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.

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.

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

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

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

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

Reduction of Pesticide Toxicity Under Field-Relevant Conditions? The Interaction of Titanium Dioxide Nanoparticles, Ultraviolet, and Natural Organic Matter.

In surface waters, the illumination of photoactive engineered nanomaterials (ENMs) with ultraviolet (UV) light triggers the formation of reactive intermediates, consequently altering the ecotoxicological potential of co-occurring organic micropollutants including pesticides due to catalytic degradation. Simultaneously, omnipresent natural organic matter (NOM) adsorbs onto ENM surfaces, altering the ENM surface properties. Also, NOM absorbs light, reducing the photo(cata)lytic transformation of pesticides. Interactions between these environmental factors impact 1) directly the ecotoxicity of photoactive ENMs, and 2) indirectly the degradation of pesticides. We assessed the impact of field-relevant UV radiation (up to 2.6 W UVA/m²), NOM (4 mg TOC/L), and photoactive ENM (nTiO2, 50 µg/L) on the acute toxicity of 6 pesticides in Daphnia magna. We selected azoxystrobin, dimethoate, malathion, parathion, permethrin, and pirimicarb because of their varying photo- and hydrolytic stabilities. Increasing UVA alone partially reduced pesticide toxicity, seemingly due to enhanced degradation. Even at 50 µg/L, nano-sized titanium dioxide (nTiO2) reduced but also increased pesticide toxicity (depending on the applied pesticide), which is attributable to 1) more efficient degradation and potentially 2) photocatalytically induced formation of toxic by-products. Natural organic matter 1) partially reduced pesticide toxicity, not evidently accompanied by enhanced pesticide degradation, but also 2) inhibited pesticide degradation, effectively increasing the pesticide toxicity. Predicting the ecotoxicological potential of pesticides based on their interaction with UV light or interaction with NOM was hardly possible, which was even more difficult in the presence of nTiO2. Environ Toxicol Chem 2020;39:2237-2246. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.