DNA-based seed intake quantification for enhanced ecological risk assessment of small mammals.

To prevent the non-acceptable effects of agrochemicals on arable fields, Environmental Risk Assessment (ERA) aims to assess and protect against a wide range of risks due to stressors to non-target species. While exposure to stress is a key factor in ERA models, exposure values are difficult to obtain and rely on laboratory studies with often debatable relevance to field situations. To improve intake estimates, data from realistic field-based scenarios are needed. We developed calibration curves relating known seed numbers of up to 20 onion and carrot seeds consumed by wild-caught wood mice (Apodemus sylvaticus) to the seed DNA content in the feces. Based on these inferred quantitative relationships, a field trial was run to determine seed intake in a natural setting using realistic levels of seed spillage. Onion DNA was detected in the fecal samples of the wood mice caught in the field, which resembled a seed intake of up to 1 onion seed. No intake of carrot seeds was detected. This is the first-ever study to quantify seed intake in a realistic field scenario using a DNA-based analysis, showing that accurate seed intake estimates can be obtained. Our approach can help to improve risk assessment models through its minimally-invasive and accurate assessment of seed intake by ERA representative and non-target species, which would otherwise be undetectable with traditional methods. Our novel approach and its results are highly relevant to studies of food intake and diet composition for basic and applied research alike.

Establishment of a faecal DNA quantification technique for rare and cryptic diet constituents in small mammals.

DNA-based approaches have greatly improved the applicability of dietary studies aimed at investigating ecological processes. These studies have provided direct insights into, otherwise difficult to measure, interactions between species and trophic levels, food web structure and ecosystem functioning. However, despite these advances, DNA-based methods have struggled to accurately quantify the whole breadth of diet constituents because of methodological biases, such as amplification bias and digestive processes. The present study is, to our knowledge, the first diet study to use droplet digital PCR to quantify diet constituents. We manipulated the diet of wild-caught wood mice (Apodemus sylvaticus) by feeding them with a known amount of small vegetable seeds (onion and carrot) and quantified the DNA traces of these diet constituents in faecal samples. The sensitivity of the technique combined with the control on the experimental design allowed mitigation of methodological bias. We were able to accurately determine DNA concentrations of small vegetable seeds in the diet of wood mice. Quantification of target DNA demonstrated significant differences in DNA content when one vs. five seeds were consumed. These differences remained significant when the age, sex and other diet constituents of the mice were altered. Different DNA markers, targeting different parts of the chloroplast, influenced onion DNA detectability. However, all onion and carrot markers showed higher DNA content for higher seed numbers. Overall, the sensitive DNA-based approach developed in this study allows for minimally invasive quantification of small diet constituents in faeces, which would otherwise be undetectable with traditional methods.

Residue Levels of Pesticides on Fruits for Use in Wildlife Risk Assessments.

The guidance document on risk assessment for birds and mammals (GD) provides generic residue values of pesticides on potential diet items for use in wildlife risk assessments. For most diet items, these values are based on a large number of residue studies. However, the default residues per unit dose (RUD; standardized for application of 1 kg substance per hectare) values for fruits were taken from a few literature trials of unclear relevance for regulatory purposes. These trials were conducted according to neither current European Union (EU) agricultural practice nor to recommendations given in the GD about how to conduct wildlife-relevant residue studies. Therefore, field study data on fruit residue levels from applications of pesticides in fruiting crops were compiled and evaluated. Corresponding studies had been conducted during the last 26 y in the EU. In the final data set, 291 studies provided 1002 residue values in different fruits, including grapes, berries (currants, raspberries, gooseberries), fruits from orchards (apple, peach, pear, lemon, mandarin, orange, apricot, cherry, plum), gourds (pumpkins, cucumbers, squash, melons), and strawberries. This data set provides a basis for revising registration-relevant RUD values for fruits as potential diet items for birds and mammals in environmental wildlife risk assessments. The objective of this study was to estimate the resulting residue levels in different fruits determined under field conditions following the application of pesticides across their growing areas within the EU in diverse climatic areas that can be used directly in wildlife risk assessments. The large data set of usually about more than 100 residue values per "fruit group," all evaluated at EU member state level, revealed significantly lower RUDs compared to the current default RUDs presented in the GD. These new RUD values for fruits should be considered for use as default values in future bird and mammal risk assessments and in respective guidance documents. Integr Environ Assess Manag 2021;17:552-561. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology Chemistry (SETAC).