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On the Use of Mixture Toxicity Assessment in REACH and the WaterFramework Directive: A ReviewK. Syberg ab; T. S. Jensen b; N. Cedergreen c; J. Rank a

a Department of Environmental, Social and Spatial Change, Roskilde University, Roskilde, Denmark b

Department of Policy Analysis, National Environmental Research Institute, Aarhus University,Aarhus, Denmark c Department of Agricultural Sciences, Faculty of Life Science, University ofCopenhagen, Copenhagen, Denmark

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To cite this Article Syberg, K., Jensen, T. S., Cedergreen, N. and Rank, J.(2009) 'On the Use of Mixture Toxicity Assessmentin REACH and the Water Framework Directive: A Review', Human and Ecological Risk Assessment: An InternationalJournal, 15: 6, 1257 — 1272To link to this Article: DOI: 10.1080/10807030903304922URL: http://dx.doi.org/10.1080/10807030903304922

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REVIEW ARTICLE

On the Use of Mixture Toxicity Assessment in REACHand the Water Framework Directive: A Review

K. Syberg,1,3 T.S. Jensen,3 N. Cedergreen,2 and J. Rank1

1Department of Environmental, Social and Spatial Change, Roskilde University,Roskilde, Denmark; 2Department of Agricultural Sciences, Faculty of Life Science,University of Copenhagen, Copenhagen, Denmark; 3Department of Policy Analysis,National Environmental Research Institute, Aarhus University, Aarhus, Denmark

ABSTRACTThis review seeks to connect the scientific theory of mixture toxicity to its imple-

mentation within different regulatory frameworks. The aim is to demonstrate howmixture toxicity assessment can be more thoroughly integrated into the Europeanchemical regulations, REACH, and the Water Framework Directive (WFD), usingthe experiences gained through other regulatory frameworks. The article consistsof (1) an examination of the scientific underpinnings of the common mixture toxi-city assessment methods; (2) a discussion of how these methods have been used inregulatory frameworks; and (3) a discussion of how the methods could be appliedwithin REACH and the WFD. It is concluded that concentration addition shouldbe applied as a default model for mixture toxicity assessment. Furthermore, it isconcluded that REACH and the WFD only include mixture toxicity assessments inspecific situations. However, it is shown that it is scientifically feasible and regula-torally practicable to integrate a more holistic mixture toxicity approach into bothlegislations. In this connection, the experience gained from the U.S. frameworkson mixture toxicity assessment could be useful. The construction of a database thatincludes data on chemicals in the European environment could be used for mixturetoxicity assessment of the chemicals with individual PEC/PNECs > 0.1.

Key Words: mixture toxicity, chemical regulation, REACH, Water Framework Di-rective, concentration addition.

Received 28 August 2008; revised manuscript accepted 27 February 2009.Address correspondence to K. Syberg, Department of Environmental, Social and SpatialChange, Roskilde University, Universitetsvej 1, Postbox 260, 4000 Roskilde, Denmark. E-mail:ksyberg@ruc.dk

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INTRODUCTION

It is commonly accepted that the effect of chemicals on ecosystems and hu-man health mainly comes from exposures to mixtures rather than from individualchemicals (Backhaus et al. 2003). It is also commonly accepted that it is practicallyimpossible to do experimental mixture assessment on all combinations of chem-icals on the market. Two mathematical models, concentration addition (CA) andindependent action (IA), have been proposed for assessing mixtures as an alter-native to testing all mixtures. The choice of model in a specific situation dependson knowledge about the chemical’s mode of action. It is, however, very difficult,and sometimes impossible, to determine the mode of action of a chemical, as it,among other things, depends on both concentration and the organism exposed.We therefore find it relevant to evaluate the scientific basis for assessing mixtures ofchemicals with similar modes of action as compared to mixtures of compounds withdifferent modes of action, to find out if it is possible to pool all kinds of chemicalsfor a joint mixture toxicity assessment.

Regardless of the importance of mixture toxicity effects, the new major Europeanlegislation that aims at regulating chemicals in the environment, that is, the WaterFramework Directive (WFD) and the Registration, Evaluation and Assessment ofChemicals (REACH), are primarily based on a single toxicity assessment approach(EC 2000; EC 2003a). Simultaneously with the elaboration of REACH, the EuropeanUnion (EU) launched an integrated strategy for the environment and health, theSCALE initiative, in which knowledge about multi-pollutant exposures is one of thefocus areas (EC 2003b). Also, in 2003, the EU adopted an action plan that focuseson, among other things, producing better and more realistic risk assessments (EC2003b). These legislative initiatives show that the importance of mixtures might bepolitically acknowledged, but that it has not yet been adequately implemented inEuropean legislation. European mixture toxicity assessment is only made in regardto very specific types of mixtures at specific stages of their life cycle (such as forpreparations that are covered by REACH).

However, since effects of mixtures are relevant in all stages of a chemicals lifecycle and for all types of toxic chemicals, it seems necessary to expand the currentregulation within the field. Mixtures should ultimately be assessed for all toxicchemicals at all stages of the life cycle.

Ideas concerning how a more holistic mixture toxicity assessment can be imple-mented in REACH and the WFD can be sought in the current legislation within thefield. If current practice can be (partly) adopted within REACH and the WFD, it willbe an advance since it would save time and resources and at the same time ensurethat the assessment is based on an approach that has been carried out in practicebefore.

The aim of this article is therefore three-fold. First, we evaluate the strength ofthe scientific evidence for using predictive models in risk assessment of mixtures asregulatory tools, and address the question about whether one model can be used asdefault. Second, we review some of the existing mixture toxicity regulatory practices,and third, these two analyses are used to suggest recommendations as to how theassessment of mixture toxicity could be further integrated into REACH and theWFD.

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MIXTURE TOXICITY PREDICTION

Much effort has been dedicated to validating two mechanistic mathematical mod-els for assessing the effect of mixtures (Arrhenius et al. 2004; Backhaus et al. 2000a, b,2003; Backhaus et al. 2004; Deneer 2000; Faust and Scholze 2004; Greco et al. 1995).These are the concentration addition (CA) and independent action (IA) models(also called the dose addition and the response addition, respectively). Theoreti-cally, the two models can estimate mixture effects of similar and dissimilar actingchemicals, respectively, from dose–response data of the individual chemicals. Bothmodels assume that interactions between chemicals in the mixture do not signifi-cantly alter the overall toxicity of the mixture (Bliss 1939). If the chemicals interactto produce an effect larger than predicted by one of the two models, it is termedsynergy (Loewe- and Bliss-synergy for deviations from CA and IA, respectively), whileif chemicals interact to produce a smaller effect than predicted, it is termed Loewe-or Bliss antagony (Greco et al. 1995).

In terms of risk assessment, synergistic interactions are often those that attractmost political attention. However, these types of mixture toxicity effects are relativelyrarely found. The largest synergistic deviations from the reference models commonlyinvolve two or maybe three chemicals, which have specific characteristics in termsof affecting either uptake, transportation, or the degradation of other chemicalsin an organism (Andersen and Dennison 2004). Data on ecotoxicological effects ofpesticide mixtures show that synergistic chemicals are not common within these com-pounds (Cedergreen et al. 2006, 2007, 2008; Altenburger et al. 1996; Faust et al. 1994).

Synergistic interactions appearing in the literature in general are surprisinglyrare, considering the potential impact of such mixtures and the resulting searchfor them. Acknowledging that synergy does exist, it is worth noting that as thenumber of chemicals in a mixture increases, the individual interactions betweenchemicals are likely to become less dominant, since their relative contribution tothe overall toxicity decreases (Warne and Hawker 1995). This proposed relationshipis called the funnel hypothesis (Warne and Hawker 1995), and seems to be verifiedby literature data on chemical mixtures (McCarty and Borgert 2006) as well asstudies of complex equipotent mixtures (Altenburger et al. 1996; Backhaus et al.2000a, 2003, Backhaus et al. 2004; Walter et al. 2002). Synergistic interactions aretherefore not likely to play a big role in an environmental context, even thoughthere might be cases where it is of importance.

The choice of reference model, CA or IA, is theoretically based on the moleculartarget site of a compound, or more broadly defined, its mode of action (Grecoet al. 1995). However, knowledge of the mode of action of most chemicals on themarket is rarely available, and it is a very time consuming task to produce such data.Apart from that, it might not be feasible at all to organize chemicals by their modeof action in regard to environmental mixture toxicity assessment, since the toxicmode of action might differ from one species to the next within the same species atdifferent developmental status or as a function of exposure concentration, and soon (Hertzberg and MacDonell 2002; Syberg et al. 2007). It can thus be impossibleto group chemicals as having similar or dissimilar modes of action unless specificmodes of action in specific organisms are chosen above all others, as is the case forexample with chemicals causing endocrine disruption.

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If the effect on an entire ecosystem is to be assessed, however, other endpointsand modes of action of the same chemicals must also be considered. Fortunately,comparative studies of CA and IA in different test systems show relatively smalldifferences in their predicted effects (Altenburger et al. 1996; Cedergreen et al.2008). For a study based on 158 binary data sets and seven test systems of compoundshaving different modes of action in their target organism, there was no significantdifference between the CA and IA predictions across studies (Cedergreen et al.2008). The only exception was for the nine studies with activated sludge bacteria,where effect data were in between the predictions of the two models. Both modelsdid, nevertheless, predict effect data within a factor of two (Cedergreen et al. 2008).It thus seems to be scientifically sound to use one of the models as a default referencemodel for assessment of all mixtures.

Several authors have argued that concentration addition should be preferred asa general reference model. Backhaus et al. (2004) found that the concentration ad-dition (CA) model predicted effects of mixtures of chemicals with dissimilar actionrather well and only deviated from the independent action (IA) model predictionby a factor of 1.4. They concluded that the CA model is the better model as a generaldefault mixture toxicity assessment model, since it is most often the most conserva-tive and thus the most protective of the two models (Backhaus et al. 2000a, 2004).In accordance with the theoretical foundation of the two models, mixtures withsimilar modes of action have been shown to be best predicted by CA (Altenburgeret al. 2000; Arrhenius et al. 2004; Cedergreen et al. 2007). Junghans et al. (2006)showed that theoretically the deviation between CA and IA never exceeds a factorof 2.5 for the test system they used. Apart from being the more conservative model,they further argued that the CA model is not too overprotective in a risk assess-ment context, where safety factors often are several magnitudes higher than thedifference between CA predictions and experimental data (Junghans et al. 2006).Deneer (2000) evaluated the predictive power of the CA model on the basis of202 literature datasets of pesticide mixture toxicity. He concluded that 92% of themixtures were predictable by the concentration addition model within a factor oftwo, thereby confirming Junghaus et al. (2006) (Deneer 2000). Thus, there is sub-stantial empirical evidence to support the use of CA as a general reference modelfor the risk assessment of mixtures, even for mixtures consisting of similar actingchemicals.

Another advantage of using the CA model for risk assessment analysis is thatthe model predictions can be based on similar EC-values obtained from databaseswithout detailed knowledge of the shape of the entire dose-response curve, which,unfortunately, is rarely reported. The IA model, on the other hand, is more relianton specific knowledge of the dose–response relationship (Berenbaum 1989; Bliss1939; Greco et al. 1995). Since the European risk assessment paradigm is based oncollecting EC-values in years to come, the data collected in the future will be bestsuited for CA assessments.

Mathematically speaking, the CA model is defined as:

n∑i=1

ci

E Cxi= 1 (1)

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where; ci is the concentration of toxicant i in a mixture that causes the effect x andECxi is the concentration of toxicant i in a single compound solution that cause thesame effect x.

It must be emphasized that this equation is true only when comparing mixturesat a predefined response level. If a mixture deviates from Eq. (1) at a predefineresponse level, it is said to behave synergistically or antagonistically in relation toCA (Greco et al. 1995). Assuming a mixture behaves according to the model, itcan be used to sum up a large number of chemical concentrations corrected fortheir individual toxicity, and from that sum of toxic units calculate a joint effectestimate with the knowledge of the shape of the dose–response curve typical forthese chemicals (Teuschler 2007).

MIXTURE TOXICITY IN CURRENT RISK ASSESSMENT PROCEDURES

Apart from the scientific foundation it is relevant to consider whether an assess-ment tool is practically manageable. It is of great advantage if the mixture toxicityassessment tool can be integrated more or less directly and in line with the currentEuropean risk assessment paradigm (the technical guidance document; TGD). Theimportance of this is stressed by the fact that REACH’s risk assessment paradigm isfundamentally the same as the one used in the pre-REACH period (ECB 2003). Inorder to evaluate whether existing approaches for mixture toxicity assessment canbe used within the two legislations (i.e ., REACH and the WFD), we evaluate howmixture toxicity assessment procedures are integrated in other legislations.

U.S. Mixture Regulation

Compared to the EU, there has been a much longer tradition at a regulatory levelin the United States for considering mixture toxicity effects. The U.S. EnvironmentalProtection Agency (USEPA) has produced several guidance documents describinghow to address mixture toxicity assessment. It is beyond the scope of this articleto review them all. One of the most relevant in this context is the cumulative riskassessment (CRA) program (USEPA 2003). For a review see Fox et al. (2002). Theframework has a very broad scope, and can be used to address many different typesof cumulative risks, not only from chemicals. The term stressor is therefore used.This is important to keep in mind when evaluating the methods applied, since thescope is much broader than the problem addressed in this article. The programdistinguishes between stressors with similar and dissimilar modes of action (USEPA2003). For stressors with dissimilar modes of action, the cumulative effect is merelythe effect of the individual stressor as if other stressors were not present. However,it is important to keep in mind that the program addresses very different types ofstressors like chemicals, heat, and noise. It makes sense not to apply an additionmodel to such different stressors. The exception to this rule is when the effectsare represented as a frequency or as probabilities for affected individuals. In thesecases the cumulative effect should be estimated by the independent action approach(USEPA 2003). For chemicals with similar mode of action concentration additionis recommended. A mixture of chemicals that deviates from additivity (synergy

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and antagoni) is defined in regard to concentration addition (Loewe synergy andantagoni).

The U.S. Agency for Toxic Substances and Disease Registry (ATSDR) has devel-oped an approach in order to assess hazards from mixtures (Wilbur 2004). The workof ATSDR aims at assessing human health hazards, which means that the approachdeviates to some extent from those used to address ecotoxicological hazards. Inhealth hazard assessment, the focus is on preventing effects on the individual hu-man, while in ecotoxicological hazard assessment the focus is on protecting systemslike populations and ecosystems. Nevertheless, the ATSDR approach is an interest-ing paradigm that holds ideas and approaches applicable in ecotoxicological riskassessment. One of those aspects is a database in which chemicals at hazardous wastesites are recorded (called HazDat). In this database chemicals at waste sites acrossthe United States were ranked in regard to their occurrence, inherent toxicity, andpotential for human exposure. This database was used to retrieve information aboutthe most important single chemicals as well as binary and trinary mixtures for thecompartments water, soil, and air (Fay and Mumtaz 1996). The database can thus beused to identify which simple mixtures that pose the overall greatest hazard, and canthus serve as a tool for prioritizing regulation and monitoring of simple mixtures.

The ATSDR determines whether it is relevant to assess the mixture toxicity byassessing the magnitude of the individual compounds hazard quotients (HQ) (inprinciple similar to PEC/PNECs used in European legislation). If a mixture containsat least two chemicals with a HQ greater than 0.1 the mixture is of concern andfurther investigations are needed (Wilbur et al. 2004). This strategy is applied fornon-cancer effects. The strategy for carcinogens is somewhat different, but since thisarticle addresses environmental risk assessment, it is beyond the scope to go intodetail with this approach.

One other interesting aspect in the ATSDR approach is the application of theweight-of-evidence (WOE) method for assessing deviations from additivity. The prin-ciple of the system is to evaluate binary mixture effects and score them in regard totype of interaction (additivity, synergy, or antagoni). This score is then multipliedwith a different score that takes mechanistic and toxicological understanding, aswell as some modifying factors like type of data (in vivo or in vitro) into account. Bydoing that for all binary mixtures of concern it is possible to construct a matrix thatindicates whether interactions are of concern (Wilbur et al. 2004).

Even though most studies show that deviations from additivity (synergy and an-tagony) seldom are of importance in a ecotoxicological context, there might still besituations where synergistic interaction could be of concern. In these scenarios, theWOE approach could serve as inspiration for a European approach.

Danish Regulation of Air Pollution

The Danish EPA has implemented the regulation of mixture toxic effects in theirguidance document for industrial air emissions (Danish-EPA 2001). The regulationapproach distinguishes between emissions with similar and dissimilar modes ofaction and interacting chemicals. The mixture effect of chemicals with similar modeof action is based on the CA model (Danish-EPA 2001). The regulation of mixtures ofchemicals with dissimilar modes of action is based on identification of the chemical

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that contributes most to the toxic effect. The regulation is then made only in regardto this chemical. The regulation of mixtures with interacting chemicals is ratherpragmatic and assumes that interactions seldom occur at concentrations belowthe individual chemical No Observable Effect Concentration (NOEC) (Danish-EPA2001) and that hazardous chemicals are not allowed at concentrations greater thanthe NOEC. In other words, interactions are seen as insignificant and are thereforenot dealt with. The approach illustrates that chemical regulation is often based onpragmatic considerations regarding what is realistic without comprehensive changesto the existing procedure.

REACH AND THE WATER FRAMEWORK DIRECTIVE

The risk assessment approach is similar in REACH and the WFD. Both legislationsfollow the guidelines of the current European risk assessment paradigm, as describedin the technical guidance document (TDG) (ECB 2003). The TGD addresses theestimation of risk from single chemicals, and does not take mixture toxicity intoaccount; with the exception of mixtures of petroleum compounds. The principleof the risk assessment procedure is to distinguish between risk scenarios of concernand those of no concern by the estimation of a PEC/PNEC ratio, that is, the ratioof the Predicted Environmental Concentration (PEC) and the Predicted No-effectEnvironmental Concentration (PNEC). The use of PEC/PNEC ratios in chemicalrisk assessment is a well-established risk assessment parameter, in which severaldifferent endpoints, organisms, and exposure scenarios are weighted together inorder to evaluate risk at a screening level. If the ratio PEC/PNEC < 1, the riskscenario is evaluated as being insignificant. If the ratio PEC/PNEC > 1, the riskmight be significant. In these situations further studies are needed to quantify therisk (ECB 2003). The estimations of the local and regional PEC and the PNEC aretherefore key values for the assessment of risk scenarios of single chemicals. In orderto evaluate how the risk assessment of mixture toxicity can be better implementedin REACH and the WFD by following the existing risk assessment paradigm, it isimportant to address how mixture PECs and PNECs can be estimated. This will beaddressed in the discussion.

REACH and the WFD also differ in several important aspects. While REACH hasthe legal status of a regulation, the WFD is a minimum directive (EC 2003a; EC2000). This is important with regard to the member states’ role in the develop-ment of future legislation, since the member states can implement initiatives thatgo further than those formulated in the WFD, but have to follow the exact wordingof REACH. This means that national initiatives can be implemented less than theWFD, but not under REACH. Such national initiatives can subsequently facilitatechanges in the EU regulatory practice, since legislation is often developed on thebasis of existing practices. The two legislations further address the managementof toxic chemicals in different ways. Risk assessments conducted under REACHcan be characterized as generic risk assessments, whereas the WFD operates withsite-specific risk assessments (EC 2003a; EC 2000). A generic risk assessment, usinggeneral and somewhat arbitrary exposure scenario, can be very complex, especiallyfor chemicals used in great amounts and for multiple purposes. In these situations

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it is very difficult and comprehensive to determine all possible/relevant mixtures.Site specific ecotoxicological risk assessments are based on the evaluation and calcu-lation of exposure scenarios on one specific location, for example, a recipient likea fjord. Since the environmental boundaries are preset, it is somewhat easier, butnot necessarily easy, to determine which chemicals to include in the mixture toxicityassessment. Finally, REACH aims at regulating specific chemicals for specific uses,whereas the WFD focus on protecting/achieving environmental quality.

Regulation of Dangerous Preparations in REACH

One of the directives replaced by REACH is the Council Directive 1999/45/ECthat relates to the classification, packaging, and labeling of dangerous preparations(European Council 1999). Directive 1999/45/EC addresses mixture toxicity, and theassessment procedure was carried over into an initial version of REACH. The initialversion thus addresses mixture toxicity in regard to preparations. The principlesof the risk assessment approach are based on concentration addition by classifyingtoxicity of preparations as the sum of toxic components (European Council 1999):

∑ (PN , R50–53LN , R50–53

)(2)

where PN , R50–53 is the percentage by weight of each hazardous chemical in thepreparation, corresponding to ci of Eq. (1), and LN , R50–53 is the threshold valuefor the environment for each hazardous chemical, corresponding to the ECxi of Eq.(1). Hazardous chemicals are those labeled risk characterizations R50–53.

The equation calculates the ratio between the amounts of the chemicals in thepreparation (equal to PECs) divided by the threshold value for toxic effects in the en-vironment (equal to PNECs). In essence it is similar to a summation of PECs/PNECs,and a sum of these toxic units greater than 1 indicates a potential risk. Eq. (2) isused to estimate risk to the environment (Annex III of Directive 1999/45/EC),but a similar equation can be found in Annex II for toxicants that are classifiedas hazardous to humans. In REACH this procedure was adopted in Annex 1b (EC2003a). It was stated that an additive effect is assumed unless other information isavailable. One important aspect of this implementation that is different from theCA models most often discussed in the scientific literature is that it is based on sum-mations of PECs/PNECs rather than on knowledge of specific modes of actions ofthe given toxicants. In REACH this procedure was adopted in annex 1b at an earlystage of REACH (European Commission 2003a), but annex 1b was removed fromthe final version of REACH (European Commission 2006). Since the equation wasnot implemented in any other part of REACH, this means the equation is no longerpresent in REACH. Mixtures are still addressed for specific scenarios in Annex 1,but to a more limited degree. For preparations, such as alloys, where the individ-ual substances might alter properties, this has to be addressed in the assessment.(EC 2006). Furthermore, it is mentioned that alternative methodologies shall betaken into consideration, when those described in the legislation is insufficient. Inprinciple this allows the use of an addition model for assessing mixture effects.

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The Water Framework Directive and Danish Waste Water Guideline

The WFD aims at protecting ecosystems in inland surface waters, transitional(estuarine) waters, and coastal waters (EC 2000). The main objective is to achieve“a good ecological status” in all waters by 2015 (Borja and Elliott 2007). In orderto determine what a good ecological status is, the water quality is compared to areference water body (Borja and Elliott 2007). This reference water body is a “non-polluted” area in which the ecosystems are relatively unharmed by human activities.The two water bodies must have similar biological and abiotic conditions in orderto be comparable. By focusing on the ecological status of the receiving water bodyinstead of a specific threshold values for the chemicals, this approach introduces afundamental change in European risk assessment.

However, the risk assessment is based on a generic risk assessment similar to thatused in REACH. A list of toxic chemicals is used to prioritize which chemicals themember states should monitor and keep at concentrations below threshold values(EC 2000). The list, which is constructed by using the principles described in theTGD, is a key element in the regulation of chemical pollution of the aquatic envi-ronment. Mixture toxicity is presently not included in the risk assessment procedureof the WFD. The definition of a good ecological quality in the WFD is, in practice,primarily based on biological indicators. However, the WFD explicitly states thatthe impact of chemicals must be sustainable in order to achieve a good ecologicalquality (UK-EPA 2005).

Even though risk assessment of mixture toxicity is not directly implemented inthe WFD, the EU member states share the option of implementing it in the nationallaws, since the directive is a framework directive. The Danish EPA’s guidelines onthe evaluation of waste water toxicity include a risk assessment of mixture effectsof emissions to the aquatic waters (Tørslev et al. 2002). Eq. (3) used is based on asimilar approach to that used for preparations in REACH:

∑ (Ci

PENCi

), (3)

where Ci is the measured concentration of chemical i in the waste water and PNECi

is the Predicted No-effect Concentration for chemical i . This equation is similar tothe one adopted in REACH for assessing mixtures in preparations. This illustratesthat mixtures can be addressed within the boundaries of the WFD.

DISCUSSION

Both REACH and the WFD have some elements of mixture toxicity assessmentintegrated. However, mixtures are only assessed in very limited situations in REACHand the WFD, for example, when in a preparation. It is important to note that useof CA for assessing preparations was removed from the final version of REACH(European Commission 2003a). This strongly indicates that mixture toxicity assess-ment has been considered, but is believed to be too complex to handle even forlimited situations such as preparations, where a former directive ensured that anapproach was already established. The Danish wastewater guideline considers mix-ture effects of chemicals in effluents, but excludes emissions from other sources and

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from chemicals that are already present in the recipient. A more holistic approachis recommendable, where relevance of a mixture is based on an evaluation of allchemicals that contribute and not only those in the effluent. The major question isthen how this can be done, and if it is even possible and feasible. Two fundamentalrequirements seem to be essential. First of all, the scientific foundation has to besolid and well documented and has to come up with solutions that are practicallymanageable. Second, there has to be political will to implement the scientific solu-tion in reality. In regard to this last requirement, it is interesting to note that theintegration of mixture toxicity assessment in regard to preparations indicates thatthere is political will to assess mixtures within REACH.

It is important to note that the approach to assess mixture toxicity in the oldpreparation directive and the WFD, the risk assessment approach is based on theCA model. As described in the first part of this article, the scientific evidence indi-cates that concentration addition is more protective than the independent actionmodel, and at least as precise (Junghans et al. 2006). Even though a full consensusabout whether concentration addition can be used regardless of the mode of actionof the chemicals at hand has not been established, the evidence to support thisconclusion is strong. It thus seems that the scientific evidence for using the concen-tration addition model to predict mixture toxicity is sufficiently well established tobe used as a tool to implement mixture toxicity into the existing European chemicalsregulation.

All mixture toxicity regulation approaches reviewed in this article use the con-centration addition model to estimate mixture effects from single substance data.(REACH only applied the approach in an early version of the legislation and not inthe final version.) Two out of five legislations apply concentration addition regard-less of mode of action, whereas the remaining three distinguish between chemicalswith similar and dissimilar modes of action (see Table 1). The Danish air regulationis based on the assumption that the mixture toxicity of chemicals with dissimilarmodes of action can be determined from the chemical with the highest toxic effect(Danish-EPA 2001). However, the scientific evidence for this approach is ambiguous.Some experiments have demonstrated that the chemicals with the highest toxic con-tribution can be used to predict the toxic effect of mixtures (McCarty and Borgert2006). However, there are other studies that demonstrate the opposite, that is, thatmixtures of dissimilar acting chemicals have toxic effects much higher than the singlehighest contributor (Backhaus et al. 2000a, 2004; Faust et al. 2003; Merino-Garcia etal. 2003; Walter et al. 2002). Which of the two approaches fit better of course dependson the composition of the pollution. However, using the first approach, as does theDanish air regulation, does not take a precautionary approach, and could lead tounderestimation of mixture toxicity from chemicals with dissimilar mode of action.

The ATSDR applies CA for chemicals with similar modes of action and IA forchemicals with dissimilar modes of action. It is, however, important to note thatATSDR addresses toxicological and not ecotoxicological risk. This is illustrated bythe focus of the program, in which chemicals are divided into non-carcinogens andcarcinogens. The first group is addressed as having similar modes of action whereasthe second is viewed as a group of chemicals with dissimilar modes of action. Hence,mode of action in this context is much broader defined than “molecular target site,”which is the original definition of mode of action in regard to mixtures (Bliss 1939).

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1267

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K. Syberg et al.

This illustrates that the definition of mode of action is not clear-cut but depends onthe context in which the toxicity is addressed.

In summary, the U.S. frameworks apply both concentration addition and inde-pendent action. This requires determination of the mode of action of all chemicalsand all endpoints relevant for the mixture assessment. We argue that it is both sci-entifically feasible and necessary from a pragmatic point of view to abandon thestrategy, and apply CA as default model when addressing mixture toxicity in theenvironment.

However, it is most likely practically impossible to evaluate mixture effects ofall chemicals in the environment. It therefore seems important that a procedurethat enables the determination of a limited number of the most toxic chemicalsfor a given mixture is agreed on. This is one of the key challenges that have to beovercome before mixture toxicity can be implemented on a broader scale in bothREACH and the WFD. A part of this challenge might be to define specific areaswhere existing chemicals in the environment is regarded as contributors to a givenpollution mixture. One way to do this could be to determine regional backgroundlevels of existing pollution in air, soil, water, and sediment, and use them in all EUcountries. In Denmark this approach is used to determine background concentra-tions of single substances under the WFD. When both occurrence and toxicity datafor the single compounds exists there is nothing that hinders an assessment of themixture toxicity effect—from a scientific point of view!

In regard to choosing which specific chemicals to include in the mixture toxicityassessment, inspiration could be found in the approach applied by ATSDR for non-carcinogens, where the relevance of mixtures is determined by the occurrence ofchemicals with a PEC/PNEC greater than 0.1. If relevance of the chemicals is evalu-ated based on their PEC/PNEC, it seems reasonable to use these values in the actualmixture prediction as well. The summation of the PEC/PNEC-ratio is a pragmaticand feasible way to implement the concentration addition model. PEC/PNECs arean integrated part of current evaluations of chemical risk, which means that dataproduced following the guidance if the TGD can be directly used in mixture toxicityassessment. However, the concentration addition model is based on the assumptionthat all concentrations contribute to the mixture effect. Hence, using a pre-definedPEC/PNEC threshold value (e .g ., 0.1) to limit the number of chemicals entering amixture toxicity evaluation, could lead to underestimations of mixture toxicity formixture with large numbers of chemicals below the selected threshold. One wayto address this problem could be to take chemicals with PEC/PNEC-ratios lowerthen 0.1 into consideration if they are present in larger numbers (e .g ., +100). Itis important to keep in mind that limiting the number of chemicals in the mix-ture toxicity assessment is mainly recommended for practical and not scientificconsiderations.

Furthermore, the determinations of the PECs are important. At present it is stillnot decided how exposure scenarios should be conducted in practice under REACH.One of the working groups established in order to make REACH manageable inpractice deals with this issue (RIP 3.2) (Atrion 2006). From a scientific point of view,it seems most reasonable to construct background values on the basis of empiricaldata, rather than estimations, even though this might take a great deal of resourcesto do so.

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Mixture Toxicity Assessment in REACH

It is important to develop approaches concerning how to analyze the chemicalsof a mixture in the complex matrices of the different environmental compartments.This seems to be one of the key challenges if mixture toxicity is to be assessed in allstages of the chemical’s life cycle in REACH and the WFD. The work of U.S. ATSDRwith the HazDat database and the scoring of mixtures (Fay and Mumtaz 1996)could serve as inspiration for this task, and the construction of a similar databasefor chemicals in the European environment could help focus monitoring programson toxicologically relevant chemicals.

Considering the WFD’s aim of protecting the ecosystems it would be a majorstep forward to consider mixture toxicity. It is recommendable that a procedure likethe one used for Danish waste water effluents, which account for mixture toxicity,is implemented in the actual directive. This would ensure that mixtures are notonly accounted for if the national member states find it important. The ecosystem-oriented approach facilitates focus on specific geographic areas (like fjords). Thechemicals present within the specific ecosystem could then be considered for themixture toxicity assessment. Since some of the most problematic chemicals tend toaccumulate in sediment and biota (Davis et al. 2007), it seems necessary to includesuch existing pollutions in mixture toxicity assessments in regard to the relevantcompartments and secondary poisoning of humans. In Denmark, knowledge aboutconcentrations of specific prioritized toxicants in water (both fresh and salt), sed-iment, and biota (fish and mussels) from aquatic recipients are collected underthe Danish national monitoring program (NERI 2004). Such information could beused as “background” contamination when toxic effects of new discharges are beingassessed.

Annex X in the WFD holds a list of chemicals that pose the highest risk (EC2000). This list could be used to choose which chemicals should be in focus whendetermining “background concentrations.” In order to choose the exact number ofchemicals to include, the U.S. ATSDR’s approach could be applied in a similar wayas discussed in relation to REACH. However, it seems important to recognize thatlocal production can lead to pollution with chemicals that are not listen in Annex X,but may still yield an important contribution to the mixture effect. Local emissionsshould therefore be included in the background concentrations together with thechemicals listed in Annex X.

CONCLUSION

Mixture toxicity assessments are primarily carried out in accordance with theprinciples of concentration addition and independent action. Since grouping envi-ronmental chemical mixtures on the basis of mode of action is unfeasible, and thescientific findings over the last decades indicate that concentration addition can beapplied regardless of mode of action, it is recommended that this model should beused as a general mixture prediction model.

Even though most chemical regulation is based on a single substance approach,mixture toxicity is implemented in many regulations including national implemen-tations of the WFD and U.S. environmental regulation. This illustrates that mixturetoxicity is acknowledged as an important aspect of chemical regulation, and can

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be addressed within the boundaries of practical chemical/environmental regula-tion. Some issues have to be addressed before a more comprehensive integrationof mixture toxicity in REACH and the WFD can be achieved. Most importantly,an approach that enables the selection of a limited number of chemicals for aspecific mixture toxicity prediction has to be agreed on. Choosing chemicals withindividual PEC/PNEC > 0.1 could be a way to approach this selection. Further-more, it seems important that knowledge about both existing contaminations ofthe environment and future emissions are documented, in order to enable a sci-entifically founded selection of the limited number of relevant chemicals. In thatregard, the USEPA and U.S. ATSDR share some pragmatic and still scientific soundapproaches, which could serve as a foundation for development of a Europeanapproach.

REFERENCES

Altenburger R, Boedeker W, Faust M, et al. 1996. Regulations for combined effects of pol-lutants: Consequences from risk assessment in aquatic toxicology. Food Chem Toxicol34:1155–7

Altenburger R, Backhaus T, Boedeker W, et al. 2000. Predictability of the toxicity of multiplechemical mixtures to Vibrio fischeri: Mixtures composed of similarly acting chemicals.Environ Toxicol Chem 19:2341–47

Andersen ME and Dennison JE. 2004. Mechanistic approaches for mixture risk assessments—Present capabilities with simple mixtures and future directions. Environ Toxicol Pharmacol16:1–11

Arrhenius A, Gronvall F, Scholze M, et al. 2004. Predictability of the mixture toxicity of12 similarly acting congeneric inhibitors of photosystem II in marine periphyton andepipsammon communities. Aquatic Toxicol 68:351–67

Atrion 2006. RIP 3.2: TGD on preparing the Chemical Safety Report. Atrion Interna-tional. Available at http://www.reachlegislation.com/index.php?option=com content&task=view&id=31&Itemid=2

Backhaus T, Altenburger R, Boedeker W, et al. 2000a. Predictability of the toxicity of a multiplemixture of dissimilarly acting chemicals to Vibrio fischeri. Environ Toxicol Chem 19:2348–56

Backhaus T, Scholze M, and Grimme LH. 2000b. The single substance and mixture toxicityof quinolones to the bioluminescent bacterium Vibrio fischeri. Aquatic Toxicol 49:49–61

Backhaus T, Altenburger R, Arrhenius A, et al. 2003. The BEAM-project: Prediction and assess-ment of mixture toxicities in the aquatic environment. Continental Shelf Res 23:1757–69

Backhaus T, Arrhenius A, and Blanck H. 2004. Toxicity of a mixture of dissimilarly actingsubstances to natural algal communities: Predictive power and limitations of independentaction and concentration addition. Environ Sci Technol 38:6363–70

Berenbaum MC. 1989. What is synergy? Pharmacological Reviews 41:93–141Bliss CI. 1939. The toxicity of poisons applied jointly. Ann Appl Biol 26:585–615Borja A and Elliott M. 2007. What does ‘good ecological potential’ mean, within the European

water framework directive? Marine Pollution Bull 54:1559–64Cedergreen N, Kamper A, and Streibig JC. 2006. Is prochloraz a potent synergist across

species? A study on bacteria, daphnia, algae and higher plants. Aquatic Toxicol 78:252Cedergreen N, Kudsk P, Matthiasen S, et al. 2007. Combination effects of herbicides: Do

species and test system matter? Pest Management Sci 63:282–95

1270 Hum. Ecol. Risk Assess. Vol. 15, No. 6, 2009

Downloaded By: [Roskilde Universitetsbibliotek] At: 14:45 19 November 2009

Mixture Toxicity Assessment in REACH

Cedergreen N, Christensen AM, Kamper A, et al. 2008. A review of independent action as areference model for binary mixtures of compounds with different molecular target sites.Environ Toxicol Chem 27:1621–32

Danish-EPA. 2001. Luftvejledningen ifølge lovbekendtgørelse nr. 753 af 25/08/2001. Copen-hagen, Denmark (in Danish)

Davis JA, Hetzel F, Oram JJ, et al. 2007. Polychlorinated biphenyls (PCBs) in San FranciscoBay. Environ Res 105:67–86

Deneer JW. 2000. Toxicity of mixtures of pesticides in aquatic systems. Pest Management Sci56:516–20

ECB (European Chemical Bureau). 2003. Technical Guidance Document (TGD) on RiskAssessment of Chemical Substances following European Regulation and Directives. Ispra,Italy

EC (European Commission). 2000. Directive 2000/60/EC of the European Parliament andof the Council establishing a framework for the Community action in the field of waterpolicy. Bruxelles, Belgium

EC. 2003a. Proposal for a Regulation of the European Parliament and of the Council concern-ing the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH),establishing a European Chemicals Agency and amending Directive 1999/45/EC and Reg-ulation (EC) {on Persistent Organic Pollutants} {SEC(2003 1171}/*COM/2003/0644final – COD 2003/0256*/. European Commission, Bruxelles, Belgium

EC. 2003b. The Environment and Health Strategy—The SCALE Initiative. Technical Work-ing Group Background. Available at http://www.environmentandhealth.org/twgim/background.php (accessed 29 May 2008)

EC. 2006. Regulation (EC) No 1907/2006 of the European Parliament and of the Council of18 December 2006 concerning the Registration, Evaluation, Authorisation and Restrictionof Chemicals (REACH), establishing a European Chemicals Agency, amending Directive1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Reg-ulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Di-rectives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. European Commission,Bruxelles

European Council. 1999. Directive 1999/45/EC of the European Parliament and of theCouncil of 31 May 1999 concerning the approximation of the laws, regulations and ad-ministrative provisions of the Member States relating to the classification, packaging andlabelling of dangerous preparations. Bruxelles, Belgium

Faust M and Scholze M. 2004 Competing concepts for the prediction of mixture toxicity: Dothe difference matter for regulatory purposes? Report, EVKI-CT1999-00012, 2-62

Faust M, Altenburger R, Boedeker W, et al. 1994. Algal toxicity of binary combinations ofpesticides. Bull Environ Contam Toxicol 53:134–41

Faust M, Altenburger R, Backhaus T, et al. 2003. Joint algal toxicity of 16 dissimilarly actingchemicals is predictable by the concept of independent action. Aquatic Toxicol 63:43–63

Fay RM and Mumtaz MM. 1996. Development of a priority list of chemical mixtures occur-ring at 1188 hazardous waste sites, using the HazDat database. Food Chem Toxicol 34:1163–5

Fox MA, Groopman JD, and Burke TA. 2002. Evaluating cumulative risk assessmentfor environmental justice: A community case study. Environ Health Perspect 110:203–9

Greco WR, Bravo G, and Parsons JC. 1995. The search for synergy: A critical review from aresponse surface perspective. Pharmacological Rev 47:332–85

Hertzberg RC and MacDonell MM. 2002. Synergy and other ineffective mixture risk defini-tions. Sci Total Environ 288:31–42

Hum. Ecol. Risk Assess. Vol. 15, No. 6, 2009 1271

Downloaded By: [Roskilde Universitetsbibliotek] At: 14:45 19 November 2009

K. Syberg et al.

Junghans M, Backhaus T, Faust M, et al. 2006. Application and validation of approaches forthe predictive hazard assessment of realistic pesticide mixtures. Aquatic Toxicol 76:93–110

McCarty LS and Borgert CJ. 2006. Review of the toxicity of chemical mixtures: Theory, policy,and regulatory practice. Regulatory Toxicol Pharmacol 45:119–43

Merino-Garcia D, Kusk KO, and Christensen ER. 2003. Joint toxicity of similarly and dissimi-larly acting chemicals to Daphnia magna at different response levels. Archives of EnvironContam and Toxicol 45:289–96

NERI (National Environmental Research Institute). 2004. Det national program forovervagning af vandmiljøet og naturen. Programbeskrivelse – del 1. pp 48. – Faglig rapprotfra DMU nr. 495 (in Danish)

Syberg K, Engell-Kofoed AE, Pedersen H, et al. 2007. Mixture toxicity of three toxicantswith similar and dissimilar mode of action to Daphnia magna. Ecotoxicol Environ Safety69:428–36

Teuschler LK. 2007. Deciding which chemical mixtures risk assessment methods work bestfor what mixtures. Toxicol Applied Pharmacol 223(2):139–47

Tørslev J, Winther-Nielsen M, Perdersen F, et al. 2002 Udledning af miljøfarlige stoffer medspildevand (in Danish). Miljøprojekt nr. 690. Copenhagen, Denmark

UK-EPA (UK Environment Agency). 2005. Ecology and the Water Framework Direc-tive. 2005. Available at http://www.environment-agency.gov.uk/commondata/acrobat/bn ecology v02 1192722.pdf

USEPA (US Environmental Protection Agency). 2003. Framework for Cumulative Risk As-sessment. EPA/600/P-02/001F, 2003. Office of Research and Development, NationalCenter for Environmental Assessment, Washington Office, Washington, DC. Availableat http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=54944

Walter H, Consolaro F, Gramatica P, et al. 2002. Mixture toxicity of priority pollutants at noobserved effect concentrations (NOECs). Ecotoxicol 11:299–310

Warne MSJ and Hawker DW. 1995. The number of components in a mixture determineswhether synergistic and antagonistic or additive toxicity predominate—The funnel hy-pothesis. Ecotoxicol Environ Safety 31:23–8

Wilbur SB, Hansen H, Pohl H, et al. 2004. Using the ATSDR Guidance Manual for theassessment of joint toxic action of chemical mixtures. Environ Toxicol Pharmacol 18:223–30

1272 Hum. Ecol. Risk Assess. Vol. 15, No. 6, 2009

Downloaded By: [Roskilde Universitetsbibliotek] At: 14:45 19 November 2009