Multiresidue analysis of bat guano using GC-MS/MS.

Bats are the second largest mammalian order and are an endangered species group with a strong need for contamination monitoring. To facilitate non-invasive monitoring of the ecological burden in bat populations, a multiresidue method for the simultaneous quantification of 119 analytes including pesticides, persistent organic pollutants (POPs), active pharmaceutical ingredients (APIs), polycyclic aromatic hydrocarbons (PAHs), UV blockers, plasticizers, and other emerging pollutants in bat guano with gas chromatography tandem mass spectrometry (GC-MS/MS) was developed. Sample preparation and clean-up were performed with a modified QuEChERS approach based on DIN EN 15662. The method uses 1.00 g bat guano as sample with acetonitrile and water for liquid-liquid extraction. Phase separation is assisted by citrate-buffered salting out agent. For clean-up of the extract, primary secondary amine (PSA) was combined with graphitized carbon black (GCB). The lower limits of quantification (LLOQ) ranged between 2.5 and 250 µg kg-1. Linearity was shown in a concentration range from the respective LLOQs to 1250 µg kg-1. The median of the mean recovery was 102.4%. Precision was tested at three concentrations. Method and injection precision were adequate with a relative standard deviation (RSD) below 20%. Furthermore, the comparative analysis with LC-MS/MS demonstrated the reliability of the results and provided a valuable extension of the analytical scope. As proof of concept, three guano samples from a German nursery roost of Myotis myotis were analysed. The results show a time-dependent change in contaminant concentration, highlighting the strong need for non-invasive contamination monitoring of whole bat populations.

A temporal perspective on aquatic subsidy: Bti affects emergence of Chironomidae.

Emerging aquatic insects serve as one link between aquatic and adjacent riparian food webs via the flux of energy and nutrients. These insects provide high-quality subsidy to terrestrial predators. Thus, any disturbance of emergence processes may cascade to higher trophic levels and lead to effects across ecosystem boundaries. One stressor with potential impact on non-target aquatic insects, especially on non-biting midges (Diptera: Chironomidae), is the widely used mosquito control agent Bacillus thuringiensis var. israelensis (Bti). In a field experiment, we investigated emerging insect communities from Bti-treated (three applications, maximum field rate) and control floodplain pond mesocosms (FPMs) over 3.5 months for changes in their composition, diversity as well as the emergence dynamics and the individual weight of emerged aquatic insects over time. Bti treatments altered community compositions over the entire study duration - an effect mainly attributed to an earlier (∼10 days) and reduced (∼26%) peak in the emergence of Chironomidae, the dominant family (88% of collected individuals). The most reasonable explanation for this significant alteration is less resource competition caused by a decrease in chironomid larval density due to lethal effects of Bti. This is supported by the higher individual weight of Chironomidae emerging from treated FPMs (∼21%) during Bti application (April - May). A temporal shift in the emergence dynamics can cause changes in the availability of prey in linked terrestrial ecosystems. Consequently, terrestrial predators may be affected by a lack of appropriate prey leading to bottom-up and top-down effects in terrestrial food webs. This study indicates the importance of a responsible and elaborated use of Bti and additionally, highlights the need to include a temporal perspective in evaluations of stressors in aquatic-terrestrial meta-ecosystems.

Stress Response of European Common Frog (Rana temporaria) Tadpoles to Bti Exposure in an Outdoor Pond Mesocosm.

The biocide Bacillus thuringiensis var. israelensis (Bti) is applied to wetlands to control nuisance by mosquitoes. Amphibians inhabiting these wetlands can be exposed to Bti multiple times, potentially inducing oxidative stress in developing tadpoles. For biochemical stress responses, ambient water temperature plays a key role. Therefore, we exposed tadpoles of the European common frog (Rana temporaria) three times to field-relevant doses of Bti in outdoor floodplain pond mesocosms (FPM) under natural environmental conditions. We sampled tadpoles after each Bti application over the course of a 51-day experiment (April to June 2021) and investigated the activity of the glutathione-S-transferase (GST) and protein carbonyl content as a measure for detoxification activity and oxidative damage. GST activity increased over the course of the experiment likely due to a general increase of water temperature. We did not observe an effect of Bti on either of the investigated biomarkers under natural ambient temperatures. However, Bti-induced effects may be concealed by the generally low water temperatures in our FPMs, particularly at the first application in April, when we expected the highest effect on the most sensitive early stage tadpoles. In light of the global climate change, temperature-related effects of pesticides and biocides on tadpoles should be carefully monitored - in particular since they are known as one of the factors driving the worldwide decline of amphibian populations.

Pesticide exposure affects reproductive capacity of common toads (Bufo bufo) in a viticultural landscape.

Amphibian populations are declining worldwide at alarming rates. Among the large variety of contributing stressors, chemical pollutants like pesticides have been identified as a major factor for this decline. Besides direct effects on aquatic and terrestrial amphibian stages, sublethal effects like impairments in reproduction can affect a population. Therefore, we investigated the reproductive capacity of common toads (Bufo bufo) in the pesticide-intensive viticultural landscape of Palatinate in Southwest Germany along a pesticide gradient. In a semi-field study, we captured reproductively active common toad pairs of five breeding ponds with different pesticide contamination level and kept them in a net cage until spawning. Toads from more contaminated ponds showed an increased fecundity (more eggs) but decreased fertilization rates (fewer hatching tadpoles) as well as lower survival rates and reduced size in Gosner stage 25, suggesting that the higher exposed populations suffer from long-term reproductive impairments. In combination with acute toxicity effects, the detected sublethal effects, which are mostly not addressed in the ecological risk assessment of pesticides, pose a serious threat on amphibian populations in agricultural landscapes.

Mosquito control actions affect chironomid diversity in temporary wetlands of the Upper Rhine Valley.

The Upper Rhine Valley, a "hotspot of biodiversity" in Germany, has been treated with the biocide Bacillus thuringiensis var. israelensis (Bti) for mosquito control for decades. Previous studies discovered Bti nontarget effects in terms of severe chironomid abundance reductions. In this study, we investigated the impact of Bti on species level and addressed the community composition of the nontarget family Chironomidae by use of community metabarcoding. Chironomid emergence data were collected in three mosquito-control relevant wetland types in the Upper Rhine Valley. For all three sites the chironomid species composition, based on operational taxonomic units (OTUs), was different to varying degrees in the Bti-treated samples versus control samples, ranging from a significant 63% OTU reduction to an OTU replacement. We assumed that predatory chironomids are less prone to Bti than filter feeders, as the latter feed on floating particles leading to direct ingestion of Bti. However, a comparable percentage of predators and filter feeders (63% and 65%, respectively) was reduced in the Bti samples, suggesting that the feeding strategy is not the main driver for Bti sensitivity in chironomids. Finally, our data was compared to a three-year-old data set, indicating possible chironomid community recovery due to species recolonization a few years after the last Bti application. Considering the currently discussed worldwide insect decline we recommend a rethinking of the usage of the biocide Bti, and to prevent its ongoing application especially in nature protection reserves to enhance ecological resilience and to prevent boosting the current biodiversity loss.

Fungicides: An Overlooked Pesticide Class?

Fungicides are indispensable to global food security and their use is forecasted to intensify. Fungicides can reach aquatic ecosystems and occur in surface water bodies in agricultural catchments throughout the entire growing season due to their frequent, prophylactic application. However, in comparison to herbicides and insecticides, the exposure to and effects of fungicides have received less attention. We provide an overview of the risk of fungicides to aquatic ecosystems covering fungicide exposure (i.e., environmental fate, exposure modeling, and mitigation measures) as well as direct and indirect effects of fungicides on microorganisms, macrophytes, invertebrates, and vertebrates. We show that fungicides occur widely in aquatic systems, that the accuracy of predicted environmental concentrations is debatable, and that fungicide exposure can be effectively mitigated. We additionally demonstrate that fungicides can be highly toxic to a broad range of organisms and can pose a risk to aquatic biota. Finally, we outline central research gaps that currently challenge our ability to predict fungicide exposure and effects, promising research avenues, and shortcomings of the current environmental risk assessment for fungicides.

Adverse effects of mosquito control using Bacillus thuringiensis var. israelensis: Reduced chironomid abundances in mesocosm, semi-field and field studies.

The ecological consequences of mosquito control using the larvicide Bacillus thuringiensis israelensis (Bti) are still a matter of debate especially when it comes to adverse effects on non-target but susceptible non-biting midges (Chironomidae). Our work aimed to assess the effects of Bti applied in operational mosquito control rates on chironomid abundances in seasonal flooded freshwater wetlands. We assessed the invertebrate community alongside with aquatic insect emergence rates in studies with increasing ecotoxicological complexity, ranging from artificial mesocosms, over a semi-field approach using enclosures to natural conditions in field studies. Study sites represented different habitats (forest, meadow, floodplain) with regularly Bti treated and untreated temporal wetlands that are located in the Upper Rhine Valley, Germany. Larval chironomids were the most affected group of organism in the altered aquatic invertebrate communities of the Bti treated pond mesocosms. The larvae of the chironomid subfamilies Chironominae and Orthocladiinae were affected by Bti field concentrations while emergence rates of Tanypodinae did not change. The Bti treatment significantly reduced the targeted mosquitoes in the mesocosm and in the field studies. At the same time, however, the application of operational Bti field rates reduced overall chironomid emergence rates to about half of the control rates. The observed abundance reductions occurred independently of the ecological complexity in the study design in artificial mesocosms as well as realistic field conditions. The considerable reduction of the abundant non-target chironomids along with mosquitoes may subsequently lead to unwanted indirect negative effects for birds, bats and other aquatic organisms feeding on midges. Hence, large-scale applications of Bti for mosquito control in seasonal wetlands should be considered more carefully. This is of special importance when these wetlands are parts of national parks, nature reserves or Natura 2000 sites that were created for the protection of nature and environmental health.

European common frog Rana temporaria (Anura: Ranidae) larvae show subcellular responses under field-relevant Bacillus thuringiensis var. israelensis (Bti) exposure levels.

Bacillus thuringiensis var. israelensis (Bti) is presumed to be an environmental friendly agent for the use in either health-related mosquito control or the reduction of nuisance associated with mosquitoes from seasonal wetlands. Amphibians inhabiting these valuable wetlands may be exposed to Bti products several times during their breeding season. Up until now, information regarding effects on the non-targeted group of amphibians has to be considered rather inconsistent. On this account, we evaluated how three repeated exposures to frequently used Bti formulations (VectoBac®12AS, VectoBac®WG) in field-relevant rates affect European common frog (Rana temporaria) larvae. In a laboratory approach, we assessed potential effects with regard to enzymatic biomarkers (glutathione-S-transferase (GST), glutathione reductase (GR), acetylcholine esterase (AChE)), development, body condition and survival until the end of metamorphosis. Although survival and time to metamorphosis were not significantly affected, larval development tended to be shortened in the Bti treated water phase. Furthermore, exposure to Bti induced significant increases of GST (37-550%), GR (5-140%) and AChE (38-137%) irrespectively of the applied formulation, indicating detoxification, antioxidant responses as well as an alteration of neuronal activity. GST activity increased twice as much after two repeatedly executed Bti applications within a time period of 6 days. The examination of several biochemical markers is needed to fully evaluate the ecotoxicological risk of Bti for amphibian populations, especially in the context of worldwide amphibian declines. Nevertheless, following the precautionary principle, it may be advisable to implement certain thresholds for application numbers and intervals in order to ensure environmentally friendly mosquito control programs, especially in areas designated for nature conservation.

Trophic effects of Bti-based mosquito control on two top predators in floodplain pond mesocosms.

Chironomid (Diptera: Chironomidae) larvae play a key role in aquatic food webs as prey for predators like amphibian and dragonfly larvae. This trophic link may be disrupted by anthropogenic stressors such as Bacillus thuringiensis var. israelensis (Bti), a biocide widely used in mosquito control. In a companion study, we recorded a 41% reduction of non-target larval chironomids abundance in outdoor floodplain pond mesocosms (FPMs) treated with Bti. Therefore, we examined the diet of two top predators in the FPMs, larvae of the palmate newt (Salamandridae: Lissotriton helveticus) and dragonfly (Aeshnidae: predominantly Anax imperator), using bulk stable isotope analyses of carbon and nitrogen. Additionally, we determined neutral lipid fatty acids in newt larvae to assess diet-related effects on their physiological condition. We did not find any effects of Bti on the diet proportions of newt larvae and no significant effects on the fatty acid content. We observed a trend in Aeshnidae larvae from Bti-FPMs consuming a higher proportion of large prey (Aeshnidae, newt, damselfly larvae; ~42%), and similar parts of smaller prey (chironomid, mayfly, Libellulidae, and zooplankton), compared to controls. Our findings may suggest bottom-up effects of Bti on aquatic predators but should be further evaluated, for instance, by using compound-specific stable isotope analyses of fatty acids or metabarcoding approaches.

Impact across ecosystem boundaries - Does Bti application change quality and composition of the diet of riparian spiders?

Emerging aquatic insects link aquatic and adjacent terrestrial food webs by subsidizing terrestrial predators with high-quality prey. One of the main constituents of aquatic subsidy, the non-biting midges (Chironomidae), showed altered emergence dynamics in response to the mosquito control agent Bacillus thuringiensis var. israelensis (Bti). As riparian spiders depend on aquatic subsidy, they may be affected by such changes in prey availability. Thus, we conducted a field study in twelve floodplain pond mesocosms (FPMs), six were treated with Bti (2.88 × 109 ITU/ha, VectoBac WDG) three times, to investigate if the Bti-induced shift in chironomid emergence dynamics is reflected in their nutritional value and in the diet of riparian spiders. We measured the content of proteins, lipids, glycogen, and carbohydrates in emerged Chironomidae, and determined the stable isotope ratios of female Tetragnatha extensa, a web-building spider living in the riparian vegetation of the FPMs. We analysed the proportion of aquatic prey in spiders' diet, niche size, and trophic position. While the content of nutrients and thus the prey quality was not significantly altered by Bti, effects on the spiders' diet were observed. The trophic position of T. extensa from Bti-treated FPMs was lower compared to the control while the aquatic proportion was only minimally reduced. We assume that spiders fed more on terrestrial prey but also on other aquatic organisms such as Baetidae, whose emergence was unaffected by Bti. In contrast to the partly predaceous Chironomidae, consumption of aquatic and terrestrial primary consumers potentially explains the observed lower trophic position of spiders from Bti-treated FPMs. As prey organisms vary in their quality the suggested dietary shift could transfer previously observed effects of Bti to riparian spiders conceivably affecting their populations. Our results further support that anthropogenic stressors in aquatic ecosystems may translate to terrestrial predators through aquatic subsidy.

A new sample preparation approach for the analysis of 98 current-use pesticides in soil and herbaceous vegetation using HPLC-MS/MS in combination with an acetonitrile-based extraction.

A simple acetonitrile-based extraction method for the determination of 98 current-use pesticides (CUPs) in soil and herbaceous vegetation using HPLC-ESI-MS/MS is reported. The method was optimized in terms of extraction time, buffer (ammonium formate) ratio, and graphitized carbon black (GCB) ratio for the clean-up of vegetation. The validated method yielded accuracy in terms of percentage recovery of 71-125% (soil) and 70-117% (vegetation) for the majority of 98 CUPs. The precision in terms of relative standard deviation was at 1-14% (soil), and 1-13% (vegetation). Matrix-matched calibration curves exhibited good linearities (R2 > 0.99). The limits of quantitation ranged between 0.008 and 21.5 µg kg-1 in soil and vegetation. The reported method was applied to soils and vegetation from 13 agricultural sites across Germany. Overall, 44 of the 98 common CUPs were detected in our samples and the qualitative load is well above the average for arable soils in the EU.

Benthic macroinvertebrate community shifts based on Bti-induced chironomid reduction also decrease Odonata emergence.

Chironomid larvae (Diptera: Chironomidae) often dominate aquatic macroinvertebrate communities and are a key food source for many aquatic predators, such as dragonfly and damselfly larvae (Odonata). Changes in aquatic macroinvertebrate communities may propagate through terrestrial food webs via altered insect emergence. Bacillus thuringiensis israelensis (Bti)-based larvicides are widely used in mosquito control but can also reduce the abundance of non-biting chironomid larvae. We applied the maximum field rate of Bti used in mosquito control three times to six mesocosms in a replicated floodplain pond mesocosm (FPM) system in spring for two consecutive years, while the remaining six FPMs were untreated. Three weeks after the third Bti application in the first year, we recorded on average a 41% reduction of chironomid larvae in Bti-treated FPMs compared to untreated FPMs and a shift in benthic macroinvertebrate community composition driven by the reduced number of chironomid, Libellulidae and Coenagrionidae larvae (Odonata). Additionally, the number of emerging Libellulidae (estimated by sampling of exuviae in the second year) was reduced by 54% in Bti-treated FPMs. Since Odonata larvae are not directly susceptible to Bti, our results suggest indirect effects due to reduced prey availability (i.e., chironomid larvae) or increased intraguild predation. As Libellulidae include species of conservation concern, the necessity of Bti applications to their habitats, e.g. floodplains, should be carefully evaluated.

Pesticide use in banana plantations in Costa Rica - A review of environmental and human exposure, effects and potential risks.

Biodiversity is declining on a global scale. Especially tropical ecosystems, containing most of the planetary biodiversity, are at risk. Agricultural monocrop systems contribute to this decline as they replace original habitats and depend on extensive use of synthetic pesticides that impact ecosystems. In this review we use large-scale banana production for export purposes in Costa Rica as an example for pesticide impacts, as it is in production for over a century and uses pesticides extensively for more than fifty years. We summarise the research on pesticide exposure, effects and risks for aquatic and terrestrial environment, as well as for human health. We show that exposure to pesticides is high and relatively well-studied for aquatic systems and humans, but hardly any data are available for the terrestrial compartment including adjacent non target ecosystems such as rainforest fragments. Ecological effects are demonstrated on an organismic level for various aquatic species and processes but are not available at the population and community level. For human health studies exposure evaluation is crucial and recognised effects include various types of cancer and neurobiological dysfunctions particularly in children. With the many synthetic pesticides involved in banana production, the focus on insecticides, revealing highest aquatic risks, and partly herbicides should be extended to fungicides, which are applied aerially over larger areas. The risk assessment and regulation of pesticides so far relies on temperate models and test species and is therefore likely underestimating the risk of pesticide use in tropical ecosystems, with crops such as banana. We highlight further research approaches to improve risk assessment and, in parallel, urge to follow other strategies to reduce pesticides use and especially hazardous substances.

Low temperatures lead to higher toxicity of the fungicide folpet to larval stages of Rana temporaria and Bufotes viridis.

Pesticides are one of the main drivers of the worldwide amphibian decline. Their actual toxicity depends on a number of factors, like the species in focus or the developmental stage of exposed individuals. As ectothermic species, the metabolism of amphibians is influenced by ambient temperature. Therefore, temperature also affects metabolic rates and thus processes that might enhance or reduce toxic effects. Studies about the interactive effect of temperature and toxicity on amphibians are rare and deliver contrasting results. To investigate the temperature-dependent pesticide sensitivity of larvae of two European species we conducted acute toxicity tests for the viticultural fungicide Folpan® 500 SC with the active ingredient folpet at different temperatures (6°C, 11°C, 16°C, 21°C, 26°C). Sensitivity of Rana temporaria and Bufotes viridis was highly affected by temperature: early larvae (Gosner stage 20) were about twice more sensitive to Folpan® 500 SC at 6°C compared to 21°C. Next to temperature, species and developmental stage of larvae had an effect on sensitivity. The most sensitive individuals (early stages of R. temporaria at 6°C) were 14.5 times more sensitive than the least sensitive ones (early stages of B. viridis at 26°C). Our results raise concerns about typical ecotoxicological studies with amphibians that are often conducted at temperatures between 15°C and 20°C. We suggest that future test designs should be performed at temperatures that reflect the temperature range amphibians are exposed to in their natural habitats. Variations in the sensitivity due to temperature should also be considered as an uncertainty factor in upcoming environmental risk assessments for amphibians.

Dermal Fungicide Exposure at Realistic Field Rates Induces Lethal and Sublethal Effects on Juvenile European Common Frogs (Rana temporaria).

Viticulture is one of the most pesticide-intensive agricultures in Europe, leading to a spatiotemporal overlap of amphibian migration and pesticide applications. Because postmetamorphic, terrestrial amphibian stages are mostly neglected in ecotoxicological studies, we investigated acute effects of viticultural fungicides on juvenile common frogs (Rana temporaria). Tadpoles from an uncontaminated pond were placed in enclosures in 8 ponds with an increasing degree of pesticide contamination in southwest Germany to represent different aquatic exposure backgrounds. After metamorphosis, juveniles were exposed to soil contaminated with 50% of the recommended field rates of the fungicides Folpan® 80 water dispersible granule (WDG) and Folpan® 500 suspension concentrate with the same amount of folpet as active ingredient and differing additives. After 48 h, effects on the survival, body mass, and behavior were investigated. No effect of the aquatic exposure background on terrestrial sensitivity could be detected. Acute terrestrial exposure led to mean mortality rates of 14% (13-17%, suspension concentrate) and 60% (17-100%, WDG) and resulted in adverse effects on locomotor activity as well as feeding behavior. Moreover, the results suggest that the toxicity of the 2 tested folpet formulations depends on their additives. Because the identified effects may result in severe impairments and thus in declines of amphibian populations, a more protective risk assessment of pesticides is needed for postmetamorphic amphibians to ensure proper conservation of amphibian populations. Environ Toxicol Chem 2021;40:1289-1297. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Co-formulants and adjuvants affect the acute aquatic and terrestrial toxicity of a cycloxydim herbicide formulation to European common frogs (Rana temporaria).

While pesticides are generally recognized as contributing to amphibian declines, there is a lack of knowledge about effects of co-formulants that are present in pesticide formulations and adjuvants which are mixed with these formulations. Since aquatic and terrestrial stages of amphibians can be exposed to these substances, adverse effects cannot be excluded. We investigated acute aquatic and terrestrial effects of the herbicide formulation Focus® Ultra, its active substance cycloxydim, its co-formulants solvent naphtha and docusate as well as the stabilizing adjuvant Dash® E.C. on larval and juvenile Rana temporaria. Aquatic toxicity was determined as 96-h LC50 values. Cycloxydim was the least toxic and solvent naphtha the most toxic substance of the formulation. The addition of Dash® E.C. increased the formulation toxicity substantially. Terrestrial toxicity was determined as lethal effects after a 48-h exposure to contaminated soil with 100% of the recommended field rate (FR) and as sublethal effects after the exposure to 10% of the recommended FR. The exposure to solvent naphtha and docusate at 100% FR led to mortalities of 42-100% probably due to their inhalation toxicity and dermal as well as eye irritation, respectively. Cycloxydim, Focus® Ultra and Dash® E.C. did not lead to any mortality. Sublethal effects on juvenile locomotor activity (i.e. moved distance) were observed for cycloxydim and the combined exposure of Focus® Ultra and Dash® E.C. Juvenile body masses declined significantly for all substances except for cycloxydim. The present results show that aquatic sensitivity does not predict terrestrial sensitivity. It was shown that pesticide toxicity for amphibians can highly depend on the presence and amount of co-formulants and added adjuvants. Therefore, substances included in pesticide formulations which are known to be toxic by inhalation or harmful to eyes or skin should be specifically considered in the environmental risk assessment for amphibians.

Bottom-up effects of fungicides on tadpoles of the European common frog (Rana temporaria).

Biodiversity is under pressure worldwide, with amphibians being particularly threatened. Stressors related to human activity, such as chemicals, are contributing to this decline. It remains, however, unclear whether chemicals exhibiting a fungicidal activity could indirectly affect tadpoles that depend on microbially conditioned leaf litter as food source. The indirect effect of fungicides (sum concentration of a fungicide mixture composed of azoxystrobin, carbendazim, cyprodinil, quinoxyfen, and tebuconazole: 100 µg/L) on tadpoles was assessed relative to leaf litter colonized by microbes in absence of fungicides (control) and a worst-case scenario, that is leached leaf litter without microbial colonization. The quality of leaf litter as food for tadpoles of the European common frog (Rana temporaria) was characterized through neutral lipid fatty acid profiles and microbial sum parameters and verified by sublethal responses in tadpoles (i.e., feeding rate, feces production, growth, and fatty acid composition). Fungicides changed the nutritious quality of leaf litter likely through alterations in leaves' neutral lipid fatty acid profiles (i.e., changes in some physiologically important highly unsaturated fatty acids reached more than 200%) in combination with a potential adsorption onto leaves during conditioning. These changes were reflected by differences in the development of tadpoles ultimately resulting in an earlier start of metamorphosis. Our data provide a first indication that fungicides potentially affect tadpole development indirectly through bottom-up effects. This pathway is so far not addressed in fungicide environmental risk assessment and merits further attention.

Direct pesticide exposure of insects in nature conservation areas in Germany.

In Germany, the decline of insect biomass was observed in nature conservation areas in agricultural landscapes. One of the main causal factors discussed is the use of synthetic pesticides in conventional agriculture. In a Germany-wide field study, we collected flying insects using Malaise traps in nature conservation areas adjacent to agricultural land. We used a multi-component chemical trace element analysis to detect 92 common agricultural pesticides in ethanol from insect traps sampled in May and August 2020. In total, residues of 47 current use pesticides were detected, and insect samples were on average contaminated with 16.7 pesticides. Residues of the herbicides metolachlor-S, prosulfocarb and terbuthylazine, and the fungicides azoxystrobin and fluopyram were recorded at all sites. The neonicotinoid thiacloprid was detected in 16 of 21 nature conservation areas, most likely due to final use before an EU-wide ban. A change in residue mixture composition was noticeable due to higher herbicide use in spring and increasing fungicide applications in summer. The number of substances of recorded residues is related to the proportion of agricultural production area in a radius of 2000 m. Therefore, a drastic pesticide reduction in large buffers around nature conservation areas is necessary to avoid contamination of their insect fauna.

Avoidance behavior of juvenile common toads (Bufo bufo) in response to surface contamination by different pesticides.

Most agricultural soils are expected to be contaminated with agricultural chemicals. As the exposure to pesticides can have adverse effects on non-target organisms, avoiding contaminated areas would be advantageous on an individual level, but could lead to a chemical landscape fragmentation with disadvantages on the metapopulation level. We investigated the avoidance behavior of juvenile common toads (Bufo bufo) in response to seven pesticide formulations commonly used in German vineyards. We used test arenas filled with silica sand and oversprayed half of each with different pesticide formulations. We placed a toad in the middle of an arena, filmed its behavior over 24 hours, calculated the proportion of time a toad spent on the contaminated side and compared it to a random side choice. We found evidence for the avoidance of the folpet formulation Folpan® 500 SC, the metrafenone formulation Vivando® and the glyphosate formulation Taifun® forte at maximum recommended field rates for vine and a trend for avoidance of Wettable Sulphur Stulln (sulphur). No avoidance was observed when testing Folpan® 80 WDG (folpet), Funguran® progress (copper hydroxide), SpinTorTM (spinosad), or 10% of the maximum field rate of any formulation tested. In the choice-tests in which we observed an avoidance, toads also showed higher activity on the contaminated side of the arena. As video analysis with tracking software is not always feasible, we further tested the effect of reducing the sampling interval for manual data analyses. We showed that one data point every 15 or 60 minutes results in a risk of overlooking a weak avoidance behavior, but still allows to verify the absence/presence of an avoidance for six out of seven formulations. Our findings are important for an upcoming pesticide risk assessment for amphibians and could be a template for future standardized tests.

Environmental and socioeconomic effects of mosquito control in Europe using the biocide Bacillus thuringiensis subsp. israelensis (Bti).

Bacillus thuringiensis subsp. israelensis (Bti) has been used in mosquito control programs to reduce nuisance in Europe for decades and is generally considered an environmentally-safe, effective and target-specific biocide. However, the use of Bti is not uncontroversial. Target mosquitoes and affected midges represent an important food source for many aquatic and terrestrial predators and reduction of their populations is likely to result in food-web effects at higher trophic levels. In the context of global biodiversity loss, this appears particularly critical since treated wetlands are often representing conservation areas. In this review, we address the current large-scale use of Bti for mosquito nuisance control in Europe, provide a description of its regulation followed by an overview of the available evidence on the parameters that are essential to evaluate Bti use in mosquito control. Bti accumulation and toxin persistence could result in a chronic expose of mosquito populations ultimately affecting their susceptibility, although observed increase in resistance to Bti in mosquito populations is low due to the four toxins involved. A careful independent monitoring of mosquito susceptibility, using sensitive bioassays, is mandatory to detect resistance development timely. Direct Bti effects were documented for non-target chironomids and other invertebrate groups and are discussed for amphibians. Field studies revealed contrasting results on possible impacts on chironomid abundances. Indirect, food-web effects were rarely studied in the environment. Depending on study design and duration, Bti effects on higher trophic levels were demonstrated or not. Further long-term field studies are needed, especially with observations of bird declines in Bti-treated wetland areas. Socio-economic relevance of mosquito control requires considering nuisance, vector-borne diseases and environmental effects jointly. Existing studies indicate that a majority of the population is concerned regarding potential environmental effects of Bti mosquito control and that they are willing to pay for alternative, more environment-friendly techniques.