&EPA

L m IJnited States Office of Research and

Environmental Protection EPA/600/R-92/080

Agency

Development September 1993

Washington DC 20460

Methods for Aquatic

Toxicity Identification

Evaluations

Phase II Toxicity Identification

Procedures for Samples

Exhibiting Acute and Chronic

Toxicity
EPA/600/R-92/080

September 1993

Methods for Aquatic

Toxicity Identification Evaluations

Phase II Toxicity Identification

Procedures for Samples Exhibiting

Acute and Chronic Toxicity

bY

E. J. Durhanl

T. J. Norberg-King’

L. P. Burkhard’

With Contributions from:

G. T. Ankleyl

M. T. Lukasewycz2

M. K. Schubauer-Berigan2

J. A. Thompson2

U.S. Environmental Protection Agency

2AScl Corporation - Contract No. 68-CO-0058

Previous Phase II Methods

by D. I. Mount and L. Anderson-Carnahan

EPA-600/3-88/035

National Effluent Toxicity Assessment Center

Technical Report 01-93

Environmental Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Duluth, MN 55804

@ Printed on Recycled Paper
Disclaimer

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

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

commercial products does not constitute endorsement or recommendation for use.

ii
Foreword

This document is one in a series of guidance documents intended to assist

dischargers and their consultants in conducting acute or chronic aquatic toxicity

identification evaluations (TIES). TIES might be required by state or federal agencies

resulting from an enforcement action or as a condition of a National Pollutant

Discharge Elimination System (NPDES) permit. The methods described in this

document will also help to determine the adequacy of effluent TIES when they are

conducted as part of a toxicity reduction evaluation (TRE).

This Phase II document is the second of a three phase series of documents

that provide methods to characterize and identify the cause of toxicity in effluents. The

first phase of the series, Phase I (EPA, 1991A; EPA, 1992), characterizes the

physical/chemical nature of the acute and chronic toxicant( thereby simplifying the

analytical work needed to identify the toxicant( Phase II provides guidance to

identify the suspect toxicants, and the last phase, Phase III (EPA, 1993A) provides

methods to confirm that the suspect toxicants are indeed the cause of toxicity. The

recent TIE documents (EPA, 1991 A; EPA, 1992; EPA, 1993A; and this document)

have been produced or revised to include chronic toxicity recommendations and

additional information or experiences we have gained since the original methods were

printed (EPA, 1988A; EPA, 1989A; EPA, 19898).

This Phase II document provides identification schemes for non-polar organic chemicals, ammonia, metals, chlorine, and surfactants that cause either acute

or chronic toxicity. The document is still incomplete in that it does not provide methods

to identify all toxicants, such as polar organic compounds. This Phase II manual also

incorporates chronic and acute toxicity identification techniques into one document.

While the TIE approach was originally developed for effluents, the methods

and techniques directly apply to other types of aqueous samples, such as ambient

waters, sediment pore waters, sediment elutriates, and hazardous waste leachates.

These methods are not mandatory protocols but should be used as general guidance

for conducting TIES.

The sections of both Phase I documents (EPA, 1991A; EPA, 1992) which

address health and safety, quality assurance/quality control (QA/QC), facilities and

equipment, dilution water, testing, sampling, and parts of the introduction are

applicable to Phase II. These sections, however, are not repeated in their entirety in

this document.

. . .

III
Abstract

This manual and its companion guidance documents describe a three phase

approach for dischargers to identify the causes of toxicity in municipal and industrial

effluents (Phase I, EPA, 1991A; EPA, 1992; and Phase III, EPA, 1993A). In 1989, the

document titled Methods fur Aquatic Toxicity ldentificatiun Evaluations: Phase II

Toxicity Mentification Procedures was published as a guidance document for identifying the cause of toxicity in acutely toxic effluents (EPA, 1989A). This new Phase II

document provides details for more types of samples, tests and test procedures that

can be used to identify the specific chemical(s) responsible for acute or chronic

effluent toxicity when the cause of toxicity is related to non-polar organic compounds,

ammonia, surfactants, chlorine, or metals. Phase I characterization and Phase III

confirmation manuals, the other guidance documents in the three phase TIE approach, have also been produced or updated to include both chronic toxicity

information and new developments made since the first set of documents were

printed. The TIE approach is applicable to effluents, ambient waters, sediment pore

waters or elutriates, and hazardous waste leachates.

iv
Contents

Page

Foreword ..*

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

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Figures . . ..,....................................................,...............................................,............... ..Vlll

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*...................................*....................................... ix

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*....................................................................... X

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

1 .I General Overview .......................................................................................... l-l

1.2 Biological Testing Considerations .................................................................. l-2

2.0 Non-polar Organic Compounds ......................................................................... ...2-I

2.1 General Overview ....................................................................................... ...2-I

2.2 Acute Toxicity: Fractionation and Toxicity Testing Procedures..................... .2-2

2.2.1

2.2.2

2.2.3

2.2.4

2.2.5

2.2.6

2.2.7

2.2.8

2.2.9

Sample Volume ................................................................................. .2-3

Filtration .............................................................................................. 2-3

Column Size ....................................................................................... 2-3

C,, SPE Column Conditioning ............................................................ 2-4

Elution Blanks .................................................................................... .2-4

Column Loading with Effluent ............................................................. 2-4

C,, SPE Column Elution .................................................................... 2-6

Blank and Effluent Fraction Toxicity Tests.. ....................................... 2-6

SPE Fractions: Concentration and Subsequent Toxicity Testing ..... ..2- 7

2.2.10 HPLC Separation ............................................................................... .2-9

2.2.11 HPLC Fraction Toxicity Tests ............................................................ .2-9

2.2.12 HPLC Fractions: Concentration and Subsequent Toxicity Testing .,.2-l 0

2.3 Chronic Toxicity: Fractionation and Toxicity Testing Procedures .............. ..2-IO

2.3.1

2.3.2

2.3.3

2.3.4

2.3.5

2.3.6

2.3.7

2.3.8

2.3.9

Sample Volume ............................................................................... .2-l 1

Filtration ............................................................................................ 2-l 1

Column Size .................................................................................... 2-12

C,, SPE Column Conditioning ......................................................... 2-13

Elution Blanks ................................................................................... 2-13

Column Loading with Effluent .......................................................... 2-14

C,, SPE Column Elution .................................................................. .2-14

Blank and Effluent Fraction Toxicity Tests ........................................ 2-14

SPE Fractions: Concentration and Subsequent Toxicity Testing ... ..2-15

2.3.10 HPLC Separation .............................................................................. 2-I 6

2.3.11 HPLC Fraction Toxicity Tests ........................................................... 2-16

2.3.12 HPLC Fractions: Concentration and Subsequent Toxicity Testing. ..2-17

2.4 GUMS Analyses ......................................................................................... 2-l 7

2.5 Identifying Suspect Toxicants ...................................................................... 2-18

2.5.1 Identifying Organophosphate Pesticides .......................................... 2-19

2.5.2 Identifying Sutfactants ...................................................................... 2-19

2.6 Alternate Fractionation Procedures ............................................................ 2-21

2.6.1 Modified Elution Method ................................................................... 2-21

2.6.2 Solvent Exchange ............................................................................. 2-21

2.6.3 Alternative SPE Sorbents and Techniques ...................................... 2-22

V
3.0

4.0

5.0

6.0

7.0

8.0

Contents (continued)

Ammonia .............................................................................................................. 3-1

3.1 General Overview ......................................................................................... .3-l

3.2 Toxicity Testing Concerns ............................................................................. .3-2

3.3 Measuring Ammonia Concentration ............................................................... 3-5

3.4 Graduated pH Test ......................................................................................... 3-5

3.4.1 pH Control: Acid/Base Adjustments .................................................... 3-5

3.4.2 pH Control: CO Adjustments ....... .

pH Control: Bufier pH Adjustments

..................................................... 3-6

3.4.3 ..................................................... 3-7

3.5 Zeolite Resin Method ..................................................................................... 3-8

3.6 Air-Stripping of Ammonia ............................................................................... 3-9

Metals .................................................................................................................. 4-1

4.1 General Overview .......................................................................................... 4-1

4.2 Analysis of Metals .......................................................................................... 4-2

4.2.1 Prioritizing Metals for Analysis ............................................................ 4-2

4.2.2 Metal Analysis Methods ..................................................................... .4-2

4.2.3 Metal Speciation ................................................................................. 4-4

4.2.4 Identification of Suspect Metal Toxicants ........................................... 4-4

4.3 Additional Toxicity Testing Methods ............................................................... 4-5

4.3.1 EDTA Addition Test ............................................................................ 4-5

4.3.2 Sodium Thiosulfate Addition Test ...................................................... 4-6

4.3.3 Metal Toxicity Changes with pH ........................................................ .4-6

4.3.4 Ion-Exchange Test .............................................................................. 4-7

Chlorine ............................................................................................................... 5-I

5.1 General Oven/iew .......................................................................................... 5-I

5.2 Tracking Toxicity and TRC Levels ................................................................ 5-l

Identifying Toxicants Removed by Filtration ....................................................... .6-l

6.1 General Overview .......................................................................................... 6-l

6.2 Filter Extraction .............................................................................................. 6-1

References .......................................................................................................... 7-l

Appendix A ......................................................................................................... A-l

vi
Tables

Number Page

2-1.

2-2.

2-3.

2-4.

2-5.

2-6.

2-7.

2-8.

3-1.

3-2.

3-3.

3-4.

3-5.

4-1.

4-2.

4-3.

A-l.

A-2.

Solid phase extraction (SPE) column fractionation information ........................ .2-4

Comparison of toxic units (TUs) in each toxic fraction to TUs of all

fractions combined and whole effluent ........................................................ 2-7

Information for concentrating SPE and HPLC fractions ..................................... 2-8

Example HPLC elution gradients for four commonly toxic SPE fractions ........ ..2- 9

Eluate volumes needed for chronic SPE fraction toxicity tests with

Ceriodaphnia dubia and Pimephales promelas ........................................ 2-I 2

Approximate effluent volumes needed for the chronic non-polar organic

identification procedures ........ ..V .................................................................. 2-12

Example HPLC elution gradient for SPE fractions from chronically toxic

effluent samples ......................................................................................... 2-16

Composition of 11 recommended fractions in modified elution scheme ........ ..2-2 1

Percent un-ionized ammonia in aqueous solutions for selected

temperatures and pH values.. .................................................................... ..3- 2

Calculated unionized ammonia LC5Os (mg/l) based on 24-h and

48-h results of a Ceriodaphnia dubia toxicity test conducted

at pH 8.0 and 25°C ....................................................................................... 3-3

Calculated un-ionized ammonia LC5Os (mg/l) based on 24-h, 48-h,

72-h, and 96-h results of a fathead minnow (Pimephales promelas)

toxicity test conducted at pH 8.0 and 25°C .................................................. 3-4

Un-ionized ammonia toxicity values for species frequently used in

effluent testing .............................................................................................. 3-4

Percent unionized ammonia in aqueous solutions at 25°C and various

TDS levels ................................................................................................... 3-7

Atomic absorption detection limits and concentration ranges ............................ 4-3

Estimated instrumental detection limits for ICP-MS and ICP-AES .................... .4-3

Metal LC5Os with respect to test pH .................................................................. 4-6

Effluent volume calculation worksheets ............................................................ A-2

Effluent volume calculation worksheets (example) ............................................ A-6

vii
Number

Figures

.

Page

2-l.

2-2.

2-3.

2-4.

2-5.

2-6.

Phase II schematic for the identification of non-polar organic toxicants .......... ..2- 1

Procedures for eluting the SPE column with a gradient of methanol/water

solutions ............................................................................................................. 2-5

Concentrating effluent on the C,, SPE column .................................................. 2-5

Procedure to concentrate toxic SPE fractions .................................................... 2-8

Procedure to fractionate acutely toxic SPE concentrates using HPLC ............ ..2- 9

Procedure to concentrate toxic HPLC fractions .............................................. .2-10 ,

. . . VIII
Abbreviations

AA

Cl,

cf3

CTAS

DO

DOC

EDTA

ERL-D

GC

GCIMS

Heppso

HPLC

lCP

ICP-AES

ICP-MS

K

L%

LC/MS

LC

MBAS

Mes

Mops

NETAC

NIST

NOEC

NPDES

PBO

Pops0

CWQC

SPE

s s

Taps

TDS

TIE

TOC

TRE

TU

YCT

Atomic Absorption

Octadecylcarbon Chain

Octylcarbon Chain

Cobalt Thiocyanate Active Substances

Dissolved Oxygen

Dissolved Organic Carbon

Ethylenediamine Tetraacetic Acid

Environmental Research Laboratory-Duluth

Gas Chromatography -

Gas Chromatography/Mass Spectrometry

N-(2Hydroxyethyl) Piperazine-N’-2Hydroxypropane Sulfonic Acid

High Performance Liquid Chromatogpphy

Inhibition Concentration Percentage *

Inductively Coupled Plasma-Atomic Emission Spectroscopy

Inductively Coupled Plasma-Mass Spectrometry

0ctanol;Water Partition Coefficient

Linear Alkylbenzene Sulfonate

Liquid Chromatography/Mass Spectrometry

Lethal Concentration

Methylene Blue Active Substances

2-(N-Morpholino) Ethane-Sulfonic Acid

3-(N-Morpholino) Propane-Sulfonic Acid

National Effluent Toxicity Assessment Center

National Institute of Standards and Technology

No Observed Effect Concentration

National Pollutant Discharge Elimination System

Piperonyl Butoxide

Piperazine-N,N’-bis (2-Hydroxypropane) Sulfonic Acid

Quality Assurance/Quality Control

Solid Phase Extraction

Suspended Solids

N-tris-(Hydroxymethyl) Methyl-3-Aminopropane Sulfonic Acid

Total Dissolved Solids

Toxicity Identification Evaluation

Total Organic Carbon

Toxicity Reduction Evaluation

Toxic Unit

Yeast-CerophyP-Trout food

ix
Acknowledgments

This document presents additional methods and improvements made to the

procedures of Methods for Aquatic Toxicity ldenfification Evaluations: Phase /I

Toxicify ldentificafion Procedures (EPA-600/3-88/035) by Donald Mount and Linda

Anderson-Carnahan. This manual reflects new information, techniques, and test

procedures developed by the National Effluent Toxicity Assessment Center (NETAC)

since the previous Phase II document was printed in 1989. This Phase II document

is based on the efforts of both federal and contract staff of the NETAC group. We

gratefully acknowledge the following individuals’ contributions to the research and , development of the methods for this document: Penny Juenemann and Shaneen

Schmitt (federal staff); Joe Amato, Lara Andersen, Steve Baker, Tim Dawson, Joe

Dierkes, Nola Englehorn, Doug Jensen, Correne Jenson, Jim Jenson, Liz Makynen,

Phil Monson, Don Mount, and Greg Peterson (contract staff).

Through the support of Rick Brandes and Jim Pendergast (EPA, Permits

Division) and Nelson Thomas (ERL-D), the NETAC staff members developed and

revised the TIE series of documents.

X
4

Section 1

Introduction

1.1 General Overview

The major objective of Phase II is to identify the

suspected toxicant in effluent samples using toxicity

identification evaluation (TIE) procedures. Some general

guidance to achieve this goal might be furnished by the

results of acute or chronic Phase I tests (EPA, 1991 A;

EPA, 1992), but for many effluents, such as those that

contain non-polar organic toxicants, both separation and

concentration steps will be needed to achieve the stated

objective. If metals are the suspect toxicants, atomic

absorption (AA) spectrometry should be sensitive enough

to measure toxic concentrations directly in the sample,

and the number of metals is small enough that toxicity

can be attributed without separating one from another.

The same principle applies to toxicants such as ammonia

and chlorine; measurements can be made without separating or concentrating the effluent. However, if non-polar

organic chemicals are suspected, separation is usually

necessary for analytical and toxicological reasons.

Because there are often many constituents within

the classes of chemicals (e.g., non-polar organics) identified in Phase I, initial efforts are most productively

directed towards separating the toxic from the non-toxic

constituents. With the need to identify the toxicant

quickly, comes the temptation to analyze too soon. Using

methods such as gas chromatography/mass spectrometry (GC/MS) one can identify many non-polar organics

that are present in the whole effluent mixture, but the

association of toxicity with compound identification is

very difficult to make for several reasons:

l There can be hundreds of compounds present in

the mixture, and to investigate all of them would

be very time consuming.

. Toxicity data for many of the chemicals identified

are usually not available; chronic data are especially scarce.

l Separate constituents are often not commercially available; therefore, their toxicities cannot

be measured and compared to effluent toxicity.

l Interactions (additivity, synergism, antagonism)

are not known for the given mixtures and one

must know interactions to apportion toxicity.

Therefore, it is suggested that the search for a

separation technique to simplify the mixture into toxic and

non-toxic ‘subsamples be the first priority, rather than

spending time investigating non-toxic components. If there

is a single suspect toxicant such as ammonia, then separation needs are limited largely by the analytical requirements. If the toxicity is caused by one constituent, the

number of other non-toxic constituents is irrelevant when

attributing toxicity. However, Phase I results do not usually lead to a single suspect toxicant and, therefore,

separation may be necessary.

When a method for separating the toxicant is

found, concentration might be an inherent part of the

procedure (as in solvent extraction) which will simplify the

problem of finding a method to concentrate the toxicity. At

each stage of the separation and concentration process,

measurement of toxicity is the best way to evaluate the

success or failure of the manipulations.

The interpretation of TIE results can be different

than in the classical research approach, where experiments are designed to either accept or reject a hypothesis. In TIE work, an experiment usually permits

acceptance but not rejection of the hypothesis. For example., if ammonia is the suspect toxicant, it can be

removed using zeolite resin. If the post-zeolite effluent is

still toxic, you can conclude that there are additional

toxicants present. If the pqst-zeolite effluent is not toxic,

you cannot conclude that there are no additional toxicants

because the zeolite might have removed other toxicants

in addition to the ammonia.

The always present question of whether or not

there is more than one toxicant immensely complicates

data interpretation. Phase I results might not give an

indication of multiple toxicants unless the toxicant classes

change over time or from sample to sample. Phase II

results are often such that one cannot tell whether the

situation is one of partial removal of a single toxicant or

l-l
toxicity resulting from multiple toxicants. The issue might

be resolved when one toxicant is identified and measured

analytically. Experience shows that the best choice is to

try to focus on the toxicant that appears easiest to identify. Usually that will be a toxicant that can be separated

from the sample (e.g., extracted or recovered from a

sorbent that reduces the toxicity) and for which there is a

broad spectrum analytical identification method. Above

all, data should always be interpreted under all probable

scenarios, i.e., one toxicant, multiple toxicants, and even

different toxicants from sample to sample.

Experience gained since the first Phase II (EPA,

1989A) document was printed has shown that effluent

toxicants are not always strictly additive. When they are

not additive, the toxicant present in the largest number of

toxic units (TUs)l will determine the toxic units of the

effluent. Non-additive toxicity will not be reduced by manipulations that remove toxicants present in fewer TUs

than the major toxicant. Two or more toxicants might be

present in approximately equal TUs, however, the ratio of

TUs might change over different sampling times so that

different chemicals determine the toxicity of the effluent.

These important problems can be dealt with in Phase III

(EPA, 1993A). In Phase II, the objective is to find which

toxicants are present in toxic concentrations. However,

failure of additivity may confuse Phase II results. Minor

toxicants might not be noticed until the major one has

been removed. In addition, additivity cannot be determined until at least one toxicant has been identified.

Usually Phase II and Phase III merge and overlap, therefore such concerns regarding non-additivity must be incorporated in Phase II, at least in the latter stages.

As effluent constituents are identified, a sorting

process begins in which a decision must be made as to

whether or not each one identified contributes to the

toxicity of the effluent. Usually, this is based on the

estimated concentration and the constituents’ toxicities.

Analytical error in quantitation might be large (1 O-fold or

more) because recoveries and instrument response factors probably will not yet have been determined on a

particular chemical. Uncertainty about toxicological data

is caused by differences in species sensitivity and water

quality effects, when literature values are available. Confidence in an acute toxicity value (LC50) will vary depending on the quality of the test, the number of times it was

repeated, and the completeness by which the results and

conditions were described. Data on chronic effect levels

are often scarce and rarely have tests been repeated.

Species sensitivity frequently varies from 1 OO-fold to 1 ,OOOfold; an error will likely be introduced when the published

‘TU calculations are described in EPA, 1992. The TUs of whole

effluent equals 100% divided by the LC50, NOEC, or ICp (IC25,KXO)

of the effluent. The TU of a specific chemical equals the concentration

of the compounded divided by the effect level of the compound.

toxicity data for species other than the test species are

used. When the uncertainty of the toxicity data is high, a

maximum of lOO-fold difference between measured concentrations and literature effect would be acceptable to

classify a chemical as a suspect. If one has good data for

the test species being used, then this difference might be

reduced (e.g., to IO-fold). Since these decisions are

always subjective, they will sometimes be wrong no matter how carefully they are made. Perhaps most important

is use of an iterative process to make these decisions.

First evaluate candidates that have concentrations higher

than or closest to their chronic or acute effect levels and if

these prove to be negative, then examine those that have

concentrations below their effect levels. Remember that

the suspected toxicant concentrations at the dilution equal

to the effect level concentration are the important concentrations to compare. At some point, a decision must be

made whether the true toxicants have not yet been identified or measured and that different sample preparation

or analyses must be used.

For some effluents, Phase I results might not

have provided any guidance for selecting the appropriate

Phase II procedures to follow. Other characterization

steps that might be helpful are solvent extraction (acidic

or basic), sample evaporation, size exclusion chromatography, lyophilization, and vacuum or steam distillation

(Jop et al., 1991; Walsh et al., 1983). We have little

experience upon which to recommend procedures in

these cases. It is most important to realize that the more

severe the effluent treatment, the more likely it is that

toxic artifacts will be created. These toxic artifacts could

then be confused with effluent toxicity; therefore, artifactual toxicity must be monitored for each technique by

using blanks.

Phase II efforts should develop into Phase III

confirmation (EPA, 1989B; EPA, 1993A) as soon as good

evidence is obtained that one or more candidates are

probable toxicants. The primary product of Phase II is the

chemical identification of the suspected toxicants to furnish the basis for Phase III testing. The techniques described in this document are useful for TIE work with

effluents as well as ambient waters (Norberg-King et al.,

1991) and sediment pore water or elutriates (EPA, 1991 B).

1.2 Biological Testing Considerations

The Phase I characterization documents (EPA,

1991 A; EPA, 1992) provide detailed discussions of various issues that are important in decision making throughout the TIE. The guidance covers use of various species,

test concentrations, effluent sample types, testing requirements for quality assurance @A), test endpoints,

frequency of changing the test solutions, and more. All of

these issues will not be discussed at length here and the

user is encouraged to refer to the acute Phase I or the

chronic Phase I as companion documents for the TIE

process. As the Phase II identification and Phase III

confirmation steps are initiated, QA requirements should

1-2
be revisited and the types of tests modified as needed.

Several of these testing concerns are addressed below.

During Phase I, the analyst is searching for an

obvious alteration in effluent toxicity, which might be

obtained by using modified acute or chronic test methods.

Confirmation testing (Phase III) conducted according to

the standard methodologies will confirm whether the suspect toxicant detected in the characterization and identification steps (Phases I and II) is the true toxicant.

In characterizing the toxicity in Phase I, factors

such as time requirements, number of tests and the test

design had to be considered and weighed against the

type of questions that are posed. EPA has published

manuals that describe the acute or chronic test methods

to determine the toxicity of effluent or receiving waters to

freshwater and marine organisms (EPA, 1991 C; EPA,

1993B; EPA, 1993C), and these tests are typically those

that indicated the presence of toxicity which the TIE

initiated. Deviations from these standard effluent testing

protocols were discussed in both the acute Phase I (EPA,

1991A) and the chronic Phase I (EPA, 1992) manuals.

For either the acute or the chronic Phase I procedures,

the test volumes, number of test concentrations, and

number of replicates were all reduced from the standard

test methods (EPA, 1991 C; EPA, 19938). Additional modifications for the short-term chronic tests (EPA, 19938)

including shorter test duration and a reduction in the

frequency of the test solution renewal are suggested.

Throughout this document the TIE procedures for

acutely toxic samples are based on the following species:

Cetidaphnia dubia, Pimephales promelas, Daphnia magna,

Daphnia puiex, Hyaiella azfeca, and Chirorwmus fenfans. Almost all acute tests have been conducted using 10 ml of test

solution in a 1 oz plastic cup (or 30 ml glass beaker). TIE

procedures with chronically toxic effluents are based on

tests using either C. dubia or larval fathead minnows

(P. promelas). In our laboratory, the chronic tests with C.

dubia generally are conducted using 10 ml of test solution

in 1 oz cups and the chronic tests with fathead minnows

are conducted using 50 ml of test solution in a 4 oz plastic

cup (10 fish per cup). Use of other species is constrained

only by availability, size, age, and adaptability to test

conditions, and the threshold levels for additives and

reagents for the other organisms must be determined.

As soon as good evidence is obtained to implicate a suspect toxicant( the procedures for performing

the toxicity tests can be changed. Therefore in Phase II,

the time to modify the tests from the way they were

conducted in Phase I may depend on when the toxicant is

identified, and generally there is more flexibility for this in

Phase II than in Phase III. The quality control (QC)

measures in Phase I were not very strict because the data

are primarily informative rather than definitive. The identity of the suspect toxicant furnishes the basis upon

which Phase III testing will be conducted, which will

require stricter QC measures.

Initially, the use of modified protocols in Phase II

may continue; however, once specific toxicant identification has been made, Phase II (and Phase Ill) testing

conditions should be similar to the methods described in

the protocol that was used to trigger the TlE. Although a

shortened version of the 7d C. dubia test (which is referred to

as the 4d test) may have been used in Phase I, the use of this

test changes in Phase II (and Phase Ill). In order to use the 4d

test in Phases I and II of the TIE, the 4d test must detect similar

trends of toxicity as the 7d test does. However, in Phase III the

711 test is required because the toxicity as measured in the 7d

test (with additional replicates, more test concentrations, addc

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

should be used to confirm the cause of toxicity. In the early

Phase II chronic toxicity evaluation steps, the qualitative evaluation of toxicii might be useful and there is no reason why a

toxicity test could not be terminated sooner than day seven, if

the answer to a particular question has been found.

Information obtained from all toxicity tests should

be maximized. For instance, in acute toxicity tests, monitoring time to mortality might be useful. In chronic toxicity

tests, time to young production of the cladocerans or the

lack of food in the stomach of the larval fish might be

useful parameters. Observations such as these made

during a test might be subtle indications and quite informative of small changes in toxicity. For example, if there

is complete mortality on day four of the baseline effluent

test, and in the EDTA addition test (Section 4) the animals

either do not reproduce or grow yet they are alive at day

seven of the exposure, the indication is that the toxicity

was reduced. These results suggest that either an additional toxicant is present or the EDTA concentration was

not sufficient to remove all cationic metal toxicity. These

types of observations in the short-term tests might be just

as useful as reductions in young production or growth.

For evaluating whether any manipulation changed toxicity, the investigator should not rely only on statistical

evaluations of test endpoints (see below and Phase I;

EPA, 1992). Some treatments may have a significant

biological effect that was not detected by the statistical

analysis. Judgement and experience in toxicology should

guide the interpretation.

In addition, for acute or chronic toxicity tests,

randomization, careful exposure time readings, use of

animals of uniform narrow-age groups (i.e., C. dubia

neonates O-6 h old rather than O-12 h old) might assist in

detecting smaller differences in tests. For example, in the

chronic C. dubia tests, it is important to use organisms of

known parentage (EPA, 19938) when the number of,

replicates is reduced from ten to five. For C. dubia, daily

renewals of the test media (as required in the chronic

manual; EPA, 1993B) might not be necessary in Phase I

or early Phase II testing as long as the toxicity of the

1-3
using 20% effluent as the high concentration and a dilution factor of 0.7, the concentrations to test would be 7%,

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

4050% (or above 50%), then the concentration series to

test might be either 25%, 50%, 75%, and 100% or 40%,

60%, 80%, and 100%. Choice of dilution factor and test

concentration range is a matter of judgement and depends on precision required and practicality.

After conducting Phase I procedures on an effluent sample, the amount of effluent available for subsequent identification work can be sufficiently reduced so

that it may be impractical to try to conduct each step as

described in this manual. This is most likely to be a

concern for the non-polar organic identification techniques

and other methods that require large volumes of effluent

to identify the toxicant. Therefore, when the volume of an

effluent sample is limited, it might be possible to track

toxicity through the non-polar identification steps without

quantifying the amount of toxicity that is being tracked.

Essentially, this means that the toxicity tests are done

without dilutions and the results would indicate only that

toxicity was present or absent; the degree of toxicity

present would not be measured. Once a suspect toxicant

is identified, it is important that the amount of toxicity

removal is known (through the use of dilutions) because

this information can be used to correlate a suspect toxicant to the effluent toxicity in the Phase III confirmation.

If the number of replicates per test concentration

is reduced, one must assume that precision is sufficient to

decipher changes in toxicity that must be measured. One

problem in using reduced replicates and low numbers of

test concentrations in chronic tests is that this smaller

data set is not amenable to all statistical requirements as

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

Section 5.8). Use of more organisms and more replicates

than in the Phase I modified tests might be preferable if

Phase I and/or Phase II data are likely to be used in

Phase III confirmation (See Sections 2.2 and 2.3).

For acute toxicity tests, usually the LC50 or EC50

is reported for the toxicity data (calculated as recommended in EPA, 1991C). Endpoints for the most commonly used freshwater short-term chronic tests are growth,

reproduction, and survival. The no effect level (the NOEC),

and the effect concentration (the lowest observed effect

concentration (LOEC)) are determined using the statistical approach of hypothesis testing to determine a statistically significant response difference between a control

group and a treatment group. The NOEC/LOEC are heavily

affected by choice of test concentrations and test design

(see Phase I; EPA, 1992). The linear interpolation method

(EPA, 1993B) provides a point estimate of the effluent

concentration that causes a given percent reduction based

on organism response. To calculate the inhibition concentration percentage (ICp), a computer program (NorbergKing, 1993; DeGraeve et al., 1988; EPA, 1989C) is

available and the assumptions for the method are not the

same as the test design requirements for hypothesisbased analyses. This point estimation method is particularly useful for analyzing the type of data obtained from

chronic TIE tests using dilutions (see Phase I; EPA,

1992). Confidence intervals are calculated using a bootstrap technique and might be useful in determining if

significant toxicity alterations have been observed. A

significant reduction in toxicity and the precision of reference toxicant tests must be determined by each laboratory for each effluent. The use of the IC50 for Phase I

TIES might be more useful in correlating the characterization test results to the effluent toxicity than an IC25.

However, there are situations when an IC50 may not be

able to be estimated while the IC25 can. Above all, it is

most important to use a consistent effect level for TIE

toxicity testing (EPA, 1992A; EPA, 1992B). When substantial toxicity reductions occur in the toxicity tests, it

may not always appear to be a significant reduction when

the IC25s are compared. In order to further evaluate

whether toxicity reductions occurred, the sample size

(number of replicates, number of concentrations) should

be increased in subsequent testing in an effort to differentiate toxicity responses from the sample size limitations.

The dose response curves should then be compared to

see if responses are similar. Once the toxicant is identified, the number of replicates should be increased, the

dilution sequence might be modified and more dilutions

used (see Phase III; EPA, 1993A). This should increase

the confidence in the IC25 (or any other ICp value chosen) estimate.

l-5
Section 2

Non-Polar Organic Compounds

2.1 General Overview

The procedures described in this section presume that the results of Phase I tests have implicated

non-polar organic compounds as the cause of acute or

chronic toxicity. Results of Phase I tests that clearly

implicate a non-polar organic toxicant typically are (1) all

toxicity is removed by the C,, Solid Phase Extraction

(SPE) column and (2) toxicity was observed in the methanol eluate test (see Section 8.6, EPA, 1991 A; and Section

6.7, EPA, 1992). In some instances, toxicity might not be

removed completely by the SPE column, but sufficient

toxicity is recovered in the methanol eluate to suggest a

non-polar organic toxicant is present. While toxicants

other than non-polar organic compounds might be removed by the column (e.g., metals), the elution for such

toxicants is unlikely to be similar to that of the non-polar

organic compounds. However, there is also the possibility

that non-polar toxicants such as surfactants will be removed by the column and not recovered in the methanol

eluate. The goal in this section is to separate the nonpolar organic toxicants from the many non-toxic components of the sample to simplify the analytical work needed

to identify the toxicant.

This section provides the general background

information on non-polar organic compounds along with

methods for concentrating and separating the toxicants

for samples with acute and/or chronic toxicity. While the

method provides a stepwise procedure, there are instances where the investigator may have to modify the

approach to achieve the best results.

Also provided in this section are procedures that

might prove helpful in less common situations. Metabolic

blockers can be used to reduce or eliminate certain

organophosphate compounds from exhibiting their toxicity (Section 2.5.1). In the instance when toxicity is not

recovered in the methanol eluate and toxicity is not evident in the post-column effluent, alternate SPE elution

procedures might be needed (Section 2.6).

A flow diagram of the general procedures followed in identifying non-polar organic toxicants is shown

in Figure 2-l. In this procedure, the C,, SPE column is

used to extract non-polar organic compounds from effluent samples. These compounds are then selectively

Figure 2-1.

Effluent Sample

I

Toxicity Test

SPE Fractionation

I

Toxicity Tests

Concentrate and Combine Toxic SPE Fractions

Toxicity Tests

I

GUMS Analysis (optional)

HPLC Fractionation

Toxicity Tests

I

Concentrate and Combine Toxic HPLC Fractions

Toxicity Tests

I

GUMS Analysis

GUMS Identification

Compare Concentrations to Toxicity Values

Phase II schematic

organic toxicants.

for the identification of non-polar

2-1
stripped off the column by eluting the C,, sorbent with

solvent/water mixtures that are increasingly less polar. As

a series, the ‘fractions” resulting from column elutidn

contain analytes that are decreasingly polar and decreasingly water soluble. Each fraction is then tested for toxicity. The fractions that exhibit toxicity are concentrated,

and chromatographed using reversed phase High Performance Liquid Chromatography (HPLC). The resulting

HPLC fractions are collected and tested for toxicity. The

toxic HPLC fractions are concentrated into methanol by

using another C,, SPE column. The concentrates are

toxicity tested as before and analyzed using gas chromatography/mass spectrometry (GCIMS). Those constituents that are identified by GC/MS are roughly quantitated,

by assuming that the identified constituents and the internal standard have the same response factor, and the

estimated concentrations are compared to available tox-

. icity values for each chemical. If this process reveals

strong suspect toxicants, mass balance testing (Phase

III; EPA, 1993A) could be started to determine whether

additional toxicants are present. If no suspect toxicants

are identified by GC’MS, a longer analysis time on the

HPLC might help the identification by increasing the

separation between toxic and non-toxic components, especially if there are many constituents present. Also,

additional constituents might be identified by increasing

the concentration factor by using larger effluent samples.

At some point, the probability that the toxicants are not

chromatographing on the gaschromatograph or the mass

spectrometer is not detecting the toxicants must be considered if no suspect toxicants are identified. Use of other

types of mass spectrometry, such as liquid chromatography/mass spectrometry (LCMS) or direct probe mass

spectrometry may be useful. Some effluents might require the SPE fractionation of several different samples

befoie good suspect toxicants are found (Burkhard et al.,

1991: Lukasewycz and Durhan, 1992).

The sorbents that we recommend for use in SPE

and HPLC columns are chemically identical. The column

packing is composed of silica gel which has been reacted

with octadecyl silane to produce a covalent bonded phase

one layer ?hick. The mechanism of extraction with C,,

sorbents is relatively simple. Extraction of effluent compounds occurs because the C,, sorbent competes for the

non-polar compounds more strongly than the surrounding

water molecules of the effluent. Sorption of non-polar

organics is also influenced by ionic strength, pH, and total

organic carbon (TOC) levels. The same compounds will

partition on both SPE and HPLC columns and the order of

elution of chemicals will be approximately the same. The

major difference between the SPE and HPLC columns is

the amount of resolution achieved. The particle size employed in HPLC columns is smaller, providing a greater

surface area and better component resolution. Despite

less resolution, SPE columns have the advantage of

possessing a higher loading capacity in general than

HPLC columns. The SPE column could be considered as

a preparatory column for sample cleanup while the HPLC

column gives far more refined and controlled separation

of sample constituents.

To elute non-polar organic toxicants extracted by

the C,, SPE column, the sorbed compounds must have a

higher affinity for the eluting solvent than for the octadecyl

functional group (C,,). Choosing a solvent for elution is

complicated because the toxicants’ identities are not

known. In general, the solvent should be less polar than

water and more polar than the C,, functional group. The

degree of solvent strength required to elute the toxicants

is also unknown. Since methanol is less polar than water,

has a very low toxicity (EPA, 1991A; EPA, 1992) and

elutes chemicals from C,, sorbents, it has been a good

solvent choice for most TIE purposes to date.

During sequential column elutions with successively increasing methanol in water concentrations, the

relatively hydrophilic, polar compounds are eluted first,

and the more hydrophobic non-polar compounds are

eluted last. Given the strength of methanol as a solvent

for non-polar compounds, it is possible that very hydrophobic (octanol water partition coefficient (log KO,) 24)

effluent compounds will not be eluted from the C,, sorbent. If toxicity caused by a very hydrophobic compound

is extracted by the SPE column but not eluted by methanol, less polar solvents might be used to elute the SPE

column (Section 2.6.1).

Once toxicity is found in one or more C,, SPE

effluent fractions, the toxic fractions can be concentrated,

then fractionated using HPLC. HPLC separation is used

to reduce the number of non-polar organic chemicals

associated with the toxicant and to simplify analytical

identification. The toxic HPLC fractions are concentrated

and then analyzed by GC/MS. The estimated concentrations of constituents in the final concentrate (based on an

internal standard) are then compared to their toxicity

values to decide which may be sufficiently high in concentration to cause toxicity. If none are found, higher concentration factors, other analytical methods (e.g., LC/MS),

and better separation are recommended.

Fractionation and toxicity testing procedures for

non-polar organic toxicants causing either acute or chronic

toxicity are presented in different sections of this chapter.

The acute toxicity (Section 2.2) and chronic toxicity (Section 2.3) sections, have similar outlines and were written

so either section could be used independently. As a

result, some details that apply to both acute and chronic

toxicity are repeated. After using Section 2.2 or Section

2.3 the investigator should then follow the identification

techniques described in Sections 2.4 and 2.5. Additional

identification techniques are included in Sections 2.5.1

and 2.5.2, and alternate fractionation methods are found

in Section 2.6.

2.2 Acute Toxicity: Fractionation and

Toxicity Testing Procedures

In the initial stages of Phase II, toxicity tests may

be conducted on C,, SPE effluent fractions and blank

fractions to detect the presence of toxicants and not to

quantify the magnitude of the toxicity in each. As in the

toxicity tests conducted during Phase I, careful measure2-2
4

ment of test solution water chemistry parameters is not

required, and duplicate exposures are not needed during

initial stages of Phase II. The major purpose of this step is

to assess whether or not acute toxicity is present in the

effluent fractions and the blank fractions. However, as

suspect toxicants are identified, quantitative toxicity measurements will be needed to compare with the analytical

measurements. If Phase II data will be used to correlate

effluent toxicity to toxicant concentrations (Phase Ill),

then more replicates per concentration, randomization of

test concentrations, careful observation of organism exposure times, and organisms of approximately the same

age should be used (Section 1.2). Also, the amount of

eluate that is collected from the SPE fractionation, SPE

concentration, and the amount of eluate used for testing

and GC/MS analysis should be measured at all steps.

The volume of eluate must be measured to determine the

actual toxicity concentration in each step of the procedure. If it is expected that the Phase II data will be needed

later, it is prudent to measure the degree of toxicity in the

eight SPE effluent fractions at the onset of testing. We

rarely see blank fraction toxicity; therefore, there is little

need to evaluate the toxicity of the blank fractions with

dilutions.

2.2.1 Sample Volume

The volume of effluent needed depends on its

toxicity, the toxicity of the chemicals causing effluent

toxicity, and the sensitivity of the analytical method. Since

only the first of these will usually be known when Phase II

begins, trial and error will dictate volume size. For acutely

toxic effluents with LC50 values in the range of 25lOO%,

2,000 ml have usually been adequate to perform one

complete Phase II procedure, i.e., C,, SPE and HPLC

fractionations, and GC/MS identification. Examples of the

variables that should be considered when deciding what

volume of effluent to fractionate are provided in Appendix

A, Tables A-l and A-2.

2.2.2 Filtration

For acute tests, glass fiber filter(s) (1 pm nominal

pore size) should be prepared as described in Section 8

of Phase I (EPA, 1991A). Both 45 mm and 90 mm

diameter filters have been used routinely, the 90 mm filter

allows about four times more effluent to be passed over

the filter than one 45 mm filter. All filters and glassware

should first be pre-rinsed with pH 3 high purity water (e.g.,

Milli-@’ Water System, Millipore Co., Bedford, MA) to

remove any metal residues followed by a high purity

water rinse which is discarded. The filter should then be

rinsed with 200 ml of dilution water (rather than with high

purity water) and a sample collected after most of the

volume has been filtered, to provide the filter toxicity

blank. In subsequent steps, a dilution water column blank

will be collected after passing the filtered dilution water

through the SPE column. The same type of dilution water

should be used for the filter blank as for the column blank

(Section 2.2.4). Usually, a reconstituted water is used for

these procedures (EPA, 199X).

The volume of effluent that can be passed through

a single filter is sample specific. If more than one filter is

needed (as is often the case) a single filter blank can be

prepared by stacking three to eight pre-rinsed filters in

one filter holder, followed by a dilution water rinse. The

filters are then separated and used one at a time to filter

the effluent sample. If samples are high in suspended

solids additional pre-filtration may be needed. Centrifugation may also be useful for reducing solids in the sample.

The decision to use a vacuum or a pressure system for

filtering should have been made during the filtration tests

of Phase I. If a volatile chemical is indicated in Phase I,

pressure filtration should be used.

Filtration equipment should be thoroughly cleaned

before use to prevent any toxicity carry-over or particle

buildup from previous samples. We have found that glass

vacuum filtering apparatus with stainless steel filter supports (for samples without pH adjustments), or plastic

pressure filtering devices are the most useful. We have

also found that if removable glass frits are used, they can

be rigorously cleaned with aqua regia for 20-40 min

followed by rinsing with copious amounts of water to

remove residual effluent particles, since glass frits may

act as a filter. The removable stainless steel filter supports do not require as rigorous cleaning as fritted glassware, and therefore are a good substitute.

A portion of the filtered sample must be reserved

for toxicity testing while the rest is used for C,, extraction.

If the filtration toxicity blank exhibits slight or complete

toxicity, but the post C,, SPE column effluent is not toxic

(and effluent toxicity was unchanged after filtration), the

blank toxicity can be ignored since the effluent toxicity

was removed (see Phase I). However, as the identification process continues, the blank toxicity will haveto be

eliminated, or it could lead to a misidentification of the

cause of toxicity.

When effluent samples are readily filtered

(-2,000 ml for one 90 mm 1 ym filter) it may be possible

to filter the effluent for the filtration test of Phase I but then

use unfiltered effluent with the C,, SPE column test and

the methanol eluate test (Phase I). Once it has been

demonstrated that filtration does not reduce toxicity, routine filtering of these effluents (before passing the effluent

through the SPE column) can be eliminated. This will

reduce the amount of toxicity testing required.

2.2.3 Column Size

Various sizes of C,, SPE columns are available

ranging from 100 mg to 10,000 mg packing material. We

routinely have used Baker@ 1,000 mg columns for 1,000

ml of effluent (J.T. Baker Chemical Co, Phillipsburg, NJ).

Volumes for a 1,000 mg C,, SPE column are used in the

following description, since this is the size most often

used for acutely toxic effluents. Other available column

sizes and the appropriate volumes to be used in their

preparation are listed in Table 2-l. Positive pressure

pumps (EPA, 1991 A) are convenient for the large volume

2-3
effluent samples because flow rate can be controlled.

Vacuum manifolds can be used for drawing the small

samples and solvents through the column. Whichever

system is used, it must be made of materials that dilute

acid and solvents do not destroy, or from which chemicals

are not leached that are toxic or that interfere with analytical measurements. Teflon, glass, and stainless steel are

all acceptable choices.

2.2.4 C,, SPE Column Conditioning

The 1,000 mg C,, SPE columns are conditioned

by pumping IO ml of 100% methanol through the column

at a rate of 5 mI/min. The pumping rate can be increased

to 40-50 ml/min when using the larger C,, SPE columns

(e.g., 5 g or 10 g). The volumes of conditioning solvent

recommended for other size columns are shown in Table

2-1. We most commonly use methanol as the conditioning solvent but other water miscible solvents such as

acetonitrile, ethanol, or isopropanol may be substituted.

Before the packing goes dry, 10 ml of high purity water

must be added. As the last of the high purity water is

passing through the column, 25 ml of filtered dilution

water is added. The last 10 ml of dilution water is collected for a dilution water column blank. After the dilution

water has been collected, pumping is continued until no

dilution water emerges from the column.

2.2.5 Nution Blanks

To generate elution blanks from a 1,000 mg

column, two successive 1.5 ml volumes of 25% methanol/

water (%v/v) are pumped sequentially through the conditioned column and collected in one analytically clean,

labeled glass vial to produce a 3 ml sample. This procedure is repeated with two successive 1.5 ml volumes of

50%, 75%, 80%, 85%, 90%, 95% and 100% methanol/

water. The column should be allowed to dry for a few

seconds between each elution with the different 3 ml

volumes of methanol/water solutions. This will result in

eight 3 ml SPE fraction blanks (Figure 2-2). The volume of

methanol solutions used for elution will vary depending on

column size as shown in Table 2-1.

2.2.6 Column Loading with Effluent

The same column is then reconditioned with IO

ml of 100% methanol and IO ml of high purity water, as

described in Section 2.2.4. Without allowing the column to

dry, 1,000 ml of filtered effluent is pumped through the

column at a rate of 5 ml/min (Figure 2-3). The pumping

rate can be increased to 40-50 ml/min when using the

larger C,, SPE columns (e.g., 5 g or IO g). Three samples

(-25 ml) of the post-C,, SPE column effluent are collected

after 25 ml, 500 ml and 950 ml of the sample has passed

through the column. Each post-column aliquot is toxicity

tested to determine the presence of acute toxicity in the

post-column effluent. This information can be used to

determine whether the toxicant is removed from the eff luent by the column. As Phase II progresses, the recommendation is to increase the volume of post-column effluent

collected to 50-60 ml so that dilutions can be made and

LC50 values obtained. Pumping is continued until no

effluent emerges from the column.

The efficiency of the C,, SPE column is determined by the extraction efficiency (i.e., how well the

column sorbent removes the effluent components) and

the elution efficiency (i.e., how well sorbed effluent compounds are removed from the column by the solvent

elution). For purposes of the TIE, “efficiency” applies only

to recovery of those compounds causing or affecting

effluent toxicity. Since most acute effluent tests do not

require large volumes of post-column effluent, the question of extraction efficiency can be determined by measuring the toxicity of the post-C,, column effluent sample.

The toxicity of each aliquot collected after different volTable 2-1. Solid Phase Extraction (SPE) Column Fractionation Information’

C,* SPE

Sorbent

Amount2 (mg)

Volume

Conditioning

Solvent (ml)

High Purity

Water

Volume (ml)

Maximum

Volume

Effluent (ml)

Minimum (500x)

Elution

Volume3 (ml)

Suggested (333x)

Elution4

Volume3 (ml)

100 2 2 100 2 x0.1 2 x 0.15

500 5 5 500 2 x 0.5 2 x 0.75

1,000 10 10 1,000 2 x 1.0 2 x 1.5

5,000 50 50 5,000 2 x 5.0 2 x 7.5

10,000 100 100 10,000 2x 10 2 x 15

‘The information is based on manufacturer’s guidance and experimental data from ERL-D.

*The smaller columns (100, 500, and 1,000 mg sorbent) are available pre-packed from J.T. Baker Chemical Co., the larger columns (5,000 and

10.000 mg sorbent) are available pre-packed from Analytichem International.

3Elution with two successive aliquots of the volume listed.

4The 333x concentration factor is most often used for acute work.

2-4
C,, SPE Column

95 6 8 % i”

I I I I I I I I

Conduct Toxicity Test on Each Fraction

Figure 2-2. Procedures for eluting the SPE column with a gradient of methanol/water solutions.

Filtered

Effluent

Cl 8 SPE COhJmfl

1x

Figure 2-3. Concentrating effluent on the C,8 SPE column.
umes of effluent have passed through the column can be

compared. If there is toxicity in these aliquots, but it is

independent of the volume of effluent previously passed

through the column, then the post-column effluent toxicity

is probably caused by toxicants that are not extracted by

the column. If toxicity increases as the volume of postcolumn effluent passed through the column increases,

the capacity of the column to sorb the toxicants was

probably exceeded.

In some post-column effluent, a biological growth

may occur during toxicity testing which may result in

artifactual toxicity. Such growth can make it appear as if

the toxicant is not removed by the column. While this

growth does not occur in all effluents, when it does occur

with one post-column effluent sample, the growth often

occurs in each subsequent post-column effluent sample

from the same preparation. The growth may appear to be

filamentous and give a milky appearance in the test

vessel. This effect has been linked to methanol stimulation of bacterial growth. Methanol is present in the postcolumn samples because a small amount of methanol is

constantly released from the column during the sample

extraction. Effluents from biological treatment plants may

develop this characteristic more readily than industrial

effluents.

Additional filtering of the post-column effluent

sample through a 0.2 pm filter before testing to remove

bacteria and eliminate the growth has been helpful. To

avoid artifactual toxicity as much as possible in the postcolumn effluent, initiate the tests with the post-column

samples on the same day the effluent is extracted even if

fractions are not tested simultaneously. For those few

effluents where we have not eliminated this type of attifactual toxicity, holding the post-column effluent is problematic in that more time is available for bacteria to cause

problems in the post-column sample matrix. When postcolumn artifactual growth is not readily eliminated, a

different solvent (e.g., acetonitrile) to condition the column (but not for eluting) may be useful in reducing the

post-column artifactual bacterial growth. This artifactual

growth has not occurred in the toxicity tests with methanol

SPE fractions (Section 2.2.8).

2.2.7 C,, SPE Column Nution

Once the effluent sample has been loaded onto

the column, elution can begin. To elute a 1,000 mg

column, two successive 1.5 ml volumes of the 25%

methanol/water mixture are pumped through the column

and collected in one labelled, analytically clean vial to

make a 3 ml sample. Subsequently, two successive 1.5

ml volumes of each of the 50%, 75%, 80%, 85%, 90%,

95% and 100% methanol/water are pumped through the

column and collected in separate vials (Figure 2-2). The

next elution volume should be added when no more of the

preceding one is emerging from the column.

tion) is

This entire procedure (conditioning through elurepeated using a second 1,000 mg C,, SPE

column for the second 1,000 ml of filtered effluent. The

dilution water column blank samples should be kept separate. The corresponding fractions from the blank and the

sample from each 1,000 ml fractionation can be combined. For example, the 3 ml 100% methanol sample

fraction from the first column and the 3 ml 100% methanol

sample fraction from the second column are combined to

produce a total of 6 ml. There will be eight 6 ml blank

fractions and eight 6 ml effluent fractions.

The vials containing the methanol/water fractions

are tested immediately or sealed with perfluorocarbon or

foil-lined caps and stored under refrigeration. These fractions represent a ‘first cut” separation of effluent components. Elution volumes will vary if columns of different

sizes are used or if the particular effluent under study or

the research question being posed dictates method modification.

2.2.8 B/ank and Effluent Fraction Toxicity

Tests * The next step is to determine the toxicity of the

blank and effluent fractions. While the choice of test

concentration depends on the toxicity of the effluent in

most instances, we have used a high test concentration of

2x or 4x (the LC50 or 100% effluent) for acutely toxic

effluents. The methanol content in the fractions limits the

concentration that can be tested, and at this point the

amount of methanol is assumed to be 100% in all the

fractions for dilution calculations; however, this is not

assumed for add-back tests (described below). Usually

120 ~1 of each blank and sample fraction (333x) is injected into separate 10 ml aliquots of dilution water to test

at 4x the 100% effluent2. This will give a 1.2% methanol

concentration which is below the methanol LC50 for both

C. dubia and fathead minnows in the 100% methanol

fraction. The resulting methanol concentration must be

adjusted for the species tested (see Section 8 of EPA,

1991 A). During the initial stages, five animals in each 10

ml aliquot are used without duplicates. Using the above

volumes, the tested solution is more concentrated (i.e., 4x ) than

100% effluent, assuming 100% extraction and 100% elution

in one fraction. These test solutions can be diluted to

provide an LC50 for each sample fraction. Blank fractions

need not be diluted, since hopefully they are nontoxic.

Individual chemicals in the fractions could be

toxic even when they are not toxic in the whole effluent,

since the concentration tested may be as high as 4x

whole effluent. Therefore, to be toxic at whole effluent

concentrations, an individual fraction must have an LC50

of 25% or less. Since there is no way to know whether the

toxicant eluted over more than one fraction or what the

percent extraction and elution efficiency are, fraction tox-

*In the Phase II document published in 1989, testing at 5x was

recommended, the methanol level was 1.5% at this concentration. In

order to lower the methanol level, this was changed to 4x in this

document.

2-6
-4

icity up to 100% (4x whole effluent) should not be disregarded.

effluent and evaluating the toxicity. This methanol addition may in turn stimulate

If toxicity occurs in any of the fraction blank tests

and it is small relative to the toxicity in the corresponding

sample fraction (e.g., 20% mortality versus 80%), the

sample fraction results should not be dismissed. If all

organisms die in the blanks and the effluent fractions,

dilutions should be tested to make sure the sample fraction is substantially more toxic than the blank. In general,

blanks should not have measurable toxicity.

Table 2-2. Comparison of Toxic Units (TUs) in Each Toxic Fraction to

TUs of All Fractions Combined and Whole Effluent

Toxic Fraction (% Methanol) TUs

75 0.5

80 1.2

85 0.6

If the SPE fractions are toxic at effluent concentrations of lx or 2x and toxicity is reduced in two of the

three post-column effluent samples, the toxicant could

still be a non-polar organic compound. If the effluent

fractions are not toxic individually and the post-column

samples are non-toxic, it is possible that the toxicity has

been spread across several fractions or has not been

recovered from the column. Combining and concentrating

fractions may be useful or other elution procedures may

be necessary. If toxicity is observed in the fractions at lx,

2x, or 4x and in the post-column effluent samples, it is

possible that not all the toxicity is caused by non-polar

compounds, that break-through of the toxicant has occurred, or that the toxicity is artifactual.

SUM 2.3

Combined Fractions 2.7

Whole Effluent 2.5

In addition to concentrating column artifacts to

toxic levels, effluent constituents present at nonlethal

levels may be concentrated to toxic levels in this test if

they have a relatively high recovery value. Actual effluent

toxicants with poor recovery may not be present in these

test solutions at toxic levels. Spurious results of this

nature will be identified in the later stages of Phase II and/

or in Phase III.

biological growth, and if this happens, the test is negated.

We have rarely used this approach since the fractions

have seldom caused more toxicity than the effluent itself.

At this point in Phase II, the effluent fractions should also

be tested in water with TOC and suspended solids which

mimics the effluent to lessen matrix effects on toxicity. As

the identification step moves into Phase Ill, it is better to

use dilution water that mimics effluent or receiving water

characteristics.

2.2.9 SPE Fractions: Concentration and

Subsequent Toxicity Testjng

The SPE fractionation provides a general separaElution efficiency may be approximated by summing the amount of toxicity (i.e., TUs) in the toxic fractions

(provided dilutions are tested) and comparing this value

to whole effluent toxicity expressed as TUs. When summing acute toxicity, it is important that all values are for

comparable endpoints (i.e., LCSOs). Adding of TUs may

be somewhat imprecise for several reasons. A single

toxicant may occur in more than one adjacent fraction, in

which case a small amount of the toxicant in one fraction

may not be detectable because it is present below the

effect concentration. For acute toxicity, this problem may

be solved by combining a portion of each effluent fraction

(and separately testing the corresponding blank fractions)

and measuring total toxicity at ix. If more than one

toxicant is present, the effluent fraction toxicity may not be

strictly additive in their toxicities, and when separated into

different fractions the sum of the fraction toxicities may be

low even if extraction and elution efficiencies were 100%.

Table 2-2 illustrates a hypothetical example. The toxicity

test results from the test with a portion of all fractions or a

few of the fractions may show somewhat greater toxicities

than those of the whole effluent. This may be caused by

enhanced toxicity due to matrix effects. When this occurs,

it may be possible to compensate for toxicity enhancement by methanol, by adding methanol to the whole

tion of non-polar organics and except in relatively

uncomplicated effluents, GC/MS analysis of the concentrates of toxic C,, SPE fractions will result in very complicated chromatograms from which the toxicant cannot

be distinguished from other effluent components. A secondary fractionation using HPLC is often needed to further simplify toxic effluent fractions prior to component

identification by GC/MS analysis.

In order to maximize the chromatographic separation capability of the HPLC, the volume of the sample

injected onto an analytical size HPLC column should be

as small as possible (i.e., ~0.5 ml); therefore, the toxic

SPE fractions (usually >I ml) must be concentrated prior

to injection onto the HPLC column. This concentration

step will provide the added benefit of an increase in

concentrations of constituents in the HPLC fractions as

well as rid the SPE fractions of water. The latter issue is

important if GC/MS analysis will be performed on the

concentrated SPE fraction prior to injection on the HPLC

(Durhan et al., 1990).

The volume of the fractions from the initial SPE

fractionation procedure and the number of fractions to be

combined will determine the size of the SPE column to

use for the concentration procedure. Table 2-3 contains

information on the column sizes we have found to be

most useful. In the procedure outlined below (Figure 2-4),

we have used a 100 mg column which is the most

commonly used size for concentrating SPE fractions of

acutely toxic effluents. Most often the toxic effluent SPE

2-7
Table 2-3. Information for Concentrating SPE and HPLC Fractions’

C,8 SPE Volume

Sorbent Conditioning

Amount (mg) Solvent (ml)

High Purity

Water

Volume (ml)

Maximum

Toxic Fraction

Volume (ml)

Maximum

Diluted Fraction

Volume (ml)

Minimum

Elution

Volume2 (ml)

Approximate

Eiuate

Volume (ml)

100 1 1 20 100 3 x0.1 0.22

200 2 2 40 200 3 x 0.2 0.44

500 5 5 100 500 3 x 0.5 1.10

1,000 10 10 200 1,000 3x 1.0 2.20

‘Concentration information is based on manufacturers guidance and experimental data from ERL-D

2Elution with three successive aliquots of the volume listed.

Diluted Toxic Fraction(s)

1

100 mg C,, SPE Column Sorption

1

(discard post-C,,

effluent)

Dry Ct8 Column with Nitrogen

(optional)

1

Elute Column with three 0.10 ml volumes

of 100% Methanol

1

Collect Eluate (Concentrate)

1

Conduct Toxicity Test

Figure 2-4. Procedure to concentrate toxic SPE fractions.

fractions are combined and diluted with high purity water

and the corresponding blank fractions are treated similarly. In cases where there are multiple non-polar toxicants, and when toxicity occurs in several fractions, it may

be more useful to concentrate each fraction separately for

subsequent HPLC separation. The percent methanol in

the diluted fraction sample should be 220% and the

volume to which the fractions can be diluted is dependent

on the amount of column packing. For example, the total

volume of the diluted fractions should not exceed 100 ml

for the 100 mg C,, SPE column (Table 2-3). No more than

three toxic fractions of 6 ml each can be combined and

concentrated on the 100 mg column. When the total

volume of combined fractions or the individual fraction

volume is above 20 ml, larger columns should be used;

consult Table 2-3 for column size and elution volume

information. The effluent and blank concentrates and the

column blank are tested for toxicity to ensure that the

toxicant is still in the concentrate and that artifactual

toxicity was not introduced by the procedure. If there is

not measurable toxicity in the concentrate, it is possible

that the percentage of methanol in the diluted fraction was

too high. The concentration procedure should then be

repeated with a new set of toxicity tested fractions diluted

to a lower methanol concentration, e.g., 10%.

Below is an example of how to prepare effluent

and blank fraction concentrates. First, a 100 mg C,, SPE

column is conditioned with 1 ml of methanol and 1 ml of

high purity water similar to the procedures described in

the SPE Column Conditioning Section (2.2.4). Column

blanks for toxicity testing are obtained by rinsing the

column with at least 20 ml of dilution water. After collecting the column blank, recondition the column with 1 ml of

methanol and rinse with 1 ml of high purity water. The

diluted blank fractions (for dilution guidance see Table 2-

3) are then drawn through the 100 mg C,, SPE column

under a pressure of 380 mm Hg using a vacuum manifold.

Unlike the first fractionation step (Section 2.2.6) the postcolumn sample cannot be tested for toxicity because of its

high methanol concentration (i.e., IO-20%). The column

is then dried for IO min using a gentle flow of nitrogen (1 O20 ml/set). Drying the column usually increases the recovery of toxicity, but sometimes toxicity is not recovered

from the column, possibly as a result of volatilization. If

this occurs the concentration procedure can be repeated

without the nitrogen drying step.

After drying the sorbent, the luer tip of the column

is fitted with a luer-lock needle and 100 yl of 100%

methanol is placed into the column using a microliter

syringe. Nitrogen is then applied to the column at a rate of

-4 ml/set to force the methanol through the sorbent. The

luer-lock needle is needed to ensure the collection of

small volumes; when using larger column sizes (e.g.,

2500 mg) this is not necessary. The first 100 ul aliquot of

methanol applied to the column will yield approximately

25 yl of eluate. Two more 100 ~_rl aliquots of 100%

methanol are also forced through the column. The final

volume of eluate collected will be approximately 220 ~1. If

desired, measure the exact volume collected (using a ul

syringe) to calculate concentration factors (Table 2-3). As

in most chromatographic separations and extractions,

three separate smaller elutions of methanol are more

efficient than one large one.

2-8
The 100 mg C,, SPE column is reconditioned

with 1 ml of methanol and rinsed with 1 ml of high purity

water. It is then used to concentrate the diluted toxic SPE

column fractions, using the same procedure used for the

blank fractions (Figure 2-4). In lieu of reconditioning the

same column, two columns can be conditioned, one used

for the diluted blank fractions and the other used for

concentrating the diluted toxic SPE fractions. The resulting column blanks should be toxicity tested separately.

The original effluent volume of 2,000 ml is now

concentrated into a 220 ~1 sample or a nominal concentration of 9,091x (ignoring the amount used for testing).

As work progresses and more quantitative results are

needed, the eluate volume must be measured to provide

the correct concentration factor. If 9 pi of concentrate is

diluted to 10 ml in dilution water, the resulting test concentration will be 8x whole effluent. Additional test concentrations (e.g., 4x, 2x, Ix ) can be prepared to determine

an LC50 of the concentrate, and toxicity recovery can be

calculated by comparing this LC50 to the LC50 of the

effluent. The concentrate toxicity might be higher than the

sum of the individual toxic fractions because some of the

toxicant may have been in adjacent fractions that were

concentrated in the first step (Section 2.2.7) but not

detectable by the toxicity test of the single fraction. The

concentrate toxicity may also be lower than expected

because of low extraction and elution efficiencies. Where

greater concentration factors are desirable, SPE fractionation should be repeated with additional volumes of effluent, followed by combining the toxic fractions before

concentration. The size of the column used for concentrating may have to be increased, along with the appropriate changes in dilution and elution volumes (Table 2-3).

The important concern here is not 100% recovery

of toxicants but enough recovery for GC/MS analyses and

to obtain measurable toxicity in the HPLC fractions. If

recovery is too low, changing or eliminating the column

drying time may help. Sometimes recovery appears to

increase with drying time while other compounds are

volatilized from the column during the drying process. For

concentrates analyzed using GC/MS, column drying to

remove water is critical to GC column performance.

2.2. IO HPL C Separation

The same column packing functionality should be

used in the HPLC column as the SPE column. At later

stages, when more is known about the toxicants, other

sorbent types may be used.

The HPLC conditions presented in this section

are general. As more information on the effluent is gathered, HPLC conditions should be modified to achieve

better separation and higher concentration factors. We

use a flow rate of 1 ml/min on an instrument equipped with

a 5 urn C, reverse phase column (250 mm x 4.6 mm i.d.).

The HPL& elution conditions will change depending on

which SPE fractions have been concentrated. The HPLC

conditions for the four most commonly toxic SPE fractions

are listed in Table 2-4. Depending on the size of the

Table 2-4. Example HPLC Elution Gradients for Four Commonly Toxic

SPE Fractions

75% or 85% SPE Fractions 85% or 90% SPE Fractions

Time (min) % Methanol/Water Time (min) % Methanol/Water

0 50 ’ 0 60

1 60 1 70

13 90 13 90

20 100 20 100

25 100 25 100

HPLC injector and column, more than one HPLC fractionation run may be required to fractionate the entire blank

concentrate. When multiple HPLC fractionations are conducted, collect all the corresponding HPLC fractions in

the same set of vials. For example, if two HPLC fractionations were performed for the blank concentrate, 25-2 ml

HPLC fractions would be obtained.

Using the HPLC equipment described above, all

of the blank concentrate remaining after toxicity testing is

injected (1500 ~1) and 25-1 ml fractions are collected in

analytically clean glass vials (Figure 2-5). The same

procedure is followed using the effluent sample concentrate. The vials should be sealed (e.g., with foil lined caps)

and stored at 4°C after use. As soon as toxicant identification is obtained by GC/MS, then HPLC conditions (gradient, fraction size, and number of fractions) can be optimized

for further fractionations.

2.2.11 HPLC Fraction Toxicity Tests

Before specific toxicants are identified, toxicity

tests on each HPLC blank fraction and sample fraction

are conducted using non-replicated exposures of five

animals each. The amount of methanol in the HPLC

fractions limits the concentration that can be tested. Assume that each fraction is 100% methanol to calculate the

necessary dilution. A methanol concentration of 1.2%

should not be exceeded for C. dubia and fathead minnow

acute toxicity tests.

Fractionate SPE Concentrate

Using HPLC

1

Collect 25-l ml HPLC

Fractions

1

Conduct Toxicity Test on Each

Fraction

Figure 2-5. Procedure to fractionate acutely toxic SPE concentrates

using HPLC.

2-s
For acute studies, when all of the SPE fraction

concentrate remaining after toxicity testing is injected

(one injection) on the HPLC (Figure 2-Q each resulting 1

ml HPLC fraction equals 2,000 ml of effluent (assuming

no loss and toxicant elution in only one fraction) or a

2,000-fold concentration. If each HPLC fraction is then

diluted for testing (80 ul to IO ml) the resultant concentration is 16x the original effluent concentration. In preliminary Phase II testing the HPLC fractions are tested without

dilutions. Only the toxic HPLC fractions are tested again

with dilutions to generate an LC50. Some loss of toxicant

tends to occur in each concentration step and the resulting toxicity may be decreased relative to the original

effluent.

The blank fractions should not be toxic. If they

are, then additional tests with dilutions must be conducted

on both blanks and toxic fractions to find out whether

there is enough additional toxicity in the sample fractions

to warrant analysis

The toxicity of the HPLC fractions should be

tested at twice (at least) the concentration at which the

original SPE column fractions were tested because recovery of toxicity and analytical measurements indicate

that up to 50% of the initial concentration of toxic compounds may be lost in this step (Durhan et al., 1990). The

amount of methanol should not exceed the amount used

in the SPE fraction tests described above (Section 2.2.8).

2.2.12 HPLC Fracfions: Concentration and

Subsequent Toxicity Testing

The HPLC fractions that exhibit toxicity and their

corresponding blank fractions must be concentrated in a

solvent suitable for GC/MS or other analytical techniques.

The procedure is identical to that described in Section

2.2.9 and is depicted in Figure 2-6. Judgement must be

used to decide whether to concentrate each toxic fraction

separately or to combine various toxic and adjacent fractions prior to concentration. If, for example, three successive fractions exhibit toxicity, there is a good chance that

the same toxicant is in all three. If there are other fractions

that show toxicity but they are separated from the first set

by several non-toxic fractions, there is high probability

that the second set contains a toxicant different from the

first three. There is also a good chance that at least one

non-toxic fraction on either side of the toxic fractions

contains some of the toxicant( The advantage of combining fractions is to reduce the work load and increase

concentration in the final concentrate. The disadvantage

is that more constituents that are not the toxicant will

also be concentrated. This decision is not always straightforward and must be based on trial and error, and experience. Blank fractions corresponding to the toxic fractions

are concentrated the same way.

The HPLC fraction and blank concentrates should

be finally checked for toxicity before GC/MS analysis.

This concentrate is now nominally 9,091x more concentrated than the effluent. If 18 ul is diluted to 10 ml, the

resultant test concentration will be 16x the original sample

Diluted HPLC Fraction(s)

1

100 mg C,, SPE Column Sorption

1

(discard post -C,,

effluent)

Dry Column with Nitrogen (optional)

1

Elute Column with three 0.10 ml

Volumes of Methanol

1

Collect Eluate (Concentrate)

1

Conduct Toxicity Test

1

Analyze Concentrate on GUMS

Figure 2-6. Procedure to concentrate toxic HPLC factions.

concentration. To quantitate toxicity and use the Phase II

data later, additional lower concentrations should be tested

(e.g., 8x, 4x, 2x); TUs of this concentrate can then be

compared to previous toxicity test results. The HPLC

concentrate should be tested at one to two times the test

concentration of the HPLC fraction tests (i.e., 16x or 32x).

This is the last opportunity to assure that the toxicant is

still present in the concentrate before it is subjected to

GClMS analysis. Whether the toxicant is detected by the

analytical detector (mass spectrometry in our laboratory)

is always a question. Since GClMS detects only about

20% of organic chemicals (EPA, 1989B), even such a

broad spectrum method is not certain to identify the

toxicant. As work progresses with more samples of the

effluent and quantitative results are needed, the amount

of eluate collected should be carefully measured and

recorded to accurately calculate the concentration factors. In addition, the volume of concentrate removed for

toxicity testing and analytical analyses should also be

recorded.

2.3 Chronic Toxicity: Fractionation and

Toxicity Testing Procedures

The chronic Phase II non-polar organic toxicity

identification follows the same general approach and

employs manipulations similar to those described for the

acutely toxic non-polar organic compounds (Section 2.2).

One major difference is that the concentration of the

eluting solvent (e.g., methanol) must be lower in the

chronic toxicity tests than in acute tests. In the initial

stages of Phase II, toxicity tests may be conducted on C,,

SPE effluent fractions and blank fractions to detect the

presence of toxicants, and not to quantify the magnitude

2-l 0
of the toxicity in each. However, as suspect toxicants are

identified, quantitative toxicity measurements will be

needed to compare with the analytical measurements. If

Phase II data will be used to correlate effluent toxicity to

toxicant concentrations (Phase Ill), then more replicates

per concentration, randomization of test concentrations,

careful observation of organism exposure times, and

organisms of approximately the same age should be used

(Section 1.2). Also the amount of eluate that is collected

from the SPE fractionation, SPE concentration, and the

amount of eluate used for testing and GC/MS analysis

should be measured at all steps. If it is expected that the

Phase II data will be needed later, it is prudent to measure

the degree of toxicity in the SPE effluent fractions (Section 23.5) at the onset of testing. We rarely see blank

fraction toxicity, therefore, there is little need to evaluate

the blank fraction toxicity with dilutions. Also, the volume

of eluate must be measured to determine the actual

toxicity concentration in each step of the procedure.

The following discussion is based on our experiences with C. dubia and fathead minnows (see Section

1.2). The use of other species will require reconsideration

of the appropriate test volumes and methanol concentration for each step. Chronic testing is more labor intensive

and generally requires more effluent sample volume than

acute testing. For the most part, in the descriptions below,

for C. dubia there are five replicates containing IO ml of

test solution and one animal per cup. For the fathead

minnow tests, two replicates of 10 animals per 50 ml and

the control are usually used (Section 1.2). Typically we

use four concentrations and a control.

As soon as the cause of toxicity has been determined to be a non-polar organic compound (e.g., methanol eluate test; EPA, 1992) it is prudent to concentrate

large volumes of effluent for the subsequent analyses. By

concentrating large amounts of the effluent it is possible

to plan the optimal usage of the amount of column eluate

available for toxicity testing.

2.3.1 Sample Volume

The volume of effluent needed depends on its

toxicity, the toxicity of the chemicals causing effluent

toxicity, and the sensitivity of the analytical method. Since

only the first of these will usually be known when Phase II

begins, the volume of effluent to process should be

considered at the beginning of the identification process

to minimize the amount of re-fractionating and re-testing

of effluent and fractions. Ideally, fractionation should provide enough volume of post-column effluent (Section

2.3.6), C,, SPE fractions (Section 2.3.8), SPE fraction

concentrates (Section 2.3.9), HPLC fractions (Section

2.3.1 I), and HPLC fraction concentrates (Section 2.3.12)

to conduct all chronic toxicity testing and chemical analyses, Because of the many factors affecting the amount of

effluent needed, a significant amount of thought should

be put into the volume of effluent to obtain and process at

one time. It is prudent for the investigator to anticipate

how many identification procedures will be done, and

then calculate the volume of effluent needed using the

particular test parameters desired, before extracting any

effluent to ensure that sufficient volume of fractions, concentrates, and post-column effluent is available for the

planned procedures. It may be best to perform these

calculations with several different effluent volumes and

test conditions to ascertain the optimal volume of effluent

to fractionate. A worksheet to assist with these calculations and an example are provided in Appendix A.

The volumes of eluate needed for chronic toxicity

testing at 2x, lx, and 0.5x are provided in Table 2-5 for

the C. dubia and fathead minnow short-term tests based

on the methanol concentration that can safely be used for

the chronic tests. The amount of SPE fractionation eluate

needed for toxicity testing is presented for the range of

tests that are commonly performed with C. dubia or

fathead minnows, these volumes can be used in the

calculation worksheets found in Appendix A (Table A-l).

The approximate volume of effluent that will be needed

for testing with C. dubia and fathead minnows is listed in

Table 2-6 for various fractionation schemes and toxicity

testing parameters. When only a portion of the TIE procedures will be used, obviously less effluent volume will be

needed. In Table A-2, the example calculations are based

upon the use of minimum elution volume for the SPE

columns (Table 2-l), concentrating only one SPE fraction

(Section 2.3.9), and taking into account the toxicity testing

(Sections 2.3.8, 2.3.9, 2.3.11, and 2.3.12) and GUMS

analysis volumes (Section 2.5). These parameters are

discussed in detail below. If additional eluate is needed,

the chronic tests must be repeated for each fractionation.

In Phase II and Phase Ill more confidence in the toxicity

estimates is needed than in Phase I, therefore tests may

require more replicates. The volumes needed for those

tests are also presented in Table 2-5. When only limited

amounts of effluent are available, one must be creative

and plan its usage very carefully to obtain meaningful

results.

2.3.2 Fi/tra tion

For filtration of chronically toxic effluents, the use

of glass fiber filters (1 urn nominal pore size) is recommended. Both 45 mm and 90 mm diameter filters have

been used routinely, but the 90 mm diameter filter allows

about four times more effluent to be passed over one filter

than the 45 mm filter. All filters and glassware should first

be pre-rinsed with pH 3 high purity water to remove any

residual metals followed by a high purity water (e.g., MilliQ@ Water System) rinse which is discarded. Low levels of

metals (e.g., ug/l) from the filters may cause toxicity

interferences and pre-rinsing the filters may provide cleaner

blanks and less contamination in effluent samples. To

collect the dilution water filter blank, first pass a volume

(-200 ml) of dilution water over the filter and discard it.

Next, collect the volume of dilution water needed to

conduct the filtration blank test. It is a good idea to prepare

excess volume, at least 500 ml for the C. dubia 7d test and 800

ml for the fathead minnow 7d test. A portion of the filtered

dilution water is collected for testing and a portion is reserved for the solid phase extraction test blank (Section

6.6; EPA, 1992).

2-11
Table 2-5. Eluate Volumes Needed for Chronic SPE Fraction Toxicity Tests with Ceriodaphnia dubia and Pimephales promelas

Test Test

Species Duration

Original

Sample&

No. Renewals

High Test

Cont. of

SPE Fraction

No.

Rep

Volume (ml) of 500x

Eluate Needed

for Testing’

Test

Concentrations

C. dubia

C. dubia

C. dubia

C. dubia

C. dubia

C. dubia

C. dubia

C. dubia

P. promelas

P. promelas

P. promelas

P. promelas

4-d

4-d

7-d

7-d

4-d

4-d

7-d

7-d

7-d

7-d

7-d

7-d

2x

2x

2x

2x

2x

2x

2x

2x

2x

2x

4x

4x

10

10

10

10

0.70 2x, lx, 0.5x

1.40 2x, lx, 0.5x

1.05 2x, lx, 0.5x

2.45 2x, lx, 0.5x

1.40 2x, lx, 0.5x

2.80 2x, lx, 0.5x

2.10 2x, lx, 0.5x

4.90 2x, lx, 0.5x

4.90 2x, lx, 0.5x

9.80 2x, lx, 0.5x

9.80 4x, 2x, lx

19.60 4x, 2x, lx

‘Test volumes per replicate are 10 ml/cup for C. dubia and 50 ml/cup for P. promelas. The fraction test solutions are prepared as one solution and

divided into aliquots for the replicates. For the 500x eluate concentration, this volume is based on the assumption that the C. dubia test solutions

are prepared as 200 ul of 500x into 50 ml for 2x, 100 pl into 50 ml for lx, and 50 pl into 50 ml for 0.5x. More volume will be needed if serial

dilutions are prepared (400 fl vs 350 pl). For the fathead minnow tests this assumes test solutions are prepared as 400 pl into 100 ml for 2x, 200

ml into 100 ml for lx, and 100 pl into 100 ml for 0.5x. More volume will be needed if serial dilutions are prepared (800 pl vs 700 J.IJ). For the 4x

fathead minnow test, 800 pl per 100 ml can be prepared in a similar manner.

Table 2-6. Approximate Effluent Volumes Needed for the Chronic Non-Polar Organic Identification Procedures’

Test

Species

Test

Duration

Original

Sample &

Number of

Renewals

High Test

Cont. in

SPE Fraction

Test

No.

Rep.

Are

Dilutions

Used ?

Volume Effluent (ml)

Needed to Conduct

SPE & GC/MS*

Analyses

Volume Effluent (ml)

Needed to Conduct

SPE & HPLC & GC/MS3

Analyses

C. dubia 4-d 4 2x 5 Yes 3,000 15,000

C. dubia 7-d 3 2x 5 Yes 2,000 15,000

C. dubia 7-d 7 2x 5 Yes 5,000 20,000

P. promelas 7-d 7 2x 2 Yes 10,000 50,000

P. promelas 7-d 7 2x 4 No 5,000 40,000

‘Calculation of toxicity testing volumes assumes that: 4x high concentration for SPE concentrate test (Section 2.3.9) 8x high concentration for

HPLC fraction test (Section 2.3.1 l), 16x high concentration for HPLC concentrate test (Section 2.3.12) concentration of only one toxic fraction

(SPE and HPLC), the maximum amount of sample is concentrated on the SPE columns and all SPE columns are eluted with the minimum elution

volume.

*TIE procedures used: SPE fractionation and GWMS of SPE concentrate,

3TIE procedures used: SPE fractionation, GC/MS of SPE concentrate, HPLC fractionation, and GC/MS of HPLC concentrates.

2-12
After the filtration blank has been obtained, the

effluent sample is filtered using the same filter, a portion

of the filtrate is collected for toxicity testing, and a portion

is set aside for concentrating on the C,, SPE column. For

some effluents, one filter will often not suffice. A technique we use to prepare several filters at once is stacking

three to eight filters together in one filter holder, followed

by sequential rinses with pH 3 high purity water, high

purity water and dilution water (using the same rinse

volumes as above). Finally, the filters are separated and

set aside, using one at a time for the effluent sample. If

the samples have high suspended solids concentration,

pre-filtering using a larger pore size filter may help, and

the appropriate blanks should be used. If the sample

cannot be effectively filtered due to the presence of many

fine particles, centrifugation may be used (of course,

blanks must be prepared).

The filter housing should be thoroughly cleaned

before use to prevent any particle build-up or toxicity

carry-over from previous samples. We have found large

filtration apparatus (1,000 ml), removable glass frits, or

plastic filtering apparatus (e.g., Millipore@) to be useful.

The glassware cleaning procedure that is described in the

acute Phase I TIE manual (EPA, 1991A) is sufficient for

chronic TIE work. The glass frits may require rigorous

cleaning (i.e., soak in aqua regia for 20-40 min) to remove

residuals that may remain after filtering, since the glass

frit may itself act as a filter. Also available are removable

stainless steel filter supports in a glass vacuum filter

apparatus (available from Millipore@). These filter supports do not require as rigorous cleaning as fritted glassware, and therefore are a good substitute.

When effluent samples are readily filtered

(-2,000 ml for one 90 mm 1 urn filter) it may be possible

to filter the effluent for the filtration test of Phase I but then

use unfiltered effluent with the C,, SPE column test and

the methanol eluate test (Phase I). Once it has been

demonstrated that filtration does not reduce toxicity, routine filtering of these effluents (before passing the effluent

through the SPE column) can be eliminated, which will

reduce the amount of toxicity testing required.

2.3.3 Column Size

Available C,, SPE column sizes and the appropriate water and solvent volumes used in their preparation

are listed in Table 2-1. Positive pressure pumps are the

most convenient to use for the large volume effluent

samples because the flow rate can be controlled. Pumps

and vacuum manifolds can both be used for eluting C,,

SPE columns. Whichever system is used, it should be

made of materials that dilute acid and solvents do not

destroy, or from which chemicals are not leached that are

toxic or interfere with analytical measurements. Teflon,

glass, and stainless steel are all acceptable.

When SPE is used for isolating non-polar organic toxicants, use the maximum volume of effluent and

the minimum elution volume for the column size selected

to optimize the concentration of toxicants in the methanol

eluates. For example, if 6,000 ml is processed, it is best to

use one 5,000 mg column with 5,000 ml and one 1,000 mg

column with 1000 ml of effluent and elute both columns

with the minimum elution volumes (Table 2-1) and combine eluates. It is always best to process the maximum

volume of effluent on each column to achieve the highest

concentration of toxicants in the eluate.

2.3.4 C,, SPE Column Conditioning

The 10,000 mg C, SPE columns (Analytichem

International, Harbor City, CA) are conditioned by pumping 100 ml of methanol through the column at a rate of 40-

50 ml/min. This size column can process 10,000 ml of

effluent and is the largest commercially pre-packed SPE

column available at this time. The example presented in

this section will be for 10,000 ml effluent using a 10,000

mg SPE column. The volumes of conditioning solvent will

change when other size columns are used, as shown in

Table 2-1. We most commonly use’ methanol as the

conditioning solvent but other water miscible solvents

such as acetonitrile, ethanol, or isopropanol may also be

used to condition columns. Before the packing goes dry

100 ml of high purity distilled water must be added. As the

last of that water is passing through, filtered dilution water

is added. The volume of dilution water needed may vary

from 250 ml to 1,200 ml depending on the species tested.

The first 100 ml is discarded and the remainder is collected for the dilution water column blank. After the dilution water has been collected, pumping is continued until

no water emerges from the column.

Low dissolved oxygen (DO) in the post-column

dilution water blanks (even in reconstituted waters) has

occurred during some chronic tests; therefore, we discard

the first 100-200 ml and collect the remainder of the postcolumn dilution water. Low DO has been a problem,

particularly in the fathead minnow growth test, and is

attributed to the small amount of methanol that bleeds

into the post-column sample. This may be alleviated by

discarding the first post-column aliquots.

2.3.5 Elution Blanks

For chronic work, we have been using seven

methanol/water fractions (50%, 75%, 80%, 85%, 90%,

95%, and 100%) rather than the eight used in acute TIES.

By eliminating 25% methanol/water fraction used in acute

work the toxicity testing workload is reduced, in turn a

reduction in separation of toxic and non-toxic components

can occur.

To collect the fraction blanks from the 10,000 mg

column, two successive 10 ml volumes of 50% methanol

in water are pumped through the conditioned column and

collected in one analytically clean labeled vial, to make a

20 ml sample. This procedure is repeated six more times

with two successive 10 ml volumes of 75%, 80%, 85%,

90%, 95% and 100% methanol/water solutions. The column should be allowed to dry for a few seconds between

each elution with the different 20 ml volumes of methanol/

water mixtures. This will result in seven 20 ml blank SPE

2-13
fractions. The volume of methanol solutions used for

elution will vary depending on column size as shown in

Table 2-1.

2.3.6 Column Loading with Effluent

The same 10,000 mg column is reconditioned

with 100 ml of 100% methanol and 100 ml of high purity

water, as described in Section 2.3.4. The sorbent must be

reconditioned when the maximum volume of dilution water has been passed over the column, otherwise the

sorbents’ capacity will be exceeded. After the high purity

water rinse and without a//owing the column to dry,

10,000 ml of filtered effluent sample is pumped through

the column at a rate of about 40 - 50 ml/min.

Discard the first 100-200 ml of post-column eff luent, to reduce the possibility of higher concentrations of

methanol in post-column samples, which may contribute

to attifactual toxicity. To evaluate the post-!,, SPE column effluent for toxicity, collect at least two alrquots (e.g.,

beginning and end) separately. If only small quantities

(~500 ml) of post-column effluent are needed for toxicity

testing (e.g., C. dubia test), several separate post-column

effluent samples may be more helpful in determining if the

toxicants are retained by the column. About 800 ml of

post-column effluent is needed for the fathead minnow

test if only one concentration (100%) of post-column

effluent is tested for toxicity. If two concentrations (100%

and 50%) are used, then the required volume for that

species increases to 1,200 ml for each post-column aliquot. As Phase II progresses, the recommendation is to

collect enough post-column effluent to conduct toxicity

tests with dilutions.

2.3.7 C,, SPE Column Nution

To elute the C,, SPE column, two successive 10

ml volumes of the 50% methanol/water mixture are pumped

through the column and collected in one labelled, analytically clean vial. Subsequently, two successive 10 ml total

volumes of each of the 75%, 80%, 85%, 90%, 95% and

100% methanol/water solutions are pumped through the

column and collected in separate vials. The next elution

volume should not be added until no more of the preceding one is emerging from the column. This results in

seven 20 ml SPE fractions. If one 5 g column and two 1 g

columns are used to concentrate 7,000 ml of effluent, the

corresponding fractions can be combined. For example,

the 10 ml eluate of the 80% fraction from the 5 g column

can be combined with the two 2 ml 80% fractions from the

two 1 g columns. This applies to both sample and blank

fractions for a total of 14 ml.

This entire procedure (conditioning through elution) is repeated using a second 10,000 mg C,, SPE

column for a second 10,000 ml of filtered effluent. The

dilution water column blank samples should be kept separate. The corresponding fractions from both the blanks

and the sample from each 10,000 ml fractionation can be

combined as described above. There will be seven 40 ml

blank fractions and seven 40 ml effluent fractions, representing 20,000 ml effluent.

The vials containing the methanol/water fractions

are sealed with perfluorocarbon or foil-lined caps, and

stored at 4°C if not tested immediately. These fractions

represent a ‘first cut” separation of effluent components.

Volumes will vary if columns of different sizes are used or

if the particular effluent under study or the research

question posed dictates method modification.

2.3.8 Blank and Effluent Fraction Toxicity

Tests

While the choice of test concentration depends

on the toxicity of the effluent (Section 1.2) in most instances we have used a concentration of 4x or 2x as the

high test concentration for testing SPE fractions. The high

test concentration of the SPE fraction is in part controlled

by the tolerance of the organisms to methanol. For chronic

testing the concentration of methanol should be less than

0.6% for C. dubia, and less than or equal to1 % for fathead

minnows (see Phase I; EPA, 1992).

If the minimum elution volumes are used, typically SPE eluates are 500x effluent concentration. For

fathead minnow testing, eluates can be toxicity tested at

4x effluent concentration by diluting 80 ~1 to 10 ml, which

results in a 0.8% methanol concentration. For C. dubia,

eluates can be toxicity tested at 2x the 100% effluent

concentration by diluting 40 ul to 10 ml which results in a

methanol concentration of 0.4%. If there is the need to

toxicity test the 500x eluate with C. dubia at 4x then the

SPE eluates can be concentrated by gently airing the

eluate down (using nitrogen) to half its original volume.

However, by using this procedure you risk losing the

toxicant because of evaporation or insolubility. Also realize that when a water and methanol mixture is aired

down, the percent methanol composition changes, because methanol will evaporate faster than water.

If toxicity occurs in any of the fraction blank tests

and it is small relative to the toxicity in the corresponding

sample fraction, the sample fraction results should not be

dismissed. If all organisms die in the blanks and effluent

fractions, dilutions of each should be tested to make sure

the sample fraction is substantially more toxic than the

blank. In general, blanks should not have measurable

toxicity.

When the post-column effluent sample is toxic

and the fractions are toxic at effluent concentrations of lx

or 2x, the toxicant could still be a non-polar organic

compound. If the fractions are not toxic individually and

the post-column sample is non-toxic, it is possible that the

toxicity is spread out among the fractions. Combining and

concentrating these fractions may be useful or other

elution procedures may be necessary (Section 2.6). If the

fractions are toxic and the post-column effluent is toxic, it

is possible that the toxicant is in the fractions, and that

either an additional toxicant is present in the post2-14
column effluent, that break-through of the toxicant occurred, or that the toxicity is artifactual. If toxicity is

recovered at lx, 2x, or 4x and in one of the post-column

effluent samples, it is possible that not all the toxicity is

caused by non-polar organic compounds or the possibility

exists of break-through in the post-column sample.

-

For the chronic TIE, the question of extraction

efficiency cannot be as readily addressed as it is for the

acute TIE (Section 2.2.8). Measuring the chronic toxicity

of the post-column effluent will be limited by the species

tested, the test volumes required for the test and the

frequency of sample replacement. Without a measure of

the toxicity in the post-column effluent, conclusions regarding extraction efficiency are difficult to make. The

limitations created by this concern are addressed in Phase

III (EPA, 1993A). Artifactual toxicity in the post-column

effluent has been a problem in chronically toxic effluents

as it was in some acutely toxic effluents. For a detailed

discussion of this artifactual toxicity that appears as a

biological growth and suggestions to avoid it please refer

to Sections 2.2.6 and 2.3.4., and EPA, 1992.

concentrate one 40 ml toxic fraction from two 10,000 mg

SPE columns. Often the toxic effluent SPE fractions are

combined and diluted with high purity water. If enough

toxicity occurs in each fraction it may be more useful to

concentrate each fraction separately for subsequent HPLC

separation. The corresponding blank fractions are similarly treated. The percent methanol in the diluted fraction

sample should be 520% and the volume to which the

fractions can be diluted is dependent on the amount of

column packing. For example, the total volume of the

diluted fraction(s) should not exceed 200 ml for the 200

mg C,, column (Table 2-3).

At this point in Phase II, the effluent fractions

should also be tested in water with TOC and suspended

solids that mimic the effluent to lessen matrix effects on

toxicity. As the identification step moves into Phase Ill, it

is better to use dilution water that mimics effluent or

receiving water characteristics.

A 200 mg C,, column is conditioned with 2 ml of

methanol and rinsed with 2 ml of water similar to the

procedures described in the SPE Column Conditioning

Section (2.3.4). The diluted blank fractions are then drawn

through the 200 mg C,, SPE column under a pressure of

380 mm Hg using a vacuum manifold. When processing

larger volumes, or using larger columns, positive pressure can be used. The solution passing through the

column cannot be tested for toxicity because of its high

methanol concentration (e.g., 1 O-20% methanol). The

column is then dried for 10 min using a gentle flow of

nitrogen (1 O-20 ml/set). Drying ?he column usually increases the recovery of toxicity, but sometimes toxicity is

not recovered from the column possibly because of volatilization. If this occurs, the concentration procedure can

be repeated without the nitrogen drying step.

2.3.9 SPE Fractions: Concentration and

Subsequent Toxicity Testing

The SPE fractionation provides a general separa2-l 5

tion of non-polar organics and except in relatively

uncomplicated effluents, GC/MS analysis of the concentrates of toxic C,, SPE fractions will result in very complicated chromatograms from which the toxicant cannot

be distinguished from other effluent components. A secondary fractionation using HPLC is often needed to further simplify toxic effluent fractions prior to component

identification by GUMS analysis.

In order to maximize the chromatographic separation capability of the HPLC, the volume of the sample

injected onto an analytical size HPLC column should be

as small as possible (i.e., ~0.5 ml); therefore the toxic

SPE fractions (usually >1 ml) must be concentrated prior

to injection onto the HPLC column. This concentration

step will provide the added benefit of an increase in

concentrations of constituents in the HPLC fractions as

well as rid the SPE fractions of water. The latter issue is

important if GUMS analysis will be performed on the

concentrated SPE fraction prior to injection on the HPLC.

After drying the sorbent, the luer tip of the column

is fitted with a luer-lock needle (to ease collection of small

volumes) and 200 ul of 100% methanol is placed into the

column using a microliter syringe. Nitrogen is then applied

to the column at a rate of -4 ml/set to force the methanol

through the sorbent, which is then collected in a small

glass vial. The first 200 ~1 aliquot of methanol applied to

the column will yield approximately 125 ul of eluate. Two

more 200 ~1 aliquots (applied separately) of 100% methanol are also forced through the column. The final volume

of eluate collected will be approximately 440 uL. Measure

the exact volume collected using a pl syringe or pipet. As

in most chromatographic separations and extractions,

three separate smaller elutions of methanol are more

efficient than one large one.

The 200 mg C,, SPE column is reconditioned

following the directions given above in Section 2.3.4. It is

then used to concentrate the diluted toxic SPE fractions,

using the same sequence used for the blank fractions

(Figure 2-4). The concentrated blank fractions will serve

as the dilution water column blank because it cannot be

obtained for chronic toxicity testing as it can for acute

testing.

The volume of the SPE fraction and the number

of toxic fractions to be combined will determine which size

SPE column will be used for the concentration procedure.

Table 2-3 contains information on column sizes and the

appropriate volume of conditioning and eluting solvents

we have found to be most useful. In the procedures

outlined below we have used a 200 mg SPE column to

When the total volume of fractions is above 40

ml, larger columns should be used; consult Table 2-3 for

column size and elution volume information. The size of

the column used for concentrating should be chosen to

maximize concentration in the eluate. Therefore, choose

the smallest column appropriate for the diluted fraction

volume.

-
If there is a large toxicity loss after the concentration step, it is possible that the percentage of methanol in

the diluted fraction was too high. The concentration procedure should then be repeated with a new set of toxicity

tested SPE fractions diluted to a lower methanol concentration (e.g., loo/,). Both the effluent and blank concentrates are toxicity tested at each step to track toxicity.

Generally we suggest that this toxicity test be at least at

two times higher than the concentration used in the first

SPE fraction test. The tests are conducted exactly as the

SPE fraction tests.

tion of the toxicants from the non-toxic components decreases. We have used a 20 min separation gradient with

the collection of 20-l ml fractions. There are many other

collection options that could be used, such as 10-2 ml

fractions or 4-5 ml fractions using the same separation

gradient. As information on the effluent is gained, HPLC

conditions s_hould be modified from the general conditions

described below, to achieve better separation and higher

concentration factors.

If an original effluent volume of 20,000 ml (using

two 10,000 mg SPE columns) is now represented by a

440 ul concentrate, then the sample is 42,670x more

concentrated than the effluent (accounting for volume removed

for toxicity testing, see Table A-2 example). If 1 ul of

concentrate is diluted to 10 ml in dilution water, the resulting

test concentration will be about 4x whole effluent. However, the 4x test solution should be prepared as one

sample before solutions are split among replicates. For

example, 5 ul is diluted to 50 ml for five replicates with the

C. dubia test described above (Table A-2). Additional test

concentrations (e.g., 2x, Ix, 0.5x) can then be prepared to

determine an IC25 or IC50 of the concentrate, and toxicity

recovery can be calculated by comparing this value to the

toxicity of the effluent. The concentrate toxicity might be

higher than the sum of the individual toxic fractions because some of the toxicant may have been in adjacent

fractions that were concentrated in the first step (Section

2.3.7) but not detectable by the toxicity test of the single

fraction. The concentrate toxicity may also be lower than

expected because of low extraction and elution .efficienties.

We use a flow rate of 1 mVmin on an instrument

equipped with a 5 m C,, reverse phase column (250 mm x 4.6

mm i.d.). The HPLC elution conditions will change depending on which SPE fractions have been concentrated. An

example of HPLC conditions for commonly toxic SPE

fractions is listed in Table 2-f. First, the blank concentrate

is injected (5500 ul) and 20-I ml fractions are collected in

analytically clean glass vials. Depending on the size of

the HPLC injector and column, more than one HPLC

fractionation run may be required to fractionate the entire

blank concentrate. When multiple HPLC fractionations

are conducted, collect and combine all the corresponding

HPLC fractions in the same set of vials. For example, if

two HPLC fractionations were performed for the blank

concentrate, 20-2 ml HPLC blank fractions would be obtained. The same procedure is followed using the effluent

sample concentrate. The vials should be sealed (e.g.,

with foil lined caps) and stored at 4°C if not tested

immediately. As soon as toxicant identification is obtained

by GUMS (Section 2.5) then HPLC conditions (gradient,

fraction size, and number of fractions) can be optimized.

The important concern here is not 100% recovery

of toxicants but enough recovery for GUMS analyses to

be successful and to obtain measurable toxicity in the

HPLC fractions. If recovery is too low, changing or eliminating the column drying time may help. Sometimes

recovery appears to increase with drying time while other

compounds are volatilized from the column during the

drying process. For concentrates analyzed using GUMS,

column drying to remove water is critical to GC column

performance.

2.3. IO HPLC Separation

The same column packing functionality should be

used in the HPLC column as is used in the SPE column,

such as C,,. At later stages, when more is known about

the toxicants, other sorbents might be more appropriate.

2.3.11 HPLC Fraction Toxicity Tests

The HPLC conditions presented in this section

are general. An important consideration of HPLC fractionation is the number of HPLC fractions to collect. Since

chronic toxicity testing is very time consuming, deciding

the appropriate number of fractions to collect is an important step. However, when choosing which collection

scheme to use, keep in mind the trade-off between separation and toxicity testing load. When the fraction volume

is increased (toxicity testing load decreases) the separa2-16

In the HPLC fraction toxicity tests for chronically

toxic effluents, the methanol in the HPLC fractions is one

of the limiting factors of the concentration of the fractions

that can be tested. Each fraction is assumed to be 100%

methanol to calculate the necessary dilution. A 0.6%

methanol concentration or less can be tested with C.

dub&, while a 1% or less methanol concentration can be

tested with fathead minnows.

In a chronic TIE with C. dub&, when all of the

SPE concentrate remaining after toxicity testing from

20,000 ml effluent is injected on the HPLC (one injection),

Table 2-7. Example HPLC Elution Gradient for

SPE Fractions from Chronically Toxic

Effluent Samples

80 or 85% SPE Fractions

Time (min) % Methanol/Water

0 80

10 90

12 100

20 100
each resulting 1 ml HPLC fraction equals 15,575 ml of

effluent (assuming the toxicant elutes in only one fraction,

see Table A-2). If 11 u-I of each HPLC fraction is then

diluted to IO ml, the test concentration is 16x the original

effluent concentration. However, the 16x solution should

be prepared as one sample before aliquots are split to

provide replicates. For instance, in the example used

above, 55 ~1 should be diluted to 50 ml, which is then

equally distributed into five test cups. Additional concentrations are prepared in a similar fashion to estimate the

IC25 or IC50 and to compare toxicity recovery to the

toxicity of the sample. Of course, some loss of toxicant

will occur in each step and the toxicity may be less.

The blank fractions should not be toxic. If they

are, then additional tests with dilutions must be conducted

on both blanks and toxic fractions to find out whether

there is enough additional toxicity in the sample fractions

to warrant analysis.

The toxicity of the HPLC fractions should be

tested at twice (at least) the concentration at which the

SPE fraction concentrates were tested because recovery

of toxicity and analytical measurements indicates that up

to 50% of the initial concentration of toxic compounds

may be lost in this step (Durhan et al., 1990). The

concentration of methanol should not exceed the amount

used in the SPE fraction tests described above (Section

2.3.8).

2.3.12 HPLC Fractions: Concentration and

Subseguen t Toxicity Testing .

The toxic HPLC fractions and their corresponding

blanks must be concentrated in a solvent suitable for GC/

MS or other analytical techniques. Use the procedure

described in Section 2.3.9, Concentration of Fractions

(Figure 2-6). Judgement must be used to decide whether

to concentrate each toxic fraction separately or to combine various toxic fractions prior to concentration. if, for

example, two successive fractions are toxic, there is a

good probability that the same toxicant is present in both.

If one toxic fraction is separated from the other by several

nontoxic fractions, there is a high probability that they

contain different toxicants. There is also a good probability that at least one nontoxic fraction on either side of the

toxic fractions contains some of the toxicant. The advantage of combining fractions is to reduce the workload and

to increase the amount of toxicant in the concentrate. The

disadvantage is that more constituents that are not the

toxicant will be included. The decision has to be based on

trial and error and experience. Blanks corresponding to

the toxic fractions are concentrated the same way.

The HPLC fraction and blank concentrates should

also be checked for toxicity before analysis on the GC/

MS. Generally, we suggest that these toxicity tests be

done at concentrations at least 2x higher than the concentration used in the previous HPLC fraction tests. Hopefully, the amount of concentrate available will be enough

to conduct the toxicity test and perform a GC/MS analysis. Dilutions of the concentrate may be useful to compare

toxicity of this concentrate to each previous toxicity test

result. The HPLC concentrate (of 20,000 ml effluent) is

now 48,495x more concentrated than the effluent (see

Table A-2). If 3 ~1 is diluted to 10 ml the resultant test

concentration will be about 16x the original sample concentration. This 16x solution should be prepared as one

solution before aliquots are removed for the replicates.

For instance, 15 ul is diluted to 50 ml for use in the

example given above, then split into five IO ml test cups.

It is prudent to verify toxicity in the HPLC concentrate

before it is subjected to GC/MS analysis. Whether the

toxicant is detected by the analytical detector is always a

question. Since GC/MS detects only about 20% of organic chemicals (EPA, 1989B), even such a broad spectrum method is no guarantee that the toxicant will be

identified.

2.4 GUMS Analyses

Procedures and methods provided in this section

are based upon our experience in performing GCiMS

analyses on fractions from numerous effluents and are

applicable to both acute and chronic toxicity identification.

In general, these procedures should be used.

A GC/MS system equipped to perform standard

chemical residue analyses is suggested; i.e., a 30 m

capillary column, electron impact ionization, scan range

of 50-500 amu, scan rate of 1 or 2 scans/set, a GC

temperature program of 50 to 300°C at 5OC/min, and a

data system with library searching capability.

Prior to GC/MS analysis, the prepared blank and

toxic fraction concentrates should be tested for toxicity

(Figure 2-6). After verification of the toxicity in the methanol concentrate, inject 1 or 2 ~1 of the concentrate (to

which an internal standard has been added) and collect

the mass spectral data. Note, methanol is not a typical

solvent for GC analysis and the injection of methanol on a

capillary column will shorten the column’s life. Therefore,

routine GClMS QA/QC procedures should be followed

closely to monitor the performance of the column.

The mass spectral data should be collected, the

chromatogram integrated, and all detected peaks library

searched. Reverse search is preferred. Concentration

estimates for all chromatographic peaks can be obtained

by using the response factor of the internal standard.

Usually the internal standard is added to a small aliquot

(1 O-20 ~1) of the concentrate prior to GC/MS analysis. The

selection of internal standard to use is an individual

choice, and many different standards are available. An

external standard method could also be used for deriving

concentration estimates.

The NIST (National Institute of Standards and

Technology, Gaithersburg, MD) mass spectral library has

been used in ERL-D for performing library searches.

Other mass spectral libraries are available, but some of

the larger libraries contain multiple spectra for some of

the compounds in the database. Library searching results

that contain multiple identifications of the same com2-17
pound are not as useful as those obtained using the NIST

library.

Once the library search results are available, the

search report for each peak must be examined to decide

whether the identification by the search is valid and

reasonable. The help of a trained GUMS chemist is

required to do this evaluation. Questions we consider in

our laboratory when performing this process include: A)

are all major ions present in the correct proportions?, B) is

this identification consistent with other information about

the fraction?, C) do forward and reverse searching provide similar fits? and 0) are the library searching fits

greater than 70%? Factor A must be met! Consistency,

factor B, considers circumstances such as “has the identified chemical been found in vastly different fractions,” or

“has the same identification been given to numerous

peaks in the same chromatogram?” Both factors C and 0

are somewhat relative and depend a great deal on the

sample and its matrix. In addition, the toxicants are often

very minor components in the GUMS total ion chromatogram and thus, the quality of the mass spectral data even

after background subtraction can lead to poor results for

factors C and 0.

considerable error may be involved in the concentration

estimate. Secondly, the toxicity data, if available, may be

for a different species than that used in the TIE. Species

differences are usually as large as lOO-fold and often

1 ,OOO-fold. Given these two sources of uncertainty and

the chance that they may reinforce one another, certainly

if the estimated concentration of a chemical accounts for

the toxicity within a factor of 100, the chemical should

remain a suspect. To the extent that data for either

quantitation or toxicity values of the compound are known

to be better, concentration differences of smaller magnitude may be used to eliminate suspects.

Once a list of suspects is available, the measurements for both concentration and toxicity should be refined. This will usually require obtaining pure compound

to make better analytical measurements and to establish

acute or chronic toxicity estimates for the species of

concern. This step requires as much separation as practical before analysis so that the list of suspects is small.

After examination of the library search results, a

list of identified chemicals is assembled and evaluated

using the methods in the following section. For the confirmation analyses we suggest EPA method 625 (EPA,

1982).

2.5 Identifying Suspect Toxicants

If one toxicant is identified, then the goal of the

At this stage, only the concentration of the suspected toxicant in the concentrate is known; until re- . covery through all the fractionation and concentration

steps is complete, suspect compound concentrations in

whole effluent are not known. Since the concentrate is

virtually devoid of suspended solids and much of the

effluent TOC, both of which may dramatically affect toxicity of non-polar organics, the toxicity of non-polar chemicals may be quite different in the fraction tests than in the _ effluent test. Therefore, the toxicity of suspects in the

fraction test should be compared to the suspect’s toxicity

in a relatively pure water, such as reconstituted water.

rest of Phase II is to determine if there are any other

toxicants contributing to effluent toxicity. Two parallel

lines of investigation should be pursued to achieve that

goal. The first is to determine whether or not the concentration of the suspect toxicant is sufficient to cause toxicity

(EPA, 1993A). The second is to estimate the proportion of

the effluent toxicity that is caused by the suspected

toxicants, so that a decision can be made as to whether

other toxicants are present in the effluent.

The first line of investigation should begin by

comparing the estimated concentrations of identified

chemicals in the SPE or HPLC concentrate to their known

toxicity values. Recovery of 100% of each effluent toxicant in the C,, SPE fractions may not be crucial, because

at this stage, only the estimated concentration of compounds in the fraction and the toxicity of the fraction are

compared. Assumptions about the concentration of

toxicant in the whole effluent are not made at this point,

nor is any statement made regarding recovery of whole

effluent toxicity in C,, SPE column fractions. In later stages

of Phase II, inferences regarding the relationship between

the concentration of the suspected toxicant in whole

effluent and the observed toxicity in the SPE fractions are

made. At this step, the compound quantification will have

been performed using an internal or external standard

response and since the compound’s recovery is unknown,

During this same stage, the steps leading to the

final concentrate should be checked for toxicity recovery.

The objective is to place a good estimate on how much of

the whole effluent toxicity is contained in the final concentrate. This is best done by testing the toxicity of the

concentrate at concentrations near those of whole effluent, correcting for volume losses due to toxicity testing

SPE column fractions (which was previously ignored). If

the toxicity of the final concentrate is similar to that of

whole effluent, allowing for losses, and if the concentration of the suspect(s) is sufficient to account for the

concentrate’s toxicity, it is time to begin Phase III (EPA,

1993A). If multiple toxicants occur, the toxic units of each

are compared to the whole effluent toxic units.

2-18

If the concentrations from quantitation and toxicity measurements are close to one another, Phase III

procedure should be started, recognizing that other toxicants may yet be identified. If no suspects are found,

more concentration, more separation, and possibly different or more sophisticated analytical methods must be

used. In some of the effluents we have tested, finding

other candidates has taken months and concentration

factors of >lOO,OOO have been required. Since few laboratories will have all the needed analytical equipment,

instrumentation from other sources should be considered.
Because artifactual toxicity that equals toxicity

due to lost or unidentified toxicants can be created, as

one progresses to Phase III the suspect toxicant should

be identified. One purpose of Phase III is to identify such

errors. Should this error occur, one must start again at the

beginning of Phase II, or even return to Phase I. If several

different effluent samples were evaluated during Phase II,

redoing Phase I on additional samples may be time well

spent since the effluent may have changed in the interim.

In practice there is no sharp boundary between

Phases II and III. In general, as soon as a probable

suspect is identified, confirmation procedures of Phase III

should begin. If a toxicant has been assumed to have

been identified when it has not, the identification of other

suspected toxicants can be hampered.

A final suggestion is to investigate the additivity of

toxicity for several constituents, if all toxicity is not accounted for. Enhancement of toxicity by methanol should

also be checked.

2.5. I Identifying Organophosphate Pesticides

Certain compounds must be metabolically activated by the test organism before they become toxic.

These activation reactions consist of oxidative metabolism by a family of enzymes collectively known as cytochrome P-450. Compounds such as piperonyl butoxide

(PBO) can block the toxicity of metabolically activated

toxicants making it a useful tool in the TIE. PBO is a

synthetic methylenedioxyphenyl compound that effectively

binds to and blocks the catalytic activii of cytochrome P-450.

Thus, when a nontoxic amount of PBO is coadministered with

the effluent or the effluent fractions that exhibited toxicity,

the toxicity of the compound requiring metabolic activation is greatly reduced or completely blocked (Ankley et

al., 1991).

Phosphorothioates are organophosphates known

to require cytochrome P-450 activation before expressing

toxicity and include common insecticides such as diazinon,

malathion, parathion, methyl parathion and fenthion. There

also are a number of organophosphates that are toxic in

the absence of metabolic activation; these include insecticides such as dichlorvos, mevinphos and chlorfenvinphos.

We have found organophosphate insecticides

present in effluents and ambient waters at acute and

chronic toxicity levels (Amato et al., 1992; Norberg-King

et al., 1991). The toxicity of most organophosphates will

be removed from the sample by the C, SPE column, and

they are typically recovered in the methanol eluates (see

EPA, 1991A; EPA, 1992). The addition of PBO to the

effluent before addition of the test organisms was used as

a subsequent test in Phase I (EPA, 1991A; EPA, 1992). In

addition to the C!, SPE column removing the toxicity, a

reduction in toxicrty with the addition of PBO would suggest the presence of metabolically activated compounds

such as organophosphates. PBO has similar utility in

Phase II of the TIE in that either SPE fractions (Sections

22.8 and 2.3.8) or HPLC fractions (Sections 2.2.11 and

2.3.11) can be tested for toxicity both in the presence and

absence of PBO. A reduction in toxicity of the test fraction

would suggest the presence of a metabolically activated

chemical, and together with chemical analyses, can provide powerful evidence along with GUMS data, for specific organophosphates as the toxicant( While PBO

should be useful for both acute and chronic TIE work,

most of our experience has been in the area of acute

toxicity. Thus, guidance presented below is based mainly

on acute tests.

Toxicity values for PBO are presented in Phase I

(EPA, 1991A; EPA, 1992). In acute toxicity tests, concentrations of PBO ranging from 250-500 ug/l have effectively blocked the acute toxicity of relatively large

concentrations of metabolically activated organophosphates to cladocerans (Ankley et al., 1991). In chronic

toxicity tests with C. dubia, PBO concentrations of 50 ug/l

have been effective in blocking toxicity in the SPE fractions.

Detailed information on stock solution preparation is presented in the Phase I documents and is not repeated

here.

When toxicity tests are conducted on SPE fractions or HPLC fractions, aliquots of the PBO solution are

added to the test solutions and mixed well before the test

organisms are added. As for any TIE manipulation, the

successful use of PBO is dependent upon the use of

appropriate controls and blanks. Effluent fractions and

blank fractions with and without the addition of PBO must

be tested simultaneously. A reduction in toxicity of the

effluent fraction occurring with the PBO added, and no

toxicity exhibited in either of the blanks, indicates that the

toxicant requires metabolic activation to exhibit toxicity. If

toxicity associated with the PBO addition is observed in

the blank fraction, either PBO was present at toxic concentrations or the methanol concentration (from fraction

and/or PBO stock addition) in the test was too high. If

toxicity is observed in the effluent fraction with PBO

added, but not in the effluent fraction without the PBO or

in either of the blank fractions, this result is essentially

meaningless. In the latter situation it is possible that the

PBO has interacted in a synergistic fashion with another

compound present in the test effluent that normally would

not be toxic.

2.5.2 Identifying Surfactants

The goal in this section of Phase II is to identify

the toxicants when surfactants are implicated by Phase I

and Phase II results. The Phase I procedures of filtration,

aeration, and C,, SPE all affect surfactant toxicity, and

effluent samples that exhibit several or all of these behaviors may contain toxic concentrations of surfactants (EPA,

1991A).

Surfactants are surface active agents that have a

molecular structure that includes a polar, hydrophilic segment (either ionic or nonionic) and a relatively large nonpolar, hydrophobic, hydrocarbon segment. Surfactants

are used for a variety of household and industrial purposes and therefore are ubiquitous in effluents, particu2-19
larly in untreated wastewater, and potentially could be

present at toxic concentrations in effluents (Ankley and

Burkhard, 1992). Some examples of.surfactants are soaps,

detergents, charged stabilization polymers, and coaguiation polymers used in chemical manufacturing processes.

The molecular structure of surfactants causes them to

congregate at interfaces between water and other phases

such as air, oily liquids and particulate matter. This

congregative characteristic is responsible for the cleansing and dispersive properties of surfactants.

There are many different kinds of surfactants and

they are classified by the nature of their polar segment.

When in aqueous solution, the polar segment of a surfactant molecule can be either nonionic (not charged) or

ionic (charged). The ionic polar segment can be either

negatively charged (anionic), positively charged (cationic),

or both negatively and positively charged (amphoteric).

Based on this, surfactants are classified into the following

major classes: nonionic, anionic, cationic, and amphoteric.

Surfactants physical/chemical properties set them

apart from both strictly polar or non-polar organic compounds and these properties uniquely influence the resuits of Phases I and II procedures for surfactants.

Experiments were conducted with a small sample

of surfactants from nonionic, anionic, and cationic categories with the Phase I procedures of filtration, aeration, and

C,, SPE (Ankley et al., 1990A). In these experiments,

filtration removed the toxicity of most of the surfactants

tested to some degree, and the degree of removal is most

probably dependent on sample matrix, especially solids

concentration. Aeration removed the toxicity of all the

surfactants tested to some degree while the C,, SPE

column removed the toxicity completely for all surfactants

regardless of class. Surfactants behave unpredictably

with regard to eiution from C,, SPE columns. For example, toxicity from surfactants of the nonionic and anionic classes, eluted in all fractions 80% to 100% methanol/

water (Ankley et al., 1990A). Elution in several fractions

rather than eluting in one or two fractions may be caused

by the polar/non-polar nature inherent in surfactants. The

toxicities from the cationic surfactants were either not

recovered in any of the fractions or were recovered to

only a small degree in the 100% methanol fraction.

Important indicators of surfactant toxicity are the

toxicity test results from aeration experiments. If volatility

can be eliminated and toxicity is reduced by aeration, this

is strong evidence that a surfactant might be contributing

to effluent toxicity (EPA, 1991 A). During aeration, surfactants are most probably removed from solution by the

process of sublation. Sublation occurs because surfactant molecules tend to congregate at the interface between the aqueous sample and the aerating nitrogen or

air bubbles and are brought to the surface of the liquid

sample by the bubbles. At the liquid surface the bubbles

break releasing the surfactant, which then adheres to the

aeration vessel walls. A compound that can be removed

by sublation is by definition a surfactant. It might be

possible to recover surfactants from glassware after the

sublation process. The glassware can be rinsed with a

solvent such as methanol, which can then be toxicity

tested and analyzed in the same manner as methanol

SPE fractions (Sections 2.2.8 and 2.3.8).

Overall, most surfactants exhibit some of the

behavior that is common to non-polar organic compounds

such as removal from the effluent by the C,, resin and

recovery in the methanol/water SPE fractions. While surfactants in general can be considered to be non-polar

organics, GC/MS analysis will probably not provide successful sutfactant identification. Most surfactants are not

readily chromatographed because of the polar segment

of the, surfactant molecule. One exception is a class of

surfactants in common use that can be analyzed directly

by GC/MS, the alkylphenol ethoxyiates. Gieger et al.

(1981), provides mass spectral data for the nonylphenol

mono-, di- and tri-ethoxylates, which can be used to help

identify these compounds. Techniques such as

derivatization can make some other specific surfactants

compatible with GC and GC/MS, but it is necessary to

know the specific identity of the surfactant.

It is difficult to positively identify an unknown

surfactant. Although there are many analytical methods

available for accurately quantifying specific surfactants,

these methods are useful only if the identity of the surfactant is known, or at least suspected. It is not reasonable or

practical to analyze a sample using numerous intricate

methods, in the hope that one of these methods will

detect the surfactant in the sample. Unfortunately, there is

no analytical technique available that can readily provide

the identity and quantity of an unknown surfactant. Environmental samples (such as municipal and industrial

effluents) contain numerous substances that can interfere

with available analytical methods. Also, pure surfactants

are actually mixtures of homologous and oligomers with

varying chain lengths and, in the case of many nonionic

surfactants, varying degrees of ethoxylation. The composition and therefore the toxicity of such a mixture might

vary. In the course of a TIE, it might become necessary

not only to identify the surfactant causing toxicity, but also

to learn which particular homologue or oligomer is the

most toxic.

One approach to reducing the complexity of identifying an unknown sutfactant is to determine whether the

unknown surfactant falls into the anionic or nonionic

class. APHA (1989) describes a method for determining

anionic surfactants as methylene blue active substances

(MBAS). MBAS method can successfully measure the

concentration of anionic surfactants of the sulfonate type,

the sulfate ester type, and sulfated nonionics type. Unless

the identity of the anionic surfactant is known, the analytical measurement is calculated and expressed in terms of

the anionic surfactant linear alkylbenzene sulfonate (LAS).

APHA (1989) also describes a method for determining

nonionic surfactants as cobalt thiocyanate active substances (CTAS). This method is applicable to a wide

2-20
range of polyether nonionic surfactants, which includes

the widely used alkyl and alkylphenol ethoxylated alcohols.

With these methods the relative amount of anionic or nonionic surfactant can be estimated, but the

exact nature or molecular composition of the unknown

surfactant will not be determined. These analyses can be

conducted on the SPE fractions, HPLC fractions, fraction

concentrates, and the whole effluent. Determining the

class can be significant progress toward identifying the

unknown surfactant. With the class known, specific analyses for the more common surfactants in that class can be

performed as a subsequent effort. Unless the identity of

the nonionic surfactant is known, the analytical measurement is expressed in terms of an arbitrarily chosen reference nonionic surfactant.

The type of discharge being processed by the

wastewater treatment plant might provide information that

would enable one to target specific surfactants for analysis. For example, industries feeding into the treatment

plant might be discharging certain surfactants or a particular kind of surfactant that is being used in the manufacturing or housekeeping processes. An analytical method

suitable for that particular surfactant could then be used

to determine whether toxic concentrations can be found in

the toxic effluent, fractions, or concentrates.

2.6 Alternate Fractionation Procedures

If toxicity is not recovered in the methanol procedures described above (Sections 2.2 and 2.3) and toxicity is not observed in the post-column effluent, alternative

elution procedures can be used. These procedures are

not as widely used as the methanol/water elutions discussed above but are effective for highly hydrophobic

compounds.

2.6.7 Modified Elution Method

The current Phase II method for fractionating

non-polar organic toxicants in aqueous samples does not

effectively fractionate compounds that are highly hydrophobic. Modifications made to the method have been

successful in overcoming this limitation (SchubauerBerigan and Ankley, 1991; Durhan et al., 1993). Hydrophobic compounds probably are more prevalent in

sediment pore waters than in treated effluents. Tracking

toxicity caused by these kinds of compounds will be more

difficult because of the potential for artifactual toxicity

from the solvents required to elute them. An elution

scheme incorporating water, methanol, and methylene

chloride has been designed that effectively fractionates

compounds over a log K,, range from 2.5 to 6.9. The

higher log Kow compounds, however, elute in the same set

of fractions. Further fractionation by HPLC might be necessary to achieve better resolution of these kinds of

compounds. Substituting other sorbents for the currently

‘used C SPE resin have also produced encouraging

results. ‘Both the C, SPE and XAD-7 (Rhom and Haas,

Philadelphia, PA) sorbents might have utility with particular kinds of toxicants.

The modified elution scheme eliminates the 100%

methanol fraction used in the original method, and replaces it with one 50% methylene chloride/methanol, and

three 100% methylene chloride fractions (v/v). The composition of the resulting eleven 3 ml (when using a

1,000 mg C,* SPE column) fractions is shown in Table 2-

8. The methylene chloride containing fractions are combined, then solvent exchange is conducted as described

below. The modified elution scheme would be used when

the original methanol/water elutions did not effectively

elute toxicity in the SPE fractions. In addition, if the

suspect toxicants were known to be highly hydrophobic,

as in sediment pore water, then the modified elution

scheme would be indicated. Blank toxicity should provide

insight concerning artifactual methylene chloride toxicity;

however, slight reductions in young production might

occur in both the blanks and sample fractions. Development of this alternate procedure for chronic toxicity is

undenrvay for the C. dubia and should be used with

caution at this time. If this procedure is used, it is important to accompany the solvent exchanged methanol blank

with a methanol only blank.

When toxicity testing SPE fractions, it is always a

concern that the matrix of the effluent has been changed

and that chemicals might become bioavailable, whereas

they were not in the original sample. If this were to

happen, the fractions might be more toxic than expected

and chemicals might be added to the suspect toxicant list

erroneously. This kind of mistake should be caught by

obtaining a good toxicity value for the suspect toxicant in

an appropriate matrix. For instance, if the suspect toxicant is highly insoluble in water, then when tested in an

effluent matrix it should have low toxicity because it is

unavailable to the organism. The alternate solvent elution

might enhance this problem because the solvent is more

likely to solubilize the more hydrophobic compounds than

Table 2-8. Composition of 11 Recommended Fractions in Modified

Eiution Scheme

Fraction

Composition of Eluting Solutions (Ok v/v)

Water Methanol Methvlene Chloride

2

3

4

5

6

7

8

9

10

11

75 25 0

50 50 0

25 75 0

20 80 0

15 85 0

10 90 0

5 95 0

0 50 5 0

0 0 100

0 0 100

0 0 100

2-21
methanol. Therefore, additional confirmation steps might

be needed to eliminate the false suspects.

2.6.2 Solvent Exchange

Since methylene chloride is quite toxic to aquatic

organisms, even at very low concentrations (NOEC for C.

dubia is 0.03%), it must be removed from SPE fractions

before the fractions can be tested for toxicity. The exchange of the methylene chloride fraction into methanol is

a relatively easy process because of methylene chloride’s

volatility. The combined fractions to be exchanged (e.g.,

15 ml) are placed in a centrifuge tube with a teflon stir bar

and an additional 15 ml of methanol. The tube is placed in

a 30°C water bath and stirred while a gentle stream of

nitrogen is passed over the solution surface. When the

volume of the solution reaches 3 ml, the sides of the tube

are carefully rinsed with an additional 3 ml of methanol,

and the solution is reduced again to a final 3 ml volume.

Adjust the volume of methanol used in this procedure to

reflect the total volume of combined fractions. The final

volume of methanol may then be tested as suggested

previously in Sections 2.2.8. and 2.3.8. It is important to

obtain and toxicity test a methanol-only blank in addition

to the solvent exchanged methanol blank.

2.6.3 Alterna five SPE Sorben ts and

Techniques

In the SPE method described above, C,, bonded

silica is used as the solid phase for fractionating and

isolating non-polar organic toxicants. C,, bonded silica

was selected because, with proper conditioning, it does

not usually contribute artifactual toxicity to sample or

sample fractions, it often achieves the required degree of

separation and isolation of non-polar organic compounds,

and it is commercially available in inexpensive, easy to

use, disposable columns. There is, however, no restriction on the solid phase that is used in the TIE procedure,

as long as it results in the isolation and separation of nonpolar organic toxicants and at the same time does not

contribute amfactual toxicity. We have evaluated several

sorbents other than C, bonded silica to use for this

purpose (Durhan et al., 6993).

We evaluated two prepurified XAD sorbents, XAD4 and XAD-7 (Rohm and Haas, Philadelphia PA) and a C,

bonded silica sorbent. Of these sorbents, only XAD-4, a

non-polar styrene-divinyl benzene copolymer performed

as well as C,, bonded silica in the fractionation of nonpolar organic compounds. One disadvantage of using an

XAD sotbent such as XAD-4 is that it is not commercially

available in prepacked disposable columns. In addition, it

is important to obtain prepurified XAD-4 sorbent that is

free of toxic artifacts, otherwise extensive, time consuming cleanup procedures are required before the sorbent

can be used in a toxicity based fractionation. We found

that on XAD-7, an acrylic ester copolymer, non-polar

organic compounds were inadequately fractionated because of resolution and co-elution problems. The C,

bonded silica yielded results that were similar but significantly inferior to those obtained with C,, bonded silica.

Traditionally, SPE is carried out with the solid

phase particles packed in a cylindrical column or cartridge. An alternative form of SPE has been developed,

the Empore TM Extraction Disk, in which C,, bonded silica

particles are enmeshed in an inert PTFE matrix which is

then formed into a disk. The manufacturer (3M, St. Paul,

MN) claims good recovery of non-polar organics with flow

rates as high as 100 ml/min, which would make this an

attractive alternative form of SPE. We have evaluated this

technique to a limited degree with acutely toxic effluents

and sediment pore waters and feel it has great potential in

a toxicity based fractionation scheme. Especially attractive is the high flow rate which would allow for large

volumes of sample to be processed quickly. However, a

procedure for eluting non-polar organics from the disk into

several fractions has not yet been developed and could

prove to be a challenge.

2-22
Section 3

Ammonia

3.1 General Overview

Unlike Phase II procedures for non-polar organic compounds or metals, the toxicant identification

methods described in this section are specific for ammonia. The procedures used in this phase of the study

assume that Phase I tests and ammonia measurements

(see below) have implicated the pH sensitive toxicant,

ammonia as causing the acute or chronic toxicity (see

Phase I; EPA, 1991A; EPA, 1992). Other compounds

with toxicities that increase directly with pH may lead to

confounding results or may give results similar to ammonia. For instance, experiments at our laboratory have

shown that C. dubia are more acutely sensitive to cadmium, nickel, and zinc in acute tests at high pH levels

(Section 4). The testing in Phase II should help to

discern the toxicity caused by ammonia from that caused

by other compounds that might also become more toxic

as pH increases. The methods described below can be

used to identify ammonia as the toxicant and these data

could also be used in Phase III confirmation.

Ammonia is relatively unique in its behavior as

pH changes. When ammonia (NH,) dissolves in water,

some of the molecules react to form the ammonium ion

NH,+, and the equilibrium between these two species is

affected by both pH and temperature (EPA, 1985A). The

term “total ammonia” refers to the sum of the un-ionized

(NH,) and the ionized (NH,+) forms and is referred to as

N+. The toxicity of ammonia to some aquatic species

appears to be primarily caused by the un-ionized form.

The equilibrium shifts to increase the un-ionized ammonia concentration with increasing pH and increasing

temperature. In a constant temperature situation, Table

3-1 shows that as pH increases by one unit, there is

nearly a lo-fold increase in the percent of un-ionized

ammonia NH, present in aqueous solutions at pH 6.0-

8.5. The data in Table 3-l are calculated using the

dissociation constants for ammonia (EPA, 1979). There

are two effects to consider for ammonia as the pH

increases; first, the concentration of NH, increases (Table

3-1) and second, the toxicity of NH, decreases (Tables

3-2 and 3-3). One possible explanation for the second

effect is that NH,+ is contributing to the toxicity (EPA,

1985A). Measuring and maintaining the

solution and understanding the effect

toxicity of ammonia are very important.

pH of the test

of pH on the

As discussed in EPA’s ammonia water quality

criteria document (EPA, 1985A), the slope of the LCXOpH curve for acute toxicity is similar for different aquatic

species (i.e., an average slope can be used for many

species). A model was developed to describe the pH

dependence of ammonia toxicity, primarily with data for

fishes and cladocerans (i.e., daphnids, fathead minnows,

rainbow trout, and coho salmon, see EPA, 1985A). This

model has been used with acute toxicity data generated

at a pH of 8 and a temperature of 25°C for both C. dubia

and fathead minnows, to predict the LC50 of NH, at other

pH values (Tables 3-2 and 3-3). It is apparent that the

toxicity of NH, is about seven times less at pH 7.0 than at

pH 6.0, but the amount of NH, is ten times greater at pH

7.0 than at pH 6.0. Similarly, at pH 8.0, NH, is three times

less toxic than at pH 7.0 but ten times more is available at

pH 8.0. Ammonia can be implicated as the cause of

toxicity if the effluent toxicity and the suspect toxicant

exhibit both of these pH effects. Acute toxicity test data

generated at ERL-D indicate that this model is not appropriate for all species. For example, the trend of pHdependence has not been observed in acute tests

conducted with the amphipod, Hyalella azteca, over a

range of pH values in reconstituted waters (EPA, 1991 B)

until the hardness is greater than 160 mg/l. In hard or very

hard waters, H. azfeca is more sensitive to NH, at higher

pHs (P. Monson, personal communication, University of

Wisconsin, Superior, WI). We recommend that the effect

of pH on the toxicity of ammonia be characterized for the

TIE organism, if it has not been done, so that accurate

predictions can be made for the organism.

It has not yet been determined whether the pH

dependence of ammonia toxicity described for acute toxicity is appropriate for chronic toxicity. The chronic toxicity

of ammonia to species typically used in effluent tests, at

temperatures similar to those used in TIES and a variety

of pHs is presented in Table 3-4. If chronic ammonia

toxicity has not been characterized with respect to pH for

the TIE species, it is prudent for the investigator to

generate the ammonia toxicity data for at least three

distinct pH levels.

Generally, three procedures are used to implicate

ammonia in addition to measuring the ammonia in the

effluent. These are 1) the graduated pH test (in place of

3-1
Table3-1. Percent Un-Ionized Ammonia in Aqueous Solutions for Selected Temperatures and pH Values1

Temperature(%)

PH 15 2 0 ___- 2 1 22 2 3 24 2 5 2 6 27

6.0 0.0274 0.0397 0.0427 0.0459 0.0493 0.0530 0.0568 0.0610 0.0654

6.1 0.0345 0.0500 0.0537 0.0578 0.0621 0.0667 0.0716 0.0768 0.0823

6.2 0.0434 0.0629 0.0676 0.0727 0.0781 0.0839 0.0901 0.0966 0.104

6.3 0.0546 0.0792 0.0851 0.0915 0.0983 0.106 0.113 0.122 0.130

6.4 0.0687 0.0865 0.107 0.115 0.124 0.133 0.143 0.153 0.164

6.5 0.0865 0.125 0.135 0.145 0.156 0.167 0.180 0.193 0.207

6.6 0.109 0.158 0.170 0.182 0.196 0.210 0.226 0.242 0.260

6.7 0.137 0.199 0.214 0.230 0.247 0.265 0.284 0.305 0.327

6.8 0.172 0.250 0.269 0.289 0.310 0.333 0.358 0.384 0.411

6.9 0.217 0.314 0.338 0.363 0.390 0.419 0.450 0.482 0.517

7.0 0.273 0.396 0.425 0.457 0.491 0.527 0.566 0.607 0.650

7.1 0.343 0.497 0.535 0.575 0.617 0.663 0.711 0.762 0.817

7.2 0.432 0.625 0.672 0.733 0.776 0.833 0.893 0.958 1.027

7.3 0.543 0.786 0.845 0.908 0.975 1.05 1.12 1.20 1.29

7.4 0.683 0.988 1.06 1.14 1.22 1.31 1.41 1.51 1.62

7.5 0.858 1.24 1.33 1.43 1.54 1.65 1.77 1.89 2.03

7.6 1.08 1.56 1.67 1.80 1.93 2.07 2.21 2.37 , 2.54

7.7 1.35 1.95 2.10 2.25 2.41 2.59 2.77 2.97 3.18

7.8 1.70 2.44 2.62 2.82 3.02 3.24 3.46 3.71 3.97

7.9 2.13 3.06 3.28 3.52 3.77 4.04 4.32 4.62 4.94

8.0 2.66 3.82 4.10 4.39 4.70 5.03 5.38 5.75 6.14

8.1 3.33 4.76 5.10 5.46 5.85 6.25 6.68 7.14 7.61

8.2 4.16 5.92 6.34 6.78 7.25 7.75 8.27 8.82 9.40

8.3 5.18 6.43 7.85 8.39 8.96 9.56 10.2 10.9 11.6

8.4 6.43 9.07 9.69 10.3 11.0 11.7 12.5 13.3 14.1

8.5 7.97 11.16 11.90 12.7 13.5 14.4 15.2 16.2 17.1

8.6 9.83 13.6 14.5 15.5 16.4 17.4 18.5 19.5 20.7

8.7 12.07 16.6 17.6 18.7 19.8 21.0 22.2 23.4 24.7

8.8 14.7 20.0 21.2 22.5 23.7 25.1 26.4 27.8 29.2

8.9 17.9 24.0 25.3 26.7 28.2 29.6 31.1 32.6 34.2

9.0 21.5 28.4 29.9 31.5 33.0 34.6 36.3 37.9 39.6

‘Data from EPA, 1979.

the equitoxic solution test as described in the first Phase II

document; EPA, 1989A); 2) use of the zeolite resin to

remove the ammonia; and 3) air-stripping the ammonia

from the sample at a high pH (i.e., pH 11). For both the

zeolite resin method and the air-stripping method, subsequent toxicity tests and ammonia measurements are performed on whole effluent and the post-treatment samples.

Depending on the presence of other toxicants in

the effluent, additional sample manipulations may be

needed before proceeding with the three basic tests. For

example, if toxic oxidants such as chlorine are also present

in the effluent, sodium thiosulfate must be added to the

sample before conducting the Phase II ammonia tests. To

date we have not seen ammonia and chlorine as cooccurring toxicants in chronic tests, probably because

chlorine degrades rapidly in a test system at 25*C, while

ammonia does not. If the additional toxicant can be

removed by the C,* SPE column, it may be possible to

conduct Phase II tests for ammonia on post-C,, SPE

column effluent sample. However, the problem of artifactual toxicity associated with the post-C,, SPE column

effluent may prevent the use of the graduated pH test

(EPA, 1992) and/or the air-stripping test (see Section 8 of

EPA, 1991A) on post-column samples.

The results of the graduated pH test, the postzeolite column test, and the air-stripping test, all will be

important in identifying ammonia as a toxicant in acutely

or chronically toxic samples. Use of pH changes where

graded responses are observed are particularly useful for

data evaluation in Phase III correlation steps. Some of the

Phase II tests for ammonia are the same steps that are

used for Phase Ill confirmation procedures; therefore,

tests such as spiking the effluent with ammonia and then

performing the graduated pH test or spiking the postzeolite effluent samples and then testing the samples

simultaneously with the Phase II tests will support the

confirmation steps in Phase III.

3.2 Toxicity Testing Concerns

A key issue in interpreting acute or chronic test

results for a pH dependent toxicant such as ammonia is

monitoring pH changes during the test period. Toxicity

differences in Phase I manipulations may be misinterpreted simply because differences in NH, toxicity can

occur with only a slight pH change. To illustrate, the

change in pH from 8.0 to 7.9 lowers the concentration of

NH, 20%, as does a change in pH from 6.1 to 6.0, but a

20% difference is much more important to the toxicity

3-2
Table 3-2. Calculated Un-ionized Ammonia LCSOs (mg/l) Based on 24-h and 48-h Results of a Ceriodaphnia dubia Toxicity

Test Conducted at pH 8.0 and 25%’

PH

Percent

Dissoc.

at 25°C

Unionized

Ammonia

Expected

24-h LC50

Total

Ammonia

24-h LCSO

Un-ionized

Ammonia

Expected

48-h LC50

Total

Ammonia

48-h LC50

6.0 0.0568 0.09

6.1 0.0716 0.12

6.2 0.0901 0.14

6.3 0.1134 0.18

6.4 0.143 0.22

6.5 0.180 0.27

6.6 0.226 0.33

6.7 0.284 0.40

6.8 0.358 0.48

6.9 0.450 0.58

7.0 0.566 0.69

7.1 0.711 0.81

7.2 0.893 0.93

7.3 1.12 1.06

7.4 1.41 1.21

7.5 1.77 1.34

7.6 2.21 1.48

7.7 2.77 1.61

7.8 3.46 1.73

7.9 4.32 1.83

8.02 5.38 1.93

8.1 6.68 2.01 ”

8.2 8.27 2.08

8.3 10.2 2.14

8.4 12.5 2.19

8.5 15.2 2.23

8.6 18.5 2.27

8.7 22.2 2.30

8.8 26.4 2.32

8.9 31.1 2.34

9.0 36.3 2.35

158

168

155

159

154

150

146

141

134

129

122

114

104

95

86

76 0

:8’

50

42

36

30

25

21

18

15

12

10

8.8

7.5

6.5

0.07 123

0.09 126

0.11 122

0.14 123

0.17 119

0.21 117

0.25 111

0.31 109

0.38 106

0.45 100

0.53 94

0.62 87

0.72 81

0.82 73

0.93 66

1.04 59

1.14 52

1.24 45

1.33 38

1.42 33

1.49 28

1.55 23

1.61 20

1.65 16

1.69 14

1.73 11

1.75 9.5

1.77 8.0

1.79 6.8

1.81 5.8

1.82 5.0

l LC5Os for each pH interval were calculated using EPA’s water quality criteria document formula (EPA, 1985A)

shown below.

Formula LC50 =

(LC50[pH = 8.0])(1.25)

I+ 1 o7.4-F

2The 24 h and 48 h LCSOs to C. dubia are 1.93 mg/l and 1.49 mg/l, respectively, at pH 8.0. The formula was used to

generated expected LCSOs for pH values above 8, though the model is not recommended above pH 8, because

generally we have found C. dubia data to track with these predictions.

expressed by the ammonia at pH 8.0 than at pH 6.0. For

this reason frequent pH monitoring (at least daily) must be

performed on tests conducted to determine the trend of

ammonia toxicity. Ideally, continuous monitoring of pH is

desired. The pH should be measured on each test concentration and each replicate. Experience has shown that

the choice of pH meters and probes is critical to produce

reliable results. The pH meter used must read accurately

to two decimal places and should lock-on the stabilized

reading after the rate of change has diminished to a

specified rate. Routine cleaning of the probe and a standardized calibration procedure should be established.

The pH values can also be recorded after an elapsed time

of 60-90 sec. The pH readings should be made using a

constant and reproducible stirring rate. The stirring should

not result in excessive loss (or gain) of CO which will of

course change the pH. The choice of the ph electrode is

important. We have found that the glass-bodied combina3-3

tion electrodes provide the most consistent pH readings.

However, these should not be left in the test solutions for

longer than is needed to obtain constant readings of pH

because ions from the electrode reference solution can

leak into the test solution, potentially causing artifactual

toxicity.

For the Phase II ammonia toxicity tests more

replicates (at least double that used in Phase I) must be

used and tighter QA/QC procedures must be adhered to

than those described in the acute or chronic Phase I

manuals (see Section 1.2). For example, a control and at

least four effluent dilutions using concentrations that more

closely bracket the effect and no effect concentrations

(that were determined in Phase I) are used. While parameters such as time to mortality or onset of symptoms in

the acute and chronic tests are not an integral part of the

tests described below, these observations may be very
Table 3-3. Calculated Un-Ionized Ammonia LCSOs (mg/l) Based on 24-h, 48-h, 72-h, and 96-h Results of a Fathead Minnow (Pimephales

promelas) Toxicity Test Conducted at pH 8:O and 25°C’

Un-ionized

Ammonia

Expected

72-h LC50

Un-ionized

Total

Ammonia at

PH 96-h LC50

Percent

Dissoc.

at 25%

Un-ionized

Ammonia

Expected

24-h LC50

Total

Ammonia

24-h LCSO

Un-ionized

Ammonia

Expected

48-h LC50

Total

Ammonia

48-h LC50

Total

Ammonia

72-h LC50

Ammonia

Expected

96-h LC50

6.0 0.0568 0.075 131 0.064 113 0.049 86 0.036 63

6.1 0 0716 0.093 130 0.080 112 0.061 85 0.045 63

6.2 0.0901 0.12 128 0.10 111 0.076 84 0.056 62

6.3 0.1134 0.14 127 0.12 109 0.094 83 0.069 61

6.4 0.143 0.18 124 0.15 107 0.12 81 0.086 60

6.5 0.180 0.22 121 0.19 104 0.14 80 0.11 58

6.6 0.226 0.27 118 0.23 102 0.18 77 0.13 57

6.7 0.284 0.32 114 0.28 98 0.21 75 0.16 55

6.8 0 358 0.39 109 0.34 94 0.26 72 0.19 53

6.9 0.450 0.47 104 0.40 90 0.31 68 - 0.23 50

7.0 0.566 0.56 98 0.48 85 0.36 64 0.27 47

7.1 0.711 0.65 91 0.56 79 0.43 60 0.31 44

7.2 0.893 0.76 85 0.65 73 0.50 56 0.36 41

7.3 1.12 0.87 77 0.75 67 0.57 51 0.42 37

7.4 1.41 0.78 55 0.67 48 0.51 36 0.38 27

7.5 1.77 1.09 62 0.94 53 0.72 D 40 0.53 30

7.6 2.21 1.20 54 1.03 47 0.79 36 0.58 26

7.7 2.77 1.30 47 1.12 40 0.85 31 0.63 23

7.8 3.46 1.39 40 1.20 35 0.91 26 0.67 19

7.9 4.32 1.48 34 1.27 29 0.97 22 0.71 17

8.0 5.38 1.56 29 1.34 25 1.02 19 0.75 14

‘LCSOs for each pH interval were calculated using EPA’s water quality criteria document formula (EPA, 1985A) shown below. The 24-h, 48-h,

72-h, and 96-h LCSOs to fathead minnows are 1.56 mg/l, 1.34 mg/l, 1.02 mg/l, and 0.75 mg/l, respectively, at pH 8.0.

Formula LC50 =

(LC50[pH = 8.0])(1.25)

1+ 1074-pH

Table 3-4. Un-Ionized Ammonia Toxicity Values for Species Frequently Used in Effluent Testing

Species Method’ ‘PH Temp (“C) LC502 (mg/l) ChW

C. dub;& S, M

C. acanthia5,6 FT, M

Simocephalus ve tulusS 6 FT, M

C. dubid SM

C. dubi& SM

Daphnia magnS SM

P. promela9 FT, M

P. promelasS FT, M

P. promel& FT, M

C. dubia’ 4d-R, M 6.03 25

C. dubia’ 4d-R, M 7.05 25

C. acanthi&6 7d-FT, M 7.0-7.5 24-25

D. magna6 NR 7.6 20.2

C. dubia7 4d-R, M 8.03 25

P. promelasS FT, M 8.0 24.0

P. promelas8 7d-R, M 7.5-7.6 25.0

P. promelas8 7d-R, M 7.5-7.7 25.0

P. promelas4 7d-R, M 8.4 25

Acute Data

6.2

7.1

7.1

7.2

8.2

8.2

7.8

8.0-8.3

8.1

Chronic Data

25 0.12

24 0.77

24 0.61

25 0.78

25 1.73

25 2.08

25.6 1.87

25.2 1.65

26.1 2.55

__

_-

__

_-

__

_-

__

_-

__

0.065

0.28

0.34

0.63

0.62

0.13

0.48

0.45

0.66

‘FT - flow-through; S = static; R = renewal of solutions at 24 or 48 h; M = measured concentration; NR = not reported.

248-h LC50 for invertebrates and 96-h LC50 for fish.

%hV = chronic value which is the geometric mean of the no observed effect concentration (NOEC) and the lowest observed effect concentration

(LOEC) or an IC25.

4Data generated at ERL-D.

5C. acanthla is equivalent to C. dubia.

“Data from EPA, 1985A.

‘Data from 4-d C. dubia tests conducted at ERL-D; the effect level is an tC25 (mg/l)

aData from Beigger, 1990.

3-4
useful in describing the identification steps used in confirming ammonia as the cause of toxicity.

3.3 Measuring Ammonia Concentration

We have found that the ammonia-selective electrode method has been satisfactory for measuring the

ammonia concentrations in most samples, (EPA, 1983;

APHA, 1992). Other methods for measuring ammonia are

available (such as distillation, nesslerization, and titration)

and can be used successfully for determining ammonia

concentrations in effluents (EPA, 1983; APHA, 1992).

The level of detection for total ammonia generally need

not be below 051.0 mg/l, since concentrations of 11.0

mg/l of total ammonia have not been found to be toxic to

fathead minnows and C. dubia. If ammonia measurements are below 1 mg/l and the sample is toxic, it is likely

that the toxicant is not ammonia and other identification

procedures should be pursued.

The most reliable ammonia measurements are

obtained on fresh samples. However, samples can be

preserved by adding concentrated sulfuric acid and storing the samples at 4°C. The pH of the preserved samples

should be in the range of 1.5 to 2.0 (EPA, 1983; APHA,

1992). In recent experiments, we have used samples that

were stored without acidification at 10°C or refrigerated at

4°C for short periods with good success.

During several effluent tests, the amount of ammonia in the test solutions (see test details below) has

decreased over the duration of the test. When levels are

in the range of O-30 mg/l, it is prudent to measure the

initial concentration of ammonia in the test solution and

again after animals were exposed.

3.4 Graduated pH Test

The purpose of the Phase II graduated pH test is

to provide more definitive toxicity test data to implicate

ammonia as the toxicant in Phase II. In turn, this data may

be used in Phase Ill to confirm the role of ammonia in the

toxicity of the effluent. More stringent pH control and pH

monitoring will be needed to interpret test results and

more precise toxicity estimates (i.e., more replicates,

more dilutions, larger number of organisms; see Section

1.2; EPA, 1991A; EPA, 1992) are needed in Phase II than

in Phase I. When it is important to predict the impact of

the toxicant in the receiving water, the pH.of the dilution

water should be maintained at receiving water pH. The

test procedures discussed below provide good pH control

for the graduated pH test. Greater detail is provided for

some of the procedures in Phase I (EPA, 1991A: EPA,

1992).

The test chamber size, number of dilutions, species

to be tested, type of test (acute or chronic), and the

degree of toxicity of the effluent will dictate the volume of

effluent needed for the graduated pH test. As a general

guide for acute toxicity tests, 300 ml of effluent should

suffice for any of three pH adjustment tests described

below. The volume for chronic tests will vary based on the

type of chronic test performed, the species used, the

number of concentrations tested, and number of solution

renewals in addition to the items discussed above (Section 1.2 and EPA, 1992).

The procedure for conducting the graduated pH

test is to evaluate and determine the toxicity of the

effluent at three different pHs (e.g., 6.0, 7.0, and 8.0). The

pH should be measured in all of the chambers. If the pH

drifts 0.2 pH units or more, the results may not be usable

and better pH control must be achieved. However, if pH

fluctuates more than 0.2 pH units and toxicity is present

only at one pH, the toxicity results may still be useful. The

pH levels selected must be within the physiological tolerance of the test species used (which generally is a pH

range of 6 to 9). We recommend use of two methods of

pH control and comparison of these results to determine

that the pH adjustment itself did not introduce an artifact

of toxicity. This type of testing may be critical to explaining

effects in Phase III (EPA, 1993A).

Regardless of the pH control method chosen, the

selection of the appropriate blank is difficult. The change

in pH of the dilution water or surface water is not comparable to that of the effluent because the composition of the

solutions are different. For some effluents, the addition of

either acid or base can be used to adjust and hold the pH

within 0.1 pH unit. If this is possible, this technique can be

used to compare the results with either the CO,-pH

controlled test or the buffer-pH controlled test. Test results should be similar and these comparisons can be

used as a basis for identifying ammonia as a toxicant.

3.4. I pH Control: Acid/Base Adjustments

The first method of pH adjustment is the acid/

base adjustment described in Phase I (EPA, 1991A; EPA,

1992). For this manipulation, the adjustment of pH is

relatively easy and quick, and the loss of volatile compounds is minimized. However, the drawbacks are that:

toxicity enhancement from the additives may occur (especially in a chronic TIE), the addition of strong acid or base

disrupts the carbonate system equilibrium, the effects of

the pH change in the blanks may not selve as a toxicity

control for the effluent, the pH stabilization time is lengthy,

and pH tends to drift in longer term tests. In the pH range

6 to 9, the amount of high quality acid or base added is

usually negligible, and the likelihood of toxicity caused by

increased salinity levels is low.

The pH of each concentration and replicate must

be frequently monitored because a constant pH during

the toxicity test must be maintained. Larger test volumes

may be useful to prevent rapid pH fluctuations. The

amount of acid and/or base added should be recorded for

each pH adjustment to track the additive amount in the

effluent samples and the blanks. If toxicity increases

dramatically, the concentrations of salts should be calculated to be sure the salinity has not increased above the

tolerance level for the TIE species.

3-5
3.4.2 pH Control: CO, Adjustments

The second method uses CO, to adjust and

control test solution pH. The pH is adjusted by varying

and controlling the CO, concentration of the gas phase

over the water or effluent sample in closed headspace

test chambers. It is necessary to maintain a constant pH

throughout the test period. The pH of most natural waters

and some effluents is controlled by the bicarbonate buffering system and surface waters normally contain <IO

mg/l of CO,. Therefore, the amount of CO, to add depends on the desired pH and the chemistry of each test

solution. The CO, adjustment has the advantages that the

pH is controlled without placing additives directly into the

effluent test solutions, the pH change is easy to make,

and the pH is generally stable for at least 24 h if the gastight container is not opened. Frequent pH measurements

are still possible because the headspace can be reflushed

with a predetermined concentration of CO,/air. The disadvantages are that toxicity can occur from the CO,, the

concentration of COJair varies for each dilution and effluent (which requires sample specific experimentation) and

the manipulations for chronic tests can be time-consuming relative to the acid/base adjustment method. We have

not observed any increased toxicity from the addition of

CO, unless the concentration in the chamber is over 10%.

Adjustments of the pH to 6.0 or 7.0 can be made

by using CO, with or without first adding HCI to the test

concentrations. The CO, is purchased in pure form through

local commercial gas suppliers, and if particular concentrations of CO,/air are frequently used, a cylinder of gas of

the desired concentration may prove to be resourceefficient. 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). Some

preliminary work is needed to determine the concentration of CO, to add to achieve the desired pH. When

dilutions of an effluent have the same hardness and initial

pH as the effluent, about the same amount of CO will

usually be needed for each dilution. Sometimes, hfgher

concentrations of CO, are needed for the higher test

concentrations. Use of a dilution water of similar hardness

as the effluent may make the CO, volume adjustments

easier. A different dilution water may only be used in

these tests if the toxicity has not been shown to be

dependent on water hardness at any pH.

In our laboratory, we have found that glass canning jars with rubber seals and metal balers work well as

a gas-tight testing chamber. The testing chamber should

be large enough to hold the desired number of test cups,

with sufficient headspace to ensure proper DO levels. For

example, a 2-quart glass canning jar lying on its side will

easily hold 6-l oz cups. We simultaneously test C. dubia

and fathead minnows in the same chamber using the test

solution volumes described in Section 1.2. Since many

plastics are permeable to CO,, glass containers are recommended. When CO,/air is flushed into the headspace

of the test chamber, the pH of the test solutions will

usually reach equilibrium in about 1 h and a reliable pH

can be achieved. Generally, as the alkalinity increases,

the concentration of CO, that is needed to maintain the

pH also increases. After 1 h, check the pH of the solutions

and flush the chambers again. Check the pH again after

2-3 h and from these data determine the concentration of

CO, to add for initial pH adjustment for the actual toxicity

test and the amount needed for reflushing after the chamber is opened for feeding or pH measurements. In most

instances, the amount of CO, produced by the test organisms will not cause further pH shifts. When testing with

fish, which usually increase in size during the test, a pH

fluctuation may occur that would require flushing with

different (e.g., slightly lower) concentrations of CO,.

When the concentration of CO, to inject for the

target pH values has been determined, prepare test solutions, add test organisms (and food if necessary) and

inject the appropriate concentration of CO, in air using a

l-liter gas tight syringe, and quickly close the test chamber. The chambers should be flushed with the CO air

mixture several times to ensure the displacement of? air

currently in the chambers. Place the chamber out of direct

laboratory light, as temperatures tend to rise out of the

desirable test range in the closed chambers.

For effluents that have initial pH values from 7.8

to 8.5, O-l 0% CO, concentration in the chamber has been

used to lower the pH to 6.0. Experiments in hard reconstituted water have shown that up to 8% CO, can be

tolerated by C. dubia and fathead minnows in acute tests,

but 8% has been toxic to C. dubia and fathead minnows in

the 7-d tests. About 2-3.5% usually will lower the pH of

most effluents to 657.0. If more than 10% CO, for acute

tests or 5% CO, for chronic tests is needed to lower the

pH of the test solutions, before adding test animals adjust

the pH with high quality acid (EPA, 1991 A; EPA, 1992)

and then flush the headspace with COdair. The necessary concentration of CO, to use must first be determined

experimentally with effluent test solutions adjusted to the

appropriate pH with acid solutions. Sometimes >5% CO

cannot be used for the dilution water pH adjustment tes?

without the CO, causing toxicity.

The use of a single enclosed test chamber for

controlling the pH at all test concentrations may allow the

transfer of volatile compounds among treatments. We

have experienced volatilization of ammonia in tests and

therefore, individual test chambers for each effluent concentration are preferable. Methods that use continuous

flow of a CO,/air mixture, such as tissue cell incubators,

may be preferable and give better pH control provided

that volatilization or cross contamination is not a problem.

At this time we have not attempted to use a continuous

flow of COdair mixture and therefore cannot recommend

a system to use.

3-6
Maintaining pH above the air equilibrium pH (generally above pH 8.3) is difficult without buffers (Section

3.4.3). The pH control in this high range is much more

difficult because the concentration of CO, must be very

low and microbial respiration can increase the CO, levels

in the test chamber. Use of CO,-free air in the headspace

may work or flushing a mixture of CO,-free air and normal

air through the headspace or test solution may be successful. Because such small CO concentrations are

needed and because CO, evolution by microorganisms or

test organisms can significantly alter the CO, concentration, frequently flushing (two to four times a day) of the

headspace in static tests will probably be required to

adequately control pH. For the chronic tests, we have not

attempted to use the CO,-free air bubbled through the

test solution, because more CO, evolution tends to occur

during the chronic tests and the need for reflushing makes

the test labor intensive.

For the CO,pH controlled tests, the pH should be

measured at least every 24 h for both acute and chronic

tests and ideally, continuously during pH controlled tests.

At each reading, flush the headspace with the COdair

mixture. A small amount of experimentation will confirm

whether the concentration of CO, previously determined

is adequate, or whether the amount required for flushing

will be less than that used for the initial pH adjustment.

For chronic tests, daily renewal solutions should

be prepared, pH adjusted with HCI if necessary, dispensed into test cups, and placed into a second glass jar

chamber and flushed with appropriate concentration of

CO,. These should be left to equilibrate at least 1-2 h.

Measure the pH quickly and transfer the animals to new

test cups and place them into the glass jar. Flush the

headspace again with the appropriate COJair mixture.

Table 3-5. Percent Un-Ionized Ammonia in Aqueous Solutions at

25°C and Various TDS Levels’

TSD pH

(m@) 6.0 7.0 8.0 9.0

0 0.0568 0.566 5.38 36.2

250 0.0521 0.519 4.96 34.3

500 0.0505 0.503 4.81 33.6

750 0.0494 0.492 4.71 33.1

1000 0.0485 0.483 4.63 32.7

1500 0.0471 0.469 4.50 32.0

2000 0.0460 0.458 4.40 31.5

3000 0.0443 0.441 4.24 30.7

‘Data from Skarheim (1973).

For the 7-d tests with fathead minnows, the chambers

must be opened once more each day to accommodate

the feeding schedule. The experimenter can take advantage of this by making a pH reading prior to placing food

into the test cups. CO,Jair must again be flushed into the

chamber. It is important to note that in the fathead minnow test, the pH most likely will be lower after 24 h than in

the C. dubia test because of the food added and the

respiration of the fish which is considerably greater than

that of C. dubia.

Measurements of pH must be made rapidly to

minimize the CO, exchange between the sample and the

atmosphere. Avoid vigorously stirring unsealed samples

because at lower pH values, the CO, lost during the

measurement can cause a substantial pH rise. If possible,

measure the DO at the same time because ammonia may

have different toxicities when DO is decreased (EPA,

1985A). Keep in mind that if the test animals have been

dead for awhile, the pH and/or DO of the test water most

likely will have changed.

The controls in the CO, chamber 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 is close to the pH of the

adjusted test solutions (at their respective LCSOs, IC25s

or IC5Os), the toxicity expressed in the two tests should

be similar. Significantly greater toxicity in the pH-adjusted

test may suggest interference from other factors such as

the ionic strength related toxicity if the pH was adjusted

with either HCI or NaOH, or possibly CO, toxicity. Dilution

water blanks at the various pH levels may or may not be

appropriate since the effluent matrix may differ from that

of the dilution water. The dilution water blank will be

useful in checking the acids and bases that are added for

artifactual toxicity. Monitoring the acid and base additions

may be useful in determining if artifactual toxicity resulted

from the increase in salt content. Monitoring conductivity

of the effluent solutions after the addition of the acids and

bases may also be helpful in determining artifactual toxicity. The ionic strength of hardwaters or saline waters

results in a decreased level of un-ionized ammonia (Table

3-5). For values of TDS from O-500 mg/l, the dissociation

constants are expected to be more accurate than values

above 500 mg/l that were based on somewhat tenuous

assumptions (Skarheim, 1973; see Table 3-5).

3.4.3 pH Control: Buffer pH Aa’justments

The third method of pH control uses the addition

of standard buffers to the effluent and dilution water to

adjust the pH. This method has the advantage in that pH

is stable with the buffer addition, the pH change during a

test is slow, frequent pH measurements are possible

because test vessels are not in air-tight chambers, and

the test method set-up is rapid. The disadvantages are

that toxicity enhancement or interference from buffers

may occur, not all buffers can be used without additional

3-7
acid/base adjustments, and the pH stabilization time may

be lengthy.

Hydrogen ion buffers are used to maintain the pH

level in the graduated pH test (EPA, 1991A; EPA, 1992).

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 three buffers were

chosen based on the work done by Ferguson et al.

(1980). These buffers are: 2-(N-morpholino) ethane-sulfonic acid (Mes) (pK, = 6.15), 3-(N-morpholino) propanesulfonic acid (Mops) (pK, = 7.15) and piperazine-N,N’-bis

(2-hydroxypropane) sulfonic acid (Popso) (pKB = 7.8). We

have also used two additional buffers: N-(2-hydroxyethyl)

piperazine-N’-2-hydroxypropanesulfonic acid (Heppso)

(pKa = 7.8) and N-tris-(hydroxymethyl) methyl-3-

amrnopropanesulfonic acid (Taps) (pK, = 8.4). The Taps

buffer is more frequently used than the Heppso buffer.

We have experienced problems of having to add an

excessive amount of base to obtain the desired pH with

the Popso buffer. The Taps buffer effectively maintains

the pH above 7.8. Keep in mind that pH is best maintained at or near the pKa of the buffer.

The acute toxicity of these buffers is low to both

C. dubia and fathead minnows (EPA, 1991A) and 4 mM

concentration or less of all five buffers has not caused

chronic toxicity to C. dubia or the fathead minnow. The

buffers are added at sublethal (e.g., NOEC) levels to

maintain the pH of test solutions. While these buffers

serve to prevent the pH from drifting during the test, pH

adjustment to the desired level is required in the preparation of the solution. A portion of the buffer compound is

weighed out and added to the aliquots of whole effluent

and dilution water, and both are then pH adjusted with

acid or base solutions to the desired pH values. Serial

dilutions are made, replicates prepared, and test organisms are added. Care should be taken to ensure equilibrium of buff erect solutions, which may take at least l-2 h.

Dilutions should also be left to equilibrate and minor pH

adjustments should be made. In certain situations, it may

be desirable to prepare the solutions the day before tests

begin. At present, we have found we can use batch

solutions prepared ahead of time for solution renewals.

Our experience also indicates that the amount of any

buffer needed to hold any pH is effluent specific. Experimentation with effluents will be required 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

test reading at all dilutions.

Use of the buffers is still being developed and the

effects caused by interferences from the buffers themselves have not been fully studied. It is possible that the

buffers may reduce the toxicity of some toxicants, but this

has not generally been seen.

3.5 Zeolite Resin Method

Zeolite is composed of naturally occurring or

synthetically created crystalline, hydrated alkali-aluminum

silicates. The general formula is M”+O*Al,O,*ySiO,*zH,O;

M = group IA or IIA element, n = +2 for group IA, +l for

group IIA, y>2, and z = the number of water molecules

contained in the interconnected voids or channels within

the zeolite (Windholz, et al., 1983). When zeolite is placed

in aqueous solutions, the positively charged group IA or

IIA elements (Mn+) of the zeolite are mobile and can

undergo exchange with other cations in the water. As

such, zeolite has frequently been employed as ion exchange resins to remove the ammonium ion (NH,+) from

aqueous solutions in TIE work (Ankley et al., 19906;

Burkhard and Jensen, 1993). Because of its ability to

exchange other cations such as heavy metals, and its use

as molecular sieves, filter adsorbents and catalysts, zeolite was not suggested for use in Phase I, except as a

subsequent test (EPA, 1991 A). Zeolite can be effective in

Phase II, if Phase I results implicate ammonia as the

toxicant and establish that other types of toxicants (such

as non-polar organics and metals) play no role in the

effluent toxicity.

For the acute TIE procedure, zeolite particles

should be screened to be in the range of 32 to 95 mm, to

ensure efficient ion exchange while preventing channeling or excessive resistance to flow. Extremely large or

small particles can be removed by screening the zeolite

with sieves or mesh screens. The zeolite column can be

prepared by taking 30 g of aquarium zeolite (Argent Chemical Laboratories, Redmond, WA) and adding it to 60 ml of

high-purity water. The zeolite slurry is poured into a

chromatography column (11 mm i.d. x 15 cm) and three

bed volumes of dilution water are passed through the

column. The last 10 ml of dilution water is collected for

use as a zeolite blank and should not be toxic. Next, 200

ml of 100% effluent is passed through the column at a

rate of 2 ml/min. The post-column effluent that is collected

will be toxicity tested and its ammonia concentration

measured. Temperature and pH should be recorded at

test initiation to provide the means to calculate both total

and un-ionized ammonia in the sample.

For chronic toxicity tests larger amounts of zeolite

should be used. This can be scaled up proportionally from

the amounts used in the acute zeolite work. The amounts

of solution needed for testing and ammonia measurements will dictate the amount of sample to prepare.

Typically a slurry of 60 g of zeolite and 120 ml of high

purity water is sufficient for levels of ammonia in the range

of 5-50 mg/l and for processing 2,000 ml of effluent. The

post-zeolite effluent is collected in aliquots, then each is

toxicity tested. In this manner, break-through of ammonia

can be measured and toxicity of the various samples with

different ammonia levels can be estimated.

3-8
Toxicity tests and ammonia measurements are

conducted on the effluent and post-zeolite column effluent. Removal of toxicity by the zeolite column and removal of the ammonia concentration will add to the

evidence implicating ammonia as the toxicant. An aliquot

of the effluent sample (not having passed through the

zeolite column) is used for ammonia analysis and the

baseline toxicity test. These data will be compared with

the same data for the post-zeolite column effluent to

determine if the post-column reduction in effluent toxicity

is consistent with ammonia removal by the zeolite. The

control for test organism survival, dilution water quality

and other test conditions will be provided through toxicity

tests on dilution water. Dilution water (at the same hardness as the effluent) should be passed through the zeolite

column, and will act as a blank for toxic artifacts leached

from the zeolite. Increased toxicity in the post-zeolite

effluent, relative to the whole effluent, indicates the presence of toxic artifacts. Since many cations will be exchanged, adding solids in the acute tests, such as the

YCT food (yeast-CerophyKtrout food) fed to C. dubia,

might improve control survival. Additional clean-up techniques for the zeolite (such as Soxhlet extraction) or

alternate uncontaminated sources of zeolite might be

needed. Column packing, effluent pH, ammonia levels,

and flow rate through the column can all affect the efficiency of the cation exchange process. Lowering effluent

pH prior to zeolite treatment and/or lowering flow rate

through the column might also result in greater removal of

ammonia. Occluded gas between zeolite particles might

also impair the column’s capacity to remove ammonia. If

this appears to be a problem, the zeolite slurry should be

degassed by using a vacuum prior to pouring it into the

column.

Zeolite columns can be regenerated, but fresh

zeolite should be used to pack columns the first time. If

the graduated pH test and the zeolite test results are

consistent with ammonia toxicity, Phase III confirmation

procedures should be started.

Once ammonia is identified and confirmation is

initiated, the post-zeolite samples can be .spiked with

ammonia at the same concentrations as are present in

the effluent. These tests are an integral part of the Phase

III confirmation process (EPA, 1993A).

3.6 Air-Stripping of Ammonia

This method of ammonia removal takes advantage of the fact that the relatively volatile un-ionized

ammonia (NH,) predominates in a solution with a pH

above 9.3. For this reason, one might expect that ammo-

,

nia would be removed during the Phase I pH 11 adjustment/aeration test (acute testing) or the pH 10 adjustment

and aeration test (chronic testing). Based on our experience ammonia is not removed by this method, most likely

because the Phase I aeration manipulation is done in a

graduated cylinder, which has a low surface-to-volume

ratio. By stirring the sample for a longer period of time

(>I h) at a high pH (pH 9.0 or higher) in a container that allows

a large surface area to volume ratio, most of the ammonia

can be removed from aqueous samples.

A measured amount of effluent for subsequent

analysis and testing is pH adjusted to IO or 11 and placed

into a large shallow glass container (e.g., 1000 ml crystallizing dish). The solutions are then agitated (stirred) continuously. The length of time the sample must be stirred is

dependent on the concentration of total ammonia in the

sample. We have found that for most samples of 10400

mg/l of total ammonia, 1-6 h is adequate to remove most

of the ammonia. After air-stripping is completed, the

volume of effluent should be measured and any appreciable loss replaced with high purity water or toxicity might

be caused by the concentration of other components in

the effluent. The ammonia concentration should be measured immediately after air-stripping and after volume

adjustment is complete to ensure ammonia levels are

reduced before toxicity tests are initiated. Toxicrty tests on

the air-stripped solution can then be conducted for both

acute and chronic TIE work. Dilution water blanks at the

various pHs may or may not be appropriate since the

effluent matrix will probably differ from that of the dilution

water. Monitoring the acid and base additions may be

useful to determine if artifactual toxicity resulted from the

increase in salt content and subsequent evaporation that

occurred during the air-stripping process. Monitoring conductivity of the effluent solutions after the addition of the

acids and bases may also be helpful in determining

artifactual toxicity. The dilution water blank should be

treated in the same manner as the effluent although it

may not serve as a true toxicity control for the effluent.

If the ammonia is decreased and the toxicity is

reduced or absent after air-stripping, ammonia is strongly

implicated as a contributing factor to the toxicity of the

effluent. The results of this test should be compared with

the aeration test results of Phase I, the baseline effluent

test and the other graduated pH tests. Other compounds

could precipitate as a result of the pH adjustment and

during the air-stripping procedure. Precipitates may form

during the air-stripping process and not dissolve after the

volume is readjusted, leaving these compounds unavailable.

3-9
Section 4

Metals

4.1 General Overview

This section contains procedures that can be

used to identify suspect metal toxicants. The initial evidence used to implicate metallic toxicants is obtained

from the Phase I characterization tests, with the results

of the EDTA (ethylenediamine tetraacetic acid) addition

test providing the best indication of the presence of a

metal toxicant. When certain cationic metal toxicants are

present, a reduction in sample toxicity with the addition

of EDTA should be obsewed. Other Phase I manipulations that remove or reduce sample toxicity and suggest

the presence of a cationic metal include the sodium

thiosulfate addition test, the use of a C,, SPE column,

and filtering the sample when combined with minor pH

adjustments. One additional indication of metal toxicity

may be when the organisms’ response in the toxicity test

is atypical of the expected dose response relationship

(i.e., partial mortalities in several test concentrations,

Schubauer-Berigan et al., 1993B).

Subsequent Phase I tests such as using ion

exchange resins might also lead one to the conclusion

that a metal is the toxicant. Toxicity removal or reduction

after a sample. is treated with an anion exchange resin

might implicate toxicants that exist as anionic oxides in

water, such as arsenic, chromium, and/or selenium.

These anionic oxides will not be specifically removed or

rendered biologically unavailable by the routine Phase I

tests. Therefore, when the Phase I tests do not seem to

show any toxicity reduction, toxicants such as these

might be suspected and subsequent tests as discussed

above could be useful (see Phase I, EPA, 1991A; EPA,

1992). These situations should be approached on an

individual basis since other classes of toxicants might

demonstrate the same behavior in Phase I (e.g., total

dissolved solids (TDS)).

Further discussion and interpretation of the Phase

I results which would lead to the conclusion that a

cationic metal toxicant was present in a sample are

provided in the Phase : TIE documents (EPA, 1991A;

EPA, 1992).

Other information, such as process details from

the discharger and infcrmation from past TREs and/or

TIES, might also help to implicate cationic metals as the

toxicants. However, this type of information should be

interpreted and used with caution as it might bias the TIE

efforts.

If the EDTA addition test in Phase I showed that

toxicity was removed or reduced one should proceed to

the metal analysis section (Section 4.2). This section

provides guidance and recommendations for analyzing

samples for metals so that a list of suspect metal toxicants can be obtained. This section also discusses clean

metal techniques, detection limits, a prioritization process

for analyzing for specific metals, dissolved vs biologically

available metals, and provides the rationale for assembling the list of suspect metal toxicants. Prioritizing metals

to analyze from Phase I results is strongly recommended

in order to save money and time in the TIE process.

If other Phase I tests implicate a metal but EDTA

does not, it may be helpful to acquire additional test

information through the use of EDTA addition tests, sodium thiosulfate addition tests, graduated pH tests, and

ion-exchange resins. This additional toxicity testing (Section 4.3) may be useful in certain situations before analyzing for metals, even when EDTA additions reduced toxicity.

These situations include: when the addition tests of EDTA

and sodium thiosulfate in Phase I were performed using a

single sample concentration (i.e., no dilutions), when the

time it takes to obtain results of metal analyses is lengthy,

or when Phase I results indicate another type of toxicant

(non-metal) is present. The data obtained from the additional testing can then be included in the prioritization

process for metals analysis. Professional judgement is

required to decide when you have sufficient and appropriate toxicity testing data to proceed to metals analysis.

After processing one sample, a list of suspects

may be generated. As future samples are evaluated, the

correlation between toxicity of a sample and the

concentration(s) of metal(s) over time may also be used

to narrow the list of suspect toxicant( In Phase III, the

suspect metal toxicant is implicated based upon the correlation of effluent toxicity and metal concentrations, reference suspect metal toxicity data, the use of additives

that chelate metal toxicants, and changes in toxicity observed during manipulations of water quality characteristics.

4-l
The procedures in this chapter are generally applicable for both acute and chronic toxicity. The main

differences between the acute and chronic procedures

are the concentrations of additives used in the EDTA and

sodium thiosulfate addition tests, lower analytical detection limits, and generating non-toxic blanks for the ion

exchange resins for chronic toxicity testing. The use of

species other than C. dubia or fathead minnows will

require consideration of appropriate test volumes and

additive concentrations.

4.2 Analysis of Metals

4.2.1 Prioritizing Metals for Analysis

Many cationic metals can be analyzed in a specific sample, but to simplify the amount of analytical effort

needed for metals analysis, we suggest a prioritizing

process be performed before analyzing any samples. The

prioritization process is more valuable when the metal

analyses are performed by AA instrumentation since each

metal requires an individual analysis. Conversely, with

ICP (inductively coupled plasma) instrumentation, numerous metals can be analyzed at once, and the prioritization

process is less valuable in this instance. With both ICP

and AA methods, a list of metals and required levels of

detection will be needed before the samples are analyzed.

This prioritization is based primarily upon acute

toxicity data with C. dubia. Its applicability to chronic

toxicity and other species is expected to be similar but

has not yet been determined. The toxicity test results from

the EDTA additions, sodium thiosulfate additions, and

graduated pH tests performed in Phase I form the basis

for prioritization. When available, Phase II results from

using the procedures in Section 4.3, should be included in

this evaluation. Because we do not have a complete

understanding of the effects of these procedures for each

metal, the following should be taken as a starting point for

metals analysis.

Information regarding historical discharge monitoring data, past or current TRE and/or TIE information, or

process information may be useful in prioritizing metals

for analysis. For example, if a discharger uses zinc in their

manufacturing process and EDTA removed the toxicity, it

would be logical to analyze for zinc first. If zinc was

present at nontoxic concentrations or at concentrations

too low to cause the observed toxicity, analysis for additional metals would be performed. If zinc was present at

concentrations high enough to cause the observed toxicity, Phase III procedures (EPA, 1993A) should then be

started to confirm zinc as the identified suspect toxicant.

When EDTA additions reduce or remove the

toxicity of the sample, initially copper, lead, cadmium,

nickel, and zinc should be measured. When sodium thiosulfate additions reduce or remove the toxicity of the

sample, copper, cadmium, and silver should be measured.

Phase I results would not normally lead to the

conclusion that an anionic toxicant was present (i.e.,

cationic metals that exist in aqueous samples as anionic

oxides). If additional Phase I tests had been performed

which characterized anionic toxicants or other specific

discharger information was available, measurements of

arsenic, chromium, and selenium should be made.

As stated above, these metals should be a starting point for metals analysis. Further interpretation of the

Phase I results could be done by including the results of

the graduated pH test and by jointly examining the results

of the EDTA addition, thiosulfate addition, and graduated

pH tests.

When multiple toxicants of different classes are

present, Phase I data are often difficult to interpret. One

should try to identify and confirm as soon as possible the

role of one toxicant when multiple toxicants are present.

By defining the role of one toxicant, efforts can be better

focused on the remaining unidentified toxicants.

4.2.2 Metal Analysis Methods

There are three types of chemical instrumentation available for the analysis of cationic elements; these

are AA, inductively-coupled plasma-atomic emission spectroscopy (ICP-AES), and inductively-coupled plasma-mass

spectrometry (ICP-MS).

EPA methods using ICP-AES, ICP-MS, and AA

(EPA, 1983; EPA, 19910) are available for quantifying

cationic metals in aqueous samples. Tables 4-l and 4-2

summarize method detection limits for the analysis of

cationic metals in aqueous samples using AA with direct

aspiration, AA with the furnace procedure, ICP-AES, and

ICP-MS.

The detection limits required in Phase II for the

identification of suspect cationic metal toxicants will be

determined by the toxicity of metals for the TIE species. In

some cases, especially for chronic toxicity, the effect level

might be lower than the detection limits listed in Tables 4-I

and 4-2. Detection limits should be improved to obtain

optimal levels of detection (i.e., at least two times lower

than the effect level).

Toxicity data for some species and test types for

many metals have not been determined, especially for 7-d

chronic toxicity tests. Therefore, to determine the needed

levels of detection, effect levels for specific metals may

have to be determined.

The required level of detection will often dictate

the method needed for performing the metal measurement. It will be beneficial for laboratories to compile a

database containing method detection limits and toxic

effect levels for cationic metals using data from their

organisms, analytical methods, and toxicity testing conditions. These data are not necessary in advance but this

4-2

-
Table 4-1. Atomic Absorption Detection Limits and Concentration Ranges’

Metal

Direct Aspiration Furnace Method2

Detection Optimum Detection Optimum

Limit Concentration Limit Concentration

(m9/l) Range( mg/l) @9/l) Range @9/j)

Aluminum 0.1

Antimony 0.2

Arsenic3 0.002

Beryllium 0.005

Cadimum 0.005

Calcium 0.01

Chromium 0.05

Cobalt 0.05

Copper 0.02

Lead 0.1

Magnesium 0.001

Manganese 0.01

Mercury4 0.0002

Molybdenum (p) 0.1

Nickel(p) 0.04

Potassium 0.01

Selenium2 0.002

Silver 0.01

Sodium 0.002

Tin 0.8

Vanadium (p) 0.2

Zinc 0.005

5 - 50

I-40

0.002 - 0.02

0.05 -2

0.05 -2

0.2 -7

0.5 - 10

0.5 -5

0.2 -5

1 -20

0.002 - 0.5

0.1 -3

0.0002 - 0.01

l-40

0.3 - 5

0.1 -2

0.002 - 0.02

0.1 - 4

0.03 - 1

10 -300

2- 100

0.85 - 1

3

3

0.2

0.1

1

0.2

2 5- 100

0.2 1 - 25

5

4

0.05

20 - 200

20 - 300

5- 100

l-30

0.5 - 10

5- 100

5-100

5- 100

5- 100

l-30

3 - 60

5 - 50

20 -300

10 - 200

0.2 - 4

‘The estimated detection limits and concentration ranges were taken frpm EPA, 1983.

211e listed furnace values are those expected when using a 20 ~1 injection and normal gas flow except in

the case of arsenic and selenium where gas interrupt is used. The symbol (p) indicates the use of pyrolytic

graphite with the furnace procedure.

* Qaseous hydride method.

‘Cold vapor technique.

Table 4-2. Estimated Instrumental Detection Limits for ICP-MS and

ICP-AES

Estimated Detection Estimated Detection

Element Limit, ICP-MS @g/I) limit, ICP-AES2 @g/l)

Aluminum 0.05 45

Arsenic 0.9 53

Antimony 0.08 32

Beryllium 0.1 0.3

Cadmium 0.1 4

Calcium 10

Chromium 0.07 7

Cobalt 0.03 7

Copper 0.03 6

Lead 0.08 42

Magnesium 30

Manganese 0.1 2

Molybdenum 0.1 8

Nickel 0.2 15

Potassium -3

Selenium 5 75

Silver 0.05 7

Sodium

0.02

29

Vanadium 8

Zinc 0.2 2

l The estimated instrumental detection limits are taken from EPA,

1991 D. They are given as a guide for instrumental limits, the actual

detection limits are sample dependent and may vary as the sample

matrix vanes.

2The estimated instrumental detection limits as shown are taken from

EPA, 1983. They are given as a guide for an instrumental limit. The

actual method detection limits are sample dependent and may vary

as the sample matrix varies.

3Highly dependent on operating conditions and plasma.

type of information will be very useful for future TIE

efforts.

When toxicity effect levels appear to be below the

detection limits of current analytical methods, the use of

“clean” analytical techniques may be required through all

steps in the analysis of the sample because background

contamination is the major cause of inadequate levels of

detection. Some general principles of clean metal techniques include the use of contamination free reagents,

acid cleaned plastic labware, acid cleaned membrane

filters (not glass fiber), class 100 benches for sample

preparation, proper sample collection, preservation, and

storage procedures; and proper QAQC procedures using

blanks, spiked matrixes, and replicate analyses. A summary of clean metal techniques and procedures for lowering the levels of detection can be found in Nriagu et al.,

1993; Patterson and Settle, 1976; and Zief and Mitchell,

1976.

Some cationic metals, such as arsenic, selenium,

and chromium, have different stable oxidation states in

aqueous samples and more importantly the different oxidation states may have different toxicities. In Section

4.23, procedures to determine the concentration of the

different oxidation states are provided.

In some TIES, a measurement of the metals

associated with the suspended solids may be needed

4-3
(Section 4.2.4). Procedures for preparing suspended solids removed by filtration for metals analysis are available;

see EPA method 200 (EPA, 1983) and EPA method

200.2 (EPA, 19910).

4.2.3 Metal Specia tion

The procedures suggested above (Section 4.2.2)

are used to determine the total concentration of a metal in

an effluent. Many metals exist in water in different forms

due to the various stable oxidation states of the metal.

Arsenic (A!$+, As5+), chromium (W+, W+), and selenium

(Se4+, Se6+) are important metals that exist in different

forms in water. Determining the speciation for these metals may be important in the TIE since the toxicities are

different for the various forms of each metal. For example,

W+ is the form that is of toxicological concern while W+

is generally not toxic (EPA, 1985D).

For chromium, methods for measuring the

hexavalent form (Ca’) such as method 218.5 (EPA, 1983)

are available. The amount of the trivalent form of chromium (less toxic form) is determined by taking the difference between the concentrations for total and hexavalent

chromium.

* For arsenic, the method of Ficklin (1983) is suggested for speciation measurements. This method uses

an anion-exchange resin to separate the arsenite (As3+)

and arsenate (As5+, more toxic form) species. Graphite

furnace atomic-absorption spectroscopy is then used to

measure the concentrations of each form.

For selenium, the method of Oyamada and lshizaki

(1986) is recommended for speciation. This method (like

that for arsenic) uses column chromatography with an

anion-exchange resin to separate the selenite (Se4+) and

selenate Se6+ (more toxic form) and graphite furnace

atomic-absorption spectroscopy to measure each form.

Ion chromatography can also be used to determine the different forms of the above metals (EPA, 1991 D),

but we have not used this technique to date.

4.2.4 Identification of Suspect Metal Toxicants

Initial implication of suspect metals based on a

comparison of total metal analyses data and effluent

toxicity test results should be made. Then analysis for

dissolved and suspended metals can be made if necessary. These metal values should be compared to available toxicity values, but tests on reference metals might

have to be conducted with matching effluent conditions,

such as pH and hardness to obtain comparable toxicity

values. Side-by-side tests with individual reference metal

standards and effluent samples might prevent being mislead by different test designs and are worth the effort.

Literature summaries of metal toxicity data are also available (EPA, 1980; EPA, 19858; EPA, 1985D; EPA, 1985E;

EPA, 1985F; EPA, EPA, 1986; EPA, 1987; EPA, 19888;

and AQUIRE, 1992). In addition to matching the hardness

and pH of the dilution water to the effluent sample by the

addition of the appropriate ratios of magnesium carbonate and calcium carbonate, it might be possible in some

cases to mimic the wastewater total suspended solids

(SS) and total organic carbon (TOC) in the water used to

test the metal. For example, TOC and SS from the

addition of the YCT food can be at levels such that the

total SS level in the dilution water might be similar to that

found in the effluent. TOC may also be modified by the

addition of humic acid. If the dilution water does not

closely match the effluent, nonstandard dose-response

relationships are observed in the toxicity test, i.e., several

test concentrations exhibit partial mortality. In addition, a

trend is noticed that as metal concentrations decrease at

the effluent LC5Os or IC25s, toxicity of the effluent increases.

If a sample is to be filtered, a membrane filter(s),

such as a 0.45 um.polycarbonate filter should be prepared by rinsing with high purity water, followed by an

appropriate volume of dilution water needed for blank

toxicity tests and analysis. The toxicity test guidance is

described in Section 1.2 and in the Phase I documents

(EPA, 1991 A; EPA, 1992). An appropriate quantity (~50

ml) of the last portion of the high purity water passing

through the filter should be collected as an analytical

blank to check for metals contamination from the filter and

the filtration apparatus. An aliquot of the effluent is then

filtered through the 0.45 urn membrane filter(s). If more

than one filter is required for the effluent, a portion of each

can be combined for testing.

The filtered and unfiltered effluent samples and

the filtration blank should be tested for toxicity to measure

the effect of filtration on sample toxicity. The toxicity test

techniques are described in the non-polar organic section

(Sections 2.2.3 and 2.3.3) unless data are needed for

Phase III confirmation and then, greater replication and

randomization will be needed (see Section 1.2). The

toxicity tests should be performed for the test species

using a dilution water (e.g., reconstituted water) of a

similar hardness and pH to that of the effluent. If toxicity is

reduced or removed upon filtration (and effluent toxicity

has not previously been affected by C,, SPE or filtration

through a glass fiber filter), it is possible that metals were

retained by the 0.45 urn filter. Analysis for metals retained

by the filter may help in interpreting sample data.

Metals analyses should be performed on the

analytical blank collected from the filter and on the filtered

and unfiltered effluent samples. The choice of metals to

measure will be determined by the prioritization process

described above. As stated previously, the level of detection for the metal of interest should be lower than the

effect concentration for the metal.

Biologically Available Metals: Traditionally, dissolved metals for aqueous samples have been defined as

those that pass through a 0.45 urn membrane filter, i:e.,

4-4
polycarbonate filter. The dissolved metals are in no way

synonymous with the biologically available metals. Other

than the use of an aquatic organism there is no technique

to determine the biologically available fraction of the total

metal. Furthermore, only rudimentary techniques are available to specifically identify the individual species of a

metal (e.g., free charged metal ions [M”+], inorganic ion

pairs or complexes such as aquoions, [M(H,O)n+m],

hydroxoions [M(OH)pn-P+], oxoions [M0,“-29’1, organtc com-

’ plexes and chelates [M x EDTA], metal species bound to

high molecular weight organic material [M x lipid] or metal

species in the form of highly dispersed colloids or sorbed

on colloids [M x clay]). Stumm and Morgan (1981) have

listed some general methods for assisting in identification

of individual species. In some cases, binding of metals to

inorganic and organic ligands in effluents will reduce the

bioavailability of the metals and cause the metal concentration at the effluent LC50, IC50, or IC25 to be larger

than the metal concentration determined in the metal

dilution water toxicity test. For a set of effluent samples

with a wide range of toxicities, better agreement should

occur between the effect concentration of the metal in a

dilution water toxicity test and the more toxic effluent

samples (where the toxicity testing matrix of the effluent

more closely matches that of the dilution water). Methods

for determining the bioavailable fraction of the total metal

are limited.

Some indication of the binding of metals to organits in the effluent may be arrived at through hexane

extraction of an aliquot of the sample (Stary, 1964).

Theoretically, metals bound to organic materials that are

soluble in hexane should be extracted from the effluent.

The hexane can then be evaporated and the residue

reconstituted and analyzed for metals. Additionally, the

loss of metals can be estimated by repeating the metal

analysis on the extracted effluent and comparing this

result to the hexane extract results. The toxicity attributed

to metals associated with organics might be estimated by

performing a toxicity test on the solvent extracted effluent.

Traces of hexane must be removed from the extracted

effluent by aeration prior to toxicity testing. The effects of

aeration on sample toxicity must also be considered in

this analysis. In any case, metals strongly suspected of

causing or contributing to sample toxicity should be tested

in dilution water as described above with the TIE test

species.

The effects of variable water quality characteristics on metal toxicity must be evaluated over the effluent

sampling period. One way to assess this is to collect

several samples over a short time span. As an example,

for an acutely toxic effluent, collect six grab samples in 24 h, and

calculate the correlation coefficient for sample metal concentration (or summed toxic units of metals) versus sample

toxicity for each sample. The set of correlation coefficients for multiple sampling events might give results less

affected by hardness, SS, and TOC, assuming that water

quality characteristics affecting metal toxicity will vary less

during short time periods. For chronic toxicity, it might be

useful to measure concentrations of metals in several

daily samples and conduct separate chronic tests on

each sample. Obviously, metal concentrations must vary

enough to provide a sufficient range for correlation. When

one reaches this stage, Phase III work should start using

Phase III methods. Symptoms, species sensitivity, spiking, water quality adjustments and correlation are all

applicable Phase III approaches to confirm the cause of

toxicity.

4.3 Additional Toxicity Testing Methods

Guidance on EDTA addition tests, sodium thiosulfate addition tests, graduated pH tests, and the use of

ion-exchange resins for use in Phase II are presented in

this section. These procedures might be used before

performing analyses for cationic metals, but most often

they will be used to refine a list of suspect metal toxicants

and to provide data to support the identified suspect in

Phase III.

In the acute Phase I, EDTA and sodium thiosulfate addition tests can be conducted by adding incremental amounts of EDTA or sodium thiosulfate to a, single

effluent concentration. To provide further evidence in

Phase II, these two tests should be conducted with effluent dilutions to assess the toxicity reduction (see EPA,

1992). The data generated from these procedures provide a powerful tool for identifying the cause of toxicity in

samples containing mixtures of cationic metals. For example, toxicity caused by either copper or zinc could be

determined by using the following test information: toxicity

of both metals would be removed by EDTA addition

(Section 4.3.1), sodium thiosulfate can remove toxicity

caused by copper but not zinc (Section 4.3.2), and copper

is more toxic at higher pH levels while zinc is not (Section

4.3.3). Depending on how the toxicity of the sample

changes with these tests, one could eliminate one of

these metals from the list of suspect metal toxicants.

Results of this type of testing will be used to

develop evidence implicating the identified suspect metal.

These tests would be performed on a number of samples

over time to demonstrate the consistency of the cause of

toxicity. In addition, when a mixture of toxicants is present,

additions of EDTA or thiosulfate could be used to remove

the cationic metal toxicity after performing other Phase II

manipulations, e.g., C,, SPE.

4.3. I EDTA Addition Test

Any reduction in effluent toxicity effected by the

addition of EDTA suggests that certain cationic metals

might be present in the effluent at toxic levels. Background information and discussion of the behavior of

EDTA and cationic metals can be found in Phase I (EPA,

1991A; EPA, 1992).

Ideally, the amount of EDTA added would be just

enough to chelate the toxicant without causing EDTA

4-5
toxicity or substantially changing the water quality. For

either C. dubia or fathead minnows, we have found it

useful to add two different EDTA concentrations to two

separate effluent tests (with dilutions). Controls without

EDTA must be included. The EDTA stock solution is

added after the effluent dilutions are prepared so that the

EDTA concentration is the same at each effluent dilution

(see Phase I, EPA, 1991A; EPA, 1992).

In Phase II, conducting simultaneous EDTA addition tests on effluent and the suspect metal in matching

test water can provide evidence supporting the suspect

metal as the toxicant if the results of these two tests are

similar. If the metal is chelated by EDTA in the dilution

water test but not in the effluent test then either there is a

strong matrix effect from the effluent or it is the incorrect

suspect metal. It is important to use the same pH in both

tests in case there is any pH effect on the metal’s toxicity.

In addition to removing toxicity caused by metals,

EDTA reduces the acute toxicity of some cationic surfactants. This reduction of toxicity might also occur in chronically toxic effluents, and the toxicity reduced by EDTA

should not be assumed to be due only to cationic metals.

4.3.2 Sodium Thiosulfate Addition Test

The acute Phase I oxidant reduction test (EPA,

1991A) or the chronic sodium thiosulfate addition test

(EPA, 1992) is used to determine to what extent constituents reduced by the addition of sodium thiosulfate

(Na,S,O,) are responsible for the effluent toxicity. Although the use of the sodium thiosulfate test was designed to determine if oxidative compounds (such as

chlorine) were responsible for effluent toxicity, experience

has also shown that thiosulfate can also form a stable

non-toxic complex with some metals. Since the complexing

ability of thiosulfate is more metal specific than EDTA, this

reagent can be used to determine if a specific metal is

responsible for the effluent toxicity. Recent work by Mount

Table 4-3. Metal LCSOs with Respect to Test pH1

LCSO (pg/l)

Metal Species pH 6.2 pH 7.2 pH 8.2

Zn C. dubia >530 360 95

P. promelas 830 333 * 502

Ni C. dubia . >200 137 13

P. promelas >4000 3360 3080

Pb C. dubia 280 >2700 >2700

P. promelas 810 >5400 >540

cu C. dubia 10 28 201

P. promelas 15 44 >200

Cd C. dubia 563 350 121

P. promelas 54 74 <5

jLC50 values were determined at 48-h for C. dubia and 96-h for

P. promelas. Data taken from Schubauer-Berigan et al., 1993A.

(1991) has shown that in acute toxicity tests with C. dubia

in moderately hard water that Cu2+, Cd2+, Hg2+, Ag+, and

Se6+ can be complexed using sodium thiosulfate (see

EPA, 1991 A for more details). This complexing ability

might not be applicable to chronic toxicity. For example, in

a C. dubia 7-d test with copper, the toxicity was not

reduced with sodium thiosulfate addition but was reduced

with EDTA addition.

If the addition of sodium thiosulfate does not

reduce the effluent toxicity thought to be related to metals,

the use of SO, (EPA, 1991A) additions followed by the

addition of sodium thiosulfate is recommended. In some

situations, the thiosulfate concentration may be reduced

by non-toxic oxidants and thus, not be available for

complexing the toxic metal. The addition of SO should

preferentially reduce these non-toxic oxidants which will

allow the now available thiosulfate to complex the toxic

metals. Depending upon the complexation ability of sodium thiosulfate for a specific metal, it might or might not

complex the toxic metal. If the suspected metal toxicant

can be complexed (e.g., cadmium, copper, selenium (as

selenate), mercury; see EPA, 1991A; EPA, 1992), then a

reduction in sample toxicity should occur with the addition

of sodium thiosulfate. If the suspected metal cannot be

complexed (e.g., zinc, lead, manganese, and nickel), then

no reduction in sample toxicity should occur with the

addition of the sodium thiosulfate.

As with the EDTA addition test, sodium thiosulfate additions should be conducted concurrently on the

effluent and on dilution water spiked with the suspect

metal toxicant. Care must be used to conduct these tests

at similar pH levels. When toxicity test results are consistent with the expected behavior, strong evidence relating

the suspected metal toxicant to the cause of the effluent

toxicity has been obtained. These results in conjunction

with the ion exchange test, analytical measurements for

toxic metals, and the EDTA addition test provide evidence

sufficient for one to proceed to toxicant confirmation,

Phase Ill, of the TIE.

Both sodium thiosulfate and EDTA can reduce

the toxicity of some metals and this information can be

helpful in identifying the toxicant. However to date, this

effect of thiosulfate/metal complexation has not been

demonstrated for chronic toxicity. Knowing which metals

are bound by both sodium thiosulfate and EDTA and

which metals are complexed with only one or the other

additive can be very helpful in narrowing down the possible toxicant.

4.3.3 Metal Toxicity Changes with pH

In Phase I, the graduated pH test is performed to

evaluate the presence of compounds whose toxicity varies with pH. For ammonia, toxicity is greatest at pH 8.5

and least at pH 6.5 for some species. Therefore, as

suggested in the first Phase II document, that for samples

in which toxicity is enhanced at elevated pH, the identification effort should focus on ammonia. However, some

4-6
effluent and sediment pore water TIES have indicated that

some toxicity caused by metals can be affected by pH

within the range of pH 6 to 9 (Schubauer-Berigan et al.,

1993A). Some metals, notably zinc, nickel and cadmium,

exhibit greater toxicity at elevated pH, which could confuse their characterization with that of ammonia (Table 4-

3), while copper and lead show elevated toxicity at pH

6.2. These pH-dependent toxicities can be used as a tool

for the identification (and confirmation) of toxicity caused

by these metals. For example, toxicity to C. dubia in a

sediment pore water sample was completely removed by

additions of EDTA. The sample also exhibited greater

toxicity at pH 6.5 than at 8.5, and metal concentrations

indicated that only copper was present at toxicologically

significant concentrations. The pH dependent toxicity of

the sample along with the EDTA addition results and

metal analysis supported the identification of copper as

the toxicant.

4.3.4 Ion-Exchange Test

Ion-exchange resins have been used in TIES for

generating supporting evidence for identifying the cause

of toxicity in effluents (Doi and Grothe, 1989; Phase II

zeolite test). For cation exchange resins, removal of toxic

cations such as NH,+, Cd*+, and Pb*+ from the effluent

occurs with the corresponding release of cations (i.e.,

counter ions) such as H+ and Na+ into solution. Similarly,

for anion exchange resins, removal of toxic anions such

as Cr,O,*- and AsO,*- from the effluent occurs with the

corresponding release of anions such as OH- and Cl- into

solution. For both cation and anion exchange resins,

charge neutrality exists between the resin and aqueous

phase and therefore, if the resins remove 5 pmoles of

Cd*+ from solution, IO umoles of H+ would be released

into solution. The exchange process is concentration

dependent and is reversible. Cations removed from the

solution may then be recovered from the exchange resin

by passing an acidic solution over the resins (e.g., 1 N

HCI for analysis of metals).

We have had limited experience with ion exchange resins but the following general guidance can be

provided. First, ion exchange resins are not chemical

specific but rather remove a wide range of cations or

anions, metallic and non-metallic. We have observed that

anion exchange resins can remove cations (e.g., Zn*+)

from solution quite efficiently. The reasoning that only

cationic materials are removed by cation ion exchange

resins is not always reliable. Experimental verification of

which materials were removed by the resin will be necessary on a case-by-case basis. Second, wide changes in

the pH of the post-column effluent can occur depending

upon the type of cation or anion released by the resin.

These changes in pH will cause problems in interpreting

toxicity tests if the pH is not adjusted prior to the toxicity

test. Third, many of the ion exchange resins are based

upon a styrene or acrylic divinylbenzene backbone and

4-7

this material can remove other types of toxicants such as

non-polar organics. Consequently, because of its nonspecificity the removal of toxicity by an ion exchange

column should not be used as the only piece of evidence

to implicate a metal as the toxicant.

Resins under evaluation and/or those which have

been used include IRA-35, IRA-68, IRA-94, IRA-900,

IRC-718, and GT-73 (Rohm and Haas, Philadelphia PA)

and aquarium zeolite (Argent Chemical Laboratories,

Redmond WA). The key to obtaining useable data from

an ion exchange test is to obtain non-toxic blanks. Since

numerous ion exchange resins exist, guidance for preparing all resins for TIE work cannot be provided. A variety of

procedures have been used in our laboratory to condition

the columns and to obtain non-toxic blanks.

Effluent volumes ranging from 1,000 to 10,000 ml

have been used, and the volume is dependent on the

hardness of the dilution water, bed volume of the column,

strength and type of the ion change resin, which ions

were being exchanged, the toxicity of the effluent, and the

species being tested. For example, for acutely toxic effluents, glass chromatography columns (11 mm i.d.) are

packed with about 10 cm of resin and the solutions are

pumped up through the column at a flow rate of 4 to 5 ml/

min. First, a small volume of high purity water (e.g., 200

ml) is passed through the column, and discarded. Next,

the dilution water (volumes are variable, i.e., 1 ,OOO5,000 ml) is passed through the column until the pH of the

post-column dilution water is above 7.0.

Following this procedure, the necessary volume

of dilution water to use for toxicity testing is passed

through the column and collected. The type of dilution

water to use is effluent specific and in general, should be

the same as the dilution water used in the toxicity test for

the effluent. The pH of the post-column dilution water

should be monitored and the pH adjusted to return the

water solution to its original pH. Toxicity tests are then

performed on the post-column dilution water sample (column blank). After obtaining non-toxic blanks for a particular batch of resin, the conditioning process can be used

on other aliquots of the resin with a similar procedure;

however, column toxicity blanks must always be tested.

To identify acute toxicity, we generally begin by

using 200 ml of effluent (filtered or unfiltered) and collect

the post-column effluent. The pH of the post-column

effluent is checked and if necessary the pH is adjusted to

that of the baseline test, and tested for toxicity. For

chronic toxicity, the volume of effluent needed for the

toxicity test will dictate the amount of resin and the size of

the column. When evaluating a new resin, use proportions of water, effluent, and resin, similar to those described above for acutely toxic effluents. New aliquots of

resins should be prepared and used for each ion exchange test. By doing so, artifactual toxicity problems
from other effluents and sample manipulations can be

avoided.

We have had limited success in the elution of the

ion exchange resin to recover the exchanged toxicant(

therefore, we cannot provide specific guidance. in theory,

cations and anions can be eluted from ion exchange

resins using a strong acidic (HCI) or basic (NaOH) solution. Performing successful toxicity testing on these solutions is extremely difficult because of artifactual toxicity

problems.

When toxicity is removed by the ion exchange

test, useful information about the toxicant may be

obtained. However, as discussed above, the removal of a

toxicant by the column may not be as straightforward as

first perceived. The use of other manipulations and analytical measurements on the pre- and post-column effluents will be required to establish the significance of the

results of the manipulation.

When toxicity is not removed by the ion exchange

test and non-toxic blanks are obtained, the conclusion

that the toxicant is not a cation or anion can be made.

However, the slight possibility exists that the resin may

not be able to exchange the toxicant because of steric

and size considerations.

4-8
Section 5

Chlorine

-

5.1 General Overview

One of the first analytical measurements recommended in the Phase I documents (EPA, 1991A; EPA,

1992) upon arrival at the laboratory is for total residual

chlorine (TRC) in each effluent sample. Chlorine is a

commonly used biocide and oxidant and is frequently

found at acutely toxic concentrations in municipal effluents (EPA, 19856). Sublethal chronic toxicity from chlorine in effluent samples is not as likely to occur due to the

degradation of chlorine (see below) with holding of the

sample. Chlorine is unstable in aqueous solutions and

decomposition is more rapid in solutions when chlorine is

present at low concentrations. From the TRC measurement and the Phase I tests (sodium thiosulfate addition

and aeration tests), further steps to identify the effects

that might be due to chlorine can be taken. Oxidants

other than chlorine occur in effluents and the removal of

toxicity by the addition of sodium thiosulfate does not

prove that chlorine was the cause of effluent sample

toxicity.

Molecular chlorine or hypochlorite dissociates

into free aqueous chlorine, hypochlorous acid, and hypochlorite ion when added to effluents. Chlorine can also

combine with ammonia to form chloramines, i.e., mono- ,

di-, and tri-chloramines and with organic compounds,

especially organic nitrogen (APHA, 1992). The measured

total residual chlorine (TRC) of an effluent is the concentration of free and combined forms (mentioned above)

added together. The portion of the TRC associated with

an individual form is matrix dependent. Chlorinated industrial and wastewater effluents normally contain only

the combined form of chlorine (APHA, 1992).

These various forms of combined chlorine may

have different effect concentrations for toxicity, and the

toxicities of these individual forms are not all known for

acute or chronic toxicity to C. dubia or fathead minnows.

However, while the TRC level in the effluent samples

may be the same, the concentration of the various forms

may be different because of the matrix inherent to the

effluent. This matrix of TRC may also be variable from

sample to sample for the same discharger.

Another complication is that current analytical

methods for measuring TRC are not chlorine specific.

Other oxidizing compounds, e.g., bromine, iodine, hydrogen peroxide, ozone, and manganese, will be quantified

as chlorine by the analytical methods for measuring TRC

and may provide the analyst with a false positive for

chlorine.

5.2 Tracking Toxicity and TRC Levels

Several methods are available for measuring total TRC (EPA methods 330.1, 330.2, 330.3, 330.4, and

330.5 (EPA, 1983)). Measurements of TRC in the effluent

upon arrival of the sample at the laboratory should always

be made. If TRC is not detected, chlorine should not be

considered a suspect toxicant since the analytical methods do not yield false negatives.

For acutely toxic effluents, grab samples both

before and after the chlorination process should be collected simultaneously (i.e., within minutes of each other).

Upon arrival of these samples at the laboratory, a baseline

toxicity test should be initiated and at pre-determined

intervals after day 1 (e.g., day 2, day 3, day 5, day 8) to

evaluate whether the toxicity is degrading. TRC determinations should be performed in conjunction with each

toxicity test.

Generally the TRC in most effluent samples stored

at 4°C degrades in 2 to 5 d after collection. Therefore, if

residual chlorine is a toxicant the toxicity of the postchlorination sample should decrease as TRC levels decrease, and pre- and post-chlorination samples should

have the same toxicity after the decay of TRC.

The toxicity of chlorine in an effluent sample will

be dependent on the matrix of the effluent and the species tested. If chlorine toxicity data does not exist for the

species being used, it will be necessary to measure the

LC50 or IC25 of chlorine using the TIE organisms and

dilution water. Using those LC50 and/or IC25 values, the

comparison of TUs of the effluent to the TUs of residual

chlorine is useful to evaluate the effects of the TRC.

When the TU comparison data and pre- and post-chlorination toxicity data indicate TRC as a suspect toxicant

Phase III procedures should be initiated.

With the measurable levels of TRC at sample

collection, the loss of toxicity with the corresponding

decreasing levels of TRC, and the pre- and post-chlorina5-I
-

tion samples exhibiting similar toxicity with the decrease the toxicant is chlorine since other oxidants will be dein TRC, Phase III confirmation should begin (EPA, 1989.6; tected by the TRC measurement techniques.

EPA, 1993A). However, these steps will not insure that

5-2
Section 6

Identifying Toxicants Removed by Filtration

6.1 General Overview

If the results of Phase I .tests indicate that the

filtration manipulation removed or reduced toxicity, the

investigator should carefully compare these results to

those of the other manipulations before trying to identify

the toxicants that might be on the filter. We have obsewed that metals, non-polar organic compounds and

volatile compounds can all be removed under certain

filtering conditions, but these observations have been

dependent on the individual effluent or the sediment pore

water samples. Other Phase I manipulations (e.g., EDTA,

C,, SPE extraction) can lead to subsequent Phase II

identification steps. However, for toxicity reductions effected by filtration, more intermediate steps of Phase I

type manipulations must be done before analytical procedures are used to identify the toxicant( In addition,

some other manipulations may provide specific information regarding the identity of toxicants that may have been

removed by filtration; these include additions of PBO

(Section 2.5.1), the graduated pH test (Phase I tests for

determining toxicity caused by ammonia, metals and

ionizable organic compounds), and the sodium thiosulfate

test (Phase I test for detecting toxicity caused by volatile

oxidants such as chlorine or metals). If one or more of

these manipulations removes toxicity, then identification

work should proceed as described in the previous sections to identify the cause of toxicity.

It is important to consider that all toxicity removed

by filtration may not be actually removed by the process

of filtering. For example, when the pH of the sample is

altered, the mechanism(s) for removal by filtration can

change. While ammonia is predominantly ionized at a

sample pH of 8.3, the ammonia would not tend to be

removed through volatilization if a vacuum was applied

for filtration purposes. Yet by adjusting the sample pH to

11, the ionized ammonia concentration decreases to 1.7%

at 25°C. When a sample is adjusted to a pH of 11,

volatilization of a toxicologically significant amount of the

un-ionized ammonia could occur and the toxicity results

would indicate that filtration removed toxicity. Also, changes

in speciation at elevated pH render many metals insoluble, which could result in their removal by the filter at

pH 11 (Schubauer-Berigan et al., 1993B).

If the toxicant is thought to be a non-polar organic

toxicant, and filtration partially removes toxicity, it may be

useful and save toxicity testing time to eliminate the

filtration step altogether before applying the sample to the

C, SPE column (discussion in Section 2.2.2 and Section

24.2).

6.2 Filter Extraction

When filtration has been the only manipulation to

affect the toxicity, then extraction of the filters and tracking the toxicity of the extracts should be attempted. In

addition, the use of other types of filters should be evaluated (i.e., nylon, teflon, and polycarbonate) to see if

toxicity removal is a function of the filter type. In using the

extraction procedures, the idea is to separate the toxic

compounds associated with the filter by extracting them

into a solvent. Next, efforts are made to concentrate the

toxic compounds in the filter extract and test them at a

concentration that can be related to the original sample

and evaluate the efficiency of the extraction. Identifying

the filter-removable contaminants without additional information can be difficult because of the lack of specificity of

the filtration process. But once a suspect candidate has

been discovered, then measurements can be made to

determine whether a toxicologically significant concentration of the suspect toxicant had indeed been removed

by filtration. If this is the case, then it may not be necessary to consider further extractions of the filters. If, however, the concentrations of the suspect toxicant are not

decreased after filtration then it may be useful to attempt

additional identifications by solvent extraction of the filters

as described below.

One technique we have used with filterable toxicity is to extract the filters with either polar or non-polar

solvents. To remove toxicants from the filter we have

used either organic solvents (methanol, methylene chloride) or pH 3 high purity water as the extraction solvent.

The solvent is then toxicity tested to track toxicity (methylene chloride must first be exchanged into methanol),

additional Phase I tests are performed to characterize the

filter extract, and then chemically analyzed using Phase II

procedures. It is important to remove all of the methylene

chloride before toxicity testing a filter extract and these

procedures are described in detail in Section 2.6.2. To

date, methanol has been used to extract toxicity from

filters used with effluent samples and methylene chloride/

methanol solutions have been used to extract filters from

6-1
sediment pore water. The experiences described below

are based on acute toxicity experiments, and efforts to

recover filterable toxicity for chronically toxic effluent have

not yet been needed.

To isolate a toxicant removed through filtration,

several filters can be combined and extracted simultaneously if necessary. The volume of sample passed

through the filters is important for calculating concentration factors, and should be recorded. The filtrate should

also be reserved for toxicity comparisons and analytical

testing. Sufficient sample should I% passed through the

filter to allow for both toxicity testing and chemical analysis on both the filtered sample and the filter-extract solution (generally >200 ml). Carefully move the filters to a

glass (acid leached) or plastic beaker, then soak the

filters (l-5) in 20 ml of solvent for 1 h. Cool water sonication is optional to attempt to recover particle-associated

compounds. Carefully remove the filters and save (store

at 4°C) in case additional extractions are necessary. If pH

3 high purity water is used as the extraction solvent, the

extract should be readjusted to the initial pH of the

sample, then toxicity tested. If methanol is used, it is

evaporated to -2 ml under a stream of nitrogen. Be

careful to rinse the sides of the containers with methanol

to ensure complete solubilization of organic compounds.

This methanol solution can then be toxicity tested using

SPE fraction testing procedures (Section 2.2.8). Alternatively, if a methylene chloride/methanol solvent is used,

the solvent should first be exchanged into pure methanol

(Section 2.6.2) then treated as the methanol extract

described above. The concentration of the solvent extract

will depend on the volume originally passed through the

filters, which depends on the desired high test concentration, and the volume of extract and filtered sample required for analytical purposes. Blank filters (through which

has been passed a volume of dilution water) should be

extracted and tested identically to the sample filters to

ensure that the solvents do not introduce artifactual toxicity.

For any of the extraction techniques, the solutions should be tested at the same time as the baseline

test (unfiltered) and filtered sample test to compare the

toxicity recovered by the filter extraction with that removed from the sample.

Another option for toxicants removed by filtration

is to try other techniques to remove the toxicants which

avoid filtration. For example, sediment pore water samples

have been centrifuged at relatively high speeds (lO,OOO20,000 g) for 30 min prior to passing the sample over the

C,, SPE column and filtration could thus be eliminated.

Filter extractions (EPA, 1991 A) have been used

in several sediment TIE studies, with procedures suggested for both non-polar organics and metals (SchubauerBerigan et al., 1990; Schubauer-Berigan and Ankley,

1991). In some effluent and pore water samples, toxicity

thought to be caused by non-polar organic compounds

(e.g., PAHs and polymers) has also been removed by

filtration. These compounds may be associated with particulate material, and be physically filtered from the sample,

or removed by association with oil and grease that sorbs

to the filter.

In some cases, binding of metals or organic

compounds to inorganic and organic ligands in effluents

or sediment pore waters will reduce their bioavailability

and when toxicity testing filter extracts, it is always a

concern that the matrix of the sample has been removed

and that chemicals might become available when they

were not in the original sample. If this were to happen, the

extracts might be more toxic than expected and chemicals might be added to the suspect toxicant list erroneously. This kind of mistake should be caught by obtaining

a good toxicity value for the suspect in an appropriate

matrix (more detailed discussion in Section 26.1). Therefore, additional confirmation steps might be needed to

eliminate the false suspects.

6-2
Section 7

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EPA. 1986. Ambient Water Quality Criteria for Nickel.

EPA-440/5-86-004. Environmental Research Laboratory, Duluth, MN.

EPA. 1987. Ambient Water Quality Criteria for Zinc. EPA44015-87-003. Environmental Research Laboratqry,

Duluth, MN.

EPA. 1988A. Methods for Aquatic Toxicity Identification

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Laboratory, Duluth, MN.

EPA. 1988B. Ambient Water Quality Criieria for Aluminum. EPA-44015-86-008. Environmental Research

Laboratory, Duluth, MN.

EPA. 1989A. Methods for Aquatic Toxicity Identification

Evaluations: Phase II Toxicity Identification Procedures. EPA/600/3-881035. Environmental Research

Laboratory, Duluth, MN.

7-2

EPA. 19898. Methods for Aquatic Toxicity Identification

Evaluations: Phase III Toxicity Confirmation Procedures. EPA-600/3-88/036. Environmental Research

Laboratory, Duluth, MN.

EPA. 1989C. Short-Term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Waters to

Freshwater Organisms. Second Edition. EPA/600/4-

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Cincinnati, OH.

EPA. 1991A. Methods for Aquatic Toxicity Identification

Evaluations: Phase I Toxicity Characterization Procedures. Second Edition, EPA/600/6-91/003. Environmental Research Laboratory, Duluth, MN.

EPA. 1991 B. Sediment Toxicity Identification Evaluation:

Phase I (Characterization), Phase II (Id.entification),

Phase III (Confirmation) Modifications of Effluent Procedures. EPA-600/6-911007. Environmental Research

Laboratory, Duluth, MN.

.

EPA. 1991 C. Methods for Measuring the Acute Toxicity of

Effluents to Freshwater and Marine Organisms. Fourth

Edition. EPA-600/4-90/027. Environmental Monitoring and Support Laboratory, Cincinnati, OH.

EPA. 1991 D. Methods for the Determination of Metals in

Environmental Samples. EPA-600/4-91/010. Environmental Monitoring and Support Laboratory, Cincinnati, OH.

EPA. 1992. Toxicity Identification Evaluations: Characterization of Chronically Toxic Effluents, Phase I. EPA600/6-91/005F. Environmental Research Laboratory,

Duluth, MN.

EPA. 1993A. Methods for Aquatic Toxicity Identification

Evaluations: Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and Chronic Toxicity. EPA-600/R-92/081. Environmental Research

Laboratory, Duluth, MN.

EPA. 19938. Short-term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Waters to

Freshwater Organisms. Third Edition. EPA-600/4-91/

002. Environmental Monitoring and Support Laboratory, Cincinnati, OH.

EPA. 1993C. Short-term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Waters to

Marine and Estuarine Organisms. Second Edition,

EPA-60014~91/021. Environmental Monitoring and

Support Laboratory, Cincinnati, OH.

Ficklin, W.H.. 1983. Separation of Arsenic (III) and Arsenic (V) in Ground Waters by Ion-exchange. Talanta.

30:371-373.
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Ferguson, W.J., K.I. Braunschweiger, W.R.

Braunschweiger, JR. Smith, J.J. McCormick, C.C.

Wasmann, N.P. Jarvis, D.H. Bell, and N.E. Good.

1980. Hydrogen ton Buffers for Biological Research.

Anal. Biochem. 104:300-310.

Flaschka, H.A. and A.J. Barnard, Jr. (Eds.). 1967. Chelates in Analytical Chemistry. Marcel Dekker, Inc.,

New York, NY. 418 p.

Giger, W., C. Stephanou, and C. Schaffner. 1981. Persistent Organic Chemicals in Sewage Effluents: I. Identifications of Nonylphenols and Nonlyphenol Ethoxylates

by Glass Capillary Gas Chromatography/Mass Spectrometry. Chemosphere. IO:1 253-l 263.

Giles, M.A. and R. Danell. 1983. Water Dechlorination by

Activated Carbon, Ultraviolet Radiation and Sodium

Sulphite. Water Res. 17:667-676.

Jop, K.M., T.Z. Kendall, A.M. Askew, and R.B. Foster.

1991. Use of Fractionation Procedures and Extensive

Chemical Analysis for Toxicity Identification of a

Chemical Plant Effluent. Environ. Toxicol. Chem.

IO:981 -990.

Lukasewycz, M.T. and E.J. Durhan. 1992. Strategies for

the Identification of Non-Polar Toxicants in Aqueous

Environmental Samples using Toxicity-Based Fractionation and Gas Chromatography/Mass Spectrometry. J. Chromatogr. 580:215-228.

Mount, D.R. 1991. A Toxicity-Based Approach to Pollutant Identification. In: Proceedings of the Thirtieth

Annual EPA Conference on Analysis of Pollutants in

the Environment, May 9 and 10, 1990. 21 W-7005.

Office of Water, Washington, D.C.

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. Toxicol. Chem. 10:891-

901.

Norberg-King, T.J. 1993. A Linear Interpolation Method to

Estimate Sublethal Toxicity: The Inhibition Concentration (ICp) Approach. Release 2.0. National Effluent

Toxicity Assessment Center Technical Report 03-93.

Environmental Research Laboratory, Duluth, MN.

Nriagu, J.O., G. Lawson, H.K.T. Wong, and J.M. Azcue.

1993. A Protocol for Minimizing Contamination in the

Analysis of Trace Metals in Great Lakes Waters. J.

Great Lakes Res. 19:175-182.

Oyamada, N. and M. lshizaki. 1986. Fractional Determination of Dissolved Selenium Compounds of

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(VI) in Environmental Water Samples. Analyt. Sci.

2:365-369.

Patterson, C.C. and D.M. Settle. 1976. The Reduction of

Orders of Magnitude Errors in Lead Analysis of Biological Materials and Natural Waters by Evaluating

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Analysis: Sampling, Sample Handling, Analysis. P.D.

LaFleur (Ed.). National Bureau of Standards Spec.

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7-3

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7-4
Appendix A

Effluent Volume Calculation Worksheets

A-l
Table A-l. Effluent Volume Calculation Worksheets

SPE Fractionation of the Effluent

1) Volume of effluent:

See Table 2-6 for initial suggestions

for the volume of effluent.

ve ml

2) SPE fractionation:

Eluate volume from the SPE column:

See Table 2-l for approximate eluate volume

or measure volume.

Concentration factor for eluate: b, = ve + a

3) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-l 5 ml/replicate (acute)

D. pulex 1 O-l 5 ml/replicate (acute)

P. piomelas 1 O-200 ml/replicate (acute)

P. pfomelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals

Highest test concentration:

Is the methanol concentration okay?’

ml eluate = f x c + b,

%methanol = ml eluate x 100 + c

4) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexf+b,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration2 directly:

i=f+f+2+f+4+f+8

j=jXcxdxe+b,

Total volume of eluate used: k = g, h, or j

5) Volume of eluate remaining after toxicity testing:

m, =a-k

a ml

h X

C ml

d

; X

g

h

ml

ml

i ml

k ml

4 ml

‘Acceptable levels of methanol for C. dubia and fathead minnows are 10.6% and l%, respectively.

2An example of using four test concentrations, the number of dilutions may vary.

A-2
Table A-l. Continued

Toxlclty Testing and GUMS Analysls of the SPE Concentrate

1) SPE Concentration of the SPE fraction:

Eiuate volume from the SPE column:

See Table 2-3 for approximate &ate volume

or measure volume.

Concentration factor for the eluate: b, = b, x m, + a

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-25 ml/replicate (acute)

D. pulex 1 O-25 ml/replicate (acute)

P. promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

initial sample + number of renewals:

Highest test concentration:

3) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexf+b,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration directly:

l=f+f+2+f+4+f+8

j=lxcxdxe+b2

Total volume of eluate used: k = g, h, or j

4) Amount of eluate used for GC/MS analysis:

5) Volume of eluate remaining after toxicity testing:

m* =a-k-l

a

h

C

d

P

g

h

ml

X

ml

X

ml

ml

ml

ml

ml

ml

A-3
Table A-l. Continued

HPLC Fractionation of the SPE Concentrate

1) HPLC Fractionation of the SPE concentrate:

HPLC Fraction volume:

See Sections 2.2.10 and 2.3.10

a ml

Concentration factor for the eluate: b, = b, x m2 + a b, X

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-25 ml/replicate (acute)

D. pulex 1 O-25 ml/replicate (acute)

P, promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals:

Highest test concentration:

ml

X

3) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexf+b,

If using 0.5 dilution factor:

h=2xcxdxexftb,

If using dilutions by spiking each concentration directly:

l=f+ft2+f+4+f+8

]=ixcxdxe+b,

Total volume of eluate used: k = g, h, or j

5) Volume of eluate remaining after toxicity testing:

m3 =a-k-l

g ml

h ml

i

k

ml

ml

ma ml

A-4
Table A-1. Continued

Concentration of the HPLC Fraction for Toxicity Testing and GUMS Analysis

1) SPE Concentration of the SPE fraction:

Eluate volume from the SPE column:

See Table 2-3 for approximate eluate volume

or measure volume.

a ml

Concentration factor for the eluate: b, = b, x m3 + a b, X

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-25 ml/replicate (acute)

D. pulex 1 O-25 ml/replicate (acute)

P. promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals:

Highest test concentration:

ml

X

3) Volume of eiuate needed for toxicity testing:

If no dilutions: g=cxdxexf+b,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration directly:

i=f+f+2+f+4+f+8

j=ixcxdxe+b,

Total volume of eluate used: k = g, h, or j

4) Amount of eluate used for GUMS analysis:

5) Volume of eluate remaining after toxicity testing:

m4 =a-k-l

g ml

h ml

ml

k ml

I ml

m4 ml

A-5
Table A-2. Effluent Volume Calculation Worksheets (Example)

SPE Fractionation of the Effluent

1) Volume of effluent:

See Table 2-6 for initial suggestions

for the volume of effluent.

ve 20,000 ml

2) SPE fractionation:

Eluate volume from the SPE column:

See Table 2-1 for approximate eluate volume

or measure volume.

Concentration factor for eluate: b, q ve + a

3) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-l 5 ml/replicate (acute)

D. pulex 1 O-1 5 ml/replicate (acute)

P. promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic) ’

Number of replicates:

Initial sample + number of renewals

Highest test concentration:

Is the methanol concentration okay?’

ml eluate = f x c + b,

%methanol = ml eluate x 100 + c

4) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexftb,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration* directly:

i=f+f+2+f+4

j=ixcxdxe+b,

Total volume of eluate used: k = g, h, or j

5) Volume of eluate remaining after toxicity testing:

9

=a-k

a 40 ml

b, 500 x

C 10 ml

d

f!

5

7

2 x

g m l

h ml

i 2.45 ml

k 2.45 m l

ml m l 37.55

‘Acceptable levels of methanol for C. dubia and fathead minnows are 10.6% and l%, respectively.

*Example uses three test concentrations.

-

A-6
Table A-2. Continued

Toxicity Testing and GC/MS Analysis of the SPE Concentrate

-

1) SPE Concentration of the SPE fraction:

Eluate volume from the SPE column:

See Table 2-3 for approximate eluate volume

or measure volume.

Concentration factor for the eluate: b, = b, x m,+ a

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-25 ml/replicate (acute)

D. pulex 1 O-25 ml/replicate (acute)

P. piomelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals:

Highest test concentration:

3) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexf+b, D

If using 0.5 dilution factor:

h=2xcxdxexftb,

If using dilutions by spiking each concentration directly:

i=f+f+2+f+4

I =ixcxdxetb,

Total volume of eluate used: k = g, h, or j

4) Amount of eluate used for GUMS analysis:

5) Volume of eluate remaining after toxicity testing:

m2= a-k-l

b*

C

d 5

e 7

f 4 x

cl

h

m*

0.44 ml

42,670 x

10 ml

ml

ml

0.057 ml

0.057 ml

0.018 ml

0.365 ml

-

A-7
Table A-2. Continued

HPLC Fractionation of the SPE Concentrate

1) HPLC Fractionation of the SPE concentrate:

HPLC Fraction volume:

See Sections 2.2.10 and 2.3.10

Concentration factor for the eluate: b, = b, x m2 -+ a

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

D. magna 1 O-25 ml/replicate (acute)

D. pulex 1 O-25 ml/replicate (acute)

P. promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals:

Highest test concentration:

3) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexf+b,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration directly:

i=f+f+2+f+4

j=ixcxdxe+b3

Total volume of eluate used: k = g, h, or j

5) Volume of eluate remaining after toxicity testing:

m,=a-k

d 5

e 7

f 8x

g ml

h ml

i

k

0.315 ml

0.315 ml

0.685 ml

A-8
Table A-2. Continued

Concentration of the HPLC Fraction for Toxicity Testing and GUMS Analysis

1) SPE Concentration of the SPE fraction:

Eluate volume from the SPE column:

See Table 2-3 for approximate eluate volume

or measure volume.

a 0.22 m l

Concentration factor for the eluate: b, = b, x m3+ a b, 48.495x

2) Testing organism and conditions:

Toxicity test volume/replicate:

C. dubia 1 O-l 5 ml/replicate (acute/chronic)

0. magna 1 O-25 ml/replicate (acute)

0. pulex 1 O-25 ml/replicate (acute)

P. promelas 1 O-200 ml/replicate (acute)

P. promelas 50-250 ml/replicate (chronic)

Number of replicates:

Initial sample + number of renewals:

Highest test concentration:

IO ml

5

7

16x

3) Volume of eluate needed for toxicity testing:

If no dilutions: g=cxdxexftb,

If using 0.5 dilution factor:

h=2xcxdxexf+b,

If using dilutions by spiking each concentration directly:

i=f+f+2+f+4

j=ixcxdxe+b4

Total volume of eluate used: k = g, h, or j

4) Amount of eluate used for GUMS analysis:

5) Volume of eluate remaining after toxicity testing:

m, = a-k-i

9

h ml

m l

0.202 ml

0.202 ml

0.018 ml

---o- ml

*U.S. GOVERNMENT PRINTING OFFICE:1993-750-002/80303

A-9