Toxicity Testing, Aquatic
M Sobanska, R Cesnaitis, and B Versonnen, European Chemicals Agency, Helsinki, Finland
Ó 2014 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by Shayne C. Gad, volume 4, pp 233–239, Ó 2005, Elsevier Inc.
Introduction
As described in the article ‘Ecotoxicology,’ ecotoxicology is the
science devoted to studying the adverse effects of various
stressors on the structure, functions, and biodiversity of
ecosystems. Although these stressors can be physical, chemical,
biological, or a combination of these, the scope of ecotoxi-
cology is often narrowed down to the effects of (man-made or
man-released) chemicals. Aquatic ecotoxicology encompasses
both the pelagic and benthic environment. In addition,
a distinction between freshwater and marine ecotoxicology is
often made. Therefore, aquatic ecotoxicicology identifies
hazards of chemicals to biotic communities of ecosystems
based on toxicity testing to aquatic animals and plants.
Aquatic ecosystems consist of thousands of different species
representing various trophic levels of living organisms,
dependent on each other and on their environment. The two
main aquatic ecosystems are the marine ecosystem, which
includes oceans, estuaries, and coastal waters, and the fresh-
water ecosystem, including lakes, ponds, rivers, streams, and
groundwater. They all create complex and very specific envi-
ronments for their inhabitants ranging from monocell plank-
tonic organisms through thousands of benthic and pelagic
invertebrate species, various fish, amphibians and reptiles, and
finally birds and mammals.
With this huge number of interrelated species, aquatic
toxicity testing can serve only as a model simplifying the
complexity and relationships taking place in the natural
ecosystems. In order to accept the extrapolation of the results
from the test model to the whole ecosystem, the study needs to
be scientifically relevant, well designed, and reliable.
In aquatic toxicity testing where the tested chemical is dis-
solved in water and the waterborne exposure of a substance is
the main route of exposure, a concentration–effect relationship
is usually determined. The environmental concentration indi-
cates the amounts of substance(s) present in the test medium
or in the environment. It is worth mentioning that the envi-
ronmental concentration depends not only on the amount of
substance added to the test medium but also on its physical–
chemical properties (e.g., water solubility, volatility, hydrolysis,
biological oxygen demand, bioaccumulation potential, etc.)
influencing the fate and behavior of that substance in the
aquatic environment.
When the substance adsorbs or bounds to sediments and
exhibits toxic effects to benthic organisms, sediment toxicity
testing plays an important role. Sediments may act as both
a sink for chemicals through the sorption of contaminants to
particulate matter and a source of chemicals through resus-
pension. Sediments integrate the effects of surface water
contamination over time and space and may present a hazard
to aquatic communities (both pelagic and benthic), which is
not directly predictable from concentrations in the water
638 Encyclopedia of Toxicology, Volume 4
column. Effects on benthic organisms are of concern because
they constitute an important link in the aquatic food chain and
play an important role in the recycling of detritus material.
Purpose of Testing
There are various purposes of aquatic toxicity testing, e.g.,
scientific, product oriented, or regulatory. As the regulatory
purpose plays an important role in decision-making processes
and assures the safe use of chemicals, this type of aquatic
toxicity testing will be further described in this chapter. Regu-
latory testing may be performed for the risk assessment
purposes, to identify hazardous substances contained in water
and to assess their impact on the living organisms exposed to
them. Another purpose is to establish water quality standards
describing the quantity of pollutants that can be safely present
in the aquatic environment without causing harm to the
surrounding ecosystem. Together with science development
and increase of precision of measuring facilities/equipment,
new or improved methods are continuously developed.
“For regulatory purposes, the so-called ‘minimum data set’
has been developed. The aim of this minimum data set is to
provide a minimum level of information that is considered
necessary for setting ‘regulatory acceptable levels.’ Although
there are differences among the different jurisdictions, basically
the minimum data set for industrial chemicals consists of acute
aquatic data from three trophic levels: algae, crustaceans, and
fish (see Ecotoxicology). In addition, chronic exposure and
specified routes of exposure should be considered depending
on the intrinsic properties of the substance and its expected
environmental concentration.
‘Aquatic toxicity’ refers to the intrinsic properties of
a substance to which an aquatic organism is exposed during
short term (so called acute) and/or long term (chronic). It is
assumed, in general, that the aquatic toxicity can be related
mainly to the external concentration of that substance dis-
solved in test water. However, there are also cases where food
uptake is the predominant route of exposure (i.e., for lipophilic
substances). Effects caused by such chemicals can be more
easily measured in dietary studies.
‘Acute toxicity’ defines a toxicity to aquatic organisms
exposed to substances within a relatively short time (hours to
days) in comparison to the duration of the life cycle of the
organisms. Mortality is the most common of the measured acute
effects in aquatic ecotoxicology. Effects are normally expressed
as median lethal or effect concentrations (LC50/EC50), which is
the test concentration at which 50% of the organisms are
affected or at which 50% effect is measured for a specifically
defined endpoint (e.g., growth rate effects on algae).
‘Chronic toxicity’ defines toxicity to aquatic organisms
exposed to substances for a prolonged period in comparison to
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the duration of the life cycle of the test organisms. Exposure
(test) duration may vary widely depending on the species used.
Observations can be made on a spectrum of sublethal effects,
such as, growth and reproduction. The highest tested concen-
tration where no effects have been observed (no observed effect
concentration (NOEC)) is the most frequently used parameter.
It may often be replaced by effect concentration at which 10%
effect is measured for a specifically defined endpoint (EC10).
These can be estimated based on the concentration–effect
relationship.
Types of Aquatic Testing
Aquatic tests may be performed in the laboratory or in the
natural conditions (field studies) and may involve one or more
test species. Information on aquatic toxicity may be obtained
from studies performed according to existing national and
international guidelines as well as from scientific literature,
where different aspects of aquatic toxicity are examined. The
tests designed for the regulatory purposes should be stan-
dardized to give results that are translatable into the regulatory
measures. The standardized tests should be precise, relatively
simple, and easily reproducible. Furthermore, the standardized
tests should assure the optimal tests conditions minimizing the
effects of other or unknown factors (stressors) potentially
leading to a wrong interpretation of the test results.
The results of aquatic toxicity tests are derived by comparing
the effects caused by a tested chemical on the test organisms to
the effects observed on control organisms kept in the same test
conditions without the presence of the tested substance.
Studies should be designed to enable sufficient statistical
differences to be established between controls and exposed test
animals. Further guidance on the number of test organisms per
replicate, the number of replicates itself, the number of
concentrations necessary for a reliable ECx, and/or NOEC/
lowest observed effect concentration determination can be
found in the different Organization for Economic Cooperation
and Development (OECD) test guidelines. For regulatory
purposes, the use of standardized tests is recommended.
The European Chemicals Agency (ECHA) has published
guidance on aquatic toxicity describing international test
guidelines available for the pelagic system. These are focused
mainly on measuring the adverse effects of substances due to
waterborne exposure in freshwater. However, there are also test
guidelines available for the testing of benthic organisms and
saltwater species.
U.S. Environmental Protection Agency (US EPA) has created
and maintains ECOTOXicology database. It consists of an
extensive collection of information on adverse effects of
chemicals on aquatic and terrestrial organisms taken from peer-
reviewed literature. Since the year 2000, the ECOTOX database
is available as a web-based information system to the general
public.
Single-Species Test
For regulatory purposes, single-species tests are often a prag-
matic and relatively easy way to gather toxicity data on chem-
icals. In single-species testing, the most sensitive representatives
Toxicity Testing, Aquatic 639
of selected trophic levels are tested under the laboratory
conditions in order to focus on a specific problem as the subject
of the test. It is important to emphasize that the sensitivity of
the tested species is very important for the interpretation of the
test results. As ecosystems have a functional resilience and self-
regulation capacity, the adverse effects of the pollutants will
firstly be seen within the sensitive species or populations while
the ecosystems will still maintain their essential functions.
Therefore, the sensitive species will show toxic effects at
significantly lower concentrations than the whole ecosystem
and generally provide an early warning on potential environ-
mental damage.
Multiple-Species and Field Data
Multiple-species and field data represent a higher experimental
tier in the hazard and fate assessment as the diversity of
organisms and their interactions cannot be adequately
modeled in simpler laboratory single-species tests. The results
of these types of tests are not equally useful for regulatory
purposes. Their applicability depends on the design of the test
and the objectives set in model ecosystem studied. A stan-
dardized guidance for conducting the simulated freshwater
lentic (standing water) tests in the form of outdoor microcosms
and mesocosms has been agreed at OECD level. A microcosm is
a miniaturized model of a natural ecosystem while a mesocosm
is a controlled field experiment. Although the primary
endpoints of these studies are the effects on aquatic organisms,
it is possible to obtain information on the fate of substances at
the same time. The test system is usually closed, and spiked
with the substance under realistic outdoor conditions, with
representative flora and fauna included.
Monitoring Data
Monitoring data may demonstrate the occurrence or removal
of contaminants from the aquatic environment. When
monitoring data are considered in the risk assessment of
substances, the data are often obtained from existing moni-
toring programs. In this case, the field or monitoring study has
not been specifically designed to fulfill regulatory needs and
therefore special care should be given to the selection of
relevant data.
In Vitro Testing
At present, there are no European Union (EU)/OECD guide-
lines for in vitro tests with relevance to aquatic toxicity. There are
ongoing efforts to develop and validate in vitro methods, which
in future might be useful in a testing strategy for acute aquatic
toxicity. The extrapolation of in vitro data to in vivo data is still
under scientific discussion in literature and in the international
research fora.
Nontesting Data
Nontesting data consist of data generated by quantitative
structure–activity relationship models, expert systems, and data
obtained by grouping approaches (analogue and chemical
category approaches).
640 Toxicity Testing, Aquatic
Marine Species Testing
There are few standardized marine species protocols available
for testing marine ecosystems for regulatory purposes. In
general, the same criteria as described for freshwater tests should
be applied for the evaluation of the tests for marine species.
Additional attention should be paid to the fact that the solu-
bility of the substance might be influenced by water salinity.
Sediment Species Testing
For testing of the sediment compartment for regulatory
purposes, the whole-sediment tests using benthic organisms
are considered to be most appropriate. By using such tests, it is
possible to adequately address all routes of exposure. Due to
the generally long-term exposure of benthic organisms to
sediment-bound substances, long-term tests with sublethal
endpoints like reproduction, growth, or emergence are most
relevant.
Testing of Difficult Substances
A significant number of chemicals are described as ‘difficult
substances,’ which the OECD classifies as difficult to test for the
purpose of determining their aquatic toxicity. Some examples
of such difficulties are maintaining substance concentrations
during the test; dissolving the substance, either due to its poor
solubility in the test medium or a multicomponent substance
where each component has a different solubility; analyzing the
concentration of the substance due to problems in developing
an analytical method, etc. The possibility of a substance being
difficult to test can often be seen from its physical–chemical
properties such as low water solubility, high volatility, or
degradability properties. This underlines how important it is to
know these parameters prior to new tests being carried out or
before reviewing a test report.
Internationally Accepted Test Guidelines
The OECD test guidelines comprise internationally agreed
testing methods for environmental effects. Tests undertaken
using these guidelines are useful for risk assessment and clas-
sification and labeling purposes. Data obtained from a test
carried out in accordance with an OECD test guideline are
covered by the principle of mutual acceptance of data, thereby
reducing the number of tests that needs to be conducted saving
both animals and money. Alternatives to the OECD test
guidelines are published by the Office of Pesticide Planning
and Toxic Substances, US EPA, EU, various national standard
methods, and organizations such as American Society for
Testing and Materials and International Organization for
Standardization.
Important Elements of Toxicity Testing Performed
for Regulatory Purposes
Test Substances
Before selecting the relevant test, it is important to be able to
accurately identify the substance to be tested. Its chemical
composition and purity should be described and where possible
identification of the impurities should be made. Toxic impurities
can influence the observed effects even if they are present at
low levels. Physical–chemical properties of the test substance
should be known and reported. For aquatic toxicity testing, the
knowledge of the water solubility of the test substance is of
special importance as the test results which occur above the limit
of water solubility should be considered in further detail.
Test Organisms
The organisms selected for standard laboratory testing are
relatively easy to maintain and keep healthy in the laboratory
conditions. Moreover, clearly defined and measurable endpoints
in (standardized) toxicity tests should be favored. The organ-
isms favored for standard chronic tests should usually have
short and simple life cycles that enable clear observations of
sublethal effects.
Nowadays, standardized and internationally accepted
aquatic toxicity test methods cover tests on major groups of
aquatic organisms. Many OECD test guidelines include the lists
of standard species for which the test method is relevant. When
reporting the results of testing for the regulatory purpose,
details on the taxonomic identity of the test organisms need to
be well documented.
Test Concentrations
In aquatic toxicity testing, ‘multiconcentration tests’ are most
widely used. In this type of study, the test organisms are
exposed to the series of concentrations of the tested chemical
over a period of time. In the result of multiconcentration tests,
the dose–response relationship is established and might be
used to deliver a quantitative estimation of the level of toxicity
of the tested chemical (e.g., LC50, EC50).
Studies where only a single (high) test concentration is used
are called a ‘limit test.’ The purpose of a limit test is to confirm the
presence or absence of toxicity. Such tests are often used when
a substance is expected to be not toxic at low concentrations.
Route and Type of Exposure
Application of the test substance is a critical factor in acute/
aquatic toxicity testing to ensure appropriate (suitable) exposure
to the test organisms. Moreover, in order to ensure stable test
conditions, the constant concentration in the test environment
during the testing period should be maintained. Depending on
the type of exposure, three main categories of tests are used in the
testing of aquatic toxicity: static, semi-static, and flow-through
tests. In static tests, the test medium is not changed during the
whole experiment and the test concentrations are not renewed in
the course of the test. In semi-static tests, the test material is
periodically renewed (usually daily). In flow-through tests, the
test material is continuously renewed at a constant rate and
concentration during the whole test. For algae, static tests are the
most commonly used. For Daphnia studies, static or semi-static
tests are used while for fish all three types of tests are applied. The
potential effect of any relevant physical–chemical properties of
the substance such as solubility, high adsorption, precipitation,
etc. should be taken into account when selecting the method of

test substance administration. In some studies, food is added
during the exposure period (e.g., green algae are added as food in
a Daphnia reproduction test). In such cases, exposure may also
occur via food for substances that adsorb to the algae. A
description of the test medium and dilution water as well as
other relevant information such as the amount of total organic
carbon, unionized ammonia, etc. should be documented. It
should also be ensured that all abiotic factors fall within the
tolerance limits of the test organisms. A proper description of
abiotic parameters, such as dissolved organic carbon concen-
tration, cations, anions, etc., can also provide information on the
speciation (i.e., availability) of the test substance, which may
influence the uptake and observed effects of certain chemicals.
Test Controls
Test controls are of special importance in toxicity testing as they
provide information about the normal functioning of a test
organism in the test setup in the absence of the test substance.
Test Results
Test results presented as the concentration of the test substance
at which the effect occurs can be stated as either ‘the nominal
concentrations’ obtained from the dilution calculations or ‘the
measured concentrations’ obtained by direct or indirect
chemicals measurements. The accuracy and precision of the
chemical method should always be documented together with
the chemical measurements.
Statistical Analyses
Statistical methods for the derivation of LC50, EC50, IC50,
NOEC values, etc. should be reported.
Quality of the Study
When assessing the quality of the study, its relevance and
reliability have to be taken into account. This issue is well
presented in ECHA’s Guidance.
‘Relevance’ of the test is the extent to which data and tests
are appropriate for a particular testing purpose. In order to
assess the relevance of the test and its results, it has to be
evaluated if the appropriate test substance has been studied, if
the suitable test organisms are selected, if the applied route of
exposure is relevant for the tested population, if appropriate
doses/concentrations are tested, and if the critical parameters
influencing the tested endpoint are adequately considered.
‘Reliability’ is the inherent quality of a test report or
a publication relating to preferably standardized methodolo-
gies and the way the experimental procedure and results are
described to give evidence of the clarity and plausibility of the
findings. It is important to distinguish between reliable
methods and reliable information.
The quality of the study depends on the selected method,
the way of reporting the results, and the drawn conclusions. In
order to assess the reliability of data from toxicological and
ecotoxicological studies, various ranking systems have been
developed. One of the most widely used is the Klimisch scoring
system, which categorizes studies into four categories.
Toxicity Testing, Aquatic 641
Studies with reliability score 1 are considered to be reliable
without restrictions. These tests are generated according to
generally valid and/or internationally accepted testing guide-
lines, preferably performed according to Good Laboratory
Practice (GLP).
Studies with reliability score 2 are considered to be reliable
with restrictions. It means they do not totally comply with the
specific testing guideline and are mostly not performed
according to GLP. However, the documented test parameters
are sufficient to accept the data as they are sufficiently well
documented and scientifically acceptable.
Studies with reliability score 3 are considered to be unreli-
able. In these studies, there were obvious problems, e.g., the
measuring system did not fit to the test substance, irrelevant
test organisms/test systems were used, unacceptable test
methods were applied, or insufficient and unconvincing
documentation was provided.
Studies with reliability score 4 are considered to be not
assignable. They do not give sufficient experimental details
and/or come from secondary literature (books, reviews, etc.).
The use of such scoring tools allows the information to be
ranked and organized for further review. This implies that
a focus is paid to the most relevant and reliable studies, taking
into account the endpoint being measured or estimated. The
scoring of information, e.g., according to Klimisch scores,
should not exclude all unreliable data from further consider-
ation by expert judgment because of possible pertinence of
these data related to the evaluated endpoints.
Uncertainties in Aquatic Toxicity Testing
Aquatic toxicity testing is accompanied with various types of
uncertainties. They can consist of the possible measurement
errors (e.g., influence of the methodology used, errors in the
analytical method used to measure chemical concentrations or
technical inadvertence), and sample uncertainties reflecting
representativeness of the data set (e.g., sample characteristics,
sample size as a small sample may not give the entire range of
values found in reality, averaging methodologies, etc.). The
application of standard tests, conducted in the certified labora-
tories according to the GLP, significantly increases the reliability
and repeatability of the test results and therefore decreases
some aspects of testing uncertainties. When the results derived
in new nonstandard tests are presented, uncertainty analysis
might be a useful part of the documentation provided to
enhance robustness of and confidence in the results.
See also: Ecotoxicology; Aquatic Ecotoxicology; Ecotoxicology,
Aquatic Invertebrates; European Centre for Ecotoxicology and
Toxicology of Chemicals; The European Chemicals Agency;
Aquatic Mesocosms and Microcosms; Multispecies
Environmental Testing Designs; REACH.
Further Reading
Council Regulation No 440/2008 of 30 May 2008 laying down test methods pursuant
to REACH Regulation No 1907/2006 – Test Methods Regulation.
ECHA, 2008. Guidance on Information Requirements and Chemical Safety Assess-
ment, Chapter R.7b: Endpoint Specific Guidance. European Chemicals Agency,
http://echa.europa.eu/.
642 Toxicity Testing, Aquatic

Environment Canada, 1999. Guidance Document on Application and Interpretation of U.S. Environmental Protection Agency, 2012. ECOTOX User Guide: ECOTOXicology
Single-Species Tests in Environmental Toxicology. ISBN: 0-660-16907-X. Database System. Version 4.0. http://www.epa.gov/ecotox/
European Parliament and of the Council Regulation No 1907/2006 of 18 December
2006 concerning the Registration, Evaluation, Authorisation and Restriction of Relevant Websites
Chemicals (REACH).
Klimisch, H., Andreae, M., Tillmann, U., 1997. A systematic approach for evaluating http://www.oecd.org/ – OECD
the quality of experimental toxicological and ecotoxicological data. Regulatory http://echa.europa.eu/ – European Chemicals Agency
Toxicology and Pharmacology 25, 1–5. http://www.epa.gov/ – US EPA
OECD, 2000. Guidance, No. 23 Testing and Assessment: Guidance Document on http://www.ec.gc.ca/default.asp?lang¼en – Environment Canada
Aquatic Toxicity Testing of Difficult Substances and Mixtures ENV/JM/MONO(2000)6, http://cfpub.epa.gov/ecotox/ – US EPA Ecotox database
http://www.oecd.org
OECD, 2006. Guidance, No. 53 Testing and Assessment: Guidance Document on
Simulated Fresh-water Lentic Field Test (Outdoor Microcosm and Mesocosm) ENV/
JM/MONO(2006)17, http://www.oecd.org/