&EPA

United States Office of

Environmental Protection

Agency

Research and Development

Washington, DC 20460

EPA/600/6-9 l/003

February 1991

Methods for Aquatic

Toxicity Identification

Evaluations

Phase I Toxicity

Characterizatidn Procedures

Second Edition

c
EPA/600/6-9 l/O03

February 1991

Methods for Aquatic

Toxicity Identification Evaluations

Phase 1 Toxicity Characterization Procedures

(Second Edition)

Edited by

T.J. Norberg-King

D.I. Mount

E.J. Durhan

G.T. An kley

L.P. Burkhard

Environmental Research Laboratory

Duluth, MN 55804

JR. Amato

M.T. Lukasewycz

M.K. Schubauer-Berigan

AScl Corporation

6201 Congdon Boulevard

Duluth, MN 55804

L. Anderson-Carnahan

Region IV - Policy Planning & Evaluation Branch

Atlanta, GA 30365

National Effluent Toxicity

Assessment Center

Technical Report 18-90

Environmental Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Duluth, MN 55804

@ Punted on Recycled Paper
Notice

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 document is one in a series of guidance documents intended to aid dischargers and

their consultants in conducting aquatic organism Toxicity Identification Evaluations (TIES)

as part of Toxicity Reduction Evaluations (TREs). Such effluent evaluations may be required

as the result of an enforcement action or as a condition of a National Pollutant Discharge

Elimination System (NPDES) permit. This document will also help to provide U.S. Environmental Protection Agency (EPA) and State Pollution Control Agency staff with the background necessary to oversee and determine the adequacy of effluent TIES proposed and

performed by NPDES permittees. While this TIE approach was developed for effluents, the

methods and techniques have direct applicability to other types of aqueous samples, such

as ambient waters, sediment pore waters, sediment elutriates, and hazardous waste

leachates.

The TIE approach is divided into three phases. Phase I (this document) contains

methods to characterize the physical/chemical nature of the constituents which cause c

toxicity. Such characteristics as solubility, volatility and filterability are determined without

specifically identifying the toxicants. Phase I results are intended as a first step in specifically

identifying the toxicants but the data generated can also be used to develop treatment

methods to removetoxicity without specific identification of the toxicants. Two EPA TRE

manuals (EPA, 1989A; 1989B) use parts of Phase I in developing those approaches.

Phase II (EPA, 1989C) describes methods to specifically identify toxicants if they are

non-polar organics, ammonia, or metals. This Phase is incomplete because methods for

other specific groups, such as polar organics, have not yet been developed. As additional

methods are developed, they will be added.

Phase III (EPA, 1989D) describes methods to confirm the suspected toxicants. It is

applicable whether or not the identification of the toxicants was made using Phases I and

II. Complete Phase III confirmations have been limited to date, but avoiding Phase III may

invite disaster because the suspected toxicant was not the actual toxicant(

Phases I and II are intended for acutely toxic effluents. However, that limitation does not

mean that effluents having chronic limits cannot be evaluated using these methods. TIE

methods to evaluate the cause of chronic toxicity in effluents are being developed (EPA,

1991A).

These methods are not mandatory but are intended to aid those who need to characterize, identify or confirm the cause of toxicity in effluents or other aqueous samples such as

ambient waters, sediments, and leachates. Where we lack experience, we have indicated

this and have suggested avenues to follow. All tests need not be done on every sample; the

tests are, in general, independent. However, experience has taught us that skipping tests

may result in wasted time, especially during the early stages of Phase I. An exception to this

is when one wants to know if only a specific substance, for example ammonia, is causing

the toxicity or if toxicants other than ammonia are involved. Otherwise, we urge the use of

, the whole battery of tests.

We welcome comments from users of these manuals so that future editions can be

improved. Comments can be sent to NETAC, ERL-Duluth, 6201 Congdon Boulevard,

Duluth, MN 55804.

.*.

III
Abstract

In 1988, the first edition “Methods for Aquatic Toxicity Identification Evaluations:

Phase I Toxicity Characterization Procedures” was published (EPA, 1988A). This second

edition provides more details and more insight into the techniques described in the 1988

document. The manual describes procedures for characterizing the physical/chemical

nature of toxicants in acutely toxic effluent samples, with applications to other types of

samples such as receiving water samples, sediment pore water or elutriate samples, and

hazardous wastes. The presence and the potency of the toxicants in the samples are

detected by performing various manipulations on the sample and by using aquatic organisms to track the changes in the toxicity. This toxicity tracking step is the basis of the toxicity

identification evaluation (TIE). The final step is to separate the toxicants from the other

constituents in the sample in order to simplify the analytical process. Many toxicants must

be concentrated for analysis.

The Phase I manipulations include pH changes along with aeration, filtration, sparging,

solid phase extraction, and the addition of chelating (i.e., ethylenediaminetetraacetate

ligand (EDTA)) and reducing (i.e., sodium thiosulfate) agents. The physical/chemical

characteristics of the toxicants are indicated by the results of the toxicity tests conducted on

the manipulated samples.

Since the first document was developed, additional options or new procedures have

been developed. For example, additional options are provided in the EDTA and sodium

thiosulfate addition tests, and in the graduated pH test. Also a discussion has been added

for testing the effluent sample over time (weekly) to measure the rate of decay of toxicity

which is used to detect the presence of degradable substances, particularly chlorine or

surfactants. Guidance for characterizing whether a toxicant removed by aeration is

sublatable is described, and techniques for characterizing filterable toxicity and adiscussion

of C,, solid phase extraction elutable toxicity has been added. Use of multiple manipulations

is dtscussed and example interpretations of the results of the Phase I manipulations are

provided.

Additional manuals describe the methods used to specifically identify the toxicants

(EPA, 19896) and to confirm whether or not the suspect toxicant is the actual toxicant

(EPA, 19890).
Contents

Page

Foreword l . .

.~..~.........~...~...............~....~.....~~............~............................................................... Ill

Abstract . . . . . . . . ..*........*..........................................*................................................*.*.......... iv

Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~..................................................................... V

Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~................................... vii

Tables . . .. . . . . . . . . . . . . . . . . . ..*..............................................*.............................*........................... VIII

Acknowledgments . . . . . . . . . . . . . ..*......................................................*..................................... ix

1.

2.

3.

4.

5.

6.

7.

8.

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

1.1 Background .................................................................................................... l-l

1.2 Conventional Approach to TIES ..................................................................... l-l

1.3 Toxicity Based Approach ............................................................................... l-3 l

Health and Safety .................................................................................................... 2-l

Quality Assurance .................................................................................................... 3-l

3.1 TIE Quality Control Plans ............................................................................... 3-l

3.2 Cost Considerations/Concessions ................................................................. 3-l

3.3 Variability ........................................................................................................ 3-2

3.4 Intra-Laboratory Communication ................................................................... 3-2

3.5 Record Keeping ............................................................................................. 3-2

3.6 Phase I Considerations .................................................................................. 3-2

3.7 Phase II Considerations ................................................................................. 3-3

3.8 Phase III Considerations ................................................................................ _ 3-3

Facilities and Equipment .......................................................................................... 4-l

-

Dilution Water........................................................................................................... 5-l

Effluent Sampling and Handling .............................................................................. 6-l

6.1 Sample Shipment and Collection in Plastic versus Glass .............................. 6-3

Toxicity Tests ........................................................................................................... 7-l

7.1 Principles ........................................................................................................ 7-l

7.2 Test Species .................................................................................................. 7-l

7.3 Toxicity Test Procedures ................................................................................ 7-2

7.4 Test Endpoints ............................................................................................... 7-3

7.5 Feeding .......................................................................................................... 7-5

7.6 Multiple Species ............................................................................................. 7-5

Phase I Toxicity Characterization Tests ................................................................... 8-l

8.1 Initial Effluent Toxicity Test ........................................................................... 8-4

8.2 Baseline Effluent Toxicity Test ...................................................................... 8-5

8.3 pH Adjustment Test ....................................................................................... 8-8

8.4 pH Adjustment/Filtration Test ...................................................................... 8-15

8.5 pH Adjustment/Aeration Test ...................................................................... 8-21

v
Contents (continued)

Page

8.6 pH Adjustment/C,, Solid Phase Extraction Test ......................................... 8-27

8.7 Oxidant Reduction Test ............................................................................... 8-33

8.8 EDTA Chelation Test .................................................................................. 8-38

8.9 Graduated pH Test ..................................................................................... 8-44

9. Time Frame and Additional Tests a ............................................................................9-l

9.1 Time Frame for Phase I Studies ................................................................... 9-1

9.2 When Phase I Tests are Inadequate ............................................................. 9-1

9.3 Interpreting Phase I Results ......................................................................... 9-2

9.4 Interpretation Examples ............................................................................... 9-3 .

10. References . . . . . . . . . . . . . . . . . . . . . ..I..........................................................*.......................... 10-l

vi
Figures

Number Page

l-l.

1-2.

6-1.

7-l.

8-l.

8-2.

8-3.

8-4.

8-5.

8-6.

8-7.

8-8.

8-9.

8-10.

8-11.

8-12.

8-13.

8-14.

8-15.

8-l 6.

8-l 7.

8-l 8.

8-l 9.

Conventional approach to TIES ......................................................................... l-2

Flow chart for toxicity reduction evaluations ...................... ..-............................. l-5

Example data sheet for logging in samples ....................................................... 6-2

Schematic for preparing effluent test concentrations using

simple dilution techniques ........................................................................... 7-4

Overview of Phase I effluent characterization tests ............................................ 8-2

Example data sheet for initial effluent toxicity test ............................................. 8-6

Example data sheet for baseline effluent toxicity test ........................................ 8-7

pE -pH diagrams for the CO,, H,O, and Mn-CO, systems (25°C) .................... .8-9

Flow chart for pH adjustment tests .................................................................. 8-11

Example data sheet for pH adjustment test ..................................................... 8-13

Oven/iew of steps needed in preparing the filter and dilution water blanks

for the filtration and/or the C,, SPE column tests ...................................... 8-17

Overview of steps needed in preparing the effluent for the filtration and/or

C,, SPE column tests ................................................................................ 8-18

Example data sheet for filtration test ............................................................... 8-20

Diagram for preparing pH adjustment/aeration test samples .......................... 8-23

Example data sheet for aeration test ............................................................... 8-24

Closed loop schematic for volatile chemicals .................................................. 8-26

Step-wise diagram for preparing the C,, SPE column samples ..................... .8-29

Example data sheet for effluent SPE test with and without pH adjustment.. . ..8-3 1

Example data sheet for the oxidant reduction test when using a

gradient of sodium thiosulfate concentrations ........................................... 8-36

Example data sheet for the oxidant reduction test when effluent

dilutions are used ...................................................................................... 8-37

Example data sheet for EDTA chelation test when using a gradient of

EDTA concentrations ................................................................................. 8-41

Example data sheet for the EDTA chelation test when

effluent dilutions are used .......................................................................... 8-42

Example of data sheet for the graduated pH test when

effluent dilutions are used .......................................................................... 8-46

l

vii
Tables

Number Page

6-1. Volumes needed for Phase I tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

8-1. Outline of Phase I effluent manipulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..‘......8-3

8-2. Acute toxicity of sodium chloride to selected aquatic organisms . . . . . . . . . . . . . . . . . . . . .8-14

8-3. Toxicity of methanol to several freshwater species . . . . . ..*.................................. 8-32

8-4. Toxicity of sodium thiosulfate to Ceriodaphnia dubia, Daphnia magna, and

fathead minnows . . . . . . . . ..~~.....~~~.~...~~..~....~~..............~....~.~~.........~.....~............ 8-34

8-5. Toxicity of EDTA to Ceriodaphnia dubia and fathead minnows in

water of various hardnesses and salinities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-39

8-6. The toxicity of the Mes, Mops, and Popso buffers to Ceriodaphnia dubia and

fathead minnows ~,,......,,.....,,,.,..~,,........,,..,,,..,.....,..,...,,..,....,.....,,..~.......... 8-48

_

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Acknowledgments

We wish to acknowledge the assistance of many people from the Environmental

Research Laboratory-Duluth (ERL-D) for their help and advice in preparing the manual. The

experience referred to throughout the document is the collective experience of the individuals in the National Effluent Toxicity Assessment Center (NETAC). During the development

of this document and the first edition, that group has consisted of Don Mount, Teresa

Norberg-King, Larry Burkhard, Liz Durhan, Gary Ankley, Shaneen Murphy (all ERL-D staff),

Linda Anderson-Carnahan, (EPA, Region IV), Joe Amato, Marta Lukasewycz, Greg

Peterson, Jim Taraldsen, Jim Jenson, Mary Schubauer-Berigan, Art Fenstad, Doug Jensen,

Steve Baker, Liz Makynen, Jo Thompson, Correne Jenson, Lara Andersen, Linda

Eisenschenk, Nola Englehorn, and Eric Robert, (all currently or formerly with AScl Corporation, Duluth).

For this revision, several individuals were assigned sections to write or re-write. This

group consisted of Don Mount, Teresa Norberg-King, Larry Burkhard, Liz Durhan, Gary

Ankley, Marta Lukasewycz, Joe Amato, and Mary Schubauer-Berigan. Teresa synthesized

all the rewrites into similar styles, added additional sections, and updated the entire

document. The assistance Debra Williams (AScl) provided to produce the graphics, to

prepare the document, and assist in all aspects is greatly appreciated. Without her input the

production of the document would have been slowed tremendously. Much of the data have

been developed by a few people: Joe Amato generated much of the laboratory data that is

discussed in this document, and others also generated the data for the tables (Jim Jenson,

Doug Jensen, Shaneen Murphy, Greg Peterson, Gary Ankley, Mary Schubauer-Berigan,

and Jo Thompson).

We also want to express our appreciation to Russ Hackett and Dave Mount (ENSR, Fort

Collins, CO) for their data on sodium thiosulfate and W. Tom Wailer (University of North

Texas, Denton, TX) for his data and his technique for sulfur dioxide dechlorination.

As stated in the first edition of the TIE characterization document, the effluent group

would not have been able to complete the work that is summarized in this report without the

support and backing of Nelson Thomas, Senior Advisor for National Programs (ERL-D). In

addition, Rick Brandes (EPA, Permits Division, Washington, D.C.) has been a strong voice

in support of all the work upon which the manual is based. The support provided from the

Off ice of Water through his impetus has enabled NETAC to become a well-established and

well-staffed center.

c

This manual is truly the result of the effort of many people. We welcome your suggestions

for improvement so that any future revision can make the methods more useful.
Section I

Introduction

1. I Background

The Clean Water Act

basis for control of toxic

(CWA, 1972) provides the

substances discharged to

waters of the United States. The Declaration of Goals

and Policy of the Federal Water Pollution Control Act of

1972 states that ”. ..it is the national policy that the

discharge of toxic pollutants in toxic amounts be prohibited.” This policy statement has been maintained in

all subsequent versions of the CWA.

It is the goal of the CWA that zero discharge of

pollutants to waters of the U.S. be achieved. Because

this goal is not immediately attainable, the CWA allows

for National Pollutant Discharge Elimination System

(NPDES) permits for wastewater discharges. The five

year NPDES permits contain technology-based effluent

limits reflecting the best controls available. Where these

technology-based permit limits do not protect water

quality, additional water quality-based limits are included

in the NPDES permit in order to meet the CWA policy

of “no toxic pollutants in toxic amounts.*’ State narrative

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

to determine the adequacy of technology-based permit

limits and the need for additional water quality-based

controls.

To insure that the CWA’s prohibitions on toxic discharges are met, EPA has issued a “Policy for the

Development of Water Quality-Based Permit Limitations for Toxic Pollutants” (Federal Register, 1984).

This national policy recommends an integrated approach

for controlling toxic pollutants that uses whole effluent

toxicity testing to complement chemical-specific analyses. The use of whole effluent toxicity testing is necessitated by several factors including a) the limitations

presented by chemical analysis methods, b) inadequate

chemical-specific aquatic toxicity data, and c) inability

to predict the aggregate toxicity of chemicals in an

effluent.

To determine the toxicity of effluents to aquatic life,

standardized methods for measuring acute and chronic

toxicity have been developed by EPA (EPA, 1985A;

EPA, 19886; EPA, 1989E). These cost-effective methods facilitate the inclusion of whole effluent toxicity

limits and biomonitoring conditions in NPDES permits

for facilities suspected of causing violations of state

water quality toxicity standards.

As a result of the increasing use of aquatic organism toxicity limits and biomonitoring conditions in permits, a substantial number of unacceptably toxic effluents have been and continue to be identified. To rectify

these problems, permittees are being required, through

permit conditions and administrative orders or other

enforcement actions, to perform effluent toxicity reducfion evaluations (TREs). The object of the TRE is to

determine which measures are necessary to maintain

the effluent’s toxicity at acceptable levels. Such evaluations, however, have often proven to be very complicated.

The goal of the TRE wilt be set by either the state

regulatory agency or EPA and will be dependent on

state standards that define acceptable levels of toxicity

in the receiving water and effluent. Because of thts, and

because specific TRE actions may also be required,

communication between the regulators and TRE investigators is crucial.

This document provides NPDES permittees with

procedures to assess the nature of effluent toxicity to

aquatic organisms. It is intended for use by those

permittees having difficulty meeting their permit for whole

effluent aquatic organism toxicity limits or permittees

required, through special conditions, to reduce or eliminate effluent toxicity. This document does not address

human health toxicity concerns such as those from

bioconcentration, water supplies and recreational uses.

The methods are applicable to identifying the cause of

toxicity for samples other than effluents which display

acute toxicity, such as ambient water samples, elutriates

and pore waters from sediments, and possibly leachates.

While we generally refer to effluents, the application of

the techniques for any aqueous sample is implied.

These methods may have applicability to effluents and

other types of samples that exhibit chronic toxicity as

well.

1.2 Conventional Approach to TIES

In order to appreciate the complexities involved in a

typical effluent toxicity identification evaluation (TIE),

one must first understand the drawbacks in what can

be considered the conventional approach to the problem of controlling toxics. The following discussion is

meant, to exemplify the need for a logical approach

which builds on the effluent data as they are being

collected.

Traditionally, when an effluent has been identified

as toxic or is suspected of being toxic to aquatic organisms, a sample of the wastewater is analyzed for the

126 ‘*priority pollutants.” The concentration of each pri-
Toxic Eff bent

Generate

Eff bent LCSO

t

Mass Balance

and

Comparison

Search Literature

for Aquatic

Toxicity

Data on Effluent

Constituents

Figure l-l. Conventional approach to TIES.

ority pollutant present in the sample is subsequently

compared to literature toxicity data for the pollutant, or

is compared to EPA’s Ambient Water Quality Criteria or

state standards for aquatic life protection for that compound. The goal of this exercise is to determine which

pollutants in the wastewater sample are responsible for

effluent toxicity (Figure l-l). Unfortunately, determining

the source of an effluent’s toxicity is rarely this straightforward.

The first problem encountered in this course is one

of effluent variability. Because toxicity is a generic response, there is no way .to determine whether the

Conduct

Priority

Pollutant

Analysis

Evaluate Effluent

Constituents and

Their

Concentrations

toxicity observed over time is consistently caused by a

single constituent or a combination of constituents or a

number of different constituents, each acting periodically to cause effluent toxicity. Experience has shown

that the latter may be a frequent occurrence especially

in publicly owned treatment works (POTW) effluents.

To further complicate the problem, the variability in

conventional effluent monitoring parameters may not

coincide with variability in the effluent toxicant( Monitoring methods for conventional parameters such as

biological oxygen demand (BOD) frequently are not

responsive to shifts in the toxicants because they are at

relatively low concentrations in the effluent or simply

l-2
because the toxicants are not amenable to analysis by

these procedures. For the conventional TIE approach

to be successful, it is crucial that the same sample be

analyzed using both chemical and biological techniques,

and that a number of samples over time be studied to

assess the variability in the toxicant(

A second problem with the conventional approach

involves the focus on the priority pollutants. These

have become known as the “toxic pollutants,” conveying an implication that they constitute the universe of

toxic chemicals but the priority pollutants are only a tiny

fraction of all chemicals. Limiting the search to these

126 compounds will result in failure to identify the

cause of toxicity in most cases.

On the surface, solving this difficulty may seem

inconsequential; the effluent analysis must include monitoring techniques for “non-priority” as well as priority

pollutants. To analyze an effluent for every chemical

would cost tens of thousands of dollars and there

would be no assurance that the detection levels would

be low enough. Determination of the composition of an

effluent is limited to the analyses used. For instance

gas chromatography/mass spectrometry (GUMS) will

not identify cadmium and Inductively Coupled Emission

Spectroscopy (ICP) may not detect it when the concentration is low. The absence of a measurable quantity of

any substance at the method detection level is often

interpreted as meaning that it is not present in the

effluent at all or not at toxic levels.

The toxicants may be present at low concentrations

because only small concentrations of highly toxic chemicals are needed to produce toxicity. If this is true, then

low concentrations must be measured. Such chemicals

are not easily found by examining system loadings. For

example, if a chemical has an LC50 of 1 ug/L, 380 g

(less than a pound per day) of the compound must be

present to cause lethality in the effluent of a 100 million

gallons per day (mgd) treatment plant. With a removal

efficiency of 99%, a loading of only 100 pounds per day

would be needed to produce a toxic effluent. Clearly

then, large loadings cannot be used to guide selection

of analytical techniques, and loads of a few pounds in a

collection system producing 100 mgd may be next to

impossible to identify by the usual methods of establishing loadings.

Many analytical methods are relatively limited in

their applicability. Even GUMS, an instrument heavily

relied upon in typical wastewater analyses, is incapable

of detecting about 80% of all synthetic organic compounds (G. Veith, personal communication, ERL-Duluth).

This limitation is related to selection and efficiency of

solvent extraction techniques, analyte volatility and thermal stability, detector specificity and sensitivity, and

analytical interferences and artifacts. The percentage

of organics detected can be improved by derivatization

but the results are much more difficult to interpret. In

general, the broader spectrum methods are less sensitive and require higher concentrations of analytes for

detection and are costly. To detect lower concentrations, more specific methods are usually more sensitive. To choose specific methods one must have knowledge of the toxicants-knowledge which does not exist,

since that is the purpose of the analyses.

Surprisingly, even with these limitations, one usually sees lengthy lists of effluent constituents when

analyses are performed on wastewater. In the case of

GUMS chromatograms, large peaks of non-toxic effluent constituents can overlap and hide smaller peaks

that may represent the toxicants of concern. When

many chemicals are present, the number of peaks that

can be identified may be small. Failure to identify a

component does not mean that the chemical is not

toxic. By using reference spectra, many peaks may be

tentatively identified as several different compounds

which serves only to increase, not decrease, the number of possibilities. No aquatic toxicity data will be

available for most of these compounds, so toxicity data

must be generated during the study. Compounds may

need to be synthesized in order to test them because

they are not available commercially. For those compounds for which aquatic toxicity data are available, the

data may not include the species used for the TIE.

Even if all this work is done, trying to pinpoint the cause

of toxicity in such a complex mixture is likelyJo fail

because this approach does not include matrix effects

and toxicant bioavailability. For example, several metals may be present in an effluent sample at concentrations well above the toxic threshold. These metals may

not be the source of the effluent’s toxicity, however,

because they are not biologically available. Characteristics such as total organic carbon (TOC), total suspended solids (TSS), ionic strength, pH, hardness and

alkalinity can change toxicity. The inability to quantitate

the effects these parameters have on toxicity further

decreases the chances for a successful TIE.

1.3 Toxicity Based Approach

The approach described in this manual uses the

responses of organisms to detect the presence of the

toxicant during the first stages of the TIE. In this way,

the number of constituents associated with the toxicants can be reduced before analyses begin and some

knowledge of physical/chemical characteristics is gained.

This approach simplifies the analytical problems and

reduces cost. Some of the problems limiting the conventional approach can be used to enhance the success of this alternate approach.

There are two main objectives in the first step of

this approach. First, characteristics of the toxicants

(e.g., solubility, volatility) must be established. This

allows them to be separated from other non-toxic constituents to simplify analyses and enhance interpretation of analytical data. Secondly, throughout the TIE,

one must establish whether or not the toxicity is consistently caused by the same substances. Failing to establish the variability related to the toxicants could lead

to control choices that do not correct the problem.

Knowledge of the physical/chemical characteristics

of the toxicants aids in choosing the appropriate anal -3
lytical method. Such information also may be useful in

selecting an effluent treatment method.

Figure l-2 is a flowchart representation of a TRE.

This document details the toxicity characterization procedures (Phase I). Phase II (toxicant identification) and

Phase III (toxicant confirmation) usually follow Phase I.

Two other EPA manuals (EPA, 1989A; 19898) can be

consulted for more information on bench scale and pilot

plant effluent toxicity treatability studies and source

control options.

Phase I tests characterize the physical/chemical

properties of the effluent toxicant using effluent manipulations and accompanying toxicity tests. Each characterization test in the Phase I series is designed to

alter or render biologically unavailable a group of toxicants such as oxidants, cationic metals, volatiles, nonpolar organics or chelatable metals. Aquatic toxicity

tests, performed on the effluent before and after the

individual characterization treatment, indicate the effectiveness of the treatment and provide information on

the nature of the toxicant( By repeating the toxicity

characterization tests using samples of a particular

effluent collected over time, these screening tests will

provide information on whether the characteristics of

the compounds causing toxicity remain consistent. These

tests will not provide information on the variability of

toxicants within a characterization group. Knowing that

the toxicants have similar physical/chemical properties

means that they can be identified in Phase II using

similar techniques. With successful completion of Phase

I, the toxicants can be tentatively categorized as cationic metals, non-polar organics, oxidants, substances

whose toxicity is pH dependent, and others. Information on physical/chemical characteristics of the toxicants will indicate filterability, degradability, volatility,

and solubility. Either of two choices is available in the

second phase of testing, i.e., toxicant treatability or

toxicant identification studies.

Toxicant identification is described in Phase II (EPA,

1989C). Phase II involves several steps, all of which

rely on tracking the toxicity of the effluent throughout

the analytical procedure. Although effluent toxicants

are partially isolated in the first phase of the study,

further separation from other compounds present in the

effluent is usually necessary. Techniques are available

to reduce the number of compounds associated with

the toxicants. Unlike Phase I procedures, Phase II

methods will be toxicant-specific. Currently available

techniques in Phase II are for identifying non-polar

organics, EDTA chelatable metals, and ammonia.

Enough information exists now to add a section for

surfactants. Additional procedures for other toxicants

will be added as they are developed. Once the toxicants have been adequately isolated from other compounds in the effluent and tentatively identified as the

causative agents, final confirmation (Phase III) can begin.

Like Phase I, Phase III (EPA, 19890) contains

methods generic to all toxicants. No single test provides irrefutable proof that a certain chemical is causing effluent toxicity. Rather, the combined results of the

confirmation tests are used to provide the “weight of

evidence” that the toxicant has been identified.

Once the toxicant has been identified, it can be

tracked through the process collection system using

chemical analyses. Toxicity cannot be used to find the

source for untreated wastes because toxicity from other

constituents that are toxic in untreated waste but removed by treatment, will confuse the results. Of course,

using bench- or pilot-scale systems and measuring

toxicity on treated waste, is feasible. c

TIES require that toxicity be present frequently

enough and endure storage (that is, the toxicity is not

rapidly degrading) so that repeated testing can char;acterize and subsequently identify and confirm the toxicants in Phases II and III. Therefore, enough testing

stiould be done to assure consistent presence of toxicity before TIES are initiated. This is done not to validate

a given test but to establish the sufficient and frequent

presence of toxicity.

The methods described herein are applicable primarily to acute toxicity. Chronic toxicity identification

methods are being developed (EPA, 1991A). In some

special cases in which toxicity can be concentrated (as

in the non-polar organic section of Phase II) one may

be able to “convert” chronic toxicity to acute toxicity by

concentration and successfully identify what is causing

the chronic toxicity.

To be successful, TIES must be conducted by

multidisciplinary teams whose members must interact

daily so that toxicologists and chemists are aware of

the many concerns that affect test results. Speed is

usually important because effluents may decay during

storage. Often subsequent tests cannot be designed

until the results of the previous ones are known. Obviously then, waiting a week for analytical or toxicological

results may preclude more work while the effluent

sample undergoes changes during the waiting period. If

this happens, one must begin again on a new sample

in which case resources are not being used effectively.

l-4
1 Effluent Sample 1

Phase I

Toxicant Characterization Tests

TREATABILITY

APPROACH or Identify Toxicant

IDENTIFY TOX

Phase II

Toxicant Identification Analyses

Phase Ill

Toxicant Confirmation Procedures

ICANT

Considerations

Control Method Selection

and Implementation

*,

Figure 1-2. Flow chart for toxicity reduction evaluations.

l-5
Section 2

Health and Safety

Working with effluents of unknown composition is

the nature of toxicity identification evaluations. Therefore safety measures must be adequate for a wide

spectrum of chemicals as well as biological agents.

From the type of treatment used one may be able to

judge probable concerns. For example, extended aeration is likely to minimize the presence of volatile chemicals and chlorinated effluents are less likely to contain

viable pathogens.

Exposure to the wastewater during collection and

its use in the laboratory should be kept at a minimum.

Inhalation and dermal adsorption can be reduced by

wearing rubber gloves, laboratory aprons or coats, safety.

glasses, and respirators, and by using laboratory hoods.

Further guidance on health and safety for toxicity testing is described in Walters and Jameson (1984).

In addition to taking precautions with effluent

samples, a number of the reagents that might be used

during Phase II toxicant identification and Phase III

toxicant confirmation studies are known or suspected

to be very toxic to humans. Analysts should familiarize

themselves with safe handling procedures for these

chemicals (DHEW, 1977; OSHA, 1976). Use of these

compounds may also necessitate specific waste disposal practices.

L

2-l
-

Section 3

Quality Assurance

Quality assurance is composed of two aspects,

quality verification and quality control. Quality verification entails a demonstration that the proposed study

plan was followed as detailed and that work carried out

was properly documented. Some of the aspects of

quality verification include chain of custody procedures,

statements on the objective of the study and what is

known about the problem at its outset, instrumental log

books, and work assignments. This aspect of quality

assurance ensures that a “paper trail” is created to

prove that the work plan has been covered completely.

The quality control aspect of quality assurance involves

the procedures which take place such as the number of

samples to be taken and the mode of collection, standard operating procedures for analyses, and spiking

protocols.

No set quality assurance program can be dictated

for a TIE; the formula to a successful study will be

unique to each situation. However, adherence to some

general guidelines in formulating a Quality Assurance

Plan (QAP) may increase the probability of success.

In preparing a QAP, enough detail should be included so that any investigator with an appropriate

background could take over the study at any time.

Cross checking of results and procedures should be

built into the program to the extent possible. Records

should be of a quality that can be offered as evidence

in court. Generally, the QAP should be provided in a

narrative form that encourages users to think about

quality assurance. To be effective, the QAP must be

more than a paper exercise simply restating standard

operating procedures (SOPS). It must increase communication between clients, program planners, field and

laboratory personnel and data analysts. The QAP must

make clear the specific responsibilities of each individual. The larger the staff, the more important this

becomes. While QAPs may seem to be ‘an inconvenience, the amount of effort they require is commensurate with the benefits derived.

3. I TIE Quality Control Plans

A successful TIE is dependent upon a strong quality control program. Obtaining quality TIE data is more

difficult because the constituents are unknown in contrast to quality control procedures for a standard analytical method for a specific chemical. In such an analysis, one knows the characteristics of the analyte and

the implications of the analytical procedure being utilized. Without knowledge of the physical/chemical char3

acteristics of the analyte, however, the impact of various analytical procedures on the compound in question

is not known. Further, quality control procedures are

specific to each compound; quality control procedures

appropriate to one analyte may be completely inappropriate to another.

The problem of quality control is further exaggerated because quality control procedures for aquatic

toxicity tests may be radically different from those required for individual chemical analyses. This additional

dimension to quality control requires a unique framework of checks and controls to be successful. The

impacts of chemical analytical procedures on sample

toxicity must be included. Likewise, procedures used to

insure quality toxicity test results should not impact

chemical analyses. For example, in performing standard aquatic toxicity tests, samples with low dissolved

oxygen (DO) are usually aerated. This practice may,

however, result in a loss of toxicity if the toxicant is

volatile or subject to oxidation.

3.2 Cost Considerations/Concessions

The quality control practices required in any given

experiment must be weighed against the importance of

the data and decisions to be based upon it. The crucial

nature of certain data will demand stringent controls,

while quality control can be lessened in other experiments having less impact on the overall outcome.

Effluent toxicant identification evaluations require a

large number of aquatic toxicity tests. The decision to

use the standard toxicity test methods described in

EPA (1985A; 19918) (involving a relatively high degree

of quality control), must be weighed against the degree

of complexity involved, the time required and the number of tests performed; all of these affect the cost of

testing. For this reason, toxicity tests used in the early

phases of the evaluation generally do not follow this

protocol, nor do they require exacting quality controls

because the data are only preliminary. Phase I, and to

a lesser extent, Phase II results are more tentative in

nature as compared to the tests performed for the

confirmation of the effluent toxicant in Phase III.

The progression towards increasingly definitive results is also reflected in the use of a single species in

the initial evaluation studies and multiple species in the

later stages. The use of several species of aquatic

organisms to assure that effluent toxicity has been

reduced to acceptable levels is necessary because

species have different sensitivities to the same pollut1
ant. Quality control must relate to the ultimate goal of

attaining and maintaining the designated uses of the

receiving water. For this reason, final effluent test results must be of sufficient quality to ensure ecosystem

protection. The use of dilution water for the toxicity

tests which mimics receiving water characteristics (in

hardness and pH) will help to ensure that the effluent

will remain non-toxic after being discharged into the

environment. In the instances where the effluent dominates the receiving water, the dilution water should

mimic the water chemistry characteristics of the effluent. This is discussed in Section 5, Dilution Water. In

addition, it is essential that the variability in the cause

of effluent toxicity be defined during the course of the

TIE so that appropriate control actions provide a final

effluent safe for discharge.

3.3 Variability

The opportunities to retest any effluent to confirm

the quality of initial TIE results will be limited at best. In

addition to the shifting chemical and toxicological nature of the diseharge over time, individual effluent

samples stored in the laboratory change. Effluent constituents degrade at unknown rates, as each compound

has its own rate of change. The change in a sample’s

toxicity over time represents the cumulative change in

all of the constituents, plus that variation resulting from

experimental error. Some guidelines for assessing and

minimizing changes in sample chemistry and toxicity

are discussed in Sections 6 and 8. Regardless of the

precautions taken to minimize sample changes, a

sample cannot be retested with certainty that it has not

changed.

3.4 lntra-Laboratory Communication

Quality control procedures in chemistry and biology

can be quite different. For example, phthalates are a

frequent analytical contaminant requiring special precautions that are not of toxicological concern. The toxicological problem presented by the zinc levels typically

associated with new glassware are of no concern to

those performing organic analyses. The difference in

glassware cleanup procedures is an example of many

differences that must be resolved. Cleaning procedures

must be established to cover the requirements of both.

Time schedules for analyses must be detailed in advance. One cannot assume compound stability; therefore, time delays between the biological and chemical

analysis of a sample cannot be tolerated.

3.5 Record Keeping

Throughout the TIE, record keeping is an important

aspect of quality verification. All observations, including

organism symptoms, should be documented. Details

that may seem unimportant during testing may be crucial in later stages of the evaluation. Investigators must

record test results in a manner such that preconceived

notions about the effluent toxicants are not unintentionally reflected in the data. TIES required by state or

federal pollution control agencies may require that some

or all records be reviewed.

3.6 Phase I Considerations

Effluent toxicity is “tracked” through Phases I, II

and III using aquatic organisms. Such tracking is the

only way to detect where the toxicants are until their

identity is known. The organism’s response must be

considered as the foundation and therefore, the toxicity

test results must be dependable. System blanks (blank

samples carried through procedures and analyses identical to those performed on the effluent sample) are

used extensively throughout the TIE in order to detect

toxic artifacts added during the effluent characterization

manipulations. With the exception of tests intended to

make the effluent more toxic, or situations in which a

known amount of artifactual toxicity has been intentionally added, sample manipulation should not cause the

eff,iuent toxicity to change.

There are many sources of toxicity artifacts in Phase

I. These include: excessive ionic strength resulting from

the addition of acid and base during pH adjustment,

formation of toxic products by acids and bases, contaminated air or nitrogen sources, inadequate mixing of

test solutions, contaminants leached from filters, pH

probes, solid phase extraction (SPE) columns, and the

reagents added and their contaminants. The appropriate toxicity data for the reagent chemicals usti in

Phase I and common aquatic test organisms are provided as needed in subsequent sections of this document.

,

Frequently toxic artifacts are unknowingly introduced. For example, pH meters with refillable electrodes can act as a source of silver which can reach

toxic levels in the solutions being measured for pH.

This is especially a problem where there is a need to

carefully maintain or track solution PH. Using pH electrodes without membranes avoids the silver problem

(which can only be detected by profuse use of blanks).

Oil in air lines or from compressors is a source of

contamination. Simple aeration devices, such as those

sold for use with aquaria are better as long as caution

is taken to prevent contamination of the laboratory air

which is taken in by the pump.

Worst case blanks should be used to better ensure

that toxicity artifacts will be recognized. Test chambers

should be covered to prevent contamination by dust

and to minimize evaporation. Since small volumes are

often used, evaporation must be controlled. Plastic disposable test chambers are recommended to avoid problems related to the reuse of test chambers. Cups from

the same lot should be spot-checked for toxicity.

Glassware used in various tests and analyses must

be cleaned not only for the chemical analyses but so

that toxicity is not introduced either by other contaminants or by residues of cleaning agents. Since the

organisms are sensitive to all chemicals at some concentrations, all toxic concentrations must be removed

and not just those for which analyses are being made.

Randomization techniques, careful observance of

organism exposure times and the use of organisms of

3-2
approximately the same age ensure quality data. Standard reference toxicant tests should be performed with

the aquatic test species on a regular basis and control

charts should be developed (EPA, 1985A; 1991B). During Phase I it will not be known how much the toxicity

of the reference toxicant varies over time compared to

the toxicant( When the toxicants are known, they

should be used as the reference toxicant. Reference

toxicant tests should be performed to coincide with the

TIE testing schedule.

3.7 Phase II Considerations

In Phase II, a more detailed quality control program

is required. Interferences in toxicant analysis are for the

most part unknown initially but as toxicant identifications are made, interferences can be determined. Likewise instrumental response, degree of toxicant separation, and detector sensitivity can be determined as

identifications proceed.

3.8 Phase 111 Considerations

In Phase III of a TIE, the detail paid to quality

control and verification is at the maximum. This phase

of the study responds to the compromises made to

data quality in Phases I and II. For this reason, confidence intervals for toxicity and chemical measurements

must be calculated. These measurements allow the

correlation between the concentration of the toxicants

and effluent toxicity to be checked for significance based

on test variability. Effluent manipulations prior to chemical analysis and toxicity testing are minimized in this

phase in an effort to decrease the chance for production of artifacts. Field replicates to validate the precision

of the sampling techniques and laboratory replicates to

validate the precision of analyses must be included in

the Phase III quality control program. System blanks

must be provided. Calibration standards and spiked

samples must also be included in the laboratory quality

control program. Because an attempt will be made to

correlate effluent toxicity to toxicant concentration, spiking experiments are important in determining recovery

for the toxicant( These procedures are feasible in

this phase of the study because the identities of the

substances being measured are known.

The toxicants being analyzed can be tested using

pure compounds, thereby alleviating the need for a

general reference toxicant. Because the test organism

also acts as an analytical detector in the correlation of

effluent toxicity with toxicant concentration, changes

in the sensitivity of the test organisms must be known.

This is best achieved by using the same cheFicals

identified for the reference toxicants.

3-3
Section 4

Facilities and Equipment

The facilities, equipment and reagents needed to

perform an effluent TIE will depend on the phase of the

study and the characteristics of the toxicant( The

equipment required for Phase I characterization tests is

described throughout Section 8. The facility and equipment needs in Phase II of the TIE will be site-specific

and will depend both on the physical/chemical characteristics of the toxicants and on the choice of the Phase

II approach.

Phase I requires only basic analytical and toxicity

testing equipment which would be available in most

laboratories where toxicity tests and the usual water

chemistry analyses are performed. Phase III requirements are largely limited by equipment found in a

typical toxicity testing lab and equipment necessary for

the analysis of the toxicant(

Because of the equipment needs and time required

to conduct the evaluations, complete on-site effluent

TIES using a mobile laboratory are generally not feasible. Measurement of the loss of toxicity over time in

several effluent samples will provide information upon

which to base acceptable storage times. Usually, with

modern rapid sample shipment methods, off-site work

is practical. The cost of shipment is usually far less

than the cost of on-site work.

Large numbers of organisms and many tests are

needed for TIES. Ready availability of test organisms is

important because often the test(s) needed are not

predictable. Only after the results of one experiment

are known can the next test be planned. It is probably

more economical to culture many of the test species

that might be used in TIES than it is to purchas#them.

A delay in testing caused by shipment time or lack of

availability of test organisms could cost far more in

work loss than it would cost to maintain cultures for

many weeks.

4-1
Section 5

Dilution Water

The choice of dilution water will change with the

purpose of the tests and therefore the choice will often

be more varied in Phases I and II than in Phase III.

Particularly for some toxicant groups in Phase II, some

very unusual dilution water is recommended in order to

achieve the desired chemical conditions. Sometimes

the water may in itsetf be toxic! Such concepts are

foreign to conventional toxicology and rightly so, but

this is not conventional toxicology.

Much of Phase I and parts of Phase II utilize

organism tolerance and relative toxicity to accomplish

the objectives of the study. Methanol, hydrogen ion

concentration, and osmotic pressure may sometimes

be near lethal levels in order to test necessary conditions. In some cases, the dilution medium may cause

complete mortality in 48 h, but the point of interest is

whether treatment causes more rapid mortality. If so,

one can say that one condition is more toxic than

another and obtain important information from the test.

The key is to run sufficient numbers of system blanks

so that the relative contribution to mortality is known

and toxicity is not attributed to an incorrect cause.

These are examples of the previous statement that

these methods “utilize tolerance and relative toxicity.”

In reality, this approach is very much like the comparison of the toxicity of two chemicals, A and B. If one

determines LC5Os for A and B and concludes that A is

twice as toxic as B, lethal conditions are being compared in order to say this. Controls are not involved in

the LC50 calculation and high control survival does not

change the data interpretation, The same concept of

relative toxicity is used here. Chemical “A,, is the blank

and chemical “B” is the treated sample and the question is, ‘,which is more toxic?“.

As these methods are built on tolerance (i.e., survival), chronic toxicity endpoints cannot be used and

that is why these methods are primarily intended for

acute toxicity. Obviously, if one wants to measure chronic

effects, the test organisms must be able to live long

enough to display chronic effects. Many of the pH

changes and other manipulations used in these methods do not allow sufficient survival time or health for

reproduction or growth. For chronic TIES, more attention has to be given to acclimation, feeding and general

living conditions (EPA, 1991 A).

Many of the additives used in the Phase I manipulations change the mixture of the effluent much more

than the dilution water. In general, for Phase I, any

water which is of a consistent quality and which will

support growth and reproduction of the test species is

suitable. We have found the use of a dilution water that

has a hardness similar to that of the effluent or the

receiving water to be beneficial. A variety of dilution

water choices are provided by EPA (1985A; 1989E)

and any of these may be used for TIES.

In Phase III, where the objective is to confirm the

true cause of toxicity, where artifacts are to be excluded to the extent possible and where absolute toxicity is more important than relative toxicity, practices

including choice of dilution water, must follow conventional toxicological methodology. Tolerance to additives

must not be necessary in order to provide the desired

response. Attention must be given to simulation of the

dilution water into which the effluent is discharged.

Some toxicant dose response relationships may be

totally different as the water quality characteristics

change. These factors must be incorporated into Phase

III where absolute toxicity is of the utmost concern. In

Phases I and II, only relative differences are being

considered.

Perhaps a cautionary note is warranted regarding

the effects of dilution water on effluent toxicity. If high

concentrations of effluent are being tested (e.g., 80%)

the physical/chemical characteristics will resemble those

of the effluent. If low concentrations are tested (e.g.,

5%) then characteristics will resemble those of the

dilution water.

Little specific information can be given about the

selection of dilution water in Phases I and II except that

the desired tested conditions will often dictate its characteristics. For example, in Section 8.6, the same column used for the blank may not be usable for the

effluent sample if receiving water is used as the dilution

water. Secondly, sufficient numbers of blanks must be

included to interpret the results. In Phase III, the choice

of the appropriate dilution water should be based on

the characteristics of the receiving water where the

discharge occurs.

5-1
.

Section 6

Eff bent Sampling and Handling

A wastewater sample may be representative only

of the discharge at the time of sampling. In effect, each

sample is a “snapshot” of the effluent’s toxicological

and chemical quality over time. To determine whether

any effluent sample is typical of the wastewater may

require the collection of a large population of samples.

Further, what constitutes a “representative” sample is a

function of the parameter of concern. Because effluents

vary in composition, sampling must be extensive enough

that one is confident that the groups of samples represent the discharge over time. Guidelines for determining the number and frequency of samples required to

represent effluent quality are contained in the “Handbook for Sampling and Sample Preservation of Water

and Wastewater” (Berg, 1982). However, since this

guidance is not based on toxicity, it should be used

with caution.

Both quantitative (change in concentration) and

qualitative (change in toxicants) variability commonly

occur in effluents and both may affect toxicity. Changes

in effluent toxicity are the result of varying concentrations of individual toxicants, different toxicants, changing water quality characteristics (affecting compound

toxicity) and analytical and toxicological error. Even if

the toxicity of an effluent to an aquatic organism is

relatively stable, this does not mean that there is only a

single toxicant causing toxicity in any given sample or

among several samples.

Determining whether a sample is typically toxic is

not as simple as comparing the conventional pollutants

of the sample to long-term effluent averages. Effluent

toxicants often do not follow the same trends as BOD,

TOC and TSS. The toxicant may be present at such

a low level that it does not significantly affect the quantity of the conventional pollutant, even though it is

present in toxic concentrations.

Conventional parameters, BOD, TSS, and other

pollutants limited in the facility’s NPDES permit, will

provide an indication of the operational status of the

treatment system on the day of sampling. For industrial

discharges, information on production levels and types

of operating processes may be helpful. The condition of

the facility’s treatment system at the time of sampling

should be determined by the individual collecting the

sample. The type of sample, time of collection, and

other general information on the facility should be recorded. An example of a page of a log book is given in

Figure 6-1.

Upon the arrival of the sample in the laboratory,

temperature, pH, toxicity, hardness, conductivity, total

residual chlorine (TRC), total ammonia, alkalinity, and

DO should be measured. Toxicity should be measured

periodically during storage to document any changes

(cf., Section 8).

Investigators should not be surprised to find that

well operated municipal and industrial treatment systems discharge unacceptably toxic wastewaters. Eff luent guideline-based limits which reflect best achievable

technology, do not prescribe limits for more than a few

chemicals. Many compounds present in effluents are

not regulated because the discharger is not required to

report their presence in permit applications or they

cannot be detected using typical methods for wastewater analysis.

For chlorinated effluents, whether sampling should

be done before chlorination depends on the question to

be answered. Sometimes the question may be whether

or not there are toxicants other than chlorine present.

Dechlorination prior to toxicity characterization may be

needed in order to distinguish toxicity from causes

other than chlorine. Usual methods of dechlorination

may remove more than toxicity from chlorine alone and

careful data interpretation is needed to understand the

results. Toxicity from more than one cause is often not

additive in effluents, so relative contributions from two

or more causes can be very hard to decipher.

The choice of grab or composite samples will depend on the specific discharge situation, (e.g., plant

retention time) questions to be answered by the TIE

and the stage of the TIE. In Phase I testing, samples

that are very different from one another give results

that are difficutt to interpret; therefore composite samples

are more similar and are easier to. use. In Phase III,

effluent variability is used to advantage; therefore, grab

samples are often best. If toxicity is low or intermittently

present, grab samples may be best during all phases.

The additional difficulty of getting flow proportional

samples should be balanced against their advantage in

each situation. While grab sampling may provide maximum effluent toxicity, it is more difficult to catch peaks

in toxicity and Phase I sampling may require more time.

EPA (1985A; 1991 B) discusses the advantages and

disadvantages of grab and composite sampling and

have also detailed methods for sampling intermittent

discharges.

6-1
Figure 6-1. Example data sheet for logging in samples.

Sample Log No.: Sample Type: Cl Grab 0 Composite

Date of Arrival: 0 Glass 0 Plastic

Date and Time

of Sample Collection:

cl

0

cl

Prechlorinated

Chlorinated

Dechlorinated

Facility:

Location: Sample Conditions Upon Arrival:

NPDES No: Temperature

PH

Contact:

Phone Number:

Sampler:

Condition of treatment system at time of sampling:

Status of process operations/production (if applicable):

iota1 Alkalinity

Total Hardness

Conductivity/Salinity

Total Residual Chlorine

Total Ammonia

l

Comments:

6-2
If the TIE analyses are not conducted on-site,

samples must be shipped on ice to the testing location.

Effluent samples should not be filtered prior to testing

unless it is necessary to remove other organisms.

Sample filtration could affect the results of the characterization tests, one of which entails filtering the effluent. Sample aeration should also be minimized during

collection and transfer. Initial sample analysis should

begin as soon as practical after effluent sampling. Phase

II and especially Phase III may require specific types of

sample containers or the addition of preservative to

aliquots of sample designated for chemical analyses.

For a single Phase I test series, 3 L of effluent are

needed for analysis if test organisms such as daphnids

or newly hatched fathead minnows are used. The exact

volume required depends on the toxicity of the effluent

and to a lesser extent, the test options chosen (cf.,

Section 8). For other species different volumes may be

necessary. Volumes frequently used in each characterization test are supplied in Table 6-l.

The extent of the analyses carried out on any

individual sample must be weighed against the cost of

Table 6-1. Volumes needed for Phase I tests

Volume for Total

Characterization Step Each Step’ Volumes2 (mL)

Chemical analyses3 _- <500

pH 3 Adjustment 30 -300

filtration 235

solid phase extraction 200

aeration 35

pH 11 Adjustment 30 -300

filtration 235

solid phase extraction’ 200

aeration 35

Unadjusted pH effluent (pH ;)” -590

initial test 40

baseline test 80

filtration 235

solid phase extraction 200

aeration 35

EDTA additions 100

sodium thiosulfate additions 100

Graduated pH . -120-1000

PH 6 40-500

PH 7 40-500

PH 8 40-500

11 Amount is dependent on effluent characteristics.

2 Total volume is -3 L; this is maximum needed, does not include

subsequent testing.

3 These include temperature, pH, hardness, conductivity, TRC,

total ammonia, alkalinity, and DO.

4 The pH is readjusted to pH 9 before it is put through the C,, SPE

column.

5 The pH i of the effluent is the initial pH of the effluent sample. It

may be important to know the pH at the point of discharge as

well as the receiving water pH and to know the pH of the

effluent at air equilibrium.

additional sampling, the stability of the sample, sample

representativeness and the need to have samples of

different toxicity. Clearly, the resources required for

such TIES are too great to expend on a single sample

or on a few samples which do not represent the effluent. Likewise, there is not a set number of samples

which should be analyzed in Phases I, II or III before

going on to subsequent phases of the study or taking

final measures to control effluent toxicity. The number

of samples analyzed in each phase will be a function of

the apparent variability in the effluent, the number of

toxicants, how persuasive the data are, the cost of the

remedial action, regulatory deadlines and finally, the

success of each study phase.

6.7 Sample Shipment and Collection in

Plastic versus Glass

Effluent samples often have been collected, shipped

and stored in various types of plastic (e.g., polyethylene) containers rather than glass. However, with a few

effluents, we have noted that samples shipped and

stored in glass were more toxic and retained their

toxicity longer than split samples shipped and stored in

plastic. This effect appeared to be due to adsorption of

certain types of toxicants (e.g., surfactants) to theslastic. For these instances the samples in glass were

more representative of the effluent, and thus for TIE

purposes were preferable to the samples in plastic.

An easy way to check whether or not there is a

difference in the toxicity of samples shipped and stored

in glass containers versus those shipped and stored in

plastic containers, is to test two or three sets of effluent

samples. Effluent should be collected in glass or stainless steel, then a portion shipped in glass and another

portion shipped in plastic. Baseline toxicity tests (cf.,

Section 8) are conducted on each, perhaps on days 4

and 7 after receipt. If the initial toxicity of the sample is

similar for both the plastic and the glass containers,

and the toxicity for samples from the two containers is

similar over time (i.e., over storage time), it is appropriate to have the effluent samples shipped and stored in

plastic containers. However, if effluent shipped and

stored in glass appears to be more toxic, and retains

the toxicity longer than the effluent sample shipped and

stored in plastic, glass containers should be used for all

shipments and storage for that particular effluent. These

same considerations also apply to the sampling/collecting equipment. Collection, shipment, and storage of

effluent samples in glass may involve more effort than

plastic containers. The use of glass containers for

samples that retain their toxicity longer might result in

more rapid and cost effective progress through the TIE

because fewer samples might be required for identification of effluent(s). Since only certain classes of compounds are expected to adsorb to plastic containers

(e.g., surfactants), if the effluent is more toxic in glass,

this can be a useful piece of information for characterizing the toxicants.

6-3
a

Section 7

Toxicity Tests

7.1 Principles

-

Acute lethality tests with aquatic organisms are

utilized throughout the toxicity characterization procedures described in this manual as well as in Phases II

and III. Using toxicity for such evaluations is logical

since toxicity triggers the TIE requirement. In these

tests the organism acts as the “detector” for chemicals

causing effluent toxicity. As such, they provide the true

response regardless of the outcome of other analyses.

The toxicity test is the only analytical procedure that

can be used to measure toxicity. Until the cause of

toxicity is known, chemical methods cannot be used to

identify and quantify the toxicants.

There are a number of consequences associated

with this reliance on toxicity. The organism responds to

every constituent, provided that it is present above a

threshold level either individually or collectively if the

constituents are additive. While this general response

to any compound presents an advantage as a broad

spectrum test for toxicants, it requires considerable

effort to determine the primary cause of toxicity because it is not specific. This non-specific response

necessitates a generic chemical/physical characterization of toxicants during Phase I testing before Phase II

identification is begun.

A further repercussion of this universal response is

the probability of artifactual toxicity. Because the analyst is reliant upon the organism’s ability to track toxicity

throughout the effluent characterization steps, sample

manipulations are constrained. While characterizing the

effluent, no manipulation should change the toxicity of

the sample in an unpredictable manner. “Toxicity-blanks

and controls” are helpful but the difficulties associated

with them are far greater than those connected with

chemical analyses because of their non-specificity. As

a result many more blanks are employed in TIE testing

than in chemical analyses or standard toxicity testing.

Negative blank toxicity cannot be assumed regardless

of past results. Quite unexpected sources of artifactual

toxicity will occur in the course of conducting an evaluation.

For some Phase I tests the corresponding blanks

(treatments on the dilution water) do not provide completely relevant information concerning the effect of the

manipulation on the effluent. For example, blanks of

the graduated pH test (Section 8.9) are not particularly

useful whether the pH is adjusted with acids, bases, or

CO,. The amount of the acid or CO, used to adjust and

maintain the same pH for an effluent sample and a

blank are often radically different due to the differences

in the buffering capacity of each of the solutions. Since

the matrix of the effluent and dilution water are different, the pH in each solution will change at different

rates during the toxicity tests. Therefore the blanks are

not representative of what is occurring in the effluent

test and the controls exposure does not provide information on the manipulation effect on the test organisms. The use of blanks for the other manipulation

steps is relevant, and they provide information on cleanliness of the acids and bases added, the air system,

filter apparatus, and SPE columns.

7.2 Test Species l

Just as different analytical methods have different

detection levels for the same chemical, different species have different sensitivities to the same toxicants.

The major difference is that the toxicity measurement is

non-specific to chemicals and so for an unknown mixture (effluent, sediment pore water) one must determine whether a different toxicity value for the sample is

caused by the organisms different sensitivity to the

same toxicant or to different toxicants.

The choice of species to use for the toxicity test

can change the conclusion reached. In addition to the

obvious need to use species of an appropriate size,

age, availability, and adaptability to test conditions,

there are other important considerations. An effluent

toxic to two species, having equal or different LCSOs

may be toxic because of different toxicants. Differences

of 1,000x in sensitivity are common and differences of

10,000x occur among species exposed to a single

chemical. Anyone involved in identifying the cause of

toxicity of an effluent will be concerned because someone has found the effluent toxic to some organism. If

that is not the case, before a TIE is begun, one should

determine to which organisms the toxicity concern is

directed.

Many effluents will be received for TIES because

they have been found toxic to the cladocerans,

Ceriodaphnia or Daphnia--species well suited to TIE

methods. TIE test species selection is obvious in these

instances. Where toxicity concern is based on species

(trout or mysid shrimp), that are not going to be the TIE

test species, one must demonstrate that the toxicity of

concern has the

by the species

depends on theI

7-1

same cause as the toxicity manifested

to be used in the TIE. The difficulty

effluent characteristics (especially tox-
icity variability), the number of TIE steps which affect

toxicity and the difference in sensitivity between the

species being compared. Since this problem has not

been one we have experienced frequently, our suggestions are certainly not all-inclusive. The final confirmation (Phase III) methods are designed to show whether

the wrong toxicant was identified. However, many resources may be consumed before reaching that stage

and earlier assurances should be obtained if reasonable, to save time and cost.

One approach is to compare the LC50 values of

whole, unaltered effluent samples for the species originally raising the toxicity concern and the selected species for the TIE. If the acute toxicity varies similarly for

each species among samples then there is evidence

that the two species are responding to the same

toxicant( If the LC50 values vary differently for the

two species, there is evidence that the toxicants are

different. If the LC50 values among samples do not

vary more than the precision of the test method this

approach is useless for that effluent. Successful application of this approach does not require equal sensitivity of the two species or the greatest toxicity of the TIE

organism, but rather sensitivity of the species to the

same toxicant.

If in Phase I, several steps (e.g., pH decrease,

aeration, solid phase extraction) all changed toxicity,

and if the direction and relative magnitude of change

was the same for both test species, then there is

evidence that both are sensitive to the same toxicant. If

one or more parameters are different, the evidence is

strong that the toxicants are different. This is not to say

that if a Phase I technique completely removes toxicity

to one species, it will remove it to the same extent for

_ the other species. Because different species have dissimilar sensitivities to the same chemical, removal of

90% of a compound in an effluent sample may lead to

a non-toxic concentration to one species while only

reducing the toxicity to another species. If the Phase I

procedures that successfully remove or reduce effluent

toxicity differ by test species, it is unlikely that toxicity is

caused by the same chemical(s).

Symptom comparisons are useful, especially if one

is comparing similar organisms. Comparing fish symptoms to Daphnia symptoms could be very misleading

but comparing symptoms of Daphnia magna to those of

Ceriodaphnia dubia should be relatively safe. If one

finds comparable symptoms, the evidence is not convincing because many toxicants cause specific symptoms but if symptoms are distinctly different, the evidence is strong that the toxicants are different. This is

true only when symptoms are compared at effluent

concentrations that are the same multiple of the LC50

for each species. For example, if two species have

LC50 values of 10% and 90%, comparing symptoms at

100% concentrations could be misleading. At 100%

effluent, the species with an LC50 of 10% might experience the symptoms so fast that their sensitivity would

appear completely different from those of the less sensitive species. Experience will reveal additional techniques that can be used.

Freshwater discharges to saline receiving water

require separate considerations. Sea salts can be added

to raise the salinity of the effluent (EPA, 1991 B) enough

so that marine species can be used in the TIE. However, the tolerances of marine organisms to the additives and effluent manipulations have not been determined. To do so is costly and time consuming and a

more efficient method may be to use a freshwater

species in Phase I and II. If this is done, data must be

gathered to show that the freshwater species chosen is

sufficiently sensitive and is responding to the same

toxicant as the marine species. The principles of

doing this are the same as described above for different freshwater species. When Phase III is reached,

marine species should be used, but in that phase,

manipulations and additives are minimal and little ancillary data are needed in order to use marine species.

For discharges with conductivities comparable to

brackish or marine water, caution is in order. Most

methods for measuring “salinity” (conductivity or refraction) are non-specific for NaCI, which is the principal

component of sea water. Marine organisms accomplish

osmotic regulation by regulating sodium and chloride. If

salinity of an effluent is not caused by NaCI, marine

species may be stressed as much as freshwater species by high concentrations of other dissolved salts.

Unless the “salinity” of an effluent is known to be

caused by NaCI, marine species cannot be used to

avoid the salinity effects.

7.3 Toxicity Test Procedures

The purpose of the toxicity test in Phase I is the

same as that of any analytical method--to measure

(detect) the presence of the toxicants. This use is quite

different than conventional toxicity testing where the

objective is to accurately and quantitatively measure

the sensitivity of the organism to known concentrations

of a chemical or effluent. For this latter purpose, removing stress (e.g., low DO) or other contaminants, and

lack of space is important because such stresses may

change the sensitivity of the organism to the contaminant of concern. In Phase I, relative sensitivity is used;

that is, we compare whether one condition is more or

less toxic than another but both may be toxic. Therefore, concern of documenting and/or removing other

stresses is not very important. It is important to be sure

that these other stresses are similar for each condition

being compared, each time the manipulation and subsequent toxicity tests are performed.

The reason for this discussion under test methods

is that effort must be made to make the tests used in

Phase I as inexpensive as possible, because for some

effluents, large numbers of tests may be needed. For

example, we have used more than 100 tests on some

effluents in Phase I. If the effort usually expended in

measuring all the required water chemistries for a whole

effluent test (EPA, 1985A) had been done for these

7-2
tests, the cost would have been prohibitive. The reader

may wonder whether data collected from such tests

can be trusted. Confidence in the data hinges on careful assurance that the stresses are similar among comparisons. For example, it does not matter if the test

organisms are acclimated to a pH change. It does

matter that stress from lack of acclimation to pH change

occurs in each treatment compared.

Sometimes, in order to achieve desired chemical

conditions, the stress from pH change cannot be made

uniform. In these situations, only gross differences in

response may be dependable. In some cases, erroneous conclusions will be reached. While these may cause

wasted effort, the error should be found in Phase III.

That is why, in Phase III, careful quality control must be

exercised and cost saving shortcuts are not acceptable

because one of the purposes of Phase III is to catch

errors or artifacts that may occur in Phases I and II.

One need not use the standard acute methods

(EPA, 1985A; 1991A) in Phase I for these reasons. The

following mechanics of performing an acute test with

cladocerans and newly hatched fathead minnows have

been found by experience to be very cost effective and

are offered as an aid to those doing Phase I testing.

Specific volumes and sizes are used in this example for

simplicity, but of course, these are varied depending on

each test purpose.

For example, arrange a set of 12 plastic

cups into six pairs. Fill 10 cups with 10 mL

of dilution water using a disposable pipette.

Add 10 mL of effluent to the two empty

cups to make the high concentration, (e.g.,

100%). Add 10 mL of effluent to the next

pair of test cups (duplicates labeled A and

B) already containing 10 mL each of the

dilution water (.Figure 7-1). The resulting

concentration is 50%. From each cup of

the 50% solution, transfer 10 mL to the

third pair of test cups to produce the 25%

concentration. Continue this process until

sufficient exposure concentrations have

been prepared. One pair of cups in the

series contains only dilution water and

serves as the control. Mixing the solutions

prior to the transfer of each aliquot is very

important. This can be accomplished by

drawing the solution into the pipette and

discharging it back into the cup several

times prior to transfer. Additional mixing of

test solutions should be done for

experiments in which reagents such as

sodium thiosulfate (Na,S,O,) and EDTA

(Phase I), or effluent methanol eluate

concentrates (Phase I and II) are added to

effluent or dilution water.

‘The need for duplicates will depend on the accuracy and precision required of the test results. Tests

requiring a measure of accuracy in the form of confidence intervals (Cls) should be run in duplicate. Tests

designed to provide only’ an indication of positive or

negative toxicity need not be run in duplicate. Beyond

the initial and baseline effluent toxicity tests (Sections

8.1 and 8.2) which are designed to define effluent

toxicity upon arrival in the laboratory and periodically

during the TIE with each effluent sample testing, respectively, Phase I toxicity tests usually do not require

duplicates.

The test organisms of uniform age should be placed

at random in each test cup to insure valid results.

Because the volume of test solution may be small, care

must be taken to minimize the volume added during

test organism transfer. If the volume of water transferred with the organism is reduced to a drop (50 pL),

only five organisms are added to the test chamber and

a 10 mL test volume is used, the resulting change in

test solution volume will be 2.5%. Minimizing the change

in volume is more critical as test solution volume is

reduced. This is particularly important in the Phase II

experiments, when limited volumes of effluent fraction

concentrates are available. Care should also be taken

to avoid chemical contamination between concentrations when test animals are being added.

We have stressed a relaxation of the usual water

chemistry requirements in these Phase I tests because

they are not as necessary here as they are in Ph&e III.

However, sometimes, in order to maintain the desired

conditions in the test (such as maintaining a specific

pH) frequent specific repetitive measurements of those

items will be necessary. The distinction drawn here is

to avoid measurements you don’t need (e.g., sample

hardness) and concentrate on those that are important

(e.g., pH). Effluents are often well buffered and pH

sometimes will change quickly if equilibrium is not already established. POTW eifluents are not in air equilibrium when discharged and as soon as they are

exposed to air, the pH will rise. A typical POTW effluent

pH is 7.2-7.4 when discharged but it will equilibrate

after contact with air and may stabilize at 8.2-8.5. If pH

is important to test interpretation, pH must be monitored throughout the test. It will also be important to

decide what the initial pH (pH i) of the effluent is since

the pH at the discharge and/or the initial pH may be

different from the pH of the effluent at air equilibrium.

7.4 Test Endpoints

Little effort should be expended in calculating LC50

values for Phase I toxicity tests. There is no need to

apply sophisticated and complex programs to the test

results. Several methods for estimating the LCSO from

the acute toxicity data are described in EPA (1985A),

however a method which is most easily and quickly

applied to the data should be used. In many cases, the

graphical method entailing interpolation may prove to

be the most convenient. Differences resulting from the

choice of data analysis method should not impair the

outcome of Phase I studies. Phase III tests may require

more sophisticated analyses.

Toxic units (TU) have a special utility in some parts

of a TIE. The TU of whole effluent is 100% divided by

the LC50 of the effluent. For specific chemicals the TU

7-3
Figure 7-l. Schematic for preparing effluent test concentrations using simple dilution techniques. Two replicates are used for initial and baseline whole

effluent toxicity test.

Effluent

aAdd 10mLtocupsAandB

for the high concentration.

0

l Add 10 mL to next A and B cups

for the second high concentration.

Add 10 mL to each

replicate except in

the high concentration

56

Mix

Dilution

Water

6B

Control

High

Cont.

* Serial Dilutions m

Waste

6A

Control
is equal to the concentration of the compound present

in the effluent divided by the LC50 of the compound

(EPA, 1991 B). For example, if the LC50 of an effluent

is 25%, the effluent contains 4 TU (100/25). If the 48-h

LC50 of compound A is 3 mg/L, a solution of 1 mg/L of

this compound contains 0.33 TU. By normalizing the

concentration term (such as the LC50) to a unit of

toxicity, the TU allows the toxicity of effluents and/or

chemicals to be “summed,” provided that the test length

and species used are the same in every test. This

cannot be done using LC5Os because chemicals and

effluents each have different toxicity, and different concentrations each equal one LC50. Phase III contains

more discussion about adding TUs; however one must

be cautious in summing them. Unless it is known that

the toxicants are strictly additive, simple summation of

TUs will be incorrect.

7.5 Feeding

Most species used in acute tests are not fed during

the test. However, the acute effluent manual (EPA,

1991 B) has modified the effluent tests to allow cladocerans to be fed before test initiation. We routinely add

food to all test waters (this includes the 100% effluent)

for all Ceriodaphnia and Daphnia tests but only at the

initiation of each test. This practice is standard in Phases

I, II, and III. However, the decision to feed will be

species specific and dependent on the characteristics

of the effluent. Consistency throughout each phase of

the TIE is most important. All tolerance data for

Ceriodaphnia given in Section 8 are based on tests in

which animals were fed the yeast-cerophyll-trout food

(YCT) mixture (EPA, 1989E: EPA, 1991C). The amount

of YCT added was 66 uL of YCT per 10 mL and 5

animals.

7.6 Multiple Species

A useful technique is to test two species together in

the same test chamber (e.g., 1 oz. plastic cup). This is

very beneficial in the initial toxicity test in order to select

the most sensitive species for the Phase I tests or in

situations where two species appear to be responding

to toxicity of the effluent differently. This type of test

also can be useful when conditions in tests with different organisms vary independently. For example, testing

C. dubia and fathead minnows ~48 h old) together

under the same pH conditions is very, helpful in evaluating the role of ammonia in an effluent’s toxicity. By

testing the species together, the experimental conditions may change but both species experience identical

fluctuations. We have tested the following sets of species together: C. dubia and fathead minnows, C. dubia

and D. magna, and C. dubia and D. pulex. B

7-5
Section 8

Phase I Toxicity Characterization Tests

The first phase of a TIE involves characterization of

the toxic effluent. The characterization information gathered in Phase I forms the basis and direction for Phase

II identification of the specific toxicants or may be

useful for treatability evaluations. In Phase I, simple

manipulations for toxicity removal or alteration are performed on the whole effluent. Acute toxicity tests utilizing aquatic organisms are used to determine whether

the toxic chemicals have certain physical or chemical

characteristics. Two objectives are accomplished during the toxicity characterization phase: a) the physical

and chemical characteristics of the toxicant are

broadly defined and b) some information is gathered to

indicate whether the toxicants are similar in effluent

samples taken over time. Several patterns of Phase I

results are indicative of certain toxicants (See Section

9.4) but otherwise Phase I only provides evidence of

characteristics of groups of chemicals that may be the

toxicants. This information can subsequently be used in

the second phase of the study, either in the development of bench-scale wastewater treatment processes

(EPA, 1989A; 19898) or in choosing separation and

analytical procedures for toxicant identification as described in Phase II.

The tests described in this section are designed

primarily for acutely toxic effluents. Methods for chronic

toxicity are being developed (EPA, 1991 A). The methods in this section are based on the use of small test

organisms such as daphnids (Ceriodaphnia, Daphnia)

and newly hatched fish (fathead minnows). If larger

species are used, modifications to these methods will

have to be made.

Analysis of samples should begin as soon as practical following collection. Until experience is gained with

the effluent, there is no way to predict how long samples

can be stored before substantial changes in toxicity

occur. In transit and in the laboratory, the bulk effluent

should be held below 4°C and kept headspace free.

Minimizing the headspace for samples shipped in glass

is not practical. Once in the laboratory, testing on individual samples of each effluent may continue indefinitely, provided that whole effluent toxicity stabilizes.

The degree of toxicity can remain similar, while the

cause of toxicity may change with age. Especially in

the early stages of the TIE, fresh samples should be

used regardless of toxicant stability. The degree to

which any single sample is analyzed should be weighed

against the cost of the analyses and the probability that

the sample is an adequate representation of typical

effluent. Obviously, when several samples show that a

single class of compounds is responsible for effluent

toxicity, Phase II procedures should be initiated.

Each of the characterization tests described in Section 8 is designed to change the toxicity of groups of

constituents (Figure 8-l). Toxicity before and after the

characterization treatment will indicate for which groups

the toxicity was changed. All but one (initial toxicity

test) of the characterization tests is performed at the

same time in order to minimize confounding effects

resulting from degradation of sample toxicity over time.

While it is not critical that each characterization manipulation be performed at exactly the same tipe, the

toxicity tests should be initiated at approximately the

same time. If more than one species is used, the

Phase I results must be interpreted separately for each

because at this stage one cannot tell whether the same

toxicant is involved for all species.

Following receipt of the effluent sample, various

steps to initiate Phase I are done (Table 8-l). Day 1 is

when the sample arrives in the laboratory. On day 1,

initial routine chemical measurements are taken for the

effluent sample and an initial toxicity test is started on

an aliquot of the sample. This LC50 is used to set the

desired exposure concentrations for subsequent Phase

I toxicity tests and is referred to as the “initial” toxicity

test to distinguish it from the “baseline” toxicity test

described below. Other aliquots of the sample are adjusted to pH 3 and 11, filtered, aerated and/or

chromatographed using a C,, SPE column. Following

these manipulations, each effluent aliquot is readjusted

to the initial pH (pH i ) of the effluent. By pH i, we

generally refer to the pH of the effluent at arrival in the

laboratory, which may or may not be the pH of the

effluent at air equilibrium. These aliquots and the remainder of the effluent are then covered to minimize

evaporation and held at 4OC overnight. However, upon

warming the solutions, supersaturation from dissolved

gases might occur. If the test organism to be used is

sensitive to supersaturation, then the supersaturation

must be removed. Generally, Ceriodaphnia are not

very “sensitive” to such situations, unlike newly hatched

fathead minnows.

Delaying the majority of the toxicity testing until the

next day (day 2) allows the test exposures to be set at

concentrations bracketing the 24-h LC50 of the day 1

initial toxicity test. This procedure also allows pH adjusted effluent aliquots more time to stabilize, and additional pH adjustments can be made as necessary.

8-l
Figure 8-1. Overview of Phase I effluent characterization tests. (Note: pH i stands for initial PH.)

t

-r

- Toxic Effluent Sample

t I I

1

EDTA

Chelation

Test (Day 2) 1

Cl8 Solid Phase

Extraction Tests I

I Oxidant

Reduction

Test (Day 2)

Graduated pH

Tests (Day 2)
Table 8-1. I effluent manipulations

Description Section

DAY 1 SAMPLE ARRIVAL:

Chemical analyses

l PH

*conductivity

*total residual chlorine (TRC)

*hardness

*temperature

*total ammonia

*dissolved oxygen (DO)

*alkalinity

6.0

Initial toxicity test

Sample Manipulations’:

l pH adjustment (pH 3, pH i, pH 11)

l pH adjustment/filtration

l pH adjustment/aeration

l pH adjustment/C,, solid phase extraction

DAY 2 TOXICITY TESTING:

8.1

8.3-8.9

8.3

8.4

8.5

8.6

Warm effluent samples from day 1 and set-up

toxicity tests’

*baseline toxicity

l pH adjustment samples

*filtration samples

*aeration samples

l C,,, solid phase extraction samples

*sodium thiosulfate addition samples

l EDTA addition samples

*graduated pH samples

Read 24-h mortality on initial toxicity test

DAYS 3 AND 4 MONlTORlNG TESTS:

8.2

8.3

8.4 .

8.5

8.6

8.7

8.8

8.9

8.1

Read 48 h mortality initial toxicity test 8.1

Read 24 h and 48 h mortality on tests from day 2 8.2-8.9

11 These manipulations and toxicity tests can be performed on day

2 after the presence of toxicity has been confirmed; see text for

details.

However, the manipulations can be performed and the

test initiated the next day (day 2) rather than on sample

arrival. This is useful when the toxicity of the effluent is

unknown, and prevents conducting a Phase I on nontoxic samples. It is important that sufficient time is

allowed so that the pH adjusted samples can stabilize

at the pH i. The following sections assume the manipulations were made on day 1.

On the second day the aliquots of whole and

manipulated effluent prepared on day 1 are diluted to

4x-, 2x-, 1 x-, and 05x the 24-h LC50 of the effluent and

subsequently tested for toxicity. This dilution series is

used so that for highly toxic effluents, smaller changes

in toxicity can be detected than would be the case if

100% effluent was used. (See Section 9 regarding

multiple toxicants and this dilution series.) A whole

effluent toxicity test is begun using unaltered effluent,

now 24 h old. The result of this test (and subsequent

whole effluent tests) is referred to as the “baseline”

effluent LC50. Other toxicity tests involving the addition

of chelating or reducing agents and less severe pH

adjustments are also conducted. For the EDTA addition

test, the time needed for the EDTA to complex any

metals present may be a function of the matrix of the

effluent. Therefore, the addition of EDTA should be

made first on day 2 and the sample held until all other

manipulations are complete before introducing the test

organisms (see Section 8.8 for details on EDTA test).

For one complete “Phase I” of a TIE as described

in this section, there are nine categories of toxicity tests

that are conducted. These are as follows: initial toxicity

test, baseline test, pH adjustment test, pH adjustment/

filtration test, pH adjustment/aeration tests, pH adjustment/C,, SPE test, EDTA addition test, sodium thiosulfate addition test, and graduated pH test. Toxicity test

results are read on subsequent testing days and depending on the outcome of the Phase I test series,

additional toxicity tests designed to further define or

confirm the nature of the toxicants are conducted. ”

For an experienced analyst the amount of time

required to conduct the sample manipulation tasks

scheduled for day 1 is about half of one day. If at 24 h,

less than 50% mortality of test organisms exposed to

the 100% day 1 effluent has occurred, the sample can

be discarded and a new sample collected with relatively little loss of resources or time. For this reason,

waiting to perform the manipulations on day 2 is useful.

Alternatively, the test can be continued to 48, 72 or

96 h at which time the effluent may produce an LC50.

In such cases, the baseline toxicity tests prepared on

the second day (day 2) following sample arrival are set

up at exposure levels of 1 OO%, 50%, 25%, 12.5%,

6.25% effluent.

For a highly toxic effluent sample with rapidly degradable toxicants, it may be prudent to override the

use of 4 x -24-h LC50 treatment level and opt for conducting the Phase I using 100% effluent. These rapidly

degradable compounds will be discovered only through

periodic testing as the sample ages.

Several Phase I characterization tests are relatively

broad in scope, intended to include more than one

class of toxicant. Therefore, if a significant change in

effluent toxicity is seen following these characterization

procedures, additional tests are needed to further delineate the nature of the toxicity. The amount of testing

beyond the initial characterization of the sample will

depend on the stability of effluent toxicity, the nature of

the toxicity, and previous Phase I results for the effluent

(i.e., observed trends in the nature of the toxicity). A

“significant reduction” in toxicity between aliquots of the

day 2 whole effluent (baseline LC50) and treated effluent must be decided based upon the laboratory’s test

precision. Usually a change in the LC50 equal to one

concentration interval can be considered significant but

when precision is good smaller differences can be

used. This suggestion is arbitrary and should not replace good judgement and experience. None of these

tests by themselves are conclusive, so the danger of

type I or type II errors is not great. Experience has

8-3
shown that for many effluents, at least one Phase I

characterization test will be successful in substantially

altering effluent toxicity. If not all toxicity is removed,

other groups of toxicants (not addressed by Phase I

procedures) may be present in the effluent or a single

toxicant may be present in the effluent at such high

concentrations that only partial toxicity removal is

achieved. Additional testing to resolve these findings

involves applying the successful Phase I test at a

higher level (i.e., increased degradation time, increased

aeration, larger C,, SPE column volume, increased

reagent concentratrons).

Another outcome of the Phase I characterization

test series may be that several tests succeed in partially removing effluent toxicity. In this situation, one

may be dealing with several toxicants, each with different physical/chemical characteristics, or a single toxicant of such a nature as to be removed by more than

one Phase I test. These results may be resolved by

treating a single aliquot of the sample with all of the

characterization tests that significantly reduced the

baseline toxicity of the effluent. If effluent toxicity removal is enhanced as compared to the reduction provided by individual characterization tests, the sample

may contain more than one type of toxicant. If the final

toxicity removal at the end of the series of characterization tests is approximately the same as that provided

by the most efficient single Phase I test, then it is likely

that all of the test methods involved are successful in

reducing the same toxicant to varying extents. This

outcome is also suggested when one or more Phase I

tests completely remove toxicity while some number of

other tests partially reduce toxicity. Phase I tests overlap somewhat in their abilities to remove groups of

toxicants. For example, increasing pH may cause a

metal to precipitate and toxicity removed and EDTA

may also remove toxicity. In any case, results of this

nature are useful in selecting Phase II options. Use of

multiple manipulations (Section 9.2) builds upon these

principles. When several treatments are applied to the

same sample, tests must be designed to ensure that

toxicity does not result from the additives used (e.g.,

acid, base, EDTA) rather than from the effluent’s

toxicant(

The assumption must not be made that toxicants

are either additive or synergistic. Our experience shows

that independent action (one or more of multiple toxicants acting independently of the rest, as though the

others were not present) is not uncommon in effluents.

Experience also shows that one should not use selected tests to confirm a suspicion that a certain toxicant is the cause of toxicity. Time and again, this leads

to wasted effort. There are so many possible causes of

toxicity that such guesses are rarely helpful and more

often channel one’s thinking and delay the final solution. On the other hand, if one wants only to know

whether a certain chemical is the toxicant, these tests

can be selected to accomplish that goal. Frequently

one needs to know whether the toxicity is due to ammonia or whether there are toxicants present other

than salt. These questions are quite different from the

former case where one is playing the “I’ll bet you the

toxicant is...” game.

No Phase I characterization test should be dropped

from use on the basis that the toxicants it is designed

to address are not likely to be present in the effluent. In

excluding any Phase I test, the analyst may be limiting

the information that can be gained on effluent toxicants.

The investigator should approach effluent characterization without a preconceived notion as to the cause of

toxicity.

There are two types of checks that can be used to

detect artifact toxicity. A “toxicity blank” consists of

performing the same (Phase I) test on dilution water

and measuring to determine whether any toxicity is

added by the test procedure. However, a toxicity blank

does poorly in identifying artifact toxicity if toxicity is

affected by the effluents’ matrix (cf., Section 7.1). For

example, the toxicity of the Phase I reagent, EDTA,

may be completely different in dilution water and in

effluent. If so, a toxicity blank is inappropriate for the 4

chelation test. A “toxicity control”, for many Phase I

steps involves a comparison of the toxicity of the manipulated test solution and the baseline effluent twicity.

In this case, the comparison must demonstrate that the

manipulated effluent test solution has not become more

toxic than the unaltered effluent (baseline test). If it has,

the test procedure has produced artifactual toxicity.

The “toxicity control” for the pH adjustment/C,, test is

the filtered effluent sample at the respective pH. For

some treatments, valid toxicity blanks or toxicity controls cannot be made. The use of toxicity blanks and

toxicity controls still requires the use of “regular” controls, which are always included to determine the performance of the test organism and dilution water. Dilution water blanks for the EDTA addition test, sodium

thiosulfate addition test, and the graduated pH test are

not relevant (see Section 7.1 for more information).

No procedure should be assumed to be free of

artifactual toxicity. Many of the Phase I toxicity tests

involve relatively severe or unorthodox effluent manipulations. Toxicity blanks and toxicity controls must be

used consistently and conscientiously to detect the

introduction of toxic artifacts or other changes to the

effluent that increase sample toxicity.

For the following sections, the guidance for the

volumes required, apparatus, and test organisms is

based on test conditions using Ceriodaphnia, Daphnia

and/or larval fathead minnows exposed in 10 mL test

volumes.

8. I lnitial Effluent Toxicity Test

Principles/General Discussion:

The major purpose of the “initial” effluent test is to

provide an estimate of the 24-h LC50 for purposes of

setting exposure concentrations in Phase I tests.

Volume Required:

Initial toxicity test is performed in duplicate using 40

mL of effluent. I

0-4
Apparatus:

Disposable 1 oz plastic cups or 30 mL glass beakers, 

species.

Procedure:

7: mL in duplicate of 1 OO%, 50%, 25%, 6.25% effluent, and a

control will suffice for most effluents. Obviously more

toxic effluents will require a lower range. If nothing is

known about the toxicity, more concentrations should

be included. A sample data sheet for the initial test is

shown in Figure 8-2.

8.2 Baseline Effluent Toxicity Test

In order to determine the effects that the various

Phase I characterization tests have on effluent toxicity,

the toxicity of the effluent sample, prior to any treatment in the laboratory, must be determined. The portion of the effluent sample, tested for toxicity the day

after it arrives in the laboratory (day will be referred

LC50

the characterization tests. Such a comparison will demonstrate whether the removal or alteration of various

groups of toxicants changes the effluent toxicity. By

comparing these results, an indication of the physical/

chemical nature of the toxicants can be obtained. If the

LC50 is substantially different

from the toxicity of the effluent when it arrived in the

laboratory (initial toxicity), one must decide whether the

schedule suggested in these methods should be revised to reduce a delay in testing.

When Phase I testing is extended to additional

days, baseline tests must be done each time on succeeding days, and used for comparison to these additional manipulation tests.

Volume Required:

The baseline toxicity test is performed in duplicate.

The total volume necessary will depend, on the 24-h

LC50mL should

be adequate..

Apparatus:

Disposable 1 oz plastic cups or 30 glass beascope (optional).

Test Organisms:

Test organisms, 60 or more, of the same age and

species.

. .

Procedure:

Day 2: Two concentration series will be used in

duplicate for the static acute toxicity test. In preparing

the test solutions for the day 2 baseline test, any

obvious physical changes (e.g., formation of precipitates, odors), which occurred during storage, should be

noted.

The first test series will have exposure levels based

on the 24-h LC50 of the initial (day 1) toxicity and will

include day 2 effluent concentrations at 4x-, 2x-, lx,

and 0.5x- the 24-h LC50. In this case, the method for

making dilutions described earlier may need to be

changed slightly. Most of the toxicity tests with the

characterization solutions will also be performed using

these same exposure concentrations. If the 24-h LC50

of the initial effluent is greater than 25%, the series

obviously begins at lOO%, and includes four exposure

concentrations. Of course, if the 24-h LC50 of the day 1

initial effluent is greater than or equal to 25%, the

second series will be unnecessary because this test

fulfills the requirements for comparison to the initial

effluent test and characterization solution toxicity test

results.

The second test series will provide exposur s at

effluent dilutions of 1 OO%, 50%, 25%, 12.5% and 6.s5%

(and lower dilutions as appropriate if the effluent is

more toxic). This series will enable a comparison of the

results of the baseline (day 2) test to the initial effluent

LC50 (cf., Section 8.1).

A sample data sheet is shown in Figure 8-3. In

order to compare the baseline toxicity and the toxicity

of the effluent aliquots subjected to characterization

tests, all of the day 2 toxicity tests should have the test

organisms added to test solutions at approximately the

same time.

The baseline toxicity test (toxicity control) must be

repeated each time additional characterization tests are

performed on the sample after the initial Phase I battery of tests. The baseline test will serve as the basis

for determining the effects produced by the additional

characterization tests, and will also provide information

on the degradation of sample toxicity. For effluents

whose initial toxicity is low (i.e., LC50 -6O-70%) and

where the baseline toxicity is greatly changed compared to the initial toxicity of the sample, it may be

advisable to discard the remaining sample and collect a

fresh one.

Interferences/Controls and Blanks:

The control treatment of animals in unaltered dilution water in this test is used for comparison to several

subsequent tests and provides an important reference

for diluent water and organism acceptability. Mortality

in these controls will negate other work.

Results/Subsequent Tests:

Baseline LCSO’s should be generated for as long

as the effluent sample is being used and a baseline

test (toxicity control) should be started every time the

8-5
Figure 8-2. Example data sheet for initial effluent toxicity test.

Test Type: Initial Emumat

Test Initiation (Date & Time):

Investigator:

Sample Log No., Name:

Date of Collection:

Species/Age:

No. Animals/No. Reps:

Source of Animals:

Dilution Water/Control:

Test Volume:

Other Info:

Cont. 0 h

(Oh Effluent) 11 pH

12.5 II

6.25 II

-

r

24 h

A 8 pH 00

Survival Readings:

zqGz+K

Comments:

8-6

-

effluent sample IS put through any characterization steps.

(Note: similar procedures should be followed in Phases

II and III.)

8.3 pH Adjustment Test

Principles/General Discussion:

The pH has a substantial effect on the toxicity of

many compounds found in effluents. Therefore pH adjustment is used throughout Phase I to provide more

information on the nature of the toxicants. Changes in

pH can affect the solubility, polarity, volatility, stability

and speciation of a compound, thereby affecting its

bioavailability as weil as its toxicity. Before describing

the pH adjustment test, some discussion on the effect

of pH on various groups of compounds is warranted.

Two major groups of compounds significantly impacted by solution pH are acids and bases. To understand how organic and inorganic compounds of this

type are affected by pH changes, one must have a

basic understanding of the thermodynamic equilibrium

acidity constant, K,, for the proton transfer reaction:

HA + H,O = H,O+ + A*

Ka = [A-][H+]

WI

H+: H,O+

HA: protonated acid

Kp: thermodynamic equilibrium constant for

the acid

For example:

HCN + H,O = H,O+ + CNKa= [CN-][H+] = 6.0 x lo-lo

WNI

The stronger the acid (i.e., the more it tends to

dissociate into its ionic state), the greater the value of

K,, and the smaller the -log,,K, or pK. In effect, the

abo.ve reaction is shifted to the right for acidic compounds. For acids in water, when the pH of the solution

equals the pK, of the compound, equivalent amounts of

the compound will exist in the ionized (A) and unionized (HA) forms. At a pH one unit lower than the pK,

of the acid, approximately 90% of the compound will be

in the un-ionized form with the remainder in the ionized

form. A solution pH two units below the acid’s pKd will

result in 99% in the un-ionized form and 1% in the

ionized form. Likewise, at one pH unit above the pK,,

90% of the acid will be present in the dissociated

(ionized) form and 10% present in the un-ionized form;

at two pH units above the pKa, 99% of the acid is in the

dissociated form while, 1% is present in the un-ionized

form. For example, at pH 4.2, the pK, of benzoic acid,

50% of the compound is present as C,H,COOH and

50% is present at C H COO-,H+. At pH 3.2, this ratio

shifts to roughly 9O*L C H COOH:lO% C H COO-, H+

while at 5.2 the ratio ne”ar’s 10% C,H,CdOk to 90%

C,H,COO-,H’.

This relationship generally holds for diprotic and

triprotic acids (i.e., acids with two and three H atoms,

respectively, that can dissociate from the molecule).

This trend is not followed by multiprotic acids with pK,‘s

less than three units apart (e.g., H,BO, with pIQ13.8

and pK, *= 12.74). The amount of each dissociated species in such cases will not always follow the 90/i 0, 99/l

rule stated above. For example, H,BO,, H,BO,-, and

HBOa2- will be present at pH 13.5.

Basic compounds function in a similar fashion.

B + H,O = BH+ + OHK, = [BH;EH-1

B: unprotonated base

K,: thermodynamic equilibrium constant for the base

For example:

C,H,NH, + H,O = C,H,NH3* + OHK,= [C,H,NH,+][OH-] = 4.2 x lo-‘O

[C,yJq

5

In the above reaction, BH+ can be considered the

“conjugate acid of the base”, that is, the protonated

form of the base. Thus, the same reaction can be

expressed as follows:

BH++ H,O = H,O+ + B

K, = W’IPI

W’l

Note: pK,+ pK,= 14

For example:

C,H,NH,+ + H

conjugate acid

0 = H,O+ + C,H,NH,

Ka= [H+][C6HSNH2] = 2.34 x 1O-5

[C,H,NH,‘l

This convention can be used to simplify dealing

with equilibrium constants for acids and bases.

As with acids, when the solution pH is equal to the

pK, of the conjugate acid of a base, equal amounts of

the base will exist in the ionized and un-ionized forms.

For example, ammonia in an aqueous solution at pH

9.25 (the pK, of ammonia) will be found as 50% NH,+

and 50% NH,. At one pH unit above the pK, (i.e.,

10.25) roughly 90% of the ammonia will be in the unionized form (NH,) and the remainder will be in the

NH,+ form. At pH 8.25, one unit below the pK, of

ammonia, approximately 90% of the ammonia will be in

the NH,+ form, and approximately 10% will be in the

NH, form.

8-8
Figure 8-4.

4 6 8 IO I2 14

PH

-

pE -pH diagram for the CO,, H,O, and Mn-CO, systems (25°C). Solid phases considered: Mn(OH) (8)

(pyrochroite), MnCO,(s) (rhodochrosite), Mn,O,(s) (hausmannite), YMnOOH (manganite), YMna,

(nsutite). (Reprinted with permission from Stumm 81 Morgan, 1981.)

The above can be summarized by the following:

PY.

Orqanic Inoroanic

PH > PK

acid RCOO-, RCO- A

base RNH, B

pH < PK

acid RCOOH, RCOH HA

base RNH,+ BH+

R = aliphatic or aromatic group

The effect of pH on the ratio of the ionized and unionized forms of acids and bases has a number of

impacts on Phase I results. First, compounds may be

more toxic in the unionized form as compared to the

ionized form. For example, un-ionized ammonia (NH,)

is generally recognized as the toxic form of ammonia

while total ammonia (NH,+) is of far less concern (EPA,

19858). A second implication of this effect relates to

toxicant solubility. Unionized forms of acids and bases

can be considered less polar than their ionized forms,

which interact to a greater extent with water molecules.

Consequently, un-ionized forms of acids and bases can

be more easily stripped from water using aeration (Section 8.5) or extraction with non-polar solvents or solid

phase column techniques (Section 8.6). Likewise,

changes in compound solubility with pH change may

mediate removal through filtration (Section 8.4).

Another implication of the pH effect involves metal

ion complexes. An example of how pH can alter the

form of a metal in a natural water system is shown in

Figure 8-4. Given a p& (the equilibrium electron activity-in a simple sense, whether the system is aerobic

or anaerobic), one can see how various forms of manganese are created and eliminated as’ pH shifts.

Each of the different forms of a metal will be

manifested differently in aquatic organism effects. Some

forms of the metal will be relatively insoluble; these

forms may not affect toxicity. Likewise, as with acids

and bases, the toxicity of the soluble forms of the metal

will be a function of the actual species present (e.g.,

the LC50 of Mn2* as compared to the LC50 of MnO:).

The actual species formed will depend, in addition to

pH and p& on the other chemical constituents present

in the water. The hydrolysis rate of organics is greatly

affected by pH, and pH changes may also alter organic

toxicity.

Regardless of the speciation effect on toxicity,

changes in solution pH may affect the toxicity of any

given compound. The pH of the test solution may affect

8-9
membrane permeability at the cell membrane as well

as the chemistry of the toxicant. One might expect that

changing the pH, only to return it to its original pH in a

short time, would not alter toxicity. Experience shows

that this is not the case and that this adjustment sometimes results in reduction, loss or increase in toxicity. If

the kinetics of the pH driven reaction (on return to the

original effluent pH) are slow or irreversible, pH adjustment alone may be effective in evidencing toxicants

affected by pH change. Some organics may also degrade due to pH change.

Another purpose of the pH adjustment test is to

provide blanks (with both dilution water and effluent) for

subsequent Phase I pH adjustment tests performed in

combination with other operations. This test will demonstrate whether toxic concentrations of ions have been

reached as a result of the addition of acid and base or

whether the reagent solutions are contaminated.

Comparable results for toxicity blanks are not obtained when the same volumes and same strengths of

acids and bases are added to the effluent. Effluents

already contain substantial concentrations of major anions and cations that are not found in dilution water.

Further, the volumes and strengths of the acid and

base necessary, for example, to lower an effluent with

a pH i of 7.6 to pH 3 and raise it back to pH 7.6, are

not likely to result in the same final pH when added to

dilution water. However, it is necessary to conduct pH

adjustment blanks to determine the cleanliness of the

acid and base solutions used for the pH manipulations.

Volume Required:

To make pH adjustments, 680 mL of whole effluent

is needed to have enough effluent for the four exposure

concentrations at each of the three pH’s. A 300 mL

aliquot of the day 1 effluent sample is raised to pH 11,

and the second 300 mL sample is lowered to pH 3. An

aliquot of the pH i effluent (used for the baseline test:

80 mL) is set aside for the duration of the manipulation

(Figure 8-5). Approximately 30 mL will be needed for

the pH adjustment test but the actual amount depends

on the 24-h LC50 of the initial effluent test. The remaining 270 mL of each of these solutions is reserved for

the “pH adjustment/filtration”, “pH adjustment/aeration”

and “pH adjustment/C,B SPE” Phase I tests.

These pH adjustments must also be done using

dilution water for toxicity blanks for each test. To make

these adjustments, approximately 295 mL of dilution

water will provide enough (and an excess) to test the

blanks with one exposure level and one replicate. One

aliquot of 105 mL is adjusted to pH 3 and another 105

mL is adjusted to pH 11. Only 10 mL is needed for the

pH adjustment blanks but excess (~10 ml) is included.

The, pH i dilution water blank is the control of the

baseline test. The remaining 85 mL of pH adjusted and

85 mL of pH i dilution water are used for the “pH

adjustment/filtration,” “pH adjustment/aeration” and “pH

adjustment/C,, SPE” toxicjty blanks.

Apparatus:

six glass stoppered bottles for acid and base solutions, pH meter and probe, 2-500 mL beakers, 2-500 mL

graduated cylinders, 30 mL beakers or 1 oz plastic

cups, stir plate, and stir bars (perfluorocarbon), automatic pipette, disposable pipette tips, eye dropper or

wide bore pipette, light box and/or microscope (optional).

Reagents:

1 .O, 0.1 and 0.01 N NaOH, 1 .O, 0.1 and 0.01 N

HCL (ACS grade in high purity water) and buffers for

pH meter calibration.

Test Organisms:

Test organisms, 40 or more, of the same age and

species.

Procedure:

Day 7: The general procedure for the pH adjustment test is shown in Figure 8-5.

Blank Preparation: The first step is to prepare the

dilution blanks. These blanks are used as the controls

for the other dilution water pH adjustment testsof

aeration, filtration, and C,, SPE separation as well as to

determine whether the acid or base solutions are contaminated. The pH i blank is the control while the pH 3

or pH 11 adjustment blanks are treated in the manner

described below under sample preparation.

Sample Preparation: Stirring constantly, 1 .O N NaOH

is added dropwise to a 300 mL aliquot day 1 effluent

until the solution pH nears 11. (Note: overshooting

results in the addition of more salts and a volume

change and should be avoided.) In order to minimize

any over-adjustment of the pH, 0.1 N NaOH is added

dropwise in the latter stages to bring the effluent aliquot

to pH 11. The solution should be allowed to equilibrate

after each incremental addition of base. The amount of

time necessary for pH equilibration will depend on the

buffering capacity of the effluent. Caution should be

taken to prevent any solution pH of greater than 11. If

pH 11 is exceeded, 0.1 N HCI must be used to lower

the pH to 11. The goal of the pH adjustment step is to

reach pH 11, while minimizing both the change in

aliquot volume and the increase in ionic strength. Volumes and strengths of base (and any acid added)

should be recorded. A 30 ml volume of effluent and

dilution water is held for the same length of time it

takes to complete other Phase I manipulations with pH

11 effluent.

Once other manipulation work has been completed

with the total volume of the pH 11 effluent, the 30 mL

volume at pH 11 is returned to the initial pH (pH i ) of

the day 1 effluent. (The other aliquots of pH 11 effluent

are also returned to pH i at this time.) This is accomplished by the slow, dropwise addition of 0.1 N HCI first

and later 0.01 N HCL as the pH of the stirred solution

nears pH i. If pH i is exceeded, the pH must be

appropriately increased with 0.01 N NaOH. Again, the

8-l 0
.

n

-I

0

I

a

94

0

I

I

I

c

0. -

5

. .

.-

3

5

a

k

z

0 0
volumes and strengths of acid and any base added

should be recorded.

The pH of the solution should be checked periodically throughout the remainder of the work day and

readjusted as necessary. Changes in the total volume

of acids and bases added should be recorded.

This procedure is repeated, except the pH is lowered to pH 3 using the second 300 mL aliquot of

effluent, and 1 .O N and 0.1 N t-U. As with the pH 11

effluent, 270 mL of the pH 3 effluent is used for the pH

adjustment/aeration, pH adjustment/filtration, and pH

adjustment/C,, SPE tests. The remaining 30 mL of the

pH 3 effluent is held until all of the work on all of the pH

3 effluent has been completed. At this point, the pH of

the 30 ml_ volume of pH 3 effluent is readjusted to pH i

by the dropwise addition of 0.1 N and 0.01 N NaOH.

Maintenance of pH i must be assured through checking and readjusting the sample periodically throughout

the work day. All volumes and strengths of acid and

base added should be recorded.

Day 2: At the beginning of the work day (the day

after the arrival of the effluent in the laboratory), the pH

of both of the 30 mL volumes is again checked to

ensure that pH i has been maintained. Any additional

pH adjustments are made and the volumes of the acid

and/or base added are recorded. The acute toxicity of

each pH-adjusted solution is tested at 4x-, 2x-, lx-,

0.5x-LCSO (the 24-h initial LC50) as described in Section 7. Test solution pH should be measured in all

exposure concentrations and recorded at least every

24 h. A sample data sheet is shown in Figure 8-6.

Interferences/Controls and Blanks:

Controls prepared for the baseline toxicity test also

act as a check on the organisms, dilution water, and

test chambers for this test.

The baseline effluent test acts as a control for the

pH adjustment test, indicating whether the addition of

NaCl in the form of the acid and base has increased

effluent toxicity. This pH adjustment test acts as the

control for other Phase I tests entailing pH adjustment.

In addition to serving as a control for other pH adjustment tests, increased toxicity following pH adjustment,

not as a result of NaCl concentration, indicates a pH

effect on toxicity or contamination of acids or bases

(see below).

Results/Subsequent Tests:

If either the pH 3 or pH 11 adjustment effluent tests

have significantly greater toxicity than the baseline effluent test, two possible sources of toxicity exist: 1) the

ions (Na*, Cl.) added by the acid and base have resulted in a solution with an ionic strength intolerable to

the test organism; or 2) chemical reactions driven by

the pW change have not reversed upon readjusting to

pH i. Neither of these phenomena would be detected

through the use of a blank (dilution water). To help

resolve this situation, the NaCl LC50 values for common test organisms are provided in Table 8-2. The

minimum concentration of NaCl in the test solution (i.e.,

not including the concentration of NaCl originally present

in the effluent) can be calculated from the volumes and

strengths of the acid and base added and final solution

volume. The data in the table can be used only as a

rough guide, however, because the toxicity of sodium

chloride depends on the other anions and cations as

well as the total osmotic pressure exerted by the dissolved substances. The toxicity of the added NaCl is

best determined by adding that amount of NaCl directly

to the effluent and to see if the addition increased

effluent toxicity.

If either the pH 3 and/or pH 11 adjustment tests

indicate in a significant decrease in effluent toxicity, it

could result from volume changes by acid and base

additions or from chemical reactions driven by the pH

change that may not have been re-established or are

irreversible. To determine if the addition of acid or base

diluted the sample due to their volume addition, add a

volume of dilution water equivalent to the total volume

of acid and base originally added td the effluent volume. If a similar loss of toxicity in the diluted wastewater occurs, the pH adjustment test should be repeated

using more concentrated acid and base solutions.

_

A reduction or loss of toxicity may also bmhe

result of the degradation of toxicant at the altered pH

values. In some cases, the toxicity could also be increased if the degradation product is more toxic than

the original compound. Both organics and inorganics

can be so changed with a probable loss in toxicity.

Inorganic and organic substances may precipitate during the process of pH adjustment. The precipitated

chemical may or may not be the toxicant. The precipitated chemical (which most often forms with the pH 11

adjustment) removes from solution via the flocculation

process, suspended solids, microbial growth, and colloids, and via the adsorption process, metals and organics. If the process of precipitation seems to remove

toxicity, it is important to realize that the precipitating

chemical might not be the toxicant, but rather that the

toxicant may have been removed by the flocculation

and/or adsorption processes. In some cases, the precipitate may dissolve with the adjustment of the effluent

back to pH i. The removal of toxicity when dissolution

of the precipitate occurs should be evaluated carefully

since the toxicant might be unavailable and/or not

completely dissolved.

For most of the Phase I combination pH adjustment

tests (i.e., pH adjustment/filtration), the pH adjustment

test will act as an equivalent or ‘Worst case” toxicity

control for changes in test solution ionic strength and

volume. In effect, most of the operations applied to the

pH adjusted effluent in the following Sections (8.4-8.6)

will either not affect pH or will drive it closer to the pH i.

This may not be the case for the pH adjustment/aeration test, however. Because pH 3 and pH 11 must be

maintained throughout the aeration process and because the loss of

towards pH i, more

these test solutions

only solutions.

volatiles may result in pH shifts

acid and/or base may be added to

as compared to the pH adjustment

8-l 2
Figure 8-6. Example data sheet for pH adjustment test.

Test Type: pH A@dment

Date of Collection:

Test Initiation (Date & Time):

Investigator:

Sample Log No., Name:

Other Info:

Species/Age:

No. Animals/No. Reps:

Source of Animals:

Dilution Water/Control:

Test Volume:

pH/Concentration 0 h 24 h

(Oh pH adjusted

effluent) pH A I-pH DO

/I 3/4x-LC50 II II

/I 3/2x-LC50

II 3/1x-LC50 II II

II 3 IO. 5x-LC50 II /I

II 3jblank II II

II 11/2x-LC50 II II

Survivai Readings:

48 h 72 h

II

96 h

Note: See baseline data sheet for controi data.

Volumes and Strength of Solutions Added:

HCI NaOH

-

300 ml pH 3

300 ml_ pH 11

30 mL pH 3

30 ml_ pH 11

Comments:

8-13
Table 8-2. Acute toxicity of sodium chloride to selected aquatic organisms

Water UfeSpecies Type 24 h

LCSO (g/L) (95% Cl)

stage 48 h 72 h 96-K

Ceriodaphnla dubia SRW’2 124 h

SRW’ 2 124 h

SRW’ 2 124 h

SRW12 524 h

VHRW’,2 524 h

Daphnia magna

Pimephales promelas

Lepomis macrochirus SRW’

NR

NR

524 h

524 h

11 wk

s24 h

l-9 g

0

iii

(2.012.6)

2.8

(-_)

3.3

(NV

6.4

(NR)

7.9

(7.0-9.0)

(4.$8,

(;;I

(-1

NR

(2.2z.6)

2.7

(27)

\FJ

(2.0-2.6)

2.8

(-_)

3.1

(NR)

(:I$

7.9

(7.0-9.0)

(4 $6)

(4ii.6)

NR

6.9

NR

4.6

‘2.8;47.3’

(7.4-7.9)

(2.ii.7,

12.9

(NW

’ Data generated at ERL-Duluth.

water (DMW)).

2 Static, unmeasured test.

3 Dowden and Bennett, 1965.

C. dubia were 424 h old at test initiation and fed. Water used was soft reconstituted water (diluted mineral

* Data generated at ERL-Duluth and values represent those from 7-d fathead minnow growth and survival tests and daily renewals.

5 Adelman et al., 1976.

* Pat&k et al., 1968.

Note: (-) = Confidence interval cannot be calculated as no partial mortality occurred. NR = Not reported; SRW = soft reconstituted water;

VHRW = very hard reconstituted water; RW = reconstituted water; LSW = Lake Superior water.

There is another factor which must be considered

when carrying out pH adjustment tests. In those manipulations where the pH is changed to pH 3 or pH 11

and then readjusted to pH i, the pH may tend to drift

over the course of the 48-h or 96-h toxicity tests. The

drift can be very dissimilar among test manipulations.

This is likely to occur even though the starting pH’s (of

samples readjusted to pH i ) may be similar. This can

lead to confusion in interpreting Phase I results if a

compound whose toxicity is pH dependent is present in

the sample. An example of a manipulation in which this

effect is encountered routinely is the pH 3 adjustment/

aeration test (Section 8.5). For instance, an aliquot of

an effluent with a pH i of 7.5 is adjusted to pH 3 and is

kept at that pH while the other manipulations are conducted. This pH 3 adjusted sample serves as a control

.for the pH 3 adjustments. Another portion of the pH 3

adjusted effluent is aerated (Section 8.5). Both aliquots

are then readjusted to pH i (75) prior to toxicity testing. The pH of the pH 3 adjustment/aeration test solution will probably not behave in a similar manner to the

baseline or pH 3 adjustment test. We have obsen/ed

the pH in this test to go unchanged or drift downward

after adjustment up to pH i of 7.5 over the course of the

toxicity test. However, the pH of the effluent in the

baseline test and the pH 11 adjustment test may drift

upwards over the course of the toxicity test, from pH i

(7.5) to as high as pH 8.5. By the end of the test, the

analyst may be confronted with interpreting the results

of tests conducted at different pH values. If a compound whose toxicity is dependent upon pH (e.g., ammonia) is present in the sample (cf., Section 8.9 for a

discussion of the effects of pH on ammonia toxicity),

the fact that pH either did not change, or even drifted

down in the pH 3 adjustment/aeration test sample (relative to the baseline and/or pH 3 adjustment test), can

complicate interpreting the test results. If ammonia were

present (which is less toxic at a low pH), the sample

would appear to have lost toxicity in the pH 3 adjustment/aeration test, when the loss in toxicity may have

been the result only of the differences in pH drift during

the toxicity tests.

The pH 3 adjustment/aeration test is not the only

manipulation that may cause differential pH drift over

the course of the toxicity test. Virtually all the manipulations in Phase I have the potential to cause this effect.

For example, with some effluents any pH 3 manipulation (Le., pH adjustment, aeration, or filtration) followed

8-l 4
by readjustment to pH i, will cause pH to behave

differently than the pH of the baseline test. Similarly,

the pH 11 manipulations (i.e., pH adjustment, aeration,

or filtration) can cause similar fluctuations, but they do

not seem to occur as frequently as with the pH 3

manipulations. Another manipulation that causes this

differential pH dnf-t is passing effluent over the C,, SPE

column at pH i, pH 3 or pH 9 (Section 8.6). Although

not as drastic as some of the effects observed with the

pH 3 adjustment/aeration tests. the sample collected

after passing the effluent over the C,, SPE column may

have a slightly lower pH by the end of the toxicity test

than the pH of the baseline test (e.g., pH 8.2 as opposed to pH 8.5). A final manipulation that has the

potential to cause acidic pH drift is the addition of

EDTA; details of this pH fluctuation are elaborated in

Section 8.8.

Although ammonia is a commonly encountered

sample toxicant whose toxicity is pH dependent, it is

not the only compound whose toxicity can be affected

by different test pH’s (cf ., Phase II). We have observed

pH dependence with several metals and the effects of

differential pH drift after various Phase I manipulations

should be considered. pH should always be monitored

and recorded whenever mortality readings are made

(e.g., 2 h, 24 h) as well as at the end of the test. It is

particularly important to record pH of the concentrations that determine the LC50, especially if greater than

5 mg/L of total ammonia is present in the sample.

Differential pH drift after manipulations can be overcome by closely monitoring the test pH, and adjusting

the pH in the manipulated samples to match the pH of

the baseline toxicity test. These adjustments are done

before animals are introduced.

8.4 pH Adjustment/Filtration lest

Principles/General Discussion:

The filtration experiment provides information on

effluent toxicants associated with fitterable material. Toxic

pollutants associated with particles may be less biologically available. However, aquatic organisms can be

exposed to these pollutants through ingestion of the

particles. This route of exposure may be significant for

cladocerans and other filter feeders ingesting bacterial

cells and other solids with sorbed toxicants. The degree to which any compound exists sorbed or in solution depends on a number of factors including particle

surface charge (or lack thereof), surface area, compound polarity and charge, solubility and the effluent

matrix. By filtering particles from the effluent, an immediate cause or a sink of toxic chemicals may be removed.

In addition to determining the effect of filtration on

the toxicity of the whole effluent, the effects of pH

adjustment in combination with filtration are also assessed with this manipulation. As discussed in Section

8.3, changes in solution pH can result in the formation

of insoluble complexes of metals (Figure 8-4). Similarly,

organic acids and bases existing in ionic form can be

transformed into the non-ionic form by pH adjustment.

Shifts in effluent pH can also act to drive dissolved

toxicants Onto particles in the effluent (e.g., shifting the

dissolved/sorbed equilibrium away from the free form).

Changes in toxicant polarity resulting from solution pH

change can make some particle/toxicant interactions

stronger. In other cases, the increase in effluent ionic

strength resulting from the shift in pH may force nonpolar organic compounds onto uncharged surfaces to a

greater extent.

By filtering pH adjusted aliquots of effluents, those

compounds typically in solution at unadjusted pH but

insoluble or associated with particles to a greater extent at more extreme pH’s, are removed. By removing

the toxicant-contaminated particles or precipitated compounds prior to readjustment of the sample to pH i,

these toxicants are no longer available for dissolution in

the effluent. The pH change may also destroy or dissolve the particles, thereby removing the sorption surfaces or driving the dissolved/sorbed equilibrium in the

opposite direction.

Positive pressure filtration is recommended. Use of

a vacuum to draw the effluent sample through the filter

may result in a loss of volatile compounds by de.gassing the solution during filtration. This problem is p@entially worsened in pH adjusted effluents if toxicants

become more volatile as a result of pH changes. If

vacuum filtration is used and effluent toxicity is reduced, subsequent tests must be performed to define

the nature of the toxicity loss. In this filtration step,

whether pressure or vacuum filtering is done, it is

important to avoid stainless steel housings for either

the pH 3 or pH 11 adjustment tests. Teflon, plastic or

glass equipment does not have the associated toxicity

that the stainless steel has under acidic or basic conditions.

The pH adjustment/C,, SPE test (Section 8.6) requires the use of filtered effluent. Without knowledge of

the effect of filtering on the effluent toxicity, it is not

possible to tell whether or not the SPE column or the

filtration removed the toxicity. Filtering may also be

useful in connection with other Phase I tests.

Volume Required:

A 235 mL aliquot of pH i effluent is filtered. Also,

235 mL each of pH 3 and pH 11 effluent aliquots

(Section 8.3) are filtered. The remaining 35 mL of each

solution is reserved for the pH adjustment/aeration tests.

A maximum volume of 30 mL of each of these three

solutions is needed to perform the filtration toxicity

tests. The exact effluent volume required for the toxicity

test will be a function of the effluent toxicity (Section 7).

Each test (pH 3, pH i, pH 11) requires four exposure

concentrations (10 mL each). The remaining filtered

effluent volumes (+200 mL) of pH 3, pH 11, and the

pH i solutions are each resewed for the C,, SPE tests

(Section 8.6). Excess volume has been Included to

cover losses occurring during the filtration operation.

For the blanks, 85 mL of dilution water is needed

for each pH test, of which 50 mi will be used in the pH

adjustment/SPE test.

8-15
Apparatus:

Six 250 ml graduate cylinders, six 250 mL beakers, six 50 mL beakers, pump with sample reservoir,

teflon tubing, in-line filter housing, ring stands, clamps.

Alternatively, vacuum flask, filter stand, clamp, vacuum

tubing, water aspirator or vacuum pump. Glass-fiber

filters (nominal size of 1 .O pm, without organic binder),

stainless steel forceps, glass stoppered bottles for acid

and base solutions, pH meter and probe, stir plate,

perfluorocarbon stir bars, automatic pipette, disposable

pipette tips, eye dropper or wide bore pipette, 30 mL

beakers or 1 oz plastic cups, light box and/or microscope (optional).

Reagents:

Solvents and high purity water for cleaning pump

reservoir and filter, 1 .O, 0.1, and 0.01 N NaOH, 1 .O, 0.1,

and 0.01 N HCI (ACS grade in high purity water),

buffers for pH meter calibration.

Test Organisms:

Test organisms, 75 or more, of the same age and

species.

Procedure:

Day 7: First, the filters must be prepared. These

steps are outlined in Figure 8-7. After the filters are

prepared, the dilution water at the appropriate pH is

filtered and collected for the toxicity blanks. Finally the

effluent samples at each of the three pH’s are filtered

(Figure 8-8). Use of glass-fiber rather than cellulosebased filters should minimize the adsorption and loss of

dissolved non-polar organic compounds from the effluent sample. Adsorption of toxic dissolved compounds

onto the filter or onto particles retained by the filter can

lead to spurious results.

filter Preparation: To prepare the 1 .O pm glassfiber filter for use, wash two 25 mL volumes of high

purity water through the filter. For the pH 3 effluent

filtration test, the filter must be washed with high purity

water adjusted to pH 3 using HCI. Likewise, the filter

used with the pH 11 effluent sample must first be

washed with high purity water adjusted to pH 11 using

a concentrated NaOH solution. Washing the filters with

water adjusted to the same pH as the effluent should

prevent sample contamination with water soluble toxicants contained on the filters.

Blank Preparation: The next step is to prepare filter

blanks using dilution water (Figure 8-7). These blanks

are used to detect the presence of any water soluble

toxicants which may remain on the filter following the

washing process. The pH i filtration blank is simply

prepared by passing 50 mL of dilution water (where the

pH is unadjusted) through a washed filter. The filtered

dilution water is collected and 30 mL of this volume is

reserved for the post-C,, SPE column toxicity blank

(Section 8.6). The remaining 20 mL is used as a filtration toxicity blank. Again, excess is included to cover

any possible loss during rinses.

To prepare the pH 3 filtration blank, 105 mL of

dilution water is adjusted to pH 3 with HCI, caution

being. taken to minimize the increase in dilution water

ionic strength. Of the pH 3 adjusted dilution water, 20

mL is for the pt-l adjustment only test, 35 mL is reserved for use as a toxicity blank in the aeration test

(Section 8.9, and 50 mC of pH 3 dilution water is

passed through a filter previously washed with pH 3

rinse water. The filtered pH 3 dilution water is collected

and 30 mL of this volume is reserved for the pH 3

filtration/C,, SPE toxicity test blank. The remaining 20

ml is readjusted to the initial pH of the dilution water

using NaOH, again taking care not to exceed the initial

pH of the dilution water during the readjustment process. This solution is used in a single exposure toxicity

test as the filtered pH adjustment toxicity blank.

The pH 11 toxicity blank sample is prepared in a

similar fashion using 105 mL of dilution water adjusted

to pH 11 with NaOH. Of the pH 11 dilution water, 20

mL is reserved for use in the pH adjustment only test

and 35 mL for the aeration test. The remaining volume

(50 mL) is filtered using the filter previously washed

with pH 11 rinse water and 30 mL of the filtered pH 11

d,ilution water is collected for use as the pH 11 filtration/

C,, SPE blank. The remaining 20 mL is readjusted to

the initial pH of the dilution water with HCI and used as

the pH 11 filtered toxicity blank using a single exposure.

_

Sample Preparation: The same filter(s) used to

prepare the pH i (or pH 3 or pH 11) dilution water

filtration blank(s) is now used to filter the pH i (or pH 3

or pH 11) effluent. First, a 235 mL aliquot of the pH i

effluent is passed through the pH i prepared filter and

collected; of which 200 mL is reserved for the C,, SPE

test. The remaining volume (approximately 30 mL) is

held for the pH adjustment/filtration toxicity tests.

Now, using the same filter used to prepare the pH

3 filtration blank, 235 ml of pH 3 effluent (see Section

8.3) is filtered and collected. The filtered pH 3 effluent

is split into two aliquots (200 mL and approximately 30

mL). The 200 mL aliquot is used in the pH 3 adjustment/C,, SPE test. The 35 mL filtered aliquot is readjusted to pH i using NaOH. Care must be taken to

minimize both an increase in aliquot volume and ionic

strength. The pH readjusted 35 mL aliquot is held for

the toxicity testing.

Finally, the filtration step is repeated using 235 mL

of the pH 11 effluent (Section 8.3) and the filter originally used to filter pH 11 dilution water. Again, 200 mL

of the pH 11 filtered effluent is used in the pH adjustment/C,,, SPE test and the filtered sample (approximately 30-35 mL) is readjusted to the pH i of the

effluent with HCI and used to conduct a toxicity test on

day 2.

In filtering effluent samples with high solids content,

it may be necessary to use more than one filter for the

235 mL of effluent. If so, the filter preparation step must

-

8-16
Figure 8-7. Overview of steps needed in preparing the filter and the dilution water blanks for the filtration and/or the C,, solid phase extraction column tests.

Preparing

the Filter

Day 1

Preparing

Dilution

Water

Blanks

__

I

Day 2

.-

NaOH

300 mL High Purity Water

HCI -

J

150mLofpH3 1

ti

Filter

1 Discard water 1

50 mL of pH i(unadj.)

(Dilution1

‘P+cNaOH

50mLofpH 11

.

Filter

Discard water

t- HCI

u50mLo;pH3 t 50mLofpH i

t l

prepared filter

NaOH --+ t

v

Through C18

SPE Column

IIC NaOH HCI -+ + - HCI

.

be repeated to provide additional filtration blanks or

several filters can be prepared at one time by stacking

them together in the filter housing. Alternativeiy, it may

be possible to centrifuge samples high in ‘suspended

solids and filter the supernatant through a single filter. If

this option is taken, the toxicity of the supernatant must

be tested and compared to the toxicity in the baseline

effluent test.

In the above procedures, either separate effluent

filtration systems should be used or the filtration system

must be cleaned between pH adjusted aliquots to prevent any carry-over of toxicity or particles. This means

all equipment should be thoroughly rinsed with 10%

HNO,, acetone, and high purity water between aliquots

of effluent with the exception of stainless steel equipment where dilute solutions should be used.

The pH of the pH adjusted blanks and effluent

aliquots, designated for day 2 toxicity tests, should be

checked periodically throughout the work day. Adjustments should be made as necessary in order to maintain the pH i of these solutions.

Day 2: Prior to initiating the toxicity tests, the pH of

the pH 3 and 11 blanks and filtered effluent aliquots

should be measured and readjusted to pH i. Toxicity

tests performed on all three (pH 3, pH i, and pH 11)

filtration blanks involve testing without dilution. Based

on the 24-h initial LC50 of the day 1 effluent, toxicity

tests performed on the effluent aliquots filtered at pH 3,

pH 11, and pH i are set up at 4x-, 2x-, 1 x-, and 0.5xLCSO as described in Section 8.2. Measurement of

exposure pH should be made daily on concentrations

around the mortality and the highest tested concentration, concurrently with survival readings. A sample data

sheet for the filtration tests is shown in Figure 8-9.

Interferences/Controls and Blanks:

Controls prepared for the baseline toxicity test serve

as a check on the quality of organisms, dilution water

and test conditions. Results of the pH adjustment test

(Section 8.3) will indicate whether or not toxic levels of

NaCl have been produced through pH adjustment only.

Results of the effluent filtration tests at each pH

should be compared with the filtration dilution water

toxicity blank performed at the corresponding pH to

determine the validity of the toxicity test outcome. No

significant mortality should occur in any of the filtration

blanks. If unacceptable mortality of organisms occurs in

either the pH 3 or pH 11 adjusted filtration blanks,

further investigation will be necessary to determine

whether lethality resulted from toxicants leached from

the filter at pH 3 and/or pH 11, or whether the increase

in dilution water ionic strength (via acid and base addition) is responsible for the problem. Additionally, if the

pH 3 and/or 11 filtration, aeration (Section 8.5) and CI,

SPE (Section 8.6) dilution water blanks have approxt-

’ mately the same final concentration of acid and base,

any ionic strength related toxicity should also be detected in them.

If a filtration toxicity blank shows unacceptable acute

toxicity but the corresponding filtered effluent is equally

or less toxic than the baseline toxicity test, it is possible

that the dilution water toxicity blank removed the final

traces of toxic filter artifacts. In some cases, the effluent matrix may have also prevented the artifacts from

leaving the filter or masked their presence. Alternatively

the observed filtered effluent toxicity may represent the

net effect of toxicant removal via filtration plus contamination by filter artifacts.

Results/Subsequent Tests:

The LCSOs for the aliquots of pH 3, pH i and pH 11

filtered effluent are compared to the baseline effluent

LCSO to determine whether any of these processes

resulted in a significant change in effluent toxicity.

If toxicity can be removed by filtration, either with or

without pH change, one has a method for separating

the toxicants from other material in the effluent. This

knowledge itself provides an important advance because further characterization and analyses will be less

confused by non-toxic constituents. Usually further characterization will be the next step. Tests must be designed to determine whether the mechanisms causing

removal are precipitation, sorption, change in equilibrium or volatilization. One necessary step is to trecover

the toxicity from the filter. If this cannot be done and the

loss is not by volatilization, then the whole experiment

may have little utility. Comparisons of pressure and

vacuum filtration may reveal if volatilization is involved.

If characterization of the toxicant is also achieved

through other tests, filtration can be used to remove the

toxicants. Then if the suspected toxicant is the true

one, its concentration should be lower or zero after

toxicity is removed by filtration.

If any or all of these pHIfiltration combinations result in less effluent toxicity (not attributable to the effects of pH adjustment alone), it may be possible to

confirm the findings of the test. A transfer of the solids

contained on the filter back into the filtrate at pH i can

be attempted by reversing the flow of the filtrate through

the filter or by rinsing the solids off the filter with filtrate.

The toxicity exhibited by this solution should be similar

to that of the original effluent, provided that the final

concentration of solids in the test solution approximates

the solids level in the sample that was filtered. For

precipitates formed as a result of pH changes or for

contamination of suspended solids facilitated by pH

adjustment, time must be allowed for the precipitate to

redissolve in the pH i filtrate or for a new equilibrium to

be set up between the contaminants on the solids and

in solution. The results of this test are not likely to be

quantitative due to the recovery problems inherent in

the process.

In order to determine whether the effluent matrix

affects the toxicity of filterable particles (e.g., its ionic

strength, dissolved organic carbon content), the filtered

material can also be added to a volume of pH i dilution

water equal to the volume of effluent that was passed

8-I 9
Figure 8-9. Example data sheet for filtration test.

Test Type: Filtration Species/Age:

Test Initiation (Date & Time): No. Animals/No. Reps:

Investigator:

Sample Log No., Name:

Date of Collection:

Source of Animals:

Dilution Water/Control:

Test Volume:

Other Info:

Note: See baseline data sheet for control data.

nt-4 ? p’I ”

pH i

pH 11

Volumes and Strength of Solutions Added:

HCI NaOH

Comments: 8-20
through the filter. The toxicity of this dilution water,

spiked with effluent solids, can be compared to the

toxicity of the unfiltered (baseline) effluent and the fiitrate spiked with its own solids.

pH The additional tests suggested herein

may or may not provide the relevant information.

to recover the toxicant from

is to sonicate with a solvent (e.g.,

 water). For some effluents,

 11 adjustment/

filtration test. These results may cause one to suspect

either cationic as theoxicant

 We have had success in recovering

toxicity from the glass fiber filter when the filter was

pH 3 adjusted dilution water. Typically, 300 mL is

sonicated in 75 mL of pH 3 dilution water, this concentrates the toxicity 4x (theoretically). The pH of the

concentrate is then readjusted to pH i before use in the

toxicity test. For some effluents the amount of a cationic metal was reduced after filtration and the toxicity

in the effluent removed after filtration. Toxicity was

recovered in the pH 3 dilution water extract of the filter.

If methanol is used, after sonication the methanol must

be concentrated before use in toxicity tests (see Section 8.6 for methanol tolerances). Additional solvents

can be used to recover toxicity off the filters (SchubauerBerigan and Ankley, 1991).

8.5 pH Ad’usfmenf/Aerafion Test

Principles/General Dlscussion:

The aeration test is designed to determine how

much effluent toxicity can be attributed to volatile,

sublatable, or oxidizable compounds. The test is performed with pH-adjusted and unadjusted (i.e., pH i)

effluent. By comparing the toxicity test results for acidic,

pH i and basic aerated samples, the toxicity may vary

and this knowledge can be used for further characterization. Some compounds can be removed or oxidized

most easily at one pH, whereas others are most easily

removed or oxidized at a different pH. Thus, the aeration is performed at several pH values.

Whether a constituent is completely removed, or

sufficiently removed to reduce toxicity, depends on many

chemical/physical conditions. At a minimum, one must

be certain that the geometry of the sparging process is

always the same for a given effluent sample and that

the duration is constant, otherwise the test is of little

value. The pH of many effluents will change, sometimes rapidly (cf., Section 8.3) during sparging and so

pH must be frequently checked and maintained during

the entire aeration period.

Oxidation can change the constituents in many

ways and one must determine if oxidation or sparging

is the mechanism before additional tests can be designed. Water soluble constituents such as ammonia

8-21

and possibly cyanide are not readily stripped using

techniques described in the Procedure below, and one

should not assume that they will be removed. Under

both air and nitrogen sparging, a removal process, in

addition to volatilization, may occur. Sparging can remove Surface active agents from solution by the process of sublation (lifting up, carrying away). Surface

active agents have a molecular structure that includes

a polar end (either ionic or nonionic) and a relatively

large non-polar, hydrocarbon end. Some examples of

surface active agents are resin acids, soaps, detergents, charged stabilization polymers and coagulation

polymers used in chemical manufacturing processes.

The process of sublation occurs because during

sparging, surface active agents congregate at the liquid/gas interface of the air or nitrogen bubbles and are

carried along with the gas bubbles to the surface of the

sparged liquid. As the bubbles break up, they are

deposited and concentrated with continuous sparging

at the surface of the sparged liquid, and the sides of

the aeration vessel. After sparging, a faint deposit may

or may not be visible on the sides of the aeration

vessel.

Air is used for sparging so that oxidation is included. Subsequent tests with nitrogen may betsed to

separate sparging from oxidation and tests described

undei Results/Subsequent Tests can be used for

sublatable compounds. We have grouped the tests to

avoid many tests initially.

Volume Requlred:

Thirty-five mL volumes of each pH 3, pH 11 (see

Section 8.3) and pH i effluent are needed for this test.

A maximum volume of 30 mL of each of' these solutions

is required for the toxicity tests on aerated solutions. An

excess volume has been provided to allow for losses

through aeration. Each toxicity test utilizes four exposure concentrations (10 mL each) without replication.

The exact volume required for the toxicity test on each

pH adjusted or unadjusted aerated solution will depend

on the toxicity of the effluent (the 24-h initial LC50).

The amount of dilution water that was used for the

toxicity blanks (35 mL) is kept the same as the effluent

for each pH. An excess volume has been provided to

allow for any volume loss through aeration (cf., Section

8.3).

Apparatus:

Aeration device or compressed air system with a

0.22 urn filter, six air flow regulators, six glass diffusers,

six 50 mL graduated cylinders with ground glass stoppers, glass stoppered bottles for acid and base solutions, pH meter and probe, stir plate(s), penluorocarbon

stir bars, automatic pipette, disposable pipette tips, eye

dropper or wide bore pipette, 30 mL beakers or 1 oz

plastic cups, light box and/or a microscope (optional).

Reagents:

1.0, 0.1, and 0.01 N NaOH, 1.0, 0.1, and 0.01 HCI

(ACS grade in high purity water), buffers for pH meter

calibration.
Test Organisms:

Test organisms, 75 or more, of the same age and

species.

Procedure :

Day 7: Six different solutions are aerated in this

test; pH 3, pH i, and pH 11 effluent, and pH 3, pH i,

and pH 11 dilution water, (cf., Sections 8.3 and 8.4,

respectively for preparation of pH adjusted effluent and

dilution water). A flow chart for the effluent samples of

the pH adjustment/aeration test is shown in Figure 8-

10. Each sample is transferred to a 50 mL cylinder

containing a small per-fluorocarbon stir bar. The diameter and length of the pH probe must be such that it

can be placed into the solution during aeration. The

taller the water column and the smaller the bubbles, the

better the stripping will be. Each solution should be

moderately aerated (approximately 500 mL air/min) for

a standard time, such as 60 min. Formation of precipitates may or not be important and should be noted.

The pH of the acidic and basic effluent and dilution

water aliquots should be checked every 5 min during

the first 30 min of aeration and every 10 min thereafter.

If the pH of any pH 3 or pH 11 solution drifts more than

0.2 pH units, it must be readjusted back to the nominal

value. The volume and concentration of additional acid

and/or base added to the solutions should be recorded

so that the final concentration of Na+ and Cl- in each

solution can be calculated following final pH readjustment. Solutions should be stirred slowly during any pH

readjustment. Again, precautions must be taken in order to minimize the amount of acid and base added.

Note that the aeration time does not include the time

intervals during which aeration is temporarily discontinued to readjust pH. A constant pH is not maintained in

the “pH i ” effluent because this solution represents the

generalized effects of aeration on the effluent without

regard to pH. Only slight changes in the pH of the

dilution water at its initial pH are expected since such

water is usually at air equilibrium before the start of the

. manipulation.

The sparged sample must be removed from the

graduated cylinder for toxicity testing so that any toxicant that may have been sublated is not redissolved in

the sparged sample. This may happen if the sample is

simply poured from the cylinder, and sublation would

never be suspected. Therefore, one way to transfer the

effluent sample is by pipetting it out of the cylinder,

exercising care to prevent any sample from contacting

the sides of the cylinder above the liquid level. For

example, when using a 100 mL graduated cylinder for

the aeration vessel, a 50 mL pipette can be used to

remove the 30 mL sample. At this point it may be

possible to recover a sublated toxicant from the sides

of the cylinder. This must be done at the end of the

aeration step; see ResuWSubsequent Tests section

below for details.

Sparging air contaminated with oil (droplets or vapor) or any other substance is unacceptable. Contami-

-

nated air is probable from air lines containing oil or in

cases where the source of the air is contaminated (e.g.,

boiler room). Small air pumps, sold for home aquaria

are adequate, but only if the room air is free of chemicals or contaminants. Chemistry laboratories where concentrated chemicals and solvents are used often might

not have suitable air quality.

Following aeration, the pH of each solution (including the 35 mL portions of pH unadjusted effluent (pH i )

and dilution water) is returned to the pH of the initial

effluent or dilution water using the necessary volumes

of NaOH and HCI. Returning all effluent solutions to the

initial pH of the wastewater will ensure that a valid

comparison can be made with the baseline LC50. The

pH of each sample must be checked periodically

throughout the remaining work day and readjusted as

necessary. If stable pH can be attained prior to toxicity

test initiation, the pH during the test is likely to change

less.

Day 2: Before initiating the toxicity tests, the pH of

all of the aerated effluent and blank solutions should be

checked and adjusted to pH i . Toxicity tests are performed on a single 100% concentration of all three

dilution water blanks (pH 3, pH i and pH 11). Tbese

dilution water blanks will provide information on toxic

artifacts resulting from aeration.

Based on the 24-h initial LC50 of the day 1 effluent,

toxicity tests are performed on each aerated effluent

solution at concentrations of 4x- (or 1 OO%), 2x-, lx-,

and 0.5x-LC50 (cf., Section 8.2). The pH of each test

concentration should be measured and recorded daily.

An example of the data sheet for the aeration test is

given in Figure 8-l 1.

Interferences/Controls and Blanks:

Dilution water controls prepared for the baseline

toxicity test also act as controls on organisms, dilution

water and test conditions for this test. Results of the pH

adjustment test (Section 8.3) will suggest whether or

not toxic levels of NaCl may have been reached as a

result of the addition of acids and bases to the effluent.

No significant mortality should occur in any of the

three aeration blanks. If there is significant mortality,

the cause must be found and corrected before the test

can be meaningful. To determine which factor(s) caused

blank toxicity, the toxicity of pH adjusted aerated dilution water can be compared to that in the same pH

adjusted, unaerated dilution water. Approximately the

same quantities and concentrations of acid and base

should be added to both of these samples of dilution

water to make them comparable. Blank toxicity in the

pH adjusted and unadjusted aerated dilution water suggests contaminated air. Other possible causes include

contaminated equipment, such as electrodes or glassware (especially where low or high pH solutions were in

contact), or the addition of too much acid or base.

Another approach to this blank question involves evaluating the pH adjustment/filtration and pH adjustment/

C,, SPE blanks (Sections 8.4 and 8.6). Assuming the

8-22
L

Y

+ r-l c9

c

8-23
Figure 8-11. Example data sheet for aeration test.

Test Type: Aeration

Test Initiation (Date & Time):

Investigator:

Sample Log No., Name:

Date of Collection:

Species/Age: a

No. Animals/No. Reps:

Source of Animals:

Dilution Water/Control:

Test Volume:

Other Info:

Note: See baseline data sheet for control data.

nH -3 pm. v

pH i

pH 11

Volumes and Strength of Solutions Added:

HCI NaOH

Comments:

8-24
concentration of acid and base in the final blank solution is approximately the same in all dilution waters for

the three tests, toxicity in the aeration blanks but not in

the filtration or C,, SPE blanks suggests that aeration

rather than pH adjustment has led to contamination.

Compare the toxicity from the effluent baseline test to

the toxicity of all three aerated effluent samples. When

the baseline toxicity is significantly less than that of any

one of the aerated samples, toxicity was added or

created during effluent manipulations. This check is

especially important because pH adjustment of aerated

effluent may have required larger quantities of acid and

base as compared to the pH adjustment test (Section

8.3).

If nitrogen was used for stripping, DO depletion is

likely to have occurred. If a relatively large surface-tovolume ratio is used (such as the 10 mL volume in a 1

oz plastic cup) during the overnight holding period, DO

should not be a problem.

Results/Subsequent Tests:

The LCSOs for the aliquots of pH 3, pH i, and pH 11

aerated effluent are compared to the baseline effluent

LC50 to determine whether any of these processes

resulted in a significant change in effluent toxicity. If a

substantial reduction in toxicity is seen for any or all of

the three aerated effluent solutions, one must next

determine whether the separation of effects was caused

by sparging, oxidation, or sublation. This is done by

repeating those tests in which toxicity was reduced,

substituting nitrogen for air in the stripping process.

Use of nitrogen eliminates oxidation as a remo.vaI process. If side-by-side effluent stripping tests with air and

nitrogen provide the same results, toxicant removal is

probably caused by the sparging process. If only the

test(s) conducted with air succeeds in reducing or removing effluent toxicity, oxidation is a probable cause.

An effluent sample may contain toxicants removed

through sparging and oxidation. An example of this is

where aeration at pH 3 and pH i reduces toxicity, but

nitrogen stripping removes the toxicity only in the pH 3

effluent. Using the Procedure described above ammonia should not be air-stripped; however, if different

aeration vessels are used and greater surface area is

used, ammonia can be reduced/stripped. If toxicity is

reduced at pH 11 compared to pH i aerated, pH 3

aerated samples, or the baseline test, measuring the

ammonia concentration after aeration can be informative.

To determine if toxicity is due to sublatable compounds, toxicant recovery can be attempted by adding

dilution water to the emptied cylinder used for sparging

(preferably graduated cylinders with ground glass stoppers), stoppering it .and shaking it vigorously, making

sure that the sides of the cylinder are thoroughly rinsed

with the dilution water. This dilution water is then tested

for toxicity. Recovery of the sublated material provides

further evidence that a surface active compound was

present. A more concentrated solution of the sublated

material can be obtained by using a larger sample

:

volume for sparging, such as 90 mL, and less dilution

water (i.e., 30 mL) to recover the toxicant( This

would result in a nominal concentration of 3x the whole

effluent concentration of the toxicant( To avoid spurious results in cases where sublated toxicity is recovered, a dilution water blank should be run. A dilution

water sample should be subjected to the sparging step

and to the toxicant recovery step. In that way, if toxicity

is inadvertently being added to samples during the

manipulation (from contaminated glassware or contaminated air or nitrogen supply) it should occur in the blank

sample. As some sublated compounds are difficuft to

recover. from the glass surface, a solvent rinse with

methanol may result in more efficient recovery. If solvents are used and because of the low concentration

factor involved, most of the solvent will have to be aired

down in order to have an adequate water concentration

to perform the toxicity test. If solvents are needed, one

is wise to scale up the volumes to obtain higher concentrations for testing. However, not all kinds of surface

active agents are removed to the same degree, and

some are not removed at all (Ankley et al., 1990). This

is probably due to factors such as matrix effects and

solubility. Recovery of a sublated toxicant can be difficult. Consequently, reduction of toxicity by sparging

with air and nitrogen can be an indication of a tUxicant

which is a surface active agent, but a lack of toxicity

removal does not rule out the presence of these compounds.

In the pH adjustment/aeration test, removal of toxicants by precipitation resulting from pH change alone

should also be detected by the pH adjustment/filtration

test. Oxidation of compounds can cause precipitation. If

oxidation is the cause, the pH adjustment/filtration test

will not change toxicity. If nitrogen sparging has removed the toxicant, the “volatile toxicant transfer” experiment described below may provide separation of

the volatile toxicant from other constituents. Our experience with this technique is limited to a few effluents. To

perform the “volatile toxicant transfer” experiment, a

closed loop stripping/trapping apparatus is used (Figure 8-12). This apparatus consists of a pump which can

circulate air or nitrogen gas, two airtight fluid reservoirs,

perfluorocarbon tubing, and diffusers. The arrangement

should be such that air or nitrogen can be passed

through the effluent in one reservoir and then through

the dilution water in the second reservoir before cycling

back to the first reservoir. The reservoir of the dilution

water serves as a trap that will collect the volatilized

toxicant( Of utmost importance to this experiment is

an air tight system. The time to equilibrium of the

volatile toxicant will be dependent upon the efficiency

of the sparging process and the rate of volatilization of

the toxicant which may be affected by pH. For example, using a glass or plastic pipette to aerate the

samples may not effectively sparge the entire volume

of sample. To optimize the toxicant recovery, use of

gas washing bottles (for example, 125 mL and 500 mL

bottles from Kontes Glass Co., Vineland, NJ) fitted with

glass frit diffusers is suggested because they sparge

the sample volumes more effectively.

8-25
l b

Figure 8-12. Closed loop schematic for volatile chemicals.

Numerous operating conditions can be selected,

each providing different information. This system should

so that if the trap is inefficient in removing the toxicant

from the nitrogen, the toxicant will not be lost from

not be operated as a conventional purge and trap the sample. 8ecause conditions to optimize transfer

system. The reason is that since one does not know

the identity of the toxicant( the conditions for trapping

cannot be selected until the chemical identity is known,

are not known. Initially, the objective should be to get

longer sparging times should be chosen,

measurable toxicity moved into the dilution water me- The first experiments should involve no pH changes

dium in the trap. This will establish that there are at if any measurable change in toxicity occurred in the

least some volatile toxicants present. At this stage the earlier tests without the pH 3 or pH 11 adjustments.

goal is not to move all the toxicant to the dilution The reason for this selection is that drastic changes in

water in the trap. If the same concentration of the pH can cause many unknown effluent changes, and

toxicant in the effluent can be transferred to the dilution artifacts are more likely to occur. Of course if pH changes

water as exists in the unaltered effluent, the data are are required to change toxicity, then pH will have to be

easiest to interpret. For this purpose the volume of altered. When pH is altered, then equilibrium objecsparged effluent should be large and the dilution water tives, mentioned above, are not possible and the entire

volume in the trap small. The nitrogen gas is recirculated process takes on characteristics of more conventional

8-26
purge and trap experiments. The usual resin traps

described in EPA methods are not suitable because

ously, relative degrees of water solubility exist, Many

the trap cannot be tested for toxicity. The trapping

highly toxic pollutants found in effluents at very low

concentrations are not considered to be water soluble

medium must be, or be able to be, made into a toxicity despite the fact that they are present at toxic concentestable water. trations.

In those instances when sparging affects toxicity

only when accompanied by a pH change (pH 3 or

pH 1 1 ), the method to be used to operate it as a

conventional purge and trap is as follows. The trap’s

dilution water volume should be small relative to the

sample volume and its pH should be opposite that of

the sample pH (e.g., if the sample pH is 3, then the trap

pH should be 11). One can no longer conclude anything about the original effluent equilibrium, and the

procedure is one of separation. Toxicity in the trap may

or may not be caused by the same substance as that

which causes the original effluent toxicity. Obviously, all

the precautions mentioned above regarding NaCl addition and other adjustments must be tracked with blanks

just as in any other experiment. We have not found

many effluents where the transfer technique is useful,

but for those effluents where it works, it is a powerful

tool. We have found the volatile toxicant transfer experiment to be useful with some samples (i.e., sediment

pore water), where two pH dependent toxicants (e.g.,

ammonia and hydrogen sulfide) are suspect. There is

sometimes an appreciable loss of toxicity after the pH 3

aeration step in samples with ammonia toxicity, yet it is

unknown whether the toxicity loss is due to volatilization of hydrogen sulfide (or some other pH dependent

toxicant) at low pH, or is an artifactual decrease of

ammonia toxicity due to a downward pH drift in the test

(cf., Section 6.3). In this case, a trap such as the one

described previously for transferring a volatile toxicant

at altered pH is useful. Water in the trap that volumetrically concentrates the toxicant at two or more times its

whole sample concentration may be successfully tested

for toxicity. We suggest, in the case of suspected hydrogen sulfide toxicity, testing the trap water at pH 6,

as the toxicity of hydrogen sulfide is enhanced at that

pH. One caution in this setup is that the volatility of

some pH dependent toxicants such as hydrogen sulfide

makes it imperative that the experiment be initiated

immediately after adjusting the pH to minimize their

loss.

8.6 pH Adjustmen t/C,, Solid Phase

ObviCompounds extracted by the C,, pH

or chloroform. The C,,

pH and a high pH,

C,, SPE column. Because C,,

column degradation, pH’s

used. To ensure column integrity, the pH

to be used on the SPE columns will be either pH

or pH pH 11) in this manipulation.

Manufacturer’s data should be consulted for tolerable

column pH ranges and for exact column conditioning

Of the 235 mL pH) filterEd

8.4), 35 mL

pH) is held for the pH

test. Now, the additional 200 mL

through the C,, (pH 3, pH i,

and pH

sure concentrations (10 mL

pH adjusted or pH i post-colu

on the toxicity of the effluent (the 24-h initial LC50).

For the blanks, 30 mL of pH adjusted (pH 3 or pH

11) and/or filtered (pH i) dilution water is needed. The

last 10 mL of the post-column water should be used for

blank toxicity tests.

Apparatus:

Six 250 mL graduated cylinders, eight 25 mL graduated cylinders, glass stoppered bottles for acid and

base solutions, pH meter and probe, stir plate,

perfluorocarbon stir bars, pump with sample reservoir,

perfluorocarbon tubing, ring stands, clamps, three 3 mL

C, SPE columns (200 mg sorbent), automatic pipette

(18 mL), disposable pipette tips (10 mL), eye dropper

or wide bore pipette, 30 mL glass beakers of 1 oz

plastic cups, light box and/or microscope (optional).

Reagents:

HPLC grade methanol, high purity water, 1 .O, 0.1,

and 0.01 N NaOH, 1.0, 0.1, and 0.01 N HCI (ACS

grade in high purity water), buffers for pH meter calibration, acetone and methanol for cleaning the pump and

reservoir, and vials to collect methanol eluate.

Test Organisms:

Test organisms, 135 or more, of the same age and

species.

a-27 .
Procedure:

Day 7: This procedure is performed with effluent

samples adjusted to the various pH’s; however, the

manipulations have three distinct steps (Figure 8-13)

which are generally the same for each PH. Prior to

attaching a new column to the apparatus, the reservoir

and pump must be cleaned with acetone, methanol,

and high purity water.

Step 1 involves conditioning the solid phase extraction columns for each pH. Column conditioning procedures may vary with the manufacturer of the column.

The procedures described below are modifications of

the conditioning steps used with Bakep C,, SPE columns (J.T. Baker Chemical Company, Phillipsburg, NJ).

Using a flow-rate of 5 mUmin, 15 mL of HPLC

grade methanol is pumped through the column and

discarded. Next 15 mL of high purity water, adjusted to

pH 3 with HCI, is placed in the sample reservoir. Care

must be taken in timing the addition of solutions after

the methanol has passed through the column. While

the mixing of methanol with subsequent solutions must

be minimized, the column must also be prevented from

going dry following the methanol wash and dilution

water or sample application. The amount of time needed

between introduction of solutions to prevent any column drying will be unique to each investigator’s apparatus. This timing should be determined before performing this procedure with actual effluent samples. If

the column dries at any time after introduction of the

methanol during conditioning, the column must be reconditioned (with methanol).

As the last volume of pH 3 high purity water is

entering the column (Step l), the pH 3 adjusted, filtered

dilution water is placed into the reservoir (Step 2).

Again, the column must not be allowed to dry before

the pH 3 dilution water enters the column. The pH 3

high purity water passing from the column should be

measured to determine the point at which the dilution

water begins to leave the column. This pH 3 high purity

water used to condition the column is discarded, Next,

30 mL of the filtered pH 3 dilution water is collected,

and the last 10 mL aliquot collected is used for the

toxicity blank to detect toxicity leached from the column. This aliquot will have to be pH re-adjusted to the

initial pH of the dilution water using NaOH, and it is

reserved for day 2 toxicity testing. Care should be

taken to minimize changes in sample volume and ionic

strength during pH readjustment.

As the last several mL of filtered pH 3 dilution water

are entering the column, the 200 mL volume of filtered

pH 3 effluent is placed in the sample reservoir (Step 3).

Again, the column sorbent must not be allowed to dry

between the dilution water blank and the effluent. Collect a 30 mL aliquot of post-column effluent after 25 mL

of the sample passes through the system. A second

post-column 30 mL aliquot is collected after a total of

150 mL of the sample passes through the column.

Collection of the first post-column sample after 25 mL

of sample has passed the column ensures that any

dilution water left in the system will not be present in

the post-column sample. The second subsample of

post-column solution provides information on column

overloading and toxicant these

30 mL pH i using th

NaOH. The total volume oNaOH

necessary for pH adjustment should be reThese

aliquots are reserved for day 2 toxicity testing. Columns

 but should be saved 

elution (see the Results/Subsequent Tests section).

Receiving water should not be used as the dilution

water because trace organic and metal contaminants

or organics (such as humic acid) may be present. If for

any reason, such a water is needed, the same column

should not be used for concentrating the toxic sample.

mL dilution water blank, 3mL

be checked for toxicity. The pH

c

For pH i, the above procedure is repeated using a

clean reservoir and pump and a new conditioneM mL

C SPE column for the filtered pH i effluent (Figure 8-

l$ The pH of the post-column dilution water and postcolumn effluent should be measured.

In the final C,, SPE manipulation, pH 9 (readjusted

from pH 11) dilution water and effluent are processed

as described above. While use of pH 11 effluent offers

the likely advantage of shifting a larger number of basic

organic compounds farther towards the predominately

unionized form, and therefore removal, the C SPE

column cannot withstand a pH above 10. For t& reason, the pH 11 filtered dilution water and sample aliquots

prepared in Sections 8.3 and 8.4 are readjusted to pH

9 with HCI before they are put through the column. The

15 mL of high purity water used to rinse the column

following methanol conditioning must also be readjusted to pH 9 with NaOH. The 10 mL aliquot of postcolumn pH 9 dilution water and both 30 mL aiiquots of

post-column pH 9 effluent are further adjusted to their

pH i’s respectively, prior to toxicity testing. The total

volume of acid added for pH readjustment is recorded.

The pH of all aliquots of the chromatographed dilution

water and effluent should be checked and readjusted

as appropriate throughout the remainder of the work

day.

Day 2: The pH of all of the post-column dilution

water and effluent aliquots should be checked and

readjusted if pH has drifted overnight. Toxicity tests are

performed on a single 100% concentration of all three

of the dilution water blanks. These blanks will provide

information on the presence of toxicity leached from the

C,, column at different pH’s.

The six 30 mL post-column effluent aliquots are

tested for toxicity using an exposure series based on

the 24-h LC50 of the original effluent. Chromatographed

effluent aliquots are tested at concentrations of 4x-,

8-28
Figure 8-13. Step-wise diagram for preparing the C,, solid phase extraction column samples.

step 1

r

Prepare three Cl8 SPE’ Columns l

l 15 mL methanol

l 15 ml_ high purity water

00 NOT LET SOABENT GO DRY

Olscard Methanol 8

Water After Rinses 1

step 2

step 3

L 4

Oilutlon Water

1 I

\

HCI +

,

*NaOH

30mLatpH3

Rltered Water

30mLatpH9

Filtered Water

Collect 10 mL Sample

I

Day I

Prepared Column2

1 If column will be eluted with 1 mL methanol (cf., Resu/&‘Subseque~ Tests), collect methanol column blank

before dilution water is passed over column. Column should go to dryness and will have to be

re-cxnditioned (Step 1) before proceeding to Step 2.

2 Use same column used with the dilution water unless receiving water is used (see text for details).

3 Same test as depicted in Figure 8-8.

8-29
2x-, lx-, 0.5x-LC50 (cf., Section 8.2). The pH of each

solutlon tested should be measured daily and recorded

along with organism survival. A sample data sheet for

the C,, SPE test is shown in Figure 8-14.

Interferences/Controls and Blanks:

Controls on test organism performance, dilution

water quality, and test conditions are provided by the

control from the baseline toxicity test. The pH adjustment and filtration tests (Sections 8.3 and 8.4) provide

information on the effects of pH adjustment and filtration on eff iuent toxicity apart from any additional changes

caused by C,, SPE. Effluent and blank test results from

these two tests must be evaluated prior to interpreting

the results of the C,, SPE test, both in terms of identifying any toxic artifacts added during filtration and pH

adjustment and in allocating toxicity reduction to the

three components potentially impacting effluent toxicants in the C,, SPE test.

Of those methods discussed so far, the C,, technique requires the greatest manipulation. More problems are likely to be encountered with toxic blanks

because in addition to those factors associated with pH

adjustment and filtering, the C,, method also involves

use of resin and methanol. Blanks for toxicity must be

chec:,-3d in the same manner as before for acid and

base addition, filter artifact toxicity, pH drift, as well as

toxicity from the C,, column. In addition to these, some

effluents behave in a peculiar way after passing through

the SPE column (cf., discussion below).

Results/Subsequent Tests:

The above unique properties of some effluents and

the potential for column blank toxicity problems make

interpretation of the test results more subjective.

If toxicity is not reduced in post-column effluent, not

too much credence should be placed on the results.

One needs to go back and sort through the possible

causes.

If none of the Phase I treatments reduced toxicity

(including the C,, SPE column) or if the toxicity was

reduced by the C,, column, it is useful to elute the

column with 100% methanol. Of course, a column blank

must also be evaluated; this is a methanol elution

following the column conditioning with methanol and

high purity water. The column must go to dryness

before collecting the blank of methanol. After the methanol blank is collected, the column must be reconditioned as described in Step 1 in Figure 8-13 and the

column must not go to dryness before starting the

dilution water over the column. If a 1 mL volume of

methanol is used (for 200 ml of effluent on a 3 mL

SPE column) and the sorption and elution efficiency

are loo%, any substances retained by the columns will

be concentrated 200x. To test the eluate, 150 PL of the

methanol fraction is diluted to 10 mL with dilution water.

The resultant methanol concentration is lS%, which is

below the 48-h or 96-h LC50 for all species given in

Table 8-3. This provides a concentration of effluent

constituents 3x whole effluent concentration. This small

a-30

amount of concentration over whole effluent allows

detection even if some loss occurred either in sorption

or elution.

If the post-column effluent is not toxic or less toxic,

and the methanol eluate is toxic, the next step is to

proceed with the Phase II C,, SPE procedure to identify

the toxicant removed by the column. At this point it

should not be assumed that toxicity removed by the C,,

SPE column is due to non-polar organic compounds.

While metals are not non-polar organic chemicals, they

can be removed from some effluents using the C,, SPE

column. However, metals generally are not eluted with

methanol and therefore the fractions are not toxic. Metals may be eluted with dilution water adjusted to pH 3

or pH 11. Surfactants can be sorbed by the C,, SPE

column just as other non-polar organics, and some

elute with methanol.

If neither the post-column effluent nor the methanol

fraction is toxic, the toxicant is probably retained by the

C,, SPE column but is not covered by the methanol

elution. When interpreting these results it is important .

to consider that when a sample is passed over the C,,

column there can be other mechanisms besides reverse phase SPE by which a toxicant can Qe removed. For example, the C!, column packing may

remove toxic compounds by filtering them out of solution, e.g., the toxicant may be associated with solids in

the effluent, and the 40 pm C,, packing material may

remove the solids. The toxic compqunds could also be

physically adsorbed or ionically bound onto the surface

of the column packing and the methanol elution cannot

recover the toxicant from the column. Perhaps the

toxicant has been removed by the reversed phase

SPE mechanism but the methanol does not recover the

toxicant because either methanol is too polar a solvent or the toxicants have too low a Volubility in methanol. In this case a different solvent system may be

needed to remove the toxicants, e.g., methylene chloride, hexane or pH adjusted water. Both hexane and

methylene chloride are much more toxic than methanol, and if hexane or any other “toxic” solvent is used,

solvent exchange or some other method must be used

to remove the solvent in order to effectively track toxicity throughout the procedure. Another possibility that

should be considered is that the toxicant has decomposed. Whatever the mechanism of toxicant removal,

the interpretation of the loss of toxicity should be evaluated carefully.

After passage through the C,, column, some effluents exhibit artifactual toxicity which is not observed in

the post-column dilution water blanks. Artifactual toxicity can arise from two sources: a) pH drift and/or b)

biological growth in the post-column effluent. (Problems

with pH drift are discussed in Section 8.3; consult that

section for further information.) Artifactual toxicity from

biological growth in the post-column effluent can be a

major problem for some effluents, particularly municipal

effluents. This growth has not been observed in all

effluents, but for the effluents where it did occur, it was

present in nearly every post-column sample. The post-

.

Te ble 8-3. Toxicity of methanol to several freshwater rpecfer

Species Ufestage

Cenodaphnia dub/a G h’

24 h

>3.0

LCSO f%. v/v (95% Cl))

48 h 72 h 96 h

A.0

124 h’ :-:

(2.6 : 2.9)

z

(2.6 : 2.9)

548 h’ 2.4 2.0

(2.2-2.6) (1.9-2.2)

Daphnla magna 124 ht NR 3.2

(2.5-3.7)

Daphnla pulex 114 h’ 2.55 NR

(2.3-2.8)

Hyaleiia azteca juvenile4

(129-5258)

NR NR NR

Salt-no gairdneri

Pimephaies promeias

juvenile’

524 h’

28-32 d6

(2.:::.7)

2.5 NR

(2.5-2.7) (2.giz.7)

4.0

(-)

3.8

;+; (3.zli.2) , @i-:.2)

NR

(3.7-3.9) (3.7-3.9) (3.63-37.9)

Lepomis macrochirus juvenile6

(2.;:;.7)

2.4 NR 1.9

(2.2-2.7) (1.8-2.3)

11 Data generated at ERL-Duluth.

unmeasured.)

C. dubia were 124 h old at test initiation and fed. (Tested in soft reconstituted water (DMW); static Qod

z Randall and Knopp, 1980. (Tested in spring water: static and unmeasured.)

3 48-h ECSO. 4 Bowman et al., 1981. (Tested in well-water: static and unmeasured.)

5 18-h LCSO. 6 Poirier et al., 1986. (Tested in Lake Superior water; flow-through and concentrations measured.)

Note. (-) = Confidence interval cannot be calculated as no partial mortality occurred; NR = Not reported.

column samples exhibited a turbid, often filamentous

growth and sometimes, lower than normal DO levels in

the toxicity tests. In one effluent, this growth was caused

by methylotrophic bacteria. Methanol occurs in postcolumn effluent samples because the methanol used in

conditioning (activating) the C,, SPE column is slowly

leached out of the column and into the effluent as it

passes through the column.

Methods for eliminating or controlling this type of

artifactual toxicity problem are currently limited. For

some effluents, the most promising method appears to

be additional filtering of the post-column effluent through

a 0.2 pm filter to remove bacteria prior to testing. Whole

effluent filtered through a 0.2 pm filter serves as a

control for the toxicity test with the post column/filtration

manipulation. Filtration is easy to perform and allows

useful post-column toxicity data to be obtained, provided it does not alter or reduce toxicity in the postcolumn effluent. If toxicity is removed in the 0.2 urn

filtered post-column effluent, but not in the 0.2 pm

filtered whole effluent, repeat the experiment filtering

the whole effluent with 0.2 pm filter and testing the

post-C,, 0.2 pm filtered effluent. The post-column (0.2

pm filtered effluent) may need to be filtered (0.2 pm)

again. If toxicity is removed by filtration, see Section

8.4 Results/Subsequent Tests. If this growth in the

post-column cannot be eliminated but toxicity occurs in

the methanol eluate, then proceed with Phase II identification. If growth is not eliminated and no toxicity

occurs in methanol eluate, then use of different solvents to condition the C,, column may reduce growth

(e.g., acetonitrile). Control of the turbid growth may

also be possible by performing daily renewals with

post-column effluent. Initially, more post-column sample

would have to be collected (60 mL rather than 30 mL),

and a portion should be refrigerated. If control of the

artifactual toxicity caused by the turbid growth cannot

be achieved, other sorbents (e.g., XADs, activated carbon) may have to be used. Another possible method of

controlling the growth may be by the use of antibiotics

but we have not investigated this approach.

Observation and judgement must be used to detect

problems occurring from artifactual toxicity and only

through experience can one recognize when they occur. Failure to recognize them will result in the conclusion that the C,, SPE column did not remove toxicity

when it in fact may have done so.

If toxicity occurred in the methanol eluate from a

POTW effluent, and the C. dubia were more sensitive

than fathead minnows, it might be cost-effective to try

adding a metabolic blocker, piperonyl butoxide (PSO),

to the effluent and eluate. We have frequently found

non-polar organics in POTW effluents and have identified organophosphate pesticides (OP’s) as the toxicantfs) _Q? a-Jc
to C. dubia (Amato et al., 1992; Norberg-King et al.,

1991). Most metabolic blockers used in aquatic toxicology have been used with fish; however, OP’s are generally less acutely toxic to most fish than to cladocerans. PBO is a synthetic methylenedioxyphenyl that can

block the toxicity of various chemicals that need to be

metabolized in the cytochrome P450 cycle to be toxic.

In tests with cladocerans, sublethal additions of PBO to

the whole effluent and/or the methanol eluate test have

been useful for implicating some metabolically activated OP’s as the toxicant (Ankley et al., 1991).

Experiments showed that for C. dubia, D. magna, and

D. pulex, PBO blocked the acute toxicity of parathion,

methyl parathion, diazinon, and malathion, but not dichlorvos, chlorfenvinphos, and mevinphos. For those

OP’s where PBO reduced the toxicity, the reduction

was greater in the first 24 h; the toxicity of the OP’s in

an effluent may be expressed after 24 h.

plexes with the thiosuffate anion (Giles and Danell,

1983). Cationic metals that appear to have this potential for complexation (based upon their equilibrium stability constants) include <cadmium (*+), copper (Cu2+),

silver (Agl*), and mercury (Hg2+) (Smith and Martell,

1981). However, the rate of formation of the complex is

specific for various metals and some cationic metals

may not be rendered non-toxic in the 48-h or 96-h

period used for the toxicity test due to a slow complexation rate.

To perform the PBO addition test for cladocerans,

a PBO water stock is prepared and microliter quantities

are added at various sublethal concentrations (final

concentrations of PBO are 500, 250, and 125 f_rg/L).

(Note: the 48-h LCSO’s for C. dubia, 0. magna, and D.

pulex are 1,000, 2,830, and 1,620 ug/L, respectively).

The PBO additions can be set up in a similar manner to

the EDTA and oxidant reduction tests (Section 8.7 and

8.8) using a 3 x 3 matrix of PBO and effluent concentrations. Toxicity reduction with the addition of PBO

would suggest the presence of toxic levels of metabolically-activated compounds such as OP’s. However if

toxicity was not changed, it does not mean those types

of compounds (i.e., OP’s) will not be present. Further

tests with PBO will be described in the second edition

of Phase II.

Recent work using C. dubia has shown that sodium

thiosulfate (and EDTA) can remove the toxicity of several cationic metals (Hackett and Mount, in preparation)

from dilution water and effluents. The toxicity of copper,

cadmium, mercury,-silver and selenium (as selenate) at

4x the 24-h LC50 of each in moderately hard reconstituted water was removed by the levels of thiosulfate

typically added in this test. Mercury toxicity was removed with the addition of thiosuffate for 24 h but not

48 h, indicating it may not have been completely

complexed by the thiosulfate. In addition, tests with

zinc, manganese, lead, and nickel and thiosulfate, indicated that the metal toxicity was not removed by thiosulfate. However, with these metals and the addition of

EDTA, the toxicity to C. dubia was complexrd (cf.,

Section 8.8, EDTA Test). Knowing which metals are

bound by both thiosulfate and EDTA, and which metals

are complexed with only one or the other additives can

be very helpful in narrowing down the possible toxicant.

8.7 Oxidant Reduction Test

Principles/General Discussion:

This test is designed to determine to what extent

constituents reduced by the addition of sodium thiosulfate (Na,S,O,) are responsible for effluent toxicity. Chlorine, a commonly used biocide and oxidant, is frequently found at acutely toxic concentrations in municipal effluents. Chlorine is unstable in aqueous solutions

and decomposition is more rapid in solutions when

chlorine is present at low concentrations. Phase I initial

aeration tests will provide information on chlorine toxicity as will the oxidant reduction test. However, this

oxidant reduction test does not simply affect chlorine

toxicity. Also neutralized in this test are other chemicals

used in disinfection (such as ozone, and chlorine dioxide), chemicals formed during chlorination (such as

mono and dichloramines), bromine, iodine, manganous

ions, and some electrophile organic chemicals. Frequently, the reduced form of the toxicant has a much

lower toxicity.

Data on the toxicity of sodium thiosulfate to

Ceriodaphnia dubia, Daphnia magna and fathead minnows are given in Table 8-4. Data generated at ERL-D

show that for Ceriodaphnia, both feeding and lower

hardness waters results in greater thiosulfate toxicity,

and this trend appears to be the same for fathead

minnows (Table 8-4). In effluents, some of the added

thiosulfate will combine with certain oxidants present,

thereby lowering the concentration of the reactive and

toxic thiosulfate. Therefore, the LCSO values indicate

that less toxicity due to thiosulfate (Table 8-4) might be

expected in effluents than in dilution water (i.e., reconstituted water) where no oxidants are present to react

with the thiosulfate. More importantly, when an effluent

concentration of 4x the LC50 is tested, toxic oxidant

levels should not be excessively high. As a result there

should not be a need to add very large amounts of

thiosulfate to neutralize toxic oxidants in the test solution.

Additions of sodium thiosulfate for this test can be

approached in either of two ways; a gradient of thiosulfate concentration can be added to several test chambers containing the same effluent concentration or as a

dilution test where a 3 by 3 matrix of effluent concentrations and thiosulfate concentrations are used.

Although the thiosulfate addition test was initially For the gradient approach, concentrations of sodesigned to determine if oxidants (such as chlorine) are dium thiosulfate equal to and lower than the thiosulfate

responsible for effluent toxicity, thiosulfate can also be LC50 for the test species being used are added to

a chelating agent for some cationic metals. Conse- several containers with effluent at the 4x-LC50 (or 100%)

quently, reductions in effluent toxicity observed with concentration (cf., Figure 8-l 5). If the test species is

this test may be due to the formation of metal com- not listed in Table 8-4, the thiosulfate LC50 will have to

8-33
be determined. Time to mortality may also be useful in

addition to observing mortality at a fixed time (i.e., 24,

48 or 96 h). Time to mortality measurements are important when no dilutions of the effluent are used.

The dilution approach has the advantage in that

LCSOs can be calculated to see how much the toxicity

was reduced. For this test a matrix of three effluent

concentrations and three levels of thiosulfate concentrations are used. The choice of the thiosulfate concentrations to add to the effluent is based on the thiosulfate

LC50 for the test species being used in an appropriate

dilution water (Table 8-4). Three sets of effluent solutions (i.e., 4x-LC50, 2x-LC50, 1 x-LCSO or 1 OO%, 50%,

25%) are prepared. To the first set, thiosulfate is added

to each test solution at one-half the thiosulfate LC50; to

the second set, thiosulfate is added at one-fourth (0.25x)

the LC50; and to the third set, thiosulfate is added at

0.125x the LC50. In this approach the concentration of

thiosulfate remains constant over each effluent dilution

series. The test results are compared to the baseline

test result to determine the amount of toxicity removal.

For cases where oxidants account for only part of

the toxicity, sodium thiosulfate may only reduce, not

eliminate the toxicity. The thiosulfate addition test is

useful even when chlorine appears to be absent in the

effluent. As discussed above, oxidants other than chlorine occur in effluents and this test should not be

omitted just because the effluent is not chlorinated.

Likewise, removal of toxicity by thiosulfate does not

prove that chlorine was the cause of effluent sample

toxicity. Refer to the Results/Subsequent Tests section

below for additional options.

Table 8-4. Toxicity of sodium thiosulfate to Ceriodaphnia dubia, Daphnla magna, and fathead minnows

Water Life LC50 (u/l I (95% Cl\

.

Species Type Stage 24 h 48 h 72 h 96 h

Ceriodaphnia dub& SRW 524 h

SRW 124 h

SRW 124 h

SRW ~24 h

MHRW 124 h

HRW 124 h

VHRW ~24 h

VHRW 524 h

VHRW 524 h

Daphnia magn$

Pimephales promelasl

SRW

SRW3

SRW’

MHRWJ

MHRW’

HRW3

HRW’

VHRWJ

VHRW’

NR

524 h

124 h

124 h

524 h

124 h

524 h

124 h

524 h

. 2.5

(12

(1.0-l .7)

1.5

(1.2-2.0)

2.0

(152.7)

(1 Z2)

6.6

(5.8-7.5)

5.0

(3.8-6.5)

6.6

(5.8-7.6)

5.0

(3.9-6.4)

7.4

(6.3-8.9)

;+j

I-_)

11.6

(9.7-l 3.9)

12.9

(11.8-14.1)

13.3

(11.8-15.7)

13.4

(11.8-15.3)

0.85

(0.72-l .O)

0.88

(0.72-l .l)

0.95

(0.83-l .l)

(0.: -80499,

0.98

(0.62-l .6)

1.6

z

(2.5i4.3)

1.8

(1.4-2.3)

(0.;;: .6)

8.4

(7.6-9.3)

(5.E.4)

(8.ti.7,

(“-“,

(7.ElLO)

12.9

(11.8-14.1)

(6.:::.2)

12.1

(10.8-l 3.4)

(7.Z.5)

7.1

(5.9-8.4)

$2)

9.8

0

(6.9-9.1)

12.5

(11.3-13.8)

6.7

(5.5-8.2)

12.1

(10.8-13.4)

(6.ki.3,

(4Z.9)

(3.ii.6)

Pi

(6.ip9.0)

11.7

(10.4-13.0)

(4.Z.9)

12.1

(10.8-13.4)

11 Data generated at ERL-Duluth; both species were 524 h old at test initiation and C. dubia were fed.

2 Dowden and Bennett, 1965.

3 Data generated at ERL-Duluth and values represent those from 7-d growth and survival tests and daily renewals.

Note: (-) = Confidence interval cannot be calculated as no partial mortality occurred; NR = Not reported; VSRW = very soft reconstituted

water; SRW = soft reconstituted water; MHRW = moderately hard reconstituted water; HRW = hard reconstituted water; VHRW = very

hard reconstituted water.

8-34
Volume Required:

A maximum volume of 100 mL effluent is required

for the oxidant reduction test. The exact volume required will depend on the 24-h initial LC50. For the

gradient addition, six effluent aliquots at 4x-LC50 or

(100%) are required, each having different thiosulfate

concentrations. For the dilution test, three sets of three

effluent solutions are prepared (Le., 4x-, 2x-, lx- 24-h

LC50) and three different concentrations of sodium

thiosulfate (e.g., 0.25, 0.5, and 1 .O g/L) are each added

to a set of dilutions.

Apparatus:

Glass stirring rods, glass volumetric flask for sodium thiosulfate, 1 mL glass pipettes, automatic pipette, disposable pipette tips, 10 - 1000 PL pipettes,

eye dropper or wide bore pipette, 30 mL beakers or 1

oz plastic cups, light box and/or microscope (optional),

pH meter and probe.

Reagents:

Regardless of whether the 1 x 6 gradient addition

test or the dilution test is to be done, the sodium

thiosulfate stock concentration should be 10x the sodium thiosulfate LC50 concentration for the test species being used.

Test Organisms:

Test organisms, 50 or more, of the same age and

species.

Procedure:

Day 2: To perform the gradient thiosuifate addition

test, transfer six 10 mL aliquots of effluent diluted to 4xLC50 (or 100%) into six test chambers. Add 1 .O, 0.8,

0.6, 0.4, and 0.2 mL of the appropriate concentration of

the thiosulfate stock to five aliquots and mix. Do not

add any to the sixth. The container receiving 1 mL of

thiosulfate should now contain the approximate concentration of sodium thiosulfate equal to the LC50 of

the test species. Figure 8-15 contains an example form

for recording the data. A suggested schedule for observing time to mortality is shown on the data form.

To perform the thiosulfate dilution addition test,

prepare three sets of effluent dilutions (i.e., 4x-LC50,

2x-LC50, lx-LCSO) and add the appropriate amount of

thiosulfate (i.e., 0.5x, 0.25x, and 0.125x thjosulfate LC50)

for the test species to each set of dilutions. Figure 8-16

is an example data sheet for the thiosulfate addition

test using this effluent dilution approach. The baseline

test conducted at the same time will provide information on effluent toxicity without thiosulfate added.

interferences/Controls and Blanks:

Controls prepared for the baseline toxicity test act

as a check on the general health of test organisms,

dilution water quality and test conditions.

When the time to mortality in the various thiosulfate

exposure concentrations in the gradient addition test is

compared to ,$he treatment without thiosulfate, one can

determine whether the addition of thiosutfate increased

the time to mortality at some thiosulfate concentration.

If, in all of the effluent exposures, the time to mortality

decreases, then thiosulfate is affecting toxicity. If all

test solutions cause mortality in the thiosuifate effluent

dilution test, but this trend does not occur in the baseline

test, the thiosulfate may be causing the toxicity. In

either case, the test should be repeated with weaker

sodium thiosutfate additions. If the toxicity is unchanged,

perhaps not enough sodium thiosutfate was added, and

the test can be repeated using a higher range of the

thiosulfate additions.

If a significant loss in effluent toxicity is apparent

over the first 24-h period after sample arrival in the

laboratory (i.e., initial LC50 c baseline LC50), it may be

necessary to conduct future oxidant reduction tests for

Phase I immediately upon arrival of the sample in the

laboratory.

Results/Subsequent Tests:

If oxidants are causing toxicity, time to mortality

should increase somewhere in the range of tested

thiosulfate additions or the toxicity should be reduced

from the baseline LC50. No change in toxicity suggests

either no oxidant toxicity or not enough thiosulfate was

added. The experiment should be repeated, increasing

the concentration of thiosulfate added.

When the LCSOs from the sodium thiosulfate addition dilution test indicate toxicity was reduced when

compared to the baseline LC50, thiosulfate has either

reduced or complexed the toxicant( If the highest

addition of thiosulfate increases the toxicity of the

sample, the thiosulfate itself may be at a toxic concentration. However, if the LC50 for the next lower additiofl

of thiosulfate of the effluent dilutions reduced and/or

removed toxicity, then more tests for oxidants or metals

should be explored.

If oxidant toxicity is evident, a measurement of free

chlorine should be made and the concentration compared to the chlorine toxicity value for the test species

used. For identification it may be necessary to measure

mono and dichloramine since they have different

toxicities than free chlorine (see Phase Iii for confirming mixtures as toxicants). A comparison of the aeration and C,, SPE test results to the oxidant reduction

test results may provide even more information on the

physical/chemical nature of the oxidants.

For those effluents where chlorine is measurable,

dechlorination may be achieved by the use of sulfur

dioxide (SO,) gas. This technique used was developed

by T. Wailer (personal communication, University of

North Texas, Denton, TX). (Note: Caution in handling

the SO, should be exercised because it is an extreme

irritant.) As with thiosuifate, SO may also reduce compounds other than chlorine. This information can be

useful when one needs to know if substances other

than chlorine are causing the toxicity.

To dechlorinate using SO,, the following procedure

is used. Place 10 mL of high quality distilled water into

8-35
Flgurm 6-15. Exrmplo data shoat for the oxldrnt toduction toet when urlng I gradlant of sodium thloaulfato

concantrrtlona.

Test Type: Oxidant Reduction

Test Initiation (Date & Time):

Investigator:

Sample Log No., Name:

Date of Collection:

Species/Age:

No. Animals/No. Reps:

Source of Animals:

Dilution Water/Control:

Test Volume:

Other Info:

4x-LC50: Of 100%

TRC:

mL Stock II II SuMal Readlngs:

72 h 96 h

ApH DO A pH DO

l *

Note: See baseline data sheet for control data.

Stock Concentration - o/L Na2S203

Cgmments:

6-36

a graduated cylinder. Bubble the SO, gas directly into

the water for about 5 min to prepare SO,-saturated

water. (Caution: This saturation procedure must be

done in a hood!) For a first attempt at the amount of

SO, to add without the TRC measured we use 2 PL of

the SO,-saturated water per 100 mL of effluent with

TRC values of O-5 mg/L). This amount of SO, is not

acutely toxic and is effective at removing most commonly encountered TRC concentrations. For measured

chlorine concentrations, proportional amounts of SO,-

saturated water have been used as follows: for 0.02

mglL TRC add 3.6 PL SO,-saturated water to 1 L of

sample; for 0.16 mg/L TRC, add 12 @_/L SO,-saturated

water; for 1.3 mg/L TRC add 39 @L SO, saturated

water: and for 2.1 mg/L TRC add 64 PUL SO -saturated water (T. Wailer, personal communication . f Another technique to remove the chlorine is being explored at present. The use of sodium bisulfite solutions

added in the same way as sodium thiosulfate solutions

are added is being explored.

In cases where both the oxidant reduction test and

EDTA chelation test reduce the toxicity in the effluent

sample, there is a strong possibility that the toxicant

may be a cationic metal(s). For example, thiosutfate

and EDTA both reduce the toxicity of copper, cadmium,

and mercury, At this point, the Phase II methods for

identification for cationic metal(s) toxicants should be

investigated.

8.8 EDTA Chelation Test

Principles/General Dlscussion:

To determine the extent to which effluent toxicity is

caused by certain cationic metals, increasing amounts

of a chelating agent (EDTA; ethylenediaminetetraacetate

ligand) are added to aliquots of the effluent sample.

The form of the metal (e.g., the aquo ion, insoluble

complex) has a major effect on its toxicity to aquatic

organisms (Magnuson et al., 1979) and specific metal

forms are more important in aquatic toxicity than the

total quantity of the metal.

EDTA is a strong chelating agent, and its addition

to water solutions produces relatively non-toxic complexes with many metals. The success of EDTA in

removing metal toxicity is a function of solution pH, the

type and speciation of the metal, other ligands in the

solution, and the binding affinity of EDTA for the metal

versus the affinity of the metal for the tissues of the

organism (Stumm and Morgan, 1981). Because of its

complexing strength, EDTA-metal complexes will often

displace other soluble forms such as chlorides and

oxides of many metals. Among the cations typically

chelated by EDTA are aluminum, barium, cadmium,

cobalt, copper, iron, lead, manganese (2+), nickel, strontium, and zinc (Stumm and Morgan, 1981). EDTA will

not complex anionic forms of metals such as selenides,

chromates and hydrochromates, and forms relatively

weak chelates with arsenic and mercury. For those

metals with which it forms relatively strong complexes,

the toxicity of the metal to aquatic organisms is frequently reduced: EDTA has been shown to chelate the

toxicity to C. dubia due to copper, cadmium, zinc,

manganese, lead; and nickel (Hackett and Mount, In

Preparation) in both dilution water and effluents. HOWever, it was also found that EDTA did not complex the

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.

Since EDTA chelates calcium and magnesium (albeit weakly) the choice of the level of EDTA to add was

originally (EPA, 1988A) based on the premise that

calcium and magnesium had to be chelated before

toxic metals would be. However, recent work has shown

that the toxicity due to cationic metals was reduced

regardless of water hardness. Therefore the mass of

chelating agent required should be approximated because excess EDTA becomes toxic when present above

a certain concentration. The range of EDTA concentrations that will adequately bind the metals but is not

toxic appears to be smaller than that for sodium thiosulfate and oxidants.

_

Table 8-5 contains LCSOs of EDTA for Ceriodaphnia

and fathead minnows at various hardness and ainity

values. Note that the concentration of EDTA tolerated

by organisms increases directly with both water hardness and salinity. By measuring the hardness and salinity of the effluent, the range of EDTA concentrations

that should not be toxic in an effluent sample can be

estimated. “Salinity” not due strictly to NaCl will have

different effects on toxicity. This calculation, for prediction of the EDTA concentration, is more involved than

is at first apparent. The data in Table 8-5 indicate that

over the physiological range of hardness and salinity,

hardness affects the toxicity of EDTA more than NaCI.

The usual methods for measurement of salinity (conductivity meter, salinometer or refractometer) do not

specifically measure sodium chloride. The choice of

EDTA concentrations should always be based first on

hardness and secondly on salinity when the salinity is

known. The particular combination of hardness and

salinity present in an effluent sample may have to be

tested to get an accurate EDTA LC50. If the salinity is

composed of ions other than sodium and chloride, the

hardness of the dilution water should be made equal to

the effluent hardness and the additional “salinity” added

in the form of other major cations and anions such as

potassium, sulfate and carbonate.

An EDTA LC50 value derived in a standard dilution

test water (such as reconstituted water) is likely to be

much lower than the LC50 of EDTA added to an effluent. For example, the values contained in Table 8-5

represent worst case conditions presented by EDTA in

dilution water. Likewise, 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. For this

reason the concentrations of EDTA added to the effluent should bracket the expected LC50 based on clean

water with a similar hardness and salinity value as per

a-38

a graduated cyiinder. Bubble the SO, gas directly into

the water for about 5 min to prepare SO,-saturated

water. (Caution: This saturation procedure must be

done in a hood!) For a first attempt at the amount of

SO, to add without the TRC measured we use 2 PL of

the SO,-saturated water per 100 mL of effluent with

TRC values of O-5 mg/L). This amount of SO, is not

acutely toxic and is effective at removing most commonly encountered TRC concentrations. For measured

chlorine concentrations, proportional amounts of SO,-

saturated water have been used as follows: for 0.02

mglL TRC add 3.6 PL SO,-saturated water to 1 L of

sample; for 0.16 mg/L TRC, add 12 plfL SO,-saturated

water; for 1.3 mg/L TRC add 39 uL/L SO, saturated

water; and for 2.1 mg/L TRC add 64 pUL SO -saturated water (T. Wailer, personal communication . f Another technique to remove the chlorine is being explored at present. The use of sodium bisulfite solutions

added in the same way as sodium thiosulfate solutions

are added is being explored.

In cases where both the oxidant reduction test and

EDTA chelation test reduce the toxicity in the effluent

sample, there is a strong possibility that the toxicant

may be a cationic metal(s). For example, thiosulfate

and EDTA both reduce the toxicity of copper, cadmium,

and mercury. At this point, the Phase II methods for

identification for cationic metal(s) toxicants should be

investigated.

8.8 EDTA Chelation Test

PrlnclplesfGeneral Discussion:

To determine the extent to which effluent toxicity is

caused by certain cationic metals, increasing amounts

of a chelating agent (EDTA; ethylenediaminetetraacetate

ligand) are added to aliquots of the effluent sample.

The form of the metal (e.g., the aquo ion, insoluble

complex) has a major effect on its toxicity to aquatic

organisms (Magnuson et al., 1979) and specific metal

forms are more important in aquatic toxicity than the

total quantity of the metal.

EDTA is a strong chelating agent, and its addition

to water solutions produces relatively non-toxic complexes with many metals. The success of EDTA in

removing metal toxicity is a function of solution pH, the

type and speciation of the metal, other ligands in the

solution, and the binding affinity of EDTA for the metal

versus the affinity of the metal for the tissues of the

organism (Stumm and Morgan, 1981). Because of its

complexing strength, EDTA-metal complexes will often

displace other soluble forms such as chlorides and

oxides of many metals. Among the cations typically

chelated by EDTA are aluminum, barium, cadmium,

cobalt, copper, iron, lead, manganese (**), nickel, strontium, and zinc (Stumm and Morgan, 1981). EDTA will

not complex anionic forms of metals such as selenides,

chromates and hydrochromates, and forms relatively

weak chelates with arsenic and mercury. For those

metals with which it forms relatively strong complexes,

the toxicity of the metal to aquatic organisms is frequently reduced, EDTA has been shown to chelate the

toxicity to C. dub& due to copper, cadmium, zinc,

manganese, lead; and nickel (Hackett and Mount, In

Preparation) in both dilution water and effluents. HOWever, it was also found that EDTA did not complex the

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.

Since EDTA chelates calcium and magnesium (albeit weakly) the choice of the level of EDTA to add was

originally (EPA, 1988A) based on the premise that

calcium and magnesium had to be chelated before

toxic metals would be. However, recent work has shown

that the toxicity due to cationic metals was reduced

regardless of water hardness. Therefore the mass of

chelating agent required should be approximated because excess EDTA becomes toxic when present above

a certain concentration. The range of EDTA concentrations that will adequately bind the metals but is not

toxic appears to be smaller than that for sodium thiosulfate and oxidants.

.

Table 8-5 contains LC5Os of EDTA for Ceriodaphnia

and fathead minnows at various hardness and s&n&y

values. Note that the concentration of EDTA tolerated

by organisms increases directly with both water hardness and salinity. By measuring the hardness and salinity of the effluent, the range of EDTA concentrations

that should not be toxic in an effluent sample can be

estimated. “Salinity” not due strictly to NaCl will have

different effects on toxicity. This calculation, for prediction of the EDTA concentration, is more involved than

is at first apparent. The data in Table 8-5 indicate that

over the physiological range of hardness and salinity,

hardness affects the toxicity of EDTA more than NaCI.

The usual methods for measurement of salinity (conductivity meter, salinometer or refractometer) do not

specifically measure sodium chloride. The choice of

EDTA concentrations should always be based first on

hardness and secondly on salinity when the salinity is

known. The particular combination of hardness and

salinity present in an effluent sample may have to be

tested to get an accurate EDTA LC50. If the salinity is

composed of ions other than sodium and chloride, the

hardness of the dilution water should be made equal to

the effluent hardness and the additional “salinity” added

in the form of other major cations and anions such as

potassium, sulfate and carbonate.

An EDTA LC50 value derived in a standard dilution

test water (such as reconstituted water) is likely to be

much lower than the LC50 of EDTA added to an effluent. For example, the values contained in Table 8-5

represent worst case conditions presented by EDTA in

dilution water. Likewise, 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. For this

reason the concentrations of EDTA added to the effluent should bracket the expected LC50 based on clean

water with a similar hardness and salinity value as per

a-38
Table 8-5. EDTA to Ceriodaphnlo and fathead minnow@ and sailnitlee

Hardness

Species

Water

Type

 as

CaCO,)

I Salinity’

(PPt) 24 h

LC50 tall) (95%

dubra

VSRW lo-13

SRW 40-48

80-100

HRW 160-180

VHRW

0

0

0

0

0

0.5

1

2

3

0

0

0

0

0

0.5

1

2

3

(O.i&)

0.12

(0.104.13)

0.23

(0.21-0.27)

(0.402SO)

0.71

(0.58-0.87)

0.05

(-)

0.12

(0.10-0.13)

0.33

(0.27-0.41)

0.44

(-)

(O.C!&4)

0.14

(0.12-0.18)

(O.E35)

(0.4Z6)

0.81

(068-0.97)

0.03

(-_)

0.11

(-)

0.22

AZ

(-)

0.41

(0.36-0.47)

0.05

(-)

0.11

(-)

0.23

(0.21-0.27)

0.32

(0.23-0.45)

0.03

(0.03-0.04) (O&&4)

0.14 0.11

(0.12-0.18)(0.08-0.14)

0.27 0.27

(0.22-0.33) (0.22-0.33)

(O.E62) (O.&O)

0.81 0.81

(068-0.97) (0.68-0.97)

(O.cz?%4)

(0.&%9,

0.25

(0.20-0.31)

(O.Zk)

0.81

(0.52-0.83)

0.11

(-)

0.17

(0.13-0.21)

0.23

(0.17-0.32)

0.37

(0.28-0.48)

l Brine from evaporated seawater used as source of salinity. All data generated at EPA ERL-Duluth. All C. dubia were 524 h old and the

fathead minnows were all 136 h old at test initiation. Ceriockphnia were fed; see section on toxicity tests for details.

Note: (-) = Confidence interval cannot be calculated as no partial mortality occurred: NR = Not reported; VSRW = very soft reconstituted

water; SRW = soft reconstituted water; MHRW = moderately hard reconstituted water; HRW = hard reconstituted water; VHRW = very

hard reconstituted water.

the above discussion. The complexation of metals with

EDTA may not be immediate after the addition of EDTA.

Therefore, it is recommended that the EDTA test solutions be set up first and these solutions allowed to sit

for the duration of pH adjustments and other manipulations before the introduction of test organisms. This is

at least 2 h.

As with the oxidant reduction test, the EDTA can

be added in two ways; a gradient of EDTA can be

added to replicate of one effluent concentration or three

concentrations of EDTA can be added to three sets of

effluent dilutions. The effluent itself is used as a control

rather than a blank based on dilution water as in the pH

adjustment test. The gradient addition test is done by

adding increasing concentrations of EDTA to several

aliquots of the effluent (4x-LC50 or 100%). The goal of

this test is to add enough EDTA to reduce metal toxicity. At some EDTA addition the metals will be chelated

and the EDTA will not be present at toxic concentrations. At lower EDTA additions the metal toxicity is not

removed; in the midrange of the EDTA additions the

metals will be rendered non-toxic by the EDTA, and at

the high end of the range of EDTA additions, the

unreacted EDTA is itself toxic. By using an effluent

concentration of 4x-LC50 (or 100% if the LC50 is greater

than 25%) the potential for exceeding the binding capability of the added EDTA is lessened, especially for

very toxic effluents (LC50 ~10%).

To conduct the EDTA test using effluent dilutions,

three addition levels of EDTA (using one stock) are

selected (based on the LC50 of EDTA for the species

of choice). Each of these three EDTA levels are then

added to effluent dilution tests in a 3 x 3 matrix. The

EDTA is added to the 4x-, 2x-, and lx-LCSO test cups

after the effluent solutions are prepared so that the

three EDTA concentrations are constant across each

a-39
set of effluent dilutions. For example, 0.2 mL of an

EOTA solution is added to test cups containing

4xLC50, 2x-LC50, lx-l_CSO of 1 OO%, 50%,

and 25%. To the next set of test cups of same effluent

dilution sequence, 0.05 mL of the EDTA stock solution

is added and likewise 0.0125 mL is added to the third

set of test cups.

To determine the amount of EDTA to add, one can

use the hardness titration, the measurement of calcium

and magnesium concentrations, or the concentration of

EDTA at the EDTA LC50 for the species of interest.

These are described in the Procedure below.

Volume Required:

A volume of 100 mL effluent usually is required for

the EDTA chelation test. The exact volume needed will

depend on the 24-h initial LC50 and the particular

option chosen to determine the EDTA addition.

Apparatus:

Glass stirring rods, glass volumetric for EDTA stock

solution, automatic pipette, disposable pipette tips, 10,

100, and 1000 uL pipettes, eye dropper or wide bore

pipette, 30 ml beakers or 1 of plastic cups, light box

and/or microscope (optional).

Reagents:

EDTA (disodium salt, Na, EDTA) stock solution

(see discussion under Procedure), reagents for determination of effluent hardness and salinity (APHA, 1980;

Methods 314 and 210).

Test Organisms:

Test organisms, 50 or more, of the same age and

species.

Procedure:

Day 2: There are three ways to determine the

concentration of EDTA stock to prepare.

The first and the most accurate approach (when it

can be used) is to measure the hardness of the 4x-24-h

LC50 effluent concentration (or 100% when the LCSO is

>25%) using the standard method for measuring hardness (APHA, 1980). The concentration of EDTA that

produced the endpoint in the hardness titration of the

effluent sample is the concentration of EDTA needed at

the 0.2 mL addition for either the EDTA gradient test or

the effluent dilution test. An example illustrates this

calculation. In a 36% effluent sample (4x-LC50), 5 mL

of 0.01 M EDTA was needed to titrate the hardness

(100 ml sample size). For the gradient test, 7 EDTA

concentrations will be added to several test chambers,

all containing one concentration of effluent (4x-LC50).

The concentration of EDTA required for the hardness

titration is the highest additive concentration. For a 10

mL test volume of 36% effluent, when 0.5 mL of 0.01 M

EDTA stock was added the resultant EDTA concentration is that which is desired at the 0.2 mL addition. To

provide this EDTA concentration at the 0.2 mL addition

(minimizing the volume addition), increase the 0.01 M

EDTA concentration (concentration of EDTA used in

titration) by 0.510.2 or 2.5x = 0.025 M EDTA stock.

(Note: Molecular weight (MW) of Na,EDTA is 372.3 g.)

The second approach is used when the hardness

measurement endpoint cannot be discerned because

of interferences. If the hardness cannot be titrated,

measure the calcium (Cal*) and magnesium (Mg2+) of

the sample using atomic absorption procedures, and

calculate the amount of EDTA needed to chelate the

calcium and magnesium. EDTA binds with both Cal+

and Mg2+ on a 1:l molar basis. The combined number

of moles of Ca2* (MW=40.1 g) and Mg2* (MW=24.3 g) in

10 mL of effluent at 4x-LC50 equals the number of

moles of EDTA needed for the 0.2 mL addition for a

10 mL sample. This calculated concentration should be

added at the 0.2 mL addition for either the gradient or

dilution test. The calcium and magnesium should be

measured at 100% effluent if the LC50 is greater than

25%.

The third approach, and the one we use most

frequently, is to use the EDTA LC50 concentration to

select addition levels. This approach allows for either a

gradient of EDTA additions to 100% (4x-LC50) solutions or EDTA additions to effluent dilution tests. Choice

of the EDTA LC50 must be based on effluent hardiness

(and salinity). It may be necessary to determine the

EDTA LCSO for the particular combination of effluent

hardness and salinity and test organism used.

After the concentration for the stock solution of

EDTA has been determined, the EDTA gradient test

can be set up. The EDTA LC50 is generally set at the

0.2 mL addition. To perform the gradient EDTA addition test, 7 aliquots of the effluent are prepared at a

concentration equal to 4x-LCSO, or 100% effluent where

the initial 24-h LC50 is greater than 25%. Next, 0.4 mL

of the appropriate EDTA stock is added to the first 10

mL aliquot of the effluent, 0.2 mL is added to the

second 10 mL sample of effluent, 0.1 mL to the third,

and so on until the sixth 10 mL effluent sample has

received 0.0125 mL. The seventh is an effluent blank

used to compare treatment effects on time to mortality

(see Figure S-17). A microliter syringe will be needed

for the smaller additions. If the effluent has a low

toxicity (LCXO = 50-100%) a series of dilution blanks

may be necessary to check for the dilution effect of the

EDTA stock addition. No more than 10% dilution of the

effluent aliquots should be allowed unless a dilution

blank series is included.

To perform the EDTA additions using the effluent

dilution test, three sets of three effluent concentrations

are prepared (1 OO%, 50%, 25%, or 4x-, 2x-, 1 x-LCSO),

while the baseline test serves as the toxicity blank.

After the concentration of stock solution of EDTA has

been established, add the EDTA using a 3 x 3 matrix.

This means that the 0.2 mL addition is added to the

loo%, 50%, 25%, 0.05 ml is added to another set, and

0.0125 mL is added to the third effluent set (see Figure

8-18). The EDTA is added after all the dilutions are

prepared. To allow the EDTA time to complex the

8-40
-

Figure 8-17. Example data sheet for EDTA chelation test when uslng a gradlent of EDTA concentrations.

Test Type: EDTA Chelation

Test Initiation (Date & Time):

Investigator:

Sample Log No., Name:

Date of Collection:

Species/Age:

No. Animals/No. Reps:

Source of Animals:

Dilution Water/Control:

Test Volume:

Other info:

4x-LC50: or 100%

.

0.4

0.2

0.1

0.05

0.025

0.0125

0.0

b

Note: See baseline data sheet for

Stock Concentration - !J/L

Comments:

control data.

EDTA

8-41

8-43

metals, these samples should be prepared first. The

test solutions should not have test organisms added

until all other manipulations are performed.

The complexation of metals by EDTA proceeds at

a rate which may vary according to the sample matrix.

In studies of some aqueous samples containing metal

toxicity, better success in demonstrating chelation of

metals by EDTA may occur if samples spiked with

EDTA remain refrigerated overnight. The next day they

are warmed to the test temperature and the pH is

adjusted to pH i before placing test organisms in the

chambers. This allows time for the EDTA to chelate

any metals which may be in the sample. The solutions

should be mixed thoroughly after spiking with EDTA

and before adding the test organisms.

For both the gradient EDTA addition and EDTA

dilution tests, the pH of the effluent after addition of

EDTA should be checked. Since EDTA is an acid,

additions of this reagent will 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. When stable pHs are obtained

and all other manipulations have been completed, test

organisms are added to the test chambers.

Interferences/Controls and Blanks:

Controls prepared for the baseline toxicity test provide quality control for test organisms, dilution water

and test conditions. Either the zero mL EDTA addition

in the gradient test or the baseline effluent test serves

as a blank for use in determining the presence of EDTA

toxicity.

For the EDTA gradient test, time to mortality may

be recorded at each EDTA addition and then compared

to the untreated effluent. If time to mortality is shorter in

all treatments than in the untreated effluent, repeat the

test using lower EDTA concentrations. If the baseline

test has less toxicity than the EDTA additions in the

dilution test, then the EDTA may be causing toxicity. If

time to mortality or toxicity is not reduced in any treatment, it may be wise to repeat the additions using a

higher range of EDTA concentrations. Erratic patterns

in mortality cannot be used, and when this occurs it

suggests that this test is not appropriate for the particular effluent being studied.

The addition of EDTA to the sample often lowers

the pH of the test solution to as low as pH 4.0. If the

presence of a pH-dependent toxicant such as ammonia

is suspected, then the results of this test must be

interpreted cautiously before attributing losses of toxicity to chelation of metals. For instance, a sample which

contains ammonia toxicity and an undetermined amount

of metal toxicity (perhaps none) may show a loss of

toxicity at some EDTA concentrations. A closer look

may reveal that pH drifted in these samples to 7.5 or

lower, rendering ammonia non-toxic in the sample, and

that metal chelation may have had no role in reducing

toxicity. In the same way, the presence of compounds

whose toxicity is exacerbated at low pH (e.g., hydrogen

sulfide) may confound interpretations of this test. In

such a sample, metal toxicity may indeed be reduced

by chelation at some EDTA additions: however, the

increased toxicity of hydrogen sulfide at the lower pH

could mask the metal toxicity loss. In cases such as

these, we have returned pH to the initial value, and

successfully employed a simple method of pH control

(e.g., closing the test vessel) to avoid misleading pHdependent toxicant interferences. This strategy may

need to be attempted to ferret out interferences and

obtain useful information from the EDTA addition test.

In certain efflueNs, EDTA may reduce the toxicity

of cationic surfactants. This reduction may appear as a

delay in time to mortality. If the EDTA test result is not

likely caused by cationic metals, other Phase I procedures, such as sublation during the aeration step, may

also indicate surfactant toxicity (cf., Section 8.3 aeration test).

Results/Subsequent Tests:

For the EDTA gradient test, if the appropriate EDTA

concentration range is utilized, the time to mortaTtty will

not change from that seen in the exposure 4x-24-h

LCSO of unaltered effluent at low additions of EDTA. In

the 0.2 mL addition, toxicity should be reduced and at

higher additions of EDTA, toxicity will be as high or

higher than the whole effluent itself due to unbound

EDTA toxicity and effluent toxicants other than chelatable metals if present. Time to mortality must be used

to detect partial toxicity removal. Toxicity may be removed at all exposures if the lowest addition of EDTA

removes metal toxicity and the highest addition does

not cause E.DTA toxicity. If toxicity is not reduced in any

treatment, either the effluent has no chelatable metal

toxicity or not enough EDTA was added. Increased

toxicity over the toxicity of untreated effluent suggests

EDTA toxicity and a lower EDTA range should be

tested.

For the EDTA dilution test, if the effluent is less

toxic (i.e., LC50 is greater than baseline LC50) in any

of the three EDTA addition dilution tests, then the

indication is that EDTA removed or reduced the toxicity

and therefore metal toxicity is present. If in all three

tests the effluent is more toxic (i.e., treatment LCSOs

are lower than baseline LC50), then the possibility

exists that EDTA itself is causing toxicity and the test

should be repeated using lower EDTA addition concentrations. If no LC50 of any of the three additions indicates less toxicity than in the baseline test, the possibility of the presence of cationic metals causing toxicity in

the effluent is low, but additions of EDTA at higher

levels may need to be explored.

If toxicity is reduced in a systematic manner, proceed to Phase II methods for specific identification of

the metal(s).
.

8.9 Graduated pH Test

Principles/General Dlscussion:

This test is designed to 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 allow sufficient differences in dissociation to occur in a physiologically tolerable pH range (pH 6-9). Also, the two forms of the

compound (ionized versus un-ionized) must have detectable toxicity differences to the TIE organism. The

ionizable compounds commonly found in municipal and

industrial discharges include ammonia, hydrogen sulfide, cyanide, and some organic compounds (e.g., pentachlorophenol). In addition, pH differences can affect

metal toxicity through changes in solubility and speciation. The effect of pH on ammonia toxicity might be

more readily observed than the effect of pH on the

levels of toxicity of metals, hydrogen sulfide, cyanide,

and ionizable ofganics.

concentration of ammonia occurring in the toxic form

with increasing pH is greater than the decrease in its

toxicity. The net result is an increased toxicity of a

given total ammonia concentration with increased pH.

Temperature also affects the dissociation of ammonia,

but since the temperature is held constant in these

toxicity tests for Phase I, it can be ignored.

Ammonia is frequently present in effluents at concentrations of 5 mg/L to 40 mg/L (and higher). (The

ammonia is measured upon arrival of the sample (Section 6) and this information will be helpful for the graduated pH test.) Levels of 5 mg/L to 40 mg/L are likely to

cause toxicity when several other effluent conditions

occur. Effluent parameters to consider are pH, temperature, DO, CO, content, and TDS. Of these parameters, pH has the largest effect on ammonia toxicity,

and for many effluents (especially with POTW effluents) the pH of a sample rises upon contact with air.

Typically, the pH at air equilibrium ranges from 8.0 to

8.5. Literature data on ammonia toxicity (EPA, 19858)

can be used only as a general guide because of the

large effect of very slight pH changes. The pH values

for most ammonia toxicity tests are usually not measured or reported fully enough to be useful.

One might expect ammonia to be removed during

the pH 11 adjustment/aeration test. Based on our experience, however, ammonia is not substantially removed

by the method described in Section 8.5. Other techniques which can be used to remove ammonia related

toxicity may also displace metals or other toxicants with

completely different physical and chemical characteristics. For example, ion exchange resins (e.g., zeolite)

removes ammonia, cationic metals, and possibly organic compounds through adsorption. For these reasons, the graduated pH test is most effective in differentiating toxicity related to ammonia from other causes

of toxicity, if it is the dominant toxicant.

Effluent toxicity related to metals may also be detected by the graduated pH test, although these effects

are less well documented in effluents than those associated with ammonia toxicity. Acidification of a sample

may increase the bioavailable portion of a metal, and in

some cases (i.e., cadmium, copper, and zinc) this is

countered by a decreasing toxicity of the metal as the

test pH decreases. It-is known, however, that aluminum

toxicity increases as pH diverges from neutral. In experiments in the pH range of 5 to 7 (Campbell and

Stokes, 1985) the toxicities of cadmium, copper and

zinc were shown to increase with increasing pH while

the toxicity of lead decreased with increasing pH. We

have found lead and copper to be more toxic to C. _

dubia at pH 6.5 than at pH 8.0 or 8.5, (in very hard

reconstituted water) and nickel, zinc, and cadmium

were more toxic at pH 8.5 than at 6.5. Since these

compounds are also chelatable by EDTA, the resuqs of

both tests (the graduated pH test and the EDTA addition test) can give information about whether it is an

ionizable compound or a pH sensitive cationic metal.

Other metals have exhibited some degree of pH dependence, but these are not as well defined. Results of

the graduated pH test should be considered in conjunction with the EDTA addition test (Section 8.8). Whether

the metal toxicity can be discerned will depend in large

part on the concentration of other pH dependent toxit

cants in the sample. In order to detect metal toxicity,

one must be cautious when selecting a dilution water

when the test solutions are at low effluent dilutions

because artifactually enhanced toxicity due to metals

may be created if the hardness of the dilution water is

much different than that of the effluent. This effect may

be magnified for metals when coupled with the pH

change. A dilution water similar in hardness to the

effluent must be used for this test to reveal metalcaused toxicity. If more than one pH dependent toxicant is present, the pH effects may either cancel or

enhance one another.

Ammonia acts as a basic compound in water. The

un-ionized, more toxic form (NH,) predominates above

pH 9.3 and the ionized, essentially non-toxic form (NH,+)

is most abundant below pH 9.3 at 25°C. Through the

pH range of 6-8.5, the percent of ammonia in the toxic

form increases 250x over this range. Importantly, as pH

increases, the percentage of the toxic form becomes

greater but the toxicity of the toxic form is less, and

conversely, as pH decreases, the percentage of ammonia (NH,) decreases, but the toxicity of the NH, increases (EPA, 19858). However, the increase in the

Hydrogen sulfide (H,S) occurs in wastewaters, and

its toxicity can be detected by the graduated pH test.

Dissolved sulfide exists in two forms, H,S and HS. The

predominant form depends on both pH and tempefature, but since temperature is held constant in these

Phase I toxicity tests it essentially can be ignored. The

un-ionized form (H,S) is more toxic to aquatic ofganisms, and at pH 6 it comprises over 90% of the dissolved sulfide, while at pH 7, 50% is unionized. At a

pH of 8.5, less than 5% of the dissolved sulfide is

present in the unionized form. Since H,S is the more

toxic form, one would expect to observe an increase in

toxicity relative to a decrease in solution pH. When

considering results of this type it is wise to check

toxicity alteration by the pH adjustment/aeration tests

8-44
(Section 8.5). H,S is readily oxidized and also removed

through volatilization; therefore if H,S is the predominating toxicant, a significant reduction of toxicity should

be observed in the pH adjustment/aeration tests.

The effects of pH on toxicity can be used to detect

the presence of these pH dependent toxicants. By

conducting three effluent tests, each at a different pH,

the effluent toxicity can be enhanced, reduced or eliminated. For a typical 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,. Similar changes in the percent ammonia as NH

for pH’s 6.5, 7.5 and 8.5 occur at other temperatures:

for example, the percentages of unionized ammonia at

20°C for pH’s 6.5, 7.5, and 8.5 are 0.130%, 1.24% and

11.2%, respectively (EPA, 1979). This difference in the

percentages of unionized 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 tests

are done at dilutions symmetrical about the LC50, one

should see toxicity differences between pH 6.5 and 8.5

(cf., Phase II discussion on equitoxic test). The effluent

LC50 (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 will depend upon the characteristics of the particular effluent

being tested. For example, if the air equilibrium pH of

the effluent at 4x the 24-h LC50 is 8.0 it may be more

appropriate to use pH’s 6.0, 7.0, and 8.0. 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). The

pH’s of many POTW effluents rise to 8.5 or higher, so a

gradient of pH’s such as 6.5, 7.5 and 8.5 is more

appropriate. In any case, it will be necessary to conduct

the test at more than one effluent concentration (4x-,

2x-, lx-, 24-h LC50) or with a different graduated pH

scheme to determine what role, if any, the pH dependent compounds play in toxicity.

Perhaps the greatest challenge faced in this graduated pH test is that of maintaining a constant pH in the

test solution. This is a necessity if the ratio of ionized to

the un-ionized form is to remain constant and the test

results are to be valid. In conducting toxicity tests on

effluents, it is not unusual to see the pH of the test

solutions with effluent concentrations of 212% drift 1 to

2 units over a 48 to 96-h period (see Procedure for

suggestions on pH control).

Volume Required:

The volume needed

sign chosen to conduct

size, number of dilutions,

will dictate this; however,

suffice for all three pH’s.

is dependent on the test dethis test. The test chamber

and the toxicity of the effluent

200 mL of test volume’should

Apparatus:

Test chambers such as 78 mm L x 50 mm W x 50

mm H or 1 oz plastic cups; Hamilton 1 L gas syringe

(Model S-1000, Reno, NV); 35 mm x 14 mm H Corning

plastic petri dish bottoms, rubber stoppers, eye droppers or wide bore pipette, 30 mL beakers or 1 oz

plastic cups, light box, and/or microscope (optional).

Reagents:

Cylinder tank of CO , 1 .O, 0.1, and 0.01 N HCI

1 .O, 0.1, and 0.01 N NaaH (ACS grade in high ‘purity

water), buffers for pH meter calibration.

Test Organisms:

Use 5 for each of three dilutions of the whole

effluent (4x-, 2x-, lx-, 24-h LC50 or 1 OO%, 50%, and

25%) and for each test pH (e.g., pH 6, 7, 8) (Figure 8-

19) as well as a control.

Procedure:

Day 2: Either CO or HCI (or the combination of

both) can be used to r’ower the pH of the sample. The

pH of most natural waters and some effluents is controlled by the bicarbonate buffering system. Surface

waters normally contain ~10 mg/L of free CO,. ,;

For the CO, pH controlled tests, the pH is adjusted

with CO, by varying CO, content of the gas phase over

the water or effluent sample. It is necessary to maintain

constant pH’s in the static acute test throughout the 48

or 96-h tests. By usiirg closed headspace test chambers, the CO, content of the gas phase can be controlled. The amount of CO, 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 constituents (e.g., dissolved solids). When

dilutions of an effluent have the same hardness and

initial pH as the effluent, the same amount of CO will

usually be needed for each dilution, but somet!mes

more is needed in the higher effluent concentrations.

Use of a dilution water of similar hardness as the

effluent makes the CO, volume adjustments easier.

In our laboratory, a rectangular chamber (measuring 78 mm L x 50 mm W x 50 mm H) with a small

diameter hole (approximately 20 mm) on one end has

worked well for the CO, graduated pH test. The test

solution volume should be about 10% of the headspace

volume to maintain a large surface to volume ratio

should be maintained. For a 20 mL test volume, with

the CO, gas flushed into air space of the test chamber,

pH’s have reached equilibrium in about 1 h. In most

instances, the amount of CO, produced by the invertebrates has not caused further pH shifts, but with larval

fathead minnows, the pH can drop from the amount of

CO, they respire as well as decomposition of food.

Therefore, in fish tests, the headspace must be reflushed

daily.

The exact amount of CO td inject for pH’s 6.0, 7.0

and 8.0 must be determined through experimentation

with each effluent before the graduated pH test begins.

8-45

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. inject the CO using a gas tight syringe

and quickly close the test chamber tightly. Place the

test chamber in a position that maximizes the surface

to volume ratio. To prepare the test solutions, use a

dilution water of a similar hardness to the effluent and

transfer the effluent solutions to the test container and

randomly add the test organisms. Then add the predetermined amount of CO, to obtain the desired pH’s

and close the container. For pH values from pH 8.5 to

6, O-10% CO, has been needed. If more than 10% CO,

is needed, adjust the solutions with acids and bases

(described below) and flush the headspace with CO,.

Again, the necessary concentration of CO, to use must ’ first be determined experimentally with effluent test

solutions already adjusted to the appropriate pH. This

may require the test to be set up one day later than the

other Phase I tests.

For some effluents adequate pH control can be

obtained by adjusting the pH with acid or base and

tightly covering the test container (no headspace pH

test). A technique that we use has the 1 oz plastic cups

covered with plastic tissue culture dishes (see Apparatus for details). This technique works well with effluents

that have adequate DO content, and’where the BOD is

not high. The procedure for using plastic cups with

tissue culture dish covers is as follows. Adjust three

aliquots of the effluent and the dilution water to the

appropriate pHs. Next, prepare the appropriate dilutions for testing (i.e., 4x-, 2x-, 1x-24-h LC50, or 1 O%,

50%, 25%) and check the pH in one-half hour. If the

pH’s have drifted, readjust them with the appropriate

acid or base. Transfer about 35 mL of each into the 1

oz plastic cups, and randomly add the test organisms.

Carefully place the cover onto the cup; care must be

exercised because some test water will be displaced by

the lid, and organisms can be lost. Ensure that no air is

trapped under the lid during the sealing process. If air

is trapped, remove the cover, count the number of

organisms, and add an additional small amount of the

appropriate pH adjusted test solution. The test organisms can readily be observed through the clear cover

or the sides of the plastic cup. The cover should be

removed only when all the animals have died as the

tight seal cannot be obtained after initially setting up

the test without adding more test water. Once animals

have died or the test is over, remove the cover and

measure the pH and DO. It is important to measure the

DO because toxicants such as ammonia have different

toxicities when DO is low (EPA, 1985B). Keep in mind

that if all of the test animals have been dead for a

while, the pH and/or DO of the test water could have

changed.

Methods that use continuous flow of a COdair

mixture, such as tissue cell incubators, may be preferable and give better pH control. 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 8.3) is difficult to achieve. The pH control

in this high range is much more difficuft because the

concentration of CO2 must be very low and the microbial respiration can Increase the CO, levels in the test

chamber. Use of CO,-free air in the headspace may

work or bubbling a mtx of CO,-free air and normal air

through the headspace or test solution may be needed.

Because such small CGZ concentrations are needed

and because CO, evolutron by microorganisms or test

organisms can significantly alter the CO, concentration,

more frequent flushing of the headspace in static tests

will be needed.

Since many plastics are permeable to CO,, glass

containers may need to be used. Measurements of pH

must be made rapidly to minimize the CO exchange

between the sample and the atmosphere. dvoid vigorous stirring of unsealed samples because at lower pH

values, the CO loss during the measurement can cause

a substantial pk rise.

For the CO, pH controlled tests, the pH should be

measured at 24, 48, 72, and 96 h and at each reading,

one may need to re-flush the headspace with CO,. A

small amount of experimentation will determiOe the

amount of CO, needed for this step. For tf?e no

headspace pH tests conducted in cups with covers, air

bubbles may start to appear after 12 h, and this can

cause the pH to change. An excess of each test solution may need to be prepared to be added to the test

cup at each 24 h interval to prevent the formation of air

pockets which contribute to pH drift.

We also have been exploring the use of hydrogen

ion buffers to maintain the pH of effluent test solutions.

Efforts to use phosphate buffers were unsuccessful

due to the toxicity of the phosphates themselves. 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). The

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).

The acute toxicity of these buffers is low to both

C. dubia and fathead minnows (Table 8-6) and sublethal levels can be added to hold the pH of test solutions. For example, 6.25 mM (1.2 g/L) of the Mes buffer

has been adequate to maintain the pH of one effluent

to within +O.l pH units. However when used in a sediment pore water, more buffer was needed (i.e., 25 mM

or 4.9 g/L) but these levels are still below the acute

toxicity for the buffer. Likewise for the Mops buffer, 6.25

mM (1.3 g/L) held the pH of the effluent at kO.1 pH

units, but 50 mM (10.5 g/L) was needed for the pore

water. The Popso buffer held the pH at 8.2 or 8.5 using

6.25 or 12.5 mM (2.3 or 4.5 g/L, respectively) of buffer

for both the effluent or pore water. The addition of

these buffers did not change the toxicity of a non-toxic

effluent or change the toxicity of a toxic effluent and

sediment pore water.
Table 8-6. The toxicity of the Mea, Mopr, and Popao buffers to Cerfodaphnlo dubia and fathead minnowa

Buffsr Specie8

Water

Type 24 h

50 @,/!_I

48 h 72 h 96 h

Mes C. dubia

Mes C. dubia

LSW

VHRW

15.0

17.4

Mops

Mops

C dubia

C. d&a

LSW

VHAW

16.1

B20.9

13.0

11.9

Pops0

Pops0

C. dubia

C. dubia

LSW

VHRW

>2.3

12.7

~2.3

8.3

Mes

Mes

P. promelas

P. promelas

LSW

VHRW

13.9

B19.5

13.9 13.9 13.9

B19.5 B19.5 z-19.5

Mops

Mops

P. promelas

P. promelas

LSW

VHRW

B20.9 17.2 16.1 16.1

>20.9 B20.9 >20.9 B20.9

Pops0

Pops0

P. promelas

P. promelas

LSW

VHRW

32.3

~36.2

32.3 27.9 27.9

~36.2 ~36.2 fl6.2

Note: The pH was held to at least f 0.1 pH unit of desired pH for all tests. Mes buffer tests were at pH 6.2, Mops buffer tests were at pH

7.2, and Popso buffer tests were at pH 8.2. LSW = Lake Superior water; VHRW = very hard reconstituted water.

While these buffers serve to prevent the pH from

drifting, their addition alone does not actually adjust the

pH value to the desired pH. The buffers are weighed

out and added to the aliquots of whole effluent and

dilution water and both are then pH adjusted with base

to the appropriate values. Serial dilutions are made,

and test organisms are added. While our experience

with the buffers is limited, we have found the amount of

any buffer needed to hold any pH is effluent specific.

Experiments will need to be done to determine the

lowest concentration of buffer needed to maintain 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.

In all graduated pH tests, the pH should be measured at least in the chambers that bracket the LC50

concentration as soon all the animals die. If the pH

drifts more than 0.2 pH units, the results may not be

usable and better pH control must be achieved.

Interferences/Controls and Blanks:

The controls in the CO, chamber or closed cup,

and the baseline test 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

test (at the LCSQ) is close to the pH of the pH adjusted

test solutions (at their respective LCZO’s), 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 pH was adjusted with either HCL or NaOH (cf.,

Section 8.3), or CO, toxicity. Dilution water blanks at

the various pH’s are not used because such blanks are

not appropriate since the effluent matrix may differ from

that of the dilution water. The cleanliness of the acids

and bases is checked in the blanks of the pH adjustment test. Other compounds with toxicities that increase directly with pH may lead to confounding resutts

or may give results similar to ammonia. Phase II contains a suggested test (called the equitoxic test) to

identify ammonia as the cause of toxicity. Monitoring

the acid and base additions may be useful to determine

if amfactual toxicity resulted from the addition of the

salts. Monitoring conductivity of the effluent solutions

after the addition of the acids and bases may also be

helpful in determining artifactual toxicity.

Results/Subsequent Tests:

For the graduated pH test, 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.

8-48
.

When ammonia is the dominant toxicant, the effluent LC50 of the pH 6.5 test solution should be higher

than in the pH 7.5 test, which in turn, should be higher

than the pH 8.5 test. However, ammonia is not the only

possible cause of toxicity. Using the pH at the baseline

effluent LC50, the relative toxicity of each pH adjusted

solution can be predicted if ammonia is the sole cause

of toxicity. For example, if in the baseline effluent toxicity test, the average pH was 8.0 in the 100% concentration in which no organisms survived and the average

pH for the 50% concentration was 7.5 and all organisms survived, the estimated pH at the LC50 (71%)

could be approximated at 7.7. One would expect greater

than 50% mortality in the pH 8 test solution and significantly less in the pH 7 solution. Therefore, if this occurs

one should proceed to Phase II to identify the pH

sensitive toxicant.

If ammonia is one of several toxicants in an effluent, this procedure may pose problems. For this reason, if effluent total ammonia levels are greater than 20

mg/L, it may be appropriate to include a pH 6 effluent

treatment interfaced with other Phase I tests (cf., Section 9). Methods for further identifying and confirming

ammonia as the toxicant can be found in Phases II and

III.

8-49
Section 9

Time Frame and Additional Tests

9.1 Time Frame fur Phase I Studies

The amount of time necessary to adequately chatacterize the physical/chemical nature of, and variability

in, an effluent’s toxicant will be discharge specific.

Among the factors affecting the length of Phase I studies for a given discharge is the appropriateness of

Phase I tests to the toxicants, the existence of long- or

short-term periodicity in individual toxicants and to a

lesser extent, 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 moved into Phase

II more quickly than an ephemerally toxic effluent with

highly variable constituents, none of which are impacted by any of the Phase I tests. The decision as to

when to go beyond Phase I should be based in part on

the regulatory implications and resources involved in

subsequent actions. Where a great amount of resources

is involved, it is crucial that Phase I results be adequate.

There are no clearly defined boundaries between

Phase I and Phase II. The section Results/Subsequent

Tests of the characterization tests in Section 8 provide

further tests to conduct and may be thought of as

intermediate studies between Phases I and II. In terms

of guidance for the time frame of the TIE, several

samples should be subjected to the Phase I characterization test battery 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. The time required to perform a complete

Phase I battery on a sample will depend on many

circumstances, not the least of which is how well organized and experienced the performing lab is at doing

TIES.

If the Phase I characterization tests needed to

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. We cannot provide a time frame or

the number of samples to evaluate. Again, judgement

will have to be used but the differences seen among

samples can be used to decide when further differences are not being found. Phase I characterization

months this may take--each problem for every discharger is unique. The LC50 of samples can be very

different but the same screening tests must be SUCcessful in removing and/or neutralizing effluent toxicity.

The individual Phase I tests which were previously

successful in changing toxicity should be used as a

starting point for Phase II identification. The first step in

Phase II will often be to reduce the number of constituents accompanying the toxicants. These efforts may

reveal more toxicants than 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. More Phase I charactarization

may then be needed. b

Phase I results will not usually provide information

on the specific toxicants. Therefore, if effluent toxicity is

consistently reduced, for example through the use of

Cre SPE, this does not prove the existence of a single

toxicant. In fact, 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. Recognizing

this lack of specificity is very important for subsequent

Phase II toxicant identification.

9.2 When Phase I Tests are Inadequate

For some effluents, the Phase I tests described

above will provide few or no clues as to the characteristics of the toxicants. For such effluents, other approaches

must be tried. Some additional approaches are given

below in much less detail than tests in Section 8 because our experience with them is limited. In addition to

these, one should not hesitate to use originality and

innovation to develop other approaches. As long as

toxicity is used to track the changes, any approach may

be helpful.

Use of Multlple Phase I Manipulations

Our experience suggests that independent action

and less than additive action are much more common

than we realize, at least in effluents. When these interactions occur, interpreting Phase I data may be difficult

and in some instances (especially with independent

action) no apparent effect on toxicity will be seen unless Phase I tests are clustered or used in a series.

These steps do not begin until all Phase I manipular

tions have been completed and the results evaluated. testing should continue until there is reasonable certainty that new types of toxicants are not appearing. No Tests are continued to further separate and concenguidance can be given as to how many weeks or trate the toxicant(

. ’

9-1
The pH of effluents plays an amazingly powerful

role in how it affects both the form of toxicants and their

toxicity. Including pH adjustments to different values

than is suggested in Phase I may be helpful. For

example, if the C, SPE column has partially removed

the toxicity, then flhase I manipulations with the postcolumn sample may be possible (cf., Section 8.6 pH

adjustment/solid phase extraction test). For this multiple manipulation, the post-C,, SPE column effluent

can be treated as whole effluent, and several of the

Phase I steps conducted on the post-column effluent

have been found to be useful in further characterizing

additional effluent toxicants. These include the EOTA

addition test, the thiosulfate addition test, and the graduated pH test. Another combined test is to test the postcolumn effluent that has been spiked with the 100%

methanol eluate to see if the toxicity is equal to that of

the whole effluent. However, this can be a tricky manipulation as the post-column effluent is not the same

as the original effluent, and spiking methanol into the

sample may lower the DO as well as cause quick

bacterial growths, which may result in erratic mortalities.

We have used aeration/fiItration/pH adjustment/C,,

SPE in various combinations to decipher the changes

that occur. The presence of more than one toxicant

may often require such combinations. For instances

where there are multiple toxicants and aeration and

EDTA have both removed some toxicity, the addition of

EDTA in the post-aeration sample may help characterize whether a metal(s) is causing the toxicity that is not

removed through aeration.

If the C,, SPE column has partially removed toxicity, it may be possible to pass 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 (cf.,

Phase II), 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 it is essential that toxicity

tests be performed after each manipulation and not just

on the multiple manipulated sample.

A special effect occurs when an effluent, which

contains two toxicants at very different concentrations,

is diluted. Suppose that toxicant A would produce an

LC50 at 50% effluent and toxicant B causes an effluent

LC50 at 5%. In most cases, only toxicant t3 will materially affect toxicity because the effect of A will be “diluted out” long before the LC50 of B is reached. Unless

the toxicity degrades rapidly, the Phase I tests for such

an effluent would be performed near the LC50 of B

(20% is 4x-LC50) in which case the toxicity of A will not

be yoticed. If one finds toxicity at effluent concentrations in the very low range (such as loo/,) additional

Phase I testing at higher effluent concentrations should

subsequently be done. Cases such as these should be

caught in Phase III, but earlier detection will be more

cost-effective.

The two objectives which usually move the TIE

along more rapidly are to separate and concentrate the

toxicant( Anything that can be done in Phase I to

achieve these goals will speed the process.

Activated Carbon

The use of carbon has been limited because it is

much less selective than ion exchange or C,, SPE

columns, and extraction is less precise and more difficult. However, carbon’s non-selectivity can be an advantage in some situations. When a rather wide array

of more specific methods have failed and the Phase I

tests above have not changed toxicity, a “chemical

sponge” may be useful. In order to start, one must be

able to alter toxicity somehow in order to tell what

changes are occurring. A second objective in early

work is a way to remove the toxicants from the sample

(i.e., to concentrate them). Carbon has a high capability

to do both. Furthermore, the knowledge about carbon

sorption and extraction is extensive and help can be

found in the literature. While it is true that carbon may

alter some chemicals, many are not affected by it. We

must recognize that other conventional methods such

as ion exchange are also not specific. Ion exchange

columns can sorb non-polar organics and C,, SPE

columns can sorb metals. *

Otfter Specific Ion Columns

_

Many other types of resin columns are available

through commercial sources. Many of these have “insurmountable” blank toxicity problems but some show

promise. Mixed bed ion exchange columns appear promising because pH is not drastically altered as the sample

passes through the resin bed and the blanks appear to

be acceptable. Of course with any of these lesser used

methods, the organism’s tolerance to dilution water

passed over the resins and the eluate(s) must be determined.

Other LIgands

EDTA reduces toxicity for only some of the cationic

metals, and other ligands may help.

9.3 interpreting Phase I Results

After the suite 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. Rarely is there no effect from

any manipulation. Frequently more than one manipulation affects toxicity. 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 about 80 different 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. In regard to multiple

toxicants, refer to the above section Use of Multiple

9-2
Phase I Manipulations, regarding complications of determining toxicant interactions in effluents.

After Phase I is completed on a sample, 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.

As a matter of principle, where multiple toxicants

are involved, experience shows that once one toxicant

is identified, identification of subsequent toxicants becomes easier because:

1. The toxicity contribution of the identified

toxicant can be established for each sample.

2. The number of Phase I manipulations that

will affect the toxicity of the known toxicant

can be determined.

3. One can determine whether the identified

and the unidentified toxicant are additive.

4. If some manipulations affect the toxicity

due only to the unidentified toxicants, some I

of their characteristics can be inferred.

5. 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 physical/chemical characteristics.

Another suggestion, is that when some Phase I

outcomes are understandable and others are not, concentrate on the one or the few that seem to be the most

clear-cut and which have a major effect on toxicity. For

example, if an effluent has 10 TU and 2 TU are removed by the addition of EDTA, 1 TU is removed by

the C,, column and 5 TU are removed by the aeration

manipulation, begin identification on the toxicity removed

by aeration. In another example, suppose the filtration

manipulation reduced the toxicity by 1 TU, both pH 3

and pH 11 adjustment tests showed that the toxicity

increased by 2 TU, the graduated pH test at pH 7

decreased toxicity by 2 TU and the post-c,, SPE column effluent (at pH i ) had 2.5 TU less toxicity than the

whole effluent. of th8 2.5 TU removed by the column,

1.7 TU could be eluted with the 100% methanol. The

next step then is to begin the Phase II identification on

the SPE extractable toxicity because:

1. Widely accepted methods are available for

analyses of many non-polar organic

compounds.

2. The method exists for both separating and

concentrating such toxicants (cf., Phase II).

3. This C, extractable toxicity manipulation

behaved as expected.

4. Many effluents have non-polar toxicity, and

based on those probabilities, that non-polar

toxicity is likely to be real.

In the latter example, the unexplainable pH and

filtration effects might be a result of the behavior of the

non-polar toxicant or could be caused by some associated artifact. If the non-polar toxicity is identified,

then the results of the pH adjustment and filtration

steps may be explainable.

The third suggestion is to concentrate on those

manipulations affecting toxicity in which the toxicant is

removed from other effluent constituents. In the above

example, the SPE column separated the toxicant

from other non-sorbable constituents. Other examples

of where the toxicant is removed from the other constituents are the filtration and the aeration manipulations.

Separating the‘toxicant(s) from non-toxicant(s), and

concentrating the toxicant are usually the most productive efforts to pursue before identification (analyses)

begins. Attempts to begin analysis for suspect toxicant

without thisstep is frequently a mistake, and can be

costly.

9.4 Interpretation Examples

In this section, various examples of Phase I results

are given with interpretation suggestions. TheseShould

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. However, as in

the example in Section 9.3, if the toxicity can be recovered in the methanol fraction (see Section 8.6, Results/

Subsequent Tests for elution and Phase II for more

details), then the theory that a non-polar toxicant is

causing the toxicity is better substantiated. Metals do

not elute with methanol and therefore do not produce

toxicity in the methanol fraction toxicity test (cf.,

Phase II).

Example 1. Nun-polar toxicant( The Phase I results implicating non-polar toxicants are:

1. All toxicity in the post-c,, SPE column

effluent was removed.

2. The toxicity removed was recovered in the

methanol elution of the SPE column.

The above discussion (cf., Section 9.3) has provided most of the interpretative rationale for these

Phase I results which are typical of non-polar organics.

As stated above, toxicants other than non-polar compounds may be retained by the SPE column but they

are less likely to be eluted sharply. Also, as discussed

9-3
Example V. 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 6). If more than 5 mg/L of total ammonia is

present, additional evaluations should be done. Sole

dependence on analyses is not advisable because there

is little or no additivity between ammonia and some

other toxicants (e.g., such as surfactants). 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:

1. The concentration of total ammonia is

5 mg/L or greater.

2. Toxicity increases as the pH increases.

3. The effluent is more toxic to fathead

minnows than to Ceriodaphnia or Daphnia.

Example VI. Oxidants. In effluents, oxidants other

than chlorine may be present. Measurement of a chlo-

-

rine residual (TRC) is not enough to conclude that the

toxicity is due to an oxidant.

In general, oxidants are indicated by the following:

1.

2.

3.

4.

The addition of sodium thiosulfate to the

effluent reduced or removed the toxicity.

Aeration without any pH adjustment

removed or reduced toxicity.

The sample is less toxic over time when

held at 4°C (type of container is not an

issue here).

Ceriodaphnia are more sensitive than

fathead minnows.

Of course, TRC greater than 0.1 mg/L at the effluent LC50 concentration (and depending on test species) would indicate chlorine as the oxidant causing the

toxicity. In addition, the dechlorination with SO, provides evidence of chlorine toxicity in the same manner

as the sodium thiosulfate addition test.

9-5
Section IO

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Aqueous Systems, E.A. Jenne, Editor. pp. 635

656.

Neilson, A.J.-, A.S. Allard, S. Fischer, M. Malmberg, and

T. Viktor. 1990. Incorporation of a Subacute Test

with Zebra Fish into a Hierarchical System for

Evaluating the effect of Toxicants in the Aquatic

Environment. Ecotox. and Environ. Safety 20:82-

97.

Norberg-King, T.J., E.J. Durhan, G.T. Ankley and E.

Robert. 1991. Application of Toxicity Identification

Evaluation Procedures to the Ambient Waters of

the Colusa Basin Drain. Environ. Tox. and Chem.

10(7):891-900.

OSHA. 1976. Occupational Safety and Health

Administration. OSHA Safety and Health Standards,

General Industry. 29 CFR 1910. OSHA 2206

(Revised).

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Relative Sensitivity of Diatoms, Snails, and Fish to

Twenty Common Constituents of Industrial Wastes.

Prog. Fish-Cult.:1 37-140.

Poirier, S.H., M.L Knuth, C.D. Anderson-Buchou, L.T.

Brooke, A.R. Lima, and P.J. Shubat. 1986.

Comparative Toxicity of Methanol and N,NDimethylformamide to Freshwater Fish and

Invertebrates. Bull. Environ. Contam. Toxicol.

37(4):615-621.

Randall, T.L. and P.V. Knopp. 1980. Detoxification of

Specific Organic Substances by Wet Oxidation. J.

Water Pollut. Control Fed. 52(8):2117-2130.

Schubauer-Berigan, M.K. and G.T. Ankley. 1991. The

Contribution of Ammonia, Metals and Non-polar

Organic Compounds to the Toxicity of Sediment

Interstitial Water from an Illinois River Tributary.

Environ. Toxicol. and Chem. 10(7):925-939.

Smith, R.M. and A.E. Martell. 1981. Critical Stability

Constants. Volume 4: Inorganic Complexes. Plenum

Press, NY.

Stumm, W. and J.J. Morgan. 1981. Aquatic Cher%try -

An Introduction Emphasizing Chemical Equilibria in

Natural Waters. John Wiley & Sons, Inc., New

York, NY.

Walters, C.I. and C.W. Jameson. 1984. Health and

Safety for Toxicity Testing. Butterworth Publ.,

Woburn, MA.

1 o-2
* L

,

TECHNICAL REPORT OATA

(Pltosr rrod lnrmrnont on fhr rtvtne brforw comulrnn~j I

1 ntpont NO. a. (

3. ALC4?lE~T’S ACCtSflON NO* I

EPA/600/6-91,'003 PEW-100 072 I

, , TITLE ANOSUB~~~L~ 9.RCrOnTOATl ii

~ktiods for -ati& mxicity Identificdion Evaluation: February 1991

Phase 1 Toxicity Characterization Procedures (Second ~.~E~~OR~INGORC~~~Z~TI~~CC=)~ I

Edition)

7 *UtHORISJ 1 3 1

Nor&p-King, T.J.I,lbbunt, D/, Durban, &,' Ank$ey,

G.T. , Burkhard, L. I Amato, J.*, Lukasewycz, M. I

0, ~R~OMHG ORGANIZATION NAME AN0 AODRESS

I

10. PROGRAM ELSMENT two,

knited States Env~onmental Protection Agency,

Environmntal Reseych Laboratory, 6201 Congdon glvd. 11. CONTRACTIGRANT NO.

Duluth, MI3 55804; AScI Carp, Duluth, MN 55804; U.S.

12. SPONSORING AGENCY NAME ANO ADOmESS

Environmental Research Labdratory

Iffice of Research and Development

13. TYPE OC REPORT AN0 PER,00 COVEAEO

14. SPONSOR1NG AGENCY COOE

j.S. Environmental Protection Agency

Iuluth, MN 55804

5. SUPPLEMENTARY NOTES

EPASeries Research Reprt .

.

EPA-600/03

.

I

ABSTRACT

f

In 1988, the first edition “Methods for Aquatic Toxicity Identification Evaluations:

Phase I Toxicity Characterization Procedyres” was publiShed (EPA, 1988A). This

second edition provides more details and ‘more insight into the techniques described in

the 1988 document. The manual describes procedures for characterizing the

physical/chemical nature of toxicants in acutely toxic effluent samples, with

applications to other types of samples such as receiving water samples, sediment

pore water or elutriate samples, and hazardous wastes. .

d

5

. &LY wOROS AN0 OOCUMINT ANALYSIS

OasCnICtOns b.tocfuT~FlliRS/OPIN ENOEO TERMS

Author(s) Contmued: c

Anderson<amahan, L.3.

Performing Org. Name and Address Continuak

Environmental Protection Agency, -on

Iv-Policy Planning and Evaluation Branch,

345 courtland, Atlanta, GA 30365.

1. ~~ST~~~UTIO~ S~ATL.MENT rs. SECURITY CLASS lthtr Report}

elease to public Unclassified ,

120. SECURITY CLASS /Thu pot*)

. COSAT4 FeldiCroup

21. NO. OF PAG

22. PRICE

)) Unclassified I

trr Pwc* ZflO-I (Rev. 4-11) rrCvlO&Jl COlT~O*) 1% omrobc~s

*U.S. co- mma: 1991.5ra-le)rabo2