Elevated Fungicide and Nutrient Concentrations Change Structure but not Function of Aquatic Microbial Communities.

Leaf decomposition is a key process in stream ecosystems within forested catchments; it is driven by microbial communities, particularly fungi and bacteria. These microorganisms make nutrients and energy bound in leaves available for wider parts of the food web. Leaf-associated microorganisms are subjected to anthropogenic pressures, such as the increased exposure to nutrients and fungicides associated with land-use change. We assessed the sensitivity of leaf-associated microbial communities with differing exposure histories, namely, from pristine (P) streams, and streams impacted by wastewater (W) and agricultural run-off (vineyards; V). In the laboratory, microbial communities were exposed to elevated nutrient (NO3-N: 0.2-18.0 mg/L, PO4-P: 0.02-1.8 mg/L) and fungicide concentrations (sum concentration 0-300 µg/L) in a fully crossed 3 × 4 × 4-factorial design over 21 days. Leaf decomposition and exoenzyme activity were measured as functional endpoints, and fungal community composition and microbial abundance served as structural variables. Overall, leaf decomposition did not differ between fungicide treatments or exposure histories. Nonetheless, substantial changes in the fungal community composition were observed after exposure to environmentally relevant fungicide concentrations. Elevated nutrient concentrations assisted leaf decomposition, and the effect size depended on the exposure history. The observed changes in the fungal community composition support the principle of functional redundancy, with highly efficient decomposers maintaining leaf decomposition. Environ Toxicol Chem 2024;00:1-12. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Bio-QSARs 2.0: Unlocking a new level of predictive power for machine learning-based ecotoxicity predictions by exploiting chemical and biological information.

Practical, legal, and ethical reasons necessitate the development of methods to replace animal experiments. Computational techniques to acquire information that traditionally relied on animal testing are considered a crucial pillar among these so-called new approach methodologies. In this light, we recently introduced the Bio-QSAR concept for multispecies aquatic toxicity regression tasks. These machine learning models, trained on both chemical and biological information, are capable of both cross-chemical and cross-species predictions. Here, we significantly extend these models' applicability. This was realized by increasing the quantity of training data by a factor of approximately 20, accomplished by considering both additional chemicals and aquatic organisms. Additionally, variable test durations and associated random effects were accommodated by employing a machine learning algorithm that combines tree-boosting with mixed-effects modeling (i.e., Gaussian Process Boosting). We also explored various biological descriptors including Dynamic Energy Budget model parameters, taxonomic distances, as well as genus-specific traits and investigated the inclusion of mode-of-action information. Through these efforts, we developed Bio-QSARs for fish and aquatic invertebrates with exceptional predictive power (R squared of up to 0.92 on independent test sets). Moreover, we made considerable strides to make models applicable for a range of use cases in environmental risk assessment as well as research and development of chemicals. Models were made fully explainable by implementing an algorithmic multicollinearity correction combined with SHapley Additive exPlanations. Furthermore, we devised novel approaches for applicability domain construction that take feature importance into account. We are hence confident these models, which are available via open access, will make a significant contribution towards the implementation of new approach methodologies and ultimately have the potential to support "Green Chemistry" and "Green Toxicology".

The influence of season, hunting mode, and habitat specialization on riparian spiders as key predators in the aquatic-terrestrial linkage.

Freshwater ecosystems subsidize riparian zones with high-quality nutrients via the emergence of aquatic insects. Spiders are dominant consumers of these insect subsidies. However, little is known about the variation of aquatic insect consumption across spiders of different hunting modes, habitat specializations, seasons, and systems. To explore this, we assembled a large stable isotope dataset (n > 1000) of aquatic versus terrestrial sources and six spider species over four points in time adjacent to a lotic and a lentic system. The spiders represent three hunting modes each consisting of a wetland specialist and a habitat generalist. We expected that specialists would feed more on aquatic prey than their generalist counterparts. Mixing models showed that spiders' diet consisted of 17-99% of aquatic sources, with no clear effect of habitat specialization. Averaged over the whole study period, web builders (WB) showed the highest proportions (78%) followed by ground hunters (GH, 42%) and vegetation hunters (VH, 31%). Consumption of aquatic prey was highest in June and August, which is most pronounced in GH and WBs, with the latter feeding almost entirely on aquatic sources during this period. Additionally, the elevated importance of high-quality lipids from aquatic origin during fall is indicated by elemental analyses pointing to an accumulation of lipids in October, which represent critical energy reserves during winter. Consequently, this study underlines the importance of aquatic prey irrespective of the habitat specialization of spiders. Furthermore, it suggests that energy flows vary substantially between spider hunting modes and seasons.

Nitrogen retention in stream biofilms - A potential contribution to the self-cleaning capacity.

Surface waters are under increasing pressure due to human activities, such as nutrient emissions from wastewater treatment plants (WWTPs). Using the retention of nitrogen (N) released from WWTPs as a proxy, we assessed the contribution of biofilms grown on inorganic and organic substrates to the self-cleaning capacity of second-order streams within the biosphere reserve Vosges du Nord/Palatinate forest (France/Germany). The uptake of N from anthropogenic sources, which is enriched with the heavy isotope 15N, into biofilms was assessed up- and downstream of WWTPs after five weeks of substrate deployment. Biofilms at downstream sites showed a significant positive linear relationship between δ15N and the relative contribution of wastewater to the streams' discharge. Furthermore, δ15N substantially increased in areas affected by WWTP effluent (∼8.5‰ and ∼7‰ for inorganic and organic substrate-associated biofilms, respectively) and afterwards declined with increasing distance to the WWTP effluent, approaching levels of upstream sections. The present study highlights that biofilms contribute to nutrient retention and likely the self-cleaning capacity of streams. This function seems, however, to be limited by the fact that biofilms are restricted in their capacity to process excessive N loads with large differences between individual reaches (e.g., δ15N: -3.25 to 12.81‰), influenced by surrounding conditions (e.g., land use) and modulated through climatic factors and thus impacted by climate change. Consequently, the impact of WWTPs located close to the source of a stream are dampened by the biofilms' capacity to retain N only to a minor share and suggest substantial N loads being transported downstream.

Physiological variables in machine learning QSARs allow for both cross-chemical and cross-species predictions.

A major challenge in ecological risk assessment is estimating chemical-induced effects across taxa without species-specific testing. Where ecotoxicological data may be more challenging to gather, information on species physiology is more available for a broad range of taxa. Physiology is known to drive species sensitivity but understanding about the relative contribution of specific underlying processes is still elusive. Consequently, there remains a need to understand which physiological processes lead to differences in species sensitivity. The objective of our study was to utilize existing knowledge about organismal physiology to both understand and predict differences in species sensitivity. Machine learning models were trained to predict chemical- and species-specific endpoints as a function of both chemical fingerprints/descriptors and physiological properties represented by dynamic energy budget (DEB) parameters. We found that random forest models were able to predict chemical- and species-specific endpoints, and that DEB parameters were relatively important in the models, particularly for invertebrates. Our approach illuminates how physiological properties may drive species sensitivity, which will allow more realistic predictions of effects across species without the need for additional animal testing.

Tracer or toxicant: Does stable isotope labeling affect central processes in aquatic food webs?

Stable isotope analysis (SIA) is an elementary technique in food web ecology, but its insights become increasingly ambiguous in complex systems. One approach to elevate the utility of SIA in such systems is the use of heavy isotope tracers (i.e., labeling). However, the fundamental assumption that the addition of such tracers does not affect in situ conditions has been challenged. This study tests if labeling is suitable for autotrophy-based and detritus-based aquatic food webs. For the former, the survival and reproduction of Daphnia magna fed with phytoplankton cultured at different levels of 15N addition were assessed. For the latter, the microbial decomposition of leaf litter was assessed at the same tracer levels. While no significant differences were observed, effect patterns were comparable to a previous study, supporting the isotopic redundancy hypothesis that postulates discrete quantum mechanical states at which the reaction speeds of metabolic processes are altered. Although physiology (reproduction) and activity (microbial decomposition) might not be altered to an ecologically significant level, labeling with heavy stable isotopes could potentially affect isotopic fractionation in biochemical processes and bias conclusions drawn from resulting SI ratios.

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.

Metal Exposure and Sex Shape the Fatty Acid Profile of Midges and Reduce the Aquatic Subsidy to Terrestrial Food Webs.

Aquatic micropollutants can be transported to terrestrial systems and their consumers by emergent aquatic insects. However, micropollutants, such as metals, may also affect the flux of physiologically important polyunsaturated fatty acids (PUFAs). As certain PUFAs have been linked to physiological fitness and breeding success of terrestrial consumers, reduced fluxes from aquatic systems could affect terrestrial populations and food webs. We chronically exposed larvae of the aquatic insect Chironomus riparius to a range of environmentally relevant sediment contents of cadmium (Cd) or copper (Cu) in a 28-day microcosm study. Since elevated water temperatures can enhance metals' toxic effects, we used two temperature regimes, control and periodically elevated temperatures (heat waves) reflecting an aspect of climate change. Cd and Cu significantly reduced adult emergence by up to 95% and 45%, respectively, while elevated temperatures had negligible effects. Both metal contents were strongly reduced (∼90%) during metamorphosis. Furthermore, the chironomid FA profile was significantly altered during metamorphosis with the factors sex and metal exposure being relevant predictors. Consequently, fluxes of physiologically important PUFAs by emergent adults were reduced by up to ∼80%. Our results suggest that considering fluxes of physiologically important compounds, such as PUFAs, by emergent aquatic insects is important to understand the implications of aquatic micropollutants on aquatic-terrestrial meta-ecosystems.

Making the Invisible Visible? Using Stable Isotope Analysis to Detect Indirect Toxicant Effects.

Although stable isotope analysis (SIA) is widely used to address ecological research questions, its application in an ecotoxicological context has been limited. Recent studies have proposed an effect of chemical stressors on an organism's isotope signature, questioning the use of SIA in food webs impacted by toxicants. Against this background, the present study investigates 1) whether trophic enrichment factors (TEFs; i.e., the offset in stable isotope signatures of a consumer to its diet) are altered by the neonicotinoid thiacloprid, and 2) whether tracking toxicant effects on an organism's diet composition (i.e., indirect effect) with SIA fits direct observations of consumption. To address the former, the amphipod Gammarus fossarum (Koch) was exposed to three levels (0, 0.75, and 5 µg L-1 ) of thiacloprid and fed with either black alder leaves or Baetis rhodani (Pictet) larvae over 6 weeks (n = 35). The thiacloprid-induced changes in TEFs that we found were statistically significant but small compared with other factors (e.g., resource quality, consumer, and physiological condition) and thus likely of minor importance. To address the latter issue, gammarids were exposed to two levels of thiacloprid (0 and 0.75 µg L-1 ) and fed with either black alder leaves, live B. rhodani larvae, or both over 2 weeks (n = 10). Dietary proportions as suggested by SIA were indeed in agreement with those derived from direct observation of consumption. The present study consequently suggests that SIA is as a robust tool to detect indirect toxicant effects especially if TEFs are assessed in parallel. Environ Toxicol Chem 2023;42:1937-1945. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Trophic Dynamics of Mercury in the Baltic Archipelago Sea Food Web: The Impact of Ecological and Ecophysiological Traits.

We investigated trophic dynamics of Hg in the polluted Baltic Archipelago Sea using established trophic magnification (TMFs) and biomagnification factors (BMFs) on a comprehensive set of bird, fish, and invertebrate species. As different ecological and ecophysiological species traits may affect trophic dynamics, we explored the effect of food chain (benthic, pelagic, benthopelagic) and thermoregulatory strategy on trophic total Hg (THg) dynamics, using different approaches to accommodate benthopelagic species and normalize for trophic position (TP). We observed TMFs and most BMFs greater than 1, indicating overall THg biomagnification. We found significantly higher pelagic TMFs (3.58-4.02) compared to benthic ones (2.11-2.34) when the homeotherm bird species were excluded from models, but not when included. This difference between the benthic and pelagic TMFs remained regardless of how the TP of benthopelagic species was modeled, or whether TMFs were normalized for TP or not. TP-corrected BMFs showed a larger range (0.44-508) compared to BMFs representing predator-prey concentration ratios (0.05-82.2). Overall, the present study shows the importance of including and evaluating the effect of ecological and ecophysiological traits when investigating trophic contaminant dynamics.

Structural and functional development of twelve newly established floodplain pond mesocosms.

Ecosystems are complex structures with interacting abiotic and biotic processes evolving with ongoing succession. However, limited knowledge exists on the very initial phase of ecosystem development and colonization. Here, we report results of a comprehensive ecosystem development monitoring for twelve floodplain pond mesocosms (FPM; 23.5 m × 7.5 m × 1.5 m each) located in south-western Germany. In total, 20 abiotic and biotic parameters, including structural and functional variables, were monitored for 21 months after establishment of the FPMs. The results showed evolving ecosystem development and primary succession in all FPMs, with fluctuating abiotic conditions over time. Principal component analyses and redundancy analyses revealed season and succession time (i.e., time since ecosystem establishment) to be significant drivers of changes in environmental conditions. Initial colonization of both aquatic (i.e., water bodies) and terrestrial (i.e., riparian land areas) parts of the pond ecosystems occurred within the first month, with subsequent season-specific increases in richness and abundance for aquatic and terrestrial taxa over the entire study period. Abiotic environmental conditions and aquatic and terrestrial communities showed increasing interpond variations over time, that is, increasing heterogeneity among the FPMs due to natural environmental divergence. However, both functional variables assessed (i.e., aquatic and terrestrial litter decomposition) showed opposite patterns as litter decomposition rates slightly decreased over time and interpond differences converged with successional ecosystem developments. Overall, our results provide rare insights into the abiotic and biotic conditions and processes during the initial stages of freshwater ecosystem formation, as well as into structural and functional developments of the aquatic and terrestrial environment of newly established pond ecosystems.

Legacy and emerging organohalogenated compounds in feathers of Eurasian eagle-owls (Bubo bubo) in Norway: Spatiotemporal variations and associations with dietary proxies (δ13C and δ15N).

The occurrence of organohalogenated compounds (OHCs) in wildlife has received considerable attention over the last decades. Among the matrices used for OHCs biomonitoring, feathers are particularly useful as they can be collected in a minimally or non-invasive manner. In this study, concentrations of various legacy OHCs -polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs)-, as well as emerging OHCs -per- and polyfluoroalkyl substances (PFAS) and organophosphate ester flame retardants (OPEs)- were determined in feathers of 72 Eurasian eagle-owls (Bubo bubo) from Norway, with the goal of studying spatiotemporal variation using a non-invasive approach. Molted feathers were collected at nest sites from northern, central and southern Norway across four summers (2013-2016). Additionally, two museum-archived feathers from 1979 to 1989 were included. Stable carbon (δ13C) and nitrogen isotopes (δ15N) were used as dietary proxies. In total, 11 PFAS (sum range 8.25-215.90 ng g-1), 15 PCBs (4.19-430.01 ng g-1), 6 OCPs (1.48-220.94 ng g-1), 5 PBDEs (0.21-5.32 ng g-1) and 3 OPEs (4.49-222.21 ng g-1) were quantified. While we observed large variation in the values of both stable isotopes, suggesting a diverse diet of the eagle-owls, only δ13C seemed to explain variation in PFAS concentrations. Geographic area and year were influential factors for δ15N and δ13C. Considerable spatial variation was observed in PFAS levels, with the southern area showing higher levels compared to northern and central Norway. For the rest of OHCs, we observed between-year variations; sum concentrations of PCBs, OCPs, PBDEs and OPEs reached a maximum in 2015 and 2016. Concentrations from 1979 to 1989 were within the ranges observed between 2013 and 2016. Overall, our data indicate high levels of legacy and emerging OHCs in a top predator in Norway, further highlighting the risk posed by OHCs to wildlife.

Herbicide-Induced Shifts in the Periphyton Community Composition Indirectly Affect Feeding Activity and Physiology of the Gastropod Grazer Physella acuta.

Herbicides are well known for unintended effects on freshwater periphyton communities. Large knowledge gaps, however, exist regarding indirect herbicide impacts on primary consumers through changes in the quality of periphyton as a food source (i.e., diet-related effects). To address this gap, the grazer Physella acuta (Gastropoda) was fed for 21 days with periphyton that grew for 15 days in the presence or absence of the herbicide diuron (8 µg/L) to quantify changes in the feeding rate, growth rate, and energy storage (neutral lipid fatty acids; NLFAs) of P. acuta. Periphyton biomass, cell viability, community structure, and FAs served as proxies for food quality that support a mechanistic interpretation of the grazers' responses. Diuron changed the algae periphyton community and fatty acid profiles, indicating alterations in the food quality, which could explain differences in the snails' feeding rate compared to the control. While the snails' growth rate was, despite an effect size of 55%, not statistically significantly changed, NLFA profiles of P. acuta were altered. These results indicate that herbicides can change the food quality of periphyton by shifts in the algae composition, which may affect the physiology of grazers.

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.

Environmentally relevant fungicide levels modify fungal community composition and interactions but not functioning.

Aquatic hyphomycetes (AHs), a group of saprotrophic fungi adapted to submerged leaf litter, play key functional roles in stream ecosystems as decomposers and food source for higher trophic levels. Fungicides, controlling fungal pathogens, target evolutionary conserved molecular processes in fungi and contaminate streams via their use in agricultural and urban landscapes. Thus fungicides pose a risk to AHs and the functions they provide. To investigate the impacts of fungicide exposure on the composition and functioning of AH communities, we exposed four AH species in monocultures and mixed cultures to increasing fungicide concentrations (0, 5, 50, 500, and 2500 µg/L). We assessed the biomass of each species via quantitative real-time PCR. Moreover, leaf decomposition was investigated. In monocultures, none of the species was affected at environmentally relevant fungicide levels (5 and 50 µg/L). The two most tolerant species were able to colonize and decompose leaves even at very high fungicide levels (≥500 µg/L), although less efficiently. In mixed cultures, changes in leaf decomposition reflected the response pattern of the species most tolerant in monocultures. Accordingly, the decomposition process may be safeguarded by tolerant species in combination with functional redundancy. In all fungicide treatments, however, sensitive species were displaced and interactions between fungi changed from complementarity to competition. As AH community composition determines leaves' nutritional quality for consumers, the data suggest that fungicide exposures rather induce bottom-up effects in food webs than impairments in leaf decomposition.

Photoactive titanium dioxide nanoparticles modify heterotrophic microbial functioning.

Nanoparticulate titanium dioxide (nTiO2) is frequently applied, raising concerns about potential side effects on the environment. While various studies have assessed structural effects in aquatic model ecosystems, its impact on ecosystem functions provided by microbial communities (biofilms) is not well understood. This is all the more the case when considering additional stressors, such as UV irradiation - a factor known to amplify nTiO2-induced toxicity. Using pairwise comparisons, we assessed the impact of UV (UV-A = 1.6 W/m2; UV-B = 0.7 W/m2) at 0, 20 or 2000 µg nTiO2/L on two ecosystem functions provided by leaf-associated biofilms: while leaf litter conditioning, important for detritivorous invertebrate nutrition, seems unaffected, microbial leaf decomposition was stimulated (up to 25%) by UV, with effect sizes being higher in the presence of nTiO2. Although stoichiometric and microbial analyses did not allow for uncovering the underlying mechanism, it seems plausible that the combination of a shift in biofilm community composition and activity together with photodegradation as well as the formation of reactive oxygen species triggered changes in leaf litter decomposition. The present study implies that the multiple functions a microbial community performs are not equally sensitive. Consequently, relying on one of the many functions realized by the same microbial community may be misleading for environmental management.

Bottom-up effects of fungicides on tadpoles of the European common frog (Rana temporaria).

Biodiversity is under pressure worldwide, with amphibians being particularly threatened. Stressors related to human activity, such as chemicals, are contributing to this decline. It remains, however, unclear whether chemicals exhibiting a fungicidal activity could indirectly affect tadpoles that depend on microbially conditioned leaf litter as food source. The indirect effect of fungicides (sum concentration of a fungicide mixture composed of azoxystrobin, carbendazim, cyprodinil, quinoxyfen, and tebuconazole: 100 µg/L) on tadpoles was assessed relative to leaf litter colonized by microbes in absence of fungicides (control) and a worst-case scenario, that is leached leaf litter without microbial colonization. The quality of leaf litter as food for tadpoles of the European common frog (Rana temporaria) was characterized through neutral lipid fatty acid profiles and microbial sum parameters and verified by sublethal responses in tadpoles (i.e., feeding rate, feces production, growth, and fatty acid composition). Fungicides changed the nutritious quality of leaf litter likely through alterations in leaves' neutral lipid fatty acid profiles (i.e., changes in some physiologically important highly unsaturated fatty acids reached more than 200%) in combination with a potential adsorption onto leaves during conditioning. These changes were reflected by differences in the development of tadpoles ultimately resulting in an earlier start of metamorphosis. Our data provide a first indication that fungicides potentially affect tadpole development indirectly through bottom-up effects. This pathway is so far not addressed in fungicide environmental risk assessment and merits further attention.

Mixture effects of a fungicide and an antibiotic: Assessment and prediction using a decomposer-detritivore system.

Antimicrobials, such as fungicides and antibiotics, pose a risk for microbial decomposers (i.e., bacteria and aquatic fungi) and invertebrate detritivores (i.e., shredders) that play a pivotal role in the ecosystem function of leaf litter breakdown. Although waterborne toxicity and diet-related effects (i.e., dietary exposure and microorganism-mediated alterations in food quality for shredders) of fungicides and antibiotics on decomposer-detritivore systems have been increasingly documented, their joint effect is unknown. We therefore assessed waterborne and dietary effects of an antimicrobial mixture consisting of the fungicide azoxystrobin (AZO) and the antibiotic ciprofloxacin (CIP) on microbial decomposers and the shredder Gammarus fossarum using a tiered approach. We compared effect sizes measured in the present study with model predictions (i.e., independent action) based on published data. During a 7-day feeding activity assay quantifying waterborne toxicity in G. fossarum, the leaf consumption of gammarids was reduced by ∼60 % compared to the control when subjected to the mixture at concentrations of each component causing a 20 % reduction in the same response variable when applied individually. Moreover, the selective feeding of gammarids during the food choice assay indicated alterations in food quality induced by the antimicrobial mixture. The food selection and, in addition, the decrease in microbial leaf decomposition is likely linked to changes in leaf-associated bacteria and fungi. During a long-term assay, energy processing, growth and energy reserves of gammarids were increased in presence of 15 and 500 µg/L of AZO and CIP, respectively, through the dietary pathway. These physiological responses were probably driven by CIP-induced alterations in the gut microbiome or immune system of gammarids. In general, model predictions matched observed effects caused by waterborne exposure on the leaf consumption, energy processing and growth of gammarids during short- and long-term assays, respectively. However, when complex horizontal (bacteria and aquatic fungi) and vertical (leaf-associated microorganisms and shredders) interactions were involved, model predictions partly over- or underestimated mixture effects. Therefore, the present study identifies uncertainties of mixture effect predictions for complex biological systems calling for studies targeting the underlying processes and mechanisms.

The Fungicide Tebuconazole Confounds Concentrations of Molecular Biomarkers Estimating Fungal Biomass.

Due to their ecological importance, fungi are suitable indicator organisms for anthropogenic stress. To estimate fungal biomass, the fungal membrane molecule ergosterol is often quantified as a proxy. Estimates based on ergosterol may, however, be distorted by exposure to demethylase inhibiting (DMI) fungicides, interfering with sterol synthesis. To test this hypothesis, we exposed ten fungal species to the DMI fungicide tebuconazole and measured concentrations of ergosterol and DNA per unit dry mass of the fungal hyphae. The latter served as alternative biomass proxy that is not specifically targeted by tebuconazole. Effects of tebuconazole on ergosterol concentrations were species-specific, while concentrations were on average reduced by 13%. In contrast, DNA concentrations were on average increased by 13%. We demonstrate that DMI fungicides - at close to field relevant levels - can distort fungal biomass estimation, complicating the use of this endpoint for environmental management.

Multiple Stressors in Aquatic Ecosystems: Sublethal Effects of Temperature, Dissolved Organic Matter, Light and a Neonicotinoid Insecticide on Gammarids.

Whether and to which extent the effects of chemicals in the environment interact with other factors remains a scientific challenge. Here we assess the combined effects of temperature (16 vs. 20°C), light conditions (darkness vs. 400 lx), dissolved organic matter (DOM; 0 vs. 6 mg/L) and the model insecticide thiacloprid (0 vs. 3 µg/L) in a full-factorial experiment on molting and leaf consumption of Gammarus fossarum. Thiacloprid was the only factor significantly affecting gammarids' molting. While DOM had low effects on leaf consumption, temperature, light and thiacloprid significantly affected this response variable. The various interactions among these factors were not significant suggesting additivity. Only the interaction of the factors temperature and thiacloprid suggested a tendency for antagonism. As most stressors interacted additively, their joint effects may be predictable with available models. However, synergistic interactions are difficult to capture while being central for securing ecosystem integrity.