New Insights to Compare and Choose TKTD Models for Survival Based on an Interlaboratory Study for Lymnaea stagnalis Exposed to Cd.

Toxicokinetic-toxicodynamic (TKTD) models, as the General Unified Threshold model of Survival (GUTS), provide a consistent process-based framework compared to classical dose-response models to analyze both time and concentration-dependent data sets. However, the extent to which GUTS models (Stochastic Death (SD) and Individual Tolerance (IT)) lead to a better fitting than classical dose-response model at a given target time (TT) has poorly been investigated. Our paper highlights that GUTS estimates are generally more conservative and have a reduced uncertainty through smaller credible intervals for the studied data sets than classical TT approaches. Also, GUTS models enable estimating any x% lethal concentration at any time (LCx,t), and provide biological information on the internal processes occurring during the experiments. While both GUTS-SD and GUTS-IT models outcompete classical TT approaches, choosing one preferentially to the other is still challenging. Indeed, the estimates of survival rate over time and LCx,t are very close between both models, but our study also points out that the joint posterior distributions of SD model parameters are sometimes bimodal, while two parameters of the IT model seems strongly correlated. Therefore, the selection between these two models has to be supported by the experimental design and the biological objectives, and this paper provides some insights to drive this choice.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68

Hormesis on life-history traits: is there such thing as a free lunch?

The term "hormesis" is used to describe dose-response relationships where the response is reversed between low and high doses of a stressor (generally, stimulation at low doses and inhibition at high ones). A mechanistic explanation is needed to interpret the relevance of such responses, but there does not appear to be a single universal mechanism underlying hormesis. When the endpoint is a life-history trait such as growth or reproduction, a stimulation of the response comes with costs in terms of resources. Organisms have to obey the conservation laws for mass and energy; there is no such thing as a free lunch. Based on the principles of Dynamic Energy Budget theory, we introduce three categories of explanations for hormesis that obey the conservation laws: acquisition (i.e., increasing the input of energy into the individual), allocation (i.e., rearranging the energy flows over various traits) and medication (e.g., the stressor is an essential element or acts as a cure for a disease or infection). In this discussion paper, we illustrate these explanations with cases where they might apply, and elaborate on the potential consequences for field populations.

How to account for the uncertainty from standard toxicity tests in species sensitivity distributions: An example in non-target plants.

This research proposes new perspectives accounting for the uncertainty on 50% effective rates (ER50) as interval input for species sensitivity distribution (SSD) analyses and evaluating how to include this uncertainty may influence the 5% Hazard Rate (HR5) estimation. We explored various endpoints (survival, emergence, shoot-dry-weight) for non-target plants from seven standard greenhouse studies that used different experimental approaches (vegetative vigour vs. seedling emergence) and applied seven herbicides at different growth stages. Firstly, for each endpoint of each study, a three-parameter log-logistic model was fitted to experimental toxicity test data for each species under a Bayesian framework to get a posterior probability distribution for ER50. Then, in order to account for the uncertainty on the ER50, we explored two censoring criteria to automatically censor ER50 taking the ER50 probability distribution and the range of tested rates into account. Secondly, based on dose-response fitting results and censoring criteria, we considered input ER50 values for SSD analyses in three ways (only point estimates chosen as ER50 medians, interval-censored ER50 based on their 95% credible interval and censored ER50 according to one of the two criteria), by fitting a log-normal distribution under a frequentist framework to get the three corresponding HR5 estimates. We observed that SSD fitted reasonably well when there were at least six distinct intervals for the ER50 values. By comparing the three SSD curves and the three HR5 estimates, we shed new light on the fact that both propagating the uncertainty from the ER50 estimates and including censored data into SSD analyses often leads to smaller point estimates of HR5, which is more conservative in a risk assessment context. In addition, we recommend not to focus solely on the point estimate of the HR5, but also to look at the precision of this estimate as depicted by its 95% confidence interval.

Key considerations to inform operational EU-specific protection goals: An example for non-target terrestrial plants.

This paper complements recent considerations of specific protection goals (SPG) to inform risk assessments for non-target terrestrial plants (NTTP) in the European Union. The SPG options in-field appear to be of the most disruptive potential from agronomic perspective and are therefore investigated in more detail. Overarching prerequisites have been identified that need to be accounted for to ensure that any of the potential SPG options remain operational in a sustainable agricultural context. As soon as crop production is considered a desired ecosystem service for the in-field, its specific requirements in the context of sustainable agriculture have to be factored in. Good agricultural practices (GAPs), potential ecosystem disservices (e.g. weeds, pests and diseases) and supporting and regulating services need to be considered to ensure a successful and sustainable delivery of the ecosystem service crop production. Concerning in-field SPG options for NTTP specifically GAPs related to integrated weed management (IWM) require detailed assessment, as they individually and in combination have the purpose of weed control. Therefore, they result in specific implications to the environment, ecosystem services and biodiversity within the context of sustainable agricultural production. When diverging in-field ecosystem services are considered for the same context, the protection goals options require an additional assessment of synergies and trade-offs between the relevant ecosystem services (e.g. crop production, climate regulation and aesthetic values), a corresponding weighing and prioritization. Similarly, for biodiversity conservation, the trade-offs and synergies between sustainable crop production and specific habitat requirements need to be accounted for. Consequently, an interdisciplinary approach can ensure that SPG are operational by integrating a broad understanding of cropping systems, the environmental impact of the tools a farmer uses and the link between habitat availability, the impact of any of the applied tools on habitat quality and the broader landscape context. Integr Environ Assess Manag 2021;17:905-910. © 2021 Bayer AG, BASF SE and Syngenta. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of partial life-cycle experiments to assess the effects of endocrine disruptors on the freshwater gastropod Lymnaea stagnalis: a case-study with vinclozolin.

Long-term effects of endocrine disruptors (EDs) on aquatic invertebrates remain difficult to assess, mainly due to the lack of appropriate sensitive toxicity test methods and relevant data analysis procedures. This study aimed at identifying windows of sensitivity to EDs along the life-cycle of the freshwater snail Lymnaea stagnalis, a candidate species for the development of forthcoming test guidelines. Juveniles, sub-adults, young adults and adults were exposed for 21 days to the fungicide vinclozolin (VZ). Survival, growth, onset of reproduction, fertility and fecundity were monitored weekly. Data were analyzed using standard statistical analysis procedures and mixed-effect models. No deleterious effect on survival and growth occurred in snails exposed to VZ at environmentally relevant concentrations. A significant impairment of the male function occurred in young adults, leading to infertility at concentrations exceeding 0.025 µg/L. Furthermore, fecundity was impaired in adults exposed to concentrations exceeding 25 µg/L. Biological responses depended on VZ concentration, exposure duration and on their interaction, leading to complex response patterns. The use of a standard statistical approach to analyze those data led to underestimation of VZ effects on reproduction, whereas effects could reliably be analyzed by mixed-effect models. L. stagnalis may be among the most sensitive invertebrate species to VZ, a 21-day reproduction test allowing the detection of deleterious effects at environmentally relevant concentrations of the fungicide. These results thus reinforce the relevance of L. stagnalis as a good candidate species for the development of guidelines devoted to the risk assessment of EDs.

A Field Study Method as a Potential Higher Tier Option to Refine Herbicide Risk Assessment for Nontarget Terrestrial Plants.

During herbicide spray application, nontarget terrestrial plants (NTTPs) growing in the off-field area need to be protected from unacceptable effects of herbicide drift. The risk of such unintended effects is assessed in order to establish whether a particular use can be approved, possibly in combination with mitigation measures. In Europe, the risk of herbicide treatment to NTTPs is assessed on the basis of tier 2 studies done under controlled conditions in greenhouses. Following the concept of a tiered testing approach, higher tier field studies under more realistic conditions could be used to refine the risk assessment. No current guideline for conducting higher tier NTTP field studies is available. We developed an NTTP higher tier field study method done on an experimental plant community established by sowing of a seed mixture. The setup was optimized in 3 pilot field studies and subsequently used for a definitive study testing effects of the herbicide iofensulfuron-sodium. Results show that the method can be regarded as a suitable higher tier option for assessing effects of herbicides on NTTPs. Growth of species from the soil seed bank cannot be avoided and has to be carefully considered when evaluating results. Adaptations of the study design may be necessary when testing different herbicides. Community-level endpoints were at the same level as single-species endpoints. Results of the field study were compared to standard greenhouse study results for the same herbicide. No observed effect rates (NOERs) in the field were about a factor of 10 higher and show that the current tier 2 risk assessment for NTTPs can be regarded as protective in this case. Whether the present field study design and the assessed endpoints can be used in higher tier risk assessment of NTTPs depends on selection of the specific protection goal and requires further discussion. Integr Environ Assess Manag 2020;16:691-705. © 2020 Bayer AG. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Predicting impacts of chemicals from organisms to ecosystem service delivery: A case study of insecticide impacts on a freshwater lake.

Assessing and managing risks of anthropogenic activities to ecological systems is necessary to ensure sustained delivery of ecosystem services for future generations. Ecological models provide a means of quantitatively linking measured risk assessment endpoints with protection goals, by integrating potential chemical effects with species life history, ecological interactions, environmental drivers and other potential stressors. Here we demonstrate how an ecosystem modeling approach can be used to quantify insecticide-induced impacts on ecosystem services provided by a lake from toxicity data for organism-level endpoints. We used a publicly available aquatic ecosystem model AQUATOX that integrates environmental fate of chemicals and their impacts on food webs in aquatic environments. By simulating a range of exposure patterns, we illustrated how exposure to a hypothetical insecticide could affect aquatic species populations (e.g., recreational fish abundance) and environmental properties (e.g., water clarity) that would in turn affect delivery of ecosystem services. Different results were observed for different species of fish, thus the decision to manage the use of the insecticide for ecosystem services derived by anglers depends upon the favored species of fish. In our hypothetical shallow reservoir, water clarity was mostly driven by changes in food web dynamics, specifically the presence of zooplankton. In contrast to the complex response by fishing value, water clarity increased with reduced insecticide use, which produced a monotonic increase in value by waders and swimmers. Our study clearly showed the importance of considering nonlinear ecosystem feedbacks where the presence of insecticide changed the modeled food-web dynamics in unexpected ways. Our study highlights one of the main advantages of using ecological models for risk assessment, namely the ability to generalize to meaningful levels of organization and to facilitate quantitative comparisons among alternative scenarios and associated trade-offs among them while explicitly accounting for different groups of beneficiaries.

Predicting impacts of chemicals from organisms to ecosystem service delivery: A case study of endocrine disruptor effects on trout.

We demonstrate how mechanistic modeling can be used to predict whether and how biological responses to chemicals at (sub)organismal levels in model species (i.e., what we typically measure) translate into impacts on ecosystem service delivery (i.e., what we care about). We consider a hypothetical case study of two species of trout, brown trout (Salmo trutta; BT) and greenback cutthroat trout (Oncorhynchus clarkii stomias; GCT). These hypothetical populations live in a high-altitude river system and are exposed to human-derived estrogen (17α­ethinyl estradiol, EE2), which is the bioactive estrogen in many contraceptives. We use the individual-based model inSTREAM to explore how seasonally varying concentrations of EE2 could influence male spawning and sperm quality. Resulting impacts on trout recruitment and the consequences of such for anglers and for the continued viability of populations of GCT (the state fish of Colorado) are explored. inSTREAM incorporates seasonally varying river flow and temperature, fishing pressure, the influence of EE2 on species-specific demography, and inter-specific competition. The model facilitates quantitative exploration of the relative importance of endocrine disruption and inter-species competition on trout population dynamics. Simulations predicted constant EE2 loading to have more impacts on GCT than BT. However, increasing removal of BT by anglers can enhance the persistence of GCT and offset some of the negative effects of EE2. We demonstrate how models that quantitatively link impacts of chemicals and other stressors on individual survival, growth, and reproduction to consequences for populations and ecosystem service delivery, can be coupled with ecosystem service valuation. The approach facilitates interpretation of toxicity data in an ecological context and gives beneficiaries of ecosystem services a more explicit role in management decisions. Although challenges remain, this type of approach may be particularly helpful for site-specific risk assessments and those in which tradeoffs and synergies among ecosystem services need to be considered.

Modelling effects of time-variable exposure to the pyrethroid beta-cyfluthrin on rainbow trout early life stages.

BACKGROUND: Available literature and regulatory studies show that the severity of effects of beta-cyfluthrin (a synthetic pyrethroid) on fish is influenced by the magnitude and duration of exposure. To investigate how the exposure pattern to beta-cyfluthrin (constant vs peak) may influence the response of the fish, we used a mechanistic effect model to predict the survival and growth of the rainbow trout over its early life stages (i.e. egg, alevin and swim-up fry). We parameterized a toxicokinetic-toxicodynamic (TKTD) module in combination with a dynamic energy budget model enabling us to describe uptake and elimination, as well as to predict the threshold concentration for survival and sublethal effects (feeding behaviour and growth). This effect model was calibrated using data from an early life stage experiment where trout was exposed to a constant concentration of cyfluthrin. The model was validated by comparing model predictions to independent data from a pulsed-exposure study with early life stages of rainbow trout. RESULTS: The co-occurrence of effects on behaviour and growth raised the possibility that these were interrelated, i.e. impairment of feeding behaviour may have led to reduced food intake and slower growth. We, therefore, included 'effect on feeding' as mode of action in the TKTD module. At higher concentrations, the constant exposure led to death. The model was able to adequately capture this effect pattern in the calibration. The model was able to adequately predict the response of fish eggs, alevins and swim-up fry, from both the qualitative (response pattern) and quantitative points of view. CONCLUSIONS: Since the model was successfully validated, it can be used to predict survival and growth of early life stages under various realistic time-variable exposure profiles (e.g. profiles from FOCUS surface water modelling) of beta-cyfluthrin.

Rearing and estimation of life-cycle parameters of the tubicifid worm Branchiura sowerbyi: application to ecotoxicity testing.

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 degrees 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.

Dynamic energy budget as a basis to model population-level effects of zinc-spiked sediments in the gastropod Valvata piscinalis.

This paper presents original toxicity test designs and mathematical models that may be used to assess the deleterious effects of toxicants on Valvata piscinalis (Mollusca, Gastropoda). Results obtained for zinc, used as a reference toxicant, are presented. The feeding behavior, juvenile survival, growth, age at puberty, onset of reproduction, number of breedings during the life cycle, and fecundity were significantly altered when the snails were exposed to zinc-spiked sediments. Dynamic energy budget models (DEBtox) adequately predicted the effects of zinc on the V. piscinalis life cycle. They also provided estimates for lifecycle parameters that were used to parameterize a demographic model, based on a Z-transformed life-cycle graph. The effect threshold for the population growth rate (lambda) was estimated at 259 mg/kg dry sediment of zinc, showing that significant changes in abundance may occur at environmental concentrations. Significant effects occurring just above this threshold value were mainly caused by the severe impairment of reproductive endpoints. Sensitivity analysis showed that the value of lambda depended mainly on the juvenile survival rate. The impairment of this latter parameter may result in extinction of V. piscinalis. Finally, the present study highlights advantages of the proposed modeling approach in V. piscinalis and possible transfer to other test species and contaminants.

A framework for predicting impacts on ecosystem services from (sub)organismal responses to chemicals.

Protection of ecosystem services is increasingly emphasized as a risk-assessment goal, but there are wide gaps between current ecological risk-assessment endpoints and potential effects on services provided by ecosystems. The authors present a framework that links common ecotoxicological endpoints to chemical impacts on populations and communities and the ecosystem services that they provide. This framework builds on considerable advances in mechanistic effects models designed to span multiple levels of biological organization and account for various types of biological interactions and feedbacks. For illustration, the authors introduce 2 case studies that employ well-developed and validated mechanistic effects models: the inSTREAM individual-based model for fish populations and the AQUATOX ecosystem model. They also show how dynamic energy budget theory can provide a common currency for interpreting organism-level toxicity. They suggest that a framework based on mechanistic models that predict impacts on ecosystem services resulting from chemical exposure, combined with economic valuation, can provide a useful approach for informing environmental management. The authors highlight the potential benefits of using this framework as well as the challenges that will need to be addressed in future work. Environ Toxicol Chem 2017;36:845-859. © 2017 SETAC.

Development and validation of an OECD reproductive toxicity test guideline with the mudsnail Potamopyrgus antipodarum (Mollusca, Gastropoda).

Mollusks are known to be uniquely sensitive to a number of reproductive toxicants including some vertebrate endocrine disrupting chemicals. However, they have widely been ignored in environmental risk assessment procedures for chemicals. This study describes the validation of the Potamopyrgus antipodarum reproduction test within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment. The number of embryos in the brood pouch and adult mortality serve as main endpoints. The experiments are conducted as static systems in beakers filled with artificial medium, which is aerated trough glass pipettes. The test chemical is dispersed into the medium, and adult snails are subsequently introduced into the beakers. After 28 days the reproductive success is determined by opening the brood pouch and embryo counting. This study presents the results of two validation studies of the reproduction test with eleven laboratories and the chemicals tributyltin (TBT) with nominal concentrations ranging from 10 to 1000 ng TBT-Sn/L and cadmium with concentrations from 1.56 to 25 µg/L. The test design could be implemented by all laboratories resulting in comparable effect concentrations for the endpoint number of embryos in the brood pouch. After TBT exposure mean EC10, EC50, NOEC and LOEC were 35.6, 127, 39.2 and 75.7 ng Sn/L, respectively. Mean effect concentrations in cadmium exposed snails were, respectively, 6.53, 14.2, 6.45 and 12.6 µg/L. The effect concentrations are in good accordance with already published data. Both validation studies show that the reproduction test with P. antipodarum is a well-suited tool to assess reproductive effects of chemicals.

Development of rearing and testing protocols for a new freshwater sediment test species: the gastropod Valvata piscinalis.

This paper aimed at proposing rearing and testing protocols for Valvata piscinalis, a new potential species for sediment toxicity testing. Such tests were developed since this species reliably represents the bio/ecological characteristics of other gastropods. It may thus be representative of their sensitivity to chemicals. V. piscinalis was successfully cultured in our laboratory for six generations. Cultures provided a high productivity for a low working time and low costs. The tests conditions we proposed seemed to be relevant for the development of reliable tests with this species. Indeed, hatching probability of egg-capsules, as well as embryo, newborn and juvenile survival rates, were close to 100%. Moreover, growth rates and fecundity were significantly higher than in field and in other laboratory studies. Partial life-cycle tests on clean sediments were achieved for various feeding levels to determine survival, growth and reproduction patterns, ad libitum feeding level and life cycle parameters values. Ad libitum feeding levels for newborn, juveniles and adults were 0.1, 0.4 and 0.8 mg Tetramin/individual/working day. Growth tests with zinc-spiked sediments provided a no-effect concentration and a lowest effect concentration of respectively 200 and 624 mg zinc/kg dry sediment. Other growth tests on spiked sediments we ran at our laboratory with second, third and fourth instars larvae of Chironomus riparius pointed out that V. piscinalis was more sensible to zinc than the chironomid, which is a routine test species in ecotoxicology. According to these results, V. piscinalis is a promising candidate species for sediment toxicity testing.

How to use mechanistic effect models in environmental risk assessment of pesticides: Case studies and recommendations from the SETAC workshop MODELINK.

Mechanistic effect models (MEMs) are useful tools for ecological risk assessment of chemicals to complement experimentation. However, currently no recommendations exist for how to use them in risk assessments. Therefore, the Society of Environmental Toxicology and Chemistry (SETAC) MODELINK workshop aimed at providing guidance for when and how to apply MEMs in regulatory risk assessments. The workshop focused on risk assessment of plant protection products under Regulation (EC) No 1107/2009 using MEMs at the organism and population levels. Realistic applications of MEMs were demonstrated in 6 case studies covering assessments for plants, invertebrates, and vertebrates in aquatic and terrestrial habitats. From the case studies and their evaluation, 12 recommendations on the future use of MEMs were formulated, addressing the issues of how to translate specific protection goals into workable questions, how to select species and scenarios to be modeled, and where and how to fit MEMs into current and future risk assessment schemes. The most important recommendations are that protection goals should be made more quantitative; the species to be modeled must be vulnerable not only regarding toxic effects but also regarding their life history and dispersal traits; the models should be as realistic as possible for a specific risk assessment question, and the level of conservatism required for a specific risk assessment should be reached by designing appropriately conservative environmental and exposure scenarios; scenarios should include different regions of the European Union (EU) and different crops; in the long run, generic MEMs covering relevant species based on representative scenarios should be developed, which will require EU-level joint initiatives of all stakeholders involved. The main conclusion from the MODELINK workshop is that the considerable effort required for making MEMs an integral part of environmental risk assessment of pesticides is worthwhile, because it will make risk assessments not only more ecologically relevant and less uncertain but also more comprehensive, coherent, and cost effective.

Using toxicokinetic-toxicodynamic modeling as an acute risk assessment refinement approach in vertebrate ecological risk assessment.

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.

Modelling survival: exposure pattern, species sensitivity and uncertainty.

The General Unified Threshold model for Survival (GUTS) integrates previously published toxicokinetic-toxicodynamic models and estimates survival with explicitly defined assumptions. Importantly, GUTS accounts for time-variable exposure to the stressor. We performed three studies to test the ability of GUTS to predict survival of aquatic organisms across different pesticide exposure patterns, time scales and species. Firstly, using synthetic data, we identified experimental data requirements which allow for the estimation of all parameters of the GUTS proper model. Secondly, we assessed how well GUTS, calibrated with short-term survival data of Gammarus pulex exposed to four pesticides, can forecast effects of longer-term pulsed exposures. Thirdly, we tested the ability of GUTS to estimate 14-day median effect concentrations of malathion for a range of species and use these estimates to build species sensitivity distributions for different exposure patterns. We find that GUTS adequately predicts survival across exposure patterns that vary over time. When toxicity is assessed for time-variable concentrations species may differ in their responses depending on the exposure profile. This can result in different species sensitivity rankings and safe levels. The interplay of exposure pattern and species sensitivity deserves systematic investigation in order to better understand how organisms respond to stress, including humans.