Antibiotics as a silent driver of climate change? A case study investigating methane production in freshwater sediments.

Methane (CH4) is the second most important greenhouse gas after carbon dioxide (CO2) and is inter alia produced in natural freshwater ecosystems. Given the rise in CH4 emissions from natural sources, researchers are investigating environmental factors and climate change feedbacks to explain this increment. Despite being omnipresent in freshwaters, knowledge on the influence of chemical stressors of anthropogenic origin (e.g., antibiotics) on methanogenesis is lacking. To address this knowledge gap, we incubated freshwater sediment under anaerobic conditions with a mixture of five antibiotics at four levels (from 0 to 5000 µg/L) for 42 days. Weekly measurements of CH4 and CO2 in the headspace, as well as their compound-specific δ13C, showed that the CH4 production rate was increased by up to 94% at 5000 µg/L and up to 29% at field-relevant concentrations (i.e., 50 µg/L). Metabarcoding of the archaeal and eubacterial 16S rRNA gene showed that effects of antibiotics on bacterial community level (i.e., species composition) may partially explain the observed differences in CH4 production rates. Despite the complications of transferring experimental CH4 production rates to realistic field conditions, the study indicated that chemical stressors contribute to the emissions of greenhouse gases by affecting the methanogenesis in freshwaters.

Chronic effects of the strobilurin fungicide azoxystrobin in the leaf shredder Gammarus fossarum (Crustacea; Amphipoda) via two effect pathways.

Fungicides pose a risk for crustacean leaf shredders serving as key-stone species for leaf litter breakdown in detritus-based stream ecosystems. However, little is known about the impact of strobilurin fungicides on shredders, even though they are presumed to be the most hazardous fungicide class for aquafauna. Therefore, we assessed the impact of the strobilurin azoxystrobin (AZO) on the survival, energy processing (leaf consumption and feces production), somatic growth (growth rate and molting activity), and energy reserves (neutral lipid fatty and amino acids) of the amphipod crustacean Gammarus fossarum via waterborne exposure and food quality-mediated (through the impact of leaf colonizing aquatic microorganisms) and thus indirect effects using 2 × 2-factorial experiments over 24 days. In a first bioassay with 30 µg AZO/L, waterborne exposure substantially reduced survival, energy processing and affected molting activity of gammarids, while no effects were observed via the dietary pathway. Furthermore, a negative growth rate (indicating a body mass loss in gammarids) was induced by waterborne exposure, which cannot be explained by a loss in neutral lipid fatty and amino acids. These energy reserves were increased indicating a disruption of the energy metabolism in G. fossarum caused by AZO. Contrary to the first bioassay, no waterborne AZO effects were observed during a second experiment with 15 µg AZO/L. However, an altered energy processing was determined in gammarids fed with leaves microbially colonized in the presence of AZO, which was probably caused by fungicide-induced effects on the microbial decomposition efficiency ultimately resulting in a lower food quality. The results of the present study show that diet-related strobilurin effects can occur at concentrations below those inducing waterborne toxicity. However, the latter seems to be more relevant at higher fungicide concentrations.

Waterborne and diet-related effects of inorganic and organic fungicides on the insect leaf shredder Chaetopteryx villosa (Trichoptera).

It is well-documented that fungicides can affect crustacean leaf shredders via two effect pathways, namely waterborne exposure and their diet (i.e., via dietary uptake of fungicides adsorbed to leaf material and an altered microorganism-mediated food quality). As a consequence of different life history strategies, the relevance of these effect pathways for aquatic shredders belonging to other taxonomic classes, for instance insects, remains unclear. Therefore, we investigated waterborne and diet-related effects in larvae of the caddisfly leaf shredder Chaetopteryx villosa (Insecta: Trichoptera) and compared our observations to previous reports on effects in adults of the crustacean leaf shredder Gammarus fossarum (Malacostraca: Amphipoda). We assessed acute waterborne effects of an organic fungicide mixture (OFM) and the inorganic fungicide copper (Cu) on the leaf consumption (n = 30) of the fourth-/fifth-instar larvae of C. villosa and their food choice (n = 49) when offered leaf material, which was either conditioned in presence or in absence of the respective fungicide(s). Moreover, the larval leaf consumption (n = 50) and physiological fitness (i.e., growth as well as lipid and protein content) were examined after subjecting C. villosa for 24 days towards the combination of both effect pathways at environmentally relevant concentrations. G. fossarum and C. villosa exhibited similar sensitivities and the same effect direction when exposed to the OFM (either waterborne or dietary pathways). Both shredders also showed the same effect direction when exposed to dietary Cu, while with regards to mortality and leaf consumption C. villosa was less sensitive to waterborne Cu than G. fossarum. Finally, as observed for G. fossarum, the combined exposure to OFM over 24 days negatively affected leaf consumption and the physiology (i.e., growth and lipid reserves) of C. villosa. While no combined Cu effects were observed for larval leaf consumption, contrasting to the observations for G. fossarum, the physiology of both shredders was negatively affected, despite partly differing effect sizes and directions. Our results suggest that C. villosa and G. fossarum are of comparable sensitivity towards waterborne and diet-related organic fungicide exposure, whereas the trichopteran is less sensitive to Cu-based waterborne fungicide exposure. However, when both pathways act jointly, organic and inorganic fungicides can affect the physiology of shredder species with completely different life history strategies. As caddisflies represent a subsidy for terrestrial consumers, these observations indicate that fungicide exposure might not only affect aquatic ecosystem functioning but also the flux of energy across ecosystem boundaries.

The relative importance of diet-related and waterborne effects of copper for a leaf-shredding invertebrate.

Copper (Cu) exposure can increase leaf-associated fungal biomass, an important food component for leaf-shredding macroinvertebrates. To test if this positive nutritional effect supports the physiological fitness of these animals and to assess its importance compared to waterborne toxicity, we performed a 24-day-bioassay in combination with a 2×2 factorial design using the amphipod shredder Gammarus fossarum and a field-relevant Cu concentration of 25 µg/L (n = 65). Waterborne toxicity was negligible, while gammarids fed leaves exposed to Cu during microbial colonization exhibited a near-significant impairment in growth (∼30%) and a significantly reduced lipid content (∼20%). These effects appear to be governed by dietary uptake of Cu, which accumulated in leaves as well as gammarids and likely overrode the positive nutritional effect of the increased fungal biomass. Our results suggest that for adsorptive freshwater contaminants dietary uptake should be evaluated already during the registration process to safeguard the integrity of detritus-based ecosystems.

Waterborne toxicity and diet-related effects of fungicides in the key leaf shredder Gammarus fossarum (Crustacea: Amphipoda).

Animals involved in leaf litter breakdown (i.e., shredders) play a central role in detritus-based stream food webs, while their fitness and functioning can be impaired by anthropogenic stressors. Particularly fungicides can affect shredders via both waterborne exposure and their diet, namely due to co-ingestion of adsorbed fungicides and shifts in the leaf-associated fungal community, on which shredders' nutrition heavily relies. To understand the relevance of these effect pathways, we used a full 2×2-factorial test design: the leaf material serving as food was microbially colonized for 12 days either in a fungicide-free control or exposed to a mixture of five current-use fungicides (sum concentration of 62.5µg/L). Similarly, the amphipod shredder Gammarus fossarum was subjected to the same treatments but for 24 days. Waterborne exposure reduced leaf consumption by ∼20%, which did not fully explain the reduction in feces production (∼30%), indicating an enhanced utilization of food to compensate for detoxification mechanisms. This may also explain the reduced feces production (∼10%) of gammarids feeding on fungicide-exposed leaves. The reduction may, however, also be caused by a decreased nutritious quality of the leaves indicated by a reduced species richness (∼40%) of leaf-associated fungi. However, compensation for these effects by Gammarus was seemingly incomplete, since both waterborne exposure and the consumption of the fungicide-affected diet drastically reduced gammarid growth (∼110% and ∼40%, respectively). Our results thus indicate that fungicide mixtures have the potential for detrimental implications in aquatic ecosystem functioning by affecting shredders via both effect pathways.

Effects of current-use fungicides and their mixtures on the feeding and survival of the key shredder Gammarus fossarum.

Fungicides are frequently applied in agriculture and are subsequently detected in surface waters in total concentrations of up to several tens of micrograms per liter. These concentrations imply potential effects on aquatic communities and fundamental ecosystem functions such as leaf litter breakdown. In this context, the present study investigates sublethal and lethal effects of organic (azoxystrobin, carbendazim, cyprodinil, quinoxyfen, and tebuconazole) and inorganic (three copper (Cu)-based substances and sulfur) current-use fungicides and their mixtures on the key leaf-shredding invertebrate Gammarus fossarum. The feeding activity of fungicide-exposed gammarids was quantified as sublethal endpoint using a static (organic fungicides; 7 d test duration) or a semi-static (inorganic fungicides; 6 d test duration with a water exchange after 3 d) approach (n=30). EC50-values of organic fungicides were generally observed at concentrations resulting in less than 20% mortality, with the exception of carbendazim. With regard to feeding, quinoxyfen was the most toxic organic fungicide, followed by cyprodinil, carbendazim, azoxystrobin, and tebuconazole. Although all tested organic fungicides have dissimilar (intended) modes of action, a mixture experiment revealed a synergistic effect on gammarids' feeding at high concentrations when using "independent action" as the reference model (∼35% deviation between predicted and observed effect). This may be explained by the presence of a synergizing azole fungicide (i.e. tebuconazole) in this mixture. Furthermore, lethal concentrations of all Cu-based fungicides assessed in this study were comparable amongst one another. However, they differed markedly in their effective concentrations when using feeding activity as the endpoint, with Cu-sulfate being most toxic, followed by Cu-hydroxide and Cu-octanoate. In contrast, sulfur neither affected survival nor the feeding activity of gammarids (up to ∼5 mg/L) but reduced Cu-sulfate's toxicity when applied in a binary mixture. Sulfur-related metabolic processes which reduce the physiological availability of Cu may explain this antagonistic effect. For both fungicide mixtures, the present study thus uncovered deviations from the appropriate reference model, while ecotoxicological effects were observed at field relevant (total) fungicide concentrations. Additionally, for more than half of the tested single substances, a potential risk for Gammarus and thus for the ecological function mediated by these organisms was evident at concentrations measured in agriculturally influenced surface waters. These results suggest that risks to the fundamental ecosystem function of leaf litter breakdown posed by fungicides may not be adequately considered during the regulation of these compounds, which makes further experimental efforts necessary.