Combined effects of herbicides and insecticides reduce biomass of sensitive aquatic invertebrates.

The structure and biomass of aquatic invertebrate communities play a crucial role in the matter dynamics of streams. However, biomass is rarely quantified in ecological assessments of streams, and little is known about the environmental and anthropogenic factors that influence it. In this study, we aimed to identify environmental factors that are associated with invertebrate structure and biomass through a monitoring of 25 streams across Germany. We identified invertebrates, assigned them to taxonomic and trait-based groups, and quantified biomass using image-based analysis. We found that insecticide pressure generally reduced the abundance of insecticide-vulnerable populations (R2 = 0.43 applying SPEARpesticides indicator), but not invertebrate biomass. In contrast, herbicide pressure reduced the biomass of several biomass aggregations. Especially, insecticide-sensitive populations, that were directly (algae feeder, R2 = 0.39) or indirectly (predators, R2 = 0.29) dependent on algae, were affected. This indicated a combined effect of possible food shortage due to herbicides and direct insecticide pressure. Specifically, all streams with increased herbicide pressure showed a reduced overall biomass share of Trichoptera from 43 % to 3 % and those of Ephemeroptera from 20 % to 3 % compared to streams grouped by low herbicide pressure. In contrast, insecticide-insensitive Gastropoda increased from 10 % to 45 %, and non-vulnerable leaf-shredding Crustacea increased from 10 % to 22 %. In summary, our results indicate that at the community level, the direct effects of insecticides and the indirect, food-mediated effects of herbicides exert a combined effect on the biomass of sensitive insect groups, thus disrupting food chains at ecosystem level.

Predicting the Combined Effects of Multiple Stressors and Stress Adaptation in Gammarus pulex.

Global change confronts organisms with multiple stressors causing nonadditive effects. Persistent stress, however, leads to adaptation and related trade-offs. The question arises: How can the resulting effects of these contradictory processes be predicted? Here we show that Gammarus pulex from agricultural streams were more tolerant to clothianidin (mean EC50 148 µg/L) than populations from reference streams (mean EC50 67 µg/L). We assume that this increased tolerance results from a combination of physiological acclimation, epigenetic effects, and genetic evolution, termed as adaptation. Further, joint exposure to pesticide mixture and temperature stress led to synergistic interactions of all three stressors. However, these combined effects were significantly stronger in adapted populations as shown by the model deviation ratio (MDR) of 4, compared to reference populations (MDR = 2.7). The pesticide adaptation reduced the General-Stress capacity of adapted individuals, and the related trade-off process increased vulnerability to combined stress. Overall, synergistic interactions were stronger with increasing total stress and could be well predicted by the stress addition model (SAM). In contrast, traditional models such as concentration addition (CA) and effect addition (EA) substantially underestimated the combined effects. We conclude that several, even very disparate stress factors, including population adaptations to stress, can act synergistically. The strong synergistic potential underscores the critical importance of correctly predicting multiple stresses for risk assessment.

Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

Citizen science shows that small agricultural streams in Germany are in a poor ecological status.

Agricultural pesticides, nutrients, and habitat degradation are major causes of insect declines in lowland streams. To effectively conserve and restore stream habitats, standardized stream monitoring data and societal support for freshwater protection are needed. Here, we sampled 137 small stream monitoring sites across Germany, 83 % of which were located in agricultural catchments, with >900 citizen scientists in 96 monitoring groups. Sampling was carried out according to Water Framework Directive standards as part of the citizen science freshwater monitoring program FLOW in spring and summer 2021, 2022 and 2023. The biological indicator SPEARpesticides was used to assess pesticide exposure and effects based on macroinvertebrate community composition. Overall, 58 % of the agricultural monitoring sites failed to achieve a good ecological status in terms of macroinvertebrate community composition and indicated high pesticide exposure (SPEARpesticides status class: 29 % "moderate", 19 % "poor", 11 % "bad"). The indicated pesticide pressure in streams was related to the proportion of arable land in the catchment areas (R2 = 0.23, p < 0.001). Also with regards to hydromorphology, monitoring results revealed that 65 % of the agricultural monitoring sites failed to reach a good status. The database produced by citizen science groups was characterized by a high degree of accuracy, as results obtained by citizen scientists and professionals were highly correlated for SPEARpesticides index (R2 = 0.79, p < 0.001) and hydromorphology index values (R2 = 0.72, p < 0.001). Such citizen-driven monitoring of the status of watercourses could play a crucial role in monitoring and implementing the objectives of the European Water Framework Directive, thus contributing to restoring and protecting freshwater ecosystems.

Revealing the cascade of pesticide effects from gene to community.

Global pesticide exposure in agriculture leads to biodiversity loss, even at ultra-low concentrations below the legal limits. The mechanisms by which the effects of toxicants act at such low concentrations are still unclear, particularly in relation to their propagation across the different biological levels. In this study, we demonstrate, for the first time, a cascade of effects from the gene to the community level. At the gene level, agricultural pesticide exposure resulted in reduced genetic diversity of field-collected Gammarus pulex, a dominant freshwater crustacean in Europe. Additionally, we identified alleles associated with adaptations to pesticide contamination. At the individual level, this genetic adaptation to pesticides was linked to a lower fecundity, indicating related fitness costs. At the community level, the combined effect of pesticides and competitors caused a decline in the overall number and abundance of pesticides susceptible macroinvertebrate competing with gammarids. The resulting reduction in interspecific competition provided an advantage for pesticide-adapted G. pulex to dominate macroinvertebrate communities in contaminated areas, despite their reduced fitness due to adaptation. These processes demonstrate the complex cascade of effects, and also illustrate the resilience and adaptability of biological systems across organisational levels to meet the challenges of a changing environment.

Glyphosate without Co-formulants affects embryonic development of the south african clawed frog Xenopus laevis.

BACKGROUND: Glyphosate (GLY) is the most widely used herbicide in the world. Due to its mode of action as an inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, an important step in the shikimate pathway, specifically in plants, GLY is considered to be of low toxicity to non-target organisms. However, various studies have shown the negative effects of GLY on the mortality and development of different non-target organisms, including insects, rodents, fish and amphibians. To better understand the various effects of GLY in more detail, we studied the effects of GLY without co-formulants during the embryogenesis of the aquatic model organism Xenopus laevis. RESULTS: A treatment with GLY affected various morphological endpoints in X. laevis tadpoles (body length, head width and area, eye area). Additionally, GLY interfered with the mobility as well as the neural and cardiac development of the embryos at stage 44/45. We were able to detect detailed structural changes in the cranial nerves and the heart and gained insights into the negative effects of GLY on cardiomyocyte differentiation. CONCLUSION: The application of GLY without co-formulants resulted in negative effects on several endpoints in the early embryonic development of X. laevis at concentrations that are environmentally relevant and concentrations that reflect the worst-case scenarios. This indicates that GLY could have a strong negative impact on the survival and lives of amphibians in natural waters. As a result, future GLY approvals should consider its impact on the environment.

Ultra-low Esfenvalerate concentrations increase biomass and may reduce competitiveness of Daphnia magna populations.

Aquatic communities are frequently exposed to pesticides at sublethal concentrations, known to affect fitness parameters such as feeding, reproduction and population growth. Beside adverse effects, beneficial responses to toxicants at low concentrations may also occur. Positive effects, however, are thought to involve trade-offs. To identify such trade-offs, we quantified the population level effects on Daphnia magna during population carrying capacity in laboratory nanocosms after exposure to a single pulse of the pyrethroid insecticide esfenvalerate, including ultra-low concentrations ≤1/30 EC50. Population abundance and biomass were monitored 3 times per week for 3 months using a non-invasive imaging technique. High concentrations ≥1/10 EC50 resulted in reduced fitness endpoints. In contrast, ultra-low concentrations in the range of 0.01 µg/L significantly increased the population abundance of small (+160 %), medium (+130 %) and large organisms (+340 %) and their combined biomass (+200 %) during the 2 months after exposure. During the first five days after exposure to 0.01 µg/L and 0.03 µg/L esfenvalerate, population biomass increased by 0.1 mg/day while staying constant in the controls. While high control mortality makes firm conclusions about population responses of D. magna to esfenvalerate difficult, we hypothesize that population increases at ultra-low concentrations may be due to a hormetic response, where reduced intraspecific competitiveness is the trade-off that enables this response.

Temporal scales of pesticide exposure and risks in German small streams.

Following agricultural application, pesticides can enter streams through runoff during rain events. However, little information is available on the temporal dynamics of pesticide toxicity during the main application period. We investigated pesticide application and large scale in-stream monitoring data from 101 agricultural catchments obtained from a Germany-wide monitoring from April to July in 2018 and 2019. We analysed temporal patterns of pesticide application, in-stream toxicity and exceedances of regulatory acceptable concentrations (RAC) for over 70 pesticides. On a monthly scale from April to July, toxicity to invertebrates and algae/aquatic plants (algae) obtained with event-driven samples (EDS) was highest in May/June. The peak of toxicity towards invertebrates and algae coincided with the peaks of insecticide and herbicide application. Future monitoring, i.e. related to the Water Framework Directive, could be limited to time periods of highest pesticide applications on a seasonal scale. On a daily scale, toxicity to invertebrates from EDS exceeded those of grab samples collected within one day after rainfall by a factor of 3.7. Within two to three days, toxicity in grab samples declined compared to EDS by a factor of ten for invertebrates, and a factor of 1.6 for algae. Thus, toxicity to invertebrates declined rapidly within 1 day after a rainfall event, whereas toxicity to algae remained elevated for up to 4 days. For six pesticides, RAC exceedances could only be detected in EDS. The exceedances of RACs coincided with the peaks in pesticide application. Based on EDS, we estimated that pesticide exposure would need a 37-fold reduction of all analysed pesticides, to meet the German environmental target to keep RAC exceedances below 1 % of EDS. Overall, our study shows a high temporal variability of exposure on a monthly but also daily scale to individual pesticides that can be linked to their period of application and related rain events.

Standard Versus Natural: Assessing the Impact of Environmental Variables on Organic Matter Decomposition in Streams Using Three Substrates.

The decomposition of allochthonous organic matter, such as leaves, is a crucial ecosystem process in low-order streams. Microbial communities, including fungi and bacteria, colonize allochthonous organic material, break up large molecules, and increase the nutritional value for macroinvertebrates. Environmental variables are known to affect microbial as well as macroinvertebrate communities and alter their ability to decompose organic matter. Studying the relationship between environmental variables and decomposition has mainly been realized using leaves, with the drawbacks of differing substrate composition and consequently between-study variability. To overcome these drawbacks, artificial substrates have been developed, serving as standardizable surrogates. In the present study, we compared microbial and total decomposition of leaves with the standardized substrates of decotabs and, only for microbial decomposition, of cotton strips, across 70 stream sites in a Germany-wide study. Furthermore, we identified the most influential environmental variables for the decomposition of each substrate from a range of 26 variables, including pesticide toxicity, concentrations of nutrients, and trace elements, using stability selection. The microbial as well as total decomposition of the standardized substrates (i.e., cotton strips and decotabs) were weak or not associated with that of the natural substrate (i.e., leaves, r² < 0.01 to r² = 0.04). The decomposition of the two standardized substrates, however, showed a moderate association (r² = 0.21), which is probably driven by their similar composition, with both being made of cellulose. Different environmental variables were identified as the most influential for each of the substrates and the directions of these relationships contrasted between the substrates. Our results imply that these standardized substrates are unsuitable surrogates when investigating the decomposition of allochthonous organic matter in streams. Environ Toxicol Chem 2023;42:2007-2018. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Width of vegetated buffer strips to protect aquatic life from pesticide effects.

Vegetated buffer strips (VBS) are an effective measure to retain pesticide inputs during rain events. Numerous studies have examined the retention effects of VBS on pesticides. However, no study has addressed on a large scale with event-related peak concentrations how wide the VBS should be to avoid ecological impacts on aquatic life. Here, we investigated for 115 lowland stream sections in Germany the relevance of environmental and physico-chemical parameters to determine the in-stream pesticide concentration and their ecological risks. Based on peak concentrations related to rain events with precipitation amount resulting in VBS relevant surface runoff for 30 of the 115 investigated stream sections (25 to 70 mm/d), we demonstrated that the average width of VBS was the main parameter (R² = 0.38) reducing the pesticide input ratio, indicating a relevant proportion of surface runoff contributing to the total in-stream pesticide concentrations. Additionally, dry ditches within agricultural fields increased pesticide input (R² = 0.31). Generally, substances classified as slightly mobile were better retained by VBS than mobile substances. Other factors including slope, land use and vegetation cover of VBS had only a minor influence. We assessed the ecological risk of in-stream pesticide concentrations by quantifying exceedances of regulatory- (RAC) and field-validated acceptable concentrations (ACfield). We then translated this ecological risk into protective VBS width by calculating the quotient of in-stream concentration and threshold (RQ). We estimate that a VBS width of 18 m is sufficient to meet the RQACfield protection goal for 95% of streams. The presence of dry ditches increased the protective VBS width to 32 m. In current agricultural practice, however, 26% of the water stretches investigated do not comply with the prescribed 5 m VBS. An extension of the VBS area to 18 m would demand 3.8% of agricultural land within the catchments. A 50% reduction in pesticide use, as required by the European green deal, would still result in 39% (RAC) and 68% (ACfield) of event-related samples being exceeded. Consequently, we see the extension of the VBS width as the most efficient mearsure to sustainably reduce pesticide concentrations in small streams.

Citizen science for assessing pesticide impacts in agricultural streams.

The majority of central European streams are in poor ecological condition. Pesticide inputs from terrestrial habitats present a key threat to sensitive insects in streams. Both standardized stream monitoring data and societal support are needed to conserve and restore freshwater habitats. Citizen science (CS) offers potential to complement international freshwater monitoring while it is often viewed critically due to concerns about data accuracy. Here, we developed a CS program based on the Water Framework Directive that enables citizen scientists to provide data on stream hydromorphology, physicochemical status and benthic macroinvertebrates to apply the trait-based bio-indicator SPEARpesticides for pesticide exposure. We compared CS monitoring data with professional data across 28 central German stream sites and could show that both CS and professional monitoring identified a similar average proportion of pesticide-sensitive macroinvertebrate taxa per stream site (20 %). CS data were highly correlated to the professional data for both stream hydromorphology and SPEARpesticides (r = 0.72 and 0.76). To assess the extent to which CS macroinvertebrate data can indicate pesticide exposure, we tested the relationship of CS generated SPEARpesticides values and measured pesticide concentrations at 21 stream sites, and found a fair correlation similar to professional results. We conclude that given appropriate training and support, citizen scientists can generate valid data on the ecological status and pesticide contamination of streams. By complementing official monitoring, data from well-managed CS programs can advance freshwater science and enhance the implementation of freshwater conservation goals.

Land use changes biomass and temporal patterns of insect cross-ecosystem flows.

Emergent aquatic insects constitute an important food source for higher trophic levels, linking aquatic to terrestrial ecosystems. Little is known about how land use affects the biomass or composition of insect emergence. Previous studies are limited to individual time points or seasons, hampering understanding of annual biomass export patterns and detection of phenological changes. Over 1 year's primary emergence period, we continuously determined the biomass, abundance, and identity of >45,000 aquatic insects and recorded land-use-related environmental variables in 20 stream sites using a paired design with upstream forested sites and downstream agricultural sites. Total insect biomass and abundance were 2-7 mg day-1  m-2 and 7-36 ind day-1  m-2 higher in agricultural than forested sites. However, we found turnover of families between forested and agricultural sites, with more insects with shorter generation time in agriculture, indicating lower sensitivity to land-use-related stress because of higher recovery potential. Except for stoneflies, biomass and abundance of major orders were higher in agriculture, but their phenology differed. For different orders, emergence peaked 30 days earlier to 51 days later in agriculture than forest, whereas total abundance and biomass both peaked earlier in agriculture: 3-5 and 3-19 days, respectively. The most important land-use-related drivers were pesticide toxicity and electrical conductivity, which were differentially associated with different aquatic insect order abundances and biomass. Overall, we found that land use was related to changes in composition and phenology of aquatic insect emergence, which is likely to affect food-web dynamics in a cross-ecosystem context.

Time between Sequential Exposures to Multiple Stress Turns Antagonism into Synergism.

Aquatic communities are exposed to repeated pulses of toxicants and environmental stressors. We hypothesize that the dose, order, and timing of stress events shape the interactions of these communities. For this, we conducted a fully-crossed, four-factorial, multiple stress exposure experiment to study the combined effects of Esfenvalerate and ultraviolet-B (UV-B) radiation related to the exposure timing and order on Daphnia magna. We revealed that initial exposure to low stress doses, independent of the stress type (UV-B or Esfenvalerate), significantly increased the resistance toward the second stressor. This beneficial effect was apparent only when the second stressor was applied immediately after the first stressor (p < 0.01). When the period between stressor applications was extended to 2 days, the antagonism between the two stressors turned into synergism. The stressor interaction could be predicted with an abstract-mechanistic model of the temporal dynamics of the early-stage stress response. With this model, the timing and order of exposures were able to successfully explain interactions observed in all treatments (model-R2 = 1.0). We conclude that especially the duration of a break between exposures and the exposure dose have a decisive influence on interactions between toxicants and environmental stressors.

Balancing effort and benefit - How taxonomic and quantitative resolution influence the pesticide indicator system SPEARpesticides.

Biological indices aim to reflect the ecological quality of streams based on the community's species or trait composition. Accordingly, the capability to predict the ecological quality depends on (i) the knowledge on the association of taxa or traits with stressors and (ii) the taxonomic and quantitative resolution of taxa. Generally speaking, a higher resolution is associated with a better linkage between environmental condition and biological response but also with higher efforts and costs. So far it is unknown how the taxonomic and quantitative resolution affect the ecological quality assessment of streams related to pesticide effects when applying the invertebrate-based indicator SPEARpesticides. We investigated the ecological quality of 101 streams considering four taxonomic levels (species, genus, family, order) and three quantitative resolutions (abundance, three abundance classes, and presence-absence). In a multiple linear regression analysis between 13 investigated stressors and SPEARpesticides, the full models' explained variance remained fairly constant with decreasing taxonomic and quantitative resolution. As expected, the highest association between pesticide pressure and SPEARpesticides was reached at a species/abundance resolution yielding an R2 of 0.43. In contrast, the lowest quantitative resolution of order level combined with presence-absence information revealed an explained variance of 0.28 R2. We suggest the family/abundance class resolution (R2 = 0.38) as the best trade-off between effort and accuracy for large-scale monitoring. Due to a comparable linear regression at family/abundance class resolution, the assigned ecological quality classes were largely congruent (69 %) to species/abundance resolution. We conclude that the ecological quality assessment with SPEARpesticides at family/abundance class resolution can be used to link pesticide contamination and invertebrate community structure with less taxonomic expertise and less quantification effort.

Potential propagation of agricultural pesticide exposure and effects to upstream sections in a biosphere reserve.

In the last decades, several studies have shown that pesticides frequently occur above water quality thresholds in small streams draining arable land and are associated with changes in invertebrate communities. However, we know little about the potential propagation of pesticide effects from agricultural stream sections to least impacted stream sections that can serve as refuge areas. We sampled invertebrates and pesticides along six small streams in south-west Germany. In each stream, the sampling was conducted at an agricultural site, at an upstream forest site (later considered as "refuge"), and at a transition zone between forest and agriculture (later considered as "edge"). Pesticide exposure was higher and the proportion of pesticide-sensitive species (SPEARpesticides) was lower in agricultural sites compared to edge and refuge sites. Notwithstanding, at some edge and refuge sites, which were considered as being least impacted, we estimated unexpected pesticide toxicity (sum toxic units) exceeding thresholds at which field studies suggested adverse effects on freshwater invertebrates. We conclude that organisms in forest sections within a few kilometres upstream of agricultural areas can be exposed to ecologically relevant pesticide levels. In addition, although not statistically significant, the abundance of pesticide-sensitive taxa was slightly lower in edge compared to refuge sites, indicating a potential influence of adjacent agriculture. Future studies should further investigate the influence of spatial relationships, such as the distance between refuge and agriculture, for the propagation of pesticide effects and focus on the underlying mechanisms.

Three reasons why the Water Framework Directive (WFD) fails to identify pesticide risks.

The Water Framework Directive (WFD) demands that good status is to be achieved for all European water bodies. While governmental monitoring under the WFD mostly concludes a good status with regard to pesticide pollution, numerous scientific studies have demonstrated widespread negative ecological impacts of pesticide exposure in surface waters. To identify reasons for this discrepancy, we analysed pesticide concentrations measured in a monitoring campaign of 91 agricultural streams in 2018 and 2019 using methodologies that exceed the requirements of the WFD. This included a sampling strategy that takes into account the periodic occurrence of pesticides and a different analyte spectrum designed to reflect current pesticide use. We found that regulatory acceptable concentrations (RACs) were exceeded for 39 different pesticides at 81% of monitoring sites. In comparison, WFD-compliant monitoring of the same sites would have detected only eleven pesticides as exceeding the WFD-based environmental quality standards (EQS) at 35% of monitoring sites. We suggest three reasons for this underestimation of pesticide risk under the WFD-compliant monitoring: (1) The sampling approach - the timing and site selection are unable to adequately capture the periodic occurrence of pesticides and investigate surface waters particularly susceptible to pesticide risks; (2) the measuring method - a too narrow analyte spectrum (6% of pesticides currently approved in Germany) and insufficient analytical capacities result in risk drivers being overlooked; (3) the assessment method for measured concentrations - the protectivity and availability of regulatory thresholds are not sufficient to ensure a good ecological status. We therefore propose practical and legal refinements to improve the WFD's monitoring and assessment strategy in order to gain a more realistic picture of pesticide surface water pollution. This will enable more rapid identification of risk drivers and suitable risk management measures to ultimately improve the status of European surface waters.

Multiple Stress Reduces the Advantage of Pesticide Adaptation.

Under global change scenarios, multistress conditions may occur regularly and require adaptation. However, the adaptation to one stressor might be associated with the increased sensitivity to another stressor. Here, we investigated the ecological consequences of such trade-off under multiple stress. We compared the pesticide tolerance of the crustacean Gammarus pulex from agricultural streams with populations from reference streams. Under optimum temperature, G. pulex from agricultural streams were considerably more tolerant to pesticides as compared to the reference populations. Here, we assume that the increased tolerance in agricultural populations is the combination of acclimation, epigenetic effect, and genetic evolution. After experimental pre-exposure to very low concentration (LC50/1000), reference populations showed increased pesticide tolerance. In contrast, pre-exposure did not further increase the tolerance of agricultural populations. Moreover, these populations were more sensitive to elevated temperature alone due to the hypothesized fitness cost of genetic adaptation to pesticides. However, both reference and agricultural populations showed a similar tolerance to the combined stress of pesticides and warming due to stronger synergistic effects in adapted populations. As a result, pesticide adaptation loses its advantage. The combined effect was predicted well using the stress addition model, developed for predicting the synergistic interaction of independent stressors. We conclude that under multistress conditions, adaptation to pesticides reduces the general stress capacity of individuals and trade-off processes increase the sensitivity to additional stressors. This causes strong synergistic effects of additional stressors on pesticide-adapted individuals.