U.S. GEOLOGICAL SURVEY CIRCULAR 1049 

Applicability of Ambient 

Toxicity Testing to 

National or Regional 

Water-Quality Assessment 
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Applicability of Ambient 

Toxicity Testing to 

National or Regional 

Water-Quality Assessment 

By JOHN F. ELDER 

U.S. GEOLOGICAL SURVEY CIRCULAR 1049 
U.S. DEPARTMENT OF THE INTERIOR 

MANUEL LUJAN, Jr., Secretary 

U.S. GEOLOGICAL SURVEY 

Dallas L. Peck, Director 

Any use of trade, product, or firm names in this publication is for descriptive 

purposes only and does not imply endorsement by the U.S. Government 

UNITED STATES GOVERNMENT PRINTING OFFICE: 1990 

Free on application to the 

Books and Open-File Reports Section 

U.S. Geological Survey 

Federal Center, Box 25425 

Denver, CO 80225 

Library of Congress Cataloging in Publication Data 

Elder, John F. 

Applicability of ambient toxicity testing to national or regional water-quality 

assessment I by John F. Elder. 

p. cm.-(U.S. Geological Survey circular : 1049) 

Includes bibliographical references (p. ) 

1. Water quality bioassay. 2. Toxicity testing. I. Title. II. Series. 

QH96.8BSE43 1990 

628.1'61-dc20 90-3171 

CJP 
CONTENTS 

Abstract 1 

Introduction 1 

Need for Biological Methods in Water-Quality Assessment 1 

Difficulties of Biological Methods in Water-Quality Assessment 2 

Purpose and Scope 2 

Acknowledgments 3 

Benefits and Limitations of Toxicity Testing 3 

Benefits 3 

Limitations 4 

Special Considerations for Large-Scale Toxicity Testing 5 

Uses of Toxicity-Test Results 6 

Procedures and Applications 6 

Test-Species Selection 6 

Acute Sensitivities of Test Species 11 

Overview of Test-Species Selection 15 

Acute and Chronic Tests 15 

Design of Test Enclosure 20 

Multispecies Tests 20 

Microcosm Approach 20 

Battery Approach 21 

Sediment Toxicity Tests 22 

Biochemical Tests 23 

Alternatives to Laboratory Toxicity Testing 25 

Overview of Test-Type Differences 25 

Field and Interlaboratory Verification of Toxicity Tests 25 

Summary 29 

References Cited 30 

Appendix: Toxicity-Test Procedures 43 

FIGURES 

1. Diagram showing taxonomic lineages of commonly used freshwater toxicity-test 

species 10 

2. Graph comparing 50-percent lethal concentration data from tables 15-19 with 

maximum and median concentration data for copper, cadmium, zinc, mercury, 

and lead in water from five selected U.S. Geological Survey waterquality monitoring sites and water-quality criteria 16 

3. Diagram showing general scheme for sequential toxicity screening 22 

TABLES 

1. Some possible uses of toxicity tests 6 

2. Criteria for species selection 7 

Contents Ill 
IV Contents 

3. Characteristics of some floral and faunal species frequently used in aquatic 

toxicity-testing procedures 8 

4. Characteristics of flora and fauna used in toxicity tests 11 

5-10. Evaluation of commonly used toxicity-test species with respect to selection 

criteria listed in table 2: 

5. Bacteria 12 

6. Algae 12 

7. Daphnia magna 13 

8. Cladocerans, excluding Daphnia magna 13 

9. Pimephales promelas 14 

10. Fish, excluding Pimephales promelas 14 

11-14. Acute toxicities of selected organic compounds to various test species: 

11. Phenol 15 

12. Pentachlorophenol 17 

13. Benzene 17 

14. Toluene 17 

15-19. Acute toxicities of selected metals to various test species: 

15. Copper 18 

16. Cadmium 18 

17. Zinc 19 

18. Mercury 19 

19. Lead 19 

20. Arguments for and against use of selected toxicity-test procedures 26 

21. Comparison of different types of toxicity tests 28 
Applicability of Ambient Toxicity Testing to 

National or Regional Water-Quality ·Assessment 

By john F. Elder 

Abstract 

Comprehensive assessment of the quality of natural 

waters requires a multifaceted approach. Descriptions of 

existing conditions may be achieved by various kinds of 

chemical and hydrologic analyses, whereas information 

about the effects of such conditions on living organisms 

depends on biological monitoring. Toxicity testing is one 

type of biological monitoring that can be used to identify 

possible effects of toxic contaminants. 

Based on experimentation designed to monitor 

responses of organisms to environmental stresses, toxicity 

testing may have diverse purposes in water-quality assessments. These purposes may include identification of areas 

that warrant further study because of poor water quality or 

unusual ecological features, verification of other types of 

monitoring, or assessment of contaminant effects on 

aquatic communities. Toxicity-test results are most effective when used as a complement to chemical analyses, 

hydrologic measurements, and other biological monitoring. However, all toxicity-testing procedures have certain 

limitations that must be considered in developing the 

methodology and applications of toxicity testing in any 

large-scale water-quality-assessment program. 

A wide variety of toxicity-test methods have been 

developed to fulfill the needs of diverse applications. The 

methods differ primarily in the selections made relative to 

four characteristics: (1) test species, (2) endpoint (acute 

or chronic), (3) test-enclosure type, and (4) test substance 

(toxicant) that functions as the environmental stress. 

Toxicity-test approaches vary in their capacity to meet 

the needs of large-scale assessments of existing water 

quality. Ambient testing, whereby the test organism is 

exposed to naturally occurring substances that contain 

toxicant mixtures in an organic or inorganic matrix, is 

more likely to meet these needs than are procedures that 

call for exposure of the test organisms to known concentrations of a single toxicant. However, meaningful interpretation of ambient test results depends on the existence 

of accompanying chemical analysis of the ambient media. 

The ambient test substance may be water or sediments. 

Sediment tests have had limited application, but they are 

Manuscript approved for publication February 10, 1989. 

useful because most toxicants tend to accumulate in 

sediments and many test species either inhabit t'1e sediments or are in frequent contact with them. Biochemical 

testing methods, which have been developing rc-'lidly in 

recent years, are likely to be among the most useful 

procedures for large-scale water-quality assersments. 

They are relatively rapid and simple, and more importantly, they focus on biochemical changes that are the 

initial responses of virtually all organisms to environmental stimuli. 

Most species are sensitive to relatively few toxicants, 

and their sensitivities vary as conditions change. Therefore, each test method has particular uses and !irritations, 

and no single test has universal applicability. Ore of the 

most informative approaches to toxicity testing is to combine biochemical tests with other test methc ds in a 11 battery of tests" that is diversified enough to cha"acterize 

different types of toxicants and different trophic levels. 

However, such an approach can be costly, an.-J if not 

carefully designed, it may not yield enough additional 

information to warrant the additional cost. 

The application of toxicity tests to large-sca1e waterquality assessments is hampered by a number of difficulties. Toxicity tests often are not sensitive enough to enable 

detection of most contaminant problems in the natural 

environment. Furthermore, because sensitivitie:::. among 

different species and test conditions can be hif'lly variable, conclusions about the toxicant problerrs of an 

ecosystem are strongly dependent on the test procedure 

used. In addition, the experimental systems use--! in toxicity tests cannot replicate the complexity or vari::\bility of 

natural conditions, and positive test results cannot identify the source or nature of a problem without accompanying chemical analyses. Finally, it is difficult to develop 

adequate control systems for toxicity tests that use ambient waters or sediments as exposure media. 

INTRODUCTION 

Need for Biological Methods in 

Water-Quality Assessment 

Protection and enhancement of water quality ultimately depend on establishment of sound ma'lagement 

Introduction 
policy on regional or national levels. Development of 

management policy is, in tum, dependent on regional or 

national programs to assess water quality-its current conditions, trends, and controlling factors. One of the particularly important and challenging needs in developing such 

large-scale assessment programs is appropriate planning of 

the collection and analysis of biological data. 

There can be little doubt as to the need for biological 

information to accurately evaluate water-quality conditions. 

The terms "pollution" and "contamination" generally refer 

to environmental occurrence of foreign substances that are 

biologically detrimental. Therefore, much of the concern 

for water-quality degradation is biologically motivated. 

The importance of biological analyses is further 

underscored by our understanding that water quality is not 

simply an expression of chemical characteristics. It is 

strongly influenced by biological activity, and, conversely, 

it strongly influences the composition and function of the 

biological community. For example, nitrogen and phosphorus concentrations in natural water systems are affected by 

uptake in algal cells (Richey, 1979; Goldman and Home, 

1983, p. 126; Schindler, 1985), and algal photosynthesis 

and biomass are conversely dependent on inputs of nitrogen 

and phosphorus (Smith, 1982; Canfield and others, 1985). 

Information from biological measurements often can be 

used to complement information from physical and chemical measurements, leading to better descriptions of waterquality conditions and improved understanding of the processes causing the conditions. 

A variety of biological assessment procedures can 

contribute to understanding of the complex relations among 

biological, physical, and chemical characteristics of an 

ecosystem. Among the most commonly used procedures to 

characterize the biological aspects of water quality are 

measurements of 

1. Distribution and abundance of floral and faunal species 

within an ecosystem (community surveys), 

2. Biological processes, such as respiration and primary 

productivity, which are common indicators of community metabolic activity, 

3. Biological products, such as chlorophyll and ATP (adenosine triphosphate), which also are common indicators 

of metabolic activity, 

4. Biogeochemical processes that influence the chemical 

character of water and sediments, 

5. Occurrence of pathogenic organisms, 

6. Biological uptake and depuration of contaminants that 

occur in the aquatic habitat, and 

7. Effects of water pollution on biota. 

The results of one or more of these types of biological 

analyses, combined with chemical and hydrologic data, can 

be used to ( 1) define and quantify biological processes that 

affect physical and chemical aspects of water quality, (2) 

determine the sanitary quality of the water, (3) determine 

the occurrence, distribution, and fate of contaminants, and 

( 4) assess the relation between the physical and chemical 

factors and the functional or structural as1Jects of the 

biological community. 

Difficulties of Biological Methods in 

Water-Quality Assessment 

Notwithstanding the obvious need for implementation 

of biological procedures in large-scale studies of water 

quality, it is clear that there are particular protiems that are 

likely to be associated with biological water-qualityassessment work. The heterogeneous nature of biological 

systems is among the most important of such problems. 

Biological variables can fluctuate widely ow~r space and 

time and are influenced by innumerable physical, chemical, 

and ecological factors (Hutchinson, 1953; Odum, 1969; 

Wallen and Botek, 1984). Furthermore, species distributions are extremely patchy (nonuniform), even within a 

single ecosystem (Odum, 1971, p. 205), and certainly over 

broad geographical areas. Different species respond very 

differently to particular environmental stimuli or stresses 

(Luoma, 1977). Biological variability severely limits universal applicability of native bioindicator organisms. It 

becomes very difficult to separate the effects of contaminants from natural variation, especially in comparisons 

among different aquatic systems. 

Problems of methodology are importrnt considerations in developing a biomonitoring program. Some 

biomonitoring methods are not well defined, tested, or 

verified. This is partly due to the biological variability and 

nonuniform species distribution already memioned. For 

some types of analyses (toxicity tests or bic~eochemical 

process measurements, for example), it is ext':'emely difficult to establish standardized procedures to be used in a 

consistent manner throughout a large-scale program. Even 

if a satisfactory procedure is available, the cos+ of applying 

it widely throughout a region can be prohit'~tive. Many 

types of biological analyses are labor intemjve. This is 

especially true for large-scale assessments, because natural 

variability requires that large amounts of data be collected 

to compensate for the variability. 

Purpose and Scope 

This report examines toxicity testing-just one of the 

different types of biological measurement that might be 

used for evaluation of water quality. The avera 11 purpose of 

the report is to evaluate the utility and feasibility of current 

toxicity-test methods for ambient water-quality assessments 

conducted on regional or national scales. Toricity testing 

has been used widely in specialized research projects, but 

certain limitations of current procedures cast some doubt on 

whether it can be applied successfully to large-scale waterquality assessments. 

2 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Specific questions addressed in this report include the 

following: 

1. What are the characteristics and applications of different 

types of toxicity tests? 

2. What are the advantages and disadvantages of different 

types of test procedures, particularly with reference to 

application in large-scale water-quality assessments? 

3. Do the results of toxicity tests accurately reflect environmental conditions and the probable effects of contaminants on biota in natural systems? 

4. Will different toxicity tests result in different conclusions about existing toxicant problems in the environment? 

5. Is there a particular type of test, with respect to selection 

of test species, test substance (ambient or artificial), and 

test medium (water or sediment), that can be applied in 

standardized format, with consistently reliable results, 

over a broad range of aquatic systems and environmental 

conditions? 

The evaluation of toxicity testing for water-quality 

assessment is based largely on review of existing information. This information includes background data about the 

current status of toxicological methods and toxicity-test 

results from published aquatic toxicological studies. Various types of toxicity-test designs are discussed, and criteria 

for selection of test organisms and testing procedures are 

identified. 

A great deal of information about procedures and 

applications of aquatic toxicity tests has been published in 

reports and technical papers in scientific literature (Kline 

and others, 1987). It is not the purpose of this report to 

review this literature exhaustively. Instead, the objectives 

are to summarize important concepts and conclusions that 

are contained in many past and current reports on toxicitytest applications and to consider the implications of these 

concepts for possible application of the methodology in 

large-scale projects. 

Detailed descriptions of methods also may be found 

in the literature. The appendix identifies some of these 

sources and includes a discussion of general methodological 

principles. 

The term "toxicity test'' as discussed in this report 

refers to any water-quality-assessment procedure that 

involves monitoring of responses of organisms to environmental stresses after exposure of the organisms to such 

stresses either in the natural environment or in controlled 

enclosures. The effects of the stresses are evaluated by 

monitoring an "endpoint" response. The endpoint may be 

mortality, or it may be a sublethal response. Toxicity tests 

have been used frequently in a wide variety of studies of 

pollutant impacts on aquatic systems. 

An "ambient" toxicity test is one in which the stress 

on the test organism is produced by exposure to a natural 

water or sediment sample, or an extract of such a sample. 

This differs from a more controlled experimental situation 

in which the test organisms are exposed to known concentrations of specific toxic agents. Ambient testing would be 

the method of choice if the results are to be used for 

assessment of existing water or sediment quality. 

The term "bioassay" commonly is used interchangeably with "toxicity test" in aquatic toxicological studies. 

Technically, the terms are not synonymous (Murty, 1986, 

p. 117). A toxicity test is used to determine the tm'icity of 

an agent to a test species. A bioassay test, like a chemical 

test, is used to measure the concentration of a chemical or 

effluent, using biological-response intensity as a means of 

quantification. By these definitions, "toxicity tesf' more 

closely signifies the procedure that is appropriate for a 

water-quality assessment in which ambient materials are 

examined for possible content of toxic agents.· Hence, 

"toxicity test" is the preferred term throughout the remainder of this report. 

Acknowledgments 

Consultation with many researchers contribu+~d substantially to the background information used for preparation of this report. Consultants included Peter Cha,man of 

EVS Consultants, Vancouver, B.C., Canadr: John 

Lawrence and Bernard Dutka of the Canadian National 

Water Research Institute; Teresa Norberg-King a'ld Alan 

Nebeker of the U.S. Environmental Protection Agency; 

John Cairns of Virginia Polytechnic Institute and State 

University; and numerous colleagues of the U.S. Geological Survey. 

BENEFITS AND LIMITATIONS OF 

TOXICITY TESTING 

Benefits 

Toxicity tests show directly how certain organisms 

respond to contaminants under certain conditions. They 

supply complementary data that can help fill sorre of the 

information gaps left by chemical analyses. 

There are some very compelling arguments for the 

use of toxicity tests in assessment of water qualit:' (Chapman and Long, 1983). Regardless of what levels of contaminants are found in the environment, their effects on 

biota are unknown without some biological measu~ements. 

Furthermore, chemical analyses, no matter how extensive, 

cannot include measurement of all possible toxic agents that 

may occur in the system. Not only do toxicity tests show the 

biological effects of specific contaminants, but t'ley also 

integrate the effects of combinations of conta111inants, 

including those that are not detected by established analytical methods. 

In most cases of environmental contamination, more 

than one toxic substance is present at concentrations greater 

Benefits and Limitations of Toxicity Teffing 3 
than background levels. The effects of combinations of 

toxic substances are likely to be different than the sum of 

their individual effects (Voyer and Heltshe, 1984). In cases 

of synergism, the total effect is greater than individual 

toxicities would suggest (Macek, 1975; Thompson and 

others, 1980; Hermens and others, 1984). Conversely, 

where antagonism occurs, the total effect is smaller than 

might be caused by the substances' individual effects 

(Bartlett and others, 1974; Christens~n and others, 1979; 

Hemelraad and others, 1987). Sequential exposure to two or 

more toxicants may sensitize biota so that they are more 

susceptible to damage after the initial exposure (Trevors 

and others, 1982). Conversely, the initial stress may stimulate protective mechanisms, such as production of 

metallothionein-like binding agents, that decrease the 

organism's susceptibility to further damage upon exposure 

to additional toxicants (Doherty and others, 1987). Mere 

detection of the toxicants reveals nothing about these kinds 

of interactions. 

Occurrences of environmental contaminants are further complicated by nonuniform spatial or temporal distribution. Intermittent releases into the environmen,t may 

occur, especially from point sources that discharge directly 

to the affected ecosystem (Elder and Dresler, 1988). Water 

concentrations of pollutants are especially subject to temporal variability because the water is mobile and contaminant inputs tend to be transported or diluted quickly. 

Sediments, as historical integrators of water quality (Feltz, 

1980), tend to accumulate substances from the overlying 

water, and are much less prone to show short-term temporal 

fluctuations in contaminant concentrations. However, sediments are likely to show considerable spatial variability of 

contaminant concentrations (Salomons and Forstner, 1984, 

p. 165). Chemical detection of contaminants is thus highly 

dependent on sampling time and frequency (in the case of 

water and suspended sediments) and sampling location (in 

the case of sediments). Certain toxicity tests, primarily 

those that are conducted in situ, may diminish this problem 

by integrating effects over time and space. 

Another reason for use of toxicity testing in waterquality assessment is the limited capability of chemical 

analysis to detect specific forms and degradation products 

of metals and organic compounds. Total toxicant concentration data can be misleading because the toxic effects can 

vary enormously depending on the speciation of the chemical (Diks and Allen, 1983; Mayes and others, 1985). 

Furthermore, chemical analyses may not show the products 

of degradation, which are likely to have different toxicological effects than those of the parent compounds (Mayes 

and others, 1985). 

Limitations 

There also are limitations to the use of toxicity tests. 

Principal among these is that it is extremely difficult, if not 

impossible, for toxicity-test models to truly mimic natural 

systems. Therefore, responses of selected tes .. organisms to 

contaminants in a controlled environment a~e unlikely to 

represent accurately the responses of a complex natural 

community to the same contaminant. Furtl ~rmore, it is 

unlikely that the biota in the natural system would be 

presented with the same simplicity of exrosure that is 

characteristic of the regulated and relatively constant conditions of a toxicity test. Because of this weatness, toxicity 

tests have questionable predictive value, and may even be 

misleading. 

Another limitation of toxicity testing is the difficulty 

of identifying cause-effect relations. Even if a test demonstrates toxicological responses of biota expos~d to ambient 

water or sediments, it cannot identify the subs<:ances or their 

concentrations that cause such responses. Ch~mical analyses are needed to identify possible toxic agents that are 

present in the system. The coupling of biological and 

chemical monitoring procedures to obtain complementary 

data has been effective in some studies (Pes~ah and Comwall, 1980; Long and Chapman, 1985). How~ver, identification of contaminant occurrence at elevated concentrations 

and simultaneous observation of abnormal responses of 

biota in bioassay tests do not necessarily demonstrate 

cause-effect relations. 

Most toxicity tests are conducted in er~losures outside the natural aquatic environment. Many of' the physical 

and chemical conditions within the test e:1.closures are 

controlled. Factors such as temperature, salinity, water 

hardness, pH, and photoperiod may vary f-om study to 

study. In some cases they are set to be ccnsistent with 

conventional experimental methods. In other cases they are 

set to mimic, as closely as possible, the natural environmental conditions of the test species. Contr,Jl of the test 

conditions is needed in order to interpret the results. 

However, the ambient conditions are like];' to have a 

significant effect on test results (Leeuwangh, 1978; Judy 

and Davies, 1979; Graney and others, 1984; Babich and 

Stotzky, 1985). Variability of uncontrolled test factors, 

such as bacterial activity, chemical speciation, and health of 

the test organisms, may increase further the variability of 

test results. 

Another cause of response variability is the wide 

variance of different species in their sensitivities and 

responses to any particular toxic substance (Plotkin and 

Ram, 1984; Phipps and Holcombe, 1985; Slooff, 1985). 

Even within a single species, there may t~ significant 

differences in sensitivities among individuals of different 

sexes, age groups, and genotypes (Adelmar and Smith, 

1976; Wright and Frain, 1981; Nebeker, Cairr<;, and Wise, 

1984; Woltering, 1984; Nebeker and others, 1985). Such 

biological non uniformity, compounded with t'--e variability 

due to test conditions, makes it very difficul .. to compare 

results from different studies. 

4 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Toxicity-test methodology generally calls for relatively standard formats for evaluating biological responses. 

In particular, the standard endpoints are concentrations that, 

in a specified time period, produce mortality in half the 

tested population ("50-percent lethal concentration," or 

LC50) or elicit an observable response in half the tested 

population ("50-percent effective concentration," or EC50). 

White and Champ (1984) criticized these endpoints, stating 

that they are arbitrarily chosen for the convenience of 

reporting results and have no demonstrated relevance to true 

hazard levels in the natural environment. Because of the 

dependence on test conditions, the 50-percent effective 

dose level may vary over several orders of magnitude. 

Hence, the toxicity-test results may have limited broadscale significance for human health or environmental preservation. However, despite the implications of their 

title-"The great bioassay hoax, and alternatives"-White 

and Champ ( 1984) did not demonstrate total uselessness of 

toxicity-test methods or applications. The authors did not 

deny that use of biological indicators can be a valuable tool 

to complement other kinds of data in an evaluation of 

environmental contamination. In fact, they suggested that 

toxicity studies can be designed and implemented so that 

they are useful, provided they meet the critical criteria of 

scientific soundness, adequate relation to natural systems, 

and relation to broad-scale processes. 

The practical and logistical difficulties of toxicity 

testing can be considerable. Most tests require an elaborate 

laboratory setup and specially trained personnel. Test 

organisms are often reared in captivity, which may itself 

introduce variables that can affect experimental results (Ten 

Berge, 1978; Goulden and others, 1982). Applications of 

toxicity-test procedures over a broad geographical area to 

assess regional or national water-quality problems require 

either the operation of numerous laboratories in different 

areas or transport of samples to a central processing 

laboratory. Either option poses logistical problems. 

One of the major difficulties with ambient tests is the 

establishment of control systems. Wong has pointed- out 

( 1984) that "a control medium can never be obtained since 

we can neither remove contaminants from ambient waters 

nor can we simulate water with identical chemistry." Even 

if simulation could be achieved, the conditions in ambient 

media are not static, and it would be impossible to simulate 

natural fluctuations. The usual solution to this problem is to 

avoid control systems altogether and depend either on serial 

dilutions ofthe ambient media (De Vries and Hotting, 1985; 

Gaur and Kumar, 1986) or on comparisons among samples 

from different sites (Long and Chapman, 1985; Mount and 

Norberg-King, 1985; Norberg-King and Mount, 1986) to 

evaluate relative toxicity. 

The limited capacity of toxicity testing to predict 

ecological effects of toxic agents within a complex and 

variable aquatic ecosystem was emphasized by Stumm and 

others ( 1983). The authors stressed a need to consider 

various processes, such as adsorption, atmc «.oheric 

exchange, microbial degradation, and chemical tran3formation, that affect the chemistry and biological availability of 

toxicants. They suggested that toxicity testing, even if 

combined with chemical monitoring, is not enough: meaningful information about environ~ental cycling of contaminants depends on modeling based on data that describe 

compound-specific variables (including solubility, vapor 

pressure, and lipophility); transformation processes, and 

spatial and temporal distribution of contaminant5' in the 

natural environment. 

Special Considerations for 

Large-Scale Toxicity Testing 

Toxicity testing on a regional or national scale would 

have special requirements distinct from those of tests 

conducted as part of small-scale, specialized stud:,~s. The 

most important consideration is that tests would be applied 

to a wide diversity of sample sites. Many different contaminants would be encountered; hence, tests would not be 

aimed at particular toxic compounds or elements. Environmental variables and biological communities als') would 

vary over broad ranges among different sites. There would 

be little value in designing a test that is representative of a 

particular community type because it would then fail for 

other community types. Single-species tests would have 

limited capacity to represent the diverse communities characteristic of the sample sites. 

The most important function of toxicity tests in a 

large-scale program would be to identify sites where indications of toxicity coincide with contaminant problems 

suggested by results of analyses and any other 1: iological 

monitoring that may- be done at the sites. The te~ts could 

serve as initial indicators, in which the results of te:'ts at any 

given site may determine whether or not more detailed 

monitoring or research at the site is advisable. 

In addition to being diverse, most of the sanple sites 

would be free of severe contamination. To assess tl ~quality 

of usable waters, the emphasis would be on natural waters 

rather than on effluents, leachates, or other dire~tly contaminated materials. For a toxicity test to be useful on 

natural water and sediment samples, it must be sensitive to 

relatively low concentrations of at least some contaminants. 

At the same time, the test should not be' so comr1ex, time 

consuming, or expensive that it could not be conducted on 

a large number of samples from widely dispersed locations. 

The broad geographical distribution of study sites in a 

large-scale project would almost certainly require shipment 

of samples to a central laboratory for analysis. Th~re would 

be a need to test for possible changes in toxicity characteristics of the samples during shipment. 

Benefits and Limitations of Toxicity Testing 5 
USES OF TOXICITY-TEST RESULTS 

Some possible uses of toxicity tests are shown in table 

1. The uses are listed in order of the probable risk of error; 

however, the absolute risk may vary considerably among 

different situations, owing to different kinds of restrictions 

presented by different cases. It is impossible to eliminate 

the risk of error entirely. Hence, there is always a dilemna 

in designing or interpreting a test. If the test is overextended 

(more is interpreted from the test results than the data can 

support), the amount of information produced may be 

impressive but there is substantial risk that much of it is 

erroneous. If the test is underextended, the risk of error is 

low but the amount of information generated may be so 

minor that the test was hardly worth the effort. 

Toxicity-test results provide information on the tox ... 

icity of particular contaminants to particular organisms 

under particular conditions. This can be valuable information if used in the proper context. However, extrapolation of 

the results to more general conditions may lead to erroneous 

or misleading interpretations. A few studies have demonstrated some of the difficulties in extrapolating toxicity-test 

results to predict toxicant effects in situations other than the 

specific tested case. Nevertheless, a certain amount of 

extrapolation may be valid. Chapman (1983) emphasized 

that existing laboratory toxicity-test data generally are 

inapplicable as precise indicators of toxic-effect levels in 

nature, although they have considerable capability for 

answering site-specific questions. Interspecific variation in 

sensitivity to toxicants should discourage most attempts to 

extrapolate results to nontested species. Nevertheless, 

LeBlanc (1984) pointed out that closely related species have 

similar sensitivities to most chemicals. It is reasonably safe 

to assume, for example, that a substance that produces a 

toxic response in bluegills will also have some toxic effect 

on large-mouth bass, but it probably would be invalid to 

assume similar toxicity to invertebrate species based solely 

on the bluegill results. As the breadth of extrapolation 

increases, so does the risk. 

Uses of toxicity-test results are determined in part by 

recognition of limitations of the tests balanced against the 

needs and possible benefits of· the tests. Because of the 

limitations, some water-quality researchers may be discouraged from including any kind of toxicity-testing procedures 

in their investigations. On the other hand, if toxicity-testing 

procedures are to be included, full awareness of their 

limitations will enable the researchers to minimize the 

detrimental effects of these limitations. 

Some of the important potential problems of toxicity 

testing can be avoided or diminished by initiating the study 

with a clear perception and statement of its purpose. The 

stated purpose should be adequately restrictive with respect 

to the possible applications of the test results shown in table 

1 so that the test is not overextended. In addition, the 

purpose should be suited to the needs and constraints of the 

Table 1. Some possible uses of toxicity tests 

[Listed in order of increasing probable risk of error] 

1. Identification of toxic conditions in waters or sediments 

without describing effects of those conditi"ns 

2. Verification of other assessment measurements 

3. Assessment of effects of toxic conditions on one or a 

few test species 

4. Prediction of effects of toxic conditions on one or 

a few test species 

5. Assess~t of effects of toxic conditions on entire 

coaunities 

6. Prediction of effects of toxic conditions on entire 

ca.unfties 

7. Establishment of environmental standards 

investigation and the study area. An appropriate statement 

of purpose, followed by execution of the study such that it 

fulfills the purpose, will do a great deal to minimize 

misinterpretation and perceptions that the te5't results are 

irrelevant or unimportant. 

PROCEDURES AND APPLICATIONS 

A wide variety of toxicity-test method' have been 

developed to fulfill the needs of diverse applic."~tions. Each 

test has particular purposes and limitations, and no test is 

universally applicable. The test methods may be distinguished primarily on the basis of four charac+~ristics: (1) 

test species, (2) endpoint (acute or chronic, arri variations 

of each), (3) test enclosure, and (4) test substan~e (toxicant) 

that acts as the environmental stress. Some asr~cts of each 

of these design characteristics are discussed here. 

Test-Species Selection 

The most important feature that distinguishes different toxicity-test methods is the selection of pla"'t or animal 

species to be used as indicators of contaminant effects. This 

is a necessary early step in nearly all toxicity-test procedures. Because of the difficulty of testing toxicity responses 

of all potentially affected organisms in the natural water 

body of interest, one or a small number of bioindicator 

species generally is used to represent a larger community. 

The selection of test species usually i~ based on 

several criteria related to the reliability of the o~ganisms as 

indicators and the feasibility of their use as captive organisms. Various authors have discussed important requirements for a species to be useful as a toxicity-te~t organism 

(Benfield and Buikema, 1980; Phillips, 198('; Nebeker, 

Cairns, Gakstatter, and others, 1984). Some requirements, 

or criteria for species selection, are shown in table 2, listed 

6 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 2. Criteria for species selection 

[Listed in order of decreasing estimated overall importance] 

1. Sensitivity: The organiSII should respond to a variety of conta11inants, 

at concentrations that 11ay be encountered in the natural environment and 

with an intensity of response that is related to conta11inant 

concentrati,on(s ). 

2. Representativeness: The organisll should respond to the contuinant in 

ways that characterize responses that could be expected fr011 a large 

nuaber of other species. It should not be prone to giving false 

positive or false negative results. 

3. Response detection: Responses or endpoints should be readily detectable 

and quantifiable. If life-cycle tests are used, life stages should be 

easy to identify. 

4. Allenabfl fty to laboratory culture: The organis11 should be adaptable to 

laboratory captivity without presenting unusual problMS for rearing or 

experi11entation. Control 110rtal ity should not be a proble11. 

5. Reproducibility of results: Repeated experi11ents should give uniform 

results, within acceptable error lf•its. There should not be a great 

deal of variability among individuals in their responses to 

contuf nants. 

6. Relevance: The organisll should have ecological or econ011ic significance 

because of its abundance, i11portance in the food web, or c01111ercfal 

importance. 

7. SiiiiPliCity of test: Toxicity-testing procedures should be simple and 

rapid. 

B. Short-duration life cycle: If life-cycle testing is to be done, the 

cycle should be short so that tests can be completed in reasonable time. 

9. Availability of background information: A data base of toxicity 

information, based on results fr011 previous work, should be available. 

10. Documented 11ethodology: There should be established and tested 

procedures for use of the species in toxicity tests. 

11. Biological uptake activity: The contuinant cannot directly affect the 

organis11 if it is not incorporated by the organis• in s011e way, either 

internally or externally. Therefore, bioaccUIIUlation or uptake rates 

should be relatively rapid. 

12. Low cost: Toxicity-testing procedures with the species should not 

preclude acc011plishing a •eaningful nu11ber of analyses. 

in order of estimated importance. Any species that does not 

meet the description given for a particular criterion is less 

than ideal as a test species with respect to that criterion. 

Among the great variety of aquatic floral and faunal 

species. a relatively small number have emerged as favorites in toxicological research. Most species simply do not 

meet enough of the requirements listed in table 2 to be 

considered as test organisms. Even among those that are 

acceptable, none of them would be considered exceptional 

with respect to all 12 criteria. Each species has particular 

characteristics that limit its use in certain applications, and 

no species clearly stands out as a "universal" indicator. 

Although the criteria are listed in table 2 in order of 

estimated overall importance, the priorities of a specific 

study may alter this order considerably. Hence, species 

selection depends to a large extent on the peculiarities and 

objectives of the study. 

Most of the species commonly used in freshwater 

toxicity tests discussed later in this report are listed in tables 

3 and 4. Their taxonomic lineages are shown in figure 1. A 

wide variety of taxonomic groups and trophic levels is 

represented. This variety of usable species enhances the 

potential usefulness of toxicity testing for characterizing 

aquatic commumttes; selection of a few test sper.ies may 

provide information about toxicant effects for a broad 

spectrum of organisms in the community. P<Jwever, 

because of practical and economic considerations, the scope 

of most studies is limited to one or two test speci,~s. 

Certain characteristics of taxonomic groups and individual species, including habitat, trophic level, economic 

importance, and tolerance ranges for environmental variables, influence the selection of toxicity-test specir.s. Information about these characteristics is given in tables 3 and 4. 

More detailed information can be found in the reference 

publications listed at the end of table 4. 

Species selections are made by toxicity-test researchers for various reasons. The reasons are not giver in most 

published reports, especially if the species is well known as 

a test organism. If there is an established preced~nt of its 

use for toxicity testing, then there is generally an implied 

assumption that its use is appropriate for the particular study 

being reported. However, many investigators do give 

explicit reasons for their choices of test species. Categorized in tables 5-10 are statements about particulrr species 

selections in a variety of published toxicity-test studies. 

The information shown in tables 5-10 originally was 

assembled to look for patterns of strengths and wr.aknesses 

of different species. For each species, it was expr.cted that 

authors would observe similar advantages and disadvantages with respect to a particular set of criteria. In other 

words, species were expected to be distinguishable in their 

patterns of strengths and weaknesses. In fact, the tables 

show little consistency in how species were rzted on a 

particular criterion. There was considerable overlap in 

stated advantages and disadvantages of species or related 

groups of species. For example, amenability to 1':\boratory 

culture (criterion 4) was considered an advantage of Daphnia magna by several authors (table 7), but an almost equal 

number of authors stated that this species was quit~ difficult 

to culture. Pimephales promelas (table 9) was se ~cted for 

many studies because of sensitivity, as expect~~d, but a 

number of other reasons were given, and no cle':\r pattern 

emerged about which of those might be most important. 

Criterion 6 (relevance) might be expected to be an important 

reason for selecting any fish species, but tables 9 and 10 do 

not give a strong suggestion that this criterion was more 

critical than many other possible reasons for sele~tion. 

Results in tables 5-10 indicate that certain criteria for 

test-species selection were considered much more frequently than others. Sensitivity (criterion 1) was nearly 

always considered, whereas other factors, such as responsedetection capability (criterion 3) or biological-uptake activity (criterion 11), were discussed infrequently. Th~refore, it 

is difficult to achieve a balanced view of the strengths and 

weaknesses of each species. Whatever the species selected, 

different authors tend to give the same reasonf for their 

selection, although there may be other important reasons 

that were not considered or mentioned. 

Procedures and Applications 7 
Table 3. Characteristics of some floral and faunal species frequently used in aquatic 

[Except for fish, common names are very general or nonexistent ("spp." indicates that various or unnamed 

indicated by "o"; absence of "o" signifies that the characteristic does not apply or that information is not 

Taxon011ic 

group 

Bacteria 

Protozoans 

Green algae 

Macrophytes 

Ne~~atodes 

01 igochaetes 

Cladocerans 

Allphipods 

Decapods 

Insects 

Mollusks 

Fish 

Scientific nue 

Photobacteriu• J!!!osphoreu• 

Spirillu• volunwns 

~spp. 

~ hydrophila 

~ capricornutu. 

Scenedes.us guaclricauda 

Chlorella stig•atophora 

Stigeocloniu• !!!!!!!! 

L"na spp. 

Eichhornia craSS!ipes 

Panaqrellus ~ 

Panaqrellus ~ 

Lf•nodrilus hoff'uisteri 

Tubifex tubifex 

Lu•briculus varieaatus 

Nais spp. 

Ilyodrilus spp. 

Daphnia !!!9!!! 

Daphnia ~!!!.!!:!. 

Daphnia pulicarta 

Daphnia laevh 

Ceriodaphnia reticulata 

~ lli!!ill!! 

Ganllarus ~!!!.!!:!. 

Hyallela ~ 

Chi ron011us ten tans 

Tanytarsus spp. 

Hexaqenia limbata 

~~ 

(or •anilensis) 

!!!!!f!!.!..i!! transversum 

Anodonta cyqnea 

Piuphales promelas 

Sal110 gairdneri 

Sa 1110 !!:!!lli 

~ill!:!!! 

Salvelinus ~ 

~~ 

~auratus 

~ punctatus 

Cypri nodon vari eaatus 

1Relative to other toxicity•test species. 

Wide to 1 erance rangel 

Sa lineo-on nue pH TetiP. D.O. ity 

parueciu. 

duckweed 

water hyacinth 

sludge worm 

water flea 

scud 

crayfish 

midge 

tanytarsus 

11ayfly 

Asiatic clu 

fingernail clu 

fathead 11innow 

rainbow trout 

brown trout 

cutthroat trout 

brook trout 

bluegill 

goldfish 

channel catfish 

sheepshead minnow 

Pred011inant trophic level 

Dec011- Auto- Hetero- l!'!rbi- Carni- Ollniposer troph troph v'lre vore vore 

2Nonll0bfle: have no anat011ical structures for loc0110tion; subject to transport by currents and waves. 

3

Epibenthic: lives and •ay 110ve about on surface of sediments. Infauna: burrows beneath sediment surface. 4

Asexual reproduction may occur. but is not necessarily the only uans of reproduction. 5

Supports other econ011ically important populations by ecological association, such as serving as food supply or creating 

shelter. 

8 Applicability of Ambient Toxicity Testing to National or Regiol!lal Water-Quality Assessment 
toxicity-testing procedures 

species of this genus are used). Characteristics of adult forms, based on the best information available, are 

available. "L" signifies larval or immature forms only. D.O., dissolved oxygen; temp., temperature] 

Habitat Reproduction Econotti c importance 

Water Benthtcl Short HUll an Support Detriaental 

life food of or 

cyc:le1 Asexua14 source resourceS nuisance 

Non- Weakly very Epf- InSc1ent1ftc: naae 110bt1e2 110bile 110bile benthic: fauna 

Photobacterf.,. l!!!osehor!!!• 0 

Sp1rf11.,.~ 0 

Pseudo!oftas spp, 0 

Aerollonas ~ 0 

9!!.!.2!!!!!!!. earaect ... 0 

Selenastru caer1comutu. 0 

Scenedes•s guadr1cauda 0 

~ stf!!llatO!!bora 

Stfgeoclon1u. !!!!!!! 

!:!!!!!! spp. 0 

~crasstees 0 

Panagrellus !:!!li!!.i'!!!! 0 

Panagre 11 us !!l!!!1.!! 0 

L f1111odrflus hoffMfsterf 0 

I!!!!!!!! tubffex 0 

Lu.brtculus varfeoatus 

Mats spp. 

Ilvodrflus spp, 

O.ehnta !!9!!! 0 

~2!!.!!!! 0 

l!!l!!!!!!! pulfcarta 

O.phn1a laevfs 

Cerfodaphnia !!!!£ 0 0 0 

~ !lli!1!.i! 0 

~I!!!!!! 0 0 

Hvallela ~ 

Orconectes .!!!!m.1! 

~tentans 

Tanvtarsus spp. 

Hexasenia li~ata 

Corbi cu 1 a !l!!!!!!!! 

(or !!!!1!!!!!1!) 0 

!!!!!sl!ll!!!! transversUII 0 0 

~eygnea 0 

!i!!P!!!!n erotelas 

!!!.!!!! sairdnerf 

!!!.!!!! !!:!!!!! 

!!!.!!!!~ 0 

Sa 1 ve 1i nus !!mll!!!!.i! 0 

.!.!1!!!!!! ucrochfrus 0 

~!!!!:lli! 0 

!E!!.!!!!:!!! punc:tatus 

Cxertnodon varteoatus 

Procedures and Applications 9 
PROCARYOTESPROTISTA (no true nucleus) 

BACTERIA 

EUCARYOTES-

(true nucleus) I 

PROTOZOANS: Chilomonas (Paramecium) 

ALGAE- CHLOROPHYTA: Selenastrum, Scenedesmus, Chlorella, Stigea~lonium 

(green algae) 

I ARTHROPODAN 

I 

CLADOCERA: Daphnia, Ceriodaphnia 

CRUSTACEA AMPHIPODA: Gammarus, Hyallela 

DECAPODA: Orconectes (crayfish) 

A 

N 

I 

M 

A 

L 

s 

v 

E INSECTA- DIPTERA: Chironomus, Tanytarsus 

R 

T 

E 

B 

R 

ANNELIDA OLIGOCHAETA: Limnadrilus, Lumbriculus, Tubifex, Nais, 1/yodri/us 

A 

T 

E 

s 

MOLLUSCA- PELECYPODA: Corbicula, Anodonta 

ATHERINIFORMES: sheepshead minnO\'' 

SALMONIFORMES: trout species 

CHORDATA- VERTEBRATA-- OSTEICHTHYES PERCIFORMES: bluegill 

CYPRINIFORMES: fathead minnow, golcfish 

SILURIFORMES: channel catfish 

PLANTS- SPERMATOPHYTAILemna 

Eichhornia 

Figure 1. Taxonomic lineages of commonly used freshwater toxicity-test species. 

The data for bacteria (table 5) are especially abundant, largely because this methodology has been developing 

rapidly and advantages are often discussed in support of this 

development. If table 5 represents an accurate appraisal of 

bacterial techniques, it is apparent why bacteria are attracting more users. Researchers are almost unanimous in 

believing that an important reason for using a bacteria test 

method is simplicity and rapidity (criterion 7). Low cost 

(criterion 12) also is mentioned frequently. Of greater 

significance, however, is the frequency with which criteria 

1 and 2 (sensitivity and representativeness) were given as 

advantages of bacteria tests. Investigations that compare the 

sensitivities of bacteria tests with those of eucaryotic 

species almost invariably show inferior sensitivity of bacteria. Nevertheless, many authors reported bacterial sensitivities for specific applications that are comparable to, or 

better than, those of other organisms. 

The frequent consideration of sensitivity in selecting 

a test species is especially intriguing. Sensitivity was 

mentioned as an advantage of the species chosen for many 

studies, regardless of what species it was, in spite of 

evidence (discussed later in this report) of wide discrepancies among species in their sensitivities to specific substances. The apparent contradictions illustrate that sensitivity evaluations should be cast in terms of the tcxic agents in 

question and that sensitivity should be compa .. ed with that 

of other species. 

Daphnia magna, for example, has been shown to be 

one of the most sensitive of common test spedes for most 

metals. However, it is not very sensitive to organic compounds, and its sensitivity varies considerabl~, among different classes of organics. Whatever the t0xicant, the 

sensitivity of Daphnia magna is based largely on comparison with sensitivities of other species to the srme toxicant. 

The outcome of this comparison is clearly dep~ndent on the 

species with which Daphnia is compared. 

The lessons from the sensitivity data in tables 5-10, 

therefore, are that (1) sensitivity is toxicant d~pendent and 

(2) sensitivity is usually assessed by comparing it with the 

sensitivity of other species rather than by comparing it with 

some absolute scale based on expected toxicant concentrations in nature. Every species listed in table 3 has been 

selected as a toxicity-test organism largely because its 

sensitivity was judged better than that of many other 

species, at least for some toxicants. Thus, the large number 

of favorable marks for criterion 1 in tables 5-10 should not 

be interpreted as an indication of good overall sensitivity to 

different types of toxicants or of good sensitivity to any 

10 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 4. Characteristics of flora and fauna used in toxicity tests 

BACTERIA 

PROTOZIWIS 

ALGAE 

NEMATOOES 

OLIGOCHAETES 

ClAIIOCEIWIS 

IIMPHIPODS 

DECAPOOS 

(Crayfish) 

INSECTS 

MOLLUSKS 

FISH 

Characteristics pertinent to use as test species 

Microscopic, unicellular, anat011ically si1111le (no true nucleus or nuclear -.brane; no 11itotic division). 

Biochl!tlistry of JUIIinescence or other endpoints is si11ilar to cytochrOIIe-linked respiratory chain e-n 

to other organisiiS. L.-inescence requires 1111ch energy; hence it is likely to be responsive to toxicants. 

Unicellular, but cellular organization is COIIPlete, like in 1111lticellular organisiiS. Cilia or flagella 

provide IIObility, but s111ll size aakes protozoans subject to transport by currents or wave action. 

~ oarnec:iUII, 110st c~ test species, injests no particulate food; utilizes dissolved organic 

utter to synthesize protoplas•ic substance. 

Cllllllonly used as indicators of water quality (Rawson, 1956; Pal~~er, 1969). l11portant ecological niche as 

priury producers at base of food web. Utilize dissolved substances, thus not affected by toxicants in 

sediments, except to eKtent that such pollutants are desorbed into water. Most frequently used test 

species is single-celled green algae (Chlorophyta). ~ capricornutUII, easy to culture, Identify, 

and quantify, is 1110ng IIOSt c-nly used of all test species. 

Larger plants, rooted or free floating. Le11111 and lli!!!!2!J!!! are both free floating, often in dense 

populations; can be nuisances by clogging waterways or causing oxygen depletions upon decay. Can also be 

beneficial as food and shelter for other organisiiS, and for contributions to photosynthesis and element 

cycling. Li11ited toxicological data available (s-tr, 1986). 

Extr-ly abundant and widely distributed in all kinds of aquatic systeiiS. Not widely used as test 

species; poor sensitivity to IIOSt toxic agents. 

Aquatic counterparts to terrestrial earthwor~~~. Many test species have been used, but none widely used. 

Relative to arthropods, tend to be 110re tolerant of pesticides, but less tolerant of toxic etalS 

(Brinkhurst and Cook, 1974). ~ ~ tolerant of unfavorable environmental conditions; hence 

usually considered a pollution indicator. 

Extr-ly c011110n in freshwater systtiiS. filter feeders. Tend to be 110re sensitive to ~~etals than to 

organics. Life cycle includes instars, separated by 110lts. Daphnia !!9!!! 1101t c-nly used of all test 

species. Ceriodaehnia ~ distinguished from other cladocerans by s111ll size, short life cycle, and 

eo1111on occurrence in a variety of freshwater habitats. 

!!!!!!!:!!! species 1110ng IIOSt c011110nly used test organisiiS, especially for sedf~~ent-toxicity tests. Life 

cycle includes instars, separated by 1101ts. 

Widely distributed, especially in Southeastern United States. Life cycle includes instar stages, separated by 

110lts. During 110lts, aniaals are 110re sensitive to toxicants (Hobbs and Hall, 1974). ~ 

i111111nis, an active burrower, inhabits sluggish streus and ponds. 

Extre..ly adaptive to all kinds of environ~~ents. Great diversity reflects environmental conditions; 

hence useful bioindicators. Chiron011idae is one of largest flllflies--widely distributed and often 

eKtrelll!ly abundant (up to 50,000 per square 11eter). Difficult to identify Chiron011id species. 

Extr- econ011ic importance, both beneficial and detrillental. Filter-feeding bivalves (cliiiS 

and 1101lusks) IIOSt e-n test species. ~ larval stages are ciliated and free swiMing, 

unlike IIOSt other bivalve species. ~ can exploit nearly any type of substrate. 

As the only vertebrates COIIIIOIIly used in toxicity tests, fish represent higher trophic levels than 

other test species. Eggs or early life stages usually 110re sensitive to toxicants than adults. 

Contain high lipid concentrations [up to 15 percent of total body weight (Nfi11i, 1983)]; hence 

hydrophobic substances, pri11arly organics, readily accu11ulate in fish tissue (Chiou, 1985). Extre~~e 

110bil ity often allows escape fro11 toxic sources in natural systtiiS. Pi!I!!J!hales prlllltlas used 

extensively as toxicity test species, frequently as basis for setting aaxi11u11 tolerance 1f11its. 

References, for 110re inforllltion: 

ward and Whipple. 1959 (invertebrates, .acrophytes) 

Meglitsch, 1967 (invertebrates) 

Prescott, 1970 (algae) 

Brinkhurst and Cook, 1974 (oligochaetes) 

Mitchell, 1974 (bacteria) 

P-ak, 1978 (invertebrates) 

Brock, 1979 (bacteria) 

Anderson, 1980 (chiron011ids) 

Arthur, 1980 (IIIPhipods) 

particular toxicant at naturally occurring or even maximum 

allowable concentrations. 

Acute Sensitivities of Test Species 

Sensitivity is the primary factor that determines the 

usefulness of a test species (table 2). However, it is not a 

simple matter to select the most sensitive indicator organism 

for every test situation. It is especially difficult for ambient 

Benfield and Buikl!tll, 1980 (invertebrates) 

Bitton, 1982 (bacteria) 

Bone and Marshall, 1982 (fish) 

Allerican Public Health Association and others, 1985 

Hobbs and Hall, 1974 (crayfish) 

Fuller, 1974 (bivalves) 

Roback, 1974 (insects) 

Rheinhei~~er, 1974 (bacteria) 

tests in which the test substance may contain several toxic 

agents. Not only is sensitivity dependent on the toxic agent, 

but there is little information available on which to base 

evaluations of the relative sensitivities of species to specifit! 

agents. This is especially true for chronic tests t'~cause of 

the wide variety of monitoring procedures and endpoints. 

Some data have been compiled in tables 11-19 to 

compare acute sensitivities of different species to certain 

Procedures and Applications 11 
Table 5. Evaluation of bacteria species commonly used in toxicity tests 

with respect to selection criteria listed in table 2 

[Data are from published studies in which indicated species were used or discussed. Ratings 

with respect to numbered criteria are based on statements by authors: "+ ," advantage of this 

species over other commonly used species; "-," disadvantage of this species; "±," 

advantageous in some cases, disadvantageous in others (for example, species may be 

sensitive to some toxicants and insensitive to others). Absence of symbol indicates that 

criterion was not mentioned. 

Toxicant codes: "var," various; "0," organics. in general; "M," metals; "E," effluents; "N," 

natural sediments or water. 

In all cases. contaminant effects are detected by measuring changes in luminescence or other 

metabolic functions] 

Species Toxicant(s) 

Bacteria (var. species) 

Bacteria (var. species) var 

Bacteria (var. species) var 

PhotobacteriUII l!hOSI!horeUII 

Photobacterillll ffscheri + 

Photobacteriull l!hOSI!horeull 0 + 

Photobacterillll I!!!OSI!horeua M,O,E ! 

Bacteria (var. species) var + 

Photobacteriu• 1!hosl!hore1111 M,O + 

PhotobacterfUII l!hOSI!horeUII M,O 

PhotobacteriUII l!hosl!horeu• 0 + 

PhotobacteriUII l!hOSI!horeUII var 

Photobacteriull l!hOSI!horeum var + 

Sl!irfllllll voluntans var + 

Photobacteriua l!hoSI!horeUII 

Pseudomonas I!Utida M,O 

Criterion nuaber 

2 3 6 8 9 10 11 12 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Reference 

Greene & others, 1985 

Berkowitz, 1979 

8ulich, 1979 

Dutka & Kwan, 1981 

Lebsack & others, 1981 

Curtis & others, 1982 

Qureshi & others, 1982 

Bitton, 1982 

Oe Zwart & Slooff, 1983 

Mcfeters & others, 1983 

Ribo & Kaiser, 1983 

Vasseur & others, 1984 

Coleten & Qureshi, 1985 

Cole~~~an & Qureshi, 1985 

Schiewe & others, 1985 

Slabbert, 1986 

Table 6. Evaluation of algae species commonly used in toxicity tests with 

respect to selection criteria listed in table 2 

[Data are from published studies in which indicated species were used or discussed. Ratings 

with respect to numbered criteria are based on statements by authors: "+,"advantage of this 

species over other commonly used species. Absence of symbol indicates that criterion was not 

mentioned. 

Toxicant codes: "var," various; "M," metals; "PC," petroleum or coal tar derivatives; "OH," 

organic herbicides; "N," natural sediments or water; "0," organics, in generaL 

In aU cases, contaminant effects are detected by measuring changes in growth, productivity. 

or other metabolic functions] 

Criterion number 

Species Toxicant(s) 1 2 3 4 5 6 7 8 9 10 11 12 Reference 

Selenastr1111 capricornutu• var Payne & Hall, 1979 

(and other species) 

Se 1 enastrum capri cornutum M 

Selenastrum capricornutum PC 

Scenedesmus guadri cauda OH 

~ capricornutum 

Selenastrum capricornutum 

Stigeocloni1111 tenue 

Selenastr1111 capricornutum OH 

Selenastru• capricornutum 

(and other species) 

organic compounds and metals in water. Despite the fact 

that methods and test conditions vary, single-species acute 

toxicity tests provide data in a standard format for specific 

toxic agents. Other kinds of tests do not produce this kind of 

comparable information. Therefore, results of chronic tests, 

or results from studies that were done with ambient subChristensen & others, 1979 

Giddings & others, 1983 

Aly & others, 1984 

Eloranta & Halttunen-Keyrilainen, 1984 

Adams, Goulding, & Dobbs, 1g95 

De Vries & Hotting, 1985 

Meyerho~f & others , 1985 

Gaur & Kumar, 1981 

stances or variable mixtures of substances (such rs leachates 

or effluents), could not be included. 

The endpoint for all animal species was mortality, 

and the results were reported as LC50, in milligrams per 

liter. There was some variation in exposure time, but in 

most cases it was 96 hours for fish, 48 hours fer cladocer12 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 7. Evaluation of Daphnia magna with respect to selection criteria 

listed in table 2 

[Data are from published studies in which Daphnia magna was used or discussed. Ratings 

with respect to numbered criteria are based on statements by authors: "+ . " advantage of this 

species over other commonly used species;"-," disadvantage of this species. Absence of 

symbol indicates that criterion was not mentioned. 

Test type codes: "A," acute; "C," chronic. 

Toxicant codes: "var," various; '"0," organics, in general; "M," metals; "OP," organic 

pesticides; "N." natural sediments or water] 

Criterion number 

Test 

type Toxicant{s) 1 2 3 4 5 6 7 8 9 10 11 12 Reference 

AC var + + 

A var + + 

AC var + + + 

A 0 + 

A var + 

AC M,OP 

A 0 

A M,O + + 

AC var + 

ACS N + + 

A M,O + 

+ + 

+ 

+ + 

+ 

- + 

+ 

Adema, 1978 

Kenaga, 1978 

Ten Berge, 1978 

Oill & others, 1982 

LeBlanc, 1980 

- Nebeker, 1982 

Barera & Adams. 1983 

8erglind and Dave. 1984 

- Mount & Norberg, 1984 

+ Nebeker & others, 1984 

lewis & Weber, 1985 

Table 8. Evaluation of Cladocerans, excluding Daphnia magna, commonly used in toxicity tests with respect to selection criteria listed in 

table 2 

[D., Daphnia; C., Ceriodaphnia. Data are from published studies in which indicated species 

were used or discussed. Ratings with respect to numbered criteria are based on statements by 

authors: "+." advantage of this species over other commonly used species; "-," disadvantage of this species. Absence of symbol indicates that criterion was not mentioned. 

Test type codes: "A," acute; "C," chronic. 

Toxicant codes: "var," various; '"0," organics, in general; "OP," organic pesticides; "M," 

metals] 

Test 

Species type Toxicant(s) 1 2 3 

Jl.~ A var + + 

var A var 

var A chroniUII + 

Daphnia spp, AC var 

Jl. pulfcaria A 0 

f. retfculata c var 

Jl. laevis A OP 

Jl. ~!!!..!!! A M.O 

f. retfculata A M.O 

ans, and either 48 or 96 hours for other invertebrates. For 

bacteria, the endpoint was 50-percent reduction in luminescence, and for algae, it was 50-percent reduction in growth 

or production. Although different from mortality, these 

standardized endpoints for bacteria and algae are well 

established and may be compared with animal LC50's for 

the purpose of comparing sensitivities. 

Criterion nu11ber 

4 5 

+ + 

+ -

+ 

6 7 8 9 10 11 12 Reference 

Kenaga. 1978 

+ leeuwangh. 1978 

+ + + + lee & Buikema. 1979 

- Buikema & others. 1980 

DeGraeve & others. 1980 

+ Mount & Norberg. 1984 

+ Foran & others. 1985 

lewis & Weber. 1985 

Elnabarawy & others. 1986 

The data in tables 11-17 indicate some patterns of 

sensitivity among different species. and they also raise 

some questions. Bacteria are apparently sensitive to organics under some conditions, but they are much less useful for 

testing metal toxicity. Mercury is a possible eJfCeption; it 

produces responses in Photobacterium phosptwreum at 

relatively low concentrations (table 18). Cladocerans are 

Procedures and Applic~tions 13 
Table 9. Evaluation of Pimepha/es promelas (fathead minnows) with 

respect to selection criteria listed in table 2 

[Data are from published studies in which Pimephales promelas was used or discussed. 

Ratings with respect to numbered criteria are based on statements by authors: "+" advantage 

of this species over other commonly used species. Absence of symbol indicates that criterion 

was not mentioned. 

Test type codes: "A," acute; "C," chronic. 

Toxicant codes: "M," metals; "0," organics, in general; "var," various; "E," effluents] 

Criterion number 

Test 

type Toxicant(s) 1 2 3 4 5 6 7 8 9 10 11 12 Reference 

AC M,O + + Adelman & Smith, 1976 

c var + + + McKim. 1977 

A 0 + + + Spehar & others, 1982 

A E + + + Keefe & others, 1983 

c toluene + + + + Devlin & others, 1985 

c E + + + + + + Norberg & Mount, 1985b 

Table 10. Evaluation of fish species, excluding Pimephales promelas, 

commonly used in toxicity tests with respect to selection criteria listed in 

table 2 

[Data are from published studies in which indicated species were used or discussed. Ratings 

with respect to numbered criteria are based on statements by authors: "+ , " advantage of this 

species over other commonly used species. Absence of symbol indicates that criterion was not 

mentioned. 

Test type codes: "A," acute; "C," chronic. 

Toxicant codes: "var," various; "M," metals; "0," organics, in general; "E," effluents] 

Test 

Fish type Toxicant(s) 1 2 3 

Rainbow trout A var 

Sal-ids (various) A M 

Rainbow trout A phenolics 

Bluegill c var 

Rainbow trout A 0 

Bluegfll A + + 

generally among the most sensitive species to metals. Some 

of the less commonly used invertebrates, such as Corbicula 

species and some of the amphipods and oligochaetes, show 

relatively high sensitivity to some substances. 

Among the peculiarities shown by the tables are the 

wide ranges of sensitivities, even within a single species. 

For example, there was a 100-fold range in sensitivity to 

benzene by Photobacterium phosphoreum in different studies (table 13). A similar range appeared in the sensitivity of 

Daphnia magna to toluene (table 14), and the overall range 

in sensitivity to toluene was nearly 3,000-fold among just 

six studies. Fish species generally are considered especially 

good indicators for organic contaminants, but this contention is not strongly supported by tables 11-14. None of the 

tables reveals a clearly superior species in terms of its 

sensitivity to that substance. 

Criterion nuaber 

4 5 6 7 8 9 10 11 12 Reference 

Kenaga. 1978 

Davies & Woodling. 1980 

Oe6raeve and others. 1980 

van der Schalie. 1980 

Di 11 & others. 1982 

Keefe & others. 1983 

Another disturbing aspect of sensitivity r1~vealed by 

tables 11-19 is that even the best sensitivities shewn are not 

indicative of truly useful bioindicators. The lowest concentrations shown for each of the toxicants represented are 

higher than any that would be encountered in m 'Jst aquatic 

systems, except for highly contaminated waters. An illustration of this point is shown in figure 2. The LC50 values 

for copper, cadmium, zinc, mercury, and lead are taken 

from tables 15-19. Thus, they represent lethal concentrations in various acute tests, using different test Sfi'!Cies. The 

actual concentration data, shown by the histograms, are the 

maximum and median total concentrations of the same 

metals at some sites around the country that have been 

monitored by the U.S. Geological Survey (USGS) for 

several years. These sites are subject to inputs from various 

industrial, agricultural, or municipal sources and may be 

14 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 11. Acute toxicities of phenol to various test species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes] 

Endpoint 

Exposure concentration 

Species time (milligrams Reference 

per liter) 

DaJ!hni a magna 48 h 6.6 Keen and Baillod, 1985 

DaJ!hnfa magna 48 h 12 leBlanc, 1980 

Photobacterium l!hOSJ!horeum 5 II 22 Qureshi and others, 1982 

Photobacteri um 1!hos2horeum 5 II 25 Lebsack and others, 1981 

Photobacterium l!hOSI!horeum 5 II 25 Bulich and others, 1981 

Photobacteri um l!hOSJ!horeum 5 II 26 Chang and others, 1981 

Photobacteri um l!hOSI!horeum 5 II 28 Dutka and others, 1983 

Da2hni a magna 48h 30 Bobra and others, 1983 

Photobacterium J!hOSI!horeum 15 II 34 Dutka and Kwan, 1981 

Photobacteri Ull J!hOSJ!horeum 5 II 39.5 Mcfeters and others, 1983 

Photobacterium l!hOSJ!horeum 5 II 40.2 Curtis and others, 1982 

Pi11e2hales 2romelas 96 h 67.5 DeGraeve and others, 1980 

Da2hnia pulfcaria 48 h >109 DeGraeve and others, 1980 

Pseud0110nas 2utida 6 h 244 Slabbert, 1986 

expected to contain higher metal concentrations than most 

natural waters. Nevertheless, nearly all the maximum concentrations of cadmium, mercury, and lead reported from 

these sites are lower than the LC50 values shown, and many 

are lower by a factor of 10 or more. Even for copper and 

zinc, the natural concentrations are lower than most LC50 

values shown. Although sublethal effects or lower percentage mortality may occur at lower concentrations than the 

LC50 values shown in figure 2, these data cast serious 

doubt that most current test procedures would have adequate sensitivity to reveal long-term toxicant conditions in 

ambient waters. 

Figure 2 also compares LC50 data with water-quality 

criteria established by the U.S. Environmental Protection 

Agency ( 1986). Acute criteria are concentrations likely to 

be detrimental to aquatic life if exceeded for a 1-hour period 

at least once during 3 years, on the average. Chronic criteria 

are concentrations likely to be detrimental to aquatic life if 

exceeded for a 4-day period at least once during 3 years, on 

the average. The acute criteria are lower than nearly all of 

the LC50 values given. The chronic criteria are even further 

below the detection ranges for the toxicity tests; they are 

less than LC50 values for all methods shown except zinc. 

Overview of Test-Species Selection 

The preceding discussion of test species and criteria 

for their selection does not lead to clear choices of species 

to use for a large-scale water-quality-assessment program or 

for any particular study within such a program. It does 

include information that should be considered before selecting species. The selections themselves would necessarily 

depend on conditions of the test and application requirements, which would vary from study to study. 

Certain criteria from table 2 are especially important 

for selection of procedures to investigate potential ambient 

toxicity problems over broad geographical areas. These 

include amenability to laboratory culture (criterion 4), 

reproducibility of results (5), simplicity of test (7), availability of background information (9), documented methodology (10), and low cost (12). All of these factors relate 

to the practicality of using the species in a standard fashion 

in a variety of different test waters, and (or) to the 

interpretability and comparability of data obtained in this 

way. Presumably, therefore, these criteria would move up 

on the scale of relative importance in selecting test species 

for a national program. 

Sensitivity (criterion 1) remains a very important 

criterion for test-species selection in large-scale ambient 

toxicity assessments. If the test is so insensitive that toxicity 

detection is not likely even with heavily contaminated 

samples, then it is of little use as a biomonitoring tool, 

regardless of its other attributes. Representativeness (criterion 2) also is a very important selection criterion if there is 

any expectation of analyzing the biological implications of 

contaminant occurrences. 

The test species selected should suit the stated purpose of the test. For example, if rainbow trout is the key 

species in the study area that might be affected by a 

contaminant input, then rainbow trout would be the logical 

choice as a test species. Most selections are likely to be far 

less obvious, but consideration of the advantages and 

disadvantages of different test species should improve the 

likelihood of success. 

In general, any toxicity-testing procedure that uses 

only one test species is not appropriate for determining the 

effects of the broad array of contaminants that may be 

present over a large geographical area. The current status of 

toxicity-test methodology and information about sensitivities of test species indicate that no single-species test has the 

general applicability and uniformly high sensitivity required 

for ambient toxicity testing on a regional or national scale. 

There are greater possibilities if more than one species can 

be used; more discussion of this option follows. 

Acute and Chronic Tests 

One of the most important methodology decisions to 

be made in designing a single-species test is whether testing 

Procedures and Applications 15 
1070 

416 477 

100 218 

10 Cu Cd Zn Hg - Pb 

a: 

w 

.... 

::::; 

a: 

w v 0.. v C/) 1.0 ~ 

< a: 

~ 

::::; 

-I v ~ v 

~ v 

v 

z 0.1 

0 I 

~ ~ 

v: ,:. 

a: - v~-

.... :_ ... vI' 

z 

w v; 

(.) -_ ; '- z A 

0 c .c- :: .i (.) 0.01 -

- v-,. 

::··· ~I I 

/ 

- v ~-

I 

_- ... v' 0.001 

I_ 

!= 

= 

~ 

-

I 

I ' v', 

v \~ 

A - - v> 

::_ v ~ 

-

-· v: ~ 

-- -, 

v 

--

- ~ I -

- ' - ~ 

A ·- I 

c - ;; 

:: ! 

=r+- ~ f v ,' 

:: ~ -

::: :;, 

= ~ ~~ - ~ 

:::- v ~ 

::: I I 

= v~ -- 1/ 

:::-- :·· ~l 

A 

v ·i . . ~ 

I 

,, 

-~ [;:: ~ 

t:; ~ [;::.: 

A /' ' .c-

- [;:: ,- -· IV ~ -

- f:::'• IV' - r:;~. t:; _I 

~ vi 

~,-, 

ff, IV I , 

-I ~ 

~:~ c 'I 

- ~> = ' - /: 

0.000012 

EX PLANA liON 

Bar Site 

shading number 

11!!1 01474500 

G 05543500 

0 07373420 

0 06438000 

Gl 11074000 

Site 

name 

Schuylkill A iver at Philadelphia, Pennsylvania 

Illinois River at Marseilles, Illinois 

Mississippi River near St. Francisville, Louisiana 

Belle Fourche River near Elm Springs, South Dakota 

Santa Ana River below 

Prado Dam, California 

Period of 

record 

10/77-9/82 

10/79-9/86 

10/lll-9/86 

10/79-9/84 

10/79-9/86 

LC50 data 

(from Tables 15-19) 

Maximum 

U.S. Geological Survey 

water-quality 

monitoring site data 

Figure 2. LCSO (50-percent lethal concentration) data from tables 15-19 compared with maximum and 

median concentration data for copper, cadmium, zinc, mercury, and lead in water from five selected 

U.S. Geological Survey water-quality monitoring sites and water-quality criteria. 

should be done by acute lethality tests or chronic exposure 

tests. 

In an acute test, the test organisms are exposed to 

relatively high concentrations of the contaminant, and the 

test is concluded in a short time (usually a few days). The 

common endpoint of such tests is mortality, measured as 

LC50, which is the minimum concentration that causes 

50-percent mortality of test organisms during a specified 

time period (usually 48 or 96 hours). The test generally is 

done with a single species in small enclosures in a laboratory. In the simplest method, the "static" acute test, the 

medium and its toxicant content are not changed during the 

experiment. Alternatives to this method are "staticrenewal," "flow-through," and "continuous-flow" tests, in 

16 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 12. Acute toxicities of pentachlorophenol to various 

test species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes] 

Endpoint 

Exposure concentration 

Species tille (milligra~~s Reference 

per liter) 

.!5!!!!!!:!!! ounctatus (catfish) g5 h 0.053 Phipps and Holcombe, 1g55 

Photobacterfu. phosphoreu• 5 II .08 Curtis and others, 1982 

Salmo gairdneri 96 h .093 McKi11 and others, 1987 

!!!!.~ 96 h .11 Chaptllan and Mitchell, 1986 

!!!!!! gairdnerf 96 h .us Thurston and others, 1985 

!:!~!!!!!! ucrochfrus 96 h .14 Phipps and Holcombe, 1985 

Daphnia !!9!!.! 48h .143 Mount and Norberg, 1984 

P!R!m!! !!9!!.! 48h .145 Thurston and others, 1985 

~ ~ (goldfish) 96 h .16 Phipps and Holc011be, 1985 

Cerfodaphnia reticulata 48h .164 Mount and Norberg, 1984 

~auratus 96 h .190 Adelman and others, 1976 

!!!!!! !!:!!!!! 24 h .2 Hattula and others, 1981 

.!:!1!!!!!! ucroch i rus 96 h .202 Thurston and others, 1985 

Ptmephales pra~~elas 96 h .203 Adel11an and others, 1976 

Daphnia P!!.!!! 48h .246 Mount and Norberg. 1984 

Pille!)hales I!!J!!!!!!! 96 h .25 Phipps and Holc011be, 1985 

£!!:!!!!!!!. auratus 96 h .264 Thurston and others, 1985 

Pi!!!l!hales pra~~elas 96 h .266 Thurston and others, 1985 

IlYOCirflus .tt!!!!!! 96 h .31 Chapman and Mitchell, 1986 

Daphnia !!9!!.! 48h .33 Lewis and Weber, 1985 

l i~rflus hoff11eisteri 96 h .33 Chapman and others, 1982a 

Tubifex tubtfex 96 h .38 Chapman and others. 1982a 

Daphnia pulex 48h .39 Lewis and Weber, 1985 

Photobactert u• phosphoreum 5 II .s Bulich and others, 1981 

Daphnia •asna 48h .68 LeBlanc, 1980 

Photobacteriu• phosphore.- 15 II .76 De Zwart and Slooff, 1983 

Photobactert • phosphoreum 5 II .94 De Zwart and Slooff, 1983 

Tanxtarsus dissi•ilis 48h 25.2 Thurston and others. 1985 

Pseudomonas fluorescens 1 h 29.2 Trevors and others, 1982 

Orconectes .!.!!!!!!!! 96 h 183 Thurston and others, 1985 

which the medium and its toxicant load are continually or 

periodically replenished as spent medium flows from the 

test enclosure. 

The LC50 concept is relatively simple and is widely 

used. Lethality is an easily monitored endpoint, and LC50 

provides a convenient, standardized, and unambiguous 

format for reporting toxicities. However, it has been argued 

that LC50 is an arbitrary and meaningless standard that is 

irrelevant to the natural environment (White and Champ, 

1984). Certainly, it is an indicator of the toxicity of a 

particular substance to a particular organism under the 

conditions of the test; however, it is not at all clear what that 

Table 13. Acute toxicities of benzene to various test 

species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. P'ldpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes] 

Endpoint 

Exposure concentration 

Species time (milligraiiiS Refert,.ce 

per liter) 

Photobacteriu• J!!!osphoreum 5 II 2.0 Bulich and others, 1981 

Photobacteriu. g!!osJ!!!oreum 5 II 4.11 McFeters and others. 1983 

Daphnia magna 48h 31.3 Bobra and ott ers, 1983 

Photobact!ri u. phosphoreu• 5 II 200 Chang and ott ers, 1981 

Oaphnt a •asna 48h 200 LeBlanc, 198(' 

Photobacteri Ull phosphore• 5 II 214 De Zwart and Slooff. 1983 

Photobactert • 1!!!2!phoreum 15. 238 De Zwart and Slooff, 1983 

means in terms of overall toxicity of the substance in the 

natural environment. Some reports show a good C'lrrelation 

between LC50 and measures of natural toxica'lt effects 

(Mount and others, 1984; Giddings and Frano1, 1985), 

whereas others show a poor relation (Rodgers and others, 

1980; Kimball and Levin, 1985). Numerous aut'--ors have 

cautioned against assumptions of community toxicity based 

on measurements with a single species or proces~ (Cairns, 

1983; Dutka and others, 1983; Blanck, 1984; Bowmer, 

1986). 

Table 14. Acute toxicities of toluene to various te~t species 

[Species listed in decreasing order of reported sensitivity (incr~asing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mezn values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured actiYity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h. hours; m, 

minutes] · 

Endpoint 

Exposure concentration 

Species time (11illigra1115 

per liter) 

Daphnia •asna 48h 11.5 

Pf!!!phales pro~~elas 96 h 26 

PhotobacteriUII ehosphoreua 5 II 50 

Pi!l!l!hales promelas 96 h 63 

(etlbryos) 

Dapnfa !!!9!!! 48h 310 

Photobacteriua phosphoreu• 5 II 33,833 

Reference 

Bobra and otr-ters , 1983 

Devlin and o":hers, 1982 

Chang and otr-ters, 1981 

Dev11n and o":llers, 1982 

LeBlanc. 1981 

Mcfeters and others, 1983 

Procedures and Applica•'ons 17 
Table 15. Acute toxicities of copper to various test species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes] 

Endpoint 

Exposure concentration 

Species time 

Ceriodaphnia reticulata 48 h 

Daphnia !!!91!! 48 h 

Corbicula manilens is 24 h 

(veliger larvae) 

Corbicula flu11inea 96 h 

Daphnia ~ 48 h 

Daphnia !!!91!! 48 h 

Se 1 enas tru11 capri cornu tum 

Chlorella sti!J!IIatophora 

Se 1 en as tru11 capri cornu tum 

Selenastru11 capricornutum 

Lumbrkulus variegatus 96 h 

Lumbriculus variegatus 48 h 

Salmo gairdneri 96 h 

Chironomus 1!n!!!!!. 96 h 

(1st instar) 

Corbicula flu11inea 24 h 

LePOtlis macrochirus 96 h 

Pseudomonas putida (bact.) 6 h 

Crangonyx pseudogracilis 96 h 

(amphipod) 

Corbicula 11anilensis 96 h 

(adult) 

Photobacteriull phosphoreum 15 11 

Spiril lu11 voluntans 5 m 

Photobacteriull phosphoreu11 5 11 

Photobacterium phosphoreum 5 111 

Asellus aquaticus 96 h 

(amphipod) 

Photobacteriull phosphoreum 5 11 

(milligrams 

per liter) 

0.017 

.02 

.028 

.04 

.053 

.054 

.054 

.07 

.07 

.085 

.15 

.23 

.25 

.30 

.59 

1.0 

1.05 

1.29 

2.6 

3.8 

7. 4 

7.4 

8.0 

9.21 

25 

Reference 

Mount and Norberg, 1984 

Qureshi and others, 1982 

Harrison and others, 1984 

Rodgers and others, 1980 

Mount and Norberg, 1984 

Mount and Norberg, 1984 

Turbak and others, 1986 

Christensen and others, 1979 

Bartlett and others, 1974 

Christensen and others, 1979 

Bailey and Liu, 1980 

Bailey and Liu, 1980 

Qureshi and others, 1982 

Nebeker, Cairns, and Wise, 

1984 

Rodgers and others, 1980 

Tho~apson and others, 1980 

Slabbert, 1986 

Martin and Holdich, 1986 

Harrison and others, 1984 

Dutka and Kwan, 1981 

Qureshi and others, 1982 

Qureshi and others, 1982 

Bulich and others, 1981 

Martin and Holdich, 1986 

Mcfeters and others, 1983 

In a chronic test, the organisms are exposed to 

nonlethal concentrations over a relatively long period of 

time. Long-term exposure may produce some mortality, but 

the endpoint of the experiment is some sublethal response 

such as decrease in growth rate, reduction of reproductive 

capacity, interference with mobility, or anatomical change. 

The results yield information about "effective" concentrations of the toxic agent rather than the lethal concentrations 

determined by acute tests. Sublethal responses might not 

occur during a short-term test. For example, Winner (1981) 

found that copper and zinc affect the longevity of Daphnia 

magna, but those effects did not appear until some 50-70 

days after initiation of the experiment. Many chronic test 

endpoints can be determined only by monitoring the complete life cycle of the organism (Goodman and others, 

Table 16. Acute toxicities of cadmium to various test 

species 

[Species listed in decreasing order of reported sensitivity (ircreasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time perioc. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes] 

Species 

Sal110 qairdneri 

Daphnia !!9!!! 

SelenastrUII capricornutu• 

Se 1 en as tr1111 capri cornutu• 

Ceriodaphnia reticulata 

Daphnia pulex 

Luabriculus varieqatus 

Daphnia magna 

Luabriculus variegatus 

~pulex 

Lianodrflus hoff11eisteri 

Tubifex tubifex 

Ga•arus pu 1 ex 

Pseud0110nas put ida (bact.) 

Carassius auratus 

Asellus aquatfcus 

(amphfpod) 

Piaephales promelas 

Cranqonyx pseudograci 1 is 

(amphfpod) 

~punctatus 

!:!P!!!!! ucrochirus 

Photobacterf 1111 phosphoreua 

Photobacteri ua phosphoreua 

Photobacterium phosphoreua 

Endpoint 

Exposure concentraUon 

time 

96 h 

48 h 

48h 

48 h 

96 h 

48h 

48h 

96 h 

96 h 

96 h 

48 h 

6 h 

96 h 

96 h 

96 h 

96 h 

96 h 

96 h 

15 II 

5 II 

5 II 

(millfgraiiS 

per liter) 

0.003 

.053 

.057 

.06 

.066 

.068 

.074 

.118 

.12 

.12 

.17 

.32 

.68 

• 72 

.748 

1.32 

1.5 

1. 70 

4.48 

6.47 

218 

416 

1,070 

Reference 

Phipps and Holcombe, 1985 

Lewis and Weber, 1985 

Turbak and others, 1986 

Bartlett and others, 1974 

Mount and Norberg, 1984 

Mount and Norberg, 1984 

Bailey and Liu, 1980 

Mount and Norberg, 1984 

Bailey and Liu, 1980 

Wright and Frain, 1981 

Chapman an~ others, 1982a 

Chapman an~ others, 1982a 

Wright and Frain, 1981 

S 1 abbert, 1986 

Phipps and Holcombe, 1985 

Martin and Holdfch, 1986 

Phipps and Holc011be, 1985 

Martin and Holdich, 1986 

Phipps and Holcombe, 1985 

Phipps and Holc011be, 1g95 

De Zwart ard Slooff, 1983 

McFeters ar·d others, 1983 

De Zwart ard Slooff, 1983 

1982). Such "life cycle" tests must be continuec for at least 

as long as one reproductive cycle. Chronic tests are potentially more informative than acute tests, not only because 

they avoid the problems of short, unrealistic exposures 

(Eaton, 1973), but also because more data are generated, 

allowing more rigorous statistical analysis (Brown, 1973). 

A number of recent studies of the effects of toxicants 

on a variety of animal species have empl· !!sized the 

improved sensitivity of chronic tests over acute tests (Eaton, 

1973; Sprague, 1976; Winner and Hrrrell, 1976; Winner, 

1981, Snarski and Olson, 1982; Chapman and Brinkhurst, 

1984; Hermens and others, 1984; Chapman and others, 

1985; Norberg and Mount, 1985b). Sublethal responses 

may be observed at toxicant concentrations C'Jnsiderably 

lower than those that produce mortality of half of the 

18 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Table 17. Acute toxicities of zinc to various test species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; d, days; 

m, minutes) 

Endpoint 

Exposure concentration 

Species time (milligrams Reference 

per liter) 

Selenastrum ca(!ricornutum 0.051 Turbak and others, 1986 

Da(!hn i a !!!9!!! 48h .068 Mount and Norberg, 1984 

Cerioda(!hnia ~ 48h .076 Mount and Norberg, 1984 

Da(!hnla I!!!!!! 48h .107 Mount and Norberg. 1984 

Sal110 gairdneri 14 d .41 Nehring and Goettl, 1974 

Sal110 !!!!ll! 14 d .64 Nehring and Goettl, 1974 

Sa 1 mo ill!:!tl 14 d .67 Nehring and Goettl, 1974 

Selenastru11 ca(!ricornutum • 7 Bartlett and others, 1974 

~ fontina11s 14 d .96 Nehring and Goettl, 1974 

Salmo gairdneri 96 h 2.2 Qureshi and others, 1982 

Photobacterium (!hOS(!horeum 5 II 2. 5 8ulich and others, 1981 

Photobacterium (!hOS(!horeum 15 II 3.5 Dutka and Kwan, 1981 

Da(!hnia ~ 48 h 5.1 Qureshi and others, 1982 

Corbicula !l.!!!!!!!!! 96 h 6.04 Rodgers and others, 1980 

lumbriculus variegatus 96 h 6.3 Bailey and liu, 1980 

PseudoiiOnas (!Utida (bact.) 6 h 7.15 Slabbert. 1986 

S(!iril lum voluntans 5 II 7.2 Qureshi and others, 1982 

lumbriculus variegatus 48 h 8.1 Bailey and liu, 1980 

Asellus aguaticus 96 h 18.2 Martin and Holdich, 1986 

(uphipod) 

Cra1190nyx (!!eUd2Qrac11i s 96 h 19.8 Martin and Holdich, 1986 

(amphipod) 

~ fluminea 24 h >40 Rodgers and others, 1980 

Photobacteri um (!hOS(!horeum 5 II 49 Qureshi and others, 1982 

Photobacteri um ehos(!horeum 5 II 477 Mcfeters and others. 1983 

population. However, this advantage of chronic testing 

must be weighed against the disadvant':lge that sublethal 

responses are often subtle and difficult to monitor. Inconsistent responses among different individuals of a population is a common problem in chronic tests (Geiger and 

Buikema, 1981). Furthermore, chronic tests may require 

complex experimental setups and long-term culturing of 

organisms. 

In part because of their sensitivity, chronic tests may 

produce more meaningful results than acute tests. Factors 

such as growth, fecundity, and feeding habits may be more 

significant indicators of contaminant impacts than lethality 

at relatively high concentrations. This argument is based on 

the presumption that environmental contamination, even in 

extreme cases of pollution, generally will be lower than 

acute lethal levels but will present the biota with long-term, 

low-level exposure. Hence, the effects are likely to be 

sublethal, but still may severely affect the community. 

Table 18. Acute toxicities of mercury to various test species 

[Species listed in decreasing order of reported sensitivity (increasing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mea:1 values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). h, hours; m, 

minutes) 

Endpoint 

Exposure concentration 

Species time (milligrams Rehrence 

per liter) 

Crangonyx eseUd2Sjraci 1 is 96 h 0.001 Martin and Holdich, 1986 

(uphipod) 

Daehn fa magna 48h .OJ QuresM and o+:hers, 1982 

PhotobacteriUII ehosehoreum 15 II .044 De Zwart and Slooff, 1983 

PhotobacterfUII ehosehoreum 511 .051 De Zwart and Slooff, 1983 

PhotobacterfUII ehosehoreum 5 II .06 Mcfeters and others, 1983 

Photobacteri Ull ehosehoreull 5. .065 Bulich and ot'lers, 1981 

PhotobacteriUII ehosehoreull 5• .08 Qureshi and others, 1982 

LUIIbrfcuJus variegatus 96 h .10 Bailey and li•1, 1980 

Lumbriculus variesatus 48 h .11 Bailey and l i•.•, 1980 

!!!!!.!!!! ~ 96 h .14 Chap!lan and ethers , 1982a 

Na fs f2!!!!!!!!ll 96 h .16 Chap!lan and lfitchell, 1986 

limnodrilus hoffmeister! 96 h .18 Chap11an and cthers, 1982a 

~ aguaticus 96 h .20 Martin and HC'ldich, 1986 

(aiiJihfpod) 

l!l!!! gairdneri 96 h .21 Qureshi and others, 1982 

Il)'Odrilus !!:!!!!!! 96 h .29 Chap!lan and r• tche 11 , 1986 

Seirf11UII voluntans 5 II 3.7 Qureshi and others, 1982 

Table 19. Acute toxicities of lead to various test species 

[Species listed in decreasing order of reported sensitivity (inc¥/'!asing order 

of concentrations needed to reach endpoint). When ranges of endpoint 

concentrations were reported by authors, only median or mean values are 

reported here. Endpoint for animal species is 50-percent mortality in 

specified exposure time period unless otherwise indicated. Endpoint for 

bacteria species is 50-percent reduction in measured activity (usually 

luminescence or mobility) in specified exposure time period. Endpoint for 

phytoplankton species is 50-percent reduction in growth rate (usually 

measured as carbon-14 uptake or oxygen production). L hours; m, 

minutes) 

Endpoint 

Exposure concentration 

Species time (11ill igrams Reference 

per liter) 

SelenastrUII caericornutUII 0.14 Christensen arrl others, 1979 

Ceriodal!hnia reticulata 48 h .53 Mount and Norberg, 1984 

Chlorella stigmato(!hora .70 Christensen and others. 1979 

lullbrfculus variegatus 96 h 1.8 Bailey and liu, 1980 

LUIIbriculus variegatus 48h 3.4 Bailey and liu, 1980 

Daphnia 11agna 48 h 4.4 Mount and Nor'lerg, 1984 

Daphnia eulex 48h 5.1 Mount and Nor'lerg, 1984 

Crangonyx (!!eud29raci lis 96 h 27.6 Martin and Ho'(jich, 1986 

(uphipod) 

PhotobacteriUII phosehoreull 15 II 30 Dutka and Kwa,, 1981 

Asellus aguaticus 96 h 64.1 Martin and Holdich, 1986 

{uphipod) 

Procedures and Applications 19 
Despite the repeated claims of the advantages of the 

chronic-test approach, the simplicity and precision of acute 

tests are significant factors in their favor. The continued use 

of acute, single-species tests is ensured by their applications 

for determining compliance of effluents with regulatory 

standards. Macek and others (1978) reported that the 

consensus among attendees at a 1977 workshop on application of aquatic toxicity methods was that acute lethality 

tests are the most useful of various types of toxicity tests. 

This was because they were judged the most practical 

means for determining relative toxicities of various chemicals, relative sensitivities of different species, and effects of 

water quality on the toxicity of chemicals. However, 

chronic-testing methodology has developed considerably 

since the time of that workshop. 

Design of Test Enclosure 

Most nonbacterial toxicity tests entail enclosure of 

test organisms in some variation of a static laboratory 

aquarium of relatively small, manageable size. When the 

test organisms are macroscopic in size, static enclosures 

generally hold just one or a few individuals. The static 

enclosure has the distinct advantage of experimental control 

and reproducibility. Extraneous variables that might affect 

test results are minimized, and responses are relatively easy 

to measure. On the other hand, it has the disadvantage of 

presenting the test organisms with a very unnatural habitat. 

Their responses to stress in such a setting may be different 

than if they were in their natural environment, surrounded 

by other species with which they interact. 

Various alternatives to static enclosures have been 

used with increasing frequency in recent years, especially 

for multispecies tests and for tests in which the principal 

endpoint is something other than acute lethality. Although 

the static-enclosure approach remains the most popular, 

other methods have been encouraged by improvements in 

toxicity-test methodology, including the development of 

complex design features and more sensitive measurement 

techniques. The selection of test-enclosure type depends 

largely on the test species used and the response endpoints 

monitored. Some species selections, such as bacteria, leave 

no choices of test enclosures. 

Multispecies Tests 

A toxicity test need not be limited to a single species. 

Multispecies tests have been used with increasing frequency 

in recent years (Hansen and Tagatz, 1980; Kaushik and 

others, 1985; Phipps and Holcombe, 1985). Their advantages have been pointed out by Maciorowski and Clarke 

(1980), Suter (1983), Cairns (1983, 1984, 1985), and 

Kimball and Levin ( 1985). The most frequent argument is 

that relative to a single-species test, the multispecies view 

offers the researcher a more complete and m'lre realistic 

picture of probable toxicant effects on the entire community. Although there may be some loss in contro1 of untested 

variables and standardization of procedures, it i~ argued that 

this is compensated for by improved realism, co'llpleteness, 

and even sensitivity (Suter, 1983; Kimball and Levin, 

1985). There is also an economical argument in support of 

multispecies testing. Costs per experiment are likely to be 

higher than single-species tests, but because the amount and 

quality of information is enhanced, the cost/benefit ratio can 

be reduced (Suter, 1983; Perez and Morrison, 1985). 

It is important to clarify the meaning of "multispecies 

testing." Two entirely different approaches may be signified 

by this term. One is a "microcosm" approach, whereby a 

number of different species are exposed simultaneously to 

the same environmental stress. Under these conditions, the 

test "species" is actually a community of spe6~s that can 

interact with each other. Such tests are conducted with the 

hope that they mimic the natural environment. Rather than 

showing how the survival or metabolic activities of only one 

species is affected, these tests are expected to indicate how 

the toxicant will affect community characteristics such as 

succession, diversity, predator-prey relations, or dominant 

taxa. The "multispecies" approach may also refer to a 

"battery of tests" in which the toxicity problem is examined 

by conducting a number of separate single-species tests. A 

different species is used in each test, and collectively they 

represent distinct trophic levels and (or) are sensitive to 

different types of toxicants. For example, a cladoceran 

species and a fish species might be used in a battery. In 

addition to representing different trophic levels, the cladoceran is likely to be more sensitive to metals, whereas 

the fish is more sensitive to organic compounds. The 

tests may be conducted simultaneously or ~equentially 

(Cairns. 1983). 

Microcosm Approach 

Considerable success has been reported by authors 

using laboratory microcosms. Portier (1985) cited evidence 

from microbial studies to support use of benctt.op microcosms as a toxicity-testing tool. Correlation coefficients 

generally greater than 0.9 were reported betwe.~n lab and 

field measurements of a number of microbial population 

parameters and metabolic activities. Harrass and Taub 

( 1985) described a standardized aquatic microcosm 

designed to be an especially replicable system. The experimental systems were treated with copper, anc responses 

were compared with responses in untreated microcosms and 

with reported results from field studies. Responses of the 

microcosms, with respect to algal/grazer interactions, species shifts in algal communities, and recov~ry of the 

systems when the toxicant treatment was termir.ated, were 

similar to those generally observed in natural systems. Levy 

and others ( 1985) compared the pelagic epilirmion of a 

20 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
California reservoir with three microcosms containing water 

from the same reservoir. No toxicants were added, but the 

effects of water agitation were examined. For 6 weeks, the 

phytoplankton and zooplankton communities of two of the 

microcosms were virtually indistinguishable from those of 

the natural system. 

Microcosms designed as "in situ" test enclosures have 

been used to mimic the natural environment as closely as 

possible. By suspending translucent enclosures on a vertical 

line, Marshall and Mellinger (1980) tested the effects of 

depth on responses of plankton to cadmium addition in Lake 

Michigan. Depth was indeed found to have an effect. The 

"limnocorral," a large in situ enclosure placed in lakes to 

represent the natural pelagic community, was used by 

Kaushik and others ( 1985) and Herman and others ( 1986) to 

examine the effects of pesticides on plankton. The limnocorral technique was described as "an important tool for 

assessment of direct and indirect impacts" of toxicants 

(Kaushik and others, 1985). 

Some general disadvantages of microcosm-type 

multispecies procedures were discussed by Mount ( 1985) 

and Slooff ( 1985). Costs and practical restrictions do not 

allow multispecies experiments to be fully representative of 

their simulated natural environments. The resulting generality in test design reduces sensitivity and predictability. 

Analysis of data from studies in which both single-species 

and multispecies tests have been done shows little difference in the results or conclusions of the different 

approaches. This led Slooff (1985) to conclude that "as long 

as there is no solid evidence that predictions made from 

single species tests are unreliable, there are no imperative 

reasons to propose expensive and time-consuming multispecies tests as additional or alternative research tools." 

Mount ( 1985) pointed out that if the primary purpose of the 

toxicological work is to examine the effects on a valuable 

resource species (for example, a sport fishery), singlespecies tests are certainly more suitable. He also suggested 

that the claims of improved realism and sensitivity may be 

misleading: " ... community sensitivity is only an expression of individual species sensitivity ... tha! there are interactions between species in multispecies tests is not a 

measure of their validity or informative value. In fact, the 

reverse could well be true!" 

Numerous specific microcosm applications have 

demonstrated weaknesses in the microcosm approach. A 

three-phase (gaseous-aqueous-sediment) microcosm was 

used by Adams, Werner, and others (1985) to simulate 

Lake Powell, Utah-Arizona, and to study effects of and fate 

of benz(a)anthracene (BA). Results in the microcosm were 

representative of simultaneous field measurements, but 

differences in physical conditions caused significant differences in BA behavior. For example, reduced light levels 

substantially diminished the rate of photooxidation. Open 

microcosms were used by Selby and others (1985) to assess 

the effects of cadmium on a stream community. Because of 

the possibility of community changes unrelatei to the 

toxicant, the method was not recommended for use as a 

screening tool. Woltering (1983) found that responses to 

toxicants in laboratory ecosystem studies were highly 

dependent on ecological factors such as predator-prey 

fluctuations, competition, energy input, and habitat availability. Predation can be an especially important controlling 

factor, and must be at least partly restricted in most 

microcosm studies (Harrass and Taub, 1985). Aging (nutrient depletion) of the microcosm can also influence toxicant 

effects on test organisms (Kindig and others, 19f 3). 

At the 1977 workshop on estimating the hazard of 

chemicals to aquatic life, where attendees evaluated various 

toxicity-test techniques (Macek and others, 1978), the 

microcosm approach was rated rather low in overall utility. 

It was considered inferior to most other techniques in 

ecological significance, scientific and legal def('nsibility, 

and simplicity and cost. Two of the participan+s in that 

workshop (Brungs and Mount, 1978) pointed out that the 

microcosm idea was basically sound, but that its implementation was still problematic because of difficulties in replicating the natural system. 

Because of the increased complexity of microcosmtest procedures, it would be difficult to use them successfully on a routine basis in a large-scale assessment program. 

However, they may be useful in small-scale ass~ssments, 

such as in studies of selected stream reaches. In snch cases, 

they would probably be used at sites where specir 1 toxicant 

problems may occur, as indicated by initial single-species 

tests and by chemical analyses. 

Battery Approach 

The precision and accuracy of any scientific study 

generally are improved both by replication of a s:ngle type 

of experiment and by sequential attack on the question using 

a variety of experiments. Given the uncertainties surrounding the assessment of biological responses to constituents in 

the aquatic environment, replication and sequent:al testing 

merit special consideration for toxicity testing. Interpretation of toxicity-test results involves comparisons of toxicant 

concentrations that elicit biological responses rnd actual 

toxicant concentrations in the natural environment. If a 

battery approach is used, there is a presumed improvement 

in the reliability of this comparison because it is l'<~sed on a 

more diverse data base than it would be if only one test were 

used. The data base will be especially diversified if the 

selected test organisms represent different tropltic levels 

(Maciorowski and Clarke, 1980). 

Several researchers have reported good results in 

applications of the battery approach. Dutka znd K wan 

( 1982) found that four bacterial screening procedures they 

tested were each characterized by particular sensitivity 

patterns and could not be readily correlated with each other. 

If only one procedure were used, it could give misleading 

Procedures and Applica+1ons 21 
information. Further evidence that independent toxicity 

tests may give misleading results when interpreted alone 

was provided by data from Lake Ontario (Dutka and others, 

1986). Plotkin and Ram ( 1984) tested the effects of sanitary 

landfill leachate on fish, daphnids, algae, and bacteria. The 

responses were very different among the different organisms, and not reliably predictable on the basis of measured 

concentrations of toxicants in the leachate. They concluded 

that toxicity assessments should be based on multiple tests 

with organisms from different trophic levels. Three test 

species-bacteria, oyster embryos, and amphipods-tested 

by Williams and others (1986) showed considerable variation in sensitivity to toxic sediments. The authors emphasized that a diversity of toxicity-testing procedures was 

important for evaluating sediment toxicity. 

Diversification of toxicity tests by using the battery 

approach may improve the reliability of statistical treatment 

by providing a broader data base. Multiple trophic levels 

may be tested, providing a more complete characterization 

of the community. The different strengths of a diverse array 

of tests may be used in complementary fashion. 

Although the benefits of diversification are recognized, there are also disadvantages. The sensitivities of 

different test species vary considerably but, as shown in 

figure 2, they may all be insufficient to permit positive 

detections of contaminant concentrations commonly found 

in natural waters. Hence, consistent negative results from 

all tests of a battery do not necessarily lead to a firm 

conclusion that there is no toxicant problem. If, on the other 

hand, some of the tests in the battery produce positive 

results while others do not, the composite result may be 

ambiguous and interpretation may be especially difficult. 

The previously discussed arguments against microcosm procedures may also apply to the battery approach. A 

multispecies approach, whether microcosm or battery, is 

more complex and more costly than a single acute-lethality 

test. Is it simply a more costly means to arrive at the same 

answer? The cost problem is an especially important consideration for designing a toxicity-test approach for largescale assessments. 

A variation of the battery approach is a sequential 

screening procedure (Slooff, 1985) (fig. 3). A rapid test, 

such as a bacterial luminescence test, is used as an initial 

screening tool. If ambient substances produce stress 

responses in the initial screening test, a second test at a 

higher level of biological organization is performed. This 

process may proceed through several levels of biological 

organization to assess toxicity effects. Cairns, who had 

earlier joined with others in advocating the sequential test 

approach (Cairns and others, 1978). later argued that 

sequential testing, if done at all, should not proceed from 

lower to higher levels of biological organization (Cairns, 

1983). He pointed out a "lack of substantive evidence that 

one can accurately predict the response at higher levels of 

biological organization from the single-species tests." 

I MICROTOX ASSAY SYSTEM I 

! 

SHORT-TERM TESTS 

ALGAE, INVERTEBRATES, FISH 

/_ ~ SHORT-TERM TESTS LONG-TERM TESTS 

OTHER SPECIES INVERTEBRATES, FISH 

l 

TOXICITY RANGE 

OF A CHEMICAL 

Figure 3. General scheme for sequential toxicity 

screening. 

Sediment Toxicity Tests 

Conventional toxicity tests involve assess'Tient of the 

effects of toxicants dissolved or suspended in water. However, large numbers of aquatic organisms, including many 

of the test species listed in table 3, reside in or on bottom 

sediments. Others are exposed directly to sedimentary 

materials because they are benthic or deposit feeders. 

Furthermore, sediments are an important rep ')Sitory for 

many contaminants that may be released to overl:·ing water. 

These factors support the argument that toxicity assessments of aquatic systems should include exposure of test 

organisms to contaminants contained in the sediments. This 

is the rationale leading to relatively recent development and 

application of sediment toxicity tests, mostly in the marine 

environment (Tsai and others, 1979; Swartz and others, 

1980; Chapman and Fink, 1984; Tietjen and Lee, 1984; 

Long and Chapman, 1985; Swartz and othprs, 1985; 

Mearns and others, 1986). Freshwater studies in~lude those 

of Prater and Anderson (1977), Laskowski-Hoke and Prater 

( 1981), Cairns and others ( 1984), Malueg and others 

(1984a, b), Nebeker and others (1984), and Schuytema and 

others ( 1984). 

Although sediment toxicity tests involve irvestigation 

of contaminants associated with the sediments, tl'~ exposure 

route is not necessarily through direct contact between 

organism and sediments. Any one of three different exposure routes are possible in the experimental design (Chapman, 1987): 

1. Exposure to whole, intact sediments. This i~ generally 

the preferred exposure route, especially if the test 

species inhabits the sediments. The test enclosure contains contaminated sediments and water. The test species 

may be either benthic or pelagic (that is, it nay inhabit 

either sediments or water). Exposure may be through 

direct contact with sediments, ingestion of s~dimentary 

materials, or contact with overlying water that carries 

desorbed or resuspended contaminants. 

22 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
2. Exposure to a sediment elutriate (suspended or liquid 

phase). The test enclosure contains water that has 

previously contacted the contaminated sediments (as in a 

sediment-water slurry) for a specified time, then is 

filtered or centrifuged. Because of desorption or resuspension, water contains contaminants previously associated with sediments. The sediments themselves are not 

added to the test enclosure. This may be the method of 

choice if it is not practical to add sediments to the test 

system, or if the toxicological response involves aqueous contact (for example, respiration; Chapman and 

Brinkhurst, 1984). 

3. Exposure to a sediment extract. The test enclosure 

contains water to which sediment extract is added. The 

extract is an organic carrier solvent containing some of 

the contaminant that has transferred to it during an 

extraction procedure similar to procedures used for 

chemical analyses. Thus, the water-extract mixture contains contaminants previously associated with sediments. The sediments themselves are not added to the 

test enclosure. Again, this method may be appropriate if 

it is not practical to add sediments to the test system. It 

is applicable only for readily extractable nonionic 

organic contaminants (Chapman, 1987). 

Comparisons of the elutriate and whole-sediment 

exposure routes by Chapman and Fink ( 1984) showed some 

discrepancies in results. Toxicity responses in some cases 

were caused only by elutriate exposure, and in other cases 

only by whole-sediment exposure. Ideally, whole-sediment 

exposure should be used in combination with either 

sediment-elutriate or sediment-extract exposure to obtain 

more complete toxicity information. 

Sediment toxicity-test procedures can be used for 

acute or chronic testing with any of the common test 

species, whether benthic or pelagic (Nebeker, Cairns, 

Gakstatter, and others, 1984). Swartz and others (1985) 

monitored mortality and sublethal responses of amphipods 

exposed directly to sediments in static test beakers. Control 

survival was 95 percent, and the organisms were quite 

sensitive to contaminants amended to the sediments. An 

example of the elutriate exposure method is the threechamber-recirculation apparatus used by LeBlanc and 

Suprenant ( 1985) to test the effects of contaminated sediments on fathead minnows, daphnids, and midges. 

Responses of test organisms were closely correlated with 

the degree of chemical contamination of sediments. 

Some verification studies of sediment tests have 

produced favorable results. Field validation by Swartz and 

others ( 1980) showed good correlations between sediment 

toxicity, as determined by amphipod responses, and 18 

biological and geochemical variables on a pollution gradient 

on the Palos Verdes Shelf (California). Mearns and others 

( 1986) conducted an interlaboratory comparison of an 

amphipod sediment toxicity test and obtained results that 

led them to recommend wider use of the test. Control 

survival was greater than 90 percent in five la':loratories, 

and the laboratories were in close agreement on toxicity 

ranking and mean responses. Acute toxicity of sediment 

extracts as determined by bacterial biolurinescence 

(Schiewe and others, 1985) correlated with total concentrations of aromatic hydrocarbons, chlorinated hydrocarbons, 

and naphthalenes in the sediments. 

The disadvantages of sediment toxicity t~sting should 

also be considered prior to incorporation in a long-term 

study plan. First, the introduction of sediments (or their 

elutriates or extracts) into the test system comrlicates the 

chemistry of the system and increases the likelihood of 

secondary variable effects. Second, although a more complex system may be more realistic, it also makes interpretation of test results more difficult. Third, the se'lsitivity of 

tests involving exposure to whole, intact sedime'lts may be 

inferior to that of conventional tests because of the likelihood that the contaminant must move through the aqueous 

or suspended phase before affecting the test organism. Most 

quality-assurance work with sediment technique~ indicates 

good sensitivity, but it has also been noted that sediments 

tend to ameliorate toxicity of contaminants in the system 

(Chapman and others, 1982b; Graney and othf!rs, 1984). 

Fourth, relatively little work with sediment toxicrty tests has 

been done, especially for freshwater systems. Therefore, 

the documentation of methods and availability of comparative data are limited. Finally, work witt sediment 

increases the complexity of collecting samples for testing 

and of performing the tests. 

The importance of sediments, both as a habitat for 

biological species and as a reservoir for many xenobiotic 

substances, suggests that sediment tests should not be 

overlooked in designing a toxicity-testing study. The most 

productive approach for most studies, provided funding and 

personnel are adequate, is to implement a suitrble combination of sediment and water tests supplem~nted with 

chemical analysis of the same sediment and water media. 

Biochemical Tests 

The toxicity of heavy metals and organic compounds 

to aquatic biota is very commonly attributable to direct or 

indirect effects of the toxicant on enzyme activit:', biochemical functions, or membrane integrity (Neff, 19(15). Therefore, it is reasonable to expect that one of the most sensitive 

indices of contaminant stress would be a chang~ in enzyme 

activity, enzyme synthesis, or biochemical composition. 

Toxicity-test methods that use this approach have been 

developing rapidly in recent years. Most of the research has 

been done with fish. 

It is logical to look at the effects of toxins on 

biochemical processes as a first step in toxkity testing. 

Biological responses to stress may be thought of as a series, 

propagating through increasingly complex levels of organization (Jenkins and Sanders, 1986). Biochemi~al changes 

Procedures and Applications 23 
are very early in the series; for practical purposes, they are 

initial responses. Furthermore, they are common to many 

different kinds of organisms. The biochemical changes may 

elicit subsequent responses at the cellular, organ, organism, 

population, and, finally, community levels. But as one 

proceeds along this scale of propagation, the variability of 

response increases because of increasing secondary effects 

due to individual tolerances and environmental factors. 

Thus, both the sensitivity and the reproducibility of 

biochemical-response measurements are likely to be greater 

than those of other types of toxicity testing. 

Biochemical changes tend to be rapid and very 

responsive to causative factors, in part because they are 

initial responses to stress. For example, a study by Kurelec 

and others (1977) showed that mixed-function oxygenase 

activities in Blennideae fish in the Adriatic Sea increased by 

nearly an order of magnitude within a few days after an oil 

spill. This kind of quick and dramatic response is not 

uncommon. 

There are many possible variations of the biochemical 

assay approach to toxicity testing. Generally, they involve 

exposure of the living organism or tissues of the organism to 

the contaminant and measurement of relatively short term 

changes in enzyme activity, biochemical composition of 

blood and tissues, or production of detoxification proteins 

such as metallothioneins or mixed-function oxygenase systems. Biochemical techniques of monitoring responses may 

be applied in either a laboratory setting (test organisms in an 

enclosure) or a community survey (capture and analysis of 

native organisms). 

Metallothioneins are proteins that have a high binding 

capacity for divalent metal cations. They have been identified in many species of fish and other animals, and they are 

thought to play an important role in detoxification of several 

metals, including silver, gold, cadmium, mercury, copper, 

and zinc (Neff, 1985). Exposure to elevated concentrations of such elements should stimulate production of 

metallothioneins (Roch and others, 1982; Thompson and 

others, 1982; Sanders and others, 1983; Sanders and 

Jenkins, 1984). 

Mixed-function oxygenase (MFO) systems might be 

considered the counterpart of metallothioneins with respect 

to detoxification of organic contaminants. MFO systems 

include a group of enzymes that initiate metabolization of 

numerous lipophilic organic compounds, rendering them 

more water soluble and therefore more available for excretion. They have also been identified in many fish species 

(Neff, 1985). Various studies have demonstrated increased 

MFO activity as a result of exposure to organics in the 

environment (Payne, 1976; Stegeman, 1978; Lech and 

others, 1982; Foureman and others, 1983). Ironically, the 

fish does not necessarily benefit from this increased MFO 

activity. Instead, there may actually be an increase in 

toxicity, owing to the production of intermediates that 

are carcinogenic (Hinton and others, 1981; Tan and 

others, 1981). 

Enzymes may be affected directly or indirectly by 

toxicants, usually resulting in an increase in enzyme activity. Increased activity in glutamate-oxaloacetat~ transaminase and glutamate-pyruvate transaminase, two indicators 

of liver pathology, may be induced by elevated concentrations of carbon tetrachloride (Casillas and othen, 1983) or 

sewage discharges (Weisner and Hinterleitner, 1~~0). Some 

enzymes are affected specifically by certain pollutants. One 

of these is delta amino levulinic acid dehydratas~ (ALAD), 

which is contained in blood erythrocytes and is important in 

the formation of hemoglobin, cytochrome, and peroxidase. 

Its activity may be sharply inhibited by lead in the blood 

(Hodson and others, 1978). Concentrations as low as 10 

f.Lg/L (micrograms per liter) can produce significant inhibition of erythrocyte ALAD in rainbow trout (Hodson and 

others, 1977). In other species, such as carp and white 

suckers, the ALAD activity was a less sensitive indicator of 

lead contamination. 

In addition to enzymes, the production and activity of 

various biochemical substances in the blood and t:ssues may 

be affected by exposure to toxicants. In fish bloo-1, some of 

the substances most frequently affected by pollutants are 

cortisol, glucose, proteins, lactic acid, pyruvic acid, and 

cholesterol. In tissues, some of the most frequently affected 

substances are glycogen, proteins, lipids, collagen, glutathione, and ascorbic acid. 

Biochemical responses to stress are common to all 

types of organisms and thus can be used as bioiniicators of 

toxicity in a wide range of environmental situations. 

Responses vary according to the type and conce'ltration of 

the causative agent (Jenkins and Sanders, 19ffJ). These 

stress-response relations currently are not well understood, 

but as more information about them becomes avrilable, the 

usefulness of biochemical testing to identify particular types 

of contaminant problems should improve. 

Biochemical analysis offers considerable promise for 

application in large-scale studies. The number of possible 

methods is almost limitless, considering the numr~r of toxic 

agents in the environment and the number of biochemical 

responses caused by those toxicants. In general, biochemical testing has the same advantages as bacterial tests and 

offers the additional advantage of greater sensitivity. 

The primary disadvantages of the biochemical 

approach are (1) limited knowledge of the correlation 

between biochemical responses and deleterious effects on 

fish populations and (2) biochemical variability caused by a 

great number of environmental variables other thr'l toxicant 

concentrations (Neff, 1985). The latter problem is significant for biochemical techniques because there ar~ so many 

factors that can have biochemical effects, and the resulting 

biochemical fluctuations can be dramatic. The stress of 

capture and handling can be an especially impcrtant controlling factor. As methodology development proceeds, 

some of these difficulties may be overcome and biochemical techniques will become increasingly useful. 

24 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 

j 
Alternatives to Laboratory Toxicity Testing 

Toxicity tests conducted in static, flow-through, or 

microcosm systems represent just some of various types of 

biological study that could fill the need for biological data to 

assess water quality. These types of tests most directly 

address the question of effects of contaminants. However, 

certain allernative approaches may be implemented to 

provide somewhat different kinds of information about 

toxicity. 

One alternative is to omit test enclosures altogether 

and conduct ecological surveys in the study basin. The sites 

for such surveys may be selected to represent a range of 

contaminant conditions in the basin. Chemical data may be 

used to make appropriate site selections. The survey data 

can provide information about community structure (species 

present and relative abundance of each), diversity (numbers 

of species relative to numbers of individuals), and biomass 

(total abundance of biota, regardless of taxa). This information provides a useful complement to chemical data 

collected at the same sites and times (Lafont, 1984; Long 

and Chapman, 1985). 

Ecological surveys avoid some of the problems of 

prolonged incubation of enclosure organisms in artificial 

enclosures. They are not dependent on representation of an 

entire system by a limited number of species, and they are 

not subject to inaccuracy due to unnatural conditions in a 

controlled environment. More analytical work (for species 

identification and organism counts) is required, but standard 

methods are generally available. The high costs of this 

analytical work may be offset by savings in labor and 

equipment costs. 

The principal disadvantage of ecological survey analysis is that the lack of control on environmental variables 

limits the ability of the researcher to infer cause-effect 

relations from the results. Even if trends are found (either 

temporal or spatial), they are not sufficient in themselves to 

show cause and effect. However, they do provide clues that 

may be used to identify areas that merit further toxicity 

testing and other biological investigation. Conversely, if no 

trend is found, the study makes a significant statement that 

would be lacking if only the chemical data were available. 

A second alternative is to install artificial substrates in 

the natural environment. As in the ecological surveys, site 

locations are selected to represent a range of contaminant 

conditions. The colonization of the artificial substrates by 

natural biota is observed and measured after being left 

undisturbed in the system for a specified time period. 

Colonization rates and species composition of the established community may reflect contaminant effects. Several 

possible variations of this general procedure are possible, 

including the incubation of natural substrates from the 

stream system or the measurement of loss of organisms 

from a substrate (such as loss due to drift of attached 

benthos). Like the ecological survey approach, this proce-

~ure has the advantages and disadvantages of being conducted in the natural environment. However, it is somewhat 

less natural and more controlled than the ecological survey 

because of the use of standardized colonization surfaces and 

a limited and rather arbitrary colonization time. 

A third alternative is to introduce test organisms in 

the natural environment in enclosures, such as c""ges, that 

prevent their escape but allow exposure to all el ~ments of 

the environment just as if they were free living. After a 

specified incubation period, mortality and (or) sublethal 

changes may be determined and compared with control 

organisms incubated in unaffected sites. This procedure is 

similar to a standard toxicity test because it uses test 

organisms selected primarily for their sensitivity and practicality for study and entails enclosure of the orgr'lisms for 

a limited incubation time. On the other hand, it is similar to 

ecological surveys or artificial substrate mea~urements 

because the organisms are exposed to natural conditions. 

Toxicity-test procedures that use relatively large test 

organisms such as mollusks may be supplemented by tissue 

analysis to determine bioaccumulation rates. If contaminants are accumulated over time in the biological tissue, the 

organisms function as integrators of time-variabh contaminant inputs and thus allow detection of substances that 

might be missed by analysis of constituents in water. 

However, bioaccumulation rates are highly dep~ndent on 

properties of the constituents and characteristicf, particularly lipid content, of the biological species. The.y are also 

susceptible to changes in environmental conditio:'ls such as 

pH, temperature, sediment characteristics, ani organic 

carbon concentrations. 

Overview of Test-Type Differences 

The differences among test types are reflect~d in their 

particular strengths and limitations. As a summ""ry of the 

preceding discussion, table 20 lists major test types and 

some of the reasons why each type might be sdected or 

deselected for water-quality-assessment purpose~. 

FIELD AND INTERLABORATORY 

VERIFICATION OF TOXICITY TESTS 

Field verification of laboratory tests is one means of 

obtaining quality-assurance information about tcxicity-test 

procedures. In some cases, additional quality assurance has 

been achieved through interlaboratory compariscns. 

A 1983 symposium (Boyle, 1985) was dedicated to 

validation of laboratory toxicity-testing meth'Jds, with 

emphasis on verification of microcosms and ma+hematical 

models by comparison with field data. Most of t~e authors 

reported favorably on their verification results (Adams and 

others, 1985; Giddings and Franco, 1985; Harrass and 

Taub, 1985; Levy and others, 1985; Portier, 1985). 

Other verification results reported at the 1983 symposium revealed some inconsistencies between laboratory 

Field and Interlaboratory Verification of Toxicity Tests 25 
Table 20. Arguments for and against use of selected 

toxicity·test procedures 

~tSfOI': Art-..ts against: 

1. Acute ttstt. sit~~le sp.cies (static or flow-thi'OIIIh). 

o GDocl ~tatiOD of eethods. o Unnatural; si119le-species responses 

o btensfwe Illata ttue of results in H•ttld enclosure do not reflect 

fi"OII clf~e SJ~t ... 

o Endpoint (110f't11fty) rlldfly 

dettctld and IIOIIitOI'ICI. 

o Results rt110rtld tn standardtzld. 

unlllltiguous LCSO fOI'Ut, 

o GDocl control and replfcuntty of 

ttst conditions, 

2. ChrODfc ttsts. st11911 sp.cfa. 

o Aelatfwely 10011 --.ntattOD of 

....... o Larp Illata llaH of r'elults froe 

clfftrle syst ... 

o GDocl control and rtPifcallflfty of 

test condfttons. 

o Slllstttvfty of sultlethal responsa 

greater t11an that of .rtaHty 

(s.-tt ... lly wc1ers of 

llll!litude). 

species interactions in natural 

COMUntty, 

o Varfallle sensitfvtty; species 

sensttfve to s- toxfCIIIts. 

insensftfvt to others. 

o Relatively long culture u ... 

r'equirld; uy cause .rtaHty or 

other prolll ... 

o Responses often sulltle; uy be 

difficult to detect or •nftor • 

o Forut for reporting results not 

well standardizld; can be 

a.lliguous. 

o Unnatural; single-species responses 

in lf•itld enclosure do not 

refltct sp.ctu interactions fn 

natural COMUntty. 

o variable sensitivity; species 

sensitive to SOM toxicants. 

insensitive to others. 

o L0119 culture ti.., reqllirld; 1111 

cause Mrtality or other 

prolll ... 

3. Lllloratory •fcrocosa. or •1tisp.ci.S tests. 

o llelatfvely good dOc:UMtltatfon of o C:O.,lfcated setup increases cost 

llethods. per test and decreases nu8ber of 

o •f• datllllse of results froe 

othel' studt11. 

0 ... dlllti'Ol and repltcallflfty ot 

tilt conditions. 

• -ltcatton of natural eo.unttya 

responses Incorporate species 

fnteractfOIIS. 

4. llctertal tests. 

0 st.,lfcfty llld rlpidfty; uny 

repltutu posstble. 

• May be conductld fn local laboratoriu; elf•fnatu neld for 

sllflllifll!l s•lu and delay 

11etwen s-11119 and testf119. 

o Avoids .. Y ......,, ... of 1111 

encloslll'el IIIII 10119 cvlture 

u .... 

o llood ,.,..,.tatfon of ,....,.., 

tufcfty ~~Hause l•fnucence 

response fs fependent on c-.t. 

bfocllt8tca1 'atllways. 

5. Sldfllllllt tests. 

o SldfMntS are natural repesttory 

of .. ,toxicants. 

o Mally aquattc ortanisa exposed to 

llftllet toxtcant COIICIIItratfons 

thi'OIIIII sedflltnts than tllrouth 

veter. 

o 5Hf8tnt-water syst• 110re 

...,....entatfve of natural 

eolldttfons. 

replicates possible. 

o llultfspeciu situation increases 

CCIIIPiexity and likelihood of 

secondary variable effects and 

prolll..s of laboratory culture. 

o Responsu often subtle; uy be 

difficult to detect or 11011ftor. 

o Very poor sensttfvfty to 80St toxic 

agents. 

o Poor reproducibility; sensitive to 

slttht changes in test conditions 

or cheractertstics of bacterial 

populations. 

o Variable pathways of exposure 

through sediMnts: need to use 

different types of tnts in 

c08bination. 

o SedfHnts fn test syst• caplicate 

chaistry; llllces interpretation 

_,.. difficult • 

o SediHnts 1111 -Horate toxicity 

Ol' decrease bioavatlabiltty of 

toxicants. 

Table 20. Arguments for and against use cf selected 

toxicity-test procedures-Continued 

~ts for: 

11. Biochaical or physiological tKts. 

Argueents agaiopst: 

o Relatively sun dah base. 

especially for frt"lhwater 

syst-. 

o More difficult sa.ple collection. 

treatMnt and labc: .. atory set-up. 

o High s-ittvity to eost toxicants: o Relatively new fteh'; ll•tted (but 

biochatcal or physiological rapidly increasinq) data base and 

changes one of ffrst responses 

to -ironeental str11ses. 

o SiiiPlfcity; usually only involves 

collection of blood s..,les that 

uy be stored or shipped wfth 

1fttle risk of deterioration. 

docueentation of nthods. 

o Especially sensitive- to secondary 

variable effects. 

o Avoids prolll- of laboratory culture. 

o Mally rep1fcate analysu possible. 

o Responsu readily detectable and uy 

be reported ill unlllbiguous 8tnner. 

7. c-nity surveys in i~~pacted and uni11pacted areas. 

o Study of natural syst•: avoids 

probl- of arttftcial culture. 

o USe of naturally occurrf119 species. 

o S111P1fcity: no laboratory setup 

required; low cost for suppltu 

and equfJIIHIIt. 

o 11o control over environeental 

variablK and IISC'dated 

secondary effects. 

o Poor reproducibiHtr over ti• 

and space because of changing 

conditions and variable s..,leo Retlec:tfon of effects of long-ttr8 collection technic'ues. 

exposure rather than H•ttld and o Tf-suefng fielc'work, speciK 

arbitrary exposure tfH. identification, ar·d individual 

counting required; high labor 

cost. 

B. Artfftcial substratK in natural enviJ"OMent. 

o Study conducted in natural syst•: 

representation of natural 

conditions. 

o USe of naturally occurring 

species. 

o Si.,Hctt.Y: no laboratory setup 

requirld; low cost for suppltes 

and equi~~~~tnt. 

o llo control over environeental true 

variables and ass«iated 

secondary effects. 

o Difficult to coeparr data over tt• 

and space because of changint 

conditions and different specin. 

o Tf..-consueing ffelc'work. species 

identification. ard individual 

counting required; hfgh labor 

cost. 

o Specin li•tted to those that can 

colonize artif'ici.-1 substrate. 

o Risk of loss or dest-ruction of test 

substrates. 

9. Incubation of tnt organisa in cages in natural syst•. 

o Study conducted in natural syst•: o llo control over enviro-tal 

true representation of natural 

conditions. 

o Test species uy be selected 180119 

80St suitable bioindicator 

speciK. 

o llo laboratory setup required; 

relatively low cost for suppltes 

and equi .... t. 

variables and assC'Ciated 

secondary effects. 

o Difficult to coeparr data over 

tf• and space bec'ause of 

changing condition. 

o SpeciK generally 1f•fted to 

those that have rrlatively large 

size and lf•ited 100bt11ty. 

o L f•fted nueber of s~fes tested: 

usually only one. 

o Risk of loss or destruction of test 

enclosures. 

26 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
and field data. Experiments by deNoyelles and Kettle 

( 1985) to determine the effects of atrazine on phytoplankton 

indicated that short-term laboratory bioassays are reasonably effective in representing natural stress responses for 

about 24 hours, but that later the responses become unnatural owing to conditions not representative of the natural 

environment. The effects of fluorine on various trophic 

levels were monitored in both laboratory static toxicity tests 

(Finger and others, 1985) and experimental ponds (Boyle 

and others, 1985). Algae and invertebrates were sensitive to 

fluorine in the laboratory enclosures, but in the ponds, two 

fish species were more sensitive than either the algae or the 

invertebrates. 

Varying degrees of support for laboratory-testing 

methods have been expressed by other researchers who 

have compared laboratory and field data. Greene and others 

(1976) reported that replication of a natural lake community 

by the algal assay test was excellent, and they derived an 

equation for predicting chlorophyll-a concentrations in the 

lake on the basis of assay results. Kallqvist (1984) also 

reported good results with algal assays, and suggested that 

the patterns of growth of experimental and control cultures 

can be used to classify natural waters into one of five 

categories of toxicant and nutritive conditions. Larsen and 

others ( 1986) compared three procedures-single-species 

tests, microcosm, and experimental pond-to examine the 

effects of atrazine on eight species of algae. Good replication was reported; 50-percent inhibition of photosynthesis, respiration, and algal biomass occurred in all 

three systems at atrazine concentrations within the range of 

100 to 150 j.Lg/L. 

A study by Mount and others ( 1984) included diverse 

procedures whose results could be used to evaluate the 

validity of ambient toxicity testing. The effects of discharges from a municipal sewage treatment plant, a refinery, and a chemical company on the Ottawa River, Ohio, 

were investigated. In addition to ambient toxicity tests of 

waters from various sites downstream from the discharge 

points, the study included effluent toxicity tests, dye studies 

to describe dilution characteristics of the effluents, and in 

situ toxicity tests with fish in cages. It also included surveys 

of periphyton, phytoplankton, zooplankton, benthos, and 

fish. Positive toxicity-test results were obtained with some 

of the ambient samples collected from sites likely to be 

affected by the effluents. The authors reported that these 

results correlated reasonably well with aquatic community 

measurements, as determined by regression analyses. However, they also acknowledged that various complexities, 

such as year-to-year variations and toxicities upstream from 

effluent inputs, made interpretation difficult. They also 

pointed out the need to collect extensive and diverse 

biological data. At least two test species from different 

trophic levels should be used, and biological surveys 

should include monitoring of many segments of the aquatic 

community. 

A similar study by the same group (I\1ount and 

Norberg-King, 1985) of an Ohio stream included additional 

comparison of ambient toxicity-test results with community 

measurements. However, no verification of the test results 

was possible because no toxicity was shown by the tests. 

The stream receives discharge from a chemical rt:"ins plant, 

but even the undiluted effluent was not appreciat'1y toxic to 

the test organisms. 

Recent studies of sediments from embayments of 

Puget Sound, Wash. (Long and Chapman, 1985; Chapman, 

1986), included three phases: (1) sediment chemical analysis to determine concentrations of three metals, polychlorinated biphenyls, and polynuclear aromatic hydrocarbons, 

(2) sediment toxicity tests, using bulk sediments and an 

amphipod test species, and (3) surveys of th~ infaunal 

communities at the sample sites. This "sediment quality 

triad" approach showed good correlation amcng results 

from all three phases of the study. However, the correspondence was not nearly as consistent on a station-by-station 

basis. The authors recognized weaknesses in the data sets, 

and complexities such as contaminant interactions, that 

contributed to inconsistencies. The general implication was 

that if there is good correspondence among toxicity, chemistry, and community measurements, conclusion~ about the 

toxicity problem can be drawn, but that lack of correspondence does not lead to converse conclusions. Th~ deviation 

from "expected" results may be attributable to single or 

combined effects of innumerable environmental factors that 

are not accounted for in the triad analysis. 

Various authors (Leeuwangh, 1978; Lee and Jones, 

1983; Sadler, 1983; Kimball and Levin, 1985; Livingston 

and Meeter, 1985; Thorp and Gloss, 1986) have pointed out 

that the reliability of laboratory tests is significantly influenced by differences between laboratory and natural systems in their physical, chemical, and ecological conditions. 

This might be termed a "secondary variable" effect, a 

biological response caused by unnatural condit:')ns in the 

laboratory environment that is not related to additions of the 

tested contaminant. One of the most important secondary 

variables is pH. Numerous studies have de'tlonstrated 

effects of pH changes on toxicity of contaminants to test 

organism (Nasu and Kugimoto, 1981; Suloway and others, 

1981; Giddings and others, 1983; Lee and Jones, 1983; 

Eloranta and Halttunen-Keyrilainen, 1984; Graney and 

others, 1984; Michnowicz and Weaks, 1984). In some 

cases, detrimental effects on the test organism caused by 

secondary variables may exceed toxic effects of the contaminant (Leeuwangh, 1978). Secondary varjables may 

affect laboratory results in either direction. Fo .. example, 

low metal methylation rates may lead to underestimates of 

toxicity in laboratory tests (Benson and Summons, 1981), 

but the inability of mobile organisms to escare in a test 

enclosure may lead to overestimates of toxicit~' (Kimball 

and Levin, 1985). 

Field and Interlaboratory Verification of Toxicity Tests 27 
Table 21 summarizes reports of field verification of 

laboratory testing procedures. No clear consensus appears 

about whether laboratory tests are good indicators of contaminant effects in a natural system. However, there ooes 

seem to be good agreement between microcosm tests and 

natural systems monitoring. 

Interlaboratory or "round robin" tests have been 

conducted on several occasions to evaluate the reliability 

and precision of particular toxicity-test procedures. Favorable results of such a test were reported by Davis and Hoos 

(1975), who compared data from seven laboratories or,. 

determination of pentachlorophenate (PCP) toxicity to 

salmonid fishes. The range of 96-hour LC50 values 

reported for rainbow trout was 48 to 100 J.Lg/L, calculated 

by the log-probit estimate, and 47 to 106 J.Lg/L, calculated 

by nomographic analysis. The results were considered by 

the authors to show good consistency or, where disparities 

occurred, to be explainable by variations in the physical 

or chemical conditions of the test. An interlaboratory 

comparison of determinations of• silver and endosulfan 

toxicity to a polychaete worm was conducted by Pesch and 

Hoffman (1983). Mean 28-day LC50 values reported were 

165±52 J.Lg/L for silver and 106±24 J.Lg/L for endosulfan. 

These results were considered to demonstrate low variability among laboratories. A s~ment toxicity test was evaluated by Mearns and others (1986), based on participation 

by five laboratories. The laboratories all reported better than 

90-percent survival in control sediments. There was also at 

least 80-percent agreement among the laboratories on the 

rank order of toxicity for three endpoints (survival, emergence, and reburial), and on the mean survival in the test 

sediments. Because of the narrow range of toxicity of the 

tested samples, the interlaboratory results did not show 

close agreement as to whether the sediments should be 

classified as toxic or nontoxic. 

Somewhat less optimistic results of interlaboratory 

comparisons were reported by Nebeker (1982). Six laboratories participated in a round-robin experiment, based on 

Daphnia magna renewal life-cycle test results for silver 

and endosulfan toxicity. Four of the laboratories reported 

48-hour EC50 values for silver within a range of 0.6 to 

2.9 J.Lg/L, but the other two laboratories reported much 

higher values (8.4 to 55 J.Lg/L). The explanation given by 

the author for this discrepancy was that the two laboratories 

reporting the higher EC50 results used harder water in their 

experiments. The range of reported EC50 values for 

endosulfan was 158 to 720 J.Lg/L. A number of difficulties 

interfered with consistent results in this test, and the author 

concluded that "the Daphnia magna renewal life-cycle test 

was not validated as a routine, easily conducted test 

method." 

A protocol for interlaboratory testing of a microcosm 

toxicity-test procedure was described by Taub (1985). 

Precaution in standardizing variables that might ~ause 

inconsistencies among different laboratories was emphaTable 21. Comparison of different types of toxicity tests 

[Symbols indicate whether study reported that there is good agreement 

("+"), poor agreement ("o"), or variable agreement (both symbols) 

between the two types of tests] 

Compared types 

Static and Static and Static and Microcosm 

Flow-through Microcosm Natural and Natural 

+ 0 

0 

+ 0 

Test types ere deffned as follows: 

Reference 

Adams and others , 1983 

Adams, Werner, a'!d others, 1985 

Cherry and others, 1980 

8iesinger and others, 1982 

Thorp and Gloss, 1g86 

Kett 1 e and other~ • 1980 

Marsha 11 , 1978 

Hansen and Gart~.,. 1982 

Kimball and Levi'!, 1985 

Davies and Wood 1i ng , 1980 

Portier, 1985 

Giddings and Fra'lco, 1985 

Greene and others, 1976 

Harrass and Taub, 1985 

Kay and others , 1984 

Larsen and others, 1986 

Levy and others, 1985 

Horberg-King anc' Mount, 1986 

Mount and others, 1984 

Maciorowski and Clarke, 1980 

Sad 1 er, 1983 

B 1 anck, 1985 

Statfc: Statfc, acute, single-species test in laboratory 

Flow-through: Continuous-flow, acute, single-species test in lab1ratory 

Mfcrocos•: Enclosed, si•lated c011111unity, in either lab or fi~ld 

llatural: Natural aquatic system. 

Data obtained by field monitoring, such as ecosystem surveys, 

11easuret11ents of couunity metabolism, etc. 

8ized. Preliminary analysis was done by comparing replicate control groups in a single laboratory. As stated by the 

author, "the ability to obtain repeatable results within a 

single laboratory is a necessary prerequisite to testing for 

reprodltCible results in different laboratories.'' These data 

could be used not only to evaluate the precision cf replicate 

experiments but also to determine if the biological activity 

in the microcosms is sufficient to assess toxic art stresses. 

The analysis indicated both good replication anct adequate 

biological activity. 

In general, interlaboratory comparisons have indicated that toxicity tests can produce better precision than 

might be expected, given the extreme natural variability that 

is characteristic of nearly all biological systems. However, 

reports of good precision do not necessarily imply high 

accuracy. For example, if a toxicant is present at concentrations below the sensitivity of the test organisms, the 

concurrence of all participating laboratories in a negative 

result for this toxicant would simply emphasize the detection limitations of the test. Interlaboratory comparison data 

camtot be used to evaluate sensitivity, representativeness, 

or relevance of the test method (criteria 1, 2, and 6 of 

table 2). 

28 Applicability of Ambient Toxicity Testing to National or Regional Water·Quality Assessment 
SUMMARY 

Contaminants in the aquatic environment are of 

concern for biological reasons. They are potentially harmful 

to native aquatic organisms and to nonaquatic organisms, 

including humans, that use the water resource in some way. 

Organisms that are not directly affected by the contaminants 

may suffer indirectly through food-web transfers. Therefore, biological analyses add breadth and relevance to a 

water-quality monitoring program. As one of several types 

of biological analysis, toxicity testing produces information 

about direct impacts of contaminants on aquatic biota. 

Combined with chemical analyses, toxicity-testing procedures may provide an opportunity to correlate biological 

variables with contaminant concentrations and chemistry. 

However, there are important limitations to application of 

toxicity-testing procedures to a large-scale assessment program. These include sensitivity limitations, difficulties in 

representing the natural environment, response variability 

among test organisms, and secondary variable effects. 

To further summarize the information in this report, I 

refer back to the five questions raised in the "Introduction." 

The questions are repeated below, along with my brief 

answers to them, based on the foregoing review. 

1. What are the characteristics and applications of different 

types of toxicity tests? 

A wide variety of test types are documented by 

published reports. Procedures can be distinguished primarily on the basis of four criteria: (1) test species, (2) 

endpoint, (3) test enclosure, and (4) test substance or 

toxicant. Ambient tests can be done with water, sediments, 

or sediment extracts. The test organisms used can be limited 

to a single known species, or may include mixed species. 

Applications vary according to test type and study objectives. Applications such as the use of test results as a 

preliminary survey for planning further toxicological 

research or as a verification of other types of water-quality 

monitoring involve relatively little risk of error. More risky 

applications are those that require extrapolation of test 

results to more complex systems. 

2. What are the advantages and disadvantages of different 

types of test procedures, particularly with reference to 

application in large-scale water-quality assessments? 

Acute tests, including bacterial tests, are straightforward and rapid, but they often have poor sensitivity and are 

not representative of natural situations. Chronic tests commonly offer better sensitivity and are more realistic, but 

endpoints are usually more subtle and more difficult to 

monitor. Chronic responses are highly susceptible to 

changes in environmental conditions. Multispecies tests, 

such as large microcosm experiments, might extend the 

general applicability and sensitivity range beyond those of a 

single-species test, but their use in large-scale studies is 

limited by their design complexities. 

Compared with laboratory toxicity te<;'ts, fieldoriented procedures more closely represent natural situations and are not as subject to error due to artificial 

experimental conditions or species selection. There are 

other important sources of error, however, including natural 

biological variability, sampling inconsistencies, and lack of 

control over environmental conditions. Where possible, it is 

advantageous to employ both Laboratory and feld procedures, in addition to chemical analyses, to provide a diverse 

data base for thorough quality assessment. 

3. Do the results of toxicity tests accurately reflect environmental conditions and the probable effects of contaminants on biota in natural systems? 

It is rare that a microcosm situation, as used in most 

toxicity tests, can truly mimic the natural system it is 

intended to represent. Although many document~d toxicity 

tests may be considered reliable measures of toxic effects 

under specified conditions, they cannot replicat1~ the complexity or variability of the natural system. A positive result 

suggests the potential for a toxicant problem in the natural 

system but does very little to predict the nat'.lre of the 

probable response. A negative result does not prove the 

absence of a problem or potential problem in the natural 

system. 

Notwithstanding the difficulty of represenfng natural 

situations and stress responses, toxicity tests can be useful 

to indicate the occurrence of a condition that may be of 

concern, and to suggest possible directions of further 

research. They may be especially applicable wh~n used in 

conjunction with other water-quality and hydrologic monitoring data. Hence, failure to closely mimic natural stress 

responses is not necessarily critical. The need to accurately 

represent a natural system depends on the in ten ied use of 

the test results. 

4. Will different toxicity tests result in different conclusions about existing toxicant problems in th~ environment? 

Yes. Because of widely varying sensitivities among 

different species and among different test conditions, two 

tests of different types are unlikely to give simi'.ar results. 

This is the principal argument for implementation of a 

battery of tests in a large-scale assessment program. Procedures included in the battery may be selected so t"'at the test 

species represent different trophic levels and the tests 

complement each other with respect to sensitivity to different toxic agents. More extensive and diverse data would be 

produced, and false negative results would be less likely 

(assuming that a positive response on just one test in the 

battery would constitute an overall positive result). However, there would be substantially greater labor and monetary expenditure than would be required for a single test. 

5. Is there a particular type of test, with respect to selection 

of test species, test substance (ambient or artificial), and 

test medium (water or sediment), that can be applied in 

Sum..,..ary 29 
standardized format, with consistently reliable results, 

over a broad range of aquatic systems and environmental 

conditions? 

No. The variety of toxicant situations and the diversity of biological communities and environmental conditions among different study sites are so great that no single 

existing test method is universally applicable and reliable. 

Many criteria are important for selection of an appropriate toxicity test. Sensitivity and capability to represent 

species responses in the natural environment are the most 

critical. Other criteria that are especially important for 

selecting tests to use in large-scale studies are reproducibility, simplicity of procedures, availability of background 

information, documented methodology, and cost. Each type 

of test has its advantages, but none meets all of the selection 

criteria adequately to be considered useful as a universal 

test. 

The best prospects for future development of a 

universally applicable test are in the area of biochemical 

assays. There are many sensitive biochemical methods, 

most of which are simple and applicable with native 

organisms. Most important of their advantages is that the 

biochemical changes monitored are initial responses to 

environmental stimuli, and the responses are common to 

nearly all organisms. 

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Toxicology, v. 11, p. 203-207. 

Trotter, D.M., and Hendricks, A.C., 1976, The use of Stigeoc/onium subsecundum (Chlorophyceae) as a bioassay organism. 

II. The use of Stigeometers in a continuous flovr system: 

Water Research, v. 10, p. 913-917. 

Tsai, D.F., Welch, J, Chang, K.W., Shaeffer, J., and Cronin, 

L.E., 1979, Bioassay of Baltimore Harbor sedime.,ts: Estuaries, v. 2, p. 141-153. 

Turbak, S.C., Olson, S.B., and McPeters, G.A., 1986, Comparison of algal assay systems for detecting waterborne herbicides and metals: Water Research, v. 20, no. 1, p. 91-96. 

U.S. Environmental Protection Agency, 1986, Quality criteria for 

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Van Coillie, R., Couture, P., and Visser, S.A., 1983, Use of 

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toxicology: New York, John Wiley and Sons, chap. 18, 

p. 487-502. 

van der Schalie, W.H., 1980, A new technique for automatic 

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References Cit "d 41 
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42 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
APPENDIX: TOXICITY-TEST PROCEDURES 

As indicated in this report, many biological species 

and different procedures are used in toxicity testing. 

Detailed descriptions of the methods for each type of test 

are not given here, largely because they would only 

duplicate descriptions that are readily available in the 

literature. Publications that contain method descriptions for 

each of various test species and test types are listed in 

appendix tables 1 and 2. 

As an aid to readers who are unfamiliar with the 

toxicity-testing approach in general, some concepts and 

common features of test procedures are discussed below. 

Most toxicity-test procedures require enclosure of 

organisms, either in the laboratory or in the natural environment. Each enclosure functions as a simple microcosm 

to demonstrate an environmental effect. Functional, anatomical, and (or) behavioral characteristics of test organisms are monitored simultaneously in each system. Changes 

of biological variables in experimental systems are compared with those in control systems. The experimental 

systems contain known concentrations of the test substance, 

varying from a concentration that is not expected to be 

bioeffective to a concentration that is equal to or greater 

than any concentration likely to be found in nature. Such a 

test gradient is intended to bracket the minimum bioeffective concentration. The control systems contain no introduced toxicants, but all other conditions are identical (or as 

close as possible) to those of the experimental systems. 

Ideally, any stress response by the test organisms will 

be attributable to the introduced toxicant rather than to other 

conditions of the experiment. Therefore, it is important to 

monitor such properties as temperature, dissolved oxygen, 

pH, water hardness, and light in the experimental and 

control media. To the extent possible, these conditions 

should be controlled to maximize ( 1) their consistency 

among all enclosures, (2) their stability throughout the 

experiment, and (3) their representation of natural conditions. 

Some species are amenable for use in virtually any 

type of toxicity test, and their responses may be monitored 

by means of any of a variety of acute or chronic endpoints. 

For other species, the choices of test type and endpoints are 

more limited. Bacteria and phytoplankton, for example, 

cannot be used in acute lethality studies because of the 

impracticality of monitoring mortality in these microscopic 

organisms. Some population metabolic rates, such as bioluminescence or primary productivity, can be readily measured by standard methods, and are used as endpoints for 

species of microbiota. 

Most applications of toxicity tests call for replication 

of experimental and control systems such that there are at 

least duplicate systems for each toxicant con~entration 

tested, and for the controls. When macroscopic animals are 

used as test organisms, there are recommended lirits to the 

number and biomass of organisms in each tes.t chamber. 

These limits vary according to species and type o-f test, but 

for small invertebrates and early life stages of fish, they are 

on the order of 1-5 giL (grams per liter) (live wr'ght) and 

10 organisms per chamber. 

The duration of the test may vary from a feY' minutes 

for some bacterial tests to several months for som~ chronic 

life-cycle tests with invertebrates and fish. Acute tests with 

invertebrates and fish commonly have specified du~ations of 

48 or 96 hours. The length of the test, and the requirements 

of the species, are important in determining whetl' ~r or not 

to feed the test organisms during the test. The question of 

feeding presents somewhat of a dilemma. Lack of feeding 

may be stressful to the organisms and increase their susceptibility to toxicant effects (Nebeker and others, 1983). 

However, feeding introduces another potential var:able that 

could affect test results. Some of the references in appendix 

table 1, in particular those marked with an "S," contain 

considerable discussion of feeding techniques and effects. 

There are various possible field microcosm or mesocosm configurations, including limnocorrals (larp~ enclosures set in a lake or other water body) and expr.rimental 

ponds. The general approach for these microcosm studies is 

si~ilar to that for laboratory studies in that the biological 

effects of an introduced toxicant in experimental systems 

are measured against comparable biological ac+ivity in 

control systems. Field microcosm units usually are very 

large and contain a multispecies community that closely 

replicates the natural community they represent. Because 

they are set in the natural environment, their physical and 

chemical conditions are not under the control of the experimenter, but they are likely to mimic conditions in the larger 

system. 

Other field toxicity studies involve biological monitoring of organisms exposed to existing conditiors in the 

natural environment, rather than to a test substance introduced by the researcher. The test organisms may be 

naturally occurring biota in their natural habitats, biota that 

colonize some artificial habitat emplaced in the natural 

system, or introduced species held in any type of enclosure 

that allows environmental exposure while preventing escape 

of the organisms. Monitoring usually is done at selected 

sites that represent a known or suspected gradient of 

toxicant concentrations. For example, sampling sitr.s might 

be located upstream and at various distances downstream 

from a point source. Chemical analyses of water and 

sediments from the same sites and times provide complementary data that are useful for interpretation of the 

biomonitoring results. 

Appendix: Toxicity-Test Procedures 43 
Appendix Table 1. Partial list of publications containing detailed descriptions of 

methods for species-specific toxicity tests. 

[Within a·taxonomic group, "X" indicates species used. Both acute and chronic test methods are applicable to 

invertebrate and fish species, and are indicated by "A" and "C," respectively. "S" indiccrtes description of 

special tools or auxiliary procedures that can facilitate or modify the method. Special feeding techniques or 

requirements are included in the special procedures] 

Reference 

Bitton, 1982 

Bu I t ch , 1979 

Burton & Lanza, 1985 

Coleman & Qureshi, 1985 

De Zwart & Slooff, 1983 

Dutka & Kwan, 1981 

Dutka & Kwan, 1982 

Dutka & others, 1983 

Freeman, 1986 

McFeters & others, 1983 

Schiewe & others, 1985 

Seyfried & Horgan, 1985 

Slabbert & Grabow, 1986 

Vasseur & others, 1984 

Reference 

Honig & others, 1980 

Slabbert & Morgan, 1982 

Reference 

Aly & others, 1984 

Bartlett & others, 1974 

Christensen & others, 1979 

De Vries & Hotting, 1985 

Freetaan, 1986 

Gaur & Kumar, 1986 

Giddings & others, 1983 

Joubert, 1983 

Kutvasnlemi & others, 1985 

Miller & others~ 1978 

Ordog, 1982 

Payne & Hall, 1979 

Trotter & Hendricks, 1976 

Van Coil lie & others, 1983 

BACTERIA 

Photobacterium 

phosphoreum 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

PROTOZOANS 

Others 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

Chi lomonas 

paramecium 

Tetrahymena 

pyriformis 

c 

c 

GREEN ALGAE 

Selenastrum Scenedesmus 

capricornutum quadricauda Others 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

x. 

X 

44 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Appendix Table 1. Partial list of publications containing detailed descriptions of 

methods for species-specific toxicity tests-Continued 

[Within a taxonomic group, "X" indicates species used. Both acute and chronic test methods are applicable to 

invertebrate and fish species, and are indicated by "A'' and "C," respectively. "S" indicates description of 

special tools or auxiliary procedures that can facilitate or modify the method. Special feeding techniques or 

requirements are included in the special procedures] 

Reference 

Bishop & Perry, 1981 

Hartman & Martin, 1985 

Kay & others, 1984 

King & Coley, 1985 

lockhart & others, 1983 

Wang, 1986 

Reference 

American Public Health 

Association & others, 1985 

Bailey & liu, 1980 

Chapman & Mitchell, 1986 

Chapman & Brinkhurst, 1984 

Chapman & others, 1982a 

Chapman & others, 1982b 

Reference 

Adams and Heidolph, 1985 

American Public Health 

Association_& others, 1985 

Barera & Adams, 1983 

Bowman & others, 1981 

Buikema & others, 1980 

Cowgill & others, 1985 

Geiger & others, 1980 

Gersich & Mayes, 1986 

Goulden & others, 1982 

Horning & Weber, 1985 

Jop & others, 1986 

Keating, 1985 

leBlanc & others, 1983 

McNaught & Mount, 1985 

Mount & Norberg, 1984 

Nebeker, 1982 

Norberg & Mount, 1985a 

Peltier & Weber, 1985 

Taylor, 1985 

MACROPHYTES 

lemna Eichhornia 

minor crassipes 

X 

X 

X 

X 

X 

X 

OLIGOCHAETES 

Others 

X 

limnoldrilus Tubifex 

hoffmeisteri tubifex Others 

AC AC 

A 

A 

A A A 

A A 

A A 

CLADOCERANS 

Daphnia Ceriodaphnia Other 

magna spp. Daphnia 

c 

AC 

A 

A 

ACS 

s 

AC 

A -

ACS 

c 

s 

s 

c 

c 

c 

AC 

s 

A A 

s 

Appendix: Toxicity-Test Procedures 45 
Appendix Table 1. Partial list of publications containing detailed descriptions of 

methods for species-specific toxicity tests-Continued 

[Within a taxonomic group, "X" indicates species used. Both acute and chronic test methods are applicable 

to invertebrate and fish species, and are indicated by "A" and "C," respectively. "S" indicates description of 

special tools or auxiliary procedures that can facilitate or modify the method. Special feeding techniques or 

requirements are included in the special procedures] 

OTHER CRUSTACEANS 

Reference Amphipods Oecapods Others 

American Public Health 

Association & others, 1985 AC 

Abel, 1980 A 

Abel & Garner, 1986 A 

Arthur, 1980 AC 

Bowman & others, 1981 

Buikema & others, 1980 

Martin & Holdich, 1986 

Prater & Anderson, 1977 

Swartz & others, 1985 

Thorp and Gloss, 1986 

Graney & Geisy, 1987 

A 

A 

AC 

A 

A 

INSECTS 

Chironomidae Hexagenia 

Reference 

Anderson, 1980 

Batac-Catalan & White, 1983 

Bowman & others, 1981 

Darvi lie & Wi I hm, 1984 

Fremling & Mauck, 1980 

Prater & Anderson, 1977 

Nebeker, Cairns, & Wise, 1984 

Reference 

Harrison & others, 1984 

Dauble & others, 1985 

Paparo & Sparks, 1977 

Rodgers & others, 1980 

Reference 

Tietjen & Lee, 1984 

Samoiloff & others, 1980 

Haight & others, 1982 

spp. spp. 

A 

c 

c 

ACS 

A 

A 

MOLLUSKS 

Corbicula Other 

spp. bivalves 

A 

c 

c 

A 

OTHER INVERTEBRATES 

NEMATODES 

X 

X 

X 

A 

AC 

A 

A 

Others 

A 

46 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 
Appendix Table 1. Partial list of publications containing detailed descriptions of 

methods for species-specific toxicity tests-Continued 

[Within a taxonomic group, "X" indicates species used. Both acute and chronic test methods are applicable to 

invertebrate and fish species, and are indicated by "A" and "C," respectively. "S" indicates description of 

special tools or auxiliary procedures that can facilitate or modify the method. Special feeding techniques or 

requirements are included in the special procedures] 

FISH 

Pi me- Salmo Lepomis Others 

phales gaird- macroReference promelas neri chirus 

American Public Health 

Association & others, 1985 AC AC 

Alexander & others, 1978 A 

Cleveland & others, 1986 AC AC AC AC 

Feder & Collins, 1982 c 

Gersich & Mayes, 1986 A 

Horning & Weber, 1985 c 

Mason, 1981 c c c 

McKim & others, 1987 c 

Nebeker & others, 1985 AC 

Norberg & Mount, 1985b c 

Peltier & Weber, 1985 A A 

Phipps & Holcombe, 1985 A A A A 

Sprague, 1973 A A A 

van der Schalie, 1980 c 

Westlake & van der Schal ie, 1977 c 

Murty, 1986 ACS ACS ACS ACS 

Appendix: Toxicity-Test Procedure~ 47 
Appendix Table 2. Partial list of publications containing detailed descriptions of 

methods for different types of toxicity tests 

SMALL LABORATORY ENCLOSURES (Acute or Chronic) 

Reference Static Flow-through 

Alexander & others, 1978 

American Public Health 

Association & others, 1985 

Bishop & Perry, 1981 

Bowman & others, 1981 

Brungs, 1973 

Bu i kema & others , 1980 tlillll" 

Geiger & others, 1980 

Gersich & Mayes, 1986 

Hansen & Tagatz, 1980 

Horning & Weber, 1985 

Mason, 1981 

Meador & others, 1984 

Mount & Brungs, 1967 

Mount & Norberg, 1984 

Nebeker, 1982 

Nebeker & others, 1984 

Norberg & Mount, 1985b 

Peltier & Weber, 1985 

Phipps & Holcombe, 1985 

Sprague, 1973 

Thurston & others, 1985 

Birge & others, 1979 

lwan & Cella, 1981 

Gruber & others, 1980 

Meador & others, 1984 

Wuerthele & others, 1973 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

LABORATORY MICROCOSMS 

Reference 

Adams, V.D., & others, 1985 

Black & others, 1973 

Giddings, 1986 

Giddings & Franco, 1985 

Graney & others, 1984 

Hansen & Tagatz, 1980 

Harrass & Taub, 1985 

Hedtke, 1984 

Honig & Buikema, 1980 

Levy & others, 1985 

Portier, 1985 

Rodgers & others, 1980 

Shriner & Gregory, 1984 

Stay & others, 1985 

Taub & Crow, 1978 

Taub & others, 1983 

Westlake & van der Schalie, 1977 

Yasuno & others, 1985 

Aquaria 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

Artificial 

streams 

X 

X 

X 

X 

X 

48 Applicability of Ambient Toxicity Testing to National or Regional Water-Quality Assessment 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 

X 
Appendix Table 2. Partial list of publications containing detailed descriptions of 

methods for different types of toxicity tests-Continued 

Reference 

deNoyelles & Kettle, 1985 

Giddings & Franco, 1985 

Hedtke & Arthur, 1985 

Herman & others, 1986 

Kaushik & others, 1985 

Kaushik & others, 1986 

Robinson-Wilson & others, 

Marshal I & Mellinger, 1980 

Wilde & Parrott, 1984 

Reference 

Beak & others, 1973 

Foe & Knight, 1987 

Rice & White, 1987 

Perkins, 1983 

leland & Carter, 1985 

Reference 

FIELD MICROCOSMS 

Experimental 

ponds and Limnostreams corrals 

X 

X 

X 

X 

X 

X 

1983 X 

FIELD INCUBATION PROCEDURES 

Artificial 

subtrates 

Caged 

organisms 

X 

X 

X 

X 

X 

OTHER TEST TYPES 

Sediment 

tests 

Biochemical and 

physiological tests 

leBlanc & Suprenant, 1985 X 

long & Chapman, 1985 X 

Malueg & others, 1984a X 

Prater & Anderson, 1977 

Schiewe & others, 1985 

Bitton, 1982 

Graney & Geisy, 1987 

Hinton & others, 1973 

Katz, 1979 

Neff, 1985 

Wong & others, 1982 

Besch & others, 1977 

Fisher & others, 1982 

Gruber & Cairns, 1981 

Gruber & others, 1980 

Kleerekoper, 1977 

Maki, 1979 

Morgan, 1977 

Poels, 1977 

van der Schalie, 1980 

X 

X 

X 

X 

X 

X 

X 

X 

Others 

X 

X 

Electronic and 

computerized 

monitoring 

X 

X 

X 

X 

X 

X 

X 

X 

X 

Appendix: Toxicity-Test Procedure~ 49 



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