905R87108
METHODS FOR TOXICITY REDUCTION EVALUATIONS
PHASE I
TOXICITY CHARACTERIZATION PROCEDURES
DRAFT - JANUARY 1987
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EPA/600/
1987
METHODS FOR TOXICITY REDUCTION EVALUATIONS
PHASE I
TOXICITY CHARACTERIZATION PROCEDURES
DRAFT - JANUARY 1987
Linda Anderson-Carnahan
U.S. EPA Region V
Water Division, Water Duality Branch
Chicago, Illinois R0604
Donald I. Mount
U.S. EPA Environmental Research Laboratory
Office of Research and Development
Duluth, Minnesota 55804
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NOTICE
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
ii
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FORWARD
This document is the first in a series of guidance documents intended to aid
dischargers in conducting effluent Toxicity Reduction Evaluations (TREs).
Such evaluations may be required as the result of an enforcement action or as
a condition of an NPDES permit. It will also provide U.S. Environmental Pro-
tection Agency (EPA) and State pollution control agency staff with the back-
ground necessary to overview and determine the adequacy of effluent TREs
proposed and performed by NPDES permittees.
This document provides methods for performing effluent toxicant characteriza-
tion studies. Because many of the techniques described in EPA's "Methods for
Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms,
3rd Edition" are cited in this document, this manual should be available for
use in conjunction with this document. In addition, individuals involved in
effluent Toxicity Reduction Evaluations may wish to consult EPA's "Technical
Support Document For Water Quality-based Toxics Control" and "Permit Writer's
Guide To Water Quality-based Permitting For Toxic Pollutants."
The procedures contained in this document are not mandatory and are intended
to be suggestions for approaching the problem of effluent toxicity reduction.
This document is expected to be revised periodically to reflect advances in
the area of toxicant characterization.
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ABSTRACT
This manual describes procedures for characterizing the physical/chemical
nature of causative toxicants in effluents. Also included are guidelines on
laboratory safety, quality assurance, equipment and reagents, dilution water,
effluent sampling and holding, toxicity testing, and data interpretation and
utilization.
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CONTENTS
Forward Ill
Preface i v
Abstract v
Fi gures vi i
Tabl es vi i i
Acknowledgments ix
1. Introduction 1
Background 1
Technical Approach to Conducting Toxicity Reduction
Evaluations 1
2. Health and Safety 6
3. Quality Assurance 7
4. Facilities and Equipment 10
5. Control and Dilution Water 11
6. Effluent Sampling and Handling 12
7. Toxicity Tests 15
Test Organisms 15
Test Endpoints 16
Acute Toxicity Tests 16
Physical/Chemical Measurements 19
8. Phase I Toxicant Characterization Tests 22
Baseline Effluent Toxicity Test 24
Degradati on Test 26
Filtration Test 29
Air Stripping Test 30
Oxidant Reduction Test '.... 37
EDTA Chelation Test 39
Solid Phase Extraction Test 41
9. Data Analysis 50
Overall Interpretation of Results 50
Variability Analysis in Phase I Testing 50
Selected References 52
Append! ces 54
A. List of Required Equipment 55
vi
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FIGURES
Number Page
1. Flow Chart of Suggested Effluent Toxiclty Reduction Evaluation ... 3
2. Example of Page From Sample Log Book 14
3. Example of a Graphical ETso Calculation For an Effluent Sample ... 18
4. Phase I Effluent Characterization Tests 23
5. Example of Data Collection Sheet For Baseline Effluent Timed
Lethality and Definitive Acute Toxicity Data 25
6. Example of Data Collection Sheet For Background-Light Or -Dark
Effluent Timed Lethality and Definitive Acute Toxicity Data 27
7. Flow Chart of Degradation Test (Background Effluent) 28
8. Effluent Timed Lethality and Definitive Effluent Filtration
Test 31
9. Flow Chart for Filtration Test 32
10. Flow Chart for Air Stripping Test 33
11. Example of Data Collection Sheet For Effluent Air Stripping
Test 35
12. Fl ow Chart for Reduction Test 38
13. Example of Data Collection Sheet For Effluent Reduction Test 40
14. Flow Chart for Chelation Test 42
15. Example of Data Collection Sheet For Effluent Chelation Test 43
16. Row Chart for Effluent Solid Phase Extraction Test 45
17. Example of Data Collection Sheet For Effluent Solid Phase
Extraction Test, Part I 47
18. Example of Data Collection Sheet For Effluent Solid Phase
Extraction Test, Part II 48
vii
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TABLES
Number Page
1. Chemical and physical test data 21
viii
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SECTION 1
INTRODUCTION
Background
This document provides NPDES permittees with procedures for analyzing the
nature of effluent toxicity. It is intended for use by those permittees
having difficulty meeting their permit whole effluent toxicity limits or
permittees required, through special conditions, to reduce or eliminate
effluent toxicity.
The regulatory basis for these requirements is described in EPA's "Policy for
the Development of Water Quality-Based Permit Limitations for Toxic Pollutants"
(Federal Register Vol. 49, No. 8, March 9, 1984). This national policy
recommends an integrated approach for controlling toxic pollutants that
utilizes whole effluent toxicity testing to complement chemical-specific
analyses. The use of whole effluent toxicity testing is necessitated by
several factors including a) the limitations presented by chemical analysis
methods, b) inadequate chemical-specific aquatic toxicity data, and c) inability
to predict the aggregate toxicity of chemicals in an effluent.
As a result of the increasing use of toxicity limits and biomonitoring condi-
tions in permits, a number of unacceptable effluent toxicity problems have
been, and continue to be, identified. To rectify these violations, permittees
are being required, through permit conditions and administrative orders or other
enforcement actions, to perform effluent Toxicity Reduction Evaluations (TRE).
The object of the effluent Toxicity Reduction Evaluation is to determine what
measures are necessary to control the effluent's toxicity to acceptable levels
The conventional TRE approach of identifying the priority pollutants in an
effluent and comparing their concentrations to literature toxicity data
frequently fails to ascertain the true cause(s) of toxicity. This ts often
the case with toxic municipal effluents and complex industrial wastewaters.
More complete chemical analysis of the effluent, identifying nonpriority
pollutants as well as priority pollutants, often results in lengthy lists of
tentatively identified chemicals for which no corresponding aquatic toxicity
data exist. It is possible, however, to focus the search for the source(s)
of effluent toxicity.
Technical Approach to Conducting Toxicity Reduction Evaluations
In any effluent TRE, there are two questions that must be answered before
measures can be taken to reduce effluent toxicity. First, the variability
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associated with the compounds that are causing effluent toxicity (causative
toxicants) must be assessed. Because toxicity is a generic measurement, the
same compound may not be responsible for effluent toxicity over a period of
time. For this reason, it is crucial that causative toxicant variability be
addressed prior to implementing effluent toxicity control measures.
In addition to the question of causative toxicant variability, the physical/
chemical characteristics of the causative toxicants need to be determined.
This information is necessary to choose the analytical method appropriate for
measurement of the causative toxicant, or alternatively, to select an effluent
treatment method for toxicant removal.
Figure 1 represents an idealized, schematic representation of an overall
effluent toxicity reduction process designed to answer these two questions.
Because all NPDES discharges are unique in nature, no single effluent TRE
procedure will apply to every case. In the approach outlined in this document,
there are two generic phases of study. The first, or screening phase, is
conducted to isolate and characterize certain physical/chemical properties of
the effluent toxicant(s) using a parallel series of relatively simple, low-cost
analyses. Each characterization test is designed to remove or render biolog-
ically unavailable a specific group of toxicants such as oxidants, metals,
nonpolar organics and metal chelates. Aquatic organism toxicity tests per-
formed on the effluent prior to and after the characterization treatment,
indicate the effectiveness of the treatment and thus provide information on
the nature of the toxicant(s). By repeating the series of toxicity character-
ization tests using samples of a particular effluent collected over a period
of time, these screening tests will also provide valuable information on the
variability associated with the type of compounds causing the effluent's
toxicity.
With completion of the initial phase of study, the investigator will have an
indication of whether effluent toxicants are likely to be cationic metals,
nonpolar organics, ammonia, and/or chlorine. Information on certain physical/
chemical aspects of the effluent toxicants (such as filterability, degradability,
volatility, and solubility) will also result from Phase I studies. The
permittee has the choice of two directions in the second phase of testing:
toxicant treatability studies or toxicant identification /source investigation
and control studies. The choice of Phase II study options is influenced by
Phase I results.
In the first option, given the information gathered in Phase I, the permittee
will have an indication of which treatment methods (Phase II, option 1) should
remove the causative toxicant(s) from the effluent. The physical/chemical
information gathered in Phase I will enable the investigator to choose the
treatment options best suited to effluent toxicants. Bench-scale studies are
used to evaluate the feasibility of treating effluent toxicity on a large
scale. In this option, the actual identity of the causative toxicant(s) is
not required; it is only necessary that enough information be available on
the toxicants' physical/chemical characteristics to predict which treatment
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Phase I
effluent samples
v v v v v v
causative toxicant characterization tests
-variability associated with causative toxicant(s)
-physical/chemical nature of causative toxicant(s)
Phase II
Option 1
I
v
bench scale and
pilot plant
effluent toxicity
treatability study
implementation
of treatment
(Option 3)
(Combination of
Options 1 and 2)
Option 2
chemical analysis method
toxicant identification
source investigation
source control
-spill control
-process modification
-substitution of
raw materials
-pretreatment
-treatment
Phase III
v
post control monitoring
-chemical
-biological
Figure 1. Flow Chart of Suggested Effluent Toxicity Reduction Evaluation.
3
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options should be studied. An analogy can be made to the approach taken
with biological oxygen demand (BOD). Treatment systems are designed to
remove BOD with little or no knowledge of the actual chemicals composing BOD.
Treatment technology is based on the nature of BOD rather than on the actual
BOD constituents.
The second option in Phase II, the toxicant identification/source investigation
and control option (Phase II, option 2) involves several steps, all of which
rely on tracking the toxicity of the effluent fractions throughout the procedure.
Although potential effluent toxicants are partially isolated in the first
phase of the study, further separation of the toxicants from other compounds
present in the effluent at nontoxic levels may be necessary. A number of
techniques are available to further reduce the number of compounds in the
toxic fraction of the effluent. These techniques will be described in the Phase
II-Toxicant Identification/Source Investigation and Control Manual. Once the
toxicants have been adequately isolated from the other compounds in the
effluent, the causative toxicant confirmation step may proceed.
One of the primary methods available for identifying the causative toxicant
utilizes the correlation between effluent toxicity and causative toxicant
concentration as both vary over time. A number of other techniques, detailed
in the Phase II Manual, can also be used in conjunction with the toxicity/
toxicant correlation procedure to further confirm the identity of the causative
toxicant(s).
If the causative toxicant can be identified in an effluent, it can be tracked
through the process line or effluent collection system to its source using
chemical analyses. Testing the toxicity of individual process or sewer lines
in order to trace effluent toxicity to its source is not recommended because of
the generic nature of the measurement. In effect, many concentrated waste-
streams may be toxic prior to dilution with the influent. It is likely to be
very difficult, however, to determine which, if any, of these wastestreams is
the source of the final effluent's toxicity. In addition, some of the influent
toxicants may be removed by the wastewater treatment operations. In other
words, the primary compounds causing influent toxicity are not necessarily
the compounds causing the toxicity of the final effluent.
Following identification of the source of the effluent toxicant, its
concentration in the effluent can be controlled using process modification,
substitution of raw materials, pretreatment of individual wastestreams, ef-
fluent treatment, and/or best management practices for spills, leaks, runoff,
etc. Post-control measures should include chemical specific monitoring of
the causative agent to insure that its concentration is below toxic levels,
and biomonitoring to insure that all other toxicants have also been adequately
addressed in the TRE.
Certain site-specific factors may make one or the other of the Phase II
options more attractive. Permittees planning to construct new treatment
facilities may wish to follow the toxicant treatability option. When
construction of additional treatment units is not an option due to cost or
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space constraints, toxicant identification and source control may be the only
viable alternative. In some cases the discharger may be faced with a single
option for control of effluent toxicity regardless of the initial choice of
Phase II approaches. As a result of the findings in the first or second
Phase of the TRE, the required control option may become obvious. For
example, the treatability approach will have to be followed if the toxicant
source cannot be adequately controlled, as is the case with municipal discharges
containing toxic levels of chlorine, ammonia, or household chemicals such as
pesticides. Industries unable to substitute raw materials or modify processes
in order to remove causative toxicants may also need to utilize treatment
methods for toxicant control. In the case of effluents exhibiting extreme
variability in the causative toxicant(s), treatment of effluent toxicity may
be the only reasonable choice, provided that the various compounds causing
toxicity are similar enough in their physical/chemical properties to be
removed by a single treatment process. In the same vein, if toxicant
identification studies reveal that a large number of physically/chemically
similar compounds are causing effluent toxicity, treatment of effluent toxicity
may be more attractive than toxicant source investigation and control.
Alternatively, if the large number of chemicals causing effluent toxicity are
extremely diverse in their treatability characteristics, it may be more cost
effective to identify and remove each at its source as opposed to relying on
multiple treatment units. Source control is also necessitated when economical
treatment techniques are incapable of removing the causative agents to nontoxic
levels. The economic feasibility of removing the causative agents to levels
necessary to achieve compliance with permit toxicity limits is likely to play
a major role in the decision to choose the toxicant identification, source
investigation and control option.
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SECTION 2
HEALTH AND SAFETY
Dealing with effluents of unknown composition is the nature of toxicity
reduction evaluations. Because the analyst cannot be aware of all the
possible toxicants in an effluent, extreme caution should be taken when
collecting and working with samples for effluent toxicity reduction evaluations.
In addition to the human pathogens potentially found in effluents containing
human wastes, threshold and nonthreshold toxicants may also be present in
wastewater samples. Because of the possibility of effluent contamination
with mutagenic, carcinogenic, and/or teratogenic substances, exposure to the
wastewater during collection and use in the laboratory should be minimized so
as to reduce to the greatest extent possible worker risk. Exposure via
inhalation and dermal adsorption can be prevented through the use of proper
safety equipment such as plastic gloves, laboratory aprons or coats, safety
glasses, respirators, and laboratory hoods. Use of a laboratory hood is
especially important for the effluent air-stripping characterization procedure
(Section 8) because of the potential release of volatile carcinogenic compounds,
hydrogen sulfide, hydrogen cyanide, etc. Further guidance on health and
safety for toxicity testing has been published by Walters and Jameson (1984).
In addition to taking precautions with effluent samples, a number of the
reagents commonly used during Phase II toxicant identification studies are
known or suspected human or mammalian carcinogens. Analysts should familiarize
themselves with safe handling procedures for these chemicals and with Office
of Health and Safety Administration regulations (DEHW, 1977, OSHA, 1976).
Use of these compounds may also necessitate specific waste disposal practices
and at the very least should prompt an effort to minimize the volume of
contaminated waste produced in the laboratory.
Other standard laboratory safety procedures should also be followed. These
include use of ground-fault interrupters in wet laboratories, laboratory
decontamination procedures, properly working safety equipment, etc. The
Selected Reference Section of this manual cites a number of laboratory safety
manuals (ACS, 1979, EPA, 1977).
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SECTION 3
QUALITY ASSURANCF
A successful effluent toxicity reduction evaluation is dependent upon a
strong quality assurance program. As a minimum, laboratories undertaking
effluent toxicity reduction evaluations must demonstrate' competency in
performing the chemical and biological tests required for the study. In the
first phase of the TRE laboratories must be capable of performing simple
chemical/physical manipulations on the effluent and acute toxicity tests with
aquatic organisms.
The second phase of the studies requires personnel trained in more specific
areas of chemistry and engineering. Depending on the Phase II option chosen
and the nature of the toxicant(s), high pressure liquid chromatographs, gas
chromatographs, mass spectrometers and/or atomic absorption analyzers may
need to be employed.
As one proceeds through the steps of an effluent toxicity reduction evaluation,
test results become more definitive in nature. In order for TREs to he
cost-effective, the results of early tests are more nonspecific and tentative
in nature. The strategy is reflected in the use of screening toxicity tests
with single species in the first phase as compared to multiple species toxicity
tests resulting in defined endpoints with confidence intervals in the second
phase.
It is important to stress that effluent toxicity is "tracked" through the
stages of a TRE using aquatic organisms. Such tracking is imperative if
accurate characterization and control of effluent toxicants is to he achieved.
For this reason, system blanks are used extensively throughout the TRE to
identify toxic artifacts added during effluent manipulation. In no case
should sample manipulation cause the effluent to become more toxic, with the
exception of tests intended to make the effluent more toxic (see Section 8,
Air Stripping Subsection Note). By preparing the system of blanks and controls
discussed in Section 8, and comparing their toxicity against the unaltered
effluent samples, the validity of the test results can be assessed. Because
effluent toxicity is the only parameter measured in Phase I, the introduction
of nontoxic artifacts to the effluent during the characterization process is
not critical, provided that they do not have an affect on the activity of the
causative toxicants.
Potential sources of toxic artifacts in Phase I include the reagents added
and their contaminants, excessive ionic strength resulting from the addition
of acids and bases, contaminated air sources used in effluent stripping
experiments, contaminants leached from filters and SPE columns, etc. Oil
sealed air compressors should not he used in the air stripping test or to
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increase sample dissolved oxygen. Instead, simple aeration devices, such as
those sold for use with aquariums, should be used. Caution should also be
taken to prevent contamination of the laboratory air (for example, from
building pesticide use or fumes from other laboratories). In addition to not
creating toxic artifacts, effluent manipulations must not spuriously remove
the causative toxicants. An example of this situation occurs when an effluent
sample containing organic toxicants is filtered through a filter coated with
an organic binder. The resulting decrease in effluent toxicity would appear
to result from the removal of filterable materials. In reality, however,
effluent toxicity is reduced as the result of toxic organic compounds in the
sample absorbing to the organic based filter, thus being removed from the
solution. Such occurrences lead to incorrect characterization of effluent
toxicants.
Other standard laboratory practices can also protect the quality of the
results. All test chambers should be covered to prevent contamination by
dust. If disposable test chambers are used, a control should be provided for
each lot. This is also true for Cja SPE columns. All glassware used in
toxicity testing should be washed with detergent, and sequentially rinsed in
10% nitric acid (to remove trace metals), hexane, acetone (to remove trace
organics), and finally high quality water. Glassware may be highly
contaminated with metals, therefore, any glassware being used in the TRE for
the first time should be soaked for three days in 10% nitric acid. Other
quality control checks routinely followed include an assessment of sample
holding time and conditions on effluent toxicity.
In performing toxicity tests for TREs it is desirable to hold all of the
parameters potentially affecting effluent toxicity constant, so that the the
toxicity of the effluent is the sole variable. This can be accomplished by
maintaining the test solution temperature and dissolved oxygen level within a
specified range. In addition to the effluent toxicity tests, a toxicity test
on control and dilution water should accompany each round of effluent tests.
As a rule, control mortality must not exceed 10% in 96 hours. All resulting
data should be graphed prior to analysis.
Standard reference toxicant tests should be performed with the aquatic test
organisms on a regular basis and accompanying control charts should be
developed (Peltier and Weber, 1985). These tests should be performed monthly
or to coincide with the characterization tests. If test organism cultures
are not maintained in the laboratory, standard reference toxicant tests
should also be performed with each group of test organisms received. Test
organisms should be positively identified to species, free from disease and
have no prior exposure to pollutants. Organism mortality during holding
should be minimal and a sensitive life stage should routinely be chosen for
testing.
If the toxicant identification option of Phase II is chosen, a more detailed
quality control program is required. Field replicates to validate the precision
of the sampling technique and laboratory replicates to validate the precision
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of the analysis should be included in the Phase II QA/QC program. In this
phase of the TRE, alterations to the effluent cannot result in toxic contaminants,
nor can they interfere with chemical analysis of the causative toxicants
(either through addition or masking). Individual chemical analysis methods
should be consulted for appropriate clean up procedures, sources of interference,
specific quality control procedures, etc. HPLC-grade solvents and ACS-grade
chemicals should be used in all analyses. System blanks (blank samples
carried through procedures and analyses identical to those performed on the
sample) must be provided. Calibration standards and spiked samples must also
be included in the laboratory quality control program. Because effluent
toxicity will eventually be correlated to causative toxicant concentration,
spiking experiments are extremely important in determining method recovery
for the causative pollutant.
Use of standard reference toxicant tests in Phase II is more critical than
in Phase I. Because the test organism also acts as an analytical detector in
the correlation of effluent toxicity with toxicant concentration, it is
important to assure that the sensitivity of the species being used is relatively
constant over time. Standard reference toxicant tests will provide information
on the uniformity of the test organism's response during the effluent testing
period.
Records keeping is a very important aspect of quality assurance. Toxicity
reduction evaluations required by State or Federal pollution control agencies
may require that some or all records be submitted for overview.
As a final note on the quality of toxicity reduction evaluations, all of the
Phase I characterization tests should be performed, even if it is unlikely
that the particular group of toxicants is present in the wastewater. Because
of the generic nature of the toxicity measurement and the inability to analyze
every compound potentially present in complex effluents, it may be advantageous
to undertake TREs without any preconceived notions as to the cause of an
effluent's toxicity.
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SECTION 4
FACILITIES AND EQUIPMENT
The facilities, equipment and reagents needed to perform an effluent toxicity
reduction evaluation will depend on the phase of the study. A list of the
equipment required for the Phase I characterization tests can be found in
Appendix A. The facility and equipment needs in the second phase of the TRE
will be site specific and will depend both on the physical/chemical
characteristics of the causative toxicants and on the choice of Phase II
approach. If the Phase II treatment option is chosen, bench scale treatment
equipment and/or pilot plant facilities will be required. The Phase II
toxicant identification option may require a range of relatively complex
pieces of equipment including gas chromatographs, mass spectrometers, high
pressure liquid chromatographs, atomic absorption spectrometers and/or
inductively coupled plasma atomic emission spectrometers.
Because of the need to study the effluent over a period of time, on-site
effluent toxicity reduction evaluations are generally not feasible. Permittees
with on-site laboratories may be able to conduct certain portions of the TRE
provided that personnel are skilled in the required methods. In most cases,
effluent samples may need to be sent to one or more laboratories for testing.
Permitees opting to conduct Phase I studies on-site should have test organism
culturing facilities (as described in Peltier and Weber, 1985) or have access
to a commercial source of test organisms. In-house culturing of the primary
species of test organism is desirable because of the need for early life
stages of the test organisms. Other test organisms used in multiple species
toxicity tests (to confirm effluent toxicant control measures) can be obtained
from commercial suppliers.
The equipment requirements of Phase I are generally standard as a result of
the generic nature of the characterization phase. When choosing laboratory
equipment for toxicity tests, some general rules should be kept in mind.
Copper, galvanized material, lead, and brass equipment should not be used in
collecting or storing effluent or control water. Relatively inert materials
such as perfluorocarbon plastics should be used whenever possible, both to
prevent contamination and adsorption of toxicants in the effluent. Use of
these materials in Phase II studies is more crucial due to the use of trace
pollutant analysis methods and the potential for sample contamination by
phthalates, etc.
Disposable one ounce test chambers are suggested for use in all Phase I acute
toxicity tests. These chambers should be rinsed once with high quality water
prior to introduction of the test solution and should be discarded after use.
Because of the definitive nature of toxicity tests performed during Phase II
studies, test chambers recommended in the above reference or in Peltier and
Weber (1985) should be used.
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SECTION 5
CONTROL AND DILUTION WATER
The type of control and dilution water used in effluent toxicity reduction
evaluations will depend on the phase of the study. Water that is acceptable
as a control and for dilution in toxicity tests may not he acceptable for the
purpose of chemical analysis.
In Phase I, control and dilution water should be from the same source. The
main requirement is that the water have an adequate life support capacity.
Water producing significant acute toxicity (greater than 10% mortality in 96
hours) should not be used. Test organisms must be acclimated to the control/
dilution water prior to test initiation.
Effluent receiving water is not recommended for use in Phase I tests unless
it is toxicity-free. NPDES permit biomonitoring requirements may specify
the use of the receiving water for effluent toxicity testing, however, TRE
toxicity tests should be performed using a toxicity-free water. Prior to
implementing toxicity control measures, the effluent should be retested using
the receiving water to assess any possible interactions. Control and dilution
water collected from surface water sources should be collected outside the
zone of impact of point and nonpoint sources of pollution. Surface water
should be filtered prior to use (?-4 mm) and should not be stored longer than
96 hours prior to use (Peltier and Weber, 1985).
Synthetic fresh waters can also be used in Phase I studies. Table 3 in EPA's
Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms, 3rd Edition, contains formulas for the preparation of soft to hard
synthetic freshwater. Caution should be taken in using laboratory water.
Peionization processes may remove essential minerals like calcium and magnesium
and introduce toxic levels of other cations. Chlorinated tap water should
not be used due to the many potential toxicants produced by chlorination.
Dechlorinated tap water should only be used as a last resort.
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SECTION 6
EFFLUENT SAMPLING AND HANDLING
The most important factor in effluent sampling is that the wastewater sample
be representative of the discharge. The condition of the facility's treatment
system at the time of sampling must be noted by the individual collecting the
sample. This can be accomplished by comparing the effluent sample concentrations
of BOD, TSS, and other pollutants (limited in the facility's NPDES permit) to
long-term historical averages and/or permitted values for those parameters. Such
a comparison will provide an indication of the operational status of the
treatment system on the day of sampling. For industrial discharges, the
production level, as it relates to treatment capacity, should be considered
when determining the representativeness of a sample.
One of the most important aspects of collecting a representative sample is
timing. Daily and seasonal changes in effluent quality, as well as those
resulting from process changes, must be considered prior to sampling. In
some cases, the frequency and location for sampling may be dictated by the
NPDES permit or by an administrative order. If not specified, the sample
should be collected at the point of discharge. Testing the toxicity of
individual wastestreams for the purpose of effluent toxicity reduction evalua-
tion is not recommended, particularly in the initial characterization phase.
Such testing is likely to reveal some toxicity in all wastestreams (due to
their concentrated nature) and will not indicate the source of the final
effluent's toxicity. In-plant wastestream analyses should only be carried out
in Phase II studies: 1) after characterization tests have suggested the effluent
toxicity can be controlled using wastestream pretreatment or 2) once the
causative toxicant can be traced in the process line or collection system,
using a chemical analysis method, to its source. Municipal samples should be
collected after chlorination in order to assess the toxicity of any compounds,
such as chloramines, created during the disinfection process.
If possible, flow proportioned composite samples of effluent should be analyzed
during Phase I to provide an integration of the toxic components in the effluent
over time. Unfortunately, such an integration frequently leads to dilution
of toxic spikes to nonlethal levels. If acute toxicity is not seen in composite
effluent samples, grab samples should be collected. While this type of discrete
sampling may provide maximum effluent toxicity, it is more difficult to catch
these peaks in effluent toxicity. Grab samples should be used in Phase II in
order to maximize the possibility of collecting samples with a wide range of
causative toxicant concentrations. A wide range in causative toxicant concen-
trations among effluent samples will allow the correlation of effluent sample
toxicity with effluent sample causative toxicant concentration. Such a correla-
tion can be used to confirm the cause of effluent toxicity. Peltier and Weber
(1985) have discussed the advantages and disadvantages of grab and composite
sampling and have also detailed methods for sampling intermittent discharges.
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Sampling methods must not introduce additional toxicants or compounds that
will interfer with chemical analyses. The sample must not be allowed to
degrade, thus changing the toxicity or chemical make-up of the wastewater.
If the TRE analyses are not conducted on-site, samples should be shipped on
ice to the testing location. Effluent samples should not be filtered unless
it is necessary to remove other aquatic organisms. Sample filtration could
effect the results of the characterization tests, one of which entails filter-
ing the effluent. Sample aeration should be minimized during collection and
transfer. Testing should begin within 36 hours of effluent sampling and in
no case should a sample older than 72 hours be used for toxicity reduction
evaluation. The Phase II toxicant identification approach may require specific
types of sample containers or the addition of preservatives to the sample. For
Phase I testing, slightly under 2 liter of effluent is needed for analysis
when invertebrates are used for testing. Approximately 2.1 liter of effluent
are needed for characterization tests with fish. A 3 to 4 liter (or 1 gal.)
sample of effluent is recommended to cover sample loss through spills, etc.
An example of an effluent sample data sheet is shown in Figure 2.
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Sample Data Sheet
Sample Log #: Sample Type
Grab
Facility: Collected: AM/PM
Date:
NPDES #: Composite:
Collected From: AM/PM
Location: Date:
To: AM/PM
Contact: Date:
Phone Number: Dilution Water:
Investigator: Control Water:
Condition of treatment system at time of sampling:
Comments:
Figure ?.. Example of Page From Sample Log Rook.
-------
SECTION 7
TOXICITY TESTS
Test Organisms
To determine the toxicity of effluents to aquatic life, standardized, cost-
effective methods for measuring acute and chronic toxicity have been developed
by EPA (Peltier and Weber, 1985). Toxicity tests using aquatic organisms are
utilized throughout the toxicity reduction procedure. In these tests, the
aquatic organism acts as the "detector" for chemicals causing effluent toxicity.
For this reason, it is critical that the test organism species be sensitive
to the toxicants in the effluent. Prior to determining the cause of an
effluent's toxicity, it is difficult to judge which aquatic species will be
the most sensitive detector of effluent toxicants. The most obvious candidate
for test organism is the identified sensitive species used to initially detect
the effluent's toxicity (through permit or 308 letter monitoring requirements,
etc.).
In addition to sensitivity, other factors, such as availability and cost must
also be considered when choosing a test organism. Because a large number of
toxicity tests will be performed during the course of the study, the preferred
organism should be easily cultured throughout the year and relatively small so
as to minimize the space requirements of the Phase I study. EPA research
personnel have been very successful in utilizing Cladocerans, particularly
Ceriodaphm'a species, in toxicity characterization studies. Toxicity tests
using these organisms require only small volumes of sample and culturing
techniques are well documented (Horning and Weber, 1985, Weber and Peltier,
1981).
To prevent unnecessary problems associated with salinity changes, the use of
marine organisms should be avoided if possible in effluent characterization
studies. Effluents found to be toxic to marine species should be retested to
determine whether use of a freshwater organism will provide adequate sen-
sitivity. If an effluent discharged to a freshwater body is high in total
dissolved solids (as may be the case with many industrial discharges), it may
be helpful to use a marine species as the test organism until effluent salinity
problems can be resolved. Use of a marine species will allow the detection
of toxicants other than salts (see Physical/Chemical Measurements Subsection).
In order to be cost-effective, throughout the initial stages of the evalua-
tion, the use of one species of test organism will be sufficient, provided
that it is relatively sensitive to the range of toxicants encountered in the
effluent. If more than one species has a demonstrated sensitivity to ef-
fluent toxicants, the species exhibiting the lowest effect level (LCsg)
should be chosen for Phase I studies, provided that the species meets the
15
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criteria previously described. In cases where use of the most sensitive
species is not feasible, other species with demonstrated sensitivity to
effluent toxicants can be used. Caution must be exercised in cases where
the most sensitive test species changes over time for a particular effluent.
A change in the relative sensitivity of test organisms to a particular effluent
over time indicates that more than one causative toxicant is present in the
effluent. In these cases, it may be advantageous to use each species exhibiting
sensitivity to the effluent. In the latter stages of the evaluation, i.e.,
during effluent toxicity treatability studies or following toxicant identifi-
cation, toxicity tests should be performed with a number of aquatic organism
species. To insure that an effluent is not toxic to aquatic organisms, EPA
recommends that a minimum of three test species (fish, macroinvertebrate, and
plant) be used in post-control toxicity testing (USEPA, 1985). Effluents
discharged to marine or estuarine waters should be tested with saltwater
species during the final phase of the TRE to insure that all effluent toxicants
have been removed.
Test Endpoints
Characterization tests described in Phase I can be used to study acutely
toxic effluents. Phase I procedures specifically designed to address effluents
exhibiting only chronic toxicity are currently being developed.
Acutely toxic effluents are more easily studied due to the inconveniences and
expense associated with the longer periods required for chronic toxicity tests.
Some of the undesirable factors associated with longer test periods include
the potential for dissolved oxygen problems, the necessity for larger effluent
samples and test volumes, and test organism feeding requirements. If significant
mortality (i.e. lethality in significant excess of observed control lethality)
typically occurs in 100% effluent within 96 hours, acute toxicity tests can
be used in the characterization phase of the study. The decision of whether
acute toxicity tests can be used in the characterization phase of the TRE
should be based on past toxicity test results for the given effluent. As a
final confirmation step in Phase I, the characterization procedures used to
remove or reduce acute effluent toxicity should also address the chronic
toxicity of the effluent.
Acute Toxicity Tests
Two types of static tests are used in effluent acute toxicity reduction
evaluations: definitive tests resulting in LCsgS and timed lethality tests.
Static timed lethality tests are used more frequently in the screening tier
of the TRE, while definitive tests are used extensively in the Phase II
study.
16
-------
In a definitive toxicity test resulting in an LCsg, the relative toxicity of
the effluent is measured and expressed in terms of the concentration necessary to
cause a specified response (50% mortality in exposed test organisms) within a
given time period. In effect, the exposure period is held constant while the
concentration of effluent that the organisms are exposed to is the variable.
A number of methods for estimating the LCijg from acute toxicity data are described
in "Methods For Measuring the Acute Toxicity of Effluents to Freshwater and
Marine Organisms, 3rd Edition" (Peltier and Weber, 1985). In order to minimize
the time and cost of TREs, test conditions used to establish the LCsg of
effluent samples deviate somewhat from the methods specified by Peltier and
Weber. It is stressed that the methods described in this Section cannot be
used for the purpose of demonstrating compliance with NPDES permit toxicity
limits.
In timed lethality tests, the concentration of effluent that the organisms
are exposed to is held constant (100%) and time of exposure is the variable.
The relative toxicity of the effluent is measured in terms of the time it
takes to elicit a given response (lethality) from the exposed test organisms.
The relative toxicity of an effluent used in a timed lethality test is expressed
as the median lethal time (ETsg). To calculate the ETsg, test organism mortality
is observed at time intervals of approximate geometric or logarithmic progession:
i.e., 10, 15, 30, and 60 minutes; 2, 4, 8, 24, 48, and 96 hours. The percentage
mortality, plotted on a probit scale, is graphed against the exposure time,
plotted on a logarithmic scale. A straight line is fitted to the graph
points and the median lethal time is found at the point where the regression
line crosses 50% mortality as shown in Figure 3. (National Academy of Science,
1972). Note that an ETsg can be calculated using data from a definitive
toxicity test resulting in an LCsg, provided that frequent lethality readings
are taken for the 100% effluent dilution.
To perform a timed lethality test, five young organisms of approximately the
same age are randomly placed in the test solution contained in a 30 ml (1
oz.) container. A definitive acute toxicity test resulting in an LCsg is
performed in a similar manner using serial dilutions (eg. 100%, 50%, 25%,
12.5%, 6.25%, etc.) of the test solution. The optimum life stage, maximum
age difference within a test group, and test temperature for recommended test
species is described by Peltier and Weber (1985). Fish and relatively large
invertebrates (>lmm) are transferred with a wide bore pipette or dip net to
10 ml of test solution; smaller invertebrates, such as daphnids are transferred
with an eyedropper to 5 ml of test solution. Because the volume of test
solution is small, care must be taken to minimize the volume added during
test organism transfer. At least one replicate of each test solution should
be examined to assure the quality of test results. Test organisms must also
be exposed to control and blank solutions as described in Sections 8.
As previously discussed, for the purpose of calculating an ETsg, observations
of test organisms should be made approximately every twenty minutes for the first
hour, every two hours thereafter for eight hours and at twenty-four and forty-
eight hours. In both the timed lethality and definitive toxicity tests, if
death does not occur within 48 hours, the test may be continued to 96 hours.
17
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981 '
95 -
90
0.1
1 10
Exposure (Hours)
100
Figure 3. Example of a Graphical ET5g Calculation For an Effluent Sample,
18
-------
It is desirable however, to avoid longer test periods, especially in the
early stages of the study, because of the many inconveniences (such as feeding,
D.O., evaporation of test solution, etc.) associated with the longer tests.
The criterion employed in establishing test organism death is lack of movement
of the body or appendages upon gentle prodding. Other general observations
that should be recorded include appearance and behavior such as erratic
swimming, loss of reflex, paralysis, discoloration, molting, condition of
young produced, etc. Such observations may provide valuable information on
the type of compound causing toxicity. For example, test organism paralysis
may indicate the presence of a neurotoxin, such as an organophosphate pesticide
(Doull, et al., 1980). This information may be especially helpful when
ruling out, as the causative toxicant, various compounds contained in the
effluent fraction exhibiting toxicity.
Physical/Chemical Measurements
Meaningful effluent characterization results are also assured through
physical and chemical measurements (Table 1). In timed toxicity tests, the
pH and temperature should be measured in at least one replicate of each test
solution and the control and/or blank at the beginning of the test and
every 24 hours thereafter. In definitive tests resulting in LCsgs and NOELs,
these measurements should be recorded daily for the control solutions and the
highest effluent concentration tested. Test solution pH should remain between
6.0 and 9.0. It may be necessary to acclimate the test organisms to effluent
sample pH if it differs from culture water pH by more than 0.5 standard
units. Acclimation techniques are described by Peltier and Weber (1985).
Sample temperature should be maintained within +_ 2°C of the temperature
recommended for the particular species. The dissolved oxygen should be measured
frequently during the first day of testing (at 2, 4, and 8 hours) and daily
thereafter in the control and one replicate of the highest concentration of
baseline and background effluent tested (see Section 8 for definitions).
The dissolved oxygen should be maintained at greater than 40% saturation (60%
if cold water species are used). While it is unlikely that the dissolved
oxygen concentrations will present a problem because of the large surface to
volume ratio provided by the test container, gentle aeration can be used to
increase sample D.O. if necessary.
Total alkalinity, total hardness, and salinity or conductivity are measured
in the 100% background and baseline effluent and in the control at the beginning
of the test. The ionic strength of the effluent, as measured by conductivity
or salinity, must be in the physiologically acceptable range for the particular
species of aquatic test organism being used. As a rule of thumb, for freshwater
species, the conductivity of the sample should be less than 5,000 umho/cm
and the salinity should be less than 3 ppt (Martin Roll, Florida Dept. of
Environmental Regulation, personal communication).
19
-------
In some cases, effluent pH or ionic strength may be indicated as the cause
of toxicity. Additional testing should be performed, however, to insure
that no other toxicants are present in the effluent sample. For example,
an effluent with a high conductivity may be toxic to marine species as well
as freshwater species if a heavy metal rather than sodium chloride is the
source of the high conductivity reading. Toxicity tests can be conducted on
these samples following neutralization of acidic or basic effluents or by
using marine species to test saline wastewaters. Note that it may be necessary
to further increase sample salinity to a value within the acceptable range
of the marine species being tested.
Other test conditions and procedures used (e.g. loading, illumination,
feeding, culturing, etc.) should be in accordance with the static acute
toxicity test procedures described by Peltier and Weber (1985) unless otherwise
specified.
20
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Table 1. Chemical and Physical Test Data1.
-------
SECTION 8
TOXICANT CHARACTERIZATION TFSTS
In the first phase of a toxicity reduction evaluation, simple toxicity
removal analyses are performed in a parallel series, using the whole effluent.
Toxicity tests utilizing aquatic organisms are used to determine whether
specific groups of chemicals are present in the effluent in toxic concentrations
Two objectives are accomplished during the screening phase; a) the physical
and chemical characteristics of effluent toxicant(s) are broadly defined,
and, b) by performing these experiments on effluent samples collected over a
period of time, the variability associated with the type of toxicant(s)
causing test organism toxicity is addressed. This information can subsequently
be used in the second phase of the study, either in the development of
bench-scale wastewater treatment processes or in choosing analytical procedures
for toxicant identification.
Upon identifying a toxic effluent, a sample for toxicity reduction evaluation
should be collected and transported according to the methods described in
Section fi. Initially, it may be advantageous to collect only one sample at a
time for analysis. As toxicity characterization proceeds, multiple samples
should be collected throughout a period during which the effluent is expected
to exhibit the most variability in its chemistry. Results from the analyses
of these samples will define toxicant-type variability and can also aid in
identification of the causative agent. Analysis of samples should begin as
soon possible following collection. Samples older than 36 hours or those
improperly handled during transport to the laboratory (e.g. allowed to warm-up)
should be replaced.
Each of the following characterization tests is designed to remove or
neutralize a specific pollutant or group of pollutants which may cause
effluent toxicity (Figure 4). Toxicity tests performed on the effluent
prior to and following the characterization treatment will indicate which
groups of toxicants are present in lethal amounts. All but one of these
tests are performed in parallel in order to prevent potential sample
degradation over time. While it is not critical that each analysis be
started at exactly the same time, the toxicity tests performed with the
characterization test solutions should be initiated at approximately the
same time. One species of test organism should be used throughout the
first phase of the TRE.
Note that the tests described in Section 8 are designed for acutely toxic
effluents. Modifications to these procedures will allow the study of effluents
exhibiting only chronic toxicity. The necessary modifications to these
procedures are being developed by EPA and will eventually be incorporated
in this manual.
22
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Baseline
Toxicity
Tests
Degradation
Test
Toxic Affluent Sample
Air Stripping
Test
Filtration
Test
Acid Rase Neutral
Reducing
Agent
Test
Chelation
Test
CIH Solid Phase
Extraction Test
Acid Base Neutral
Figure 4. Phase I Effluent Characterization Tests.
23
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Baseline Effluent Toxlcity Test
In order to determine the effects the various characterization tests have on
effluent toxicity, the toxicity of the effluent sample, prior to any treatment
in the laboratory, must be determined. The portion of the effluent sample
not undergoing any characterization treatment will be referred to as the
"baseline effluent". The ETso and LCso of acutely toxic effluents should be
recorded. Both of these endpoints can be calculated using the same static
acute test by simply making more frequent lethality readings in the 100%
effluent concentration. Again, the decision as to whether acute toxicity
tests are appropriate for use in the characterization phase of the TRE will
depend on past toxicity test results for the particular effluent. An acute
toxicity test on control water (Section 5) must be run in conjunction with
the baseline effluent toxicity test in order to assure the quality of the
results. A sample data collection sheet for acutely toxic baseline effluents
is shown in Figure 5.
In order to compare the baseline effluent toxicity and the toxicity of the
effluent aliquots subjected to characterization tests, all toxicity tests
must be initiated at approximately the same time. Immediately following the
sample's arrival in the laboratory, a portion of the effluent is used in each
of the characterization tests. Carrying aliquots of the effluent sample
through each characterization test will generally take up most of the first
eight hours of testing. So that early and frequent toxicity measurements can
be made, toxicity tests on the baseline effluent and effluent aliquots subjected
to the various characterization tests should be initiated on the second day
of testing. Test solutions and the aliquot of effluent sample to be used in
the baseline toxicity test should be stored under lighted conditions at room
temperature overnight.
For acutely toxic effluents, the median lethal time (ETsg) resulting from the
baseline effluent is compared to the ETsgs resulting for the aliquots of effluent
used in each characterization test. A significant difference in the ETsgs will be
demonstrated by a 100% or greater increase in the ETsg of the treated effluent
aliquot as compared to the baseline effluent ETsg. In effect, the median lethal time
must at least double following a characterization treatment. If calculation
of an ETso is not possible, timed lethality data for the baseline and treated
effluent solutions can be directly compared. Such a comparison will demonstrate
whether the removal or neutralization of various groups of toxicants lessens
or eliminates effluent toxicity. Thus, by comparing these results, an indication
of the physical/chemical nature of the causative toxicants can be obtained.
Degradation Test
In this test, the stability of the effluent toxicants will be considered.
The results of the test will indicate whether the causative toxicants in the
effluent are biodegradable, photolyzable, and/or oxidizable. In some cases, a
decrease in effluent sample toxicity over time may also be the result of pre-
cipitation of effluent pollutants. Formation of effluent sample precipitates
should be noted.
24
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Baseline Effluent Timed Lethality and Definitive Acute Toxicity Test Data
PARAMETER
Dissolved Oxygen (mg/L )
pH (std. units)
Temperature (°C)
Conductivity (uhmo /cm )/Salinity (ppt)
Total Hardness (mg/L as CaCO^ )
Total Alkalinity (mg/1 as CaCO^ )
100% Effluent rep:
TIME (hours)
0
2
X
X
X
X
X
4
X
X
X
X
X
8
X
X
X
X
X
24
X
X
X
48
X
X
X
Control rep:
TIME (hours)
0
2
X
X
X
X
X
4
X
X
X
X
X
8
X
X
X
X
X
24
X
X
X
48
X
X
X
Sample Log #
Dilution Water_
Control Water ~
# Animals/Replicate
Test Initiation
Date Time
Test Organism
Source
% Mortality
Effluent
Concentration
100% a
b
50% a
b
25% a
b
12% a
b
6% a
b
3% a
b
Control a
b
0.33
0.67
Tirm
1
? (h<
2
JUTS)
4
6
8
XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
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xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
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24
48
Other
Observations
Baseline
Baseline
Comments:
Figure 5. Example of Data Collection Sheet for Baseline Effluent Timed
Lethality and Definitive Acute Toxicity Tests.
-------
While the degradation test does not provide information that can be used in
choosing an analytical method for toxicant identification, it may provide
toxicant treatability information. It should be noted, however, that it is
unlikely that effluent samples with limited populations of microorganisms, such
as disinfected wastewaters, will undergo biodegradation during this test.
Furthermore, no significant decrease in effluent toxicity over the 24 hour
test period can be expected for highly treated effluents. Changes in the
toxicity of effluent samples over time have been observed however, even when
the samples have been stored under refrigeration. Because the majority of
the effluent aliquots will have been stored for 24 hours after the arrival of
the effluent in the laboratory, it is important to assess any change that has
taken place in effluent toxicity during this period. More importantly, the
results of the degradation test will provide valuable information on allowable
effluent holding time and acceptable effluent storage conditions. This
information is crucial in Phase II of the TRE.
Upon receiving the sample in the laboratory (day 1 of the study), four aliquots
of the effluent are used in an acute toxicity test; two of the aliquots are
tested under normal laboratory illumination, the other two portions are
tested in complete darkness. The toxicity tests performed on these aliquots
of the effluent sample will be nearly identical to the tests performed on
the baseline effluent (i.e. day 2 effluent). The only difference will be
the lighting conditions used for two of the aliquots and the age of the
effluent. These four day 1 aliquots of the sample will be referred to as the
"background" effluent (background-light, background-dark). For all practical
purposes, the sample data collection sheets for the background effluent
(Figure 6) are the same as those used to record acute toxicity data for the
baseline effluent. A flow chart for the degradation test is shown in Figure 7.
The blank for the degradation test entails performing a toxicity test in com-
plete darkness using control water (i.e. along side the "background-dark"
aliquot test). No significant mortality, as defined in Section 5, should
occur.
The results of the background effluent toxicity tests are compared to the
results of the baseline effluent toxicity test. For acutely toxic effluents,
if the baseline ETsg is at least twice as large as the background-light ET5Q,
the effluent sample toxicants are considered to be degradable. In other words,
if, on the average, the test organisms in the baseline effluent live at least
twice as long as the test organisms in the background-light effluent, wastewater
sample toxicants have degraded to nontoxic byproducts. It is also possible
that the baseline effluent ETRQ may be significantly less than the background
ETsgs (i.e. the effluent sample becomes more toxic rather than less toxic during
the 24 hour holding time). Such factors must be considered in interpreting
the results of the other toxicity tests.
If the baseline ETso is significantly greater than the background-light
the degradation process can be further characterized by including the back-
ground-dark ETso in the comparison. Similar background-light and background-dark
indicate that photolysis does not play a role in effluent sample toxicant
26
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Background Effluent Timed Lethality and Definitive Acute Toxicity Test Data
Test: LIGHT DARK
PARAMETER
Dissolved Oxygen (mg/L )
pH (std. units )
Temperature (°C )
Conduct ivity(uhmo /cm )/Salinity (ppt )
Total Hardness (mq/L as CaC(h )
Total Alkalinity (mg/1 as CaCO^ )
100% Effluent rep:
TIME (hours)
0
2
X
X
X
X
X
4
X
X
X
X
X
8
X
X
X
X
X
24
X
X
X
48
X
X
X
Control rep:
TIME (hours)
0~
2
X
X
X
X
X
4
X
X
X
X
X
8
X
X
X
X
X
24
X
X
X
48
X
X
X
Sample Log #
Dilution Water_
Control Water "
Animals/Replicate
Test Initiation
Date Time_
Test Organism
Source
% Mortality
Effluent
Concentration
100% a
b
50% a
b
25% a
b
12% a
b
6% a
b
3% a
h
Control a
b
0.33
0.67
Tim<
1
? (h(
2
jurs
4
)
6
8
XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
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xxxxxxxxxxxxxxxxxxxxxxxxxxxxx
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24
48
[Other
Observations
Background ETij0:
Light Dark
Comments:
Background
Light Dark
Figure 6. Example of Data Collection Sheet for Background-Light or -Dark
Effluent Timed Lethality and Definitive Acute Toxicity Tests.
27
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DEGRADATION TEST
day 1 effluent
fcr~— *~-^4
light dark
\.
definitive definitive
toxicity toxicity
test test
LC50 F.T50
CONTROL
control water
light dark
\
definitive definitive
toxicity toxicity
test test
LC50 ET50
Figure 7. Flow Chart For Degradation Test (Background Effluent),
28
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breakdown. If the baseline effluent is less toxic than the background-light
effluent, which is in turn less toxic than the background-dark effluent (i.e.,
background-dark ETso < background-light ET5g < baseline ETjjo), photolysis, while
not being the sole cause, does play a role in the degradation of effluent
sample toxicants to nontoxic products. If the background-light ETsg and
baseline ETso are similar in magnitude and the background-dark ETsg is much
smaller (i.e., the background-dark effluent is more toxic than the background-light
and baseline effluents), photolysis plays the major role in degradation of
effluent sample toxicants to nontoxic byproducts.
The role that oxidation and/or volatilization plays in the degradation of
effluent toxicants can be assessed by comparing the background and baseline
ETsgs to the aeration-neutral ETso (consult the Air Stripping Subsection for
further details).
Filtration Test
The filtration experiment provides information on effluent toxicants associated
with filterable material. As with the degradation test, the information
provided by the filtration test may be more useful in the toxicant treatability
option of Phase II as opposed to the toxicant identification/source investigat ^
and control option. These results may, however, provide an indication of how
the effluent sample will need to be prepared for chemical analysis (eg. solvent
extraction of filtered solids). Toxicants associated with solids are generally
not biologically available; however, the effect of filtration on effluent
toxicity must be determined because the solid phase extraction characterization
test utilizes filtered effluent. It may be advisable to use filtered effluent
in all of the characterization tests if high levels of effluent solids
interfere with toxicity tests. In such cases, it is imperative that the
effect of filtration be considered when interpreting test results.
To perform this test, the effluent sample is filtered using a glass-fiber
filter, nominal size 1.0 urn to 1.2 urn (without organic binder). The use of
glass-fiber rather than cellulose-based filters should prevent the adsorption
and loss of dissolved organic compounds from the effluent sample. Adsorption
of toxic effluent organics onto the filter could lead to spurious results.
To prepare the glass-fiber filter, place it on the membrane filter apparatus,
apply vacuum and wash it with three successive 20-mL volumes of distilled
water. Continue to apply vacuum until all traces of water have left the filter.
Following preparation of the filter, discard the washings, and wash the
suction flask as described in Section 3. To further insure that no toxic
artifacts enter the sample from the filter, pass 2X5 (invertebrates) or 10
(fish) ml aliquots of control water through a prepared filter, collect the
filtrate and use it in timed lethality tests. No significant organism mortality
(greater than 10% in 96 hours) should be noted.
29
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Under vacuum, filter 800 ml of well-mixed sample through a prepared glass
fiber filter. Transfer the filtrate to a one liter flask and repeat the
procedure with a second 800 ml of well-mixed effluent. (If filtered effluent
is to be used in all of the characterization tests, 2.5 L of effluent is
filtered). It may be necessary to change filters during this procedure
because of solids build up. Alternatively, effluent samples high in suspended
solids can be centrifuged. The supernatant is then filtered using the 1.0 or
1.2 urn glass-fiber filter.
For acutely toxic effluents, two 5 ml (for invertebrates), or 10 ml (for
fish) aliquots of the filtered effluent will be used in timed lethality
tests. The remainder of the filtered effluent will be used in the C\Q solid
phase extraction test. An example of a data collection sheet for the effluent
filtration characterization test is shown in Figure 8. If the ETsg of the
baseline (i.e., unfiltered) effluent is less than half of the filtered effluent
ET5Q (i.e., the baseline effluent is substantially more toxic than the filtered
effluent) then the sample's toxicity is at least partially caused by toxicants
associated with suspended solids. If no mortality occurs in the filtrate,
then the toxicity of the effluent sample is solely related to compounds
associated with filterable solids.
To confirm these results, filter 5 (invertebrates) or 10 (fish) ml of well
mixed effluent using a fresh filter. Transfer the solids contained on the
filter to 5 ml (invertebrates) or 10 ml (fish) of control water. The toxicity
exhibited by this sample should be similar to that seen in the unfiltered
effluent. If it -is not, dissolved toxicants in the effluent sample may have
become adsorbed on the filter (i.e., sample toxicity is not actually related
to suspended solids in the sample). The flow chart for the acute toxicity
filtration test is shown in Figure 9.
Air Stripping Test
The air stripping test is designed to determine how much of the effluent
sample's toxicity can be attributed to volatile or oxidizable compounds. The
test is performed with pH-adjusted and unadjusted effluent (Figure 10-). By
comparing the toxicity test results for aerated acidic, neutral and basic
samples, it may be possible to characterize the compounds causing effluent
toxicity.
In preparing the pH-adjusted effluent samples, precaution must be taken to
minimize changes in sample ionic strength and volume. Using a pH meter to
monitor sample pH, 50 mL of acutely toxic effluent is acidified to pH 3 by
the dropwise addition of 1.2 N hydrochloric acid (10% concentrated HC1). To
prevent excess acidification, as the pH of the sample nears 3, final adjustments
may be made using 0.12 N HC1. Another 50 ml aliquot of the acutely toxic
effluent is likewise brought to pH 11 by the dropwise addition of 1.0 and 0.1
N sodium hydroxide (NaOH). For reference purposes, the volumes of acid and
base used to adjust the effluent pH are recorded. Sample volume should not
be increased by more than 10% in order to prevent dilution of the effluent's
30
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Filtered Effluent Timed Lethality Test Data
PARAMETER
pH (std. units)
Temperature (°C)
Filtered Effluent rep:
TIME (hours)
0
24
48
Filtration Blank rep:
TIME (hours)
0
24
48
Sample Log #
Control Water
# Animals/Replicate
Test Initiation
Date
Test Organism _
Source
Time
Mortality
Filtered a
Effluent b
Filtration a
Blank b
0.33
0.67
1
Time
2
> (he
4
)urs
6
8
24
48
Other
Observations
Filtration
Comments:
Figure 8. Example of Data Collection Sheet for Effluent Filtration Test.
31
-------
FILTRATION BLANK
2 x 5 or 10 mL
control
water
timed
lethality
test
FILTERED EFFLUENT TEST
2 x 800 mL
effluent
*1580 or 1590 mL-—X^g SPE experiment
timed
x 5 or 10 mL >lethality
test
EFFLUENT
SOLIDS
TEST
10 or 20 mL
effluent
V control water timed
filter--->solids > lethality
test
timed
•>lethality
test
V
filtrate-
Figure 9. Flow Chart for Filtration Test.
32
-------
Effluent
adjust 25 ml adjust
50 mL no pH 50 mL
to pH 3 adjustment to pH 11
>4
25 ml
?5 ml
Air Stripping
25 mL
i
25 mL
I
V V V V V
-Adjust To Initial pH of Effluent-'
I I I I I
V V V V V
Timed Lethality Tests
Figure 10. Flow Chart for Air Stripping Test,
33
-------
toxicity. Depending on the individual effluent more or less concentrated
solutions of HC1 and NaOH may be needed. Twenty-five ml each of the basic
and acidic effluent is held without aeration for use as test blanks.
The two 25 ml samples of acidic and basic effluent and 25 ml of pH-unadjusted
effluent are each transferred to clean 100 ml graduated cylinders and
moderately aerated (approx. 7ml air/min.) for 60 minutes. Air pumps utilizing
oil should not be used for air stripping in order to prevent sample contami-
nation. Small aeration devices such as those sold for use with aquariums are
satisfactory. Compressed air systems can be used provided that the air is
first passed through a molecular sieve to remove any impurities. The pH of
the acidic sample is checked every five to ten minutes during aeration. If
the pH of the sample has drifted, it should be readjusted back to pH 3 (i.e.,
sample pH should be maintained at pH 3 throughout aeration). The volume of
additional acid and/or base added to the solution should be recorded. The
same volume of acid and/or base is added to the 25 mL pH 3 unaerated effluent
sample. This procedure is also carried out with the basic effluent samples
(i.e., sample pH maintained at pH 11 throughout aeration, etc.).
Following air stripping, the pH of each solution (including the 25 ml portions
of unaerated acidic and basic effluent) should be returned to that of the
initial effluent using the necessary volumes of NaOH and HC1. The pH of each
sample should be periodically checked and readjusted as necessary during the
holding period prior to toxicity test initiation (day 2). It is critical
that a stable pH be maintained prior to toxicity test initiation. Once an
acceptable stable pH has been achieved, timed lethality tests are performed
on two 5 or 10 ml aliquots of each sample. An example of a data collection
sheet for the air stripping experiment is shown in Figure 11. The average
ETso of each aerated effluent sample is compared to the ETso resulting from the
timed lethality test on the baseline effluent. Test organism mortality in the
three aerated solutions should not be greater than the mortality in the baseline
effluent or the mortality in the corresponding unaerated solutions. Higher
relative mortality in the aerated versus unaerated solutions indicates that
sample contamination via aeration has occurred or that effluent components have
become more toxic following oxidation. The ETsgs of the 25 ml samples
of acidic and basic unaerated effluent should be approximately the same as
the baseline effluent ETso ^ excessive ionic strength is not a problem.
If the toxicity reduction in all three aerated effluent samples is equivalent
(ETsos are the same), then sample toxicity may be at least partially the result
of volatile organic compounds whose volatility is unrelated to pH. This also
suggests that the Henry's Law constant for the compound is greater than 10~3
(Kavanaugh and Trusself, 1980).
To understand why different levels of toxicity reduction occurs between
acidic, basic and neutral effluent samples, one must have a basic understanding
34
-------
A1r Stripping Timed Lethality Test Data
Effluent Sample
Acidified - Aerated rep:
>H unadjusted Aerated rep:
Baslfled - Aerated reo:
Acidified - Unaerated rep:
Raslfled - Unaerated rep:
pH
Ul
0
(St
m
24
.d
48
Temj
('
0
leral
•c).
24
lure
48
Sample Log i
Dilution Water
Control Water
I Animals/Replicate
Test Initiation
Date Time
Test 0>gan1sm
Source
t Mortality
Effluent Sample
Acidified a
Aerated F"
Basified a
Aerated b
pH unadjusted a
Aerated b
Acidified a
Unaerated b
Basified a
Unaerated b
0.33
0.67
1
1
rime
2
(hoi
4
irs)
6
8
?4
48
Other
Observations
Aerated Samples
Aerated - Acid ETso:
Aerated - Neutral ET50
Aerated - Base ETjg:
Aerated Samples
Effluent pH (initial):
Unaerated Samples
Unaerated - Acid
Unaerated - Base
Effluent pH after aeration:
Adjustment*:
Acid pH**:
Base pH**:
Unaerated Samples
Acid pH:
Base pH:
lit. 1.2N HC1 added:
nt l.ON NaOH added:
rt. l.ON NaOH added:
nt 1.2N HC1 added:
nt 1.2N HC1 added:
roL l.ON NaOH added:
lit. l.ON NaOH added:
nt 1.2N HC1 added:
nt 0.12N HC1 added:
mL 0.1N NaOH added:
nt 0.1N NaOH added:
mL 0.12N HC1 added:
nt 0.12N HC1 added:
ml 0.1N NaOH added:
nt 0.1N NaOH added:
mL 0.12N HC1 added:
*nt add or based added to return aerated effluent to Initial pH
** 1) same volumes of acid and base are added to corresponding unaerated samples;
2) if resulting pHs of unaerated samples are greater than 0.5 units
from initial effluent pH, readjust to Initial effluent pH and record
total volumes of acid and base added.
Comments:
Figure 11. Example of Data Collection Sheet for
Effluent A1r Stripping Test.
35
-------
i I
of the thermodynamic equilibrium acidity constant, Ka, for the proton transfer
reaction...
HA + S = HS+ + A" Ka = 'ArjrHSl S: soivent, such as
Kg: thermodynamic equilibrium
[HAJ
^ a'
constant
e.g. HCN + HoO = H^O"1" + CM' K., =
^ J a
The stronger the acid, the greater the value of Ka, and the larger the
Ka or PKa« *n effect, the reaction is shifted to the left for acidic compounds.
For acids in water, when the pH of the solution equals the pK» of the compound,
equivalent amounts of the compound will exist in the ionized (H*, A') and
unionized (HA) forms. At a pH one unit lower than the pKa of the add, 90% of
the compound will be in the unionized form, 10% will be in the ionized form.
A solution pH two units below the acid's pKa will result in 99% of the unionized
form and 1% of the ionized form. The significant point is that, given a
certain degree of compound solubility and volatility, the unionized form of the
acid can be stripped from the solution, whereas the ionized form will remain.
Basic compounds function in a similar fashion. When the solution pH is equal
to the pKa of a base, equal amounts of the base will exist 1n the Ionized and
unionized forms. For example, ammonia in an aqueous solution at pH 9.25 (pKa
of ammonia) will be found as 50% NH4+ and 50% NH3. At one pH unit above the
pKa (i.e., 10.25) 90% of the ammonia will be in the unionized, volatile form
(NHj) and the remainder, will be in the Nfy* form which 1s not strippable.
At pH 11.15, two units above the p«a of ammonia, 99% of the ammonia will be
in the NH3 form, 1% will be in the NH4* form.
A reduction in the toxicity of the acidified, aerated effluent sample, relative
to the other aerated and baseline effluent samples, indicates the presence of
volatile acidic compounds with a pKa at least one to two units higher than pH
3. This group includes H2S (pKa=7.0) HCN (pKa=9.2), phenol (pKa=10.0), and other
relatively volatile acidic compounds (organic and Inorganic) which are, for
the most part, protonated and therefore volatile at pH 3.
A reduction 1n the toxicity of the aerated basified effluent, relative to
the other aerated and baseline effluent samples, indicates the presence of
volatile basic compounds with a pka at least one to two units lower than
pH 11. This group includes ammonia (pKa=9.25) and analine (pKa=4.6) and other
relatively volatile basic compounds (organic and inorganic) which are for the
most part unprotonated (and therefore volatile) at pH 11. Thus, by comparing
the relative reduction in toxicity among the effluent samples tested in this
experiment, it may be possible to roughly estimate the pKa and acidic or
basic nature of the toxicant.
36
-------
Note: If the toxicity of the basic effluent sample is reduced during air strip-
ping, a further characterization test may be performed. Six 5 or 10 ml aliquots
of the effluent are adjusted to pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively
The pH of these aliquots must be carefully monitored and readjusted as necessary
until a stable pH is maintained. Upon attaining a stable pH in each solution,
timed lethality tests are conducted with these solutions. If the ETso of the
solutions increases with decreasing pH (i.e. effluent at pH 8.5 is more toxic
than the effluent at pH 8.0, effluent at pH 8.0 is more toxic than the effluent
at pH 7.5, etc.) ammonia may be causing at least some of the toxicity in the
sample.
Oxidant Reduction Test
This test is designed to determine to what extent oxidants or electrophiles
are responsible for effluent toxicity. Chlorine, a commonly used biocide and
oxidant, is frequently found in acutely toxic concentrations in municipal and
domestic effluents. Other oxidants used in disinfection (such as ozone and
chlorine dioxide), oxidants formed during chlorinatlon, (such as mono- and
dichloramines), and other oxidizing compounds (such as bromine, iodine, and
manganous ion) are also neutralized in this analysis. Although the presence
of such oxidants in the effluent may not be likely, this test should not be
omitted from the screening tfer. Many oxidants are toxic to aquatic organisms
at very low levels and could be present in otherwise unoxldlzed effluents
simply as the result of the use of chlorinated process water. Oxldant-related
toxicity in unchlorinated municipal effluent has also been observed. While
chlorine is infrequently the sole cause of municipal and domestic effluent
toxicity, it frequently adds to the toxicity of these effluents. In the case
of municipal and domestic effluents, therefore, it is very likely that the
reducing agent test will at least partially remove effluent toxicity.
In this test varying amounts of reducing agent are added to aliquots of
the effluent (Figure 12). By covering a range of reducing agent concentra-
tions, a level high enough to reduce effluent oxidants but low enough to
prevent reducing agent toxicity is obtained. When no oxidants are expected
to be present in the effluent, 10~4 and 10~3N Na2s2°3 are -Ysed- Mnen °«3 to
3 mg/1 of residual chlorine is measured in the sample, 10~3N Na^oOg should
be used as the reducing agent. Effluent residua] chlorine concentrations in
excess of 3 mg/1 require the use of 10~z and 10~1N tta2^2^39 If chlorine is
suspected to be present in the effluent (as in the case of most municipal
wastewaters), the appropriate range of reducing agent concentrations can be
predicted by measuring the chlorine (Std. Methods, 15th Ed., 1980, 408A
lodometric Method I). When in doubt, use stronger rather than weaker solutions
of the reducing agent to account for other oxidants in the solution that are
not measured by the lodometric Method. If reducing agent toxicity is apparent
in all samples, the test can be repeated with weaker NagS^S solutions (i.e.
ET5Q of at least one sample should be greater than or equal to the baseline
effluent
To prepare a 0.1 N sodium thiosulfate stock solution, dissolve 25 grams
N^2S2°3*H2° 1n 1>0 L of fresnly boiled distilled water. The 10" , 10 , and
ICr2 if sodium thiosulfate solutions can be prepared as needed by diluting the
stock solution.
37
-------
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Once the appropriate reducing agent normality has been determined and the
Na2s2°3 solutions made, three sets of 5 ml portions, of effluent are prepared.
If fish species are used as the test organism, three sets of ten 10 ml portions
of effluent are prepared. For invertebrate tests, 0.05 ml (one drop) of
reducing agent (1 x 10~4, 1 x 10~J, or 1 x 10'2 N Na2S203 depending on the
effluent residual chlorine concentration) is added to the first 5 ml aliquot
of effluent using a buret or eyedropper. To the second 5 ml sample of effluent,
0.015 mL (3 drops) is added, to the third, 0.025 ml (5 drops), and so on
until the tenth 5 ml effluent sample has received 0.95 ml or 19 drops. Repeat
the procedure with the second set of ten 5 ml aliquots of effluent using the
10X stronger ^$203 solution. To insure that effluent toxicity is not being
reduced through dilution, add the same volumes (i.e. 0.05 ml, 0.15 ml, 0.25 ml,
0.95mL) of control water to a third series of ten 5 ml effluent samples.
For fish tests, 0.1 ml of the reducing agent is added to the first 10 ml
aliquot of effluent; 0.3 ml is added to the second 10 ml aliquot of effluent,
etc. The final effluent sample in the first series of ten receives 1.9 ml of
the N32S203 solution. The second set of ten 10 ml effluent samples receives
like volumes of the 10X stronger ^28303 solution. The third set of ten 10 ml
effluent samples will act as the blank; each receives the corresponding
amount of control water (i.e. 0.1, 0.3, 0.5 .... 1.9 ml control water). Five
test organisms are placed in each solution and the ETso is recorded
(Figure 13).
If oxidants are present in the effluent at toxic levels, the ET5Q of at least
one of the series of twenty effluents samples receiving the reducing agent should
be significantly greater than the ETcg of the baseline effluent and the ETKQ of
the effluent sample receiving an equivalent amount of control water. In the two
series of ten effluent samples receiving varying amounts of reducing agent,
ETso should increase in a systematic pattern if oxidant toxicity is occurring.
It may not be unusual, however, to observe the ETsg of this series increase to
a point and then progressively decrease. Such a pattern results when the reducing
agent itself becomes toxic after neutralizing the available oxidants.
EDTA Chelation Test
To determine to what extent effluent toxicity is caused by cationic toxicants,
such as heavy metals, a chelating agent (EOTA) is added in varying amounts to
aliquots of the effluent.
The form that a metal is in (eg. as the aquo ion, insoluble complex, etc.)
has a major effect on its toxicity to aquatic organisms (Magnuson et al.,
1979). Addition of the ethylenediaminetetraacetate ligand (EDTA), a strong
chelating agent, will produce relatively nontoxic complexes with many metals.
The success of EDTA in removing metal toxicity, however, is a function of
several factors including system pH, the type and speciation of the metal,
and other ligands in the solution (Stumm and Morgan, 1981). EDTA will not
complex anionic forms of metals (eg. selenium, arsenic, chromate) and has
only limited success in reducing the toxicity of mercury (Glass, 1977).
Despite these drawbacks, the addition of EDTA to some toxic effluents has
reduced or prevented toxicity to aquatic organisms.
39
-------
Reduced Effluent Timed Lethality Test Data
PARAMETER
pH (std. units)
temperature (UC)
Effluent*
rep:
Ti me( h rs . )
0 ?4 48
81 ank*
rep:
Time(hrs.)
0 24 48
Sample Log # _
Dilution Water
Control Water ~
# Animals/Replicate
Test Initiation
Date
Time
Test Organism
Source
Total Residual Chlorine in Effluent (mg/L)_
% Mortality
N Na2S2<>3
N Na2$203
Control
Water
mL/drops added
0.33
0.67
1
Tim<
2
? (h<
4
surs
6
)
8
24
48
Other
Observations
Comment s:
* Measure water quality parameters in only one of the series of test containers,
Figure 13. Example of Data Collection Sheet for Effluent Reduction Test.
40
-------
In this experiment successive small measured amounts of EDTA arp added to
aliquots of toxic effluent (Figure 14). Because EDTA will complex relatively
nontoxic metals (eg. calcium, magnesium) as well as many toxic heavy metals,
fairly concentrated solutions are needed. It is suggested that investigators
begin with 0.1 and 0.01 M EDTA solutions. A 0.1M solution of EDTA is pre-
pared by dissolving 37.23 grams of disodium ethylenedi ami netetraacetatedi hydrate
in 1 L of distilled water. Note that this solution should be stored in
polyethylene or borosilicate glass (Std Methods, 15th ed. , 1980, Method 314).
To prepare the 0.01M EDTA solution, 100 ml of this 0.1M EDTA solution is
brought to 1L with distilled water. For extremely hard effluents, a more
concentrated solution of EOTA may be needed; for extremely soft effluents, a
less concentrated solution of EDTA may be needed.
If an invertebrate species is used as the test organism three sets of ten
5 ml aliquots of effluent are prepared; one of these sets will act as a blank
to account for dilution of effluent toxidty. To the first of the ten 5 ml
portions of effluent in the test series, 0.05 ml (1 drop) of 0.10M EDTA is
added using a buret or eyedropper. To the second sample of effluent, 0.15 ml
(3 drops) is added, to the third, 0.25 ml (5 drops), and so on until the
tenth effluent sample in the series has received 0.95 mis (19 drops) of 0.1M
EDTA. Repeat this process with the second set of ten 5 ml aliquots of effluent
using 0.01 M EDTA. To the third, or blank, series of ten 5 ml effluent
samples, add the same volumes (i.e. 0.05 ml, 0.15 ml, 0.25 ml. ..0.95 ml) of
control water to account for effluent toxicity dilution. If a fish species 1s
used as the test organism, three sets of ten 10 ml portions of effluent are
prepared. The first ten samples receive 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3,
1.5, 1.7, and 1.9 ml of 0.1 M EDTA respectively. This procedure 1s repeated
with the second set of ten samples using 0.01M EDTA. The final set of ten
samples receives corresponding volumes of control water. Five test organisms
are placed in each of the 30 samples and mortality is observed over time
(Figure 15).
If the ETso of at least one of the effluent samples in the test series is
significantly greater than the ETsg of the corresponding effluent sample in
the blank series and the baseline effluent ETsp, , then it is likely that
cations are present in the effluent sample in toxic concentrations. As in the
reducing agent test, the ET5QS for the series of effluent samples receiving
increasing amounts of EDTA should increase in a systematic pattern if toxic cations
metals are present. Again, it is not unusual to observe an initial increase
in ETsos with increasing concentrations of EDTA followed by an eventual decrease
in ET5QS that can be attributed to EDTA toxicity. The level at which EDTA
becomes toxic is a function of the sample. EDTA 1s less toxic in samples with
relatively high hardness as compared to samples of soft water.
Solid Phase Extraction Test
The solid phase extraction test is designed to determine the extent of
effluent toxicity caused by non-polar organic compounds, or those organic
41
-------
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-------
Chelated Effluent Timed Lethality Test Data
PARAMETER
pH (std. units)
Temperature (UC)
Effluent*
rep:
Ti me( h rs . )
0 24 48
Bl ank*
rep:
Time(hrs.)
0 24 48
Sample Log # _
Dilution Water
Control Water ~
# Animals/Replicate
Test Initiation
Date Time
Test Organism
Source
% Mortality
M EDTA
M EDTA
Control
Water
mL/drops added
0.33
0.67
1
Tirm
2
» (h<
4
surs
6
8
24
48
Other
Observations
Comments:
* Measure water quality parameters in only one of the series of test containers
Figure 15. Example of Data Collection Sheet for Effluent Chelation Test.
43
-------
compounds that can be made relatively non-polar thrdugh manipulation of effluent
pH. While this technique may not be successful in removing some groups of or-
ganics, such as those that are highly water soluble, it has been used to remove
toxicity from a large fraction of the effluent samples studied thus far.
In this analysis, the effluent is passed through a small column packed with
an octadecyl sorbant (Figure 16). As in liquid chromatography, the functional
groups of compounds in the effluent interact through solubility and polarity
with the functional groups of the sorbant and are, in essence, extracted from
the effluent onto the sorbant. In other words, nonpolar compounds in the
effluent are partitioned onto the CIQ bonded phase through the Interaction of
the nonpolar functional groups of both sample components and sorbant. (J. T.
Baker, 1984). This type of chromatography (i.e. extraction of non-polar
compounds from very polar solvents such as water) 1s known as reversed-phase
solid phase extraction (SPE).
Compounds extracted by the Cjg sorbant from a neutral aqueous solution have
an apparent molecular weight of less than 2000 (molecular size: approx. fiO
A) and include those organics and metal chelates which are soluble In hexane
or chloroform. Certain types of other organic compounds can also be extracted
by "weakening" the solvent strength (i.e. by making the sorhant more attractive
to these compounds than is the solvent). This can be accomplished 1n a number
of ways which include buffering the effluent, changing its pH, or adding salt
to it (J. T. Baker, 1984). Weakening the solvent decreases the solubility or
polar interaction of the compounds of interest, thus favoring Interactions
with the octadecyl sorbant. The options for weakening the solvent are limited
by the fact that toxicity tests will be used to analyze the effluent after it
has passed through the column. In general, only pH changes will be used
since they are "reversible" (i.e., pH-adjusted effluent can be brought back
to neutral following solid phase extraction).
As in the air-stripping test, by lowering effluent pH one to two units below
the pKa of an organic acid, the majority of the acid (90 to 99%) will exist
in the unionized form. The unionized organic adds in the effluent will now
interact with the C\Q sorbant rather than the water. In a similar manner, by
raising the effluent pH one to two units above the pKa of an organic base, the
majority of the base (90 to 99%) will be in the unionized form and should be
amenable to C.\Q solid phase extraction. Obviously, the pKas of organic acids
and bases in the effluent are unknown. However, by adjusting the effluent to
a low pH and a high pH, a large number of these compounds are likely to be in
the unionized form. Because of Cjg column degradation, the use of pHs above 10
and below 2 are discouraged. Therefore, to insure column integrity, effluent pH
will be lowered to 3 and raised to 9 in this analysis.
To perform the Cjg SPE test, 1.54 I of filtered effluent (see Filtration Sub-
section) is needed. The presence of a fritted-disc (pore size: 20 urn) at
the top of the column necessitates the use of filtered effluent. Adjust the
pH of 520 ml of the filtered effluent to 3 using 1.2N HC1 (switching to 0.12N
HC1 as the effluent pH nears 3). A second 520 ml portion of the effluent 1s
adjusted to pH 9 using l.ON NaOH and 0.1N NaOH. For the purpose of reference,
the volumes of acid and base added to each sample of effluent should be recorded.
44
-------
TEST
Effluent
adjust
520 mL
to pH 3 —
I 20
V
Cl8
olumn
500 mL
no
ad jus
mL
pH
jtment
20
V
'1
8
column
adjust
520 mL
/. — to
mL
pH 9
V
f
column
Adjust to initial pH of the effluent
I I I I I
V V V V V
V V V
-TIMED LETHALITY TESTS-
BLANK*
Control Water
adjust 500 mL adjust
520 mL no
to pH 3=-^ adjus
1 >v
| 20 mL
1
\l
Cl8
column
1
\
pH 520 mL
tment ^ — to pH 9
20 mL
/
cia
column
1
V
HI 3
col
umn
V V V V V
Adjust to initial pH of the effluent
I I I I I
v v v v v
TIMED LETHALITY TESTS
*0nce per lot of columns
Figure 16. Flow Chart for Effluent Solid Phase Extraction Test,
45
-------
To prepare the Cjs SPE column for use, follow the instructions for column
conditioning provided by the manufacturer. Note that, following the final
column wash with methanol, the column sorbant should not be allowed to dry
(i.e., leave some of the water on the column at all times).
Draw 500 ml of the effluent (no pH adjustment) through the prepared Cjg column
at a rate of approximately 10 mL/min. Collect 10 or 20 ml aliquots of the
column effluent when 50, 250 and 450 ml of the effluent sample has passed
through. By collecting samples for toxicity testing during different stages
of the elution, possible column overloading and compound breakthrough can be
detected. If this is not happening, the ETsgs of the samples collected at
50, 250, and 450 ml should be similar (not decreasing). Using a second con-
ditioned GIS column, draw 500 ml of the acidified effluent sample through the
column, again collecting 20 ml aliquots of the column effluent as 50, 250 and 450
ml of effluent passes through it. The 20 ml portion of acidified effluent
not drawn through the column will act as a blank for ionic strength changes.
Repeat this procedure with a third conditioned column using 500 ml of the
basified sample, again holding a 20 ml portion back as a blank. The toxicity
of the samples of acidified and hasified effluent (not chromatographed)
should be no greater than the toxicity of the baseline effluent. If the Tjg
column appears to be successful in removing effluent toxicants, the used
columns should be retained for solvent extraction and chemical analysis 1n
the later stages of Phase I,
To insure that no toxic artifacts are leaching off the column into the effluent,
column quality should be checked once per lot of columns. Repeat the above
procedure with 520 mL each of neutral (pH at effluent pH), acidic (pH = 3
using HC1 ) and basic (pH = 9 using NaOH) control water. These blank solutions
are also used in timed lethality tests. No significant toxicity should be
seen in control water samples.
The acidic and basic 20 mL aliquots off the column (effluent and control water)
and the 20 mL portions of acidic and basic effluent held back from the column
are returned to the pH of the initial effluent prior to performing the timed
lethality tests. It is critical that the volume of these samples be changed
as little as possible (<10%). The pH of the acidic samples is raised using
0.01 N NaOH initially and 0.001 N NaOH as sample pH approaches that of the
effluent. The pH of the basic samples is lowered using 0.012 N and 0.0012 N
HC1. The pH of these samples should be checked periodically during the hold-
ing period prior to toxicity test initiation. If necessary, sample pH should
be readjusted. The volume and normality of acid and base added to each sample
should be recorded. The strength of the acids and bases used may depend on
the sample. Following the necessary pH adjustment, all samples (effluent and
control water) are used in timed lethality tests (Figures 17 and 18).
If the ETsQS of any of the CIQ column effluent samples are significantly greater
than the E^Q of the baseline effluent (i.e., if any of the samples from the column
are significantly less toxic than the unchromatographed effluent), effluent
sample toxicity is at least partially due to organic compounds. By comparing
the ETsos of the acidified, basified and neutral effluent, it may be possible
46
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SPE Effluent Timed Lethality Test Data
Acidified-Chromatographed Effluent rep:
Basifled-Chromatographed Effluent rep:
Neutral Chromatographed Effluent rep:
Acidified Effluent rep:
Basified Effluent rep:
Acidified-Chromatographed Blank* rep:
Basified-Chromatographed Blank* rep:
Neutral Chromatographed Blank* rep:
Acidified Effluent rep:
Basified Effluent rep:
pH (std.
units)
0
I
2
4
(Temperature
(°C)
8
24
48
Effluent pH (initial):
Acid pH:
Sample at 50 ml:
Sample at 250 ml:
Sample at 450 ml:
Pre-column sample:
Base pH:
Sample at 50 ml:
Sample at 250 ml:
Sample at 450 ml:
Pre-column sample:
Blank* pH (initial):
Acid pH:
Sample at 50 ml:
Sample at 250 ml:
Sample at 450 ml:
Pre-column sample:
Base pH:
Sample at 50 ml:
Sample at 250 ml:
Sample at 450 ml:
Pre-column sample:
Effluent pH (post-column):
Adjustments**:
mL 0.12N HC1 added:
nt 0.01N NaOH added:
ml 0.01N NaOH added:
nt' 0.01N NaOH added:
mL 0.01N NaOH added:
nt 0.1N NaOH added:
mL 0.012N HC1 added:
nt 0.012N HC1 added:
mL 0.012N HC1 added:
nt 0.012N HC1 added:
Control pH (post-column):
Adjustments**:
mL 0.12N HC1 added:
nt 0.01N NaOH added:
nt 0.01N NaOH added:
nt 0.01N NaOH added:
mL 0.01N NaOH added:
nt 0.1N NaOH a.dded:
nt 0.012N HC1 added:
nt 0.012N HC1 added:
mL 0.012N HC1 added:
nt 0.012N HC1 added:
nt 0.012N HC1 added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.01N NaOH added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
nt 0.012N HC1 added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.001N NaOH added:
nt 0.01N NaOH added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
nt 0.0012N HC1 added:
* Once per lot of columns.
** nt acid or base added to return post-column effluent to initial pH
of effluent sample (if necessary).
Figure 17. Example of Data Collection Sheet for Effluent Solid Phase
Extraction Test, Part I.
47
-------
SPE Effluent Timed Lethality Test Data
Sample Log #
Dilution Water
Control Water
Test Initiation
Time
# Animals/Replicate
Date • '
Test Organism
Source
% Mortality
Effluent-Chromatographed
Acidified 50 mL
250 nt
450 mL
Basified 50 mL
250 mL
450 mL
Neutral 50 mL
250 mL
450 mL
Effluent-Mot Chromatographed
Acidified a
b
Basified a
b
Blank -Chromatographed*
Acidified 50 mL
250 mL
450 mL
Basified 50 mL
250 mL
450 mL
Neutral 50 mL
250 mL
450 mL
Blank -Not Chromatographed*
Acidified a
b
Basified a
b
Time i hours)
0.33
XX
XX
XX
XX
0.67
XX
XX
XX
XX
1
X
X
X
X
?.
X
X
X
X
4
X
X
X
X
6
y
X
X
X
8
)(
X
X
X
24
XX
XX
XX
XX
48
XX
XX
XX
XX
ETiin
XX
XX
XX
XX
Other
Ohservatio
Comments:
* Once per lot of columns.
Figure 18. Example of Data Collection Sheet for Effluent Solid P
Extraction Test, Part II.
48
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to approximate the pKa of the toxicant(s). If the E'T50s of the neutral effluent
are greater than the ET50s the acidified of basified effluent, toxicants are
relatively non-polar at the pH of the effluent. If the ET50s of the acidified
effluent are greater than the ET^s of the neutral and hasified effluent, effluent
sample toxicants are essentially unionized at that pH (i.e., acidic in nature).
If the ETsos of the basified effluent are greater than the ETsos of the acidic
and neutral effluent, effluent toxicants are for the most part unionized at
pH 9 (I.e., slightly basic in nature). If ET5os of both acidic and basic
effluent are greater than the neutral effluent ET^, toxicity reduction may
be the result of organic compounds being "salted out" of solution onto the Cia
column (i.e., the higher ionic strength of the acidified and basified effluent
relative to the unaltered effluent may drive non-polar organic compounds into
the nonpolar column sorbant). Alternatively, both acidic and basic organic
toxicants may be present in the effluent sample.
49
-------
SECTION 9
DATA ANALYSIS
Overall Interpretation of Results
Results of Phase I characterization tests should provide a reasonable
description of the sources of an effluent's toxicity. There may be some
situations in which none of the screening tier experiments remove or neutra-
lize the effluent's toxicity. Such results do provide information regarding
effluent toxicants (i.e., what groups of compounds are not likely to be
causing toxicity) and indicate other possible groups of compounds to be
investigated using more definitive procedures. In other words, if character-
ization tests fail to remove effluent toxicity, the cause of toxicity is
likely to be a group not covered by Phase I tests such as anions or polar
organics.
Results of all the characterization analysis for a particular effluent sample
should be studied together. When two or more analysis each remove sample
toxicity completely, the possible causes of sample toxicity are narrowed down
even further. For example, if effluent toxicity is removed by both neutral
air-stripping and the CIQ column, it is likely that the compounds causing
toxicity are volatile, non-polar organics. An in-depth discussion and
interpretation of the Phase I results will be provided in the Phase II TRE
manual.
The interpretation of data generated in Phase I (and in Phase II for that
matter) depends on what might be called the "weight of evidence". No single
piece of data is sufficient to make a positive identification of the causes
of effluent toxicity. Results of screening tests should also be used in the
selection of a Phase II option. If the screening test results indicate that
the effluent toxicant may be easily identified (e.g., inorganic or relatively
volatile organics) the identification/source investigation option should be
chosen. If the results indicate the effluent toxicity can be removed through
reasonable treatment (eg., chlorine), then the treatment option should be
followed. In many situations, both options may need to be investigated prior
to the final decision on effluent toxicity control.
Variability Analysis in Phase I
In order to take the most appropriate and cost effective steps towards
reduction of effluent toxicity, the variability associated with compounds
causing effluent toxicity must be addressed. Obviously, toxicity reduction
measures must be based on the results from more than one or two samples. The
duration and frequency of Phase I testing will be site-specific.
50
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As specified earlier, samples should be collected over a period when effluent
variability is expected to.be greatest. At the same time, samples should be
collected and tested in an expeditious manner so as to bring about toxicity
control as quickly as possible. These two factors (i.e., rapid corrective
measures and the need to consider potential temporal variability) need to be
balanced. While the regulatory agencies involved are obliged to expedite
toxicity reduction evaluations, if effluent toxicity returns after control
measures have been taken, the TRE will have to be continued.
As a rule of thumb, if the toxicity of the first three samples is consistently
removed by the same characterization test or series of characterization tests,
there would appear to be minimal variability in the character of the effluent
toxicants. This of course, is provided that the compounds in the effluent
samples are representative of the type of effluent toxicants encountered over
time.
If there appears to be little variability in the character of the compounds
causing effluent toxicity, randomly collected samples (at least three) should
be periodically screened using only the characterization tests previously
proven to remove effluent toxicity. In order to confirm the initial results,
these characterization tests must continue to be successful in eliminating
effluent sample toxicity. The LC$Q of the effluent can be highly variable in
this case, however, the same screening tier experiments must be successful in
removing and/or neutralizing effluent toxicity.
For a facility at which the screening tests needed to remove or neutralize
effluent toxicity change with the sample, testing duration and frequency will
need to be increased. The frequency of testing should be such that any
temporal patterns related to the type of compound causing effluent toxicity
are identified. Such patterns may provide valuable information for identifying
the source of effluent toxicants. Phase I toxicant characterization testing
should continue until there is reasonably certainty that all sources of
effluent toxicity have been addressed. The characterization tests that have
been used to eliminate effluent sample toxicity during the Phase I study
should be documented for use in Phase II. The nature of the toxicant variability
may indicate the need for one or the other Phase II approach (i.e., treatment
or toxicant identification and control).
51
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SELECTED REFERENCES
American Chemistry Society. 1979. Safety in Academic Chemistry Laboratories,
ACS Publication. Committee on Chemical Safety. 3rd Edition.
American Public Health Association, American Water Works Association
Water Pollution Control Federation. 1980. Standard Methods for
the Examination of Water and Wastewater. 15th Edition.
Doull, Klaassen, and Amdur. 1980. Casarett and Doull's Toxicology, The
Basic Science of Poisons, 2nd Edition. Macmillan Publishing Co., Inc.
Glass, G. E. 1977. Identification and Distribution of Inorganic
Components in Water: What to Measure? Annals of the New York
Academy of Sciences. Vol. 298. pp. 31-46.
Gordon, Arnold and Richard Ford. 1972. The Chemist's Companion.
John Wiley & Sons, Inc.
J. T. Baker Chemical Company. 1984. Solid Phase Extraction; Sample
Preparation. 222 Red School Lane, Phillipsburg, NJ 08865.
M. C. Kavanaugh and R. R. Trusself. 1980. Design of Aeration Towers to
Strip Volatile Contaminants From Drinking Water. Journal of the
American Water Works Association. 72, pp. 684-692.
Magnuson, V. R., D. K. Harriss, M. S. Sun, D. K. Taylor, G. E. Glass.
1979. Relationships of Activities of Metal-Ligand Species to
Aquatic Toxicity. ACS Symposium Series, No. 93. Chemical
Modeling in Aquious Systems, E. A. Jenne, Editor, pp. 635-656.
Marking, L. L. and V. K. Dawson. 1973. Toxicity of Quinaldine Sulfate
Fish. Invest. Fish Contr. No. 48., U.S. Fish and Wildlife Service,
Department of the Interior, Washington, D.C.
52
-------
1 f
National Academy of Sciences. 1972. Water Quality Criteria 1972. A Report
of the Committee on Water Quality Criteria. Environmental Studies Board.
Washington, D.C.
Occupational Safety and Health Administration. 1976. OSHA Safety and
Health Standards, General Industry. 29 CFR 1910. OSHA 2206 (Revised).
Peltier, W. And C. I. Weber. 1985. Methods for Measuring the Acute
Toxicity of Effluents to Freshwater and Marine Organisms. 3rd
Edition. Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio. March 1985.
EPA 600/4-85/013.
Strumm, and Morgan. 1981. Aquatic Chemistry. John Wiley and Sons, Inc.
U.S. Department of Health, Education and Welfare. 1977. Carcinogens-
Working With Carcinogens. Public Health Service, Centers for Disease
Control, National Institute of Occupational Safety and Health. Publica-
tion No. 77-206.
U.S. Environmental Protection Agency. 1977. Occupational Health and Safety
Manual. Office of Planning and Management, Washington, D.C.
U.S. Environmental Protection Agency. 1985. Technical Support Document
for Water Quality-Based Toxic Control. Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 1987. Permit Writer's Guide to Water
Quality-based Permitting for Toxic Pollutants, Office of Water, Washing-
ton, D.C.
Walters, C. I. and C. W. Jameson. 1984. Health and Safety for Toxicity
Testing. Butterworth Publ., Woburn, Ma.
Weber, C. I. and W. Peltier. 1981. Effluent Toxicity Screening Test
Using Daphnia and Mysid Shrimp. Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency,
Cincinnati, Ohio.
53
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I t
APPENDIX A
LIST OF REQUIRED EQUIPMENT
-------
I I
Reagents, Facilities and Equipment
General:
- 30 ml (1 oz.) plastic sample cups
- eyedropper
- wide bore pipettes (for fish)
- pH meter, probe and buffers
- D.O. meter, probe, membranes and reagents
- conductivity meter or refractometer, standards
- reagents for hardness analysis (buffer, EDTA, indicator)
- thermometer
- microscope
- test organism culture
- control water
- water bath or environmental chamber
- refrigerator (for sample storage)
- water purification system
- hexane
- acetone
- detergent
- 10% HN03
- sampling equipment (discrete or composite)
Filtration:
- glass fiber filters, 1.0 urn nominal size (Gelman type A/E,
1.0 urn, Whatman grade GF/D, 1.2 urn*)
- glass filtration apparatus and filter holder suitable for
filter selected
- vacuum flask, 1 L capacity.
- water aspirator, small vacuum pump or house vacuum
- vacuum tubing
- distilled water
centrifuge)
centrifuge tubes)
Air Stripping:
- bench scale aeration device (such as those sold commericially
for use in small aquariums)
- air stones
- tubing (Teflon*)
- six-100 ml graduate cylinders
- four-150 ml beakers
- magnetic stir bars (Teflon*)
- magnetic stirrer
- pH meter
- 0.12N and 0.012 N HC1
- 0.1N and 0.01 N NaOH
55
-------
i I
Reducing Agent:
- 0.1N Na2S203, stock solution
- 1 x 10-* N, 1 x 10-3 N or 1 x 1Q-4 N
N32S203, titrant
- reagents as specified in Standard Methods, 15th Ed.,
498A. lodometric Method I (where residual chlorine is
suspected to be present)
- eyedropper or buret
- volumetric flasks, 1 L and two-100 ml
Chelating Agent:
- Ethylenediaminetetraacetate, sodium salt, ACS
- two-borosilicate glass or polyethylene storage bottles, 1 L
- eyedropper or buret
- two-lL volumetric flasks
C18 SPE:
- small vacuum pump, water aspirator, or house vacuum
- GIS solid phase extraction columns, 3 ml, Baker*
- tubing (Teflon*)
- graduate cylinders, 10 ml, 500 ml
- four-1000 ml beakers
- magnetic stir bars (Teflon*)
- magnetic stirrer
- pH meter
- 1.2 N, 0.12 N and 0.012 N HC1
- 1.0 N, 0.1 N and 0.01 N NaOH
- 500 ml reservoir (for introduction of sample onto column)
- vacuum flask
- methanol, HPLC-grade
- distilled water in a water bottle
* or equivalent
56
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