Assessing the Mixture Effects in In Vitro Bioassays of Chemicals Occurring in Small Agricultural Streams during Rain Events.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity; activation of the estrogen, aryl hydrocarbon, and peroxisome proliferator-activated receptors; and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street runoff were detected. The unexpectedly high estrogenic effects in many samples indicated the impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (<8%) of the measured effects. The experimental effects of the designed mixtures of detected chemicals that were expected to dominate the mixture effects of detected chemicals were consistent with predictions for concentration addition within a factor of two for 94% of the mixtures. Overall, the burden of chemicals and effects was much higher than that previously detected in surface water during dry weather, with the effects often exceeding proposed effect-based trigger values.

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.

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.

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.

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.

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.

Risk from pesticide mixtures - The gap between risk assessment and reality.

Pesticide applications in agricultural crops often comprise a mixture of plant protection products (PPP), and single fields face multiple applications per year leading to complex pesticide mixtures in the environment. Restricted to single PPP, the current European Union PPP regulation, however, disregards the ecological risks of pesticide mixtures. To quantify this additional risk, we evaluated the contribution of single pesticide active ingredients to the additive mixture risk for aquatic risk indicators (invertebrates and algae) in 464 different PPP used, 3446 applications sprayed and 830 water samples collected in Central Europe, Germany. We identified an average number of 1.3 different pesticides in a single PPP, 3.1 for complete applications often involving multiple PPP and 30 in stream water samples. Under realistic worst-case conditions, the estimated stream water pesticide risk based on additive effects was 3.2 times higher than predicted from single PPP. We found that in streams, however, the majority of regulatory threshold exceedances was caused by single pesticides alone (69% for algae, 81% for invertebrates). Both in PPP applications and in stream samples, pesticide exposure occurred in repeated pulses each driven by one to few alternating pesticides. The time intervals between pulses were shorter than the 8 weeks considered for ecological recovery in environmental risk assessment in 88% of spray series and 53% of streams. We conclude that pesticide risk assessment should consider an additional assessment factor to account for the additive, but also potential synergistic simultaneous pesticide mixture risk. Additionally, future research and risk assessment need to address the risk from the frequent sequential pesticide exposure observed in this study.

Small streams-large concentrations? Pesticide monitoring in small agricultural streams in Germany during dry weather and rainfall.

Few studies have examined the exposure of small streams (< 30 km2 catchment size) to agriculturally used pesticides, compared to large rivers. A total of 105 sites in 103 small agricultural streams were investigated for 76 pesticides (insecticides, herbicides, fungicides) and 32 pesticide metabolites in spring and summer over two years (2018 and 2019) during dry weather and rainfall using event-driven sampling. The median total concentration of the 76 pesticides was 0.18 µg/L, with 9 pesticides per sample on average (n = 815). This is significantly higher than monitoring data for larger streams, reflecting the close proximity to agricultural fields and the limited dilution by non-agricultural waters. The frequency of detection of all pesticides correlated with sales quantity and half-lives in water. Terbuthylazine, MCPA, boscalid, and tebuconazole showed the highest median concentrations. The median of the total concentration of the 32 metabolites exceeded the pesticide concentration by more than an order of magnitude. During dry weather, the median total concentration of the 76 pesticides was 0.07 µg/L, with 5 pesticides per sample on average. Rainfall events increased the median total pesticide concentration by a factor of 10 (to 0.7 µg/L), and the average number of pesticides per sample to 14 (with up to 41 in single samples). The concentration increase was particularly strong for 2,4-D, MCPA, terbuthylazine, and nicosulfuron (75 percentile). Metabolite concentrations were generally less responsive to rainfall, except for those of terbuthylazine, flufenacet, metamitron, and prothioconazole. The frequent and widespread exceedance of the regulatory acceptable concentrations (RAC) of the 76 pesticides during both, dry weather and rainfall, suggests that current plant protection product authorization and risk mitigation methods are not sufficient to protect small streams.

Pesticides are the dominant stressors for vulnerable insects in lowland streams.

Despite elaborate regulation of agricultural pesticides, their occurrence in non-target areas has been linked to adverse ecological effects on insects in several field investigations. Their quantitative role in contributing to the biodiversity crisis is, however, still not known. In a large-scale study across 101 sites of small lowland streams in Central Europe, Germany we revealed that 83% of agricultural streams did not meet the pesticide-related ecological targets. For the first time we identified that agricultural nonpoint-source pesticide pollution was the major driver in reducing vulnerable insect populations in aquatic invertebrate communities, exceeding the relevance of other anthropogenic stressors such as poor hydro-morphological structure and nutrients. We identified that the current authorisation of pesticides, which aims to prevent unacceptable adverse effects, underestimates the actual ecological risk as (i) measured pesticide concentrations exceeded current regulatory acceptable concentrations in 81% of the agricultural streams investigated, (ii) for several pesticides the inertia of the authorisation process impedes the incorporation of new scientific knowledge and (iii) existing thresholds of invertebrate toxicity drivers are not protective by a factor of 5.3 to 40. To provide adequate environmental quality objectives, the authorisation process needs to include monitoring-derived information on pesticide effects at the ecosystem level. Here, we derive such thresholds that ensure a protection of the invertebrate stream community.