The prediction of combined effects based on the effects of the individual components of mixtures by using the pharmacological concepts of concentration addition and independent action might be a ...promising tool for the risk assessment of pollutant mixtures. To analyze and compare the predictive capabilities of the reference concepts for similarly acting chemicals, the overall toxicity of a multiple mixture was determined in a bioluminescence inhibition assay with Vibrio fischeri. The mixture was composed of 16 similarly and specifically acting chemicals, anticipated to have a common mode of action via weak acid respiratory uncoupling of oxidative phosphorylation. Results show that the observed mixture toxicity is rather well predicted by both concepts. Concentration addition shows an excellent predictive power; the median effective concentration (EC50) of the mixture is predicted with an error of about 10%. Independent action, in contrast, underestimates the EC50 of the mixture by a factor of a little more than three. With respect to risk assessment procedures, it may be concluded that concentration addition gives a valid estimation of the overall toxicity for multiple mixtures with similar and specific mechanisms of action of the mixture components in this type of biotest.
A promising tool for the risk assessment of chemical mixtures is the prediction of their toxicities from the effects of the individual components. For that purpose, concentration addition is ...uniformly regarded as valid for mixtures of similarly acting chemicals. Whether this concept or the competing notion of independent action is more appropriate for mixtures of dissimilarly acting chemicals is still in dispute. Therefore, the presented study analyzed and compared the predictive capabilities of both concepts for a multiple mixture designed of strictly dissimilarly acting compounds. Experimental investigations were conducted using a long‐term bioluminescence inhibition assay with Vibrio fischeri. Results show an excellent predictive power of independent action, while concentration addition overestimates the mixture toxicity. Thus, the precise prediction of mixture toxicities depends on a valid assessment of the similarity/dissimilarity of the mixture components. However, concentration addition underestimates the EC50 of the mixture only by a factor of less than three. As the similarity of components is often unknown for mixtures found in the environment, it is concluded that concentration addition may give a realistic worst case estimation of mixture toxicities for risk assessment procedures.
We investigated the ability of a mixture of three androgen receptor antagonists to induce disruption of male sexual differentiation after perinatal exposure. The aim was to assess whether the joint ...effects of vinclozolin, flutamide, and procymidone can be predicted based on dose-response data of the individual chemicals. Chemicals were administered orally to pregnant Wistar rats from gestational day 7 to postnatal day 16. Changes in reproductive organ weights and of androgen-regulated gene expression in prostates from male rat pups were chosen as end points for extensive dose-response studies. With all end points, the joint effects of the three antiandrogens were dose additive. Histological evaluations showed that dysgenesis and hypoplasia of prostates, seminal vesicles, and epididymis were seen with the highest mixture doses. No changes were observed in any single-compound low-dose group for these lesions, nor were there histopathological changes in the testes. Pronounced dysgenesis of external genitals was observed with all doses of the mixture, and severe dysgenesis was seen with a mixture for which the individual compounds caused no effects. A combination of doses of each chemical that on its own did not produce significant reductions in the weights of seminal vesicles and PBP C3 expression induced a marked mixture effect. Thus, antiandrogens cause additive effects on end points of various molecular complexities such as alterations at the morphological and the molecular level. Exposure to antiandrogens, which appears to exert only small effects when judged on a chemical-by-chemical basis, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.
Risk assessments of toxic chemicals currently rely heavily on the use of no‐observed‐effect concentrations (NOECs). Due to several crucial flaws in this concept, however, discussion of replacing ...NOECs with statistically estimated low‐effect concentrations continues. This paper describes a general best‐fit method for the estimation of effects and effect concentrations by the use of a pool of 10 different sigmoidal regression functions for continuous toxicity data. Due to heterogeneous variabilities in replicated data (i.e., heteroscedasticity), the concept of generalized least squares is used for the estimation of the model parameters, whereas a nonparametric variance model based on smoothing spline functions is used to describe the heteroscedasticity. To protect the estimates against outliers, the generalized least‐squares method is improved by winsorization. On the basis of statistical selection criteria, the best‐fit model is chosen individually for each set of data. Furthermore, the bootstrap methodology is applied for constructing confidence intervals for the estimated effect concentrations. The best‐fit method for the estimation of low‐effect concentrations is validated by a simulation study, and its applicability is demonstrated with toxicity data for 64 chemicals tested in an algal and a bacterial bioassay. In comparison with common methods of concentration‐response analysis, a clear improvement is achieved.
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
μM for swimming and 0.039
μM for lethargy) and FE the least (EC
10=0.29
μM for swimming and 0.26
μM 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.
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.
Testing of single chemicals with single species is common ecotoxicological practice in contrast to contaminated environments where highly diverse biological communities are exposed to highly diverse ...mixtures of chemical compounds. We, therefore, investigated whether mixture toxicity approaches that have been used successfully for single species, might also be applied on a community level of biological complexity. Twelve inhibitors of photosystem II, selected by QSAR and chemometrical approaches as the structurally most similar from a congeneric group of phenylurea herbicides, were tested singly and as mixtures on two types of marine microalgal communities, periphyton and epipsammon. Inhibition of photosynthesis was measured in short-term tests using incorporation of radiolabelled carbon (
14
C
) to estimate photosynthetic rates. Two basic concepts, concentration addition (CA) and independent action (IA), were used to predict the toxicities of the mixtures. Congeneric and similar-acting substances such as the phenylureas are expected to comply with CA rather than IA. The aim of the present study was to evaluate whether these concepts can be used to predict mixture toxicity also to periphyton and epipsammon photosynthesis, i.e. at the level of natural communities. We found that deviations between observed and predicted mixture toxicity were relatively small but that CA predictions were the more accurate ones. The predictions proved to be robust, when based on single substance information even from different seasons, years, and sites. We conclude that the concept of CA for predicting mixture toxicity applies also at the community level of algal testing; at least when a physiological short-term effect indicator is used that matches the mechanism of action of the substances.
Amphibians are declining and fertility/fecundity are major drivers of population stability. The development of non-destructive methods to assess reproductive health are needed as destructive measures ...are fundamentally at odds with conservation goals for declining species. We investigated the utility of body size, nuptial pad size and forelimb width as non-destructive biomarkers of internal reproductive physiology, by analysing correlations with commonly used destructive methods in adult male toads (Bufo bufo) from a low human impact and a high human impact site. Principal component analyses revealed that size was the most important variable for explaining inter-individual differences in other measured endpoints, both non-destructive and destructive, except for hormone levels and nuptial pad, which were independent of size. Toads from the LI and the HI site differed in almost all of the measured endpoints; this was largely driven by the significantly smaller size of toads from the HI site. Correlational analyses within sites revealed that size was correlated with several reproductive endpoints in toads from the HI site but not the LI site, indicating a possible limiting effect of size on reproductive physiology. Intersex was observed in 33 % of toads from the HI site and incidence was not related to any other measured endpoint. In conclusion, we provide evidence that size is associated with reproductive physiology and that nuptial pad/hormone levels have potential as additional markers due to their independence from size. We also show that human activities can have a negative effect on reproductive physiology of the common toad.
Most toxicological studies with PAHs investigate their impact on aquatic organisms only at very specific levels of organization, either at molecular and cellular levels via biomarkers, or at higher ...integral endpoints such as reproduction and behavior. The link between both has received less attention in science. The aim of this multi-response study was to investigate the relationship between specific molecular processes (induction of biotransformation enzymes and oxidative stress) and the behavioral performance of fish. We performed two concentration-effect studies with juvenile gilthead seabream (
Sparus aurata), at which fish were exposed for 4 days to phenanthrene (PHE) (0.11 to 0.56 μM). Groups of five fish per aquarium were recorded for changes in the patterns of their movement and social interactions. Biomarkers analyzed were ethoxyresorufin-
O-deethlylase (EROD), total glutathione-
S-transferase (GST), phenanthrene-type metabolites in bile, lipid peroxidation (LP), superoxide dismutase (SOD) and catalase (CAT). The physiological status of the fish was determined by the liver somatic index. In general, PHE changed the overall behavioral performance of fish, all behavior activities were affected in a dose-response way. The incidence of lethargic fish was strongly increased (up to 39%), as the fish activities were reduced. The changes in the individual swimming activity had influenced negatively the social behavior of fish groups, i.e. the more fish in the group were lethargic, the less the social interactions were marked. The biomarkers responded to PHE differently, with an increase of EROD activity at low exposures (72.25 pmol min
−
1
prot
−
1
), but an inhibition at high concentrations (42.60 pmol min
−
1
prot
−
1
). For GST, we observed the reverse pattern. Together with the strong increase of PHE-type metabolites in bile, we conclude that both biotransformation enzymes are involved in the metabolism of PHE in liver. We found indications for oxidative stress already at low PHE concentrations, as LP levels were increased in the liver. However, higher exposures provoked less pronounced levels, but elevated activities of the antioxidants CAT and SOD (up to 37% and 17%, respectively). We conclude that especially the enzymatic activations at high-PHE exposures might have required additional energetic costs for the chemical detoxication that lead to the marked changes in the fish behaviors, i.e. demonstrating a “trade-off” between detoxication processes via the biliary–hepatic system and the fish activity. Thus, the strong increases in lethargy might be the consequence of higher energetic demands for the PHE detoxication. This illustrates how an integrated use of biomarkers can contribute to our understanding of the impact of PAHs at increasing levels of biological complexity.
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