reconsidering environmental effects assessment of chemicals: proposal for a dynamic testing strategy

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Basic and Applied Ecology 9 (2008) 356–364 Reconsidering environmental effects assessment of chemicals: Proposal for a dynamic testing strategy Marion Junghans a , Maike Schaefer b , Wiebke Drost a , Enken Hassold a , Frauke Stock c , Matthias Du¨nne c , Tanja Juffernholz b , Wiebke Meyer a , Johannes Ranke c, a Department of Biology and Chemistry, Institute for Cell Biology, Biochemistry and Biotechnology, University of Bremen, Germany b Department of General & Theoretical Ecology, Centre for Environmental Research and Technology (UFT), University of Bremen, Germany c Department of Bioorganic Chemistry, Centre for Environmental Research and Technology (UFT), University of Bremen, Germany Received 20 July 2007; accepted 25 August 2007 Abstract Certain substances may be hazardous to ecosystems. To be able to preserve the structures and functions of ecosystems, knowledge is required to qualify and quantify such hazards. To this end, biotests are indispensable tools. For the development and/or choice of biotests, special attention has to be drawn to conflicts between scientific demands and practical constraints. From a purely scientific point of view, experiments should be designed to maximise the ecological relevance of the obtained results. However, this often collides with the limited resources (budget, time, manpower) available. Furthermore, societal issues (e.g. animal welfare) have to be taken into account. Thus, it is necessary to develop a scientifically sound testing approach that avoids unnecessary animal testing, keeps the costs low, and can be performed within a short timeframe. The different perspectives of ecology, environmental toxicology, and environmental chemistry should be integrated into a balanced ecotoxicological approach. Accordingly, we propose a dynamic testing strategy, which is adapted to the substance (or substance group) in question and its mode(s) of action. r 2007 Gesellschaft fu¨r O ¨ kologie. Published by Elsevier GmbH. All rights reserved. Zusammenfassung Bestimmte chemische Substanzen ko¨nnen eine Gefahr fu¨r O ¨ kosysteme darstellen. Fu¨r den Schutz der Strukturen und Funktionen in O ¨ kosystemen, mu¨ssen diese Gefahren qualifiziert und quantifiziert werden. Biotests liefern dafu¨r die entscheidenden Ergebnisse. Fu¨ r die Entwicklung und/oder Wahl solcher Biotests, muss den Konflikten zwischen wissenschaftlichen Anspru¨ chen und praktischen Beschra¨nkungen besondere Aufmerksamkeit geschenkt werden. Aus rein wissenschaftlicher Sicht sollten die Experimente so gestaltet sein, dass die o¨ kologische Relevanz der Ergebnisse maximiert wird. Dies sto¨sst jedoch ha¨ufig an die Grenzen der verfu¨gbaren Ressourcen (Budget, Zeit, Arbeitskraft). Zusa¨tzlich mu¨ssen gesellschaftliche Aspekte beru¨cksichtigt werde (z.B. der Tierschutz). Daher ist es notwendig, einen wissenschaftlich stimmigen Test-Ansatz zu wa¨hlen, der unno¨ tige Tierversuche vermeidet, die Kosten gering ha¨lt und innerhalb kurzer Zeit durchgefu¨ hrt werden kann. Die unterschiedlichen Aspekte von O ¨ kologie, Umwelttoxikologie und Umweltchemie sollten zu einem ausgewogenen o¨kotoxikologischen Ansatz zusammengefu¨hrt werden. Daher www.elsevier.de/baae 1439-1791/$ - see front matter r 2007 Gesellschaft fu¨r O ¨ kologie. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.baae.2007.08.011 Corresponding author. Tel.: +4942121863373; fax: +494212187645. E-mail address: [email protected] (J. Ranke).

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Basic and Applied Ecology 9 (2008) 356–364 www.elsevier.de/baae

Reconsidering environmental effects assessment of chemicals:

Proposal for a dynamic testing strategy

Marion Junghansa, Maike Schaeferb, Wiebke Drosta, Enken Hassolda, Frauke Stockc,Matthias Dunnec, Tanja Juffernholzb, Wiebke Meyera, Johannes Rankec,�

aDepartment of Biology and Chemistry, Institute for Cell Biology, Biochemistry and Biotechnology, University of Bremen, GermanybDepartment of General & Theoretical Ecology, Centre for Environmental Research and Technology (UFT),

University of Bremen, GermanycDepartment of Bioorganic Chemistry, Centre for Environmental Research and Technology (UFT), University of Bremen, Germany

Received 20 July 2007; accepted 25 August 2007

Abstract

Certain substances may be hazardous to ecosystems. To be able to preserve the structures and functions ofecosystems, knowledge is required to qualify and quantify such hazards. To this end, biotests are indispensable tools.For the development and/or choice of biotests, special attention has to be drawn to conflicts between scientificdemands and practical constraints. From a purely scientific point of view, experiments should be designed to maximisethe ecological relevance of the obtained results. However, this often collides with the limited resources (budget, time,manpower) available. Furthermore, societal issues (e.g. animal welfare) have to be taken into account. Thus, it isnecessary to develop a scientifically sound testing approach that avoids unnecessary animal testing, keeps the costs low,and can be performed within a short timeframe. The different perspectives of ecology, environmental toxicology, andenvironmental chemistry should be integrated into a balanced ecotoxicological approach. Accordingly, we propose adynamic testing strategy, which is adapted to the substance (or substance group) in question and its mode(s) of action.r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Zusammenfassung

Bestimmte chemische Substanzen konnen eine Gefahr fur Okosysteme darstellen. Fur den Schutz der Strukturenund Funktionen in Okosystemen, mussen diese Gefahren qualifiziert und quantifiziert werden. Biotests liefern dafurdie entscheidenden Ergebnisse. Fur die Entwicklung und/oder Wahl solcher Biotests, muss den Konflikten zwischenwissenschaftlichen Anspruchen und praktischen Beschrankungen besondere Aufmerksamkeit geschenkt werden. Ausrein wissenschaftlicher Sicht sollten die Experimente so gestaltet sein, dass die okologische Relevanz der Ergebnissemaximiert wird. Dies stosst jedoch haufig an die Grenzen der verfugbaren Ressourcen (Budget, Zeit, Arbeitskraft).Zusatzlich mussen gesellschaftliche Aspekte berucksichtigt werde (z.B. der Tierschutz). Daher ist es notwendig, einenwissenschaftlich stimmigen Test-Ansatz zu wahlen, der unnotige Tierversuche vermeidet, die Kosten gering halt undinnerhalb kurzer Zeit durchgefuhrt werden kann. Die unterschiedlichen Aspekte von Okologie, Umwelttoxikologieund Umweltchemie sollten zu einem ausgewogenen okotoxikologischen Ansatz zusammengefuhrt werden. Daher

e front matter r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

ae.2007.08.011

ing author. Tel.: +4942121863373; fax: +494212187645.

ess: [email protected] (J. Ranke).

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364 357

schlagen wir eine dynamische Teststrategie vor, welche sich an die jeweilige Substanz (oder Substanzgruppe) und ihreWirkmechanismen anpasst.r 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.

Keywords: Effects assessment; Structure–activity relationships; Biotests; ACR; Substance groups; Test battery; Multispecies tests;

Time; Budget

Table 1. Scientific demands and practical constraints in

effects assessment within ecological risk assessment

Scientific demands Practical constraints

Gain of knowledge Resources: time, budget, lab

resources, technical expertise

Ecological/ biological

relevance, e.g.

Culturability of test organisms

Test organism(s)

representative for species at

risk

Societal aspects, e.g. animal

welfare

Ecosystem structure

Reproducibility Legislative aspects, e.g.

decisiveness

Sensitivity

Generalisability Socio-economic considerations

Introduction

Scientific demands and practical constraints

In order to preserve the structure and functioning ofecosystems, environmental protection and managementstrategies rely on the estimation of the hazard a chemicalmay pose to ecosystems (Forbes & Forbes, 1994).Environmental effects assessments are based to a largeextent on ecotoxicological tests (biotests), which qualifyand quantify the hazard of chemicals under standar-dised conditions (European Commission, 2003, 2004;van Leeuwen et al., 1996). The results of an effectsassessment serve as a basis to decide whether a risk ofadverse effects in the environment is likely to occur. In asubsequent risk assessment usually the predicted noeffect concentration (PNEC; the lowest no observedeffect concentration (NOEC) from a set if biotestsmultiplied by a given factor that should account for theuncertainty associated with the extrapolation from labto environment) is compared to a predicted environ-mental concentration that derives from an exposureassessment for the chemical (European Commission,2003, 2004).

The challenge of effects assessments is to accuratelyextrapolate the results of ecotoxicological testing tovarious organisms, populations and ecosystems (Forbes& Calow, 2002). Thus, biotests should be designed toreflect the environmental situation as realistically aspossible, while concurrently being sensitive, reproduci-ble, and interpretable. The selected biotests should beable to account for specific environmental interactionswhile covering effects on the environment as a wholeand thus being generalisable. This dilemma of ecotox-icologists has been addressed in several publications(e.g. Chapman, 2002; Forbes & Calow, 2002; Mathes,1997; Rieß, Manthey, & Grimme, 1993). Ideally, testdurations should be adapted to whole life cycles of thetest organisms as different life stages might differ in theirsensitivity towards a chemical. Furthermore, knowledgeof exposure pathways is necessary to ensure that the testspecies are representative for the ecosystem at risk.

However, the more complex and realistic a study, thegreater is the chance that it will collide with the practicalconstraints like budget and time resources. Moreover,other practical constraints have to be considered in thechoice of the biotest(s), such as legislative and societal

requirements. An overview of practical constraints andscientific demands is given in Table 1. Ideally theselected biotests should be quick, cost-effective, easy tohandle, restrict harming of animals to a minimum, andultimately give clear results that can be used to definedecision criteria for regulatory action.

Aims and approaches

With the above-named difficulties in mind, wepropose a pragmatic but scientifically sound approachfor an environmental effects assessment of chemicals,which attempts to balance scientific demands andpractical constraints. For this purpose, already existingconcepts were adopted or further developed andsubsequently combined into a testing strategy. The maingoals of this strategy are the integration of chemicalknowledge of the investigated substances and a dynamicprocedure for selecting only the appropriate biotests.This approach is compared to existing regulatoryframeworks for prospective effects assessments and isset into the current scientific and regulatory contexts.

Proposal for a dynamic testing strategy

The implementation of test strategies is necessary forgenerating sound prospective hazard assessments (e.g.Breitholz, Ruden, Hansson, & Bengtsson, 2006; Rießet al., 1993). A testing strategy commonly includes a

chemical class

reactivity

toxic mechanisms

(Q)SAR

immission / mobility /

distribution / degradation /

persistence

exposure-profilein vitro biotest battery

type of action / toxic mechanism

enzyme, cell and microbiotests for diverse

known types of action or toxic mechanisms

qualitative hazard

profile (QHP)

specific or unspecific action?

which environmental compartment?

biomagnification expected?

quantitative

ecotoxicity profile (QEP)

toxic data from mono- and

multispecies-tests

multi-species-system

representative of ecosystem

focus: interactions and processes

defined exposure scenarios:

e.g. lowest NOEC from in-vivo test-battery

predicted environmental concentration (PEC)

In vivo biotest battery

generic ecosystem / type of action

representative of the environmental compartment

monospecies-tests (div. taxa and trophic levels)

specific action: “focused selection”

unspecific action “broad selection”

concentration-response-analysis

legislative-

administrative

risk assessment

acute-

chronic-

scheme

(see Fig.2)

validation in the environment

(e.g. site-specific

assessment, TIE-approach,

monitoring)

retrospective studies

Fig. 1. Dynamic testing strategy for the effects assessment of chemicals (see text for explanation). (Q)SAR: (quantitative) structure–

activity relationship; NOEC: no observed effect concentration; TIE: toxicity identification and evaluation – determination of the

toxic components in a water sample by fractionation and biotesting.

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364358

variety of biotests and a tiered ‘‘decision tree’’ for bio-testing (Breitholz et al., 2006). Our aim is to integratethe results from tests on molecular, single species andpopulation levels to gain insight into the potentialhazards a given chemical may pose to the structure andfunctioning of an exposed ecosystem. Aiming towards asubstance group directed analysis, the integration ofadditional substance properties like partitioning beha-viour and reactivity is important. In Fig. 1 we illustratethe proposed testing strategy, consisting of two majorcharacterisation steps resulting in (i) a qualitativehazard profile (QHP) and (ii) a quantitative ecotoxicityprofile (QEP).

In a first step, an in vitro test and analysis of thechemical structure should give information on thepossible modes of action. Additionally, by generatingan exposure profile, ecosystem compartments with highexposure should be detected. The resulting QHP shouldserve as a means to decide for which species theecotoxicity needs to be quantified, i.e. in which specieslikely to be exposed to the substance, the detected modesof action of the chemical might occur. Based on theseconsiderations single and multispecies biotests forconcentration-effect assessments are selected. With theresulting information an ecotoxicity profile is estab-lished, which can be used for risk assessments. The

dynamic character of the testing strategy lies in theadaptation to evolving ecotoxicological knowledge: ifimproved exposure analysis or retrospective studiesreveal undetected ecotoxicological effects, this can easilybe accounted for by extending the research that leads tothe qualitative hazard profile.

Qualitative hazard profile

The QHP is intended to characterise the ecotoxicolo-gical properties of the substance. The aim of the profileis to gather as much information as possible on thesubstance’s mode(s) of action, possible bio-magnifica-tion, and the environmental compartment considered.Information on the mode(s) of action is derived fromstructure–activity relationships (SARs) and an in vitro

biotest-battery. Information on the chemical class, thereactivity of the substance, and a comparison withknown structural alerts may give initial insights intopossible modes of action (e.g. Veith, Call, & Brooke,1983; Verhaar, van Leeuwen, & Hermens, 1992; von derOhe et al., 2005). The experimental analysis with anin vitro biotest battery gives additional evidence on themode(s) of toxic action. This battery includes cellularand subcellular tests. Examples for such a mode of

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364 359

action-based test battery for the detection of ecotox-icological hazards can be found in, e.g. Schweigert,Eggen, Escher, Burkhardt-Holm, and Behra (2002) orEscher, Bramaz, Eggen, and Richter (2005).

Exposure profiles give information on possible bio-magnification as well as on the chemical’s environmentalfate, such as emission, mobility, distribution, degrada-tion, and persistence. Additionally, exposure profilescan focus on possible exposure pathways, potentiallyaccumulating environmental media, and specific geo-graphic areas.

Information about the possible mode of action gives afirst insight into the hazard potential of a chemical, andthe exposure profile detects the possible groups oforganisms and types of ecosystems to be considered.The information gathered in the QHP is used in the nextstep, the quantification of the effects.

Quantitative ecotoxicity profile

In a second step the analysed effects have to bevalidated and quantified in vivo. For this purpose theQHP is to be used as a guideline, to decide whichorganisms and which processes will be most likelyaffected by the substance and hence which test designswill give the most relevant results. This leads to thepossibility to restrict the test battery to these groups oforganisms. Based on these test designs, a QEP can beestablished. It will comprise both the relevant acute andthe chronic toxicity data from mono- and multispecies-tests (Fig. 1) and can serve as a basis for legislative andadministrative risk assessment.

In the following the in vivo test battery and the designof multi-species tests will be introduced.

In vivo test battery

In the in vivo test battery the effects quantificationshould be focused on species that represent the affectedenvironmental compartment(s) as indicated by theexposure profile. Furthermore, the efficiency of identify-ing the most sensitive taxonomic group should beincreased by the results of the (Q)SAR analysis andthe in vitro test battery. If the QHP suggests that specificeffects are only likely to occur in certain taxonomicgroups, in vivo tests should be selected accordingly,avoiding tests with organisms for which only baselinetoxicity (the minimal toxicity of a given chemical causedby its partitioning into membranes; see Verhaar, Busser,& Hermens, 1995) is expected (e.g. testing animal speciescan be avoided when for structurally similar substancesthe only detected specific effect was photosynthesisinhibition, and testing plant species is irrelevant whenstructurally similar substances exhibit endocrine disrup-tion only). If no specific mode of action has beendetected, the composition of the test battery should be

more diverse, i.e. tests should be made using organismsfrom different taxonomic groups and trophic levels. Ifthe specific mode of action was not confirmed in thebattery of in vivo biotests, the test battery should beenlarged with tests selected according to the samecriteria. Ideally, the QHP should report every possiblemode of action. Although there are in vitro biotestsreporting several modes of action, there are still testsmissing for some modes (Escher et al., 2005). However,additional information from the (Q)SAR analysis cannarrow down possible blind spots in the in vitro testbattery. In case there is still concern about anundetected mode of toxic action, the in vivo test batterymay have to be broadened. However, the goal for thefuture would be to erase the blind spots.

We emphasise the importance of concentration–response analysis for the prediction of mixture toxicityin a subsequent risk assessment. Numerous studies havereported successful predictions of mixture toxicitiesbased on dose–response curves of the components, evenfor realistic exposure scenarios (Junghans, Backhaus,Faust, Scholze, & Grimme, 2006 and references therein).Hence, the complete concentration–response relation-ship should be analysed and fully documented in everyselected test, to avoid cost-intensive effects assessmentsfor every mixture of concern later on.

Substance group directed analysis of chronic toxicity

Before any tests for chronic toxicity are carried out,the extrapolation of chronic data from acute data, asproposed by several authors (e.g. Ahlers et al., 2006;Lange, Hutchinson, Scholz, & Solbe, 1998; Roex, vanGestel, van Wezel, & van Straalen, 2000) should beconsidered as a time and cost-effective solution.

Lange et al. (1998) analysed acute to chronic ratios(ACR) for various types of substances using theECETOC aquatic toxicity (EAT) database. They useda probabilitic approach, in which the ACR is calculatedas the 90%-ile of the distributions of the ratios betweenthe acute EC50 value(the concentration that causes halfthe maximal effect) and the corresponding chronicNOEC. Other authors suggest using a regression toextrapolate between acute and chronic toxicity (Elme-gaard & Jagers op Akkerhuis, 2000), to be able toaccount for a dependency of the ACR on the toxicity.The ACRs were estimated individually for each sub-stance and species available. The authors concluded thatACRs are dependant on the substance category(halogenated compounds, aromatic hydrocarbons, me-tals/organometals, other organics, and other inorganics)as well as on their intended use (e.g. pesticide activeingredients). Especially metals, organometals and pesti-cides show highly variable ACRs.

Based on the above approach we developed a decisiontree for a substance group directed assessment ofchronic toxicity (Fig. 2), which may allow for both a

no

yes

grouping(a) physico-chemical features

(b) mode of action

acute chron. tox.

(ACR ~ 1)

knowledge sufficient?

ACR is constant or can be estimated from

physico-chemical descriptorsACR is variable

biotest(s) on acute toxicitybiotest(s) on acute and

chronic toxicity

direct assessment of the acute toxicity

extrapolation of chronic toxicity from acute toxicity

uncertainty analysis

direct assessment of both

acute and chronic toxicity

substance class analysis• acute toxicity

• chronic toxicity

according to primary scientific

aspects

Fig. 2. Decision tree for a substance group directed assessment of the chronic toxicity from acute toxicity using acute to chronic

ratios (ACR).

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364360

scientifically sound assessment of the chronic toxicityand the avoidance of unnecessary consumption ofresources and test organisms.

As a start we suggest a brief investigation of thecompound regarding its chemical structure and mode ofaction, in order to classify the chemical. If currentknowledge is not sufficient for a classification, an acuteto chronic extrapolation is not possible and chronictoxicity tests are called for.

In case of classifiable substances, the chronic toxicitycan be extrapolated from empirical acute toxicity data ifthe acute–chronic ratio is low and rather constant withina group of chemicals (e.g. for baseline toxicants). Afurther possibility for extrapolation may be available if acorrelation between ACRs and available (Q)SARdescriptors can be found. In the case of baselinetoxicants, the ACR can be low and rather constant,provided that there is no metabolisation or switch toanother mode of action (Leslie, Kraak, & Hermens,2004). Both the acute and chronic toxicity have to bedetermined empirically if the variability of the ACR istoo high within the group (e.g. heavy metals) or ifstructural alerts indicate a high and variable ACR(Ahlers et al., 2006). According to the acute chronicscheme, a decision can be made whether the chronictoxicity might be estimated from acute toxicity data andACRs, or whether chronic tests are necessary (Fig. 2).The uncertainty analysis for the chronic toxicity valuedepends on the method with which chronic toxicity was

extrapolated from the acute toxicity, e.g. if regression isused to estimate the relationship between acute andchronic toxicity an uncertainty analysis can be calcu-lated by means of the confidence limit (Elmegaard &Jagers op Akkerhuis, 2000).

Multispecies test

As in the single species biotest-battery, the QHPshould also form the basis of the design of themultispecies test: It should be representative of theecosystem in which the test substance most likely willoccur (determined by the exposure profile). While thein vivo biotest-battery focuses on the effects of thechemical on individual species, the multispecies testsystem should be designed to report its effects oninteractions and processes within the ecosystem. Thusthe selection of species must not be restricted to speciesfor which a specific mode of action is expected.Exposure concentrations should be chosen in a wayallowing for determination of concentration–responsecurves. If available, concentration–response relation-ships from the in vivo battery of biotests could be usedfor guidance.

Retrospective studies

Applying the proposed test strategy, the knownadverse effects can be detected. However, as in every

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364 361

effects assessment, some adverse effects may remainundetected. Therefore, we consider the coupling of theprospective effects assessment with environmental sam-pling as mandatory for a scientifically sound assessmentof chemicals. The release of the substance into theenvironment should be monitored to validate the resultsof the testing strategy in the field. This may be done, e.g.by ecological monitoring, a site-specific assessment, ortoxicity identification and evaluation (TIE) procedures(e.g. Burgess, 2000). Additionally, the dynamic testingstrategy should be frequently adapted to account fornewly detected modes of action.

A scientifically sound effects assessment of

chemicals – general discussion

Possible pitfalls of the dynamic testing strategy

The proposed dynamic testing strategy is based on theassumption that the most sensitive biotests are selectedfor the quantification of ecotoxicity. However, it can beargued that the selection of test species according to a

prior mode of action assessment narrows down thechance of detecting the most sensitive species.

When applying the proposed dynamic testing strategyfour scenarios may be envisaged regarding the mode ofaction: (i) neither the QHP nor the in vivo test battery,composed of biotests from diverse taxonomic groups,gives evidence for a specific mode of action, (ii) a specificmode of action determined in the QHP was notconfirmed in the in vivo battery of biotests andsubsequent testing of the substance in a broader selectedbattery of biotest did not reveal any other specific modesof toxic action, (iii) the QHP has not reported a specificmode of action, but in some in vivo biotests a specifictoxicity is observed, and (iv) the QHP has reported aspecific mode of action which was confirmed in the in

vivo battery of biotests. In scenarios (i) and (ii) the mostsensitive biotest is the one, which intrinsically shows thehighest baseline toxicity. In the two latter scenarios, theprobability that the most sensitive biotest was applieddepends on species sensitivity distributions within theaffected taxonomic group(s) (for differences in speciessensitivity also refer to Schmitt-Jansen, Veit, Dudel &Altenburger, 2007). A further source of uncertainty forall scenarios lies in the possibility of undetected modesof toxic action occurring in untested groups of organ-isms. For the derivation of PNECs however, this wouldonly cause a problem, if they occur at lower effectconcentrations than the detected mode(s) of action,since PNECs are usually based on the most sensitivedata. The probability of such a case has to be studiedempirically by analysing existing eco-toxicity data sets:(i) how many specific modes of action has a single

substance and (ii) what is the quantitative differenceregarding the effect concentrations. The possibility ofthe occurrence of such a case makes retrospective studiesmandatory.

Though many authors have dealt with the extrapola-tion of acute to chronic data (Ahlers et al., 2006;Elmegaard & Jagers op Akkerhuis, 2000; Heger, Jung,Martin, & Peter, 1995; Hunt et al., 2002; Kenaga, 1982;Lange et al., 1998; Roex et al., 2000) no clear picture ofACR has evolved so far. However, there is a generalagreement that ACR depends on the family of substance,the mode of action as well as on the test species.

So far the availability of good experimental data for asound ACR model is limited (Ahlers et al., 2006), hencemore research is needed. In-depth physiological knowl-edge could give more insight into the factors governingthe quantitative relationship between acute and chronictoxicity.

One method discussed by Douboudin, Ciffroy, andMagaud (2004), the acute to chronic transformation(ACT), includes the variability of species sensitivity andthus circumvents the problem of varying ACRs fordifferent invertebrate taxa. This methodology is basedon the extrapolation of acute species sensitivity dis-tribution (SSD) to chronic SSD (Douboudin et al.,2004). According to the authors it is too early to decidewhether this approach can be used operationally in thecontext of risk assessments. Promising data werepublished by Chevre et al. (2006). With a similarapproach, they were able to predict SSDs for chronictoxicity data for two chemical classes of photosynthesis-inhibiting pesticides.

Despite the partially contradictory results and theremaining uncertainties the ACR approach is a promis-ing tool, because toxicants are assessed including theirspecific chemical and biochemical properties and costand time consuming chronic tests may be avoided.

Comparison to current regulatory frameworks

To highlight advances/advantages and drawbacks ofthe proposed testing strategy regarding practical con-straints and scientific demands (Table 1), it will becompared to existing regulatory frameworks for aprospective effects assessment of chemical substances.The pesticide guideline (European Commission, 2004),the technical guidance document on risk assessment ofnew notified substances, existing substances and biocidalproducts (TGD; EU Commission, 2003) and the newEU chemicals policy REACH (European Council, 2005)are used as references.

Test strategy

In the current TGD a standard minimum set of in vivo

biotests is required. With the same biotests, both hazard

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364362

identification and effect quantification are to beachieved, and both steps are mainly uncorrelated. Incontrast, in the proposed testing strategy hazardidentification (QHP) and quantification (QEP) are twoclosely related steps. This combination has advantagesregarding the practical constraints as well as thescientific demands. The restriction of the effectsquantification to only the relevant in vivo biotests mayhelp to avoid unnecessary sacrifice of test organisms.Furthermore, the focus on the mode(s) of action byusing an in vitro battery of biotests is advantageous forgeneralisability: knowledge on the mode(s) of action ofthe test chemical allows to predict effects in ecosystems.

Multispecies tests

Multispecies tests for effect quantification are com-pletely missing in the TGD, are not mandatory inREACH, and are only required by the pesticide guide-line if expert judgement deems a mesocosm testnecessary.

However, with a specifically tailored multispecies testimportant ecotoxicological information can be gained –while balancing scientific demands and cost effective-ness. Therefore we regard a specifically designed multi-species test as a necessary feature in an effectsassessment aiming for ecologically relevant results.

In vitro tests

Current regulatory frameworks for ecotoxicologicaleffects assessment do not include in vitro tests. However,there is an ongoing discussion of replacing in vivo fishtests with fish egg tests. In human health risk assessmentseveral in vitro tests as well as strategies for thereplacement of in vivo tests are discussed (Blaauboer,2002). This might be realistic for distinct, mechanisti-cally understood endpoints such as skin sensation. Yetwe regard the total replacement of in vivo tests for highlyintegral endpoints such as fish toxicity to be moredifficult, since interactions within organisms and, evenmore important, effects on ecosystem structure andfunctioning are not taken into account. However, as aninput for a QHP in vitro tests can be valuable tools tofocus the effect quantification with in vivo toxicitytesting. To our knowledge this is a novel approach.

Acute – chronic ratios

Extrapolation from acute to chronic toxicity isnecessary because chronic data are often not available,neither for existing substances nor for new substances.According to the TGD, extrapolation from acute tochronic toxicity is already being applied in the riskassessment procedure. If the data are limited to acutedata and the so-called base set of species (algae, daphniaand fish) an arbitrarily chosen assessment factor of 100is used to extrapolate between acute and chronictoxicity. In the pesticide guideline, chronic tests are

mandatory and thus an extrapolation is not necessary.Like the TGD approach, our approach aims atminimising the effort for testing, avoiding chronic testswhenever possible by using the ACR. However, oursuggested procedure might be a more scientificallysound alternative to an arbitrarily chosen applicationfactor, because toxicants are treated with regard to theirspecific chemical and biochemical properties. Fixedapplication factors imply that the relationship betweenacute and chronic toxicity is independent of both the testspecies and the compound, while extrapolation bymeans of substance-specific ACR has a scientific basisand therefore meets external as well as scientificdemands. Furthermore, it seems to be necessary to baseacute to chronic extrapolation on a scientific approach,because Ahlers et al. (2006) showed that currentapplication factors may not be high enough to coveracute–chronic ratios for more than 10% of industrialchemicals.

Retrospective studies

Retrospective studies based on chemical analysis arecommon practice for many soil and drinking watercontaminants, and for contaminated sites. Retrospectiveeffect monitoring is to our knowledge not yet imple-mented in other regulatory frameworks. However, forthe aquatic environment, the assessments of theecological status as demanded in the water frameworkdirective of the European Union (European Commis-sion, 2000) may report adverse effects of chemicalswhich were not detected during the hazard assessmentprocess. A prominent example of such an undetectedadverse effect is endocrine disruption, which was firstretrospectively detected in the field and not prospec-tively by standard laboratory tests (Calow & Forbes,2003). This example stresses the need for monitoringstudies. In this context, the use of biomarkers as earlywarning systems could be useful (Calow & Forbes, 2003;Eggen, Behra, Burkhardt-Holm, & Escher, 2004). Also,TIE procedures should be included to bridge exposureand effect assessment (Eggen et al., 2004; Eggen &Segner, 2003).

The proposed testing strategy and current directions in

ecotoxicology

Putting more ‘eco’ into ecotoxicology as proposed byBaird, Maltby, Greig-Smith, and Douben (1996), recentdirections indeed aim towards more ecological ap-proaches, but do also include the demand for amechanistic understanding of underlying processes.Thus, a broad range of challenges must be met, e.g.low concentrations, long exposure times, complexmixtures, multiple stress and ecosystem effects (Eggenet al., 2004). Breitholtz, Ruden, Hansson, and Bengtsson(2006) identified 10 additional challenges for ecotoxico-logical testing: representative test organisms, regional

M. Junghans et al. / Basic and Applied Ecology 9 (2008) 356–364 363

relevance, sensitive life stages, population genetic effects,general mechanistic understanding, endocrine disrup-tion, relevance of exposure routes, ethical considera-tions, validating testing strategies, and science-basedprecaution. Ideally, these aspects should be all includedin a testing strategy.

According to several authors (Breitholtz et al., 2006;Calow & Forbes, 2003; Eggen et al., 2004) more effortshould be put into the extrapolation of effects betweendifferent endpoints, different levels of organisation,different time scales and account for differences inspecies sensitivity. The proposed acute–chronic scheme(Fig. 2) is based on the same idea. Furthermore, resultsobtained with our testing strategy might help to betterunderstand the influence of these parameters on theeffect in vivo and might thus help to find extrapolationfactors.

The integration of several types of information inorder to optimise the gain of information while retainingcost efficiency is a widely accepted goal (e.g. Breitholtzet al., 2006; Davoren & Fogarty, 2004; Jos et al., 2005).Resource efficiency becomes even more pressing, whenconsidering the testing effort that has to be undertakenunder the new EU chemicals policy REACH (EuropeanCouncil, 2005). Although not developed for REACH,our testing strategy includes several requirementsaddressed in the new REACH legislation such as useof in vitro test, QSARs and evaluation of groups ofsimilar chemicals (grouping approach, see Annex 9 ofthe REACH legislation).

The experimental validation of the proposed ap-proach of a testing strategy is one goal to be strived forin the future.

A dynamic testing strategy needs many disciplines

As discussed above, a dynamic testing strategy isindispensable when the toxicity of chemicals is to beassessed under the premises of cost and time efficiencyand ecological relevance.

A successful dynamic strategy for effects assessmentrequires the basic knowledge of different disciplines suchas environmental chemistry, biochemistry, ecology,toxicology, terrestrial and aquatic ecotoxicology inorder to understand the behaviour of chemicals in theenvironment and to assess their effects. However, it isalso necessary to consider the implementation of thisknowledge into politics and legislation. Fenner andEscher (2006) have pointed out that communicationbetween scientists and regulators is not ideal. Thus, it isnecessary to involve regulators, social scientists, lawyersand politicians in the development of test strategies foran effects assessment of chemicals. Only interdisciplin-ary hand-in-hand work of scientists belonging to all

disciplines makes the effects assessment scientificallysound.

Acknowledgements

The contributions of Dr. T. Backhaus and Dr. T.Frische to the discussions that finally lead to this paperare gratefully acknowledged. We would also like tothank Dr. J. Rombke and Prof. Dr. J. Filser, Prof. Dr.K. Hovemeyer as well as two anonymous reviewers forhelpful comments on earlier versions of the manuscript.

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