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We thank BASF, Bayer CropScience, CEFIC LRI, INRA, Makhteshim-Agan, Sumitomo Chemical, and Syngenta for providing funds to support the MODELINK workshop. We also thank our colleagues at that workshop for their discussions and inputs. We are particularly grateful to Daniela Jolli and Alf Aagaard for their contribution to the discussions on this case study. Finally, we thank 2 anonymous reviewers for their help in improving this manuscript.; Recent guidance identified toxicokinetic-toxicodynamic (TK-TD) modeling as a relevant approach for risk assessment refinement. Yet, its added value compared to other refinement options is not detailed, and how to conduct the modeling appropriately is not explained. This case study addresses these issues through 2 examples of individual-level risk assessment for 2 hypothetical plant protection products: 1) evaluating the risk for small granivorous birds and small omnivorous mammals of a single application, as a seed treatment in winter cereals, and 2) evaluating the risk for fish after a pulsed treatment in the edge-of-field zone. Using acute test data, we conducted the first tier risk assessment as defined in the European Food Safety Authority (EFSA) guidance. When first tier risk assessment highlighted a concern, refinement options were discussed. Cases where the use of models should be preferred over other existing refinement approaches were highlighted. We then practically conducted the risk assessment refinement by using 2 different models as examples. In example 1, a TK model accounting for toxicokinetics and relevant feeding patterns in the skylark and in the wood mouse was used to predict internal doses of the hypothetical active ingredient in individuals, based on relevant feeding patterns in an in-crop situation, and identify the residue levels leading to mortality. In example 2, a TK-TD model accounting for toxicokinetics, toxicodynamics, and relevant exposure patterns in the fathead minnow was used to predict the time-course of fish survival for relevant FOCUS SW exposure scenarios and identify which scenarios might lead to mortality. Models were calibrated using available standard data and implemented to simulate the time-course of internal dose of active ingredient or survival for different exposure scenarios. Simulation results were discussed and used to derive the risk assessment refinement endpoints used for decision. Finally, we compared the "classical" risk assessment approach with the model-based approach. These comparisons showed that TK and TK-TD models can bring more realism to the risk assessment through the possibility to study realistic exposure scenarios and to simulate relevant mechanisms of effects (including delayed toxicity and recovery). Noticeably, using TK-TD models is currently the most relevant way to directly connect realistic exposure patterns to effects. We conclude with recommendations on how to properly use TK and TK-TD model in acute risk assessment for vertebrates. Integr Environ Assess Manag 2016; 12: 32-45. (C) 2015 SETAC

Toxicokinetic-toxicodynamic (TKTD) modeling offers many advantages in the analysis of ecotoxicity test data. Calibration of TKTD models, however, places different demands on test design compared with classical concentration-response approaches. In the present study, useful complementary information is provided regarding test design for TKTD modeling. A case study is presented for the pond snail Lymnaea stagnalis exposed to the narcotic compound acetone, in which the data on all endpoints were analyzed together using a relatively simple TKTD model called DEBkiss. Furthermore, the influence of the data used for calibration on accuracy and precision of model parameters is discussed. The DEBkiss model described toxic effects on survival, growth, and reproduction over time well, within a single integrated analysis. Regarding the parameter estimates (e. g., no-effect concentration), precision rather than accuracy was affected depending on which data set was used for model calibration. In addition, the present study shows that the intrinsic sensitivity of snails to acetone stays the same across different life stages, including the embryonic stage. In fact, the data on egg development allowed for selection of a unique metabolic mode of action for the toxicant. Practical and theoretical considerations for test design to accommodate TKTD modeling are discussed in the hope that this information will aid other researchers to make the best possible use of their test animals.

Endocrine disruptors are known to alter endogenous free and esterified levels of androgenic and estrogenic steroid hormones in aquatic mollusks. The origin of steroids in these animals, however, remains controversial. In the present study, free and esterified testosterone concentrations were measured in the hermaphroditic aquatic gastropod Lymnaea stagnalis exposed to molecules known for their androgenic (testosterone and tributyltin), anti-androgenic (cyproterone-acetate), and estrogenic (chlordecone) properties, by reference to their mode of action in vertebrates. In parallel, snail oviposition and fecundity were followed over a 21-d exposure period. Testosterone exposure resulted in increased esterified testosterone levels, whereas free testosterone concentrations remained stable. In contrast, cyproterone-acetate significantly increased the free form of testosterone with no changes in the esterified form, whereas chlordecone showed a tendency to reduce (though not significantly) esterified testosterone concentrations without changing free testosterone levels. Finally, tributyltin did not alter testosterone homeostasis. The production of egg clutches and eggs was significantly reduced only in the snails exposed to the highest concentrations of chlordecone (19.6 mu g/L) and tributyltin (94.2ng Sn/L). Overall, the present study demonstrates that uptake of testosterone from the exposure medium occurs in L. stagnalis. Moreover, it shows that cyproterone-acetate and, to a lesser extent, chlordecone can alter endogenous testosterone levels in this freshwater snail. However, the relationship between hormonal changes and snail reproduction has not been established. Environ Toxicol Chem 2013;32:1740-1745. (c) 2013 SETAC

Regulation mostly relies upon the results of standard toxicity tests that have been validated for a reduced set of species and endpoints. This pragmatic strategy ensures the reliability of toxicological data. Yet, current practices might not appropriately cover the broad range of toxicant mode of action (e.g., endocrine disruptors - EDCs). In particular, the standard approach may fail to provide realistic risk forecasts when the test design is not sensitive enough to highlight toxicity and when the various standard tests provide conflicting results. Through two case studies, we explain why the standard approach may not allow assessing properly the risk of reprotoxic pesticides in invertebrates. The first case study illustrates current issues in risk assessment of EDCs. In this context, we show how non-standard partial life-cycle (PLC) tests allow identifying sensitive development phases/endpoints to EDCs (e.g., anti-androgens) along the life-cycle of Lymnaea stagnalis, a candidate species for the development of forthcoming OECD test guidelines. We also show how non-standard data analysis methods, such as mixed effect models, can be used to avoid underestimation of EDCs toxicity. The second case study illustrates how a non-standard approach can contribute to provide complementary data when standard approaches led to conflicting risk forecasts. In this context, we repeatedly exposed L. stagnalis to pulses of diquat and monitored life-cycle traits. We show how non-standard full life-cycle (FLC) tests allow assessing effective diquat concentrations more realistically and why non-standard models, such as TK-TD models, are relevant to characterise the time-course of biological responses to complex exposure patterns. For each case study, we show how to derive relevant effects criteria and include them in the regulatory risk assessment process. We then investigate whether including this additional data might modify current risk forecasts for the studied compounds. This study illustrates how non-standard test procedures of different nature may contribute to ecological risk assessment of chemicals, when standard procedure are lacking or provide conflicting results. Most importantly, the use of some simple but relevant PLC and FLC tests methods with L. stagnalis should be promoted when standard approaches are suspected to provide unrealistic risk forecasts.

Extrapolation methods in aquatic effect assessment of time-variable exposures to pesticides

This paper provides original collection, acclimatizing, rearing and toxicity test methods for the freshwater worm Branchiura sowerbyi, an alternative species to Tubifex tubifex for ecotoxicity evaluation of sediments. Influence of the substrate, type of food, and feeding level on individual performances was assessed in short-term tests, in order to set up optimal culture and test conditions. Low-size particles and high organic matter content favoured the growth and reproduction of B. sowerbyi. The relative contribution of sediments and fish food to the individual food intake was assessed using a foraging efficiency model based upon the dynamic energy budget theory. Individual performances were optimal when the substrate plus fish food provided the energy equivalent to 5 mg Tetramin® per worm per day, which is the ad libitum food level for adults at 21 °C. The life-cycle of the worm was fully characterized using a life-cycle test conducted under the previously defined optimal conditions. Hatching rates were low (32%), whereas newborn and juveniles exhibited high survival (> 80%) and growth (2.4 mg/day in juveniles) rates. Age at puberty was low (60 days) when compared to the maximal life span (1100 days) as predicted using a Weibull model. Adults reproduced every other month with a constant fecundity (0.16 cocoon/worm/day). The mean values of the life-cycle parameters and their variability and reproducibility among laboratory studies were discussed in order to identify relevant endpoints to be used in ecotoxicity tests. Survival, juvenile growth, and fecundity may constitute suitable test endpoints, whereas hatching rate and adult growth should not be used as endpoints in B. sowerbyi.

We sought to improve the ecological relevance of the existing effect assessment methods for benthic invertebrate communities. We have thus developed a method, which permits to build test batteries that are representative of the functioning of natural communities. It is based on the statistical analysis of the combinations of 22 biological and ecological traits for 479 western European invertebrate species. We used this method in order to build a test battery that represents the functioning of benthic communities that inhabit fine sediments of lowland rivers and lakes in Western Europe. This battery gathers five species: Chironomus riparius, Valvata piscinalis, Branchiura sowerbyi, Lumbriculus variegatus and Sericostoma personatum. We have then sought to characterize the biological response to zinc of lab populations for C. riparius, V. piscinalis and B. sowerbyi. For these species, we developed handling and breeding methods and described their life cycle at the lab in clean sediments. Then, we ran toxicity tests to characterize the effects of zinc on individual survival, growth and reproduction. Test data were first analyzed at the individual level using mechanistic models based on the perturbation of energy budgets by the toxicant. On the basis of both the data from toxicity tests and the effects models, we estimated the effects of zinc at the population level using matrix population models. Finally, we used population level results to derive a no effect concentration for the benthic community. The study of population effects of zinc on C. riparius, V. piscinalis and B. sowerbyi allowed us to estimate (at the lab) a no effect concentration for the benthic community, which is reliable when compared to field results. The validation and the adaptation the methods we used for routine effect assessment of chemicals represents the major prospects of our work., Nous avons cherché à améliorer la pertinence écologique des méthodes existantes d'évaluation des effets des substances chimiques pour les communautés d'invertébrés benthiques. Nous avons tout d'abord mis au point une méthode qui permet de construire des batteries de test représentatives du fonctionnement des communautés naturelles grâce à l'analyse statistique des combinaisons de 22 caractéristiques biologiques et écologiques pour 479 espèces d'invertébrés rencontrées en Europe de l'Ouest. Nous avons utilisé cette méthode afin de construire une batterie de test représentative de la communauté d'invertébrés inféodés aux sédiments de zone aval de cours d'eau et de lacs. Cette batterie est constituée de cinq espèces: Chironomus riparius, Valvata piscinalis, Branchiura sowerbyi, Lumbriculus variegatus et Sericostoma personatum. Nous avons alors caractérisé la réponse biologique au zinc des populations de laboratoire pour C. riparius, V. piscinalis et B. sowerbyi. Pour ce faire, nous avons mis au point des méthodes d'élevage, décrit le cycle de vie en laboratoire en milieu propre et réalisé des tests de toxicité afin de caractériser les effets d'une contamination du sédiment par le zinc sur la survie, la croissance et la reproduction des organismes. Ces effets ont été analysés au niveau individuel grâce à des modèles basés sur la perturbation de la gestion de l'énergie par le toxique. A partir de données individuelles, nous avons estimé les effets attendus au niveau des populations en couplant ces modèles d'effets sur les individus avec des modèles biodémographiques matriciels basés sur l'étude du cycle de vie des organismes. L'étude de la sensibilité au zinc des populations de C. riparius, V. piscinalis et B. sowerbyi nous a permis d'estimer une concentration sans effet du zinc sur les communautés benthiques qui est relativement fiable par rapport aux résultats observés sur le terrain. La validation et l'adaptation des outils proposés en vue d'une utilisation en routine pour l'évaluation des effets des substances chimiques représentent les perspectives majeures de la thèse.

Sediment particles provide habitat and food to many aquatic invertebrates during their whole development or during part of it. The amounts of chemicals accumulated in the sediment from surface water are of great concern for benthic communities. Consequently, sediment toxicity assessment has become a critical issue. Acute and chronic effects of sediment binding chemicals are commonly studied via bioassays involving Chironomidae. The measurement of deleterious effects on individual endpoints generally constitutes the basis of sediment toxicity evaluation. Several studies have shown that the ecological relevance of this evaluation could be improved by studying toxic effects at the population level. We thus set up a model which allows to infer the effects expected at the population level from the effects modelled at the individual level. Individual-scaled models were based on mechanistic hypotheses concerning the use of energy during chironomids` life cycle and its perturbation by the toxicant. Individual to population models were based on basic population dynamics principles and permitted to predict equilibrium density under toxic stress. Our models were applied to the evaluation of the toxicity of zinc-spiked sediment to populations of C. riparius. Presence of zinc above 1794 mg/kg caused behavioural effects and a significant decrease of survival in second instar larva. There were no acute effects on survival in third and fourth larval instars, but chronic effects on survival were significant for these latter instars above 1382 mg/kg. A significant decrease of growth was observed for every instar when zinc concentration in the sediment were higher than 523 mg/kg. Mean emergence time was significantly increased above 953 mg/kg, thus reducing mating probability in exposed organisms. When mating occurred, fecundity was significantly reduced above the first tested concentration: 523 mg/kg. Effects on populations were modelled on the basis of these experimental data. No effects on population density were observed under 300 mg/kg (NOEC). EC30 was 661.3 mg/kg: above this level, chironomid populations might be significantly endangered by zinc contamination.

Test species for bioassays are commonly selected on the basis of their breeding and handling facilities. These species thus provide hardly toxicological and ecological relevance to laboratory data. Biological and ecological traits of species are involved (1) in the structure and functioning of communities, (2) in species sensitivity to toxicant, and (3) in toxicity mechanisms. We thus believe that the analysis of bio/ecological trait patterns of taxa may be a relevant basis for test battery building. Using fuzzy coded information on 11 biological traits and 9 ecological traits for 700 West European macroinvertebrate taxa, we (1) selected 109 potential representatives of benthic communities inhabiting potamic environments in lowland rivers (2) assembled selected taxa in successively larger faunal groups with 'similar' suites of traits, using co-inertia analyses and a classification procedure (3) selected a species, which were the best representatives of each functional group and included them in an "ecologically relevant" test battery. The selected taxa were Branchiura sowerbyi, Lumbriculus variegatus (Oligochaeta), Valvata piscinalis (Gasteropoda), Chironomus riparius (Diptera), and Sericostoma personatum (Trichoptera). This test battery accounts for the functional diversity of lowland river benthic communities, and also for various routes of exposure and toxicant modes of action at individual and population levels. Our original species selection method makes it possible to intrinsically integrate ecological knowledge into a toxicologically designed tool and can easily be applied to any aquatic ecosystem. It should therefore provide relevant insights into improvement of risk assessment methods.

Considering the huge variability of sensitivity (up to 106) to a toxicant among species, the choice of the tested organism can dramatically affect toxicity evaluation. The variability of sensitivity among taxa has thus to be accounted for in risk assessment methods. To fulfil this need, between-taxa differences of sensitivity to toxicants have to be better understood. We hypothesize that some biological and ecological traits can explain a large part of the between-taxa variability of sensitivity to a compound. On this hypothesis, 11 biological and 9 ecological traits that we believe to be directly involved either with the exposure level or with toxicity mechanisms at the individual scale have been selected. Using multivariate analyses and classification methods, we clustered 109 benthic invertebrate species (previously selected for their habitat characteristics from faunal lists) in 6 functional groups according to their similarities in terms of combinations of the selected biological and ecological traits. The clustering of species into functional groups allows us (1) to consider the organisms within a group as relevant combinations of biological and ecological traits, permitting us to link sensitivity with these traits; (2) to set up a rational basis for species selection on a toxicity testing perspective. The resulting taxa selection includes Chironomus riparius, Branchiura sowerbyi, Lumbriculus variegatus, Valvata piscinalis and Sericostoma personatum. Testing protocols will be developed for the non standard testing species., Nous proposons une sélection d'une batterie de tests de toxicité sur invertébrés qui se fonde sur un regroupement selon 9 critères écologiques et 11 critères biologiques pour rendre compte en partie des différences de sensibilité entre espèces. Nous sommes parvenus à la sélection de 6 groupes avec comme représentants : Chironomus riparius, Branchiura sowerbyi, Lumbriculus variegatus, Valvata piscinalis et Sericostoma personatum.

Considering the huge variability of sensitivity (up to 106) to a toxicant among species, the choice of the tested organism can dramatically affect toxicity evaluation. The variability of sensitivity among taxa has thus to be accounted for in risk assessment methods. To fulfil this need, between-taxa differences of sensitivity to toxicants have to be better understood. We hypothesize that some biological and ecological traits can explain a large part of the between-taxa variability of sensitivity to a compound. On this hypothesis, 11 biological and 9 ecological traits that we believe to be directly involved either with the exposure level or with toxicity mechanisms at the individual scale have been selected. Using multivariate analyses and classification methods, we clustered 109 benthic invertebrate species (previously selected for their habitat characteristics from faunal lists) in 6 functional groups according to their similarities in terms of combinations of the selected biological and ecological traits. The clustering of species into functional groups allows us (1) to consider the organisms within a group as relevant combinations of biological and ecological traits, permitting us to link sensitivity with these traits; (2) to set up a rational basis for species selection on a toxicity testing perspective. The resulting taxa selection includes Chironomus riparius, Branchiura sowerbyi, Lumbriculus variegatus, Valvata piscinalis and Sericostoma personatum. Testing protocols will be developed for the non standard testing species., Nous proposons une sélection d'une batterie de tests de toxicité sur invertébrés qui se fonde sur un regroupement selon 9 critères écologiques et 11 critères biologiques pour rendre compte en partie des différences de sensibilité entre espèces. Nous sommes parvenus à la sélection de 6 groupes avec comme représentants : Chironomus riparius, Branchiura sowerbyi, Lumbriculus variegatus, Valvata piscinalis et Sericostoma personatum.

Integrated environmental assessment and management 12(1), 21-31 (2016). doi:10.1002/ieam.1704, Published by Wiley, Hoboken,NJ