Evaluation of pesticides and metals in fish of the Dniester River, Moldova

The Dniester River is one of the largest rivers in Eastern Europe and receives input from numerous anthropogenic activities. In October 2001, fish samples (Abramis brama, Rutilus rutilus heckeli, Perca fluviatilis, Chondrostoma nasus, Carassius auratus gibelio, and Cyprinus carpio) were collected and analyzed for metals and various pesticides. Maximum total DDT pesticide concentrations were between 27.7 and 34.2 ng/g wet weight in muscles and gonads, respectively. DDE isomers comprised up to 70% of total DDT measurement in fish tissues, indicating historical rather than recent DDT pollution. Chlordane and lindane were also detected in amounts up to 37 and 76 ng/g wet weight in gonads, respectively. Among organophosphorous pesticides, malathion showed extremely high concentrations--3 microg/g wet weight in muscles and 46 microg/g wet weight in gonads, indicating recent exposure. Copper concentrations in muscles (1.8-7.2 microg/g) exceeded Threshold Effect Concentrations for copper in fish. Detection of high concentrations of malathion and copper in fish tissues indicated potential anthropogenic stress for more sensitive aquatic biota, likely from agricultural sources in the Dniester River.     

Assessment of copper and zinc levels in fish from freshwater ecosystems of Moldova

Purpose

The aim of this work was to assess the levels of copper and zinc in fish from the main freshwater ecosystems of Moldova, in relation with species, habitat, age, sex, season, and development stage.

Methods

Fish from Cyprinidae and Percidae families (Cyprinus carpio, Carassius auratus gibelio, Rutilus rutilus heckeli, Abramis brama, Aristichthys nobilis, Hypophtalmichthys molitrix, Sander lucioperca) were collected from Prut and Dniester rivers, Cuciurgan, Dubasari, and Costesti-Stanca reservoirs, and ponds of farms in the Dniester delta. The Cu and Zn content of fish tissues (skeletal muscles, liver, gonads, gills, skin, and scales) was determined by flame atomic absorption spectrophotometer AAS-3, of water by graphite furnace HGA 900 of AAnalist 400.

Results

The level of heavy metals accumulation in muscles of immature fish follows their dynamics in water. The highest concentration of zinc was registered in the gonads of mature fish, and of copper??in the liver. The lowest Cu and Zn contents were recorded in the muscles and are in the United Nations Food and Agriculture Organization safety-permissible levels for human consumption. Cu and Zn contents in muscles of fish depend on specimen age. Their level in fish gonads was sharply increasing during pre-spawning period. During the early developmental stages, the metal concentration in fish eggs and larvae varies within wide limits, but the accumulation pattern is similar in the investigated species.

Conclusions

The fish represent one of the most indicative factors for the estimation of trace metals pollution in freshwater systems and this is important not only for monitoring purposes, but also for the fish culture ones.     

Assessment of oxidative stress and metabolic changes in common carp (Cyprinus carpio) acutely exposed to different concentrations of the fungicide tebuconazole

The aim of this research is to determine the lethal concentration (LC(50-96 h)) of fungicide tebuconazole for common carp (Cyprinus carpio) and to investigate the occurrence of oxidative stress and metabolic changes after acute exposure to different concentrations of tebuconazole. For this purpose, several parameters were assessed as whole-body levels of thiobarbituric acid-reactive substance (TBARS), protein carbonyl, catalase, superoxide dismutase, glutathione S-transferase, nonprotein thiols, ascorbic acid, glycogen, glucose, lactate, protein, amino acids and ammonia in tebuconazole-exposed fish. The calculated LC(50-96 h) was 2.37 mg L(-1). Fish exhibited significant increase in TBARS levels in all concentrations used while the enzymatic and nonenzymatic antioxidants were decreased. Among the metabolic parameters, glycogen and glucose increased at 1.19 mg L(-1) and protein levels decreased at 1.78 and 2.37 mg L(-1). In conclusion, the fish health was adversely affected by exposure to tebuconazole, and those changes can compromise animal survival in the natural environment. The results indicate that the some of the parameters measured like a possible biomarkers of exposure to tebuconazole for this species of fish.     

Suppression of aromatase activity in populations of bream (Abramis brama) from the river Elbe, Germany

Aromatase activity was determined in brain and gonads of wild bream collected along the river Elbe, Germany, and correlated with other endocrine and reproductive endpoints such as plasma sex steroid concentrations, secondary sex characteristics (STI), plasma vitellogenin, gonad size (GSI), and maturation stages of germ cells (MS) that were reported for the same fish in a previous study. Furthermore, regional patterns of aromatase activity were correlated to a number of environmental factors such as exposure to environmental contaminants and parasitism. While aromatase activity was not detectable in the gonads of male and female fish with the assay used, fish of both genders revealed relatively great brain enzyme activities. As for most of the endocrine and reproductive parameters, with the exception of plasma testosterone (T), aromatase activities were significantly less in fish from a river stretch characterized by elevated exposures to organic contaminants and metals. Brain aromatase activity was positively and significantly correlated with plasma estradiol (E2) and MS in females, and showed a similar trend with plasma 11-ketotestosterone (11KT) and STI in males. No comparable trend occurred for T. This decrease of the reproductively relevant hormones 11KT and E2 may be indicative of a disruption of the last step in sex hormone synthesis, a hypothesis that was supported for E2 by the strong (R2=0.78, p<0.05) linear regression between aromatase activity and E2 in female bream. It is also hypothesized that the effects on brain aromatase activity were likely to be related to the disruption of other reproductive parameters including sexual maturity and expression of secondary sex characteristics. Although a number of factors such as exposure to pollutants and prevalence of the tapeworm Ligula intestinalis correlated with the suppression of aromatase activity, the exact causes for the regional decrease in brain aromatase activity remain unclear due to inconsistencies of these correlations between sampling events or gender.     

Decontamination trends in the aquacultured fish gilthead seabream (Sparus aurata) after feeding long-term a PCDD/F spiked feed

Aquacultured fish gilthead seabream (Sparusaurata), previously exposed to low levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) trough the diet for a 13 months period, were fed on a clean feed for another 22 months. Gilthead seabream is a protandrous hermaphrodite species and this “decontamination” period coincided with the stages of sex differentiation, maturity and reproduction of the fish. PCDD/F levels in the fish tissues (i.e. muscle, liver, perivisceral fat and gonads), expressed in pg WHO-TEQ g⁻¹ fresh weight, showed a general decreasing trend during the “decontamination” period. However, this general trend varied among tissues and was also dependent on sex and lipid contents. Toxicological effects affecting fish behaviour and hepatic marker responses were also evaluated. The results pointed out that exposure to PCDD/Fs did not have an impact on fish development and reproduction, since the proportion of sexes found after the sex reversal process was within the normal range described for this species. In addition, long-term exposure to low PCDD/F levels did not significantly affect the response of most of the biochemical markers considered. On the contrary, some of them (e.g. EROD activity) showed variations in their responses during the sex differentiation process and onwards. Finally, the hepatic AhR mRNA levels increased during dioxin exposure but they returned to values typical for non-exposed fish after the “decontamination” period.     

Evidence of endocrine alteration in the red mullet, Mullus barbatus from the NW Mediterranean

Red mullet (Mullus barbatus) were collected from different sampling sites (NW Mediterranean) in spring and autumn, with the aim of assessing potential alterations of the endocrine system. Alkylphenols were measured in fish bile as an indicator of estrogenic exposure. Key enzymatic activities involved in both synthesis (ovarian 17beta-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism of steroids were assessed together with histological alterations of the gonads. During the spring sampling, delayed gamete maturation, intersexuality, fibrosis, and depressed ovarian P450 aromatase activity were observed in organisms from the most polluted sites. During the autumn sampling, those effects were less evident, indicating that fish might be more susceptible to endocrine disrupting chemicals during the reproductive period. Nonetheless, enhanced glucuronidation of testosterone and estradiol was observed. Overall, this work provides first evidences of significant alterations in the endocrine system of red mullet from highly impacted areas in the NW Mediterranean.     

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: a partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F(0), 14 d post-fertilization, dpf) were exposed via the water for 70 d to 0 (control), 10, 50 and 250 microg L(-1) PFOS, followed by a further 30 d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70 d experiment. Embryos and larvae (F(1)) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 microg L(-1) PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30 d in clean water. The triiodothyronine (T(3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality.     

Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.     

Histopathological and biochemical changes in the liver of a teleost fish, Channa punctatus (Bloch) induced by a mercurial fungicide

The chronic toxic effects of a prolonged exposure of 6 months, from January to June, to a 'safe dose' (0.20 mg litre(-1)) of a commercial organomercurial fungicide, Emisan (methoxy ethyl mercuric chloride: MeEHgCl), on the histophysiology of liver in adults and young (yearlings) of Channa punctatus, are described in this investigation. In both of the experimental groups, liver histology exhibited various abnormalities, including hyperplasia, nuclear pyknosis, fatty necrosis, and degeneration of hepatocytes leading to tumour and syncytium formation, which are indicative of carcinogenesis. Apart from this, blood vessel congestion and oedema were also observed in young treated fish. In the livers of both exposed groups, corresponding with this cellular damage, a marked reduction in hepatosomatic index, levels of total protein and lipid, and an elevation in cholesterol and acid and alkaline phosphatase contents were recorded. However, these alterations were more pronounced in young than in adult fish. These Emisan-induced, histopathological and biochemical changes are suggestive of severe hepatic dysfunction, which may result in the impairment of the physio-metabolic process, in this species.     

Microsomal monooxygenase activity in Tilapia (Oreochromis mossambicus) exposed to a bleached kraft mill effluent using different exposure systems.

Bleached kraft pulp and paper mill effluents (BKMEs) are known to have adverse effects on aquatic organisms. One of the effects of BKMEs is its ability to induce cytochrome P4501A activity in exposed fish. 7-Ethoxyresorufin O-deethylase (EROD) activity is the most common biomarker used to measure the mixed-function monooxygenase activity. In this study, Tilapia were exposed to BKMEs using different exposure systems and their hepatic EROD activity, as well as liver/somatic index (LSI), were determined. In the Phase I study, Tilapia treated with betaNF and a whole (100%) BKME using a static, non-renewal system exhibited statistically significant EROD induction, but LSI values were not altered. In the Phase II study, fish were either caged in the mill's fishpond with the whole effluent passing through or cultured in tanks receiving 100% of the BKME continuously using a flow-through system in the laboratory. Their EROD activities were then compared with the non-exposed fish (control). The EROD activities in both groups of fish were elevated significantly with the greatest induction being observed in the field-exposed group. The LSI values in all of the field-exposed fish were significantly greater than the control Tilapia. The EROD assay was sensitive in detecting biological changes in fish exposed to the BKME. Further studies are warranted to better understand the impacts of BKMEs on aquatic organisms in Taiwan.     

Stable isotope determination in wild and farmed gilthead sea bream (Sparus aurata) tissues from the western Mediterranean

Stable isotopes of carbon and nitrogen (delta(13)C and delta(15)N) have been determined in wild and farmed gilthead sea bream (Sparus aurata) samples of white and red muscle, liver, gills and gonads. First, delta(13)C and delta(15)N values were determined in samples with and without lipid removal to check the possible effect of lipid content on the stable isotope values of the different tissues studied. Differences were found for delta(13)C in all tissues studied apart from white muscle of wild fish, the tissue with the lowest lipid content. For delta(15)N values no differences were found in wild fish tissues. Liver from farmed fish showed lower delta(15)N value after lipid removing. Further conclusions were based on results obtained from lipid-free samples. delta(13)C of cultured fish tissues showed a mean depletion of 2.9+/-0.4 per thousand compared to wild specimens, suggesting different sources of carbon in the diet, probably due to the feed used during sea-cage culture. Cultured gilthead sea bream tissues were significantly more enriched in nitrogen than wild specimens by an average of 1.5+/-0.2 per thousand in white muscle, indicating a slight increase in the trophic level. Determination of stable isotope signatures of gilthead sea bream tissues allows clear discrimination between wild and cultured sea bream, and characterisation of differences in diet and feeding conditions in any tissue studied.     

Unexplained gonad alterations in whitefish (Coregonus spp.) from Lake Thun, Switzerland: levels of persistent organic pollutants in different morphs

Since 2000, a surprisingly high number of macroscopical gonad alterations has been reported in whitefish (Coregonus spp.) from Lake Thun, Switzerland. This unique phenomenon is still unexplained and has received much public attention. As one possible trigger for these effects, the presence of persistent, bioaccumulative and toxic compounds acting as endocrine disruptors in the lake has been discussed. In this study, concentrations of selected persistent organic pollutants were examined in two morphs of whitefish from Lake Thun and their link to the observed abnormalities was investigated. Analyzed compound classes included polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated naphthalenes, polybrominated diphenyl ethers and hexabromocyclododecanes. The target substances were identified in all samples and concentrations of the analyzed compounds were highly correlated among each other. These correlations show that the analyzed substances have the same distribution pattern throughout the lake and that uptake, accumulation and elimination processes are similar. Significant differences in contaminant levels within the samples existed between the two analyzed morphs of whitefish, most likely due to different age, food patterns and growth rate. No difference in contaminant levels was observed between fish with abnormal gonads and fish with normal gonads, suggesting no causal link between the investigated lipophilic organohalogen compounds present in fish and the observed gonad abnormalities in whitefish from Lake Thun. A comparison to existing data shows that concentrations in Lake Thun whitefish are at the lower bound of contaminant levels in whitefish from Swiss lakes or from European waters.     

Assessing the biological status of fish in a river receiving pulp and paper mill effluents

This study compared the use of sentinel species- and community-based field approaches for assessing the biological status of fish living in a river receiving pulp and paper mill effluents. Three approaches were compared. Two approaches used sentinel species. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index (HAI) and the other involved biochemical measurements of hepatic mixed function oxidase (MFO) activity and plasma steroid levels. The third approach characterized the fish community structure according to an index of biotic integrity (IBI). The comparison focused on how the methods respond to the hypothesis that recent process modifications/effluent treatment changes, resulting in demonstrable improvements in effluent quality, have beneficial effects on fish. Neither of the approaches using sentinel fish indicated clear mill-related influences either before or after the process modifications/effluent treatment changes. There was no evidence of depressed plasma steroids and increased MFO activity in fish frequently associated with mill effluent exposure in previous studies. While the HAI was higher at stations downstream from two mills, this could not be linked to effluent exposure alone. In contrast, the study of community structure showed a substantial improvement in fish assemblages at all the mill sites.     

Metal accumulation in the smooth toadfish, Tetractenos glaber, in estuaries around Sydney, Australia

This study determined the metal levels in sediments and tissues of a common estuarine fish, Tetractenos glaber (smooth toadfish), from two metal contaminated and two reference estuaries near Sydney, Australia. Metal levels were highest in sediments and fish from contaminated estuaries. Gonads contained the highest metal levels followed by muscle, gill and liver. Metal accumulation was gender-dependant (e.g. male gonads were >20 times higher in As than females; female gills were >30 times higher than males for Pb). Cadmium, Pb and Ni levels in fish tissues reflected sediment levels, indicating sediment and/or dietary metal uptake. Levels of As, Co, Cd & Pb in gills showed similar patterns to other tissues, suggesting that metals may have been taken up by gills through contaminated water. Similar metal patterns in tissues and sediments suggest more than one uptake pathway. This study indicates that multiple factors influence metal accumulation in fish.     

Importance of fish gender as a factor in environmental monitoring of mercury

Total mercury concentrations were determined in the gonadal tissues of 15 female and 10 male European perch (Perca fluviatilis) from one location of the stream “Jevanský potok” located about 30 km from Prague (Czech Republic). Tissue samples were frozen at ?26 °C in polypropylene containers until further processing, which was carried out using an Advance Mercury Analyser (single purpose atomic absorption spectrometer). Mercury concentrations were present in all analysed gonad samples, and ranged from 2.3 to 12.7 μg/kg wet weight. However, we determined a mean Hg concentration (9.45 μg/kg) in male gonads that was 2.4 times greater than that of female gonads (3.9 μg/kg). This is an important finding when taking into account fish sex in environmental pollution monitoring (especially for mercury contamination).     

Investigation of acute toxicity of chlorpyrifos-methyl on guppy Poecilia reticulata

Static bioassays were made to determine acute toxicity of chlorpyrifos-methyl, a wide spectrum organophosphorus insecticide and potential toxic pollutant of aquatic ecosystem, Guppy fish (Poecilia reticulata). Bioassays were made at a regulated temperature of 22+/-1 degrees C and were repeated three times. Lethal doses of the insecticides were determined using LC50 software programme of U.S. EPA based on Finney's Probit Analysis statistical method. The 96 h LC50 value and 95% confidence limit of chlorpyrifos-methyl for Guppy was estimated as 1.79 (1.47-2.10) mg/l. The fish exposed to chlorpyrifos-methyl exhibited behavioral changes in the form of neurotoxin toxicity: less general activity than control group, loss of equilibrium, erratic swimming and staying motionless at a certain location generally at mid-water level for prolonged periods. The 1 mg/l (lowest) concentration had similar behavior (NOEC) with the control group.     

Recovery of mercury-contaminated fisheries

In this paper, we synthesize available information on the links between changes in ecosystem loading of inorganic mercury (Hg) and levels of methylmercury (MeHg) in fish. Although it is widely hypothesized that increased Hg load to aquatic ecosystems leads to increases in MeHg in fish, there is limited quantitative data to test this hypothesis. Here we examine the available evidence from a range of sources: studies of ecosystems contaminated by industrial discharges, observations of fish MeHg responses to changes in atmospheric load, studies over space and environmental gradients, and experimental manipulations. A summary of the current understanding of the main processes involved in the transport and transformation from Hg load to MeHg in fish is provided. The role of Hg loading is discussed in context with other factors affecting Hg cycling and bioaccumulation in relation to timing and magnitude of response in fish MeHg. The main conclusion drawn is that changes in Hg loading (increase or decrease) will yield a response in fish MeHg but that the timing and magnitude of the response will vary depending of ecosystem-specific variables and the form of the Hg loaded.     

Changes in plasma thyroid hormones and cortisol levels in crucian carp (Carassius auratus) exposed to the extracted microcystins

The endocrine response of crucian carp injected intraperitoneally with extracted microcystins (MC) was investigated in this study. Fish were injected intraperitoneally either with 0.75% NaCl (control) and Microcystis extract corresponding to 150 and 600 microg microcystins per kg body weight. The plasma levels of triiodothyronine (T(3)), thyroxine (T(4)), free triiodothyronine (FT(3)), free thyroxine (FT(4)), and cortisol were determined at 0, 1, 3, 12, 24, and 48h post-administration of MC-containing extract. Treated fish displayed abnormal behaviors, such as a startle response and disoriented swimming, as well as changes in ventilation rates. Plasma cortisol concentrations of fish in both dose groups significantly increased after administration of extracted MC and remained high throughout the experiment, which suggested that MC elicited a stress response in treated fish. The profiles of cortisol changes in treated fish appeared to be dose dependent, indicating that fish in the high dose group experienced greater MC-induced disturbance. Mortality occurred after 12h in the high dose group. Plasma levels of T(4), T(3), FT(4), and FT(3) did not vary significantly between the control fish. In contrast to this, fish exposed to MC-containing extract showed significant declines in T(3), FT(4), and FT(3) levels in a dose-dependent manner throughout the experiment. Plasma T(4) levels, however, did not vary significantly in the low dose group, whereas they decreased significantly at 48h post injection in the high dose group. This study demonstrates that administration of microcystins-containing extract causes a stress response and reduces the plasma levels of thyroid hormones in crucian carp. These results illustrate that microcystins exerted potent effects on the endocrine system of crucian carp, through activating their hypothalamus-pituitary- interrenal axis and disturbing thyroid function.     

Uptake and depuration of the anti-depressant fluoxetine by the Japanese medaka (Oryzias latipes)

The selective serotonin reuptake inhibitor (SSRI) class of anti-depressants is among the most widely prescribed groups of pharmaceuticals. Consequently, aquatic ecosystems impacted by municipal wastewater discharges are predicted to receive substantial annual loadings of these compounds. Although SSRIs have been detected in fish tissues, little is known of their uptake and depuration in freshwater fish species. In this study, Japanese medaka (Oryzias latipes) were exposed to fluoxetine at a nominal concentration of 0.64 microg L(-1) for 7d and subsequently allowed to depurate in clean water over a 21d period. Fluoxetine uptake by medaka was observed within the first 5h of exposure and the biologically active metabolite, norfluoxetine, was also detected in medaka tissues during this timeframe. A maximum fluoxetine concentration was measured in medaka by the third day of the uptake phase, yielding an uptake rate constant (k(1)) of 5.9+/-0.5 (d(-1)). During the depuration phase of the experiment, a half life of 9.4+/-1.1d was determined for fluoxetine. Using these data, bioconcentration factor (BCF) values of 74 and 80 were estimated for fluoxetine and a pseudo-BCF (the ratio of the concentration of norfluoxetine in medaka and the aqueous fluoxetine concentration) of 117 was calculated for norfluoxetine. These results indicate longer persistence and greater potential for the bioaccumulation of fluoxetine and norfluoxetine in fish tissues than would be predicted from prior half life estimates derived using mammalian species.     

Effect of vegetable oil factory effluent on the gonads of a freshwater teleost, Heteropneustes fossilis (Bl.) --a biochemical study

The effects of three sublethal concentrations (5,10,15% v/v) of vegetable oil factory effluent on biochemical changes in the gonads (ovaries/testes) of a freshwater teleost were studied. The results reveal a significant decline in the total lipid, cholesterol, phospholipid, and glycerine levels, but an increase in the free fatty acid levels, both in the ovaries and the testes of H. fossilis, following 150 days (mid-January through mid-June) of exposure to 10% and 15% concentrations of the effluent. This decrease/increase in the biochemical parameters was more or less dependent on the concentration of the effluent. The functional significance of any decrease/increase in the biochemical parameter(s) has been discussed.