10-32 BIOLOGICAL EXAMINATION (10000)

10300 PERIPHYTON*

10300 A. Introduction

1. Definition and Significance The use of periphyton in assessing water quality often is
hindered by the lack of suitable natural substrata† at the desired
Microorganisms growing on stones, sticks, aquatic macro- sampling station. Furthermore, it often is difficult to collect
phytes, and other submerged surfaces are useful in assessing the quantitative samples from these surfaces. To circumvent these
effects of pollutants on lakes, streams, and estuaries. Included in problems artificial substrata have been used to provide a uniform
this group of organisms, here designated periphyton,1,2 are the surface type, area, and orientation.3
zoogleal and filamentous bacteria, attached protozoa, rotifers,
and algae, and the free-living microorganisms that swim, creep, 2. References
or lodge among the attached forms.
Unlike the plankton, which often do not respond fully to the 1. ROLL, H. 1939. Zur Terminologie des Periphytons. Arch. Hydrobiol.
influence of pollution in rivers for a considerable distance down- 35:39.
stream, the periphyton show marked responses immediately be- 2. YOUNG, O.W. 1945. A limnological investigation of periphyton in
low pollution sources. Examples are the beds of Sphaerotilus and Douglas Lake, Michigan. Trans. Amer. Microsc. Soc. 64:1.
other “slime organisms” commonly observed in streams below 3. SLÁDEČKOVÁ, A. 1962. Limnological investigation methods for the
periphyton (“Aufwuchs”) community. Bot. Rev. 28:286.
discharges of organic wastes. Because the abundance and com-
position of the periphyton at a given location are governed by the
water quality at that point, observations of their condition gen-
erally are useful in evaluating conditions in bodies of water. † Although the terms “substrate” and “substratum” often have been used inter-
changeably, technically it is more correct to use “substratum” in connection with
periphyton. A substrate (plural substrates), in biochemical usage, is the substance
acted upon by an enzyme and the source of energy. In contrast, a substratum
* Approved by Standard Methods Committee, 2001. (plural substrata) as defined in biological usage is the base or material on which
Joint Task Group: Robert G. Wetzel (chair), Hamish Duthie, Gordon Goldsbor- a nonmotile organism lives or grows (i.e., the submerged surfaces used for
ough, Michael K. Hein, Stanford Loeb, Alena Sládec̆ková. periphyton colonization).

10300 B. Sample Collection

1. Station Selection concentrically, locate stations in areas adjacent to a waste outfall

and in unaffected areas. Use control stations similar to the
In rivers, locate stations a short distance upstream and at one affected ones (e.g., similar in water depth and distance from
or more points downstream from the suspected pollution source shore).
or intended study area in the areas of central mixing. In large
rivers, sample both sides of the stream in main flow areas.
Because the effects of a pollutant depend on the assimilative 2. Sample Collection
capacity of the stream and on the nature of the pollutant, pro-
gressive changes in water quality downstream from the pollution a. Natural substrata: Collect qualitative samples by scraping
source may be caused entirely by dilution and cooling—as in the submerged stones, sticks, pilings, and other available substrata.
case of nutrients, toxic industrial wastes, and thermal pollu- Many devices have been developed to collect quantitative sam-
tion— or by gradual mineralization of degradable organic com- ples from irregular surfaces. Appropriate techniques for the
pounds. Cursory examination of shoreline and bottom periphy- removal of periphyton from both living and nonliving surfaces
ton growths on natural substrata downstream from an outfall have been described.1– 4
may indicate conspicuous zones of biological response to water b. Artificial substrata: The most widely used artificial substra-
quality that will be useful in determining appropriate sites for tum is the standard, plain, 25- by 75-mm glass microscope slide,
sampling stations. When an intensive sampling program is not but other materials such as clear vinyl plastic also are suitable.
feasible, a minimum of three sampling stations, one in a refer- Do not change substratum type during a study because coloni-
ence area upstream from a pollution source and the others in the zation varies with substratum. In small, shallow streams and in
community downstream from the source, where complete mix- the littoral regions of lakes and reservoirs where light penetrates
ing with the receiving water has occurred, will provide minimal to the bottom, place slides or other substrata vertically in frames
data on the periphyton community. anchored to the bottom. In large, deep streams or standing-water
In lentic waters (e.g., lakes, reservoirs, ponds) and other stand- bodies where turbidity varies widely, place slides vertically with
ing-water bodies where zones of pollution may be arranged the slide face at right angles to the prevailing current. A floating
PERIPHYTON (10300)/Sample Collection 10-33

rack, as shown in Figure 10300:1,* is suitable. Expose several Section 10200B.2). Gluteraldehyde (2 to 5%) also is an excellent
slides (minimally five; three for biomass, one for species, and preservative, and in some ways is superior because it impacts
one backup for each time interval) for each type of analysis to cell membranes less severely.
assure collecting sufficient material and to determine variability Preserve slides intact in bottles of suitable size or scrape into
in results caused by normal differences in colonization of indi- containers in the field. Air-dry slides for dry and ash-free dry
vidual slides. In addition to effects of pollutants, length of weight in the field and store in a 3.0- ⫻ 7.7-cm glass bottle. Place
substratum exposure and seasonal changes in temperature and slides for chlorophyll analyses in acetone or methanol in the field
other natural environmental conditions may have a profound or collect and freeze with trichlorotrifluoroethane† (or alterna-
effect on sample composition. No community on an artificial tive) or CO2. Ethanol (95%) is an excellent solvent for chloro-
substratum is representative of the natural community. phyll extraction and yields greater extraction, is less toxic, and is
Place, expose, and handle all artificial substratum samplers in less expensive than acetone or methanol.5,6 The specific absorp-
conditions as nearly identical as possible, whether they are tion coefficient for chlorophyll a in 95% ethanol is 83.4
replicate samplers at a particular sampling location or samplers L/(g䡠cm). Alternatively, hold on dry ice until returned to the
at different locations. Sampler type and/or construction cause laboratory. If samples are frozen in the field, remove from the
changes in surrounding physical conditions that in turn affect substratum and concentrate (by filtration or other means) before
periphyton growth. Variations of 10 to 25% between sample freezing. Store all samples in the dark. For pollution and eu-
replicates are common. Therefore, to reduce sampling error and trophication studies using periphyton biota as indicators, do not
increase interpretive power, reduce the magnitude of all possible preserve samples. Enclose substrata with periphyton in contain-
test variables and use sufficient replication. ers filled with water, and transport and analyze them immedi-
c. Exposure period: Colonization on clean slides proceeds at ately. Also see Section 10300E.
an exponential rate for the first 1 or 2 weeks and then slows.
Because exposures of less than 2 weeks may result in very sparse 4. References
collections, and exposures of more than 2 weeks may result in
loss of material due to sloughing, sample for 2 weeks during the 1. SLÁDEČKOVÁ, A. 1962. Limnological investigation methods for the
summer. This exposure period precludes collecting sexually periphyton (“Aufwuchs”) community. Bot. Rev. 28:286.
mature thalli of larger, slow-growing filamentous algae such as 2. GOUGH, S.B. & W.J. WOELKERLING. 1976. On the removal and quan-
Cladophora and Stigeoclonium. To obtain optimum growth dur- tification of algal aufwuchs from macrophyte hosts. Hydrobiologia
ing the winter, use a longer exposure period. For the most 48:203.
exacting work, determine the optimum exposure period by test- 3. BOOTH, W.E. 1981. A method for removal of some epiphytic diatoms.
ing colonization rates over a period of about 6 weeks. Botanica Marina 24:603.
Secondary problems associated with macroinvertebrate infes- 4. DELBECQUE, E.J.P. 1985. Periphyton on nymphaeids: An evaluation of
tation and grazing may occur, often within 7 to 14 d. To reduce methods and separation techniques. Hydrobiologia 124:85.
5. SARTORY, D.P. & J.U. GROBBELAAR. 1984. Extraction of chlorophyll a
the confounding influence of grazing, increase substratum sam-
from freshwater phytoplankton for spectrophotometric analysis. Hy-
pling area and expose for 7 to 10 d. drobiologia 114:177.
6. JESPERSEN, A.-M. & K. CHRISTOFFERSEN. 1987. Measurements of chlo-
3. Sample Preservation rophyll a from phytoplankton using ethanol as extraction solvent.
Arch. Hydrobiol. 109:445.
Preserve samples that are taken for counting and identification
in 5% neutralized formalin, Lugol’s iodine, or merthiolate (see 5. Bibliography

* Wildlife Supply Co., 301 Cass St., Saginaw, MI 48602, or equivalent. COOKE, W.B. 1956. Colonization of artificial bare areas by microorgan-
isms. Bot. Rev. 22:613.
HOHN, M.H. 1966. Artificial substratum for benthic diatoms— collec-
tion, analysis, and interpretation. In K.W. Cummings, C.A. Tryon,
Jr. & R.T. Hartman, eds. Organism-Substratum Relationships in
Streams. Spec. Publ. No. 4, p. 87. Pymatuning Lab. Ecology, Univ.
Pittsburgh, Pittsburgh, Pa.
KEVERN, N.R., J.L. WILHM & G.M. VAN DYNE. 1966. Use of artificial
substrata to estimate the productivity of periphyton communities.
Limnol. Oceanogr. 11:499.
ARTHUR, J.W. & W.B. HORNING. 1969. The use of artificial substrata in
pollution surveys. Amer. Midland Natur. 82:83.
TIPPETT, R. 1970. Artificial surfaces as a method of studying populations
of benthic micro-algae in fresh water. Brit. Phycol. J. 5:187.
ERTL, M. 1971. A quantitative method of sampling periphyton from
rough substrata. Limnol. Oceanogr. 16:576.
ANDERSON, M.A. & S.L. PAULSON. 1972. A simple and inexpensive
woodfloat periphyton sampler. Progr. Fish-Cult. 34:225.
Figure 10300:1. Periphyton sampler. Floating sampler with upstream de-
flecting baffle and transparent, removable slide rack hold-
ing up to eight microscope slides. † Freon or equivalent.
10-34 BIOLOGICAL EXAMINATION (10000)

NORTH AMERICAN BENTHOLOGICAL SOCIETY. 1974 –1991. (Annual) Cur- STEVENSON, R.J. 1984. How currents on different sides of substrata in
rent and Select Bibliographies on Benthic Biology. Springfield, Ill. streams affect mechanisms of benthic algal accumulation. Int. Rev.
MARKER, A.F.H., C.A. CROWTHER & R.J.M. GUNN. 1980. Methanol and ges. Hydrobiol. 69:241.
acetone as solvents for estimating chlorophyll a and phaeopigments VYMAZAL, J. 1984. Short-term uptake of heavy metals by periphytic
by spectrophotometry. Arch. Hydrobiol. Ergebn. Limnol. 14:52. algae. Hydrobiologia 119:171.
NEROZZI, A. & P. SILVER. 1983. Periphytic community analysis in a small AUSTIN, A. & J. DENISEGER. 1985. Periphyton community changes along
oligotropic lake. Proc. Penn. Acad. Sci. 57:138. a heavy metals gradient in a long narrow lake. Environ. Exper. Bot.
WETZEL, R., ed. 1983. Periphyton of Freshwater Ecosystems. Develop- 25:41.
ments in Hydrobiology 17. Dr. W. Junk BV Publishers, The Hague, FLOWER, R.J. 1985. An improved epilithon sampler and its evaluation in
The Netherlands. two acid lakes. Brit. Phycol. J. 20:109.
LAMBERTI, G.A. & V.H. RESH. 1985. Comparability of introduced tiles
HAMILTON, P.B. & H.C. DUTHIE. 1984. Periphyton colonization of rock
and natural substrata for sampling lotic bacteria, algae, and macro-
surfaces in a boreal forest stream studied by scanning electron
invertebrates. Freshwater Biol. 15:21.
microscopy and track autoradiography. J. Phycol. 20:525.
PIEKARCZYK, R. & E. MCARDLE. 1985. Pioneer colonization and interac-
NIELSEN, T.S., W.H. FUNK, H.L. GIBBONS & R.M. DUFFNER. 1984. A
tion of photosynthetic and heterotrophic microorganisms on an
comparison of periphyton growth on artificial and natural substrata artificial substratum of polyurethane foam in E.J. Beck Lake, Illi-
in the Upper Spokane River, Washington, USA. Northwest Sci. nois, USA. Trans. Ill. State Acad. Sci. 78:81.
58:243. CATTANEO, A. & G. ROBERGE. 1991. Efficiency of a brush sampler to
PIP, E. & G.G.C. ROBINSON. 1984. A comparison of algal periphyton measure periphyton in streams and lakes. Can. J. Fish. Aquat. Sci.
composition on 11 species of submerged macrophytes. Hydrobiol. 48:1877.
Bull. 18:109. CATTANEO, A. & M.C. AMIREAULT. 1992. How artificial are artificial
POULIN, M., L. BERARD-THERRIAULT & A. CARDINAL. 1984. Benthic substrata for periphyton? J.N. Amer. Benthol. Soc. 11:244.
diatoms from hard substrata of marine and brackish waters of STEVENSON, R.J., M.L. BOTHWELL & R.L. LOWE, eds. 1996. Algal Ecol-
Quebec Canada 3. Fragilarioideae, Fragilariales, Fragilariaceae. ogy: Freshwater Benthic Ecosystems. Academic Press, San Diego,
Nat. Can. (Que). 111:349. Calif.

10300 C. Sample Analysis

1. Sedgwick-Rafter Counts N ⫻ At ⫻ Vt
Organisms/mm2 ⫽
Ac ⫻ Vs ⫻ As
Remove periphyton from slides with a razor blade and rubber
policeman. Disperse scrapings in 100 mL or other suitable vol- where:
ume of preservative with vigorous shaking, or use a blender. N ⫽ number of organisms counted,
Transfer a 1-mL portion to a Sedgwick-Rafter cell, and make a At ⫽ total area of chamber bottom, mm2,
strip count as described in Section 10200F.2a. If material in the Vt ⫽ total volume of original sample suspension, mL,
Sedgwick-Rafter cell is too dense to count directly, discard and Ac ⫽ area counted (strips or fields), mm2,
replace with a diluted sample. Vs ⫽ sample volume used in chamber, mL, and
Sedgwick-Rafter cells do not permit examination at magnifi- As ⫽ surface area of slide or substratum, mm2.
cations higher than 200⫻. The Palmer cell,1 a thinner version of
the S-R cell, permits examination at 400 to 500⫻ with a standard Separation of periphyton from silt and detritus may be en-
compound microscope. hanced by adding a drop or less of a saturated iodine solution to
Express counts as cells or filaments per square millimeter of the counting chamber just before counting. This method is es-
substratum area, calculated as in ¶ C.2. pecially useful when Chlorophyta are the predominant organ-
isms because iodine stains starch food reserves blue. Iodine can
be added even to preserved samples.
2. Inverted Microscope Method Counts
3. Diatom Species Counts
Using an inverted microscope for periphyton counts permits
magnifications higher than those possible with the Sedgwick- Preparation of permanent diatom mounts from periphyton
Rafter cell. If an inverted microscope is not available, use one of samples differs from preparation of mounts from plankton sam-
the available alternative methods with a standard compound ples because of the need to remove extracellular organic matter
microscope.2,3 Remove periphyton quantitatively from slides (such as gelatinous materials). If this organic matter is not
with a razor blade and policeman. Transfer a measured portion, removed it will produce a thick brown or black carbonaceous
after serial dilution if necessary, into a standardized plankton deposit on the cover glass when the sample is incinerated. Clear
sedimentation chamber. After a suitable period of settling (see organic matter by incineration by placing a small, known volume
Section 10200C.1), count organisms in the settling chamber by of sample (⬍ 1 mL) directly on a cover slip. Let water evaporate
counting all organisms within a known number of strips or and ash at 525°C (not more) for 6 to 10 min. Mount cover slip
random fields. Calculate algal density per unit area of substratum for direct examination of diatom frustules. Alternatively, decom-
as follows: pose organic substances by oxidation with ammonium persulfate
PERIPHYTON (10300)/Sample Analysis 10-35

or with HNO3 or 30% H2O2 and K2Cr2O7 (see Section ple into a thin ring of mounting medium* on a slide. Mix the
10200D.3) before mounting sample. To oxidize with persulfate xylene suspension and medium with a spatula until the xylene
place a measured sample of approximately 5 mL in a disposable has evaporated. Warm the slide on a hot plate at 45°C and cover
10-mL vial. Let stand 24 h, withdraw supernatant liquid by sample with a cover slip.
aspiration, replace with a 5% solution of (NH4)2S2O8, and mix Count diatoms on the prepared slides using the magnification
thoroughly. Do not exceed a total volume of 8 mL. Heat vial to most appropriate to the desired level of taxonomic identification.
approximately 90°C for 30 min. Let stand 24 h, withdraw su- Count strips or random fields. Calculate diatom density per unit
pernatant liquid, and replace with reagent-grade water. After area of substratum:
three changes of reagent-grade water, with a disposable pipet
transfer a drop of the diatom suspension to a cover glass, N ⫻ At ⫻ Vt
evaporate to dryness, and prepare and count a mount as de- Organisms/ area sampled2 ⫽
Ac ⫻ Vs ⫻ As
scribed for plankton (Section 10200). Count as least 500 frus-
tules and express results as relative numbers or percentage of
each species per unit area. Counts of more than 500 frustules where the terms are as defined in 10300C.2.
may be needed, depending on the questions being addressed.4
5. Biovolume

4. Stained Sample Preparation and Counting Cell volume (biovolume) provides a much more accurate
evaluation of cellular biomass because of the great differences in
Staining periphyton samples permits distinguishing algae from cell dimensions among species and sometimes seasonally within
detritus and “live” from “dead” diatoms. This distinction is the same species under different growth conditions. Cell vol-
especially important because periphyton often contains many umes, based on cell dimensions, are calculated for each species
dead diatoms of planktonic as well as periphytic origin. from formulas for solid geometric shapes that most closely
In the first method, cells are exposed to a vital stain to evaluate match the cell shape. A comprehensive set of geometric shapes
the percentages of live, senescent, and dead algae, particularly and mathematical equations for calculating biovolume of more
diatoms, by estimating relative metabolic activities. The color- than 850 pelagic and benthic freshwater and marine microalgal
less tetrazolium violet is reduced in the cytochrome system of genera has been compiled.6
metabolically active cells to form violet-colored triphenyl-
formazan. When cells are senescent or dead, the reaction fails. 6. Dry and Ash-Free Weight
Make tetrazolium violet solution by adding 2.0 g tetrazolium
violet to 1.0 L water. The solution may be buffered to a pH of 7.5 Collect at least three replicate slides for weight determina-
to 7.7 with tris-hydroxymethyl amine. Add 1 mL tetrazolium tions.7 Slides air-dried in the field can be stored indefinitely if
violet solution to 9 mL sample and incubate 2 to 4 h at room protected from abrasion, moisture, and dust. Use slides expressly
temperature. Count diatom frustules and other cells (at least designated for dry and ash-free weight analysis.
a. Equipment:
300/sample) and place into the following categories: a) active:
1) Analytical balance, with a sensitivity of 0.1 mg.
violet precipitate observed within the cell or mitochondria; b)
2) Drying oven, double-wall, thermostatically controlled to
senescent: chlorophyll present, but no violet precipitate; c) dead:
within ⫾1°C.
no chlorophyll or violet precipitate present.
3) Electric muffle furnace with automatic temperature control.
In the second method, all algal components of periphyton may
4) Crucibles, porcelain, 30-mL capacity.
be studied in one preparation, without sacrificing detailed diatom
5) Single-edge razor blades or rubber policeman.
taxonomy.5 This method yields permanent slides for reference
b. Procedure:
collections.
1) Dry slides to constant weight at 105°C, and ignite for 1 h
Thoroughly mix preserved samples in the preservative solu- at 500°C. If weights are to be obtained from field-dried material,
tion. Prepare acid fuchsin stain by dissolving 1 g acid fuchsin in re-wet dried material with reagent-grade water and remove from
100 mL reagent-grade water, adding 2 mL glacial acetic acid, slides with a razor blade or rubber policeman. Place scrapings
and filtering. Place a measured sample in a centrifuge tube with from each slide in a separate prewashed, prefired, tared crucible;
10 to 15 mL acid fuchsin stain. Mix sample and stain several dry to constant weight at 105°C; cool in a desiccator and weigh;
times during a 20-min staining period; centrifuge at 1000 g for and ignite for 1 h at 500°C.
20 min. 2) Re-wet ash with reagent-grade water and dry to constant
Decant stain, being careful not to disturb sediment, or siphon weight at 105°C. This reintroduces water of hydration of clay
off supernatant. Add 10 to 15 mL 90% propanol, mix, centrifuge and other minerals, which is not driven off at 105°C but is lost
for 20 min, and decant supernatant. Repeat using two washes of during ashing. If not corrected for, this water loss will be
100% propanol and one wash of xylene. Centrifuge, decant recorded as volatile organic matter.8
xylene, and add fresh xylene. At this stage, store sample in c. Calculations: Calculate mean weight from slides and report
well-sealed vials or prepare slides. as dry weight [(crucible ⫹ sample weight at 105°C) minus (tare
Slides for periphyton examinations require random dispersion
of a known amount of xylene suspension. Use a microstirrer to
break up clumps of algae before removing sample portion from * Naphrax威, Northern Biological Supply, 3 Betts Avenue, Martlesham Heath,
xylene suspension. Count a number of drops of suspended sam- Ipswich IP5 7RH, U.K., or equivalent.
10-36 BIOLOGICAL EXAMINATION (10000)

weight of crucible)] per square meter of exposed surface. If 25- 8. References

by 75-mm slides are used, then
1. WETZEL, R.G. & G.E. LIKENS. 2000. Limnological Analyses, 3rd ed.
g/slide (average) Springer-Verlag, New York, N.Y.
g/m ⫽
2
0.003 75 2. CRUMPTON, W.G. 1987. A simple and reliable method for making
permanent mounts of phytoplankton for light and fluorescence mi-
Calculate ash weight for sample [(crucible ⫹ sample weight croscopy. Limnol. Oceanogr. 32:1154.
at 500°C) minus (tare weight of crucible)]. Subtract ash 3. STEVENSON, R.J. 1984. Procedures for mounting algae in a syrup
weight from dry weight to obtain ash-free weight, and report medium. Trans. Amer. Microsc. Soc. 103:320.
as ash-free weight per square meter of exposed surface. 4. STEVENSON, R.J. & R.L. LOWE. 1986. Sampling and interpretation of
algal patterns for water quality assessments. In B.G. Isom, ed. Ra-
7. Chlorophyll and Pheophytin tionale for Sampling and Interpretation of Ecological Data in the
Assessment of Freshwater Ecosystems, p. 118. STP 894, American
The chlorophyll content of attached algal communities is a Soc. Testing & Materials, Philadelphia, Pa.
useful index of the phytoperiphyton biomass. Quantitative chlo- 5. OWEN, B.B., JR. 1977. The effect of increased temperatures on algal
rophyll determinations require the collection of periphyton from communities of artificial stream channels. Ph.D. dissertation, Univ.
Alberta, Edmonton.
a known surface area. Extract the pigments with aqueous acetone
6. HILLEBRAND, H., C.-D. DÜRSELEN, D. KIRSCHTEL, U. POLLINGHER & T.
or methanol (see Section 10200H) and use a spectrophotometer ZOHARY. 1999. Biovolume calculations for pelagic and benthic mi-
or fluorometer for analysis. If immediate pigment extraction is croalgae. J. Phycol. 35:403.
not possible, samples may be stored frozen for as long as 28 d if 7. NEWCOMBE, C.L. 1950. A quantitative study of attachment materials
kept in the dark.9 The ease with which chlorophylls are removed in Sodon Lake, Michigan. Ecology 31:204.
from cells varies considerably with different algae; to achieve 8. NELSON, D.J. & D.C. SCOTT. 1962. Role of detritus in the productivity
complete pigment extraction disrupt the cells mechanically with of a rock outcrop community in a piedmont stream. Limnol. Ocean-
a grinder, blender, or sonic disintegrator, or freeze them. Grind- ogr. 7:396.
ing is the most rigorous and effective of these methods. 9. GRZENDA, A.R. & M.L. BREHMER. 1960. A quantitative method for the
The Autotrophic Index (AI) is a means of determining the collection and measurement of stream periphyton. Limnol. Oceanogr.
5:190.
trophic nature of the periphyton community (see Section
10200H). It is calculated as follows:
9. Bibliography
Biomass (ash-free weight of organic matter), mg/m2
AI ⫽ EATON, J.W. & B. MOSS. 1966. The estimation of numbers and pigment
Chlorophyll a, mg/m2
content in epipelic algal populations. Limnol. Oceanogr. 11:584.
MOSS, B. 1968. The chlorophyll a content of some benthic algal com-
Normal AI values range from 50 to 200; larger values indicate
munities. Arch. Hydrobiol. 65:51.
heterotrophic associations or poor water quality. Nonviable or- CRIPPEN, R.R. & J.L. PERRIER. 1974. The use of neutral red and Evans
ganic material affects this index. Depending on the community, blue for live-dead determinations of marine plankton. Stain Tech-
its location and growth habit, and method of sample collection, nol. 49:97.
there may be large amounts of nonliving organic material that OWEN, B.B., M. AFZAL & W.R. CODY. 1978. Staining preparations for
may inflate the numerator and produce disproportionately high phytoplankton and periphyton. Brit. Phycol. J. 13:155.
AI values. Nonetheless, the AI is an approximate means of OWEN, B.B., M. AFZAL & W.R. CODY. 1979. Distinguishing between live
describing changes in periphyton communities between sam- and dead diatoms in periphyton communities. In R.L. Weitzel, ed.
pling locations. Methods and Measurements of Periphyton Communities: A Re-
a. Equipment and reagents: See Section 10200H. view. STP 690, American Soc. Testing & Materials, Philadelphia,
Pa.
b. Procedure: In the field, place individual glass microscope
WETZEL, R.G., ed. 1983. Periphyton of Freshwater Ecosystems. Devel-
slides used as substrata directly into 100 mL of a mixture of 90% opments in Hydrobiology 17. Dr. W. Junk BV Publishers, The
acetone (water with 10% saturated MgCO3 solution). Immedi- Hague, The Netherlands.
ately store on dry ice in the dark. (NOTE: Vinyl plastic is soluble DELBECQUE, E.J.P. 1985. Periphyton on Nymphaeids: An evaluation of
in acetone. If vinyl plastic is used as the substratum, scrape methods and separation techniques. Hydrobiologia 124:85.
periphyton from it before solvent extraction.) If extraction can- TREES, C.C., M.C. KENNICUTT & J. M. BROOKS. 1985. Errors associated
not be carried out immediately, freeze samples in the field and with the standard fluorometric determination of chlorophylls and
keep frozen until processed. phaeopigments. Mar. Chem. 17:1.
Rupture cells by grinding in a tissue homogenizer and steep in
acetone for 24 h in the dark at or near 4°C.
To determine pigment concentration, follow the procedures
given in Section 10200H.
c. Calculation: After determining pigment concentration in the
extract, calculate amount of pigment per unit surface area of
sample as follows:
C a⫻volume of extract, L
mg chlorophyll a/m2 ⫽
area of substrate, m2
where Ca is as defined in Section 10200H.
PERIPHYTON (10300)/Primary Productivity 10-37

10300 D. Primary Productivity

The productivity of periphyton communities is a function of confining this community briefly in bottles, bell jars, or other
water quality, substrata, and seasonal patterns in temperature and chambers. In contrast, the metabolism of organisms in flowing
solar illumination. Measurements of biomass accrual rates can be water is highly dependent on current velocity and cannot be
useful indicators of pollution and eutrophication, but periphyton determined with precision under static conditions. Productivity
biomass accrual is not a measure of productivity. Productivity estimates for flowing waters and those for standing waters
may be estimated from the rate of oxygen evolution or carbon present different problems; therefore, separate procedures are
uptake by the community.1 given.
Productivity and respiration of epilithic and epipelic periphy-
1. Biomass Accumulation ton in littoral regions of lakes and ponds can be determined by
inserting transparent and opaque bell jars or open-ended plastic
a. Ash-free dry weight: The accumulation rate of organic chambers into the substratum along transects perpendicular to
matter on artificial substrata by attachment, growth, and repro- the shoreline.5,6 Chambers are left in place for one-half the daily
duction of colonizing organisms has been used widely to esti- photoperiod. The DO concentration in a chamber is determined
mate the productivity of streams and reservoirs.2,3 To use this at the beginning and end of the exposure period. Gross produc-
method, expose several replicate clean substrata for a predeter- tivity is the sum of the net gain in DO in the transparent chamber
mined period, scrape the accumulated material from the slides, and the oxygen used in respiration. Values obtained are doubled
and ash as described previously. to estimate productivity for the entire photoperiod. Alternatively,
determine the proportion of the incubation period of the total
mg ash-free weight/slide
P ⫽ insolation during the photoperiod more accurately by measuring
tA the insolation of the incubation period as a percentage of the total
daily insolation. Both these methods assume that photosynthesis
where:
is proportional to irradiance (i.e., not light saturated and no
P ⫽ net productivity, mg ash-free weight/m2/d,
t ⫽ exposure time, d, and photoinhibition).
A ⫽ area of a slide, m2. Failure to account for changes in DO in chambers caused by
phytoplankton photosynthesis and respiration may cause serious
Obtain estimates of seasonal changes in biomass of estab- errors in the estimates of periphyton metabolism. It is essential
lished communities by placing many replicate substrata at a that these values be obtained at the time the periphyton is studied
sampling point and then retrieving a few at a time at regular by using the light- and dark-bottle method (see Section 10200J).
intervals. Replace removed slides with new clean slides. The a. Equipment and reagents:
recommended collection interval ranges from 2 to 4 weeks for a 1) Clear and darkened glass or plastic* chambers, approxi-
year or longer.2 Gain in ash-free weight per unit area from one mately 20 cm in diameter and 30 cm high, with a median lateral
collection period to the next is a measure of net production. port, sealed with a serum bottle stopper for removal of small
b. ATP estimates: Measurement of adenosine triphosphate water samples for DO analyses or for the insertion of an oxygen
(ATP) has been used in recent years to estimate microbial probe. Fit the chamber with a small, manually operated, propel-
biomass in water. This technique is applicable to periphyton.4 It ler-shaped stirring paddle.
provides an additional tool for assessing the magnitude and rate 2) Dissolved oxygen probe, or equipment and reagents re-
of biomass accumulation on substrata in natural waters. At quired for Winkler dissolved oxygen determinations: See Section
present, the procedure should be limited to communities colo- 4500-O.
nizing artificial substrata. b. Procedure: At each station place both a transparent and an
1) Equipment and reagents—See Section 10200I.6a. opaque chamber over the substratum at sunrise or mid-daylight
2) Procedure—Either scrape periphyton from an exposed ar- and leave in place for one-half the daily photoperiod. In ex-
tificial substratum or, if standard glass microscope slides are tremely productive environments or to define the hourly primary
used, place them in polyethylene slide mailers containing pre- productivity changes throughout the day, use incubation periods
heated (99°C) Tris buffer. Immerse in a boiling water bath for 10 shorter than one-half the photoperiod. The minimum incubation
min to extract ATP. If samples are not assayed immediately, period giving reliable results is 2 h. Determine DO concentration
freeze at ⫺25°C; they may be stored in a freezer for up to several at the beginning of the incubation period.
months. Complete analysis as directed in Section 10200I.6b. Include a set of Gaarder-Gran light- and dark-bottle produc-
Slides exposed in waters containing high turbidity may collect tivity and respiration measurements with each set of chambers to
substantial amounts of particulates including clays. ATP sorbs to obtain a correction for phytoplankton metabolism. Incubate for
these materials; the sorption results in a quenching effect. the same time period as the chambers. See Section 10200J.
3) Calculations—See Section 10200I.6c. At end of exposure period, carefully mix the water in the
chambers and determine DO concentration.
2. Standing Water Productivity Measured by Oxygen
Method
* Plexiglas or equivalent. Normal glass is opaque to UV-A and UV-B radiation
Hourly and daily rates of oxygen evolution and carbon uptake and could have differential effects on photosynthesis of periphyton, whereas
by periphyton growing in standing water can be studied by plastic of this type is UV transparent.7
10-38 BIOLOGICAL EXAMINATION (10000)

c. Calculations: When the exposure period is one-half of the t p关V c共C⬘fc ⫺ C⬘ic兲 ⫹ V o共C⬘io ⫺ C⬘fo兲兴
photoperiod, calculate gross primary productivity of the periphy- PG ⫽
tA
ton community as:
where:
2关V c共C⬘fc ⫺ C⬘ic兲 ⫹ V o共C⬘io ⫺ C⬘fo兲兴 tp ⫽ length of the daily photoperiod, h.
PG ⫽
A Community respiration and net production calculations for
incubation periods other than one-half the photoperiod are not
where: changed.
PG ⫽ gross production, mg O2/m2/d12h,
Vc ⫽ volume of clear chamber, L, 3. Standing Water Productivity Measured by Carbon-14
C⬘fc and C⬘ic ⫽ final and initial concentrations, respectively, of Method
DO in the clear chamber, mg/L, corrected for
phytoplankton metabolism, The approach is similar to that described above for the oxygen
Vo ⫽ volume of opaque chamber, L, method. Transparent and opaque chambers are placed over the
C⬘io and C⬘fo ⫽ initial and final concentrations, respectively, of substratum, carbon-14-labeled Na2CO3 is injected into the cham-
DO in the opaque chamber, mg/L, corrected for ber by syringe, mixed well, and allowed to incubate with the
phytoplankton metabolism, and periphyton for one-half the photoperiod. The concentration of
A ⫽ substratum area, m2. dissolved inorganic carbon available for photosynthesis is deter-
Correct for the effects of phytoplankton metabolism in the mined by titration. At the end of the incubation period, the
overall oxygen change in the clear chamber by the following periphyton is removed from the substratum and assayed for
equations: carbon-14.5
a. Equipment and reagents:
1) Incubation chamber: See ¶ 2a above.
C⬘fc ⫽ C fc ⫺ C flb 2) Special equipment and reagents: See Section 10200J.
3) Carbon-14-labeled solution of sodium carbonate, having a
C⬘ic ⫽ C ic ⫺ C ilb known specific activity of approximately 10 ␮Ci/mL.
4) Other equipment and reagents: See Section 4500-CO2.
C⬘fo ⫽ C fo ⫺ C fdb b. Procedure: At each station place a transparent and opaque
chamber over the substratum and add approximately 10 ␮Ci
C⬘io ⫽ C io ⫺ C idb carbon-14/L of chamber volume. Mix water in the chambers
well, taking care to avoid disturbing the periphyton. Determine
where: concentration of dissolved inorganic carbon as described in
Cfc ⫽ final DO concentration in clear chamber, mg/L, Section 2320. At end of exposure period, remove surface centi-
Cflb ⫽ final DO concentration in light bottle, mg/L, meter of periphyton and sediment enclosed in the chamber,
Cic ⫽ initial DO concentration in clear chamber, mg/L, freeze, and store frozen in a vacuum desiccator.
Cilb ⫽ initial DO concentration in light bottle, mg/L, Immediately before analysis, expose sample to fumes of HCl
Cfo ⫽ final DO concentration in opaque chamber, mg/L, for 10 to 15 min to drive off all inorganic carbon-14 retained in
Cfdb ⫽ final DO concentration in dark bottle, mg/L, the periphyton. Combust sample (or portion) by the Van Slyke
Cio ⫽ initial DO concentration in opaque chamber, mg/L, and method6 or oxidize by heating in a closed system. Collect all
Cidb ⫽ initial DO concentration in dark bottle, mg/L. CO2 for radioassay either by flushing CO2 into a two-vial train of
ethanolamine (2-aminoethanol) or alternative CO2 absorber,
Calculate periphyton community respiration by: such as methoxyethanol (1:7)8 or flushing CO2 produced by
combustion into a gas-flow counter or electrometer. Alterna-
24V o共C⬘io ⫺ C⬘fo兲 tively, extract known amounts of periphyton biomass with a
R ⫽ tissue solubilizer,† using, for example, 1.0 mL in closed vials at
tA
60°C for 48 h.9 Radioassay subsamples (100-␮L) by liquid
scintillation.
where: c. Calculations:
R ⫽ community respiration, mg O2/m2/d24h, and
t ⫽ length of exposure, h. 14
C assimilated ⫻ conversion factors
P N ⫽ 12 C available ⫻
Determine the net periphyton community productivity (PN) as 14
C available (added)
the difference:
共a兲共b兲共d兲共e兲
PN ⫽
共c兲
PN ⫽ PG ⫺ R
where:
If the incubation time is different from one-half the photope-
riod, modify the daily gross production calculation as follows: † Beckman BTS-450 or equivalent.
PERIPHYTON (10300)/Primary Productivity 10-39

PN ⫽ net primary productivity per unit area of substratum per

unit time, mg C/m2/d,
a⫽ C available ⫽ dissolved inorganic carbon, mg 12C/L ⫽
12

(total alkalinity ⫺ phenolphthalein alkalinity) ⫻ 0.2406

⫽ mg 12C/L,
b ⫽ 14C assimilated ⫽ [(radioactivity of sample in light
chamber ⫻ k1) ⫺ (background activity of dark chamber
⫻ k2)] ⫻ (isotope effect, 1.06). Express radioactivity as
disintegrations per second (dps), i.e., counts per second
corrected to 100% radioassay counter efficiency.
k1 ⫽ correction factor to convert individually different
light-chamber volumes to 1 L,
k2 ⫽ correction factor to convert individually different
dark-chamber volumes to 1 L,
1.06 ⫽ isotope effect to correct for slightly greater mass of
14
C than of 12C, which results in a 6% slower
assimilation rate,
c ⫽ 14C available ⫽ 14C activity added ⫽ (␮Ci 14C added) ⫻
(disintegrations of 14C/s/␮Ci) ⫽ 3.7 ⫻ 104 ␮Ci 14C added,
mL,
d ⫽ a dimensional factor to convert area of substratum sampled
to m2, and
e ⫽ factor to expand incubation period to the total daylight
period. After integration by planimetry or electronic digi-
tizer of the total amount of insolation for the day, determine
percentage of total represented by the incubation period.

4. Flowing Water Productivity Measured by Oxygen Method

Primary productivity of the periphyton community in a stream

or river ecosystem can be related to changes in DO. These
changes are the integrated effects of photosynthesis, affected by
light levels and turbidity, that occur during the photoperiod by
stream phytoplankton, periphyton, and the submerged portions
of macrophytes. Respiration results from metabolism of plant
communities, aquatic animals, and attached and free-floating
microbial heterotrophs. Water depth, turbulence, and water tem-
perature all influence the process of reaeration. Oxygen also can
enter by accrual of groundwater and surface waters. Daily fluc-
tuations in photosynthetic production of oxygen are imposed on
the relatively steady demand of respiratory activity. However,
this latter process may fluctuate greatly in streams receiving a
significant load of organic wastes, particularly under intermittent
loads such as oxygen demand from urban stormwater runoff. Figure 10300:2. Component processes in the oxygen metabolism of a
Respiration rates also may vary diurnally under certain condi- section of a hypothetical stream during the course of a
tions, but the factors involved are not well understood. cloudless day. Production, respiration, and diffusion are
The rate of change in stream DO (q) in grams per cubic meter given on an areal basis. The combined effect of these rate
per hour is represented by the following function of the photo- processes for a stream 1 m deep is given in mg/L/h (q). The
synthetic rate (p), respiration (r), reaeration (d), and accrual from actual oxygen values that would result in a stream with a
groundwater inflow and surface runoff (a):10 long homogeneous community are given in the lowermost
curve. Source: ODUM, H.T. 1956. Primary production in
flowing waters. Limnol. Oceanogr. 1:102.
q ⫽ p ⫺ r ⫹ d ⫹ a

If the equation is multiplied through by depth in meters (z), the separated into components due to respiration and primary pro-
resulting values are in terms of grams oxygen per square meter duction. The metabolic rates are the sum of the activity of the
per hour. Figure 10300:2 illustrates this conceptual relationship entire stream community. Planktonic productivity and respira-
between q, primary productivity, and respiration of the stream tion can be separated from overall community activity by the use
plant community. of the light- and dark-bottle oxygen technique (see Section
The procedure measures the time-variable oxygen concentra- 10200J). However, in most small streams planktonic production
tions in a stream over a 24-h period. Compensations are made for is insignificant. The component of production and respiration
oxygen changes due to physical factors (accrual and reaeration) due to macrophytes is very difficult to separate from periphytic
and the rate of oxygen change due to biological activity that is metabolic activity in systems where vascular plants are common.
10-40 BIOLOGICAL EXAMINATION (10000)

Because periphyton attach to plant surfaces as well as nonliv- 10200J.2. Incubate light and dark bottles for the same time
ing substrata, radiotracer techniques are required to separate the interval as the chambers.
component of production due to macrophytes from that due to Make several measurements during the photoperiod to define
attached algae.11 When vascular plants are present use tech- daily primary productivity. In addition, collect sufficient natural
niques discussed in Section 10400 to estimate their contribution substratum samples of the study reach to estimate periphyton
to net primary productivity. biomass (see Section 10300C). At end of incubation period
Respiration by fish and benthic fauna also is difficult to harvest enclosed periphyton and determine ash-free biomass (see
quantitate directly and usually is not separated from periphyton Section 10300C.6).
respiration. If compartmentalized animal metabolism is required, 2) Free-water diurnal curve methods—Measure, hourly or
calculate this contribution from laboratory respiration rates ex- continuously, DO concentration and water temperature for a
trapolated to the field situation based on animal population 24-h period at one or two stations, depending on stream condi-
sizes.12,13 tions, precision desired, and availability of equipment. If similar
Estimate primary productivity in flowing water by either the conditions exist for some distance upstream from the reach being
free water demand method or the chamber method.14,15 The first studied, diurnal measurements of DO at a single station are
does not introduce artificiality to the system; however, it is sufficient to estimate productivity. Where upstream conditions
difficult to separate the components of metabolic activity except are significantly different from those in the reach being studied,
for the contribution due to plankton. The chamber method mea- make measurements at the upstream and downstream limits of
sures periphyton activity alone.16 –20 the reach.
Depending on the hydrologic characteristics of the stream If the single-station method is used, measure depth at several
system, accrual and reaeration may be significant. Accrual can points along the study reach to define average depth. Map and/or
be accounted for by simple mixing equations if estimates of the make physical surveys to estimate magnitude of possible sources
accrued flow and its oxygen concentration are known. In prac- of accrual via effluents or tributary streams and springs. If the
tice, select for study reaches that do not incur significant accrual. two-station method is used, measure the wetted cross-sectional
Measure reaeration rates either directly16 –19 or by estimation stream area as well as current velocity at several points to define
from physical and hydrodynamic features of the stream it- flow (in cubic meters per second) and average cross-sectional
area. Correct for phytoplankton activity by light- and dark-bottle
self.18,19
measurements (see Section 10200J.2).
a. Equipment:
3) Direct measurement of reaeration17—Under special cir-
1) BOD bottles, for light- and dark-bottle measurements. See
cumstances it may be desirable to estimate reaeration directly
Section 10200J.
although the results may not be more accurate than those of the
2) DO meter and probe for measurement of DO.
empirical formulations usually used. The tracer gas technique is
3) Bottom chamber, 60 ⫻ 20 ⫻ 10 cm, with 32-cm length-
satisfactory, but is difficult and requires sophisticated equipment
wise dividing baffle, rheostat-controlled submersible pump, tem-
not routinely available. Use this method with care and with full
perature thermistor, and DO probe.15 Use clear and opaque
recognition of its restrictions. Depending on stream flow, release
plastic sleeves for covering chamber and petri dishes or other 10 to 250 ␮Ci 85Kr with 5 to 125 ␮Ci 3H at the upstream end of
means of placing periphyton within chambers. the reach together with sufficient fluorescent dye to produce a
4) Current meter, capable of detecting water current veloci- concentration of 10 ␮g/L when completely mixed across the
ties ranging from 0.03 to 3 m/s in water depths as shallow as river cross section. Make fluorometric measurements at the
0.3 m. downstream end of the reach until the dye peak appears, then
5) Tape measure (30 m) and depth staff, or similar equipment, collect water samples to measure the 85Kr/3H ratio by liquid
as required to measure stream cross sections. scintillation techniques. Record time of travel for the dye peak
6) Fluorometer, capable of detecting fluorescent dye concen- from the injection point.
tration at 0.5 to 100 ␮g/L (required only if direct measurement c. Calculations:
of reaeration is made). 1) Chamber method—Calculation is analogous to that used
7) Liquid scintillation counter, capable of sensitive detection for the bell jar technique discussed in Section 10300D.2.
of 85Kr and 3H (required only if direct measurement of reaeration
is made).
V c共C⬘fc ⫺ C⬘ic兲B
b. Procedure: Pn ⫽
1) Light- and dark-chamber method—Grow samples of typi- tW c
cal periphyton communities on artificial substratum or collect
natural material. Transfer identical portions to both clear and where:
opaque chambers, taking care to use sufficient periphyton to Pn ⫽ hourly rate of net primary production, mg O2/m2/h,
make the ratio of chamber volume to periphyton area equivalent Vc ⫽ volume of clear chamber, L,
to the ratio of stream volume to periphyton substratum area. B ⫽ average periphyton biomass estimated for the study
reach, mg/m2,
Measure current in the stream and match the circulation rate in
t ⫽ incubation period, h,
the clear and opaque chambers to the current. Measure DO Wc ⫽ total biomass of periphyton contained in clear chamber,
concentrations initially in both clear and opaque chambers and mg,
after 1 to 3 h to estimate the rate of oxygen increase or decrease. C⬘fc ⫽ final oxygen concentration in clear chamber, corrected
Make concurrent measurements of phytoplankton activity using for phytoplankton metabolism, mg/L:
light- and dark-bottle techniques as described in Section C⬘fc ⫽ Cfc ⫺ Cflb
PERIPHYTON (10300)/Primary Productivity 10-41

Cfc ⫽ final DO in clear chamber,

Cflb ⫽ final DO in light bottle, and
C⬘ic ⫽ initial oxygen concentration in clear chamber corrected
for light-bottle measurement, mg/L:
C⬘ic ⫽ Cic ⫺ Cilb
Cic ⫽ initial DO in clear chamber, and
Cilb ⫽ initial DO in light bottle.

V o(C⬘io ⫺ C⬘fo)B
r⫽
tW o

where:
r ⫽ hourly periphyton respiration rate, mg O2/m2/h,
Vo ⫽ volume of opaque chamber, L,
B ⫽ average periphyton biomass for the study reach, mg/m2,
Wo ⫽ total biomass of periphyton contained in opaque cham-
ber, mg,
C⬘io ⫽ initial oxygen concentration in opaque chamber, cor- Figure 10300:3. Gross periphytic primary production (PG) determined
rected for phytoplankton respiration, mg/L: by the O’Connell-Thomas Chamber. PG is the area under
C⬘io ⫽ Cio ⫺ Cidb the curve obtained by graphical integration planimetry.
Cio ⫽ initial DO in opaque chamber, mg/L, Each point is the run Pg ⫽ Pn ⫹ r for incubation periods 1,
Cidb ⫽ initial DO in dark bottle, mg/L, and 2, and 3, which are denoted by the indicated lines.
C⬘fo ⫽ final oxygen concentration in opaque chamber, mg/L:
C⬘fo ⫽ Cfo ⫺ Cfdb
Cfo ⫽ final DO in opaque chamber, mg/L, and ⫺ 1 (C Kr/C H)d
K Kr ⫽ ln
Cfdb ⫽ final DO in dark bottle, mg/L. t (C Kr/C H)u

For each pair of chamber measurements, and

Pg ⫽ Pn ⫹ r K Kr
k2 ⫽
0.83
where:
where:
Pg ⫽ hourly gross periphytic primary production, mg O2/m2/h.
k2 ⫽ reaeration coefficient (base e), d⫺1,
PG is the area under the curve of primary production per hour KKr ⫽ base e transfer coefficient for 85Kr, d⫺1,
through the photoperiod, mg O2/m2/d (see Figure 10300:3). T ⫽ time of travel, d,
Also, (CKr/CH)u ⫽ ratio of released radioactivities (␮Ci/mL) 85Kr to
3
H at the upstream station, and
(CKr/CH)d ⫽ ratio of radioactivities (␮Ci/mL) 85Kr to 3H at the

冢 冣
冘
n downstream station.
rn
I The reaeration coefficient also can be calculated from an
R⫽ ⫻ 24
n equation relating the rate of energy dissipation in a stream to
k2.17,18
where:
⌬h
k2 ⫽ K
R ⫽ total periphyton community respiration, mg O2/m2/d, and t
n ⫽ number of observations.
where:
Thus,
K ⫽ escape coefficient,
PN ⫽ PG ⫺ R ⌬h ⫽ change in water surface elevation in a stream reach, and
t ⫽ time of flow through a stream reach.
where: This can be expressed in terms of hydrodynamic and physical
data:
PN ⫽ net periphytic production, mg O2/m /d. 2

2) Free water methods ⌬H

k 220 ⫽ K⬘ ⫻V
a) Calculation of reaeration or diffusion for both the single ⌬X
and upstream-downstream methods—Calculate k2 from radio-
tracer data as follows: where:
10-42 BIOLOGICAL EXAMINATION (10000)

K⬘ ⫽ 28.3 ⫻ 103 s/m 䡠 d for stream flows between 0.028 and

0.28 m3/s; 21.3 ⫻ 103 s/m 䡠 d for stream flows between
0.28 and 0.56 m3/s; and 15.3 ⫻ 103 s/m 䡠 d for stream
flows above 0.56 m3/s,
k220 ⫽ reaeration coefficient, d⫺1, at 20°C,
⌬H
⫽ slope, m/km, and
⌬V
V ⫽ velocity, m/s.

Convert k220 to the temperature of the stream by the following

equation:

k 2t ⫽ k 220 共1.024兲 共T⫺20兲

where:

k2t ⫽ k2 at ambient water temperature, d⫺1, and

T ⫽ ambient water temperature, °C.

Convert to D in mg/L/h:

k 2t共C s ⫺ C兲
D⫽
24

where:

Cs ⫽ oxygen concentration at saturation at ambient stream tem-

peratures, mg/L, and
C ⫽ measured oxygen concentration, mg/L.

b) Single-station method—Calculation of primary productivity and

respiration from diurnal oxygen and temperature measurements at a
single station is summarized in Figure 10300:4 and Table 10300:I.
Tabulate hourly DO measurements and temperatures. Determine
Cs (DO of air-saturated H2O at each temperature from Table 4500-
Figure 10300:4. Calculation of gross primary production at a single
O:I) and compute uncorrected DO consumption, milligrams per liter
station. Pg, g O2/m2/h ⫽ area of corrected rate of change
per hour, for each period: curve integrated for the length of the photoperiod multi-
plied by average water depth, z, for the reach in meters.
⌬DOhours 1 to 2⫽DOhour 2⫺DOhour 1

Plot on the half hour, as shown in Figure 10300:4b. for a reach, z meters, to obtain PG in grams per square meter
Calculate the net primary production and respiration of phy- per day. Calculate community respiration:
toplankton as shown in Section 10200J. Determine the 24-h
R ⫽ 24 z F
average hourly plankton respiration,共 冘1 r p兲/n in milligrams per
n

liter per hour every half hour. Calculate the hourly net phyto- where:
plankton production and tabulate for the approximate hours
during the photoperiod. Plot as shown on Figure 10300:4c. R ⫽ community respiration, g/m2/d,
Calculate and tabulate k2t and substitute D for each Cs, as outlined z ⫽ depth, m, and
in ¶ a), above. Plot as shown in Figure 10300:4c. F ⫽ average hourly ⌬DO for the dark period (without regard to
Correct each ⌬DO for diffusion and phytoplankton metabolism: sign), mg/L/h.

Calculate net primary productivity PN as:

⌬DOcorrected, mg/L/h⫽⌬DOuncorrected ⫺ D ⫺ P p ⫺ R p
PN ⫽ PG ⫺ R
Plot each point as shown in Figure 10300:4d.
Gross primary productivity of the benthic and attached algal c) Upstream-downstream method—Calculation of primary
populations is computed as the area under the curve in Figure productivity and respiration for a stream reach from upstream
10300:4d from sunrise to sunset. This is primary production in and downstream pairs of diurnal curves of oxygen and water
grams per cubic meter per day. Multiply by the average depth temperature is summarized in Figure 10300:5 and Table
PERIPHYTON (10300)/Primary Productivity 10-43

TABLE 10300:I. SAMPLE CALCULATION LEDGER FOR COMPUTATION OF CORRECTED RATE OF OXYGEN CHANGE FROM A SINGLE-STATION DIURNAL CURVE
Uncorrected Corrected
Time DO Water Temp. Cs* ⌬DO† Pp‡ Rp§ k2 D ⌬DO㛳
h mg/L °C mg/L mg/L/h mg/L/h mg/L/h d⫺1 mg/L/h mg/L/h

Midnight
0030
0100
0230
䡠
䡠
䡠
Noon
1230
1300
䡠
䡠
䡠
Midnight
* DO concentration at 100% saturation for a given water temperature, from Table 4500-O:I.
† Hourly rate of change of DO. For example, for noon to 1300, DO1200 –1300 ⫽ DO1300 ⫺ DO1200; plot at 1230.
‡ Phytoplankton net production.
§ Phytoplankton respiration rate.
㛳 ⌬DOcorrected ⫽ ⌬DOuncorrected ⫺ D ⫺ Pp ⫺ Rp

10300:II. Alternatively, calculate as below, with oxygen temperature for the reach at each hour. Calculate ⌬DO be-
change expressed as the difference between stations rather tween upstream and downstream stations for each hour as
than as change per hour. The calculations are analogous.
Multiply the area under a curve of oxygen change between
⌬DO⫽DOdownstream⫺DOupstream
two stations, corrected for diffusion and plankton metabolism
and expressed in milligrams per liter, by the discharge in
cubic meters per hour, and divide by the water surface area Tabulate Cs and determine the planktonic activity. Correct for
between the two stations. This, multiplied by 24, yields gross planktonic respiration by relating average hourly dark bottle
primary productivity in grams per square meter per day. DO change to the time of travel in the stream reach; correct
To compute gross primary productivity by this method, for planktonic production by the hourly change in DO in the
tabulate upstream and downstream DO and average water light bottle times the time of travel (see Table 10300:II).

TABLE 10300:II. SAMPLE CALCULATION LEDGER FORCOMPUTATION OF CORRECTED RATES OF OXYGEN CHANGE FROM THE UPSTREAM-DOWNSTREAM DIURNAL
CURVES OF OXYGEN CONCENTRATION AND TEMPERATURE
DO
Uncorrected Corrected
mg/L
Time ⌬DO Water Temp. Cs* Pp † Rp‡ k2 ⌬DO㛳
h Upstream Downstream mg/L °C mg/L mg/L mg/L d⫺1 mg/L

Midnight
0100
0200
䡠
䡠
䡠
Noon
1300
䡠
䡠
䡠
Midnight
* DO concentration at 100% saturation for a given water temperature, from Table 4500-O:I.
† Change in oxygen concentration in the light bottle per hour multiplied by travel time between the upstream and downstream station.
‡ Change in oxygen concentration in the dark bottle multiplied by travel time between the upstream and downstream station.
㛳 ⌬DOcorrected ⫽ ⌬DOuncorrected⫺D⫺Pp⫺Rp
10-44 BIOLOGICAL EXAMINATION (10000)

Net production P N ⫽ P G ⫺ R

Metabolism is thus estimated21,22 with the difference in the

upstream-downstream data by the graphical technique in Fig-
ure 10300:5 as:

Net metabolism ⫽ ⌬ DOlight⫺reaeration

Dark metabolism ⫽ ⌬ DOdark⫺reaeration

Gross community primary productivity (GPP) then equals net

metabolism minus respiration (light), and community respi-
ration (CR24) is average night respiration scaled for 24 h.

5. References

1. VOLLENWEIDER, R.A., ed. 1969. A Manual on Methods for Measur-

ing Primary Production in Aquatic Environments. IBP Handbook
No. 12. F.A. Davis Co., Philadelphia, Pa.
2. SLÁDEČEK, V. & A. SLÁDEČKOVÁ. 1964. Determination of periphyton
production by means of the glass slide method. Hydrobiologia 23:125.
3. KING, D.L. & R.C. BALL. 1966. A qualitative and quantitative measure
of aufwuchs production. Trans. Amer. Microsc. Soc. 82:232.
4. CLARK, J.R., D.I. MESSENGER, K.L. DICKSON & J. CAIRNS, JR. 1978.
Extraction of ATP from aufwuchs communities. Limnol. Oceanogr.
23:1055.
5. WETZEL, R.G. 1963. Primary productivity of periphyton. Nature 197:1026.
6. WETZEL, R.G. 1964. A comparative study of the primary production
of higher aquatic plants, periphyton, and phytoplankton in a large
shallow lake. Int. Rev. ges. Hydrobiol. 49:1.
7. KAHN, W.E. & R.G. WETZEL. 1999. Effects of microscale water level
fluctuations and altered ultraviolet radiation on periphyton micro-
flora. Microbial Ecol. 38:253.
8. LOEB, S.L. 1981. An in situ method for measuring the primary
productivity and standing crop of the epilithic periphyton commu-
Figure 10300:5. Calculation of gross periphytic primary productivity nity in lentic systems. Limnol. Oceanogr. 26:394.
from upstream-downstream diurnal curves. P is the area 9. BEER, S., A.J. STEWART & R.G. WETZEL. 1982. Measuring chloro-
under the corrected rate of change graph. phyll a and 14C-labeled photosynthate in aquatic angiosperms by
use of a tissue solubilizer. Plant Physiol. 69:54.
10. ODUM, H.T. 1956. Primary production in flowing waters. Limnol.
Calculate or tabulate k2 and convert it to the total oxygen Oceanogr. 1:102.
diffusion for the reach. Because diffusion, D, is expressed as 11. ALLEN, H.L. 1971. Primary productivity, chemo-organotrophy, and
milligrams per liter per hour, multiply it by the travel time to nutritional interactions of epiphytic algae and bacteria or macro-
obtain the diffusion correction. phytes in the littoral of a lake. Ecol. Monogr. 41:97.
12. HALL, C.A.S. 1972. Migration and metabolism in a temperate
Correct each hourly upstream-downstream ⌬DO as shown
stream ecosystem. Ecology 53:585.
in Table 10300:II. Integrate the area under this ⌬DO curve 13. NIXON, S.W. & C.A. OVIATT. 1974. Ecology of a New England salt
from sunrise to sunset to give P as in Figure 10300:5d. marsh. Ecol. Monogr. 43:463.
14. MCINTIRE, C.D., R.L. GARRISON, H.K. PHINNEY & C.E. WARREN. 1964.
Q Primary production in laboratory streams. Limnol. Oceanogr. 9:92.
P G, g/m2/d ⫽ P 15. THOMAS, N.A. & R.L. O’CONNELL. 1966. A method for measuring
A primary production by stream benthos. Limnol. Oceanogr. 11:386.
16. COPELAND, B.J. & W.R. DUFFER. 1964. Use of a clear plastic dome
where: to measure gaseous diffusion rates in natural waters. Limnol. Ocean-
Q ⫽ flow, m3/h, and ogr. 9:494.
A ⫽ reach area, m2 (average reach width ⫻ reach length). 17. TSIVOGLOU, E.C. & L.A. NEAL. 1976. Tracer measurement of reaera-
tion. III. Predicting the capacity of inland streams. J. Water Pollut.
Control Fed. 48:2669.
⌬DOdark ⫻ Q ⫻ 24
Respiration, R, g O2/m2/d ⫽ 18. GRANT, R.S. 1976. Reaeration-coefficient measurements of 10 small
A streams in Wisconsin. U.S. Geol. Surv. Water Resources Publ. 76-96.
19. ODUM, H.T. & C.M. HOSKIN. 1958. Comparative studies of the
and metabolism of marine water. Publ. Inst. Mar. Sci. Univ. Tex. 4:115.
PERIPHYTON (10300)/Interpreting and Reporting Results 10-45

20. BOTT, T.L., J.T. BROCK, C.E. CUSHING, S.V. GREGORY, D. KING & R.C. WHITFORD, L.A. & G.J. SCHUMACHER. 1964. Effect of a current on
PETERSEN. 1978. A comparison of methods for measuring primary respiration and mineral uptake in Spirogyra and Oedogonium.
productivity and community respiration in streams. Hydrobiologia 60:3. Ecology 45:168.
21. MARZOLF, E.R., P.J. MULHOLLAND & A.D. STEINMAN. 1994. Improve- DUFFER, W.R. & T.C. DORRIS. 1966. Primary productivity in a southern
ments to the diurnal upstream-downstream dissolved oxygen Great Plains stream. Limnol. Oceanogr. 11:143.
change technique for determining whole-metabolism in small MCINTIRE, C.D. 1966. Some factors affecting respiration of periphyton
streams. Can. J. Fish. Aquat. Sci. 51:1591. communities in lotic environments. Ecology 47:918.
22. YOUNG, R.G. & A.D. HURYN. 1998. Comment: Improvements to the CUSHING, C.E. 1967. Periphyton productivity and radionuclide accumu-
diurnal upstream-downstream dissolved oxygen change technique lation in the Columbia River, Washington, USA. Hydrobiologia
for determining whole-stream metabolism in small streams. Can. J. 29:125.
Fish. Aquat. Sci. 55:1784. HANSMANN, E.W., C.B. LANE & J.D. HALL. 1971. A direct method of
measuring benthic primary production in streams. Limnol. Ocean-
ogr. 16:822.
SCHINDLER, D.W., V.E. FROST & R.V. SCHMIDT. 1973. Production of
6. Bibliography epilithiphyton in two lakes of the experimental lakes area, north-
western Ontario. J. Fish. Res. Board Can. 30:1511.
POMEROY, L.R. 1959. Algal productivity in salt marshes. Limnol. Ocean- NORTH AMERICAN BENTHOLOGICAL SOCIETY. 1974 –1990 (annual). Current
ogr. 4:386. and Select Bibliographics on Benthic Biology. Springfield, Ill.
CASTENHOLZ, R.W. 1961. An evaluation of a submerged glass method of WETZEL, R.G. & G.E. LIKENS. 2000. Limnological Analyses, 3rd ed.
estimating production of attached algae. Verh. Int. Ver. Limnol. 14:155. Springer-Verlag, New York, N.Y.

10300 E. Interpreting and Reporting Results

Although several systems have been developed to organize abundance of individual species are used to calculate a Mean
and interpret periphyton data, no single method is universally Saprobial Index. Results also may be expressed by the truncated-
accepted. The methods may be qualitative or quantitative. Qual- log normal distribution of diatom species13,14 as well as the
itative methods deal with the taxonomic composition of the Autotrophic Index (AI).15
communities in zones of pollution, whereas quantitative methods Multivariate techniques provide an excellent way to analyze
deal with community structure using diversity indices, similarity and present periphyton community composition data with re-
indices, and numerical indices of saprobity. spect to pollution.16 –19 The importance of replication and statis-
tical analysis, particularly in the use of multivariate techniques,
1. Qualitative Methods (Indicator Species and has been noted.20
Communities)
3. Water Quality Applications
The saprobity system developed by Kolkwitz and Marsson is
a widely used method of interpreting periphyton data. This
Qualitative analyses of periphyton communities can be used
scheme divides polluted stream reaches into polysaprobic, ␣ and
␤ mesosaprobic, and oligosaprobic zones, and lists the charac- for indication of pollution, eutrophication, and hygienic prob-
teristics of each. The system has been refined1,2 and enlarged by lems in the monitoring of drinking water quality.21 Water quality
Fjerdingstad3,4 and Sládeček.5–7 surveillance can be assisted by bioassays on different types of
Evaluation of the saprobity system requires microscopic eval- artificial substrata in which changes and differences in species
uation of living indicator biota, particularly for the sensitive composition are determined.22 In addition to the indicator value
of individual species, the rates of biomass accrual during per-
sessile protozoans. Glass slides and other transparent substrata
iphytic colonization on exposed artificial substrata can serve as a
are advantageous because they permit direct microscopic exam-
further criterion of water quality. Simple screening assays of
ination and identification. Removal of periphyton from slides
periphyton are useful for the classification of biological stability
and preservation for subsequent examination may be acceptable
of water in treatment and distribution systems.23
for diatoms, but observation of preserved material is not accept-
In wastewater treatment, qualitative periphyton analyses cou-
able for most flagellated organisms.
pled with saprobiological evaluations may be used for classifi-
cation of waste treatment plant efficiency and for monitoring of
2. Quantitative Methods
treatment plant effluents.24 The utilization of periphyton growing
on exposed artificial substrata for the reduction of nutrients in
These methods use cell counts or biomass estimations per unit water supplies also has been proposed for water management
area of substratum and numerical indices of pollution or water practices.25
quality. Considerable data on cell densities and species compo-
sition of periphyton collected on glass slides in polluted rivers in
England are available.8 4. References
Other indices include the Shannon-Wiener,9 Simpson’s,10 and
Pinkham-Pearson.11 The saprobity system12 also may be used 1. KOLKWITZ, R. 1950. Oekologie der saprobien. Ver Wasser-, Boden,
where code numbers assigned for the saprobial value and the Lufthyg. Schriftenreihe (Berlin) 4:1.
10-46 BIOLOGICAL EXAMINATION (10000)

2. LIEBMANN, H. 1951. Handbuch der Frischwasser und Abwasserbi- 24. SLÁDEČKOVÁ, A. 1994. The role of periphyton in waste treatment
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3. FJERDINGSTAD, E. 1964. Pollution of streams estimated by benthal 25. SLÁDEČKOVÁ, A. & D. MATULOVÁ. 1998. Periphyton as bioelimina-
phytomicroorganisms. I. A saprobic system based on communities tor. Verh. Int. Ver. Limnol. 26:1777.
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