A two years field experiment to assess the impact of two fungicides on earthworm communities and their recovery.

Recent EFSA (European Food Safety Authority) reports highlighted that the ecological risk assessment of pesticides needed to go further by taking more into account the impacts of chemicals on biodiversity under field conditions. We assessed the effects of two commercial formulations of fungicides separately and in mixture, i.e., Cuprafor Micro® (containing 500 g kg-1 copper oxychloride) at 4 (C1, corresponding to 3.1 mg kg-1 dry soil of copper) and 40 kg ha-1 (C10), and Swing® Gold (50 g L-1 epoxiconazole EPX and 133 g L-1 dimoxystrobin DMX) at one (D1, 5.81 10-2 and 1.55 10-1 mg kg-1 dry soil of EPX and DMX, respectively) and ten times (D10) the recommended field rate, on earthworms at 1, 6, 12, 18 and 24 months after the application following the international ISO standard no. 11268-3 to determine the effects on earthworms in field situations. The D10 treatment significantly reduced the species diversity (Shannon diversity index, 54% of the control), anecic abundance (29% of the control), and total biomass (49% of the control) over the first 18 months of experiment. The Shannon diversity index also decreased in the mixture treatment (both fungicides at the recommended dose) at 1 and 6 months after the first application (68% of the control at both sampling dates), and in C10 (78% of the control) at 18 months compared with the control. Lumbricus terrestris, Aporrectodea caliginosa, Aporrectodea giardi, Aporrectodea longa, and Allolobophora chlorotica were (in decreasing order) the most sensitive species to the tested fungicides. This study not only addressed field ecotoxicological effects of fungicides at the community level and ecological recovery, but it also pinpointed some methodological weaknesses (e.g., regarding fungicide concentrations in soil and statistics) of the guideline to determine the effects on earthworms in field situations.

Soil dissipation and bioavailability to earthworms of two fungicides under laboratory and field conditions.

The representativeness of laboratory studies of the fate of pesticides in soil in field conditions is questionable. This study aimed at comparing the dissipation and bioavailability to earthworms of two fungicides, dimoxystrobin (DMX) and epoxiconazole (EPX), over 12 months under laboratory and field conditions. In both approaches, the fungicides were applied to the same loamy soil as a formulated mixture at several concentrations. We determined total DMX and EPX concentrations in the soil using exhaustive extraction, their environmental availability using mild extraction and their bioavailability through internal concentrations in exposed earthworms. The initial fungicide application appeared as much better controlled in terms of dose and homogeneity in the laboratory than in the field. One year after application, a similar dissipation rate was observed between the laboratory and field experiments (ca 80% and 60% for DMX and EPX, respectively). Similarly, the ratio of available/total concentrations in soil displayed the same trend whatever the duration and the conditions (field or lab), EPX being more available than DMX. Finally, the environmental bioavailability of the two fungicides to earthworms was heterogeneous in the field, but, in the laboratory, the bioaccumulation was evidenced to be dose-dependent only for DMX. Our findings suggest that the actual fate of the two considered fungicides in the environment is consistent with the one determined in the laboratory, although the conditions differed (e.g., presence of vegetation, endogeic earthworm species). This study allowed better understanding of the fate of the two considered active substances in the soil and underlined the need for more research dedicated to the link between environmental and toxicological bioavailability.

An energy-based model to analyze growth data of earthworms exposed to two fungicides.

The pesticide risk assessment for earthworms is currently performed using standardized tests, the model species Eisenia fetida, and the analyses of the data obtained are performed with ad hoc statistical tools. We assessed the impact of two fungicides on the entire growth pattern of the earthworm species Aporrectodea caliginosa, which is highly representative of agricultural fields. Individuals of three different ages (from hatching to 56 days old) were exposed to Cuprafor micro® (copper oxychloride) and Swing® Gold (dimoxystrobin and epoxiconazole). Data were analyzed with an energy-based toxicodynamic model coupled with a toxicokinetic model. The copper fungicide caused a drastic growth inhibition once the no effect concentration (NEC), estimated at 65 mg kg-1 of copper, was exceeded. The Swing® Gold negatively affected the growth with NEC values estimated at 0.387 mg kg-1 and 0.128 mg kg-1 for the dimoxystrobin and the epoxiconazole in this fungicide formulation, respectively. The time-profile of the effects on A. caliginosa individuals was fully accounted for by the model, whatever their age of exposure. Furthermore, toxicity data analyses, supported by measurements of fungicide concentrations in earthworm at the end of the experiment, allowed bettering understanding of the mechanisms of action of the fungicides towards earthworm growth.

Earthworms Mitigate Pesticide Effects on Soil Microbial Activities.

Earthworms act synergistically with microorganisms in soils. They are ecosystem engineers involved in soil organic matter degradation and nutrient cycling, leading to the modulation of resource availability for all soil organisms. Using a soil microcosm approach, we aimed to assess the influence of the earthworm Aporrectodea caliginosa on the response of soil microbial activities against two fungicides, i.e., Cuprafor Micro® (copper oxychloride, a metal) and Swing® Gold (epoxiconazole and dimoxystrobin, synthetic organic compounds). The potential nitrification activity (PNA) and soil enzyme activities (glucosidase, phosphatase, arylamidase, and urease) involved in biogeochemical cycling were measured at the end of the incubation period, together with earthworm biomass. Two common indices of the soil biochemistry were used to aggregate the response of the soil microbial functioning: the geometric mean (Gmean) and the Soil Quality Index (SQI). At the end of the experiment, the earthworm biomass was not impacted by the fungicide treatments. Overall, in the earthworm-free soil microcosms, the two fungicides significantly increased several soil enzyme and nitrification activities, leading to a higher GMean index as compared to the non-treated control soils. The microbial activity responses depended on the type of activity (nitrification was the most sensitive one), on the fungicide (Swing® Gold or Cuprafor Micro®), and on the doses. The SQI indices revealed higher effects of both fungicides on the soil microbial activity in the absence of earthworms. The presence of earthworms enhanced all soil microbial activities in both the control and fungicide-contaminated soils. Moreover, the magnitude of the fungicide impact, integrated through the SQI index, was mitigated by the presence of earthworms, conferring a higher stability of microbial functional diversity. Our results highlight the importance of biotic interactions in the response of indicators of soil functioning (i.e., microbial activity) to pesticides.

Effects of two common fungicides on the reproduction of Aporrectodea caliginosa in natural soil.

The use of pesticides in agroecosystems can have negative effects on earthworms, which play key roles in soil functioning such as organic matter decomposition. The aim of this study was to assess the effects of two fungicides (Cuprafor micro®, composed of copper oxychloride, and Swing Gold®, composed of epoxiconazole (EPX) and dimoxystrobin (DMX)) on earthworm reproduction by exposing adults and cocoons. First, adult Aporrectodea caliginosa individuals were exposed for 28 days to 3.33, 10 and 30 times the recommended dose (RD) of Cuprafor micro® corresponding to 25.8, 77.5 and 232.5 mg kg-1 dry soil of copper, respectively, and 0.33, 1 and 3 times the RD of Swing Gold® (corresponding to 5.2 × 10-2 mg DMX kg-1 + 1.94 × 10-2 mg EPX kg-1, 1.55 × 10-1 mg DMX kg-1 + 5.81 × 10-2 mg EPX kg-1 and 4.62 × 10-1 mg DMX kg-1 + 1.74 × 10-1 mg EPX kg-1 respectively), in addition to a control soil with no fungicide treatment. Cocoon variables (production, weight, hatching success, hatching time) were monitored. Second, "naïve" cocoons produced by uncontaminated earthworms were exposed to soils contaminated by the same concentrations of the two fungicides, and we assessed hatching success and hatching time. In the first experiment, cocoon production was halved at the highest copper concentration (232.5 mg Cu kg-1 of dry soil) as compared to the control. Cocoons took 5 more days to hatch, and the hatching success decreased by 35% as compared to the control. In the Swing Gold® treatments, cocoon production was reduced by 63% at 3 times the RD, and the hatching success significantly decreased by 16% at the RD. In the second experiment, only the hatching success of cocoons was impacted by Swing Gold® at 3 times the RD (30% less hatching). It is concluded that the cocoon stock in the soil is crucial for the renewal of populations in the field. The most sensitive endpoint was the hatching success of the cocoons produced by exposed adults. This endpoint and the effects observed on the "naïve" cocoons could be taken into account in pesticide risk assessment.

Aporrectodea caliginosa, a relevant earthworm species for a posteriori pesticide risk assessment: current knowledge and recommendations for culture and experimental design.

Ecotoxicological tests with earthworms are widely used and are mandatory for the risk assessment of pesticides prior to registration and commercial use. The current model species for standardized tests is Eisenia fetida or Eisenia andrei. However, these species are absent from agricultural soils and often less sensitive to pesticides than other earthworm species found in mineral soils. To move towards a better assessment of pesticide effects on non-target organisms, there is a need to perform a posteriori tests using relevant species. The endogeic species Aporrectodea caliginosa (Savigny, 1826) is representative of cultivated fields in temperate regions and is suggested as a relevant model test species. After providing information on its taxonomy, biology, and ecology, we reviewed current knowledge concerning its sensitivity towards pesticides. Moreover, we highlighted research gaps and promising perspectives. Finally, advice and recommendations are given for the establishment of laboratory cultures and experiments using this soil-dwelling earthworm species.

How to assess the feeding activity in ecotoxicological laboratory tests using enchytraeids?

The risk assessment of pesticides on soil fauna is an issue to protect agroecosystem sustainability. Enchytraeids are recognized as relevant soil bioindicators of chemical stress in agroecosystems. In laboratory, the reproduction test was found to be sensitive to reveal chemical impacts on enchytraeids. However, it does not allow to assess the impacts on ecological functions in which enchytraeids are involved. The objectives of this study were (i) to explore the feasibility of the bait-lamina test with enchytraeids under laboratory conditions and (ii) to compare its sensitivity with the Enchytraeid Reproduction Test. We exposed individuals of Enchytraeus albidus to two commercial formulations of fungicides widely used in Europe. The EC50 reproduction for the Swing® Gold (50 g L-1 epoxiconazole and 133 g L-1 dimoxystrobin) and the Cuprafor micro® (50% copper oxychloride) were respectively estimated at 1.66 ± 0.93 times the recommended dose and > 496 mg kg-1 of copper. However, no impact was found on the feeding activity of enchytraeids. The bait-lamina test thus appeared less sensitive than the Enchytraeid Reproduction Test to the tested fungicides. Despite that, this test which is achievable under laboratory conditions and allows assessing indirect effects of pesticides is quick, cheap, and easy to perform. It would deserve to be used to explore longer-exposure effects through the repeated addition of bait-lamina sticks.

Short-term effects of two fungicides on enchytraeid and earthworm communities under field conditions.

Because of the wide use of pesticides in agriculture, there is still a need of higher-tier field studies to assess ecotoxicological effects of commercial formulations on a wider variety of non-target soil organisms such as soil annelids. We here tested the effects of different concentrations of two fungicide formulations, i.e., Cuprafor Micro® (composed of 500 g kg-1 copper oxychloride) and Swing Gold® (composed of 50 g l-1 epoxiconazole and 133 g l-1 dimoxystrobin) on two families of terrestrial oligochaetes (Lumbricidae and Enchytraeidae) after 1 month of exposure. We also assessed the feeding activity of soil organisms using the bait lamina method. Along with the feeding activity, the enchytraeid density, diversity and communities were not different in the control and the contaminated plots. By contrast, epigeic earthworms were absent and earthworm diversity and densities of anecic species decreased significantly in the plots contaminated at ten times the recommended dose of the Swing Gold® formulation. The copper fungicide (at 0.75 and 7.5 kg Cu ha-1) and the treatment with the pesticide mixture (Cuprafor Micro® at 0.75 kg Cu ha-1 and Swing Gold® at the recommended dose) did not affect Oligochaeta communities compared with the control, except the Shannon index for earthworms in the mixture of both fungicides. Responses of the two annelid families to the tested pesticides were different with higher effects observed on the diversity and the community structure of earthworms compared with enchytraeids. This study allowed detecting early changes on oligochaete populations after pesticide application.

Differences in sensitivity between earthworms and enchytraeids exposed to two commercial fungicides.

The use of pesticides in crop fields may have negative effects on soil Oligochaeta Annelida, i.e., earthworms and enchytraeids, and thus affect soil quality. The aim of this study was to assess the effects of two commercial fungicide formulations on the earthworm Aporrectodea caliginosa and the enchytraeid Enchytraeus albidus in a natural soil. The fungicides were Cuprafor micro® (copper oxychloride), commonly used in organic farming, and Swing Gold® (epoxiconazole and dimoxystrobin), a synthetic fungicide widely used in conventional farming to protect cereal crops. Laboratory experiments were used to assess the survival, biomass loss and avoidance behaviour. No lethal effect was observed following exposure to the copper fungicide for 14 days, even at 5000mgkg-1 of copper, i.e. 650 times the recommended dose (RD). However, a significant decrease in biomass was observed from 50mgkg-1 of copper (6.5 times the RD) for A. caliginosa and at 5000mgkg-1 of copper (650 times the RD) for E. albidus. These sublethal effects suggest that a longer period of exposure would probably have led to lethal effects. The EC50 avoidance for the copper fungicide was estimated to be 51.2mgkg-1 of copper (6.7 times the RD) for A. caliginosa, and 393mgkg-1 of copper (51 times the RD) for E. albidus. For the Swing Gold® fungicide, the estimated LC50 was 7.0 10-3mLkg-1 (6.3 times the RD) for A. caliginosa and 12.7 10-3mLkg-1 (11.0 times the RD) for E. albidus. No effect on biomass or avoidance was observed at sublethal concentrations of this synthetic fungicide. It was concluded that enchytraeids were less sensitive than earthworms to the two commercial fungicides in terms of mortality, biomass loss and avoidance behaviour. Therefore we discuss the different strategies possibly used by the two Oligochaeta species to cope with the presence of the pesticides were discussed, along with the potential consequences on the soil functions.

Transgenerational Adaptation to Pollution Changes Energy Allocation in Populations of Nematodes.

Assessing the evolutionary responses of long-term exposed populations requires multigeneration ecotoxicity tests. However, the analysis of the data from these tests is not straightforward. Mechanistic models allow the in-depth analysis of the variation of physiological traits over many generations, by quantifying the trend of the physiological and toxicological parameters of the model. In the present study, a bioenergetic mechanistic model has been used to assess the evolution of two populations of the nematode Caenorhabditis elegans in control conditions or exposed to uranium. This evolutionary pressure resulted in a brood size reduction of 60%. We showed an adaptation of individuals of both populations to experimental conditions (increase of maximal length, decrease of growth rate, decrease of brood size, and decrease of the elimination rate). In addition, differential evolution was also highlighted between the two populations once the maternal effects had been diminished after several generations. Thus, individuals that were greater in maximal length, but with apparently a greater sensitivity to uranium were selected in the uranium population. In this study, we showed that this bioenergetics mechanistic modeling approach provided a precise, certain, and powerful analysis of the life strategy of C. elegans populations exposed to heavy metals resulting in an evolutionary pressure across successive generations.

An individual-based model of zebrafish population dynamics accounting for energy dynamics.

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level.

Energy-based modelling to assess effects of chemicals on Caenorhabditis elegans: a case study on uranium.

The ubiquitous free-living nematode Caenorhabditis elegans is a powerful animal model for measuring the evolutionary effects of pollutants which is increasingly used in (eco) toxicological studies. Indeed, toxicity tests with this nematode can provide in a few days data on the whole life cycle. These data can be analysed with mathematical tools such as toxicokinetic-toxicodynamic modelling approaches. In this study, we assessed how a chronic exposure to a radioactive heavy metal (uranium) affects the life-cycle of C. elegans using a mechanistic model. In order to achieve this, we exposed individuals to a range of seven concentrations of uranium. Growth and reproduction were followed daily. These data were analysed with a model for nematodes based on the Dynamic Energy Budget theory, able to handle a wide range of plausible biological parameters values. Parameter estimations were performed using a Bayesian framework. Our results showed that uranium affects the assimilation of energy from food with a no-effect concentration (NEC) of 0.42 mM U which would be the threshold for effects on both growth and reproduction. The sensitivity analysis showed that the main contributors to the model output were parameters linked to the feeding processes and the actual exposure concentration. This confirms that the real exposure concentration should be measured accurately and that the feeding parameters should not be fixed, but need to be reestimated during the parameter estimation process.

A physiologically based toxicokinetic model for the zebrafish Danio rerio.

Zebrafish (Danio rerio) is a widely used model for toxicological studies, in particular those related to investigations on endocrine disruption. The development and regulatory use of in vivo and in vitro tests based on this species can be enhanced by toxicokinetic modeling. For this reason, we propose a physiologically based toxicokinetic (PBTK) model for zebrafish describing the uptake and disposition of organic chemicals. The model is based on literature data on zebrafish, other cyprinidae and other fish families, new experimental physiological information (volumes, lipids and water contents) obtained from zebrafish, and chemical-specific parameters predicted by generic models. The relevance of available models predicting the latter parameters was evaluated with respect to gill uptake and partition coefficients in zebrafish. This evaluation benefited from the fact that the influence of confounding factors such as body weight and temperature on ventilation rate was included in our model. The predictions for six chemicals (65 data points) yielded by our PBTK model were compared to available toxicokinetics data for zebrafish and 88% of them were within a factor of 5 of the corresponding experimental values. Sensitivity analysis highlighted that the 1-octanol/water partition coefficient, the metabolism rate, and all the parameters that enable the prediction of assimilation efficiency and partitioning of chemicals need to be precisely determined in order to allow an effective toxicokinetic modeling.

Effects of bisphenol A on different trophic levels in a lotic experimental ecosystem.

Bisphenol A (BPA) is commonly used by manufacturers and can be found in many aquatic ecosystems. Data relative to BPA ecotoxicity are only available for studies in laboratory conditions on macro-invertebrates and fish. There is thus a lack of information for other trophic levels such as macrophytes. Moreover, the impacts of BPA within an ecosystem context, i.e. with populations from different trophic levels studied at long term in environmental conditions, have never been assessed. We carried out a long-term lotic mesocosm study in 20 m long channels under three exposure concentrations of BPA (nominal concentrations of 0, 1, 10 and 100 µg/L) delivered continuously for 165 days. Three trophic levels were followed: macrophytes, macro-invertebrates (with a focus on Radix balthica) and fish (Gasterosteus aculeatus). Significant effects were shown at 100 µg/L BPA on the three trophic levels. BPA had a direct impact on macrophyte community structure, direct and indirect impacts on macro-invertebrates and on fish population structure. Gonad morphology of fish was affected at 1 and 10 µg/L of BPA, respectively for female and male sticklebacks. In addition to these ecotoxicity data, our results suggest that fish are good integrators of the responses of other communities (including macro-invertebrates and macrophytes) in mesocosm systems.

Consequences of a multi-generation exposure to uranium on Caenorhabditis elegans life parameters and sensitivity.

The assessment of toxic effects at biologically and ecologically relevant scales is an important challenge in ecosystem protection. Indeed, stressors may impact populations at much longer term than the usual timescale of toxicity tests. It is therefore important to study the evolutionary response of a population under chronic stress. We performed a 16-generation study to assess the evolution of two populations of the ubiquitous nematode Caenorhabditis elegans in control conditions or exposed to 1.1 mM of uranium. Several generations were selected to assess growth, reproduction, survival, and dose-responses relationships, through exposure to a range of concentrations (from 0 to 1.2 mM U) with all endpoints measured daily. Our experiment showed an adaptation of individuals to experimental conditions (increase of maximal length and decrease of fecundity) for both populations. We also observed an increase of adverse effects (reduction of growth and fertility) as a function of uranium concentration. We pointed out the emergence of population differentiation for reproduction traits. In contrast, no differentiation was observed on growth traits. Our results confirm the importance of assessing environmental risk related to pollutant through multi-generational studies.

A non-invasive method based on head morphology to sex mature three-spined stickleback (Gasterosteus aculeatus L.) in rearing conditions.

A mathematical model to distinguish mature female and male three-spined sticklebacks Gasterosteus aculeatus L. 1758 is proposed. This method is based on sexual dimorphism in the head morphology. The discrimination was established on five distances of interest on the head, adjusted by the standard length of fish. The parameters were estimated based on a training set composed of 102 fish with an equilibrium sex ratio and validated on a test set composed of 69 fish. The model estimates the relationship between the percentage of fish that can be sexed with our model and the percentage of fish correctly sexed. For instance, to reach 1% of error in the sex determination, only 53% of the fish should be considered, whereas to reach 5% of error, 90% of the fish can be used. Compared to other available methods to sex G. aculeatus, the model is non invasive, not expensive, rapid, replicable, and can be calibrated outside of the breeding period.

Prediction of dose-hepatotoxic response in humans based on toxicokinetic/toxicodynamic modeling with or without in vivo data: a case study with acetaminophen.

In the present legislations, the use of methods alternative to animal testing is explicitly encouraged, to use animal testing only 'as a last resort' or to ban it. The use of alternative methods to replace kinetics or repeated dose in vivo tests is a challenging issue. We propose here a strategy based on in vitro tests and QSAR (Quantitative Structure Activity Relationship) models to calibrate a dose-response model predicting hepatotoxicity. The dose response consists in calibrating and coupling a PBPK (physiologically-based pharmacokinetic) model with a toxicodynamic model for cell viability. We applied our strategy to acetaminophen and compared three different ways to calibrate the PBPK model: only with in vitro and in silico methods, using rat data or using all available data including data on humans. Some estimates of kinetic parameters differed substantially among the three calibration processes, but, at the end, the three models were quite comparable in terms of liver toxicity predictions and close to the usual range of human overdose. For the model based on alternative methods, the good adequation with the two other models resulted from an overestimated renal elimination rate which compensated for the underestimation of the metabolism rate. Our study points out that toxicokinetics/toxicodynamics approaches, based on alternative methods and modelling only, can predict in vivo liver toxicity with accuracy comparable to in vivo methods.

Comparison of species sensitivity distributions based on population or individual endpoints.

Species sensitivity distributions (SSDs) developed from individual and population endpoints were compared based on simulations and a case study. The simulations were performed with five invertebrate species accounting for the diversity of benthic macroinvertebrate communities in large European lowland rivers and for five benthic invertebrates used as laboratory species. Population growth rate 10% effective concentration (EC10) values were, in most of the simulations, higher than the lowest of the EC10 values at the individual level. However, for the set of ecologically representative species, the fifth percentile level of this distribution (HC5) was more protective for population endpoints than for individual endpoints. This was the opposite for the set of laboratory species. Population and individual SSDs were also compared based on existing data on Cu for the five laboratory invertebrate species. In this case, the calculated population HC5 value was almost twice the individual value, and the authors showed much reduced variability between species sensitivities at population level compared with individual level. They conclude that population-based HC5 would generally be more protective than individual-based HC5. However, the change of level could reveal higher homogeneity at population level than at individual level, supporting the use of population-based HC5 to avoid overprotection. The authors thus advise the derivation of population-based HC5, as soon as it is possible, to derive such value with a relevant panel of species.

Individual sensitivity distribution evaluation from survival data using a mechanistic model: implications for ecotoxicological risk assessment.

Two main alternatives are typically used to model mechanistically dose-survival relationship in ecotoxicity tests. Effects are related to a concentration of concern, for instance body concentration, and, to account for their differences relative to time-to-death, individuals have either different concentration thresholds for death ("individual tolerance approach"), or equal probability to die, with death occurring randomly ("stochastic death approach"). A general framework to unify both approaches has recently been proposed. We derived a model from this framework to analyse five datasets (daphnids exposed to selenium, guppies exposed to dieldrin and second, third and fourth instars chironomids exposed to copper), by extending the standard stochastic death approach. We showed the possibility to estimate properly the toxicity parameters together with inter-organisms differences of sensitivity for at least one of these parameters (here the threshold for effect). For the daphnids, there was no improvement of using the extended model, which confirms the expected low variability among genetically identical individuals. For all the other datasets, our model outperformed the standard approach without accounting for differences of sensitivity. We estimated coefficients of variations in the distribution of the logarithm of the threshold from 44% to 4% and showed, for chironomids, a decrease of inter-individual differences of sensitivity with the age of the larvae. All standard threshold estimates were close but above the medium value of the distribution in the new approach, which means that a concentration equal to the standard threshold would ultimately result in the death of more than half of the exposed organisms. A more relevant parameter, such as the concentration protecting 95% of the population, would be 2-4 times inferior to the standard threshold.

A multi-scale modeling framework for individualized, spatiotemporal prediction of drug effects and toxicological risk.

In this study, we focus on a novel multi-scale modeling approach for spatiotemporal prediction of the distribution of substances and resulting hepatotoxicity by combining cellular models, a 2D liver model, and whole body model. As a case study, we focused on predicting human hepatotoxicity upon treatment with acetaminophen based on in vitro toxicity data and potential inter-individual variability in gene expression and enzyme activities. By aggregating mechanistic, genome-based in silico cells to a novel 2D liver model and eventually to a whole body model, we predicted pharmacokinetic properties, metabolism, and the onset of hepatotoxicity in an in silico patient. Depending on the concentration of acetaminophen in the liver and the accumulation of toxic metabolites, cell integrity in the liver as a function of space and time as well as changes in the elimination rate of substances were estimated. We show that the variations in elimination rates also influence the distribution of acetaminophen and its metabolites in the whole body. Our results are in agreement with experimental results. What is more, the integrated model also predicted variations in drug toxicity depending on alterations of metabolic enzyme activities. Variations in enzyme activity, in turn, reflect genetic characteristics or diseases of individuals. In conclusion, this framework presents an important basis for efficiently integrating inter-individual variability data into models, paving the way for personalized or stratified predictions of drug toxicity and efficacy.