Exposure pathway-dependent effects of the fungicide epoxiconazole on a decomposer-detritivore system

Highlights

• Fungicides provoke complex effects on aquatic decomposer-detritivore systems.

• Waterborne exposure causes stronger effects than food-related pathways.

• Waterborne and food-quality related effect pathways of fungicides interact.

• Asellus insufficiently compensates for increased energy demands.

• Essential fatty acid composition may explain physiological effects.

Abstract

Shredders play a central role in the breakdown of leaf material in aquatic systems. These organisms and the ecological function they provide may, however, be affected by chemical stressors either as a consequence of direct waterborne exposure or through alterations in food-quality (indirect pathway). To unravel the biological relevance of these effect pathways, we applied a 2 × 2-factorial test design. Leaf material was microbially colonized for 10 days in absence or presence of the fungicide epoxiconazole (15 μg/L) and subsequently fed to the shredder Asellus aquaticus under exposure to epoxiconazole (15 μg/L) or in fungicide-free medium over a 28-day period (n = 40). Both effect pathways caused alterations in asselids' food processing, physiological fitness, and growth, although not always statistically significantly: assimilation either increased or remained at a similar level relative to the control suggesting compensatory behavior of A. aquaticus to cope with the enhanced energy demand for detoxification processes and decreased nutritional quality of the food. The latter was driven by lowered microbial biomasses and the altered composition of fatty acids associated with the leaf material. Even with increased assimilation, direct and indirect effects caused decreases in the growth and lipid (fatty acid) content of A. aquaticus with relative effect sizes between 10 and 40%. Moreover, the concentrations of two essential polyunsaturated fatty acids (i.e., arachidonic acid and eicosapentaenoic acid) were non-significantly reduced (up to ~ 15%) in asselids. This effect was, however, independent of the exposure pathway. Although waterborne effects were generally stronger than the diet-related effects, results suggest impaired functioning of A. aquaticus via both effect pathways.

Introduction

In low order stream ecosystems with forested catchments, detritus from the riparian vegetation (e.g. leaf material) is often the primary source of carbon (C) that fuels aquatic food web productivity (Fisher and Likens, 1973). To incorporate these detrital resources into the food web, the leaf material has to be broken down by microbial decomposers and leaf-shredding macroinvertebrates (i.e., detritivores; Cummins and Klug, 1979). In this process, microbial colonization (i.e., conditioning; in particular by aquatic hyphomycetes) enhances the palatability and nutritional quality of leaf material for detritivores by incorporating nutrients from the water phase and increasing the amount of proteins and lipids such as fatty acids (FA; Arce Funck, J, et al., 2015, Bärlocher, F., 1985, Danger, M and Chauvet, E, 2013). Therefore, feeding on microbially conditioned leaf material allows shredders to maintain survival, growth rate and reproductive output (Graça, 2001).

Anthropogenic stressors like fungicides, however, can affect the ecological integrity of such decomposer-detritivore systems (as reviewed in Feckler et al., 2015). On the one hand, direct effects on shredders have been reported when subjected to fungicides at field-relevant concentrations via the water phase (e.g., Zubrod et al., 2014). Additionally, fungicides may cause toxic effects when sorbed to leaf material (Dimitrov et al., 2014) and are co-ingested during consumption, as already reported for other pesticides (Bundschuh et al., 2013). On the other hand, fungicides may affect shredders indirectly by inhibiting the microbial conditioning process and lowering the palatability of the leaf material (e.g. Zubrod et al., 2015a). Fungicide-induced effects via this indirect pathway, however, have only been studied for two amphipod species within the diverse functional group of shredders, namely Echinogammarus berilloni (Flores et al., 2014) and Gammarus fossarum (Zubrod, JP, et al., 2011, Zubrod, JP, et al., 2015a). Although the abundance of Gammarus can be high in undisturbed stream sections, abundance can be relatively low in streams affected by nutrient pollution (e.g., agricultural streams; Whitehurst, 1991). In such impacted streams, the species Asellus aquaticus (L.) (Crustacea: Isopoda) often becomes the predominant crustacean shredder (Whitehurst, 1991). A. aquaticus thus provides the energy for local and downstream communities by its leaf shredding activity, a function typically attributed to Gammarus (Graça et al., 1993). Despite the suggested higher tolerance of A. aquaticus towards nutrient stress, its behavior and survival is affected by pesticides (e.g., Beketov, MA and Liess, M, 2008, Bundschuh, M, et al., 2012). This highlights the importance of assessing the effect(s) of pesticides on A. aquaticus via direct and indirect effect pathways.

In this context, the objectives of the present study were (i) to assess fungicide-induced shifts in the leaf-associated microbial community and (ii) to unravel the biological relevance of food-quality and waterborne related toxicity of a fungicide on the food processing (consumption and excretion) as well as the physiological fitness (FA composition, lipid content; cf., Koop et al., 2011) and growth of A. aquaticus. Because triazole fungicides can negatively affect both fungi and animals (Stenersen, 2004), epoxiconazole was chosen as a model fungicide at a concentration inducing sublethal effects on the test organism A. aquaticus (i.e., 15 μg/L; EC₂₀ = 15.7 μg/L, see Supplementary data). The use of epoxiconazole at this particular concentration is further motivated by its extensive usage for crop protection (Hillocks, 2012) and its frequent detection in surface waters (e.g., Passeport et al., 2013) and edge-of-field run-offs at maximum concentrations up to 6 μg/L (Liess and von der Ohe, 2005) and 20 μg/L (Neumann et al., 2002), respectively. Using a 2 × 2-factorial design, epoxiconazole was applied either during the microbial conditioning of the leaf material or during the bioassay with Asellus similar to the method in Zubrod et al. (2015a). Under this test design, A. aquaticus was affected either indirectly via the food, directly over the water phase, or combined via both pathways (i.e., food and water phase). To characterize the fungicide-induced effects on the conditioning status and thus the nutritional quality of the leaf material, fungal biomass, bacterial abundance, microbial C usage, and FA composition were assessed. Moreover, changes in food processing as well as the physiological fitness and growth of A. aquaticus, induced by the different effect pathways, were determined. We hypothesized that epoxiconazole modifies the leaf-associated microbial community, ultimately affecting the physiology of the model species (indirect effect pathway). Additionally, we hypothesized direct effects of epoxiconazole on A. aquaticus via the water phase based on the results of the preliminary experiment (direct effect pathway; Supplementary data). Based on the results of Zubrod et al. (2015a), it was further assumed that both pathways interact when applied in combination.