Environmental Stress Increases Synergistic Effects of Pesticide Mixtures on Daphnia magna.

Some widely used pesticide mixtures produce more than additive effects according to conventional combined effect models. However, synergistic effects have been so far generally observed at unrealistically high pesticide concentrations. Here, we used Daphnia magna as a test organism and investigated how food limitation-a common ecological stressor-affects the mixture toxicity of a pyrethroid insecticide and azole fungicide. We also compared three models regarding the prediction of mixture effects, including concentration addition (CA), effect addition (EA), and stress addition model (SAM). We revealed that especially under low food, the strength of synergism between esfenvalerate and prochloraz increased with an increasing concentration of prochloraz independent of the null model. Under high food conditions and at concentrations of prochloraz ≥32 µg/L, we observed a marginal synergistic effect with a model deviation ratio (MDR) = 2.1 at 32 µg/L prochloraz and 2.2 at 100 µg/L prochloraz when using CA as the null model. In contrast, the combination of both pesticides and food stress caused synergistic effects shown by an MDR = 10.9 even at 1 µg/L of prochloraz that is frequently detected in the environment. The combined effects of pesticides and food stress could be predicted best with the SAM that showed the lowest mean deviation between effect observation and prediction (mean deviation SAM = 16 [SD = 28], EA = 1072 [2105], CA = 1345 [2644]). We conclude that common environmental stressors can strongly increase the synergistic effects of toxicants. This knowledge is especially relevant considering current efforts to include the additional risk of pesticide mixtures and environmental stressors into the environmental risk assessment of pesticides.

Environmental stressors can enhance the development of community tolerance to a toxicant.

Ecosystems are subject to a combination of recurring anthropogenic and natural disturbances, such as climate change and pesticide exposure. Biological communities are known to develop tolerance to recurring disturbances due to successive changes at both the community and organismal levels. However, information on how additional stressors may affect the development of such community tolerance is scarce to date. We studied the influence of hydrological disturbance on the reaction of zooplankton communities to repeated insecticide pulses in outdoor microcosms. The communities were exposed to three successive pulses of the insecticide esfenvalerate (0.03, 0.3, and 3 µg/L) and to the gradual removal of water and its subsequent replacement over three cycles or to a constant water level. Except at the highest esfenvalerate concentration, the communities developed tolerance to the toxicant, as indicated by their decreasing reaction to subsequent insecticide applications, and this development was enhanced by hydrological disturbance. The pronounced decline of the key taxa Daphnia spp. through the combined action of the two stressors was identified as the main mechanism responsible for the increase in community tolerance under a fluctuating water level. Under a constant water level, the abundance of Daphnia spp. did not decrease significantly without the insecticide treatment, indicating that other mechanisms were responsible for the observed community tolerance. The present study shows that additional stressors can facilitate the development of community tolerance and that such facilitation is propagated through community-level mechanisms.

A systems-based analysis to rethink the European environmental risk assessment of regulated chemicals using pesticides as a pilot case.

A growing body of scientific literature stresses the need to advance current environmental risk assessment (ERA) methodologies and associated regulatory frameworks to better address the landscape-scale and long-term impact of pesticide use on biodiversity and the ecosystem. Moreover, more collaborative and integrative approaches are needed to meet sustainability goals. The One Health approach is increasingly applied by the European Food Safety Authority (EFSA) to support the transition towards safer, healthier and more sustainable food. To this end, EFSA commissioned the development of a roadmap for action to establish a European Partnership for next-generation, systems-based Environmental Risk Assessment (PERA). Here, we summarise the main conclusions and recommendations reported in the 2022 PERA Roadmap. This roadmap highlights that fragmentation of data, knowledge and expertise across regulatory sectors results in suboptimal processes and hinders the implementation of integrative ERA approaches needed to better protect the environment. To advance ERA, we revisited the underlying assumptions of the current ERA paradigm; that chemical risks are generally assessed and managed in isolation with a substance-by-substance, realistic worst-case and tiered approach. We suggest optimising the use of the vast amount of information and expertise available with pesticides as a pilot area. It is recommended to as soon as possible adopt a systems-based approach, i.e. within the current regulatory framework, to spark a step-wise transition towards an ERA framed at a system level of ecological and societal relevance. Tangible systems-based and integrative steps are available. For instance, the rich sources of existing data for prospective and retrospective ERA of pesticides could be used to reality-benchmark existing and new ERA methods. To achieve these goals, collaboration among stakeholders across scientific disciplines and regulatory sectors must be strengthened.

Intraspecific competition increases toxicant effects in outdoor pond microcosms.

Competition is a ubiquitous factor in natural populations and has been reported to alter the ecological impact of xenobiotics. We investigated conditions that mirror the natural variation of environmental factors. For this, different treatments were applied to 96 outdoor pond microcosms by shading the ponds and harvesting the communities. Then, the effect of esfenvalerate (0.03, 0.3, and 3 µg/L) on populations of Daphnia spp. was investigated. The pesticide effect and the sensitivity of Daphnia spp. in the context of a zooplankton community was increased by intraspecific competition 11 days after contamination. This relationship was most pronounced at 0.03 and 0.3 µg/L esfenvalerate, which were the concentrations that led to partial mortality. In contrast, interspecific interaction did not significantly alter the effect of the toxicant on Daphnia spp. Modelled concentration-response curves showed that the negative effects of the pesticide differed by a factor of up to 100 depending on the strength of intraspecific competition. In addition, a wider range of concentrations led to negative effects at high levels of intraspecific competition than at low levels. We argue that increased intraspecific competition reduces the availability of resources at the individual level and thereby increases the effect of contaminants. This knowledge about the interaction between competition and the response to toxicants is important in assessing the effects of these factors under field conditions.

Interspecific competition delays recovery of Daphnia spp. populations from pesticide stress.

Xenobiotics alter the balance of competition between species and induce shifts in community composition. However, little is known about how these alterations affect the recovery of sensitive taxa. We exposed zooplankton communities to esfenvalerate (0.03, 0.3, and 3 µg/L) in outdoor microcosms and investigated the long-term effects on populations of Daphnia spp. To cover a broad and realistic range of environmental conditions, we established 96 microcosms with different treatments of shading and periodic harvesting. Populations of Daphnia spp. decreased in abundance for more than 8 weeks after contamination at 0.3 and 3 µg/L esfenvalerate. The period required for recovery at 0.3 and 3 µg/L was more than eight and three times longer, respectively, than the recovery period that was predicted on the basis of the life cycle of Daphnia spp. without considering the environmental context. We found that the recovery of sensitive Daphnia spp. populations depended on the initial pesticide survival and the related increase of less sensitive, competing taxa. We assert that this increase in the abundance of competing species, as well as sub-lethal effects of esfenvalerate, caused the unexpectedly prolonged effects of esfenvalerate on populations of Daphnia spp. We conclude that assessing biotic interactions is essential to understand and hence predict the effects and recovery from toxicant stress in communities.

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.

Long-term effects of a catastrophic insecticide spill on stream invertebrates.

Accidental spills or illegal discharges of pesticides in aquatic ecosystems can lead to exposure levels that strongly exceed authorized pesticide concentrations, causing major impacts on aquatic ecosystems. Such short-term events often remain undetected in regular monitoring programs with infrequent sampling. In early spring 2015, we identified a catastrophic pesticide spill with the insecticide cypermethrin in the Holtemme River, Germany. Based on existing pre-event macroinvertebrate community data, we monitored the effects and recovery of the macroinvertebrate community for more than two years after the spill. Strong short-term effects were apparent for all taxa with the exception of Chironomidae and Tubificidae. Effects could also be observed on the community level as total abundance, taxa number and biomass strongly decreased. Total abundance and taxa number showed a fast recovery. Regarding long-term effects, the total biomass remained substantially below the pre-contamination level (76%) until the end of the study. Also the abundances of three taxa (Gammarus, Leuctra, Limnius Ad.) did not return to levels prior to the spill even after 26 months. This lack of the taxon-specific recovery was likely due to their long generation time and a low migration ability due to a restricted connectivity between the contaminated site and uncontaminated stream sections. These factors proved to be stronger predictors for the recovery than the pesticide tolerance. We revealed that the biological indicators SPEARpesticides and share of Ephemeroptera, Plecoptera and Trichoptera (EPT) are not suitable for the identification of such extreme events, when nearly all taxa are eradicated. Both indicators are functioning only when repeated stressors initiate long-term competitive replacement of sensitive by insensitive taxa. We conclude that pesticide spills can have significant long-term effects on stream macroinvertebrate communities. Regular ecological monitoring is imperative to identify such ecosystem impairments, combined with analytical chemistry methods to identify the potential sources of spills.

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.

Species occurrence relates to pesticide gradient in streams.

Freshwater communities are threatened worldwide, with pesticides being one of the main stressors for vulnerable invertebrates. Whereas the effects of pesticides on communities can be quantified by trait-based bioindicators such as SPEARpesticides, single species' responses remain largely unknown. We used the bioindicator SPEARpesticides to predict the toxic pressure from pesticides in 6942 macroinvertebrate samples from 4147 sites during the period 2004 to 2013, obtained by environmental authorities in Germany, and classified all samples according to their magnitude of pesticide pressure. Along this gradient of pesticide pressure, we quantified the occurrence of 139 macroinvertebrate species. We identified 71 species characterized by decreasing occurrence with increasing pesticide pressure. These 'decreasing species', mainly insects, occurred at a frequency of 19.7% at sites with reference conditions and decreased to 1.7% at sites with the highest pesticide pressure. We further determined 55 'nonspecific species' with no strong response as well as 13 'increasing species', mainly Gastropoda, Oligochaeta and Diptera, which showed an increase of frequency from 1.8% at sites with reference conditions to 11.4% at sites with the highest pesticide pressure. Based on the change in frequency we determined the pesticide vulnerability of single species, expressed as Pesticide Associated Response (PARe). Furthermore, a trait analysis revealed that species' occurrence may additionally depend on oxygen demand and, to a lesser extent on substrate preference, whereas no significant effect of feeding and respiration type could be found. Our results provide the first extensive pesticide vulnerability ranking for single macroinvertebrate species based on empirical large-scale field data.

Predicting low-concentration effects of pesticides.

We present a model to identify the effects of low toxicant concentrations. Due to inadequate models, such effects have so far often been misinterpreted as random variability. Instead, a tri-phasic relationship describes the effects of a toxicant when a broad range of concentrations is assessed: i) at high concentrations where substantial mortality occurs (LC50), we confirmed the traditional sigmoidal response curve (ii) at low concentrations about 10 times below the LC50, we identified higher survival than previously modelled, and (iii) at ultra-low concentrations starting at around 100 times below the LC50, higher mortality than previously modelled. This suggests that individuals benefit from low toxicant stress. Accordingly, we postulate that in the absence of external toxicant stress individuals are affected by an internal "System Stress" (SyS) and that SyS is reduced with increasing strength of toxicant stress. We show that the observed tri-phasic concentration-effect relationship can be modelled on the basis of this approach. Here we revealed that toxicant-related effects (LC5) occurred at remarkably low concentrations, 3 to 4 orders of magnitude below those concentrations inducing strong effects (LC50). Thus, the ECx-SyS model presented allows us to attribute ultra-low toxicant concentrations to their effects on individuals. This information will contribute to performing a more realistic environmental and human risk assessment.

Indication of pesticide effects and recolonization in streams.

The agricultural use of pesticides leads to environmentally relevant pesticide concentrations that cause adverse effects on stream ecosystems. These effects on invertebrate community composition can be identified by the bio-indicator SPEARpesticides. However, refuge areas have been found to partly confound the indicator. On the basis of three monitoring campaigns of 41 sites in Central Germany, we identified 11 refuge taxa. The refuge taxa, mainly characterized by dispersal-based resilience, were observed only nearby uncontaminated stream sections and independent of the level of pesticide pressure. Through incorporation of this information into the revised SPEARpesticides indicator, the community structure specifically identified the toxic pressure and no longer depended on the presence of refuge areas. With regard to ecosystem functions, leaf litter degradation was predicted by the revised SPEARpesticides and the median water temperature at a site (R2 = 0.38, P = 0.003). Furthermore, we designed the bio-indicator SPEARrefuge to quantify the magnitude of general recolonization at a given stream site. We conclude that the taxonomic composition of aquatic invertebrate communities enables a specific indication of anthropogenic stressors and resilience of ecosystems.

Predicting the synergy of multiple stress effects.

Toxicants and other, non-chemical environmental stressors contribute to the global biodiversity crisis. Examples include the loss of bees and the reduction of aquatic biodiversity. Although non-compliance with regulations might be contributing, the widespread existence of these impacts suggests that for example the current approach of pesticide risk assessment fails to protect biodiversity when multiple stressors concurrently affect organisms. To quantify such multiple stress effects, we analysed all applicable aquatic studies and found that the presence of environmental stressors increases individual sensitivity to toxicants (pesticides, trace metals) by a factor of up to 100. To predict this dependence, we developed the "Stress Addition Model" (SAM). With the SAM, we assume that each individual has a general stress capacity towards all types of specific stress that should not be exhausted. Experimental stress levels are transferred into general stress levels of the SAM using the stress-related mortality as a common link. These general stress levels of independent stressors are additive, with the sum determining the total stress exerted on a population. With this approach, we provide a tool that quantitatively predicts the highly synergistic direct effects of independent stressor combinations.

Forested headwaters mitigate pesticide effects on macroinvertebrate communities in streams: Mechanisms and quantification.

Pesticides impact invertebrate communities in freshwater ecosystems, leading to the loss of biodiversity and ecosystem functions. One approach to reduce such effects is to maintain uncontaminated stream reaches that can foster recovery of the impacted populations. We assessed the potential of uncontaminated forested headwaters to mitigate pesticide impact on the downstream macroinvertebrate communities in 37 streams, using the SPEARpesticides index. Pesticide contamination was measured with runoff-triggered techniques and Chemcatcher® passive samplers. The data originated from 3 field studies conducted between 1998 and 2011. The proportion of vulnerable species decreased significantly after pesticide exposure even at low toxicity levels (-4

Elevated temperature prolongs long-term effects of a pesticide on Daphnia spp. due to altered competition in zooplankton communities.

Considerable research efforts have been made to predict the influences of climate change on species composition in biological communities. However, little is known about how changing environmental conditions and anthropogenic pollution can affect aquatic communities in combination. We investigated the influence of short warming periods on the response of a zooplankton community to the insecticide esfenvalerate at a range of environmentally realistic concentrations (0.03, 0.3 and 3 µg L(-1) ) in 55 outdoor pond microcosms. Warming periods increased the cumulative water temperature, but did not exceed the maximum temperature measured under ambient conditions. Under warming conditions alone the abundance of some zooplankton taxa increased selectively compared to ambient conditions. This resulted in a shift in the community composition that had not recovered by the end of the experiment, 8 weeks after the last warming period. Regarding the pesticide exposure, short-term effects of esfenvalerate on the community structure and the sensitive taxa Daphnia spp. did not differ between the two temperature regimes. In contrast, long-term effects of esfenvalerate on Daphnia spp., a taxon that did not benefit from elevated temperatures, were observed twice as long under warming than under ambient conditions. This resulted in long-term effects on Daphnia spp. until 4 months after contamination at 3 µg L(-1) esfenvalerate. Under both temperature regimes, we identified strength of interspecific competition as the mechanism determining the time until recovery. However, enhanced interspecific competition under warming conditions was prolonged and explained the delayed recovery of Daphnia spp. from esfenvalerate. These results show that, for realistic prediction of the combined effects of changing environmental factors and toxicants on sensitive taxa, the impacts of stressors on the biotic interactions within the community need to be considered.

Two stressors and a community: effects of hydrological disturbance and a toxicant on freshwater zooplankton.

Climate change models predict an increase in the frequency and intensity of extreme fluctuations in water level in aquatic habitats. Therefore, it is necessary to understand the combined effects of hydrological fluctuations and toxicants on aquatic biological communities. We investigated the individual and combined effects of the insecticide esfenvalerate and recurring fluctuations in water level on zooplankton communities in a system of 55 outdoor pond microcosms. The communities were exposed to esfenvalerate contamination as a single pulse (at 0.03, 0.3, or 3µg/L) and gradual removal of water and its subsequent replacement over three cycles and monitored until 84 days after contamination. The results showed that the sensitivities of the community and its constituent populations to the toxicant were increased by the hydrological stress. Specifically, for both the community structure and abundance of Daphnia spp. the lowest-observed-effect concentrations (LOEC) were 0.03 and 0.3µg/L for the series with fluctuating and constant water levels, respectively. Despite these differences in sensitivity, the interactive effects of the two stressors were found to be additive for both the community structure and the abundance of the most affected species. Presumably, it was not possible to detect synergism due to the strong individual effects of the water level fluctuations. Recovery times in the series exposed to the highest pesticide concentration were 64 and 55 days under fluctuating and constant water level regimes, respectively. Competition and water quality are suggested to be the major factors that underlie the observed effects of fluctuations in the water level. For the ecological risk assessment of toxicants, the present results suggest that (i) community sensitivity may vary substantially, depending on the environmental context, and (ii) this variability can be assessed experimentally to derive safety factors (coefficients used to avoid unexpected effects and define safe concentrations of toxicants) based on empirical findings. This contrasts with the current approach where such factors are usually defined arbitrarily.

Environmental context determines community sensitivity of freshwater zooplankton to a pesticide.

The environment is currently changing worldwide, and ecosystems are being exposed to multiple anthropogenic pressures. Understanding and consideration of such environmental conditions is required in ecological risk assessment of toxicants, but it remains basically limited. In the present study, we aimed to determine how and to what extent alterations in the abiotic and biotic environmental conditions can alter the sensitivity of a community to an insecticide, as well as its recovery after contamination. We conducted an outdoor microcosm experiment in which zooplankton communities were exposed to the insecticide esfenvalerate (0.03, 0.3, and 3 µg/L) under different regimes of solar radiation and community density, which represented different levels of food availability and competition. We focused on the sensitivity of the entire community and analysed it using multivariate statistical methods, such as principal response curves and redundancy analysis. The results showed that community sensitivity varied markedly between the treatments. In the experimental series with the lowest availability of food and strongest competition significant effects of the insecticide were found at the concentration of 0.03 µg/L. In contrast, in the series with relatively higher food availability and weak competition such effects were detected at 3 µg/L only. However, we did not find significant differences in the community recovery rates between the experimental treatments. These findings indicate that environmental context is more important for ecotoxicological evaluation than assumed previously.