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The present document corresponds to the final report (deliverable No. 7) of the specific agreement (SA 2) "To determine the incidence and epidemiological characteristics of ciguatera cases in Europe" within the Framework Partnership Agreement GP/EFSA/AFSCO/2015/03 "Risk characterization of ciguatera food poisoning in Europe". This report summarises the activities performed, results and conclusions obtained in the specific agreement. The objective of the SA2 was to estimate the incidence and to describe the epidemiological characteristics of ciguatera in the EU/EEA. A protocol for a harmonized ciguatera surveillance in the European Union (EU)/Economic European Area (EEA) was elaborated. The protocol includes a ciguatera case definition and questionnaires for collecting information on ciguatera cases or outbreak. Around half of the countries answered the data call for reporting cases. Four countries reported 34 outbreaks from 2012 to 2019. Spain and Portugal reported outbreaks due to consumption of fish captured in the Canary Islands and Madeira (autochthonous outbreaks), mainly due to consumption of Seriola spp. and Ephinephelus spp. In more than half of these outbreaks, the fish was captured by sport fishing. Germany and France reported outbreaks due to consumption of fish imported from outside the EU (imported outbreaks, mainly Lutjanus spp). Spain, Germany and France reported outbreaks in travellers to tropical endemic territories (travel related outbreaks). All the outbreaks cases presented neurological symptoms, most of them had also gastrointestinal symptoms and few outbreak cases mentioned cardiovascular symptoms. Six countries (Austria, France, Germany, Portugal, Spain and Switzerland) reported 34 single cases. The incidence rate in the EU/EEA was very low (0.0054 cases per 100.000 inhabitants per year). The incidence rate in the Canary Islands was higher 0.47 cases/100.000 inhabitants. [ABSTRACT FROM AUTHOR]

The present document corresponds to Deliverable No. 10: "Final report (1st of April 2016‐31stOctober2020)." of the specific agreement "EVALUATION OF CTXs IN SEAFOOD AND THE ENVIRONMENT for the RISK ASSESSMENT OF CIGUATERA FISH POISONING (CFP), with the consequent OBTENTION OF REFERENCE MATERIAL" within the Framework Partnership Agreement GP/EFSA/AFSCO/2015/03 "Risk characterization of ciguatera food poisoning in Europe"© Institute for Research and Technology in Food and Agriculture (IRTA), Universidad de Las Palmas de Gran Canarias (ULPGC), Canary Health Service (ServicioCanario de la Salud, SCS), Instituto Português do Mar e da Atmosfera I.P (IPMA), State General Laboratory (SGL) / Ministry of Health, Aristotle University of Thessaloniki (AUT), 2021 [ABSTRACT FROM AUTHOR]

The examined fish intoxicated with aflatoxin B1 (AFB1) showed several non-specific signs, including inadequate growth, significant losses in aquaculture industry especially resulting from aflatoxicosis. The study using Zinc oxide (ZnO) and Hematite (α-Fe2O3) nanoparticles (NPs) to assess the effect of using as on their own negative impacts of AFB1 3 ppm/Kg for 13 weeks was studied 360 healthy fingerlings of Nile tilapia, were divided into eight groups (15 fish/group) with three replicates, it has been applied in 24 aquariums. Group1 (G1) fed only a basal diet as control. While, G2, G3, and G4 were fed on diets supplemented with 2 g/kg diet of ZnO NPs, Hematite NPs and combination of both respectively. Group5 (G5) was fed on diets contamination with AFB1 (3 ppm/kg diet), while G6, G7 and G8 were fed on diets containing 2 g/kg diet of (ZnO NPs, Hematite NPs and combination of both) plus AFB1 (3 ppm/kg diet) respectively. Experimental treatments affected significantly on the Final Body Weight (FBW), Daily Gain (DG), Feed Conversion Ratio (FCR) and mortality Table (1, 2(. FBW and DG reduced to 37.856 g and 0.227 g in fish feed containing AFB1, when compared with groups AFB1 which supported with feed additives (ZnO, Hematite and combination of both) NPs, which reported final body weight 45.507, 47.007, 49.762 g and daily gain 0.313, 0.328, 0.360 g respectively. The highest FBW and DG were determined in group treated with feed additives combination of (ZnO and Hematite) nanoparticles, keep it track of by the fish group fed diet supplemented with Hematite NPs then those fed diet supplemented with ZnO NPs and the recorded values were for final body weight 55.857, 61.500, 63.874 g daily gain 0.427, 0.488, 0.515 g respectively than that of the control group G1 52.784, g FBW and 0.393 g DG. The highest survival rate was obtained in fish group treated with combination of (ZnO and Hematite) nanoparticles, The survival rate was (95.556 %) in fish fed diets combination of (ZnO and Hematite) NPs, while it was 57.778 % in fish group G5 fed AFB1, which enhanced in aflatoxicated group G8 which supplemented with combination of (ZnO and Hematite) NPs 84.444 %, and in aflatoxicated group G7 which supplemented with Hematite NPs 77.778%, and in aflatoxicated group G6 which supplemented with ZnO NPs 75.556%. The best FCR was obtained in same order in fish group treated with combination of ZnO NPs and Hematite NPs, Hematite NPs then ZnO NPs 1.187, 1.170, 1.253 compared to the control group which recorded 1.462. On the other hand, aflatoxicated groups significantly affected feed conversion impaired significantly affected, aflatoxicated group recorded 1.950 but when supported with ZnO NPs, Hematite NPs and combination of both recorded 1.541, 1.525 and 1.502 respectively. [ABSTRACT FROM AUTHOR]

Highlights • Doehlert design and desirability function allow optimize fish samples dissolution. • Ultrasound energy was used to increase the efficiency of the TMAH dissolution. • The method presents good analytical characteristics allowing simultaneous treatment. • Calcium, Mg, Fe and Zn were determinate from fillet fish samples using FAAS. Abstract Doehlert design and desirability function were used in the optimization of an ultrasound assisted dissolution method of fish fillet samples with tetramethyl ammonium hydroxide (TMAH) for the determination of Ca, Fe, Zn and Mg by flame atomic absorption spectrometry. The variables values after optimization were 620 μL (volume of TMAH), 25 min (sonication time) and 46 °C (bath temperature). The quantification limits for Ca, Mg, Fe and Zn were 56, 107, 23 and 2.9 μg g−1, respectively. Accuracy was evaluated by comparing the results generated from the analysis of the samples treated by the proposed method and by wet digestion. Additionally, accuracy for Fe and Zn was assessed by the analysis of certified reference materials Tort-3 (Lobster hepatopancreas), ERM-CE 278 (mussel tissue) and Dolt-4 (Fish liver). There were no significant differences between the results obtained at a 95% confidence level when results were evaluated by t -test application. [ABSTRACT FROM AUTHOR]

Highlights • Polish and Ukraine strains of R. raciborskii differ in in vitro toxicity. • Extracts of all strains increased oxidative stress indices in carp hepatocytes. • Extracts of Polish strains increased acetylcholinesterase activity in carp brain. • Further studies are required to determine metabolites implicated in toxicity. Abstract As yet European strains of Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) have not been found to produce known cyanotoxins although their extracts have caused adverse effects in mammals, as shown using in vitro and in vivo experimental models. The present study investigated whether R. raciborskii isolated from Western Poland and Ukraine can affect fish cells using in vitro exposures of hepatocytes and red blood cells (RBC), and brain homogenates obtained from common carp (Cyprinus carpio) to 1.0% and 0.1% extracts of 7 strains. The studied extracts evoked different responses of catalase activity in hepatocytes with both increase and decrease observed under low and high concentrations. The cellular thiol pool was also altered with most extracts inducing a decrease in the activity of glutathione-S-transferase, and Ukrainian strains leading to an increase in glutathione level and a decrease in metallothionein content. All the studied extracts induced comparable reactive oxygen species formation, lipid peroxidation, protein carbonylation and DNA fragmentation in hepatocytes, and all but one increased the activity of caspase-3. Only one extract caused lysosomal membrane destabilization as measured by neutral red retention in RBC. In contrast to extracts of Ukrainian isolates, exposure of brain homogenates to extracts of Polish strains induced an increase in acetylcholinesterase activity suggesting the neurotoxic action of their exudates. The results indicate that both Polish and Ukrainian strains of R. raciborskii may pose a toxicological risk to freshwater fish, and further, that Polish strains may produce compound(s) evoking neurotoxic effects. [ABSTRACT FROM AUTHOR]

Pesguard FG161™, a mixture of d-tetramethrin and cyphenothrin (1:3 ratio), is extensively used to achieve rapid control of adult dengue vector, Aedes aegypti, during the disease outbreaks. Both d-tetramethrin and cyphenothrin are synthetic pyrethroids that are known to have adverse effects on non-mammalian organisms such as fish. The present study intended to use zebrafish embryo toxicity model to investigate the toxic effect of the above binary mixture on fish. Particularly, zebrafish embryo toxicity model provides an alternative to acute fish toxicity tests in terms of animal welfare perspective as the embryos are not considered live until 5 days after fertilization. The zebrafish embryos (2 hrs after fertilization) were exposed to a binary mixture of pyrethroids at different concentrations (d-tetramethrin: 0.01 – 1.20 μmolL-1 and cyphenothrin: 0.03 – 3.20 μmolL-1) for 24, 48, and 72 hrs at room temperature (26°C) according to the OECD guideline no. 236. Percentage mortality of embryos were calculated by observing the lethal endpoints and LC50 values were calculated for each time interval employing the probit analysis. This binary mixture was highly toxic to zebrafish embryos and was found to be concentration and time dependent. LC50 values at 24 hrs (d-tet: 0.58 μmolL-1, cyp: 1.74 μmolL-1) were significantly reduced in 48 hrs (d-tet: 0.11 μmolL-1, cyp: 0.33 μmolL-1) and 72 hrs (d-tet: 0.03 μmolL-1, cyp: 0.09 μmolL-1). Coagulation of embryos was the most common lethal effect observed and lack of somite formation and lack of heartbeat were also observed. The present study revealed that the binary mixture is highly toxic to zebrafish embryos even when based on nominal concentrations. Hence, extensive use of these pesticides could be detrimental to fish population and integrated vector control methods which involve the minimum use of insecticides are recommended. Further, this study highlights the applicability of zebrafish embryo toxicity model as an alternative method to investigate the toxicity of pyrethroids to fish. [ABSTRACT FROM AUTHOR]

Abstract Fish consumption is an important route of exposure to persistent organic pollutants (POPs) in dolphins as well as humans. In order to assess the potential risks associated with these contaminants, 39 whole fish and 37 fillets from fish representing species consumed by dolphins and humans captured from Charleston Harbor and tributaries, South Carolina, USA, were measured for a suite of POPs. Polychlorinated biphenyls (PCBs) were the predominant contaminant with concentrations ranging from 5.02 to 232.20 ng/g in whole fish and 5.42–131.95 ng/g in fillets (weight weight ww) followed by total organochlorine pesticides (OCPs) and polybrominated diphenyl ethers (PBDEs). Total POPs levels varied by location and species with general trends indicating significantly higher levels in fish from the Cooper (93.4 ng/g ww) and Ashley Rivers (56.2 ng/g ww) compared to Charleston Harbor (31.6 ng/g ww). Mullet and spot were found to have significantly higher PCBs, OCPs and total POPs, 2–3 times higher than red drum; mullet were also significantly higher in OCPs compared to seatrout. PCB concentrations in whole fish and fillets exceeded EPA human screening values for cancer risk in all fish sampled. For PCBs in fillets, all samples had values of maximum allowable meals per month that were less than the EPA, FDA guidelines for recommended fish meals per month, suggesting lower (more stringent) allowable fish meals per month. All fish exceeded PBDE wildlife values and all fish except two exceeded the level where 95% of the dolphin population would have tissue levels below the health effect threshold. Considering that POP concentrations in fish potentially consumed by humans exceed human health effect thresholds levels, consumption advisories should be considered as a prudent public health measure. Graphical abstract fx1 Highlights • Higher contaminant levels in fish from Cooper/Ashley Rivers than Charleston Harbor. • Mullet and spot were significantly higher in PCBs and pesticides than red drum. • PCBs were the predominant contaminant in whole fish and fillets from Charleston SC. • PCBs in all fillets exceed EPA human health values, consumption advisories warranted. • Whole fish PCBs, PBDEs exceed wildlife screening values, a concern for local dolphins. [ABSTRACT FROM AUTHOR]

Liquid chromatography (LC) coupled to mass spectrometry (MS) is being widely applied as an analytical tool in the field of marine biotoxins both for regulated and for new and emerging compounds. LC-MS/MS recently became the reference method for the control of lipophilic toxins in the European Union, and new methods are being developed and optimized to extend the applicability of this technique to other toxin groups. In this work, conditions for the analysis of ciguatoxins (CTXs) by LC-MS/MS were investigated using standard solutions of CTX1B and CTX3C, which are structurally representative compounds for the rest of the main congeners of Pacific group toxins (P-CTXs). Preliminary studies were carried out for the selection of precursor and product ions used for multiple reaction monitoring. Two transitions based on the chemical structures of CTXs were set up, and mass spectrometer parameters were adjusted for selected reactions monitored. The electrospray ionization source has been carefully optimized through a design of experiments that consisted of a two-level fractional factorial design of resolution IV for the screening of adequate source conditions and of response surface designs for optimization of the main interactions between factors. The statistical approach allowed maximizing the sensitivity on the MS analyzer that provides a good specificity in P-CTX detection, which can be also used for confirmation purposes. [ABSTRACT FROM AUTHOR]

Abamectin (ABA) toxicity in fish, amphibian and mammals was already proven, but its effect on birds is almost unknown. Thus, the aim of our study is to assess the impact of exposure to water with ABA for 40 days at predicted environmentally relevant concentrations on the behavior of female Japanese quails ( Coturnix coturnix japonica ). The three following experimental groups (n = 10 each) were set: “control”, quails exposed to drinking water, without ABA, “EC1x” and “EC1000x” (0.31 mg a.i./L and 310.0 a.i./L, respectively; via commercial formulation Kraft® 36EC). The open field test showed anxiolytic response in birds exposed to ABA. These birds did not show locomotor changes or aggressive behavior in the aggressiveness test. Quails exposed to the pesticide did not react to the introduction of an object in the experimental box during the object recognition test, and it suggested perception deficit due to ABA. Moreover, these birds did not recognize the cat ( Felix catus ) and the vocalization of a hawk ( Rupornis magnirostris ) as potential predatory threats. These responses also suggest anti-predatory behavior deficit due to the pesticide. Thus, our study is pioneer in showing that water with ABA, at tested concentrations, influences the behavior of C. coturnix japonica , as well as in highlighting the potential impacts of this pesticide on this group of birds. [ABSTRACT FROM AUTHOR]

Herein, we propose models for predicting fish early-life stage (ELS) toxicity from acute Daphnia magna toxicity and various molecular descriptors. Specifically, eight models were developed with fathead minnow (Pimephales promelas) data and were validated against Japanese medaka (Oryzias latipes) data because the quantity of available Japanese medaka data is much smaller than the quantity of fathead minnow data. The training data set for the models consisted of ELS fathead minnow toxicity data for 77 chemicals; data for 67 of the 77 chemicals originated from the OPP Pesticide Ecotoxicity Database of the US Environmental Protection Agency. The training data were biased toward pesticides. A simple quantitative activity-activity relationship (QAAR) model based on the correlation between fish ELS and acute Daphnia magna toxicities showed good predictivity for the chemicals in the external validation data set relative to the predictivities of the other models in this study. However, goodness-of-fit and robustness were better for quantitative structure-activity-activity relationship (QSAAR) models that included molecular descriptors (such as pesticide-related atoms and substructures as well as molecular weight and three-dimensional-structure-based parameters). A battery approach involving the use of both the QAAR and the QSAARs might enhance the reliability of the estimated values and prevent underestimates. [ABSTRACT FROM AUTHOR]

The former lead-zinc mine at Mestersvig, Greenland, continues to contaminate the surrounding environment despite its operations ceasing over 50 years ago. Elevated concentrations of heavy metals are found in water, sediment and biota in the terrestrial, freshwater and marine environments. To shed light on the present contamination and its potential effects on local fish we investigated gill and liver histology of sculpins ( Myoxocephalus spp.) around the former mining area. Two species of sculpins were caught; shorthorn sculpins ( M. scorpius ; n = 16) and fourhorn sculpins ( M. quadricornis ; n = 17) at a contaminated site, Nyhavn, and shorthorn sculpins ( M. scorpius ; n = 25) at the reference site. In a previous study we found histopathological changes in the tissues of the sculpins, and we suspected this to be related to elevated heavy metal tissue concentrations. Concentrations of Fe, Hg, Mn, Pb, Se and Zn were significantly higher in sculpins at Nyhavn compared to the reference site. Reference NOED and LOEC thresholds for biochemistry, tissue lesions, growth, survival and reproduction for hepatic Hg, As, Cd and Pb from the ERED database were exceeded in both sculpin species. Histopathological investigations of the sculpins gills revealed significant increases in the prevalence of hyperplastic epithelium, inflammation, intensity of neutral and total mucus cells and chloride cells along with an increased infection of colonial Peritricha. At the contaminated Nyhavn site, fourhorn sculpins had a significantly higher prevalence of chondroplastic tissue and intensity of neutral, mixed and total mucus cells in the gills compared to the shorthorn sculpins. The data indicate that both sculpin species could be useful indicator species for environmental monitoring of metal pollution in Arctic areas. However, confounding effects of gender and species should be investigated further. Effects on other biomarkers as well as baseline measurements should be included in future environmental monitoring efforts around mining activities in Greenland. [ABSTRACT FROM AUTHOR]

The decline in marine and freshwaters catches in recent years in Colombia has led to a change in dietary habits, with an increase in the purchase and consumption of imported fish. This is of particular concern as fish are sometimes caught in mercury-contaminated waters, and are subsequently sold canned or uncanned. In addition, canned tuna has received little attention as it is widely assumed that concentrations are low. In this study, total mercury (THg) and methylmercury (MeHg) concentrations were evaluated in three imported fish species marketed in Colombia, Prochilodus lineatus, Prochilodus reticulatus, and Pangasianodon hypophthalmus, plus four brands of canned tuna and one of sardines. One brand of tuna showed the highest mean concentrations of THg (0.543 ± 0.237 μg/g, wet weight, ww) and MeHg (0.518 ± 0.337 μg/g ww), while concentrations in P. hypophthalmus were approximately 30 times lower (≈0.02 µg/g ww). The estimated weekly intake (EWI) in children was above the provisional tolerable weekly intake (PTWI) of MeHg established by the Joint FAO/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) in 2007, 1.6 μg/kg body weight (bw) per week, for all the canned tuna brands. Values for adults were below PTWI, whereas for women of childbearing age, values were above PTWI only for brand D of canned tuna. The estimate of the potential risk indicated that MeHg levels in canned tuna can generate negative effects in vulnerable groups, while the EWI of fresh fish did not pose a threat to the general population. Therefore, establishing strategies to address the high consumption of canned tuna, and continuous monitoring to control commercial food, are recommended to decrease Hg exposure. [ABSTRACT FROM AUTHOR]

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), α-HCH and β-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods. [ABSTRACT FROM AUTHOR]

The suggested link between β- N -methylamino-L-alanine (BMAA) and the onset of neurodegenerative diseases and the detection of this cyanotoxin in aquatic organisms has prompted research into the potential human exposure risk associated with sourcing food items from eutrophied water bodies worldwide. The Hartbeespoort Dam reservoir in the North West province of South Africa has persistent cyanobacterial blooms and is used extensively by anglers, many of whom consume their catch. The commercial sale of fish species harvested from this reservoir as part of a recent biomanipulative remediation strategy may pose an additional hazard. BMAA and Microcystins (MC) were detected in fish sourced from this reservoir. BMAA levels of up to 1630 ng g −1 dry weight and MC concentrations of up to 29.44 ng g −1 dry weight were detected in fish sourced during an extensive bloom episode, with a clear correlation between the total amount of BMAA detected in the fish muscle tissue and their relative position in the Hartbeespoort Dam reservoir food web. Interestingly, fish sourced from this reservoir in winter when dense cyanobacterial blooms were lacking contained BMAA levels of up to 3055 ng g −1 dry weight. We also comment on the observed seasonal variations of BMAA levels in phytoplankton and fish sourced from this water body as well as the potential exposure risks associated with harvesting food items from this reservoir. [ABSTRACT FROM AUTHOR]

The effects of emerging environmental contaminants on human and environmental health is of high concern, especially those potentially induced by mixtures. The main goal of the present study was to assess the chronic effects of mixtures of citrate stabilized ≈5 nm gold nanoparticles (AuNP) and 1–5 μm microplastics (MP) on Daphnia magna . A 21-day bioassay was carried out. The effect criteria were parental mortality, somatic growth and several reproductive parameters. AuNP induced parental mortality, reduced the total offspring and caused immobile juveniles and aborted eggs. MP induced parental mortality, delayed the first brood release, decreased the number of broods released, the total offspring, and caused immobile juveniles. All the mixtures caused higher toxicity than AuNP and MP alone. Based on parental mortality, evidences of antagonism between AuNP and MP were observed at low concentrations of both mixture components, whereas evidences of synergism at high concentrations were found. Chronic (21-day) exposure of D. magna to AuNPs, MP, and their mixtures can impair development, reproduction, ultimately leading to death. [ABSTRACT FROM AUTHOR]

In this work a sample treatment based on a modified QuEChERS method combined with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was proposed to determine the residues of five common tetracyclines in fish muscle samples. The separation was achieved in less than 4 min and the analytes were detected in electrospray ionization in positive mode (ESI+) with multiple reaction monitoring mode. Parameters affecting the extraction step, such as the amount of sample and EDTA-McIlvaine buffer and extraction solvent volumes, were optimized by means of experimental design. In order to obtain the lowest matrix effect, parameters affecting the clean-up step by dispersive solid phase extraction (dSPE), were also studied. Under optimum conditions, matrix effect was lower than │15│% in all cases. Limits of quantification were lower than 4.4 μg kg −1 for the compounds in the selected samples, being in compliance with the current legislation. The precision, expressed as relative standard deviation, was below 18.5% and the recoveries for fortified fish samples (salmon and panga) higher than 80%. These results revealed that the proposed method is simple, rapid, cheap and environmentally friendly, being successfully applicable for the determination of these residues in fish samples. [ABSTRACT FROM AUTHOR]

Human-driven environmental change has increased the occurrence of harmful cyanobacteria blooms in aquatic ecosystems. Concomitantly, exposure to microcystin (MC), a cyanobacterial toxin that can accumulate in animals, edible plants, and agricultural soils, has become a growing public health concern. For accurate estimation of health risks and timely monitoring, availability of reliable detection methods is imperative. Nonetheless, quantitative analysis of MCs in many types of biological and environmental samples has proven challenging because matrix interferences can hinder sample preparation and extraction procedures, leading to poor MC recovery. Herein, controlled experiments were conducted to enhance the use of ultra-performance liquid-chromatography tandem-mass spectrometry (UPLC–MS/MS) to recover MC-LR and MC-RR at a range of concentrations in seafood (fish), vegetables (lettuce), and environmental (soil) matrices. Although these experiments offer insight into detailed technical aspects of the MC homogenization and extraction process (i.e., sonication duration and centrifugation speed during homogenization; elution solvent to use during the final extraction), they centered on identifying the best (1) solvent system to use during homogenization (2–3 tested per matrix) and (2) single-phase extraction (SPE) column type (3 tested) to use for the final extraction. The best procedure consisted of the following, regardless of sample type: centrifugation speed = 4200 ×  g ; elution volume = 8 mL; elution solvent = 80% methanol; and SPE column type = hydrophilic–lipophilic balance (HLB), with carbon also being satisfactory for fish. For sonication, 2 min, 5 min, and 10 min were optimal for fish, lettuce, and soil matrices, respectively. Using the recommended HLB column, the solvent systems that led to the highest recovery of MCs were methanol:water:butanol for fish, methanol:water for lettuce, and EDTA-Na 4 P 2 O 7 for soils. Given that the recommended procedures resulted in average MC-LR and MC-RR recoveries that ranged 93 to 98%, their adoption for the preparation of samples with complex matrices before UPLC–MS/MS analysis is encouraged. [ABSTRACT FROM AUTHOR]

Abstract: The fish acute toxicity test method is foundational to aquatic toxicity testing strategies, yet the literature lacks a concise sample size assessment. Although various sources address sample size, historical precedent seems to play a larger role than objective measures. We present a novel and comprehensive quantification of the effect of sample size on estimation of the median lethal concentration (LC50), covering a wide range of scenarios. The results put into perspective the practical differences across a range of sample sizes, from n = 5/concentration up to n = 23/concentration. We also provide a framework for setting sample size guidance illustrating ways to quantify the performance of LC50 estimation, which can be used to set sample size guidance given reasonably difficult (or worst‐case) scenarios. There is a clear benefit to larger sample size studies: they reduce error in the determination of LC50s, and lead to more robust safe environmental concentration determinations, particularly in cases likely to be called worst‐case (shallow slope and true LC50 near the edges of the concentration range). Given that the use of well‐justified sample sizes is crucial to reducing uncertainty in toxicity estimates, these results lead us to recommend a reconsideration of the current de minimis 7/concentration sample size for critical studies (e.g., studies needed for a chemical registration, which are being tested for the first time, or involving difficult test substances). Environ Toxicol Chem 2018;37:1565–1578. © 2018 SETAC [ABSTRACT FROM AUTHOR]

Polychlorinated naphthalenes (PCNs) were introduced to market about a century ago and their production is thought to have ceased by the early 1980s. However, relatively limited knowledge exists on their abundance in the edible portion of a variety of Great Lakes fish to aid in understanding their potential risk to human consumers. We studied levels, patterns, trends and significance of PCNs in a total 470 fillet samples of 18 fish species collected from the Canadian waters of the Great Lakes between 2006 and 2013. A limited comparison of fillet and wholebody concentrations in Carp and Bullhead was also conducted. The ∑ PCN ranged from 0.006–6.7 ng/g wet weight (ww) and 0.15–190 ng/g lipid weight (lw) with the dominant congeners being PCN-52/60 (34%), -42 (21%) and -66/67 (15%). The concentrations spatially varied in the order of the Detroit River > Lakes Erie > Ontario > Huron > Superior. PCN-66/67 was the dominating congener contributing on average 76–80% of toxic equivalent concentration (TEQ PCN ). Contribution of TEQ PCN to TEQ Total (TEQ Dioxins + Furans + dioxin-likePCBs + PCNs ) was mostly < 15%, especially at higher TEQ Total, and PCB-126 remains the major congener contributing to TEQ Total . The congener pattern suggests that impurities in PCB formulations and thereby historical PCB contamination, instead of unintentional releases from industrial thermal processes, could be an important source of PCNs in Great Lakes fish. A limited temporal change analysis indicated declines in the levels of PCN-66/67 between 2006 and 2012, complemented by previously reported decrease in PCNs in Lake Ontario Lake Trout between 1979 and 2004. The whole body concentrations were 1.4–3.2 fold higher than the corresponding fillets of Carp and Bullhead. Overall, the study results suggest that only targeted monitoring of PCNs in Great Lakes fish, especially at the Detroit River, Lake Erie and Lake Ontario, is necessary to assess continued future improvements of this group of contaminants of concern. [ABSTRACT FROM AUTHOR]

Abstract: Offspring of wild adult brown trout exposed to a range of Se concentrations were reared in a laboratory setting to primarily assess effects on survival and deformities. Maternal whole‐body Se concentrations ranged from 4.7 to 22.6 mg/kg dry weight for wild fish. Corresponding Se concentrations in embryos ranged from 6.2 to 40.3 mg/kg dry weight. Significant relationships were found between embryo and whole‐body tissue concentrations. Increasing egg Se concentrations were correlated with decreasing survival; however, hatch success was not significantly correlated with increasing embryo Se. The best fit effect concentration, 10% (EC10) for survival in the hatch to swim‐up period was 20.6 mg/kg dry weight, and the EC10 for hatch to test termination at 88 d was 20.5 mg/kg dry weight egg Se. The best fit model for deformities was based on a baseline‐adjusted severity index and resulted in an EC10 of 21.8 mg/kg dry weight egg Se. Both the best fit model EC10s represent more sensitive values than the published range of trout species EC10s. An egg to whole‐body tissue conversion factor derived from the paired data resulted in a conversion factor for brown trout of 1.46, which resulted in a whole‐body tissue EC10 of 14.04 mg/kg dry weight at an egg tissue EC10 of 20.5 mg/kg dry weight. Environ Toxicol Chem 2018;37:1396–1408. © 2018 SETAC [ABSTRACT FROM AUTHOR]

Notothenioid fish and invertebrate samples from Antarctica were collected in the austral summer of 2009, and analyzed for persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs), and polybrominated diphenylethers (PBDEs), as well as δ13C and δ15N stable isotopes for trophic level determination. In this study, the POP levels in the Antarctic biota samples were found to be ranked in the following order: OCPs > PAHs >> PBDEs. The POP levels in notothenioid fish and krill correlate to trophic levels; however, the POP concentrations in intertidal benthic invertebrates are higher than in notothenioid fish implying that specific biogeochemical factors may affect bioaccumulation in the Antarctica ecosystem. Biomagnification of POPs may have a smaller role than bioconcentration in Antarctica environment. In addition to the source, transport, exposure, and absorption for each group of POPs in the short food chain in Antarctica, the biological variation among species, interaction habitats, diet and metabolism are also factors for future studies on contaminant bioaccumulation. [ABSTRACT FROM AUTHOR]

As the worldwide application of silver nanomaterials in commercial products increases every year, and concern about the environmental risks of such nanoparticles also grows. A clear understanding of how different characteristics of nanoparticles contribute in their toxic behavior to organisms are imperative for predicting and control nanotoxicity. Within our research, we investigated the toxic effect of two types of silver nanoparticles (spherical and flat Ag nanoparticles) on zebrafish ( Danio rerio ) embryos. Particular interest was paid to proper characterization of Ag nanoparticles initially and during the experiment. A proper test medium was found and used for ecotoxicity evaluation. The behavior of flat silver nanoparticles with respect to embryos of zebrafish was analyzed and compared to the ecotoxicity of silver ionic form (AgNO 3 ). Both types of nanoparticles showed a more pronounced toxic effect to Danio rerio embryos than silver ions (AgNO 3 ), while silver nanoplates were more harmful than Ag nanospheres. While previous investigations showed that toxicity of Ag nanoparticles can be explained by the presence of Ag + in solution of silver nanoparticles, our results demonstrate that the harmful effects of nanosilver may be associated with silver nanoparticles themselves than with ionic silver released into solution. [ABSTRACT FROM AUTHOR]

Ill-defined, multi-component mixtures of steroidal pharmaceuticals are present in the aquatic environment. Fish are extremely sensitive to some of these steroids. It is important to know how fish respond to these mixtures, and from that knowledge develop methodology that enables accurate prediction of those responses. To provide some of the data required to reach this objective, pairs of fish were first exposed to five different synthetic steroidal pharmaceuticals (one estrogen, EE2; one androgen, trenbolone; one glucocorticoid, beclomethasone dipropionate; and two progestogens, desogestrel and levonorgestrel) and concentration-response data on egg production obtained. Based on those concentration-response relationships, a five component mixture was designed and tested twice. Very similar effects were observed in the two experiments. The mixture inhibited egg production in an additive manner predicted better by the model of Independent Action than that of Concentration Addition. Our data provide a reference case for independent action in an in vivo model. A significant combined effect was observed when each steroidal pharmaceutical in the mixture was present at a concentration which on its own would produce no statistically significant effect (something from ‘nothing’). Further, when each component was present in the mixture at a concentration expected to inhibit egg production by between 18% (Beclomethasone diproprionate) and 40% (trenbolone), this mixture almost completely inhibited egg production: a phenomenon we term ‘a lot from a little’. The results from this proof-of-principle study suggest that multiple steroids present in the aquatic environment can be analysed for their potential combined environmental risk. [ABSTRACT FROM AUTHOR]

Background: We have come across the plant secondary metabolites having pesticidal properties being used in the form of pesticides. In this study, we used one of newly available and plant metabolite used as pesticide in wide agricultural fields - Karanjin, obtained from seeds of plant Pongamia pinnata. Methods: The study was conducted during the month of March 2016 at the Department of Zoology, Osmania University. The fish common carp- Cyprinus carpio was exposed to sub-lethal concentration of karanjin i.e., 1/10th of 96 h LC50 value (0.28 ppm) for a period of 21 d. Moreover, at 24 h, 7 d, 14 d and 21 d, the peripheral blood of both control and exposed group fishes were studied for the presences of micronuclei and other nuclear abnormalities. Results: The micronuclei were completely absent, i.e., were not seen during the sub-lethal exposure period. There were seen nuclear abnormalities such as blebbed nuclei (BN), notched nuclei (NN), differently shaped nuclei (DSN), pear-shaped nuclei (PSN), circular nuclei (CN), lobed nuclei (LN) and Karyolysed nuclei (KN). The control group showed few nuclear abnormalities. Conclusion: During the sub-lethal exposure, as the days of exposure increased, the types of aberrations and their number also increased. [ABSTRACT FROM AUTHOR]

Abstract: In 2013 the Organisation for Economic Co‐operation and Development (OECD) test guideline (236) for fish embryo acute toxicity (FET) was adopted. It determines the acute toxicity of chemicals to embryonic fish. Previous studies show a good correlation of FET with the standard acute fish toxicity (AFT) test; however, the potential of the FET test to predict AFT, which is required by the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) and the Classification, Labelling and Packaging (CLP) Regulation (EC 1272/2008), has not yet been fully clarified. In 2015 the European Chemicals Agency (ECHA) requested that a consultant perform a scientific analysis of the applicability of FET to predict AFT. The purpose was to compare the toxicity of substances to fish embryos and to adult fish, and to investigate whether certain factors (e.g., physicochemical properties, modes of action, or chemical structures) could be used to define the applicability boundaries of the FET test. Given the limited data availability, the analysis focused on organic substances. The present critical review summarizes the main findings and discusses regulatory application of the FET test under REACH. Given some limitations (e.g., neurotoxic mode of action) and/or remaining uncertainties (e.g., deviation of some narcotic substances), it has been found that the FET test alone is currently not sufficient to meet the essential information on AFT as required by the REACH regulation. However, the test may be used within weight‐of‐evidence approaches together with other independent, relevant, and reliable sources of information. The present review also discusses further research needs that may overcome the remaining uncertainties and help to increase acceptance of FET as a replacement for AFT in the future. For example, an increase in the availability of data generated according to OECD test guideline 236 may provide evidence of a higher predictive power of the test. Environ Toxicol Chem 2018;37:657–670. © 2017 SETAC [ABSTRACT FROM AUTHOR]

Abstract: Testing for acute fish toxicity is an integral part of the environmental safety assessment of chemicals. A true replacement of primary fish tissue was recently proposed using cell viability in a fish gill cell line (RTgill‐W1) as a means of predicting acute toxicity, showing good predictivity on 35 chemicals. To promote regulatory acceptance, the predictivity and applicability domain of novel tests need to be carefully evaluated on chemicals with existing high‐quality in vivo data. We applied the RTgill‐W1 cell assay to 38 fragrance chemicals with a wide range of both physicochemical properties and median lethal concentration (LC50) values and representing a diverse range of chemistries. A strong correlation (R2 = 0.90–0.94) between the logarithmic in vivo LC50 values, based on fish mortality, and the logarithmic in vitro median effect concentration (EC50) values based on cell viability was observed. A leave‐one‐out analysis illustrates a median under‐/overprediction from in vitro EC50 values to in vivo LC50 values by a factor of 1.5. This assay offers a simple, accurate, and reliable alternative to in vivo acute fish toxicity testing for chemicals, presumably acting mainly by a narcotic mode of action. Furthermore, the present study provides validation of the predictivity of the RTgill‐W1 assay on a completely independent set of chemicals that had not been previously tested and indicates that fragrance chemicals are clearly within the applicability domain. Environ Toxicol Chem 2018;37:931–941. © 2017 SETAC [ABSTRACT FROM AUTHOR]

The levels of 20 metals (aluminum, boron, barium, calcium, cadmium, cobalt, chromium, copper, iron, potassium, lithium, magnesium, manganese, molybdenum, sodium, nickel, lead, strontium, vanadium, and zinc) were analyzed in muscle and liver tissue of Trachurus picturatus marketed in the Canary Islands (Spain) by using inductively coupled plasma optical emission spectrometry. In the liver samples, the mean concentrations in milligrams per kilogram wet weight (wt) of Al (14.7), B (0.99), Ba (1.64), Ca (314), Cd (2.52), Co (0.15), Cu (4.07), Fe (106), Li (3.89), Mn (0.85), Mo (0.16), Na (1510), Ni (0.51), Pb (0.36), Sr (3.54), V (0.78), and Zn (23.13) were higher than those detected in the muscle samples in milligrams per killogram wet wt, which were as follows: Al (8.76), B (0.07), Ba (0.30), Ca (210), Cd (0.01), Co (0.01), Cu (1.51 ), Fe (7.33), Li (1.08), Mn (0.12), Mo (0.01), Na (697), Ni (0.11), Pb (0.04), Sr (1.45), V (0.01), and Zn (4.69). The mean concentrations of Cr, K, and Mg (0.14, 1,904, and 243 mg/kg wet wt, respectively) were higher in muscle than in liver (0.05, 1,333 and 236 mg/kg wet wt, respectively). The mean concentrations of Cd and Pb (0.01 and 0.04 mg/kg wet wt) in muscle did not exceed the maximum limits established by a European Commission regulation (0.1 mg of Cd/kg and 0.3 mg of Pb/kg, respectively). Considering a mean daily consumption of fish muscle for the adult population of 31.9 g/day published in the report on food consumption by the Spanish Ministry of Agriculture and Fisheries, Food and Environment, Mg made the highest contributions to the intake (2.58% for adult women of 60 kg and 2.22% for men of 70 kg), and the estimated intakes of Al (0.35 to 0.46 mg/day), Cd (0.55 to 0.74 mg/day) and Pb (1.66 to 5.53 mg/day) were below the respective established tolerable intakes. In conclusion, the results of this study show that the consumption of muscle from this benthopelagic species can be considered safe in terms of maximum legal limits, while consumption of liver is discouraged as a major source of exposure to toxic metals, such as Al, Cd, and Pb. [ABSTRACT FROM AUTHOR]

Our research reports polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (CP-PCBs) concentrations and age-corrected trends for lake trout and walleye in the Great Lakes over the 2004-2014 period. We determined that age -- contaminant corrections are required to accurately report contaminant trends due to significant lake trout age structure changes. The age-trend model (ATM) described here uses a lake-specific age -- contaminant regression to mitigate the effect of a fluctuating lake trout age structure to directly improve the log -- linear regression model. ATM results indicate that half-life (t1/2) and percent decreases for PCDD/Fs, CP-PCBs, and toxic equivalence (TEQ) (average -56 to 70%) were fairly uniform and consistent across the Great Lakes over the 2004-2014 period. The vast majority of TEQ. associated with all Great Lakes lake trout and walleye samples is due to the nonortho CP-PCBs (average = 79%) as compared with PCDD/Fs (average = 21%). On average, CP-PCB_126 individually accounted for over 95% of the total CP-PCB TEQ. A retrospective analysis (1977-2014) of 2378-TCDF and 2378-TCDD raw concentrations in Lake Ontario lake trout revealed decreases of 94% and 96%, respectively. Tissue residue guidelines for wildlife protection based on lake trout and walleye total TEQ. were uniformly exceeded in all the Great Lakes. [ABSTRACT FROM AUTHOR]

Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there is 2- to 27-fold variation in toxicity within species, there was a 1740-fold difference between the least and most toxic species. Consequently, production of CTX or CTX-like compounds is more dependent on the species present than on the random occurrence of high or low toxicity strains. Seven of the eight species tested (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, Gambierdiscus ribotype 2, G. silvae and F. ruetzleri) exhibited low toxicities, ranging from 0 to 24.5 fg CTX3C equivalents cell-1, relative to G. excentricus, which had a toxicity of 469 fg CTX3C eq. cell-1. Isolates of G. excentricus from other regions have shown similarly high toxicities. If the hypothesis that G. excentricus is the primary source of ciguatoxins in the Atlantic is confirmed, it should be possible to identify areas where CFP risk is greatest by monitoring only G. excentricus abundance using species-specific molecular assays. [ABSTRACT FROM AUTHOR]

Amberjack Seriola dumerili (Carangidae) and yellowtail S. quinqueradiata are important commercial fish species in Japan; however, they are susceptible to skin and gill monogeneans. Recently, a treatment of 75 ppm hydrogen peroxide (H 2 O 2 ) for 30 min was found to effectively eliminate these monogeneans. However, histological analyses of the host fish subjected to treatments with lower H 2 O 2 concentrations have not been performed to determine the influence of these treatments on fish. Therefore, we investigated the influence of a 100 ppm H 2 O 2 treatment at 25 °C on the survival, swimming behavior, feeding behavior and the histopathology of S. dumerili and S. quinqueradiata compared with the 300 ppm treatment at 25 °C, which is the conventional concentration. We then evaluated the susceptibility of both species to infestations of the skin monogenean Neobenedenia girellae after the administration of a 75 ppm H 2 O 2 bath treatment relative to the susceptibility after the administration of the conventional procedure (300 ppm for 3 or 4 min) at 25 °C. Both species suffered severe effects when exposed to 300 ppm H 2 O 2 ; however, when exposed to 100 ppm H 2 O 2 for 60 min, the survival, appetite, and swimming behavior were unaffected. Histopathological changes were not observed during the examination of the skin, gills, and internal organs of the fish in the control group and the group treated with 100 ppm H 2 O 2 for 60 min, whereas histopathological changes were observed in the skin, gills and liver of the fish treated with 300 ppm H 2 O 2 for 60 min, and the changes in S. dumerili became more pronounced with increasing exposure time. Furthermore, the fish treated with 75 ppm H 2 O 2 baths for 30 or 40 min did not show increased N. girellae infestation levels relative to the untreated controls; however, the fish treated with the 300 ppm H 2 O 2 baths for 4 min showed significantly increased N. girellae infestation levels relative to the untreated controls ( P < 0.05). These results show that treating fish with 75 ppm H 2 O 2 for 30 min, which effectively eliminates the monogeneans infecting S. dumerili , would be presumably safe for the use in the higher water temperatures; however, the exposure time at higher H 2 O 2 concentrations, i.e., 300 ppm, must be strictly managed. Statement of relevance To eliminate B. seriolae and N. girellae , Z. japonica , fish may be fed praziquantel-supplemented feed, submerged in a freshwater bath for 2- to 5- min, or placed in a hydrogen peroxide bath. However, none of these measures simultaneously dislodge B. seriolae , N. girellae , and Z. japonica . Orally administered praziquantel is ineffective against N. girellae infestations in Seriola species, and the freshwater treatment is not effective when the fish are infected with Z. japonica . Additionally, H 2 O 2 treatment at a concentration of 300 ppm (conventional concentration in Japan) negatively affects fish health and viability, especially in the summer when water temperatures are higher and the parasite doubling and maturation rates are increase. Thus, any parasite control measure must be performed frequently during the summer. A bath treatment with 75 ppm H 2 O 2 for 30 min effectively eliminates these parasites and the fish survival, appetite and swimming behavior were unaffected when exposed to 75 ppm H 2 O 2 for 6 h at 25 °C. Therefore, treatment with 75 ppm H 2 O 2 for 30 min is not expected to adversely affect S. dumerili . However, histological analyses of lower H 2 O 2 concentrations have not been performed to determine the effects on the host fish, and experimental proof of the safety of different concentrations is important for the use of this treatment in aquaculture. [ABSTRACT FROM AUTHOR]

The present study reports total mercury (THg) and methylmercury (MeHg) concentrations in 32 different lean fish species from the Western Mediterranean Sea, with a special focus on the Balearic Islands. The concentrations of THg ranged between 0.05 mg/kg ww and 3.1 mg/kg ww (mean 0.41 mg/kg ww). A considerable number of the most frequently fish species consumed by the Spanish population exceed the maximum levels proposed by the European legislation when they originate from the Mediterranean Sea, such as dusky grouper (100% of the examined specimens), common dentex (65%), conger (45%), common sole (38%), hake (26%) and angler (15%), among others. The estimated weekly intakes (EWI) in children (7–12 years of age) and adults from the Spanish population (2.7 µg/kg bw and 2.1 µg/kg bw, respectively) for population only consuming Mediterranean fish were below the provisional tolerable weekly intake (PTWI) of THg established by EFSA in 2012, 4 µg/kg bw. However, the equivalent estimations for methylmercury, involving PTWI of 1.3 µg/kg bw, were two times higher in children and above 50% in adults. For hake, sole, angler and dusky grouper, the most frequently consumed fish, the estimated weekly intakes in both children and adults were below the maximum levels accepted. These intakes correspond to maximum potential estimations because fish from non-Mediterranean origin is often consumed by the Spanish population including the one from the Balearic Islands. [ABSTRACT FROM AUTHOR]

Exposure to contaminants in fish may be associated with adverse health outcomes even as fish consumption is generally considered beneficial. Risk assessments conducted to support regulatory analyses rely on quantitative fish consumption estimates. Here we report the results of a national survey of high-frequency fish consumers (n = 2099) based on a survey population statistically representative of ~17.6 million U.S. individuals consuming three or more fish meals per week. The survey was conducted during 2013 using an on-line survey instrument. Total fish consumption averaged 111 g/day from market, restaurant and self-caught sources. Depending on the season, the incidence of individuals reporting consumption of self-caught species ranged between 10–12% of our high-frequency fish consuming demographic, averaging approximately 30 g/day and comprising 23% of total fish consumption from all sources of fish. Recreational or self-caught consumption rates vary regionally and are poorly understood, particularly for high-frequency consumers, making it difficult to support national-scale assessments. A divergence between sport-fishing and harvesting of fish as a food-staple is apparent in survey results given differences in consumption patterns with income and education. Highest consumption rates were reported for low income respondents more likely to harvest fish as a food staple. By contrast, the incidence of self-caught fish consumption was higher with income and education although overall consumption rates were lower. Regional differences were evident, with respondents from the East-South Central and New England regions reporting lowest consumption rates from self-caught fish on the order of 12–16 g/day and those from Mountain, Pacific and Mid-Atlantic regions reporting highest rates ranging from 44 to 59 g/day. Respondent-specific consumption rates together with national-level data on fish tissue concentrations of PCBs, MeHg, and PFOS suggest that 10–58% of respondents reporting self-caught fish consumption are exposed to concentrations of these contaminants that exceed threshold levels for health effects based on a target hazard index of one, representing 2.3 M to 19 M individuals. The results of this nationwide survey of high-frequency fish consumers highlights regional and demographic differences in self-caught and total fish consumption useful for policy analysis with implications for distributional differences in potential health impacts in the context of both contaminant exposures as well as protective effects. [ABSTRACT FROM AUTHOR]

Fish are known to respond to a wide range of irritant chemicals, displaying clear behavioural changes after exposure to potentially noxious stimuli. Recent evidence shows that these agents can have an impact on larval forms of fish. However, very little information is available on the effects of carbon dioxide (CO 2 ) on these stages. Therefore, five days post-fertilisation zebrafish were exposed to a preparation containing CO 2 (10% soda water, 7.53 mmol/l CO 2 ) for 10 min and the behavioural response of the fish (percentage time spent active) recorded. In addition, exposure to the analgesic compounds aspirin, lidocaine, morphine and flunixin via immersion was investigated. Larvae exhibited a significant reduction in activity from the pre- to the post-stimulation period when exposed to the soda water (reduced by 18.9%), whereas lidocaine 5 mg/l and morphine 48 mg/l ameliorated this response (reduced by 0.1 and 0.8%, respectively). These results show for the first time that CO 2 induced behavioural effects on larval fish that can be reduced using analgesics. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the levels of airborne biological agents, such as bacteria, fungi, endotoxin, and (1→3)-β-d-glucan in university fish toxicity laboratory every month for one year and assessed the associated environmental factors. A single-stage viable cascade impactor connected with a pump was used for culturable bacteria and fungi. An analysis of airborne endotoxin and (1→3)-β-d-glucan was performed using kinetic Limulus amebocyte lysate assays. Levels of culturable bacteria and fungi were the highest in summer, whereas levels of endotoxin and (1→3)-β-d-glucan were the highest in winter and spring, respectively. Human activity was correlated with culturable bacteria and fungi, and culturable fungi were also associated with culturable bacteria. Although additional studies based on advanced analyzing technology are required, simultaneous sampling with biomarkers of bacteria is required to further elucidate the characteristics of biological agents. [ABSTRACT FROM PUBLISHER]

Disinfection to protect human health occurs at drinking water and wastewater facilities through application of non-selective oxidants including chlorine. Oxidants also transform organic material and form disinfection by-products (DBPs), many of which are halogenated and cyto- and genotoxic. Only a handful of assays have been used to compare DBP toxicity, and researchers are unsure which DBP(s) drive the increased cancer risk associated with drinking chlorinated water. The most extensive data set employs an in vitro model cell, Chinese hamster ovary cells. Traditionally, most DBP research focuses on the threat to human health, but the effects on aquatic species exposed to DBPs in wastewater effluents remain ill defined. We present the developmental toxicity for 15 DBPs and a chlorinated wastewater to a model aquatic vertebrate, zebrafish. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides > acetic acids > acetonitriles ~ nitrosamines, which agrees well with previously published mammalian in vitro data. Di- and tri-halogenated acetonitriles were more toxic than their mono-halogenated analogues, and bromine- and iodine-substituted DBPs tended to be more toxic than chlorinated analogues. No zebrafish development effects were observed after exposure to undiluted or non-concentrated, chlorinated wastewater. We find zebrafish development to be a viable in vivo alternative or confirmatory assay to mammalian in vitro cell assays. [ABSTRACT FROM AUTHOR]

EU chemicals legislation requires the use of all available information for hazard and risk assessment before new tests on vertebrates are proposed or conducted. In this context, extrapolation approaches for avoiding chronic fish testing on the basis of existing data have been explored. Simple linear relationships and interspecies sensitivity ratios betweenDaphniaand fish were calculated and acute-to-chronic relationships and ratios were calculated for fish, taking into consideration the mode of action. The best fitted relationships for the prediction of chronic fish toxicity are obtained based on acute fish andDaphniadata. Chemicals acting by unspecific reactivity and non-polar narcosis give the strongest acute and chronicDaphnia-to-chronic fish relationship. With acute fish data, strong relationships are obtained for all mode of action.Daphniawas found to be more sensitive than fish to several aniline derivatives and pesticides acting through cholinesterase inhibition, and less sensitive than fish to known endocrine disruptors. Extreme (i.e. <1 or >100) interspecies sensitivity ratios were mainly evident for chemicals acting by polar narcosis and specific reactivity. The safety factor of 100 commonly applied in environmental risk assessment does not seem to be equally protective for each mode of action. [ABSTRACT FROM AUTHOR]

Moray eels (Gymnothorax species) from tropical waters have long been known to be high-risk species, and the consumption of particularly the viscera or ungutted eels can result in severe ciguatera (known as Gymnothorax or moray eel poisoning), characterized by prominent neurological features. In this review, the main objective was to describe the risk and severity of ciguatera caused by eating moray eels in different parts of the world. Moray eels can accumulate very high ciguatoxin (CTX) levels in the flesh and particularly the liver. Therefore, even the smaller ones can be toxic and the consumption of an average portion (particularly liver) can result in severe or fatal ciguatera. Moray eels (particularly when ungutted) must never be served in gatherings since they can cause mass poisoning because of their large sizes and high CTX levels. Apart from regulatory measures restricting or excluding access, the public should be repeatedly warned to avoid eating moray eels. [ABSTRACT FROM AUTHOR]

Background: Methylmercury (MeHg) is a highly toxic compound that traverses the blood–brain barrier with deleterious effects to the central nervous system. Exposure is generally through the ingestion of contaminated fish. Fish are a main source of MeHg. Goals and methods: The aim of this study was to determine the dependence of MeHg concentrations on fish species and age, the percentage of MeHg in total mercury (THg) and risk assessment depending on the size of fish. Assays of THg and MeHg were performed on the muscle tissues of 18 species of fish. Results: The investigations indicated there were differences in the mercury concentrations depending on fish size. THg and MeHg concentrations in the muscles of fish species that have a wide length distribution were strongly, positively correlated with fish length. However, concentrations of MeHg were strongly, positively correlated with those of THg in all the fish species investigated. Variation in the percentage share of MeHg in THg in the muscles of fish of large sizes was also noted within species, but this correlation was not noted in small-sized fish. The dose of MeHg in small-sized fish species was estimated and the risk posed to consumer health was assessed using mean MeHg concentrations determined for different fish species. Conclusions: For species of fish that occur within a wide length distribution, the dose of MeHg should be assessed separately in different length classes. Fish consumption of small-sized species poses no health risk. [ABSTRACT FROM AUTHOR]

In Australia, trace organic contaminants (TrOCs) and endocrine active compounds (EACs) have been detected in rivers impacted by sewage effluent, urban stormwater, agricultural and industrial inputs. It is unclear whether these chemicals are at concentrations that can elicit endocrine disruption in Australian fish species. In this study, native rainbowfish ( Melanotaenia fluviatilis ) and introduced invasive (but prevalent) mosquitofish ( Gambusia holbrooki ) were exposed to the individual compounds atrazine, estrone, bisphenol A, propylparaben and pyrimethanil, and mixtures of compounds including hormones and personal care products, industrial compounds, and pesticides at environmentally relevant concentrations. Vitellogenin (Vtg) protein and liver Vtg mRNA induction were used to assess the estrogenic potential of these compounds. Vtg expression was significantly affected in both species exposed to estrone at concentrations that leave little margin for safety (p < 0.001). Propylparaben caused a small but statistically significant 3× increase in Vtg protein levels (p = 0.035) in rainbowfish but at a concentration 40× higher than that measured in the environment, therefore propylparaben poses a low risk of inducing endocrine disruption in fish. Mixtures of pesticides and a mixture of hormones, pharmaceuticals, industrial compounds and pesticides induced a small but statistically significant increase in plasma Vtg in rainbowfish, but did not affect mosquitofish Vtg protein or mRNA expression. These results suggest that estrogenic activity represents a low risk to fish in most Australian rivers monitored to-date except for some species of fish at the most polluted sites. [ABSTRACT FROM AUTHOR]

Lethal concentration ( LC50) and sub-lethal effects of NO2-N on the endemic viviparous fishes Skiffia multipunctata (Pellegrin, 1911) ( SM; considered threatened under Mexican regulations) and Goodea atripinnis (Jordan, 1880) ( GA; without conservation status) were determined. LC50/96 hr values were 0.002 and 0.359 mg NO2-N/L for SM and GA, respectively. Sub-lethal exposure resulted in swelling of the gill epithelium, hyperplasia, fusion of the primary and secondary lamellae, erythrocyte pyknosis, and aneurysms with respiratory stress responses at 0.0012 and 0.244 mg NO2-N/L for SM and GA. The study demonstrates that sensitivity of both species is strongly related to the conservation status and acceptable nitrite concentrations, which are clearly below those permitted by the Mexican national water quality norm for wildlife preservation. Therefore, this norm is not adequate protection for these species, and where other threatened species may also be affected. Adequate criteria for nitrites in Mexican waters should include the data of all threatened endemic species. [ABSTRACT FROM AUTHOR]

Arsenic (As) is found in waters such as seawater, warm springs, groundwater, rivers, and lakes. In aquatic environments, As occurs as a mixture of arsenate and arsenite, with arsenate usually predominating. The unrestricted application of As pesticides, industrial activities, and mining operations has led to the global occurrence of soluble As above permissible levels of 0.010 mg/L. Continuous exposure of freshwater organisms including fish to low concentrations of As results in bioaccumulation, notably in liver and kidney. As a consequence As induces hyperglycemia, depletion of enzymatic activities, various acute and chronic toxicity, and immune system dysfunction. Here we review arsenic chemistry, the occurrence of arsenic in aquatic system, the transformation and metabolism of arsenic; arsenic bioaccumulation and bioconcentration; behavioral changes; and acute and other effects such as biochemical, immunotoxic, and cytogenotoxic effects on fish. [ABSTRACT FROM AUTHOR]

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10-100 μg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 μg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals. [ABSTRACT FROM AUTHOR]

The ichthyotoxic dinoflagellate Cochlodinium polykrikoides poses a significant threat to the Korean finfish aquaculture industry, thus calling for effective mitigation strategies. We here report for the first time on investigations to assess the efficacy of routine application of clay to a naturally occurring Cochlodinium polykrikoides bloom off Namhae Island in South Korea during August-September 2015. Applications of conventional Korean loess reduced Cochlodinium cell concentrations from a maximum of 4600 mL −1 to levels considered safe for aquaculture operations (<1000 cells mL −1 ). However, at the same time, two clay application episodes increased ichthyotoxicity in water samples (measured by the gill cell line RTgill-W1 assay) by up to 32% compared to non-treated areas. Simulated laboratory culture experiments identified clay mediated Cochlodinium cell lysis as the probable cause. Lysed cells maintained cytotoxicity for up to 48 h (gill cell viability reduced to 39 ± 7% after 2.5 h exposure), but application of a finely-milled Korean loess completely eliminated ichthyotoxicity. Our results suggest that the fish-killing mechanism of Cochlodinium is comprised of both a stable (up to 48 h) and a transient fraction (decay within minutes). The latter indicates potential involvement of reactive oxygen species (ROS). We offer recommendations to fine-tune existing clay application regimes in Korean waters by focusing not on Cochlodinium cell removal, but on the adsorptive properties of clays to eliminate ichthyotoxicity. [ABSTRACT FROM AUTHOR]

A fast, sensitive and environmental-friendly method was developed to determine 13 organochlorine pesticides in fish tissue through gas chromatography with electron capture detection. Sample was extracted initially by acetonitrile and then concentrated through dispersive liquid–liquid microextraction based on solidification of floating organic droplet (DLLME–SFO). Dispersive solid phase extraction (d-SPE) was not a separate step but integrated to DLLME-SFO procedure. In order to separate the d-SPE sorbent, water and organic phase well, a modification to the glass centrifuge tube was made to achieving high centrifugal speed. The optimized method was validated with recoveries ranging from 88.1% to 121.2% (with relative standard deviations <15%) at three spiked levels for all of the pesticides. Good linearity was achieved at seven concentration levels from 6.25 to 625 μg L −1 , which corresponded to 0.001–0.100 mg kg −1 in the sample. The limits of quantification and the limits of detection for 13 OCPs ranged from 1.94×10 −3 to 4.93×10 −3 mg kg −1 and 6.50×10 −4 to 1.58×10 −3 mg kg −1 , respectively. The method was successfully applied to analyze real fish samples. [ABSTRACT FROM AUTHOR]

Pufferfish, Lagocephalus sceleratus, L. suezensis and L. spadiceus, are widely distributed Lessepsian species along the Turkish and other eastern Mediterranean coasts. L. sceleratus is considered one of the worst invasive species in the Mediterranean Sea and recently caused human intoxication by accidental consumption. Therefore, methods to assess pufferfish toxicity and accurately identify toxic pufferfish are important for food safety. Firstly, 20 specimens of L. sceleratus were collected from the eastern Mediterranean coasts of Turkey. Tetrodotoxin (TTX) content was determined among different tissues (muscle, liver, gonad, kidney, intestine and skin) by liquid chromatography-tandem mass spectrometry. All of the specimens contained TTX. The highest TTX concentration was detected in the ovary (80.0 µg TTX/g), followed by the intestine (48.8 µg TTX/g), kidney (34.0 µg TTX/g) and liver (25.4 µg TTX/g). The flesh and testis contained detectable amounts of TTX (3.4 µg TTX/g and 2.6 µg TTX/g, respectively). TTX analogs were also detected in the tissues, with 4,9-anhydro-5,6,11-trideoxyTTX and 5,6,11-trideoxyTTX being the major analog. A species-specific PCR method was developed to identify three species of genus Lagocephalus based on the nicotinamide adenine dinucleotide dehydrogenase subunit 2 or cytochrome oxidase subunit I regions of the mitochondrial DNA of L. sceleratus, L. suezensis, and L. spadiceus. The designed primers amplified only DNA of target species and not those of other fish and shellfish species. The species-specific PCR method successfully identified these pufferfish species and could be applied to thermally treated products of the pufferfish with only 1 ng of extracted DNA. [ABSTRACT FROM AUTHOR]

We study the non-parametric estimation of a continuous distribution functionFbased on the partially rank-ordered set (PROS) sampling design. A PROS sampling design first selects a random sample from the underlying population and uses judgement ranking to rank them into partially ordered sets, without measuring the variable of interest. The final measurements are then obtained from one of the partially ordered sets. Considering an imperfect PROS sampling procedure, we first develop the empirical distribution function (EDF) estimator ofFand study its theoretical properties. Then, we consider the problem of estimatingF, where the underlying distribution is assumed to be symmetric. We also find a unique admissible estimator ofFwithin the class of nondecreasing step functions with jumps at observed values and show the inadmissibility of the EDF. In addition, we introduce a smooth estimator ofFand discuss its theoretical properties. Finally, we expand on various numerical illustrations of our results via several simulation studies and a real data application and show the advantages of PROS estimates over their counterparts under the simple random and ranked set sampling designs. [ABSTRACT FROM AUTHOR]

The present study reports the isolation of Clostridium perfringens from samples comprising of freshwater fish and fish products from Kolkata city of India, and determining their genotypes and antibiogram profile. A total of 102 samples consisting of intestinal and gill samples from fresh water fish (n=69) and fish products including fish pickles, fish curry and fried fish (n=33) were collected randomly from retail shops and restaurants. On cultural isolation and biochemical characterization, 24 (23.52%) samples [17 (24.63%) from fresh water fish (n=69) and 07 (21.21%) from fish products (n=33)] were presumptively identified as C. perfringens positive. The genotyping of the recovered C. perfringens isolates was done by amplifying species specific 16S rRNA gene and four major lethal toxin genes viz., alpha toxin gene (cpa), beta toxin gene (cpb), epsilon toxin gene (εtx) and iota toxin gene (ιtx). Apart from these, enterotoxin gene (cpe) and beta2 toxin gene (cpb2) were also targeted. In PCR assays, all the 24 (100%) isolates [17 (70.83%) from fresh water fish and 07 (29.17%) from fish products] were found to harbor species specific 16S rRNA and cpa toxin gene, however, 17 (70.83%) cpa positive isolates [12 (70.58%) from fresh water fish and 05 (71.42%) from fish products] were also found to harbor additional cpb2 toxin gene, while none of the isolates were found to be positive for cpb, εtx, ιtx and cpe genes. Based on these results, all the isolates were confirmed as C. perfringens type A (containing only alpha toxin gene). On antibiotic sensitivity testing, 76.47% of the isolates were found to be multidrug resistant with ciprofloxacin and amoxicillin/clavulanic acid as being the most sensitive drugs. Report regarding isolation and molecular characterization of C. perfringens from fish and fish products especially from Kolkata does not exist as well as from other regions of India are very scanty. The present findings suggest that fish may be considered as a potential source of C. perfringens type A infection to human populations through food chain and the high antibiotic resistance observed may pose serious public health concerns. Further detailed molecular epidemiological and antibiogram studies are suggested for designing and adapting appropriate prevention and control strategies for countering this important pathogen and its food-borne zoonotic concerns. [ABSTRACT FROM AUTHOR]

Long-term effect assessments in ecotoxicological investigations are important, yet there is a lack of suitable exposure systems for these experiments that can be used for riverine species. A cost-efficient miniature circulatory system was developed that was evaluated for its applicability in long-term exposures in 2 stream-dwelling species: brown trout ( Salmo trutta) and an amphipod ( Gammarus roeseli). In an egg-to-fry exposure of S. trutta, the toxicity of 2 reverse osmosis concentrates was investigated as examples. Control hatching rate of yolk sac fry was 75 ± 7% and thus complies with the Organisation for Economic Co-operation and Development validity criterion (≥66%). The reverse osmosis concentrates did not impair the hatching rate in any tested concentration. In G. roeseli, mortality rates remained below 20% during a 21-d cultivation, fulfilling the common validity criterion in ecotoxicological testing. Mortality was significantly lower when the species was fed with conditioned alder leaves instead of an artificial shrimp food. Finally, a toxicity test on G. roeseli using copper as the test substance revealed median lethal concentration (LC50) values of 156 μg/L after 96 h and 99 μg/L after 264 h, which is in line with literature findings using other accepted exposure units. In conclusion, the miniature circulatory system provides a novel and cost-efficient exposure system for long-term investigations on riverine species that may also be applicable for other species of fishes and macroinvertebrates. Environ Toxicol Chem 2016;35:2827-2833. © 2016 SETAC [ABSTRACT FROM AUTHOR]

The need for alternative approaches to the use of vertebrate animals for hazard assessment of chemicals and pollutants has become of increasing importance. It is now the first consideration when initiating a vertebrate ecotoxicity test, to ensure that unnecessary use of vertebrate organisms is minimized wherever possible. For some regulatory purposes, the use of vertebrate organisms for environmental risk assessments has been banned; in other situations, the number of organisms tested has been dramatically reduced or the severity of the procedure refined. However, there is still a long way to go to achieve a complete replacement of vertebrate organisms to generate environmental hazard data. The development of animal alternatives is based not just on ethical considerations but also on reducing the cost of performing vertebrate ecotoxicity tests and in some cases on providing better information aimed at improving environmental risk assessments. The present Focus article provides an overview of the considerable advances that have been made toward alternative approaches for ecotoxicity assessments over the last few decades. Environ Toxicol Chem 2016;35:2637-2646. © 2016 SETAC [ABSTRACT FROM AUTHOR]

Jellyfish sting has become a worldwide issue of critical concern to human health and safety in coastal areas in recent decades. Cyanea nozakii is one of the dominant blooming species and dangerous stingers in China. However, it remains unclear how many and what types of toxins are present in the venom. So, we used a combined transcriptomics and proteomics approach to investigate the venom composition of jellyfish C. nozakii . In total 4,608,524 Illumina valid reads were obtained to de novo assemble to 40,434 unigenes in the transcriptomics analysis. And, a total of 311,635 MS/MS spectra with 12,247 unique MS/MS spectra were generated to 1556 homologous proteins in the proteomics analysis. 174 potential toxin proteins were identified, with 27 proteins homology to the toxins from venomous animals, including phospholipase A2, zinc metalloproteinase-disintegrin agkistin, serine protease inhibitor, plancitoxin-1, alpha-latrocrustotoxin-Lt1a, etc. This study described the transcriptomics and venom proteomics of jellyfish C. nozakii for the first time. Our findings provide a comprehensive understanding of the venom composition of C. nozakii . Furthermore, the results may also be very helpful for the discovery of novel bioactive proteins, as well as the development of effective treatments for jellyfish sting in the future. Biological significance Jellyfish Cyanea nozakii is one of the most dangerous stingers in the coast of china. Hundreds of thousands of people would be stung every year and victims suffered a severe pain, itch, swelling, inflammation, wheal and even more serious consequence. However, it remains unclear how many and what types of toxins are present as well as the relationship between the clinical symptoms and toxins. Our combined transcriptomics and proteomics findings can provide a comprehensive understanding of the venom composition of C. nozakii and will also be helpful for the development of effective treatments for jellyfish sting in the future. [ABSTRACT FROM AUTHOR]