Prediction and assessment of mixture toxicity of compounds in antifouling paints using the sea-urchin embryo-larval bioassay

The ecotoxicological assessment of alternative "booster" biocides is urgently needed in order to develop environmentally acceptable antifouling paints. However, research has focused mainly on single compounds, and there is still a lack of data on their mixture toxicity. The present study investigated the single and mixture toxicity of three of the most widely used antifouling biocides: zinc pyrithione, chlorothalonil and Sea-Nine, using the sea-urchin (Paracentrotus lividus) embryo-larval bioassay. Also, the predictive ability of the concentration addition (CA) and independent action (IA) concepts for antifouling mixtures was evaluated. Both concepts failed to accurately predict the toxicity of the antifouling mixtures, with the exception of the zinc pyrithione and Sea-Nine mixture, which was accurately predicted by the IA concept, suggesting a dissimilar mode of action of those substances. In general, CA predicted consistently higher toxicity than IA; however, CA overestimated the toxicity of the studied mixtures by a factor of only 1.6, representing a reasonable worst-case approach to be used in the predictive hazard assessment of antifouling mixtures. Finally, the present study demonstrates that the risk of antifouling mixtures for the early developmental stages of sea urchin is higher than the risk of each single substance, and therefore, the inclusion of mixture considerations in the development of water quality criteria for antifouling compounds is strongly recommended.     

Application and validation of approaches for the predictive hazard assessment of realistic pesticide mixtures

In freshwater systems located in agricultural areas, organisms are exposed to a multitude of toxicologically and structurally different pesticides. For regulatory purposes it is of major importance whether the combined hazard of these substances can be predictively assessed from the single substance toxicity. For artificially designed multi-component mixtures, it has been shown that the mixture toxicity can be predicted by concentration addition (CA) in case of similarly acting substances and by independent action (IA), if mixtures are composed of dissimilarly acting substances. This study aimed to analyse whether these concepts may also be used to predictively assess the toxicity of environmentally realistic mixtures. For this purpose a mixture of 25 pesticides, which reflects a realistic exposure scenario in field run-off water, was studied for its effects on the reproduction of the freshwater alga Scenedesmus vacuolatus. The toxicity of the tested mixtures showed a good predictability by CA. This is consistent with the finding that the toxicity was dominated by a group of similarly acting photosystem II inhibitors, although the mixture included substances with diverse and partly unknown mechanisms of action. IA slightly underestimated the actual mixture toxicity. However, the EC(50) values that can be derived from each prediction, according to CA respectively IA, only differed by a factor of 1.3. The finding of such a small difference is partly explainable by the fact that only few components dominate the mixture scenario in terms of so-called toxic units (TUs). This connection is established by developing an equation that allows to calculate the maximum possible ratio between corresponding predictions of effect concentrations by IA and CA for any given ratio of the TUs of mixture components, irrespective of their individual concentration-response functions and independent from their mechanisms of action. To evaluate whether small quantitative differences between EC(50) values predicted by CA and IA are an exception or rather the rule for agricultural exposure scenarios, this calculation was applied on an additional set of 18 pesticide exposure scenarios that were taken from the literature. For these scenarios, EC(50) values predicted by IA can never exceed those predicted by CA by more than a factor of 2.5. The findings of this study support the view that CA provides a precautious but not overprotective approach to the predictive hazard assessment of pesticide mixtures under realistic exposure scenarios, irrespective of the similarity or dissimilarity of their mechanisms of action.     

The photosynthetic responses of marine phytoplankton, periphyton and epipsammon to the herbicides paraquat and simazine

Short-term toxicity tests using photosynthesis (incorporation of 14C) as a test parameter were performed in order to compare the sensitivities of three marine microalgal communities (phytoplankton, periphyton and epipsammon) to two herbicides, paraquat and simazine. Thirty minutes of pre-exposure to simazine were sufficient to obtain the full effect in all communities, while for paraquat 4 h was required. The bioavailability of paraquat and simazine was not limited by adsorption to sediment in the epipsammon samples. Simazine was more toxic than paraquat for the three communities at similar concentrations. Phytoplankton was slightly more sensitive for both herbicides (EC50 ranges of 9--23 mu m for paraquat and 0.37--0.99 mu m for simazine) than periphyton and epipsammon. These attached communities exhibited different results for each toxicant, periphyton being more sensitive to paraquat (EC50 range 9--21 mu m) and epipsammon to simazine (EC50 range 0.44--1.17 mu m). The three communities presented EC ranges comparable to those found in single species tests, suggesting that different levels of biological organization can exhibit a similar sensibility to toxicants, thus indicating that natural communities are suitable for use in these kinds of toxicity tests     

Toxicity of binary mixtures of metals and pyrethroid insecticides to Daphnia magna Straus. Implications for multi-substance risks assessment

Two different concepts, termed concentration addition (CA) and independent action (IA), describe general relationships between the effects of single substances and their corresponding mixtures allowing calculation of an expected mixture toxicity on the basis of known toxicities of the mixture components. Both concepts are limited to cases in which all substances in a mixture influence the same experimental endpoint, and are usually tested against a "fixed ratio design" where the mixture ratio is kept constant throughout the studies and the overall concentration of the mixture is systematically varied. With this design, interaction among toxic components across different mixture ratios and endpoints (i.e. lethal versus sublethal) is not assessed. In this study lethal and sublethal (feeding) responses of Daphnia magna individuals to single and binary combinations of similarly and dissimilarly acting chemicals including the metals (cadmium, copper) and the pyrethroid insecticides (lambda-cyhalothrin and deltamethrin) were assayed using a composite experimental design to test for interactions among toxic components across mixture effect levels, mixture ratios, lethal and sublethal toxic effects. To account for inter-experiment response variability, in each binary mixture toxicity assay the toxicity of the individual mixture constituents was also assessed. Model adequacy was then evaluated comparing the slopes and elevations of predicted versus observed mixture toxicity curves with those estimated for the individual components. Model predictive abilities changed across endpoints. The IA concept was able to predict accurately mixture toxicities of dissimilarly acting chemicals for lethal responses, whereas the CA concept did so in three out of four pairings for feeding response, irrespective of the chemical mode of action. Interaction effects across mixture effect levels, evidenced by crossing slopes, were only observed for the binary mixture Cd and Cu for lethal effects. The analysis of regression residuals showed that interaction effects across mixture ratios were restricted to feeding responses in binary mixtures that included Cu. These results indicate that the ability of the CA and IA concept to predict mixture toxicity effects varies from lethal to sublethal endpoints irrespective of their primary mode of action. This suggests that when considering complex responses, the pharmacological notion of mode of action should be extended to encompass an ecotoxicological mode of action, based on the concentration at which various toxicological effects become operative in the biological system under consideration.     

Contrasting effects of organic and inorganic toxicants on freshwater periphyton

This study analyses the factors influencing the response of periphyton communities to toxicants in order to test the validity of the short-term physiological method. The effects of two model compounds, atrazine and zinc, on the photosynthesis of periphyton from different lotic systems were analyzed using short-term toxicity tests. Periphyton communities from seven sites belonging to a relatively small area were used to explore the ecological parameters affecting toxicity in flowing waters. Furthermore, time course studies of zinc toxicity on intact and suspended periphyton were carried out in order to evaluate the significance of periphyton structure (thickness) on toxicity. Atrazine toxicity varied slightly (0.42-2.42 microM) in tests with photosynthesis as an endpoint. Only environmental light was detected to lead to systematic changes in sensitivity of field collected communities. In contrast, the range of EC(10) values for zinc was very broad (7->1000 microM), and mainly related to algal biomass, while also water chemistry (alkalinity) was important. The results of time-course experiments indicated that the penetration of zinc into thick biofilms was limited. The model compounds tested in the present study may exemplify two different situations. In the case of atrazine, the effect on periphyton is easy to predict from the physiological tests used here. On the other hand, zinc toxicity assessed using photosynthesis of periphyton is much affected by many biological and chemical variables. It is concluded that toxicity tests of metals and other organic and inorganic compounds should be based on longer times of exposure.     

A novel model integrated concentration addition with independent action for the prediction of toxicity of multi-component mixture

Concentration addition (CA) and independent action (IA) have been used to describe the mixture of components having similar and dissimilar mode of action (MOA), respectively. Environmentally relevant mixture does, however, not follow the strictly similar or dissimilar MOA. A novel model, which integrated CA with IA based on the multiple linear regression (ICIM), was proposed for predicting the toxicity of noninteractive mixture. The predictive power of the ICIM model was validated by data set 1 including 13 mixtures of nine components and data set 2 including six mixtures of six components. For data set 1, ten uniform design with fixed concentration ratio ray (UDCR) mixtures was used as a training set to build an ICIM model, and the model was used to predict the toxicity of the test set consisting of three equivalent-effect concentration ratio (EECR) mixtures. For data set 2, the ICIM model based on four UDCR mixtures was used to predict the remaining two EECR mixtures. It is concluded that the ICIM model shows a strong predictive power for the mixture toxicities in the two data sets, and its prediction is better than CA and IA where the two models deviate from the concentration-response data of the mixtures. Thus, ICIM model is a powerful tool to evaluate and predict mixture toxicity, and maybe offer an important approach in risk assessment of mixture toxicity.     

Cadmium,lead and their mixtures with copper: <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> embryotoxicity assessment,prediction, and offspring quality evaluation

The aim of this research was to assess the combined effects of three heavy metals (copper, lead, cadmium) on the fertilization and offspring quality of the sea urchin Paracentrotus lividus at EC50, NOEL, and EC1 concentrations. The observed data were compared with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA) in order to evaluate the proper prediction of the observed mixture toxic effect. The P. lividus embryotoxicity of trace metals decreases as follows: Cu > Pb > Cd at all toxicity concentration tested. EC50 mixture revealed less toxic only than Cu; EC50 was 0.80 (±0.07) mg/l, the offspring malformations were mainly P1 type (skeletal alterations) up to 20% mixture concentration, and P2 type from 70% concentration. The NOEL and EC1 mixtures evidenced that all compounds contribute to the overall toxicity, even if present at low concentrations: the former EC50 was 0.532 (±0.058) mg/l and the latter was 1.081 (±0.240) mg/l. The developmental defects observed were mainly P1 type in both mixtures. Both CA and IA models did not accurately predict mixture toxicity for EC50 and NOEL mixtures. Instead, EC1 mixture effects seemed well represented by the IA model. The protective action of the CA model, although quite accurate when applied to simple biological systems like algae and bacteria, but failed to represent the worst-case in this study with more complex organisms. It would be useful to introduce in the models one or more factors that take into account the complexity of these biological systems.     

Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action

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.     

Assessing combined toxicity of estrogen receptor agonists in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes

The presence of highly complex mixtures of chemicals in the environment challenges our ability to assess single chemical effects and the interaction that occurs with cellular receptor targets and regulation of endocrine processes. In this study concentration addition (CA) and independent action (IA) prediction models were used to assess the combined toxicity of mixtures of environmental relevant estrogen receptor (ER) agonists (hormones and anthropogenic pollutants) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of vitellogenin (Vtg) as a biological marker (biomarker) for estrogenicity. Nine of the eleven tested chemicals induced the production of Vtg and the parameters from the fitted concentration-response curves were used to model four mixtures containing four (17β-estradiol, estrone, estriol and diethylstilbestrol), five (musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene), seven (17β-estradiol, estrone, estriol, diethylstilbestrol, 4-tert-octylphenol, bisphenol A and o,p′-DDT) and nine compounds (17β-estradiol, estrone, estriol, diethylstilbestrol, musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene). The CA and IA prediction model proved to be a good estimation for the combined effect of mixtures of ER agonists at low relative mixture concentration (e.g. relative to the maximum mixture concentrations used), but a deviation from the prediction models was observed when exposing hepatocytes to high relative mixture concentrations. The CA and IA prediction models’ ability to predict the combined estrogenic effect of complex mixtures, especially in the low concentration-response range, is of ecological relevance since organisms in the environment generally encounter low concentrations of chemicals from a wide array of chemical groups that may not elicit estrogenic effects on their own.     

Assessment of toxic interactions of heavy metals in multi-component mixtures using sea urchin embryo-larval bioassay

The toxicities of copper, lead, zinc and cadmium ions and various concentrations of mixtures of them were studied using sea urchin (Strongylocentyotus intermedius) embryo-larval bioassay. Toxic unit analysis was used to determine type of joint action for each mixture combination (binary, ternary and quaternary). For the majority of the binary combinations, the interactions were of synergistic nature, but in ternary or quaternary mixtures, the joint action was mainly concentration additive, while antagonism was only observed for two mixtures (Cu + Pb and Zn + Cd) among all the 11 combinations. Two prevailing theoretical models: the concentration addition (CA) model and the independent action (IA) model were used to predict the mixture toxicities. The weak correlation obtained (R ? 0.55) indicated that the hypotheses of mode of action involved in the two models to some extent failed to describe the behavior of the mixture system. Then a novel bio-concentration factor-based model was developed and was successful to predict the toxicities of mixtures, with an obtained R of 0.92. This model indicated that in a mixture system of heavy metals, the joint toxicity was mainly determined by the combined action of bio-concentrations of metals other than the simply similar (CA) or dissimilar (IA) modes of action of the mixture components.     

Life-history responses of Daphnia magna Straus to binary mixtures of toxic substances: pharmacological versus ecotoxicological modes of action

Two prevailing theoretical models: concentration addition (CA) and independent action (IA), predict mixture toxicity on the basis of known toxicities of the mixture components. To date, both models have been in most occasions evaluated using unicellular in vivo responses or biochemical in vitro responses. However, when considering more complex models such as the whole organism physiology or life-history traits, the dominant ecotoxicological mode of action, based on the exposure concentrations at which various toxicological effects become operative at the level of whole organism, should be considered. Offspring production in Daphnia magna is driven by the resources acquired from food, and the number of live offspring produced by an organism is the result of two independent factors: the number of eggs produced and the percentage of eggs that survive egg development. In this study joint toxicity effects on offspring production in D. magna were tested using binary mixtures of toxic contaminants known to specifically impair food acquisition (lambda-cyhalothrin and cadmium) or to cause egg mortality during development (3,4 dichloroaniline). Tests were performed using a simplified 10-day reproduction assay initiated with gravid females. The results obtained indicate that irrespective of their primary pharmacological mode of action, the joint toxicity of cadmium and lambda-cyhalothrin was predicted by the CA model, whereas the joint toxicity of cadmium and 3,4 dichloroaniline was predicted by the IA model. Thus, the results obtained indicate that for life-history traits such as reproduction responses, the dominant ecotoxicological rather than the pharmacological mode of action should be used for predicting joint mixture effects.     

In vitro combined cytotoxic effects of pesticide cocktails simultaneously found in the French diet

Although human populations may be constantly exposed to complex pesticide mixtures through their diet, the human health risk of pesticide exposure is currently assessed on the basis of toxicity data on individual compounds. To investigate the combined toxic effects of pesticide cocktails previously identified in the French diet, we first studied the cytotoxicity induced by seven cocktails composed of two to six pesticides on human hepatic (HepG2) and colon (Caco-2) cell lines using the MTT and neutral red uptake assays. Secondly, we challenged to assess the combined effects of the two most cytotoxic cocktails by comparing the measured effects of the mixtures with the predictions based on additive effects on two concepts—independent action (IA) and concentration addition (CA). For the cocktail composed of dichlorodiphenyltrichloroethane (DDT) and dieldrin, the cytotoxicity of the equimolar cocktail proved greater than the additive effect estimated by the two concepts. Furthermore, apoptosis induction was higher in equimolar cocktail than predicted by summing the effects of DDT and dieldrin. Thus, some supra-additive toxicity was found in the DDT-dieldrin cocktail. Nevertheless, if IA and CA models could reveal combined effects of pesticide cocktails, an accurate evaluation remains challenging.     

Acute effects of binary mixtures of Type II pyrethroids and organophosphate insecticides on <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Pyrethroid and organophosphate insecticides have been used for more than 20 years worldwide to control a variety of insect pest in different settings. These pesticides have been detected in a variety of environmental samples, including surface waters and sediments and therefore there is significant concern about their potential toxic effects on non-target organisms. Mixtures of compounds from these groups of pesticides have been found to frequently show enhanced toxicity but it has been a challenge to predict whether or not enhanced toxicity will occur for a given combination of compounds. This study therefore studied the effects of binary pyrethroid-organophosphate mixtures using cypermethrin, deltamethrin and dimethoate in an acute toxicity test system with Oreochromis niloticus. The 96?h LC50s for individual insecticides were 9.13?µg/l, 9.42?µg/l and 45.52?mg/l for cypermethrin, deltamethrin and dimethoate respectively. These showed that the pyrethroid insecticides were highly toxic to Oreochromis niloticus and were far more toxic than dimethoate. All mixtures were also more toxic than single insecticides throughout the concentration-response curve with mixtures resulting in mortality at concentrations which the individual pesticides in the mixture were below their respective NOECs. In addition, observed mixture toxicities deviated from the predicted mixture effects based either on the Concentration Addition (CA) or Independent Action (IA) models independent of mixture ratio. However, the extent of observed mixture mortality deviation was dependent on the effect level. Significant deviations (MDR?>?2.0) were observed at lower concentrations indicating synergistic effects at lower and possibly environmentally relevant concentrations. This is not unexpected since organophosphate insecticides are known to inhibit acetylcholinesterase as well as inactivate esterase, resulting in reduced detoxification of pyrethroid insecticides and consequently greater toxicity than would be expected. This has important implications for risk assessment of mixtures since the risk of pyrethroid-organophosphate mixtures may be underestimated if either the CA or IA model is employed.     

Application of multielectrode array (MEA) chips for the evaluation of mixtures neurotoxicity

Cortical neurons grown on multielectrode array (MEA) chips have been shown to be a valuable alternative method to study electrophysiological properties of the central nervous system neurons and to perform functional toxicological screening. Here we studied the effects of binary mixtures on neuronal networks cultured on MEAs. We have considered compounds with similar and different mode-of-action (MoA) to characterize and assess their combined effects. Individual and binary mixture dose-response curves based on spontaneous neuronal activity have been generated and the IC(50) has been considered as the end-point for neurotoxicity assessment. The two classical approaches of mixtures toxicity studies: concentration addition (CA) and independent action (IA) have been applied to compare calculated and experimental results. Nuclear magnetic resonance (NMR) spectroscopy has been employed to confirm no chemical reaction or complexation between mixtures components. The results suggest that both CA and IA are able to predict the toxicity of the mixture and that the combination of in vitro test methods with theoretical dose-response models has a strong potential as an alternative tool for the prediction of mixtures neurotoxicity.     

Vitellogenin induction by a mixture of steroidal estrogens in freshwater fishes and relevant risk assessment

The study method on combined effects of environmental contaminant mixture and ecological risk assessment was discussed. Batch tests were conducted to assess the in vivo potency of binary mixtures of estrogens using plasma vitellogenin concentrations in male crucian carp as the endpoint. The estrogenic potencies of 17β‐estradiol (E₂) and 17α‐ethynylestradiol (EE₂) were determined following 14 day exposure to the individual chemicals and equipotent binary mixtures. A Nonlinear regression was obtained and 95% confidence limits of effect concentration were achieved using the bootstrap method. Concentration‐response curve for fixed ratio binary mixtures of E₂ and EE₂ was compared with those for individual chemicals, using the biomathematical models of concentration addition (CA) and independent action (IA). A complete overlap was found for the CA predictions with the 95% confidence interval of the best‐fit regression line of the observed responses, and the IA predictions was shown lower than the observations. The observed mixture effects were considerably higher than those of the hormone alone and far exceeded the 95% confidence interval of the estrogen regression lines. The predicted effects of binary mixtures at different mixture ratios indicated that the potential impact of components on mixture would depend predominantly on its concentration, the mixture ratio and its relative potency. Results suggested that E₂ and EE₂ acted together in an additive manner and the combined effects can be accurately predicted in whole range of exposure concentration by the models of CA and IA, the model of CA might be realistic, but more useful for ecological risk assessment. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Mixture toxicity effects of chloramphenicol,thiamphenicol, florfenicol in Daphnia magna under different temperatures

Acute toxicities of chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FLO) and their mixtures on Daphnia magna under two representative temperatures of the aquatic environment (20 and 25 °C) have been examined. Their toxicities depicted with an order of 72-h EC₅₀ values were as follows: CAP > FLO > TAP and CAP ≈ FLO > TAP under 20 and 25 °C, separately. Furthermore, the acute toxicity significantly increased with the rise of temperature from 20 to 25 °C in nearly all separate and mixture phenicol antibiotics. Meanwhile, the most toxic combination under two different temperatures was diverse. The nature of toxicological interactions of phenicol antibiotic mixtures was analyzed by Combination Index (CI) equation. In general, a dual synergism-antagonism effect was dominant in nearly all mixtures at both temperatures. The prediction suitability of Concentration Addition (CA), Independent Action (IA) models, and CI method was compared, suggesting that the CI equation seems to be more appropriate for predicting the toxicity values of phenicol drugs than CA and IA models. In brief, phenicol antibiotic mixtures with temperature variation may pose more significant hazards and risks to aquatic organisms; hence, the environment.

     

Toxicity prediction of binary combinations of cadmium, carbendazim and low dissolved oxygen on Daphnia magna

Environmental contamination is often characterised by a combination of stress factors of various sources (biological, physical and chemical). The predictability of their joint effects is an important stage in environmental risk assessment procedures. In this study, the two main conceptual models for mixture evaluation based on the effect of individual compounds, concentration addition (CA) and independent action (IA) and deviations to synergism/antagonism, "dose ratio" and "dose level" dependency were used. The single and combined effects of cadmium, carbendazim and low dissolved oxygen levels were assayed for life-cycle parameters (survival and feeding) of the water flea Daphnia magna Straus. The results of single exposures revealed an increase of acute and chronic toxicity as concentrations of cadmium and carbendazim increases. At low dissolved oxygen levels both survival and feeding parameters were significantly affected (P< or =0.05). In the acute mixture exposure of cadmium and carbendazim a "dose ratio" dependency was observed with a higher toxicity when cadmium was dominant whereas at high concentrations of carbendazim a lower effect on survival was observed. At chronic exposures an antagonistic deviation from IA model was observed for this mixture. The IA model showed to be adequate for toxicity prediction on acute exposure combinations with low DO levels where a synergistic behaviour was observed. However, at sublethal exposures IA and CA models failed by underestimation. Validation from toxicokinetic and toxicodynamic modelling studies should be made in the future as a way to understand toxicological pathways involved in complex mixture/combination exposures.     

Prediction of the joint toxicity of five organophosphorus pesticides to Daphnia magna

The individual toxicities of five organophosphorus pesticides (dichlorvos, parathion, methyl parathion, malathion and dimethoate) to Daphnia magna were investigated in 24-h immobilization experiments. Using these toxicity data, their combined toxicities were measured in pesticide mixtures designed using either ‘equivalent-effect concentration ratios’ or ‘uniform-design concentration ratios’. The toxicities of mixtures of similarly or dissimilarly acting toxicants are often predicted from the individual toxicities of the component compounds, using one of two distinct biometric models: concentration addition (CA) or independent action (IA). The relative accuracies of the CA and IA models were assessed using the model deviation rate (MDR), which represents the difference between the effect predicted from the individual pesticide concentrations and the observed effect. The mean MDR value of CA was 0.93 (range 0.75–1.31) and the mean value obtained by IA was 3.13 (range 2.52–4.37). We conclude that the CA model is better able to predict the joint toxicities of mixtures of organophosphorus pesticides to D. magna.     

Genotoxic mixtures and dissimilar action: concepts for prediction and assessment

Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.