1060

Collection and Preservation of Samples

Reviewed by Standard Methods Committee, 2011. Editorial revisions, 2021. 2011 revisions by L. Malcom Baker, Rodger B. Baird, Nilda B. Cox, Andrew D. Eaton. Joint Task
Group: Lawrence H. Keith (chair), Clifford G. Annis, Gary L. DeKock, Carleton P. Edmunds, Scott J. Mickelson, Mark Wyzalek.

1060 A. Introduction

The result of any testing method can be no better than the sam- because of varied purposes and analytical procedures. Detailed
ple on which it is performed. However, it is beyond the scope of information may be presented in specific methods and that infor-
Standard Methods for the Examination of Water and Wastewater mation is to be followed when available. This section presents
to specify detailed procedures for the collection of all samples general considerations for the collection and preservation of

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Not for distribution or resale except by copyright holder only.
 1060 A. Introduction

s­ amples applicable primarily to chemical analyses. See appropri- Composite samples can be obtained by collecting over a
ate sections for samples to be used in toxicity testing (Section period of time, depth, or at many different sampling points. The
8020) and microbiological (Sections 9020 and 9060), biological details of collection vary with local conditions, so specific rec-
(Part 10 000), and radiological (Section 7010) examinations. ommendations are not universally applicable. Sometimes it is
The objective of sampling is to collect a portion of material more informative to analyze numerous separate samples instead
small enough in volume to be transported conveniently and yet of one composite so variability, maxima, and minima can be
large enough for analytical purposes while still accurately rep- determined.
resenting the material being sampled. This objective implies that Because of the inherent instability of certain properties and
the relative proportions or concentrations of all pertinent compo- compounds, composite sampling for some analytes is not recom-
nents are the same in the samples as in the material being sampled mended where quantitative values are desired (examples include
and that the samples are handled in such a way that no significant oil and grease, acidity, alkalinity, carbon dioxide, chlorine residual,
changes in composition occur before the tests are made. iodine, hexavalent chromium, nitrite, volatile organic compounds,
Frequently, the objective of sampling and testing is to demon- radon-222, dissolved oxygen, ozone, temperature, and pH). In cer-
strate whether continuing compliance with specific regulatory tain cases, such as when a composite sample is required by a reg-
requirements has been achieved. Samples are presented to the ulatory agency, the composite sample is refrigerated throughout
laboratory for specific determinations, with the sampler being the collection process to limit the instabilities of a compound and
responsible for collecting a valid and representative sample. its properties.
Because of the increasing importance placed on verifying the Sample carefully so that analytical results are as representative
accuracy and representativeness of data, greater emphasis is as possible of the actual sample composition. Important factors
placed on proper sample collection, tracking, and preservation affecting results are the presence of suspended matter or turbid-
techniques. Often, laboratory personnel help plan a sampling ity, the method chosen for removing a sample from its container,
program in consultation with the user of the test results. Such and the physical and chemical changes brought about by storage
consultation is essential to ensure selected samples and analytical or aeration. Detailed procedures are essential when processing
methods provide a sound and valid basis for answering the ques- (blending, sieving, filtering) samples to be analyzed for trace con-
tions that prompted the sampling and that meet regulatory and stituents, especially metals and organic compounds. Some deter-
project-specific requirements. minations can be invalidated by contamination during processing.
This section addresses the collection and preservation of water Treat each sample individually with regard to the substances to be
and wastewater samples; the general principles also apply to the determined, the amount and nature of turbidity present, and other
sampling of solid or semisolid matrices. conditions that may influence the results.
Carefully consider the technique for collecting a representative
1. General Requirements sample and define it in the sampling plan. For metals, it often
is appropriate to collect both a filtered and an unfiltered sample
Obtain a sample that meets the requirements of the sampling to differentiate between total and dissolved metals present in the
program, and handle it so it does not deteriorate or become con- matrix. Be aware that some metals may partially sorb to filters.
taminated or compromised before it is analyzed. Beforehand, determine the acid requirements to bring the pH to
Ensure that all sampling equipment is clean and quality-assured < 2 on a separate sample for each sample location and type. Add
before use. Always use sample containers that are clean and free this same amount of acid to the samples collected at the location
of contaminants. Bake all bottles to be used for organic-analysis and of the same type. Use ultrapure acid preservative to prevent
sampling at 450 °C. contamination. Be sure that the dilution caused by acidifying is
Fill sample containers that are clean and free of contaminants negligible or sufficiently reproducible for a dilution correction
directly with the sample. Do not prerinse the container with factor. When filtered samples are collected, filter them in the
sample because this results in the loss of any pre-added preser- field, if possible, or at the point of collection before preservation
vative and sometimes can bias results high when certain sample with acid. Filter samples in a laboratory-controlled environment
components adhere to the sides of the container. Depending on if field conditions could cause error or contamination. In this
the determinations to be performed, fill the container full (most case, filter as soon as possible after returning these samples to the
organic compound determinations) or leave space for aeration, laboratory. Often, slight turbidity can be tolerated if experience
mixing, etc. (microbiological and inorganic analyses). If a bottle shows that it causes no interference in gravimetric or volumetric
already contains preservative, take care not to overfill the bottle, tests and that its influence can be corrected in colorimetric tests,
as preservative may be lost or diluted. Except when sampling for where it has potentially the greatest interfering effect. Sample
the analysis of volatile organic compounds or radon, leave an air collector must state whether or not the sample has been filtered.
space of at least 1% of the container’s volume to allow for thermal Make a record of every sample collected and identify every bottle
expansion during shipment, or as may otherwise be required by a with a unique sample number, preferably by attaching an appropri-
method to ensure proper mixing (by inverting or shaking) before ately inscribed tag or label. Document sufficient information to pro-
sample withdrawal for analysis. vide positive sample identification at a later date, including the unique
Special precautions (discussed below) are necessary for sam- sample identification number, the name of the sample collector, the
ples containing organic compounds and trace metals. Because date, hour, exact location, and, if possible, sample type (e.g., grab or
many constituents may be present at low concentrations (micro- composite) and any other data that may be needed for correlation,
grams or nanograms per liter), they may be totally or partially lost such as water temperature, weather conditions, water level, stream
or easily contaminated when proper sampling and preservation flow, and post-collection conditions. If all pertinent information does
procedures are not followed. not fit on a label or attached tag, record the information in a bound

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1060 ANALYTICAL AND DATA QUALITY SYSTEMS: Collection and Preservation of Samples

sample log book at the sampling site at the time of sample collection. collect samples beneath the surface in quiescent areas and open
Use waterproof ink to record all information (preferably with black, the sampling container below the surface with the mouth directed
nonsolvent-based ink). Fix sampling points by detailed description toward the current to avoid collecting surface scum unless oil and
in the sampling plan, by maps, or with the aid of stakes, buoys, or grease is a constituent of interest; then collect water at the surface.
landmarks in a manner that will permit their identification by other If composite samples are required, take measures to prevent the
persons without reliance on memory or personal guidance. Global loss of sample constituents during compositing. If samples will be
positioning systems (GPS) also can supply accurate sampling posi- analyzed for organic constituents, refrigerate composited portions.
tion data. Particularly when sample results are expected to be involved Do not composite samples for VOC analysis because some of the
in litigation, use formal chain-of-custody procedures (see 1060 B.2), components will be lost through volatilization.
which trace sample history from collection to final reporting.
Before collecting samples from distribution systems, flush lines 2. Safety Considerations
with 3 to 5 pipe volumes (or until water is being drawn from the
main source) to obtain a representative sample from the supply, Because sample constituents may be toxic, take adequate pre-
taking into account the volume of the pipe to be flushed and the cautions during sampling and sample handling. Toxic substances
flow velocity. If the distribution system volume is unavailable, can enter through the skin and eyes and, in the case of vapors,
flush with tap fully open for at least 2 to 3 min before sampling. An also through the lungs. Ingestion can occur via direct contact of
exception to these guidelines (i.e., collecting a first-draw sample) is toxic materials with foods or by adsorption of vapors onto foods.
when information on areas of reduced or restricted flow is desired Precautions may be limited to wearing gloves or may include cov-
or when samples for lead in drinking water are being collected. eralls, aprons, or other protective apparel. Often, the degree of
Although well-pumping protocols depend on the objectives of an protection provided by chemical protective clothing (CPC) is spe-
investigation and other factors, such as well characteristics and avail- cific for different manufacturers and their product models1; verify
able equipment, a general rule is to collect samples from wells only that the clothing chosen offers adequate protection. Always wear
after the well has been purged sufficiently (usually with 3 to 10 well eye protection (e.g., safety glasses with side shields or goggles).
volumes) to ensure that the sample represents the groundwater. Purg- When toxic vapors may be present, sample only in well-ventilated
ing stagnant water is critical. Sometimes it is necessary to pump at a areas, or use an appropriate respirator or self-contained breathing
specified rate to achieve a characteristic drawdown, if this determines apparatus. In a laboratory, open sample containers in a fume hood.
the zones from which the well is supplied. Record the purging rate Never have food in the laboratory, near samples, or near sampling
and drawdown, if necessary. By using methods with minimal draw- locations. Always wash hands thoroughly before handling food.2
down, purging volumes can be reduced significantly. Always prohibit eating, drinking, or smoking near samples,
When samples are collected from a river or stream, observed sampling locations, and in the laboratory. Keep sparks, flames,
results may vary with depth, stream flow, and distance from each and excessive heat sources away from samples and sampling
shore. Selection of the number and distribution of sites at which locations. If flammable compounds are suspected or known to
samples should be collected depends on study objectives, stream be present and samples will be refrigerated, use only specially
characteristics, available equipment, and other factors. If equip- designed explosion-proof refrigerators.2
ment is available, take an integrated sample from top to bottom Collect samples safely, avoiding situations that may lead to
in the middle of the main channel of the stream or from side to accidents. When in doubt as to the level of safety precautions
side at mid-depth. If only grab or catch samples can be collected, needed, consult a knowledgeable industrial hygienist or safety
preferably take them at various points of equal distance across the professional. Samples with radioactive contaminants may require
stream; if only one sample can be collected, take it in the middle other safety considerations; consult a health physicist.
of the main channel of the stream and at mid-depth. Integrated Label adequately any sample known or suspected to be haz-
samples are described further in 1060 B.1c. ardous because of flammability, corrosivity, toxicity, oxidizing
Rivers, streams, lakes, and reservoirs are subject to consider- chemicals, or radioactivity, so appropriate precautions can be
able variations from normal causes (e.g., seasonal stratification, taken during sample handling, storage, and disposal.
diurnal variations, rainfall, runoff, and wind). Choose the location,
depth, and frequency of sampling depending on local conditions References
and the purpose of the investigation.
Use the following examples for general guidance. Avoid areas of 1. Forsberg K, Keith LH. Instant Gloves and CPC Database. Austin
excessive turbulence because of potential loss of volatile constitu- (TX): Instant Reference Sources, Inc.; 1998.
ents and potential presence of denser-than-air toxic vapors. Avoid 2. Water Pollution Control Federation. Removal of hazardous wastes
sampling at weirs, if possible, because such locations tend to favor in wastewater facilities—halogenated organics; manual of practice
retrieval of lighter-than-water immiscible compounds. Generally, FD-11. Alexandria (VA): Water Pollution Control Federation; 1986.

1060 B. Collection of Samples

1. Types of Samples seconds or minutes). Thus, they represent a “snapshot” in both

space and time of a sampling area. Discrete grab samples are col-
a. Grab samples: Grab samples are single samples collected lected from a single selected location, depth, and time. Depth-
at a specific spot at a site over a short period of time (typically integrated grab samples are collected over a predetermined area

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 1060 B. Collection of Samples

(integrated for a horizontal spatial representation) or the entire pH. Changes in components, such as dissolved oxygen or carbon
depth of a water column (integrated for a vertical spatial represen- dioxide, pH, or temperature, may produce secondary changes in
tation), at a selected location and time in a given body of water. certain inorganic constituents such as iron, manganese, alkalin-
A grab sample represents the composition of its source only ity, or hardness. Some organic analytes also may be changed by
at the time and place of collection. However, when a source is changes in the foregoing components. Use time-composite sam-
known to be relatively constant in composition over an extended ples only for determining components that can be demonstrated
period of time or over substantial distances in all directions, then to remain unchanged under the conditions of sample collection,
the sample may represent a longer time period or a spatially preservation, and storage.
larger volume than the specific time and location at which it was Collect individual portions in a wide-mouth bottle every hour (in
collected. In such circumstances, a source may be represented some cases, every half hour or even every 5 min) and mix at the end
adequately by single grab samples. Examples of this condition of the sampling period or combine in a single bottle as collected. If
include protected groundwater supplies, water supplies receiv- preservatives are used, add them to the sample bottle initially so all
ing conventional treatment, and some well-mixed surface waters. portions of the composite are preserved as soon as collected.
Rarely can wastewater streams, rivers, large lakes, shorelines, Automatic sampling devices are available; however, do not use
estuaries, and groundwater plumes be considered as being tempo- them unless the sample is preserved as described below. Com-
rally or spatially represented by single grab samples. posite samplers running for extended periods (weeks to months)
When a source is known to vary with time, grab samples col- should undergo routine cleaning of containers and sample lines to
lected at suitable intervals and analyzed separately can document minimize sample growth and deposits.
the extent, frequency, and duration of these variations. Choose c. Integrated (discharge-weighted) samples: For certain pur-
sampling intervals on the basis of the expected frequency of poses, the information needed is best provided by analyzing
changes, which may vary from 5 min to 1 h or more. Seasonal mixtures of grab samples collected from different points simul-
variations in natural systems may necessitate sampling over taneously, or as nearly so as possible, using discharge-weighted
months. When the source composition varies in space (i.e., from methods such as equal-width increment (EWI) or equal dis-
location to location) rather than time, collect samples from appro- charge-increment (EDI) procedures and equipment. An example
priate locations that meet the objectives of the study (for example, of the need for integrated sampling occurs in a river or stream
upstream and downstream from a point source). that varies in composition across its width and depth. To evaluate
The same principles apply to sampling wastewater sludges, average composition or total loading, use a mixture of samples
sludge banks, and muds, although these matrices are not specifically representing various points in the cross-section, in proportion to
addressed in this section. Take every possible precaution to obtain their relative flows. The need for integrated samples also may exist
a representative sample or one conforming to a sampling program. if combined treatment is proposed for several separate wastewater
b. Composite samples: Composite samples should provide a streams, the interaction of which may have a significant effect on
more representative sampling of heterogeneous matrices in which treatability or even on composition. Mathematical prediction of
the concentration of the analytes of interest may vary over short the interactions among chemical components may be inaccurate
periods of time and/or space. Composite samples can be obtained or impossible to perform, and testing a suitable integrated sample
by combining portions of multiple grab samples or by using spe- may provide more useful information.
cially designed automatic sampling devices. Sequential (time) Both lakes and reservoirs show spatial variations of composi-
composite samples are collected by using continuous, constant tion (depth and horizontal location). However, there are condi-
sample pumping or by mixing equal water volumes collected tions under which neither total nor average results are especially
at regular time intervals. Flow-proportional composites are col- useful, but local variations are more important. In such cases,
lected by continuous pumping at a rate proportional to the flow, examine samples separately (i.e., do not integrate them).
by mixing equal volumes of water collected at time intervals that Preparation of integrated samples usually requires equipment
are inversely proportional to the volume of flow, or by mixing designed to collect a sample water uniformly across the depth
volumes of water proportional to the flow collected during or at profile. Knowledge of the volume, movement, and composi-
regular time intervals. tion of the various parts of the water being sampled usually is
Advantages of composite samples include reduced costs of required. Collecting integrated samples is a complicated and spe-
analyzing a large number of samples, more representative sam- cialized process that must be described adequately in a sampling
ples of heterogeneous matrices, and larger sample sizes when plan.
amounts of test samples are limited. Disadvantages of compos-
ite samples include a loss of analyte relationships in individual 2. Chain-of-Custody Procedures
samples, the potential dilution of analytes below detection levels,
increased potential analytical interferences, and an increased pos- The process of tracing the possession and handling of a sample
sibility of analyte interactions. In addition, the use of composite from the time of collection through analysis and final disposition
samples may reduce the number of samples analyzed below the is referred to as the sample’s chain of custody. Properly designed
required statistical need for specified data quality objectives or and executed chain-of-custody forms document the sample integ-
project-specific objectives. rity and that proper handling has occurred from sample collection
Do not use composite samples with components or charac- to data reporting. Sample chain of custody is required to demon-
teristics subject to significant and unavoidable changes during strate sample control when the data are to be used for regulation
storage. Analyze individual samples as soon as possible after col- or litigation. Where litigation is not involved, chain-of-custody
lection and preferably at the sampling point. Examples are dis- procedures are useful for routine control of samples and provide
solved gases, residual chlorine, soluble sulfide, temperature, and documentation for quality assurances reviews.

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1060 ANALYTICAL AND DATA QUALITY SYSTEMS: Collection and Preservation of Samples

A sample is considered to be under a person’s custody if it is sample custody documentation. Ensure that samples are accompa-
in the individual’s physical possession, in the individual’s sight, nied by a completed chain-of-custody record and a sample analysis
secured and tamper-proofed by that individual, or secured in an request sheet. Deliver the samples to the sample custodian.
area restricted to authorized personnel. The following procedures g. Receipt and logging of sample: In the laboratory, the sample
summarize the major aspects of chain of custody. More detailed custodian inspects the condition and seal of the sample and rec-
discussions are available.1,2 onciles label information and seal against the chain-of-custody
a. Sample labels (including bar-code labels): Use labels to record before the sample is accepted for analysis. After accep-
prevent sample misidentification. Gummed paper labels or tags tance, the custodian assigns a laboratory number, logs the sample
generally are adequate. Include at least the following information: in the laboratory log book or computerized laboratory informa-
a unique sample number, sample type, name of collector, date and tion management system, and stores it in a secured storage room
time of collection, place of collection, and sample preservative. or cabinet or refrigerator at the specified temperature until it is
Also include date and time of preservation for comparison to date assigned to an analyst.
and time of collection. Affix tags or self-adhesive labels to sample h. Assignment of sample for analysis: The laboratory supervi-
containers before, or at the time of, sample collection. sor usually assigns the sample for analysis. Once the sample is in
b. Sample seals: Use sample seals to detect unauthorized tamper- the laboratory, the supervisor or analyst is responsible for its care
ing with samples up to the time of analysis. Use self-adhesive paper and custody.
seals that include at least the following information: sample num- i. Disposal: Hold samples for the prescribed amount of time
ber (identical with number on sample label), collector’s name, and for the project or until the data have been reviewed and accepted.
date and time of sampling. Plastic shrink seals also may be used. Document the disposition of samples. Dispose of samples in
Attach a seal so that it must be broken to open the sample con- accordance with local, state, and U.S. EPA approved methods.
tainer or the sample shipping container (e.g., a cooler). Affix the
seal to the container before the sample leaves the custody of sam- 3. Sampling Methods
pling personnel.
c. Field log book: Record all information pertinent to a field a. Manual sampling: Manual sampling involves minimal
survey or sampling in a bound log book. As a minimum, include equipment but may be unduly costly and time-consuming for
the following in the log book: purpose of sampling; location of sam- routine or large-scale sampling programs. It requires trained field
pling point; name and address of field contact; producer of material technicians and is often necessary for regulatory and research
being sampled and address (if different from location); type of sam- investigations for which critical appraisal of field conditions and
ple; and method, date, and time of preservation. If the sample is complex sample-collection techniques are essential. Manually
wastewater, identify the process producing the waste stream. Also collect certain samples, such as waters containing oil and grease.
provide suspected sample composition, including concentrations; b. Automatic sampling: Automatic samplers can eliminate
number and volume of samples taken; description of sampling human errors in manual sampling, can reduce labor costs, may
point and sampling method; date and time of collection; collector’s provide the means for more frequent sampling,3 and are used
sample identification numbers; sample distribution and manner of increasingly. Be sure that the automatic sampler does not con-
transport; references such as maps or photographs of the sampling taminate the sample. For example, plastic components may be
site; field observations and measurements; and signatures of per- incompatible with certain organic compounds that are soluble in
sonnel responsible for observations. Because sampling situations the plastic parts or that can be contaminated (e.g., from phthalate
vary widely, it is essential to record sufficient information to recon- esters) by contact with them. If sample constituents are generally
struct the sampling event without reliance on the collector’s mem- known, contact the manufacturer of an automatic sampler regard-
ory. Protect the log book and keep it in a safe place. ing potential incompatibility of plastic components.
d. Chain-of-custody record: Fill out a chain-of-custody record to Program an automatic sampler in accordance with sampling
accompany each sample or group of samples. The record includes needs. Carefully match pump speeds and tubing sizes to the type
the following information: sample number; signature of collector; of sample to be collected.
date, time, and address of collection; sample type; sample preser- c. Sorbent sampling: The use of solid sorbents, particularly
vation requirements; signatures of persons involved in the chain of membrane-type disks, is becoming more frequent. These methods
possession; and inclusive dates and times of possession. offer rapid, inexpensive sampling if the analytes of interest can be
e. Sample analysis request sheet: The sample analysis request adsorbed and desorbed efficiently and the water matrix is free of
sheet accompanies samples to the laboratory. The collector com- particulates that plug the sorbent.
pletes the field portion of the form, which includes most of the per-
tinent information noted in the log book. The laboratory portion of
the form is to be completed by laboratory personnel and includes: 4. Sample Containers
name of person receiving the sample, laboratory sample number,
date of sample receipt, condition of each sample (if it is cold or The type of sample container used is of utmost importance. Test
warm, whether the container is full or not, color, if more than one sample containers and document that they are free of analytes of
phase is present, etc.), and determinations to be performed. interest, especially when sampling and analyzing for very low ana-
f. Sample delivery to the laboratory: Deliver the samples to the lyte levels. Containers typically are made of plastic or glass, but
laboratory as soon as practicable after collection, typically within one material may be preferred over the other. For example, silica,
2 d. If shorter sample holding times are required, make special sodium, and boron may be leached from soft glass, but not plas-
arrangements to ensure timely delivery to the laboratory. If samples tic, and trace levels of some pesticides and metals may sorb onto
are shipped by a commercial carrier, include the waybill number in the the walls of glass containers.4 Thus, hard glass containers (Pyrex

46 • Part 1000
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 1060 B. Collection of Samples

or equivalent) are preferred. For samples containing organic com-

pounds, do not use plastic containers except those made of fluori-
nated polymers, such as polytetrafluoroethylene (PTFE).3
Some sample analytes may dissolve (be absorbed) into the
walls of plastic containers; similarly, contaminants from plastic
containers may leach into samples. Avoid plastics wherever pos-
sible because of potential contamination from phthalate esters.
Container failure due to the breakdown of the plastic is possible.
Therefore, use glass containers for all organics analyses, such as
volatile organics, semivolatile organics, pesticides, PCBs, and oil
and grease. Some analytes (e.g., bromine-containing compounds
and some pesticides, and polynuclear aromatic compounds) are
light-sensitive; collect them in amber-colored glass containers
to minimize photodegradation. Container caps, typically plastic,
also can be a problem. Do not use caps with paper liners. Use foil
or PTFE liners but be aware that metal liners can contaminate
samples collected for metals analysis, and they may also react
with the sample if it is acidic or alkaline. Serum vials with PTFE-
lined rubber or plastic septa are useful.
In rare situations, it may be necessary to use sample contain-
ers not specifically prepared for use, or otherwise unsuitable for
the particular situation; thoroughly document these deviations.
Documentation should include type and source of container and
the preparation technique (e.g., acid washed with reagent water
rinse). For QA purposes, the inclusion of a bottle blank may be
necessary.

5. Number of Samples Figure 1060:1. Approximate number of samples required in estimating

a mean concentration. Source: Methods for the examination of waters
Because of variability from analytical and sampling proce- and associated materials: general principles of sampling and accuracy of
dures (i.e., population variability), a single sample is insuffi- results. London, England: Her Majesty’s Stationery Office, 1980.
cient to reach any reasonable desired level of confidence. If an
overall standard deviation (i.e., the standard deviation of com-
analyst is left with only the published error of the measurement
bined sampling and analysis) is known, the required number of
system (typically obtained by using a reagent water matrix under
samples for a mobile matrix, such as water, may be estimated
the best analytical conditions).
as follows:4
More accurate equations are available.5 These are based on the
Z distribution for determining the number of samples needed to
 ts 2 estimate a mean concentration when variability is estimated in
N ≥  
U  absolute terms using the standard deviation. The coefficient of
variation [relative standard deviation (RSD)] is used when vari-
where: ability is estimated in relative terms.
The number of random samples to be collected at a site can
N = number of samples,
be influenced partly by the method that will be used. The values
t = Student t test statistic for a given confidence level,
for SD or RSD may be obtained from each of the methods or
s = overall standard deviation, and
in the literature.6 However, calculations of estimated numbers
U = acceptable level of uncertainty.
of samples needed based only on this information will result in
To assist in calculations, use curves such as those in Figure 1060:1. underestimated numbers of samples because only the analytical
As an example, if s is 0.5 mg/L, U is ± 0.2 mg/L, and a 95% confi- variances are considered and the typically larger variances from
dence level is desired, approximately 25 to 30 samples must be taken. the sampling operations are not included. Preferably, deter-
The above equation assumes that total error (population vari- mine and use SDs or RSDs from overall sampling and analysis
ability) is known. Total variability consists of all sources of vari- operations.
ability, including the distribution of the analytes of interest within For estimates of numbers of samples needed for systematic
the sampling site; collection, preservation, preparation, and analy- sampling (e.g., drilling wells for sampling groundwater or for
sis of samples; and data handling and reporting. In simpler terms, systematically sampling large water bodies, such as lakes), equa-
error (variability) can be divided into sampling and analysis com- tions are available7 that relate number of samples to shape of grid,
ponents. Sampling error due to population variability (includ- area covered, and space between nodes of grid. The grid spacing
ing heterogeneous distribution of analytes in the environmental is a complex calculation that depends on the size and shape of any
matrix) usually is much larger than analytical error components. contaminated spot (such as a groundwater plume) to be identified,
Unfortunately, sampling error usually is not available and the in addition to the geometric shape of the sampling grid.

 Part 1000 • 47
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1060 ANALYTICAL AND DATA QUALITY SYSTEMS: Collection and Preservation of Samples

See individual methods for types and numbers of quality the ­analytical needs of sampling procedures as they pertain to the
assurance (QA) and quality control (QC) samples [e.g., for nor- goals and data quality objective of an investigation.
mal-level (procedural) or low-level (contamination) bias or for Do not use samples from the same container for multiple testing
precision] involving sampling or laboratory analysis (either over- requirements (e.g., organic, inorganic, radiological, bacteriological,
all or individually). Estimates of numbers of QC samples needed and microscopic examinations) because methods of collecting and
to achieve specified confidence levels also can be calculated. handling are different for each type of test. Always collect enough
Rates of false positives (type I error) and false negatives (type sample volume in the appropriate container in order to comply with
II error) are useful parameters for estimating required numbers sample handling, storage, and preservation requirements.
of QC samples. A false positive is the incorrect conclusion that
an analyte is present when it is absent. A false negative is the References
incorrect conclusion that an analyte is absent when it is present.
If the frequency of false positives or false negatives desired to be 1. U.S. Environmental Protection Agency. Test methods for evaluating
detected is less than 10%, then solid waste: physical/chemical methods, 3rd ed.; Pub. No. SW-846.
Washington DC: Office of Solid Waste and Emergency Response,
U.S. Environmental Protection Agency; 1986.
ln α 2. U.S. Environmental Protection Agency. NEIC policies and pro-
n=
ln (1 − Y ) cedures; EPA-330/9/78/001/-R (rev. 1982). Washington DC: U.S.
Environmental Protection Agency, 1982.
3. Water Pollution Control Federation. Removal of hazardous wastes
where:
in wastewater facilities—halogenated organics; Manual of Practice
α = (1 - desired confidence level) and FD-11. Alexandria (VA): Water Pollution Control Federation; 1986.
Y = frequency to detect (<10%). 4. Methods for the examination of waters and associated materials:
general principles of sampling and accuracy of results. London,
If the frequency that is desirable to detect is more than 10%, England: Her Majesty’s Stationery Office; 1980.
iterative solution of a binomial equation is necessary.5,8 5. Keith LH, Patton GL, Lewis DL, Edwards PG. Determining numbers
and kinds of analytical samples, Chapter 1. In: Principles of envi-
6. Sample Volumes ronmental sampling, 2nd ed. Washington DC: American Chemical
Society; 1996 (ACS Professional Reference Book).
6. Keith LH. Compilation of EPA’s sampling and analysis methods, 2nd
Collect a 1-L sample for most physical and chemical analy- ed. Boca Raton (FL): CRC Press; 1996.
ses. For certain determinations, larger samples may be neces- 7. Gilbert RO. Statistical methods for environmental pollution monitor-
sary. Table 1060:1 lists general guidance for volumes ordinarily ing. New York (NY): Van Nostrand Reinhold, 1987.
required for analyses, but it is strongly recommended that the lab- 8. Grant EL, Leavenworth RS. Statistical quality control, 6th ed. New
oratory staff who conduct the analyses also be consulted to verify York (NY): McGraw-Hill, Inc.; 1988.

1060 C. Sample Storage and Preservation

Complete and unequivocal preservation of samples, whether potential, and dissolved gases in situ and pH, specific conduc-
domestic wastewater, industrial wastes, or natural waters, is a tance, turbidity, and alkalinity immediately after sample collec-
practical impossibility because complete stability for every con- tion. Many organic compounds are sensitive to changes in pH and
stituent never can be achieved. At best, preservation techniques temperature resulting in reduced concentrations during storage.
only retard chemical and biological changes that inevitably con- Changes in the pH-alkalinity-carbon dioxide balance may
tinue after sample collection. cause calcium carbonate to precipitate, decreasing the values for
calcium and total hardness.
1. Sample Storage before Analysis Iron and manganese are readily soluble in their lower oxida-
tion states but relatively insoluble in their higher oxidation states;
a. Nature of sample changes: Some determinations are more therefore, these cations may precipitate or they may dissolve from
affected by sample storage than others. Certain cations are sub- a sediment, depending on the redox potential of the sample. Micro-
ject to loss by adsorption to, or ion exchange with, the walls of biological activity may affect the nitrate-nitrite-ammonia content,
glass containers. These include aluminum, cadmium, chromium, phenol or BOD concentration, or the reduction of sulfate to sulfide.
copper, iron, lead, manganese, silver, and zinc, which are best Residual chlorine is reduced to chloride. Sulfide, sulfite, ferrous
collected in a separate clean bottle and acidified with nitric acid iron, iodide, and cyanide may be lost through oxidation. Color,
to a pH below 2.0 to minimize precipitation and adsorption on odor, and turbidity may increase, decrease, or change in quality.
container walls. Also, some organics may be subject to loss by Sodium, silica, and boron may be leached from the glass container.
adsorption to the walls of glass containers. Hexavalent chromium may be reduced to trivalent chromium.
Temperature changes quickly; pH may change significantly in a The biological activity in a sample may change the oxidation
matter of minutes; dissolved gases (oxygen, carbon dioxide) may state of some constituents. Soluble constituents may be converted
be lost. Because changes in such basic water quality properties to organically bound materials in cell structures, or cell lysis may
may occur quickly, determine temperature, reduction-oxidation result in release of cellular material into solution. The well-known

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 1060 C. Sample Storage and Preservation

Table 1060:1. Summary of Special Sampling and Handling Requirementsa

Minimum
Sample Size Sample Maximum Storage
Determination Container (mL) Type Preservationb Recommended Regulatoryc
Acidity P, G(B), FP 100 g Cool to ≤ 6°C 24 h 14 d
Alkalinity P, G, FP 200 g Cool to ≤ 6°C 24 h 14 d
BOD P, G, FP 1000 g, c Cool to ≤ 6°C 6h 48 h
Boron FP (PTFE), G 1000 g, c HNO3 to pH < 2 28 d 6 months
(quartz)
Bromide P, G, FP 100 g, c None required 28 d 28 d
Carbon (organic G(B), P, FP 100 g, c Analyze immediately or cool to ≤ 6°C; and 7d 28 d
and total) add HCl, H3PO4, or H2SO4 to pH < 2
Carbon dioxide P, G 100 g Analyze immediately 15 min N.S.
COD P, G, FP 100 g, c Analyze as soon as possible, or add 7d 28 d
H2SO4 to pH < 2; Cool to ≤ 6 °C
Chloride P, G, FP 50 g, c None required N.S. 28 d
Chlorine (total P, G 500 g Analyze immediately 15 min 0.25 h
and residual)
Chlorine dioxide P, G 500 g Analyze immediately 15 min N.S.
Chlorophyll P, G (amber) 500 g Unfiltered, dark, 48 h; 28 d if algae N.S.
≤ 6 °C. Filtered, removed from
dark, –20 °C (Do not water having pH >
store in frost-free freezer) 6 onto glass fiber
filter, placed in
airtight plastic bags
and frozen
Color P, G, FP 500 g, c Cool to ≤ 6 °C 24 h 48 h
Specific P, G, FP 500 g, c Cool to ≤ 6 °C 28 d 28 d
conductance
Cyanide
Total P, G, FP 100 g Add NaOH to pH 12 for SDWA 48 h 14 d; 24 h
compliance and pH 11 for all other if >50
samples. If sample is to be stored, cool mg/L
to ≤ 6 °C in dark. Remove interferences sulfide
according to method specific present
instructions
 Amenable to P, G, FP 500 g Remove interferences according to 48 h 14 d; 24 h if
chlorination method specific instructions; add >50 mg/L
NaOH to pH 11 and cool to ≤ 6 °C sulfide
present
Free P, G, FP 100 g Remove interferences according to 48 h 14 d
method specific instructions; add
NaOH to pH 11 and cool to ≤ 6 °C
 Weak and P, G, FP 100 g Add NaOH to pH 12 for SDWA compliance 48 h 14 d; 24 h
dissociable and pH 11 for all other samples. If if >50
(WAD) sample is to be stored, cool to ≤ 6 °C in mg/L
dark. Remove interferences according to sulfide
method specific instructions present
Fluoride P 100 g, c None required 28 d 28 d
Hardness P, G, FP 100 g, c Add HNO3 or H2SO4 to pH < 2 6 months 6 months
Hydrogen peroxide P, G 50 g Analyze immediately 10 min after collection
Iodine P, G 500 g Analyze immediately 15 min. N.S.
Metals P(A), G(A), 1000 g, c For dissolved metals filter immediately; 6 months 6 months
FP(A) add HNO3 to pH < 2
Chromium VI P(A), G(A), 250 g Cool to ≤ 6 °C, pH 9.3–9.7, ammonium 28 d 28 d
FP(A) sulfate buffer preservative as specified
in method 3500-Cr to extend to 28 d HT
Copper by _a _ g, c _ _
colorimetry
Ferrous iron P(A), G(A), 500 g Add 2 ml conc HCL to aliquot. Add 20 mL 15 min
FP(A) phenanthroline solution and 10 mL
NH2C2H3O2 to 50 mL portion of
acidified sample. Analyze immediately

 Part 1000 • 49
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1060 ANALYTICAL AND DATA QUALITY SYSTEMS: Collection and Preservation of Samples

Table 1060:1. Continued

Minimum
Sample Size Smple Maximum Storage
Determination Container (mL) Type Preservationb Recommended Regulatoryc
Mercury P(A), G(A), 500 g, c Add HNO3 to pH < 2, Cool ≤ 6 °C 28 d 28 d
FP(A)
Nitrogen
Ammonia P, G, FP 500 g, c Analyze as soon as possible or add 7d 28 d
H2SO4 to pH < 2. Cool to 6 °C
Nitrate P, G, FP 100 g, c Analyze as soon as possible; cool to 48 h 48 h (14 d for
≤ 6 °C chlorinated
samples)
Nitrate + nitrite P, G, FP 200 g, c Add H2SO4 to pH < 2; cool to ≤ 6 °C 48 h 28 d
Nitrite P, G, FP 100 g, c Analyze as soon as possible; cool to none 48 h
≤ 6 °C
Organic, Kjeldahl P, G, FP 500 g, c Cool to ≤ 6 °C. Add H2SO4 to pH < 2 7d 28 d
Total P, G. FP 100 g, c Cool to ≤ 6 °C, add H2SO4, or HCl to 7d 28 d
pH ≤ 2
Odor G 500 g Analyze as soon as possible; cool ≤ 6 °C 6h 24 h (EPA
Manual
drinking
water)
Oil and Grease G (wide-mouth 1000 g Add HCl or H2SO4 to pH < 2, Cool to 28 d 28 d
calibrated) ≤ 6 °C
Organic Compounds
MBAS P, G, FP 250 g, c Cool to ≤ 6 °C 48 h 48 h
Pesticidesa G(S, PTFE-lined 1000 g, c Cool to ≤ 6 °C. Add 1000 mg/L ascorbic 7d 7 d until
cap) acid if residual chlorine present (0.008% extraction;
sodium thiosulfate in CFR 136) 40 d after
extraction
Phenols (5530) G 500 g Cool to ≤ 6 °C. Add H2SO4 to pH < 2 See footnotea 28 d until
extraction,
2 d after
extraction
Phenols (6420) G (PTFE-lined 1000 g, c Cool to ≤ 6 °C. add 0.008% sodium 7d 7 d until
cap) thiosulfate if chlorine is present extraction;
40 d after
extraction
 Purgeablesa G (PTFE-lined 2 × 40 g Cool, ≤ 6 °C; add HCl to pH < 2; add 7d 14 d
(purge and trap) cap) 1000 mg/L ascorbic acid if residual
chlorine present (0.008% sodium
thiosulfate in CFR 136)
 Base/neutrals G(S, amber) 1000 g, c Cool to ≤ 6 °C; add 0.008% sodium 7d 7 d until
and acids thiosulfate in CFR 136 if chlorine is extraction;
present 40 d after
extraction
Oxygen G-, P-BOD bottle 300 g
(dissolved)
Electrode Analyze immediately 15 min 15 min
Winkler Titration may be delayed after 8h 8h
acidification
Ozone G 1000 g Analyze immediately 15 min N.S.
Peracetic acid G, P 50 g Analyze immediately 10 min after
collection
pH P, G 50 g Analyze immediately 15 min 15 min
Phosphate G(A) 100 g For dissolved phosphate filter 48 h 48 h
immediately. Cool to ≤ 6 °C
Phosphorus, total P, G, FP 100 g, c Add H2SO4 to pH < 2 and cool to ≤ 6 °C 28 d 28 d
Salinity G (wax seal) 240 g Analyze immediately or use wax seal 6 months N.S.
Silica FP (PTFE), G 200 g, c Cool to ≤ 6 °C; do not freeze 28 d 28 d
(quartz)

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 1060 C. Sample Storage and Preservation

Table 1060:1. Continued

Minimum
Sample Size Smple Maximum Storage
Determination Container (mL) Type Preservationb Recommended Regulatoryc
Sludge digester gas G, gas bottle — g — N.S.
Solids1 P, G 200 g, c Cool to ≤ 6 °C 7d 2–7 d; see
reference 1
Sulfate P, G, FP 100 g, c Cool to ≤ 6 °C 28 d 28 d
Sulfide P, G, FP 100 g, c Cool to ≤ 6 °C. Add 4 drops of 2 N zinc 48 h 7d
acetate per 100 mL. Add NaOH to pH
>9
Temperature P, G, FP — g Analyze immediately 15 min 0.25 h

Turbidity P, G, FP 100 g, c Analyze same day. Store in dark up to 24 h. 24 h 48 h

Cool to ≤ 6 °C
Abbreviations: c = composite; g = grab; G = glass; G(A) or P(A) = rinsed with 1 + 1 HNO3; G(B) = glass, borosilicate; G(S) = glass, rinsed with organic solvents or baked; FP
= fluoropolymer (PTFE); N.S. = not stated in cited reference; P = plastic (polyethylene or equivalent).
a
For determinations not listed, use glass or plastic containers; preferably refrigerate during storage and analyze as soon as possible.
b
Cool = storage at, > 0 oC, ≤ 6 °C (above freezing point of water); in the dark; analyze immediately = analyze usually within 15 min of sample collection.
c
See reference 2 for possible differences regarding container and preservation requirements.
Note 1: Some drinking water (DW) and treated wastewater (WW) matrices may be subject to positive interference as a result of preservation. If such interference is demonstra-
ble, samples should be analyzed as soon as possible without preservation. Do not hold for more than 15 minutes without demonstrating that cyanide (CN) is stable for longer
periods in a specific matrix.
Note 2: This table is intended for guidance only. If there is a discrepancy between this table and the method, the information in the current method takes precedence.
If performing the method for compliance purposes, be aware that alternative preservation and holding-time requirements may exist. If so, the regulatory require-
ments should be used.
References
1. U.S. Environmental Protection Agency. 2007. 40 CFR Part 136, Table II.
2. U.S. Environmental Protection Agency. 1992. Rules and Regulations. 40 CFR Parts 100–149.

nitrogen and phosphorus cycles are examples of biological influ- methods limit the elapsed time between sample collection and
ences on sample composition. analysis (see Table 1060:1). Changes caused by growth of micro-
Zero headspace is important in the preservation of samples organisms are greatly retarded by keeping the sample at a low
with volatile organic compounds and radon. Avoid a loss of vol- temperature (< 6 °C but above freezing). When the interval
atile materials by collecting samples in a completely filled con- between sample collection and analysis is long enough to produce
tainer. Achieve this by carefully filling a bottle so the top of the changes in either the concentration or physical state of the con-
meniscus is above the top of the bottle rim. It is important to avoid stituent to be measured, follow the preservation practices given
spillage or air entrapment if preservatives, such as HCl or ascor- in Table 1060:1. Record the time elapsed between sampling and
bic acid, have already been added to the bottle. After capping or analysis, and which preservative, if any, was added.
sealing the bottle, check for air bubbles by inverting and gently
tapping it; if one or more air bubbles are observed then, if prac- 2. Preservation Techniques
tical, discard the sample and repeat, refilling the bottle with the
new sample until no air bubbles are observed (this cannot be done To minimize the potential for volatilization or biodegradation
if bottle contained preservatives before it was filled). between sampling and analysis, keep samples as cool as possible
Serum vials with septum caps are particularly useful in that a without freezing. Preferably pack samples in crushed or cubed
sample portion for analysis can be taken through the cap by using ice or commercial ice substitutes before shipment. Avoid using
a syringe,1 although the effect of pressure reduction in the head- dry ice because it will freeze samples and may cause glass con-
space must be considered. Pulling a sample into a syringe under tainers to break. Dry ice also may effect a pH change in samples.
vacuum can result in low bias data for volatile compounds and the Keep composite samples cool with ice or a refrigeration system
resulting headspace precludes taking further subsamples. set at ≤ 6 °C during compositing. Analyze samples as quickly as
b. Time interval between collection and analysis: In general, possible on arrival at the laboratory. If immediate analysis is not
the shorter the time that elapses between the collection of a sam- possible, preferably store at ≤ 6 °C.1
ple and its analysis, the more reliable are the analytical results. No single method of preservation is entirely satisfactory;
For certain constituents and physical values, immediate analysis choose the preservative with due regard to the determinations
in the field is required. For composited samples, it is common to be made. Use chemical preservatives only when they do not
practice to use the time at the end of composite collection as the interfere with the analysis being made. When they are used, add
sample-collection time. them to the sample bottle initially so all sample portions are
Check with the analyzing laboratory to determine how much preserved as soon as collected. Because a preservation method
elapsed time may be allowed between sample collection and anal- for one determination may interfere with another one, samples
ysis. This depends on the character of the sample and the stability for multiple determinations may need to be split and preserved
of the target analytes under storage conditions. Many regulatory separately. All preservation methods may be inadequate when

 Part 1000 • 51
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1080 ANALYTICAL AND DATA QUALITY SYSTEMS: Reagent Water

applied to suspended matter. Do not use formaldehyde as a degree, the dependability of an analytical determination rests
preservative for samples collected for chemical analysis because on the experience and good judgment of the person collecting
it affects many of the target analytes. the sample. Numbers of samples required for confidence levels
Preservation methods are relatively limited and are intended in data quality objectives, however, rely on statistical equations,
generally to retard biological action, retard hydrolysis of such as those discussed earlier.
chemical compounds and complexes, and reduce volatility of
constituents. Reference
Preservation methods are limited to pH control, chemical addi-
tion, the use of amber and opaque bottles, refrigeration, filtration, 1. Water Pollution Control Federation. Removal of hazardous wastes
and freezing. Table 1060:1 lists preservation methods by constit- in wastewater facilities—halogenated organics; Manual of Practice
uent. See Section 7010 B for sample collection and preservation FD-11. Alexandria (VA): Water Pollution Control Federation; 1986.
requirements for radionuclides.
The foregoing discussion is by no means exhaustive and com- Bibliography
prehensive. Clearly, it is impossible to prescribe absolute rules for
preventing all possible changes. Additional advice will be found Keith LH. Principles of environmental sampling, 2nd ed. Washington DC:
in the discussions under individual determinations, but to a large American Chemical Society; 1996 (ACS Professional Reference Book).

https://doi.org/10.2105/SMWW.2882.009
52 • Part 1000
Copyright © 2023 American Public Health Association, American Water Works Association, Water Environment Federation.
Not for distribution or resale except by copyright holder only.