Fish Plasma Research Articles

Structural features of the low-molecular-weight plasma fraction in far eastern redfins of the genus Tribolodon and other cyprinid fishes

The organization of the low-molecular fraction (LMF) of proteins from fish plasma was studied in Far Eastern redfins of the genus Tribolodon and other representatives of the family Cyprinidae. The common principle of the organization of the plasma LMF was established. According to this principle, the LMF includes two subfractions, one of which consists of oligomeric and the other of monomeric proteins. During the pre-spawning period, a decrease in the apparent molecular weight of proteins as a result of changes in their oligomeric structure was observed in the former subfraction; a reduction of the heterogeneity of the proteins occurred in the latter one. The analysis of these rearrangements allows one to differentiate two main types of the LMF: “basic” and “plastic.” The former type is characterized by a low level of metabolic processes; the latter one, by their activation during the pre-spawning period. By using MALDI mass spectrometry, polymeric forms of apolipoproteins, fetuin, and albumin-like protein were identified within the oligomeric subfraction; hemopexin and inhibitors of proteinases were found within the monomeric subfraction.

Tissue-specific bioaccumulation of human and veterinary antibiotics in bile, plasma, liver and muscle tissues of wild fish from a highly urbanized region

We investigated the bioaccumulation of antibiotics in bile, plasma, liver and muscle tissues of wild fish from four rivers in the Pearl River Delta region. In total, 12 antibiotics were present in at least one type of fish tissues from nine wild fish species in the four rivers. The mean values of log bioaccumulation factors (log BAFs) for the detected antibiotics in fish bile, plasma, liver, and muscle tissues were at the range of 2.06–4.08, 1.85–3.47, 1.41–3.51, and 0.48–2.70, respectively. As the digestion tissues, fish bile, plasma, and liver showed strong bioaccumulation ability for some antibiotics, indicating a different bioaccumulation pattern from hydrophobic organic contaminants. Human health risk assessment based on potential fish consumption indicates that these antibiotics do not appear to pose an appreciable risk to human health. To the best of our knowledge, this is first report of bioaccumulation patterns of antibiotics in wild fish bile and plasma.

Exposure and body burden of polychlorinated biphenyls (PCB) and metals in a historically contaminated community

There are many small villages where environmental contamination is substantial due to historical industrial activities. The aim of the present study was to investigate if long-term or current consumption of local foods, as reported in food frequency questionnaires, co-vary with measured concentrations of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), lead (Pb), cadmium (Cd) and mercury (Hg) in blood, urine and hair from a population living in a historically contaminated village.Blood, urine and hair were provided by men (n=38) and women (n=57), who had participated in a previous case–control study in the contaminated area, and were analyzed for PCB, OCPs, Pb, Cd and Hg. A detailed food frequency questionnaire, used in the previous epidemiological study, was repeated, and up-dated information of life-style, exposure factors and other covariates was collected. Associations between reported consumption of local foods and exposure biomarkers were explored in relation to age, gender, life-style factors and other covariates.A large part of the population in the area reported consumption of local food, and thus, was potentially exposed to the contaminants. Despite the limited number of participants and other weaknesses described, it was possible to link reported consumption of different foods to biomarker concentrations.Reported consumption of local vegetables, forest berries and mushrooms co-varied with urinary Cd, indicating an influence from the contaminated area on the Cd exposure. We found no associations between PCB plasma concentrations with reported consumption of local fish, but with consumption of herring (non-local sea fish) which is typically high in PCB. Pesticide (HCB, p,p′-DDE, trans-nonachlor) exposure was mainly associated with agricultural work and having a private well the first five years of life, but we found no associations between pesticide concentrations in plasma and consumption of local vegetables or fish. Exposure to Hg was associated with consumption of fish, both local and non-local, and Pb exposure was associated with the consumption of game.Overall, the contaminant concentrations measured in blood, urine and hair varied substantially among study participants, but on average, the concentrations were similar to concentrations measured in other groups of the general Swedish population in the same age range. Larger studies are needed to evaluate health risks (and causality) associated with historical environmental contamination.

Application of solid phase microextraction for quantitation of polyunsaturated fatty acids in biological fluids.

Development of a straightforward strategy for simultaneous quantitative analysis of nonesterified fatty acids (NEFA) species in biofluids is a challenging task because of the extreme complexity of fatty acid distribution in biological matrices. In this study, we present a direct immersion solid phase microextraction method coupled to a liquid chromatography-mass spectrometry platform (DI-SPME- HPLC-ESI -MS) for determination of unconjugated fatty acids (FA) in fish and human plasma. The proposed method was fully validated according to bioanalytical method validation guidelines. The LOD and LOQ were in the range of 0.5-2 and 5-12 ng/mL, respectively, with a linear dynamic range of 100 fold for each compound. Absolute and relative matrix effects were comprehensively evaluated and found to be in the acceptable range of 91-116%. The affinity constant (Ka) of individual FAs to protein albumin was determined to be 9.2 × 10(4) to 4.3 × 10(5) M(-1). The plasma protein binding (PPB%) was calculated and found to be in the range of 98.0-99.7% for different polyunsaturated fatty acids (PUFAs). The PUFAs under study were found at a high concentration range in fish plasma, whereas only a few were within quantification range in control human plasma. The method was successfully applied for monitoring PUFA changes during the operation in plasma samples obtained from patients undergoing cardiac surgery with the use of cardiopulmonary bypass (CPB). The most significant contribution induced by surgery was noticed in the concentration level of α-linolenic acid (18:3, ALA), arachidonic acid (20:4, AA), and docosahexanoic acid (22:6, DHA) soon after administration of CPB in all cases.

Bioaccumulation and trophic dilution of human pharmaceuticals across trophic positions of an effluent-dependent wadeable stream.

Though pharmaceuticals are increasingly observed in a variety of organisms from coastal and inland aquatic systems, trophic transfer of pharmaceuticals in aquatic food webs have not been reported. In this study, bioaccumulation of select pharmaceuticals was investigated in a lower order effluent-dependent stream in central Texas, USA, using isotope dilution liquid chromatography-tandem mass spectrometry (MS). A fish plasma model, initially developed from laboratory studies, was tested to examine observed versus predicted internal dose of select pharmaceuticals. Pharmaceuticals accumulated to higher concentrations in invertebrates relative to fish; elevated concentrations of the antidepressant sertraline and its primary metabolite desmethylsertraline were observed in the Asian clam, Corbicula fluminea, and two unionid mussel species. Trophic positions were determined from stable isotopes (δ(15)N and δ(13)C) collected by isotope ratio-MS; a Bayesian mixing model was then used to estimate diet contributions towards top fish predators. Because diphenhydramine and carbamazepine were the only target compounds detected in all species examined, trophic magnification factors (TMFs) were derived to evaluate potential trophic transfer of both compounds. TMFs for diphenhydramine (0.38) and carbamazepine (1.17) indicated neither compound experienced trophic magnification, which suggests that inhalational and not dietary exposure represented the primary route of uptake by fish in this effluent-dependent stream.

Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals.

Human and veterinary drug development addresses absorption, distribution, metabolism, elimination and toxicology (ADMET) of the Active Pharmaceutical Ingredient (API) in the target species. Metabolism is an important factor in controlling circulating plasma and target tissue API concentrations and in generating metabolites which are more easily eliminated in bile, faeces and urine. The essential purpose of xenobiotic metabolism is to convert lipid-soluble, non-polar and non-excretable chemicals into water soluble, polar molecules that are readily excreted. Xenobiotic metabolism is classified into Phase I enzymatic reactions (which add or expose reactive functional groups on xenobiotic molecules), Phase II reactions (resulting in xenobiotic conjugation with large water-soluble, polar molecules) and Phase III cellular efflux transport processes. The human-fish plasma model provides a useful approach to understanding the pharmacokinetics of APIs (e.g. diclofenac, ibuprofen and propranolol) in freshwater fish, where gill and liver metabolism of APIs have been shown to be of importance. By contrast, wildlife species with low metabolic competency may exhibit zero-order metabolic (pharmacokinetic) profiles and thus high API toxicity, as in the case of diclofenac and the dramatic decline of vulture populations across the Indian subcontinent. A similar threat looms for African Cape Griffon vultures exposed to ketoprofen and meloxicam, recent studies indicating toxicity relates to zero-order metabolism (suggesting P450 Phase I enzyme system or Phase II glucuronidation deficiencies). While all aspects of ADMET are important in toxicity evaluations, these observations demonstrate the importance of methods for predicting API comparative metabolism as a central part of environmental risk assessment.

Quantitative cross-species extrapolation between humans and fish: the case of the anti-depressant fluoxetine.

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.

Exposure and food web transfer of pharmaceuticals in ospreys (Pandion haliaetus): Predictive model and empirical data.

The osprey (Pandion haliaetus) is a well-known sentinel of environmental contamination, yet no studies have traced pharmaceuticals through the water-fish-osprey food web. A screening-level exposure assessment was used to evaluate the bioaccumulation potential of 113 pharmaceuticals and metabolites, and an artificial sweetener in this food web. Hypothetical concentrations in water reflecting "wastewater effluent dominated" or "dilution dominated" scenarios were combined with pH-specific bioconcentration factors (BCFs) to predict uptake in fish. Residues in fish and osprey food intake rate were used to calculate the daily intake (DI) of compounds by an adult female osprey. Fourteen pharmaceuticals and a drug metabolite with a BCF greater than 100 and a DI greater than 20 µg/kg were identified as being most likely to exceed the adult human therapeutic dose (HTD). These 15 compounds were also evaluated in a 40 day cumulative dose exposure scenario using first-order kinetics to account for uptake and elimination. Assuming comparable absorption to humans, the half-lives (t1/2 ) for an adult osprey to reach the HTD within 40 days were calculated. For 3 of these pharmaceuticals, the estimated t1/2 in ospreys was less than that for humans, and thus an osprey might theoretically reach or exceed the HTD in 3 to 7 days. To complement the exposure model, 24 compounds were quantified in water, fish plasma, and osprey nestling plasma from 7 potentially impaired locations in Chesapeake Bay. Of the 18 analytes detected in water, 8 were found in fish plasma, but only 1 in osprey plasma (the antihypertensive diltiazem). Compared to diltiazem detection rate and concentrations in water (10/12 detects, <method detection limits [MDL]-173 ng/L), there was a lower detection frequency in fish (31/233 detects, <MDL-2400 ng/L); however when present in fish, all values exceeded the maximum diltiazem concentration found in water. Diltiazem was found in all 69 osprey plasma samples (540-8630 ng/L), with 41% of these samples exceeding maximum concentrations found in fish. Diltiazem levels in fish and osprey plasma were below the human therapeutic plasma concentration (30000 ng/L). Effect thresholds for diltiazem are unknown in ospreys at this time, and there is no evidence to suggest adverse effects. This screening-level exposure model can help identify those compounds that warrant further investigation in high-trophic level species.

Rice Distillers Dried Grain Is a Promising Ingredient as a Partial Replacement of Plant Origin Sources in the Diet for Juvenile Red Seabream (&lt;i&gt;Pagrus major&lt;/i&gt;)

This study was designed to test the effects of dietary distillers dried grain (DDG) level on the growth performance, feed utilization, body composition and antioxidant activity of juvenile red seabream (Pagrus major). Six isonitrogenous and isocaloric diets were formulated to contain 0%, 5%, 10%, 15%, 20%, and 25% DDG from rice (designated as DDG0, DDG5, DDG10, DDG15, DDG20, and DDG25), respectively. Juvenile red seabream averaging 10.1±0.05 g were randomly distributed into 400-L tanks in a flow through systems. Three replicate groups of fish were fed one of the experimental diets to visual satiation two times a day for 10 weeks. Survival, weight gain, feed efficiency, protein efficiency ratio and hepatosomatic index of fish were not affected by dietary DDG levels (p>0.05). Proximate and amino acid composition of whole body in juvenile red seabream were not affected by dietary DDG levels (p>0.05). Plasma content of total protein, glucose, cholesterol, glutamic-pyruvic transaminase, phospholipid and triglyceride were not affected by dietary DDG levels (p>0.05). 1, 1-Diphenyl-2-picryl-hydrazyl radical and alkyl radical scavenging activities in plasma and liver of fish were not affected by dietary DDG levels (p>0.05). The results of this experiment suggest that DDG has the potential to replace plant origin ingredients such as wheat flour and corn gluten meal and could be used up to 25% in diet without incurring negative effects on the growth performance of juvenile red seabream.

Endocrine disrupting compounds affecting corticosteroid signaling pathways in Czech and Swiss waters: potential impact on fish.

This study investigated the occurrence of corticosteroid signaling disruptors in wastewaters and rivers in the Czech Republic and in Switzerland. 36 target compounds were detected using HPLC-MS/MS, with up to 6.4 μg/L for azole antifungals that indirectly affect corticosteroid signaling. Glucocorticoid receptor (GR)-mediated activity was determined using the GR-CALUX bioassay with dexamethasone equivalent concentrations ranging from <LOD-2.6, 19-37, and 78-542 ng/L for river water, treated, and untreated wastewater, respectively. For most samples, the chemically predicted GR-mediated response was higher than that determined by the bioassay. Correspondingly, antiglucocorticoid activity was observed in some fractions. The fish plasma model (FPM), which predicts plasma concentrations, was applied to evaluate the potential of detected pharmaceuticals to cause receptor-mediated effects in fish. With one exception, medroxyprogesterone, the FPM applied to individual compounds predicted fish plasma concentrations to be below the level of human therapeutic plasma concentrations. To account for the activity of the sum of GR-active compounds, we introduce the "cortisol equivalents fish plasma concentration" approach, through which an increase in fish glucocorticoid plasma levels comparable to 0.9-83 ng/mL cortisol after exposure to the analyzed river waters was estimated. The results suggest that these chemicals may impact wild fish.

Effects of low-voltage constant direct current on plasma biochemical profiles and gene expression levels in crucian carp Carassius carassius

This study investigated the plasma biochemical profiles and gene expression levels of crucian carp Carassius carassius subjected to anesthetization with low-voltage constant direct current (DC) and tricaine methanesulfonate (MS222). Fish in the MS222 treatments and the DC treatments were exposed to either two concentrations of MS222 (100 and 200 mg/l) or constant DC with a voltage of 26 V, at a voltage gradient of 0.68 V/cm, respectively. The results showed that low-voltage constant DC immobilized fish more quickly, resulting in significant elevations of plasma cortisol levels compared to exposure of high or low doses of MS222. Electronarcosis led to an increased expression of heat shock proteins 70 (HSP70) as well as HSP90 compared to the treatment group with MS222. Also, electronarcosis enhanced mitochondrial respiratory chain gene (atp6, cox1 and nd5) and antioxidant enzyme gene (CAT, SOD and GSH-px1) expression levels. In conclusion, low-voltage constant DC can disturb fish plasma biochemical profiles and gene expression levels.

Physiological and biochemical strategies for withstanding emersion in two galaxiid fishes

The galaxiid fishes of the Southern hemisphere display variable tolerance to aerial exposure. Brown mudfish (Neochanna apoda), for example, pseudoaestivate, inhabiting moist soil for months at a time, whereas inanga (Galaxias maculatus) emerse under unfavourable water conditions, but only for periods of a few hours. This study sought to identify the physiological and biochemical strategies that determine emersion tolerance in these species. Nitrogenous waste excretion was measured before and after an experimental emersion period (14days for mudfish, 6h for inanga). Both species showed significantly elevated ammonia “washout” upon return to water, but no increase in plasma or muscle ammonia. Post-emersion urea levels were elevated in plasma and muscle in both fish, however the extent of the accumulation did not indicate significant de novo urea production. This was supported by the lack of carbamoyl phosphate synthetase activity in tissues. Consequently, mudfish metabolism was examined to determine whether changes in parameters such as oxygen consumption, carbon dioxide excretion, and/or altered metabolic costs (represented by the key ionoregulatory enzyme Na+, K+-ATPase; NKA) could explain emersion tolerance. Oxygen consumption rates, already very low in immersed mudfish, were largely maintained over the course of emersion. Carbon dioxide excretion decreased during emersion, and a small, but significant, decrease in NKA was noted. These data suggest that the extended emersion capacity of mudfish may result from a generally low metabolic rate that is maintained throughout aerial exposure via cutaneous gas exchange, and which limits the production of potentially toxic nitrogenous waste.

Connections between dark fish intake, lipidomics and plasma triglycerides in the framingham heart study

Connections between dark fish intake, lipidomics and plasma triglycerides in the framingham heart study

Quantitative liquid chromatography-tandem mass spectrometry method for determination of microcystin-RR and its glutathione and cysteine conjugates in fish plasma and bile.

A rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of microcystin-RR (MC-RR) and its glutathione and cysteine conjugates (MC-RR-GSH and MC-RR-Cys, respectively) in fish plasma and bile. The analytes were extracted using methanol, followed by an Oasis mixed-mode cation-exchange polymeric sorbent. The separation was performed on a reversed-phase Waters XBridge C18 column with the gradient mobile phase, consisting of water and acetonitrile (both acidified with 0.5‰ formic acid). Mean recoveries of MC-RR, MC-RR-GSH and MC-RR-Cys ranged from 80.7 to 93.7%, 81.1 to 93.1% and 80.3 to 93.2%, respectively, at three concentrations (0.2, 1.0 and 5.0 μg mL(-1)). Limits of detection (LODs) for MC-RR, MC-RR-GSH and MC-RR-Cys were 6, 12 and 9 ng mL(-1), respectively. Limits of quantification (LOQs) were 15, 30 and 22.5 ng mL(-1) for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. This method makes it feasible for the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in limited and complex biological fluid samples (such as plasma and bile, typically 50 μL), which were previously excluded or difficult to study due to the relatively large sample volumes.

Biochemical Parameters of Common Carp ( Cyprinus carpio ) Exposed to Crude Leaf Extract of Cannabis Sativa

The effect of sub-lethal concentrations (1.88, 3.75, 7.50, 15.00 and 30.00 mg/L) of crude leaf extract of Cannabis sativa was determined in the plasma, liver and gill biochemical parameters of Common carp, Cyprinus carpio (mean weight of 15.05±0.05g) after 56-day exposure period in static renewable bioassay system. During the experiment, some physicochemical parameters were monitored while at the end of the experiment, the selected biochemical parameters were determined in the plasma, liver and gill of the test fish. The biochemical parameters determined were alkaline phosphatase (ALP), Lactate dehydrogenase (LDH), total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, total bilirubin (TB), albumin (ALB), urea acid (UA) and cholesterol (CHOL). The monitored pH showed significant difference (p 0.05) in C. sativa test set-ups compared to the control during the experimental period. There were significant difference (p<0.05) in some of the determined biochemical parameters of the test fish exposed to C. sativa compared to the control. Therefore, it can be deduce from the study that prolonged exposure of C. carpio fingerlings to crude leaf extract of C. sativa affected some determined biochemical parameters in the tissue/organs of the test fish.

An integrated approach for prioritizing pharmaceuticals found in the environment for risk assessment, monitoring and advanced research

Numerous active pharmaceutical ingredients (APIs), approved prior to enactment of detailed environmental risk assessment (ERA) guidance in the EU in 2006, have been detected in surface waters as a result of advancements in analytical technologies. Without adequate knowledge of the potential hazards these APIs may pose, assessing their environmental risk is challenging. As it would be impractical to commence hazard characterization and ERA en masse, several approaches to prioritizing substances for further attention have been published. Here, through the combination of three presentations given at a recent conference, “Pharmaceuticals in the Environment, Is there a problem?” (Nîmes, France, June 2013) we review several of these approaches, identify salient components, and present available techniques and tools that could facilitate a pragmatic, scientifically sound approach to prioritizing APIs for advanced study or ERA and, where warranted, fill critical data gaps through targeted, intelligent testing. We further present a modest proposal to facilitate future prioritization efforts and advanced research studies that incorporates mammalian pharmacology data (e.g., adverse outcomes pathways and the fish plasma model) and modeled exposure data based on pharmaceutical use.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Degradation, Residual Determination, and Cholinesterase Activity of Triclorfon in Piaractus mesopotamicus Holmberg (PACU) 1887

The aim of this study was to determine the no-observable-adverse-effect concentration (NOAEC) for trichlorfon, an antiparasitic agent used in aquaculture, in Piractus mesopotamicus (pacu) using acetylcholinesterase (AChE) activity as an end point. Fish were exposed 24 h/d for 15 d to different concentrations of trichlorfon in tanks of water for which a curve of dissipation was previously determined. Analysis of trichlorfon in water and fish plasma using gas chromatography with electron capture detection (GC-ECD) enabled measurement of limit of detection (LOD) and limit of quantification (LOQ), respectively, to be 3 and 10 ppb. Thirty-six hours after trichlorfon dilution in water, the concentration was below the LOD, and data showed that plasma concentrations did not exceed the LOQ. Apart from the 6.25 μg/L, all concentrations of trichlorfon significantly inhibited plasma and brain AChE activity compared to controls. The AChE activity levels returned to control values in 7 d. These data may be useful to determine the concentration of trichlorfon that destroys parasites without producing adverse effects in fish.

Investigating the Utility of Measuring 11α‐Ketotestosterone and Vitellogenin in Surface Mucus as an Alternative to Plasma Samples in Assessments of the Reproductive Axis of White Bass

AbstractSex steroids and vitellogenin measured in plasma have been used in a variety of fish species to investigate the reproductive system. Surface mucus has been shown to be an effective substitute for plasma in several species of fish. The goal of this study was to investigate the utility of mucus sampling as an approach to investigating the hypothalamic–pituitary–gonadal (HPG) axis in White Bass Morone chrysops. We measured 11α‐ketotestosterone and vitellogenin in both surface mucus and plasma samples from White Bass collected from a wild population during their annual spawning migration. Mucus and plasma samples were collected from male and female White Bass that were treated to stimulate the HPG axis (carp pituitary extract, 10 mg/kg; estradiol, 10 mg/kg) or treated with saline injections. Mucus and plasma concentrations of both vitellogenin and 11α‐ketotestosterone were positively correlated with one another. Treatments with estradiol led to higher amounts of vitellogenin in both plasma and mucus in both male and female fish. Assessments of 11α‐ketotestosterone in mucus correctly identified the sex of 90% of spawning White Bass, as did vitellogenin in 94%. These data demonstrate the potential utility of mucus sampling as a tool to less invasively assess the annual reproductive cycle, identify stimulation of the HPG axis, and identify fish sex in hatchery populations of White Bass.

Effect of prochloraz fungicide on neopterin and biopterin concentrations in blood plasma of common carp

Neopterin and biopterin are often used as markers of cell mediated immunity. The aim of this study was to evaluate the effect of prochloraz on neopterin and biopterin concentrations in blood plasma of common carp as possible indicators for monitoring perturbations of the immune system caused by xenobiotics like pesticides. Prochloraz is a widely used imidazole fungicide in horticulture and agriculture. A total of 60 juvenile common carp were divided into four groups of 15 fish and exposed to prochloraz at concentrations of 0, 50, 150 and 380 µg·l-1, respectively. The total length of exposure was 28 days. Neopterin and biopterin concentrations were determined by reverse phase high performance liquid chromatography with fluorescence detection. Our results indicated that concentrations of neopterin (25 ± 7.6 nmol·l-1) and biopterin (190 ± 29 nmol·l-1) in plasma of untreated common carp were comparable with those in mammals. Neopterin concentrations significantly (P &lt; 0.01) increased after exposure to prochloraz in comparison to non-exposed fish, while biopterin concentrations were not influenced by exposure to prochloraz. This study contains new data on neopterin and biopterin concentrations in fish plasma and investigates neopterin and biopterin in their non-traditional role as markers of cell mediated immunity of fish associated with pesticide exposure.