.07 0.10 0.11 0.11 0.14 0.15
1 x 103 0.14 0.20 0.22 0.21 0.26 0.28
1 x 104 0.48 0.66 0.74 0.58 0.73 0.79
1 x 105 0.63 0.87 0.97 0.71 0.90 0.97
1 x 106 0.65 0.90 1.00 0.73 0.92 1.00
1 x 107 0.65 0.90 1.00 0.73 0.92 1.00

1.2000

1.0000
Fraction removed

0.8000
s=0.65
0.6000
s=0.9
0.4000 s=1

0.2000

0.0000
0 2 4 6 8
log(Kd)

Figure 4.8. Overall fraction of polyquaternium removed as a function of the partition coefficient
KD. Plot of removal fraction for varying solids removal rates.

4.4.5. Discussion
It has been the practice in the risk assessment to assume that non-ionic, cationic, and
amphoteric polymers with molecular weight > 1000 will partition to solids, with 90%
removal relative to influent concentration. The 90% figure is based on USEPA
estimates of typical solids removal in WWTPs. The remaining 10% is assumed to be
discharged to receiving waters in the form of a polymer bound to sludge solids
(Boethling and Nabholz 1997). With the solids removal for Oxley i.e. 65%, the
maximum removal fraction is 65% (PST only) and 73% (including WAS). If the
solids removal via primary sludge is the 90% assumed by Boethling and Nabholz
(1997), the maximum removal fraction is 90% (PST only ) and 92% (including
WAS). This maximum is approached for a theoretical polyquaternium with log
KD ≥ 5, significantly higher than the level found for polyquaterniums in this study.

107
At the values of KD determined in this study, the maximum removal percentage in
biosolids of a polyquaternium with KD ≈ 400 is 7-14% (PST only) and 11-20%
(including WAS). If the solids removal is the 90% assumed by Boethling and Nabholz
(1997), the maximum removal is 10-20% (PST only) and 13-26% (including WAS).
Allowing an underestimate of one order of magnitude for the partition coefficients in
this study, removal between 48 and 73% only could be expected. It would appear that
only with KD ≥ 106, and 100% solids removal via primary sludge would it be possible
to expect the 99% removal with solids claimed for Luviquat Care (NICNAS 2001).

4.5. Predicted Environmental Concentration

The purpose of determining the partitioning behaviour of chemicals such as
polyquaterniums is to determine in which compartment(s) the chemical will end up,
and how much of it will be there. This will enable estimation of the likely impact
resulting from, or risk associated with, its release. It is often not possible to predict
actual environmental concentrations, as these vary spatially and temporally
(Samiullah 1990). However, it should be possible to estimate the likely concentrations
that could occur in selected circumstances, and thereby test the assumption inherent in
the NICNAS/DEW method of 90% removal in WWTP and 90% dilution in receiving
waters.

It has been estimated earlier in this Section (4.2) that the import/manufacture/use
volume of polyquaterniums for PCPs is most likely between 30 and 60 tonnes, but
possibly as high as 80 tonnes or as low as 20 tonnes. There is no way of estimating
from this data what the mix of polyquaterniums might be nor further, of knowing
what the range of charge density and molecular weight for these polyquaterniums
might be. Therefore, the calculations that follow will be based on a theoretical
‘surrogate’ polyquaternium with properties typical of those used in PCPs.

With the assumption of 90% removal in WWTP and 10:1 dilution in receiving waters,
as used by NICNAS/DEW in assessment of new chemicals, the predicted
environmental concentration (PEC) of cosmetic polyquaterniums would be between
0.14 and 0.55 µg/L (Table 4.11). However, actual removal rates in WWTP may be
significantly less, as suggested by the modelling in the previous section.

108
Table 4.11. Determination of PEC for environmental risk assessment using NICNAS/DEW
method.
Import/Manufacture Volume 20 40 60 80
tonnes tonnes tonnes tonnes
Usage volume (kg/day) 55 110 164 219
Population (x106) 20 20 20 20
Water volume (L/day) 200 200 200 200
Influent concentration (µg/L) 14 27 41 55
Effluent concentration (µg/L) 1.4 2.7 4.1 5.5
Dilution to receiving waters (µg/L) 0.14 0.27 0.41 0.55

Taking a value for KD, which would appear to be reasonably representative of our
polyquaternium sample (1000), and two biosolids removal rates, 0.65 and 0.9, and
applying these to the influent concentrations in Table 4.11, effluent concentrations
based on these removal rates (21% and 26%) are potentially an order of magnitude
higher than predicted by the standard model. For an import volume of 60 tonnes, the
predicted effluent concentration would be between 0.034 and 0.032 µg/L. Assuming
the KD value could be underestimated by an order of magnitude, and removal rates are
the higher values (58 and 73% for s = 0.65 and 0.9 respectively), the effluent
concentration for an import volume of 60 tonnes, would be between 1.8 and 1.2 µg/L
(Table 4.12).

Table 4.12 Effluent discharge polyquaternium concentrations (µg/L) for a range of import
volumes and WWTP removal rates.

Fraction Removed Import/Manufacture volume

20 tonnes 40 tonnes 60 tonnes 80 tonnes
0.21 1.1 2.3 3.4 4.6
0.26 1.1 2.1 3.2 4.3
0.58 0.61 1.2 1.8 2.4
0.73 0.39 0.78 1.2 1.6
0.90 (for comparison) 0.14 0.29 0.43 0.58

The assumed dilution to receiving waters is based on discharge to large coastal rivers
and estuaries. A significant portion of outfall from WWTPs in Australia does occur to
major rivers with significant flows for most of the year. Sydney Water, for example,
has 18 WWTPs servicing > 600,000 people equivalents discharging > 250 ML/day
into rivers in 6 catchments (Sydney Water 2007). This represents almost 15% of
Sydney Water’s total discharge by volume (and people equivalents serviced). In
Victoria, 80% of discharge by volume, 1040 ML/day, is discharged to coastal waters,
12% (156 ML/day) is discharged to inland waters, and 8% (104 ML/day) is recycled
(EPA Victoria 1995). In Queensland, total discharge for 2001 was 339 GL/year, of

109
which 11.2% was recycled and 23 GL (around 8%) was discharged to non-tidal water
bodies (Beeton et al. 2006).

Consequently, between 8 and 15% of total effluent is discharged into vulnerable

inland waterways, where dilution is likely to be less than 10:1. Victorian regulations
require that the dilution be at least 5:1. Queensland and NSW regulations are based on
water quality outcomes and regulated in licensing agreements. Of course, dilution
values could be less than 5:1 or more than 10:1; in fact, for discharge into ocean
outfall (the majority of WWTP discharge in Australia) it will be greater than 10:1.
The range of environmental concentrations likely under 10:1 and 5:1 dilution regimes
is given in Table 4.13. Without even considering the possibility of zero dilution
(effectively the effluent concentration) on discharge, a fifteen-fold difference in the
PEC can occur for some possible import volumes. The application of the model has
produced a range or distribution of possible concentrations. All these concentrations
are lower than the concentrations predicted by the NICNAS/DEW method (Table
4.11), assuming no adsorption in WWTP, but higher that that assumed by adopting
the USEPA default of 90% loss to solids in WWTPs.

110
Table 4.13 PECs of polyquaterniums for different import volumes and environmental dilution
ratios (µg/L). The effluent concentration would also represent the case where there was zero
dilution.
Import volume Fraction Removed Effluent Dilution Dilution
(tonnes) % Concentration 5:1 10:1
(µg/L)
0.21 1.1 0.22 0.11
0.26 1.1 0.21 0.11
20 0.58 0.61 0.12 0.061
0.73 0.39 0.08 0.039
0.9 0.14 0.028 0.014
0.21 2.3 0.46 0.23
0.26 2.1 0.43 0.21
40 0.58 1.2 0.24 0.12
0.73 0.78 0.16 0.078
0.9 0.29 0.058 0.029
0.21 3.4 0.68 0.34
0.26 3.2 0.64 0.32
60 0.58 1.8 0.36 0.18
0.73 1.2 0.23 0.12
0.9 0.43 0.08 0.043
0.21 4.6 0.91 0.46
0.26 4.3 0.85 0.43
80 0.58 2.4 0.48 0.24
0.73 1.6 0.31 0.16
0.9 0.58 0.12 0.058

Some assumptions have, at this stage been left in place, for example, the per capita
volume of water at 200 L per person per day. The water use volume used by
NICNAS/DEW in estimated PEC has been increasing over the years, in line with
domestic consumption. Water restrictions have reversed that trend. Per capita
domestic consumption of water in SE Queensland, for example, is currently around
134 L/ day (Lucas 2007).

At each stage in the determination of PEC, a range of possibilities have been

considered, for example, import volume between 20 and 80 tonnes, WWTP
proportion removed between 7 and 73%, dilution ratios between zero (the effluent
concentration) and 10:1. Consequently, a range of PEC values is produced, which will
need to be considered in the risk characterisation.

Further to the variation that has been described for the PEC here, it is important to
remember that toxicants are not uniformly distributed over time and space, even
within the immediate zone of a point source. Although there are many models of

111
dilution effects as a function of distance from release, mixing zones and patch effects
can also occur (Spromberg et al. 1998; Landis and McLaughlin 2000). No boundaries
are specified for the PEC, which can be assumed to be applicable only in the
proximity of the WWTP outfall. The process of adsorption of the polyquaternium to
DOM continues to occur once the WWTP effluent has been released to receiving
waters, and removal to sediments in the downstream area will occur. In addition to the
removal of residual cationic polyelectrolytes to DOM and clay in the receiving waters,
removal of soluble polyelectrolyte-DOM complexes by adsorption to clay particles
can occur (Bennett et al. 2000). While these factors are often critical in the assessment
of contaminated sites, they are generally outside the scope of the risk assessment of a
new chemical. However, the partition coefficients determined in this study have
implications for the extent of removal of the polyquaterniums in receiving waters, as
well as the extent of removal in WWTP. Sorption to solids in receiving waters may be
similarly lower than expected.

4.6. Conclusion
The import volume for cosmetic polyquaterniums has been estimated using two
different methods. The results of these estimates resulted in a similar range of values.
The experimentally determined KD for the polyquaterniums was less than expected,
and significantly less than that for the monoquaternary surfactant, cetylpyridinium
chloride, that was also tested. Consequently, the fraction removed in WWTP would be
expected to be significantly less than the default value for polymers that is generally
used in the risk assessment of cosmetic polyquaterniums. The PEC calculated has a
range of approximately 0.01 to 1.0 µg/L between the highest and lowest estimates.
Therefore, considerable uncertainty will need to be considered in the risk
characterisation.

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5. Aquatic Toxicology – Effects Assessment of
Polyquaterniums
5.1. Introduction
The assessment of the effects of a chemical is often regarded as comprising two
components; hazard identification and dose (concentration)–response assessment
(ECB 2003). Hazard relates to the capacity of a specific agent to produce a particular
type of adverse health or environmental effect, for example the capacity of benzene to
cause cancer or the capacity of solar radiation to cause skin cancer (EnHealth 2002).
Dose-response assessment explores the relationship between the amount of exposure
to the hazard, and the level of effect observed (Neely 1994), which can be estimated
or determined from toxicological studies, epidemiological studies and, for
environmental impacts, ecological studies. Thus, effects assessment is a two step
process comprising the identification of the hazard, and the capacity of the hazard to
cause the response, that is the level of exposure, or dose required (EnHealth 2002). In
practical terms, a substance is labelled as hazardous if it meets certain criteria. While
these criteria may be based on physico-chemical properties of the chemical, for
example if it is corrosive, determination of a hazard classification is generally
determined by the toxic (dose) response in a standard test (NOHSC 1999, OECD
2001).

In regulatory chemical risk assessment, the identification of the hazard is generally

determined by criteria set out in the appropriate act. The Industrial Chemicals
(Notification and Assessment) Act (ICNA), for example, has previously categorised
polymers according to criteria that determine low concern (Polymers of Low Concern,
PLC), and all other chemicals solely on volume imported or manufactured. More
recently, criteria for identifying all chemicals of low concern have been developed
(Low Regulatory Concern Chemicals, LRCC). The development of such criteria is
usually only possible after years of operation of the assessment process allows the
identification of suitable criteria (NICNAS 2004a). The process, therefore, is not
about identifying a hazard, but of screening out those chemicals that are unlikely to be
hazardous. The data required for assessment is also established in the Act or
regulations. For new chemicals that are regarded as potentially hazardous, the
minimum data requirement for hazard assessment for the aquatic compartment
comprises short-term toxicity data for three species from three trophic levels, algae,

113
Daphnia, and fish, and a bacteria (respiratory inhibition test) for sludge-based
organisms (EC 2003; NICNAS 2004b). However, further information may be
requested by the regulatory authorities, and notifiers are expected to provide all
available information above the minimum requirement if such data exists (NICNAS
2004b). Waivers may also be granted for some or all of the required tests, allowing
substitution of analogue data, or complete waiver in cases where the testing is not
technically possible, for example, readily hydrolysed compounds.

As exposure assessment is the process leading to the predicted environmental

concentration (PEC), for the purposes of this study effects assessment is the process
leading to the establishment of a Predicted No Effect Concentration (PNEC). A PNEC
is regarded as a concentration below which an unacceptable effect will most likely not
occur (ECB 2003). In practice, the PNEC is calculated by dividing the short-term
median effective concentration (EC50) or long-term no observed effect level (NOEC)
value by an appropriate assessment or safety factor. A safety factor is generally a
margin of safety applied to a NOEC level to produce a value below which exposures
are assumed to be safe (Bascietto 1990). Assessment factors were developed for the
process of reviewing premanufacture notices and are applied to acute toxicity values,
and take into account the uncertainties due to such variables as test species’
sensitivities to acute and chronic exposures, laboratory test conditions, and age-group
susceptibility (Bascietto 1990). The EC50 required for the determination of the PNEC
in new chemicals assessment by National Industrial Chemicals Notification and
Assessment Scheme/Department of Environment and Water Resources
(NICNAS/DEW) is minimally required to be the lowest of the three trophic levels
required for regulatory assessment (ECB 2003, NICNAS 2004b). The level of
assessment factors used in determining the PNEC is dependent on the availability of
test results above the minimum requirement. Therefore, in this study, fish,
invertebrate and algae tests are reported. The three species selected were Gambusia
holbrooki, Artemia sp., and Chlorella sp. 12. As polyquaterniums are released with
wastewater across a variety of aquatic environments, relevance of species to the
ecosystem of interest was not critical. However, Gambusia are found widely in
freshwater streams in eastern Australia (McDowall 1996). As large releases of
wastewater to ocean outfall occur in coastal areas, and little data is available on the
toxicity of polyquaterniums to marine species, a marine species was included. As

114
there was little data available on sensitivity of species to suggest whether the species
selected would be sensitive to polyquaterniums, Gambusia and Artemia were chosen
primarily because of their availability and ease of handling.

The aim of the following experimental section is to establish if:

• the cosmetic polyquaterniums are toxic to the three species, G. holbrooki,

Artemia spp. and Chlorella sp12;

• If the polyquaterniums are toxic to any species, to determine if the formation

of the polymer-surfactant aggregate in cosmetic formulations has any impact
on the toxicity;

• If the polyquaterniums are toxic to any species, to investigate if the presence

of humic acid has any effect on the toxicity;

• To use the toxicity data in the hazard assessment of the risk assessment
process.

5.2. Methods
5.2.1. Experimental Design
The initial step in the experimental design was to determine the toxicity of the
cosmetic polyquaterniums to the test species. The toxicity of the anionic surfactant
SDS was also tested. For comparison, the toxicity of the water treatment polymer
poly(DADMAC) and the cationic surfactant cetyl pyridinium chloride were also
tested. As these tests would allow the comparison of the toxicity of the cosmetic
polyquaterniums to the published values for water treatment polycations and other
cationic surfactants, the inclusion of poly(DADMAC) and cetyl pyridinium chloride
would ensure that differences found were not due to the methods adopted in this
study.

If the polyquaternium(s) were toxic to the test species, and the anionic surfactant was
either not classifiable as toxic, or significantly less toxic than the polyquaternium, it
would then be possible to test the toxicity of the polymer-surfactant complex.

In addition to direct comparison of the toxicity of cosmetic polyquaterniums to the

published toxicity values for water treatment, this would determine if any differences
in the toxicity of water treatment polycations and cosmetic polyquaterniums could be
attributed to the factors resulting from the formulation of cosmetic products, that is,

115
the presence of the anionic surfactant. If any of the polyquaterniums were classifiable
as toxic, it was proposed to test the polyquaternium in the presence of
environmentally relevant concentrations of humic acid to determine if this resulted in
any change in the toxicity of the polyquaternium.

As metachromatic colloid titration had not proved amenable to determining the

concentration of polyquaterniums in the test solutions, the concentrations quoted are
nominal rather than actual. However, choice of all equipment in test should take into
account the tendency of polyquaterniums to adsorb to certain surfaces.

At the beginning of testing, it was not known if all polyquaterniums would have
similar toxicities, or if differences in the structure of the polymers would result in
determinable differences in toxicity. As testing was limited to polyquaterniums that
manufacturers were prepared to provide, it was determined that testing would begin
with all polyquaterniums that were obtained. However, if the behaviour of
polyquaterniums were not discernibly different, or if groupings became apparent, the
choice of polyquaterniums for the remaining tests would be refined.

5.2.2. Test Solutions

All chemicals used were as described previously in Section 3.3.1. Stock solutions of
polyquaterniums were prepared in Milli-Q water at a concentration of 10 g/l, and
diluted to make working solutions of 1 g/l. For tests with humic acid, the soluble
fraction separated as described in the previous chapter was used. The concentration
was determined from the organic carbon concentration of the supernatant and the
organic carbon content of the humic acid (51%). The humic acid was added to the test
solution before the toxicant, at concentrations of 5 and 10 mg/L humic acid. The
relative concentration was determined from the organic carbon content of the
supernatant and the organic carbon content of the humic acid (51%).

5.2.3. Fish
Gambusia holbrooki were trapped in a small dam on a private property in the
Beenleigh area in SE Queensland (Figure 5.1). The dam collects runoff from an area
that is largely rural (hobby farms). Fish were collected on a fortnightly basis, and kept
for a minimum of 12 days prior to testing. G. holbrooki are dimorphic, so only female
fish between 1.5 and 3 cm in length, with no obvious gravid spot, were used in the
test. This group was the most numerous in the field collections in autumn and winter.
Larger female fish are often survivors from the previous season (i.e. were older than

116
one year), and generally did not meet the weight requirement to ensure stocking
standards were not exceeded. In preliminary tests with poly(DADMAC), male fish
were found to be more sensitive to the polyquaterniums, but were also more prone to
stress and injury in handling during transport, sorting and testing. In addition, very
large numbers of fish would have needed to be trapped to ensure adequate numbers of
males for testing. Female fish, generally ten, were used at each test concentration,
though in late season testing the number of fish was reduced to ensure stocking rates
(1 g fish/L) were not exceeded (OECD 1992).

Figure 5.1 The small dam on a private property in the Beenleigh area in SE Queensland where
the Gambusia used in the toxicity testing were caught. The dam collected runoff from an area
that is largely rural (hobby farms).

Test Conditions: Tests were conducted in a mix of de-chlorinated tap and

demineralised water, to match the condition of the dam water from which the fish
were collected (conductivity 180-200 µS cm-1, salinity 80-90 ppm), as this gave the
best survival rates in acclimatisation tanks. The tests were conducted in 1.5 litres of
water in 2 litre capacity polypropylene containers (Figure 5.2). Air was introduced via
an inverted plastic transfer pipette set to bubble just below the surface. The fish were
not fed for 24 hours before the test or during the test. The tests were static, and the
duration was 96 hours. Water parameters (pH, temperature, DO, conductivity,
salinity) were recorded before and after the test; acceptable parameters are given in
Table 5.1. The test area was in a laboratory with good natural light, and no artificial
light was provided. A minimum of five concentrations and one control were used in
each test. For the testing of the polymer-surfactant complex (PSC), an additional

117
control for SDS only at the maximum SDS concentration was included, and a
complete parallel test of the polyquaternium was also conducted. Original plans to
include poly(DADMAC) as a positive control were abandoned due to the lack of
differentiation between the NOEC and EC50 in preliminary tests with
poly(DADMAC). Acceptable mortality in the controls was ≤ 10%, in accordance with
the Organisation for Economic Cooperation and Development (OECD) guidelines
(OECD 1992).

Table 5.1 Water parameters before and after exposure of fish to the polyquaternium of PSC for
an acceptable test with G. holbrooki.
Parameter Acceptable before exposure Acceptable after exposure
pH 7.5 ± 0.3 ≤ 8.0
DO 90% ≥ 65%
Temperature 21 ± 1.0°C 21 ± 1.0°C

Figure 5.2 Gambusia in the polypropylene containers set up for a range-finding test. The three
tests in the foreground contain humic acid.

Test Concentrations: The test concentrations used in the test were determined from
rage-finding tests, and modified in subsequent tests based on the results. The anionic
surfactant SDS was also tested in the concentrations 0, 1, 2.5, 5, 10 mg/L.

118
Table 5.2 Concentrations of polyquaterniums used in fish toxicity tests.
Test Concentrations (mg/L)
Cetyl Pyridinium Chloride 0, 0.1, 0.25, 0.5, 0.75
poly(DADMAC) 0, 0.1, 0.25, 0.5, 1, 1.25
Gafquat® 440 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2.0
Gafquat® 734 0, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
Gafquat® HS100 (A) 0, 1.0, 1.33, 1.66, 2.0, 2.33
Gafquat® HS100 (B) 0, 0.3, 0.6, 1.0, 1.33, 1.66
Gafquat® HS100 (C) 0. 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
UCareTM JR125 (A) 0, 0.66, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
UCareTM JR125 (B) 0, 0.33, 0.66, 1.0, 1.33, 1.66
UCareTM JR125 (C) 0.33, 0.66, 1.0, 1.33, 1.66
UCareTM JR30M (A) 0, 0.66, 1.0, 1.33, 1.66, 2.0
UCareTM JR30M (B) 0, 0.1, 0.2, 0.33, 0.66, 1.0, 1.33
UCareTM JR400 0, 0.01, 0.02, 0.33, 0.66, 1, 1.33
UCareTM LK 0, 1, 3.33, 6.66, 10, 33, 66, 100
UCareTM LR30M 0, 1, 3.3, 6.6, 10, 33, 50
UCareTM LR400 (A) 0, 5, 10, 22, 48, 100
UCareTM LR400 (B) 0, 5, 10, 22, 48, 100
Conditioneze® NT-20 (A) 0, 0.3, 0.6, 1.0, 3.0, 5.0
Conditioneze® NT-20 (B) 0, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
Styleze® W-20 (A) 0, 0.05, 0.1, 0.25, 0.5, 1.0
Styleze® W-20 (B) 0, 0.1, 0.25, 0.5, 1, 2, 2.5

Table 5.3 Concentration of polyquaternium as polymer-surfactant complex used in the toxicity

tests.
Test Concentrations
Cetyl Pyridinium Chloride 0, 0.1, 0.25, 0.5, 0.75
poly(DADMAC) 0.1, 0.25, 0.5, 1.0
Gafquat® 440 0.66, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66, 5, 10.0
Gafquat® 734 0, 1, 1.33, 1.66, 2, 2.33
Gafquat® HS100 1, 1.33, 1.66, 2, 2.33, 2.66, 5
UCareTM JR125 0, 0.66, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
UCareTM JR30M 0, 0.66, 1.0, 1.33, 1.66, 2.0
UCareTM JR400 0, 0.66, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66
UCareTM LK 5, 10, 22, 48, 100
UCareTM LR30M 5, 10, 22, 48, 100
UCareTM LR400 5, 10, 22, 48, 100
Conditioneze® NT-20 0, 0.66, 1.0, 1.33, 1.66, 2.0, 2.33
Styleze W-20 0, 0.1, 0.25, 0.5, 1.0, 2.0, 2.5

Ethical Considerations: Under the Queensland Animal Care and Protection Act 2001,
Section 92, it is an offence to use animals, including fish and some macro-
invertebrates, to conduct the experiment commonly known as the classical LD50 test,
or a similar test; or use an animal for a scientific purpose if the use involves a
cosmetic (Queensland Government 2001). Compliance with the Australian Code of

119
Practice for the Care and Use of Animals for Scientific Purposes (NHMRC 2004) is
also required. The Code requires that death as an endpoint be avoided wherever
possible. To meet the requirements of the Griffith University Animal Ethics
Committee, changes to the standard OECD Test Guideline 203 Fish, Acute Toxicity
Test were made with respect to endpoint and ranging of the test (OECD 1992):

a) a non-lethal endpoint would be used;

b) only environmentally relevant concentrations would be tested.

As the mode of action of polyquaterniums was believed to result from the polymer
binding to the surface of the gill, leading to suffocation, and as Gambusia is able to
gulp air at the water surface in low oxygen situations, it was thought that this gulping
action would be an early indication of respiratory distress and therefore an appropriate
endpoint. Actual environmental concentrations were not known at the time ethics
approval was sought, therefore it was agreed that tests were to be conducted only in
the range that would result in a classification of Acute Toxicity under the Harmonised
Integrated Classification System For Human Health And Environmental Hazards Of
Chemical Substances And Mixtures i.e. up to, and not exceeding, 100 mg/L (OECD
2001).

5.2.4. Brine Shrimp

Brine shrimp (Artemia sp.) were purchased from a local supplier < 1week post-
hatching. The shrimp were acclimatised in artificial seawater prepared with
Coralife™ scientific grade marine salt for 24 hours and fed once before testing. Tests
were static and conducted in 100 ml of the same artificial seawater, (salinity
18 ± 0.5 ppk, pH 8.3, temperature 22 ± 1°C) in 250 ml plastic specimen jars. Air was
supplied as a gentle stream to the surface of the container. A minimum of 7
concentrations and a clean water control were used for each test. For tests of the PSC,
additional controls containing only the polyquaternium were included. Two replicates
of 10 shrimp were used for each concentration. The test duration was 48 hours and the
shrimp were not fed during that time. The shrimp were checked every 24 hours for
motility and morbidity, and dead shrimp removed from the container. The number of
surviving shrimp was counted at the end of the test. For a valid test, losses of < 10%
in the controls and DO ≥ 60% were required (OECD 1992).

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Test chemicals and concentrations: For the brine shrimp tests, the polyquateriums
UCareTM JR125, UCareTM JR30M, Gafquat® 440, Gafquat® 734 and
poly(DADMAC) were selected. The anionic surfactant, SDS, and the water treatment
polyquaterium, poly(DADMAC) were also tested. With this combination, both
cellulosic (UCare™) and synthetic polyquateriums (Gafquat®) are represented. In
addition, two charge densities are represented in the Gafquat® polyquateriums, and
two molecular weights by the UCareTM. This combination would enable any
differences found in the toxicity of the polyquateriums to be explained, or explored
with further testing.

Table 5.4 Concentrations of polyquaterniums, SDS and SDS as polyquaternium-surfactant

complexes used in brine shrimp tests.
Test Concentrations mg/L
SDS 0, 2.5, 5, 10, 15, 20, 25, 30
Polyquateriums 0, 1.5, 3.125, 6.25, 12.5, 25, 50, 100
Polyquaternium-surfactant 5, 10, 15, 20, 25, 30, SDS control 25,
complex polyquaternium control 100

5.2.5. Algae
The algal species, Chlorella sp 12, was obtained from the Centre for Environmental
Contaminants Research, CSIRO Land and Water, Lucas Heights, Sydney. The culture
and test procedures used are outlined in Franklin et al. (2002), and are summarised in
Table 5.5. Algae were counted using a Neubauer haemocytometer and microscope, at
24, 48 and 72 hours. To minimise adsorption loss of the test substance to the bioassay
test container, the flasks were pre-silanised with Coatasil™ (Ajax Finechem, Sydney)
(Stauber 1995). Coatasil had no effect on algal growth over 72 hours.

Table 5.5 Conditions for the culture and testing of algae.

Light intensity 0.96 - 1x104 lux cool-white light
Temperature 27° C
pH 7.5 ± 0.2
Culture Medium 1/5th Jarworky’s Medium
Test Medium Synthetic soft water (hardness 80-90 mg/L as
CaCO3), with 0.5 ml 26 mM NaNO3 and 0.05 ml 1.3
mM KH2PO4
Culture Vessel 250 ml glass screw-top culture flask
Test Vessel 250 ml silanised wide-mouth Erlenmeyer flask
loosely capped with plastic top.

Test chemicals and concentrations. For the algal tests, the polyquateriums UCareTM
JR125, UCareTM 400, UCareTM JR30M, Gafquat® 440, Gafquat® 734 and

121
poly(DADMAC) were selected. The anionic surfactant, SDS, and the water treatment
polyquaterium, poly(DADMAC) were also tested. Again, this combination would
enable any differences found in the toxicity of the polyquateriums to be explained, or
explored with further testing. Only one test was performed with the polyquaternium-
surfactant complex, as the anionic surfactant damages the Coatasil™ surface on the
test containers. It was not possible to test the effect of humic acid on the toxicity of
the polyquaterniums to algae. Humic acid produces a coloured solution which would
interfere with the light, and consequently the growth rate of the algae. Further, the
humic acid would hinder the counting of the algae with a microscope.

Table 5.6 Test concentrations of chemicals used in algal toxicity tests

Test Concentrations (mg/L)
poly(DADMAC) 0, 0.005, 0.01, 0.04, 0.06, 0.1
UCareTM JR125 0, 0.005, 0.01, 0.05, 0.1, 0.175
UCareTM JR400 0.0, 0.005, 0.01, 0.1, 0.1, 0.2
UCareTM JR30M .000, 0.005, 0.010, 0.050, 0.100, ,175, 0.250
Gafquat® 440 0.000, 0.005, 0.010, 0.050, 0.100, ,175, 0.250
Gafquat® 734 .000, 0.005, 0.010, 0.050, 0.100, ,175, 0.250
SDS 0, 0.0013, 0.975, 1.95
UCareTM JR125-SDS Complex 0, 0.01, 0.025, 0.05, 0.1, 1.0

5.3. Results, Observations and Data Analysis

5.3.1. Fish
The expected gulping of air by Gambusia at the surface of the water did not occur,
indicating either that morbidity did not result from suffocation or that this
compensatory mechanism was not invoked in this situation. Gulping is a response to
low oxygen levels in the environment, and has been reported as a response of
Gambusia to the blocking of the uptake of oxygen following exposure to the piscicide
rotenone (Nunes 2005). The signs observed in this work included lethargy (or
quiescence) – the fish ‘rested’ at the bottom of the container, sometimes with the tail
elevated; some bloating of the abdominal area, followed by loss of equilibrium (loe),
characterised by rolling and loss of control of mobility, and death. The lowest
observed effect level (LOEC) was defined as the lowest concentration at which these
symptoms occurred, and mortality greater than the mortality in the control. The
NOEC is thus the concentration immediately below that LOEC (if any). Any fish
displaying loe were removed from the container during inspection. Therefore the
EC50, where it was possible to establish, is defined as the EC50(loe).

122
Acute toxicity curves for Gambusia were very steep. Although the range of
concentrations was generally less than one order of magnitude (commonly 0.66 mg/L
to 2.66 mg/L), the LOEC concentration and the concentration at which ≥ 50%
mortality occurred were often consecutive test concentrations, and even in some
cases, the same concentration. This all-or-none type of lethal toxicity has also been
reported for the cationic surfactant didecyldimethylammonium chloride (DDAC)
(Juergensen et al. 2000), and for quaternary ammonium halides generally (Farrell et
al. 1998).

This steepness of the dose-response curve (Figure 5.3) presents some difficulties for
statistical analysis. The standard method of analysis for typical dose-response curves,
probit, ignores upper and lower concentrations where mortality is 0 or 100%, reducing
the degrees of freedom in the analysis. The data becomes too sparse to test for
goodness of fit, for example with Pearson’s test for chi squared distribution of
deviance (SAS 2002). Nevertheless, probit analysis does give an EC50 in the range
suggested by examination of the data, although fiducial limits (confidence intervals)
were not calculated for all tests. The toxicity (as EC50) of the cosmetic
polyquaterniums ranged from > 100 mg/L for the low charge density cellulosic
polyquaterniums UCareTM LR30M, LR400 and LK, to 0.5 mg/L for the high charge
density polyquaternium Styleze W-20. The latter was the only cosmetic
polyquaternium to have a toxicity approaching that of the water treatment
polyquaternium, pDADMAC and the quaternary surfactant cetyl pyridinium chloride.
For most of the cosmetic polyquaterniums tested, the EC50 was between 1.0 and
2.5 mg/L.

123
 120

100
80
Mortality

60
40
20
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration mg/L
(a)

120

100

80
% Mortality

60

40

20

0
0 0.5 1 1.5 2 2.5 3
Concentration mg/L
(b)
Figure 5.3 Typical plots of concentration vs. mortality for two polyquaterniums, poly(DADMAC)
(a) and Conditioneze® W-20 (Polyquaternium-55) (b) demonstrating the steepness of the curve in
this all-or-nothing toxicity to fish.

The results of the aquatic toxicity testing of the polyquaternium and the PSC are given
in Table 5.7 and Table 5.8 respectively. The EC50 values were determined by probit
analysis using SAS software. Tests of significance were also performed using SAS
software (SAS 2002). The EC50 values for the polyquaterniums and the PSCs were
highly correlated (Pearsons Correlation Coefficient (r) = 0.99494; Pr = < 0.0001). In a
paired t-test there was no significant difference between the EC50 for polyquaterniums
and PSCs (t Value = 0.14: Pr > |t| = 0.8867) (Table 5.9).

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Table 5.7 Result of probit analysis of data from 96 hour fish tests using G. holbrooki with
polyquaterniums and the monoquaternium cetyl pyridinium chloride without surfactant. Where
more than one test has been performed, the repeat results are indicated by the letters in
parenthesis.
NOEC LOEC EC50 range EC50 by 95%
mg/L mg/L mg/L Probit Fiducial
mg/L Limits
Cetyl Pyridinium 0.1 0.25 0.1-0.25 0.26 -
Chloride
poly(DADMAC) 0.1 0.25 0.25-0.5 0.35 -
Gafquat® 440 0.8 1.0 Not 1.0 -
determined
Gafquat® 734 < 1.0 ≤ 1.0 1.33-1.66 1.4 -
Gafquat® HS100 (A) < 1.0 ≤ 1.0 1.0-1.33 1.2 0.87
1.4
Gafquat® HS100 (B) 1.33-1.66 1.7 -
Gafquat® HS100 (C) 1.0-2.5 2.0 1.8
2.2
UCareTM JR125 (A) 0.66 1.0 1.0-1.33 1.2 -
UCareTM JR125 (B) 0.66 1.0 1.0-1.33 0.96 0.82
1.1
UCareTM JR125 (C) 0.33 0.66 1.0-1.33 1.3 -
UCareTM JR30M (A) 0.66 1.0 1.33-1.66 1.5 1.4
1.7
UCareTM JR30M (B) 0.66 1.0 ≥ 1.33 2.4 -
UCareTM JR400 0.66 1.0 2.0-2.33 2.1 -
UCareTM LK > 100 > 100 > 100 not
determined
UCareTM LR30M 10 33 > 100 not
determined
UCareTM LR400 (A) 10 22 > 100 not
determined
UCareTM LR400 (B) 5.0 10.0 48-100 52 32
120
Conditioneze® NT- 0.3 0.6 1.33-1.66 1.5 -
20 (A)
Conditioneze® NT- 1.0 1.33 1.33-1.66 1.5 1.3
20 (B) 1.72
Styleze® W-20 (A) 0.1 0.25 0.25-0.5 0.43 0.31
0.60
Styleze® W-20 (B) 1.0 0.25 0.5-1.0 0.52 0.39
0.71

125
Table 5.8 Result of probit analysis of data from 96 hour fish tests using G. holbrooki with PSC
and the monoquaternium cetylpyridinium chloride and SDS.
NOEC LOEC EC50 EC50 by 95% Fiducial
mg/L mg/L range Probit Limits
mg/L mg/L
Cetyl Pyridinium 0.1 0.25 0.1- 0.17 0.11
Chloride 0.25 0.24
poly(DADMAC) <0.1 0.1 0.25- 0.26 0.16
0.5 0.37
Gafquat® 440 0.66 1.0 5.0- 5.3 4.2
10.0 7.5
Gafquat® 734 1.0 1.33 1.0- 1.3 1.1
1.33 1.4
Gafquat® HS100 1.0 1.33 2.0- 2.1 2.0
2.33 2.3
UCareTM JR125 0.66 1.0 1.33- 1.4 1.2
1.66 1.5
UCareTM JR30M < 0.66 0.66 0.66- 1.59 1.4
1.0 2.0
UCareTM JR400 0.66 1.0 1.66- 1.72 1.4
2.0 2.1
UCareTM LK - - >100 not
detected
UCareTM LR30M - - - not
detected
UCareTM LR400 10 22 22-48 45 30
74
Conditioneze® NT- 1.0 1.33 1.0- 1.3 1.2
20 1.33 1.5
Styleze W-20 0.1 0.25 1.0-2.0 0.83 0.53
1.3

Table 5.9 Results of paired t-test for fish toxicity studies.

Analysis Variable : diff
N Mean Std Dev Lower 95% Upper 95% t Value Pr > |t|
CL for Mean CL for Mean
18 -0.0665328 1.9515180 -1.0369999 0.9039342 -0.14 0.8867

Humic acid, at concentrations of 5 and 10 mg/L reduced mortality to zero for

2.5 mg/L of UCareTM JR125, Conditioneze® NT-20 and Gafquat® HS100 and their
surfactant aggregates. Mortality was also reduced to zero by humic acid for
poly(DADMAC) and poly(DADMAC) with SDS at concentrations of 1.0 mg/L, and
Styleze® W-20 and W-20 with SDS at 0.5 mg/L. However, at concentrations of
1.0 mg/L, mortality for W-20 and W-20 with SDS was between 10 and 20%even in
the presence of humic acid.

126
 5.3.2. Brine Shrimp
The polyquaterniums tested were not classifiable as toxic to brine shrimp using
Globally Harmonised System for Classification and Labelling of Chemicals (GHS)
criteria (OECD 2001), as the median lethal concentration (LC50) was > 1000 mg/L.
The NOEC was < 100 mg/L. Sub-lethal effects noted were impaired motility,
resulting from loss of motion of the pleopods (also called swimmerets; the small
swimming appendages). Clumping of waste material occurred in the higher
concentrations (above the NOEC), with entrapment of individual shrimp with the
material. The LC50 for SDS was 16.6 mg/L (probit result 2.5 mg/L > EC50 <25 mg/L
range test). The observed effect of the surfactant was a blackening of the
metepipodites, which are ion-secreting epithelial structures on the individual thoracic
swimming appendages (phyllopodia).

The PSCs had non-standard toxicity curves. The maximum toxicity occurred at a
concentration of approximately 100 mg/L SDS (approximately 25 mg/L polymer),
indicating that the polymer had some mitigating effect on the toxicity of the
surfactant. Above that concentration, the mortality either declined (JR polymers)
(Figure 5.4) or stabilised (Gafquat® polymers) (Figure 5.5). At these higher
concentrations, clouding of the solution was observed, indicating association of the
polymer-surfactant aggregates.

80
70

60
% Mortality

50
40
30
20

10
0
0 20 40 60 80 100 120
[SDS] mg/L

Figure 5.4 Toxicity to Artemia (% mortality) for SDS complexed with UCareTM JR125. The non-
standard toxicity curves show a reduced mortality at higher concentrations of the complex.

127
 70

60

50
% Mortality

40

30

20

10

0
0 20 40 60 80 100 120
[SDS] mg/L

Figure 5.5 Toxicity (% mortality) for SDS complexed with Gafquat® 734. The non-standard
toxicity curves show a reduced mortality at higher concentrations of the complex.
5.3.3. Algae
The EC50 for algal growth inhibition was approximately one order of magnitude lower
than the fish toxicity value (Table 5.10). Surprisingly, the natural backbone JR
polymers were more toxic than the synthetic Gafquat® polymers, however this
difference was not significant if unequal variance was taken into account (Table 5.11).
Regression analysis of algae toxicity (EC50) and charge density was not significant
(F = 1.03; Pr > F 0.3567; R2 = 0.1709). In Dunnett’s test (Table 5.12) for difference
between test concentrations and control, the two lowest concentrations (0.005 and
0.01 mg/L) for the two Gafquat® polymers were not significantly different from the
controls. However, all other concentrations for these polymers, and all concentrations
for the remaining polymers were different to the controls.

Table 5.10 Results of analysis of data from algal growth inhibition test for polyquaterniums and
for the PSC for UCareTM JR125 only.
EC10 EC50 EC90
mg/L mg/L mg/L
poly(DADMAC) . 0.03 0.06
UCareTM JR125 . 0.04 0.09
UCareTM JR400 0.013 0.05 0.09
UCareTM JR30M 0.002 0.05 0.09
Gafquat® 440 0.037 0.12 0.21
Gafquat® 734 0.024 0.08 0.14
UCareTM JR125 + SDS 0.012 0.05 0.09

128
Table 5.11 Results of statistical test for difference in algal toxicity between the samples of UCare
JR125, JR400 and JR30M (Polyquaternium-10) and Gafquat® 440 and 734 (Polyquaternium-
11).
Method Variances DF t Value Pr > |t|
Pooled Equal 3 3.44 0.0413
Satterthwaite Unequal 1.06 2.63 0.2205
Test for Equality of Variances
Method Num DF Den DF F Value Pr > F
Folded F 1 2 24.00 0.0785

Table 5.12 Results of Dunnett's test for difference between test concentrations and controls in
algal growth inhibition test.
F value Pr > F R2 CV
Gafquat® 440 292.78 <0.0001 0.992966 10.66611
Gafquat® 734 367.02 <0.0001 0.994381 9.129007
UCareTM JR30M 553.40 <0.0001 0.996266 9.384088
UCareTM JR400 20.61 <0.0001 0.907302 27.91962
UCareTM JR125 147.53 <0.0001 0.984795 13.53072
poly(DADMAC) 120.62 <0.0001 0.982992 19.96053
UCareTM JR125 + SDS 44.32 <0.0001 0.954654 26.16208

5.4. Discussion
Toxic responses fall into two broad categories, reactive or specific toxicity and non-
reactive toxicity or narcosis. Reactive toxicity involves a specific chemical interaction
such as chemical reaction with an enzyme or inhibition of a metabolic pathway. Non-
reactive toxicity is not associated with a specific mechanism, but rather a physical
property which may be shared by chemicals of very different structural types, and is
related directly to the amount of toxicant acting on the organism. The toxicity of
chemicals with a reactive mode of action is primarily dependent on specific features
of the chemical structure of the toxicant. The aquatic toxicity of chemicals with a non-
reactive mode of action is generally dependent on the partitioning of the chemical
from water to the biophase and is generally related to the solubility of the toxicant in
lipids (Blum 1990).

Aquatic specific toxicity can be further divided according to the type of reaction it
involves, and the resulting manifestation of toxicity that occurs in the fish. McKim et
al. (1987) identified four types of response based on discriminant analysis of a range
of behavioural and morphological responses of fish to toxicants. In addition to the
narcotic response of non-reactive toxicants, three Fish Acute Toxicity Syndromes
(FATS) were identified: respiratory uncoupling, AChE inhibition, and gill irritation
(Table 5.13). The identification of FATS and the mechanism underlying them is an

129
important step in the development of Quantitative Structure-Activity Relationships
(QSAR) for predicting the toxicology of industrial chemicals. Although QSARs must
be developed for each mode of action, their development allows for rapid predictive
screening of hazardous chemicals for further study (McKim et al. 1987).

Table 5.13 Fish Acute Toxicity Syndromes (FATS) as described by McKim et al. (1987)
Mode of Respiratory- Plasma ion Gill Damage Behavioural
Action cardiovascular regulation Observations
Response
Narcosis Decline in pH, No increase in hypoactive,
O2; haemocrit concentrations under-reactive
and of plasma ions to outside
haemoglobin stimuli,
increased (in respiration
response to shallow and
hypoxia rapid,
discolouration
(dark,
blackish)
Respiratory pH, haemocrit, [K+], [Ca2+], No gill hyperactive
uncouplers haemoglobin and [Mg2+] damage and over-
and CO2 increased reactive, most
remained die without
unchanged exhibiting
numerous
signs of stress
AChE Heart rate and [Ca2+] and No gill generally
inhibitors O2 uptake is [Mg2+] damage for hypoactive;
reduced increased 80% of over-reactive
significantly survival time to stimuli;
high incidence
of abnormal
swimming;
deformities;
numerous
signs of stress
Gill irritants O2, CO2 and [Na+] and [Cl-] Histological initial increase
pH decreased; decreased damage to gill in cough rate,
haemocrit moderate-to-
increased low increase in
steadily, ventilation rate
haemoglobin
remained
constant

In the fish tests with both the polyquaternium and the PSC, no coughing was observed
to indicate mechanical clogging of the gill such as occurs with exposure to suspended

130
solids (Jobling 1995). No respiratory compensation such as gulping at the surface
occurred. This response has previously been reported for rotenone, which reportedly
acts by blockage of cellular oxygen uptake (McKim et al. 1987) in the study of the
toxicity of rotenone to Gambusia spp (Willis and Ling 2000). The reported response
to the rotenone exposure, however, included hyperactivity, stress and colour change
from silver to almost black. The hyperactivity exhibited in response to rotenone
suggests that this compound fits the category of respiratory uncoupler as described by
McKim et al. (1987), and the gulping action observed is a response to this
mechanism.

From the description of gill damage upon exposure to polyquaterniums (Boethling

and Nabholz 1997), the measurement of decreased blood pH (increase in [H+]), and
decreased concentrations of Na+ and Cl- (Muir et al. 1997), it would appear that
polyquaterniums, like many other ionic compounds, are gill irritants. Epithelial and
endothelial integument of the fish is tough and relatively impermeable, so that toxic
substances tend to be taken up by fish either by ingestion or through the gills
(Metcalfe 2000). The gill is the site of transport mechanisms that regulate osmotic and
ionic gradients, plasma pH and nitrogenous waste excretion, and so performs many of
the functions of the kidney in terrestrial animals (Evans et al. 2005). In addition to
entering the body via the gills, toxins can cause damage to the gill membrane itself
(Jobling 1995). Ions cannot diffuse through lipid membranes, and require active
transport via amino acids or protein ligands to penetrate membranes (Metcalfe 2000).
In conjunction with the findings of Muir et al. (1997) it appears the polyquaterniums
exert this toxic action without penetrating beyond the gill membrane. Other chemicals
known to be gill irritants include heavy metals, ozone (Wedemeyer et al. 1979),
acrolein and benzaldehyde (McKim et al. 1987).

All the reactive mechanisms described by McKim et al. (1987) cause alterations to
blood osmolity, but only gill irritation syndrome also results in a decline in blood pH.
That the observed decrease in sodium concentration occurs with increased
concentrations of H+ (decreasing pH) is probably the result of the coupling of Na+
uptake with proton efflux via V-H+-ATPase in the fish brachial epithelium (McKim et
al. 1987). In freshwater teleosts, V-H+-ATPase is located in respiratory pavement
cells of the brachial epithelium (Clairborne et al. 2002), and has also been described

131
for tight epithelia such as frog skin and distal portions of mammalian nephron tissue
(McKim et al. 1987).

The symptoms displayed by Gambusia in tests with polyquaterniums in this study

included lethargy with an onset of < 24 hours, followed by loss of equilibrium and
death, usually between 48 and 72 hours. It is likely, therefore, that the cause of death
was not suffocation, but acidosis resulting from the disruption of sodium uptake, and
consequently, acid-base regulation mechanisms.

The brine shrimp, in contrast to the fish, exhibited a toxic response to the anionic
surfactant SDS rather than to the polyquaterniums. The visible effect was the
discolouration (blackening) of the metepipodites, similar to the staining effect
reported by Holliday et al. (1990) following exposure to silver nitrate. However, as
the observed effect in this study followed exposure to the anionic surfactant SDS, it is
unlikely that the discolouration is associated with sites of chloride efflux as suggested
for silver nitrate staining. The metepipodites function like the gill in marine teleosts,
and have high levels of Na+/K+-ATPase indicating a role in osmoregulation (Holliday
et al. 1990). Although the similarity in function could suggest that marine teleosts
may also be less sensitive than fresh water teleosts, no difference in the sensitivity of
fresh and marine invertebrates and fish was found in a study of the cationic surfactant
ditallowdimethylammonium chloride (Lewis and Wee 1983). In published data by
Beim and Beim. (1994), fresh water shrimp were consistently less sensitive to cationic
polyelectrolytes than fish (or daphnids), with EC50 values of 70, 650 and 1160 mg/L
to three cationic flocculants (Appendix 2). However, shrimp were as sensitive or more
sensitive than fish to one of two cationic polyelectrolytes (Superfloc 330) in Biesinger
et al. (1976), and one of 12 cationic polyelectrolytes in Biesinger and Stokes (1986).
The difference in sensitivity between shrimp and fish to cationic polyelectrolytes
would therefore appear to be the result of differences in structure of the fish gill and
the metepipodite.

Toxicity of the polyquaterniums to algae follows the same steep dose-response curve
as observed for fish, suggesting again that a reactive mechanism of toxicity is
involved. Surfactants are known to bind and denature proteins on the cell wall of
algae, altering the permeability of the membrane to nutrients and chemicals (Pelczar
et al. 1993). Algal cell walls differ in thickness and chemical composition, which can
influence the severity of the toxic response to surface active agents (Lewis 1990).

132
This difference may account for exceptions reported by Boethling and Nabholz (1997)
to the general trend of cationic polyelectrolytes being six times more toxic to algae
than fish. In this study, algal EC50 values were between 2.5 and 11% of the fish EC50,
suggesting that in the case of these two species, Chlorella and Gambusia,
polyquaterniums are approximately an order of magnitude more toxic to the algae
than the fish.

5.4.1. Charge Density

The results for fish toxicity give no support to the relationship between charge density
and EC50. Of the polyelectrolytes in this study, only poly(DADMAC) has a charge
density high enough to suggest that it may be asymptotic according to the criteria of
Boethling and Nabholz (1997). Overall, the correlation between fish toxicity and
charge density in the data is not significant (r = -37441, p = 0.2566). The model for
fish toxicity proposed by Boethling and Nabholz (1997) (Equation 5.1 and Equation
5.2) is not particularly successful at predicating the toxicity of the polyelectrolytes in
this study, and especially not for the polyelectrolytes with charge density in the higher
or lower ranges of % a-N. For example, the toxicity of polyelectrolytes UCareTM LK,
LR400 and LR30M is significantly overestimated, while the toxicity of Styleze® W-
20 is underestimated.

Log[fish 96-hour LC50] = 1.209-0.462*% a-N Equation 5.1

for % a-N ≤ 3.5

Fish 96-hour LC50 = 0.28 mg/L Equation 5.2

for %a-N > 3.5

The UCareTM LR/LK polyelectrolytes have both low charge density and low toxicity.
These are cellulosic polyelectrolytes (natural backbone). Their low toxicity accords
with the results for Gendriv 162 (cationic guar gum) in Biesinger et al. (1976), and
with the low toxicity of the low charge density cellulosic polyelectrolytes in the
Office of Pollution Prevention and Toxics (OPPT) data (Boethling and Nabholz 1997)
discussed in Section 2.5.4. Clearly, charge density does not appear to be the
controlling factor here, as UCareTM LR30M and LR400 have similar charge density to
Gafquat® HS100 and 440. Nor does it appear to simply be the result of the cellulosic
backbone, as the higher charge density UCareTM JR polyelectrolytes have similar
toxicity to the synthetic carbon polyelectrolytes with similar charge density. It would

133
appear, therefore, that low charge density cellulosic polyquaterniums are less toxic
than their synthetic analogues, but high charge density cellulosic polyquaterniums are
not.

In a plot of the toxicity against charge density, most of the polymers lie in a cluster,
having similar charge densities and EC50s (Figure 5.6). In addition to the UCareTM
LR/LK polymers already mentioned, two other polymers are distinctive; Styleze® W-
20 (Polyquaternium-55), a high charge density, low molecular weight polymer used in
hair gels, and the low mw homopolymer poly(DADMAC) (Polyquaternium-6). To
further examine the role of charge density, it is possible to convert the EC50 from
mass units to equivalence units (Figure 5.7). Most of the polyelectrolytes still occur in
a cluster, but the two outliers, Styleze® W-20 and poly(DADMAC) are now
separated on both axes. These two polymers appear to be equally toxic when toxicity
is expressed in mass units (≈ 0.5 mg l-1), but are quite distinct when the toxicity is
expressed in equivalence units. In fact, in terms of equivalence units,
poly(DADMAC) is less toxic than all except the UCareTM LR/LK polyquaterniums,
while Styleze® W-20 remains the most toxic (Figure 5.7).

2.5

2
EC50 (mg/L)

1.5

0.5 W20
p(DADMAC)

0
0 0.001 0.002 0.003 0.004 0.005 0.006
Polymer charge density

Figure 5.6 Plot of EC50 for fish in mass units (mg/L) against charge density (eq/g) for eight
cosmetic polyquaterniums (Gafquat® 440, 734, and HS100; UCareTM JR125, JR30M, JR400;
Styleze® W-20) and polyDADMAC.

134
 3.E-03

3.E-03

2.E-03
EC50 (eq/L)
p(DADMAC)

2.E-03

1.E-03

5.E-04 W20

0.E+00
0 0.001 0.002 0.003 0.004 0.005 0.006
Polymer charge density

Figure 5.7 Plot of EC50 for fish in equivalence units (eq/L) against charge density (eq/g) for eight
cosmetic polyquaterniums (Gafquat® 440, 734, and HS100; UCareTM JR125, JR30M, JR400;
Styleze® W-20) and polyDADMAC.

As mentioned earlier, the UCareTM JR/LR/LK polymers are quaternised polymers of

quaternised hydroxyethyl cellulose and poly(DADMAC) is a synthetic carbon
homopolymer with its charged centre located in the polymer backbone. The remaining
polymers are all copolymers of 1-ethenyl-2-pyrrolidinone. Polyquaternium-11
(Gafquat® 755 and 440) and Polyquaternim-28 (Gafquat® HS100 and Conditioneze®
NT-20) are very similar acrylic copolymers. Polyquaternium-28 is polymerised with a
quaternary charged monomer, while Polyquaternium-11 is quaternised after reaction
with diethyl sulphate. Polyquaternium-28 has its trimethyl charged centre attached to
a three carbon pendant separated from the backbone by a nitrogen, while
Polyquaternium-11 has a dimethylethyl charged centre at the end of a two carbon
pendant separated from the backbone by an oxygen. The two cationic polymers also
have different counterions. These differences do not seem to have an impact on the
toxicity of the polyquaterniums to fish. Polyquaternium-55 (Styleze® W-20),
however is a highly cross-linked terpolymer. It has a tertiary ammonium group on a
short (3) carbon chain separated from the backbone by a nitrogen, but its quaternary
group, also three carbons attached via a nitrogen, has a dodecyl chain in place of one
methyl group on its charge centre. Polyquaternium-55 has a similar backbone to
Polyquaternium-28, but is only partly quaternised; however the quaternisation
involves addition of the hydrophobic long chain dodecyl group. It would appear,
therefore, that the important factor in toxicity may be the hydrophobicity of the
polymer, which appears to be the result of both charge density and polymer structure.
As was noted in Section 3.2.7, the presence of structural features that contribute to

135
hydrophobicity can result in deviations from 1:1 stoichiometry in metachromatic
polyelectrolyte titration, however, it is not clear that a similar mechanism is operating
in the toxic action.

5.4.2. Polymer-surfactant Complex

The low rates of deposition of the polyquaterniums on hair in the presence of SDS
reported by Andre et al. (1999) suggests that the aquatic toxicity of the PSC might be
lower than the toxicity of the polyquaternium alone, as mechanism of toxicity is
thought to be binding to the surface of the fish gill (Biesinger et al. 1976). This is
because binding to hair and sorption to gill surfaces are believed to be the result of the
same mechanism, i.e. coulombic binding with anionic sites on the surface of the gill
cells or hair. In addition, it has been suggested that the PSC formed at 1:1
stoichiometry is not as soluble as the polyquaternium alone, or aggregates formed at
other stoichiometries.

Phase diagrams produced by Goddard et al. (1975) suggest a precipitation zone at 1:1
stoichiometry, with areas of cloudiness, slight precipitation or turbidity. Clear zones
occur with either large excess of polyquaternium, or large excess of surfactant. At the
point of phase separation, it has been reported that the surface tension of the solution,
which begins to lower above the CAC of the polymer-surfactant system, continues to
drop, and does not level out until the critical micelle concentration (CMC) of the
surfactant is reached. This accords with the description by Koetz et al. (1996) that a
system in which two ions are of vastly different molecular weights will tend to form
films rather than flocs. The PSC being formed in these systems is surface active and
accumulates at surfaces, continuing to do so until the surfactant in solution begins to
form micelles in the bulk phase.

There was no clouding observed in the solution at the effective concentrations in any
of the fish or algae tests in this investigation. The solution concentration for SDS was
in the range 5 x 10-7 to 1.5 x 10-6 M which was well below the CMC of 8 x 10-3 M,
and possibly below the CAC, expected to be 2-3 orders of magnitude lower than the
CMC. Further, the effective polyquaternium concentration employed here in this work
is 2-3 orders of magnitude below the concentration at which many studies of PSC
behaviour are studied, generally 0.01 to 0.1%. According to Manuszak-Guerrini et al.
(1997), at concentrations less than 0.01%, a fixed concentration of SDS is required to

136
precipitate the complexes. Thus at the effective concentration, below 0.01%
polyquaternium, the neutral PSC may not precipitate.

The results of this work suggest that at the low concentrations which may be found in
the environment, the PSC could remain in solution long enough to effect the toxic
action and no mitigation of the toxicity of the polyquaternium resulting from
precipitation following association with an anionic surfactant such as SDS can be
expected. The toxicity of the monoquaternary surfactant cetylpyridinium chloride was
also unaffected by the presence of SDS at a 1:1 ratio. For the singly charged species,
formation of an ion pair would be expected to decrease the solution activity of the
cationic surfactant, which could be a factor in mitigating toxicity. The lack of effect
on the monoquaternary surfactant indicates that the reduction in solution activity at a
1:1 ratio and environmentally relevant concentrations of the cationic test substances
does not mitigate toxicity, and reduced toxicity may not be expected unless the
anionic surfactant concentration is sufficiently high to lead to precipitation of the
PSC.

5.4.3. Humic Acid

The mitigating effect of humic acid on the toxicity of polyquaterniums to fish has
been demonstrated to occur even in the presence of the anionic surfactant SDS. The
full extent of the mitigation was not measured in this work because of ethical
considerations mentioned previously. Qualitatively however, the mitigation is as
expected based on published studies. The extent of the mitigating effect has been
shown to be between one and two orders of magnitude at 10 mg/L humic acid, (Cary
et al. 1987; Goodrich et al. 1991; Hall and Mirenda 1991), and between two and three
orders of magnitude at 50-60 mg/L humic acid (Goodrich et al. 1991; Hall and
Mirenda 1991).

It has been recommended by Nabholz et al. (1993), for regulatory testing, that fish
toxicity tests of cation polyelectrolytes be separately conducted in clean dilution water
with total organic carbon (TOC) < 2 mg/L, and also with 10 and 20 mg/L humic acid.
The toxicity of the polyelectrolyte in the presence of 10 mg/L TOC can then be
determined from regression of the three toxicity values against TOC level. A
‘mitigating factor’ can then be determined by dividing the LC50 in 10 mg/L by the
LC50 in clean water. According to Nabholz et al. (1993), the mitigating factor for
cationic surfactants is expected to be less than 20. Based on an unpublished study of

137
eight polymers, Nabholz et al. (1993) suggested that the reduction in toxicity is about
94 times for high charge density cationic polyelectrolytes (3.3% a-N) in 10 mg/L
TOC equivalent to 27.6 mg/L humic acid, sodium salt (Aldrich).

The need to determine the mitigating effect in terms of TOC rather than humic acid
concentration is apparent due to differences in the TOC content of the humic acids
used in the published studies. The TOC content of humic acid used by Nabholz et al.
(1993) is approximately 37%, whereas Hall and Mirenda (1991) reported a TOC
content of approximately 16%. Neither Cary et al. (1987; 1989) nor Goodrich et al.
(1991) state the TOC content of the humic acid used in their mitigation studies. It has
been suggested that the use of Aldrich humic acid may result in an overestimation of
the effect of dissolved organic matter, as humic substances account for only 50 to
75% of dissolved organic carbon (Haitzer et al. 1998).

The difference in the binding capacity of humic acids of different sources is relevant
because of the assumption that the mitigating effect of humic acid is due to
preferential binding of cationic polyelectrolytes to dissolved organics resulting in
decreased availability of the polyelectrolyte to aquatic organisms (Goodrich et al.
1991). However, Matthews et al. (1995) has suggested that the amount expected to be
bound to humic acid or dissolved humic material may not be enough to explain the
reduction in toxicity. Humic acid has surfactant properties and can affect
physiological responses in living cells. By increasing the permeability of the
membrane in microorganisms, humic acid can result in an increase in
bioconcentration of some organic chemicals (Visser 1985). The mitigating effect of
humic acid on the toxicity of cationic surfactants has been shown to be dependent of
the chain length of the surfactant. According to Versteeg and Shorter, (1992) humic
acid did not affect the toxicity of quaternary ammonium surfactants to fish if the alkyl
chain length of the surfactant was 14 carbons or shorter, suggesting that the
interaction between the cation and humic acid was hydrophobic rather than
coulombic.

The results of this work show that the presence of dissolved organic material such as
humic acid can lead to a reduction in the toxicity of polyquaterniums and other cation
polyelectrolytes and surfactants, and this needs to be considered in the risk assessment
process. However, the determination of mitigation factor for individual polymers and
surfactants based on toxicity levels at various concentrations of humic acid requires a

138
level of testing which is difficult to justify on ethical grounds (let alone economic
ones). As the reduction in toxicity as reported by Hall and Mirenda (1991) and Cary et
al. (1987) seems to be reasonably consistent at between one and two orders of
magnitude regardless of the source of humic material, it may be appropriate to take
this into account when determining the assessment factors used in determining the
PEC, however, the presence of such concentrations of humic acid in natural water
cannot always be assumed, and therefore the use of a mitigation factor may not be
justifiable on environmental grounds.

5.5. PNEC
The lowest toxicity values in this study were the EC50 for algal growth inhibition, in
the order of 0.03 to 0.12 mg/L for the polyquaterniums tested. This accords with the
statement by Nabholz et al. (1993) that algae are the most sensitive species to cationic
polyelectrolytes. Although the algal test is technically a chronic test, covering several
generations, it is regarded as an acute test for the purposes of risk assessment (ECB
2003).

A number of uncertainties exist in extrapolating from laboratory toxicity data to

natural ecosystems, and these have been summarised as

a) intra- and inter-laboratory variation of toxicity data;

b) intra- and inter-species variations (biological variance);

c) short-term to long-term toxicity extrapolation;

d) laboratory data to field impact extrapolation (additive, synergistic and

antagonistic effects from the presence of other substances may also play a role
here).

The assessment factors recommended depend on the confidence with which the PNEC
can be derived, and are outlined in Table 5.14. When only short-term data are
available, as in this study, an assessment factor of 1000 will be applied to the lowest
L(E)C50. While the algal growth inhibition test is a multigenerational test, it is treated
as a short-term toxicity value for the purposes of determining appropriate assessment
factors (ECB 2003). Accordingly, the assessment factor to be applied in the
calculation of the PNEC for the polyquaterniums based on this study is 1000.
Applying the assessment factor to the algal EC50, therefore, gives a PNEC of between
0.03 and 0.12 µg/L.

139
Table 5.14 Recommended assessment factors to be applied in determining the PNEC are
dependent on the confidence that can be attributed to the available data, which is dependent on
the amount and types of toxicity data available.
Dataset Assessment Factor
At least one acute L(E)C50 from each of 1000x
the three trophic levels (fish,
invertebrates and aquatic plants)
One chronic NOEC (either fish or 100x
invertebrates)
Chronic NOECs from species 50x
representing different trophic levels
(fish, invertebrates and/or aquatic plants)
Chronic NOECs from at least three 10x
species (normally fish, invertebrates and
algae) representing three trophic levels
Species sensitivity distribution (SSD) 5-1
method (to be justified case by case)
Field data or model ecosystem review on a case by case basis

The mitigating effect of humic acid is generally not taken into account in the
determination of the PNEC. In determining the fate of polyquaterniums, it has been
assumed that the PEC will be fully mitigated by sorption to solids as, for example, in
the assessment of Merquat 2001 (NICNAS 2002a) which states

‘However, receiving waters usually contain suspended colloidal matter of

both mineral and organic origin (for example, humic material), and the
interaction of cationic polymers with this material has been demonstrated
to dramatically reduce the toxicity of the polymers. In addition, most of
the polymer will become associated with sewage sludge during passage
through the sewer mains and the sewage treatment plants and will not be
released to receiving waters.’

However, weight of evidence from published data gives reasonable certainty to a

reduction of one order of magnitude, at environmental concentrations of DOM, at
least in the case of fish. For this reason it may be possible to argue that this should be
taken into account in determining the assessment factors to be applied in determining
the PNEC. Such a strategy would reduce the assessment factors being applied in this
case to 100, and give a PNEC between 0.3 and 1.2 µg/L.

This strategy may be appropriate when the hazard of a group or class of cationic
polyelectrolytes is being considered, as in a Priority Existing Chemical assessment.
However, for the assessment of new cationic polymers, the extrapolation of possible

140
additive effects presents an additional factor that is not generally taken into account,
and the assessment factors based on the available data, without consideration of humic
acid effects, should probably be applied.

5.6. Conclusions
The polyquaterniums in this study were toxic to fish and algae. The lowest EC50, for
algae is ≤ 1 mg/L, therefore classification of Acute I under GHS is generally required
for many of these polymers. Establishing toxicity for polymers is made difficult by
the variation that can occur in the structure of the polymer, without changing the
identity of the polymer, as noted in Section 2.5.3. This has been demonstrated here in
the range of EC50 (fish) values for Polyquaternium-10, from 1.2 mg/L for UCareTM
JR125 to > 100 mg/L for UCareTM LK. There was no difference between the toxicity
to fish of a polyquaternium and the corresponding PSC.

The mechanism of toxicity of the polyquaterniums to fish would appear to be

disruption of osmoregulation following binding of the polyquaternium to the gill
surface. Knowledge of the toxic mechanism may be as important as the chemical
structure/behaviour in determining toxic responses, and further knowledge of the
interaction between the polyquaternium and the gill surface may be required before
QSARs can be developed. Further, an understanding of the different interactions that
occur with fresh and marine species may be useful in predicting toxicity in marine
environments.

The characteristic of polyquaterniums which has the most influence on the toxicity of
the chemical would appear to be hydrophobicity. Hydrophobicity is a function of both
the charge density and structural features of the polyquaternium such as chain length.
Greater understanding of the structural elements that contribute to hydrophobicity,
and the role of hydrophobicity in toxicity, may help to clarify some of the apparent
inconsistencies in the published toxicity data on cationic polyelectrolytes, as well as
assist in the development of QSARs.

The PNECs for the cosmetic polyquaterniums examined in this study are in the range
between 0.03 and 0.12 µg/L in the absence of humic acid.

141
6. Risk Characterisation
6.1. Introduction
The risk characterisation process combines the information obtained from the effects
and exposure assessments to evaluate the nature of the potential risk (Molak 1997). It
is the final process of the four-step paradigm as originally described in ‘Risk
Assessment in the Federal Government: Managing the Process’ (NRC 1983),
commonly referred to as ‘the red book’ and further developed in ‘Science and
Judgement in Risk Assessment’ (NRC 1994) (known as ‘the blue book’) as methods
for determining health risks to human populations, and later adapted for ecological
risk assessment. The development of the process of risk characterisation is reflected in
the changing definition of risk characterisation since the original publication of the
red book (Table 6.1).

142
Table 6.1 Definitions of risk characterisation published in various guidelines and articles since
the introduction of the four-step paradigm in 1983.
The description of the nature and often the magnitude of (NRC 1983)
human risk, including attendant uncertainty.
The description of the nature and often the magnitude of risk (Ramamoorthy and
to human health with associated levels of uncertainty. Baddaloo 1991)
Risk characterisation involves the integration of information (NRC 1994)
from the first three steps to develop a qualitative or
quantitative estimate of the likelihood that any of the
hazards associated with the agent of concern will be realised
in exposed people.
Risk characterisation consists of integrating the results from (Covello and
release assessment, exposure assessment, and consequence Merkhoffer 1993)
assessment, to produce a quantitative measure of health and
environmental risks.
Risk characterisation consists of evaluating and combining (Molak 1997)
dose-response relationship data with an exposure
assessment.
Risk characterisation integrates information from the (USEPA 2000)
preceding components of the risk assessment and
synthesises an overall conclusion about risk that is complete,
informative, and useful for decision makers.
Risk characterisation provides a qualitative or quantitative (EnHealth 2002)
estimate, including attendant uncertainties, of the nature,
severity and potential incidence of effects in a given
population based on the hazard identification, dose-response
and exposure assessments.
Risk characterisation is the estimation of the incidence and (ECB 2003)
severity of the adverse effects likely to occur in a human
population or environmental compartment due to actual or
predicted exposure to a substance, and may include ‘risk
estimation’, i.e. the quantification of that likelihood.

Although there seems to have been reasonable consistency as to what the risk
characterisation process is, there has been no definition of risk included in either of
the National Research Council (NRC) publications mentioned above. In fact, many
guidelines for risk characterisation have been published without including a definition
of risk itself, for example, the United States Environment Protection Agency
(USEPA) Guidelines for Carcinogen Risk Assessment (USEPA 1996b), Guidelines
for Reproductive Toxicity Assessment (USEPA 1996a), and even the Risk
Characterisation Handbook (USEPA 2000). A definition of risk was included in the
ERA Guidance for Superfund (USEPA 1997a), and is included in Table 6.2 with other
recent attempts to define the entity being measured in the risk characterisation
process.

143
Table 6.2 Definitions of risk published in various guidelines and articles since the introduction of
the four-step paradigm in 1983.
The probability that, in a certain timeframe, and adverse outcome (EnHealth
will occur in a person, group of people, plants, animals and/or the 2002)
ecology of a specified area that is exposed to a particular dose or
concentration of a hazardous agent, i.e. it depends on both the
level of toxicity of the agent and the level of exposure.
Actual risk is the probability or chance of an occurrence of (Frantzen and
adverse outcomes (sickness or death) to human health or Ackerman
environmental functioning at some frequency and some level of 2002)
intensity.
Perceived Risk is a risk as defined by its importance and
significance to individuals, community and society.
A measure of the probability of the introduction of an exotic (Molak 1997)
disease and the seriousness of such an outcome in the context of
the importance of animal or animal products.
The expected frequency or probability of undesirable effects (Molak 1997)
resulting from exposure to known or expected stressors.
A characteristic of a situation or action wherein two or more (USEPA
outcomes are possible, the particular outcome that will occur is 1997a)
unknown, and at least one of the possibilities is undesired.
The probability that a hazard (H) will have the unwanted effect {Malmfors,
(E) on a vulnerable object (V) under certain given conditions (C), 2002 #61}
which can be expressed as R(hvec)

The lack of clarity in the definition of risk is evident in the various meanings that are
assigned to the word risk in the scientific literature, for example: Risk = hazard;
Risk = likelihood, probability; Risk = damage, loss, consequence; Risk = relative
frequency (Malmfors and Rosing 2002). According to Malmfors and Rosing (2002),
this can lead to ambiguity in the outcome of the risk assessment, and difficulties in
interpretation and communication of the risk.

In addition to the problems of defining and identifying risk, measuring risk also
presents difficulties. Generally, it is considered that risk can be characterised
quantitatively (as a number) or qualitatively (in categories, e.g. high, medium, low).
In situations where no toxicological and exposure data are available, only qualitative
estimates of risk are possible. This occurs frequently in Australia in new chemical risk
assessment, where some chemical classes, or chemicals imported at low volumes, are
exempt from providing toxicological data (NICNAS 2004b). An example of this type
of qualitative estimation of risk is the Full Public Report (FPR) for Gafquat® HS-100
(NICNAS 1999).

144
One of the most common methods of risk characterisation is the quotient method, the
ratio between predicted no effect concentration (PNEC) and predicted environmental
concentration (PEC). Often considered a numerical method, descriptions of this
method are given in many risk assessment handbooks. The process of calculating the
quotient is intended to be iterative, with ambiguous results resulting from a quotient
≈ 1 necessitating further investigation of either the toxicology or environmental fate in
order to arrive at a more definitive result. An alternative method, developed to meet
the demand for rapid assessment required for the European Chemicals Bureau (ECB)
review of industrial chemicals, Registration, Evaluation and Authorisation of
Chemicals (REACH) involves a screening process based on an Environmental
Threshold of No Concern (ETNC). The ETNC is a level of environmental exposure to
chemicals below which no significant risk is expected to exist, and may be determined
for chemicals or classes of chemicals (de Wolf et al. 2005). Both these methods
provide a point estimate of risk. A third method, Probabilistic Risk Assessment
(PRA), uses statistical methods to describe the uncertainty in the risk estimate. Using
techniques such as Monte Carlo Simulation, the likelihood of each risk is
quantitatively characterised in the risk estimate (USEPA 2004).

In this chapter, three methods, ETNC, PNEC/PEC ratio, and a probabilistic version of
the PNEC/PEC ratio using Monte Carlo Simulation will be applied to the risk
characterisation of cosmetic polyquaterniums in Australia. Further, the risk
characterisation methods will be reviewed to assist in determining the usefulness,
advantages and shortcomings of the process with respect to the regulatory risk
assessment of new and existing chemicals.

6.1.1. Monte Carlo Simulation

Monte Carlo Simulation is a technique for characterising the uncertainty and
variability in risk estimates by repeatedly sampling the probability distributions of the
risk equation inputs and using these inputs to calculate a range of risk values (USEPA
2001). This technique will be used in this thesis to examine the probabilities and
uncertainties in the model presented in Section 6.2, and the risk characterisation in
Section 6.4. In Monte Carlo Simulation, the same calculations used in a point estimate
model are performed many times, with the input variables resampled for each
calculation from a probability distribution selected for each variable. The probability
distribution, or probability density function (pdf) of each variable can either be

145
determined directly from the data if sufficient data points are available, from
statistical parameters such as mean and standard deviation if they are known, or
estimated from the most likely value of the variable, the Central Tendency Estimates
(CTE) and a reasonable worst case or conservative estimate, the Reasonable
Maximum Estimate (RME). The RME is the value most often used in estimates. The
result of the Monte Carlo Simulation is a probability distribution for the output
variable (the forecast). Monte Carlo Simulation thus gives a range of output values,
with their associated probabilities.

6.2. The Environmental Threshold of No Concern

Method
One method for evaluating the potential risk of a chemical is the threshold of
toxicological concern (TTC), which is based on establishing an exposure threshold
value below which no significant risk to human health and/or the environment is
expected. In this method, the emphasis is not on the hazard of individual chemicals,
but in establishing a de minimis value for any chemical or class of chemicals that
could be applied even if no toxicological data is available. The method has been
proposed in response to the requirements of the REACH legislation in the EU, and
would allow the legislative requirements to be met while reducing animal testing and
focusing resources on the substances likely to pose the greatest risk (De Wolf et al.
2005).

Specifically for environmental concerns, the TTC takes the form of an Exposure
Threshold of No Concern (ETNC) for each environmental compartment (De Wolf et
al. 2005). In the aquatic compartment, the application of ETNCaq relies on a
classification of chemicals by mode of action as proposed by Verhaar et al. (2000).
Similar to the classification scheme for fish toxicity discussed in Chapter 5, this
scheme has four classifications base on mode of action (MOA): MOA1 inert
chemicals, MOA2 less inert chemicals, MOA3 reactive chemicals, and MOA4
specifically reacting chemicals. The first two of these correspond approximately to the
narcotic and polar narcotic substances of McKim et al. (1987). In the scheme
proposed by Verhaar et al. (2000), the specific mechanism of reactive chemicals is not
considered. Chemicals classed as MOA4 tend to be pharmaceuticals and pesticides
whose toxicity is based on specific interactions with receptor molecules, while those

146
classed as MOA3 react unselectively with certain chemical structures commonly
found in biomolecules or can be metabolised to more toxic species.

It has been suggested, based on analysis of the available toxicity data on the European
Union New Chemicals Database, that there is no evidence to suggest that a de minimis
ETNCaq(MOA1-3) of 0.1 µg/L is an unacceptable value, and that this value can be used
as a first approximation for comparison with PEC values for screening-level risk
assessments (de Wolf et al. 2005). No similar consensus has been reached on a default
value for de minimis ETNCaq(MOA4), (de Wolf et al. 2005), although a value of
0.01 µg/L has been suggested (Straub 2002). In Chapter 5, it was suggested that the
PNEC for polyquaterniums was nominally ≥ 0.01 µg/L, and approximately between
0.01 and 0.1 if a one order of magnitude mitigating effect by DOM was considered.
This result is consistent with polyquaterniums having a specific, targeted mode of
action (MOA4) in vulnerable species. The incorrect classification of an MOA4
chemical as MOA1, MOA2 or MOA3 can lead to the underestimation of its potential
toxicity (De Wolf et al. 2005). However, as the mode of action of polyquaterniums is
not certain, and the mitigating effect of DOM needs to be considered, model estimates
will be derived for concentrations of 0.1 and 0.01 µg/L.

Advocates of the ETNC approach have suggested three areas where the application of
the method may be appropriate:

a) as a first approximation in screening-level assessment by chemical producers

in the early stages of product development;

b) site-specific assessments for downstream users;

c) selecting chemicals for including in environmental monitoring.

The concept is useful in the context of this work too. The model used in Chapter 4 to
predict Wastewater Treatment Plant (WWTP) effluent levels from given influent data
can be reversed to determine an influent concentration corresponding to an ETNCaq if
appropriate physico-chemical data are known or can be estimated. This method allows
the estimation of a trigger value of import/manufacture that would justify further
investigation of the fate and aquatic toxicity of the chemical concerned.

6.2.1. Method
Returning to the fugacity model and representative wastewater treatment plant used in
Chapter 4 to determine the PEC, it should be possible to estimate, from the ETNCaq,

147
the input flux that leads to the threshold concentration. For polyquaterniums, as in the
model in Chapter 4, only sorption and advection are considered. However, in contrast
to the simple model in Chapter 4 which required only the partition coefficient and
fluxes, this application of the model requires the determination of the fugacity
capacity constant, Z for dissolved polyquaternium (ZW) and also for polyquaternium
sorbed to biosolids (ZB).

The value for ZW can be estimated from Henry’s Law Constant as

1
ZW =
H Equation 6.1

where ZW is the fugacity capacity constant for the dissolved polyquaternium

in µg/L.Pa
H is Henry’s Law Constant

and

P
H = Equation 6.2
CW
where P is pressure
and CW is concentration in water.

Therefore the fugacity capacity constant is

CW
ZW = μg / L.Pa Equation 6.3
P

For the purposes of this exercise, the water solubility is estimated as 107 µg/L, which
is the highest concentration dissolved in preparation of solutions during this project.
The vapour pressure used was 1 x 10-10 Pa, which was one of the lowest measured
vapour pressures found in the literature (for octochlorodibenzodioxin). Therefore, by
Equation 6.2, for a given polyquaternium (PQ-X), HPQ-X = 10-17 L.Pa/µg and by
Equation 6.3, ZW = 1017 µg/L.Pa. This approach of using an arbitrarily small vapour
pressure has previously been employed by Holysh et al. (1986) when investigating the
fate of linear alkylbenzenesulphonate surfactants. It is necessary because
fundamentally, fugacity requires a chemical species to exert a vapour pressure.
Another alternative would be to use the concept of ‘aquivalence’ (Diamond 1999), but
since there was a good precedent for the former approach, it was adopted.

It has been demonstrated (Tan et al. 2007) that the value of ZB changes at various
sequences in the process train, and is concentration dependent. The value of ZB,

148
therefore, must be estimated for each process. This can be accomplished from the
experimentally determined value of KD and ZW.

ZB
KD = Equation 6.4
ZW
where ZB is the fugacity Capacity Constant for the polyquaternium in
biosolids in µg/kg Pa.

For the purposes of demonstrating the utility and application of the model, an ETNCaq
of 1 µg/L will be used. This will be assumed to be the concentration (dissolved) of the
polyquaternium (PQ-X) in effluent, without dilution to receiving waters. Using the
parameter estimate of KD ≈ 400 L/kg, implies that Csorbed in effluent is ≈ 400 µg/kg
(Equation 6.5).

Csorbed μg / kg
K D ≈ 400 l / kg = Equation 6.5
Cwater μg / l

The mass balance parameters based on Oxley WWTP also remain as used in the
previous model in Chapter 4. As the effluent flow rate, from the water balance, is
571 x 103 L/h, the effluent flux (dissolved) is 5.71 x 105 µg/h. The effluent flow rate
(solids) is 5.71 kg/h and the effluent concentration (sorbed) for PQ-X is 400 µg/kg.
The effluent flux (sorbed) for PQ-X is 2.3 x 103 µg/h and the total PQ-X effluent flux,
dissolved and sorbed, is 5.73 x 105 µg/h.

Final Settling Tank (FST)

D5 Effluent

D4

Figure 6.1 Representation of the mass balance in the Final Settling Tank of the WWTP.

For the final settling tank, the advection rate constants, D can be estimated from a
mass balance

149
D5 f PQ − X = Effluent flux + D4 f PQ − X Equation 6.6

where

D5 = Q5W ZW + Q5 B Z B Equation 6.7

where Q is the volumetric flow rate of the phase (m3/h)
and

D4 = Q4W Z + Q4 B Z B Equation 6.8

The mass balance equation can be rearranged to find fPQ-X in the final settling tank
(Equation 6.9)

( D5 − D4 ) f PQ − X = Effluent flux Equation 6.9

Finally, the total flux into the final settling tank, and the return activated sludge flux
can be estimated from

total flux in = D5 ∗ f PQ − X Equation 6.10

flux ( RAS ) = D4 ∗ f PQ − X Equation 6.11

where flux(RAS) is the return activated sludge flux

Bioreactor

D3 D5

D4

Figure 6.2 Representation of the mass balance in the bioreactor of the WWTP.

The mass balance for the bioreactor is given as

( D3 + D4 ) f PQ− X = D5 f PQ− X Equation 6.12

The rate constants D4, D5 and the fugacity fPQ-X, have been calculated above, and

150
D3 = Q3W ZW + Q3 B Z B Equation 6.13

Primary Settling Tank (PST)

D1 D3

D2

Figure 6.3 Representation of the mass balance in the Primary settling tank of the WWTP.

The mass balance for the primary settling tank is given by

D1 f PQ − X = ( D2 + D3 ) f PQ − X Equation 6.14

The rate constants D3 and the fugacity fPQ-X, have been calculated above, and

D1 = Q1W ZW + Q1B Z B Equation 6.15

and

D2 = Q2W ZW + Q2 B Z B Equation 6.16

As the sorption-desorption process occurs prior to arrival at the WWTP, the influent
flux can be subdivided into its dissolved and sorbed components (assuming
equilibrium)

Total flux = Q1W CW + Q1B Csorbed Equation 6.17

and

Csorbed Equation 6.18

= KD
Cwater

therefore

151
Csorbed = K D ∗ CW Equation 6.19

The model parameters used here are based on the flow rates of only one of four waste
streams in the Oxley WWTP. To convert to a population usage volume for PQ-X, it is
necessary to multiply the influent flux by four (assuming equal flow in all four
streams) before converting to a tonnes per year volume usage. The people equivalent
capacity for Oxley WWTP is 240,000 (Tan et al. 2007). Based on Brisbane water
usage of 62% domestic, a rounded figure of 60% of the people equivalent capacity is
assumed to come from domestic sources. The critical usage volume is calculated from
the Oxley WWTP influent flux in tonnes/year as per Equation 6.20.

flux Equation 6.20

Total import volume = × total population
pec × df

where flux: total influent flux for Oxley

pec people equivalent capacity for Oxley WWTP (240,000)
df fraction of pec assumed to be from domestic sources (0.6)
total population of Australia, 20, 000,000
6.2.2. Results
With the assumption that the ETNCaq is an in-stream concentration, diluted by a factor
of 10 from WWTP effluent concentration, and that the partition coefficient for
polyquaterniums is 400 L/kg, the approximate value for the JR polyquaterniums, or
1000 L/kg as for the Gafquat® polyquaterniums, the point estimates of critical usage
volume for risk assessment purposes is approximately 3 tonnes per annum if the mode
of action is non-specific (MOA3) and an order of magnitude less if the mode of action
is targeted (MOA4). This would translate to a personal use of 0.1 and 2.0 grams per
person per day of product containing 0.25% polyquaternium (Table 6.3). For
comparison, the results assuming a KD of 10,000 are also reported. The point estimate
was calculated in an Excel spreadsheet (Appendix 3b).

152
Table 6.3 Estimates of input volume and product usage for various ETNCaq for polyquaterniums,
calculated for KD values of 400 (UCareTM JR125) and 1000 (Gafquat® 755) and 10000.
ETNCaq Effluent KD Influent flux Influent flux National As product
µg/L Conc. Oxley Oxley Use Volume at 0.25%
µg/L µg/h kg/year tonnes/year g/person day
0.01 0.1 400 2.46E+05 2.16 0.26 0.1
0.1 1 400 2.46E+06 21.59 2.63 1.0
0.01 0.1 1000 2.70E+05 2.36 0.29 0.2
0.1 1 1000 2.70E+06 23.63 2.88 2.0
0.01 0.1 10000 4.81E+05 4.22 0.51 0.3
0.1 1 10000 4.81E+06 42.16 5.13 3.0

In a Monte Carlo simulation of the model using Crystal Ball® (Decisioneering 2007)
the influent concentration was shown to have a lognormal distribution. The model was
run separately for the two values of ETNCaq. The model variable KD was assumed to
have a Maximum Extreme Distribution (Gumbel) with the input parameters, Likeliest
and Scale, set at 1000 and 400 respectively. This setting gave a distribution which
included values up to approximately 3000 in the extreme tail (Figure 6.4), This
positively skewed distribution suggests that while the KD values determined
experimentally in Chapter 4 are the most likely values, higher values such as those
reported by Podoll and Irwin (1988) are also possible. This probability density
function for KD was used in all following simulations. All other parameters relating to
the flows, water and solids balance in Oxley WWTP were assumed to be normally
distributed, as no data was available to suggest an alternative distribution. The results
for sample simulations are given in Figure 6.5. The mean concentration in the
simulation is close to the value determined in the point estimates above, indicating
that the model gives a Central Tendency Estimate of the influent flux. Any
conservatism in the estimate is therefore contained in the estimate of the ETNCaq.
Sensitivity analysis suggests that the modelled influent flux of polyquaternium is most
sensitive to the solids balance in the final settling tank, with KD having a significantly
smaller effect.

The importance of choosing the correct mode of action, and consequently the correct
ETNCaq is apparent in the point estimate result, as shown in both the point estimate
and probabilistic results. The one order of magnitude difference in the ETNCaq
translates directly into an order of magnitude difference in the trigger value for the
import/manufacture. By contrast, an incorrect estimation of the partition coefficient is
far less significant. A change of one order of magnitude in KD results in a difference

153
in the trigger value of only a factor of about two. All the results, up to and including
the extreme value of KD, result in a per capita use of shampoo or equivalent product
that is less than the default guideline value of 2.3 g/day in the EU guidance manual
(ECB 2003).

Figure 6.4 Probability density function for the input variable partition coefficient KD used in the
Monte Carlo Simulation of the ETNCaq model.

Figure 6.5 Forecast Probability Density Function (left) and sensitivity analysis (right) for the
influent flux of polyquaternium, from the Monte Carlo simulation of the ETNCaq model.

6.3. The PEC/PNEC Ratio (The Quotient Method)

The PEC/PNEC ratio, sometimes called the Risk Quotient (Verdonck et al. 2007) or
Hazard Quotient (Hull and Swanson 2006) is perhaps the most common method of
conducting the risk characterisation in the four-step paradigm associated with risk
assessment. In the quotient method, the exposure value, PEC, is directly compared to
the hazard value, PNEC. The greater the quotient is above one, the higher the
probability that an adverse effect will occur (Bascietto 1990). Although the values of
the quotient are said to correspond to levels of risk, it is important to note that the
quotient does not actually measure probability (Tannenbaum 2005a).

154
 6.3.1. Method
It was noted in Chapter 4 that it was necessary for the PNEC and PEC to be expressed
in units that allowed their ratio to be determined. Although both have been derived in
units of concentration (mg/L or µg/L), the PEC has been estimated for all cosmetic
polyquaterniums combined, while the PNEC has been estimated for a sub-set of those
polyquaterniums. While there are possible methods of estimating market share, it is
not clear if this will improve the certainty of any predicted PNEC/PEC ratio.
Importantly, it is not known to what degree either the toxicities of, or the risk from,
polyquaterniums may be additive.

The PNEC/PEC ratio has been estimated in only one assessment of new
polyquaterniums by National Industrial Chemicals Notification and Assessment
Scheme/Department of Environment and water Resources (NICNAS/DEW) (Table
6.4), viz the nominally high volume import Merquat 2001 (NICNAS 2002a). In the
assessment of PQ-28 Gafquat® (NICNAS 1999) the import volume is given as 5
tonnes, but the PEC has been calculated on an import volume of 1 tonne. The
remaining assessments are all based on import volumes of less that one tonne per
year, resulting in a PECinfluent of 1 µg/L using the method outlined in Section 1.2. In
only one assessment was the PEC predicted for receiving waters (NICNAS 2002a).

Based on these reports however, data generated in this study can be used to predict a
PNEC/PEC ratio for polyquaterniums in Australia. The calculations will assume that
the volumes in the NICNAS reports, 1, 5 and 16 tonnes are typical
import/manufacture volumes for polyquaterniums. PNEC/PEC ratio will be calculated
for these typical volumes, as an estimate of the possible risk of individual
polyquaterniums. A PNEC/PEC ratio will also be calculated for the total volumes
estimated in Chapter 4, to estimate the risk from polyquaterniums if the toxicities/risk
are additive.

155
Table 6.4 Analysis of the PEC/PNEC estimates in NICNAS assessments. Note that different
assumptions regarding population size and water use volumes may apply in these assessments.
Assessment Lowest PNEC PECinfluent PECreceiving_waters PEC/PNEC
Toxicity
Value
NA475 0.68 mg/L Not 1.01 µg/L - Not
PQ-34 (Daphnia) estimated estimated,
but safety
margin of
134 assumed
to exist
NA533 1 mg/L Not 1 µg/L - Not
Luviquat (algae) estimated estimated,
hold estimated but a safety
from fish margin of
toxicity of 1000
10 mg/L assumed to
exist
NA896 0.73 mg/L Not 15.4 µg/L 1.54 µg/L 0.5 (based
Merquat (algae) estimated on applying
2001 the safety
factor of
1000 to the
ratio, rather
than the
PNEC.
NA961 1 µg/L Not 0.95 µg/L 0.0048 µg/L Not
Luviquat (algae) estimated estimated,
Care estimated but safety
from fish margin of
toxicity 1000
data assumed to
exist
NA89 None Not 1 µg/L - Not
Gafquat® available estimated (based on estimated
HS-100 1 tonne
pa,
although
import
volume is
up to 5
tonnes pa).
6.3.2. Results
The PEC/PNEC ratios in data calculated in this work involve PEC values which are
based on the highest and lowest fraction removed via sludge modelled in Chapter 4
(21 and 73%), and on the default removal rate of 90% recommended by Nabholz et al.
(1993), and include a 1:10 dilution in receiving waters. The highest and lowest PNEC

156
data estimated in Chapter 5 have also been used. In line with the recommendation of
the USEPA review (2004), the PEC/PNEC ratio is reported to one significant figure
only. As can be seen from Table 6.5, even at the minimum import/manufacture
volumes (< 1 tonne), the PEC/PNEC ratio may exceed one if the fraction removed is
low (21%). At the higher import volumes the risk quotient can be markedly > 1.

Table 6.5 Estimated PEC/PNEC for a polyquaternium at the modelled WWTP fraction removed
(21-73%) from Chapter 4 and the default assumption of 90% removal via sludge.
Import Volume Percent PEC PNEC (PEC/PNEC)
Removal µg/L µg/L ratio
21 0.06 0.03 2
21 0.06 0.12 0.5
73 0.02 0.03 0.6
1 tonne
73 0.02 0.12 0.2
90 0.01 0.03 0.2
90 0.01 0.12 0.08
21 0.28 0.03 9
21 0.28 0.12 2
73 0.10 0.03 3
5 tonnes
73 0.10 0.12 0.8
90 0.04 0.03 1
90 0.04 0.12 0.3
21 0.91 0.03 30
21 0.91 0.12 8
73 0.31 0.03 10
16 tonnes
73 0.31 0.12 3
90 0.12 0.03 4
90 0.12 0.12 1

The estimate of the risk associated with all polyquaternium use from cosmetic
applications is difficult to estimate, as the total import/manufacture volume is
unknown, although estimates of Australian usage were provided in Table 4.4 and
Table 4.5. Further, as has been demonstrated, not all polyquaterniums are equally
toxic, although the PNEC for most cosmetic polyquaterniums falls within a narrow
range. By using the volume estimates from Chapter 4, and a representative toxicity
value from Chapter 5 (say, UCareTM JR125), it is possible to estimate a maximum
PEC/PNEC for cosmetic polyquaternium use in Australia (Table 6.6). As this would
be a high-end estimate for polyquaterniums as a class, it may be reasonable to allow a
mitigating factor of one order of magnitude for the effect of DOM. However, even in
this scenario, the risk quotient is ≤ 1 only if the default 90% sorption to and removal

157
with sludge in a WWTP is assumed, and not at the removal rates estimated by the
models used in this work.

Table 6.6 Estimated PEC/PNEC for all polyquaternium use in Australia, assuming additive
toxicities, at the modelled WWTP removal rates (21-73%) from Chapter 4 and the default
assumption of 90% removal via sludge.
Import Volume (tonnes) removal rate % PEC µg/L PNEC µg/L PEC/PNEC
21 4.50 0.04 110
80 73 1.60 0.04 40
90 0.58 0.04 10
21 3.20 0.04 80
60 73 1.20 0.04 30
90 0.43 0.04 10
21 2.30 0.04 60
40 73 0.77 0.04 19
90 0.29 0.04 7

6.4. Probabilistic Risk Characterisation

Risk analysis has been defined as a methodology that derives a probability of an
adverse effect on an agent (Molak 1997). More specifically, Malmfors and Rosing
(2002) defined risk as the probability that a hazard (H) will have the unwanted effect
(E) on a vulnerable object (V) under certain given conditions (C), which can be
expressed as R(hvec). It is not surprising then, that there are increasing calls for risk
assessors to report risk in terms of probability (Williams 2002). Probabilistic Risk
Assessment is a term for risk assessments that use a variety of probabilistic models to
characterise uncertainty in risk estimates, or to quantify the variability in a population
that results in different levels of risk (USEPA 2001). The application of PRA methods
has been facilitated by the development of statistical sampling techniques and the
availability of software to undertake the complex calculations required (USEPA
2001). According to Williams et al. (2002), PRA addresses the question ‘What is the
likelihood (i.e. the probability) that risks to an exposed individual will exceed a
regulatory level of concern’ and provides an answer along the lines ‘It is estimated
that there is a 10% probability of an individual exposed under these circumstances has
a risk exceeding 10-6’. While conventional measures of risk, such as PEC/PNEC
provide an estimate of the extent of possible effects, PRA goes a step further and
estimates a probability of the effect occurring.

6.4.1. Method
The Monte Carlo simulations were performed using Crystal Ball® software
(Decisioneering 2007). The simulations correspond to the point estimates calculated

158
in the previous section (PEC/PNEC), for import levels of 1 tonne, 16 tonnes and for
the total import volume model. To determine the probability distribution for the
quotient, it is first necessary to determine a probability distribution for the PEC and
PNEC. For the PEC estimate, several parameters were assigned distributions and
assumptions, and forecasts were made for PEC (all simulations) and for influent and
effluent concentrations (for import volume 16 tonnes and all polyquaterniums). With
the exception of import volume, the same distributions were used for each simulation.
Where the parameter estimate is usually a conservative value in point estimates of
risk, a Maximum Extreme Description or Minimum Extreme Description was used.
These distributions are positively and negatively skewed respectively, and the
distribution is defined by two parameters, Likeliest and Scale. A Central Tendency
Estimate (CTE) was used as Likeliest, and the scaling factor adjusted to give an
appropriate range, with the conservative default estimate in the long tail. For example,
the default value for the percent of the import value that is released to sewer is
designed to factor in the amount of the product that is generally left in the retail
container and disposed to landfill. This is generally assumed to be between 10%
(highly viscous material such as paint) and 1% (low viscosity liquids). The default
includes the assumption that all products are used maximally, which may not be true.
The Maximum Extreme Distribution used for this variable has 90% as the Likeliest
value, and a scaling factor of 5 (Table 6.7). The distribution is therefore skewed
towards a high value and has a range from 100% to as low as 65% at the low extreme
(Figure 6.6a). Similarly, the default value for the amount removed in the WWTP is
90%, whereas the modelling in this study suggests it could be as low as 20%. The
distribution of water use per day is scaled to include the default value of 200 L/day
and the value of 350 L/day evident in the Oxley WWTP based model in Chapter 4
(Table 6.7). The distribution also includes the extreme values of domestic use that are
used in as default values in risk assessment (150 L/person day) (Figure 6.6b). A
Monte Carlo Simulation of the percentage removal model using an approximate
distribution of KD described in Chapter 4, with all other Oxley parameters normally
distributed suggests that percentage removal has a gamma distribution. However, the
Minimum Extreme Distribution used here takes in the default value of 90% used by
NICNAS/DEW (Figure 6.6c). Similarly, the distribution for dilution to receiving
waters encompasses both the default options 10 (ocean) and 1 (rivers), while allowing
that the dilution to ocean outfall may be somewhat higher (Figure 6.6d). No

159
probability distribution was assigned to the variables of population or days per year
when release occurs. The values for these parameters were set at 20 million and 365
respectively.

Table 6.7 Probability density functions of variables used in all Monte Carlo Simulation of the
PEC calculation.
Model Variable Point Probability Density Distribution
Estimate Function Parameters
(Default)
Percent of polyquaternium 100 Minimum Extreme Likeliest: 90;
expected to be released to Distribution Scale: 5
sewer
Water Use per person (L/day) 200 Minimum Extreme Likeliest: 300;
Distribution Scale: 30
Removal within STP (fraction) 0.9 Maximum Extreme Likeliest: 0.26;
Distribution Scale: 0.12
Dilution Factor 10 Maximum Extreme Likeliest: 10;
Distribution Scale: 5

(a) (b)

(c) (d)
Figure 6.6 Probability density functions for variables common to all simulations of probabilistic
risk assessment (a) proportion of polyquaternium released to sewer; (b) water use per person; (c)
proportion of polyquaternium removed in WWTP; and (d) dilution to receiving waters.

Different pdfs were used for import volume in each of the simulations (Table 6.8). For
the simulations of import volumes < 1000 kg and ≤ 16 tonnes, distributions that could
take into account the maximum and minimum limits of the NICNAS notification
categories were selected, that is, uniform and beta respectively. The uniform
distribution assumes that any value between the set minimum and maximum is
equally likely (Figure 6.7a). The beta distribution also allows a minimum and
maximum value to be set, but allows for a scaling of the distribution away from
normal, in this case, towards the maximum value of 16 tonnes (Figure 6.7b). For the

160
simulation of all cosmetic polyquaterniums, no minimum or maximum constraint is
set. However, the volume is unlikely to be less than the import volume for any one
polyquaternium. Therefore a log normal distribution was selected (Figure 6.7c),
limiting lower end estimates. The mean and standard deviation (50 and 10 tonnes
respectively) were selected to cover the possible range of import volumes estimated in
Chapter 4.

(a) (b)

(c)
Figure 6.7 Probability density functions for input volumes for the three simulations of the PEC
(a) import volume < 1000 kg; (b) import volume < 16 tonnes; and (c) estimated total import
volume for all cosmetic polyquaterniums.

The result of the Monte Carlo Simulation is a pdf for the PEC for each of the three
import scenarios. This pdf was then combined with the pdf determined from the
toxicity data to determine the probability that the PEC/PNEC was ≤ 1. Although algae
were the most sensitive species, there was not sufficient data to create a pdf in Crystal
Ball® for algal toxicity. However, a pdf was created for the fish data, which was
found to have a log normal distribution with a mean of 2.46 µg/L and a standard
deviation of 3.85 µg/L (Figure 6.8a). The toxicity of polyquaterniums to algae was
approximately one order of magnitude higher, and for the purposes of this simulation,
could be assumed to follow the same distribution as fish toxicity. However, the
purpose of risk assessment is to protect vulnerable, not average, species and
individuals, as indicated by the selection of the most sensitive species toxicity value
for the risk characterisation, and the pdf in a includes the values of median effective
concentration (EC50) for the low toxicity polyquaterniums UCareTM LR400 and
LR30M. This inclusion skews the mean toxicity away from values that would protect
sensitive species from more toxic polyquaterniums. The value selected for this

161
variable may still have some uncertainty, however and a probability function can be
estimated. In this case, the extreme maximum distribution was again selected. Based
on the algal data in this study, the Likeliest value was determined to be 0.12 µg/L, and
the scale factor 0.02 selected to include the highest toxicity value (lowest EC50) of
0.03 µg/L, as determined in Chapter 5 (Figure 6.8b). For more general assessments,
the probability function could be set to include the ETNCaq, with 1.0 as Likeliest, and
the scale adjusted to include the MOA4 value of 0.01 in the tail.

Table 6.8 Probability density functions of import volumes used in Monte Carlo Simulation for
three simulations of the PEC calculation.
Import Volume Probability Distribution Distribution
Function Parameters
<1000 kg Uniform Minimum: 100 kg;
Maximum: 1000 kg.
≤ 16 tonnes Beta Minimum: 1 tonne;
Maximum: 16
tonnes;
α: 4; β: 2
All cosmetic Log normal Mean: 50
polyquaterniums. Standard deviation:
10

(a) (b)
Figure 6.8 Fish probability distribution function from data (a), and assumed for the Monte Carlo
Simulation (b)

6.4.2. Results
For the simulation of PEC for the < 1 tonne import volume, the probability that the
PEC ≤ 0.03 µg/L is 0.9, at a confidence level of 99% (Figure 6.9). The simulation of
the PEC was most sensitive to the variables import volume (51.3%) and dilution to
receiving waters (-38.3%) (Figure 6.9). The probability distribution function for PEC
input into the Monte Carlo Simulation of the PEC/PNEC was a log normal
distribution with a mean of 0.02 and a standard deviation of 0.08 (Figure 6.10). The
probability that the ratio (PEC/PNEC > 1) was between 0.94 and 0.99, with an
average 0.95 at 95% level of certainty in five successive runs of the simulation. An
example of the output is shown in Figure 6.11.

162
Figure 6.9 Probability Density Forecast (left) and sensitivity analysis (right) from a Monte Carlo
Simulation of the PEC for a polyquaternium at an import volume of < 1tonne.

Figure 6.10 Probability Density Function for the PEC input into PEC/PNEC Monte Carlo
Simulation for import volume < 1 tonne, a log normal distribution with mean = 0.02; stddv = 0.08

Figure 6.11 Probability density forecast from a Monte Carlo Simulation for the PEC/PNEC for a
polyquaternium import volume of < 1 tonne

At an import volume of 16 tonnes, the mean forecast PEC was 0.32 µg/L (Figure
6.12). At 95% level of confidence, the PEC at p = 0.9 was 0.6 µg/L. The variables to
which the PEC simulation was most sensitive were dilution factor (73.2%), import
volume (-21.2%) and removal in the WWTP (-13.5%) (Figure 6.12). The probability
distribution function for PEC input into the Monte Carlo Simulation of the
PEC/PNEC was a log normal distribution with a mean of 0.34 and a standard
deviation of 1.13 (Figure 6.13). In five successive simulations, the probability that the
PEC/PNEC > 1 was 0.5 at 95% level of certainty (Figure 6.14).

163
Figure 6.12 Probability density forecast and sensitivity analysis from the Monte Carlo Simulation
of PEC for a polyquaternium at an import volume of 16 tonnes.

Figure 6.13 Probability Density Function for PEC input into Monte Carlo Simulation of the
PEC/PNEC for import volume of < 16 tonnes, a log normal distribution with mean = 0.34; stddv
= 1.13.

Figure 6.14 Probability density forecast from a Monte Carlo Simulation of PEC/PNEC for
polyquaternium at an import volume of < 16 tonnes.

In the simulation of the PEC estimate for all cosmetic polyquaterniums, the forecast
mean PEC was 1.51 µg/L (Figure 6.15). At 95% level of confidence, the PEC at
p = 0.9 was 2.68 µg/L. The simulation of the PEC was again most sensitive to the
variables dilution factor (-69.3%), import volume (-14.0%) and removal in the
WWTP (10.9%) (Figure 6.15). The probability distribution function for PEC input
into the Monte Carlo Simulation of the PEC/PNEC was a log normal distribution with
a mean of 1.51 and a standard deviation of 1.17 (Figure 6.16). In five successive
simulations, the probability that the PEC/PNEC > 1 was 1.0 at 95% level of certainty
(Figure 6.17).

164
Figure 6.15 Probability density forecast (right) and sensitivity analysis (left) from a Monte Carlo
Simulation of the PEC for an estimated total cosmetic polyquaternium import volume.

Figure 6.16 Probability Density Function of PEC for input into PEC/PNEC Monte Carlo
Simulation for import volume for all polyquaterniums, a log normal distribution with
mean = 1.51; stddv = 1.17.

Figure 6.17 Probability density forecast from a Monte Carlo Simulation of the PEC/PNEC for
estimated total cosmetic polyquaterniums import volume.

6.5. Discussion
6.5.1. Is This Risk?
Three methods of characterising ‘risk’ for the purpose of regulating chemicals have
been employed here to examine the possible impacts of polyquaterniums from
cosmetic uses. The PEC/PNEC ratio method is a widely accepted method of
estimating the risk associated with the release of new chemicals and the review of
existing chemicals. The ETNCaq, method, however, is a new method proposed to meet
the increased demand for the assessment of existing chemicals required by REACH.
Probabilistic Risk Assessment (PRA) is becoming increasingly used to quantify the

165
uncertainty in risk assessments of contaminated sites by USEPA (USEPA 2001), but
has not yet been applied to regulatory new or existing chemical risk assessment in
Australia. It is necessary to examine, however, exactly how these methods have
characterised the risk, and how they advance the risk assessment process for
regulatory chemical risk assessment.

The ETNCaq, does not combine the PNEC and PEC to produce a single numeric
indicator in the way the ratio method does. It is claimed by its advocates to provide an
indication of risk by purporting to be a critical value of the PEC at which risk
approaches zero. Rather than putting a value on the risk per se, it is intended to be a
screening tool, to determine if further risk assessment is required by identify those
cases in which the risk may be significantly greater than zero. The method, therefore,
has only two possible outcomes. Either the risk approaches zero (negligible risk) or
the risk is not close to zero and therefore further assessment is required. The
application of the method requires, in addition to the ETNCaq, a good estimate of the
usage volumes, and possibly release and fate parameters. In assessment of new
chemicals, the usage volume is known, though fate parameters may not be well
established, and measured environmental concentrations are generally not available
(or at least, not for the jurisdiction under consideration). In assessment of existing
chemicals, usage data can generally be sought under the regulatory framework, but
fate parameters and measured environmental concentrations may or may not be
available. In the proposed application of the method for REACH, if the usage volume
indicated that the PEC may be greater than the ETNCaq, further assessment of the fate
parameters and release data may be required (De Wolf et al. 2005). As it has been
applied here, where actual usage volumes are not known but some information on the
fate parameters is available, it has been used to determine the import/manufacture
volume at which further assessment of the fate of the polyquaternium may need to be
examined, probably around three tonnes per annum.

As the PEC/PNEC ratio gives a numerical result, it is generally regarded to be a

quantitative measure of risk (ECB 2003). However, it has also been suggested that the
PEC/PNEC is not a measure of risk at all (Tannenbaum 2005a), and indeed, it is
sometimes referred to as Hazard Quotient rather than Risk Quotient (DeMott et al.
2005; Hull and Swanson 2006). Further, the quotient method has been criticised for
the tendency of risk assessors to view the value of one as a threshold value, with

166
quotients of 1.1 requiring progression to the next stage of risk assessment (DeMott et
al. 2005). This assumption of precision led to the USEPA guideline of one significant
figure for the PEC/PNEC ratio referred to in the previous section.

Alternative methods of interpreting the result from the PEC/PNEC ratio method exist.
One example that has been used is the USEPA ecotoxicological assessment criteria
for pesticides, shown in Table 6.9 (Bascietto 1990). Note that in this method, no
assessment factors are applied to the median lethal concentration (LC50), and the
equivalent of the PEC is called the Estimated Environmental Concentration (EEC). As
risk assessment can be defined as an estimate of risk (Rowe 1992), the use of this term
perhaps goes some way to addressing the problem of perceived precision in numerical
expressions of risk.

Table 6.9 Ecotoxicological assessment criteria for pesticides used by USEPA to estimate the
hazard potential of pesticides to non-target aquatic organisms (Bascietto 1990).
EEC <1/10 LC50 Presumption of no hazard
1/10 LC50 ≤EEC < ½ LC50 Presumption of hazard that may be
mitigated by restricted use
EEC ≥ ½ LC50 Presumption of unacceptable hazard

Similarly, the European Commission directive for risk assessment of new substances
has four possible outcomes from the quotient method (Figure 6.18). These are (1) no
further testing (PEC/PNEC ≤ 1), (2) further testing at 10 tonnes production threshold,
(3) immediate further testing, or (4) immediate risk reduction measures. Where the
PEC/PNEC ratio is between 10 and 100, the decision to go to further testing
immediately or at the 10 tonne production threshold is made on the basis of factors
such as

a) indication of bioaccumulation potential;

b) the shape of the toxicity/time curve in ecotoxicity testing;

c) data on structurally analogous substances.

167
Figure 6.18 Decision scheme for aquatic risk characterisation of new chemicals (ECB 2003). The
fourth possibility, no further assessment if PEC/PNEC is < 1 is not shown on the diagram.

In comparison to its own criteria for the assessment of pesticides, and the EU criteria
for the assessment of new chemicals as described above, the USEPA staff paper’s
proscription against more than one significant figure as a way of addressing the
assumption of precision in the risk characterisation seems barely adequate. By this
method, a point estimate of the PEC/PNEC of 0.9 is acceptable, but 2 is not. Even in
the EU and pesticide methods, the interpretation of certain values as ‘bright lines’
exists, and this still seems to be giving the impression of a level of certainty in these

168
assessments that may not be justified. It is suggested therefore, that although the point
estimate of PEC/PNEC uses numerical methods, it is, in fact, only a qualitative
method of characterising risk, and only three outcomes are possible from this method:
PEC/PNEC << 1 (acceptable risk); PEC/PNEC >> 1 (unacceptable risk); or
PEC/PNEC ≈ 1 (indeterminate risk or risk requiring intervention). The decision of
how much greater or less than one is significant is a policy decision, not a scientific
one, and will be addressed in Section 6.5.4.

In contrast to the point estimate of the PEC/PNEC, the results of the PRA include a
distribution of forecast results, the probabilities associated with the forecast results,
and analysis of the sensitivity of the forecast to each input variable. Not only was it
possible to fit probability distributions to both the PEC and the PNEC, but it was also
possible to determine importance of the variables in determining the forecast. The
essential difference is that with PRA, it can be stated that ‘At the level of 95%
confidence the probability that the PEC/PNEC for an import volume of 16 tonnes will
be < 1 is 0.5’, rather than ‘A conservative estimate is that the PEC/PNEC for an
import volume of 16 tonnes is likely to be < 1’. While the probability estimate, or
more specifically, the sensitivity analysis that accompanies it, is helpful in
determining where it might be necessary to focus further data collection and research,
it does not account for all the uncertainty in the risk characterisation, nor overcome all
the limitations of the PEC/PNEC method. Further, the method has not dealt with the
issue of the appropriateness of the PEC/PNEC as a measure of risk. For example, the
PRA gives us no answer to the problem of the assessment factors used to account for
uncertainties in toxicological methods (effects of incremental dosages, differences
between laboratory test and field populations, indirect effects of toxicant such as food
chain interactions, other ecosystem effects including predator-prey relationships,
community metabolism, structural shifts) (Bascietto 1990). Further, the reliability of
the PRA is dependent to a large extent on the appropriateness and/or suitability of the
pdfs assigned to the variables, which can only be validated by data from field studies.

6.5.2. Uncertainty in PNEC and PEC

The red book (NRC 1983) describes uncertainty as ‘pervasive’ and the ‘dominant
analytical difficulty’ in risk assessment. Uncertainty and variability exist in all risk
assessments (USEPA 2004) to an often unknown degree (Malmfors and Rosing
2002). An examination of the sources of uncertainty, either qualitatively or

169
quantitatively, can be a productive means of refining a risk assessment, or to find
solutions to the problems at issue (Verdonck et al. 2007), and is of interest in this
work.

In many discussions of uncertainty and variability in risk characterisation, the

distinction between the two terms is often blurred, and the terms even used
interchangeably. Alternative terms have also been used, for example, Wilson and
Shlyakhter (1997) describes ‘stochastic uncertainties’ and ‘uncertainties of fact’,
while Verdonk et al. (2007) uses ‘epistemological uncertainty’ and ‘ontological
uncertainty’ for uncertainty and variability respectively. Variability (stochastic
uncertainty) is generally considered to be the measured and therefore known variation
among members of a defined population potentially leading to variations in risk
(Wilson and Shlyakhter 1997). For Verdonk et al. (2007), ontological uncertainty
(variability) is the inherent randomness in the parameter, but also includes variability
in human behaviour and variability in societal factors such as the development of
technological systems. Variability is a fundamental property of nature and is
irreducible through further measurement. Uncertainty (epistemological uncertainty,
uncertainty of fact), however, arises from a lack of knowledge which can be reduced
through additional investigation.

In this study, a large amount of the uncertainty in the risk characterisation results
because the import/manufacture volume of the polyquaterniums is unknown and
therefore uncertain (reducible uncertainty). If it was possible to determine the usage
volume by asking the importers and manufacturers how much is manufactured or sold
each year, there would still be variability in the usage volume. The volume may
change from year to year, market share may be won from or lost to alternative
products, formulation of specific products may change, new products may be
developed, all contributing to variation in the usage volume. In new chemicals risk
assessment, the notifier is required to state the maximum annual import volume for
the first five years. There is no uncertainty in this volume; though there may be some
variability (the notifier may not reach their sales target). However, after 5 years, when
the chemical is publicly listed on the Australian Inventory of Chemical Substances
(AICS), no import restrictions exist, and usage volume becomes uncertain and
variable.

170
Increasing the data available for risk assessment, however, does not necessarily
reduce uncertainty. For example, it has been regarded as reasonably certain in risk
assessments by NICNAS/DEW and in the literature that the adsorption of
polyquaterniums in WWTP was close to 100% due to the high affinity of the cationic
sites on the polymer for the anionic sites on biosolids. Based on this assumption, the
default value of 90% reduction in concentration in WWTPs has been used as a
‘conservative’ estimate. However, this work with cosmetic polyquaterniums has
suggested that removal of polyquaterniums in WWTP may be significantly lower, due
to the relatively high aqueous solubility of the cosmetic polyquaterniums and the
relatively low concentrations of solids in wastewater. It has also been assumed that
the desorption of polymers generally, and therefore polyquaterniums, is unlikely to
occur for reasons outlined in Section 2.4.2, and this view has been maintained in risk
assessment, despite some evidence to the contrary. The ability of polyquaterniums to
desorb (as mentioned previously) is considered one of their advantages in hair and
skin care applications, and evidence to this effect is freely available on the
manufacturers’ websites.

Uncertainty in risk assessment can arise from many factors, and has been broadly
classified into three main areas (USEPA 1992):

a) scenario uncertainty, arising from missing or incomplete information e.g.

descriptive errors, aggregation errors, errors in professional judgement, and
incomplete analysis;

b) parameter uncertainty, which affects a particular parameter e.g. measurement

errors, sampling errors, variability, and use of generic or surrogate data;

c) model uncertainty, arising from the scientific theory affecting the ability of a
model to make predictions.

Uncertainty in the derivation of the PEC and PNEC in this study contains
uncertainties that fall into each of these groups. Greater uncertainty exists in the PEC,
the sources of which are described in Table 6.10. In each of the scenarios modelled
(< 1, < 16 and ≈ 50 tonnes import volume), the difference between the CTE (mean
estimate) value and the RME (conservative estimate) was two orders of magnitude. In
sensitivity analysis, several factors were found to contribute significantly to the
uncertainty the PEC, notably import volume, in-stream dilution of WWTP effluent,

171
removal within the WWTP, and to a lesser extent per capita water use and the
proportion expected to be released to sewer. Generally, there was greater sensitivity to
those variables with a large amount of scenario uncertainty, where data was missing
or incomplete.

Table 6.10 Factors contributing to uncertainty in the estimation of the PEC of polyquaterniums
in Australia.
Parameter Type of Comments
Uncertainty
Import/manufacture Parameter Very little data available on which to make an
volume uncertainty estimate, particularly for total polyquaternium
use. Even for scenarios representing new
chemical assessment, the right of notifiers to
keep the true volume from publication
contributes to uncertainty in the prediction of
PEC. The reliability of pre-market estimates
may also a problem.
Emission estimate Scenario Assumed 100% release of the imported
uncertainty volume to waste water. In some assessments,
90% release would be used, assuming 10%
remained in container and is disposed of to
landfill.
Partition coefficient Parameter Arising from measurement using
uncertainty metachromasy, and from the surrogate use of
humic acid.
WWTP model Model Assumption that Oxley WWTP is typical of
uncertainty all WWTPs.
Lack of measured data to validate the model.
PEC calculation Parameter Arising from estimates of water usage
uncertainty estimate, and dilution to receiving waters.

Uncertainty in the PNEC is considerably less than the PEC, with the highest and
lowest value determined varying only by a factor of four, from 0.03 to 0.12 µg/L.
However, this lower variation arises in part as a result of ignoring those
polyquaterniums with lower toxicity (higher EC50) on the basis that they present no
risk to aquatic organisms at any conceivable environmental concentration. A wider
variation of aquatic toxicities exists in the literature, but the values determined in this
study are a reasonable fit to published values. The species differences in toxicity of
polyquaterniums covers approximately three to four orders of magnitude (Appendix
2), very similar in range to cationic surfactants (Madsen et al. 2001). Although some
of the uncertainty in the PNEC is addressed by the assessment factors discussed in
Chapter 5, other factors can apply in individual studies. Some possible sources of
uncertainty resulting from the methods used in this study are included in Table 6.11.

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A further difficulty in examining the uncertainty in the toxicity of polyquaterniums
stems from the inability to allocate a specific toxicity value to a specific polymer,
because the toxicity may vary with the charge density and to a lesser extent molecular
weight, of the polyquaternium, as demonstrated with the range of values for
Polyquaternium-10.

Table 6.11 Factors contributing to uncertainty in the estimation of the PNEC of polyquaterniums
in Australia.
Parameter Type of Uncertainty Comments
Toxicity testing Parameter uncertainty Arising from the use of
procedures nominal rather than actual
concentrations; selection
of test organisms,
selection of test medium;
use of non-lethal
endpoint.
Mitigation study Parameter uncertainty Use of humic acid as a
surrogate for DOM.
Calculation of the PNEC Model uncertainty Arising from use of
assessment factors.

In the point estimate of PEC/PNEC, the uncertainties in critical parameters have been
retained and aggregated in each stage of the study. This method often results in
uncertainties that are too large to result in meaningful conclusions (Bascietto 1990).
The combining of the highest and lowest values of the PEC and PNEC has resulted in
ratios that range from << 1 to >> 1, a result that would appear to be unhelpful in
resolving the risk characterisation of polyquaterniums. However, the use of
uncertainty analysis, even qualitatively, can be useful in understanding the risk
characterisation. In this study, the qualitative characterisation of uncertainty supports
and enhances the sensitivity analysis from the Monte Carlo Simulation.

6.5.3. Conservatism and Precaution

An alternative approach to aggregating the uncertainties in point estimates of
PEC/PNEC is to use default assumptions, models and surrogate data as substitutes for
the missing data. According to the USEPA staff paper (USEPA 2004), the approach
adopted should ‘not underestimate risk in the face of uncertainty and variability’. The
default values used should, according to the staff paper, ‘guard against
underestimating risk while also being scientifically plausible given existing
uncertainty.’ However, this conservative approach has been widely criticised among
industry stakeholders, earning such titles as ‘unnecessary conservatism’ (Conolly et

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al. 1999) and ‘compounding conservatism’ (Stahl et al. 2005). The use of
‘intentionally protective’ assumptions results in ‘biased’ risk characterisation (DeMott
et al. 2005), and has the potential to mask uncertainty in the risk characterisation
(Verdonck et al. 2007). The overstating of risks in conservative risk assessments is
thought to lead to inappropriate risk management decisions (Stahl et al. 2005), unless
risk managers are aware that the risk characterisation is not a real risk measure, but a
measure to avoid false negatives (unsafe chemicals that are assessed as safe)
(Verdonck et al. 2007). Further, the method may lead to more stringent regulatory
controls and standards than are necessary (Conolly et al. 1999). The use of PRA,
however, does not completely overcome the conservatism of most point estimate
calculations, as the conservatism can simply be built into the probability distribution
of the variable.

Other stakeholders may take the position that the conservative approach is not
protective or precautionary enough. As the USEPA acknowledges in the staff paper
(USEPA 2004), it is possible that in individual cases the final overall risk estimate
will underestimate the true risk, even though the tendency is for the estimate to be
higher than the true risk rather than lower. In contrast to the position taken by industry
stakeholders, it has been argued that uncertainty in the data and scientific evidence in
the risk assessment process may create a bias in favour of regulatory inaction (Latin
1997; Wilson and Shlyakhter 1997), as regulators may be reluctant to enforce
restrictions or controls without good science and a degree of certainty.

According to Gullett (2000), a distinction needs to be made between ‘prevention’ and

‘precaution’. While prevention deals with avoiding uncertain outcomes which may or
may not be harmful, precaution, as embodied in the precautionary principle,
encompasses not only the preventative aspects of traditional regulatory approaches,
but also provides justification for acting in advance of knowledge where outcomes are
uncertain. Gullet (2000) defines the central elements of the precautionary principle as:

a) Being confident about predictions of future environmental effects of activities

before allowing them;

b) Not waiting for conclusive proof of environmental harm before adopting

appropriate remedial measures.

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Although the precautionary principle has been included in some environmental
legislation in Australia, including the Environment Protection and Biodiversity
Conservation Act, (1999), and in amendments to Great Barrier Reef Marine Park Act,
(1975) and the Fisheries Management Act, (1991), the definition of the precautionary
principle in legislation is often problematic and indistinct from the conventional
conservative approach (Gullett 2000). According to the EU, the ‘prudential’
(conservative) approach is an integral part of the scientific opinion delivered by the
risk evaluators, while the precautionary principle becomes a risk management option
‘when scientific uncertainty precludes a full assessment of the risk and when decision-
makers consider that the chosen level of environmental protection or of human,
animal and plant health may be in jeopardy’ (EC 2000).

In the application of new chemicals risk assessment, conservatism does not usually
contribute to a significantly inflated risk. For example, the assumption of 100%
release for dispersed uses such as personal care products, is not particularly inflated
compared to the assumption of between 1% and 10% percent loss (depending on
viscosity) to landfill with the container. In the sensitivity analysis, this variable
contributed only about 1% of uncertainty in the PEC. In other situations, such as the
use of a large volume at an industrial site, however, this variable may contribute more
to uncertainty. According to Stahl et al. (2005), most companies making pre-
manufacture notice (PMN) applications under the Toxic Substances Control Act
(TSCA) are aware of the extrapolations and default assumptions used in the process.
Their preference is for a quick response to the application. If the PMN cannot be
approved due to uncertainty in either the PEC/PNEC ratio or structure-activity
relationships, the decision to generate the required data or withdraw the application is
a business decision, not a scientific one.

However, the conservative approach applied to the point estimate of risk in an

assessment such as this one, where the usage volume is not known, generates a
quotient significantly greater than one, and results in a characterisation of significant
risk. The criticism that high-end estimates mask uncertainty, therefore, would be
applicable in this case.

6.5.4. Science, Policy, Assessment and Management

The conservative stance taken by risk assessors in organisations such as USEPA and
NICNAS/DEW is of course, a policy position rather than a scientific one. According

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to the USEPA staff paper (USEPA 2004), policy provides the fundamental framework
of the risk assessment, and informs the default assumptions used within the
framework. These policy positions are developed outside the risk assessment. An
alternative view, however, would place a greater emphasis on science rather than
policy in determining the risk assessment process. For example Conolly et al. (1999)
suggests that toxicologists should refine existing approaches and develop new tools so
that ‘toxicological science’ rather than ‘science policy’ becomes the primary basis for
risk assessment. Stahl et al. (2005) have argued that a greater reliance on scientific
information, rather than plausible conservatism in selecting defaults should be
possible due to increasing experience in ‘the field’. However, Tannenbaum (2005b)
has suggested that toxicological science lacks the ability to identify true biological
harm when and if it occurs in the field.

It is not universally accepted, however, that the role of policy is one of framing and
informing the risk assessment process, or of filling in the gaps left by scientific
knowledge. Science and policy are sometimes viewed as belonging to risk assessment
and risk management respectively. According to Verdonk et al. (2005), risk
assessment is a science-based process which identifies and characterises hazard,
exposure and risk (including uncertainty), while risk management is a ‘decision-
making process used for deciding between policy alternatives in consultation with
stakeholders, taking into account both the estimated risk and its uncertainty, the
precautionary principle, the cost and other factors (e.g. social, economic and legal
considerations)’. Risk assessments, in this framework, should ‘aspire to the greatest
extent to be objective scientific exercises that seek realistically to estimate risk’ while
subsequent risk management ‘must be fully and transparently distinguished from risk
assessment if the practice of risk assessment is to have scientific credibility’ (ACC
2003). According to Stahl et al. (2005), this position is reflected in the policy directive
from the Executive Office of the President (USA), which stated ‘EPA risk
assessments must not intermingle important policy judgements within the scientific
assessment of risk. Rather, the choice of an appropriate margin of safety should
remain the province of responsible risk-management officials, and should not be pre-
empted through biased risk assessments.’ However, this position does not appear to
have been maintained in subsequent publications from the Executive Office, for

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example in the Updated Principles of Risk Assessment (EOP 2007), which takes a
position more in line with the traditional USEPA approach.

In the European Union, two separate bodies are involved in the scientific and policy
aspects of risk assessment. The Committee for Toxicology, Ecotoxicology and the
Environment (CSTEE) provides independent advice on scientific issues relevant to
regulatory decision making. After a risk assessment has been peer reviewed by
CSTEE, a separate policy body, Competent Authorities (CA), recommends follow up
to the conclusion of the assessment ( Munn and Hansen 2002). There are three
possible outcomes of the EU risk assessment, viz

a) recommend further information be generated to reduce uncertainties;

b) recommend no further exposure reduction measures, based on the
consideration that an ‘unacceptable risk’ has not been demonstrated;
c) recommend further exposure reduction measures, based on the consideration
that an ‘unacceptable risk’ cannot be ruled out.
According to Munn and Hansen (2002), a science-based conclusion will lean towards
(a) or (b), while a policy-based conclusion will be influenced by socio-economic
factors in the choice between (a) and (c). Assessments that take into account practical
considerations including the scientific uncertainties and the needs of risk managers are
described by Verdonk et al. (2007) as being based on regulatory science rather than
research science. This suggests that the policy-science distinction that plagues the US
system of risk assessment has been resolved in the EU as a science in its own right,
distinct from the normal research science from which it draws much data and
methodology, by its ability to incorporate the social and legal aspects of the risk
assessment, management and communication processes.

The approach adopted by the EU suggests that issues such as evidence and uncertainty
in risk assessment can be treated as they would be in science, and this is, in fact, what
PRA is expected to do. However, the risk assessment process is to a large extent
shaped by the ‘requirements, constraints and adversarial climate of regulation’, rather
that the disciplinary norms of science (Latin 1997), and uncertainty and evidence do
not have the same meaning in law and science (Gawlack et al. 1987). A scientist,
using an uncertainty analysis approach, may view the addition of components, each
with its own uncertainty, as leading to an increase in uncertainty. A lawyer weighing
the same evidence in a ‘sum of evidence’ approach would regard uncertainty as

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reduced by the addition of each piece of evidence (Gawlack et al. 1987). It should not
be surprising then, that a regulatory risk assessment can be regarded as giving too
little weight to uncertainty by some reviewers, and being too conservative or cautious
by others. The two views represent, according to Gawlack et al. (1987) diverging
scientific and legal opinions.

6.5.5. Characterising the Risk to the Aquatic Environment from

Cosmetic Polyquaternium Use in Australia
It is necessary then, to approach the uncertainty in the risk characterisation of
cosmetic polyquaterniums in Australia from the perspective of the applicability of the
questions of science and policy, and risk assessment and risk management. For
example, one major source of uncertainty in the risk characterisation of cosmetic
polyquaterniums identified above is the import volume. There is no system for the
generation of statistics on the production and use of chemicals in Australia that is
nationally uniform, consistent and comparable (DEH 1998). Data collected by the
Australian Bureau of Statistics (ABS) is designed to be useful for the study and
understanding of trade, not safety or environment, as evidenced by the collection of
some categories in terms of monetary value rather than quantity. As mentioned in
Section 4.2, the actual import or manufacture volume of a new chemical can be kept
confidential at the request of the importer. Data on volume and use can be collected
by NICNAS as part of a Priority Existing Chemical assessment, even in the initial
stage of review. However, there are approximately 40,000 existing chemicals on
AICS, and as at September 2007, only 29 Priority Existing Chemical reports have
been produced since the inception of NICNAS in 1989. There are 7 assessments
currently in progress; 43 chemicals are currently on the candidate list of chemicals for
assessment with a further 12 on a standby list. To date, 13 nominated chemicals have
been rejected for assessment. The majority of chemicals nominated as priority
existing chemicals are nominated on public health and occupational health and safety
(OHS) grounds, a priority that is understandable in light of the enormity of the task.
The main reasons stated for the rejection of candidate chemicals are either that
NICNAS was unable to ascertain any usage in Australia, or the usage determined was
very low (<< 1 tonne). However, two assessments currently in progress are based to a
large extent on environmental concerns; Sodium Cyanide, a chemical used in gold
extraction, and Triclosan, an antibacterial agent used in personal care and household
products. The reasons for the nomination of Sodium Cyanide include; widespread use

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provides a number of ways for the chemical to enter the environment; it is acutely
toxic to animals, fish and birds, may cause death and low growth rates in plants; and
bird kills had been reported following possible exposure at tailings dams (NICNAS
2002c). Triclosan was nominated on the basis of its toxicity to aquatic species,
particularly algae; the number of ways it can enter environment; the possibility that it
may be persistent and bioaccumulate; and because disposal by incineration of wastes
containing Triclosan may result in formation of dioxins (NICNAS 2003).

While the collection of more extensive data on chemical use and release would seem
desirable, considerable obstacles to such collection exist. For example, importers of
household and personal care products may not know the formula of the product; the
number of different products imported is very large; and the collation of the data once
collected is likely to be time consuming and expensive. Further, while standardisation
of names exists for cosmetic products, and for industry applications requiring Material
Safety Data Sheets (MSDS), the same does not apply to ingredients in household and
other consumer products. These problems not only make the collection and collation
of data difficult, they also contribute to difficulties in the enforcement of compliance
with the Industrial Chemicals (Notification and Assessment) Act (ICNA). Although
mandatory reporting of volume is required in some circumstance, for example for
chemicals on the HVICL, and for some categories of permits and certificates, any
attempt to collect usage data on a wider scale is likely to be resisted by industry.

A further source of uncertainty identified above concerned the fate of the

polyquaterniums in WWTPs and dilution to receiving waters. In the risk
characterisation of polyquaterniums by NICNAS/DEW, default assumptions of 90%
removal and 1:10 dilution are used. Although the partitioning behaviour of the
polyquaterniums is an important factor here, also significant is the extent of solids
removal in the WWTP. However, it would appear from this study that these
assumptions, instead of being conservative, may be optimistic. In the risk
characterisation process for new and existing chemicals, where field studies are not
part of the process, assessors are, to a significant degree, reliant on
information/feedback from risk managers for information and updates on the
parameters affecting these variables. In the Australian regulatory system, the risk
assessors, NICNAS/DEW, are departments of the Commonwealth Government, while
the risk managers are the State and Territory departments of environment, health and

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occupational health and safety (OHS). The relationship between the Commonwealth,
as risk assessor, and the States and Territories, as risk managers, is subject to a
Memorandum of Understanding (MOU), established in 1991. As defined in the MOU,
the role of the States and Territories is to implement recommendations from
assessment reports published by the Director of NICNAS. The States and Territories
must also inform the Director of NICNAS of any consequential action taken in respect
of any recommendations, and can raise issues of concern regarding chemicals with the
Commonwealth agencies. Due to the confidentiality provisions of ICNA, the risk
managers receive the Full Public Report, which may not contain the actual volume. It
is therefore essential that the risk be well characterised within the report. However, it
is not at all clear from the assessments of polyquaterniums that the risk assessors have
adequate information on WWTP processes and receiving waters relevant to
polyquaterniums to make an adequate characterisation of the risk and recommend
adequate control measures. Risk managers may, for example, be better placed to
determine the extent to which the toxicity of polyquaterniums is mitigated by the
presence of solids in the receiving waters into which they discharge, than risk
assessors who can only apply a generalised determination.

In 2007, the States and Commonwealth signed a National Framework for Chemicals
Environmental Management (NChEM), which may well address these issues to some
degree. It includes in its recommendations the investigation of the feasibility of a
centralised national approach to gathering, storing and utilising information on
industrial chemicals that have been made available for use, and consultation with state
departments during the assessment process. The agreement also recommends further
development of the environmental monitoring database to ensure that it incorporates
information on environmental impacts and usage.

However, in order for data on polyquaterniums to be included on a database of

environmental impacts, there must first be some way of measuring such impacts, and
as has been discussed previously, there is no reliable method available for measuring
concentrations of polyquaterniums in environmental matrices such as water or
sediment. The assessment of new chemicals does require that the notifier provide
details on methods of identification of the chemical, for example, an infra-red
spectrum, however, like the import volume, this information is covered by the
confidentiality provisions, and may not be included in the Full Public Report. The

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purpose of this data requirement is perhaps more to facilitate identification of the
assessed chemical if required in compliance matters. It may be time, however, in the
on-going regulatory review of the Act and its implementation, to consider requesting
that notifiers provide a method of extracting and quantifying the notified chemical in
environmental samples, if release to the environment is likely to occur.

6.6. Conclusion
As complex as the chemistry and toxicology of this class of polymers is, the
difficulties faced in this assessment are as much about the process as the subject.
Uncertainty exists in several variables in the risk characterisation – both in the PEC
and PNEC. Further scientific studies may help. For example, further toxicological
studies would be valuable in determining the mechanism of toxicity, species
sensitivity distribution, and polymer architecture effects. Such studies may also help
in further understanding the toxicity of cationic surfactants generally. However,
considerable data already exists, published and unpublished, to establish that, except
in a few cases of very low charge density natural polyquaterniums, these polymers are
very toxic to some aquatic organisms. There is probably already enough information
to begin screening of these polymers on the basis of chemistry, without recourse to
any further toxicity testing. The issue of mitigation by DOM, total organic carbon
(TOC) or humic acid is a wider issue, and needs to be addressed for all organics as
part of the differences between laboratory testing and field effects. Further, it may be
better addressed in risk management than risk assessment. It is difficult, based on the
partitioning results of this study, to make a special case for polyquaterniums. A
method of determining the concentration of polyquaterniums in environmental
samples, particularly water and sediment, would also be useful, as an alternative to a
policy commitment to monitor import volumes. But the development of an analytical
method is likely to be expensive, and the question of who would be responsible for
the development of the method would need to be resolved. To require industry to
provide an appropriate test again requires a policy commitment.

Even assuming the problems of uncertainty in critical variables can be addressed, it is

far from clear that the risk assessment methodology is able to quantify and/or
characterise risk in a way that is meaningful to, and useful for, risk managers. The
methodology adopted in regulatory chemical risk assessment has been adopted from
the methodologies developed for the ecological risk assessment of contaminated sites,

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where the basis of the risk assessment is measured environmental concentrations
generally taken in response to an observed adverse effect on the site ecology. The
applicability of the method even to contaminated site assessment has been the subject
of some criticism. It has been suggested that ‘ERA, from its inception until the present
day, remains devoid of a risk assessment tool. A greater tragedy may be that
ecological risk assessors and risk managers do not seem to have this realization’
(Tannenbaum 2005a). A review of the methodology, and possible alternatives, for the
environmental risk assessment of new industrial chemicals is needed.

Polyquaterniums, a class of polymer that are known to be toxic in the aquatic

environment, are being introduced into Australia, in unknown quantities, for use in
applications that result in wide, dispersive release through WWTPs. They do not
degrade, but partition between anionic solids (including biota) and water, yet there is
no way of reasonably determining their fate, or of measuring their concentration in
environmental samples. Uncertain and unproven default assumptions are used in
assessing the risk of these compounds in the aquatic environment, and no assessment
has been attempted of possible risk in sediments. Only limited risk assessment is
carried out when new polyquaterniums are introduced, and not in the context of the
total amount of polyquaternium released. This assessment has looked at the risk of
polyquaterniums from only one source, cosmetics, and found that the risk may be
significant.

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7. Overall Conclusions and Future Research
The work presented in this thesis has examined the environmental fate and aquatic
toxicology of polyquaterniums from cosmetic uses in Australia, within the framework
of the four-step risk assessment paradigm. This work has made a significant and
original contribution to our understanding of the fate and hazard of cosmetic
polyquaterniums and the potential risk to the Australian environment from their wide
dispersive release. This chapter presents the main conclusions arising from this thesis,
and identifies topics requiring further research.

7.1. Conclusions
7.1.1. Analysis
In order to investigate polyquaternium environmental fate and behaviour, a reliable
analytical method is necessary. A suitable technique was not available.
Metachromatic Polyelectrolyte Titration (Colloid Titration) was developed from
earlier studies for the purposes of the work and proved to be a reliable method for the
determination of concentration of polyquaterniums in clean water and was further
developed to work in the presence of the anionic surfactant, SDS. Visual titration of
the polyquaternium alone and in the presence of SDS was possible to concentrations
as low as 10-4 N. Spectrophotometric titration, coupled with reliable mathematical
methods for determining the breakpoint of the titration curve, enabled determination
at concentrations as low as 10-5 N. Although the effectiveness of the method was
limited to polyquaterniums in simple matrices, it was also possible to determine the
concentration in the supernatant following sorption experiments with humic acid. All
the cosmetic polyquaterniums had a low charge density, relative to polyDADMAC,
and some, such as UCareTM LR30M, LR400 and LK were significantly lower.

7.1.2. Environmental Fate

Data were not readily available to determine the volume of cosmetic polyquaternium
usage in Australia. Estimates made on the basis of the number of polyquaterniums on
the Australian Inventory of Chemical Substances (AICS), and information contained
in Full Public Reports of new polyquaterniums published by the National Industrial
Chemicals Notification and Assessment Scheme (NICNAS) were in reasonable
agreement with estimates made on the basis of emission scenarios based on EU
guidelines. The import volume was estimated at between 20 and 60 tonnes.

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Wastewater Treatment Plant (WWTP) biosolids are an important phase in the
environmental fate of polyquaterniums. Partition Coefficients, KD, determined for
nine of the twelve polyquaterniums were somewhat lower than expected, and
significantly lower than that determined for the cationic surfactant cetyl pyridinium
choired. They were also lower than those determined for Busan 77 (Polyquaternium-
42) (Mathews et al. 1995), but in general agreement with those determined for tertiary
cationic oligomers (Podoll and Irwin 1988).

The magnitude of the partition coefficient has important implications for the fate of
polyquaterniums. As a consequence of the low partition coefficients between water
and biosolids, KD, the modelled partitioning of polyquaterniums from the dissolved
phase to the sediment phase, was significantly lower than the default value of 90%
removal in WWTP for polymers which is generally applied in risk assessment
(Boethling and Nabholz 1997).

As a result of the lack of data available to determine the import volume, considerable
uncertainty remains in the predicted environmental concentration. The predicted
environmental concentration (PEC) was estimated to be between 0.014 and 4.6 µg/L.

7.1.3. Toxicology
Of the twelve samples, six of Polyquaternium-10 from Amerchol (The Dow Chemical
Company), two samples each of Polyquaternium-11 and Polyquaternium-28, and one
of Polyquaternium-55 from ISP, and polydimethyldiallyl ammonium chloride
(poly(DADMAC), Polyquaternium-6), only the very low charge density cellulosic
polyquaternium UCareTM LK (Polyquaternium-10) would not be classified as toxic
according the Globally Harmonised System for Classification and Labelling of
Chemicals (GHS) criteria (median effective concentration for fish (EC50(fish))
≤ 100 mg/L) based on testing with G. holbrooki (Table 1.4). Two other samples,
UCareTM LR400, LR30M (Polyquaternium-10) could be classified as category Acute
III (EC50(fish) > 10 - ≤ 100 mg/L). Five of the polyquaterniums could be classified as
category Acute I (EC50 ≤ 1 mg/L) based on the results of testing with Chlorella sp12.
There was no difference between the toxicity of a polyquaternium and the toxicity of
its complex with SDS. The polyquaterniums were not toxic to the marine invertebrate
species Artemia, however the anionic surfactant SDS was moderately toxic to
Artemia. There did not appear to be any relationship between charge density and
toxicity. The high charge density polyDADMAC was the most toxic polyquaternium

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when EC50 was expressed in mass units, but one of the least toxic if EC50 was
expressed in terms of equivalence. It is probable that the toxicity of the
polyquaternium is dependent on hydrophobicity, which is in turn dependent on
multiple structural features of the polymer. Algae were the most sensitive of the three
species tested. With the application of assessment factors, the predicted no effect
concentration (PNEC) for the toxic polyquaterniums was estimated to be 0.03 and
0.12 µg/L. This PNEC does not take into account possible mitigation of the toxicity of
polyquaterniums by humic acid.

7.1.4. Risk Characterisation

A PEC/PNEC ratio > 1 is possible at even low volumes if the fraction of
polyquaternium removed in WWTP is in the lower range modelled in this work. At
higher volumes, > 5 tonnes, the PEC/PNEC ratio can exceed 1 even at the default
removal of 90% in WWTPs. Monte Carlo Simulation highlighted the sensitivity of the
calculations to those variables for which a large data gap exists – import volume and
dilution to receiving waters. Consequently, the greatest uncertainty in the risk
characterisation results from those aspects of the PEC calculation.

7.2. Implications and Future Research

The most significant difficulty for the risk assessment of polyquaterniums is most
likely the lack of a viable method of identifying and quantifying polyquaterniums
(and cationic polyelectrolytes generally) in environmental samples. However, there
are inherent problems in the analysis of polymers due to their distribution of
molecular size, and in the analysis of cations due to interference of other cations, that
combined make the development of a suitable analytical method difficult. A method
of tagging polyquaterniums, such as that proposed by Bennett et al. (2000) could at
least enable the verification of fate models such as those used in this work, if the
method of tagging can be shown to have no effect on the behaviour of the
polyquaternium in laboratory tests. As an interim measure pending a method of
identification quantification of polyquaterniums in environmental samples, the
tracking of tagged polyquaterniums would be a worthwhile area for future research.

In terms of the ecotoxicology, uncertainty still exists with regard to the mechanism of
toxicity, and species sensitivity distribution. Neither of these is of significant
consequence for the risk assessment process, and further toxicity studies are unlikely
to contribute to the risk characterisation of polyquaterniums. It is not possible to

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assign a toxicity to a particular polyquaternium in the absence of specific testing, due
to variations in structure that may contribute to toxicity without altering the ‘identity’
of the polymer, nor is it possible to draw generalised conclusions regarding particular
characteristics such as ‘natural’ versus ‘synthetic’ polyquaterniums. However,
sufficient data exists to enable reasonable prediction of toxicity based on chemistry.
While some very low charge density cellulose (and probably guar) based polymers
have significantly lower toxicity than either ‘synthetics’ or higher charge ‘natural’
polyquaterniums, generally polyquaterniums should be assumed to be very toxic to
aquatic organisms. The toxicity of individual polyquaternium samples appears to be
the result of a complex mix of those characteristics that contribute to the
hydrophobicity of the polymer, and this would seem to be a possible direction for
future research.

An understanding of the mechanism of toxicity may be important as part of a more

general understanding of the toxicity of cationic species generally. Further, the
mitigating effect of humic acid needs to be considered in the wider consequence of
the differences between laboratory studies and field effects, and the role of DOM in
mitigating the toxic effects of anthropogenic chemicals. Without addressing wider
questions, such as the release of other toxic cations from DOM on binding of
polyquaterniums, there is little justification for the special treatment of humic acid
mitigation in the risk assessment of cationic polyelectrolytes without reliable
information on DOM concentrations in the receiving environment, and the role of
DOM in the mitigation of toxicity of organic chemicals more generally.

The lack of available data on the volume usage of polyquaterniums (and many other
chemicals) presents a significant challenge for researchers and risk managers. Even
where data is collected, it is not necessarily freely available, such as the confidential
NICNAS data, nor available in a useful format, such as the Australian Bureau of
Statistics (ABS) import figures in dollar values. This deficiency has been critical in
the case of polyquaterniums, where no method of measuring environmental
concentration exists. However, even for other chemicals released with wastewater,
volume usage data may enable issues of spatial and temporal distribution to be
addressed. Without creating additional data collection and collation schemes, the data
available for monitoring and research could be improved by addressing the form in
which data is collected, and the extent to which it is made public.

186
In this study, probability distribution functions were generated using the ‘reasonable’
and ‘conservative’ values that may be used in point estimate risk assessments. The
determination of appropriate distributions of parameters such as per capita water use
and dilution to receiving waters could be better determined if appropriate feedback
from risk managers to risk assessors was available. Much of this data could easily be
generated from information already held by various water authorities and state EPAs.
The collection and collation of this data and the description of realistic pdfs would
greatly assist in any move from point estimate to probabilistic risk assessment of
chemicals in Australia. Further, as hazard assessment endeavours to protect the most
vulnerable species, realistic probability distribution functions would enable
characterisation of the risk to the most vulnerable environments.

Of the scientific and knowledge gaps identified above, NICNAS already has the
authority to collect the information from importers and manufacturers of new and
existing chemicals. Volume data is a requirement for all new chemical notifications
(NICNAS 2004), and can be requested for existing chemicals under Section 48 of the
Industrial Chemicals (Notification and Assessment) Act (ICNA) (CofA 1989).

Notifiers of new chemicals are also required to provide details of a method of

identification of the notified chemical or polymer. The requirement is usually met
with the provision of an infra-red spectrum of the pure substance. As a method of
identification the infra-red spectrum may not be adequate to identify even the
chemical or polymer in the form/matrix in which it is imported into Australia for
regulatory compliance purposes, and even less useful for the monitoring of
environmental, public or occupational health and safety (OHS). It may be more
appropriate in the case of novel chemicals to interpret the data requirement as the
requirement of a method of identification of the chemical/polymer in samples taken
for compliance, environmental, public or health monitoring purposes. However, such
a requirement would be problematic for a class of compound such as
polyquaterniums, where a large number are already widely available. For some data
requirements of ICNA, additional fees are charged where data is either not available or
the notifier otherwise seeks a variation to the data requirements. The decision to
proceed with the notification by meeting the testing requirement, or payment of the
fee, would be a commercial one for the notifier as suggested by Stahl et al. (2005).
The possibility of imposing release controls, which can be applied in the case of

187
industrial chemicals whose use is limited to specific sites, cannot be applied to
cosmetics.

7.3. Concluding Comments

Polyquaterniums are a class of polymers with an already extensive use pattern, and in
the case of cosmetic uses, with a dispersive release to the environment through the
wastewater system. The risk assessment of polyquaterniums is characterised by a set
of default assumptions that do not appear to be supported either by limited
environmental studies of their fate, or by studies of the behaviour of polyquaterniums
in cosmetic applications. There is no known method of reliably measuring or
determining the concentration of polyquaterniums in environmental samples. With
few exceptions, polyquaterniums are very toxic to some aquatic species; and tend to
have steep toxicity curves. The extent of polyquaternium usage in cosmetic products
is not known, however, limited available data suggests that sufficient quantities of
polyquaterniums may currently be released through the wastewater system in
Australia to present a significant risk to more vulnerable waterways that receive
WWTP effluent.

188
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 Appendix 1. Database of cosmetic polyquaterniums
Registry Number: 75345-27-6
CA Index Name: Poly[(dimethyliminio)-2-butene-1,4-diyl chloride], α-[4-[tris(2-
hydroxyethyl)ammonio]-2-butenyl]-ω-[tris(2-hydroxyethyl)ammonio]-,
dichloride (9CI)
Other Names: Onamer M; Onyxsperse 12S; Polidronium chloride; Polyquad;
Polyquaternium 1
Formula: (C6 H12 N)n C16 H36 N2 O6 . 3 Cl
Alternate Formula: (C6 H12 N . Cl)n C16 H36 N2 O6 . 2 Cl
Polymer Class Term: Polyionene
INCI Name: POLYQUATERNIUM-1
Function: antistatic agents/film formers
Confidentiality Public
Status:
AICS: No
Commercial Profile: Onamer M = contact lens solution; Polyquad (in Tears Naturale)
Structure:
HO CH 2 CH 2 Me
+
HO CH 2 CH 2 N+ CH 2 CH CH CH 2 N CH 2 CH CH CH 2
n
HO CH 2 CH 2 Me

· 3 Cl -

CH 2 CH 2 OH
+
N CH 2 CH 2 OH

CH 2 CH 2 OH

i
Registry Number: 63451-27-4
CA Index Name: Poly[oxy-1,2-ethanediyl(dimethyliminio)-1,3-propanediyliminocarbonylimino-
1,3-propanediyl(dimethyliminio)-1,2-ethanediyl dichloride] (9CI)
Other Names: Bis(2-chloroethyl) ether-1,3-bis[3-(dimethylamino)propyl]urea, SRU; KA 1092;
Mirapol A 15; PAQ 3; Polyquaternium 2; Poly[iminocarbonylimino-1,3-
propanediyl(dimethyliminio)-1,2-ethanediyloxy-1,2-ethanediyl(dimethyliminio)-
1,3-propanediyl dichloride]
Formula: (C15 H34 N4 O2)n . 2 Cl
Alternate (C15 H34 N4 O2 . 2 Cl)n
Formula:
INCI Name POLYQUATERNIUM-2
Polymer Class Polyether, Polyionene, Polyurea
Term:
Confidentiality Public
Status:
AICS: Yes
Other CANADA: DSL (January 26, 1991); PHILIPPINES: PICCS (2000.); USA:
Regulatory: FIFRA, TSCA (January 2003)
Commercial Rhodia (Rhone-Poulenc) (Mirapol A-15)
Profile:
Structure:
Component Registry Number: 52338-87-1
Formula: C11 H26 N4 O
O

Me 2 N (CH 2 ) 3 NH C NH (CH 2 ) 3 NMe 2

Component Registry Number: 111-44-4

Formula: C4 H8 Cl2 O
ClCH 2 CH 2 O CH 2 CH 2 Cl

ii
Registry Number: 92183-41-0
CA Index Name: Cellulose, 2-hydroxyethyl ether, polymer with N,N-dimethyl-N-2-propenyl-2-
propen-1-aminium chloride (9CI)
Other Names: 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, polymer with
cellulose 2-hydroxyethyl ether (9CI); Celquat H 100; Celquat L 200; Celquat
LOR; Polyquaternium 4
Formula: (C8 H16 N . C2 H6 O2 . Cl . x Unspecified)x
Polymer Class Term: Manual component, Polyother, Polyvinyl
INCI Name: POLYQUATERNIUM-4
Function: antistatic agents/film formers; Hair fixative/conditioning agent

AICS: No
Commercial Profile: Celquat-L200, L230M, H-100 (National Starch)
Structure:
Component Registry Number: 7398-69-8 (48042-45-1)
Formula: C8 H16 N . Cl
Me
+
H2 C CH CH 2 N CH 2 CH CH 2

Me

· Cl -

Component Registry Number: 9004-62-0

Formula: C2 H6 O2 . x Unspecified
No structure diagram available
Component Registry Number: 9004-34-6
Formula: Unspecified
No structure diagram available
Component Registry Number: 107-21-1
Formula: C2 H6 O2
HO CH 2 CH 2 OH

iii
Registry Number: 26006-22-4
CA Index Name: Ethanaminium, N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-propenyl)oxy]-,
methyl sulphate, polymer with 2-propenamide (9CI)
Other Names: 2-Propenamide, polymer with N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethanaminium methyl sulphate (9CI); Acrylamide, polymer with
choline methyl sulphate methacrylate (8CI); …..Acrylamide-[2-
(methacryloyloxy)ethyl]trimethylammonium methyl sulphate polymer; Calgon
K 400; Catamer Q; Hercofloc 812; Hercofloc 849; Kayafloc C 599-1F;
Polyquaternium 5; Reten 1104; Reten 1105; Reten 1106; Reten 210; Reten
220; Reten 230; Reten 240; Reten 260; Reten 420; Reten SPX 1098
Formula: (C9 H18 N O2 . C3 H5 N O . C H3 O4 S)x
Polymer Class Term: Polyacrylic, Polyother
INCI Name: POLYQUATERNIUM-5
Function: antistatic agents/film formers
AICS: Yes (1996)
Other Regulatory: TSCA 2003 (EPA: XU); DSL 1991; ENCS No 6-538; ECL 1997; PICCS 2000
Commercial Profile: Reckitt Benckeser (Calgon); Hercules (Reten 220); Ondeo Nalco (Merquat 5)
Structure:
Component Registry Number: 79-06-1
Formula: C3 H5 N O
O

H2 N C CH CH 2

Component Registry Number: 6891-44-7

Formula: C9 H18 N O2 . C H3 O4 S
No structure diagram
Component Registry Number: 33611-56-2
Formula: C9 H18 N O2
O CH 2

Me 3 + N CH 2 CH 2 O C C Me

Component Registry Number: 21228-90-0

Formula: C H3 O4 S
Me O SO 3 -

iv
Registry Number: 26062-79-3
CA Index Name: 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, homopolymer
(9CI)
Other Names: Ammonium, diallyldimethyl-, chloride, polymers (8CI); 261LV; Additol
VXT 3529; Agefloc WT 20; Alcofix; Aronfloc (polymer); Bufloc 536;
Calgon, DMDACC, E 904, E 905, E 921; Calgon Polymer 261; Cartafix
VXT; Cat-Floc; Certrex 340; CinFix RDF; CM 100; CM 100 (onium
compound); Conductive Polymer 261; Croscolor; DADMAC polymer;
Danfix 707; Danfix F; Diallyldimethylammonium chloride homopolymer;
Diallyldimethylammonium chloride polymer; Dimethyldiallylammonium
chloride homopolymer; Dimethyldiallylammonium chloride polymer; E 261;
ECCat 2020; Floerger FL; Hydraid 2010, 2020; Jayfloc 842; Kufloc; KZ
106K; KZ 63K; Lectrapel; M 40176; Mackernium 006; Magnafloc 1697, 368;
Magnifloc 585C, 587C, 589C, 591C; Merck 261; Merquat 100; Mirapol 100;
Mobil ED 87/04; N,N-Diallyl-N,N-dimethylammonium chloride
homopolymer; Nalco 2010; PAS 10L; PAS-H 10; PAS-H 10L; PAS-H 1L;
PAS-H 35L; PAS-H 35S; PBK 1; PBK 1 (quaternary compound); PCL 2;
PDMDAAC; PDMDAC 50; Percol 1620; Percol 1697; Percol 368; Percol
406; Percol 406F; PKB 1;… Poly-DMDAAC; Polydadmac; Polydadmac 570;
Polymer 261; Polymer 261LV; Polypure C 318; Polyquat; Polyquaternium 6;
Ponilit CP 1; Quaternium 40; Reten 203; Salcare SC 30;
Formula: (C8 H16 N . Cl)x
Polymer Class Term: Polyvinyl
INCI Name: POLYQUATERNIUM-6
Function: Conditioner, moisturiser (L&G:1999)
AICS: Yes
Other Regulatory: TSCA 2003 (EPA:XU); DSL 1991; ENCS; ECL 1997; PICCS 2000
Commercial Profile: Ondeo-Nalco (Merquat 100); 3V (Conditioner P6)
Structure:
Component Registry Number: 7398-69-8 (48042-45-1)
Formula: C8 H16 N . Cl
Me
+
H2 C CH CH 2 N CH 2 CH CH 2

Me

· Cl -

v
Registry Number: 26590-05-6
CA Index Name: 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, polymer with 2-
propenamide (9CI)
Other Names: 2-Propenamide, polymer with N,N-dimethyl-N-2-propenyl-2-propen-1-
aminium chloride (9CI); Acrylamide, polymer with
diallyldimethylammonium chloride (8CI); … Acrylamide-DADMAC
copolymer; … Agequat 500, 5008, C 3204; Betz 2651; CV 5380;
Diallyldimethylammonium chloride-acrylamide copolymer;
Dimethyldiallylammonium chloride-acrylamide copolymer; E 949; ECCat
777; Himacs SC 100; Hydraid 777; Kayacryl EC 315, M-N, Resin M-N;
Lipoflow MN; Mack K 007; Mackernium 007, 007S; ME Polymer 09W;
Merquat 2200, 500, 550, 550L, S; Mirapol 550; Nalco 1470, 8105; PAS-J 11;
PAS-J 41; PAS-J 81; PDMDAAC-AM; Poly(acrylamide-
dimethyldiallylammonium chloride); Polyquaternium 7; Quaternium 41;
Salcare SC 10; Salcare Super 7; WT 2575, 2640, 2860, 5504; XB 54-15-1;
XQ 550
Formula: (C8 H16 N . C3 H5 N O . Cl)x
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-7
Function: Function: antistatic agents/film formers; Condition, moisturise, smooth;
rheology builder (L&G:1999)
AICS: Yes (1996)
Other Regulatory: TSCA 2003 (EPA:XU); DSL 1991; ENCS; ECL 1997; PICCS 2000
Commercial Profile: Ondeo Nalco (Merquat); Rhodia (Mirapol)
Structure:
Component Registry Number: 7398-69-8 (48042-45-1)
Formula: C8 H16 N . Cl
Me
+
H2 C CH CH 2 N CH 2 CH CH 2

Me

· Cl -

Component Registry Number: 79-06-1

Formula: C3 H5 N O
O

H2 N C CH CH 2

vi
Registry Number: 146189-14-2
CA Index Name: 2-Propenoic acid, 2-methyl-, 2-(dimethylamino)ethyl ester, polymer with
methyl 2-methyl-2-propenoate and octadecyl 2-methyl-2-propenoate,
compd. with dimethyl sulphate (9CI)
Other Names: 2-Propenoic acid, 2-methyl-, methyl ester, polymer with 2-
(dimethylamino)ethyl 2-methyl-2-propenoate and octadecyl 2-methyl-2-
propenoate, compd. with dimethyl sulphate (9CI); 2-Propenoic acid, 2-
methyl-, octadecyl ester, polymer with 2-(dimethylamino)ethyl 2-methyl-2-
propenoate and methyl 2-methyl-2-propenoate, compd. with dimethyl
sulphate (9CI); Sulphuric acid, dimethyl ester, compd. with 2-
(dimethylamino)ethyl 2-methyl-2-propenoate polymer with methyl 2-
methyl-2-propenoate and octadecyl 2-methyl-2-propenoate (9CI);
Polyquaternium 8
Formula: (C22 H42 O2 . C8 H15 N O2 . C5 H8 O2)x . x C2 H6 O4 S
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-8
Function: Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Structure:
Component Registry Number: 77-78-1
Formula: C2 H6 O4 S MeO−S(O2)−OMe
Component Registry Number: 41510-85-4
Formula: (C22 H42 O2 . C8 H15 N O2 . C5 H8 O2)x
Component Registry Number: 32360-05-7
Formula: C22 H42 O2
O CH 2

Me (CH 2 ) 17 O C C Me

Component Registry Number: 2867-47-2

Formula: C8 H15 N O2
O CH 2

Me 2 N CH 2 CH 2 O C C Me

Component Registry Formula:

C5 H8 O2Number: 80-62-6
H2 C O

Me C C OMe

vii
Registry Number: 130291-58-6
CA Index Name: 2-Propenoic acid, 2-methyl-, 2-(dimethylamino)ethyl ester, homopolymer,
compd. with bromomethane (9CI)
Other Names: Methane, bromo-, compd. with 2-(dimethylamino)ethyl 2-methyl-2-
propenoate homopolymer (9CI); Polyquaternium 9
Formula: (C8 H15 N O2)x . x C H3 Br
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-9
Function: Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Other Regulatory:
Structure:
Component Registry Number: 74-83-9
Formula: C H3 Br
Br CH 3

Component Registry Number: 25154-86-3

Formula: (C8 H15 N O2)x
Component Registry Number: 2867-47-2
Formula: C8 H15 N O2
O CH 2

Me 2 N CH 2 CH 2 O C C Me

viii
Registry Number: 81859-24-7
CA Index Name: Cellulose, 2-hydroxyethyl 2-[2-hydroxy-3-
(trimethylammonio)propoxy]ethyl 2-hydroxy-3-(trimethylammonio)propyl
ether, chloride (9CI)
Other Names: Amerchol JR 400; Catinal HC 100; Catinal HC 35; Catinal LC 100; Celquat
SC 230M; Celquat SC 240C; JR 1; JR 125; JR 30M; JR 400; KG 30M;
Leogard G; Leogard GP; Leogard LP; Leogard P; LR 300M; LR 400;
Polymer JR 400; Polymer KG 30M; Polymer LR 30M; Polyquaternium 10;
Quaternium 19; Ritaquat 3000; Ritaquat 400KG; Ucare JR 125; Ucare JR
400; UCARE Polymer JR; UCARE Polymer JR 125; UCARE Polymer JR
30; UCARE Polymer JR 30M; UCARE Polymer JR 400; UCARE Polymer
JR 40M; UCARE Polymer KG 30M; UCARE Polymer LR; UCARE
Polymer LR 30M; UCARE Polymer LR 400; UCARE Polymer SR 10
Formula: C8 H20 N O3 . x C6 H16 N O2 . x C2 H6 O2 . x Cl . x Unspecified
Polymer Class Term: Manual registration
INCI Name: POLYQUATERNIUM-10
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: Yes (1996)
Other Regulatory: ECL 1997; PICCS 2000;
Commercial Profile: Akzo Noble (Leogard); National Starch (Celquat); Amerchol (Dow
Corning) (UCARE)
Structure:
Component Registry Number: 170553-71-6
Formula: C8 H20 N O3 . x C6 H16 N O2 . x C2 H6 O2 . x Unspecified
OH

Me 3 + N CH 2 CH CH 2 O CH 2 CH 2 OH

Component Registry Number: 170344-46-4

Formula: C8 H20 N O3
OH

HO CH 2 CH CH 2 N + Me 3

Component Registry Number: 44814-66-6

Formula: C6 H16 N O2 No Structure Diagram Available
Component Registry Number: 9004-34-6
Formula: Unspecified
Component Registry Number: 107-21-1
Formula: C2 H6 O2
HO CH 2 CH 2 OH

ix
Registry Number: 53633-54-8
CA Index Name: 2-Propenoic acid, 2-methyl-, 2-(dimethylamino)ethyl ester, polymer
with 1-ethenyl-2-pyrrolidinone, compd. with diethyl sulphate (9CI)
Other Names: 2-Pyrrolidinone, 1-ethenyl-, polymer with 2-
(dimethylamino)ethyl 2-methyl-2-propenoate, compd. with
diethyl sulphate (9CI); Sulphuric acid, diethyl ester, compd.
with 2-(dimethylamino)ethyl 2-methyl-2-propenoate
polymer with 1-ethenyl-2-pyrrolidinone (9CI); Celquat 200;
Copolymer 755; Gafquat 734; Gafquat 755; Gafquat 755N-
P; HC Polymer 2L; Luviquat PQ 11; N,N-
Dimethylaminoethyl methacrylate-vinylpyrrolidone
copolymer diethyl sulphate salt; Polyquat 11;
Polyquaternium 11; Quaternium 23
Formula: (C8 H15 N O2 . C6 H9 N O)x . x C4 H10 O4 S
Alternate Formula: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-11
Function: antistatic agents/film formers
Hair conditioner/fixative resin (L&G:1999)
Confidentiality Status: Public
AICS: Yes (1996)
Other Regulatory: TSCA 2003; DSL 1991; ECL 1997; PICCS 2000
Commercial Profile: ISP (Gafquat 755, 755N, 734, 440); BASF (Luviquat PQ 11 N)
Structure:
Component Registry Number: 64-67-5
Formula: C4 H10 O4 S
EtO−S(O2)−OEt
Component Registry Number: 30581-59-0 (co-polymer of 2867-47-2 & 88-12-0
Formula: (C8 H15 N O2 . C6 H9 N O)x
Component Registry Number: 2867-47-2
Formula: C8 H15 N O2
O CH 2

Me 2 N CH 2 CH 2 O C C Me
Component Registry Number: 88-12-0
Formula: C6 H9 N O
CH CH 2

N
O

x
Registry Number: 68877-50-9
2-Propenoic acid, 2-methyl-, [(1R,4aR,4bR,10aR)-1,2,3,4,4a,4b,5,6,10,10a-
decahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenyl]methyl ester,
polymer with 2-(diethylamino)ethyl 2-methyl-2-propenoate and ethyl 2-
CA Index Name: methyl-2-propenoate, compd. with dimethyl sulphate (9CI)
Formula: (C24 H36 O2 . C10 H19 N O2 . C6 H10 O2)x . x C2 H6 O4 S
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-12
AICS: No
Other Regulatory: TSCA 2003 (EPA:XU); NDSL 1998
Structure:
Component Registry Number: 77-78-1
Formula: C2 H6 O4 S
Component Registry Number: 68877-49-6
Formula: (C24 H36 O2 . C10 H19 N O2 . C6 H10 O2)x
Component Registry Number: 68877-48-5
Absolute stereochemistry.
Formula: C24 H36 O2
Pr-i

Me
R

R
H
R
O R
Me H
Me
O

CH 2
MeO−S(O2)−OMe
Component Registry Number: 105-16-8
Formula: C10 H19 N O2
H2 C O

Me C C O CH 2 CH 2 NEt 2

Component Registry Number: 97-63-2

Formula: C6 H10 O2
H2 C O

Me C C OEt

xi
Registry Number: 68877-47-4
CA Index Name: 2-Propenoic acid, 2-methyl-, 2-(diethylamino)ethyl ester, polymer with ethyl
2-methyl-2-propenoate and (9Z)-9-octadecenyl 2-methyl-2-propenoate,
compd. with dimethyl sulphate (9CI)
Other Names: 2-Propenoic acid, 2-methyl-, (9Z)-9-octadecenyl ester, polymer with 2-
(diethylamino)ethyl 2-methyl-2-propenoate and ethyl 2-methyl-2-
propenoate, compd. with dimethyl sulphate (9CI) ... Sulphuric acid,
dimethyl ester, compd. with 2-(diethylamino)ethyl 2-methyl-2-propenoate
polymer with ethyl 2-methyl-2-propenoate and (9Z)-9-octadecenyl 2-
methyl-2-propenoate (9CI); … Polyquaternium 13
Formula: (C22 H40 O2 . C10 H19 N O2 . C6 H10 O2)x . x C2 H6 O4 S
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-13
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Other Regulatory: TSCA 2003 (EPA:XU); NDSL 1998
Structure:
Component Registry Number: 77-78-1
Formula: C2 H6 O4 S
Component Registry Number: 68877-46-3
Formula: (C22 H40 O2 . C10 H19 N O2 . C6 H10 O2)x
Component Registry Number: 13533-08-9
Double bond geometry as shown.
Formula: C22 H40 O2
CH 2

O Me
Me (CH 2 ) 8 Z (CH 2 ) 7

Component Registry Number: 105-16-8

Formula: C10 H19 N O2
H2 C O

Me C C O CH 2 CH 2 NEt 2

Component Registry Number: 97-63-2

Formula: C6 H10 O2
H2 C O

Me C C OEt

xii
Registry Number: 27103-90-8
CA Index Name: Ethanaminium, N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-propenyl)oxy]-,
methyl sulphate, homopolymer (9CI)
Other Names: AETAC; Choline, methyl sulphate, methacrylate, polymers (8CI);
Methacrylic acid, ester with choline methyl sulphate, polymers (8CI);
(Methacryloyloxyethyl)trimethylammonium methosulphate polymer; 
Akromidan LK; Hercofloc 828; Jayfloc 911; ... Poly(2-
methacryloyloxyethyltrimethylammonium methyl sulphate);
Poly(methacryloylethyl trimethylammonium methylsulphate; ...
Polyquaternium 14; Poly[( -methacryloyloxyethyl)trimethylammonium
methyl sulphate]; ... Reten 300; [2-
(Methacryloyloxy)ethyl]trimethylammonium methosulphate polymer
Formula: (C9 H18 N O2 . C H3 O4 S)x
Polymer Class Term: Polyacrylic, Polyother
INCI Name: POLYQUATERNIUM-14
Function: antistatic agents/film formers
AICS: Yes
Other Regulatory: TSCA 2003 (EPA:XU); DSL 1991; ENCS; ECL 1997; PICCS 2000
Commercial Profile: Vulcan (Jayfloc); Hercules (Reten)
Structure:
Component Registry Number: 33611-56-2
Formula: C9 H18 N O2
O CH 2

Me 3 + N CH 2 CH 2 O C C Me

Component Registry Number: 21228-90-0

Formula: C H3 O4 S
Me O SO 3 -

xiii
Registry Number: 35429-19-7
CA Index Name: Ethanaminium, N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-propenyl)oxy]-,
chloride, polymer with 2-propenamide (9CI)
Other Names: 2-Propenamide, polymer with N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethanaminium chloride (9CI);
(Methacryloxyethyl)trimethylammonium chloride-acrylamide polymer;
Acrylamide dimethylaminoethyl methacrylate methyl chloride quaternised
salt polymer; ... Alcostat 684; Aronfloc C 325; Binaquat P 100; Clarifloc C
316; Flocogil G 1090; Hercofloc 859; Hiset C 721; Kayafloc C 599-2P;
Percol 757; Polyquaternium 15; Polyquaternium 32; Praestol 423; Praestol
434K; Rohagit KF 720F; Salcare SC 92; Sanfloc C 509P; Sanfloc C 809P;
Sanfloc C 909P; Sanfloc CH 839P; Sequex PC; Sunrez PC; Zetag 76
Formula: (C9 H18 N O2 . C3 H5 N O . Cl)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-15/POLYQUATERNIUM-32
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: Yes
Other Regulatory: TSCA 2003 (XU); DSL 1991; ENCS; ECL 1997; PICCS 2000
Structure:
Component Registry Number: 5039-78-1 (33611-56-2)
Formula: C9 H18 N O2 . Cl
O CH 2

Me 3 + N CH 2 CH 2 O C C Me

· Cl -

Component Registry Number: 79-06-1

Formula: C3 H5 N O
O

H2 N C CH CH 2

xiv
Registry Number: 95144-24-4
CA Index Name: 1H-Imidazolium, 1-ethenyl-3-methyl-, chloride, polymer with 1-ethenyl-2-
pyrrolidinone (9CI)
Other Names: 2-Pyrrolidinone, 1-ethenyl-, polymer with 1-ethenyl-3-methyl-1H-
imidazolium chloride (9CI); 1-Ethenyl-2-pyrrolidinone-imidazolimine
compd. with chloromethane; 1-Vinyl-3-methylimidazolinium chloride-1-
vinylpyrrolidone copolymer; Luviquat FC 370; Luviquat FC 500; Luviquat
FC 550; Luviquat FC 905; Luviquat FC 9059; Luviquat HM 550; Luviquat
HM 552; Luviquat SC 370; Polyquaternium 16
Formula: (C6 H9 N2 . C6 H9 N O . Cl)x
Polymer Class Term: Polyvinyl
INCI Name: POLYQUATERNIUM-16
Function: antistatic agents/film formers; Substantive conditioner, film former
Confidentiality Status: Public
AICS: Yes
Other Regulatory: DSL 1991; ECL 1997; PICCS 2000
Commercial Profile: BASF (Luviquat FC 370, Luviquat FC 550, Luviquat HM 552, Luviquat
style, Luviquat Excellence)
Structure:
Component Registry Number: 13474-25-4 (45534-45-0)
Formula: C6 H9 N2 . Cl

*** FRAGMENT DIAGRAM IS INCOMPLETE ***

Me

CH CH 2
Cl-
Component Registry Number: 88-12-0
Formula: C6 H9 N O
CH CH 2

N
O

xv
Registry Number: 148506-50-7
CA Index Name: Poly[oxy-1,2-ethanediyl(dimethyliminio)-1,3-propanediylimino(1,6-dioxo-
1,6-hexanediyl)imino-1,3-propanediyl(dimethyliminio)-1,2-ethanediyl
dichloride] (9CI)
Other Names: Mirapol AD 1; Polyquaternium 17
Formula: (C20 H42 N4 O3)n . 2 Cl
Alternate Formula: (C20 H42 N4 O3 . 2 Cl)n
Polymer Class Term: Polyamide, Polyether, Polyionene
INCI Name: POLYQUATERNIUM-17
Function: antistatic agents/film formers; Hair and skin conditioner
AICS: No
Other Regulatory PICCS 2000
Commercial Profile: Rhodia (Mirapol AD-1)
Structure

Me O O Me
+ +
CH 2 CH 2 N (CH 2 ) 3 NH C (CH 2 ) 4 C NH (CH 2 ) 3 N CH 2

Me Me

· 2 Cl -

CH 2 O

xvi
Registry Number: 113784-58-0
CA Index Name: Poly[oxy-1,2-ethanediyl(dimethyliminio)-1,3-propanediylimino(1,9-dioxo-
1,9-nonanediyl)imino-1,3-propanediyl(dimethyliminio)-1,2-ethanediyl
dichloride] (9CI)
Other Names: Mirapol AZ 1; Polyquaternium 18
Formula: (C23 H48 N4 O3)n . 2 Cl
Alternate Formula: (C23 H48 N4 O3 . 2 Cl)n
Polymer Class Term: Polyamide, Polyether, Polyionene
INCI Name: POLYQUATERNIUM-18
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Commercial Profile: Rhodia (Mirapol AZ-1)

Me O O Me
+ +
CH 2 CH 2 N (CH 2 ) 3 NH C (CH 2 ) 7 C NH (CH 2 ) 3 N CH 2

Me Me

· 2 Cl -

CH 2 O

xvii
Registry Number: 110736-85-1
CA Index Name: Polyquaternium 19 (9CI)
Other Names: Arlatone PQ 220
Formula: Unspecified
Polymer Class Term: Manual Registration
INCI Name: POLYQUATERNIUM-19
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Commercial Profile: Arlatone = Uniqema
Structure: No Structure Diagram Available

Registry Number: 110736-86-2

CA Index Name: Polyquaternium 20 (9CI)
Other Names: Arlatone PQ 225
Formula: Unspecified
Polymer Class Term:
INCI Name: POLYQUATERNIUM-20
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Commercial Profile: Arlatone = Uniqema
Structure: No Structure diagram available

xviii
Registry Number: 53694-17-0
CA Index Name: 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, polymer with 2-
propenoic acid (9CI)
Other Names: 2-Propenoic acid, polymer with N,N-dimethyl-N-2-propenyl-2-propen-1-
aminium chloride (9CI); Acrylic acid-diallyldimethylammonium chloride
copolymer; Acrylic acid-diallyldimethylammonium chloride polymer; Acrylic
acid-dimethyldiallylammonium chloride copolymer; Floc Aid 34; Merquat
280; Merquat 295; OF 280; Polyquaternium 22
Formula: (C8 H16 N . C3 H4 O2 . Cl)x
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-22
Function: conditioner, moisturiser (L&G:1999)
AICS: Yes
Commercial Profile: Ondeo Nalco (Merquat 280, 280 dry, 281, 295)
Structure:
Component Registry Number: 7398-69-8 (48042-45-1)
Formula: C8 H16 N . Cl
Me
+
H2 C CH CH 2 N CH 2 CH CH 2

Me

· Cl -

Component Registry Number: 79-10-7

Formula: C3 H4 O2
O

HO C CH CH 2

xix
Registry Number: 98616-25-2
CA Index Name: Cellulose, ether with α-[3-(dodecyldimethylammonio)-2-hydroxypropyl]-ω-
hydroxypoly(oxy-1,2-ethanediyl) chloride (9CI)
Other Names: Amerchol LM 200; LM 200; Polyquaternium 24; Quatrisoft; Quatrisoft LM
200; Quatrisoft Polymer LM 200
Formula: (C2 H4 O)n C17 H38 N O2 . x Cl . x Unspecified
Polymer Class Term: Manual registration, Polyether, Polyother, Polyother only
INCI Name: POLYQUATERNIUM-24
Function: Multifunctional substantive conditioner for skin and hair products. Adds
mild surfactancy. Efficient thickener (L&G:1999)
Confidentiality Status: Public
AICS: Yes
Commercial Profile: Amerchol
Structure:
Component Registry Number: 169102-72-1
Formula: (C2 H4 O)n C17 H38 N O2 . x Unspecified
Component Registry Number: 168810-59-1
Formula: (C2 H4 O)n C17 H38 N O2
OH Me Me

N Me
HO O + (CH 2 ) 11
n

Component Registry Number: 9004-34-6

Formula: Unspecified
No Structure Diagram

Registry Number: 178535-77-8

CA Index Name: Polyquaternium 26 (9CI)
Formula: Unspecified
Polymer Class Term: Manual Registration
INCI Name: POLYQUATERNIUM-26
AICS:: No
Commercial Profile:: Amerchol
Structure: No Structure diagram available

xx
Registry Number: 132977-85-6
CA Index Name: Hexanediamide, N,N'-bis[3-(dimethylamino)propyl]-, polymer with N,N'-
bis[3-(dimethylamino)propyl]urea and 1,1'-oxybis[2-chloroethane], block
(9CI)
Other Names: Ethane, 1,1'-oxybis[2-chloro-, polymer with N,N'-bis[3-
(dimethylamino)propyl]hexanediamide and N,N'-bis[3-
(dimethylamino)propyl]urea, block (9CI); Urea, N,N'-bis[3-
(dimethylamino)propyl]-, polymer with N,N'-bis[3-
(dimethylamino)propyl]hexanediamide and 1,1'-oxybis[2-chloroethane],
block (9CI); Mirapol 175; Mirapol 9; Mirapol 95; Polyquaternium 27
Formula: (C16 H34 N4 O2 . C11 H26 N4 O . C4 H8 Cl2 O)x
Polymer Class Term: Polyamide, Polyether, Polyionene, Polyionene formed, Polyurea
INCI Name: POLYQUATERNIUM-27
Function: antistatic agents/film formers
conditioner and thickener with smectite clays)
Confidentiality Status: Public
AICS:: No
Commercial Profile:: Rhodia (Mirapol 9, 95,17)
Structure:
Component Registry Number: 52338-87-1
Formula: C11 H26 N4 O
O

Me 2 N (CH 2 ) 3 NH C NH (CH 2 ) 3 NMe 2

Component Registry Number: 45267-17-2

Formula: C16 H34 N4 O2
O O

Me 2 N (CH 2 ) 3 NH C (CH 2 ) 4 C NH (CH 2 ) 3 NMe 2

Component Registry Number: 111-44-4

Formula: C4 H8 Cl2 O
ClCH 2 CH 2 O CH 2 CH 2 Cl

xxi
Registry Number: 131954-48-8
CA Index Name: 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propenyl)amino]-,
chloride, polymer with 1-ethenyl-2-pyrrolidinone (9CI)
Other Names: 2-Pyrrolidinone, 1-ethenyl-, polymer with N,N,N-trimethyl-3-[(2-methyl-1-
oxo-2-propenyl)amino]-1-propanaminium chloride (9CI); (3-
Methacrylamidopropyl)trimethylammonium chloride-N-vinyl-2-pyrrolidone
copolymer; Conditioneze NT 20; Gafquat HS 100;
Methacrylamidopropyltrimethylammonium chloride-N-vinylpyrrolidone
copolymer; Polyquaternium 28; Trimethylammoniopropylmethacrylamide
chloride-N-vinylpyrrolidone copolymer
Formula: (C10 H21 N2 O . C6 H9 N O . Cl)x
STN Files: CAPLUS, CA, CHEMLIST, CIN, MEDLINE, PROMT, TOXCENTER,
USPAT2, USPATFULL
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-28
Function: antistatic agents/film formers
Confidentiality Status Public
AICS: Yes, assessed NA/89
Commercial Profile: ISP (Gafquat HS-100, Condioneze NT-20)
Structure:
Component Registry Number: 51410-72-1 (51441-64-6)
Formula: C10 H21 N2 O . Cl
O CH 2

Me 3 + N (CH 2 ) 3 NH C C Me

· Cl -

Component Registry Number: 88-12-0

Formula: C6 H9 N O
CH CH 2

N
O

xxii
Registry Number: 148880-30-2
CA Index Name: Polyquaternium 29 (9CI)
Other Names: Lexquat CH
Formula: Unspecified
Polymer Class Term: Manual registration
INCI Name: POLYQUATERNIUM-29
Function: antistatic agents/film formers
Confidentiality Status Public
AICS No
Structure No Structure diagram available

Registry Number: 147398-77-4

CA Index Name: Ethanaminium, N-(carboxymethyl)-N,N-dimethyl-2-[(2-methyl-1-oxo-2-
propenyl)oxy]-, inner salt, polymer with methyl 2-methyl-2-propenoate (9CI)
Other Names: 2-Propenoic acid, 2-methyl-, methyl ester, polymer with N-(carboxymethyl)-
N,N-dimethyl-2-[(2-methyl-1-oxo-2-propenyl)oxy]ethanaminium inner salt
(9CI); Mexomere PX; Polyquaternium 30
Formula: (C10 H17 N O4 . C5 H8 O2)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-30
Function: antistatic agents/film formers
Confidentiality Status Public
AICS No
Structure:
Component Registry Number: 62723-61-9
Formula: C10 H17 N O4
Me O CH 2
-O C +
2 CH 2 N CH 2 CH 2 O C C Me

Me

Component Registry Number: 80-62-6

Formula: C5 H8 O2
H2 C O

Me C C OMe

xxiii
Registry Number: 136505-02-7
CA Index Name: 2-Propenenitrile, homopolymer, hydrolysed, block, reaction products with
N,N-dimethyl-1,3-propanediamine, di-Et sulphate-quaternised
Formula: Unspecified
Polymer Class Term: Manual registration
INCI Name: POLYQUATERNIUM-31
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Structure: No structure diagram available
Registry Number: 69418-26-4
CA Index Name: Ethanaminium, N,N,N-trimethyl-2-[(1-oxo-2-propenyl)oxy]-, chloride,
polymer with 2-propenamide (9CI)
Other Names: 2-Propenamide, polymer with N,N,N-trimethyl-2-[(1-oxo-2-
propenyl)oxy]ethanaminium chloride (9CI); Acrylamide-(2-
acryloxyethyl)trimethylammonium chloride copolymer; ...
Acryloyloxyethyltrimethylammonium chloride-acrylamide copolymer; Betz
2680; Himoloc MP 284; Kayafloc C 599-1R; LBN 66; Magnafloc 292;
Magnifloc 491C, 492C, 494C, 496C; Nalco 1460; Percol 140; Percol 455;
Polyquaternium 33; Salcare SC 93; Zetag 32; Zetag 57, 63, 64, 89; ;
Kayafloc C 599-1R; LBN 66; Magnafloc 292; Magnifloc 491C, 494C,
496C; Nalco 1460; Percol 140, 455; Polyquaternium 33; Salcare SC 93;
Zetag 32, 57, 63, 64, 89
Formula: (C8 H16 N O2 . C3 H5 N O . Cl)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-33
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: Yes
Other Regulatory: TSCA 2003 (XU); DSL 1991; ENCS; ECL 1997; PICCS 2000
Commercial Profile: Ciba (Magnafloc); Cytec (Magnifloc); Applied Polymerics (Percol)
Structure:
Component Registry Number: 44992-01-0 (20284-80-4)
Formula: C8 H16 N O2 . Cl
O

Me 3 + N CH 2 CH 2 O C CH CH 2

· Cl -

Component Registry Number: 79-06-1

Formula: C3 H5 N O
O

H2 N C CH CH 2

xxiv
Registry Number: 189767-68-8
CA Index Name: Polyquaternium-34 (9CI)
Other Names:
Formula: Unspecified
Polymer Class Term: Manual Registration
INCI Name: POLYQUATERNIUM-34
Function: antistatic agents/film formers
Confidentiality Status Public
AICS No
Other Regulatory
Structure No Structure diagram available
OR??????
Registry Number: 143747-73-7
CA Index Name: Polyquaternium 34 (9CI)
Other Names:
Formula: Unspecified
Polymer Class Term: Manual Registration
INCI Name: POLYQUATERNIUM-34
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: NA/475

xxv
Registry Number: 189767-69-9
CA Index Name: Polyquaternium 35 (9CI)
Other Names:
Formula: Unspecified
INCI Name: POLYQUATERNIUM-35
Function: Function: antistatic agents/film formers
AICS: No
Structure: No Structure Diagram

Registry Number: 60494-40-8

CA Index Name: 2-Propenoic acid, 2-methyl-, 2-(dimethylamino)ethyl ester, polymer with
methyl 2-methyl-2-propenoate, compd. with dimethyl sulphate (9CI)
Other Names: 2-Propenoic acid, 2-methyl-, methyl ester, polymer with 2-
(dimethylamino)ethyl 2-methyl-2-propenoate, compd. with dimethyl
sulphate (9CI); Sulphuric acid, dimethyl ester, compd. with 2-
(dimethylamino)ethyl 2-methyl-2-propenoate polymer with methyl 2-
methyl-2-propenoate (9CI); Plex 4739L; Polyquaternium 36
Formula: (C8 H15 N O2 . C5 H8 O2)x . x C2 H6 O4 S
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-36
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: No
Structure:
Component Registry Number: 77-78-1
Formula: C2 H6 O4 S
MeO−S(O2)−OMe
Component Registry Number: 26222-42-4
Formula: (C8 H15 N O2 . C5 H8 O2)x
Component Registry Number: 2867-47-2
Formula: C8 H15 N O2
O CH 2

Me 2 N CH 2 CH 2 O C C Me
Component Registry Number: 80-62-6
Formula: C5 H8 O2
H2 C O

Me C C OMe

xxvi
Registry Number: 26161-33-1
CA Index Name: Ethanaminium, N,N,N-trimethyl-2-[(2-methyl-1-oxo-2-propenyl)oxy]-,
chloride, homopolymer (9CI)
Other Names: Choline, chloride, methacrylate, polymers (8CI); Methacrylic acid, ester
with choline chloride, polymers (8CI);
(Methacryloxyethyl)trimethylammonium chloride polymer; Alcostat 567;
Evagrowth C 104G; Flocogil C 4; Himoloc MP 173H; Kayafloc C 599;
Methacryloxyethyltrimethylammonium chloride homopolymer; … Praestol
444K; Sanfloc C 009P; Shallol DM 283P, 663P; Synthalen CR;
Trimethylaminoethyl methacrylate chloride polymer;
Trimethylammonioethyl methacrylate chloride polymer; Zetag 88N; [2-
(Methacryloyloxy)ethyl]trimethylammonium chloride polymer
Formula: (C9 H18 N O2 . Cl)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-37
Function: antistatic agents/film formers
Confidentiality Status: Public
AICS: Yes (1996)
Other Regulatory: DSL (1991); ENCS; ECL (1997); PICCS (2000); TSCA (2003)
Structure:
Component Registry Number: 5039-78-1 (33611-56-2)
Formula: C9 H18 N O2 . Cl
O CH 2

Me 3 + N CH 2 CH 2 O C C Me Cl -

xxvii
Registry Number: 25136-75-8
CA Index Name: 2-Propen-1-aminium, N,N-dimethyl-N-2-propenyl-, chloride, polymer with 2-
propenamide and 2-propenoic acid (9CI)
Other Names: 2-Propenamide, polymer with N,N-dimethyl-N-2-propenyl-2-propen-1-
aminium chloride and 2-propenoic acid (9CI) ... Acrylamide, polymer with
acrylic acid and diallyldimethylammonium chloride (8CI); Acrylic acid,
polymer with acrylamide and diallyldimethylammonium chloride (8CI);
Ammonium, diallyldimethyl-, chloride, polymer with acrylamide and acrylic
acid (8CI); … ECCat 7951; Merquat 3300, 3330, 3331, Plus 3300, Plus 3330,
Plus 3331; Polyquaternium 39; XQ 3330
Formula: (C8 H16 N . C3 H5 N O . C3 H4 O2 . Cl)x
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-39
Function: antistatic agents/film formers
AICS Yes
Other Regulatory TSCA 2003; DSL 1998; EPA: XU; EPA Pesticide Inert Ingredients, List 3:
Inerts of unknown toxicity.
Structure:
Component Registry Number: 7398-69-8 (48042-45-1)
Formula: C8 H16 N . Cl
Me
+
H2 C CH CH 2 N CH 2 CH CH 2
Cl -
Me
Component Registry Number: 79-10-7
Formula: C3 H4 O2
O

HO C CH CH 2

Component Registry Number: 79-06-1

Formula: C3 H5 N O
O

H2 N C CH CH 2

xxviii
Registry Number: 31512-74-0
CA Index Name: Poly[oxy-1,2-ethanediyl(dimethyliminio)-1,2-ethanediyl(dimethyliminio)-
1,2-ethanediyl dichloride] (9CI)
Other Names: Poly[oxyethylene(dimethyliminio)ethylene(dimethyliminio)ethylene
dichloride] (8CI); Armoblen NPX; BL 2142; Bualta; Bubond 60; Bulab 6002;
Busan 1507; Busan 77; KA 1700; MBC 115; Polixetonium chloride;
Polyquaternium 42;
Poly[oxyethylene(dimethylamino)ethylene(dimethylamino)ethylene
dichloride]; TB 66; WSCP
Formula: (C10 H24 N2 O)n . 2 Cl
Alternate Formula: (C10 H24 N2 O . 2 Cl)n
Polymer Class
Term: Polyether, Polyionene
INCI Name: POLYQUATERNIUM-42
Function: antistatic agents/film formers, algicide
Confidentiality
Status Public
AICS No
Other Regulatory ECL 1997; Giftliste 1 (Toxic Category 4).
Commercial Profile Buckman Laboratories (Busan 77); generic contact lense solution
(Polixetonium chloride)
Structure:

Me Me
+ +
O CH 2 CH 2 N CH 2 CH 2 N CH 2 CH 2

Me Me n

· 2 Cl -

Registry Number: 336879-27-7

CA Index Name: Polyquaternium 43 (9CI)
Formula: Unspecified
INCI Name: POLYQUATERNIUM-43
AICS No Structure Diagram available
Structure No Structure Diagram available

xxix
Registry Number: 150599-70-5
CA Index Name: 1H-Imidazolium, 1-ethenyl-3-methyl-, methyl sulphate, polymer with 1-
ethenyl-2-pyrrolidinone (9CI)
Other Names: 2-Pyrrolidinone, 1-ethenyl-, polymer with 1-ethenyl-3-methyl-1H-
imidazolium methyl sulphate (9CI); 3-Methyl-1-vinylimidazolium methyl
sulphate-N-vinylpyrrolidone copolymer; Luviquat Care; Luviquat MS 370;
Polyquaternium 44
Formula: (C6 H9 N2 . C6 H9 N O . C H3 O4 S)x
Polymer Class Term: Polyother, Polyvinyl
INCI Name: POLYQUATERNIUM-44
Confidentiality Status:
AICS: Yes NA/961
Commercial Profile: BASF (Luviquat Care)

Structure:
Component Registry Number: 88-12-0
Formula: C6 H9 N O
CH CH 2

N
O

Component Registry Number: 26591-72-0

Formula: C6 H9 N2 . C H3 O4 S
Component Registry Number: 45534-45-0
Formula: C6 H9 N2
*** FRAGMENT DIAGRAM IS INCOMPLETE ***
Me

CH CH 2

Component Registry Number: 21228-90-0

Formula: C H3 O4 S
Me O SO 3 -

xxx
Registry Number: 174761-16-1
CA Index Name: 1H-Imidazolium, 1-ethenyl-3-methyl-, methyl sulphate, polymer with 1-
ethenylhexahydro-2H-azepin-2-one and 1-ethenyl-2-pyrrolidinone (9CI)
Other Names: 2-Pyrrolidinone, 1-ethenyl-, polymer with 1-ethenylhexahydro-2H-
azepin-2-one and 1-ethenyl-3-methyl-1H-imidazolium methyl sulphate
(9CI); 2H-Azepin-2-one, 1-ethenylhexahydro-, polymer with 1-ethenyl-
3-methyl-1H-imidazolium methyl sulphate and 1-ethenyl-2-
pyrrolidinone (9CI); Luviquat Hold; Polyquaternium 46
Formula: (C8 H13 N O . C6 H9 N2 . C6 H9 N O . C H3 O4 S)x
Polymer Class Term: Polyother, Polyvinyl
INCI Name: POLYQUATERNIUM-46
AICS: Yes (2003) NA/533
Commercial Profile: BASF (Luviquat Hold)
Structure:
Component Registry Number: 2235-00-9
Formula: C8 H13 N O
CH CH 2
O
N

Component Registry Number: 88-12-0

Formula: C6 H9 N O
CH CH 2

N
O

Component Registry Number: 26591-72-0

Formula: C6 H9 N2 . C H3 O4 S
Component Registry Number: 45534-45-0
Formula: C6 H9 N2
*** FRAGMENT DIAGRAM IS INCOMPLETE ***
Me

CH CH 2

xxxi
Registry Number: 197969-51-0
CA Index Name: 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propenyl)amino]-,
chloride, polymer with methyl 2-propenoate and 2-propenoic acid (9CI)
Other Names: 2-Propenoic acid, methyl ester, polymer with 2-propenoic acid and N,N,N-
trimethyl-3-[(2-methyl-1-oxo-2-propenyl)amino]-1-propanaminium chloride
(9CI); 2-Propenoic acid, polymer with methyl 2-propenoate and N,N,N-
trimethyl-3-[(2-methyl-1-oxo-2-propenyl)amino]-1-propanaminium chloride
(9CI); Acrylic acid-3-methacryloylaminopropyltrimethylammonium chloride-
methyl acrylate copolymer; Merquat 2000, 2001, 2001N; Polyquaternium 47
Formula: (C10 H21 N2 O . C4 H6 O2 . C3 H4 O2 . Cl)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-47
AICS: No NA/896
Commercial Profile: Ondeo Nalco (Merquat 2001, Merquat 2001N)
Structure:
Component Registry Number: 51410-72-1 (51441-64-6)
Formula: C10 H21 N2 O . Cl
O CH 2

Me 3 + N (CH 2 ) 3 NH C C Me Cl -

Component Registry Number: 96-33-3

Formula: C4 H6 O2
O

MeO C CH CH 2

Component Registry Number: 79-10-7

Formula: C3 H4 O2
O

HO C CH CH 2

xxxii
Registry Number: 125275-25-4
CA Index Name: 3,5,8-Trioxa-4-phosphaundec-10-en-1-aminium, 4-hydroxy-N,N,N,10-
tetramethyl-9-oxo-, inner salt, 4-oxide, polymer with butyl 2-methyl-2-
propenoate (9CI)
Other Names: 2-Propenoic acid, 2-methyl-, butyl ester, polymer with 4-hydroxy-
N,N,N,10-tetramethyl-9-oxo-3,5,8-trioxa-4-phosphaundec-10-en-1-aminium
inner salt 4-oxide (9CI); 2-Methacryloyloxyethylphosphorylcholine-butyl
methacrylate copolymer; Butyl methacrylate-2-(methacryloyloxy)ethyl-2'-
(trimethylammonio)ethyl phosphate copolymer; Butyl methacrylate-2-
methacryloyloxyethylphosphorylcholine copolymer; Lipidure PMB; n-Butyl
methacrylate-2-methacryloyloxyethylphosphorylcholine
Formula: (C11 H22 N O6 P . C8 H14 O2)x
Polymer Class Term: Polyacrylic
INCI Name: POLYQUATERNIUM-51
Confidentiality Status: Public
AICS: No
Other Regulatory: TSCA 2003 (EPA:XU); ENCS No.: 6-2367
Structure
Component Registry Number: 67881-98-5
Formula: C11 H22 N O6 P

O- O CH 2

Me 3 + N CH 2 CH 2 O P O CH 2 CH 2 O C C Me

Component Registry Number: 97-88-1

Formula: C8 H14 O2
O CH 2

n-BuO C C Me

xxxiii
Registry Number: 306769-73-3
CA Index Name: 1-Dodecanaminium, N,N-dimethyl-N-[3-[(2-methyl-1-oxo-2-
propenyl)amino]propyl]-, chloride, polymer with N-[3-
(dimethylamino)propyl]-2-methyl-2-propenamide and 1-ethenyl-2-
pyrrolidinone (9CI)
Other Names: 2-Propenamide, N-[3-(dimethylamino)propyl]-2-methyl-, polymer with N,N-
dimethyl-N-[3-[(2-methyl-1-oxo-2-propenyl)amino]propyl]-1-
dodecanaminium chloride and 1-ethenyl-2-pyrrolidinone (9CI); 2-
Pyrrolidinone, 1-ethenyl-, polymer with N-[3-(dimethylamino)propyl]-2-
methyl-2-propenamide and N,N-dimethyl-N-[3-[(2-methyl-1-oxo-2-
propenyl)amino]propyl]-1-dodecanaminium chloride (9CI); Polyquaternium
55; Styleze W 20
Formula: (C21 H43 N2 O . C9 H18 N2 O . C6 H9 N O . Cl)x
Polymer Class Term: Polyacrylic, Polyvinyl
INCI Name: POLYQUATERNIUM-55
AICS No
Commercial Profile: ISP (Styleze NT-20)
Structure
Component Registry Number: 126758-30-3 (129684-48-6)
Formula: C21 H43 N2 O . Cl
H2 C O Me
+
Me C C NH (CH 2 ) 3 N (CH 2 ) 11 Me
Cl -
Me
Component Registry Number: 5205-93-6
Formula: C9 H18 N2 O
O CH 2

Me 2 N (CH 2 ) 3 NH C C Me

Component Registry Number: 88-12-0

Formula: C6 H9 N O
CH CH 2

N
O

xxxiv
Registry Number: 25988-97-0
CA Index Name: Methanamine, N-methyl-, polymer with (chloromethyl)oxirane
Other Names: Dimethylamine-epichlorohydrin copolymer
Dimethylamine, polymer with 1-chloro-2,3-epoxypropane (8CI); Oxirane,
(chloromethyl)-, polymer with N-methylmethanamine (9CI); Propane, 1-
chloro-2,3-epoxy-, polymer with dimethylamine (8CI); Agefloc A 50, A
50LV, B 50, B 50LV; Amerfloc 425E, 485; Bufloc 186; CA 250; CA 260;
Callaway 4000; Catiomaster PD 10; Cysep 349, 572, 573, 577;;
Dimethylamine-epichlorohydrin polymer; DMA-epichlorohydrin copolymer;
ednAgefloc A 50LV; Epichlorohydrin-dimethylamine copolymer;
Epichlorohydrin-dimethylamine polymer; Fixogene CXF; Flocmaster 5310;
Floxan 5062; Glokill pQ; HP 142A; HP 182A; Jetfix 36N; Kufloc 100A,
200A; Nalco 7655, N 7655; Neofix RE; Polyfix 601, 610; Polyplus 1290;
Polypure C 309; Proset 1810, 1820; PRP 2350; PRP 2449; PRP 2850;
Refaktan K; Ultrafloc 5000; Weisstex T 101
Formula: (C3 H5 Cl O . C2 H7 N
Polymer Class Term: Polyionene, Polyionene formed
INCI Name: none
AICS Yes
Structure
Component Registry Number: 124-40-3
Formula: C2 H7 N
H3 C NH CH 3

Component Registry Number: 106-89-8

Formula: C3 H5 Cl O
O

CH 2 Cl

xxxv
Registry Number: 68039-13-4
CA Index Name: 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-
propenyl)amino]-, chloride, homopolymer (9CI)
Other Names: Clairquat 1; Poly MAPTAC;
Poly(methacrylamidopropyltrimethylammonium) chloride;
Poly(methacryloylamidopropyltrimethylammonium chloride);
Polycare 133; Poly[(3-methacrylamidopropyl)trimethylammonium
chloride]; Poly[[3-(2-methylpropenamido)propyl]trimethylammonium
chloride]; [(Methacrylamido)propyl]trimethylammonium chloride
homopolymer
Formula: (C10 H21 N2 O . Cl)x
STN Files: CAPLUS, CA, CHEMCATS, CHEMLIST, CSCHEM,
TOXCENTER, USPATFULL
Deleted Registry Number(s): 111547-42-3
Alternate Formula:
Polymer Class Term: Polyacrylic
INCI Name: POLYMETHACRYLAMIDOPROPYLTRIMONIUM
CHLORIDE
Function: Function: antistatic agents/film formers
Confidentiality Status Public
AICS Yes 1996
Other Regulatory TSCA 2003 (EPS:XU); DSL 1991; PICCS 2000
Structure
Component Registry Number: 51410-72-1 (51441-64-6)
Formula: C10 H21 N2 O . Cl
O CH 2

Me 3 + N (CH 2 ) 3 NH C C Me

· Cl -

xxxvi
Registry Number: 65497-29-2
CA Index Name: Guar gum, 2-hydroxy-3-(trimethylammonio)propyl ether, chloride (9CI)
Other Names: Cosmedia Guar 261N; Cosmedia Guar C 261; Cosmedia Guar C 261N; Guar
hydroxypropyltrimonium chloride; HI-Care 1000; J-C 13S; Jaguar C 13;
Jaguar C 13S; Jaguar C 14S; Jaguar C 15; Jaguar C 15S; Jaguar C 17; Jaguar
CP 13; Jaguar Excel; Rhaball Gum CG-M 8M; Rhaball Gum CGM; cationic
guar gum
Formula: C6 H16 N O2 . x Cl . x Unspecified
Polymer Class Term: Manual registration
INCI Name: GUAR HYDROXYPROPYLTRIMONIUM CHLORIDE
AICS Yes
Commercial Profile: Hercules (N-Hance, N-Hance 3000, N-Hance 0.72 = CD 0.72)
Structure
Component Registry Number: 67034-33-7
Formula: C6 H16 N O2 . x Unspecified
Component Registry Number: 44814-66-6
Formula: C6 H16 N O2
OH

HO CH 2 CH CH 2 N + Me 3

Component Registry Number: 9000-30-0

Formula: Unspecified
No Structure Diagram

xxxvii
OTHER POLYQUATERNIUMS
Registry Number: 58561-79-8
Name: Magnifloc 570C
AICS: no
Reference: Biesinger et al. (1976)

Registry Number: 99675-02-2

CA Index Name: Magnifloc 573C
AICS: no
Use: Flocculant
Reference: Haarhoff and Cleasby (1989)

Registry Number: 58561-78-7

Name: Magnifloc 521C
AICS: no
Use: Biesinger et al. (1976)
Reference:
Registry Number: 39434-69-0
Name: Primafloc C-7
AICS: no
Use Rohm and Haas
Reference:
Reference Bhattacharjya et al. (1975)

Registry Number: 61008-34-2

Name: Ionac
AICS: no
Use
Reference:
Reference Bhattacharjya et al. (1975)

Registry Number: 39355-17-4

Name: Nalco-600
AICS: no
Use Ondeo-Nalco
Reference:
Reference Bhattacharjya et al. (1975)

Registry Number: 55838-93-2

Name: Purifloc C31
AICS: no
Use Chance and Hunt
Reference: Flocculant Coagulent
Reference Biesinger et al. (1976)

Registry Number: 67828-15-3

Name: 1,3-Propanediamine, N,N-dimethyl-, polymer with (chloromethyl)oxirane and
N-methylmethanamine (9CI); Hydrotriticum QL, QM, QS
AICS: no
Use Dragan et al. (2002)
Reference:
Registry Number: 130381-06-5
Name: Protein hydrolysates, wheat germ, [3-(dodecyldimethylammonio)-2-
hydroxypropyl], chlorides; Hydrotriticum QL, QM, QS
AICS: yes
Use Croda
Reference: Hair conditioner
Reference Nguyen et al. (1992)

Registry Number: 130381-05-4

xxxviii
Name: Protein hydrolysates, wheat germ, [3-(dimethyloctadecylammonio)-2-
hydroxypropyl], chlorides; Hydrotriticum QL, QM, QS
AICS: yes
Use Croda
Reference: Hair conditioner
Reference Nguyen et al. (1992)

Registry Number: 130381-04-3

Name: Protein hydrolysates, wheat germ, [3-(cocalkyldimethylammonio)-2-
hydroxypropyl], chlorides; Hydrotriticum QL, QM, QS
AICS: yes
Use Croda
Reference: Hair conditioner
Reference Nguyen et al. (1992)

Registry Number: 124046-49-7

Name: Keratins, hydrolysates, C12-alkyl-quaternised; Croquat WKP
AICS: yes
Use Croda
Reference: Hair conditioner
Reference Nguyen et al. (1992)

Registry Number: 96526-34-0

Name: 1-Octanaminium, N,N-dimethyl-N-[2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethyl]-, bromide, homopolymer (9CI)
AICS: no
Use Nagai and Ohishi (1987)
Reference:
Registry Number: 105058-33-1
Name: 1-Hexadecanaminium, N,N-dimethyl-N-[2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethyl]-, bromide, homopolymer (9CI)
AICS: no
Use Nagai and Ohishi (1987)
Reference:
Registry Number: 96526-36-2
Name: 1-Dodecanaminium, N,N-dimethyl-N-[2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethyl]-, bromide, homopolymer (9CI)
AICS: no
Use Nagai and Ohishi (1987)
Reference:
Registry Number: 107310-72-5
Name: 1-Butanaminium, N,N-dimethyl-N-[2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethyl]-, bromide, homopolymer (9CI
Reference Nagai and Ohishi (1987)

Registry Number: 96526-37-3

Name: 1-Tetradecanaminium, N,N-dimethyl-N-[2-[(2-methyl-1-oxo-2-
propenyl)oxy]ethyl]-, bromide, homopolymer (9CI)
AICS: no
Use Nagai and Ohishi (1987)
Reference:
Registry Number: 58561-66-3
Name: Superfloc 330
AICS: no
Use Cytec
Reference: Flocculant
Reference Biesinger et al. (1976)

Registry Number: 58561-66-3

Name: Calgon M-500

xxxix
AICS: no
Use Reckitt
Reference: Flocculant
Reference

Registry Number: 9000-30-0

Name: Gendriv 162
AICS: yes
Use Flocculant
Reference: yes

Registry Number: None allocated

Name: Lauryldimonium hydroxypropyl hydrolysed casein
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

Registry Number: None allocated

Name: Lauryldimonium hydroxypropyl hydrolysed keratin
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

Registry Number: None allocated

Name: Lauryldimonium hydroxypropyl hydrolysed silk
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

Registry Number: None allocated

Name: Lauryldimonium hydroxypropyl hydrolysed soy protein
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

Registry Number: None allocated

Name: Lauryldimonium hydroxypropyl hydrolysed wheat protein
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

Registry Number: None allocated

Name: Linoleamidopropyl Hydroxypropyl Dimonium Hydrolysed Oat Protein
AICS: no
Use Antistatic agent, hair and skin conditioner
Reference: INCI

xl
 Appendix 2. Published toxicity values for Cationic Polyelectrolytes
Polymer Species Test type Endpoint mg L-1 Reference
Busan 77 Fish 24 hour 0.66 Tooby et al.
48 hour 0.32 (1975)
96 hour 0.17
Superfloc 330 Rainbow trout Static 48 hr 2.35 (Biesinger
CAS 58561-88-9 TL50* et al. (1976)
Lake trout 96 hr TL50 2.12
48 hr TL50 2.90
Mysis relicta 96 hr TL50 2.85
Limnocalanus 96 hr TL50 0.50
macrurus 48 hr TL50 0.35
96 hr TL50 0.29
Daphnia Magna Dynamic 48 hr TL50 0.34
Rainbow trout 14 d TL50 0.34
Lake trout 12 d TL50 0.31
M. relicta 14 d TL50 <0.06
D. magna 7 d TL50 <0.19
Calgon M-500 cationic Rainbow trout Static 48 hr TL50 6.50 (Biesinger
CAS 58561-66-3 96 hr TL50 6.15 et al. (1976)
Lake trout 48 hr TL50 >8.00
96 hr TL50 5.70
M. relicta 96 hr TL50 >4.00
L. macrurus 48 hr TL50 2.00
96 hr TL50 2.00
D. Magna 48 hr TL50 42.00
Gendriv 162 Rainbow trout Static 96 hr TL50 218.00 (Biesinger
CAS 9000-30-0 D. Magna 48 hr TL50 42.00 et al. (1976)
(Guar Gum) 96 hr TL50 <6.20
Magnifloc 521C Rainbow trout Static 48 hr TL50 8.70 (Biesinger
CAS 58561-79-8 96 hr TL50 8.70 et al. (1976)
D. Magna 48 hr TL50 3.70
96 hr TL50 2.10
Rainbow trout Dynamic 14 d TL50 1.10
Polymer A Pimephales Static 96 hour 0.23 Devore and
Epichlorohydrin-amine promelas 48 hour 0.07 Lyons
condensate D. magna (1986)
molecular weight
20,000 amu
Polymer B P. promelas Static 96 hour 0.2 Devore and
Homopoly(diallyldimet D. magna 48 hour 0.23 Lyons
hyl ammonium (1986)
chloride)
molecular weight
300,000 amu
Polymer C P. promelas Static 96 hour 0.22 Devore and
Epichlorohydrin-amine D. magna 48 hour 0.29 Lyons
condensate (1986)
molecular weight
400,000 amu
Polymer D P. promelas Static 96 hour <0.5 Devore and
Acrylamide, vinyl D. magna 48 hour <1.0 Lyons
quaternary amine (1986)
copolymer
Polymer A P. promelas 96 hour >100 Biesinger et
D. magna 48 hour >100 al. (1986)

xli
Polymer Species Test type Endpoint mg L-1 Reference
Polymer B P. promelas 96 hour >100 Biesinger et
D. magna 48 hour 0.77 al. (1986)
Paratanytarsus
parthenogenetics 48 hour >100
Gammarus
pseudolimnaeus 96 hour 31.6
Polymer C P. promelas 96 hour >100 Biesinger et
D. magna 48 hour >100 al. (1986)
Polymer D P. promelas 96 hour 7.4 Biesinger et
D. magna 48 hour >100 al. (1986)
P. 48 hour >100
parthenogenetics 96 hour 102.9
G. pseudolinaeus
Polymer E P. promelas 96 hour 0.88 Biesinger et
D. magna 48 hour 1.2 al. (1986)
P. 48 hour 26.9
parthenogenetics 96 hour 22.8
G. pseudolinaeus
Polymer F P. promelas 96 hour 2.87 Biesinger et
D. magna 48 hour 0.24 al. (1986)
P. 48 hour 50.0
parthenogenetics 96 hour >100
G. pseudolinaeus
Polymer G P. promelas 96 hour 1.0 Biesinger et
D. magna 48 hour 0.32 al. (1986)
P. 48 hour <6.25
parthenogenetics 96 hour 8.1
G. pseudolinaeus
Polymer H P. promelas 96 hour 2.46 Biesinger et
D. magna 48 hour 0.71 al. (1986)
P. 48 hour >100
parthenogenetics 96 hour >100
G. pseudolinaeus
Polymer I P. promelas 96 hour 3.74 Biesinger et
D. magna 48 hour 0.13 al. (1986)
P. 48 hour >100
parthenogenetics 96 hour >100
G. pseudolinaeus
Polymer J P. promelas 96 hour 9.47 Biesinger et
D. magna 48 hour 6.78 al. (1986)
G. pseudolinaeus 96 hour 112.25
Polymer K P. promelas 96 hour 6.82 Biesinger et
D. magna 48 hour 0.09 al. (1986)
G. pseudolinaeus 96 hour >100
Polymer L P. promelas 96 hour 5.7 Biesinger et
D. magna 48 hour 1.84 al. (1986)
G. pseudolinaeus 96 hour 33.4
Polymer M P. promelas 96 hour 2.18 Biesinger et
D. magna 48 hour 12.9 al. (1986)
P. 48 hour >100
parthenogenetics 96 hour 21.0
G. pseudolinaeus
Polymer N P. promelas 96 hour 2.72 Biesinger et
D. magna 48 hour 70.71 al. (1986)
G. pseudolinaeus 96 hour 85.2
Polymer O P. promelas 96 hour 1.05 Biesinger et
D. magna 48 hour 0.50 al. (1986)
G. pseudolinaeus 96 hour 12.5

xlii
Polymer Species Test type Endpoint mg L-1 Reference
Polymer A P. promelas 96 hour 0.16 Cary et al.
molecular weight ≈ D. magna 48 hour 0.21 (1987)
2,000 amu
% activity 2.19
Polymer B P. promelas 96 hour 0.17 Cary et al.
molecular weight ≈ D. magna 48 hour 0.08 (1987)
1,200,000 amu
% activity 0.41
Polymer C P. promelas 96 hour 0.25 Cary et al.
molecular weight ≈ D. magna 48 hour 0.08 (1987)
100,000 amu
Polymer D P. promelas 96 hour 0.46 Cary et al.
molecular weight ≈ D. magna 48 hour 0.20 (1987)
25,000 amu
% activity 0.96
Quaternised ? 0.9 Cary et al.
polyethanolamine (1989)
molecular weight ≈
2000 amu;
% activity 2.19
Dimethyldiallyl 0.47 Cary et al.
ammonium chloride (1989)
molecular weight
25,000 amu;
% activity 0.96
Poly(dimethylvinyl- 0.17 Cary et al.
pyridinium) chloride (1989)
molecular weight ≈
1,200,000 amu;
% activity 0.41
Dimethylamine- 0.3 Cary et al.
epichlorohydrin (1989)
copolymer
molecular weight
100,000 amu
; % activity 3.65
Epichlorohydrin/amine 0.24 Cary et al.
polymer (1989)
molecular weight 2-
3000 amu;
% activity 3.72
Epichlorohydrin/amine 0.18 Cary et al.
polymer (1989)
molecular weight 2-
3000 amu;
% activity 4.53
A-1 Rainbow trout Static 24 hour 1.386 Goodrich et
Epichlorohydrin/dimeth 48 hour 0.762 al. (1991)
ylamine 72 hour 0.646
molecular weight 96 hour 0.592
10,000 amu Dynamic 48 hour 0.0544
72 hour 0.0544
96 hour 0.0426
28 day 0.0017

xliii
Polymer Species Test type Endpoint mg L-1 Reference
A-2 Rainbow trout Static 24 hour 0.767 Goodrich et
Epichlorohydrin/dimeth 48 hour 0.356 al. (1991)
ylamine 72 hour 0.275
molecular weight 96 hour 0.271
50,000 amu Dynamic 24 hour 0.361
48 hour 0.281
72 hour 0.127
96 hour 0.0397
A-3 Rainbow trout Static 24 hour 1.062 Goodrich et
Epichlorohydrin/dimeth 48 hour 0.885 al. (1991)
ylamine 72 hour 0.82
200,000-250,000 amu 96 hour 0.779
Dynamic 24 hour 0.349
48 hour 0.323
72 hour 0.254
96 hour 0.156
28 day 0.1387
B-1 Rainbow trout Static 96 hour 1.733 Goodrich et
Acrylamide/2-(N,N,N)- al. (1991)
trimethyl ammonium
ethylacrylate chloride
Charge density 10%
B-2 Rainbow trout Static 24 hour 0.705 Goodrich et
Acrylamide/2-(N,N,N)- 48 hour 0.675 al. (1991)
trimethyl ammonium 72 hour 0.661
ethylacrylate chloride 96 hour 0.661
Charge density 39% Dynamic 48 hour 0.406
72 hour 0.384
96 hour 0.384
28 day 0.3036
Cationic Emulsion 1 P. promelas Static 96 hour 4.70 Hall and
(Metac) D. pulex 48 hour 0.22 Mirenda
molecular weight (1991)
4,500,000 amu;
Charge density 10%
Cationic Emulsion 2 P. promelas Static 96 hour 1.41 Hall and
(Metac) D. pulex 48 hour 0.19 Mirenda
molecular weight (1991)
6,000,000 amu;
Charge density 25%
Cationic Emulsion 3 P. promelas Static 96 hour 1.41 Hall and
(Metac) D. pulex 48 hour 0.06 Mirenda
molecular weight (1991)
5,000,000; Charge
density 45%
Cationic Emulsion 4 P. promelas Static 96 hour 0.52 Hall and
(Metac) D. pulex 48 hour 0.26 Mirenda
molecular weight (1991)
7,000,000 amu;
Charge density 45%
Cationic Emulsion 5 P. promelas Static 96 hour 0.58 Hall and
(Metac) D. pulex 48 hour 0.08 Mirenda
molecular weight (1991)
5,000,000 amu;
Charge density 75%

xliv
Polymer Species Test type Endpoint mg L-1 Reference
Cationic Emulsion 6 P. promelas Static 96 hour 11.60 Hall and
(Aetac) D. pulex 48 hour 0.17 Mirenda
molecular weight (1991)
7,500,000 amu;
Charge density 2%
Cationic Emulsion 7 P. promelas Static 96 hour 13.49 Hall and
(Aetac) D. pulex 48 hour 0.06 Mirenda
Molecular weight (1991)
7,000,000 amu;
Charge density 6%
Cationic Emulsion 8 P. promelas Static 96 hour 4.49 Hall and
(Aetac) D. pulex 48 hour 0.15 Mirenda
Molecular (1991)
weight5,000,000 amu;
Charge density 10%
Cationic Emulsion 9 P. promelas Static 96 hour 1.45 Hall and
(Aetac) D. pulex 48 hour 0.20 Mirenda
molecular weight (1991)
7,000,000 amu;
Charge density 25%
Cationic Emulsion 10 P. promelas Static 96 hour 1.05 Hall and
(Aetac) D. pulex 48 hour 0.21 Mirenda
molecular weight (1991)
6,000,000 amu;
Charge density 35%
Cationic Emulsion 11 P. promelas Static 96 hour 1.30 Hall and
(Aetac) D. pulex 48 hour 0.19 Mirenda
molecular weight (1991)
3,000,000 amu;
Charge density 45%
Cationic Emulsion 12 P. promelas Static 96 hour 2.81 Hall and
(Aetac) D. pulex 48 hour 0.32 Mirenda
molecular weight (1991)
6,000,000; Charge
density 45%
Cationic Emulsion 13 P. promelas Static 96 hour 1.17 Hall and
(Aetac) D. pulex 48 hour 0.98 Mirenda
molecular weight (1991)
7,000,000 amu;
Charge density 45%
Cationic Emulsion 14 P. promelas Static 96 hour 0.81 Hall and
(Aetac) D. pulex 48 hour 0.57 Mirenda
molecular weight (1991)
8,000,000 amu;
Charge density 45%
Cationic Solution 1 P. promelas Static 96 hour 0.86 Hall and
(EPI/DMA) D. pulex 48 hour 0.26 Mirenda
molecular weight (1991)
100,000 amu;
Charge density 100%
Cationic Solution 2 P. promelas Static 96 hour 0.68 Hall and
(EPI/DMA) D. pulex 48 hour 0.16 Mirenda
molecular weight (1991)
500,000 amu;
Charge density 100%

xlv
Polymer Species Test type Endpoint mg L-1 Reference
Cationic Solution 3 P. promelas Static 96 hour 0.74 Hall and
(DADMAC) D. pulex 48 hour 0.77 Mirenda
molecular weight (1991)
50,000 amu;
Charge density 100%
Cationic Solution 4 P. promelas Static 96 hour 0.88 Hall and
(DADMAC) D. pulex 48 hour 2.00 Mirenda
molecular weight (1991)
200,000 aamu;
Charge density 100%
Cationic Solution 5 P. promelas Static 96 hour >170 Hall and
(Melamine D. pulex 48 hour 12.31 Mirenda
formaldehyde) (1991)
molecular weight
10,000 amu;
Charge density 75%
Cationic Solution 6 P. promelas Static 96 hour 3.29 Hall and
(Mannich) D. pulex 48 hour 51.71 Mirenda
molecular weight (1991)
3,000,000 amu;
Charge density 70%
Cationic Solution 7 P. promelas Static 96 hour 1.48 Hall and
(Mannich) D. pulex 48 hour 41.58 Mirenda
molecular weight (1991)
4,000,000 amu;
Charge density 70%
Cationic Solution 8 P. promelas Static 96 hour 1.04 Hall and
(Mannich) D. pulex 48 hour 45.96 Mirenda
molecular weight (1991)
5,00,000 amu; Charge
density %
Cationic Solution 9 P. promelas Static 96 hour 1.36 Hall and
(Mannich) D. pulex 48 hour 70.08 Mirenda
molecular weight (1991)
6,500,000 amu;
Charge density 70%
Cationic Solution 10 P. promelas Static 96 hour 1.19 Hall and
(Mannich) D. pulex 48 hour 46.24 Mirenda
molecular weight (1991)
8,000,000 amu;
Charge density 70%
Zetag 64 Baicalobia guttata 96 hour >100 Beim et al.
D. magna 96 hour 2.05 (1994)
Eulimnogammaru
s verrucosus 96 hour 1160.0
Phoxinus phoxinus 96 hour 2.82
L.
Sanfloc CH009P B. guttata 96 hour 1.63 Beim et al.
D. magna 96 hour 0.08 (1994)
E. verrucosus 96 hour 650.0
P. phoxinus L. 96 hour 141.0
Catfloc D. magna 96 hour 0.08 Beim et al.
E. verrucosus 96 hour 70.0 (1994)
P. phoxinus L. 96 hour 2.24
Polymer I Ceriodaphnia 48 hour 0.11 Fort and
Highly cationic, low dubia Stover
molecular weight (1995)
polyquaternary amine

xlvi
Polymer Species Test type Endpoint mg L-1 Reference
Polymer II C. dubia 48 hour 0.08 Fort and
Highly cationic, Stover
medium molecular (1995)
weight EPI/DMA
Polymer III C. dubia 48 hour 0.12 Fort and
Moderately cationic, Stover
medium molecular (1995)
weight quaternary
amine
Polymer IV C. dubia 48 hour 0.07 Fort and
Highly cationic, high Stover
molecular weight (1995)
polyquaternary amine

xlvii
 Appendix 3 Partitioning Models
a. Percent Removal Model.
Excel Spreadsheet (in Format Auditing View) of the model used in Section 4.4 to
estimate the percent removal for a compound with a given partition coefficient KD.
The result can then be applied to the removal in WWTP parameter in various methods
of determining the PEC for given import/manufacture volumes.
A1 B C D E
2 KD 400 L/kg Input parameter
3 Solubility 10000000 µg/L Estimate
Estimate, based on lowest pub
4 Vapour Pressure 0.0000000001 Pa (for OCHARGE DENSITY D)
Henry's Law L.Pa/
5 Const. =C4/C3 µg Vapour Pressure/Solubility
proportion of
solids removal
6 (s) 0.9
7
8 Q2Biosolids 105 kg/h From WWTP mass balance
9 Q3Biosolids 56.7 kg/h
10 Q2water 2600 L/h
11 Q3water 580000 L/h
12 QWASW 12500
13 QWASb 48.7
14
15 Ignoring WAS
Removal in =(((0.96*C6)*(C8+C9)/0.04)+(C6*(C8+C9)*C2))/((C11+C10)+((C8+C9)*C
16 Biosolids 2))
17
Taking into
18 account WAS
Removal in
19 Biosolids
20 Part a =(0.96*C6*(C9+C8))/0.04
21 Part b =C6*(C8+C9)*C2+C12+(C13*C2)
22 nominator =C21+C20
23 denominator =C10+(C8*C2)+C11+(C9*C2)+C12+(C13*C2)
24 fraction =C22/C23
25 percent =C24*100

Formula for Cell C16 ⎛ 0.96 ⎞

⎜ + K D ⎟(Q2 B + Q3 B )
0.04
p= ⎝ ⎠
Q2W + Q3W + (Q2 B + Q3 B )K D

Formula for cells C20 to C24

⎛ 0.96 × s × (Q3B + Q2 B ) ⎞
⎜ + s(Q2 B + Q3B )K D + QWASW + QWASB K D ⎟
p =⎜ 0.04 ⎟
⎜ Q2W + Q2 B K D + Q3W + Q3B K D ⎟
⎜ ⎟
⎝ ⎠

xlviii
b. Input Flux Model.
Excel Spreadsheet (in Format Auditing View) of the model used in Section 6.2 to
estimate the input flux resulting in the ETNCaq for a compound with a given partition
coefficient KD. The result can then be applied to estimate the import/manufacture
volume which may result in the ETNCaq being exceeded.
A B C D
1 ETNC 0.01 Input parameter
2 Effluent =B1*10 µg/L Dilution to receiving waters
3 K(D) 400 L/kg Input parameter
4 Solubility 10000000 µg/L Estimate
5 Estimate, based on lowest published
Vapour Pressure 0.0000000001 Pa (for OCHARGE DENSITY D)
6 Henry's Law Const. =B5/B4 L.Pa/µg Vapour Pressure/Solubility
7
8 Effluent
9 Oxley Parameters
10 Effluent Flow rate (W) 571000 L/h From water balance
11 Effluent Flow rate (S) 5.71 kg/h From solids balance
12 Sludge Density 1 Assumed
13 Q5water 1163000 L/h From WWTP mass balance
14 Q5solids 2330 kg/h From WWTP mass balance
15 Q4water 583000 L/h From WWTP mass balance
16 Q4solids 2273 kg/h From WWTP mass balance
17
18 Csorbed =B3*B2 µg/kg from KD = Csorbed/Charge density issolved
19 Effluent Flux (dissolved) =B10*B2 µg/h Cwater * Effluent Flow Rate (W)
20 Effluent Flux (sorbed) =B18*B11 µg/h Csorbed * Effluent Flow Rate (S)
21 Total Effluent Flux =SUM(B19:B20) µg/h Sum of Effluent fluxes
22
23 Zwater =1/B6 µg/L.Pa 1/Henry's Law Constant
24 Zsorbed =B23*B3 µg/kg.Pa From KD = Zsolids/Zwater
25
26 D(5) =(B13*B23)+(B14*B24) μg/h.Pa D = Q*Zwater + Q*Zsorbed
27 D(4) =(B15*B23)+(B16*B24) μg/h.Pa D = Q*Zwater + Q*Zsorbed
28
29 Final Settling Tank
30 ƒ(PQ-X) - (D5-D4) = Total Effluent
ƒ(PQ-X) =B21/(B26-B27) Pa Flux
31 Dissolved Flux PQ-X =B30*B26 µg/h D(5) * ƒ(PQ-X)
32 Sorbed Flux PQ-X =B30*B27 µg/h D(4) * ƒ(PQ-X)
33
34 Bioreactor
35 Q3water 580000 L/h From WWTP mass balance
36 Q3solids 56.7 kg/h From WWTP mass balance
37
38 D(3) =(B35*B23)+(B36*B24) μg/h.Pa D = Q * Zwater + Q * Zsorbed
39
40 Dissolved Flux (PQ-X) =B38*B30 µg/h D(3) * ƒ(PQ-X)
41
42 Primary Settling Tank
43 Q1water 583000 L/h From WWTP mass balance
44 Q1solids 162 kg/h From WWTP mass balance
45 Q2water 2630 L/h From WWTP mass balance

xlix
26 Q2solids 105 kg/h From WWTP mass balance
47
48 D (1) =(B43*B23)+(B44*B24) μg/h.Pa D = Q*Zwater + Q*Zsorbed
49 D (2) =(B45*B23)+(B46*B24) μg/h.Pa D = Q*Zwater + Q*Zsorbed
50
51 Dissolved Flux PQ-X =B48*B30 µg/h D(3) * ƒ(PQ-X)
52 Sorbed Flux PQ-X =B49*B30 µg/h D(2) * ƒ(PQ-X)
53
54 Influent
55 Cwater =B51/(B43+(B44*B3)) µg/L
56 Csorbed =B55*B3 µg/kg
57
58 Dissolved Flux PQ-X =B55*B43 µg/h
59 Sorbed Flux PQ-X =B56*B44 µg/h
60 Total Flux =SUM(B58:B59) µg/h * vol flow rate for total influent conc.
61
62 Dissolved PQ-X =(B58/(B58+B59))*100 %
63 Sorbed PQ-X =(B59/(B59+B58))*100 %
64
65 Overall
66 Total Flux in =B58+B59 µg/h
67 Total Flux out =B21 µg/h
68
69 Removed =(B66-B67)/B66*100 %
70
71 Dissolved in Effluent =(B19/B67)*100 %
72 Sorbed in Effluent =(B20/B67)*100 %