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.

Emerging Midges Transport Pesticides from Aquatic to Terrestrial Ecosystems: Importance of Compound- and Organism-Specific Parameters.

Emerging aquatic insects have the potential to retain aquatic contaminants after metamorphosis, potentially transporting them into adjacent terrestrial food webs. It is unknown whether this transfer is also relevant for current-use pesticides. We exposed larvae of the nonbiting midge, Chironomus riparius, to a sublethal pulse of a mixture of nine moderately polar fungicides and herbicides (logKow 2.5-4.7) at three field relevant treatment levels (1.2-2.5, 17.5-35.0, or 50.0-100.0 µg/L). We then assessed the pesticide bioaccumulation and bioamplification over the full aquatic-terrestrial life cycle of both sexes including the egg laying of adult females. By applying sensitive LC-MS/MS analysis to small sample volumes (∼5 mg, dry weight), we detected all pesticides in larvae from all treatment levels (2.8-1019 ng/g), five of the pesticides in the adults from the lowest treatment level and eight in the higher treatment levels (1.5-3615 ng/g). Retention of the pesticides through metamorphosis was not predictable based solely on pesticide lipophilicity. Sex-specific differences in adult insect pesticide concentrations were significant for five of the pesticides, with greater concentrations in females for four of them. Over the duration of the adults' lifespan, pesticide concentrations generally decreased in females while persisting in males. Our results suggest that a low to moderate daily dietary exposure to these pesticides may be possible for tree swallow nestlings and insectivorous bats.

Phenotypic adaptation of Chironomus riparius to chronic Bti exposure: effects on emergence time and nutrient content.

Insects with aquatic larval and terrestrial adult life stages are a key component of coupled aquatic-terrestrial ecosystems. Thus, stressors applied to water bodies adversely affecting those larvae have the potential to influence the riparian zone through altered emergence, with differences in prey availability, timing, or nutrition. In this study, the common model organism Chironomus riparius, a species of Chironomidae (Diptera), was used. This selection was further motivated by its wide distribution in European freshwaters and its importance as prey for terrestrial predators. A stressor of high importance in this context is the globally used mosquito control agent Bacillus thuringiensis var. israelensis (Bti) which has been shown to affect Chironomidae. Here, we investigated the ability of chironomid populations to adapt to a regularly applied stressor, leading to a reduced impact of Bti. Therefore, the initial sensitivity of laboratory populations of C. riparius was investigated under the influence of field-relevant Bti treatments (three doses × two application days) and different food sources (high-quality TetraMin vs. low-quality Spirulina). Following a chronic exposure to Bti over six months, the sensitivity of pre-exposed and naïve populations was re-evaluated. Food quality had a strong impact on emergence timing and nutrient content. In addition, alterations in emergence time as well as protein and lipid contents of chronically exposed populations indicated a selection for individuals of advantageous energetics, potentially leading to a more efficient development while combating Bti. Signs of adaptation could be confirmed in five out of 36 tested scenarios suggesting adaptation to Bti at the population level. Adaptive responses of one or several species could theoretically (via eco-evolutionary dynamics) result in a community shift, favouring the prevalence of Bti-tolerant species. (In)direct effects of Bti and the adaptive responses at both population and community levels could affect higher trophic levels and may determine the fate of meta-ecosystems.

How non-target chironomid communities respond to mosquito control: Integrating DNA metabarcoding and joint species distribution modelling.

The conservation and management of riparian ecosystems rely on understanding the ecological consequences of anthropogenic stressors that impact natural communities. In this context, studies investigating the effects of anthropogenic stressors require reliable methods capable of mapping the relationships between taxa occurrence or abundance and environmental predictors within a spatio-temporal framework. Here, we present an integrative approach using DNA metabarcoding and Hierarchical Modelling of Species Communities (HMSC) to unravel the intricate dynamics and resilience of chironomid communities exposed to Bacillus thuringiensis var. israelensis (Bti). Chironomid emergence was sampled from a total of 12 floodplain pond mesocosms, half of which received Bti treatment, during a 16-week period spanning spring and summer of 2020. Subsequently, we determined the community compositions of chironomids and examined their genus-specific responses to the Bti treatment, considering their phylogenetic affiliations and ecological traits of the larvae. Additionally, we investigated the impact of the Bti treatment on the body size distribution of emerging chironomids. Our study revealed consistent responses to Bti among different chironomid genera, indicating that neither phylogenetic affiliations nor larval feeding strategies significantly contributed to the observed patterns. Both taxonomic and genetic diversity were positively correlated with the number of emerged individuals. Furthermore, our findings demonstrated Bti-related effects on chironomid body size distribution, which could have relevant implications for size-selective terrestrial predators. Hence, our study highlights the value of employing a combination of DNA metabarcoding and HMSC to unravel the complex dynamics of Bti-related non-target effects on chironomid communities. The insights gained from this integrated framework contribute to our understanding of the ecological consequences of anthropogenic stressors and provide a foundation for informed decision-making regarding the conservation and management of riparian ecosystems.

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.

Does biocide treatment for mosquito control alter carbon dynamics in floodplain ponds?

Shallow lentic aquatic ecosystems, such as ponds, are important repositories of carbon (C) and hotspots of C cycling and greenhouse gas emission. Tube-dwelling benthic invertebrates, such as chironomids, may be key players in C dynamics in these water bodies, yet their role in the C-budget at ecosystem level remains unclear. We tested whether a 41 % reduction in chironomid abundance after application of the mosquito control biocide Bacillus thuringiensis israelensis (Bti) had implications for the C-fluxes to the atmosphere, C-pools, and C-transformation (i.e. organic matter decomposition) in ponds. Data were collected over one year in the shallow, deep and riparian zones of 12 experimental floodplain pond mesocosms (FPMs), half of them treated with Bti. C-fluxes were measured as CO2 and CH4 emissions, atmospheric deposition, and emerging insects. C-pools were measured as dissolved inorganic and organic C in surface and porewater, sediment organic C, C in plant and in macroinvertebrate biomass. Despite seasonal variability, treated FPMs, for which higher CH4 emissions have been reported, showed a trend towards less dissolved organic C in porewater, while no effect was observed for all remaining components of the C-budget. We attribute the effect of Bti on the C-budget to the reduction in macroinvertebrates biomass, the increase in CH4 emissions, and the input of C from the Bti excipients. This finding suggests that changes in tube-dwelling macroinvertebrates have a weak influence on C cycling in ponds and confirms the existence of long-lasting effects of Bti on specific components of C-budgets.

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.

Subsidy Quality Affects Common Riparian Web-Building Spiders: Consequences of Aquatic Contamination and Food Resource.

Anthropogenic stressors can affect the emergence of aquatic insects. These insects link aquatic and adjacent terrestrial food webs, serving as high-quality subsidy to terrestrial consumers, such as spiders. While previous studies have demonstrated that changes in the emergence biomass and timing may propagate across ecosystem boundaries, the physiological consequences of altered subsidy quality for spiders are largely unknown. We used a model food chain to study the potential effects of subsidy quality: Tetragnatha spp. were exclusively fed with emergent Chironomus riparius cultured in the absence or presence of either copper (Cu), Bacillus thuringiensis var. israelensis (Bti), or a mixture of synthetic pesticides paired with two basal resources (Spirulina vs. TetraMin®) of differing quality in terms of fatty acid (FA) composition. Basal resources shaped the FA profile of chironomids, whereas their effect on the FA profile of spiders decreased, presumably due to the capacity of both chironomids and spiders to modify (dietary) FA. In contrast, aquatic contaminants had negligible effects on prey FA profiles but reduced the content of physiologically important polyunsaturated FAs, such as 20:4n-6 (arachidonic acid) and 20:5n-3 (eicosapentaenoic acid), in spiders by approximately 30% in Cu and Bti treatments. This may have contributed to the statistically significant decline (40%-50%) in spider growth. The observed effects in spiders are likely related to prey nutritional quality because biomass consumption by spiders was, because of our experimental design, constant. Analyses of additional parameters that describe the nutritional quality for consumers such as proteins, carbohydrates, and the retention of contaminants may shed further light on the underlying mechanisms. Our results highlight that aquatic contaminants can affect the physiology of riparian spiders, likely by altering subsidy quality, with potential implications for terrestrial food webs. Environ Toxicol Chem 2023;42:1346-1358. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Fungal-fungal and fungal-bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity.

Fungi produce a variety of extracellular enzymes, making recalcitrant substrates bioavailable. Thus, fungi are central for the decomposition of dead organic matter such as leaf litter. Despite their ecological importance, our understanding of relationships between fungal species diversity and ecosystem functioning is limited, especially with regard to aquatic habitats. Moreover, fungal interactions with other groups of microorganisms such as bacteria are rarely investigated. This lack of information may be attributed to methodological limitations in tracking the biomass of individual fungal species in communities, impeding a detailed assessment of deviations from the overall performance expected from the sum of individual species' performances, so-called net diversity effects (NDEs). We used fungal species-specific biomolecular tools to target fungal-fungal and fungal-bacterial interactions on submerged leaves using four cosmopolitan aquatic fungal species and a stream microbial community dominated by bacteria. In microcosms, we experimentally manipulated fungal diversity and bacterial absence/presence and assessed functional performances and fungal community composition after 14 d of incubation. Fungal community data were used to evaluate NDEs on leaf colonization. The individual fungal species were functionally distinct and fungal cultures were on average more efficient than the bacterial culture. In absence of bacteria, NDEs correlated with growth rate (negatively) and genetic divergence (positively), but were predominantly negative, suggesting that higher fungal diversity led to a lower colonization success (niche overlap). In both absence and presence of bacteria, the overall functional performances of the communities were largely defined by their composition (i.e., no interactions at the functional level). In the presence of bacteria, NDEs correlated with genetic divergence (positively) and were largely positive, suggesting higher fungal diversity stimulated colonization (niche complementarity). This stimulation may be driven by a bacteria-induced inhibition of fungal growth, alleviating competition among fungi. Resulting feedback loops eventually promote fungal coexistence and synergistic interactions. Nonetheless, overall functional performances are reduced compared to bacteria-free cultures. These findings highlight the necessity to conduct future studies, investigating biodiversity-ecosystem functioning relationships using artificial systems, without exclusion of key organisms naturally co-occurring in the compartment of interest. Otherwise, study outcomes might not reflect true ecological relationships and ultimately misguide conservation strategies.

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.

Aquatic testing guidelines insufficiently control the influence of dilution water toc and hardness on cationic polymer toxicity - A proposal to improve standardized test procedures.

Cationic polymers (CPs) are widely used chemicals for wastewater treatment applications and in various "down-the-drain" household products. The aquatic toxicity of CPs results from an electrostatic interaction with negatively charged cell surfaces. These effects are greatly mitigated by the binding affinity of CPs to total organic carbon (TOC) in surface water. Consequently, baseline aquatic toxicity tests of CPs using clean lab water (TOC < 2 mg/L) typically overestimate toxicity and risk which is greatly mitigated at higher environmentally relevant OC levels. However, the point at which mitigation begins is not well defined and low-level TOC in lab water may influence the baseline toxicity outcome. Similarly, divalent cations, quantified as water hardness, may modulate the electrostatic binding between OC and CP. Although standard guidelines define limits for lab water hardness and TOC, the consequences of variability within those limits on test outcome is unknown. We investigated the impact of part-per-billion (ppb) additions of TOC to lab water at different hardness levels on CP acute toxicity to Daphnia magna and Raphidocelis subcapitata. In both species, the acute toxicities of CPs with different molecular weight and charge density varied by > 10-fold in response to slight changes in TOC and water hardness, although parameters were maintained within guideline limits. When determining the baseline aquatic toxicity of CPs, the lab water should be standardized at the lowest biologically tolerable hardness and TOC at a reliably measurable level (>1 - < 2 mg/L) to reduce variability and increase the reliability of the toxicity estimate.