Reproductive toxicity in birds predicted by physiologically-based kinetics and bioenergetics modelling.

Effects on the growth and reproduction of birds are important endpoints in the environmental risk assessment (ERA) of pesticides. Toxicokinetic-toxicodynamic models based on dynamic energy budget theory (DEB) are promising tools to predict these effects mechanistically and make extrapolations relevant to ERA. However, before DEB-TKTD models are accepted as part of ERA for birds, ecotoxicological case studies are required so that stakeholders can assess their capabilities. We present such a case-study, modelling the effects of the fluopyram metabolite benzamide on the northern bobwhite quail (Colinus virginianus). We parametrised a DEB-TKTD model for the embryo stage on the basis of an egg injection study, designed to provide data for model development. We found that information on various endpoints, such as survival, growth, and yolk utilisation were needed to clearly distinguish between the performance of model variants with different TKTD assumptions. The calibration data were best explained when it was assumed that chemical uptake occurs via the yolk and that benzamide places stress on energy assimilation and mobilisation. To be able to bridge from the in vitro tests to real-life exposure, we developed a physiologically-based toxicokinetic (PBK) model for the quail and used it to predict benzamide exposure inside the eggs based on dietary exposure in a standard reproductive toxicity study. We then combined the standard DEB model with the TKTD module calibrated to the egg injection studies and used it to predict effects on hatchling and 14-day chick weight based on the exposure predicted by the PBK model. Observed weight reductions, relative to controls, were accurately predicted. Thus, we demonstrate that DEB-TKTD models, in combination with suitable experimental data and, if necessary, with an exposure model, can be used in bird ERA to predict chemical effects on reproduction.

Acute toxicity of pesticide mixtures to honey bees is generally additive, and well predicted by Concentration Addition.

Understanding the frequency of non-additive effects of pesticides (synergism and antagonism) is important in the context of risk assessment. The goal of this study was to investigate the prevalence of non-additive effects of pesticides to honey bees (Apis mellifera). We investigated a large set of mixtures including insecticides and fungicides of different chemical modes of action and classes. The mixtures included represent a relevant sample of pesticides that are currently used globally. We investigated whether the experimental toxicity of the mixtures could be predicted based on the Concentration Addition (CA) model for acute contact and oral adult bee toxicity tests. We measured the degree of deviation from the additivity predictions of the experimental toxicity based on the well-known Mixture Deviation Ratio (MDR). Further, we investigated the appropriate MDR thresholds that should be used for the identification of non-additive effects based on acceptable rates for false positive (alpha) and true positive (beta) findings. We found that a deviation factor of MDR = 5 is a sound reference for labeling potential non-additive effects in acute adult bee experimental designs when assuming a typical Coefficient of Variation (CV%) = 100 in the determination of the LD50 of a pesticide (a factor of 2× deviation in the LD 50 resulting from inter-experimental variability). We found that only 2.4 % and 9 % of the mixtures evaluated had an MDR > 5 and MDR < 0.2, respectively. The frequency and magnitude of deviation from additivity found for bees in this study are consistent with those of other terrestrial and aquatic taxa. Our findings suggest that additivity is a good baseline for predicting the toxicity of pesticide mixtures to bees, and that the rare cases of synergy of pesticide mixtures to bees are not random but have a mechanistic basis.

Risk from unintentional environmental mixtures in EU surface waters is dominated by a limited number of substances.

Unintentional environmental mixtures happen when multiple chemicals co-occur in the environment. A generic mixture assessment factor (MAF), has been proposed to account for this. The MAF is a number by which safe exposure levels for single chemicals are divided to ensure protection against combined exposures to multiple chemicals. Two key elements to judge the appropriateness of a generic MAF are (1) defining the scope of mixtures that need to be addressed by a MAF (i.e.: simple mixtures vs complex mixtures), and (2) the existence of common risk drivers across large spatial scales. Simple mixtures with one to three risk drivers can easily be addressed by chemical-by-chemical regulatory action. Our work provides evidence on the prevalence and complexity of cumulative risk in EU freshwaters based on chemical monitoring data from one of the largest databases in the EU. With 334 chemicals being monitored, low complexity mixtures (one to 3 three risk drivers) dominated. After excluding metals, only 15 out of 307 chemicals (5 %) were most frequent chemical risk drivers. When these 15 chemicals were excluded from the analysis, 95 % of all monitoring site - year combinations did not pose a concern for cumulative risk. Most of these 15 chemicals are already banned or listed in various priority lists, showing that current regulatory frameworks were effective in identifying drivers of single chemical and cumulative risk. Although the monitoring data do not represent the entirety of environmental mixtures in the EU, the observed patterns of (1) limited prevalence of truly complex mixtures, and (2) limited number of overall risk drivers, argue against the need for implementing a generic MAF as a regulatory tool to address risk from unintentional mixtures in EU freshwaters.

Energetic basis for bird ontogeny and egg-laying applied to the bobwhite quail.

Birds build up their reproductive system and undergo major tissue remodeling for each reproductive season. Energetic specifics of this process are still not completely clear, despite the increasing interest. We focused on the bobwhite quail - one of the most intensely studied species due to commercial and conservation interest - to elucidate the energy fluxes associated with reproduction, including the fate of the extra assimilates ingested prior to and during reproduction. We used the standard Dynamic Energy Budget model, which is a mechanistic process-based model capable of fully specifying and predicting the life cycle of the bobwhite quail: its growth, maturation and reproduction. We expanded the standard model with an explicit egg-laying module and formulated and tested two hypotheses for energy allocation of extra assimilates associated with reproduction: Hypothesis 1, that the energy and nutrients are used directly for egg production; and Hypothesis 2, that the energy is mostly spent fueling the increased metabolic costs incurred by building up and maintaining the reproductive system and, subsequently, by egg-laying itself. Our results suggest that Hypothesis 2 is the more likely energy pathway. Model predictions capture well the whole ontogeny of a generalized northern bobwhite quail and are able to reproduce most of the data variability via variability in (i) egg size, (ii) egg-laying rate and (iii) inter-individual physiological variability modeled via the zoom factor, i.e. assimilation potential. Reliable models with a capacity to predict physiological responses of individuals are relevant not only for experimental setups studying effects of various natural and anthropogenic pressures on the quail as a bird model organism, but also for wild quail management and conservation. The model is, with minor modifications, applicable to other species of interest, making it a most valuable tool in the emerging field of conservation physiology.

A generic avian physiologically-based kinetic (PBK) model and its application in three bird species.

Physiologically-based kinetic (PBK) models are effective tools for designing toxicological studies and conducting extrapolations to inform hazard characterization in risk assessment by filling data gaps and defining safe levels of chemicals. In the present work, a generic avian PBK model for male and female birds was developed using PK-Sim and MoBi from the Open Systems Pharmacology Suite (OSPS). The PBK model includes an ovulation model (egg development) to predict concentrations of chemicals in eggs from dietary exposure. The model was parametrized for chicken (Gallus gallus), bobwhite quail (Colinus virginianus) and mallard duck (Anas platyrhynchos) and was tested with nine chemicals for which in vivo studies were available. Time-concentration profiles of chemicals reaching tissues and egg compartment were simulated and compared to in vivo data. The overall accuracy of the PBK model predictions across the analyzed chemicals was good. Model simulations were found to be in the range of 22-79% within a 3-fold and 41-89% were within 10- fold deviation of the in vivo observed data. However, for some compounds scarcity of in-vivo data and inconsistencies between published studies allowed only a limited goodness of fit evaluation. The generic avian PBK model was developed following a "best practice" workflow describing how to build a PBK model for novel species. The credibility and reproducibility of the avian PBK models were scored by evaluation according to the available guidance documents from WHO (2010), and OECD (2021), to increase applicability, confidence and acceptance of these in silico models in chemical risk assessment.

Predicted Dermal and Dietary Exposure of Bats to Pesticides.

Wild birds and mammals that feed in agricultural habitats are potentially exposed to pesticides through various routes. The European Food Safety Authority (EFSA) recently published a statement which concluded that the current EFSA risk assessment scheme for birds and mammals does not adequately cover bats (Chiroptera). In the present study, we take a more detailed look at the EFSA statement and assumptions made regarding direct (dermal) and indirect (dietary) exposure of bats to pesticides in terms of their realism and the potential implications for risk assessment outcomes. Regarding dietary exposure, errors in the residue per unit dose (RUD) values for flying insects (bat food), proposed in the EFSA bat statement, were identified and corrected. Lower RUD values based on a much broader data base are proposed. Using these more realistic RUD values, together with current assumptions regarding toxicity and exposure, the acute and long-term risk to bats appears to be within the range of those calculated for birds and ground-dwelling mammals under the current risk assessment scheme. Depending on the assumptions made, some uncertainties may remain and should be investigated further. According to the EFSA bat statement, dermal exposure of bats is the most significant route of exposure, resulting in the highest predicted daily doses. We demonstrated that the dermal exposure models in the EFSA bat statement predict much higher residues for bats than those measured for other flying organisms that have larger surface area to volume ratios, and thus would be expected to have the reverse relationship. We also illustrated that the amounts of spray liquid required to achieve the predicted dermal exposures of bats are implausibly high, with bats carrying an amount of spray liquid that exceeds their body weight many fold. It is recommended that a bat risk assessment framework should be based on realistic, sound science, allowing resources to be focused on those scenarios that are not already covered by the existing bird and mammal framework. Therefore, a quantitative risk assessment scheme should not be implemented until the many scientific uncertainties within the EFSA bat statement are addressed. Environ Toxicol Chem 2022;41:2595-2602. © 2022 Cambridge Environmental Assessments. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Quantitative Morphometric, Physiological, and Metabolic Characteristics of Chickens and Mallards for Physiologically Based Kinetic Model Development.

Physiologically based kinetic (PBK) models are a promising tool for xenobiotic environmental risk assessment that could reduce animal testing by predicting in vivo exposure. PBK models for birds could further our understanding of species-specific sensitivities to xenobiotics, but would require species-specific parameterization. To this end, we summarize multiple major morphometric and physiological characteristics in chickens, particularly laying hens (Gallus gallus) and mallards (Anas platyrhynchos) in a meta-analysis of published data. Where such data did not exist, data are substituted from domesticated ducks (Anas platyrhynchos) and, in their absence, from chickens. The distribution of water between intracellular, extracellular, and plasma is similar in laying hens and mallards. Similarly, the lengths of the components of the small intestine (duodenum, jejunum, and ileum) are similar in chickens and mallards. Moreover, not only are the gastrointestinal absorptive areas similar in mallard and chickens but also they are similar to those in mammals when expressed on a log basis and compared to log body weight. In contrast, the following are much lower in laying hens than mallards: cardiac output (CO), hematocrit (Hct), and blood hemoglobin. There are shifts in ovary weight (increased), oviduct weight (increased), and plasma/serum concentrations of vitellogenin and triglyceride between laying hens and sexually immature females. In contrast, reproductive state does not affect the relative weights of the liver, kidneys, spleen, and gizzard.

Quantitative Comparison of Avian and Mammalian Physiologies for Parameterization of Physiologically Based Kinetic Models.

Physiologically based kinetic (PBK) models facilitate chemical risk assessment by predicting in vivo exposure while reducing the need for animal testing. PBK models for mammals have seen significant progress, which has yet to be achieved for avian systems. Here, we quantitatively compare physiological, metabolic and anatomical characteristics between birds and mammals, with the aim of facilitating bird PBK model development. For some characteristics, there is considerable complementarity between avian and mammalian species with identical values for the following: blood hemoglobin and hemoglobin concentrations per unit erythrocyte volume together with relative weights of the liver, heart, and lungs. There are also systematic differences for some major characteristics between avian and mammalian species including erythrocyte volume, plasma concentrations of albumin, total protein and triglyceride together with liver cell size and relative weights of the kidney, spleen, and ovary. There are also major differences between characteristics between sexually mature and sexually immature female birds. For example, the relative weights of the ovary and oviduct are greater in sexually mature females compared to immature birds as are the plasma concentrations of triglyceride and vitellogenin. Both these sets of differences reflect the genetic "blue print" inherited from ancestral archosaurs such as the production of large eggs with yolk filled oocytes surrounded by egg white proteins, membranes and a calciferous shell together with adaptions for flight in birds or ancestrally in flightless birds.

Assessing the Portion of Diet Taken by Birds and Mammals from a Pesticide-Treated Area-Proposal for a Joint Way Forward.

An environmental risk assessment for birds and mammals is an important part of any application package for potential registration of pesticides in Europe. A realistic estimation of the exposure of a certain species to the pesticide under field conditions is a refinement factor in long-term higher-tier risk assessments. Although guidance on how to collect specific exposure data is provided, there is not yet a harmonized view on how to use and assess existing and newly recorded field data. Exposure is defined as the portion of diet taken from a pesticide-treated crop field over time (the PT factor), and it is set equal to the time a (tracked) individual is active (= foraging) in the crop of concern at the crop growth stage relevant to the pesticide application being assessed. While the PT factor is set to 1 in a first tier, it can be refined in higher tiers. Differences in the methodologies influencing the robustness of a data set and intraindividual variance in PT data are currently not considered. Our study, based on data recording in accordance with the European Food Safety Authority's recommendations, shows this variance for different species in different crops for both breeding and nonbreeding periods, with tracking sessions showing both interindividual as well as intraindividual variation across several days. We discuss PT data quality and how it can be used, considering intraindividual variance in a manner that is protective and conforms to current European guidance. The present study intends to support the planned revision of the current European guidance on how to conduct environmental bird and mammal risk assessments, and its further purpose is to open constructive discussions about how and which kind of PT data can be used. Environ Toxicol Chem 2022;41:1344-1354. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Dissipation of plant protection products from foliage.

In the environmental risk assessment for plant protection products in the European Union a default foliar dissipation half-life (DT50) of 10 d is used for the risk assessment of herbivorous birds and mammals feeding on sprayed foliage. This generic DT50 of 10 d is based on a compilation of residue trials conducted over 30 yr ago, in which many compounds and formulations were considered that are not used anymore. We therefore reassessed foliar dissipation based on a data set of 396 residue trials covering 30 compounds. Foliar DT50s were calculated, and potential factors determining dissipation were analyzed, such as crop group, residue zone, and rainfall. The strongest source of variability was found between individual trials. Other factors, including the residue zone and crop group, did not have a significant impact on dissipation. Only heavy rainfall (>6.5 mm/d, i.e., the 95th percentile of rainfall) had a statistically significant influence, although rainfall explained only approximately 5% of the overall variability. Moderate rainfall (≤6.5 mm/d) did not significantly correlate with dissipation and explained only 0.1% of the overall variability. Most importantly, the differences in DT50s between crops and residue zones were neither marked nor statistically significant; hence, trials from different residue zones and crops could be pooled to obtain more robust half-lives. Over all compounds, trials, zones, and crops the geometric mean DT50 was 3.2 d (90th percentile 7.9 d). Environ Toxicol Chem 2018;37:1926-1932. © 2018 SETAC.

Appropriate exposure estimates for wildlife risk assessments of crop protection products based on continuous radio telemetry: A case study with woodpigeons.

The registration of pesticides follows guidance published by the European Food Safety Authority (EFSA). As a default, the EFSA guidance document on risk assessment for birds and mammals assumes that animals feed exclusively on pesticide-treated fields. However, the guidance document suggests refining the risk via the proportion of food animals obtain from a treated field or specific crop (expressed via the portion of diet obtained from a treated area [PT value]). The EFSA guidance equalizes the portion of food taken from a treated area per day with the portion of time spent potentially foraging over the course of a day within this area. Therefore, radiotracking is commonly used to gather species-, crop-, and season-specific PT data, and radio telemetry of continuously tracked farmland species can deliver individual PT values for a given day, crop, and species. In the present study the authors introduce a way of calculating long-term PT values based on empirically recorded data via telemetry field studies for the most appropriate use in wildlife risk assessment of pesticides. The novel aspect of the proposal is that the authors follow the prerequisite given by EFSA to cover the long-term risk by introducing 21-d PT values that aim to cover both intra- and inter-individual variability of foraging focal farmland species in cropped habitats. Currently, the intra-individual variability is not taken into account for PT calculations. The authors demonstrate this approach and discuss EFSA guidance input requirements for PT values recorded in field studies, based on a PT field study conducted with woodpigeons (Columba palumbus) radiotracked in an agricultural landscape in the United Kingdom. The results indicate that a 21-d PT value considering intra-individual variability gives a more appropriate PT value for long-term risk assessments. Environ Toxicol Chem 2017;36:1270-1277. © 2016 SETAC.

Narrow pH Range of Surface Water Bodies Receiving Pesticide Input in Europe.

Fate and toxicity of the active ingredients (AI's) of plant protection products in surface waters is often influenced by pH. Although a general range of pH values is reported in literature, an evaluation targeting aquatic ecosystems with documented AI inputs is lacking at the larger scale. Results show 95% of European surface waters (n = 3075) with a documented history of AI exposure fall within a rather narrow pH range, between 7.0 and 8.5. Spatial and temporal variability in the data may at least be partly explained by the calcareous characteristics of parental rock material, the affiliation of the sampling site to a freshwater ecoregion, and the photosynthetic activity of macrophytes (i.e., higher pH values with photosynthesis). Nonetheless, the documented pH range fits well with the standard pH of most ecotoxicological test guidelines, confirming the fate and ecotoxicity of AIs are usually adequately addressed.

An example of population-level risk assessments for small mammals using individual-based population models.

This article presents a case study demonstrating the application of 3 individual-based, spatially explicit population models (IBMs, also known as agent-based models) in ecological risk assessments to predict long-term effects of a pesticide to populations of small mammals. The 3 IBMs each used a hypothetical fungicide (FungicideX) in different scenarios: spraying in cereals (common vole, Microtus arvalis), spraying in orchards (field vole, Microtus agrestis), and cereal seed treatment (wood mouse, Apodemus sylvaticus). Each scenario used existing model landscapes, which differed greatly in size and structural complexity. The toxicological profile of FungicideX was defined so that the deterministic long-term first tier risk assessment would result in high risk to small mammals, thus providing the opportunity to use the IBMs for risk assessment refinement (i.e., higher tier risk assessment). Despite differing internal model design and scenarios, results indicated in all 3 cases low population sensitivity unless FungicideX was applied at very high (×10) rates. Recovery from local population impacts was generally fast. Only when patch extinctions occured in simulations of intentionally high acute toxic effects, recovery periods, then determined by recolonization, were of any concern. Conclusions include recommendations for the most important input considerations, including the selection of exposure levels, duration of simulations, statistically robust number of replicates, and endpoints to report. However, further investigation and agreement are needed to develop recommendations for landscape attributes such as size, structure, and crop rotation to define appropriate regulatory risk assessment scenarios. Overall, the application of IBMs provides multiple advantages to higher tier ecological risk assessments for small mammals, including consistent and transparent direct links to specific protection goals, and the consideration of more realistic scenarios.

Exposure reduction of seed treatments through dehusking behaviour of the wood mouse (Apodemus sylvaticus).

BACKGROUND, AIM AND SCOPE: Seed treatments are widely used on cereals and other annual crops throughout Europe. Most of the formulated pesticide is found on the outside of the seed, the husk. Risk assessments of seed treatments are especially needed for granivorous mice living in the agricultural landscape e.g. for registration using the guidance for risk assessment for birds and mammals (EFSA 2009). The dehusking of seeds before consumption is a known behaviour of these mammals, but so far, no quantitative data on the reduction of exposure of seed treatments by dehusking were published. Therefore, we aimed at providing a first quantitative estimate of this behaviour-related exposure reduction for the wood mouse (Apodemus sylvaticus) with different seed types. MATERIALS AND METHODS: We evaluated the efficiency of dehusking behaviour of 20 wood mice captured in the wild for four different seeds (wheat, barley, maize and sunflower). One experimental setup used a fungicide seed treatment where the remaining seed husks of consumed seeds were analysed with a HPLC-MS/MS technique. In the second setup, we measured generic pigment present in a blank seed treatment formulation and determined the leftover pigment in the husks with a photometric technique. RESULTS: The exposure reduction was similar for the fungicide and the pigment design where the same seed types were studied. We could demonstrate exposure reductions ranging from around 60% for cereals to almost 100% for sunflower seeds as a result of the dehusking behaviour. DISCUSSION: Since exposure reduction was similar in both approaches, working with pigments would be a generic way to estimate the impact of dehusking behaviour on seed treatment exposure. This behaviour can result in a substantial exposure reduction and should, therefore, be considered in a seed-type specific way in the risk assessment of pesticide seed treatments. CONCLUSIONS: It is proposed to include a seed-specific dehusking factor in the calculations of estimated theoretical exposure of seed treatments for granivorous mice. The approach of accounting for a dehusking-related exposure reduction by field relevant wild mammal species seems a more promising way to advance the risk assessment instead of using generic species and neglecting behavioural traits. The pigment approach could be used to gather data for exposure reduction for other species and seed types. Its advantage is that it is harmless to the test species and comparatively cheap since no chemical analysis is involved. RECOMMENDATIONS AND PERSPECTIVES: Seed treatments are used for most of the cereal crops grown in Europe today. Their advantages usually include a lower application rate and the reduction of drift compared to a conventional spraying regime. However, there is a potential risk especially for granivorous mice, and its assessment is challenging in case of a high residue concentration on the dressed seeds. The concept of a dehusking factor in the risk assessment scheme for seed treatments for granivorous mice is a valid approach to account for the behavioural exposure reduction, and generic data could be easily generated also for other wild mammal species and other seed types, possibly analysing the pigment in commercial seed treatment formulations.