Molecular Mechanisms in Ecotoxicology: An Interplay between Environmental Chemistry and Biology

A close collaboration between environmental chemistry and biological sciences is required for a complete understanding of ecotoxicological effects. Bioavailability and uptake of pollutants cannot be regarded as isolated chemical or biological questions. Knowledge of the effective concentrations in the organism or at the target site(s) is essential to link the fate and effects of a chemical and is a prerequisite for quantitative investigation of the modes of toxic action. These modes of action need to be unraveled using whole-organism or in vitro systems in order to be able to develop specific biomarkers and biosensors that can be applied as early warning systems. Our mode-of-action-based approaches, in which chemical and biological analytical tools are combined, should improve the understanding of ecotoxicological effects and should be implemented in the future in risk assessment

Expression of Zebra Fish Aromatase cyp19a and cyp19b Genes in Response to the Ligands of Estrogen Receptor and Aryl Hydrocarbon Receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17β-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERα, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligand

Stream microbial communities and ecosystem functioning show complex responses to multiple stressors in wastewater

Multiple anthropogenic drivers are changing ecosystems globally, with a disproportionate and intensifying impact on freshwater habitats. A major impact of urbanization are inputs from wastewater treatment plants (WWTPs). Initially designed to reduce eutrophication and improve water quality, WWTPs increasingly release a multitude of micropollutants (MPs; i.e., synthetic chemicals) and microbes (including antibiotic-resistant bacteria) to receiving environments. This pollution may have pervasive impacts on biodiversity and ecosystem services. Viewed through multiple lenses of macroecological and ecotoxicological theory, we combined field, flume, and laboratory experiments to determine the effects of wastewater (WW) on microbial communities and organic-matter processing using a standardized decomposition assay. First, we conducted a mensurative experiment sampling 60 locations above and below WWTP discharges in 20 Swiss streams. Microbial respiration and decomposition rates were positively influenced by WW inputs via warming and nutrient enrichment, but with a notable exception: WW decreased the activation energy of decomposition, indicating a "slowing" of this fundamental ecosystem process in response to temperature. Second, next-generation sequencing indicated that microbial community structure below WWTPs was altered, with significant compositional turnover, reduced richness, and evidence of negative MP influences. Third, a series of flume experiments confirmed that although diluted WW generally has positive influences on microbial-mediated processes, the negative effects of MPs are "masked" by nutrient enrichment. Finally, transplant experiments suggested that WW-borne microbes enhance decomposition rates. Taken together, our results affirm the multiple stressor paradigm by showing that different aspects of WW (warming, nutrients, microbes, and MPs) jointly influence ecosystem functioning in complex ways. Increased respiration rates below WWTPs potentially generate ecosystem "disservices" via greater carbon evasion from streams and rivers. However, toxic MP effects may fundamentally alter ecological scaling relationships, indicating the need for a rapprochement between ecotoxicological and macroecological perspectives

Agricultural pesticides pose a continuous ecotoxicological risk to aquatic organisms in a tropical horticulture catchment

Pesticides are used throughout the tropics in large amounts to protect crops against pests and weeds. These chemicals may be lost to the aquatic environment and impair its ecological status and the water quality for human consumption. Despite intensive use of pesticides in many developing countries, the knowledge of pesticide levels in aquatic ecosystems is often very limited. In this study, we try to fill this gap for an horticultural catchment of 35.9 km2 in Costa Rica, which is intensively used by small-holder farming for potatoes and vegetable production. We continuously monitored pesticides in the Tapezco river during two consecutive rainy seasons with passive sampling systems and screened for a broad set of polar and non-polar pesticides. Spatially distributed measurements revealed high pesticide concentrations of many fungicides, herbicides and insecticides throughout the watershed. Concentration ranges revealed little spatio-temporal variation. From an ecotoxicological point of view, the insecticide levels – notably chlorpyrifos and cypermethrin - were most critical. The observed concentration levels exceeded chronic environmental quality standards more than 100-fold at all sites. These high insecticide levels were partially reflected in the community composition of the macroinvertebrates. Available data revealed a poor status at two upstream locations according to the Costa Rican Biological Monitoring Working Party (BMWP-CR) Index and the SPEARpesticides index. However, the indices indicated a good quality at most downstream sites despite their high pesticide levels. The wide-spread occurrence of high pesticide levels demonstrated that the relevant sources and pathways existed throughout the catchment. Field observations and survey data showed the relevance of point sources due to poor pesticide handling as well as diffuse losses from fields, which are strongly enhanced by the steep terrain and linear structures like gullies connecting fields with the stream. Mitigation measures to reduce pesticide losses have to account for these different source-flowpath combinations.ISSN:2666-765

Dose-dependent effects of morphine on lipopolysaccharide (LPS)-induced inflammation, and involvement of multixenobiotic resistance (MXR) transporters in LPS efflux in teleost fish

Opioid drugs, such as morphine (MO), detected in aquatic environments worldwide, may harm fish due to their semi-persistence and ability to potently interact with molecular targets conserved across vertebrates. Here, we established a waterborne bacterial lipopolysaccharide (LPS) challenge assay with zebrafish embryos as a model to investigate chemically-induced disruption of the innate immune system, and used it to study the effects of MO exposure. Exposure to 1 mg/L MO resulted in pronounced immunosuppression, reflected in downregulation of several inflammation-related genes, including myd88, trif, traf6, p38, nfκb2, il-1β, il-8 and ccl34a. Fish exposed to 1 mg/L MO accumulated 11.7 ng/g (wet weight) of MO, a concentration comparable to that reported in blood of chronic drug abusers subject to higher infection rates. Surprisingly, exposure to lower MO concentrations (100 ng/L–100 μg/L) led to exacerbation of LPS-induced inflammation. Two ATP-binding cassette (ABC) transporters known to be involved in the xenobiotic efflux - abcb4 and abcc2, also known as multixenobiotic resistance (MXR) transporters - were downregulated at 100 ng/L MO. We hypothesized that ABC/MXR transporters could modulate the severity of inflammation by being involved in efflux of LPS, thus regulating its accumulation in the organism. Indeed, we could demonstrate that blocking of ABC/MXR transporters by an inhibitor, cyclosporine A, results in stronger inflammation, coinciding with higher LPS accumulation, as visualized with fluorescently labeled LPS. Our work demonstrates that MO can disrupt fish innate immune responses at environmentally relevant concentrations. We also provide evidence for a role of ABC/MXR transporters in LPS efflux in fish. These finding may be applicable across other taxa, as ABC transporters are evolutionary conserved. Since diverse environmentally present chemicals are known to interfere with ABC/MXR transporters' expression or activity, our discovery raises concerns about potential adverse effects of such compounds on the immune system responses in aquatic organisms

Impact of wastewater on the microbial diversity of periphyton and its tolerance to micropollutants in an engineered flow-through channel system

Wastewater treatment plants (WWTPs) play an important role in retaining organic matter and nutrients but to a lesser extent micropollutants. Therefore, treated wastewater is recognized as a major source of multiple stressors, including complex mixtures of micropollutants. These can potentially affect microbial communities in the receiving water bodies and the ecological functions they provide. In this study, we evaluated in flow-through channels the consequences of an exposure to a mixture of stream water and different percentages of urban WWTP effluent, ranging from 0% to 80%, on the microbial diversity and function of periphyton communities. Assuming that micropollutants exert a selective pressure for tolerant microorganisms within communities, we further examined the periphyton sensitivity to a micropollutant mixture extracted from passive samplers that were immersed in the wastewater effluent. As well, micropollutants in water and in periphyton were comprehensively quantified. Our results show that micropollutants detected in periphyton differed from those found in water, both in term of concentration and composition. Especially photosystem II inhibitors accumulated in periphyton more than other pesticides. Although effects of other substances cannot be excluded, this accumulation may have contributed to the observed higher tolerance of phototrophic communities to micropollutants upon exposure to 30% and 80% of wastewater. On the contrary, no difference in tolerance was observed for heterotrophic communities. Exposure to the gradient of wastewater led to structural differences in both prokaryotic and eukaryotic communities. For instance, the relative abundance of cyanobacteria was higher with increasing percentage of wastewater effluent, whereas the opposite was observed for diatoms. Such results could indicate that differences in community structure do not necessarily lead to higher tolerance. This highlights the need to consider other wastewater constituents such as nutrients and wastewater-derived microorganisms that can modulate community structure and tolerance. By using engineered flow-through channels that mimic to some extent the required field conditions for the development of tolerance in periphyton, our study constitutes a base to investigate the mechanisms underlying the increased tolerance, such as the potential role of microorganisms originating from wastewater effluents, and different treatment options to reduce the micropollutant load in effluents.ISSN:0043-1354ISSN:1879-244

Wastewater microorganisms impact microbial diversity and important ecological functions of stream periphyton

Effluents of wastewater treatment plants can impact microbial communities in the receiving streams. However, little is known about the role of microorganisms in wastewater as opposed to other wastewater constituents, such as nutrients and micropollutants. We aimed therefore at determining the impact of wastewater microorganisms on the microbial diversity and function of periphyton, key microbial communities in streams. We used a flow-through channel system to grow periphyton upon exposure to a mixture of stream water and unfiltered or ultra-filtered wastewater. Impacts were assessed on periphyton biomass, activities and tolerance to micropollutants, as well as on microbial diversity. Our results showed that wastewater microorganisms colonized periphyton and modified its community composition, resulting for instance in an increased abundance of Chloroflexi and a decreased abundance of diatoms and green algae. This led to shifts towards heterotrophy, as suggested by the changes in nutrient stoichiometry and the increased mineralization potential of carbon substrates. An increased tolerance towards micropollutants was only found for periphyton exposed to unfiltered wastewater but not to ultra-filtered wastewater, suggesting that wastewater microorganisms were responsible for this increased tolerance. Overall, our results highlight the need to consider the role of wastewater microorganisms when studying potential impacts of wastewater on the receiving water body

What agro-input dealers know, sell and say to smallholder farmers about pesticides: a mystery shopping and KAP analysis in Uganda

ISSN:1476-069

Environmental context and magnitude of disturbance influence trait‐mediated community responses to wastewater in streams

Human land uses and population growth represent major global threats to biodiversity and ecosystem services. Understanding how biological communities respond to multiple drivers of human‐induced environmental change is fundamental for conserving ecosystems and remediating degraded habitats. Here, we used a replicated ‘real‐world experiment’ to study the responses of invertebrate communities to wastewater perturbations across a land‐use intensity gradient in 12 Swiss streams. We used different taxonomy and trait‐based community descriptors to establish the most sensitive indicators detecting impacts and to help elucidate potential causal mechanisms of change. First, we predicted that streams in catchments adversely impacted by human land‐uses would be less impaired by wastewater inputs because their invertebrate communities should be dominated by pollution‐tolerant taxa (‘environmental context’). Second, we predicted that the negative effects of wastewater on stream invertebrate communities should be larger in streams that receive proportionally more wastewater (‘magnitude of disturbance’). In support of the ‘environmental context’ hypothesis, we found that change in the Saprobic Index (a trait‐based indicator of tolerance to organic pollution) was associated with upstream community composition; communities in catchments with intensive agricultural land uses (e.g., arable cropping and pasture) were generally more resistant to eutrophication associated with wastewater inputs. We also found support for the ‘magnitude of disturbance’ hypothesis. The SPEAR Index (a trait‐based indicator of sensitivity to pesticides) was more sensitive to the relative input of effluent, suggesting that toxic influences of wastewater scale with dilution. Whilst freshwater pollution continues to be a major environmental problem, our findings highlight that the same anthropogenic pressure (i.e., inputs of wastewater) may induce different ecological responses depending on the environmental context and community metrics used. Thus, remediation strategies aiming to improve stream ecological status (e.g., rehabilitating degraded reaches) need to consider upstream anthropogenic influences and the most appropriate indicators of restoration success