Effects of diclofenac on sentinel species and aquatic communities in semi-natural conditions.

Due to the potential hazard of diclofenac on aquatic organisms and the lack of higher-tier ecotoxicological studies, a long-term freshwater mesocosm experiment was set up to study the effects of this substance on primary producers and consumers at environmentally realistic nominal concentrations 0.1, 1 and 10 µg/L (average effective concentrations 0.041, 0.44 and 3.82 µg/L). During the six-month exposure period, the biovolume of two macrophyte species (Nasturtium officinale and Callitriche platycarpa) significantly decreased at the highest treatment level. Subsequently, a decrease in dissolved oxygen levels was observed. High mortality rates, effects on immunity, and high genotoxicity were found for encaged zebra mussels (Dreissena polymorpha) in all treatments. In the highest treatment level, one month after the beginning of the exposure, mortality of adult fish (Gasterosteus aculeatus) caused effects on the final population structure. Total abundance of fish and the percentage of juveniles decreased whereas the percentage of adults increased. This led to an overall shift in the length frequency distribution of the F1 generation compared to the control. Consequently, indirect effects on the community structure of zooplankton and macroinvertebrates were observed in the highest treatment level. The No Observed Effect Concentration (NOEC) value at the individual level was < 0.1 µg/L and 1 µg/L at the population and community levels. Our study showed that in more natural conditions, diclofenac could cause more severe effects compared to those observed in laboratory conditions. The use of our results for regulatory matters is also discussed.

Analysis of real-time mixture cytotoxicity data following repeated exposure using BK/TD models.

Cosmetic products generally consist of multiple ingredients. Thus, cosmetic risk assessment has to deal with mixture toxicity on a long-term scale which means it has to be assessed in the context of repeated exposure. Given that animal testing has been banned for cosmetics risk assessment, in vitro assays allowing long-term repeated exposure and adapted for in vitro - in vivo extrapolation need to be developed. However, most in vitro tests only assess short-term effects and consider static endpoints which hinder extrapolation to realistic human exposure scenarios where concentration in target organs is varies over time. Thanks to impedance metrics, real-time cell viability monitoring for repeated exposure has become possible. We recently constructed biokinetic/toxicodynamic models (BK/TD) to analyze such data (Teng et al., 2015) for three hepatotoxic cosmetic ingredients: coumarin, isoeugenol and benzophenone-2. In the present study, we aim to apply these models to analyze the dynamics of mixture impedance data using the concepts of concentration addition and independent action. Metabolic interactions between the mixture components were investigated, characterized and implemented in the models, as they impacted the actual cellular exposure. Indeed, cellular metabolism following mixture exposure induced a quick disappearance of the compounds from the exposure system. We showed that isoeugenol substantially decreased the metabolism of benzophenone-2, reducing the disappearance of this compound and enhancing its in vitro toxicity. Apart from this metabolic interaction, no mixtures showed any interaction, and all binary mixtures were successfully modeled by at least one model based on exposure to the individual compounds.

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.

Multiple exposures to indoor contaminants: Derivation of benchmark doses and relative potency factors based on male reprotoxic effects.

Semi-Volatile Organic Compounds (SVOCs) are commonly present in dwellings and several are suspected of having effects on male reproductive function mediated by an endocrine disruption mode of action. To improve knowledge of the health impact of these compounds, cumulative toxicity indicators are needed. This work derives Benchmark Doses (BMD) and Relative Potency Factors (RPF) for SVOCs acting on the male reproductive system through the same mode of action. We included SVOCs fulfilling the following conditions: detection frequency (>10%) in French dwellings, availability of data on the mechanism/mode of action for male reproductive toxicity, and availability of comparable dose-response relationships. Of 58 SVOCs selected, 18 induce a decrease in serum testosterone levels. Six have sufficient and comparable data to derive BMDs based on 10 or 50% of the response. The SVOCs inducing the largest decrease in serum testosterone concentration are: for 10%, bisphenol A (BMD10 = 7.72E-07 mg/kg bw/d; RPF10 = 7,033,679); for 50%, benzo[a]pyrene (BMD50 = 0.030 mg/kg bw/d; RPF50 = 1630), and the one inducing the smallest one is benzyl butyl phthalate (RPF10 and RPF50 = 0.095). This approach encompasses contaminants from diverse chemical families acting through similar modes of action, and makes possible a cumulative risk assessment in indoor environments. The main limitation remains the lack of comparable toxicological data.

Comparative genotoxic potential of 27 polycyclic aromatic hydrocarbons in three human cell lines.

Polycyclic Aromatic Hydrocarbons (PAHs) form a family of compounds that are generally found in complex mixtures. PAHs can lead to the development of carcinogenesis. The Toxicity Equivalent Factor (TEF) approach has been suggested for estimating the toxicity of PAHs, however, due to the relative weakness of available data, TEF have not been applied for the risk characterization of PAHs as food contaminants in Europe. The determination of new TEFs for a large number of PAHs could overcome some limitations of the current method and improve cancer risk assessment. The present investigation aimed at deriving new TEFs for PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a genotoxicity assay (γH2AX) with three human cell lines to analyze the genotoxic properties of 27 selected PAHs after 24 h treatment. For 11 compounds, we did not detect any genotoxic potential. For the remaining 16 PAHs, the concentration-response for genotoxic effect was modelled with the Hill equation; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. We developed for each compound, in each cell line, Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were similar in all cell lines and generally higher than the TEF usually used. These new equivalent factors for PAHs should improve cancer risk assessment.

Perspectives for integrating human and environmental risk assessment and synergies with socio-economic analysis.

For more than a decade, the integration of human and environmental risk assessment (RA) has become an attractive vision. At the same time, existing European regulations of chemical substances such as REACH (EC Regulation No. 1907/2006), the Plant Protection Products Regulation (EC regulation 1107/2009) and Biocide Regulation (EC Regulation 528/2012) continue to ask for sector-specific RAs, each of which have their individual information requirements regarding exposure and hazard data, and also use different methodologies for the ultimate risk quantification. In response to this difference between the vision for integration and the current scientific and regulatory practice, the present paper outlines five medium-term opportunities for integrating human and environmental RA, followed by detailed discussions of the associated major components and their state of the art. Current hazard assessment approaches are analyzed in terms of data availability and quality, and covering non-test tools, the integrated testing strategy (ITS) approach, the adverse outcome pathway (AOP) concept, methods for assessing uncertainty, and the issue of explicitly treating mixture toxicity. With respect to exposure, opportunities for integrating exposure assessment are discussed, taking into account the uncertainty, standardization and validation of exposure modeling as well as the availability of exposure data. A further focus is on ways to complement RA by a socio-economic assessment (SEA) in order to better inform about risk management options. In this way, the present analysis, developed as part of the EU FP7 project HEROIC, may contribute to paving the way for integrating, where useful and possible, human and environmental RA in a manner suitable for its coupling with SEA.