Hazards of current concentration-setting practices in environmental toxicology studies.

The setting of concentrations for testing substances in ecotoxicological studies is often based on fractions of the concentrations that cause 50% mortality (LC50 or LD50) rather than environmentally relevant levels. This practice can result in exposures to animals at test concentrations that are magnitudes of order greater than those experienced in the environment. Often, such unrealistically high concentrations may cause non-specific biochemical or morphologic changes that primarily reflect the near-lethal health condition of the animal subjects, as opposed to effects characteristic of the particular test compound. Meanwhile, it is recognized that for many chemicals, the toxicologic mode of action (MOA) responsible for lethality may differ entirely from the MOAs that cause various sublethal effects. One argument for employing excessively high exposure concentrations in sublethal studies is to ensure the generation of positive toxicological effects, which can then be used to establish safety thresholds; however, it is possible that the pressure to produce exposure-related effects may also contribute to false positive outcomes. The purpose of this paper is to explore issues involving some current usages of acute LC50 data in ecotoxicology testing, and to propose an alternative strategy for performing this type of research moving forward. Toward those ends, a brief literature survey was conducted to gain an appreciation of methods that are currently being used to set test concentrations for sublethal definitive studies.

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.

It's a hard knock life for some: Heterogeneity in infection life history of salmonids influences parasite disease outcomes.

Heterogeneity in immunity occurs across numerous disease systems with individuals from the same population having diverse disease outcomes. Proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae, is a persistent parasitic disease negatively impacting both wild and farmed salmonids. Little is known of how PKD is spread or maintained within wild susceptible populations. We investigated an aspect of fish disease that has been largely overlooked, that is, the role of the host phenotypic heterogeneity in disease outcome. We examined how host susceptibility to T. bryosalmonae infection, and the disease PKD, varied across different infection life-history stages and how it differs between naïve, re-infected and persistently infected hosts. We investigated the response to parasite exposure in host phenotypes with (a) different ages and (b) heterogeneous infection life histories. Among (a) the age phenotypes were young-of-the-year (YOY) fish and juvenile 1+ fish (fish older than one) and, for (b) juvenile 1+ infection survivors were either re-exposed or not re- exposed to the parasite and response phenotypes were assigned post-hoc dependant on infection status. In fish not re-exposed this included fish that cleared infection (CI) or had a persistent infection (PI). In fish re-exposed these included fish that were re-infected (RI), or re-exposed and uninfected (RCI). We assessed both parasite-centric (infection prevalence, parasite burden, malacospore transmission) and host-centric parameters (growth rates, disease severity, infection tolerance and the immune response). In (a), YOY fish, parasite success and disease severity were greater and differences in the immune response occurred, demonstrating an ontogenetic decline of susceptibility in older fish. In (b), in PI and RI fish, parasite success and disease severity were comparable. However, expression of several adaptive immunity markers was greater in RI fish, indicating concomitant immunity, as re-exposure did not intensify infection. We demonstrate the relevance of heterogeneity in infection life history on disease outcome and describe several distinctive features of immune ontogeny and protective immunity in this model not previously reported. The relevance of such themes on a population level requires greater research in many aquatic disease systems to generate clearer framework for understanding the spread and maintenance of aquatic pathogens.

Immunotoxicity of Xenobiotics in Fish: A Role for the Aryl Hydrocarbon Receptor (AhR)?

The impact of anthropogenic contaminants on the immune system of fishes is an issue of growing concern. An important xenobiotic receptor that mediates effects of chemicals, such as halogenated aromatic hydrocarbons (HAHs) and polyaromatic hydrocarbons (PAHs), is the aryl hydrocarbon receptor (AhR). Fish toxicological research has focused on the role of this receptor in xenobiotic biotransformation as well as in causing developmental, cardiac, and reproductive toxicity. However, biomedical research has unraveled an important physiological role of the AhR in the immune system, what suggests that this receptor could be involved in immunotoxic effects of environmental contaminants. The aims of the present review are to critically discuss the available knowledge on (i) the expression and possible function of the AhR in the immune systems of teleost fishes; and (ii) the impact of AhR-activating xenobiotics on the immune systems of fish at the levels of immune gene expression, immune cell proliferation and immune cell function, immune pathology, and resistance to infectious disease. The existing information indicates that the AhR is expressed in the fish immune system, but currently, we have little understanding of its physiological role. Exposure to AhR-activating contaminants results in the modulation of numerous immune structural and functional parameters of fish. Despite the diversity of fish species studied and the experimental conditions investigated, the published findings rather uniformly point to immunosuppressive actions of xenobiotic AhR ligands in fish. These effects are often associated with increased disease susceptibility. The fact that fish populations from HAH- and PAH-contaminated environments suffer immune disturbances and elevated disease susceptibility highlights that the immunotoxic effects of AhR-activating xenobiotics bear environmental relevance.

ABC transporters in gills of rainbow trout (Oncorhynchus mykiss).

Fish gills are a structurally and functionally complex organ at the interface between the organism and the aquatic environment. Gill functions include the transfer of organic molecules, both natural ones and xenobiotic compounds. Whether the branchial exchange of organic molecules involves active transporters is currently not known. Here, we investigated the presence, diversity and functional activity of ATP-binding cassette (ABC) transporters in gills of juvenile rainbow trout. By means of RT-qPCR, gene transcripts of members from the abcb, abcc and abcg subfamilies were identified. Comparisons with mRNA profiles from trout liver and kidney revealed that ABC transporters known to have an apical localization in polarized epithelia, especially abcc2 and abcb1, were under-represented in the gills. In contrast, ABC transporters with mainly basolateral localization showed comparable gene transcript levels in the three organs. The most prominent ABC transporter in gills was an abcb subfamily member, which was annotated as abcb5 based on the synteny and phylogeny. Functional in vivo assays pointed to a role of branchial ABC transporters in branchial solute exchange. We further assessed the utility of primary gill cell cultures to characterize transporter-mediated branchial exchange of organic molecules, by examining ABC transporter gene transcript patterns and functional activity in primary cultures. The gill cultures displayed functional transport activity, but the ABC mRNA expression patterns were different to those of the intact gills. Overall, the findings of this study provide evidence for the presence of functional ABC transporter activity in gills of fish.

Biomonitoring of aromatic hydrocarbons in clam Meretrix meretrix from an emerging urbanization area, and implications for human health.

Pollution with total petroleum hydrocarbons (TPHs) is a global concern and particularly in coastal environments. Polycyclic aromatic hydrocarbons (PAHs) are regarded as the most toxic components of TPHs and they can also be derived from other sources. Fangcheng Port is considered as a representative emerging coastal city in China, but the status, sources, and hazards to organisms and humans with respect to contamination with PAHs and TPHs are unknown in the coastal regions of this area. Therefore, in this study, we cloned cytochrome P450 family genes (CYP1A1, CYP3A, and CYP4) and heat shock protein 70 gene (HSP70) in the clam Meretrix meretrix as well as optimizing the method for measuring the 7-ethoxyresorufin O-deethylase activity. These molecular indicators and four specific physiological indexes were found to be appropriate biomarkers for indicating the harmful effects of PAHs and TPHs on clams after exposure to the crude oil water-soluble fraction. In field monitoring surveys, we found that the 2- and 3-ring PAHs were dominant in the clams whereas the 4- to 6-ring PAHs were dominant in the sediments at each site. The PAH levels (3.63-12.77 ng/g wet weight) in wild clams were lower, whereas the TPH levels (13.25-70.50 µg/g wet weight) were higher compared with those determined previous in China and elsewhere. The concentrations of PAHs and TPHs in the sediments (19.20-4215.76 ng/g and 3.65-866.40 µg/g dry weight) were moderate compared with those in other global regions. Diagnostic ratio analysis demonstrated that the PAHs were derived mainly from pyrogenic sources. The TPHs may have come primarily from industrial effluents, land and maritime transportation, or fishing activities. The Integrated Biomarker Response version 2 indexes indicated that the clams collected from site S5 exhibited the most harmful effects due to contamination by PAHs and TPHs. Human health risk assessments demonstrated that the risks due to PAHs and TPHs following the consumption of clams can be considered acceptable. Our results suggest that continuous monitoring of contamination by PAHs and TPHs is recommended in this emerging coastal city as well as assessing their human health risks.

Aryl Hydrocarbon Receptor Signaling Is Functional in Immune Cells of Rainbow Trout (Oncorhynchus mykiss).

The arylhydrocarbon receptor (AhR) is an important signaling pathway in the immune system of mammals. In addition to its physiological functions, the receptor mediates the immunotoxic actions of a diverse range of environmental contaminants that bind to and activate the AhR, including planar halogenated aromatic hydrocarbons (PHAHs or dioxin-like compounds) and polynuclear aromatic hydrocarbons (PAHs). AhR-binding xenobiotics are immunotoxic not only to mammals but to teleost fish as well. To date, however, it is unknown if the AhR pathway is active in the immune system of fish and thus may act as molecular initiating event in the immunotoxicity of AhR-binding xenobiotics to fish. The present study aims to examine the presence of functional AhR signaling in immune cells of rainbow trout (Oncorhynchus mykiss). Focus is given to the toxicologically relevant AhR2 clade. By means of RT-qPCR and in situ hybdridization, we show that immune cells of rainbow trout express ahr 2α and ahr 2ß mRNA; this applies for immune cells isolated from the head kidney and from the peripheral blood. Furthermore, we show that in vivo as well as in vitro exposure to the AhR ligand, benzo(a)pyrene (BaP), causes upregulation of the AhR-regulated gene, cytochrome p4501a, in rainbow trout immune cells, and that this induction is inhibited by co-treatment with an AhR antagonist. Taken together, these findings provide evidence that functional AhR signaling exists in the immune cells of the teleost species, rainbow trout.

Feed contamination with zearalenone promotes growth but affects the immune system of rainbow trout.

To investigate the effects of feed contamination with zearalenone (ZEN) at the current European Commission (EC) guidance value (2 mg⋅kg-1 feed) on the growth and health of rainbow trout, we performed a long-term feeding trial under aquaculture conditions. It started with the external feeding of the fish larvae, and continued for 96 weeks, at which point the fish had reached market size. To assess the growth of fish and their feeding efficiency throughout this period, the fish were regularly weighed and measured, and their feed consumption was monitored. Additionally, to investigate potential health effects, after 72 weeks of the exposure to ZEN, the fishes' blood was analyzed for major hematological and biochemical indices, and their head kidney, spleen, and liver were examined for morphological, histopathological, cytological, and molecular changes. Finally, to gain insight into the metabolism and distribution of ZEN in fish, the content of free and glucuronidated forms of ZEN and its major metabolites was measured in the intestine, liver, and muscles of the exposed fish. The feed-borne exposure of rainbow trout to ZEN at a dose of 2 mg⋅kg-1 feed resulted in higher feeding efficiency and growth rate, most probably due to the anabolic properties of the ZEN metabolite. Importantly for the consumers of fish, despite absorption and metabolism of ZEN in the digestive system of the fish that had been exposed for 72 weeks, the residuals of ZEN were not transferred to the fishes' muscles, which rules out a potential risk to human health related to the consumption of fish meat. However, the increased growth of fish fed with the contaminated feed may come at some cost, as the exposure to ZEN was associated with modulation of key components of the adaptive and innate immune systems. Moreover, the trunk kidney of ZEN-fed fish showed massive inflammation that was likely caused by pathogen infection. These findings raise concerns about fish health under the current recommended EC guidance values.

Antimicrobial activity and mechanisms of multiple antimicrobial peptides isolated from rockfish Sebastiscus marmoratus.

Pathogenic disease is a major factor affecting the aquaculture of the rockfish Sebastiscus marmoratus, an important commercial species inhabiting the nearshore waters of the Western Pacific Ocean. Antimicrobial peptides (AMPs), as critical components of innate immunity, have been considered as promising antibiotic substitutes. The aims of this study were 1) to identify major AMPs in the rockfish, 2) to assess their antimicrobial activity and 3) to evaluate their potential therapeutic application. Six AMPs were identified, Hepcidin 1, liver-expressed antimicrobial peptide 2 (LEAP-2), Piscidin, Moronecidin, NK-lysin and ß-defensin through analysis of the liver transcriptome of S. marmoratus. The transcriptional expression profiles of these AMPs were investigated by real-time quantitative PCR (RT-qPCR). These AMPs showed tissue-specific distribution patterns, and S. marmoratus displays a time-, dose- and tissue-dependent expression of AMPs in response to lipopolysaccharide (LPS) challenge. While the synthetic peptides of LEAP-2 and Moronecidin exerted broad-spectrum antimicrobial activity against important aquatic pathogens in vitro by directly disrupting microbial membrane, and no cytotoxicity against murine hepatic cells was observed at the effective concentrations from 5 µM to 40 µM. The existence of multiple AMPs and their distinct tissue distribution patterns and inducible expression patterns suggests a sophisticated, highly redundant, and multilevel network of antimicrobial defensive mechanisms of S. marmoratus. Therefore, S. marmoratus-derived AMPs appear to be potential therapeutic applications against pathogen infections in aquaculture.

Thyroid Hormone Disruptors Interfere with Molecular Pathways of Eye Development and Function in Zebrafish.

The effects of thyroid hormone disrupting chemicals (THDCs) on eye development of zebrafish were investigated. We expected THDC exposure to cause transcriptional changes of vision-related genes, which find their phenotypic anchoring in eye malformations and dysfunction, as observed in our previous studies. Zebrafish were exposed from 0 to 5 days post fertilization (dpf) to either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or tetrabromobisphenol-A (TBBPA), which interacts with thyroid hormone receptors. Full genome microarray analyses of RNA isolated from eye tissue revealed that the number of affected transcripts was substantially higher in PTU- than in TBBPA-treated larvae. However, multiple components of phototransduction (e.g., phosphodiesterase, opsins) were responsive to both THDC exposures. Yet, the response pattern for the gene ontology (GO)-class "sensory perception" differed between treatments, with over 90% down-regulation in PTU-exposed fish, compared to over 80% up-regulation in TBBPA-exposed fish. Additionally, the reversibility of effects after recovery in clean water for three days was investigated. Transcriptional patterns in the eyes were still altered and partly overlapped between 5 and 8 dpf, showing that no full recovery occurred within the time period investigated. However, pathways involved in repair mechanisms were significantly upregulated, which indicates activation of regeneration processes.

Temperature-related parasite infection dynamics: the case of proliferative kidney disease of brown trout.

Climate change, in particular rising temperature, is suspected to be a major driver for the emergence of many wildlife diseases. Proliferative kidney disease of salmonids, caused by the myxozoan Tetracapsuloides bryosalmonae, was used to evaluate how temperature dependence of host-parasite interactions modulates disease emergence. Brown trout (Salmo trutta fario) kept at 12 and 15 °C, were experimentally infected with T. bryosalmonae. Parasite development in the fish host and release of spores were quantified simultaneously to unravel parasite transmission potential from the vertebrate to the invertebrate host. A change to a stable plateau in infection intensity of the kidney coincided with a threshold at which spore shedding commenced. This onset of parasite release was delayed at the low temperature in accordance with reaching this infection intensity threshold, but the amount of spores released was irrespective of temperature. The production of parasite transmission stages declined with time. In conclusion, elevated temperature modifies the parasite transmission opportunities by increasing the duration of transmission stage production, which may affect the spread and establishment of the parasite in a wider range of rivers.

Immune-Specific Expression and Estrogenic Regulation of the Four Estrogen Receptor Isoforms in Female Rainbow Trout (Oncorhynchus mykiss).

Genomic actions of estrogens in vertebrates are exerted via two intracellular estrogen receptor (ER) subtypes, ERα and ERß, which show cell- and tissue-specific expression profiles. Mammalian immune cells express ERs and are responsive to estrogens. More recently, evidence became available that ERs are also present in the immune organs and cells of teleost fish, suggesting that the immunomodulatory function of estrogens has been conserved throughout vertebrate evolution. For a better understanding of the sensitivity and the responsiveness of the fish immune system to estrogens, more insight is needed on the abundance of ERs in the fish immune system, the cellular ratios of the ER subtypes, and their autoregulation by estrogens. Consequently, the aims of the present study were (i) to determine the absolute mRNA copy numbers of the four ER isoforms in the immune organs and cells of rainbow trout, Oncorhynchus mykiss, and to compare them to the hepatic ER numbers; (ii) to analyse the ER mRNA isoform ratios in the immune system; and, (iii) finally, to examine the alterations of immune ER mRNA expression levels in sexually immature trout exposed to 17ß-estradiol (E2), as well as the alterations of immune ER mRNA expression levels in sexually mature trout during the reproductive cycle. All four ER isoforms were present in immune organs-head kidney, spleen-and immune cells from head kidney and blood of rainbow trout, but their mRNA levels were substantially lower than in the liver. The ER isoform ratios were tissue- and cell-specific, both within the immune system, but also between the immune system and the liver. Short-term administration of E2 to juvenile female trout altered the ER mRNA levels in the liver, but the ERs of the immune organs and cells were not responsive. Changes of ER gene transcript numbers in immune organs and cells occurred during the reproductive cycle of mature female trout, but the changes in the immune ER profiles differed from those in the liver and gonads. The correlation between ER gene transcript numbers and serum E2 concentrations was only moderate to low. In conclusion, the low mRNA numbers of nuclear ER in the trout immune system, together with their limited estrogen-responsiveness, suggest that the known estrogen actions on trout immunity may be not primarily mediated through genomic actions, but may involve other mechanisms, such as non-genomic pathways or indirect effects.

20 Years of fish immunotoxicology - what we know and where we are.

Despite frequent field observations of impaired immune response and increased disease incidence in contaminant-exposed wildlife populations, immunotoxic effects are rarely considered in ecotoxicological risk assessment. The aim of this study was to review the literature on immunotoxic effects of chemicals in fish to quantitatively evaluate (i) which experimental approaches were used to assess immunotoxic effects, (ii) whether immune markers exist to screen for potential immunotoxic activities of chemicals, and (iii) how predictive those parameters are for adverse alterations of fish immunocompetence and disease resistance. A total of 241 publications on fish immunotoxicity were quantitatively analyzed. The main conclusions included: (i) To date, fish immunotoxicology focused mainly on innate immune responses and immunosuppressive effects. (ii) In numerous studies, the experimental conditions are poorly documented, as for instance age or sex of the fish or the rationale for the selected exposure conditions is often missing. (iii) Although a broad variety of parameters were used to assess immunotoxicity, the rationale for the choice of measured parameters was often not given, remaining unclear how they link to the suspected immunotoxic mode of action of the chemicals. (iv) At the current state of knowledge, it is impossible to identify a set of immune parameters that could reliably screen for immunotoxic potentials of chemicals. (v) Similarly, in fish immunotoxicology there is insufficient understanding of how and when chemical-induced modulations of molecular/cellular immune changes relate to adverse alterations of fish immunocompetence, although this would be crucial to include immunotoxicity in ecotoxicological risk assessment.

Who needs the hotspot? The effect of temperature on the fish host immune response to Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease.

Proliferative kidney disease (PKD) of salmonids, caused by Tetracapsuloides bryosalmonae may lead to high mortalities at elevated water temperatures. However, it has not yet been investigated how temperature affects the fish host immune response to T. bryosalmonae. We exposed YOY (young of the year) rainbow trout (Oncorhynchus mykiss) to T. bryosalmonae at two temperatures (12 °C and 15 °C) that reflect a realistic environmental scenario and could occur in the natural habitat of salmonids. We followed the development of the parasite, host pathology and immune response over seven weeks. We evaluated the composition and kinetics of the leukocytes and their major subgroups in the anterior and posterior kidney. We measured immune gene expression profiles associated with cell lineages and functional pathways in the anterior and posterior kidney. At 12 °C, both infection prevalence and pathogen load were markedly lower. While the immune response was characterized by subtle changes, mainly an increased amount of lymphocytes present in the kidney, elevated expression of Th1-like signature cytokines and strong upregulation of the natural killer cell enhancement factor, NKEF at week 6 P.E. At 15 °C the infection prevalence and pathogen burden were ominously greater. While the immune response as the disease progressed was associated with a Th2-like switch at week 6 P.E and a prominent B cell response, evidenced at the tissue, cell and transcript level. Our results highlight how a subtle, environmentally relevant difference in temperature resulted in diverse outcomes in terms of the immune response strategy, altering the type of interaction between a host and a parasite.

Stress differentially affects the systemic and leukocyte estrogen network in common carp.

Both systemic and locally released steroid hormones, such as cortisol and estrogens, show immunomodulatory actions. This research gives evidence that circulating and leukocyte-derived estrogens can be involved in the regulation of the immune response in common carp, during homeostasis and upon restraining stress. It was found that stress reduced level of blood 17ß-estradiol (E2) and down-regulated the gene expression of components of the "classical" estrogen system: the nuclear estrogen receptors and the aromatase CYP19, in the hypothalamus, the pituitary and in the ovaries. In contrast, higher gene expression of the nuclear estrogen receptors and cyp19a was found in the head kidney of stressed animals. Moreover, stress induced changes in the E2 level and in the estrogen sensitivity at local/leukocyte level. For the first time in fish, we showed the presence of physiologically relevant amounts of E2 and the substrates for its conversion (estrone - E1 and testosterone - T) in head kidney monocytes/macrophages and found that its production is modulated upon stress. Moreover, stress reduced the sensitivity of leukocytes towards estrogens, by down-regulation the expression of the erb and cyp19 genes in carp phagocytes. In contrast, era expression was up-regulated in the head kidney monocytes/macrophages and in PBLs derived from stressed animals. We hypothesize that, the increased expression of ERα, that was observed during stress, can be important for the regulation of leukocyte differentiation, maturation and migration. In conclusion, these results indicate that, in fish, the estrogen network can be actively involved in the regulation of the systemic and local stress response and the immune response.

The emergence of epitheliocystis in the upper Rhone region: evidence for Chlamydiae in wild and farmed salmonid populations.

We present the first study comparing epitheliocystis in a wild and farmed salmonid in Europe. Sampling three tributaries to the Lake Geneva, including one from headwaters to river mouth, revealed an unequal distribution of epitheliocystis in brown trout (Salmo trutta). When evaluated histologically and comparing sites grouped as wild versus farm, the probability of finding infected trout is higher on farms. In contrast, the infection intensities, as estimated by the number of cysts per gill arch, were higher on average and showed maximum values in the wild trout. Sequence analysis showed the most common epitheliocystis agents were Candidatus Piscichlamydia salmonis, all clustering into a single clade, whereas Candidatus Clavichlamydia salmonicola sequences cluster in two closely related subspecies, of which one was mostly found in farmed fish and the other exclusively in wild brown trout, indicating that farms are unlikely to be the source of infections in wild trout. A detailed morphological analysis of cysts using transmission electron microscopy revealed unique features illustrating the wide divergence existing between Ca. P. salmonis and Ca. C. salmonicola within the phylum Chlamydiae.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Benzo(a)pyrene Metabolism and EROD and GST Biotransformation Activity in the Liver of Red- and White-Blooded Antarctic Fish.

Climate change and anthropogenic pollution are of increasing concern in remote areas such as Antarctica. The evolutionary adaptation of Antarctic notothenioid fish to the cold and stable Southern Ocean led to a low plasticity of their physiological functions, what may limit their capacity to deal with altered temperature regimes and pollution in the Antarctic environment. Using a biochemical approach, we aimed to assess the hepatic biotransformation capacities of Antarctic fish species by determining (i) the activities of ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), and (ii) the metabolic clearance of benzo(a)pyrene by hepatic S9 supernatants. In addition, we determined the thermal sensitivity of the xenobiotic biotransformation enzymes. We investigated the xenobiotic metabolism of the red-blooded Gobionotothen gibberifrons and Notothenia rossii, the hemoglobin-less Chaenocephalus aceratus and Champsocephalus gunnari, and the rainbow trout Oncorhynchus mykiss as a reference. Our results revealed similar metabolic enzyme activities and metabolic clearance rates between red- and white-blooded Antarctic fish, but significantly lower rates in comparison to rainbow trout. Therefore, bioaccumulation factors for metabolizable lipophilic contaminants may be higher in Antarctic than in temperate fish. Likewise, the thermal adaptive capacities and flexibilities of the EROD and GST activities in Antarctic fish were significantly lower than in rainbow trout. As a consequence, increasing water temperatures in the Southern Ocean will additionally compromise the already low detoxification capacities of Antarctic fish.

Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR.

BACKGROUND: Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. RESULTS: We describe a qPCR technique based on the single copy gene ß' DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. CONCLUSIONS: This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.