Reliable Field Assessment of Proliferative Kidney Disease in Wild Brown Trout, Salmo trutta, Populations: When Is the Optimal Sampling Period?

Proliferative kidney disease (PKD), caused by the myxozoan parasite Tetracapsuloides bryosalmonae, is suspected to contribute to the decline of wild brown trout Salmo trutta populations. Different factors need to be taken into consideration for PKD outbreaks. Among them, water temperature appears as a main driver of the disease. To understand the epidemiology and impact of the disease on wild fish populations, reliable sampling approaches to detect the presence of T. bryosalmonae-infected fish are needed. This study aimed to characterize the seasonal variation of the prevalence of T. bryosalmonae-infected fish in brown trout populations in two small streams with differing temperature regimes between upstream and downstream sites. As water temperature is known to influence PKD manifestation in brown trout, we hypothesized that the number of T. bryosalmonae-positive fish, as well as their seasonal distribution, will vary between upper and downstream parts of the two streams. Since, in field studies, results can strongly vary across years, we extended the study over a 3-year-period. The number of infected fish and the intensity of infection were assessed by histology. The results confirmed the hypothesis of pronounced temporal- and site-related differences in the percentage of PKD-positive fish and the intensity of the infection. Comparison of water temperatures (total degree days as well as the number of days with a daily mean temperature ≥15 °C) with PKD data indicated that temperature was the driving factor for the temporal development and the intensity of the infection. A mean of 1500 degree days or 30 days with a daily mean temperature ≥15 °C was required before the infection could be detected histologically. From our findings, recommendations are derived for a water temperature-driven sampling strategy campaigns that enables the detection of PKD infection and prevalence in wild brown trout populations.

HEALTH ASSESSMENT OF CAPTIVE AND FREE-LIVING EUROPEAN POND TURTLES (EMYS ORBICULARIS) IN SWITZERLAND.

The highly endangered European pond turtle (Emys orbicularis) was reintroduced in Switzerland in 2010. Up until 2019, no routine medical examinations have been carried out prior to its release or during recapture events. The aim of this study was to assess the health status of captive and free-living Emys orbicularis populations in Switzerland, taking into account the most important and frequently occurring health threats to freshwater turtles. A total of 141 European pond turtles, including captive (n = 89) and free-living (n = 52) individuals, underwent clinical examination (n = 136), choanal and cloacal swab collection for microbiology investigation (n = 140), blood sampling (n = 121), fecal examination for parasitology (n = 92), radiography (n = 84), and ultrasound (n = 46). Microbiology investigation included conventional PCR for herpesvirus, ranavirus, and Mycoplasma spp. Blood was used for the establishment of reference values for hematocrit, leukocyte count, and differential blood count as well as for biochemistry parameters tested with the VetScan VS2. An emydid Mycoplasma was detected in 40% (n = 56/140; 95%CI: 31.82-48.61%) of the turtles, including one individual with upper respiratory signs. Four animals positive for Mycoplasma arrived dead or were euthanized during the study period. Their necropsies revealed no evidence of respiratory disease. No ranavirus or herpesvirus was detected in any of the tested turtles. Two presumptively fatal infections with spirorchiid trematodes were reported during the study period. Endoparasites were detected in only 7.94% of the samples examined. This study provides comprehensive data on the current health status of the largest sample size of captive and free-living populations of Emys orbicularis ever assessed to date and serves as a baseline for future research investigations and management recommendations in this species.

Toward sustainable environmental quality: Priority research questions for Europe.

The United Nations' Sustainable Development Goals have been established to end poverty, protect the planet, and ensure prosperity for all. Delivery of the Sustainable Development Goals will require a healthy and productive environment. An understanding of the impacts of chemicals which can negatively impact environmental health is therefore essential to the delivery of the Sustainable Development Goals. However, current research on and regulation of chemicals in the environment tend to take a simplistic view and do not account for the complexity of the real world, which inhibits the way we manage chemicals. There is therefore an urgent need for a step change in the way we study and communicate the impacts and control of chemicals in the natural environment. To do this requires the major research questions to be identified so that resources are focused on questions that really matter. We present the findings of a horizon-scanning exercise to identify research priorities of the European environmental science community around chemicals in the environment. Using the key questions approach, we identified 22 questions of priority. These questions covered overarching questions about which chemicals we should be most concerned about and where, impacts of global megatrends, protection goals, and sustainability of chemicals; the development and parameterization of assessment and management frameworks; and mechanisms to maximize the impact of the research. The research questions identified provide a first-step in the path forward for the research, regulatory, and business communities to better assess and manage chemicals in the natural environment. Environ Toxicol Chem 2018;37:2281-2295. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The SOLUTIONS project: challenges and responses for present and future emerging pollutants in land and water resources management.

SOLUTIONS (2013 to 2018) is a European Union Seventh Framework Programme Project (EU-FP7). The project aims to deliver a conceptual framework to support the evidence-based development of environmental policies with regard to water quality. SOLUTIONS will develop the tools for the identification, prioritisation and assessment of those water contaminants that may pose a risk to ecosystems and human health. To this end, a new generation of chemical and effect-based monitoring tools is developed and integrated with a full set of exposure, effect and risk assessment models. SOLUTIONS attempts to address legacy, present and future contamination by integrating monitoring and modelling based approaches with scenarios on future developments in society, economy and technology and thus in contamination. The project follows a solutions-oriented approach by addressing major problems of water and chemicals management and by assessing abatement options. SOLUTIONS takes advantage of the access to the infrastructure necessary to investigate the large basins of the Danube and Rhine as well as relevant Mediterranean basins as case studies, and puts major efforts on stakeholder dialogue and support. Particularly, the EU Water Framework Directive (WFD) Common Implementation Strategy (CIS) working groups, International River Commissions, and water works associations are directly supported with consistent guidance for the early detection, identification, prioritisation, and abatement of chemicals in the water cycle. SOLUTIONS will give a specific emphasis on concepts and tools for the impact and risk assessment of complex mixtures of emerging pollutants, their metabolites and transformation products. Analytical and effect-based screening tools will be applied together with ecological assessment tools for the identification of toxicants and their impacts. The SOLUTIONS approach is expected to provide transparent and evidence-based candidates or River Basin Specific Pollutants in the case study basins and to assist future review of priority pollutants under the WFD as well as potential abatement options.

Optimizing the aquatic toxicity assessment under REACH through an integrated testing strategy (ITS).

To satisfy REACH requirements a high number of data on chemical of interest should be supplied to the European Chemicals Agency. To organize the various kinds of information and help the registrants to choose the best strategy to obtain the needed information limiting at the minimum the use of animal testing, integrated testing strategies (ITSs) schemes can be used. The present work deals with regulatory data requirements for assessing the hazards of chemicals to the aquatic pelagic environment. We present an ITS scheme for organizing and using the complex existing data available for aquatic toxicity assessment. An ITS to optimize the choice of the correct prediction strategy for aquatic pelagic toxicity is described. All existing information (like physico-chemical information), and all the alternative methods (like in silico, in vitro or the acute-to-chronic ratio) are considered. Moreover the weight of evidence approach to combine the available data is included.

Integrated testing strategy (ITS) for bioaccumulation assessment under REACH.

REACH (registration, evaluation, authorisation and restriction of chemicals) regulation requires that all the chemicals produced or imported in Europe above 1 tonne/year are registered. To register a chemical, physicochemical, toxicological and ecotoxicological information needs to be reported in a dossier. REACH promotes the use of alternative methods to replace, refine and reduce the use of animal (eco)toxicity testing. Within the EU OSIRIS project, integrated testing strategies (ITSs) have been developed for the rational use of non-animal testing approaches in chemical hazard assessment. Here we present an ITS for evaluating the bioaccumulation potential of organic chemicals. The scheme includes the use of all available data (also the non-optimal ones), waiving schemes, analysis of physicochemical properties related to the end point and alternative methods (both in silico and in vitro). In vivo methods are used only as last resort. Using the ITS, in vivo testing could be waived for about 67% of the examined compounds, but bioaccumulation potential could be estimated on the basis of non-animal methods. The presented ITS is freely available through a web tool.

Human and ecological risk assessment of a crop protection chemical: a case study with the azole fungicide epoxiconazole.

Conventional risk assessments for crop protection chemicals compare the potential for causing toxicity (hazard identification) to anticipated exposure. New regulatory approaches have been proposed that would exclude exposure assessment and just focus on hazard identification based on endocrine disruption. This review comprises a critical analysis of hazard, focusing on the relative sensitivity of endocrine and non-endocrine endpoints, using a class of crop protection chemicals, the azole fungicides. These were selected because they are widely used on important crops (e.g. grains) and thereby can contact target and non-target plants and enter the food chain of humans and wildlife. Inhibition of lanosterol 14α-demethylase (CYP51) mediates the antifungal effect. Inhibition of other CYPs, such as aromatase (CYP19), can lead to numerous toxicological effects, which are also evident from high dose human exposures to therapeutic azoles. Because of its widespread use and substantial database, epoxiconazole was selected as a representative azole fungicide. Our critical analysis concluded that anticipated human exposure to epoxiconazole would yield a margin of safety of at least three orders of magnitude for reproductive effects observed in laboratory rodent studies that are postulated to be endocrine-driven (i.e. fetal resorptions). The most sensitive ecological species is the aquatic plant Lemna (duckweed), for which the margin of safety is less protective than for human health. For humans and wildlife, endocrine disruption is not the most sensitive endpoint. It is concluded that conventional risk assessment, considering anticipated exposure levels, will be protective of both human and ecological health. Although the toxic mechanisms of other azole compounds may be similar, large differences in potency will require a case-by-case risk assessment.

Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction.

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment.

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Assessment of estrogenic exposure in brown trout (Salmo trutta) in a Swiss midland river: integrated analysis of passive samplers, wild and caged fish, and vitellogenin mRNA and protein.

This field study examined the vitellogenin (VTG) biomarker response under conditions of low and fluctuating activities of environmental estrogenicity. The present study was performed on immature brown trout (Salmo trutta) exposed to the small river Luetzelmurg, which is located in the prealpine Swiss midland region and receives effluents from a single sewage treatment plant (STP). To understand better factors influencing the relationship between estrogenic exposure and VTG induction, we compared VTG levels in caged (stationary) and feral (free-ranging) fish, VTG levels in fish from up- and downstream of the STP, and two different methods for quantifying VTG (enzyme-linked immunosorbent assay vs real-time reverse transcription-polymerase chain reaction), and we used passive samplers (polar organic chemical integrative sampler [POCIS]) to integrate the variable, bioaccumulative estrogenic load in the river water over time. The POCIS from the downstream site contained approximately 20-fold higher levels of bioassay-derived estrogen equivalents than the POCIS from the upstream site. In feral fish, this site difference in estrogenic exposure was reflected in VTG protein levels but not in VTG mRNA. In contrast, in caged fish, the site difference was evident only for VTG mRNA but not for VTG protein. Thus, the outcome of VTG biomarker measurements varied with the analytical detection method (protein vs mRNA) and with the exposure modus (caged vs feral). Our findings suggest that for environmental situations with low and variable estrogenic contamination, a multiple-assessment approach may be necessary for the assessment of estrogenic exposure in fish.

Screening and testing for endocrine disruption in fish-biomarkers as "signposts," not "traffic lights," in risk assessment.

Biomarkers are currently best used as mechanistic "signposts" rather than as "traffic lights" in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect concentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38-55% (comparable to published CVs of 19-58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the "biomarkers as signposts" approach is a pragmatic way forward in the current risk assessment of EDCs.

The potential of mechanism-based bioanalytical tools in ecotoxicological exposure and effect assessment.

The current challenge to ecotoxicology is to develop tools that allow rapid and cost-efficient detection of those environmental chemicals or their combinations that are responsible for sublethal, chronic toxic effects in exposed organisms. Bioanalytical tools may meet these challenges, particularly if they are mechanism-based. Technically, bioanalytical tools allow rapid and cost-efficient analysis of environmental matrices. Mechanism-based, bioanalytical tools, however, do not only indicate that certain chemicals are there, but-and this is the major advantage of mechanism-based bioanalytical tools (MBBTs)-they indicate that chemicals with a specific mode of toxic action or a specific toxic potential are there. In this way MBBTs bridge exposure and effect assessment and help in a faster identification of the causative agent(s). Several principles of MBBTs, including immunoassays, enzyme inhibition assays, receptor assays and gene induction assays are briefly discussed and their application in processes such as bioassay-directed fractionation is illustrated. The focus of this manuscript is the analytical power of MBBTs in exposure and effect assessment. MBBTs have, however, a much broader potential and can support research on other challenges in ecotoxicology such as mixture effects or multiple effects caused by single pollutants or by various stresses simultaneously.