Concept Of Concentration Addition Research Articles

Short-term exposure to environmental levels of nicotine and cotinine impairs visual motor response in zebrafish larvae through a similar mode of action: Exploring the potential role of zebrafish α7 nAChR

The current view is that environmental levels of nicotine and cotinine, commonly in the ng/L range, are safe for aquatic organisms. In this study, 7 days post-fertilization zebrafish embryos have been exposed for 24 h to a range of environmental concentrations of nicotine (2.0 ng/L-2.5 μg/L) and cotinine (50 pg/L–10 μg/L), as well as to a binary mixture of these emerging pollutants. Nicotine exposure led to hyperactivity, decreased vibrational startle response and increased non-associative learning. However, the more consistent effect found for both nicotine and cotinine was a significant increase in light-off visual motor response (VMR). The effect of both pollutants on this behavior occurred through a similar mode of action, as the joint effects of the binary mixture of both chemicals were consistent with the concentration addition concept predictions. The results from docking studies suggest that the effect of nicotine and cotinine on light-off VMR could be mediated by zebrafish α7 nAChR expressed in retina. The results presented in this study emphasize the need to revisit the environmental risk assessment of chemicals including additional ecologically relevant sublethal endpoints.

Unravelling Relationships between In Vivo Effects on Plants and Detected Pesticide Mixtures in Freshwaters of a South-European Agro-Ecosystem

The multiple benefits agriculture provides to society depend on the long-term sustainable management of water resources, including the preservation of a good ecological and good chemical status of the water bodies. Presently, this good chemical status has not been reached in the majority of European river basins. Implemented monitoring strategies are targeted to identify the presence and magnitude of the ecological impacts that come from mixtures of chemicals but fail to give information on the causes of the ecosystem disruptions. This work aims to contribute to assessing the quality of surface waters used for irrigation in the LGVFX agricultural area (Central Portugal) by applying non-conventional in vivo phytotoxicity tests on three primary producers, a monocotyledon (Sorghum saccharatum) and two dicotyledons (Lepidium sativum and Sinapsis alba), complemented by chemical screening and mixture-risk modelling with component-based methods (summation of risk quotients) based on the classic concept of concentration addition (CA). Although inhibition percentages of the phytotoxicity parameter germination and root and shoot growth may be related to the presence of mixtures of pesticides, it was not possible to establish the fingerprinting of the detected compounds with the observed biological effects, mostly due to the large gap of ecotoxicological data on terrestrial plants exposed to contaminated water. In addition, pesticides can interact within the plant, leading to antagonism and synergism phenomena.

Short-term interactive effects of copper and cadmium on barley growth, metal uptake, and oxidative damage

The present study aimed at investigating the response of spring barley (Hordeum vulgare L.) across different endpoints (morphological, physiological, and biochemical) to short-term single and combined copper (Cu) and cadmium (Cd) exposure. Barley seedlings were exposed for 5 days to Cd and Cu (0.1–100 mg L−1) individually and in binary combinations. Binary mixture toxicity was evaluated using the concentration addition concept and toxic unit approach. Single metal treatment reduced barley height and biomass, altered the content of photosynthetic pigments, and induced lipid peroxidation. Plant growth (measured as plant height and biomass) and lipid peroxidation showed high sensitivity to combined Cu–Cd treatment and Cu–Cd mixture induced greater than additive effect on these endpoints. The uptake and accumulation of Cu and Cd were considerably influenced by the interaction among these metals. Cu reduced Cd accumulation in plants, while Cd had no influence on Cu accumulation in barley roots and shoots. Analysis of mixture effects across different endpoints revealed that mixture effects are endpoint dependent and different types of interactions were manifested. Overall, the present study shows that the same test design for single and mixture toxicity assessment for several endpoints cannot always be used to predict mixture effects and type of interaction.

Transcriptome-Wide Prediction and Measurement of Combined Effects Induced by Chemical Mixture Exposure in Zebrafish Embryos.

Background:Humans and environmental organisms are constantly exposed to complex mixtures of chemicals. Extending our knowledge about the combined effects of chemicals is thus essential for assessing the potential consequences of these exposures. In this context, comprehensive molecular readouts as retrieved by omics techniques are advancing our understanding of the diversity of effects upon chemical exposure. This is especially true for effects induced by chemical concentrations that do not instantaneously lead to mortality, as is commonly the case for environmental exposures. However, omics profiles induced by chemical exposures have rarely been systematically considered in mixture contexts.Objectives:In this study, we aimed to investigate the predictability of chemical mixture effects on the whole-transcriptome scale.Methods:We predicted and measured the toxicogenomic effects of a synthetic mixture on zebrafish embryos. The mixture contained the compounds diuron, diclofenac, and naproxen. To predict concentration- and time-resolved whole-transcriptome responses to the mixture exposure, we adopted the mixture concept of concentration addition. Predictions were based on the transcriptome profiles obtained for the individual mixture components in a previous study. Finally, concentration- and time-resolved mixture exposures and subsequent toxicogenomic measurements were performed and the results were compared with the predictions.Results:This comparison of the predictions with the observations showed that the concept of concentration addition provided reasonable estimates for the effects induced by the mixture exposure on the whole transcriptome. Although nonadditive effects were observed only occasionally, combined, that is, multicomponent-driven, effects were found for mixture components with anticipated similar, as well as dissimilar, modes of action.Discussion:Overall, this study demonstrates that using a concentration- and time-resolved approach, the occurrence and size of combined effects of chemicals may be predicted at the whole-transcriptome scale. This allows improving effect assessment of mixture exposures on the molecular scale that might not only be of relevance in terms of risk assessment but also for pharmacological applications. https://doi.org/10.1289/EHP7773

Gray water footprint assessment for pesticide mixtures applied to a sugarcane crop in Brazil: A comparison between two models

The concept of gray water footprint (GWF) was introduced as an indicator of the volume of freshwater required to dilute the total pollutant load, generated from a particular production process, to maintain water quality standards. In crop production, for example, the theoretical calculation of the GWF can be employed as a sound tool for assessing the environmental impacts due to the use of pesticides. In this context, we compared two models for estimating the GWF of pesticide mixtures applied to the soil in a sugarcane crop, in Pernambuco, Brazil. One model was proposed by Hoekstra et al. (2011) and calculates the GWF based on the maximum concentration limit acceptable in water. The other one was designed by Paraiba et al. (2014) and employs the concept of Concentration Addition. This latter model also takes into account the effect of toxicity of pesticide mixtures on aquatic organisms and water quality, considering the characteristics of the soil and cultivation data obtained through the field and physical-chemical tests pesticides used in agricultural activities. The model of Paraiba et al. (2014) has shown to be more conservative in determining the GWF of the pesticide mixtures (2.00 × 107 m³ ha−1) in the aquatic ecosystem, than the model of Hoekstra et al. (2011) (6.25 × 105 m³ ha−1 for Sulfentrazone). The results presented and discussed in this study reinforce the use of the GWF as an indicator of water quality for agricultural sustainability. Our work also contributes to a better understanding of the advantages and limitations of each model approach in the assessment of the volume of freshwater needed to dilute the contaminant load in agriculture activities as, for instance, in sugarcane crops.

Validating the Use of a Toxicity Database for Prediction of Plant Cover and Biodiversity in Multi-Metal Mining-Impacted Soils.

The validity of soil toxicity databases for predicting ecological impacts in the field is rarely explored. The present study was set up to test whether laboratory toxicity data and the combined concepts of metal availability and mixture toxicity can predict ecological impact in mining-affected soils. Metal and As contamination gradients were sampled approximately 5 different mines in Mexico where plant cover and abundances exhibited clear dose-related responses. Soils were analyzed for total and isotopically exchangeable (labile) concentrations of Ni, Cu, Cd, Pb, and As and for soil properties affecting the availability of these elements. Six different indices of toxic doses were compared to evaluate their accuracy in describing the field response expressed as relative abundance and cover. Each index was based on a different method to calculate the sum of toxic units ( TUs) in soil, with 1 toxic unit equal to the concentration of the element in soil yielding 50% adverse effect on plants with median sensitivity as recorded in a recent database of salt-spiked soils. Toxic concentrations in the mine-impacted soils were dominated by Zn and As. In the field, 50% reduced cover or abundance was found at 10 to 13 TUs if these were based on total soil concentrations and thresholds derived from freshly spiked soils, indicating a largely overestimated toxic effect. If thresholds were corrected for differences in availability among freshly spiked soils and spiked and laboratory-aged soils, the overestimation of field toxicity was 5- to 6-fold, irrespective of the consideration of soil properties. Finally, the TU calculated only with labile metals and As overestimated the field toxicity by factors 1.1 to 1.6 (95% confidence interval 1-7; i.e., rather accurate and indicating some Zn-As antagonism as confirmed in experimental studies). That latter index of dose yielded a bell-shaped response on species richness peaking at approximately 1.6 TU. Overall, the present study shows that the current toxicity databases of metals can predict the impact of metal contamination on plant communities within factor 2, expressing the dose as soil-labile concentrations and using the concentration addition concept in these mixed polluted environments. Environ Toxicol Chem 2020;39:1826-1838. © 2020 SETAC.

Mixture toxicity in the Erft River: assessment of ecological risks and toxicity drivers

BackgroundA vast number of chemical substances are released into the aquatic environment, leading to complex chemical mixtures in surface waters. Current water quality assessments, however, are based on the risk assessment of single substances. To consider potential mixture effects in water quality assessments, the North Rhine Westphalian State Agency for Nature, Environment and Consumer Protection (LANUV), Germany started a project assessing mixture toxicity in surface waters. This article summarises the mixture evaluation of chemical data collected by the Erftverband during a water sampling campaign in the Erft River in 2016/2017. Altogether, 153 substances were included in the analysis, of which 98 were detected. Two different approaches based on the concept of concentration addition were used to analyse the data. The results were compared to findings based on datasets from LANUV surveillance monitoring according to the EU Water Framework Directive.ResultsAcute and chronic mixture risk calculations indicated risks for 32% and up to 90% of the samples, respectively. The greatest acute toxic pressure was identified for the aquatic flora due to continuous exposure to varying pesticides, whereas the greatest chronic mixture risk was identified for fish as result of a ubiquitous presence of the pharmaceuticals diclofenac and ibuprofen. Overall, only a limited number of substances significantly contributed to the calculated mixture risks. However, these substances varied seasonally and regionally. When mixture risks were calculated based on different datasets, the monitoring design markedly affected the outcome of the mixture risk assessment. Data gaps of both ecotoxicological and exposure data lead to high uncertainties in the mixture risk assessment.ConclusionsEcotoxicological effects on aquatic organisms caused by chemical mixtures can be expected along the Erft River throughout the year. Both mixture risk assessment approaches can be used for a conservative assessment of mixture risks and characterise the aquatic pollution in the river more realistically than single substance assessments. For the integration of mixture risk evaluations into the assessment and management of the chemical water quality, a two-staged assessment combining both approaches is suggested. To improve future risk assessments, the accessibility and exchange of high-quality ecotoxicological data should be enhanced.

Prospective environmental risk assessment of mixtures in wastewater treatment plant effluents – Theoretical considerations and experimental verification

The aquatic environment is continually exposed to a complex mixture of chemicals, whereby effluents of wastewater treatment plants (WWTPs) are one key source. The aim of the present study was to investigate whether environmental risk assessments (ERAs) addressing individual substances are sufficiently protective for such coincidental mixtures. Based on a literature review of chemicals reported to occur in municipal WWTP effluents and mode-of-action considerations, four different types of mixtures were composed containing human pharmaceuticals, pesticides, and chemicals regulated under REACH. The experimentally determined chronic aquatic toxicity of these mixtures towards primary producers and the invertebrate Daphnia magna could be adequately predicted by the concept of concentration addition, with up to 5-fold overestimation and less than 3-fold underestimation of mixture toxicity. Effluents of a municipal WWTP had no impact on the predictability of mixture toxicity and showed no adverse effects on the test organisms. Predictive ERAs for the individual mixture components based on here derived predicted no effect concentrations (PNECs) and median measured concentrations in WWTP effluents (MCeff) indicated no unacceptable risk for any of the individual chemicals, while MCeff/PNEC summation indicated a possible risk for multi-component mixtures. However, a refined mixture assessment based on the sum of toxic units at species level indicated no unacceptable risks, and allowed for a safety margin of more than factor 10, not taking into account any dilution of WWTP effluents by surface waters. Individual substances, namely climbazole, fenofibric acid and fluoxetine, were dominating the risks of the investigated mixtures, while added risk due to the mixture was found to be low with the risk quotient being increased by less than factor 2. Yet, uncertainty remains regarding chronic mixture toxicity in fish, which was not included in the present study. The number and identity of substances composing environmental mixtures such as WWTP effluents is typically unknown. Therefore, a mixture assessment factor is discussed as an option for a prospective ERA of mixtures of unknown composition.

Is there synergistic interaction between fungicides inhibiting different enzymes in the ergosterol biosynthesis pathway in toxicity tests with the green alga Raphidocelis subcapitata?

Products used for plant protection or as biocides often contain more than one active substance together with numerous formulation additives. The environmental risk assessment for such commercial mixtures applies as default the concept of concentration addition. There is remaining regulatory concern, however, that underestimation of risks can occur if components in the mixture interact synergistically, i.e., elicit effects greater than those predicted by concentration addition. While cases of true synergism appear to be rare, the combination of substances targeting different steps in the same biosynthesis pathway was pointed out as one potential case of synergistic interaction although mechanistic explanations are lacking. The present study aimed to verify this hypothesis using the green alga Raphidocelis subcapitata as the regulatory standard test organism for which such synergism had been indicated earlier. Algal growth inhibition tests were conducted with mixtures of ergosterol biosynthesis inhibitors (tebuconazole, fenpropidin, and fenpropimorph). The fungicides were first tested individually to derive reliable data for a mixture toxicity prediction. The here determined toxicity estimates for two of the fungicides were considerably lower than the endpoints in the regulatory dossiers, which had been used for earlier mixture toxicity predictions. Experimentally observed toxicity estimates for the mixtures deviated <2.6-fold from the predicted values. Hence, the hypothesis of synergistic interaction between fungicides targeting different enzymes in the ergosterol biosynthesis was clearly not confirmed for the green alga R. subcapitata. Overall, the present study demonstrates the importance of reliable and correct input data for mixture toxicity predictions in order to avoid erroneous conclusions on non-additive (synergistic) interactions.

Kinetic interpretation of log-logistic dose-time response curves.

A Hill-type time-response curve was derived using a single-step chemical kinetics approximation. The rate expression for the transformation is a differential equation that provides an interpolation formula between the logistic growth curve and second order kinetics. The solution is equivalent to the log-logistic cumulative distribution function with the time constant expressed in terms of a kinetic rate constant. This expression was extended to a full dose-time-response equation by postulating a concentration dependence for the rate constant. This was achieved by invoking a modified form of Haber’s law that connects an observed toxic effect with the concentration of the active agent and the elapsed exposure time. Analysis showed that the concept of Concentration Addition corresponds to a special case where the rate constant for the overall transformation rate is proportional to the sum of the rate constants that apply when the agents act individually. Biodiesel “survival” curves were measured and used to test the applicability of the empirical model to describe the effects of inhibitor dosage and binary inhibitor mixtures. Positive results suggest that the proposed dose-response relationship for the toxicity of agents to organisms can be extended to inanimate systems especially in cases where accurate mechanistic models are lacking.

Mixture toxicity of six sulfonamides and their two transformation products to green algae Scenedesmus vacuolatus and duckweed Lemna minor

Since humans and ecosystems are continually exposed to a very complex and permanently changing mixture of chemicals, there is increasing concern in the general public about the potential adverse effects they may cause. Among all “emerging pollutants”, pharmaceuticals in particular have raised great environmental concern. For these reasons the aim of our study was to evaluate the mixture toxicity of six antimicrobial sulfonamides (SAs) and their two most commonly identified degradation products – sulfanilic acid (SNA) and sulfanilamide (SN) - to limnic green algae Scenedesmus vacuolatus and duckweed Lemna minor. The ecotoxicological data for the single toxicity of SNA and SN towards selected organisms are presented. The concept of Concentration Addition (CA) was applied to estimate the effects, and less than additive effects were observed. In general terms, it seems sufficiently precautionary for the aquatic environment to consider the toxicity of a sulfonamide mixture as additive. The Concentration Addition model proves to be a reasonable worst-case estimation. Such a comparative study on the mixture toxicity of sulfonamides and their transformation products has been presented for the first time.

From single chemicals to mixtures—Reproductive effects of levonorgestrel and ethinylestradiol on the fathead minnow

The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants—each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day ‘pair-breeding’ assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.

Personal care product preservatives: Risk assessment and mixture toxicities with an industrial wastewater

The aquatic toxicity of eight preservatives frequently used in personal care products (PCPs) (iodopropynyl butylcarbamate, bronopol, diazolidinyl urea, benzalkonium chloride, zinc pyrithione, propylparaben, triclosan and a mixture of methylchloroisothiazolinone and methylisothiazolinone) was assessed by means of two different approaches: a battery of bioassays composed of single species tests of bacteria (Vibrio fischeri and Pseudomonas putida) and protozoa (Tetrahymena thermophila), and a whole biological community resazurin-based assay using activated sludge. The tested preservatives showed considerable toxicity in the studied bioassays, but with a marked difference in potency. In fact, all biocides except propylparaben and diazolidinyl urea had EC50 values lower than 1 mg L−1 in at least one assay. Risk quotients for zinc pyrithione, benzalkonium chloride, iodopropynyl butylcarbamate and triclosan as well as the mixture of the studied preservatives exceeded 1, indicating a potential risk for the process performance and efficiency of municipal sewage treatment plants (STPs). These four single biocides explained more than 95% of the preservative mixture risk in all bioassays. Each individual preservative was also tested in combination with an industrial wastewater (IWW) from a cosmetics manufacturing facility. The toxicity assessment was performed on binary mixtures (preservative + IWW) and carried out using the median-effect principle, which is a special case of the concept of Concentration Addition (CA). Almost 70% of all experiments resulted in EC50 values within a factor of 2 of the values predicted by the median-effect principle (CI values between 0.5 and 2). The rest of the mixtures whose toxicity was mispredicted by CA were assessed with the alternative concept of Independent Action (IA), which showed higher predictive power for the biological community assay. Therefore, the concept used to accurately predict the toxicity of mixtures of a preservative with a complex industrial wastewater depends on degree of biological complexity.

Environmental risk assessment of pesticide mixtures under regulation 1107/2009/EC: a regulatory review by the German Federal Environment Agency (UBA)

This paper does review and reflect on the state of implementation of mixture risk assessment in the guidance documents for environmental risk assessment of plant protection products under regulation 1107/2009/EC. In the past years and still on-going, the specific guidance documents for the relevant assessment areas (birds and mammals, aquatic organism, bees and wild pollinators, non-target terrestrial arthropods, soil organism, non-target terrestrial plants) have been under revision. This revision process—being led by the European Food Safety Agency (EFSA)—has substantially brought forward the consideration of the state of the science in mixture hazard and risk assessment in the recently updated guidance documents and proposals for new guidance, respectively. The application of component-based approaches is now advocated as essential new methodology; these do allow for calculating the expectable joint toxicity from toxicity data available for individual mixture components, with the reference concept of concentration addition suggested as reasonable default (tier 1) assumption within tiered approaches for mixture risk assessment. Further elements newly proposed—although differing in the degree considered in the distinct assessment areas—include the counter-checking of measured and calculated mixture toxicity as well as the identification of “drivers” of mixture toxicity/risk. The German Federal Environment Agency (UBA) does acknowledge the progress made in implementation of mixture risk assessment during the past years and is generally committed to the recently updated EFSA guidance documents and proposals for future guidance documents, respectively.

AhR-mediated activities of polycyclic aromatic compound (PAC) mixtures are predictable by the concept of concentration addition

Risk assessments of polycyclic aromatic hydrocarbons (PAHs) are complicated because these compounds exist in the environment as complex mixtures of hundreds of individual PAHs and other related polycyclic aromatic compounds (PACs). In this study, the hypothesis that concentration addition (CA) can be used to predict the aryl hydrocarbon receptor (AhR)-mediated activity of PACs in mixtures containing various combinations of PACs was tested. AhR-mediated activities of 18 mixtures composed of two to 23 PACs, which included PAHs, azaarenes and oxygenated PAHs, were examined by the use of the AhR-based H4IIE-luc bioassay. Since greater AhR-mediated activities have been observed in soils contaminated by PAHs, investigations were done to test whether soil extract matrix or the presence of non-effect PACs might affect responses of the H4IIE-luc bioassay. Our results showed that AhR-mediated activities of mixtures of PACs could be predicted by the use of concentration addition. Additive activities of PACs in multi component mixtures along with the insignificant effect of the soil matrix support the use of concentration addition in mass balance calculations and AhR-based bioassays in risk assessment of environmental samples. However, independent action (IA) could not be used to predict the activity of mixtures of PACs.

Predicting acute and chronic effects of wood preservative products in daphnia magna and pseudokirchneriella subcapitata based on the concept of concentration addition

The current European legislation requires that combined effects of the active substances and any substance of concern contained in biocidal products are taken into account in environmental risk assessment. The hypothesis whether the consideration of active substances together with all formulation additives that are labeled as presenting an environmental hazard is sufficient for a reliable environmental risk assessment was tested in the present study by investigating 3 wood preservative products. Relevant single substances in the products, some of their generic mixtures, the biocidal products themselves, and aqueous eluates prepared from the products (representing potential environmental mixtures) were tested for effects on algal growth and Daphnia acute immobilization as well as reproduction. Predictions for the products and the eluates were based on the concept of concentration addition and were mostly found to provide reliable or at least protective estimates for the observed acute and chronic toxicity of the mixtures. The mixture toxicity considerations also indicated that the toxicity of each product was dominated by just 1 of the components, and that assessments based only on the dominating substance would be similarly protective as a full-mixture risk assessment. Yet, there remained uncertainty in some cases that could be related to the toxicity of transformation products, the impact of unidentified formulation additives, or synergistic interaction between active substances and formulation additives.

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.

Screening level mixture risk assessment of pharmaceuticals in STP effluents

We modeled the ecotoxicological risks of the pharmaceutical mixtures emitted from STP effluents into the environment. The classic mixture toxicity concept of Concentration Addition was used to calculate the total expected risk of the analytically determined mixtures, compare the expected impact of seven effluent streams and pinpoint the most sensitive group of species. The risk quotient of a single, randomly selected pharmaceutical is often more than a factor of 1000 lower than the mixture risk, clearly indicating the need to systematically analyse the overall risk of all pharmaceuticals present. The MCR, which is the ratio between the most risky compound and the total mixture risk, varies between 1.2 and 4.2, depending on the actual scenario and species group under consideration. The mixture risk quotients, based on acute data and an assessment factor of 1000, regularly exceed 1, indicating a potential risk for the environment, depending on the dilution in the recipient stream. The top 10 mixture components explain more than 95% of the mixture risk in all cases.A mixture toxicity assessment cannot go beyond the underlying single substance data. The lack of data on the chronic toxicity of most pharmaceuticals as well as the very few data available for in vivo fish toxicity has to be regarded as a major knowledge gap in this context. On the other hand, ignoring Independent Action or even using the sum of individual risk quotients as a rough approximation of Concentration Addition does not have a major impact on the final risk estimate.

Toxicity assessment of the maize herbicides S-metolachlor, benoxacor, mesotrione and nicosulfuron, and their corresponding commercial formulations, alone and in mixtures, using the Microtox® test

The Microtox® test, using the prokaryote Vibrio fischeri, was employed to assess the toxicity of the maize herbicides S-metolachlor, benoxacor, mesotrione and nicosulfuron, and their formulated compounds: Dual Gold Safeneur®, Callisto® and Milagro®; alone and in mixtures. For each compound we obtained original IC50 values, with consistent higher toxicities for formulated compounds compared to active ingredients alone. Mixtures of the four herbicides, prepared according to application doses encountered in agriculture, were found to be toxic at a lower concentration than single molecules. Mesotrione and nicosulfuron mixture appeared to be highly toxic to V. fischeri, however, this recommended post-emergence combination for maize crops got its toxicity decreased in formulated compound mixtures, suggesting that chemical interactions could potentially reduce the toxicity. Data comparisons to theoretical models showed a good prediction of mixture toxicity by Concentration Addition concept. Results seemed to exclude any synergistic effects on V. fischeri for the tested herbicide mixtures. Additional work coupling these bioassay data to ecosystemic level studies (aquatic and soil compartments) and data on additives and degradation products toxicity, will help to fill the gap in our knowledge of the environmental impact of these xenobiotics and in the choice of a more sustainable use of pesticides.

Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo

Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study used the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO4 (NIS-inhibitor) dosed at a fixed ratio of EC10 that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes.