EPA 

United States 

Environmental Protection 

Agency 

Office of Research and 

Development 

Washington, DC 20460 

EPA/600/6-91/005F 

May 1992 

Toxicity Identification 

Evaluation: 

Characterization of 

Chronically Toxic Effluents, 

Phase I 
EPA/600/6-91/005F 

May, 1992 

Toxicity Identification Evaluation: 

Characterization of Chronically Toxic Effluents, Phase I 

T.J. Norberg-King 

Environmental Research Laboratory 

Duluth, MN 55804 

D.I. Mount, 

J.R. Amato, 

D.A. Jensen, 

and 

J.A. Thompson 

AScl Corporation 

Duluth, MN 55804 

National Effluent Toxicity 

Assessment Center 

Technical Report 02-92 

Environmental Research Laboratory 

Office of Research and Development 

U.S. Environmental Protection Agency 

Duluth, MN 55804 

Printed on Recycled Paper 
Disclaimer 

This document has been reviewed in accordance with U.S. Environmental Protection 

Agency policy and approved for publication. Mention of trade names or commercial 

products does not constitute endorsement or recommendation for use. 

ii 
Foreword 

This guidance document has been prepared to assist dischargers and/or their 

consultant laboratories in conducting chronic aquatic toxicity identification evaluations 

(TIES). TIES may be required by the state or federal agencies as a result of enforcement 

actions or as a condition of the discharger’s National Pollutant Discharge Elimination 

System (NPDES) permit or may be conducted voluntarily by permittees. This document 

will assist the state and federal agencies and permittees in overseeing and determining 

the adequacy of the TIE in toxicity reduction evaluations (TREs). 

This document discusses methods to characterize the chemical/physical nature of 

the constituents in effluents which cause their chronic toxicity. The general approach for 

toxicity identification evaluations is described in the document Methods for Aquatic 

Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures (EPA, 

1988A; EPA, 1991A), hereafter referred to as the “acute Phase I manual.” The acute 

Phase I manual provides much of the basis for the statements and guidance provided in 

this chronic Phase I characterization document. This chronic TIE manual and the acute 

Phase I manual should be used as companion documents, because all the guidance of 

the acute Phase I manual is not repeated here. 

The general approach for the chronic characterization is divided into Tier 1 and Tier 

2. Tier 1 consists of the EDTA and sodium thiosulfate additions, the graduated pH test, 

aeration and filtration manipulations, and the use of the C,, solid phase extraction (SPE) 

resin. For Tier 1, the tests are all done using the effluent sample without any pH 

adjustments (i.e., at the initial pH (pH i) of the effluent). Tier 2 manipulations are added 

when Tier 1 tests are not definitive in characterizing the toxicity. Tier 2 includes the 

aeration, filtration, and C,, SPE steps of Tier 1 performed at pH 3 and pH 10 and returned 

to pH i prior to testing. 

The chronic Phase I procedures should provide information on whether the toxicants 

are volatile, chelatable, filterable, reducible, non-polar, or pH sensitive. These characteristics are indicated by comparing the results of toxicity tests conducted using unaltered 

and manipulated effluent samples. As with the acute TIE, the characterization results 

from the chronic TIE can be used for the treatability approach in a TRE (EPA, 1991A). 

These chronic TIE methods are not written as rigid, required protocols, but rather as 

general guidance for conducting TIES with effluents. These acute and chronic methods 

should also be applicable to samples from ambient waters, sediment pore and elutriate 

waters, and leachates. The methods to identify (Phase II; EPA, 1989A) and confirm 

(Phase III; EPA, 19898) the cause of toxicity in effluent samples evaluated with the acute 

Phase I procedure are also applicable to effluent samples evaluated with this chronic 

Phase I procedure. The identification and confirmation documents are being revised 

(EPA, 1992A; EPA, 19928) to reflect additional information from this manual and the 

revised acute Phase I manual (EPA, 1991A) to discuss the aspects of TIES for both acute 

and chronic toxicity. 

In September of 1991, we solicited peer-review comments until January 31, 1992 

from all persons who obtained the document from any of the following locations: EPA’s 

Office of Water, Washington, D.C., each EPA Regional Water Division Office, EPA’s 

Environmental Research Laboratory-Duluth, MN, or EPA’s Center for Environmental 

Research Information (CERI), Cincinnati, Ohio. Appropriate technical comments were 

incorporated into this manual. 

iii 
Abstract 

This manual is intended to provide guidance to aid dischargers in characterizing the 

type of toxicants that are causing chronic toxicity in industrial and municipal effluents. In 

a regulatory context, a toxicity identification evaluation (TIE) may be required as part of 

the National Pollutant Discharge Elimination System (NPDES) permit or as an enforcement action. TIES may also be conducted by permittees on a volunteer basis to 

characterize their discharge toxicity. 

The Phase I chronic toxicity methods are modified from those described in the acute 

Phase I TIE manual (EPA, 1988A; EPA, 1991A) and additional techniques are incorporated. This chronic Phase I manual describes procedures for characterizing the physicat/ 

chemical nature of toxicants in effluents that exhibit chronic toxicity to freshwater species, 

although many of the principles and procedures are similar for TIES on marine species. 

Aliquots of effluent samples are manipulated and the resulting effect on toxicity measured. The objective is to characterize the toxicants so that appropriate analytical 

methods can be chosen to identify the toxicants. 

The general approach to the chronic toxicity characterization is a two tiered approach, where usually Tier 1 is applied before proceeding to Tier 2. Tier 1 consists of 

filtration, aeration, use of additives to chelate or reduce the toxicants, minor pH adjustments, and use of a separation technique with the C,, solid phase extraction (SPE) resin. 

Each effluent is characterized in Tier 1 by performing the manipulations at the initial pH 

(pH i) of the effluent. Tier 2 consists of the Tier 1 manipulations combined with pH 

adjustments of additional aliquots of the effluent sample, and the Tier 2 characterization 

steps include aeration, filtration, and the C,, solid phase extraction of effluent samples 

adjusted to pH 3 and pH 10. 

The Phase I characterization methods were developed for the short-term “chronic” 

test methods using two species, Ceriodaphnia dubia and the fathead minnow (Pimephales 

promelas) (EPA, 1989C). Chronic threshold levels for the various additives (sodium 

thiosulfate, EDTA, methanol) used in some of the characterization tests are provided for 

these species. Although developed for these species, the characterization techniques 

should be applicable to other species as well, provided threshold levels are established. 

The guidance provided in this manual is intended to be supplemental to that given in 

the acute Phase I manual (EPA, 1991A). Sections of this chronic Phase I TIE manual 

discuss quality assurance, effluent handling, facilities and equipment, health and safety, 

dilution water, principles of the chronic TIE testing, and the Phase I characterization tests 

as a two tiered approach. The use of the whole effluent test as a baseline test (in 

manner similar to the acute Phase I characterization procedure), the appropriate treatment of dilution water for blanks and the toxic levels of the additives for two species are 

described. Use of short-cuts, reduced test volumes, reduced test duration, and a small 

number of replicates are discussed. The importance of sample type, frequency of 

sample collection and renewal, and descriptions of all manipulations are discussed, along 

with a section on the application of combining several of the characterization tests. 
Contents 

Page 

Foreword ................................................................................................................. iii 

Abstract ................................................................................................................. iv 

Contents .................................................................................................................. v 

Figures ................................................................................................................ vii 

Tables ............................................................................................................... viii 

Acknowledgments ................................................................................................... ix 

1. Introduction .................................................................................................... l-1 

2. Quality Assurance, Health and Safety, and Facilities and Equipment.. ....... .2-l 

2.1 Quality Assurance .............................................................................. ...2- 1 

2.2 QA/QC Cost Considerations and Testing Requirements .................... .2-l 

2.3 QA/QC and Chronic Testing Considerations.. ..................................... .2-2 

2.4 QA/QC Blanks and Artifactual Toxicity ................................................ .2-3 

2.5 Health and Safety Issues.. .................................................................... 2-3 

2.6 Facilities and Equipment.. ..................................................................... 2-3 

3. Dilution Water ................................................................................................ 3-l 

4. Effluent Samples ........................................................................................... .4-l 

5. Toxicity Testing .............................................................................................. 5-l 

5.1 

5.2 

5.3 

5.4 

5.5 

5.6 

5.7 

5.8 

Principles ............................................................................................. 5-l 

Test Species ........................................................................................ .5-l 

Toxicity Test Procedures ................................................................... ...5- 1 

Concentrations to Test.. ....................................................................... .5-2 

Renewals ............................................................................................. .5-3 

Toxicity Blanks ..................................................................................... .5-3 

Renewal of Manipulated Samples ....................................................... .5-4 

Test Endpoints and Data Analysis ...................................................... .5-4 

6. Characterization Tests .................................................................................. .6-l 

6.1 Baseline Test ....................................................................................... .6-3 

6.2 EDTA Addition Test ............................................................................. .6-4 

6.3 Sodium Thiosulfate Addition Test ........................................................ .6-6 

6.4 Aeration Test ........................................................................................ .6-8 

6.5 Filtration Test ....................................................................................... .6-9 

6.6 Post C,, Solid Phase Extraction Column Test .................................. ..6-11 

6.7 Methanol Eluate Test .......................................................................... 6-15 

6.8 Graduated pH Test ............................................................................ .6-17 

6.9 Tier 2 Characterization Tests ............................................................ .6-20 

6.10 pH Adjustment Test ............................................................................ 6-21 

6.1 1 Aeration and pH Adjustment Test ...................................................... 6-22 

6.12 Filtration and pH Adjustment Test ...................................................... 6-23 

6.13 Post C,, Solid Phase Extraction Column and pH Adjustment Test. . ..6-2 3 

6.14 Methanol Eluate Test for pH Adjusted Samples ............................... .6-24 

6.15 Toxicity Characterization Summary .................................................... 6-24 

6.16 Use of Multiple Characterization Tests.. ............................................ .6-25 

V 
Contents (continued) 

Page 

7. Interpreting Phase I Results . . . 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1 

8. References . . . . . . . . . . . . . . . . . ..,..,............................,................................................. 8-1 

vi 
Figures 

Number Page 

4-1. Example data sheet for logging in samples ........................................ .4-2 

6-1. Overview of characterization tests.. ..................................................... .6-2 

6-2. Tier 1 sample preparation and testing overview ................................. .6-2 

6-3. Tier 2 sample preparation and testing overview ............................... .6-20 

vii 
Tables 

Number Page 

6-l. 

6-2. 

6-3. 

6-4. 

6-5. 

6-6. 

6-7. 

6-8. 

6-9. 

6-10. 

Outline of Phase I effluent manipulations Tier 1 and Tier 2 . . . . .._.........__. 6-3 

Chronic toxicity of EDTA (mg/l) to C. dubia and P. promelas 

in various hardness waters using the 7-d tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5 

Concentrations of EDTA to add for chronic TIES. Values given 

are the final water concentration in ms/l.................................................. 6-5 

The chronic toxicity of zinc (ps/I) to C. dubia in very hard 

reconstituted water and the toxicity of zinc when EDTA is added . . . . . . . ...6-6 

Chronic toxicity of sodium thiosulfate (ms/l) to C. dubia and 

P. promelas in various hardness waters using the 7-d tests . . . . . . . . . . . . . . . . 6-7 

Concentrations of sodium thiosulfate to add for chronic TIES. 

Values given are the final exposure concentration in mg/l . . . . . . . . . . . . . . . . . . . . . 6-8 

Factors to consider for the size of available pre-packed C,, SPE 

columns. Appropriate volumes of sample to apply to each column 

with respect to maximum volumes of sample and minimum elution 

volumes, and elution volumes frequently used in the TIE process . . . . ...6-12 

Test volume of eluate needed for methanol eluate test with C. dubia 

or P. promelas. Volumes described are based on minimum elution 

volumes recommended and the highest test concentration possible 

with the methanol level at an acceptable concentration ..,..................... 6-13 

Chronic toxicity of methanol (%) to C. dubia and P. promelas 

using the 7-d tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,............... . . . . . . . . . . . . . 6-13 

Chronic toxicity of sodium chloride (g/l) to C. dubia 

and P. promelas in various hardness waters using the 7-d tests . . . . . . ...6-22 
Acknowledgments 

Many people at the National Effluent Toxicity Assessment Center (NETAC) at the 

Environmental Research Laboratory-Duluth (ERL-Duluth) have provided assistance to 

produce this manual by performing the toxicity tests, chemical analyses, and data 

analyses as well as providing advice based on experience. This document is the result of 

the input by the NETAC group which has consisted of both federal and contract staff 

members, and includes Gary Ankley, Larry Burkhard, Liz Durhan, Don Mount, Shaneen 

Murphy, and Teresa Norberg-King (federal staff), and Joe Amato, Lara Andersen, Steve 

Baker, Tim Dawson, Nola Englehorn, Doug Jensen, Correne Jenson, Jim Jenson, Marta 

Lukasewycz, Liz Makynen, Greg Peterson, Mary Schubauer-Berigan, and Jo Thompson 

(contract staff). The toxicity test data generated for this document and the biological data 

upon which this report is based was produced by Doug Jensen, Jo Thompson, Tim 

Dawson, Greg Peterson, Nola Englehorn, Shaneen Murphy, Mary Schubauer-Berigan, 

Joe Amato, and Jim Jenson. The skillful assistance and dedication of Debra Williams 

and Jane Norlander (NETAC) in producing this document are gratefully acknowledged. 

Comments were received from the following people and organizations: Charles 

Carry and LeAnne Hamilton for the County Sanitation Districts of Los Angeles County, 

Whittier, CA.; David Mount and J. Russell Hackett for ENSR Consulting and Engineering, 

Fort Collins, CO; Norman LeBlanc for Hampton Roads Sanitation District, Virginia Beach, 

VA; Charlie Webster, State of Ohio Environmental Protection Agency (EPA), Columbus, 

OH.; Michael Gallaway, State of Ohio EPA, Columbus, OH; Robert Berger for the 

Association of Metropolitan Sewerage Agencies (AMSA), Washington, D.C.; and Kerrie 

Schurr for EPA Region 10, Seattle, WA. We want to thank those individuals or 

organizations for reviewing the report, and in turn improving the document with their 

comments. 

In the review comments, a suggestion was made to summarize all the effort on TIES 

by government, state, academia, contract laboratories, and industries to date. While the 

TIES at Duluth can be summarized, data from all the possible sources are difficult if not 

impossible to obtain. Contract laboratories and industrial data are protected for confidentiality and proprietary reasons, and information about the kinds of toxicants, the types of 

discharges, the time-frame for the TIE, and the costs are difficult to obtain. Numerous 

toxicity problems have been resolved as TIES are initiated because of better plant 

operation. In fact, during a workshop (Aquatic Habitat Institute, 1992) held March 17 and 

18, 1992 in Richmond, CA, these issues were discussed, and presenters of chronic TIE 

data indicated chronic TIES have been much more successful than expected. 

This work was supported in part by the Office of Water, Permit Division, Washington, 

D.C., through the backing of Rick Brandes and Jim Pendergast, who have provided 

strong support for the whole effluent water quality-based approach. 

ix 
Section 1 

Introduction 

The United States Federal Water Pollution Control 

Act Amendments (commonly referred to as the Clean 

Water Act (CWA); (Public Law 92-500 of 1972) states 

that the discharge of toxic pollutants in toxic amounts is 

prohibited. In the CWA, the National Pollutant Discharge Elimination System (NPDES) was established; 

this system provides a mechanism whereby point source 

wastewater discharges are permitted. NPDES permits 

contain effluent limits that require baseline use of treatment technologies (best available technology). The 

technology-based limits are independent of receiving 

water impact, and additional water quality-based limits 

may be needed in order to meet the goal of the CWA of 

“no toxics in toxic amounts.” State narrative and state 

numerical water quality standards are used in conjunction with EPA’s water quality criteria and other toxicity 

databases to determine the adequacy of the technology-based permit limits and the need for any additional 

water quality-based controls. 

When limits were first written into the permits, they 

were based primarily on physical factors such as biological oxygen demand (BOD), suspended solids (SS), 

and color. Additional components were added in subsequent amendments to the CWA; for example, the list 

of 126 “priority pollutants” of which many or most were 

required to be monitored by the permittees. Water 

quality criteria were used to develop the water qualitybased limits for these pollutants. However, water quality criteria or discharge limits exist for only a few of the 

thousands of chemicals in use. 

An important objective of the NPDES program is 

the control of toxicity of discharges and to accomplish 

this objective, EPA uses an integrated water qualitybased approach. Published water quality criteria are 

converted to standards that consist of both chemicalspecific numeric criteria for individual toxics and narrative criteria. The states’ narrative water quality criterion 

generally requires that the waters be free from oil, 

scum, floating debris, materials that will cause odors, 

materials that are unsightly or deleterious, materials 

that will cause a nuisance, or substances in concentrations that are toxic to aquatic life, wildlife or human 

health. Use of toxicity testing and whole effluent toxicity limitations is based on a state’s narrative water 

quality criterion and in some cases, a state numeric 

criterion for toxicity. 

EPA, in 1984, issued a policy statement (Federal 

Register, 1984) that recommends an “integrated approacti’ for controlling toxic pollutants. This integrated 

approach is referred to as the water quality-based approach and is described in detail in the Technical Support Document (hereafter referred to as the TSD; EPA, 

1985A; EPA, 1991 B). The control regulations for EPA 

(Federal Register 23868, 1989) establish specific requirements that the integrated approach be used for 

water quality-based toxics control. This integrated approach results in NPDES permit limits to control toxic 

pollutants through the use of both chemical-specific 

and whole effluent toxicity limitations as a means to 

protect both aquatic life and human health. This combination of chemical specific and whole effluent toxicity 

limitations is essential to the control of toxic pollutants. 

Once the permit limits are set, compliance is established through routine monitoring of effluent quality. In 

this manner, water quality-based limits (when following 

EPA, 1991 B) will protect water quality and prevent the 

state water quality standards from being violated. 

The whole effluent toxicity limitation aspect involves 

using acute and chronic toxicity tests to measure the 

toxicity of wastewaters. Acute toxicity refers to toxicity 

that occurs in a short period of time, operationally 

defined as 96 h or less. Chronic toxicity occurs as the 

result of long exposures in which sublethal effects (fertilization, growth, reproduction) are measured in addition to lethality. The chronic test is used to measure 

the effects of long-term exposure to chemicals, wastewaters, and leachates to aquatic organisms. True chronic 

toxicity tests include the life-cycle of the organism. For 

fish, the life-cycle test is infrequently conducted (NorbergKing, 1989A), and abbreviated test methods have been 

used to estimate chronic toxicity. These tests are the 

7-d growth and survival test (EPA, 1989C), or the 32-d 

embryo-larval earty life stage test (Norberg-King, 1989A). 

These tests rely on the most sensitive life-cycle stages 

(i.e., embryos and larval fish) to estimate chronic toxicity (McKim, 1977; Woltering 1983; Norberg-King, 1989A). 

Hereafter, chronic tests refer to the short-term tests 

that are described in the EPA manuals (EPA, 1992C; 

EPA, 1992D; EPA, 1989C; EPA, 1985C). 

Toxicity is a useful parameter to protect receiving 

waters from potential impacts on water quality and 

designated uses caused by the mixture of toxic pollutants in wastewaters. EPA has published manuals 

which provide test methods for use of freshwater and 

marine organisms to determine acute and chronic toxicity of effluents. These manuals have been available 

since 1978 and 1985, respectively (EPA, 1978; EPA, 

l-l 
19858; EPA, 1985C; EPA, 19888; EPA, 1989C) and 

have been recently revised (EPA, 1991 C; EPA, 1992C; 

EPA, 1992D). These methods are used by federal, 

state and local governments to assess toxicity and 

determine compliance of permitted point source discharges. Since the late 1970’s, toxicity has been measured in wastewaters; permit writers began using toxicity limits in the early 1980’s. With the increased use of 

toxicity testing, substantial numbers of unacceptably 

toxic effluents have been identified. Now, some permittees are required to perform toxicity reduction evaluations (TREs) as a condition of the NPDES permit. The 

TSD defines a TRE as “a site specific study conducted 

in a stepwise process designed to identify the causative agents of effluent toxicity, isolate the sources of 

toxicity, evaluate the effectiveness of toxicity control 

options, and then confirm the reduction in effluent toxicity.” Toxicify identification evaluations (TIES), which 

are a part of the TRE, consist of methods to characterize (Phase I; EPA, 1988A; EPA, 1991A; EPA, 1991D), 

identify (Phase II; EPA, 1989A; EPA, 1992A), and confirm (Phase III; EPA, 19898; EPA, 19928) the cause of 

acute and chronic toxicity in effluents, 

The TIE approach (EPA, 1988A; EPA, 1991A) relies on the use of organisms to detect the presence of 

toxicants in the effluent. Information about the physicaVchemical characteristics of the effluent’s toxicity is 

gained (by the various manipulations) and if possible 

the number of constituents in the effluent is reduced 

before any analyses begin. Using this approach, analytical problems can be simplified and the costs reduced. Toxicity throughout the TIE must be tracked to 

determine if the toxicity is consistently being caused by 

the same substance. Once the physicaVchemical characteristics of toxicants are known, a better choice of 

analytical methods can be made. Knowledge of physical/chemical characteristics of any effluent is used for 

the treatability approach to TRE’s (EPA, 1989D; EPA, 

1989E). 

As with the acute Phase I TIE approach, the chronic 

Phase I TIE is based on manipulations designed to 

alter a group of toxicants (such as oxidants, cationic 

metals, volatiles, or non-polar organics) so that toxicity 

is changed. Chronic toxicity tests are conducted after 

each manipulation to indicate the effect on the toxicity 

of the effluent. Based upon the manipulations that 

change toxicity, inferences about the chemical/physical 

characteristics of the toxicants can be made. Using 

several samples of the effluent for these characterization steps provides information on whether the nature 

of compounds causing the chronic toxicity remains consistent. The tests do not provide information on the 

variability of toxicants within a characterization group. 

From these data the toxicant characteristics can be 

identified as pH sensitive, filterable, volatile, soluble, 

degradable, reducible, or EDTA chelatable. Such information indicates how samples must be handled for 

analyses and which analytical methods should be used. 

The recommended procedure is to concentrate on 

the characterization steps that are most clean-cut and 

have the major effect of reducing the toxicity in the 

effluent. If toxicity in every effluent sample is not 

caused by the same toxicant( the characterization 

tests should indicate if the type of toxicant is the 

same or different. Once identification is initiated, and 

suspects identified, the varying causes of toxicity can 

be evaluated because the concentration of toxicants 

should be tracking with the toxicity. In the earlier 

version of this document (EPA, 19910) we suggested 

that samples be subjected to Phase I techniques until 

no additional responses are found (which was suggested to be at least three samples). After conducting 

several Phase I evaluations for chronic toxicity, we 

have determined that if the effluents’ toxicity is readily 

characterized after Phase I even with one sample it 

may be prudent to proceed with Phase II (EPA, 1992C) 

to measure the toxicant( Use of toxicity patterns as 

the TIE progresses can be helpful if patterns are tracked, 

beginning with the first samples. Following characterization, a decision is made to proceed with identification (Phase II; EPA, 1989A; EPA, 1992A) and confirmation (Phase III, EPA, 19898; EPA, 19928) or to 

conduct treatability studies where the identification of 

the specific toxicants (cf., acute treatability procedures 

(EPA, 19890; EPA, 1989E)) is not made. 

Chronic toxicity must be present frequently enough 

so that an adequate number of toxic samples can be 

obtained. Enough routine toxicity testing should be 

done on each effluent before a TIE is initiated (EPA, 

1991B), to ensure that toxicity is consistently present. 

It is not important that the same amount of toxicity is 

present in each sample; in fact, variable levels of toxicity can assist in determining the cause of toxicity. If 

toxicity is not consistently present, when it occurs the 

toxicity can be pursued and if a toxicant is suspected, the non-toxic samples may be used to eliminate suspects. One cannot assume that if the effluent 

showed acute toxicity and a TIE was completed, identifying the cause(s) of acute toxicity and action taken to 

remove the acute toxicant from the effluent, that the 

sublethal toxicity exhibited is due to the same compound. 

1-2 
Section 2 

Quality Assurance, Health, and Safety, and Facilities and Equipment 

2.1 Quality Assurance 

The quality assurance plan (QAP), as described in 

Standard Methods for the Examination of Water and 

Wastewater (APHA, 1989) (describes standards to conduct performance evaluations) is primarily for analytical 

analyses. A QAP for toxicity testing can be developed, 

but determining the recovery of known additions for 

toxicity testing is not possible. For TIES the combination of chemistry and biology requires a level of checks 

and balances not typically used under other situations. 

A step-by-step QAP for all steps of a TIE is not always 

possible due to the unknown toxicant requiring various follow-up testing and analytical procedures; however as a TIE progresses, additional or different tests 

may be needed and many aspects of the TIE QAP can 

be addressed as the TIE proceeds. Adhering to the 

general guidelines of a strong QAP is important however, and should increase the probability of the TIE 

succeeding. As additional steps are recognized, the 

details should be added to the QAP. 

Specific quality control (QC) procedures for aquatic 

toxicity tests are different than the specific QC procedures for chemical analytical methods. Both procedures have common goals that are to know that reliable 

data are generated, to recognize and eliminate unreliable data, and to have methods which assist investigations in resolving problems for future work. The quality 

assurance (QAIQC) guidance given by EPA (1989C) 

for the short-term tests lists numerous items of concern 

for toxicity testing. These are: (a) effluent sampling/ 

handling, (b) test organisms, (c) facilities, equipment 

and test chambers, (d) analytical methods, (e) calibration and standardization, (f) dilution water, (g) test conditions, (h) test acceptability, (i) test precision, (j) replication and test sensitivity, (k) quality of organisms, (I) 

quality of food, (m) control charts, and (n) record keep 

ing and data evaluation. Many of these should be 

closely followed, and the reader is encouraged to review the guidance in relation to QAIQC in both the 

short-term effluent test manual (EPA, 19896; EPA, 

1992C) and the acute Phase I manual (EPA, 1991A). 

2.2 C?A/C?C Cost Considerationsand Testing 

Requirements 

For the chronic TIE, cost considerations are important and concessions in the requirements of the QC 

may have to be made. In some instances, the data will 

demand stringent control while in others, the QC can 

be lessened without impact to the overall endpoint of 

the TIE. 

TIES can require a great number of toxicity tests. 

The use of all aspects of the standard test protocols 

(EPA, 1989C; EPA, 1991C) is not necessary in Phase 

I. The factors of time requirements, number of tests 

and the test design (i.e., five replicates versus ten, four 

dilutions versus five) must be considered and weighed 

against the type of questions that are posed. For 

example, the need for water chemistry data are specific 

for each Phase I test. The testing requirement (EPA, 

1989C) according to the permit requirement most likely 

included pH, daily measurements of DO, temperature, 

conductivity, alkalinity, and hardness measurements in 

the low, middle, and high concentrations for the five 

test dilutions of the effluent. However, hardness measurements are not pertinent for the methanol eluate 

collected from a solid phase extraction column. The 

post C,, SPE column effluent samples are more similar 

to the effluent and a concern for low dissolved oxygen 

(DO) exists, while the test solutions of the methanol 

eluate are more similar to the dilution water and the 

possibility of low DOS is not as great a concern. In 

contrast, frequent pH measurements on all test concentrations are needed to determine the impact of pH 

sensitive compounds. 

As TIES are reliant on a strong QAP, there are 

several aspects of a QA/QC program for chronic TIES 

that should be delineated. In regard to test organism 

quality, there are steps for culturing organisms that 

should help provide the necessary QC verification that 

is needed to ensure the animals are representative in 

their sensitivity. These steps are simply routine items 

such as monitoring and recording the young production 

(for cladocerans) of the culture brood animals once a 

month, conducting monthly reference toxicant tests (including maintaining control charts), monitoring the preparation dates for the reconstituted waters used, and 

monitoring the types and age of the foods fed (NorbergKing, 19898). For fathead minnows, it is useful to 

monitor the survival of the breeding stock, and the 

percent hatchability of the embryos, to verify that new 

genetic stock is introduced on a regular basis, and to 

conduct monthly reference toxicant tests (Norberg-King 

and Denny, 1989; Denny, 1988). Similar parameters 

for other species that are used are also desirable. 

Since toxicity tests in the early part of the chronic 

Phase I do not generally follow all the effluent testing 

2-1 
requirements (EPA, 1989C), the QC measures are not 

as strict because the data are primarily informative 

rather than definitive. When Phases II (identification) 

and III (confirmation) are initiated, then OC aspects 

should be reconsidered and the tests modified. Phase 

I procedures frequently use one species and later stages 

of the TIE (Phase Ill) use more than one species to 

determine whether the cause of toxicity is the same for 

other species of the aquatic community. 

Reference toxicant tests are not conducted with 

each set of Phase I manipulations because of the 

amount of labor and large numbers of animals required 

for testing. In general, the utility of the reference 

toxicant test is to know that the organisms are responding as expected. Since only relative differences are 

needed at this stage (Phase I), reference toxicant data 

are much less useful for the characterization interpretation but are important for the knowledge of the quality 

of the test organisms and general test procedures. For 

various manipulations of the TIE, organism responses 

are compared to either the baseline test (see Section 

6) or the response of organisms in the dilution water 

treatments. Monthly reference toxicant tests should 

provide the necessary information about the quality of 

the organisms for the laboratory conducting the TIE. 

When a toxicant has been identified (Phase II) and 

tests for Phase III confirmation indicate it is the 

toxicant( that chemical should become the reference 

toxicant with the species used in the TIE. 

Using receiving water as the dilution water in Phase 

III confirmation will help ensure that receiving water 

effects are properly considered (see Section 3, Dilution 

Water). The variability of the effluent, by nature of the 

TIE, is defined during the TIE, and this information will 

aid in determining the appropriate control option in 

order that the final effluent is safe upon discharge. 

2.3 QA/QC and Chronic Testing 

Considerations 

An inherent problem with effluents is that no effluent test can be repeated to assure that the toxicity is 

the same and that the toxicants are the same. However, repeated baseline tests (Section 6) can be done 

with the same effluent sample to determine how long 

that effluent sample can be used. The chemical and 

toxicological nature of the effluent shifts as an effluent 

is discharged or as an effluent sample is stored. Effluent constituents degrade (at unknown rates) and each 

constituent has its own rate of change. Analysis of 

each sample should be initiated as soon as the sample 

is received in the testing laboratory (generally ~24 h). 

Until an effluent sample has been tested several times, 

there is no way to predict how long a sample can be 

stored before the toxicity changes. Testing of each 

sample can be done provided the toxicity remains and/ 

or stabilizes; however this cannot be determined at the 

beginning of the Phase I battery of tests and will be 

known only through testing several samples a few times. 

Even though the toxicity remains, it is possible that the 

toxicant may change with time. The number of samples 

to evaluate and the number of tests to conduct must be 

weighed against the cost of the effort and how representative each effluent sample is of the effluent. Effluents that have low and non-persistent toxicity may 

need to be approached with the Tier 1 and Tier 2 

characterization steps applied simultaneously (see Section 6). 

In a chronic TIE, information obtained from a test 

should be maximized. This may mean paying particularly close attention to details such as small differences 

in the number of neonates the cladocerans are producing or the lack of food in the stomach of the larval fish. 

These parameters and any other observed characteristics during a test may be subtle indicators and quite 

informative about small changes in toxicity. For example, if all the animals exposed to the whole effluent 

die on day 4, and in some characterization test the 

animals don’t reproduce or grow but are alive at day 7 

of the exposure, that characterization manipulation reduced the toxicity, but did not remove it completely. 

Observations such as these may be just as useful as 

reductions in young production or growth. 

While some abbreviations in the test design are 

made, the general principles for toxicity testing still 

apply. For example, all animals must be added to test 

solutions randomly. Animals must be placed in a test 

chamber one at a time. For the fathead minnows, use 

of an intermediate vessel to hold all 10 animals is 

preferable to ensure that animals are assigned randomly and that the volume of water added with the fish 

is minimized (l-2 ml). Also, transferring animals may 

require separate pipettes for each concentration or cleaning of the pipettes between concentrations to prevent 

cross contamination. However, we have observed that 

C. dubia do not have to be placed under the water; 

they can be added or transferred by dropping the water 

droplet containing the animal into the test solution. The 

problem frequently observed with D. pulex where animals are caught at the surface of the test solution 

(called ‘floaters”) does not occur with C. dubia. Randomization, careful exposure time readings, use of animals of uniform narrow-age groups (i.e., Ceriodaphnia 

neonates O-6 h old rather than O-l 2 h old) should assist 

in quality data generation. 

Standard operating procedures (SOPS) should be 

developed for each Phase I test, for preparing the 

reconstituted waters, preparing the foods for the test 

organisms, calibration and standardization for all measurements (temperature, DO, pH, conductivity, alkalinity, hardness, ammonia, chlorine), and other general 

routine practices. 

An important aspect of TIES is accurate and thorough data recording. All observations should be documented. Items that were not thought to be important at 

first may be useful in later stages of analysis and 

actually assist in the confirmation of the toxicant( 

These observations can be as simple as large bubbles 

produced during the aeration and filtration manipulations, large particles present in whole effluent, and low 

2-2 
pH upon arrival. It is best to record data so that any 

preconceived ideas of the toxicants are avoided. Data 

records should include records of test organisms (species, source, age, date of receipt, history and health), 

calibration records, test conditions, results of tests, and 

summaries of data. Once a control chart is developed 

using point estimates for reference toxicant tests, 1 out 

of 20 reference toxicity test results will be predicted to 

fall outside the acceptable limits if the 95% confidence 

intervals are used to develop the control chart (EPA, 

1991 C). If TIES are conducted during such a period, 

the TIE data generated must be used with caution, and 

the investigator must carefully examine the TIE data to 

determine if the results are usable. The decision may 

be based on consistency of the concentration response 

data, control blank performance, and the consistency of 

the TIE results with those obtained with the same 

effluent sample. 

2.4 CWQC Blanks and Artifactual Toxicity 

Throughout the TIE, dilution water samples are 

subjected to most of the procedures and analyses performed on the effluent sample (see Section 5.6). This 

is done to detect toxic artifacts (i.e., toxicity due to 

anything other than the effluent constituents causing 

toxicity) that are created during the effluent characterization manipulations (see Section 6). These manipulations can make QC/QA verifications difficult, as the 

use of such blanks for interpreting toxicity results is not 

standard toxicology. For example, typically organism 

responses from any toxicity test in standard aquatic 

toxicology are compared to the performance of control 

organisms which were in dilution water only. In the 

TIE, controls are used to judge organism performance 

(Section 5) and toxicity controls and blanks are used to 

evaluate whether a manipulation affected the toxicant( 

therefore the results of all characterization tests are not 

necessarily compared to the baseline test. For instance, post-column effluent samples that are collected 

and tested following concentration on a resin column 

have been filtered first. Therefore it is only logical to 

compare the post-column effluent toxicity (post C,, SPE 

column test; Section 6.6) to the toxicity observed In the 

filtered effluent sample (filtration test; Section 6.4) rather 

than to the unfiltered whole effluent (baseline test; Section 6.1) (see Section 5). 

Artifactual toxicity can occur in several of the manipulations, particularly from the major pH adjustment 

manipulation (Tier 2). Toxicity results from tests relying 

on the addition of the reagents (EDTA, sodium thiosulfate, acids/bases) must be interpretable. Addition of 

both the acid (HCI) and the base (NaOH) can form a 

toxic product (e.g., NaCI). The addition of the acid and 

base may interfere with the growth and reproduction of 

the test organisms for the short-term chronic test, at 

lower levels than cause mortality in the acute test. 

Whether additives act in an additive, synergistic, or 

independent manner with the compounds in the effluent must be determined during the TIE but this is not 

likely to be clear during Phase I. Artifactual toxicity 

can occur in the aeration process, where contaminated 

air can be introduced. Also, contaminants can be 

leached from solid phase extraction (SPE) columns, 

and methanol leaching off the column can cause bacterial growth that will confound the results in the postcolumn blankand post C,, SPE column tests. Originality and judgement are needed to devise tests that will 

reveal artifactual toxicity (see Section 6) and some of 

these methods to deal with artifactual toxicity will be 

effluent specific. 

2.5 Health and Safety Issues 

For the toxicity identification work, hazards present 

in any effluent may not be known until Phase II identification steps have been started. Therefore, safety requirements for working with effluents (or other samples) 

of unknown composition must follow safety procedures 

for a wide spectrum of chemical and biological agents. 

Because all of the hazards in an effluent sample may 

not be known when a toxicant is identified, effluent 

samples should be treated as hazards of unknown 

composition throughout the TIE. Knowledge of the 

types of wastewater treatment applied to each effluent 

can provide some insight for the possible hazards. For 

example, unchlorinated primary treatment plant effluents containing domestic waste may contain pathogens. Chlorinated secondary effluents are less likely to 

contain such agents. Effluents from activated sludge 

treatment plants are less likely to contain volatile toxicants. 

Because effluent characteristics are unknown, personnel should follow the guidelines for hazardous materials (EPA, 1991A; 1991C). Also, if any sample 

contains human waste, personnel should be immunized for diseases such as hepatitis B, tetanus, polio, 

and typhoid fever. 

Each laboratory should provide a safe and healthy 

work place. All laboratories should develop and maintain effective health and safety programs (APHA, 1989; 

EPA, 1991C). Each program should consist of: (a) 

designated health and safety officers, (b) formal written 

health and safety plans, (c) on-going training programs, 

and (d) periodic inspections of emergency equipment 

and safety violations, Further guidance on safety practices is provided in other documents (APHA, 1989; 

EPA, 1991A; 1991C). 

2.6 Facilities and Equipment 

The laboratory facilities and equipment needed to 

conduct TIES are discussed in the acute Phase I manual 

(EPA, 1988A; EPA, 1991A). Most of the equipment for 

conducting the short-term tests are delineated elsewhere (EPA, 1989C; EPA, 1992C). The reagents used 

for the chronic Phase I characterization are identical to 

those described in the acute Phase I manual (EPA, 

1991A). Compressed air systems with oil-free compressors and air filters to provide high purity air are 

very important (EPA, 1991A). All glassware should be 

rigorously cleaned, and the glassware used for filtering 

must be rigorously cleaned to remove residual contaminants from the glass frit(s). Filtering equipment may 

2-3 
need to be made of plastic to avoid leaching of metals and cause toxicity. Ultra pure acids and bases (e.g., 

or other toxicants from glass when acid washes are SuprapuP, E. Merck, Darmstadt, Germany) should be 

used (see Section 6). Use of stainless steel frits can used to prevent impurities in the acids/bases from interbe used provided pH adjustments are not made since fering in the toxicity results. 

metals will rinse off the stainless steel at extreme pH’s 

2-4 
Section 3 

Dilution Water 

Dilution water used for chronic TIE’s must meet 

several requirements. Obviously it must support adequate performance of the test animals in regard to 

growth, survival, and reproduction since these are the 

effects measured in the tests. Secondly, it must not 

substantially change the animals’ response to the sample 

toxicants. Because the characteristics of the toxicants 

are not known, there is no way to be sure which dilution 

water characteristics are important. Hardness and alkalinity are most often used to select the dilution water 

but these parameters are generally of little importance 

for non-polar organics. Rarely is the organic matter 

content considered and yet for both non-polar organics 

and metals, organic matter has more effect on toxicity 

than hardness. Experience in the acute TIE work has 

shown pH to be the single most important water quality 

characteristic for characterizing the cause of toxicity. 

The most important consideration, in addition to 

those mentioned above, is that the water be consistent 

in quality and not contain contaminants that could produce artificial toxicity. For example, if there was a 

nontoxic concentration of a non-polar organic present 

in the dilution water, when samples are concentrated, it 

might be toxic and this can confound the identification 

of the components causing toxicity in the effluent. The 

best policy is to use a high purity reconstituted water or 

a well water of known suitability. Receiving water 

should not be used until Phase III, when it is the water 

of choice to evaluate the toxicant in the receiving water 

system (see Section 2.2). 

A reconstituted water of similar pH, hardness and 

alkalinity to that of the effluent is a first approximation 

of an appropriate water; however, organic matter is 

hard to duplicate. Experience has shown that for the 

Ceriodaphnia test, the addition of food’ to the water 

has been helpful to provide some organic material. 

With food added, traces of contaminants can be less 

toxic. If higher concentrations of effluent are to be 

used, the choice of the dilution water is less important 

because the characteristics of the effluent dilution mixture will resemble those of the effluent. As information 

is gained about the toxicant characteristics, the choice 

of dilution water can be improved. 

’ Food added for the C. dubia tests are the yeast-cerophyll-trout food 

(YCT) and the algae (Selenastrvm capncomufum) at a rate of 0.1 ml/ 

15 ml (EPA, 1989C). Although at ERL-Duluth the algae has been 

addedattherateof0.05ml/l5mluntilMayof 1991 when weincreased 

the level (EPA, 1989C). 

The impact of dilution water choice depends on the 

IC25 (see Section 5.8) of the effluent. If toxicity changes 

substantially from sample to sample, but the dilution 

water selected does not match the effluent in water 

characteristics yet is kept the same throughout several 

samples for Phase I, then the effect of the effluent in 

the dilution water can also vary across samples. As 

the TIE progresses into Phase II, attributing relative 

toxicity to various constituents must be more refined. 

For instance, suppose the suspect toxicant is a cationic 

metal whose toxicity is hardness dependent. Also, 

suppose that the whole effluent has a hardness of 

300 mg/l as CaCO, (very hard water) but the dilution 

water has a hardness of 40 mg/l as CaCO,. In this 

case, the hardness in each of the test dilutions will be 

different from that of either the whole effluent or the 

dilution water. Provided the cationic metal concentrations vary over the course of the TIE period, the amount 

of toxicity (as toxic units2? TUs) due to a particular metal 

concentration will also vary depending upon the effect 

concentration in the effluent. If the first whole effluent 

sample contains 160 pg/I of zinc (for this example, 160 

ps/l is 1 .O TUc in very hard water) and the test is 

conducted using a dilution water of 40 mg/l as CaCO, 

(soft water), the no effect concentration would be 100% 

where hardness is 300 mg/l and the effluent would 

have cl TUO. The second whole effluent sample contains 480 &I of zinc. One would expect this sample to 

possess 3 TUs (480 pg/l + 160 J.@). The toxicity due 

to the second effluent sample would likely contain more 

than 3 TUs because the hardness at the effect level 

(<lOO%) would be much lower than at 100% effluent 

(where hardness is 300 mg/l as CaCO,). The effect 

2 TUs is a means of normalizing the concentration term (i.e., LC50, 

NOEC, IC25 as percent effluent; see Section 5.8) lo a unit of toxicity. 

The use of the TUs approach allows effluent toxicity to be compared 

(provided test species and test duration ara the same) to a suspect 

toxicanls toxicity. The toxicity of an effluent and a chemical are 

different and different concentrations of each equal one LC50 (1 TU). 

TUs of an effluent can be calculated for either acute or chronic toxicity 

endpoints. The number of acute TUs in the effluent is 100% + LC50 

= TU and the chronic TUs in the effluent is 100% + NOEC = TU or 

100; + IC25 = TU, (EPA, 19918). For specific chemicals the Td is 

equal to the conce’ntration of the compound present in the effluent 

divided by the acute test LC50 for TUd or the chronic test NOEC or 

IC25 for the TU.. The assignment of TU. is necessary for the 

correlation step (Phase Ill) when effluent toxkity TUs are compared 

to suspect toxicant TUs 

3-1 
level would be near 20-25% effluent where hardness water for the diluent, the hardness might change drawould be 400 mgil as CaCO, and 1 TU of zinc would matically and confound calculation of TlJ’s in a like 

be 460 pg/l. In addition, if one were to use receiving manner if the effect concentration was 400% effluent. 

3-2 
Section 4 

Eff bent Samples 

To determine whether an effluent sample is typical 

of the wastewater discharge may require a number of 

samples to be tested. Experience has shown that the 

use of several samples spanning two to three months 

has been successful in characterizing many effluents. 

TIE work on atypical samples may be problematic and 

these TIE procedures were not developed for one-time 

episodic events. However, the very nature of atypical 

samples may provide valuable assistance in the TIE 

effort by identifying the type of toxicant that previously was not suspect. This is probably more likely 

when an atypical sample has greater toxicity than the 

other samples. In addition, the atypical toxic sample 

may aid a discharger in recognizing wastewater treatment plant upsets and assist the discharger in implementing prevention procedures or generally improve 

and maintain better wastewater plant housekeeping 

efforts, which in turn may eliminate the episodic toxicity 

problems. 

The acute Phase I manual discusses the quantitative and qualitative changes in effluents (EPA, 1988A; 

EPA, 1991A) that may affect toxicity. Varying concentrations of toxicants, different toxicants, water quality 

characteristics, and analytical and toxicological error 

are all factors in determining the toxicity of an effluent. 

Although the toxicity of an effluent over time appears 

unchanged, there may be more than one toxicant involved in each sample, and not necessarily the same 

ones. 

At the same time a sample is collected, information 

on the facilities treatment system (normal operation; 

aberrant processes) may be useful. When dealing with 

industrial discharges, details of the process being used 

may be helpful. These details and others should be 

recorded and provided to the laboratory conducting the 

TIE at the time of sample shipment. When samples 

are received, temperature, pH, chronic toxicity, hardness, conductivity, total residual chlorine (TRC), total 

ammonia, alkalinity and DO should be measured. Figure 4-1 provides a typical format to record such information. 

Since most TIES are not performed on-site, the 

effluent samples must be shipped on ice to the testing 

location. The samples should be cooled to 4°C or less 

prior to shipment and they should be shipped in sturdy 

ice chests to prevent either temperature increases or 

container breakage during shipment. Primary requirements of the TIE are that toxicity occurs frequently in 

the effluent samples and that the toxicity of each sample 

(held at 4°C) remains in the effluent sample for a 

sufficient period of time. If samples repeatedly lose 

their toxicity after shipment, steps should be taken to 

preserve toxic fractions (Section 6.7) for later testing 

and analysis. For example, if the initial characterization 

tests indicate the presence of non-polar organics, one 

tool to use is to concentrate large volumes (5-10 L) of 

effluent when the sample arrives (see Section 6). Use 

of the Phase II (EPA, 1992A) non-polar fractionatiorl 

procedure is the preferred way to concentrate the nonpolar toxicants for subsequent analysis and testing. 

While efforts must be expended on this procedure, it 

can be a crucial step to aid in identifying potential 

toxicants (in instances where toxicity is present and lost 

in the effluent). The information on when toxicity degrades or is lost may become useful as the toxicant 

is identified (see Section 9; EPA, 1991A). Filterable 

toxicants which degrade quickly in the effluent may be 

recovered from the filters with solvent and stored for 

future use (cf., fikration test; Section 6.4). 

For one chronic Phase I TIE, a typical volume of 

effluent needed to ship is 19 L (5 gal) but of course this 

will depend on the options chosen for the TIE (Section 

6) and 38 L (10 gal) may be more helpful once identification and confirmation begin on any sample. The 

second edition of the acute Phase I TIE manual (EPA, 

1991A) recommends that samples be initially collected 

and stored in both glass and plastic to determine whether 

effluent stored in either container affects the toxicity. 

Some compounds (such as surfactants) are less toxic if 

water samples containing them are stored in plastic 

containers. Prior to initiating the characterization it 

may be useful to collect and test several preliminary 

samples to determine which containers to use during 

the TIE to provide samples that are the most representative of the effluent (see Phase I, Section 6 (EPA, 

1991A) for more details). Less volume (52 L) is needed 

for these tests. 

Composite samples should be used for Phase I. 

Later, in Phases II and III, where variability is desired, 

grab samples should be used. Samples that are consistent (i.e., composite samples) give results that are 

easier to interpret and lead more rapidly to identification (Phase II) and confirmation (Phase Ill) of the cause 

of toxicity. Grab samples can provide the maximum 

effluent toxicity; however, it is more difficult to catch 

4-l 
Figure 4-1. Example data sheet for logging in samples. 

Sample Log No.: 

Data of Arrival: - 

Date and Time 

of Sample Collection: 

Facility: 

Location: 

NPDES No.: 

Contact: 

Phone No. 

Sampler: 

Condition of treatment system at time of samplrng: 

Status of process operations/production (if applicable): 

Comments: 

intermittent peaks of toxicity (such episodic events may the same toxicant may not be present in each sample, 

not be caused by the same toxicant that causes routine or it is present in varying concentrations and other 

toxicity). toxicants may appear. 

Multiple effluent samples in each test should not be 

used in Phase I as is done for permit testing (EPA, 

19896). We have found that using only one composite 

sample for each set of Phase I characterization tests is 

adequate. If several effluent samples are used for 

renewals during the chronic Phase I TIE and the toxicants are different or change in their ratios one to 

another, the interpretation of Phase I will be nearly 

impossible. Indeed such variability must be identified 

but it should be done after at least one or preferably 

most of the toxicants are known. The use of one 

sample is more important in Phase III, (EPA, 19928) 

where toxicity data are correlated to the measured 

concentrations in the effluent. If multiple samples are 

used, this correlation can not be readily done because 

Existing routine toxicity test data should be examined. If one notes a sudden response such as death in 

the middle to the end of the test period and especially if 

it is associated with a new sample, the effect being 

measured may actually be acute rather than chronic 

and if so the approach may be switched to an acute 

TIE approach. The investigative approach should be 

adjusted to respond to such situations. When the 

permit test is conducted and the test fails, it may be 

desirable to try to identify the toxicants in those permit 

compliance samples. This can be done by collecting 

the appropriate volume needed for a chronic TIE of 

either the daily samples or the three samples used for 

the short-term toxicity test (EPA, 1992C). Additional 

short-term toxicity tests can be conducted on each 

Sample Type: 0 Grab 0 Composite 

0 Glass 0 Plastic 

0 Prechlorinated 

0 Chlorinated 

9 Dechlorinated 

Sample Conditions Upon Arrival: 

Temperature -__- 

PH 

Total Alkalinity 

Total Hardness 

Conductivity/Salinity 

Total Residual Chlorine--_- 

- 

4-2 
sample prior to any TIE tests on each sample or prefer- to demonstrate that the effluent is toxic in less than the 

ably additional short-term tests would be initiated on full 7-d of the C. dubia or fathead minnow tests. When 

each new sample during the 7d test to evaluate whether the toxicity that occurs in 548 h (C. d&a) or 196 h 

it is the cumulative toxicity from all samples or whether (fathead minnow) with any one of the samples from the 

one or two samples are driving the toxicity. We have permit compliance samples or any sample collected for 

observed in several effluent tests that the toxicity dur- the TIE, is observed as >50% mortality, acute TIE 

ing the short-term chronic test can be caused by one or procedures can be applied to more quickly characterize 

two samples and these samples cause the chronic test the toxicant( 

4-3 
Section 5 

Toxicity Testing 

5.1 Principles 

The test organism is used as the detector of chemicals causing chronic toxicity in effluents and other aqueous media. The response to toxic levels of chemicals 

is a general one; however the organism is the only tool 

that can be used specifically to measure toxicity. Only 

when the cause of toxicity is characterized can chemical analytical methods be applied to identify and quantify the toxicants. 

Chronic TIE’s will usually be triggered by the use of 

the toxicity test methods as found in the short-term 

chronic toxicity test manuals (EPA, 1989C; EPA, 1992C). 

These methods rely on sublethal endpoints as the indicator of chronic toxicity for the Phase I manipulations, 

therefore conducting the tests strictly as detailed in 

those manuals is not always necessary and sometimes 

not possible. Modifications have been developed and 

these include: (a) reduced test volumes, (b) shorter 

test duration, (c) smaller number of replicates, (d) reduced number of test concentrations, and (e) reduction 

in the frequency of the test solution renewal. In addition, the frequency of preparation of manipulated 

samples for test solution renewal must be established 

and this issue is discussed in the following section. 

Any loss of test precision due to these modifications is 

not as critical during Phase I characterization as it is in 

Phase II and Phase III (EPA, 1992A; EPA, 19928). 

During Phase I the analyst is searching for an obvious 

alteration in effluent toxicity, which may be obtained 

using modified chronic test methods. Confirmation 

testing (Phase Ill) conducted according to the standard 

methodologies will confirm whether the toxicant detected in the characterization and identification steps 

(Phases I and II) is the true toxicant. 

5.2 Test Species 

In most cases, freshwater effluents will be subjected to this evaluation because they have been found 

to be chronically toxic to the cladoceran, C. dubia, or to 

the fish, fathead minnow (P. promelas), or possibly to 

the cladocerans, D. magna or D. pulex. Freshwater 

effluents discharged into marine environments are evaluated for toxicity using marine species or may be assessed with freshwater species (EPA, 19910). TIE 

guidance for the marine species will be forthcoming in 

the fall of 1992 (George Morrison, personal communication, ERL-Narragansett, RI). 

The species which detected the toxicity which in 

turn triggered the TIE, is the first choice for the TIE 

species. When an alternative species is chosen one 

must prove that it is being impacted by the same 

toxicant as the species which initially detected the 

toxicity. The species need not have the same sensitivity to the toxicant( but each species’ threshold must 

be at or below the toxicant concentration(s) present in 

the effluent. One method of proving that the species 

are being affected by the same compound(s) is to test 

several samples of the effluent over time to both species. If the effluent possesses sufficient variability, and 

the two species IC25s (see Section 5.8 below for a 

description of the IC25) change in proportion to one 

another, the analyst may assume that the organisms 

are reacting to changing concentrations of the same 

compound. Further proof that the two species are 

responding to the same toxicant should surface during 

Phase III. If the toxicant is the same for both species, 

then characterization manipulations which alter toxicity 

to one species should also alter toxicity to the second 

species. The extent to which toxicity is altered for each 

will depend upon the efficiency of the manipulation and 

the organism’s sensitivity to the toxicant. Steps applied 

in Phase III will confirm whether the two species are 

indeed sensitive to the same toxicant in the effluent. 

Extensive time and resources may be wasted if one 

discovers during Phase III that the organism of choice 

is not responding to the same toxicant as the species 

which triggered the TIE. 

For the above mentioned reasons, we recommend 

when at all possible to use the organism which prompted 

the TIE. Our chronic TIE experience has been based 

on tests with C. dubia and/or larval fathead minnows. 

Obvious constraints on the use of other species are 

availability, size, age, and adaptability to test conditions. Also, the threshold levels for additives and reagents must be determined for other species. 

5.3 Toxicity Test Procedures 

Measures to conserve time and resources required 

to conduct a chronic Phase I must be used in order to 

make the procedures cost-effective. The application of 

all aspects of the standard short-term chronic tests to 

Phase I in terms of replicates, routine water chemistries, test duration, and volume is not practical due to 

time constraints and expense. Variations of the procedures need to be implemented whenever possible. 

As mentioned above, smaller test volumes can be 

used in all tests with C. dubia and in most instances 

with fathead minnows. For example, 10 ml in a 1 oz 

5-1 
plastic cup (or 30 ml glass beaker) has been adequate 

for C. dubia and 50 ml in a 4 oz plastic cup (10 fish per 

cup) has been used successfully to test the fathead 

minnows (or 100 ml in a 400 ml glass beaker). There 

are two precautions to watch for in the chronic TIE 

tests-l) evaporation of test solutions and 2) transfer 

of toxicants while moving the animals. If evaporation 

reduces test volumes, efforts to reduce the evaporation 

must be made or larger volumes must be used. The 

volume of water added with each transfer should be 

minimized, because the volume used in the test is 

small, and the resultant test concentration could be 

diluted, thereby reducing toxicity. Using the same size 

test chambers and consistent volumes should be maintained in Phase I; when Phases II and III are initiated, 

tests should be conducted following the test protocol 

that was used to trigger the TIE. This may be important in Phase I to be as sure that the oxygen requirements for the test species are met and that toxicity is 

not due to physical restrictions of the test procedure. 

If a reduction in the number of replicates per test 

concentration is used, one must assume that precision 

is sufficient enough to decipher changes in toxicity that 

must be measured. For the C. dubia test, five animals 

per concentration (one per cup) and for the fathead 

minnow test, two replicates per concentration and 10 

fish per replicate have been found to be adequate for 

interpreting the changes in toxicity. However this smaller 

data set is not amenable to all statistical requirements 

as described for the short-term tests (EPA, 1989C; see 

Section 5.8). Use of more organisms and more replicates may be preferable if Phase I data are likely to be 

used in Phase III confirmation (see Sections 2.2 and 

2.3). 

A shortened version of the 7-d C. dubia test, referred to as the 4-d test, may be useful in the TIE. The 

4-d test does not have to be as sensitive as the 7-d 

test, just sensitive enough that the toxicity changes 

occurring in Phases I and II of the TIE (using 4-d tests) 

would be the same as the 7-d tests. The 4-d day test 

was found to produce similar results for single chemicals (Oris et al., 1991), but in tests in our laboratory 

with effluents, the 4-d test has not been as sensitive for 

all effluents tested as the 7-d test in determining the 

effects on young production and survival. Masters et 

al. (1991) tested C. dubia to one effluent (three times), 

three surfactants, three metals, and three organic compounds with the 4-d and 7-d exposures. They found 

that for the most part the effluent toxicity was similar for 

the 4-d and 7-d test results but for the surfactants the 

7-d test was more sensitive. For the metals (cadmium, 

lead, and zinc), ethylene glycol, and pentachlorophenol, the chronic toxicity values for both tests were very 

similar while the 4-d test was more sensitive for phenol. 

In the 4-d test, when animals are initially exposed 

at 72 h they are ready to produce their first brood. 

Therefore, toxicity can be underestimated because these 

animals are predisposed to produce their first brood, 

unlike the animals exposed as neonates (24 h old). The 

exposure during a 4-d test may miss their most sensitive life stage. However for the Phase I where the 

purpose is to detect differences following various manipulations, this issue is not as important as the ability 

to rapidly conduct the characterization. Use of the 

shorter term test will decrease the cost of Phase I 

TIE’s. In the confirmation of toxicity (Phase Ill), the 7-d 

test is required because the toxicity as measured in the 

7-d test (with more replicates, more dilutions, more 

volume) was used to detect toxicity for the permit, and 

should be used to confirm the cause of toxicity. 

To conduct a 4-d test with C. dubia, neonates (O-12 

h old) are placed in the dilution water that will be used 

to conduct the TIE. At present these animals are held 

in groups of three, two or individually in test containers 

(with 15 ml of culture water) and fed daily until they are 

72 h (+6 h) old in a similar test fashion (Oris et al., 

1991). The animals are then transferred to the baseline 

test solutions or the various characterization test solutions. The test is then continued for 4-d using the 

endpoint of three broods. 

The use of known parentage (EPA, 1989C) for the 

C. dubia test is important when the number of replicates is reduced, and helpful for Phase I, II or III tests 

and in routine tests as well (EPA, 1992C). For Phase I, 

this known parentage approach allows the young of 

one female to be used across one replicate of all 

dilutions and the control (i.e., 5 animals), the young 

from another female for the next replicate set of dilutions and control, and so on until all test cups contain 

one young animal. By this technique, animals from a 

given female that later appear atypical in appearance 

or movement or produce no young when others in the 

same test concentration are producing normally can 

legitimately be dropped from the data set without statistical bias (Norberg-King et al., 1989). The ability to 

discard such data without bias improves precision. Precision will be better when n 2 7 per treatment for 

C. dubia or n 2 4 for the fathead minnow test. 

5.4 Concentrations to Test 

The level of toxicity for any given discharger most 

likely will have been established with some degree of 

certainty from previous tests that were conducted on 

the effluent that triggered the TIE. 

Therefore during Phase I of the TIE, we have found 

that four effluent dilutions and a control are adequate to 

define the toxicity of the sample while reducing the cost 

of the tests. Now for the TIE, the key to choosing the 

concentrations to test is to select those that will assist 

in the detection of small changes in toxicity, which is 

essential in the chronic TIE. For example, if the NOEC 

(from a previous data set) is 12% (or IC25 is 10%) 

then a concentration series such as 6.3%, 12.5%, 25%, 

and 50% would be logical; or perhaps closer concentration intervals may be desired. Using 20% as the 

high concentration and a dilution factor of 0.7, would 

mean the concentrations to test would be 7%, lo%, 

14%, and 20%. If the NOEC (from historical data) is 

40-50% (or above 50%), then the concentrations to test 

should be, for example, 25%, 50%, 75%, and 100% or 

5-2 
40%, 60%, 80%, and 100%. Choice of dilution factor 

and test concentration range is a matter of judgement 

and depends on needed precision and practicality. 

In nearly all examples in this document, the concentrations of 12.5%, 25%, 50%, and 100% are used. 

We are assuming that if effluents have ICp (or NOEC) 

values below 1 O%, the effluent is likely to show acute 

toxicity and if so, an acute TIE approach should be 

used. If chronic work is to be done on a highly toxic 

effluent, the same recommendations given in the acute 

manual should be used; that is, use concentrations of 

4x, 2x, lx and 0.5x the lC25 or IC50 value (see Section 

5.8 for which value to select). For example, if the IC25 

is 5% effluent, we would suggest using a range such as 

20%, lo%, 5% and 2.5% for the various tests. It is 

best to use the same dilution sequence within a series 

of tests (Tier 1) when tests are to be compared to each 

other for differences in toxicity. 

5.5 Renewals 

For C. dubia, daily renewals of the test media (as 

required in the chronic manual, EPA, 1989C) are not 

necessary in Phase I as long as the toxicity of the 

effluent can be measured with one or two renewals. 

Because available sample volume is limiting in some 

manipulations, fewer renewals are desirable. As with 

the test duration (4-d vs. 7-d) the acceptability of less 

frequent renewals must be established by comparison 

with whichever test duration is selected. However in 

Phase III, tests must be conducted similarly to the 

routine biomonitoring test. For the fathead minnow test 

the frequency of sample replacement must be daily to 

maintain adequate water quality because the live food 

organisms (brine shrimp, Artemia salina) die 2-8 h after 

being added to the freshwater test solutions. A baseline 

test (see Section 6) is always conducted when the 

sample is received. The suitability of reduced renewal 

frequency can efficiently be evaluated at this time by 

conducting comparative baseline tests simultaneously 

with different renewal frequencies. 

The number and types of chemical measurements 

taken initially and at the renewal intervals (referred to 

as finals) should be based on the need for these measurements and their usefulness (see Section 2). Initially, little judgement about the value of the.se can be 

made, but as toxicant characteristics are identified, the 

usefulness of various measurements can be judged. 

Initially, the usual measurements (hardness, alkalinity, 

conductivity; EPA, 1989C) should be made but some of 

these can be dropped as the TIE progresses. For 

example, if non-polar toxicity is found, then hardness 

and alkalinity need not be closely monitored. However 

if a metal is suspected, then these measurements are 

important. Low levels of dissolved oxygen in the 

fathead minnow test are a greater concern than in the 

C. dubia test, and the pH between the two tests will be 

dissimilar after 24 h of exposure. The pH measurement is frequently needed and for toxicants such as 

ammonia it is extremely important (EPA, 1992A). If an 

effluent contains greater than 5.0 ms/l of ammonia, the 

pH should be carefully measured at least daily (or more 

often) in all test concentrations. Since ammonia is a 

highly pH dependent toxicant, one must be aware of 

variable pH drift in the Phase I treatments which may 

lead to erroneous conclusions. One generalization, 

however, can be made. For characteristics that are 

unlikely to change, such as conductivity and hardness, 

both initial and final measurements need not be made 

once is enough. 

5.6 Toxicity Blanks 

A risk of the reliance on a toxicity response in the 

characterization step of TIES is the probability that 

artifactual toxicity is created during sample manipulations (see Section 2.4). While a particular manipulation 

may cause some degree of artifactual toxicity, if the 

toxicity is predictable the test may still retain its validity. 

Since chronic tests are more sensitive to artifactual 

toxicity, lower concentrations of additives or less severe conditions must be used as compared to the acute 

test. 

The presence of artifactual toxicity caused by contaminated acids, bases, air, filters and columns and by 

intentional additives are detected by treatment blanks 

and toxicify controls. A blank is dilution water manipulated the same as the effluent, and then it is toxicity 

tested to determine if the manipulation added any toxicity. The toxicify control is the reference used to judge 

the impact of a manipulation. Sometimes the toxicify 

control is the baseline test, at other times it will be a 

characterization test. For example, the toxicity control 

for the EDTA addition test is the baseline test while the 

toxicity control for the post C, SPE column test is the 

filtration test (filtered whole effiuent). Treatment blanks 

for either the EDTA addition test or the sodiurn thiosulfate addition test are not appropriate as the testing of 

these additives in clean dilution water is not representative of the effluents’ characteristics. The toxicity control must be distinguished from the control treatment 

(animals in standard culture or dilution water; also described as “performance controls”) which is always 

used. Controls provide information on the health of 

the test organism and the test conditions while the 

blanks provide information on the cleanliness of the 

acids and bases, the aeration system, the filter apparatus, the C,, SPE column, and other apparatus used. 

Although arlifactual toxicity may appear in the dilution water blanks, artifactual toxicity in the effluent 

matrix may not be observed. One must decide whether 

the test results from that manipulated sample are meaningful. For example, if the aeration manipulation caused 

toxicity in the dilution water blank but aeration removed 

the effluents’ toxicity then the conclusion that aeration 

was an effective treatment is valid. However, if the 

dilution water blank was toxic and it appeared aeration 

did not remove the effluent’s toxicity then one cannot 

conclude that aeration was not effective without further 

investigation. 

5-3 
5.7 Renewal of Manipulated Samples 

One must decide whether a manipulated sample to 

be used for renewal during the test should be prepared 

(e.g., aerated or passed over a C,, SPE column) as a 

batch sample for the entire test or prepared separately 

for each renewal. This choice may be dependent on 

the persistence of the effluent toxicity, but whether daily 

samples are prepared or batch samples are prepared 

and used for renewals of the tests should be decided 

by the investigator, and the same methods should be 

performed consistently throughout the TIE. As a general guideline, we have chosen to discuss these Phase 

I steps as though one aliquot of effluent samples prepared for the characterization tests is used for all renewals. However for either daily or batch samples, the 

same techniques should be used for all the manipulations. For example, a sample for the fillration test 

(Section 6) may be batch prepared on day 1. Then on 

day 2, a batch sample for the aeration test should be 

prepared. Yet for the EDTA and sodium thiosuffate 

addition tesfs, these additives should be added to the 

effluent dilutions on the day of each renewal as batch 

solutions for each dilution (e.g., add EDTA to 50 ml of 

100% effluent, let sample sit and dispense to test 

cups). This is true for the methanol addition and the 

graduated pH manipulations as well. To test the post 

C,, SPE column samples for some effluents, daily 

samples may need to be prepared because of bacterial 

growth problems in samples stored for several days. 

Since Phase I TIE work is often concerned with the 

qualitative evaluation of toxicity, rather than quantitative, there is no reason why a test could not be terminated sooner than 7 d, if the answer to the particular 

question posed has been found. For example, if the 

baseline test with a sample indicates a complete inhibition of C.dubia reproduction by day 5 of a 7-d test, and 

one of the manipulated samples (e.g., aeration) shows 

normal reproduction, there may be little point in continuing that test, because toxicity was altered. This 

type of judgmental decision is harder to make in a 

chronic fathead minnow test based on growth; however, by careful observation of factors such as survival 

or behavior, the trend of the toxicity response may be 

discerned earlier than 7 d. Sufficient measurable growth 

of the fathead minnows may have been achieved by 

5-d. Experiments with fish exposed to zinc and selenium for 5-d and 7-d indicated that sufficient growth 

differences could distinguish the toxic effect even at 5-d 

(Norberg-King, 1989). However, if this information is 

needed in Phase III, it is important to correlate the 

same type of data and terminating the test early may 

require additional tests later on. 

Because the chronic test is longer and requires 

more laboratory work than the acute test, loss of toxicity of any effluent sample is more troublesome when it 

occurs. If the presence of toxicity is not measured in 

the whole effluent before Phase I tests begin, much 

work will be wasted if the sample is non-toxic initially. 

On the other hand, to delay by waiting for the test may 

also result in the loss of toxicity. The best approach is 

to examine existing data sets for evidence of toxicity 

loss due to storage of samples. If there are none then 

start a baseline test, and upon the onset of chronic 

toxicity (e.g., 60% mortality, no reproduction by day 5 in 

high test concentrations of a 7-d test, absence of food 

in the gut of the fishes), additional follow-up manipulations of Phase I tests should be started. Toxicity 

degradation can be a useful tool in identification and 

confirmation (cf., Section 2). Once it has been determined that the sample toxicity degrades quickly, Tier 1 

and Tier 2 steps should be started on the day of arriial. 

Removal of headspace in effluent storage containers 

may help minimize the loss of toxicity. 

5.8 Test Endpoints and Data Analysis 

For evaluating whether any manipulation changed 

toxicity, the investigator should not rely on statistical 

evaluations only. Some treatments may have a significant biological effect that was not detected by the 

statistical analysis. Judgement and experience in toxicology should guide the interpretation. 

Endpoints for the most commonly used freshwater 

short-term chronic tests are growth, reproduction, and 

survival. Historically, the effect and no effect concentrations have been determined using the statistical approach of hypothesis testing to determine a statistically 

significant response difference between a control group 

and a treatment group. The no effect level, called the 

no observed effect concentration (NOEC), and the effect concentration, called the lowest observed effect 

concentration (LOEC), are then statistically defined endpoints. The NOEC/LOEC are heavily affected by choice 

of test concentrations and test design. For example, 

these effect levels are dependent not only on the concentration intervals (dilution sequence) chosen, but the 

number of organisms, the number of replicates used, 

and the choice of the statistical analysis for the data 

(i.e., parametric or non-parametric). The minimum significant difference detected in hypothesis tests can be 

quite variable (e.g., 10% or 50%; Stephan and Rogers, 

1985) and yet this difference is used to determine the 

NOEC. In the chronic testing manual (EPA, 1989C), 

the minimum number of replicates (a relatively large 

number), organisms, and dilutions for the C. dubia and 

fathead minnow short-term tests are needed to meet 

the hypothesis testing requirements. When less replicates, fewer numbers of dilutions and fewer test organisms are used (as in the chronic TIE) the hypothesis 

tests will not be able to detect smaller differences that 

are needed for chronic TIES. Therefore, hypothesis 

testing is not suitable for Phase I purposes and a point 

estimation method must be used. 

The linear interpolation method described in the 

supplement to the freshwater chronic manual (EPA, 

1989C) calculates a point estimate of the effluent concentration that causes a given percent reduction based 

5-4 
on the organisms response. The inhibition concentration (ICp3) program (Norberg-King, 1989; DeGraeve et 

al., 1988; EPA, 19896) was developed for the purpose 

of analyzing data from the short-term tests. This method 

of analysis is not as dependent on the test design as 

hypothesis analysis and is particularly useful for analyzing the type of data obtained from Phase I testing. 

When analyzing data for the ICp estimates, only one 

test endpoint is determined. For C. dubia all the data 

are used. If all animals have died, the data are entered 

as zeros and if some animals have some young but the 

adult dies, the partial brood values are used. We have 

found with some effluents that when the 4d test is 

routinely applied during a chronic TIE, often the first 

brood is produced and then the adult dies. In other 

cases we have observed no adult mortality in the 4-d or 

7-d test, but at the same effluent exposure concentrations the 7-d test animals will not produce any young 

while the 4-d test animals produce their first brood. 

The dose response from this 411 test is not typical in 

the 7-d test, and the production of young can be problematic in data interpretation and analysis since mortality also occurred. For example, when analyzing the 

data using the ICp program, the effects of survival and 

young production are incorporated into one estimate for 

the IC50 and IC25. Yet there is no doubt that O-40% 

survival is a significant reduction in survival that indiThe ICp program (Release 1.1) calculates confidence intervals which 

are limiting when the sample size is SI andthese confidence intervals 

are less than 95% in version I .t (R. Regal, personal communication, 

University of Minnesota, Duluth, MN). This is being corrected in the 

revision of the program now underway (for more information, contact 

Teresa Norberg-King). The ICp program is available by sending a 

formatted disk to Teresa Norberg-King. EPA, 6201 Congdon Boulevard, Duluth, MN 55604. 

cates toxicity, and would cause a routine test to fail 

(EPA, 1989C). Therefore when this occurs, to track 

toxicity in the TIE, it may require calculating the IC25/ 

IC50 for young production and survival and then recalculating the IC25/IC50 for survival alone. For the 

fathead minnow test in the routine monitoring test and 

the TIE tests, the weights are calculated as mean 

weight per original fish rather than mean weight per 

surviving fish (EPA, 1992C). Also the program allows 

direct comparison of results from tests conducted using 

different concentration intervals. The level of inhibition 

(p) used as an endpoint (e.g., 25 or 50%) is not criiical, 

although the IC25 is generally suggested as an equivalent for the NOEC (EPA, 1991 B). Confidence intervals 

are calculated using a bootstrap technique, and these 

confidence intervals can be used to determine the significance of toxicity afterations observed in Phase I. A 

“significant reduction” in toxicity must be determined by 

each laboratory for each effluent and in combination 

with the precision of reference toxicant tests that the 

performing laboratory achieves. The use of the IC50 

for Phase I TIES may be more useful when trying to 

correlate the characterization test results to the effluent 

toxicity. However, an IC50 may not be able to be 

estimated while the IC25 can; use of a consistent 

endpoint effect level is important for subsequent TIE 

work (EPA, 1992A: EPA, 19928). We have observed 

substantial toxicity reductions in characterization tests, 

yet it does not always appear to be a significant reduction when only the IC25s are compared. When this 

happens the sample size should be increased with 

subsequent testing in order to more clearly differentiate 

the toxicity and the dose response curve should be 

studied. Once the toxicant is identified, the number of 

replicates is increased and more dilutions are used 

(Phase III; EPA, 1992B), which increases the confidence in the IC25. 

5-5 
Section 6 

Characterization 

The chronic Phase I manipulations follow the same 

approach and employ the same type of manipulations 

used in the acute TIE (EPA, 1991A). These include 

aeration, filtration, C,, SPE extraction and chromatography, chelation with EDTA, oxidant reduction and/or 

complexation with sodium thiosulfate, and toxicity testing at different pH values (Figure 6-l). The main 

differences between the acute and chronic techniques 

are that the concentrations of additives must be lower 

and the test conditions must be less severe in a chronic 

TIE because the chronic test organisms are more sensitive to these conditions. The pH adjustment procedures in Tier 2 are changed from the acute Phase I 

because we found that consistent, representative blanks 

with reconstituted water could not be obtained at higher 

pH’s. 

The following characterization steps are all based 

on the use of Ceriodaphnia or fathead minnows. Obviously, use of other species will require consideration of 

appropriate test volumes and additive concentrations. 

As discussed in the acute manual, if the TIE is done 

with species different from the species used in the 

permit, one must demonstrate that both species are 

sensitive to the same toxicant (see Section 5). 

More than one effect is measured in chronic tests 

(reproduction or growth and survival) and because partial effects are more frequent in short-term chronic tests 

than in acute tests, a graded response with concentration is often seen. A graded response allows one to 

better judge small changes in toxicity-an advantage 

not often available in acute tests. Also, effects (initial 

mortality, delayed mortality, aborted young, reduced 

young, poor growth) can be observed and used in 

interpreting the results as can the time to onset of 

effect be used. Such effects can be useful in distinguishing the response to different toxicants. 

For acute TIES, tests are quick and relatively inexpensive, so the need to maximize their usefulness is 

lessened. The chronic test is more work not only 

because the test is longer and more complex, but also 

because more sample volume is needed. For example, for tests such as the sublation test (a subsequent step in the aeration rest (Section 6.4)) sample 

size can be very restricting. In addition, if an effluent is 

not always toxic, a decision has to be made as to 

whether to test for the presence of toxicity first, before 

manipulations are started. If the effluent is not toxic 

and all the manipulations are set up, the results may be 

Tests 

of no value. On the other hand, if the presence of 

toxicity is first established, often a week will have passed 

and by the time manipulations are tested, the toxicity 

may have degraded. Unfortunately, there is no clear 

answer to which way to proceed. When there are data 

for effluent toxicity for preceding months, examination 

of these data may assist in the decision. 

In the acute TIE, the initial test (EPA, 1991C) is 

used to set the range of concentrations to test. However in the chronic TIE, an equivalent of the initial test 

is not practical, therefore historical data must be used 

to make such judgements. Lacking historical data, a 

judgement will have to be made to set the test range 

and guidance for this is given in Section 5.4. 

For chronic Phase I characterization, the use of two 

tiers of characterization tests is suggested (Figure 6-l). 

Tier 1 is done without major pH adjustments. Experience with acute TIES has shown that major pH adjustments are usually not needed. Tier 2 is performed 

only when Tier 1 does not provide sufficient information, and consists of filtration, aeration and the C,, 

separation technique of Tier 1 with an effluent sample 

adjusted to both pH 3 and pH 10. Therefore when the 

characterization tests indicate Tier 2 is not required, 

resources needed to conduct the TIE are significantly 

reduced.4 Each characterization test used in the Tier 1 

or Tier 2 has as its foundation the information in the 

acute Phase I manual (EPA, 1988A; EPA, 1991A). 

The principles, methods, and interpretation of results 

are based on the acute manual, and the tests for Tier 1 

(Figure 6-2) are discussed in Sections 6.1-6.8. All tests 

within a Tier (1 or 2) should be started on the same 

day. Starting chronic tests involves more effort than 

acute tests, and logistics must be planned (for instance, available animals of the appropriate age for the 

chronic test, sufficient food supply for more chronic 

tests, adequate supply of dilution water for all test 

renewals). Tests need to be started on the same day 

in order to compare results of each manipulation test to 

others and to the baseline test (Section 6.3) results 

(Table 6-l). Once the Tier 1 data are generated, they 

are compared, and interpretations are made to see 

which inferences can be drawn concerning the nature 

of the toxicants. Usually, multiple manipulations and a 

retest of selected manipulations will be effective in 

’ A recent estimate of the cost of the Tier 1, Phase I for chronic toxicity 

was equivalent to the full Phase I acute TIE (Aquatic Habitat Institute, 

1992). 

6-l 
Figure 6-l. Overwew of characterization tests. 

l Baseline whole effluent test 

l EDTA addition test 

l Sodium thiosulfate addition test 

l Filtration test 

l Aeration test 

l Post C,, sofid phase extraction (SPE) column 

test 

l Methanol eluate test 

l Graduated pH test 

l Baseline whole effluent test 

l pH adjustment test 

l Filtration and pH adjustment test 

l Aeration and pH adjustment test 

l Post C,, SPE column and pH adjustment test 

l Methanol eluate test 

Figure 6-2. Tier 1 sample preparation and testing overview. 

Tier 1 Y 

yielding information concerning the nature of toxicants 

before additional effluent samples are tested (see Sections 6.15,6.16 and acute Phase I manual, EPA 1991A). 

Sample Preparation for the Characterization 

Tests 

As for acute TIE tests, we suggest doing certain 

chemical measurements and the manipulations on one 

day and then starting the tests the next day (Table 6-l). 

This schedule balances the work load more evenly. 

When the sample is received (day l), various measurements (Section 4) are taken and some preparatory 

manipulations for the Tier 1, Phase I are done. 

First, the routine chemical measurements are taken 

as discussed in Section 4. DO, conductivity, and pH 

should be measured on the 100% effluent to ensure 

that the values are in the physiologicalfy tolerable range 

for the test species. If these are at levels that could be 

toxic (EPA, 19896) there is little point to test the 

effluent sample without some sample manipulation. In 

addition, the water hardness and alkalinity should be 

measured so that the appropriate dilution water can be 

selected (see Section 3, Dilution Water). As the TIES 

have progressed, we have begun to match both the 

hardness and the alkalinity of the dilution water to 

similar values for the effluent. 

EDTA 

Toxicity (4 

Tests 

EDTA 

Additions 

Thiosulfate 

Toxicity Nd 

Sodium Thiosuffate 

Tests 

Additions 

Methanol Toxicity 

Elutions Test 

Post-Column 

Sample(s) 

Toxicity 

Test 

Eluates 

6-2 
Table 6-1. Outline of Phase I effluent manipulations Tier 1 and Tier 2. 

Description 

DAY 1 SAMPLE ARRIVAL: 

Section 

Measure 4.0 

l temperature 

l conductivity 

l PH 

l DO 

l alkalinity 

l hardness 

l total ammonia 

l total residual chlorine 

Perfon Sample Manipulations 6.0 

l filter effluent 6.4 

. perform solid phase extraction (SPE) 

l collect effluent 

l collect methanol eluate 

6.6 

6.7 

DAY 2 TOXICITY TESTING: 

Warm aliquot of whole effluent and aliquots 

of filtered effluent, post C,, SPE column effluent, 

and methanol eluates. 

Initiate Tier 1 Tests 

l baseline toxicity test 6.1 

9 EDTA addition test 6.2 

l sodium thiosulfate addition test 6.3 

l aeration test 6.4 

l fihation test 6.5 

l post C,, SPE column test 6.6 

l methanol ehate test 6.7 

l graduated pH test’ 6.6 

ADDITIONAL TESTING ON SUBSEQUENT DAYS ?: 

Tier 2 Tests 

l pH adjustment test 6.10 

l aeration and p/f adjostrnen~ test 6.11 

l filtration and pH adjustment test 6.12 

l post C,, SPE column and pH adjustment test 6.13 

l methanol eluate test for pH adjusted samples 6.14 

’ Experimentation may be needed for this test (see text for details). 

2 Tier 2 is primarily for those effluents where the results from Tier 1 

did not indicate any clear pattern of toxicity change following 

manipulation (see text for details). 

The initial pH of effluent upon arrival at the testing 

laboratory is referred to as pH i, which is not necessarily the pH of the effluent at air equilibriums. The pH of 

the sample after being warmed, may be selected as 

5 EPA suggests that toxicity must be prevented under worst case 

scenarios(EPA, 1991 B) which may mean the routine monitoring tests 

were conducted at high pH’s. 

pH i rather than the pH upon arrival. The important 

point is to use the same pH i for all subsequent tests. 

As an effluent warms to 25°C in an open container, 

CO, escapes and the pH may rise from 7.2-7.6 to 8- 

8.5. In some tests, once the food is added the pH may 

rise faster or in some cases (e.g., the fathead minnow 

growth test), once the food has been in the test solution 

for a period of time, the pH may be lower (e.g., 7.5- 

7.6). These changes may be important for interpreting 

the data in a chronic TIE, and pH should be measured 

in the test dilutions that determine the test endpoint. Of 

course, since the endpoint may be unknown, pH is 

typically measured in all test concentrations. 

Since samples are cooled for shipping and storage, 

upon warming to 25”C, some of the samples are apt to 

be supersaturated. Supersaturation can usually be 

monitored by measuring DO. If DO is too high, it 

should be reduced to acceptable levels as described by 

EPA (1989C) for the routine monitoring test or by maximizing surface-to-volume ratio of the container to facilitate more rapid exchange of equilibrium of the sample 

and atmospheric oxygen. Ceriodaphnia are less sensitive to supersaturation than newly hatched fathead minnows. For chronic Phase I tests, routine water chemistry measurements (such as DO, pH, temperature) are 

more important than in acute Phase I tests. 

The manipulations performed the day the sample 

arrives are filtering, extraction on the C,, SPE column, 

and collection of the methanol eluates (see Sections 

6.5 and 6.7 below). The aliquots of filtered effluent and 

post-column effluent will be held until the next day (day 

2) to start the tests. Of course these samples should 

be stored in the refrigerator at 4 (+ 2°C). This sample 

preparation schedule is particularly convenient for laboratories who rely on courier services to deliver samples, 

which typically occurs late in the morning. 

On day 2, the EDTA addition test should be prepared first so that compounds that are EDTA chelatable, yet may require an equilibration time for complexation, can be chelated (see Section 6.4). Then the rest 

of the manipulations (aeration, sodium thiosulfate additions, graduated pH adjustments) should be started. 

For the laboratory that is experienced in chronic toxicity 

testing, the amount of time required to conduct the 

Tier 1 sample manipulations and set up the toxicity 

tests is about 6-10 h. 

6.1 Baseline Test 

General Approach: To determine the effects of 

Phase I manipulations on the toxicity of the effluent, its 

inherent toxicity must be determined. The toxicity measured in this test is used to gauge toxicity changes 

caused by some manipulations and to detect changes 

in the sample’s toxicity during storage. Baseline tests 

must be repeated each time additional manipulation 

tests are started. 

Methods: The baseline test will be initiated using 

concentrations based on the historical data for each 

particular discharger. For the TIE, use of four (and 

6-3 
three) dilutions have been sufficient for defining toxicity 

(Section 5.4). If the toxicity is low, in order to draw 

distinctions between the concentrations used in the test 

for the various characterization tests, the dilutions may 

need to be set closer, for example, 40%, 60%, 80%, 

100%. In this test, and all subsequent characterization 

tests, the test concentrations, test volumes and number 

of replicates should be kept the same as described in 

Section 5, Toxicity Testing. 

On day 2, an aliquot of the effluent is warmed 

slowly in a warm water bath to test temperature (25°C). 

The various test concentrations are prepared using the 

appropriate hardness reconstituted water. Next, routine chemistries are measured (initial pH, temperature, 

DO). The use of dilution water controls is not required 

for every manipulation but at least two sets of controls 

should be included to estimate reproducibility. In addition, the tests are conducted using one C. dubia per 

one 10 ml test volume in a 1 02 plastic cup (or glass 

beaker) and five animals per treatment. For the fathead 

minnow tests, two replicates per treatment, 10 fish in 

50 ml in a 4 oz plastic cup, or 100 ml in a 400 ml 

beaker, are assumed. 

Interpretation of Results/Subsequent Tests: The 

baseline tests serve as the basis for determining the 

effects produced by various characterization tests. This 

test serves as the toxicity control for some of the other 

tests. If baseline tests done on subsequent days with 

additional manipulations indicate that the toxicity of the 

effluent is decreasing, either every effort should be 

expended to characterize the toxicity more quickly (i.e., 

Phase II identification or Tier 2 tests) or another sample 

should be obtained. The “shelf life” of the toxicity can 

be determined after a few samples have been evaluated. 

Special Considerations/Cautions: The controls 

in this test will provide information on the health of the 

test organisms, the dilution water, the test glassware 

and equipment used to prepare the test solutions and 

the cleanliness of the test chambers. This baseline test 

serves as the toxicity control for some subsequent Tier 

1 or Tier 2 tests. 

6.2 EDTA Addition Test 

General Approach: This test is designed to detect 

effluent toxicity caused by certain cationic metals. The 

addition of EDTA to water and effluent solutions can 

produce non-toxic complexes with many cationic metals. Loss of toxicity with EDTA addition(s) suggests 

that cationic metals are causing toxicity. 

EDTA is a strong chelating agent and because of 

its complexing strength, it will often displace other soluble 

forms (such as chlorides and oxides) of many metals. 

The ability of EDTA to chelate any metal is a function of 

pH, the type and speciation of the metal, other ligands 

in the solution, and the binding affinity of EDTA for the 

metal. And the complexation of metals by EDTA may 

vary according to the sample matrix. The specific form 

of metal that causes toxicity in the water matrix may be 

more important than the total concentration of the metal. 

Cations strongly chelated by EDTA include aluminum (‘+), cadmium, copper, iron, lead, manganese (2+), 

nickel, and zinc (Stumm and Morgan, 1981). EDTA 

weakly chelates barium, calcium, cobalt, magnesium, 

strontium, and thallium (Flaschka and Barnard, 1967). 

EDTA can form relatively weak chelates with arsenic 

and mercury and anionic forms of metals (selenides, 

chromates and hydrochromates) will not be chelated. 

For some cationic metals for which EDTA forms 

relatively strong complexes, the acute toxicity to C. 

dubia is reduced (Mount, 1991; Hackett and Mount, In 

Preparation). EDTA was shown to chelate the metal 

causing the acute toxicity (at 4x the LC50) for copper, 

cadmium, lead, manganese (?+), nickel, and zinc to C. 

dubia in both dilution water and effluents. However, 

they also found that EDTA did not remove/reduce the 

acute toxicity of silver, selenium (either as sodium selenite or sodium selenate), aluminum (AI(OH chromium (either as chromium chloride or potassium dichromate), or arsenic (either sodium m-arsenite or sodium arsenate) when tested using moderately hard 

water and C. dubia (Hackett and Mount, In Preparation). 

In the acute Phase I manual (EPA, 1988A), the 

recommended amount of EDTA to be added was high 

because the authors thought calcium and magnesium 

had to be complexed in order to complex toxic metals 

(D. Mount, personal communication, NETAC, Duluth, 

MN). The mass of EDTA required was approximated 

by the amount needed for the titration of hardness or 

the measurement of calcium and magnesium when 

titration was not possible due to interferences. A third 

choice was to use 0.5x the EDTA LC50 for the test 

species (EPA, 1991A). Ideally the amount of EDTA to 

add would be just enough to chelate the toxicant 

without causing toxicity or otherwise changing the matrix of the effluent. Without knowing how much toxicant 

must be chelated, the amount of EDTA to add must be 

estimated. Recently, the role of calcium and magnesium was tested in our laboratory. Acute toxicity tests 

with C.dubia were conducted in moderately hard and 

very hard reconstituted water using copper, cadmium, 

and zinc at 4x, 2x, and lx the LC50 of each. When 

one metal and EDTA were present at approximately a 

1 :l molar basis, all the toxicity was removed regardless 

of water hardness (J. Thompson, personal communication, NETAC, Duluth, MN). These results indicate that 

calcium and magnesium concentrations do not affect 

the levels of EDTA needed to remove the acute cationic metal toxicity. Whether toxicity reduction using 

the 1 :l molar ratio is true for chronic toxicity has not yet 

been evaluated in a likewise manner (cf., interpretation 

of Results/Subsequent Tests below). However, EDTA 

and nitrotriacetic acid (NTA) were effective in chelating 

the toxicity of one concentration of either cadmium or 

copper to C. dubia at molar ratios of less than 1:l 

(Zuiderveen and Birge, 1991). However, NTA pos6-4 
sesses the characteristic of increasing the toxicity of 

some metals therefore NTA is limited in its usefulness 

for the TIE. 

The threshold levels for C. dubia and fathead minnows to EDTA were determined using 7-d tests in 

different hardness waters and the results are given in 

Table 6-2. For C. dubia, the chronic toxicity of EDTA is 

not water hardness dependent, but for fathead minnows the sublethal toxicity appears to be greater in 

softer waters. This is in contrast to the acute toxicity of 

EDTA to Ceriodaphnia which indicated that EDTA 

toxicity decreased with increased water hardness (Phase 

I; EPA, 1991A). Natural waters and effluents have 

many constituents in addition to those added to reconstituted waters, and the behavior of EDTA in effluents 

(or receiving waters) could be different than in simple 

reconstituted water. 

Methods: The goal is to add enough EDTA to 

reduce metal toxicity, without causing EDTA toxicity or 

substantially changing the water quality. The toxicity of 

EDTA as determined in clean reconstituted water is 

Table 6-2. Chronic toxicity of EDTA (mg/l) to C. dubia and 

P. promelas in various hardness waters using the 7-d 

tests. 

Species 

Water IC50 IC25 

Type 95% C.I. 95% Cl. NOEC LOEC 

C. tibia VSRW 

SRW 

MHRW 

HRW 

VHRW 

VHRW 

P. promelas SRW 

MHRW 

HRW 

VHRW 

4.5 3.0 

3.6-6.0 2.1-3.9 

7.5 4.9 

6.2-6.3 3.7-5.7 

6.6 

4.7-13 

5.9 

3.4-10 

7.5 5.5 

6.2-9.6 0.98-6.9 

7.6 6.1 

6.7-6.6 4.0-6.6 

12 6.3 

10-14 4.2-10 

136 

130-139 

103 

94-110 

163 

150-166 

236 

227-246 

132 

123-144 

-1 

267 230 

269400 203-247 

2.5 5.0 

3.1 

5.0 

5.0 

5.0 

7.5 

100 

100 

200 

200 

6.3 

10 

10 

10 

15 

200 

200 

400 

400 

I Value could not be determined, value would be less than lowest 

test concentration. 

Note: Ct. = confidence interval; VSRW = very soft reconstituted 

water; SRW = soft reconstituted water; MHRW = moderately 

hard reconstituted water: HRW I hard reconstituted water; 

VHRW = very hard reconstituted water. 

likely to be higher than the toxicity of EDTA added to 

an effluent. Therefore, the EDTA toxicity values contained in Table 6-2 represent maximum toxicity in any 

effluent. The toxic concentration of EDTA in one effluent will probably not be the same as the concentration 

causing toxicity in a different effluent or even a different 

sample of the same effluent. To be safe, the concentrations of EDTA added to any effluent should be less 

than the expected effect concentration of EDTA in clean 

water. For either species, two EDTA concentrations 

are added to two sets of two effluent dilutions. EDTA 

stock solution is added after the effluent dilutions are 

prepared so that the EDTA concentrations for each 

addition are constant across each set of effluent dilutions. A stock solution of EDTA (ethylenediaminetetraacetic acid, disodium salt dihydrate) is prepared in distilled water. This EDTA stock solution 

should be prepared so that only microliter amounts of 

the stock are needed to minimize effluent dilution. No 

more than 5% dilution of the effluent aliquot by EDTA 

stock should occur. 

To perform the effluent dilution test, two sets of 

effluent dilution concentrations are prepared (e.g, lOO%, 

50%, 25%) and each set receives one of two addition 

levels of EDTA (Table 6-3). By using non-toxic concentrations of EDTA, there is less chance for artifactual 

toxicity; since the total amount of metal to be chelated 

is probably low for most chronically toxic effluents, 

there is no reason to add high levels of EDTA. The 

additive levels are based on the assumption that the 

calcium and magnesium need not be chelated in order 

to chelate the toxic metals, although the amount of 

EDTA added is most likely still an excess. 

An EDTA stock solution of 2500 mS/I can be prepared. For the C. dubia tests, 0.06 ml is added to three 

separate 50 ml aliquots in the first effluent dilution set 

(i.e., 25%, 50%, 100%) to obtain a 3.0 mg/l final EDTA 

concentration. In the second dilution set, 0.16 ml is 

added to the other set of 50 ml effluent aliquots for a 

final concentration of 8.0 mg/l. For the fathead minnow 

tests, the same concentration of an EDTA stock solution can be used but the volume of stock additions 

must be doubled for the 100 ml test volume/concentration. 

Table 6-3. Concentrations of EDTA to add for chronic TIES. Values 

given are the final exposure concentration in mg4. 

Species 

C. dubia 

and 

P. promelas 

Water Type’ Concentrations 

(mdn) 

~___ 

SRW. MHRW, HRW, VHRW 3.0 6.0 

’ In very soft water, the final concentrations of EDTA must be lower 

in order to not have EDTA induced toxicity, for example 1 .O mq/l 

and 5.0 mg/l. 

Note: SRW = soft reconstituted water, MHRW = moderately hard 

reconstituted water: HRW = hard reconstituted water: VHRW 

= very hard reconstituted water. 

6-5 
To allow the EDTA time to complex the metals, 

solutions should be set up on day 2 and all solutions 

containing EDTA are allowed to equilibrate while other 

manipulations are being prepared before test organisms are introduced. A minimum of 2 h equilibration 

time should elapse before organisms are added. 

Since EDTA is an acid, the pH of the effluent after 

addition of EDTA should be checked, although additions at these low levels should not lower the pH of the 

effluent. The amount of change in solution pH will 

depend upon the buffering capacity of the effluent and 

the amount of reagent added. If the pH of the effluent 

has changed, readjustment of the test solution pH to 

pH i should be performed. 

The EDTA is not added to one batch of effluent on 

day 2; rather at each renewal EDTA is added to the 

renewal test solutions prior to dispensing into the test 

chambers in the identical way that the test solution was 

first made (allowing equilibration time). 

Interpretation of Results/Subsequent Tests: Toxicity may be removed at all exposures provided the 

addition of EDTA does not cause toxicity. If the effluent 

is less toxic (i.e., EDTA addition IC50 (or IC25) shows 

less toxicity than baseline test IC50 (or IC25)) in either 

of the EDTA addition dilution tests, then EDTA removed or reduced the toxicity and cationic metal toxicity is probably present. If, in either test, the effluent is 

more toxic than in the baseline test, EDTA itself may be 

causing toxicity and the test should be repeated using 

lower EDTA concentrations. If toxicity is not reduced 

below the baseline test, the probability of cationic metals causing toxicity in the effluent is low and higher 

concentrations of EDTA can be tried, although this may 

or may not be useful. 

Table 6-4 shows the results of a chronic zinc test 

and the reduction of the toxicity by the addition of 

EDTA. When C. dubia were tested in very hard reconstituted water, zinc was chronically toxic at 55 pg/I and 

EDTA was chronically toxic at 15 mg/I. When EDTA 

Table 6-4. The chronic toxicity of zinc (pg/l) to C. dubia in very hard 

reconstituted water and the toxicity of zinc when EDTA 

is added. 

Zinc’ Mean Young per Female 

Cont. EDTA Additions (mg/l) 

I@ 0 2.5 5.0 7.5 15 

0 19.2 18.6 17.5 17.6 6.8 

3.4 19.4 2 - - - 

14 17.8 22.0 23.2 20.8 1.8 

55 8.2 20.8 19.0 16.6 5.3 

- 

’ Measured values. 

2 EDTA not added to this zinc concentration. 

was added to solutions of 55 f.@l zinc at 2.5, 5.0, and 

7.5 mgIl EDTA respectively, the toxicity of the zinc was 

removed but at 15 mg/l EDTA, EDTA itself was toxic. 

Such trends may be similar to the toxicity reduction 

observed in effluents. If toxicity is reduced in a systematic manner, such as in the example, proceed to Phase 

II methods for identification of those metal(s) which are 

chelated by EDTA. Additions of EDTA at 3 mg/l and 

8 mg/I removed the toxicity of copper to C. dubia in a 7- 

d two-renewal test with hard reconstituted water at 

levels of 210 us/I and 105 us/l of copper. In addition to 

removing toxicity due to metals, EDTA reduces the 

acute toxicity of some cationic surfactants. This reduction of toxicity may also occur in chronically toxic effluents, and the toxicity reduced by EDTA should not be 

assumed to be due only to cationic metals. See Section 6.4 Aeration Test for subsequent tests to conduct if 

cationic metals are not present in the effluent at chronically toxic levels but EDTA reduced toxicity. 

Special Considerations/Cautions: If pH in the 

EDTA tests is greatly different from that in the baseline 

test, the test might need to be redone. There is no way 

to distinguish the effect of pH change on the toxicity of 

a pH sensitive toxicant (e.g., ammonia) from toxicity 

changes caused by EDTA. A change of 0.1 pH unit 

can cause substantial errors if ammonia is involved. 

Before the test is reinitiated, data from the graduated 

pH test should be examined to evaluate whether the 

toxicity is pH dependent. This test data may be useful 

in deciding whether the EDTA addition test should be 

redone. EDTA additions to dilution water are not relevant controls for the EDTA additions to effluent; therefore, the roxiciry control is the baseline rest. The 

control of the baseline rest serves as the QC for the 

health of the test organisms, the quality of the dilution 

water, and general test conditions. 

If all dilutions where EDTA is added should cause 

mortality, one possibility is that the stock solution of 

EDTA is contaminated and the stock solution should be 

checked by conducting another test with a new EDTA 

stock. 

6.3 Sodium Thiosulfate Addition Test 

General Approach: Oxidative compounds (such 

as chlorine) and other compounds (such as copper and 

manganese) can be made less toxic or non-toxic by 

additions of sodium thiosulfate (Na,S,O,). Toxicity from 

bromine, iodine, ozone, and chlorine dioxide is also 

reduced. Sodium thiosulfate has been routinely used 

to reduce the toxicity of substances such as chlorine 

(EPA, 1989C). 

Reductions in effluent toxicity observed with sodium thiosulfate additions may also be due to the formation of metal complexes with the thiosulfate anion 

(Giles and Danell, 1983). The ability of sodium thiosulfate to form a metal complex is rate dependent and 

metal dependent (Smith and Martell, 1981) and sodium 

thiosulfate is not a particularly strong ligand for metal 

complexation. Cationic metals that appear to have this 

potential for complexation, based upon their equilibrium 

6-6 
stability constants, include cadmium, copper, silver, and 

mercury (*+) (Smith and Martell, 1981). The rate of 

complexation is specific for various metals and some 

cationic metals may remain toxic in the 24-h or 48-h 

renewal period of the chronic toxicity test due to the 

slow rate of complexation or the stability of the complex. The thiosulfate anion is not very stable, and the 

ability of sodium thiosulfate to complex the compound(s) 

causing chronic toxicity without daily renewals has not 

been tested completely. 

Recent findings have shown that the acute toxicity 

of certain cationic metals may be reduced by levels of 

sodium thiosulfate added in the acute Phase I tests 

(EPA, 1988A; EPA, 1991A). The acute toxicity of 

several cationic metals was shown to be removed by 

sodium thiosulfate in standard laboratory water. The 

acute toxicity at 4x the LC5Os of copper, cadmium, 

mercury, silver, and selenium (as selenate) to C.dubia 

was removed by sodium thiosulfate additions at levels 

suggested in the acute Phase I manual. However, for 

zinc, manganese, lead, and nickel, the acute toxicity 

was not removed by the sodium thiosulfate additions 

(Mount, 1991; Hackett and Mount, In Preparation). The 

toxicity of mercury with the addition of sodium thiosulfate was reduced for 24 h but not 48 h which indicates 

it may not have been completely complexed by the 

thiosulfate. If the acute toxicity of metals can be reduced or complexed by sodium thiosulfate, the same 

may be true for chronic toxicity. However, for C. dubia 

7-d tests with hard reconstituted water, sodium thiosulfate levels of 5 mg/l and 10 mg/l did not remove or 

reduce the chronic toxicity of copper at the same concentrations where EDTA complexed the toxicity (cf., 

Section 6.2). 

The test animals will probably tolerate more sodium 

thiosulfate than would ever be needed to render oxidants or metals non-toxic in effluent samples, especially the fathead minnows in comparison to the C. 

dubia (Table 6-5). The presence of oxidants or 

complexable metals will reduce the concentrations of 

sodium thiosulfate below the nominal concentrations 

added. 

Table 6-5 gives the toxicity values in various reconstituted waters. The effect concentrations for C. dubia 

and fathead minnows were measured in waters of different hardnesses (soft, moderately hard, hard, and 

very hard water (EPA, 1989C)). For Ceriodaphnia, the 

results indicate that the sublethal toxicity is unchanged 

regardless of the water type (Table 6-5). The toxicity 

tests with sodium thiosulfate and fathead minnows (7-d 

growth test) indicate that the toxicity due to sodium 

thiosulfate is greater in softer waters. 

Methods: Two sets of effluent dilutions (such as 

25%, 50%, 100%) each set with a different level of 

thiosulfate concentration (Table 6-6) are prepared regardless of whether C. dubia or fathead minnows are 

used as the TIE test organism. The concentration of 

thiosulfate remains constant across one set of effluent 

concentrations within a series (identical to EDTA addition rest). Small volumes (microliter) of the sodium 

thiosulfate stock solution should be added to minimize 

the dilution (5% of total volume). Non-toxic concentrations of sodium thiosulfate are used to reduce the proTable 6-5. Chronic toxicity of sodium thiosulfate (mgA) to C. dubia and /? promelas in various hardness waters using the 7d tests. 

Species 

Water 

Tvw 

IC50 IC25 

95% C.I. 95% C.I. NOEC LOEC 

C. dubia SRW 39 26 30 60 

30-42 15-33 

HRW 38 27 30 60 

26-44 20-36 

VHRW 43 34 30 60 

37-44 21-37 

P. pivmelas SRW 1.070 

1,041-1.1005 

MHRW 2,001 

1,891-2.161 

HRW 4,871 

4,633-5,051 

VHRW 8,522 

8,053-8.704 

820 750 l/=0 

785-859 

720 750 1.500 

550-l ,528 

3,590 3,ooo 6.0’33 

3,226-3,800 

6,780 6,(330 12,000 

6,065-7,073 

Note: Cl. = confidence interval; SRW = soft reconstituted water; MHRW = moderately hard reconstituted water; HRW = hard reconstituted 

water; VHRW = very hard reconstituted water. 

6-7 
Table 6-6. Concentrations of sodium thiosulfate to add for chronic 

TIES. Values given are the final exposure concentration 

in mgk 

Species Water Type’ Concentrations 

b-f@) 

c. ckhia 

and 

P. ptvmelas 

SRW, MHRW, HRW, VHRW 10 25 

1 In very soft water, the final concentrations of sodium thiosulfate 

must be lower in order to not have sodium thiosulfate induced 

toxicity, for example 1 .O mg/l and 5.0 mg/f. 

Note: SRW = soft reconstituted water, MHRW = moderately hard 

reconstituted water; HRW = hard reconstituted water, VHRW 

= very hard reconstituted water. 

bability of artifactual toxicity, yet sufficient concentrations are needed to remove/reduce oxidants. 

For a C. dubia test, to the first effluent dilution set 

(i.e., 25%, 50%, 1 OO%), 0.20 ml of sodium thiosulfate 

stock (2500 mg/l) is added to each 50 ml dilution to 

obtain final concentrations of sodium thiosulfate of 10 

mg/l. To the second effluent dilution set, 0.50 ml of the 

same stock solution is added to 50 ml of each test 

dilution to obtain final concentrations of 25 mg/l (Table 

6-6). 

The fathead minnow test is similar except that 

twice the volume of the same thiosulfate stock is needed 

(because of 100 ml test volumes) to achieve the same 

final concentrations (Table 6-6). 

The sodium thiosutfate is not added to a batch of 

the effluent on day 2; rather, at each renewal, sodium 

thiosulfate is added to the renewal test solutions in a 

manner identical to the way they were first prepared. 

interpretation of Results/Subsequent Tests: The 

results of the sodium thiosulfate addition tests are 

compared to one another and to the baseline rest 

results to determine whether or not toxicity reduction 

occurred. Toxicity may be completely reduced, partially reduced, or not reduced. If toxicity appears to be 

reduced and/or removed, then more tests to determine 

whether the toxicity is due to an oxidant or to some 

metal should be performed. When chlorine concentrations are ? 0.1 mg/l total residual chlorine (TX), there 

may be a toxicity problem for C. dubia. A significant 

drop in the chlorine level in the whole effluent may 

occur in the first 24-h period after sample collection and 

testing. Therefore, tests repeated on an aged sample 

may give different results if an oxidant is involved but 

may give the same results if a metal is involved. 

For cases where oxidants account for only part of 

the toxicity, sodium thiosulfate may only reduce, not 

eliminate, the toxicity. Yet the sodium thiosulfate addition rest is useful even when chlorine appears to be 

absent in the effluent. Oxidants other than chlorine 

occur in effluents, and even if the effluent is not chlorinated this test should not be omitted. Both thiosulfate 

and EDTA reduce the toxicity of some metals and this 

information can be helpful in identifying the toxicant. 

(However, this effect of thiosulfate/metal complexation 

has not been demonstrated for chronic toxicity.) In 

cases where both the sodium thiosulfate addition rest 

and the EDTA addition rest reduce the toxicity in the 

effluent sample, there is a possibility that the toxicant 

may be a cationic metal(s). Many oxidants are reduced 

by aeration but if aeration does not reduce toxicity, 

Phase II methods for identification of cationic metal(s) 

toxicants should be investigated. No change in toxicity 

suggests either no oxidants or certain metals. 

Special Considerations/Cautions: The general 

test conditions, quality of the dilution water, and health 

of the test organisms are tracked by the controls in the 

baseline test. Additions of sodium thiosulfate to dilution water are not relevant confrols for thiosulfate additions to effluent to determine if the thiosulfate was toxic. 

Therefore the toxiciv control is the baseline test. 

If all dilutions where sodium thiosulfate is added 

should exhibit mortality, one possibility is that the stock 

solution of sodium thiosulfate is contaminated and this 

phenomena should be checked by conducting another 

test. 

6.4 Aeration Test 

General Approach: Changes in toxicity due to 

aeration at pH i may be caused by substances that are 

oxidizable, spargeable, or sublatable. The chemicaU 

physical conditions of the aeration process will also 

affect whether or not the toxicity is reduced or removed. 

Sparging of samples is done using air which includes oxidation as a means of toxicity removal. In our 

experience, typically volatile compounds that are highly 

water soluble (such as ammonia) will not be air-stripped 

at pH i by this method. If aeration is one of the 

mechanisms that removes the toxicity, then additional 

tests must be performed to identify which mechanism is 

removing the toxicity. Subsequent tests with nitrogen 

can be used to determine if toxicity reduction was due 

to oxidation. Also, air or nitrogen sparging can cause 

surface active agents to subl#e. As bubbles break at 

the surface, sublatable compounds will be deposited on 

the sides of the aeration vessel. Sublatable toxicity 

identification requires special sample removal and rinsing (see below). A visible deposit does not indicate the 

presence or absence of such toxicants. 

Methods: For the aeration process, the volume 

of effluent and dilution water aerated is kept the same 

even though all of the dilution water volume is not 

needed for the aeration blank. The flow rate, bubble 

size, geometry of apparatus and time of aeration should 

be consistent among treatments. Taller water columns 

and smaller bubbles should ensure better stripping; 

therefore, the aeration vessel should be half-full or 

greater for this process. Each aliquot (effluent and 

dilution water) should be moderately aerated for a standard length of time (60 min). Use of gas washing 

bottles (Kontes Glass Co., Vineland, NJ) fitted with 

6-6 
glass frit diffusers located at the bottom of the vessel 

for aeration is suggested because they sparge the 

sample effectively. During aeration, the pH of the 

effluent is not maintained at “pH i.” 

The volume of effluent aerated should be the same 

for either a 4-d C. dubia test or a 7-d C. dubia two 

renewal test (four dilutions, five replicates for each 

dilution; see Section 5), although there is excess of 

solutions for the 4-d test. Use of 300 ml of effluent (or 

dilution water) in a 500 ml gas washing bottle or 500 ml 

in a 1 L bottle and a flow-rate of 5OOmUmin is suggested. Any loss of volume and any formation of 

precipitates should also be recorded. 

Interpretation of Results/Subsequent Tests: If 

the aerated effluent has less toxicity than the baseline 

test, and the aeration blank is not toxic, aeration was 

effective in reducing toxicity. If the toxicity of the 

aerated effluent is less than the baseline test, even 

though the aeration blank is toxic, the results indicate 

that aeration is an effective removal technique. If the 

effluent toxicity is not reduced or it is more toxic after 

aeration than in the baseline test (and the aeration 

blank was non-toxic, then either toxicity was concentrated during the aeration process or toxicity was added 

or created during the aeration process (see Special 

Considerations/Cautions below). 

Typically, using this aeration technique, ammonia 

is not air-stripped from the sample at pH i. However, if 

total ammonia is at least 10 mg/l or higher and the pH 

is above 8.0, ammonia measurements in the aerated 

sample may be useful if the aeration manipulation resulted in a toxicity reduction. 

If a substantial reduction in toxicity is observed, 

then the mechanism for the toxicity removal must be 

determined. To determine if the reduction is due to 

oxidation, sparging, or sublation, the air should be replaced by nitrogen. The flow of nitrogen through the 

sample must be the same as for air. If nitrogen sparging 

as well as air sparging removes or reduces the toxicity, 

then oxidation as the removal process is eliminated. If 

aeration only succeeds in reducing toxicity, then oxidation may be involved. It is possible that a toxicant can 

be removed through sparging and oxidation in which 

case air should reduce toxicity more than nitrogen. 

The presence of sublatable substances can be 

determined (whether air or nitrogen is used) by removing the aerated sample from the aeration vessel by 

siphoning or pipetting without contact with the sides of 

the aeration vessel. The geometry of the aeration 

vessel (i.e., at least a half-full cylinder) must remain the 

same as in the initial aeration experiment but the recovery of sublated compounds can be difficult. Dilution 

water added to the aeration vessel is used as a rinse to 

remove the sublate residue on the walls. To attempt 

this recovery, use of graduated cylinders with ground 

glass stoppers has been successful for acute testing 

(EPA, 1991A) because the water can be shaken vigorously to contact all surface areas to recover the 

sublatables. This sublation procedure is effective for 

dissolved surfactants, and while sewage particles adsorb surface active particles tightly, the actual sublation 

process may take some time (i.e., ~1 h) (AHPA, 1989). 

If toxicity is not recovered from the vessel walls, the 

presence of such compounds cannot be ruled out. 

Specific procedures, for the larger volumes needed in 

the chronic tests, have not yet been developed. 

In some instances, sublatable toxicants may not be 

removed by dilution water, and the use of solvents 

(e.g., methanol) may be needed for better recovery. 

However, the solvent will have to be reduced in volume 

(aired down) in order to have an adequate concentration factor in the test solution and a sufficiently low 

concentration of solvent for the subsequent toxicity 

tests (see Sections 6.7 and 6.8 for methanol toxicity 

information). Of course, dilution water blanks must 

also be subjected to all steps to check for artifactual 

toxicity. 

Special Considerations/Cautions: Removal of 

compounds by precipitation can occur through oxidation. However, the filtration test should not change 

toxicity of the effluent if oxidation is involved but filtration might also remove the toxicity of some sublatable 

compounds absorbed to particles and therefore the 

results of the aeration test can be compared to the 

filtration test. 

Use of nitrogen to sparge the sample is likely to 

drastically reduce the DO. For instance, 1 h of nitrogen 

sparging has caused the DO to drop below 4 mgIl. To 

increase the DO before initiating the test after a sample 

has been sparged with nitrogen, transfer the sample to 

a container with a large surface area to water volume 

ratio. The DO should rise to >5 mg/l without additional 

aeration. 

The baseline test serves as the toxicify control and 

the aeration of the dilution water (aeration blank) provides information on the system apparatus. The general test conditions, quality of the dilution water, and 

health of the test organisms are tracked by the controls 

in the baseline test. No significant toxicity should occur 

in the aeration blank. Toxicity in the aeration blank 

implies toxic artifacts from the aeration process, the 

glassware, or a dilution water problem. If the aeration 

blank is toxic, check the results of the test of the 

filtration blank. If both blanks are toxic, then most likely 

there is a problem with the dilution water but if only the 

aeration blank is toxic, artifactual toxicity arose during 

that manipulation. 

6.5 Filtration Test 

General Approach: Filtration of the effluent sample 

provides information on whether the toxicity is filterable 

yet provides relatively little specific information about 

which class of toxicant may be causing the toxicity. 

Reductions in the toxicity caused by fittering alone may 

imply toxicity associated with suspended solids or removal of particle-bound toxicants. Whether compounds 

in the effluent are in solution or sorbed to particles is 

6-9 
dependent on particle surface charge, surface area, 

compound polarity and charge, solubility, and the matrix of the effluent. If particles are removed, other 

compounds may be bound to them and are not available to cause toxicity. The way the toxicant is bound to 

the particulates is probably more important when using 

fifter feeders as the toxicity test organism in short-term 

chronic tests. This is primarily a route of exposure for 

filter feeders as compared to the fathead minnow. Toxicity can also be reduced by filtering if a toxicant is 

not particle-associated; we have observed that some 

chemicals in a dilution water stock are removed by 

filtering (e.g., DDT). 

The filtration step also serves an important purpose 

for another Phase I manipulation, the solid phase extraction (SPE) (Section 6.6), where aliquots of the effluent typically must be filtered before application to the 

SPE sorbent (see Interpretation of ResuWSubsequent 

Tests below). If many particles are present in the 

sample, the sorbent may act as a filter itself or the 

column will become plugged. 

Methods: The use of a positive pressure filtration 

system is superior to the use of a vacuum filter because volatile compounds may be removed by vacuum 

filtering and hence confuse the effect of filtering (see 

lnterprefafion of ResuWSubsequent Tests). 

As in the acute Phase I, prepare the filters (typically 

1 urn glass fiber filters without organic binder) by passing an appropriate volume (approximately one-fourth of 

effluent volume to be filtered) of high purity water over 

the filter(s) in the filter housing. This water is discarded, a small aliquot of the dilution water is filtered 

(prepare excess, at least 500 ml for the C. dubia 7-d 

test and 800 ml for the fathead minnow 7-d test) and 

discarded (100 ml) and the rest collected. A portion of 

the filtered dilution water is collected and used for 

testing and a portion reserved for the posf C,, SPE 

column test b/an& (Section 6.6). For example, the last 

400 ml of the filtrate is collected for the C. dubia 7-d 

filtration blank and post C,, SPE column blank tests. 

Next the effluent sample is filtered using the same 

filter, and a portion of the filtrate is collected for toxicity 

testing and a portion set aside that will be concentrated 

on the C,, column. When filtering the effluent, fitter 

enough sample for this test and enough sample (>l L) 

to use for the SPE step described below. For some 

effluents, one filter will not suffice. A technique we use 

is to prepare several filters at once by stacking 5-8 

filters together followed by rinses of high purity water 

and dilution water using the same rinse volumes as 

above. Then the filters are separated, and set aside, 

using one at a time for the effluent sample. If the 

samples measure quite high in total suspended solids, 

pre-filtering using a larger pore size filter may help. 

Again, appropriate blanks must be obtained for any 

pre-filtering. Low levels of metals on the glassware or 

the filters could cause interferences in toxicity interpretation. Pre-rinsing the filters and glassware with high 

purity water adjusted to pH 3 may provide consistently 

clean blanks and possibly less contamination in effluent 

samples. If the sample cannot be effectively/easily 

fiftered due to many fine particles, centrifuging may be 

better (again blanks must be prepared). 

The filter housing should be thoroughly cleaned 

between effluent samples to prevent any particle buildup or toxicity carryover. We have found large fitter 

apparatus (1 L), removable glass frits, or plastic filtering 

apparatus (Millipore@) to be useful. The glassware 

cleaning procedure that is described in the acute Phase 

I TIE manual should be sufficient for chronic TIE work 

(EPA, 1991A). The glass frits may require rigorous 

cleaning (i.e., soak in strong acid (10% v/v) for 20-40 

min) to remove residuals that may remain after filtering, 

since the glass frit may itself act as a filter, 

Interpretation of Results/Subsequent Tests: If 

toxicity in the whole effluent is reduced by filtration, a 

method for separating the toxicants from other constituents in the effluent has been achieved. This should 

advance the characterization considerably because any 

subsequent analysis will be less confused by non-toxic 

constituents. If appropriate, one should determine if 

toxicity loss was due to volatilization. Comparisons of 

pressure filtering and vacuum filtering should indicate if 

volatilization is involved. For further characterization, 

the mechanism of removal should be determined (precipitation, sorption, changes in equilibrium or volatilization). 

Identification efforts should be focused on the residue on the filter after testing indicates that the toxicant 

is not volatile. To recover the toxicity from the filter(s), 

use of acidic and basic water as well as various organic 

solvents can be tried. The recovery achieved by these 

various methods provides information about pK and 

water solubility of the toxicants. Filtration has reduced 

the quantity of total cationic metals present in some 

effluents. The recovery of the metal and acute toxicity 

was successful when dilution water adjusted to pH 3 

was used to extract the filter (EPA, 1991A). Filter 

extraction into smaller volumes than that of the effluent 

sample filtered will give a higher concentration of toxicant, perhaps allowing the use of acute test endpoints. 

However, evidence then must be gathered to be sure 

the toxicants causing acute toxicity are the same as 

those causing chronic toxicity. Use of solvents will 

require solvent reduction or solvent removal (exchange) 

before testing (see Phase II; EPA, 1992A). Sonication 

of filters is another approach but the manipulation must 

be accompanied by proper blanks in similar fashion to 

those needed for the pH 3 extraction of the filter extraction step described above. 

If large volumes of an effluent (-2 L over one 1 pm 

filter) can be readily filtered, the effluent should be 

filtered for the filtration test and unfiltered effluent can 

be passed over the C,, SPE column (see Section 6.6; 

Post C,, SPE column tesf). Once it has been demonstrated that filtration does not reduce toxicity in the 

effluent, and the toxicity is recovered in the methanol 

eluafe test the routine filtering can be eliminated. By 

6-10 
this approach the amount of testing to be done is 

decreased, yet the tracking of toxicity is possible. We 

have infrequently experienced any effluents that have 

low amounts of filterable solids where the effluent could 

be concentrated without filtering. If any effluent sample 

has reduced toxicity in the filtration test and toxicity is 

not observed in the methanol eluate rest, characteristics of the toxicant will be described as filterable and 

not C,, recoverable. 

If the toxicity cannot be recovered from the filter, 

was not volatile (see Section 6.4 aeration test) and no 

other manipulations changed toxicity, use of Tier 2 is a 

good subsequent step. Toxicity could have been removed by the glass frit, and use of a plastic filter 

apparatus or stainless steel frits may assist in identifying that the toxicant removed is on the frit or filter. 

Filter-removable toxicity in Tier 2 is more difficult to 

identify (because of the radical pH adjustments) because of irreversible reactions and potential for artifactual toxicity (see Section 6.12 below). 

Special Considerations/Cautions: The filtered 

dilution water and filtered effluent sample also serve as 

the toxicity blank and toxicity control respectively for 

the post C,, SPE column test (see Section 6.6). The 

results of the effluent filtration test should be compared 

with the filtration blanks and no major change in the 

trend of young production, growth or survival should 

occur in the filtration blanks in comparison to the controls in the baseline fesf. If the filtration blanks are 

acceptable, then the results of the filtration test and the 

baseline rest should be compared. 

As a toxicity blank for the SPE tests, if the filtration 

blank is either slightly or completely toxic, but the post 

C,, SPE column effluent is not toxic (and effluent toxicity was unchanged after filtration), the filtration blank 

toxicity can be ignored since the effluent toxicity was 

removed. However, as work proceeds to identification, 

the blank toxicity will have to be eliminated or else it 

could introduce an artifact and lead to a misidentification 

of the cause of toxicity. 

6.6 Post C,, Solid Phase Extraction Column 

Test 

General Approach: The C,, SPE column is used 

to determine the extent of the effluent’s toxicity that is 

due to compounds that are removed or sorbed onto the 

column at pH i (cf., post C,, SPE column and pH 

adjustment test; Section 6.13 below). By passing effluent through a SPE column, non-polar organics, some 

metals, and some surfactants are removed from the 

sample. In addition, these columns may also behave 

as a filter (see filtration test above). 

Compounds in effluent samples interact with the 

C,, and depending upon the polarity and solubility of 

the compounds, the sorbent may extract the chemicals 

from the water solution/effluent onto the column. Extraction occurs when the compounds have a higher 

affinity for sorbent than for the aqueous phase. Nonpolar organic chemicals are extracted because the C,, 

sorbent is very non-polar in comparison to the polar 

water phase; this extraction process is referred to as 

reverse phase chromatography. 

The effluent that passes over the column is collected and the post-column effluent is toxicity tested in 

order to determine if the column removed toxicity. If 

the toxicity of the post-column sample is decreased, 

removal of toxicant by the column is probable but if it 

is not, artifactual toxicity may be obscuring the removal. 

Steps to deal with this are given below in lnferpretafion 

of Results/Subsequent Tests. If the post-column sample 

is highly toxic, the capacity of the column to extract the 

toxicants may be exceeded or the column may have 

been inadequately conditioned. 

Because toxicity may be retained by the C,, column, efforts to recover the toxicity are necessary. After 

a sample is passed over the C,, column, many of the 

compounds extracted by the sorbent at a neutral pH 

should be soluble in less polar solvents than water (i.e., 

hexane, methylene chloride, methanol, chloroform). 

However, most of the non-polar solvents are highly 

toxic to aquatic organisms. Sorbed non-polar organics 

are eluted from the column because they have higher 

affinity for the non-polar solvent than the C,, sorbent. 

The methanol eluafe test (Section 6.7) is designed to 

determine if toxicants are non-polar. 

Methods: The toxicity of the effluent, the type of 

test to be conducted, and the frequency of the solution 

renewal affect how much effluent must be filtered and 

passed over the C,, SPE column. First, the concentrations and the volume of the eluate needed for the 

methanol eluate test (Section 6.7) to test at 2x or 4x 

the whole effluent concentrations should be determined 

(keeping in mind that the methanol test level must be 

below the chronic threshold level for the species used; 

Section 6.7). However, limiting factors of the maximum 

volume to apply to a column, the minimum elution 

volume required, and the concentration that can be 

obtained within these confines must be calculated 

(Tables 6-7 and 6-8). 

For example, our procedure has been to pass 1000 

ml of 100% effluent over a 1 g (6 ml) column and elute 

with 3 ml of methanol which results in a theoretical 

333x concentrate. The 1000 ml is the limit of sample 

volume over a 1 g (6 ml) column and the 3 ml methanol 

elution is slightly more than the minimum elution volume required (Table 6-7). However to test C. dubia at 

4x, and to have the methanol concentration at a nontoxic chronic level (Table 6-9), the 3 ml must be further 

concentrated to 1.5 ml (now 666x whole effluent concentration). At present 3 ml of the eluate is concentrated in graduated centrifuge tubes to 666x by using a 

gentle stream of nitrogen gas over the surface of the 

methanol eluate in a warm water bath (2530°C) to 

concentrate the 333x eluate to a final volume of 1.5 ml. 

For five replicates of 10 ml each, 0.30 ml of the eluate 

can be added to 50 ml of dilution water and the resultant effluent concentration is 4x and the methanol concentration is 0.6%. However the 1.5 ml eluate from the 

6-l 1 
Table 6-7. Factors to consider for the size of available pm-packed C,, SPE columns. Appropriate volumes of sample to apply to each 

column with respect to maximum volumes of sample and minimum elution volumes, and elution volumes frequently used in the 

TIE process. 

Size (ml) 

Conditioning 

Volume (ml) 

Maximum Minimum 

Volume (ml) Elution 

of Effluent Volume2 

Methanol 

Elution 

Used (ml)3 

No. 

Methanol 

Fractions4 

Eluate 

Concentration 

6 1 10 1.m 2.0, 2.4 35.2.4 3 333x5, 417x 

12 2 24 2wJ 4.8 3 3 417x 

20 5 40 5,~ 12 6 3 417x 

60 10 120 10,000 24 12 3 417x 

I 1 g columns are available from J.T. Baker Chemical Company, Phillipsburg N.J. (1 g, 6 ml columns have been extensively used at ERLDuluth). 1 g. 2 g. 5 g, and 10 g columns are available from Analytichem International, Mega Bond Elut R(, Harbor City, CA. Pumping rates 

for each column are proportional to volume based on 1 L at 5 mllmin; therefore 2 L at 10 mllmin, 5 L at 25 mUmin, and 10 L at 50 ml/min. 

We are currently evaluating the minimum elution volumes to determine if less etuting solvent can be used. Pumping rates for 5 L and 10 L 

may need to be slower when eluting each column, Yet how much the pump should be slowed will be a function of the toxicants. The 

contact time of the elution solvent with C,, sorbent may need to be increased if toxicity is not recovered in the methanol eluates. 

z Minimum elution volume as recommended by the manufacturers. For the 1 g column, J.T. Baker recommends 2.0 ml and Mega Bond 

Elut”” recommends 2.4 ml, but 2.0 ml is probably adequate. 

3 Elution of two one-half volume aliquots is better for optimizing the elution efficacy 

4 For each fractionation of any size column, collect three separate 100% methanol fractions to use in mefhanol hate test to attempt 

recovery of the non-polar toxicants (see text for more details). 

5 This procedure has been routinely used for acute TIES. To maximize concentration and minimize methanol levels in concentration and 

minimize methanol levels in toxicity tests it is best to use the minimum elution volumes recommended by the manufacturer. 

1 L fractionation will allow testing of 4x, 2x, lx only if 

two solution renewals are used (Table 6-8). Daily 

renewals for a 7-d C. dubia test require a total of 3.7 ml 

at a water concentration of 0.6% methanol (which means 

3 L of effluent must be fractionated to obtain 9 ml of 

333x eluate which is concentrated to 4.5 ml to test at 

4x) (Table 6-8). 

To test at 2x using a 417x eluate from a 2.4 ml 

elution, 0.048 ml in 10 ml will result in the 2x test 

concentration. For a 7-d, daily renewal test at 2x, lx, 

0.5x, 3.0 ml is needed (5 replicates of 10 ml each) 

which will require 1 L of effluent to be concentrated 

(Table 6-8). By this procedure the final methanol concentration is 0.48%. The 417x concentrate can also be 

concentrated to 834x and use 0.048 ml00 ml to test 

the eluate at 4x. 

For the 7-d fathead minnow test using 50 ml per 

replicate and two replicates, a total of 7.4 ml of a 

methanol eluate is needed for test initiation and six 

renewals, which requires fractionation of 3 L of effluent. 

This assumes the methanol test concentration between 

species are kept the same. Actually the fathead minnows could probably be tested at methanol concentrations of -l%, and using 0.96 ml of the 417x eluate per 

100 ml will result in 4x effluent test concentration and a 

1% methanol concentration (Table 6-9). 

The methods below assume one effluent volume 

(usually the 100%) is concentrated and the post column effluent sample collected and used for all solution 

renewals during the test (Table 6-8). The procedure 

described below is an overview of the steps needed to 

prepare the column, collect methanol blanks, recondition the column, collect post-column effluent, and collect methanol eluate (steps needed for this test and the 

next test-Section 6.7). All steps are detailed in the 

acute Phase I manual (EPA, 1991A), and the major 

difference for the chronic Phase I is that fewer postcolumn samples (one or two versus three) are collected. 

The general technique for conditioning and using 

the prepackaged SPE columns is as follows. Using a 

pump system with a reservoir for the effluent sample 

and teflon tubing, first pump lo-120 ml of HPLC grade 

methanol over the column to condition the sorbent 

(Table 6-7). This methanol is discarded. Without 

letting the column go to dryness, 1 O-120 ml of high 

purity water is passed over the column and discarded. 

Next, before the methanol blank is collected, the column is allowed to go to dryness. For 1 L of sample and 

a 1 g (6 ml) column, two 1.5 ml aliquots of 100% 

methanol are collected, combined, and tested as the 

blank. The elution is more efficient when two aliquots 

of 1.5 ml are collected in contrast to one elution of 3 ml. 

The collection of three 100% methanol eluates (2.4 or 3 

ml each) has been more helpful for tracking toxicity 

than only one 100% methanol eluate sample. The use 

of three 100% methanol elutions is replaced when the 

Phase II fractionation procedures are applied. These 

100% methanol eluates may need to be concentrated 

prior to testing (see Section 6.7). The containers to 

collect the methanol should be acid leached, hexane 

and acetone rinsed, and allowed to dry before use. 

After the methanol blank is collected, the column must 

6-12 
Table 6-8. Test volume of eluate needed for methanol &ate test with C. dubia or f. promelas. Volumes described are based on minimum 

elution volumes recommended (Table 6-7) and the highest test concentration possible with the methanol level at an acceptable 

concentration. 

Test Test 

Species Duration 

No. Renewals High No. Volume of Eluate Test Minimum 

8 Original Test Cont. Rep. Needed for Testing at: Concentrations Volume (L) 

Sample 333x’ 417x2 of Effluent’ 

C. dubia 411 2 

C. dubia 4-d 4 

C. tibia 7d 3 

C. tibia 7-d 7 

C. tibia 4-d 

C. tibia 4d 

C. dubia 7-d 

C. dubia 7-d 

P. promelas 

P. promelas 

P. promelas 

P. pmmelas 

7-d 

7d 

7-d 

7-d 

2x 

2x 

2x 

2x 

2x 

2x 

2x 

2x 

2x 

2x 

4x 

4x 

10 

10 

10 

10 

2 

4 

2 

4 

1.05 

2.10 

1.56 

3.66 

0.64 2x, lx, 0.5x 

1.66 2x. 1x. 0.5x 

1.26 2x; 1x, 0.5x 

2.94 2x, IX, 0.5x 

2.10 1.66 2x, IX, 0.5x 

4.10 3.36 2x, lx, 0.5x 

3.16 2.52 2x, IX, 0.5x 

7.35 5.66 2x, lx, 0.5x 

7.35 5.66 2x, IX, 0.5x 

14.70 11.76 2x, IX, 0.5x 

14.70 11.76 4x, a, 0.5x 

29.40 23.52 4x. a, 0.5x 

1 

1 

1 

2 

1 

2 

2 

3 

3 

5 

5 

10 

’ For the 333x eluate concentration, this volume is based on the assumption that the C. dubia test solutions are prepared as 300 pl of 333x 

into 50 ml for 2x, 150 pl into 50ml for lx, and 75 pl into 50 ml for 0.5x. More volume will be needed if serial dilutions are prepared (600 pl 

vs 525 ~1). For the fathead minnow tests this assumes test solutions are prepared as 600 ~1 into 100 mL for 2x, 300 PI into 100 mL for lx, 

and 150 1.11 into 100 mL for 0.5x. More volume will be needed if serial dilutions are prepared (1200 pl vs 1050 ~1). 

2 For the 417x eluate concentration, this volume is based on the assumption that the C. dubia test solutions are prepared as 240 @ of 333x 

into 50 ml for 2x, 120 pl into 5Oml for lx, and 60 @ into 50 ml for 0.5x. More volume will be needed if serial dilutions are prepared. For the 

fathead minnow tests this assumes test solutions are prepared as 460 ~1 into 100 ml for 2x. 240 fl into 100 mL for lx, and 120 fl into 100 

ml for 0.5x. More volume will be needed if serial dilutions are prepared. For the 4x fathead minnow test, 960 pl per 100 ml must be 

prepared for the 4x solution. 

3 Volume is based on high test concentration (2x or 4x) tested without concentration to obtain eluate twice as concentrated. If further 

concentration is needed. twice as much effluent will be needed. 

Table 6-9. Chronic toxicity of methanol (%) to C. dubia and P. promelas using the 7-d tests. 

Water Test IC50 IC25 

Species Type Renewal 95% C.I. 95% C.I. NOEC LOEC 

C. dubia SRW daily 1.2 

1.1-1.2 

0.45’ <0.5 - 

0.35-l .o 

SRW twice 1.4 0.45’ <0.5 - 

- 0.36-0.70 

SRWz twice 1.2 0.59 0.75 1.5 

0.69-l .7 0.29-0.95 

SRW twice 1.3 0.63 0.75 1.5 

- 0.34-l .o 

P. promelas SRW daily 2.1 1.34 1.3 2.5 

2.0-2.2 0.27-l .5 

’ Value is extrapolated. 

z Tests all conducted independently. 

Note: C.I. = confidence interval; SRW I soft water 

6-13 
be reconditioned with lo-120 ml of methanol (which is 

discarded). Without allowing the column to go to dtyness, follow the methanol with an aliquot (1 O-l 20 ml) of 

high purity water, immediately followed by an aliquot of 

filtered dilution water. The amount of filtered dilution 

water needed will be dependent on the species and 

type of test to be conducted. The initial aliquot of the 

post-column water should be discarded (-200 ml) and 

the remainder of the post column dilution water should 

be collected. This post-column dilution water sample 

will serve as the dilution water blank for the post C,, 

SPE column test. 

In order to optimize concentration of an effluent 

sample and not exceed the specifications of the sorbent capacity, when the maximum volume (Table 6-7) 

of a sample is passed over a column, the sorbent must 

be reconditioned following the collection of the post 

column dilution water. For example if 1.2 L of dilution 

water is needed and 5 L of effluent is to be concentrated on a 5 g column, without reconditioning the 

column between the dilution water and the effluent, the 

sorbent’s capacity is likely to be exceeded. Toxicity 

might be observed in the post C, SPE column resr 

because of the excessive volume d dilution water and 

5 L of effluent. The procedures for conditioning the 

column are similar to those above. The appropriate 

amount of methanol (Table 6-7) is used to condition the 

sot-bent and the methanol is discarded. Before the 

column goes to dryness, follow the methanol with an 

aliquot (lo-120 ml) of high purity water, immediately 

followed by the volume of filtered effluent to be concentrated. Again, collect about 200 ml of the post-column 

effluent and discard it. This is discarded to reduce the 

possibility of higher background concentrations of methanol in the post-column sample which might contribute 

to artifactual toxicity. Collect remainder of post-column 

effluent as a batch or in aliquots. If small quantities 

(~500 ml) of post-column effluent are needed for toxicity testing, separate post-column effluent samples may 

help determine if toxicity breakthrough occurred, and 

concentration factors will be different for the lower volumes. 

Interpretation of Results/Subsequent Tests: The 

extraction efficiency of the column is evaluated by comparing the toxicity in the post C, SPE column rest to 

the Warion test data. This post C!,, SPE column rest is 

most useful when there is no post-column toxicity, and 

filtration did not reduce toxicity. 

When toxicity in the post-column effluent is reduced or removed, then the next step is to compare the 

results with the methanol e/u&e test. If toxicity was 

recovered in the methanol eluates (see Section 6.7 

below), then efforts to identify the toxicants (Phase II) 

should be initiated immediately. 

If the post-column effluent toxicity was removed or 

reduced, but toxicity was not recovered in the methanol 

eluates (see below), it is possible that the toxicant is 

not eluded into 100% methanol and the C,, SPE column 

contains the toxicant. Use of the gradient of methanol 

and water fractions should be tried as well as testing 

the eluate at higher concentrations than 2x (i.e., 4x or 

8x). If those tests do not indicate toxicity present in the 

eluates (see below) alternate elution schemes (EPA, 

1992A) must be tried to recover the toxicant. It is 

important to recognize that the toxicity removed by the 

C,, SPE column is not necessarily due to non-polar 

compounds. Metals can be removed from some effluents via the C,, SPE sorbent. However, metals are not 

efficiently eluted in methanol or other organic solvents. 

Acid adjusted (pH 3) dilution water may be needed to 

elute toxicant from the column. If this is done, the 

pumping rate of the pH-adjusted water should be slowed 

(perhaps by one-fourth of original pumping rate) to 

allow adequate contact time to elute the compound 

from the sorbent. In addition, compounds such as 

polymers or surfactants may be sorbed onto the column and some will elute with methanol while others do 

not. 

The column can act as a filter itself and the various 

solvents used do not elute the toxicant. To check 

whether the C,,column is acting as a filter, unfiltered 

effluent can be passed over the C,, column and toxicity 

test results compared to those from the filtered effluent 

sample simultaneously. When effluent samples are 

readily filtered (e.g., 21.5 L for one 1 pm filter) filter the 

effluent to conduct the filtration test and use unfiltered 

effluent for the post C SPE column test and the 

methanol eluare test. VI&en toxicity can be recovered 

in the methanol eluate, the toxicant is most likely to 

be non-polar and since filtration can be eliminated for 

subsequent identification steps the amount of testing is 

subsequently reduced. 

If the post-column toxicity was reduced and/or removed but not recovered in the methanol eluare rest, 

the possibility exists that the toxicant has degraded or 

decomposed during the manipulation and the toxicant 

was not concentratable. 

As mentioned above, when no toxicity occurs in the 

post-column effluent (or the toxicity is reduced), and yet 

the methanol eluare test did not exhibit toxicity, metals 

may be involved or a non-polar that was not recovered 

in the solvent may be involved (discussed above). To 

check for cationic metal toxicity, the post C SPE 

column tesr should be combined with the EDTladdirion test and the sodium rhiosulfare addition rest to 

characterize the post-column toxicity (see Section 6.16, 

multiple characterization rests). 

For effluents that have shown that the toxicant is 

C,, recoverable, but the degradation of toxicity occurs 

fairly rapidly (i.e., the effluent sample is non-toxic in l-2 

weeks), it may be prudent to concentrate additional 

volumes of effluent immediately after the effluent arrives at the testing laboratory. Non-polar toxicants may 

not degrade in the methanol fractions as quickly in the 

effluent samples. Collect the methanol fractions (three 

100% fractions) or the various methanol/water fractions 

as described in Phase II (EPA, 1992A) and hold them 

at 4°C for analysis as the TIE proceeds. Similarly, 

6-14 
once the cause of toxici& has been determined to be 

non-polar (C, extractable) it might be more appropriate 

to immediately concentrate 10 to 20 L of effluent and 

for the elution step, replace the three 100% methanol 

elutions with the methanol/water procedures (EPA, 

1992A). For chronic work, we have been using seven 

water/methanol fractions (50%, 75%, 80%, 85%, 90%, 

95%, and 100%) rather than the eight used in acute 

TIES because the toxicity has never recovered in the 

25% fraction and by eliminating if the testing workload 

is reduced. It may be prudent to try two additional 

100% methanol fractions following the seven fractions 

as well or follow it with alternate elution schemes (cf., 

Phase II; EPA, 1992A). By immediately concentrating 

the effluent, it is possible to optimize the amount of 

methanol available for testing and subsequent concentration for analysis and the post-column samples can 

be tested at one time. This eliminates duplication of 

effort that is required when additional methanol eluate 

is needed for subsequent work in Phase II. 

Artifactual toxicity in the test containers may appear as a biological growth in the 100% post-column 

effluent and the effluent dilutions during the test. Effluents from biological treatment plants may develop this 

characteristic more readily than physical-chemical treatment plant effluents. This growth can negate actual 

toxicant removal by the column. While this growth 

does not occur in all effluents, when it does occur with 

one post-column effluent sample, the growth often occurs in each subsequent post-column effluent sample. 

The growth appears as a filamentous growth and gives 

a milky appearance in the test vessel. This growth has 

been linked to methanol stimulation of bacterial growth. 

Methanol is present in the post-column samples because methanol is constantly released from the sorbent 

during the sample extraction. Additional filtering of the 

post-column effluent sample through a 0.2 pm filter 

before testing to remove bacteria and eliminate the 

growth, has not been particularly successful. Artifactual toxicity from the post-column effluent may be 

avoided if the tests with the post-column samples are 

initiated on the same day the effluent is concentrated. 

To date, when we have collected the post-column 

samples and tested them on the same day, we have 

not experienced less artifactual toxicity than we found 

in those effluents where artifactual toxicity consistently 

has been a problem. However, less time elapses 

before animals are exposed to the test solution, therefore less time is available for bacteria to cause problems in the post-column sample matrix. Another option 

is to perform daily concentration of the effluent and 

extraction of the column during the 7d test, as fresh 

post-column samples may minimize the artifactual toxicity. 

When post-column artifactual growth is not readily 

eliminated, then a different solvent (acetonitrile) to prepare the column (but not for eluting) may be useful in 

reducing the post-column artifactual bacterial growth. 

Acetonitrile causes narcotic effects in toxicity tests, and 

is recommended only to condition the columns to avoid 

toxic concentrations. This technique has been successful on a limited number of effluents. 

Special Considerations/Cautions: Careful observations and judgement must be exercised in detecting problems in the post C,, SPE column resr. Low DO 

levels can occur in these samples. Through testing 

experience, the investigator will know whether toxicity 

appears as artifactual (i.e., growth, low DO) as opposed to the presence of the sample toxicity. If artifactual toxicity is not recognized, then a conclusion that 

the C, SPE column did not remove toxicity can erroneously be made. For this reason if the post-column 

effluent is toxic, the methanol eluate must be tested 

(Section 6.7). This avoids the arlifactual toxicity issue 

and the error can be avoided by determining the toxicity of the eluate. 

The methanol elution process does not always produce predictable results with the same effluent sample. 

When toxicity is removed by the column but no toxicity 

occurs in the 100% methanol eluates, it does not indicate that the toxicity is nor due to a non-polar toxicant( 

To check this possibility, immediately test the series of 

methanol/water fractions at concentrations of 4x or 8x. 

Not all non-polar organic compounds elute into 100% 

methanol as well as they do into lower methanol/water 

concentrations. Also toxicants may smear across the 

fractions and when ~100% recovery of toxicity from the 

column is not lOO%, toxicity may not be observed at 2x 

or lx. 

General test conditions will be tracked (dilution water, health of test animals) by the controls in the baseline 

rest. The post-column dilution water blanks should be 

compared to those controls to determine if the column 

imparted toxicity. If the post-column dilution water 

blank was toxic, but no toxicity or artifactual toxicity 

occurred in the post-column effluent sample, the toxic 

blank can be ignored. 

Results of the post C,, SPE column effluent rest(s) 

must be compared to the results of the filtration rest to 

determine if the manipulations effectively reduced toxicity. When the post C,, SPE column rest is plagued by 

artifactual toxicity, the Importance of the methanol eluare rest increases. The results of the post C,, SPE 

column rest must also be compared to the baseline 

rest to determine if toxicity was removed by the C,, 

SPE column. 

6.7 Methanol Eluate Test 

General Approach: In order to elute toxicants 

from the C , SPE sorbent, a relatively non-polar solvent 

is used. dexane, one of the most non-polar solvents, 

can be used to remove highly non-polar compounds 

from the C,, SPE column. Yet hexane is one of the 

most toxic solvents to aquatic organisms and has a low 

miscibility with water. Methanol is more polar than 

hexane, but is much less toxic and will elute many 

6-15 
compounds, The use of methanol has been adopted 

as the eluant for the acute TIE (EPA, 1991A; EPA, 

1989A) and the chronic TIE because of its low toxicity 

(Table 6-9) and its usually adequate ability to elute 

chemicals from the C,, SPE column. 

Methods: The conditioning and elution steps are 

described in detail in the post C,, SPE column test 

above (see Section 6.6). For this test, we assume that 

the column extraction efficiency and elution efficiency 

are 100%. 

If a 1 g (6 ml) SPE column was used with 1 L of 

100% effluent, and a 3 ml methanol eluate was collected, the methanol eluate is a 333x concentrate of the 

original effluent (Table 6-7). Depending on the amount 

of effluent toxicity, this eluate may have to be concentrated further in order to test at a sufficient concentration (i.e., 4x) and have methanol concentrations in the 

test lower than the methanol effect concentration. In 

Table 6-9 the toxicity data for methanol toxicity to 

C. dubia and fathead minnows are given. The toxicity 

of methanol is slightly greater for C. dubia when the 

test solutions were renewed daily but not significantly 

for this characterization stage of the TIE. From these 

data, one can decide how much methanol can be 

added and how concentrated the eluant must be to 

achieve 2x or 4x the original effluent concentration. 

The choice of test concentration depends on the toxicity of the effluent; for example, if the effluent is toxic at 

-25%, one may not need to achieve a 4x concentration. Some methanol toxicity can be present, as long 

as sufficient toxicity from the effluent is present to be 

measurable. As discussed in the post C SPE column 

test, the fathead minnows can be teste 1 at 4x using 

only 0.96 ml of a 417x methanol eluate but the methanol concentration is about l%, which cannot be tolerated by C dubia. 

Interpretation of Results/Subsequent Tests: If 

toxicity occurs in the methanol eluafe fest at any concentration tested, Phase II should be initiated. This 

step would include the use of a gradient of methanol/ 

water eluant solutions to elute additional columns and 

conduct the toxicity tests on each fraction (Phase II; 

EPA 1989A; EPA, 1992A). Toxicants other than nonpolar compounds may be retained by the SPE column 

but they are less likely to be eluted sharply or eluted at 

all (see Section 6.6). Non-polar toxicity can in some 

instances be distinguished from post-column artifactual 

toxicity if the eluate is checked for toxicity. Some 

toxicants (such as some surfactants) may not elute 

from the SPE column with methanol, but if toxicity is 

not recovered in the eluate, it does not exclude the 

possibility of a non-polar toxicant or metal (see Section 

6.6 for additional discussion). Dilution water adjusted 

to pH 3 or pH 9 may be useful in eluting a toxicant 

from the column. Some experimentation will be needed 

to determine the volumes of water to pump over the 

column. The pumping rate should be slowed considerably to allow sufficient contact time on the column (see 

details in Section 6.6 and Table 6-8). 

At this time, we have not been successful in tracking chronic non-polar toxicity using the acute test endpoint with the methanol eluates, rather chronic tests 

have been needed to track the chronic toxicity. 

A subsequent test that may be useful is to assess 

whether the toxicant must be metabolically-activated by 

the test organism before exhibiting toxicity. These 

activation reactions consist of oxidative metabolism by 

a family of enzymes collectively known as cytochrome 

P-450. Some toxicants require cytochrome P-450 activation before expressing toxicity. Piperonyl butoxide 

(PBO) is a synthetic methylenedioxyphenyl compound 

that effectively binds to, and blocks the catalytic activity 

of cytochrome P-450. When a non-toxic amount of 

PBO is added to an effluent test solution which contains a toxicant that requires metabolic activation, 

the toxicity of the effluent can be reduced or completely 

blocked (EPA, 1991A). The relative specificity of PBO 

for blocking the toxicity of metabolically-activated organic compounds makes this test a useful part of the 

subsequent testing in the TIE. For example in the 

acute Phase I (EPA, 1991A) as a subsequent test, we 

suggest that PBO may be added directly to the effluent 

before adding the organisms. The 48 h LC50 of PBO 

is 1 mg/l for C. dubia and we have used 0.250 to 

0.500 mg/l to effectively block the acute toxicity of 

metabolically-activated compounds for C. dubia in the 

effluent and the methanol eluate. The NOEC and the 

IC25 for PBO and C. dubia was determined as 63 ps/l 

and 89 us/l, respectively. Low concentrations of PBO 

have reduced the chronic toxicity in the methanol eluate test and levels of 100 cr 50 ug/l have been useful in 

chronic tests with C. dubia. The PBO should be added 

using a minimal amount of methanol as a carrier solvent since the level of methanol present in conjunction 

with the methanol eluate is present. Since PBO is not 

readily soluble in water, a superstock of 20 g/l is prepared by dissolving PBO in reagent grade methanol. 

An aliquot of the superstock is mixed in the standard 

laboratory dilution water to produce a stock solution at 

a concentration of 25 mg/l and aliquots of this stock 

solution are added to the test cups after addition of the 

methanol eluate, and the solution thoroughly mixed. 

This test should be conducted in similar fashion to the 

EDTA addition test. Appropriate blanks must be used, 

for example both the methanol blank and the methanol 

eluate must be tested with and without PBO. If toxicity 

occurs in the methanol blank fraction with the PBO 

additions, either PBO was present at toxic concentrations or the methanol concentration in the test was too 

high. If toxicity is observed in the methanol eluafe with 

the PBO addition, but not in the methanol eluate without the PBO or either of the blank eluates (with PBO 

and without PBO), this result is not very informative. It 

is possible that the PBO has interacted in a synergistic 

fashion with another compound present in the test 

effluent that normally would not be toxic. 

Compounds that are sparingly soluble in water may 

not be eluted from the column with methanol. If this 

6-16 
occurs, less polar solvents will have to be tried, but this 

technique will require solvent exchanges to avoid toxic 

solvent concentrations and other solvents may recover 

chemicals not toxic in the effluent due to solubility 

problems. At this time, we have not used solvent exchanges for chronic toxicity tests, but are exploring the 

use of methylene chloride. The 48 h LC50 of methylene chloride to C. dubia is 0.13% and the chronic 

toxicity to C. dubia is 10.03%. Therefore it cannot 

readily be used as the primary solvent, but rather as 

the exchange solvent and may be of limited use for this 

effort. Additional work on the appropriate solvent exchange for chronic TIES is on-going (EPA, 1992A). 

Special Considerations/Cautions: The baseline 

test serves as the toxicity control, and the methanol 

blank serves as a comparison of the effects of methanol alone in water. The health of the test animals, the 

viability of the dilution water and general test conditions 

are evaluated by the baseline controls. If the effluent 

methanol eluate is non-toxic at 2x or 4x but the methano/ blank is toxic, the blank toxicity can be ignored 

since no non-polar toxicity is recovered. 

If effluent dilutions are set at lOO%, 80%, 60%, and 

40%, it might be useful to test the eluate at a multiple of 

these concentrations, i.e., 2x, 1.6x, 1.2x, 0.8x or concentrate them to 4x, 3.2x, 2.4x, or 1.6x to compare the 

baseline toxicity with the toxicity in the methanol eluate 

tests. The artifactual growth observed in the post C1# 

SPE column test from the methanol has not occurred In 

our methanol eluate tests. This is most likely due to 

the differences in how the methanol degrades/behaves 

in dilution waters which are low in methanol-oxidizing 

bacteria and other organic matter in contrast to effluent 

samples (even post-column effluents). 

6.8 Graduated pH Test 

General Approach: This test will determine 

whether effluent toxicity can be attributed to compounds 

whose toxicity is pH dependent. The pH dependent 

compounds of concern are those with a pK, that allows 

sufficient differences in dissociation to occur in a physiologically tolerable pH range (pH 6-9). The toxicity 

depends on the form that is toxic (ionized versus unionized). Metal toxicity can be affected by pH differences through changes in solubility and speciation. pH 

dependent toxicity is likely to be affected by temperature, DO and CO, concentrations, and total dissolved 

solids (TDS). The graduated pH test is most effective 

in differentiating substantial toxicity related to ammonia 

from other causes of toxicity. 

Ammonia is an example of a chemical that exhibits 

different ionization states and subsequently pH dependent toxicity. Ammonia is also frequently present in 

effluents at concentrations of 5 mg/l to 200 m9/l (or 

higher). Measuring the total ammonia in the sample 

upon its arrival will be helpful to assess the potential for 

ammonia toxicity. pH has a great effect on ammonia 

toxicity. For many effluents (especially with municipal 

effluents) the pH of a sample rises upon contact with 

air, typically the pH of effluents at air equilibrium ranges 

from 8.0 to 8.5. Literature data on ammonia toxicity 

(EPA, 19850) can be used only as a general guide 

because the pH values for most ammonia toxicity tests 

as reported in the literature are usually not measured 

or reported fully enough to be useful in TIE tests. 

Additional data on ammonia toxicity for C. dubia and P. 

promelas is provided in the revised Phase II (EPA, 

1992A). The acute Phase I manual has a lengthy 

description of the toxicity behavior of ammonia (EPA, 

1991A) and Phase II provides additional information 

(EPA, 1992A). 

One might expect ammonia to be removed during 

the Tier 2 aeration and pH adjustment test at basic pH 

(described in Section 6.11). Based on our experience, 

however, ammonia is not substantially removed by the 

methods used to aerate the sample described in this 

manual. (If a larger surface to volume ratio is used, 

this manipulation can reduce ammonia levels; see Interpretation of Results/Subsequent Tests below and 

Phase II; EPA, 1992A.) Other techniques which can be 

used to remove ammonia may also displace metals or 

other toxicants with completely different physical and 

chemical characteristics. For example, ion exchange 

resins (e.g., zeolite) remove ammonia, cationic metals, 

and possibly organic compounds through adsorption. 

Toxicity related to metals may also be detected by 

the graduated pH test, although these effects are less 

well documented in effluents (and for chronic toxicity) 

than those associated with ammonia toxicity. The toxicity may change for both pH increases and decreases 

from neutral pH (pH 7). Such behavior is characteristic 

of aluminum and cadmium. Acute toxicity test experiments with C. dubia in clean dilution waters indicate 

lead and copper were more acutely toxic at pH 6.5 than 

at pH 8.0 or 8.5 (in very hard reconstituted water), 

while nickel and zinc were more toxic at pH 8.5 than at 

6.5 (EPA, 1991A). In recent experiments during a 

chronic TIE, we have found that chromium is pH dependent on an acute basis for C. dubia, but not water 

hardness dependent. The pH dependence was not 

observed in acute tests unless food (YCT) (EPA, 1992C) 

was added during the 48 h acute test at test initiation. 

Therefore, caution must be exercised in interpreting the 

chronic toxicity results with effluents, because the 

toxicant may behave in certain ways that are not 

documented in the literature. 

By conducting tests at different pHs, the effluent 

toxicity may be enhanced, reduced or eliminated. For 

example (at 25°C) where ammonia is the primary toxicant, when the pH is 6.5, 0.180% of the total ammonia 

in solution is present in the toxic form (NH,). At pH 7.5, 

1.77% of the total ammonia is present as NH, and at 

pH 8.5, 15.2% is present as NH,. This difference in the 

percentages of un-ionized ammonia is enough to make 

the same amount of total ammonia about three times 

more toxic at pH 8.5 as at pH 6.5. Whether or not 

toxicity will be eliminated at pH 6.5 and the extent to 

which toxicity will increase at pH 8.5 will depend on the 

total ammonia concentration. If the graduated pH test 

is done at two pHs using the same dilutions, one 

6-17 
should see toxicity differences between pH 6.5 and 8.5. 

The effluent effect level (expressed as percent effluent) 

should be lower at pH 8.5 than pH 6.5 if ammonia is 

the dominant toxicant. 

The most desirable pH values to choose to test for 

the graduared pH test will depend upon the characteristics of the effluent being tested. The graduation scheme 

that includes the air equilibrium (the pH the effluent 

naturally drifts to) will allow a comparison of treatments 

to unaltered effluent (i.e., baseline test). For example, 

if the air equilibrium pH of the effluent is pH 8.0, it may 

be more appropriate to use pHs 6.5, 7.3 and 8.0. The 

pHs of many municipal effluents rise to 8.2 to 8.5 (or 

higher), so pHs such as 6.5, 7.5 and 8.5 may be more 

appropriate. In any case, it will be necessary to conduct the test at more than one effluent concentration 

(e.g., 1 OO%, 50%, 25%) to determine what role, if any, 

the pH dependent compounds play in toxicity. 

The challenge of the graduated pH test is to maintain a constant pH in the test solution. This is a 

necessity if the ratio of ionized to the un-ionized form of 

a pH sensitive toxicant is to remain constant and the 

test results are to be valid. However, in conducting 

either acute or chronic toxicity tests on effluents. it is 

not unusual to see the pH of the test solutions change 

1 to 2 pH units over a 24-h period. 

Methods: To lower the pH of the samples, either 

CO air mixtures or HCI additions (or the combination of 

bot r( ) are used. The pH should be maintained throughout the 4-d or 7-d test with little variation (+0.2 pH 

units). 

When CO /air (without any acid addition) is used to 

control the p& the pH of the effluent samples is adjusted by varying the COJair content of the gas phase 

over the water or effluent samples. By using closed 

headspace test chambers, the CO, content of the gas 

phase can be controlled. The amount of CO,/air needed 

to adjust the pH of the solution is dependent upon 

sample volume, the test container volume, the desired 

pH, the temperature, and the effluent characteristics 

(e.g., dissolved solids). The exact amount of COiair to 

inject for a desired pH must be determined through 

experimentation (on day 1) with each effluent sample 

before the graduated pH test begins. Therefore, the 

test may have to be set up later than the other Phase I 

tests (e.g., day 3) unless experimentation was initiated 

on day 1. The amount of CO, added to the chamber 

assumes that the liquid volume to gas volume ratio 

remains the same. Generally, as the alkalinity increases, the concentration of CO, that is needed to 

maintain the pH also increases. For adjusting pH’.s 

downward from pH 8.5 to 6, 0.5-5% CO, has been 

used. If more than 5% CO is needed, adjust the 

solutions with acids (HCI) and then flush the headspace 

with no more than 5% COdair. With appropriate volumes of effluent, experiments with variable amounts of 

COdair and equilibrated for about 2 h, are used to 

select the needed CO concentration. More than 5% 

CO, is not recommen&d as CO, toxicity is likely to be 

observed. When dilutions of an effluent have the same 

hardness (or alkalinity) and initial pH as the effluent, 

the same amount of CO is usually needed for each 

dilution, but sometimes &fferent amounts are needed 

in the higher effluent concentrations. Use of a dilution 

water of similar hardness (or alkalinity) as the effluent 

makes the CO, volume adjustments easier. When 

tests are conducted in these CO, controlled environments, dilution water controls for each pH should be 

included. 

Acid is used first to adjust pH’s when the amount of 

COdair needed to adjust to the desired pH is greater 

than 5% COdair. Again experimentation is needed to 

determine how much COdair is needed. Techniques 

for acid adjustment are described in Section 6.10 below 

and also in the acute Phase I manual (EPA, 1991A). 

For adding a mixture of COdair to the headspace 

of the test compartments, a 1 L gas syringe (Hamilton 

Model S-1000, Reno, NV) is used. In most instances, 

the amount of CO, produced by the invertebrates has 

not caused further pH shifts, but with larval fathead 

minnows, the pH may drop from the additional amount 

of CO, respired by the fish bacterial metabolic CO, 

released. 

For the pH controlled tests, the pH should be measured at least two to three times for each 24 h period 

when readings of survival and/or young production are 

made. If samples are not renewed daily (as may be 

the case for the C. dubia tests), then the headspace 

should be re-flushed with COdair after the animals are 

fed. Again, some experimentation may be needed to 

determine the amount of COJair needed for this step. 

In all graduated pH rests, the pH should be measured 

in all the chambers. If the pH drifts as much as 0.2 pH 

units, the results may not be usable and better pH 

control must be achieved. However, if pH fluctuates 

more than 0.2 pH units and toxicity is gone at one pH 

and not another, the toxicity results may be useful (see 

lnrerprerarion of ResuWSubsequenr Tests below). 

Measurements of pH must be made rapidly to minimize the CO, exchange between the sample and the 

atmosphere. Avoid vigorous stirring of unsealed 

samples because at lower pH values, the COz loss 

during the measurement can cause a substantial pH 

rise. In addition, measure the DO because toxicants 

such as ammonia have different toxicities when DO is 

decreased (EPA, 1985D). Keep in mind that if the test 

animals have been dead for awhile, the pH and/or DO 

of the test water most likely will have changed. Therefore, pH measurements should be made as soon as 

possible if animals die rapidly. 

Methods that use continuous flow of a COdair 

mixture, such as tissue cell incubators, may be preferable and give better pH control. A pH feedback system 

can be used to control the CO,-mix to the incubators. 

At this time we have not attempted to use a continuous 

flow of CO, and cannot recommend a system to use. 

Maintaining pH above the air equilibrium pH (generally above pH 8.3) is difficult to achieve because the 

6-16 
concentration of CO, must be very low, and microbial 

respiration can increase the CO, levels in the test 

chamber. Frequently we use a dilution water that has a 

higher pH (i.e., very hard reconstituted water) to prevent pH drift downward. 

Interpretation of Results/Subsequent Tests: For 

the graduated pH rest, the pHs selected must be within 

the physiological tolerance range for the test species 

used (which generally is a pH range of 6 to 9). In this 

pH range, the amount of acid or base added is negligible, and therefore the likelihood of toxicity due to 

increased salinity levels is low. 

When ammonia is the dominant toxicant, the toxicity at pH 6.5, should be less than in the pH 8 test. 

However, ammonia is not the only possible cause of 

toxicity. Using the pH of the baseline rest, the relative 

toxicity of each pH adjusted solution can be predicted if 

ammonia is the sole cause of toxicity (EPA, 1989A; 

EPA, 1992A). 

However, if ammonia is only one of several toxicants in an effluent, this procedure will be hard to 

interpret. For this reason, if total ammonia concentrations in the 100% effluent are greater than 20 mg/l, 

include a pH 6 (rather than 6.5) and pH 7.3 (rfr0.2) 

effluent treatment interfaced with other Phase I tests. 

Complicating effects of metal toxicity may be reduced 

by adding EDTA to the test solutions. However, the 

ability of EDTA to detoxify metals may also change with 

pH, although we have not experienced this effect yet. 

Other metals may exhibit some degree of pH dependence, but these are not as well defined. Whether 

the metal toxicity can be discerned will depend in large 

part on the concentration of other toxicants in the 

sample. In order to detect metal toxicity, one must be 

cautious when selecting a dilution water if the test 

solutions are low effluent concentrations. Artifactual 

toxicity due to metals may be created if the hardness of 

the dilution water is much different from that of the 

effluent (see Section 3). This effect may be magnified 

for metals when coupled with the pH change. A dilution water similar in hardness to that of the effluent 

must be used for this test to reveal metal-caused toxicity. If more than one pH dependent toxicant is present, 

the pH effects may either cancel or enhance one another. 

In the acute TIES, we have suggested the use of 

hydrogen ion buffers to maintain the pH of effluent test 

solutions and to compare these test results to those 

from CO, adjusted samples. Three hydrogen ion buffers were used by Neilson et al. (1990) to control pH in 

toxicity tests in concentrations ranging from 2.5 to 4.0 

mM. These buffers were chosen based on the work 

done by Ferguson et al. (1980). These buffers are: 2- 

(N-morpholino) ethane-sulfonic acid (Mes) (pK, = 6.15), 

3-(N-morpholino) propane-sulfonic acid (Mops) 

(pK,=7.15), and piperazine-N,N’-bis (2-hydroxypropane) 

sulfonic acid (Popso) (pK,=7.8). We have replaced the 

Popso buffer with another buffer which is more readily 

soluble in order to achieve better pH control around the 

pH 8.0 range. This buffer is N-tris(hydroxymethyl) 

methyl-3-amino propanesulfonic acid (Taps) (pK1 = 8.4) 

and has been used primarily for the chronic C. dubia 

tests at this time. 

The acute toxicity of these Mes, Mops, and Popso 

buffers is low to both C. dubia and fathead minnows 

(Phase I; EPA, 1991A) (48-h and 96-h LC5Os for all 

buffers are 525 mM for both species). Sublethal levels 

of the buffer are added to hold the pH of test solutions 

for the acute Phase I tests (see EPA, 1991A). Chronic 

toxicity results using these three buffers indicated that 

16 mM did not cause reduced survival or growth for the 

fathead minnow 7-d test. For C. dubia, 4mM of all four 

buffers has not caused reduced survival or reproduction in either the 4-d or 7-d tests. Use of the buffers is 

preliminary and the effects due to interferences from 

the buffers themselves have not been studied. It is 

possible that the buffers may reduce the toxicity of 

some toxicants. 

The buffers must be weighed and then added to 

aliquots of the effluent dilutions and control water as 

batches. Then adjust to desired pH with acid and base 

to the selected values and add the test organisms. 

Solutions should be left for several hours to equilibrate, 

especially for the Popso buffer which has low solubility 

in water (in contrast to other buffers). While our experience with the buffers is limited, we have found the 

amount of any buffer needed to hold a pH is effluent 

specific. Once the pH is adjusted to the desired pH, 

the test solutions need not be covered tightly to maintain pH; however pH should be measured at each 

survival reading at all dilutions. The test results with 

the buffers should mimic those of the earlier graduated 

pH rest if ammonia is the suspect toxicant. 

The methods described in Phase II can be used to 

add identify ammonia as the pH sensitive toxicant. Use 

of the air-stripping method to remove ammonia from 

the sample at high pH’s should help evaluate whether 

toxicant other than ammonia are present (Phase II, 

1992A). The results of this air-stripping test should be 

compared with the aeration rest results of Phase I, the 

baseline effluent rest and the graduated pH rest. If the 

ammonia concentration is decreased and the toxicity is 

reduced or absent, more evidence that ammonia is 

playing a role in the toxicity of the effluent has been 

generated. Other compounds could precipitate with the 

pH adjustment and concentration during air-stripping 

and when water is added back into the solution, they 

may not be available. 

Special Considerations/Cautions: The controls 

in the CO, controlled chambers for each pH and the 

baseline rest act as checks on the general health of the 

test organisms, the dilution water and most test conditions. If the effluent pH in the baseline rest is close to 

that of the pH adjusted test solutions, the toxicity expressed in the two tests should be similar. Significantly 

greater toxicity may suggest interference from other 

factors such as the ionic strength related toxicity (if the 

6-19 
pH was adjusted with HCI) or CO toxicity. Dilution 

water tested at the various pH’s does not serve as 

blanks, as the effluent matrix may differ from that of the 

dilution water. However, if acids and bases are added 

(with or without CO, additions) then roxiciry blanks with 

the same amounts of acid/base added need to be 

tested to determine the cleanliness and effects of the 

acids and bases. Other compounds with toxicities that 

increase directly with pH may lead to confounding results or may give results similar to ammonia. Monitoring the conductivity of the effluent solutions after the 

addition of the acids and bases may also be helpful in 

determining amfactual toxicity. 

6.9 Tier 2 Characterization Tests 

Two tiers are used in the chronic TIE approach 

primarily because in our experience, radical pH adjustment often is not needed. Only when the manipulations in Tier 1 do not indicate clear patterns is Tier 2 

conducted. Tier 1 manipulations do not involve the use 

of drastic pH manipulations to characterize the toxicity 

of the sample. The pH adjustments are used to affect 

toxicity when the Tier 1 tests are not adequate or to 

assist in providing more information on the nature of 

the toxicants (Figure 6-3). 

Changes in pH can affect the solubility, polarity, 

volatility, stability, and speciation of a compound. These 

can change the bioavailability of the compounds, and 

also their toxicity. The Phase I acute manual (EPA, 

1991A; EPA, 1988A) discusses the effect of pH on 

groups of compounds at length, therefore only an abbreviated discussion of pH effects will be covered in 

this document. 

Unionized forms of chemicals are generally less 

polar than the ionized form, and the ionized forms 

interact with water molecules to a greater extent. Compounds may be more toxic in the unionized form, as 

was discussed above in Section 6.8 graduated pH rest 

Unionized forms may be easily stripped from water 

using aeration, or extracted with SPE techniques and 

subsequent elution with non-polar solvents. Also, 

changes in solubility with pH change may cause compounds to be removed by filtration. The form of metals 

can be altered by pH and organic compounds can be 

degraded at extreme pH values. 

Even if the chemical species are unchanged, 

changes in the pH of the solution may affect the toxicity 

of a given compound. The cell membrane permeability 

and the chemistry of the toxicant may be affected. 

Figure 6-3. Tier 2 sample preparation and testrng overview. 

l-let2 

Test 

pH Adjustments of the 

Effluent Sample 

Toxicity 

Test ’ 

readjust 

pH3 

Toxicity 

PH ’ Test 

A 

Toxicity 

Test ( readjust 

Filtrate PH 

I readjust 1 

Toxicity 

Test rezA;“-’ 

Post-Column 

Sample(s) II 

3c)ll” 

nL-__ Toxicity 

Test 

Post-Column 

Sample(s) 

6-20 
Changing pH and returning it to pH i after a short time 

(-1 h) will not always change the toxicity. However, 

this adjustment may result in a reduction, loss or increase in the toxicity. Sometimes only the pH adjustment in combination with a manipulation (e.g., filtering, 

solid phase extraction) changes toxicity when the same 

pH unadjusted manipulation test did not. 

6.10 pH Adjustment Test 

General Approach: For this Tier 2 test, the effluent is adjusted to either pH 3 or pH 10, and left at those 

pHs until other manipulations (aeration, filtration, and 

C18 SPE post-column effluent samples) are ready to be 

readjusted to pH i. The pH adjustment alone may not 

change toxicity, if equilibrium is slow. Satisfactory 

blanks in chronic tests with various reconstituted waters adjusted to pH 11 have not been consistently 

produced, but acceptable blanks have been obtained at 

pH 10 (and pH 3) while pH 11 adjustments have not 

been problematic in some effluent matrices. Since 

pH 11 was subjectively chosen, we recommend adjustment to pH 10 for chronic TIE’s The pH adjustment 

test serves as a toxicity control for the pH adjustments 

combined with aeration, filtration and the C18 SPE column manipulation. As described in Tier 1 and the 

acute Phase I manual, pH may drift very differently 

during the toxicity tests following these more severe pH 

manipulations. Therefore, monitoring and control of 

test pH is necessary. 

Methods: An aliquot of effluent is pH adjusted to 

pH 3 and another aliquot is adjusted to pH 10, along 

with dilution water samples which will serve as blanks. 

Enough sample and dilution water are pH adjusted to 

provide the necessary volumes for the aeration and pH 

adjustment test, the filtration and pH adjustment test, 

and the post C18 SPE column and pH adjustment test. 

Minimal dilution of the effluent should occur, and the 

use of 0.01 N, 0.1 N, and/or 1.0 N solutions of acids/ 

bases (Suprapur®, E. Merck, Darmstadt, Germany) to 

adjust pH are suggested. The volumes and strengths 

of the acid/base additions should be recorded as this 

information may be useful in determining if artifactual 

toxicity should be expected. This information can be 

helpful when subsequent testing is conducted and knowledge of the volumes of acid/base added to the previous 

samples assists in making the pH adjustments more 

rapidly. 

Interpretation of Results/Subsequent Tests: A 

decrease in toxicity compared to the baseline test should 

be pursued to detect the mechanism of toxicity reduction. Often precipitation occurs after drastic pH change. 

If precipitation does occur, then the filtration and pH 

adjustment test will likely remove the toxicant and efforts should be focused on recovery and identification 

from the filter. Similarly, if the C18 SPE column or 

aeration changes toxicity, these manipulations should 

be pursued. If toxicity is only reduced by pH change, 

(which is not common) not much can be made of the 

information, and clustering of several manipulations as 

well as adding additional techniques such as ion exchange should be explored. Dilution from the acid and 

base additions should also be checked. Degradation of 

toxicity is a possibility also, but is nearly impossible to 

detect at this stage. 

The adjustment of pH (to pH 3 or pH 10 and back 

to pH i) may cause toxicity problems. Just the addition 

of the NaOH or HCI may be the cause of the toxicity 

and may also occur in the dilution water blanks or only 

in the effluent sample. The effect on effluent toxicity of 

the Na+ and Cl- additions, depends on the TDS concentration of the effluent. The acid/base additions are 

typically more toxic in dilution water than in effluent, 

unless the effluent TDS concentration is high, and the 

additional concentrations of acid/base result in toxic 

TDS concentrations. These effects are of more concern in chronic TIE’s. The effect of NaCl additions on 

TDS can be tracked by measuring conductivity. Appreciable increases in conductivity should be a warning to 

evaluate TDS toxicity caused by acid and base addition. 

Increases in toxicity compared to the baseline test 

may be a result of either an increase in TDS or toxicant 

changes. TDS as a toxicant may be eliminated by 

calculating the TDS at the ICp value. Effluents that 

have high toxicity require high dilution to determine the 

ICp, and at such great dilution the TDS is subsequently 

diluted sufficiently to remove TDS as a candidate. If 

this is not the case, NaCl can be added to an aliquot of 

effluent to see if the acid/base additions could have 

caused the increased toxicity. Table 6-10 provides 

chronic toxicity information for NaCl in various hardness waters for C. dubia and fathead minnows. 

Precipitates can remove toxicity through sorption of 

such chemicals as non-polar organics. In this case the 

precipitate is only the mechanism of removal, not the 

toxicant itself. The C18 SPE column is likely to remove 

the toxicity in such cases; however, in Tier 2 a pH 

change can also desorb toxicants from particles and 

make them bioavailable and therefore toxic. 

Different pH drift during the baseline toxicity test 

and those after manipulations has been discussed (EPA, 

1991A). For a valid test, the pH during the test must be 

known and maintained the same as in the pH i test. If 

the drift of the pH varies considerably, confusion in 

interpreting the results can arise if a compound whose 

toxicity is pH dependent is present in the sample. If 

good pH control is not maintained incorrect conclusions 

are likely to be made and mislead the TIE process. 

Special Considerations/Cautions: The addition 

of acids and bases to the effluent does not give comparable results of acids and bases added to the dilution 

water. The amount of acid and base added to each 

sample will more than likely be dissimilar. However, 

dilution water toxicity blanks to assess the additions of 

the acid and base are needed to determine whether 

toxic concentrations of ions have been reached and to 

determine the cleanliness of the acid and base solutions that are used in this manipulation and subsequent 

pH manipulation tests. The controls from the baseline 

test provide information on the health of the test organ6-21 
Table 6-10. Chronic toxicity of sodium chloride (g/t) to C. dubia and P. promelas in various hardness waters using the 7-d tests. 

Water IC50 IC25 

Species Type 95% C.I. 95% Cl. NOEC LOEC 

C. dubia SRW 1.3 0.93 0.63 1.3 

1.2-1.5 0.76-0.96 

MHRW 1.6 1.3 1.0 2.0 

1.4-1.7 0.24-1.3 

HRW 1.5 1.2 1.0 2.0 

1.3-1.6 1.0-1.3 

VHRW 1.4 1.0 1.0 2.0 

1.1-1.6 0.58-1.2 

SRW 0.64 0.67 0.50 1.0 

0.76-1.1 0.63-0.77 

SRW 1.3 0.93 0.63 1.3 

1.2-1.5 0.76-0.96 

MHRW 1.5 1.2 1.0 2.0 

1.4-1.6 1.1-1.2 

HRW 3.2 2.3 2.0 4.0 

2.9-3.3 2.0-2.5 

VHRW 4.5 3.2 2.0 4.0 

3.9-4.9 2.4-3.5 

P. promelas 

Note: C.I. = confidence interval; SRW = soft reconstituted water; MHRW = moderately hard reconstituted water, HRW = hard reconstituted 

water, VHRW - very hard reconstituted water, laboratory test conditions. The pH adjustment test serves as the toxicity control (or 

perhaps the "worst case” toxicity control) for the subsequent pH adjustment/characterization tests. 

isms, dilution water, and laboratory test conditions. The 

pH adjustment test serves as the toxicity control (or 

perhaps the “worst case” toxicity control) for the subsequent pH adjustment/characterization tests. 

6.11 Aeration and pH Adjustment Test 

General Approach: Aeration at pH 3 or pH 10 

may make toxicants oxidizable, spargeable or sublatable, 

that are not so at pH i. If this does occur, avenues are 

then available to characterize and identify, similar to 

the procedures described for aeration at pH i in Tier 1. 

For this test, two effluent aliquots which were adjusted 

to pH 3 and pH 10 in the pH adjustment test are each 

aerated for a period of time, for example, 1 h. The 

aeration process can concentrate compounds due to 

loss of volume, and caution should be exercised in this 

aeration process and lost water may need to be replaced with dilution water. 

Methods: The steps for this procedure should be 

identical to those used in the non-pH adjusted sample 

aeration (Section 6.4). The pH of the effluent may drift 

during the aeration, and it should be checked at 30 min 

intervals and readjusted to the original pH (pH 3 or 10) 

if it has drifted more than 1 pH unit. The amount of 

NaCl added from the acid/base additions may be different in aerated samples than for pH adjustment test and 

proper compensation for this difference must be made 

as described above. The volume of effluent aerated 

should be compared to the amount of original sample 

volume prepared. 

After aeration is completed, adjustments back to 

pH i should be made on all samples at the same time. 

The formation of any precipitates should be noted, but 

the importance of precipitates (if any) will not be known 

at this point in the characterization. 

Interpretation of Results/Subsequent Tests: lf 

aeration with either pH adjustment removes or reduces 

the toxicity, additional tests must be performed to identify whether sparging, sublation, or oxidation removed 

the toxicity, as described in Tier 1 (Section 6.4). If 

toxicity is reduced because of precipitation, the results 

for this test and the filtration and pH adjustment test 

should be similar, but if oxidation is a problem, pH 

adjustment and filtration will not affect the toxicity of the 

effluent. At pH 10 the total ammonia levels can be 

reduced by aeration. However, the geometry of the 

aeration technique (i.e., small surface area) for this pH 

adjustment and aeration test described here is not 

particularly conducive to ammonia removal. However, 

if aeration at pH (10) reduces toxicity compared to the 

toxicity in the aeration test at pH i and the baseline test, 

measure the total ammonia level in the sample to 

determine if it was stripped from the effluent. 

Special Considerations/Cautions: The results of 

this test should be compared to the toxicity control ( pH 

6-22 
adjustment test) and the baseline test. The aeration 

and pH adjustment blank should be compared to the 

pH adjustment blank. If the effluent toxicity is reduced 

in the effluent following pH adjustment/aeration, and 

the blank is toxic, the blank can be ignored and the 

results indicate toxicity removal. However, if toxicity is 

the same or greater, artifactual toxicity cannot be ruled 

out and further tests must be conducted. Compare the 

results of the aeration and pH adjustment blank to the 

filtration and pH adjustment blank and the pH adjustment blank (Sections 6.10 and 6.12). If all have toxicity, then artifactual toxicity occurred from the pH adjustment, while if only the aeration and pH adjustment 

blank has toxicity, then the artifactual toxicity crept in 

during the aeration manipulation and the test should be 

repeated. 

6.12 Filtration and pH Adjustment Test 

General Approach: Since a pH change can 

cause toxicants to precipitate or cause solubilized toxicants to sorb on particles, filtration at altered pH values 

can be used as a tool in characterizing the effluent. 

Therefore, by filtering pH adjusted effluent, compounds 

that were in solution without a pH adjustment may no 

longer be in solution or any toxicants associated with 

particles may be removed by the filtration process. 

Differences in the toxicity caused by filtering (at pH 3 

compared to the pH adjustment rest (Section 6.10) may 

imply toxicity associated with suspended solids. If pH 

affects the filterability of the toxicants, solubility changes 

are implied at those pH values. Once the toxicants are 

filtered, the particles may be recoverable from the filter 

if toxicity has not degraded. 

Methods: Details of preparing filters are generally 

the same as described in Tier 1 (Section 6.5), except 

the high purity water used to rinse the filters must be 

pH adjusted to the appropriate pH, as should the dilution water for the blank. 

Effluent samples adjusted to pH 3 or pH 10 (Section 6.10) are filtered, readjusted to pH i, and the filtrate 

toxicity tested. Stainless steel filter housings are not to 

be used for this step, because stainless steel will frequently bleed metals when a pH 3 solution being 

filtered is in contact with the stainless steel. An inert 

plastic or properly cleaned glass housing should be 

used. 

Interpretation of Results/Subsequent Tests: The 

results of the filtration and pH adjustment rest are 

compared to the toxiciry contro/+the baseline rest and 

the pH adjustment test. If the effluent is more toxic 

after filtration and contamination is not the cause, the 

breaking of an emulsion might be involved. If the 

toxicity is removed or reduced by the filtration step and 

dilution is not the cause, then toxicants have been 

separated from the whole effluent and efforts should 

focus on identifying the compounds filtered out. The 

next step is to recover the toxicity as described in 

Tier 1 filtration rest. This may be accomplished using a 

pH adjusted sample of water, perhaps using the pH 

opposite of that used in the filtration process. 

Special Considerations/Cautions: The pH adjusted and filtered dilution water serves as a blankand 

the pH adjusted and filtered effluent sample serves as 

a toxiciry control for the solid phase extraction step 

(Section 6.13). The results of the fih-ation and pH 

adjustment test should be compared to the effluent pH 

adjustment test and the baseline rest. The fih-ation 

blank should be compared to the baseline control, the 

aeration blank, and pH adjustment blank. Toxicity in 

the filtration blank implies toxic artifacts from the filtration process, the glassware, the pH adjustment or a 

dilution water problem. If the baseline control performance is acceptable, the blanktoxicity was most likely 

created during the pH adjustment or filtration. If the 

aeration and pH adjustment blank is non-toxic, and if 

the fillration blank is toxic, and the filtered effluent 

sample is still toxic or more toxic, artifactual toxicity 

cannot be ruled out. To check if it occurred during the 

manipulation, the experiment must be repeated. If the 

filtration blank is toxic, yet the filtered pH adjusted 

effluent indicates that toxicity is reduced/eliminated, the 

toxicity in the blankcan be ignored. 

6.13 Post C,, Solid Phase Extraction (SPE) 

Column and pH Adjustment Test (pH 3 

and pH 9) 

General Approach: Shifting the ionization equilibria at high and low pHs may cause the C,, SPE column 

to extract different compounds than at pH i. pH adjusted and filtered effluent is passed over a prepared 

C,, SPE column to remove non-polar organic compounds (cf., post C SPE column test, Section 6.6 

above). Organic acl *a s and bases may be made less 

polar by shifting their equilibrium to the un-ionized species. By adjusting the effluent samples to a low pH and 

a high pH, some compounds that are in the un-ionized 

form should sorb onto the column. However, the C 

packing degrades at high pH, so pH 9 (rather than p# 

10 or pH 11) is used in this manipulation. Specific 

manufacturer’s data should be checked for acceptable 

pH range. We have had no experience in eluting 

toxicants off the C,, SPE column that would be sorbed 

only at an altered pH, and therefore we can only provide general rules to follow in these cases except those 

inferred from how ionizable compounds behave in regard to pH change. 

Methods: All of the procedures for this manipulation and the use of the C,, SPE column are the same 

as is described in Tier 1 for the SPE extraction at pH i 

(Section 6.6) with one exception. All water passed 

through the column (rinse, blank and effluent) should 

be acidified or rendered basic depending on which pH 

is under investigation (see Section 6.12). The potential 

for bacterial growth and artifactual toxicity in the postcolumn samples remain the same as for pH i 

6-23 
Interpretation of Results/Subsequent Tests: The 

extraction efficiency of the column is assessed by comparing the results of the post C,, SPE column and pH 

adjustment test (pH 3 and pH 10) to the filtration and 

pH adjustment test, and the pH adjustment test. Again 

postcolumn test results are the most interpretable when 

there is no artifactual toxicity and toxicity was removed. 

When the toxicity is removed, compare the results 

of the test with the methanol eluare rest below (Section 

6.14). If toxicity is removed that was not removed 

under pH i and recovered in the methanol eluate, efforts to identify the toxicants should be started. If 

methanol does not recover toxicity, a pH adjusted water should be tried. For further discussions of the 

interpretation of the results, see Section 6.6 above. 

Special Considerations/Cautions: Careful observations and judgement must be exercised in detecting problems in the post C,, SPE column and pH 

adjustment rest Low DO levels can occur in these 

samples (cf., Section 6.6). Through testing experience, 

the investigator will know whether toxicity appears as 

artifactual (i.e., growth, low DO) or as lack of toxicity 

removal. If artifactual toxicity is not recognized, then 

an erroneous conclusion that the C,, SPE column did 

not remove toxicity can be made. 

General test conditions (dilution water, health of 

test animals) will be tracked by the controls in the 

baseline rest. The post-column dilution water blanks 

should be compared to those controls to determine if 

the column imparted toxicity. If the post-column dilution water blank was toxic, but no toxicity or artifactual 

toxicity occurred in the post-column effluent sample the 

toxic blankcan be ignored. 

Results of the post-column effluent rest(s) must be 

compared to the results of the filtration andpH adjustment rest to determine if the manipulations effectively 

reduced toxicity. When the post C,, SPE column rest 

data is plagued by artifactual toxicity, the importance of 

the methanol eluate rest increases. 

6.14 Methanol Nuate Test for pH Adjusted 

Samples 

General Approach: This test is essentially the 

same as the methanol eluate rest in Section 6.7, except 

that the columns were prepared with pH adjusted waters/effluents (see Section 6.13). 

Methods: These are identical to those in Section 

6.7, except the pH of the rinse water, blank and effluent 

sample has to be adjusted to pH 3 or pH 9 (lowered 

from pti 10). 

Interpretation of Results/Subsequent Tests: If 

the toxicity is recovered in the eluate, identification 

should be initiated. Refer to Sections 6.6, 6.7, and 

6.13 for more information. 

Special Considerations/Cautions: The baseline 

rest serves as the foxiciry control, and the methanol 

blank (for pH adjusted samples) serves as the toxicity 

control for the effects of methanol in water. The health 

of the test animals, the viability of the dilution water and 

general test conditions are evaluated by the controls. 

The artifactual growth observed in the post C,, SPE 

column rest (with and without pH adjustments) from the 

methanol has not occurred in merhanol eluare rests. 

This is most likely due to the differences in how the 

methanol degrades/behaves in dilution waters which 

are low in bacteria and other organic matter in contrast 

to effluent samples (even post-column effluents). 

6.15 Toxicity Characterization Summary 

Phase I will not usually provide information on the 

specific toxicants. If effluent toxicity is consistently 

reduced, for example, through the use of the C,, SPE 

column, this does not prove the existence of a single 

toxicant because several non-polar organic compounds 

may be causing the toxicity in the effluent over time, 

but use of the C,, SPE technique in Phase I detects the 

presence of these compounds as a group. This lack of 

specificity is very important to understand for subsequent Phase II toxicant identification. Efforts should 

concentrate on those manipulations affecting toxicity in 

which the toxicant is isolated from other effluent constituents, such as the SPE column, filtration and aeration. 

After the Tier 1 group of Phase I tests has been 

completed, the results will usually show that some 

manipulations increased toxicity, some decreased it, 

and others effected no change. In some instances, 

Tier 1 results allow the researcher to proceed immediately into the Phase II identification, and sometimes 

Phase I (Tier 1 and/or Tier 2) and Phase II combinations are needed to determine the cause of toxicity (cf., 

EPA, 1992A). Of course, new approaches are frequently devised as more Phase I TIES are completed. 

Toxicity may be changed by two or more tests, and 

if so, then more conclusive inferences might be possible than when only one manipulation changed the 

toxicity. 

If all of the toxicity is not removed, it is possible that 

other toxicants could be present in the effluent so that 

only partial removal was obtained. Frequently more 

than one manipulation affects toxicity but only infrequently is there no effect from any manipulation. Even 

if toxicity is affected by only one manipulation, one still 

does not know whether or not there are multiple toxicants. When several manipulations affect toxicity, it 

still does not ensure that there are multiple toxicants. 

There is also no way to tell at this stage if there are 

multiple toxicants, whether or not they are additive, 

partially additive or independent. In our experience 

with acutely toxic effluents, we have not found synergism, but independent action has commonly been found. 

Some toxicants identified in effluents have been additive, but more often these have been only partially 

additive. 

6-24 
The two objectives which usually move the TIE 

along more rapidly are to separate and concentrate the 

toxicant( Therefore, the first step in Phase II (EPA, 

1989A) will often be to reduce the number of constituents accompanying the toxicants. These efforts may 

reveal more toxicants than are suggested by Phase I 

testing. In Phase II one may discover that toxicants of 

quite a different nature are also present but were not in 

evidence in Phase I and if this is the case, different 

Phase I characterizations may then be needed. Once 

the analytical methods to identify one or more of the 

toxicants is found, efforts to confirm the cause should 

be initiated immediately (EPA, 19898; EPA, 19928). 

As discussed earlier, the amount of time necessary 

to adequately characterize the physical/chemical nature and variability of the toxicity will be discharge 

specific. For a given discharge, the factors that will 

affect the length of time it takes to move through Phase 

I is the appropriateness of Phase I tests to the toxicants, the existence of long- or short-term periodicity in 

individual toxicants and the variability in the magnitude 

of toxicity. An effluent which consistently contains toxic 

levels of a single compound that can be neutralized by 

more than one characterization test should be identified 

and moved into Phase II more quickly than an ephemerally toxic effluent with highly variable constituents, 

few of which or none of which are impacted by any of 

the Phase I tests. Several samples should be subjected to the Phase I characterization tests but not all 

manipulations have to be done on all subsequent 

samples. The decision to do subsequent tests on these 

samples to confirm or further delineate initial results is 

a judgement call and will depend on whether or not the 

results of Phase I are clear-cut. Sometimes it may be 

reasonable to start Phase II and Phase III on the first 

sample. 

If the Phase I characterization tests that remove or 

neutralize effluent toxicity vary by the sample, the number of tested samples must be increased and the frequency of testing should be sufficient to include all 

major variability. The differences seen among samples 

can be used to decide when further differences are not 

being found. Phase I characterization testing should 

continue until there is reasonable certainty that new 

types of toxicants are not appearing. No guidance can 

be given as to how long this may take-each problem 

for every discharger is unique. While the toxicity of 

samples can be very different, the same characterization tests must be successful in removing and/or neutralizing effluent toxicity. 

Often the next step of the TIE is obvious; at other 

times the outcome of Phase I will be confusing and the 

next step will not be obvious. In our experience with 

acutely and chronically toxic effluents, once one toxicant is identified, identification of subsequent toxicants 

becomes easier because: (a) the toxicity contribution 

of the identified toxicant can be established for each 

sample; (b) the number of Phase I manipulations that 

will affect the toxicity of the known toxicant can be 

determined; (c) one can determine whether the identified and the unidentified toxicant are additive; (d) if 

some manipulations affect the toxicity due only to the 

unidentified toxicants, some of their characteristics can 

be inferred; and (e) one can determine if the relative 

toxicity contributions of identified and unidentified toxicants varies by sample. Such information can be used 

to design tests to elucidate additional physicaVchemical 

characteristics of the toxicants that cause chronic toxicity. 

6.16 Use of Multiple Characterization Tests 

Type and amount of testing is dependent on the 

toxicity persistence in the effluent, the nature of the 

toxicity, and reassessment of previous Phase I results 

(observed trends in the characteristics can be very 

important). Several tests could each partially remove 

the effluent toxicity because several compounds are 

causing the toxicity, or that one toxicant can be removed by several Phase I steps. FL+r example, if 

several toxicants are acting to cause the toxicity, then 

the graduated pH test and the post C,, SPE column 

test both might result in a partial toxicity reduction. If 

sodium thiosulfate and EDTA both reduce toxicity, cationic metals might be suspect. 

In the acute Phase I (EPA, 1991A), the use of 

multiple manipulations (combining two of the Phase I 

tests) was advocated and this same concept is also 

useful for the chronic TIE as well. For effluents with 

multiple toxicants, especially if they are not additive, 

multiple manipulations are helpful. Especially when no 

single manipulation removes all the toxicity, multiple 

manipulations should be tried. 

When the C,, SPE column only partially removes 

toxicity, Phase I manipulations with the post-column 

sample should be tried. For this multiple manipulation, 

the post C,, SPE column effluent can be treated as 

whole effluent, and several of the Phase I steps can be 

conducted on the post-column effluent such as the 

EDTA addition test, the thiosulfate addition test, and 

the graduated pH test. However, these combinations 

are useful only with the post-column effluent provided 

that no artifactual toxicity is present. 

If the C,, SPE column partially removes toxicity, 

pass an aliquot of the post-column effluent over an ion 

exchange column to determine the characteristics of 

the remaining toxicity. If a non-polar toxicant and 

ammonia are suspected, then passing the sample over 

the C,, SPE column and then over zeolite may assist in 

accounting for all of the toxicity. Likewise, passing the 

effluent over zeolite and then over the C,, SPE column 

may provide additional insight. To gain this knowledge 

toxicity tests must be performed after each manipulation and not just on the multiple manipulated sample. 

Effluent characterization must be approached without any preconceived notion or bias about the cause of 

6-25 
toxicity because many constituents are present in efflu- times the answer being sought is only whether or not a 

ents and their chemistry is often unknown, resulting in specific substance is causing toxicity. Obviously in 

circumstantial evidence that is frequently misleading. such cases testing is specifically selected to answer 

Certainly all available information and experience should that question and therefore not all manipulations need 

be used to guide the investigative effort but temptations to be performed. 

to reach conclusions too soon must be resisted. Some6-26 
Section 7 

Interpreting Phase I Results 

After Phase I on one sample or several samples is 

completed, the investigator must carefully evaluate the 

data, draw conclusions, and make decisions about the 

next steps that are needed. Sometimes the next step 

is obvious, at other times the outcome will be confusing 

and the next step will not be obvious. Several general 

suggestions, based on our experience to date, may 

provide some help. 

In this section, various examples of Phase I results 

are given with interpretation suggestions. This discussion is repeated from the acute Phase I characterization manual (EPA, 1991A), and not all aspects have 

been evaluated for chronic TIES yet. These examples 

should be used only as guides to thinking and not as 

definitive diagnostic characteristics. Since almost any 

toxicant can be present in effluents, clear-cut logic is 

not totally dependable in interpreting results. Rather, 

one must use the weight of evidence to proceed, and 

be aware that artifacts cannot at this point always be 

identified. 

One should avoid making categorical assumptions 

to every extent possible. For example, to assume that 

the toxicity is due to a non-polar toxicant because 

the toxicity in the post C,, SPE column effluent was 

removed, often is an error. Metals may also be the 

toxicant adsorbed by the SPE column; we have observed zinc, nickel, and aluminum concentrations remaining on the C,, SPE column. However, if the 

toxicity can be recovered in the methanol fraction, then 

the theory that a non-polar toxicant is causing the 

toxicity is better substantiated, because metals do not 

elute with methanol and therefore do not produce toxicity in the methanol fraction toxicity test (cf., Phase II). 

Example 1. Non-polar toxicant( The Phase I 

results implicating non-polar toxicants are: 

l Toxicity in the post C,, SPE column test 

was absent or reduced. 

l Toxicity was recovered in the methanol 

eluate test. 

Toxicants other than non-polar compounds may be 

retained by the SPE column but they are less likely to 

be eluted sharply. Also, artifactual post-column toxicity 

can occur, but non-polar toxicity is typically distinguished 

from the artifactual toxicity when the eluate is checked 

for toxicity. Some toxicants (metals, some sutfactants) 

may not elute from the SPE column with methanol and 

so failure to recover the toxicity in the eluate does not 

exclude the possibility of a non-polar toxicant. Recovery of toxicity in the eluate at pH i is less likety to be an 

artifact than recovery only at pH 3 or pH 9. For those 

instances where methanol does not recover C,,-removable toxicity, other solvents may be needed to elute the 

toxicants (see Phase II; EPA, 1992A). 

Example 2. Cationic Metals. This group of metals 

has varied chemicaVphysical behaviors which result in 

less definitive Phase I results. The following characteristics can be used only in a general way to point to 

metals as the cause of the toxicity: 

l The toxicity is removed or reduced in the 

EDTA addition test. 

l The toxicity is removed or reduced in the 

post C,, SPE column test. 

l The toxicity is removed or reduced in the 

filtration test, especially when pH 

adjustments and filtration are combined. 

l The toxicity is removed or reduced in the 

sodium thiosulfate addition test. 

. Erratic dose response curve observed. 

No single characteristic is definitive, with the possible exception of EDTA. In addition, toxicity may be 

pH sensitive in the range at which the graduated pH 

test is performed but may become more or less toxic at 

low or high pH depending on the particular metals 

involved. This characteristic for chronic toxicity has not 

yet been demonstrated to the extent it was for the 

acute toxicity of several metals (EPA, 1991A). 

Example 3. Total dissolved solids (TDS). TDS 

consists of a group of common cations and anions 

&a 2+, Mg2+, Na*, K+, SO;, NO;! Cl-, CO?-) and in parts 

of the United States, this group IS called ‘salinity.” TDS 

is usually measured by conductivity, density or refraction, none of which measure specific compounds or 

ions. The toxicity of any given amount of TDS will 

depend on the specific composition. TDS behaves as 

a mixture of toxicants, which do not cause toxicity 

through osmotic stress. Evidence of this is that the 

LC5Os of the individual salts expressed in motes, are 

quite different. If osmotic stress were the mode of 

action, the concentration in moles at the LC5Os would 

be similar (EPA, 1991A). One cannot use marine 

organisms to circumvent TDS unless NaCl is by far the 

7-l 
dominant TDS. Marine organisms regulate Na+ and Cl 

but like freshwater organisms, they too are sensitive to 

non-NaCI TDS. 

For these reasons, only very general relationships 

exist between toxicity and TDS. Because of their 

varied nature, they do not sort out clearly in Phase I. 

Rather, unless conductivity is very high (e.g., 10,090 

pmhos/cm), one might suspect TDS when nothing else 

is indicated. For example, if high TDS were present 

and caused by calcium sulfate (CaSO,), toxicity is likely 

to be removed in the pH adjustment test at pH 10 or in 

the filtration and pH adjustment test at pH 10, whereas 

if the TDS were due to NaCI, toxicity would likely not be 

affected. 

As a general guide, when conductivity exceeds 

1,000 and 3,000 umhos/cm at the effect concentration 

for Ceriodaphnia and fathead minnows, respectively, 

TDS toxicity might be suspect. The conductivity of 

100% effluent is not the relevant reading, but rather the 

conductivity at the concentrations bracketing the effluent no effect and effect concentrations. 

Following are some Phase I general indicators that 

TDS might be a suspect: 

l No pH adjustments changed the toxicity, 

unless a visible precipitate occurs upon pH 

adjustment, pH adjustment and filtration, 

and pH adjustment and aeration. 

l No loss of toxicity in the post Clcr SPE 

column test, or a partial loss of toxrctty but 

no change in conductivity measurements. 

l No change in toxicity with the EDTA addition 

test, sodium thiosulfate addition test or in 

the graduated pH test. 

In addition, there are two tests that can be used 

that are not included in Phase I but may help to characterize the toxicity: 

l Use acid/base ion exchange resins (EPA, 

1992A). When toxicity is removed or 

reduced, the toxicity could be due to TDS. 

l Use of activated carbon to remove toxicity 

(EPA, 1992A). When no toxicity is removed 

by passing the effluent over carbon, TDS 

could be responsible for toxicity. 

An additional caution is that where TDS is 

marginally high, the addition of NaCl from pH manipulations can increase TDS enough to produce artifactual 

TDS toxicity. The conductivity of the solutions before 

and after the pH adjustments should be monitored 

closely to avoid this. 

Example 4. Surfactants. There are three main 

groups of surfactants and/or flocculants (anionic, cationic and nonionic) that may occur in effluents. The 

Phase I behavior of these types of compounds may 

vary depending on which particular groups are present. 

The general Phase I results implicating a 

surfactant(s) as the toxicant are: 

Toxicity is reduced or removed in the 

filtration test. 

Toxicity is reduced or removed by the 

aeration test. In some cases, the toxicity is 

recoverable from the walls of the aeration 

vessel after removing the aerated effluent 

sample. 

Toxicity is reduced or removed in the post 

C,, SPE column test. The toxicity may or 

may not be recovered in the methanol eluate 

test. 

Toxicity is reduced or removed in the post 

C,, SPE column test using unfiltered effluent. 

Toxicity reduction/removal is similar to that 

observed in the filiration test and toxicity 

may or may not be recovered in methanol 

eluate test or the extraction of the glass 

fiber filter. 

Toxicity degrades over time as the effluent 

sample is kept in cold storage (4°C). 

Degradation is slower when effluent is 

stored in glass containers rather than plastic 

containers. 

Example 5. Ammonia. Ammonia concentrations 

can be measured easily, and because it is such a 

common effluent constituent, determining the total ammonia concentration in the whole effluent is a good first 

step (see Section 4). If more than 5 mgIL of total 

ammonia is present, additional evaluations should be 

done. Sole dependence on analyses is not advisable 

because the chronic effects of ammonia and some 

other toxicants (e.g., such as surfactants) is not well 

known. Even though the ammonia concentration is 

sufficient to cause toxicity, other chemicals may be 

present to cause toxicity if the ammonia is removed. 

Three indicators of ammonia toxicity are: 

l The concentration of total ammonia is 5 

mg/L or greater. 

. In the graduated pH test the toxicity 

increases as the pH increases. 

. The effluent is more toxic to fathead 

minnows than to Ceriodaphnia. 

Example 6. Oxidants. In effluents, oxidants other 

than chlorine may be present. Measurement of a 

chlorine residual (TRC) is not enough to conclude that 

the toxicity is due to an oxidant. In general, oxidants 

are indicated by the following: 

l The toxicity is reduced or removed in the 

sodium thiosulfate addition test. 

l Toxicity is removed or reduced in the 

aeration test. 

7-2 
l The sample is less toxic over time when Of course, TRC greater than 0.1 mg/L in 100% 

held at 4°C (and the type of container does 

not affect toxicity). 

effluent might indicate chlorine as the oxidant causing 

_ 

the toxicity. In addition, the dechlorination with SO, 

. Ceriodaphnia are more sensitive than 

provides evidence of chlorine toxicity in the same manf 

fathead minnows. ner as the sodium thiosulfate addition test. 

7-3 
Section 8 

References 

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and 18, 1992, Richmond, CA. 

APHA, 1989. Standard Methods for the Examination of 

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EPA. 1978. Methods for Measuring the Acute Toxicity 

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012. Environmental Monitoring and Support 

Laboratory, Cincinnati, OH. 

EPA. 1979. Aqueous Ammonia Equilibrium - Tabulation 

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091. Environmental Research Laboratory, Duluth, 

MN. 

EPA. 1985A. Technical Support Document for Water 

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EPA. 19858. Methods for Measuring the Acute Toxicity 

of Effluents to Freshwater and Marine Organisms. 

Third Edition. EPA/600/4-85/013. Environmental 

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EPA. 1985C. Short-Term Methods for Estimating the 

Chronic Toxicity of Effluents and Receiving Waters 

to Freshwater Organisms. EPAi600/4-851014. 

Environmental Monitoring and Support Laboratory, 

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EPA. 1985D. Ambient Water Quality Criteria for 

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Standards Division, Washington, D.C. 

EPA. 1988A. Methods for Aquatic Toxicity Identification 

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Research Laboratory, Duluth, MN. 

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600/2-88/062. Water Engineering Research 

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Fourth Edition. EPA/600/4-90/027. Environmental 

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Phase I. EPA/600/6-911005. Environmental 

Research Laboratory, Duluth, MN. 

EPA. 1992A. Methods for Aquatic Toxicity ldentifiition 

Evaluations: Phase II Toxicity Identification 

8-1 
Procedures. EPA/600/R-92/080. Environmental 

Research Laboratory, Duluth, MN. 

EPA. 19928. Methods for Aquatic Toxicity Identification 

Evaluations: Phase III Toxicity Confirmation 

Procedures. EPA/600/R-92/081, Environmental 

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Chronic Toxicity of Effluents and Receiving Waters 

to Freshwater Organisms. Third Edition. EPA16001 

4-91/002. Environmental Monitoring and Support 

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to Marine and Estuarine Organisms. Second 

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DeGraeve, G.M., J.D. Cooney, B.H. Marsh, T.L. Pollock 

and N.G. Reichenbach. 1989. Precision of the 

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lnterlaboratory Study. Electric Power Research 

Institute, (EPRI), Palo Alto, CA, Report EN-6469. 

Denny, J.S. 1988. Guidelines for Culture of Fathead 

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Pollutants; National Policy. EPA, Volume 49, No. 

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Neilson, A.J., A.S. Allard, S. Fischer, M. Malmberg, and 

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fathead minnows, (Pimephales promelas): 

Supplemental Report for Video Training Tape. EPA/ 

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Oris, J.T., R.W. Winner, and M.V. Moore. 1991. A 

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lo:21 7-224. 

Smith, R.M. and A.E Martell. 1981. Critical Stability 

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Plenum Press, NY. p. 87. 

Stephan, C.E. and J.W. Rogers. 1985. Advantages of 

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American Society for Testing and Materials, 

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Stumm, W. and J.J. Morgan. 1981. Aquatic Chemistry 

- An Introduction Emphasizing Chemical Equilibria 

in Natural Waters. John Wiley & Sons, Inc., New 

York, NY. 583 p. 

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Chronic and Early Life Stage Toxicity Tests: A 

Critical Review. Aquat. Toxicol. 5:1-21. 

8-2 
Zuiderveen, J.A. and W.J. Birge. 1991. A Comparison 

of Metal Chelators for Use in the TIE/TRE Chronic 

Toxicity Tests with Ceriodaphnia dubia. Poster, 

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Toxicol. and Chem., November 1991. 

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