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Chemicals in the environment occur in mixtures rather than as individual entities. Environmental quality monitoring thus faces the challenge to comprehensively assess a multitude of contaminants and potential adverse effects. Effect-based methods have been suggested as complements to chemical analytical characterisation of complex pollution patterns. The regularly observed discrepancy between chemical and biological assessments of adverse effects due to contaminants in the field may be either due to unidentified contaminants or result from interactions of compounds in mixtures. Here, we present an interlaboratory study where individual compounds and their mixtures were investigated by extensive concentration-effect analysis using 19 different bioassays. The assay panel consisted of 5 whole organism assays measuring apical effects and 14 cell- and organism-based bioassays with more specific effect observations. Twelve organic water pollutants of diverse structure and unique known modes of action were studied individually and as mixtures mirroring exposure scenarios in freshwaters. We compared the observed mixture effects against component-based mixture effect predictions derived from additivity expectations (assumption of non-interaction). Most of the assays detected the mixture response of the active components as predicted even against a background of other inactive contaminants. When none of the mixture components showed any activity by themselves then the mixture also was without effects. The mixture effects observed using apical endpoints fell in the middle of a prediction window defined by the additivity predictions for concentration addition and independent action, reflecting well the diversity of the anticipated modes of action. In one case, an unexpectedly reduced solubility of one of the mixture components led to mixture responses that fell short of the predictions of both additivity mixture models. The majority of the specific cell- and organism-based endpoints produced mixture responses in agreement with the additivity expectation of concentration addition. Exceptionally, expected (additive) mixture response did not occur due to masking effects such as general toxicity from other compounds. Generally, deviations from an additivity expectation could be explained due to experimental factors, specific limitations of the effect endpoint or masking side effects such as cytotoxicity in in vitro assays. The majority of bioassays were able to quantitatively detect the predicted non-interactive, additive combined effect of the specifically bioactive compounds against a background of complex mixture of other chemicals in the sample. This supports the use of a combination of chemical and bioanalytical monitoring tools for the identification of chemicals that drive a specific mixture effect. Furthermore, we demonstrated that a panel of bioassays can provide a diverse profile of effect responses to a complex contaminated sample. This could be extended towards representing mixture adverse outcome pathways. Our findings support the ongoing development of bioanalytical tools for (i) compiling comprehensive effect-based batteries for water quality assessment, (ii) designing tailored surveillance methods to safeguard specific water uses, and (iii) devising strategies for effect-based diagnosis of complex contamination., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ill-defined, multi-component mixtures of steroidal pharmaceuticals are present in the aquatic environment. Fish are extremely sensitive to some of these steroids. It is important to know how fish respond to these mixtures, and from that knowledge develop methodology that enables accurate prediction of those responses. To provide some of the data required to reach this objective, pairs of fish were first exposed to five different synthetic steroidal pharmaceuticals (one estrogen, EE2; one androgen, trenbolone; one glucocorticoid, beclomethasone dipropionate; and two progestogens, desogestrel and levonorgestrel) and concentration-response data on egg production obtained. Based on those concentration-response relationships, a five component mixture was designed and tested twice. Very similar effects were observed in the two experiments. The mixture inhibited egg production in an additive manner predicted better by the model of Independent Action than that of Concentration Addition. Our data provide a reference case for independent action in an in vivo model. A significant combined effect was observed when each steroidal pharmaceutical in the mixture was present at a concentration which on its own would produce no statistically significant effect (something from 'nothing'). Further, when each component was present in the mixture at a concentration expected to inhibit egg production by between 18% (Beclomethasone diproprionate) and 40% (trenbolone), this mixture almost completely inhibited egg production: a phenomenon we term 'a lot from a little'. The results from this proof-of-principle study suggest that multiple steroids present in the aquatic environment can be analysed for their potential combined environmental risk., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Psychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggests that they may elicit mode of action-mediated effects in fish as they do in humans, and the key open question is at what exposure concentrations these effects might occur. In the present study, the authors investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 d of waterborne exposure to 0.8 μg L -1 , 4.7 μg L -1 , and 30.6 μg L -1 . Successively, they explored the relationship between the internal concentrations of oxazepam and the effects on fish exploratory behavior quantified by performing 2 types of behavioral tests, the novel tank diving test and the shelter-seeking test. The highest internal concentrations of oxazepam were found in brain, followed by plasma and liver, whereas muscle presented the lowest values. Average concentrations measured in the plasma of fish from the 3 exposure groups were, respectively, 8.7 ± 5.7 μg L -1 , 30.3 ± 16.1 μg L -1 , and 98.8 ± 72.9 μg L -1 . Significant correlations between plasma and tissue concentrations of oxazepam were found in all 3 groups. Exposure of fish to 30.6 µg L -1 in water produced plasma concentrations within or just below the human therapeutic plasma concentration (H T PC) range in many individuals. Statistically significant behavioral effects in the novel tank diving test were observed in fish exposed to 4.7 μg L -1 . In this group, plasma concentrations of oxazepam were approximately one-third of the lowest H T PC value. No significant effects were observed in fish exposed to the lowest and highest concentrations. The significance of these results is discussed in the context of the species-specific behavior of fathead minnow and existing knowledge of oxazepam pharmacology. Environ Toxicol Chem 2016;35:2782-2790. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC., (© 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.)

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic environmental pollutants that pose a potential risk to marine biota. Although they occur as mixtures in the marine environment, only little information exists about their joint action on fish behavior. In 4-day tests with juvenile gilthead seabream (Sparus aurata) concentration-response analyses were performed for three PAH compounds--fluorene (FE), phenanthrene (PHE), and pyrene (PY). Responses of fish to these compounds were assessed by recording visually the changes in their locomotory activities and social behaviors. Based on these concentration-response data, mixture effects were predicted by applying the model of concentration addition. The mixture was tested using a fixed-ratio design, and the resulting effects were compared to the predictions. The single compounds and the mixture were accumulated in fish muscle and produced a clear change in the overall behavioral performance of fish, as all individual parameters were affected in a dose-response way. For lethargy and swimming, we determined regression fits for all single compounds, with PY the most potent (EC(10)=0.031 microM for swimming and 0.039 microM for lethargy) and FE the least (EC(10)=0.29 microM for swimming and 0.26 microM for lethargy). Also, changes in the number of lethargic fish were always the most sensitive parameter and social interaction the least. The mixture study revealed for lethargy and swimming a good agreement between the predicted and observed effect changes, and statistically significant deviations could not be identified.

Background: The potential impact of natural and synthetic estrogens on aquatic ecosystems has attracted considerable attention because it is currently accepted that their joint effects are more severe when they are present in mixtures. Although it is well-known that they occur as mixtures in the marine environment, there is little information about the combined effects of estrogenic chemicals on marine biota., Objective: In 14-day tests with juvenile sea bass, we analyzed singly and in combination the estrogenic activity of estradiol (E(2)), ethynylestradiol (EE(2)), and bisphenol A (BPA) using vitellogenin induction as an end point., Methods: Fish were exposed to each compound, and on the basis of these concentration-response data, we predicted mixture effects by applying the model of concentration addition. The mixtures were tested using a fixed-ratio design, and the resulting mixture effects were compared to the predictions., Results: EE(2) was the most potent steroid, with an EC(50) (median effective concentration) of 0.029 microg/L, 3.6 times more potent than E(2) (EC(50) = 0.104 microg/L); BPA was the least potent chemical, with an EC(50) of 77.94 microg/L. The comparative assessment yielded a good agreement between observed and predicted mixture effects., Conclusions: This study demonstrates the potential hazard of these compounds to seawater life by their ability to act together in an additive manner. It provides evidence that concentration addition can be used as a predictive tool for assessing the combined effects of estrogenic chemicals in marine ecosystems.

For endocrine disrupting chemicals in the environment, concerns arise primarily from the effects that may be induced in wildlife. A well studied example is estrogenic chemicals in the aquatic environment and their effects on fish. Directly measuring effects, in fieldwork studies, is an expensive and time-consuming approach that is fraught with many difficulties, ranging from study design right through to data analysis and interpretation. An alternative approach would be to predict the scale of effect(s) using suitable modeling techniques. We have attempted to do this using estrogenic chemicals as an example. We chose this group of aquatic pollutants because of the current considerable interest in them and the wealth of biological data available on them. Using the established GREAT-ER hydrological model,we have first predicted the concentrations and then the estrogenic effects on fish, of estrone, estradiol, ethinyl estradiol, and nonylphenol individually throughout an entire river catchment. We then show that knowledge of the biological responses of fish to mixtures of these chemicals can be used to predict the effect of environmentally realistic mixtures of them. To determine the degree of risk posed by this group of chemicals, it was necessary to take into account mixture effects: assessment on a chemical by chemical basis led to underestimations of the risk. Finally, we show that the approach can be used to predict how the risk will be affected by changes in the concentration of one chemical in the mixture. Although we have used only one endpoint (vitellogenin induction as an estrogenic response) and one group of similarly acting chemicals, we suggest that this general approach could prove extremely useful to regulatory authorities and other parties charged with protecting aquatic wildlife from adverse effects caused by chemicals in their environment.

The toxicity of three antifoulants (Sea-Nine, Irgarol, and TBT) was determined individually and in mixtures in two tests with microalgae. Effects on periphyton community photosynthesis and reproduction of the unicellular green algae Scenedesmus vacuolatus were investigated. The tested antifoulants were highly toxic in both tests. Observed mixture toxicities were compared with predictions derived from two concepts: Independent Action (IA), assumed to be more relevant for the tested mixtures that were composed of dissimilarly acting substances, and Concentration Addition (CA), regarded as a reasonable worst-case approach in predictive mixture hazard assessment. Despite the corresponding mechanistic basis, IA failed to provide accurate predictions of the observed mixture toxicities. Results show the same pattern in both assays. Mixture effects at high concentrations were slightly overestimated and effects at low concentrations were slightly underestimated. Maximum observed deviations between observed and IA-predicted concentrations amount to a factor of 4. The suggested worst-case approach using CA was protective only in effect regions above 20%. Nevertheless, the application of any concept that accounts for possible mixture effects is more realistic than the present chemical-by-chemical assessment.

Photosynthesis-inhibiting phenylurea derivatives, such as diuron, are widely used as herbicides. Diuron concentrations clearly exceeding the predicted-no-effect concentration have been regularly measured in European freshwater systems. The frequently observed exposure to mixtures of phenylureas additionally increases the hazard to aquatic primary producers. Fluctuating numbers and concentrations of individual toxicants make experimental testing of every potential mixture unfeasible. Thus, predictive approaches to the mixture hazard assessment are needed. For this purpose, two concepts are at hand, both of which make use of known toxicities of the individual components but are based on opposite mechanistic suppositions: Concentration addition is based on the idea of similar mechanisms of action, whereas independent action assumes dissimilarly acting mixture components. On the basis of pharmacological reasoning, it was therefore anticipated that the joint algal toxicity of phenylurea mixtures would be predictable by concentration addition. Indeed, we could demonstrate a high predictive power of concentration addition for these combinations. Surprisingly, however, the opposite concept of independent action proved to be equally valid, because both concepts predicted virtually identical mixture toxicities. This exceptional case has previously been derived from theoretical considerations. Now, the tested phenylurea mixtures serve as an example for the practical relevance of this situation for multicomponent mixtures.

For a predictive assessment of the aquatic toxicity of chemical mixtures, two competing concepts are available: concentration addition and independent action. Concentration addition is generally regarded as a reasonable expectation for the joint toxicity of similarly acting substances. In the opposite case of dissimilarly acting toxicants the choice of the most appropriate concept is a controversial issue. In tests with freshwater algae we therefore studied the extreme situation of multiple exposure to chemicals with strictly different specific mechanisms of action. Concentration response analyses were performed for 16 different biocides, and for mixtures containing all 16 substances in two different concentration ratios. Observed mixture toxicity was compared with predictions, calculated from the concentration response functions of individual toxicants by alternatively applying both concepts. The assumption of independent action yielded accurate predictions, irrespective of the mixture ratio or the effect level under consideration. Moreover, results even demonstrate that dissimilarly acting chemicals can show significant joint effects, predictable by independent action, when combined in concentrations below individual NOEC values, statistically estimated to elicit insignificant individual effects of only 1%. The alternative hypothesis of concentration addition resulted in overestimation of mixture toxicity, but differences between observed and predicted effect concentrations did not exceed a factor of 3.2. This finding complies with previous studies, which indicated near concentration-additive action of mixtures of dissimilarly acting substances. Nevertheless, with the scientific objective to predict multi-component mixture toxicity with the highest possible accuracy, concentration addition obviously is no universal solution. Independent action proves to be superior where components are well known to interact specifically with different molecular target sites, and provided that reliable statistical estimates of low toxic effects of individual mixture constituents can be given. With a regulatory perspective, however, fulfilment of both conditions may be regarded as an extraordinary situation, and hence concentration addition may be defendable as a pragmatic and precautionary default assumption.

The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.

Environmental exposure situations are often characterised by a multitude of heterogeneous chemicals with ambiguous or unknown modes of action present at low concentrations. While multiple exposure is widely acknowledged, arguments are raised that adverse combined effects might not be evoked by mixtures of substances with dissimilar modes of action and being present at only low concentrations. In this study the combined effect of a multiple mixture composed of structurally dissimilar priority pollutants with mostly unknown modes of action has been investigated using an algal biotest. The concentrations of the components in the mixture equalled statistically estimated, individual no observed effect concentrations (NOECs). The observed mixture toxicity was not only clearly higher than expected for any single substance alone, but also well predictable using the concept of independent action.

Herbicidal s-triazines are widespread contaminants of surface waters. They are highly toxic to algae and other primary producers in aquatic systems. This results from their specific interference with photosynthetic electron transport. Risk assessment for aquatic biota has to consider situations of simultaneous exposure to various of these toxicants. In tests with freshwater algae we predicted and determined the toxicity of multiple mixtures of 18 different s-triazines. The toxicity parameter was the inhibition of reproduction of Scenedesmus vacuolatus. Concentration-response analyses were performed for single toxicants and for mixtures containing all 18 s-triazines in two different concentration ratios. Experiments were designed to allow a valid statistical description of the entire concentration-response relationships, including the low concentration range down to EC1. Observed effects and effect concentrations of mixtures were compared to predictions of mixture toxicity. Predictions were calculated from the concentration-response functions of individual s-triazines by applying the concepts of concentration addition and independent action (response addition) alternatively. Predictions based on independent action tend to underestimate the overall toxicity of s-triazine mixtures. In contrast, the concept of concentration addition provides highly accurate predictions of s-triazine mixture toxicity, irrespective of the effect level under consideration and the concentration ratio of the mixture components. This also holds true when the mixture components are present in concentrations below their individual NOEC values. Concentrations statistically estimated to elicit non-significant effects of only 1% still contribute to the overall toxicity. When present in a multi-component mixture they can co-operate to give a severe joint effect. Applicability of the findings obtained with s-triazines to mixtures of other contaminants in aquatic systems and consequences for risk assessment procedures are discussed.