Chapter 7 _ Ecotoxicity testing

Prévia do material em texto

Bioindicators and biomonitors 
B.A. Markert, A.M. Breure, H.G. Zechmeister, editors 
�9 2003 Elsevier Science Ltd. All rights reserved. 221 
Chapter 7 
Ecotoxic i ty testing 
Hans Toni Ratte, Monika Hammers-Wirtz 
and Michael Cleuvers 
Abstract 
Ecotoxicity tests are required in the context o f national legislation and regulation of pesti- 
cides, other chemicals and environmental quality. Among the OECD (Organisation for 
Economic Cooperation and Development) countries the requirements differ only marginally 
as do also the current guidelines for the conduct of ecotoxicity tests. Ecotoxicity testing can 
be divided in lower-tier and higher-tier testing. In lower-tier tests single cohorts o f a test 
species from different trophic levels and environmental compartments are examined in the 
laboratory under standardised conditions. International standards and guidelines have been 
developed by CEN (ComitO EuropOen de Normalisation), ISO (International Organization for 
Standardization) and OECD. Depending on the selected test, the effect of the test material on 
one to several endpoints is investigated, mainly variables of survival, reproduction, growth 
and metabolism. The special characteristics, endpoints considered and problems of current 
lower-tier tests are described and discussed. Mainly two statistical designs are performed: the 
point-estimation approach to compute the EC x (effective concentration for an effect o f x%) as 
toxicity parameter and the hypothesis-testing approach to determine the NOEC (no-observed 
effect concentration). Sometimes a so-called limit test is conducted, in which the effect o f only 
one high concentration is investigated and statistically tested. Hypothesis testing is problem- 
atic since there is often the danger of a false-negative result, i.e. statistical test reveals "no 
effect" but there is one. Therefore, the hypothesis-testing approach has been increasingly crit- 
icised during the past decade, so that OECD and ISO decided on a phasing-out o f the NOEC 
as toxicity parameter, which at this point appears to be not followed unequivocally. 
In higher-tier testing more flexible and complex ecotoxicity tests are performed, a stan- 
dardisation of which is impossible. They are required if a chemical substance fails to meet 
the trigger values in lower-tier risk assessment. These tests range from experiments with addi- 
tional species, populations, small communities in the laboratory to larger-scaled outdoor 
mesocosms. The general aim is to research fate and effects o f a test substance under more 
realistic environmental conditions, i.e. fate processes can take place and the tested popula- 
tions o f the test community undergo biotic and abiotic interactions. 
Keywords: Ecotoxicity tests, lower-tier, higher-tier, authorisation of chemicals, guidelines, 
statistical design 
1. Introduct ion 
Ecotoxicity tests are biological experiments with various test species under presence 
of chemical substances or environmental samples. They are required for risk assess- 
ment of new and existing chemicals as well as for monitoring the environmental 
quality (e.g. of effluents, sediment and soil samples). In each test the response of one 
222 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
or more characteristic endpoints are measured. The term "endpoint" (also called "res- 
ponse variable") refers to the biological parameter measured/observed, e.g. survival, 
number of eggs, size or weight, enzyme level. The terms "effect" and "response" 
describe the change in the endpoint considered. For quantal endpoints (e.g., mortality; 
see "Statistical Treatment" section) an effect is defined in terms of a change in the 
number of animals affected, for continuous endpoints (e.g., growth rate) it is defined 
in terms of a percentage change in the average level of the endpoint, for both quantal 
and continuous endpoints in comparison to the controls. 
An important requirement for ecotoxicity tests is their reproducibility and repeata- 
bility, because the results have to be justiciable. Therefore, the tests are run under the 
principles of Good Laboratory Practice (GLP; OECD 1998b) and using internation- 
ally accepted guidelines and standards, such as developed within the OECD 
(Organisation of Economic Cooperation and Development), CEN (Comit6 Europ6en 
de Normalisation) and ISO (International Organization for Standardization). 
To bring new chemical substances onto the market, in Europe ecotoxicity testing is 
prescribed by national and European directives (e.g., 91/414/EEC, 1991). According 
to the EEC directive, a tiered approach for identifying potential risks for aquatic 
species is performed. Effects on biota are being characterised by acute and chronic 
laboratory studies (so-called Lower-tier Tests) using various aquatic organisms, e.g. 
fish, invertebrates, algae and macrophytes. As effect parameters effect concentrations 
or threshold concentrations (e.g., EC50s and NOECs, respectively) from these studies 
are compared with predicted environmental concentrations (PECs) to obtain toxicity 
exposure ratios (TERs). In Annex II (91/414/EEC, 1991), trigger values for these TERs 
of 100 and 10 are applied to acute and chronic studies, respectively. If the ratio for an 
active ingredient exceeds these trigger values, no further biotesting is required. If the 
ratio is below these trigger values, so-called Higher-tier studies are required for a 
refined risk assessment. If a chemical substance fails to meet the trigger values, a range 
of higher-tier studies have been recommended by the HARAP workshop (Campbell 
et al. 1999). The results from these studies can be used to demonstrate acceptability 
of the chemical substance. In addition, Annex II of Directive 91/414 EEC (1991) 
suggests aquatic microcosm or mesocosm studies as higher-tier tests. 
With respect to monitoring of environmental samples only lower-tier tests are used 
to determine a dilution which is regarded as tolerable. The toxicity parameters deter- 
mined are either a low EC~ (e.g., EC20, EC10) or the Lowest Ineffective Dilution 
(LID). In some countries these sort of tests forms the base for the legal control of efflu- 
ents from e.g. industrial waste water treatment plants (e.g., in Germany according to 
the "Wasserhaushaltsgesetz (WHG)" and "Abwasserabgabengesetz (AbwAG)"). 
Depending on the results companies can be sentenced by court to pay for compensa- 
tion measures. 
Compared with the more than five million species on earth only very few species 
are used as test organisms in ecological risk assessment to assess effects on the 
ecosystem. Important criteria for the selection of ecotoxicological test species are 
sensitivity, representation of the ecosystem that may receive the impact, abundance 
and availability of the species, the ecological importance, as well as practical aspects 
like easy handling and culturing of species in the laboratory. 
Furthermore background information on a species (i.e., its physiology, genetics, and 
Ecotoxicity testing 223 
behaviour) is helpful for interpreting the test results. Because of the variation in sensi- 
tivity between different species, the test species used has a great impact on the 
assessment of a chemical. The generally most sensitive test species does not exist 
because the sensitivity of a species depends on the chemical exposed to and the test 
parameter recorded. Therefore it appears to be important to investigate several species 
exhibiting different sensitivities. 
The use of test species belonging to different trophic levels has been established in 
risk assessment. Representative species traditionally used for risk assessment of chem- 
icals are various freshwater species representing algae, invertebrates and fishes. Some 
green algae like Scenedesmus subspicatus, Chlorella vulgaris or Pseudokirchneriella 
subcapitata are in use as standard test organisms representing primary producers, while 
Daphnia magna or otherover one or 
several life cycles (e.g., Taylor et al., 1992; Maund et al., 1992; Hammers-Wirtz and 
Ratte, 2000). The assessment of effects on population growth is described in, e.g., Van 
Straalen and Kammenga (1998). 
Also models of the effects of pesticides on populations including those of Daphnia 
and fish have been developed (e.g., Kooijman and Metz, 1984; Barnthouse et al., 1987; 
Gumey et al., 1990; De Angelis et al., 1991; Hommen et al., 1993; Ratte et al., 1994; 
Hallam and Lassiter, 1994; Acevedo et al., 1995; Sibly, 1996; Maund et al., 1997; 
Calow et al., 1997). The advantage of models is seen in the wide range of scenarios 
which can be evaluated. 
3.3. Indoor multi-species tests 
HARAP divided the indoor multi-species tests as follows: 
1. Simple indoor multi-species tests to study specific interactions/processes (e.g., 
bioavailability, population responses). 
2. Indoor defined microcosm tests comprising well-described assemblages of organ- 
isms at different trophic levels to assess critical ecological threshold levels. 
3. Indoor semi-realistic microcosms comprising complex natural assemblages which 
can be used to define directly an EAC. The uncertainty factor to be applied to such 
an EAC needs to be assessed on a case-by-case basis, based on the uncertainty and 
acceptability of the test. 
3.3.1. Simple indoor multi-species tests 
A few selected species are studied with regard to their interactions that are suspected 
to be of importance under realistic field conditions, in order to illustrate the influence 
of particular biotic factors on pesticide effects (e.g., the impact of the presence of 
macrophyte or algal biomass on the bioavailability and toxicity of an insecticide to 
fish or Daphnia; transfer of the pesticide via the food). Some examples of simple 
indoor multi-species tests are described in Day and Kaushik (1987), Gomez et al. 
(1997), Hamers and Krogh (1997) and Klfittgen et al. (1996). 
3.3.2. Indoor defined microcosm tests 
Relatively small test systems are inoculated with a well-described assemblage of small 
organisms but characterised by several trophic levels (e.g., primary producers, 
consumers, decomposers). These microcosms may be seeded with organisms from 
244 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
stock cultures or field or even standard test species (e.g., Taub, 1969, 1974; Leffler, 
1981; Kersting 1991). 
For a number of pesticides, both defined microcosms tests and outdoor micro- 
cosm/mesocosm tests have been performed (for references see Campbell et al., 1999) 
showing that the ecological threshold levels (i.e., the concentration above which effects 
on population densities and functional endpoints become apparent) of pesticides 
observed in indoor defined microcosms may be comparable to those measured in 
complex outdoor experimental ecosystems. 
3.3.3. Indoor semi-realistic microcosms tests 
This type of system intends to represent natural assemblages of organisms charac- 
terised by several trophic levels. They are constructed directly with samples of natural 
ecosystems. Species covering a wide range of sensitivities and biological diversity can 
be included. In general, indoor semi-realistic microcosms can include populations of 
microorganisms, planktonic, periphytic and benthic algae, zooplankton, meiofauna, 
macroinvertebrates and, when large enough, also macrophytes (for examples see 
Breneman and Pontasch, 1994; Fliedner et al., 1997; Van den Brink et al., 1997). 
Among the advantages of indoor semi-realistic laboratory microcosm tests over 
outdoor field tests are experimenting throughout the year, easier control over the exper- 
imental conditions, and less set-up costs. In contrast, disadvantages are seen in 
unrealistic population densities of large organisms (e.g., fish, newts, frogs and nymphs 
of larger insects), disturbance by larger organisms, lack of natural fluctuations, diffi- 
culty to investigate long-term effects and recovery of species with complex life cycles, 
limited number of microhabitats, and possibility of disturbing certain populations by 
sampling (e.g., macroinvertebrates and macrophytes). 
3.4. Field studies 
If laboratory studies (lower- and higher-tier) indicate potential risks, field studies 
(micro-/mesocosm) are required, in order to test specific hypotheses about ecological 
effects, i.e. population-level and community-level effects. The aim is to derive an EAC. 
This means that for certain taxa or end-points, effects observed in a field study may 
be considered acceptable, if with appropriate expert ecological .judgement, it is consid- 
ered that they would not pose significant ecological risks to natural aquatic ecosystems. 
However, if a keystone, indicator or protected species is substantially affected, this is 
considered as not acceptable. HARAP recommends that the results of field studies 
should be accompanied by clear explanations as to why a given observed effect should 
be considered ecologically significant or acceptable when they are presented to regu- 
latory authorities. Furthermore, wherever possible, such studies should be reviewed by 
an expert panel to provide the least-biased advice. 
Every field study is designed with a specific purpose and is unique in at least some 
respects. Therefore, it has to be designed accordingly. Guidance for conducting field 
studies was developed during several workshops held at Monks Wood (SETAC, 1991), 
Wintergreen (SETAC/RESOLVE, 1991), and Potsdam (SETAC, 1992), resulting in 
an OECD (1998a) draft guidance document for field studies. 
Ecotoxicity testing 245 
The test system is usually a "naturally" developed aquatic community maintained 
under outdoor conditions which usually contains naturalised sediment and appropriate 
organisms such as zoo- and phytoplankton, pelagic and benthic macroinvertebrates, 
and macrophytes (inclusion of fish is not generally recommended). It may not be neces- 
sary to include macrophytes if the study objectives only concern phytoplankton and 
zooplankton. Organisms are identified to "the lowest practical taxon" (arthropods: 
usually species or genus; algae: sometimes only to class). For a powerful statistical 
analysis of community structure, species-level identification is preferable (e.g., use of 
Principal Response Curves and Similarity Analysis; see e.g., Van den Brink and Ter 
Braak, 1999). 
It was already recognised at the HARAP workshop, that aquatic microcosms and 
mesocosms are highly complex test systems, for which the ecological interpretation 
and use in risk assessment needed further discussion and guidance. Therefore, the 
CLASSIC Workshop (Community Level Aquatic System S tud ies - Interpretation 
Criteria; Giddings et al., 2002) was organised to provide such a forum. Among the 
most important recommendations of CLASSIC were: an exposure-response experi- 
mental design as the treatment regime (including the maximum PEC if possible; based 
on the expected effects); preference for the "toxicological approach" (chemical is 
uniformly dosed into the water) over the "simulation approach" (dosing simulates field 
exposure such as due to run-off or spray drift; application of the test substance in 
spring (generally higher species richness and abundance; potentially sufficient time to 
study recovery); taxonomic resolution as high as scientifically justified or practically 
feasible (special efforts for those groups that are identified in laboratory studies as 
potentially the most sensitive); higher aquatic plants (macrophytes) and fish in general 
not the principal endpoints; univariate statistical methods in analysis of population- 
level effects, multivariate methods for community-level effects; EAC as toxicity 
parameter (highest concentration with no ecologically significant effects, some statis- 
tically significant effects at the EAC possible if considered as ecologically 
insignificant); structural and functional endpoints of the same importance (species 
structure the principalprotection aim, functional endpoints alone not considered as 
appropriate); with full population recovery an initial effect is not regarded as ecolog- 
ically significant; with incomplete recovery requirement of additional tools (e.g. further 
laboratory studies) to address the remaining uncertainty; development of ecological 
extrapolation models for extrapolation recommended; EACs from a well-designed and 
appropriately performed microcosm or mesocosm study representative for common 
field situations in isolated, static water bodies (databases on the abiotic and biotic 
conditions of surface water needed to aid interpretation and extrapolation between 
different waters and regions); landscape ecology important for evaluating the uncer- 
tainty of mesocosm results (water bodies in agricultural landscapes often not isolated 
and/or completely exposed); application of an uncertainty factor to the EAC only in 
exceptional cases; long-term persistence of populations of non-target organisms deriv- 
able from mesocosm data in regulatory risk assessment; additional guidance, training 
and tools needed by those conducting and evaluating microcosm or mesocosm studies. 
Examples of micro/mesocosm studies can be found in Hill et al. (1994), Jak et al. 
(1994), Graney et al. (1994), Leeuwangh et al. (1994), and Van den Brink and Ter 
Braak (1999). 
246 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
4. Concluding remarks 
In recent years, the design of higher-tier toxicity tests, the statistical analysis and the 
interpretation of the results was the subject of a series of workshops which carefully 
produced guidance for the experimenter and the risk assessor as well. At this point, 
practical experience is developed, which can be used in future to refine the recom- 
mendations and guidance on the conduct and interpretation of higher-tier toxicity tests. 
Because the measurement endpoints in these tests (population and community data) 
are much closer to the assessment endpoint (persistence of the community structure 
and function) than in lower-tier test, uncertainty about the real effects in the field can 
be substantially reduced. 
In contrast, in view of the authors the concept behind lower-tier testing should be 
carefully reconsidered. A large number of tests have been developed in the past, for 
which guidelines and standards have been established. However, it appears that one 
of the current "philosophies" in lower-tier testing, that representative species of the 
trophic levels are subjected to toxicity testing and the results can be extrapolated to 
other members of the same trophic level (OECD, 1993; Fent, 1998; Shaw and 
Chatwick, 1999), can be dangerous and not protective to the community, if the current 
risk assessment practices are applied. Ratte and Hammers-Wirtz (2001) give examples 
which point to severe shortcomings in the current approach: In outdoor mesocosm 
experiments, by which two fungicides were studied, Daphnia magna, the "backbone" 
of testing trophic level 2 in aquatic systems, was less and similarly affected by the 
fungicides. However, tremendous sensitivity differences between Daphnia magna and 
some rotifers were observed. With Fungicide 1, the ECs0 of the most sensitive rotifer 
(Brachionus spec.) was nearly two orders of magnitude lower than in Daphnia magna, 
whereas with Fungicide 2 the difference between the highly sensitive Keratella 
quadrata and Daphnia magna was even about three orders of magnitude. According 
to OECD (1992) a safety factor of 1000 is used, if data from one or two acute tests 
are available, it is 100, in case the acute LCs0 or ECs0 are available for the base set 
(alga, daphnid, fish) and 10, if the chronic NOEC is available for the complete base 
set. For the above example, the magnitude of sensitivity difference cannot be balanced 
even by the highest safety factor. 
As a consequence, risk assessment cannot be based on Daphnia magna as the lone 
representative of the herbivorous trophic compartment in stagnant freshwater systems. 
The example supports a change from the trophic approach (i.e. choosing the test organ- 
isms according to the trophic level) to the taxonomic approach (i.e. inclusion of enough 
representatives from the dominant species of a community). The rationale behind is 
that the sensitivity of a species depends on its physiological properties, being more 
related to the taxonomic rather than the trophic position of a species. Therefore, we 
recommend to include more (not all) species from different taxonomic groups, which 
play a major role in the considered community, into toxicity testing, such as repre- 
sentatives from rotifers, molluscs, oligochaetes. 
Also the design of lower-tier toxicity tests and the selection of appropriate endpoint 
needs to be reconsidered. Besides the fact that the statistical design of many tests is prob- 
ably poor, the suite of endpoints considerably differs among the various test and there is 
a lack of endpoints for the population level. The population in the field rather than the 
Ecotoxicity testing 247 
individual organism is the real assessment endpoint, hence more populat ion endpoints 
should be measured in a test. Even in the Daphnia reproduct ion test, which is commonly 
seen as a real l ife-cycle test, important effects on the next generat ion are disregarded, 
since the quality of offspring produced by the treated mothers is generally not assessed. 
It could be shown that this can lead to a strong underes t imat ion of the effects on the pop- 
ulation (Hammers-Wir tz and Ratte, 2000; Ratte and Hammers-Wir tz , 2001). 
Some of these shortcomings probably could have been avoided, if there would have 
been a similar supervising by expert workshops as has been done in higher-tier testing. 
Together with the development o f an improved theoretical concept behind lower-tier 
testing we r e c o m m e n d also the use of powerful s imulat ion tools, by which the optimal 
endpoints and statistical design can be found and the applied statistical methods can 
be verified (for examples see Ratte, 1996; Ratte et al., 1998; Scholze et al., 2001). 
Such type of computer simulations should be applied before ring-tests are conducted 
and a guideline is developed. 
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primary consumers or arthropods in general. Furthermore different fish species repre- 
sent the trophic level of the secondary consumers or top-predators. 
The oldest and widely used test species are freshwater species, while species repre- 
senting soil organisms are currently becoming more important in ecotoxicology. Up 
until now, among the terrestrial species mainly used in risk assessment of chemicals 
are some higher plants and the earthworm Eisenia fetida. 
2. Lower-tier testing 
2.1. Ecotoxicological parameters and investigated level of organisation 
We distinguish between two types of ecotoxicological biotests: the acute tests and the 
prolonged tests. In acute tests normally quantal responses like mortality or immobili- 
sation are observed. The prolonged tests focus on the effects on reproduction, growth 
or other physiological processes. Almost in all of the acute tests organisms are inves- 
tigated, while in the prolonged studies different levels of organisation (organisms, 
populations) are considered. In the algal growth inhibition test the population growth 
is determined by measuring the density of algal cells; here several generations of algae 
are involved during the 72 h test duration. In contrast in the Daphnia reproduction test 
the effect on reproduction is measured on organism level rather than on population 
level, since the offspring number is only measured in one generation. 
The reasons for choosing the organism level in ecotoxicological tests are mostly 
the duration of the test and the handling of those tests. There are first steps in devel- 
oping tests on sub-organism level, which are helpful tools in screening and monitoring 
the environmental quality. However, their ecological relevance is more problematic 
than with tests on organism or population level. 
Even if the tests are conducted on organism level, we have to accept that not all 
population-relevant aspects are considered. In the Daphnia reproduction test for 
example the number of offspring is the parameter used for risk assessment. In this test 
however the quality of this offspring is not considered although it can also be affected 
by the test material and is of great importance for daphnids population growth 
(Hammers-Wirtz and Ratte, 2000). The parameters investigated in standardised labo- 
ratory tests are always a compromise between practicability and ecological relevance. 
224 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
But we have to be aware which level of organisation is investigated and which conclu- 
sions can be drawn from this level for the population in the field. 
2.2. Standardisation 
Ecotoxicological tests are used in the legal control of chemicals and the quality assess- 
ment of effluents. Therefore an ecotoxicological test has to give approximately the 
same result in different laboratories and in the same laboratory at differem times, i.e. 
the results of such biotests have to be reproducible and repeatable. To reach a high 
repeatability and reproducibility the test conditions need to be strictly regulated. Thus 
since the 1980s various organisations (e.g. OECD, CEN, ISO, ASTM) are developing 
guidelines for the relevant ecotoxicity tests in order to standardise the test and evalu- 
ation methods. 
Of course, when a test will be repeated several times or will be conducted at one 
time in different places, the results won't be exactly the same, due to biological vari- 
ability of the test organisms involved. The major potential sources of variation in 
ecotoxicological tests are genetic and environmental ones. In the tests with the water- 
flea Daphnia magna the genetic variation can be eliminated because the daphnids 
normally produce genetically identical neonates due to parthenogenetic reproduction. 
Hence these test organisms provide an opportunity to distinguish between genetic vari- 
ability and environmental variability. Different studies with several clones of Daphnia 
magna showed that some genotypes were more tolerant to a toxicant than others, but 
the results depended on the toxicant applied (Baird and Barata 1999, Baird et al. 199 l, 
Baird et al. 1990, Soares and Calow 1993). There exist no generally sensitive or 
tolerant genotypes. Thus, when we use a clone in ecotoxicological studies we can 
reduce the genetic variability, but we have to be cognisant that this clone is possibly 
not as sensitive as another genotype of this species in the field. 
The environmental variability can be reduced by strictly regulating the ambient 
conditions. Some environmental factors can easily be controlled such as temperature, 
and lighting regimes. But there are many factors, first of all the supply of live food of 
approximately the same quality, which is difficult to control and has great impact on 
the development of the test organisms and therefore possibly on the toxicant's effect 
(Soares and Calow 1993). In most of the test systems the variability measured is a com- 
bination of both genetic and environmental variation. To determine the level of varia- 
tion in reproducibility and repeatability so-called "ring tests" were performed where 
different laboratories investigate one or several toxic compounds several times. The 
more complex the test system is and the less the number of test organisms involved, 
the higher is usually the variability of the test system. We need a high reproducibility 
and a high repeatability in toxicological tests, but on the other hand in view of the prac- 
titioner and those paying for them, a test should be practicable, preferably little labour- 
intensive and cheap (B6deker et al. 1992). Furthermore, the test organisms have to be 
easy to rear and nevertheless highly sensitive and representative for the biotic com- 
munity because only few species are tested as representatives for the biocenosis. 
From the above considerations it becomes clear that any ecotoxicity test is a 
compromise between practicability and cost efficiency on the one hand and the repre- 
sentativeness and sensitivity of the species investigated on the other. 
Ecotoxicity testing 225 
2.3. Test species 
A multitude of different species is used worldwide in scientific studies evaluating the 
risk of chemicals or effluents, but only a few of this species have been established as 
standard test organisms of worldwide use. This chapter will mainly focus on standard 
test organisms in ecotoxicology more than on species tested for special aspects. In the 
following standard test species and the principle of the tests with these species will be 
presented. Furthermore shortcomings of these standard tests will be discussed. Other 
test species of less importance are only shortly mentioned. An overview of the stan- 
dardised test guidelines for the different trophic levels is given in Table 1. 
2.3.1. Bacteria 
Bacteria play a decisive role in the ecosystem as decomposer of organic material. 
Furthermore they are of great importance for the biodegradation of organic compounds 
in wastewater treatment plants, sewage sludge and soils. Bacteria are commonly used 
test organisms in the Respiration inhibition test (OECD, 1984f) with activated sludge 
used to assess effects of chemicals or effluents on the aerobic bicoenosis of microor- 
ganisms in sewage treatment plants. 
Another bacterium used as test species is the marine luminescent bacterium Vibrio 
fischeri, formerly known as Photobacterium phosphoreum. Luminescent bacteria 
transmit a bioluminescence as metabolism product. The enzyme luciferase catalyses 
the oxidation of reduced riboflavin phosphate which is accompanied by emission of 
light (Kaiser, 1998). This process is linked with the microbial metabolism, and there- 
fore a reduction in the natural bioluminescence of Vibrio fischeri is an indication of a 
toxic effect. In the bioassay with Vibrio fischeri the inhibitory effect of chemicals or 
effluents on the light emission is determined.The bacteria are incubated with the toxi- 
cant or effluent for 30 minutes. Thus the duration of this bioassay is very short in 
contrast to other acute bioassays with a duration of 24 to 96 h. The bacteria used for 
the test can be taken from freshly prepared, liquid-dried or freeze-dried stock cultures 
which have to be prepared differently. For this reason the ISO standard 11348 consists 
of three parts. Due to the possibility that the bacteria stock culture can be kept frozen 
and easily prepared this bioassay is less labour-intensive than other biotests where a 
stock culture has permanently to be maintained. The field of main application of this 
test is the testing of wastewater. 
2.3.2. Algae and plants 
2.3.2.1. Alga growth inhibition test 
The ecological importance of aquatic plants can hardly be overestimated. For example, 
approximately 70% of the world's atmospheric oxygen is generated by phytoplankton 
(Reynolds, 1984). Furthermore, phytoplankton as well as benthic and epiphytic 
attached microalgae are the primary energy source for a lot of aquatic ecosystems. 
Another crucial function, which is currently discussed regarding the greenhouse effect 
and the "global warming" phenomenon, is the ability of algae to bind carbon dioxide 
and thus reducing the amount of atmospheric green house gases. 
Table 1. Existing test guidelines (OECD) and standards (ISO) for the different trophic levels; please find the references of actual versions under 
http://www.oecd.org and http://iso.org, respectively. 
t,9 
Trophic level Species Test guidelines 
Decomposer Marine luminescent bacterium Vibrio 
fischeri (formerly known as 
Photobacterium phosphoreum) 
Activated sludge microorganisms 
ISO 11348-1/-2/-3 (1998) 
ISO 15522 (1999) 
Primary producer 
Primary consumer 
Green algae 
Scenedesmus subspicatus 
Chlorella vulgaris 
Pseudola'rchneriella subcapitata 
(= Selenastrum capricornutum) 
Marine algae 
Skeletonema costatum 
Phaeodactylum tricornutum 
Duckweeds 
Lemna minor 
Lemna gibba 
Terrestrial plants 
Freshwater crustacean 
Daphnia magna 
OECD 201" Algal Growth Inhibition Test (Updated Guideline, adopted 
1984) 
ISO 8692" Algal Growth Inhibition Test (1989) 
ISO 10253" Algal Growth Inhibition Test (1995) 
OECD 221 (New Guideline, 2000): Lemna growth inhibition test 
OECD 208: Growth test (Original Guideline, adopted 1984) 
OECD 208 A: Seedling Emergence and Seedling and Seedling Growth 
(Draft Updated Guideline, 2000) 
OECD 208 B: Vegetative Vigour Test (Draft Updated Guideline, 2000) 
OECD 202: Acute Immobilisation Test (Updated Guideline, adopted 
1984) 
ISO 6341: Inhibition of the mobility (1996) 
OECD 211: Reproduction Test (Original Guideline, adopted 1998) 
ISO 10706: Long term toxicity (2000) 
Table 1. (continued) 
Trophic level 
Secondary consumer 
Species Test guidelines 
Marine crustaceans 
Acartia tonsa 
Tisbe battagliai 
Nitocra spinipes 
Chironomids 
Chironomus tentans 
Chironomus riparius 
Earthworm 
Eisenia fetida 
Eisenia fetida/andrei 
Eisenia fetida 
Enchytraeid worm 
Enchytraeus sp. 
ISO 14669: Acute lethal Toxicity (1999) 
OECD 218: Sediment-Water Chironomid Toxicity Test (using spiked 
sediment)/OECD 219 (using spiked water) (Draft New Guideline, 
2000) 
OECD 207: Acute Toxicity Test (Original Guideline, adopted 1984) 
ISO 11268-1" Acute Toxicity (1993) 
OECD: Earthworm Reproduction Test (Draft New Guideline, 2000) 
ISO 11268-2: Reproduction Test (1998) 
OECD 220: Reproduction Test (Draft New Guideline, 2000) 
Fishes 
e.g. Danio rerio 
e.g. Danio rerio, 
Oncorhynchus mykiss, 
Pimephales promelas 
e.g. Danio rerio, 
Oncorhynchus mykiss, 
Pimephales promelas 
e.g. Danio rerio 
Oncorhynchus mykiss 
OECD 203: Acute Toxicity Test (Updated Guideline, 1992) 
ISO 7346-1/-3: Acute Toxicity (1996) 
OECD No. 212 (Short-term Toxicity Test on Embryo and Sac-Fry 
Stages) (Original Guideline, 1998) 
OECD No. 210 (Early-Life Stage Toxicity Test) (Original Guideline, 
1992) 
OECD 204" Prolonged Toxicity Test (Original Guideline, 1984) 
ISO 10229: Prolonged Toxicity Test (1994) 
~,,d. 
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t , ,~ . 
0~ 
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bO 
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228 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
Various parameters of natural algal communities have been monitored to estimate 
the quality of their habitat. Shubert (1984) has presented a summary about the use of 
algae as ecological indicators in environmental studies. Due to their significance it was 
a logical step to use algae routinely in toxicity tests designed to provide information 
on the environmental safety of chemicals and effluents. The first standard methods, 
the marine and freshwater bottle tests, which were developed in the early 1970s (US 
EPA 1971, 1974), have been followed by more additional methods published by stan- 
dard writing organisations and various regulatory agencies (Payne and Hall 1979, FDA 
1982, Homing and Weber 1985, APHA 1985). The most important international guide- 
lines currently in use are the OECD guideline 201 (OECD 1984), the ISO standard 
8692 (ISO 1989) and the guideline published by the European Commission (EEC 
1993), which differ in form and content only in minor aspects. 
Test species: Regarding the algal test species the long lasting expert-knowledge of 
Scandinavian (e.g. Skulberg 1967), American (e.g. Miller et al. 1976) and German 
(e.g. Bringmann and Kiihn 1956) scientists was useful during the development of test 
protocols. Unicellular protococcale green algae like Chlorella, Scenedesmus and 
Selenastrum proved to be particularly suitable. Scenedesmus subspicatus (new name: 
Desmodesmus subspicatus) and Selenastrum capricornutum (= Pseudola'rchneriella 
subcapitata) are the most common test species for algal growth inhibition tests (ISO 
8692). 
Standard test design: The base of each evaluation of algal tests is the growth curve 
of the population in a defined test duration (normally 72 hours). Algae are grown in 
a test medium with a defined nutrient content and different concentrations of the test 
substance and in the control samples (test medium without test substance). The initial 
cell density should be 10,000 cells/ml. The algae came from an exponentially growing 
pre-culture, which has been set up 72 hours before the start of the test under the same 
conditions as in the test. For each tested concentration, three replicates and six repli- 
cates for the control are incubated. Under the prescribed light and temperature 
conditions the algae population grows exponentially during the test (at least in the 
control). 
Mean cell densities are measured after 24, 48 and 72 hours. Plotting the mean cell 
density versus the test duration leads to the growth curves of the population in the 
control as well as in the various test material concentrations. 
The inhibition of the algal growth caused by the toxicity of a test substance can be 
derived from different variables. There is an ongoing debate on the whether the cell 
number, biomass integral or growth rate should be used in risk assessment. Some 
prefer the cell number (or the derived variable "biomass integral", which is the area 
under the growth curve (Nusch, 1982, 1983)), while the other favour the average 
growth rate (Nyholm, 1985, 1990, 1994; Dorgerloh, 1997). The pros and cons of the 
various response variables are discussed thoroughly in Ratte et al. (1998). 
Surrogate toxicity data-sensitivity" In the past it was proposed to extrapolate phyto- 
toxicity from data derived with fishes or daphnia (Stephan et al., 1985), because algae 
were considered less sensitive against chemicals than animal species, as some studies 
Ecotoxicity testing 229 
indicated (Kenaga and Moolenar, 1979; Kenaga, 1982). But many publications came 
to a different result (Patrick et al., 1968, Shehata and Nawar, 1979; Bringmann and 
Kiihn, 1980; Walsh et al., 1980, 1982; Giddings et al., 1983; Sloff et al., 1983; Adema 
et al., 1983; Walsh and Merrill, 1984; Karpinska-Smulikovska, 1984; Miller et al., 
1985), withalgae being more sensitive than fish or daphnids in many cases. Generally, 
there is no universally sensitive test species or group of species, since the sensitive was 
found to be chemical- and species-specific. Two studies which have used different data- 
bases for notifications of substances came to the congruent result that algae were more 
sensitive than animal species in 50-60% and less sensitive in about 30% (Benenati, 
1990, Weyers et al., 2000). Thus, it can be seen as confirmed that algal toxicity is not 
predictable from animal test species (Lewis, 1990, 1995) and that phytotoxicity data 
are essential for a reliable risk assessment. But in any case it would be reasonable to 
use more than the two standard test species Scenedesmus and Selenastrum because the 
sensitivity to chemicals and effluents was found to be strongly species specific 
(Bringmann and Kiihn, 1978; Sloff et al., 1983; Stratton, 1987; Swanson et al., 1991; 
Lewis, 1995) and can differ clearly even between different test strains or geographical 
races of the same species (Venkatamaran and Rajyalakshmi, 1972; Millie and Hersh, 
1987; Riedel, 1989). Consequently, it was demanded on various occasions to use more 
algal species and also cyanophytes in a test battery (Blanck et al., 1984; W/ingberg and 
Blanck, 1988; Swanson et al., 1991; Cleuvers, 2001). 
Difficult substances: Difficult substances (ISO, 1997, OECD, 1999) are e.g. poorly 
soluble materials, volatile compounds, adsorbing or complexing substances, substances 
that degrade in the test system and coloured substances like dyes. While the former 
have an effect on generally all test species, in the latter case primarily algae are 
affected. Coloured substances are a sizeable part of substances applied for notification 
of new substances in the European Union. The difficulty for interpretation results from 
the fact, that dyes are able to inhibit the growth of algae in two basically different 
ways. First, coloured substances absorb light with the result that the light intensity util- 
isable for algae is diminished and additionally also the spectral quality of light is 
changed. If light saturation is not achieved, the growth of algae will be inhibited due 
to a physical property of the tested substance. Second, the substance can act toxically 
in a narrower sense, due to its chemical effect on algae and the determination of this 
toxic action is the important point, while the European Commission stipulates that inhi- 
bition due to the shading effect of the coloured substance shall not be mentioned. 
Because both effect occur concomitantly, is it a priori not possible to make a state- 
ment about the extent of the single effects. Some attempts were made to clear this 
point (Memmert et al., 1994; Comber et al., 1995) and recently this topic was thor- 
oughly discussed by Cleuvers and Ratte (2002), who showed that by increasing the 
light intensity to a level above saturation and reducing the culture volume of algae to 
shorten the light path the shading effect of different dyes even at very high concen- 
trations could be completely eliminated. 
2.3.2.2. Macrophytes- Lemna growth inhibition test 
Macrophytes are used less frequently than algae in toxicity tests (Lewis, 1995). In one 
literature survey, only 7% of 528 reported phytotoxicity tests used macrophytic species 
230 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
(Blanck et al., 1984). When macrophytes have been used, the duckweeds (Lemna spp.) 
have been the species of choice and they are often used as a representative species for 
other vascular plants. Lemnaceans are monocotyledon, angiosperm aquatic plants 
which belong within the subclass Arecidae to the Arales. It is a fast growing higher 
plant which is widespread worldwide in lenthic waters from the tropics to the temper- 
ate and arctic zone. The propagation takes place mainly vegetatively by the creation of 
daughter fronds (fronds = structure analogous to leaves). Like algae and cyanophytes 
as primary producers lemnaceans belong to the base of the food web. 
From the family of lemnaceans are mainly Lemna-species like L. minor (Wall- 
bridge, 1979; Wang, 1986; Taraldsen and Norberg-King, 1990; Jenner and Janssen- 
Mommen, 1993; Lomagin and Ulyanova, 1993; Weltje et al., 1997) and L. gibba (Holst 
and Ellwanger, 1982; Hughes et al., 1988; Cowgill and Milazzo, 1989; Wang, 1990; 
Cowgill et al., 1991; Lakatos et al., 1993; Day and Hodge, 1996), and less often also 
L. perpusilla and Spirodela polyrhizza (APHA et al., 1989) in use for toxicity testing. 
L. triscula was recommended too because of its easy culture and rapid growth (Huebert 
et al., 1990). 
Various national guidelines are currently in use (AFNOR, 1990; ASTM, 1991; 
APHA et al., 1995; SIS, 1995; US EPA, 1996; EC, 1998; DIN, 2001), which differ 
regarding the preferred test species, the used culture medium, the test duration and, 
most important, the observed endpoint. Thus, the comparability of results obtained 
with different test protocols is restricted. In 2000, an international guideline and stan- 
dard was drafted by OECD and ISO, respectively. In both drafts the test duration is 
set to seven days. All tests start with a definite number of fronds in each vessel, 
normally ten or twelve, whereas only young plants with two or three fronds are used. 
The plants were inspected in a defined time scheme, at day 0, 3, 5 and 7. Beneath the 
number of fronds either the total frond area, or, at the end of the test, the dry weight 
or the fresh weight must be measured. Furthermore, changes in the development of 
the plants ought to be noted, like the occurrence of chlorosis, necrosis or changes in 
the length of the rhizoid. A useful tool in this context is the digital image analysis, 
which facilitates the evaluation of test results clearly and enables the investigator to 
record further parameters, e.g. the distribution pattern of fronds in different size 
classes. The choice of the endpoint has a strong influence on the toxicity data; Cleuvers 
(2001), e.g. found differences of more than factor 300 between EC~0's calculated from 
inhibition of the average growth rate on the one hand and either total frond area or 
dry weight on the other hand. 
Regarding the sensitivity one could guess, that Lemna is less sensitive than algae, 
because it is often used to remove toxicants and nutrients from sewage waters and it 
shows indeed a considerable tolerance against metals (Tripathi and Chandra, 1991). 
But a general trend does not exist (Lewis 1995). In a study with 16 herbicides Fairchild 
et al. (1997) found Lemna in eight cases to be more sensitive and in eight cases to be 
less sensitive than the green algae Selenastrum capricornutum. Peterson et al. (1997) 
confirmed the findings, that green algae are not generally more or less sensitive than 
Lemna. In a study with eight pesticides, Lemna was either as sensitive as or more 
sensitive than the tested green algae (Grossman et al., 1992). 
In any case, a Lemna growth inhibition test is very useful as an additional source 
of information about phytotoxicity, whereas generally a extension of test species, e.g. 
Ecotoxicity testing 231 
by using other macrophytes, was frequently recommended (Swanson et al., 1991; 
Lewis, 1995; Fairchild et al., 1997). 
2.3.3. Aquatic invertebrates 
2.3.3.1. Daphnia 
The freshwater cladoceran Daphnia magna is one of the oldest and widely used test 
organisms in aquatic toxicology (Baudo, 1987; Enserink, 1995). The genus Daphnia 
is an important link in freshwater trophic chains as dominant consumer of primary 
producers and as food for both invertebrate and vertebrate predators. The species of 
the genus Daphnia differ in their world distribution. Daphnia magna is a pond species 
with limited geographical range (Eurasia), whereas Daphnia pulex is a more wide- 
spread species (North and South America, Greenland and Europe) (Baudo, 1987; 
F16fJner, 2000). Therefore in the USA the species D. pulex andanother cladoceran 
Ceriodaphnia dubia are also used as test organisms representing the filter-feeding 
zooplankton (Mark and Solbe, 1998). 
The choice of Daphnia magna as standard test species was influenced by several 
advantageous characteristics. It is of small size (compared to fish, molluscs or macro- 
crustaceans) and easy to culture in the laboratory. Its parthenogenetic reproduction 
under non-stressed conditions allows the testing of clones, which enhances the repro- 
ducibility and repeatability of the test results. Furthermore Daphnia magna is relatively 
sensitive to chemicals compared with other freshwater invertebrates (Mark and Solbe, 
1998; Baird et al., 1989; Radix et al., 1999; Versteeg et al., 1997) and its relatively 
short life-span and reproductive cycle are favourable for the chronic testing. The 
ecology of Daphnia has long been studied so that we have a lot of background infor- 
mation on its biology and ecology. 
Thus Daphnia magna is the most commonly tested freshwater species in acute as 
well as in chronic tests (Baird et al., 1989; Mark and Solbe, 1998; Baird and Barata, 
1999). 
Acute test: In acute tests with Daphnia magna young daphnids, aged less than 
24 hours, are exposed to different concentrations of a test substance in a static system 
for a period of 48 hours. After 24 and 48 hours the immobilisation of the test animals 
is recorded. Those animals are considered as immobilised that are not able to swim or 
to move the appendages or the postabdomen after gentle agitation of the liquid. During 
the acute test the daphnids were not fed. With the percentage of immobilised daphnids 
an EC50 at 24 h and 48 h should be calculated. The test procedure is highly standard- 
ised. There exist different test methods that differ mainly in age of test organisms and 
test medium used. A comparison of the commonly international guidelines for testing 
the acute toxicity of Daphnia is given in Versteeg et al. (1997). 
In acute tests with Daphnia magna usually clones are tested in order to reduce the 
variability due to genetic differences. An important source of variation for both acute 
and chronic tests with Daphnia magna are the food supply and the culture conditions 
of the daphnids in the stock culture (e.g. Baird and Barata, 1999). The feeding of the 
mothers and their density in the stock culture have a great impact on the size, weight 
and fat content of their neonates (Cowgill et al., 1985; Enserink et al., 1990; Goser, 
232 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
1997), and the "quality" of the neonates introduced in the test influences the sensi- 
tivity of these test organisms (Enserink et al., 1990; Goser, 1997). The lower the food 
supply of the mothers, the larger and fatter is the offspring (Boersma, 1997a, 1997b; 
Enserink et al., 1995; Cowgill et al., 1985; Guisande and Gliwicz, 1992; Glazier, 1998; 
Sokull-K1/ittgen, 1998) and higher densities in the culture lead to larger and fatter 
neonates (Cleuvers et al., 1997; Goser, 1997). In acute tests larger neonates have been 
shown to be less sensitive than smaller ones exposed to cadmium (Enserink, et al., 
1990), bromide (Naylor et al., 1992), or dichloraniline (Baird et al., 1989; Naylor et 
al., 1992; Goser, 1997). Although the culture conditions influence the test results, the 
culture conditions particularly the feeding and the density of the culture are up to now 
not regulated in the test guidelines. Here it is only stated that the culture conditions 
shall be similar to those in the test and that the daphnids shall be from a healthy stock 
showing no signs of stress such as high mortality, presence of males or ephippia, 
discoloured animals or delay in first reproduction. To get neonates of similar quality 
the culture conditions have to be specified in detail. 
Another factor affecting the results of acute tests is the age of the test animals at 
the start of the test. The test animals inserted shall be younger than 24 h at the begin- 
ning of the test. Klein (2000) showed recently that also in this range of 0 to 24 h 
age-dependent differences in sensitivity can occur. Animals with an age of 20 to 24 h 
were up to 4-fold more sensitive to potassium dichromate than younger animals. But 
for other chemicals this trend was not confirmed (B6gi, 1998). Furthermore the age- 
dependent sensitivity was different at exposure times of 24 and 48 h. Thus, on the 
basis of these facts it seems not to be useful to change the age of the test animals. 
Daphnia reproduction test." The chronic test with Daphnia magna is conducted to 
assess the effect of a test material on the reproductive output of Daphnia magna. 
Young daphnids less than 24 h old are exposed for a period of 21 days to a test 
substance, industrial or sewage effluents. The test can be performed in a static or a 
flow-through system. The flow-through design has to be used if the test substance is 
volatile or not stable over a period of three days, i.e. the concentration of the test 
substance falls below 80% of the initial measured concentration within three days. The 
effect of a test material on the reproduction of Daphnia magna can be influenced by 
various biotic and abiotic factors. The abiotic conditions during the test are highly 
standardised, while the food quantity and quality fed to the test organisms is not exactly 
regulated. Different green algae (Chlorella ssp., Pseudokirchneriella subcapitata, 
Scenedesmus subspicatus) can be used as food for the daphnids and the quantity of 
food shall be between 0.1 and 0.2 mg C per animal and day. The food quantity and 
quality are known to have a great impact on the reproduction of Daphnia magna 
(Green, 1954; Cowgill et al., 1985; Boersma, 1997a, 1997b; Kilham et al., 1997). 
Therefore a more exact regulation of feeding would reduce that variation in repro- 
ducibility and repeatability. 
The parameters recorded in the chronic test are the survival of the females together 
with the number of living offspring per live female at the end of the test. The mean 
number of live offspring per female in each exposure concentration is compared to the 
control mean by statistical tests in order to determine the LOEC and NOEC. 
Additionally the EC50 can be calculated. 
Ecotoxicity testing 233 
In the Daphnia reproduction test the number of offspring in the treatments is not 
always reduced but can also be enhanced compared to control. Such increasing effects 
normally induced by low concentrations of some chemicals, so called hormetic effects, 
occur in various species and different test endpoints (Calabrese and Baldwin, 1998). 
In the Daphnia reproduction test increased offspring number compared to control 
have been observed several times induced by different chemicals (Biesinger and 
Christensen, 1972; Francis et al., 1986; Van Leeuwen et al., 1987; Van der Hoeven, 
1990; Baldwin et al., 1995; Klfittgen et al., 1996; Brown et al., 1998; Hammers-Wirtz 
and Ratte, 2000; Wollenberger et al., 2000). Those increases in offspring number also 
occurred in solvent controls induced by organic solvent added to the test medium 
(Shurin and Dodson, 1997; Brown et al., 1998; Sanchez et al., 2000). Up to now those 
"positive" effects on reproduction are not considered in risk assessment. But one exper- 
iment showed definitely that the increase in offspring number was coupled with a 
reduction in offspring quality (Hammers-Wirtz and Ratte, 2000). A reduction of 
offspring quality has a great impact on the beginning and extent of reproduction, thus 
on the fitness of the population, which is the one that should be protected. Therefore 
it must be questioned if increases in offspring number can further be ignored in risk 
assessment, because the increases in offspring numbers can be coupled with a decrease 
in neonate fitness. 
Beyond it several chronic studies with Daphnia magna showed that the quality of 
neonates was affected by chemicals even if the offspring number was reduced. 
Although theoffspring size is not routinely recorded in chronic tests with daphnids 
several authors observed a reduction in neonate size induced by several chemicals like 
copper (Flickinger et al., 1982), cadmium (Bodar et al., 1990), lead (Enserink et al., 
1995), the pesticide carbaryl (Hanazato and Dodson, 1995), and a dispersant 
(Hammers-Wirtz and Ratte, 2000). These facts clearly indicate that the measurement 
of offspring number alone is not sufficient to detect ecological relevant effects on 
population level, the fitness of the neonates also has to be considered. Measuring the 
body size of the neonates can indicate their fitness, but body size is not always corre- 
lated to the fitness. To determine the neonates fitness either an additional test with 
these neonates is necessary or instead of the chronic test an population test has to be 
carried out where the chemical effect on subsequent generations is recorded. If only 
the offspring number is considered in risk assessment the effect on population level 
can be underestimated (Hammers-Wirtz and Ratte, 2000). 
2.3.3.2. Further aquatic invertebrates 
Another test species used as representative for the zooplankton is the cladoceran 
Ceriodaphnia dubia. This test species is commonly used in the USA, but of minor 
interest in Europe. The important advantage of Ceriodaphnia compared to Daphnia is 
the short generation time submitting the reduction of the chronic test duration to seven 
days in contrast to 21 days in the Daphnia reproduction test. A comparative study of 
toxicity tests with Daphnia magna and Ceriodaphnia dubia demonstrate the C. dubia 
are on average more sensitive than Daphnia magna in acute toxicity tests and of similar 
sensitivity in chronic toxicity tests (Versteeg et al., 1997). 
The rotifer Brachionus calyciflorus is a further invertebrate of interest as ecotoxi- 
cological test species because of its short generation time. Brachionus calyciflorus 
234 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
reproduces by parthenogenesis and is able to hatch several times during 48 h under 
favourable conditions. These animals are primary consumers and serve as prey for 
several invertebrates and fishes. In the test with Brachionus calyciflorus the popula- 
tion growth over a period of 48 h is determined. This test is not yet standardized but 
there exist a Working Draft of ISO. An advantage of this test is its short duration of 
only two days especially for testing of instable effluents. But first investigations with 
Brachionus showed that this test is on average three times less sensitive than the 
Daphnia reproduction test (Radix et al., 1999). 
2.3.4. Terrestrial invertebrates 
2.3.4.1. Earthworm 
The earthworm Eisenia fetida is one of the mostly used test organism for the terres- 
trial field. This terrestrial species representing the soil fauna is used in acute tests as 
well as in reproduction tests. The acute toxicity of a chemical on the earthworm can 
be determined in two different ways. Either the mortality is determined after the earth- 
worms were exposed for hours to an filter spiked with the test substance or the 
mortality is recorded after 7 or 14 days rearing in an artificial soil merged with the 
chemical. In the reproduction test adult worms were exposed to a chemical and lethal 
and sublethal effects were recorded over a period of 8 weeks. After four weeks growth 
and mortality effects were recorded and then adults were removed from the soil. The 
effects on reproduction were assessed after further four weeks by counting the number 
of offspring present in the soil. For the reproductive output an NOEC (no observed 
effect concentration) and, if possible, an EC50 is calculated. 
2.3.4.2. Enchytraeids 
Although the earthworm Eisenia fetida/E, andrei is recommended in various guide- 
lines as standard test organism for the terrestrial environment (R6mbke and Moser 
1998), the use of enchytraeids as standard test organisms is dicussed in the recent 
years. Soil-inhabiting enchytraeids are more ecologically relevant than E. fetida/E. 
andrei occurring almost in compost heaps. Furthermore enchytraeids are important 
members of the soil biocoenosis in many different habitats, especially where earth- 
worms are rare (Didden 1993). The test handling with enchytraeids is more practicable 
than with earthworms because the enchytraeus species are easy to handle and breed, 
their generation time is significantly shorter than for earthworms (test duration only 4 
to 6 weeks) and the volume of the test vessels is significantly smaller than for the 
earthworm test (amount of soil: 20 g vs. 500 g). 
In the enchytraeid reproduction test 10 adult worms of the test species Enchytraeus 
albidus or enchytraeus sp. are exposed to chemical spiked artificial soil (artificial soil 
according to OECD (1984)). The tests are conducted at 20~ and the worms fed 
weekly with rolled oats. After 3 weeks the adult worms are removed and after 6 weeks 
the juveniles hatched counted. The variables recorded in this test are mortality and 
behaviour of the adult worms (after 3 weeks) and the number of juveniles hatched 
(after 6 weeks). 
Ecotoxicity testing 235 
Table 2. Listing of the current test guidelines with fishes; please find the references of actual 
versions under http://www.oecd.org and http://iso.org, respectively. 
Guideline Title 
OECD 203 
OECD 204 
OECD 210 
OECD 212 
ISO 7346 (1996) 
ISO 10229 (1994) 
ISO 12890 (1999) 
Fish, Acute Toxicity Test (Updated Guideline, adopted 1992) 
Fish, Prolonged Toxicity Test: 14-Day Study (Original 
Guideline 1984) 
Fish, Early-Life Stage Toxicity Test (Original Guideline, 
adopted 1992) 
Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages 
(Original Guideline, adopted 1998) 
Water quality- Determination of the acute lethal toxicity of 
substances to a freshwater fish (Brachydanio rerio 
Hamilton-Buchanan (Teleostei, Cyprinidae)) 
Part 1: Static method 
Part 2: Semi-static method 
Part 3: Flow-through method 
Water quality- Determination of the prolonged toxicity of 
substances to freshwater f ish- Method for evaluating the 
effects of substances on the growth rate of rainbow trout 
(Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae)) 
Water quality. Determination of toxicity to embryos and 
larvae of fresh water fish - Semi-static method. 
2.3.4.3. Collembola 
Another invertebrate used as test organism for the terrestrial environment is the 
collembola Folsomia candida. CoUembola are representing the detrivorous consumers. 
Folsomia candida shows different characteristics advantageous for test organisms: it 
is easy to breed, has a short generation time and a high reproduction rate and repro- 
duces pathenogenetically. This collembola species has been used for a long time in 
risk assessment of pesticides in acute tests as well as in reproduction tests. 
2.3.5 Fish 
Fish are used as test organisms in aquatic toxicology because of their top-position in the 
trophic chain and their role as food for humans. The acute and chronic fish tests are used 
to assess effects of chemicals or effluents on the survival or sub-lethal parameters. An 
overview about the guidelines with fish as test organism is shown in Table 2. 
Different test species are used as test organisms representing the aquatic vertebrates. 
Commonly used test species are the rainbow trout Oncorrhynchus mykiss, the Zebra- 
fish Danio rerio, the Common carp Cyprinus carpio, the fathead minnow Pime- 
phales promelas, and the guppy Poecilia reticulata. In principle, also salt water fish 
species can be used in these tests assumed the species can be held under satisfactory 
236 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
conditions in the laboratory. Up to now the main database of fish toxicity data exist 
for freshwater species. Comparative studies of toxicity data of freshwater fish and 
marine species show that marine species were more sensitive than freshwater species 
for the majority of substances tested,but for 91% of all substances the sensitivity ratios 
between freshwater and saltwater fish were within a factor of 10 (Hutchinson et al., 
1998). Thus an extrapolation from freshwater fish to marine fish seems to be possible, 
the more so as the sensitivity between different freshwater species may vary by orders 
of magnitude (Nagel and Isbemer, 1998). 
2.3.5.1. Acute tests 
In acute fish tests usually juvenile or adult fish are exposed to chemicals or effluents 
preferably for a period of 96 hours (e.g. OECD guideline 203). After 24 h, 48 h or 
96 h the mortality in the treatments is recorded in order to calculate the LC50. The 
test species used for the test can be chosen by the testing laboratory, but the choice 
may be focus on practical criteria (availability throughout the year, ease of mainte- 
nance, convenience for testing) as well as relevant economic, biological an ecological 
factors. Several species are recommended as test species in the OECD guideline. The 
temperatures used in the tests and further test conditions depend on the test species 
chosen. All fish must be obtained and held in the laboratory for at least 12 days before 
they are used for testing. During this time the fishes were fed at least three times a 
week, but during the test period the fish were not fed. At least seven fish must be used 
at each test concentration and in the controls. The validity criteria for the test are less 
than 10% mortality in the controls and a dissolved oxygen concentration higher than 
60 per cent of the air saturation value throughout the test. 
Since acute fish toxicity tests are in conflict with current Animal Rights Welfare 
legislation possible alternative methods on sub-organism level were developed. 
Possible alternatives of acute fish test might be acute toxicity tests with embryo of 
zebrafish Danio rerio and cytotoxicity tests with fish cells. In the acute embryo test 
of zebrafish fertilized eggs were exposed to a test substance for 48 h. After 48 h of 
static exposure different lethal and sublethal parameters were recorded (Schulte and 
Nagel 1994). Different comparative studies demonstrate that the toxicity data derived 
from the embryo test are in good accordance with data from corresponding acute toxi- 
city tests with juvenile or adult fish (Nagel and Isberner 1998). Tests investigating the 
effect of several industrial effluents showed that the embryo toxicity test was as sensi- 
tive or even more sensitive than the conventional acute fish test (Friccius et al. 1995). 
Thus the embryo test is a promising alternative to conventional acute fish toxicity tests 
in routine waste water control (Nagel and Isberner, 1998). For the testing of effluents 
this test recently has been standardized as German DIN-Norm DIN 38415-6. 
In cytotoxicity tests with fish cells the permanent cell line RTG-2 derived from 
rainbow trout gonads was commonly used. Comparative studies with different chem- 
ical compounds showed that in most cases the zebrafish embryo test was more sensitive 
than both the acute toxicity test and the RTG-2 cell test (Nagel and Isberner, 1998). 
2.3.5.2. Short-term toxicity test on embryo and sac-fry stages 
This test is a short-term test in which the life stages from the newly fertilized egg to 
the end of the sac-fry stage are exposed to a chemical. This embryo and sac-fry test 
Ecotoxicity testing 237 
can be performed as semi-static or as flow-through test. Several fish species can be used 
for this tests, but most experience has been with the freshwater fish Danio rerio. Test 
conditions and test duration will depend on test species used. In this test lethal, and to 
a limited extent, sub-lethal effects are investigated to determine the lowest observed 
effect concentration and the no observed effect concentration, or to estimate LC/EC x 
values. The parameters observed in this type of test are survival, hatching, abnormali- 
ties in appearance and behaviour, as well as length and weight at the end of the test. 
This embryo and sac-fry test is a link between lethal and sub-lethal fish tests. It can 
be used as screening test for the full Early Life Stage test or for chronic toxicity tests. 
2.3.5.3. Chronic fish tests 
In chronic fish test lethal and sub-lethal effects are investigated for a period of 14 to 
28 days. The parameters recorded are changes in morphology (in colour), food uptake 
and swimming behaviour. 
2.3.5.4. Prolonged toxicity tests 
In the prolonged toxicity tests fish are exposed to several concentrations of a chemical 
for at least 14 days, but can be extended by one or two weeks. This test can be per- 
formed under semi-static conditions or in flow-through systems. Several test species 
are recommended for this test as in the acute fish test. During this test fish were fed at 
least daily. Parameters observed in this test beside mortality are any other than lethal 
effects like effects on behaviour (swimming behaviour, food intake), changes in appear- 
ance of fish or changes in length or body weight. The results are used to determine the 
lowest observed effect concentration (LOEC) and the no observed effect concentration 
(NOEC). Unfortunately, in most prolonged toxicity tests mortality was the crucial test 
endpoint for the determination of LOEC and NOEC (Nagel and Isberner, 1998). 
2.3.5.5. Early life stage toxicity test 
In the early life stage toxicity test different life stages of fishes like fertilized eggs, 
embryo stages, and sac fry stages are tested, because these stages are highly sensitive 
to chemicals and other test material. Fertilised fish eggs are exposed to a range of test 
concentrations preferably under flow-through conditions at least until all the control 
fish are free-feeding. There are different species recommended for this test. The test 
conditions and test duration as well as feeding and handling of the different life stages 
are depending on the test species chosen for this test. 
Several parameters are recorded during this test: observations on hatching and 
survival, abnormalities of body form in larvae or fish, abnormalities in behaviour (e.g. 
hyperventilation, uncoordinated swimming, atypical quiescence, atypical feeding 
behaviour). Furthermore the weight and length of all surviving fish are recorded at the 
end of the test. Lethal and sub-lethal effects are assessed in order to determine the 
lowest observed effect concentration and the no observed effect concentration. 
In comparative studies the early life stage test proved to be more sensitive than the 
prolonged fish test (28 days) for the majority of the chemicals tested (Nagel and 
Isberner, 1998). Due to its sensitivity and its higher ecological relevance the early life 
stage test represents a promising alternative to the conventional prolonged fish test 
(Nagel and Isberner, 1998). 
238 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
2.3.5.6. Life cycle tests 
In life cycle tests sub-lethal effects on fish were recorded over the whole life cycle in 
order to assess effects on reproduction and offspring quality. For complete life cycle 
tests only small, rapidly growing warm water fish such as zebrafish may be used to 
complete this test within a reasonable period of time. Life cycle tests with zebrafish 
usually start with fertilised eggs until the larvae of the second generation have been 
developed. This type of test requires a lot of time (25 weeks) and money. As alterna- 
tive to complete life cycle tests partial life cycle tests, early life stage tests and 
extrapolation from acute toxicity data to chronic data have been discussed (Nagel and 
Isbemer, 1998). Partial life cycle tests as well as early life stage tests are no adequate 
altematives for the full life cycle test since in these test effects on the second gener- 
ation which are often the most sensitive endpoint can not be considered. The method 
of acute-to-chronic ratio (ACR) is no suitable altemative because this ratio shows a 
high variability for different chemicalcompounds and might only carefully be extrap- 
olated to other species (Nagel and Isbemer, 1998). Furthermore in chronic studies the 
effects on populations and not on individuals should be detected. Thus if we want to 
study effects on population level complete life cycle tests have to be carried out. 
2.4. Statistical analysis 
The type of statistical methods applied in ecotoxicity tests is of prime importance for 
the interpretation of results and conclusions from these tests, and consequently for the 
associated policy decisions. The statistical treatment of biotest data aims to generate 
parameters that adequately describe the toxicity of a test material. During the last 
decade an intense discussion arised on the use of appropriate statistical methods (Pack, 
1993; SETAC, 1995; Hoekstra and Van Ewijk, 1993; Kooijman and Bedaux, 1996; 
Laskowkj, 1995; Chapman et al., 1996; OECD, 1998c; ASTM, 2000; Newman, 1994; 
Sparks, 2000). Many of the authors claimed the lack of statistical guidance given in 
guidelines and standards. As a consequence, a combined ISO (ISO TC147/SC5/WG 
10) and OECD expert group has just started to work on an agreed general guidance 
document on the statistical treatment of ecotoxicity test results. The current section 
does not intend to anticipate the results and conclusions of this guidance document, 
but some general aspects of the statistical evaluation will be dealt with below. 
A first important point is that the selection of an appropriate statistical method is 
determined by the type of response variable measured or observed in an ecotoxicity 
test. Sokal and Rohlf (1981) divide variables as follows: 
Measurement variables (quantitative variables) 
- Continuous variables (metric variables, unbounded; e.g. weight, length, experi- 
mental time). 
- Discontinuous variables (discrete variables, unbounded; e.g. count data such as 
number of offspring, number of eggs). 
Ranked variables (discrete variables, unbounded) 
These data are ordered or ranked by their magnitude; differences in ranks cannot 
be interpreted as metric distances as it can be done with measurement variables. 
These types of data are rarely found in ecotoxicity tests; however, measurement 
Ecotoxicity testing 239 
variables are sometimes transformed into ranked variables, in case rank-based 
statistical tests are to be applied. 
Attributes (nominal variables, discrete variables, bounded) 
Among the relevant variables for ecotoxicity tests are mortality (two states: dead 
or alive), fertility (two states: fertile or non-fertile), emergence (two states: emerged 
or not emerged). These variables are mostly termed "quantal" or "incidence data", 
i.e. "a number out of another number". So far as we can see, variables with more 
than two states are not used in laboratory ecotoxicity testing. 
Derived variables 
These are mainly either ratios or rates and can be based on a measurement vari- 
able or an attribute (percentages, growth rate, metabolic rate, etc.). 
A second important aspect is that generally two types of statistical methods are existing 
to determine toxic effect concentrations: hypothesis testing and point estimation. 
2.4.1. Point estimation 
A point estimate of a toxicity parameter is a concentration value obtained from inter- 
polation or extrapolation of a concentration/response relationship, such as an ECx, LCx, 
ET x. EC x is the concentration of the test material in water (e.g. in mg/1) or soil or sedi- 
ment (e.g. in mg/kg) that is estimated to cause some defined toxic effect to x% of the 
test organisms. The duration of exposure must be specified, as EC x is a function of 
time. ET x is the time at which an effect of x% is expected when the test organisms 
are exposed to a given concentration of test material (in water or sediment or soil). 
ET x is a function of the concentration of exposure which therefore has to be specified. 
EC x (ETx) is termed LC x (LTx) in case the observed variable is survival. 
To obtain a sound point estimate, a data set showing a clear concentration-effect 
relation, a concentration-response model (with ET x a time-response model) and an 
appropriate fitting method is needed. Currently, the most-used dose/response functions 
are the normal sigmoidal (probit), logistic (logit) and Weibull together with a 
weighted-maximum-likelihood regression approach as described in Finney (1978). 
This approach compensates for the heterogeneity of variances in quantal variables. An 
advantage of this method appears to be the determination of only two parameters 
(slope, intercept) and easy computing of the confidence interval around the fitted func- 
tion (and the ECx). 
The aforementioned functions are applied for both the quantitative and qualitative 
variables, being transformed to ratios relative to the control and relative frequencies, 
respectively. In case of qualitative variables (e.g., mortality) at least three treatments 
should result in partial kills. Problems arise with less than three partial kills. In these 
cases, the effect concentrations are often determined by means of the Moving Averages 
(Thompson, 1947) or Trimmed Spearman-K/irber method (Hamilton et al., 1977). 
With quantitative variables it is often disregarded that with these functions a modi- 
fied weighting in regression is required because of a differing variance structure of 
quantitative variables (for details e.g. Christensen, 1984 and Christensen and Nyholm, 
1984) and in many cases fitting of these functions is problematic, as hormesis effects 
240 H.T. Ratte, M. Hammers-Wirtz, M. Cleuvers 
(e.g. promotion of growth at lower concentrations of the test material) are sometimes 
occurring and the test material undergoes some alteration during the prolonged test 
period. Hence, deviations from the normal sigmoidal (probit), logistic (logit) and 
Weibull are often observed. In these cases the application of more flexible concentra- 
tion/response functions with more parameters are recommended. 
Among the most recommended functions is the four-parameter logistic, which 
forms also the base for modified equations to account for hormetic effects (Brain and 
Cousens, 1989, Van Ewijk and Hoekstra, 1993). In a recent paper Scholze et al. (2001) 
describe and validate a general best-fit method for the estimation of effect concentra- 
tions and recommend the use of a pool of 10 different sigmoidal regression functions 
for continuous toxicity data. Due to heterogeneous variabilities in replicated data (i.e., 
heteroscedasticity), the concept of generalized least squares is used for the estimation 
of the model parameters in combination with smoothed variance spline function 
describing the heteroscedasticity. The best-fit model is chosen individually for each 
set of data. A bootstrap method is applied for constructing confidence intervals for the 
estimated effect concentrations. At present, this approach appears to be the method of 
choice for concentration/response modelling with ecotoxicity tests. 
2.4.2. Hypothesis testing 
Statistical testing is mainly used to determine the LOEC/NOEC out of a series of test 
concentrations. In case of a so-called limit test, only one high concentration is 
compared with a control by a pairwise-test, whereas the LOEC is determined using a 
multiple statistical test, i.e. a test which keeps the experiment-wise error at the chosen 
significance level (e.g., a = 0.05). With continuous measurement variables the 
powerful ANOVA procedures are applied, if the data distribution does not substan- 
tially deviates from a normal distribution and variances are homogeneous. In these 
cases the multiple t-tests of Dunnett (Dunnett, 1964; Dunnett and Tamhane, 1991; 
Tamhane et al., 1996) or Williams (Williams, 1971, 1972) belong to the most powerful 
ones. If the aforementioned requirements are not fulfilled and cannot be established 
by an appropriate data transformation (e.g., log, square root, reciprocal), a rank-based 
test replaces the multiplet-tests, such as the Bonferroni-U-test after Holm (Holm, 
1979). With rank-based tests, measurement data are ordered by increasing size and the 
test uses their ranks rather than their metric values. Hence, there is some loss of infor- 
mation and consequently of statistical power. If a limit-test was conducted, the Student 
t-test or the Mann-Whitney U-test is applied. So far as we can see, NOECs are not 
determined for quantal responses. 
The NOEC concept has increasingly been criticised in recent years (e.g. Moore and 
Caux, 1997; Bruce and Versteeg, 1992; Chapman et al., 1996; Hoekstra and Van 
Ewijk, 1993; Laskowskj, 1995). The NOEC is seen as problematic as it is determined 
from hypothesis testing, which often cannot be performed with sufficient statistical 
power. It has to be admitted that the current design of the majority of ecotoxicity tests 
is determined by optimising cost-effectiveness rather than statistical power. The NOEC 
depends on the choice of a significance level, the statistical test selected, and the 
experimental design. Poor experimental design, such as small sample size, improper 
concentration spacing, and large experimental variability, is rewarded, since it tends 
Ecotoxicity testing 241 
to increase the NOEC. In addition, information about the concentration-effect rela- 
tionship is disregarded and confidence intervals cannot be calculated. The NOEC 
suggests that there is no or only a minimum effect. However, in practice this does not 
hold. Even in the alga growth inhibition test, where the variability is relatively low, it 
was shown that on average (38 tests) the NOEC was in the range of the EC20; in 
single case it exceeded the EC50 (Ratte et al., 1998). In other words, it is to be expected 
that false-negative results ("no-effect" is assumed but there is one) are frequent. 
As a consequence, there has been an ISO resolution (ISO TC147/SC5/WG10 
Antalya 3) as well as an OECD recommendation (OECD, 1998c) that the NOEC 
should be phased out from international standard. However, up until now NOECs are 
used by regulatory authorities and probably will be used also in future, since it appears 
that these decisions will not be followed strictly. 
If the NOEC is intended to be used further, measures have to be taken to lessen the 
aforementioned problems. Among these are: setting of a reasonable effect threshold 
that should be detected (e.g. 20% reduction relative to control), reducing variability, 
controlling the statistical power (e.g./3 ~