Metallothionein as bioindicator of freshwater metal pollution: European eel and brown trout

The aim of this work was to evaluate the potential of metallothionein (MT) as a bioindicator of heavy metal pollution in brown trout and European eel in the field situation. River Ferrerias (North Spain) provided a good gradient of metal contamination: concentrations of heavy metals were elevated both in water and in sediments at the downstream (polluted) site and were low at the upstream (unpolluted) site. MT levels of brown trout exhibited statistically significant differences between sites. Although European eel at the polluted site had a higher MT content, differences were not significant. It is concluded that MT is a good bioindicator of heavy metal pollution in brown trout.     

Eel (Anguilla anguilla) and brown trout (Salmo trutta) target species to assess the biological impact of trace metal pollution in freshwater ecosystems

Copper, lead, and cadmium pollution were measured in water and sediments of two Northern Spanish rivers: Piles and Pigüeña. Liver contents of these heavy metals were analyzed in two fish species (eel, Anguilla anguilla, and brown trout, Salmo trutta) collected from the same locations. Significant levels of heavy metal pollution were found in a 38% of fish that are potential catches for sport fishermen. The results indicate that adult eel could be a good metal bioindicator if sampled at a homogenous age. In contrast, brown trout could be considered as a bioindicator only during the first year of life.     

Brown Trout and European Minnow as Target Species for Genotoxicity Tests: Differential Sensitivity to Heavy Metals

Brown trout (Salmo trutta) and European minnow (Phoxinus phoxinus) were evaluated as target species to carry out genotoxicity tests. Assessment was made of their relative abundance in wild; their distribution areas; and their sensitivity to heavy metals, intraperitoneally exposing individuals of both species to a low dose (1.7 mg/kg body weight) of different heavy metals. Micronuclei were scored in renal erythrocytes 24 h after treatment. Cadmium chloride significantly induced micronuclei in both species whereas mercury nitrate induced micronuclei increase only in brown trout. Brown trout is abundant, present in all studied freshwater ecosystems, and more sensitive to toxic heavy metals than minnow; therefore it is presented as a target species for studies on heavy metal genotoxicity.     

A Real-Time PCR Method for the Quantification of the Two Isoforms of Metallothionein in Lake Trout (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>)

Metallothioneins (MTs) are low-molecular-weight proteins whose physiologic roles are the regulation of essential metals Cu and Zn, sequestration of heavy metals, and free radical scavenging. Induced production of MTs in a wide variety of organisms exposed to heavy metals has made them popular exposure indicators. While it has been postulated that the three different isoforms of MT play different physiologic roles, methods to discern induction separately have not been available. The development of real-time polymerase chain reaction (real-time PCR) primers and TaqMan probes to measure the two MT isoforms found in salmonid fish are described. Assuming a high degree of homology between the isoforms and within different groups of salmonids, the sequences for MT-I and MT-II from rainbow trout were used to develop primers and probes for lake trout using the Primer3 program. Two sections of each isoform that varied by only a few nucleotides were targeted. SYBR Green validated the primer specificity, and melt curve analysis further ensured that only one product was amplified. Analysis of archived samples from fish captured in unmanipulated reference lakes or from lakes experimentally treated with cadmium or ethynylestradiol (EE2) afforded an examination of seasonal and contaminant influences on MT-I and MT-II mRNA expression.     

Micronucleus test in freshwater fish species: an evaluation of its sensitivity for application in field surveys

Brown trout, Salmo trutta, European eel, Anguilla anguilla, and European minnow, Phoxinus phoxinus, three fish species inhabiting European freshwater ecosystems, were evaluated for their use as in situ pollution biomarkers using the micronucleus test in renal erythrocytes. Experimental exposure (by immersion) to different concentrations of cyclophosphamide, colchicine, and cadmium showed that brown trout are more sensitive to the three compounds than minnows and eels. In situ surveys of wild freshwater ecosystems with different levels of pollution showed that minnows and eels living in polluted sites do not present higher micronuclei averages than those caught in clean rivers systems, whereas micronuclei are induced in brown trout inhabiting polluted sites. Our results demonstrated the suitability of brown trout for in situ biomonitoring of freshwater ecosystems as well as for laboratory tests using the micronucleus test.     

Seasonal comparison of wild and farmed brown trout (Salmo trutta forma fario L., 1758): crude lipid, gonadosomatic index and fatty acids

Brown trout is one of the most preferred wild freshwater fish species in the east Black Sea region (Turkey) due to its nutritional value and palatable aroma as well as being popular for sport fishing. In this research, seasonal variations in the crude lipid, gonadosomatic index and fatty acid composition of wild and farmed brown trout were investigated. The spawning period of wild and farmed brown trout appears to be from August to October and from October to January, respectively. The mean crude lipid content in farmed brown trout (3.62%) was significantly higher (P≤0.05) than that of wild brown trout (2.80%). Significant seasonal differences (P≤0.05) in crude lipid content were observed in both fish. The percentage of total saturated fatty acids was similar (P≥0.05) in both fish. Total polyunsaturated fatty acids were higher (P≤0.05) in the wild brown trout compared with the farmed brown trout, while its total monounsaturated fatty acids content was lower (P≤0.05). The muscle lipids of wild fish contained significantly (P≤0.05) higher percentages of C16:1n-7, C17:1n-7, C18:3n-3, C20:2n-6, C20:4n-6, C20:5n-3 and C22:2n-6 fatty acids and contained lower percentages of C14:0, C18:1n-9, C18:2n-6, C20:1n-9, C24:1n-9 and C22:6n-3 fatty acids than farmed fish. The total amounts of n-3 fatty acids in wild fish were higher than in farmed fish, but total amounts of n-6 fatty acids in farmed fish were higher than in wild fish. The n3/n6 proportion in wild fish was higher than that of farmed fish.     

Induction of micronuclei in eel (Anguilla anguilla L.) by heavy metals

Micronucleus test was performed in situ on eels (Anguilla anguilla) from river sites with different levels of heavy metal pollution (cadmium and mercury). Cadmium content in eel liver but not micronuclei averages in kidney were associated with cadmium content in sediments. Mercury content in liver was not significantly associated with mercury content in sediments. Both cadmium and mercury induced micronuclei expression in eels when injected, the concentration tested being 1.7 mg metal/kg body weight and the micronuclei induction being 2.64 and 2.35 micronuclei per 1000 cells for cadmium and mercury respectively. It was concluded that these heavy metals are genotoxic for European eel, that eel liver metal content is a sensitive indicator for environmental monitoring of cadmium pollution, and that the micronuclei scores in eels are not a sensitive method to detect heavy metals pollution in freshwater ecosytems.     

European eel (Anguilla anguilla L.) metallothionein, endocrine, metabolic and genotoxic responses to copper exposure

This study investigated Anguilla anguilla (European eel) physiological and genotoxic responses to copper (Cu) and their relation with metallothionein (MT) protection. Eels were exposed during 7 days to Cu 0.2 micromol/L. MT induction was assessed in gill and liver, revealing significant response only in liver. Endocrine responses displayed a plasma free triiodothyronine (T3) and cortisol significant decrease, though the thyroid-stimulating hormone (TSH) and free thyroxine (T4) concentrations were unaltered. A significant plasma glucose increase was observed whereas lactate was significantly decreased. Despite the absence of DNA integrity decrease in blood, gill, liver and kidney, erythrocytic nuclear abnormalities (ENA) frequency significantly increased in Cu exposed group. MT induction was insufficient to prevent endocrine and metabolic alterations as well as genotoxicity/clastogenicity in blood. However, MT protection was evident in liver by preventing DNA integrity loss. Globally, it was demonstrated that Cu environmentally realistic levels may pose a serious ecological risk to fish.     

广州市场食用鱼中5种重金属含量分析及评价

目的:评价市场上常见食用鱼的重金属(锌、铅、镉、铜、铬)污染状况和食用安全性。方法:采用硝酸-高氯酸消化和火焰原子吸收法对广州市场21种鱼中重金属含量进行分析及评价。结果:21种鱼中重金属含量符合以下规律:必需微量元素锌、铜、铬含量较高,非必需微量元素铅含量较低,镉未检出;根据《NY 5073-2006无公害食品水产品中有毒有害物质限量》,所测样品的铅、镉、铜未超标;鳗鱼中的铬含量超过"人体消费卫生标准"。结论:鳗鱼中的铬含量较高,经常食用存在潜在危害,应引起有关部门重视。     

Metal Distribution in the Tissues of Two Benthic Fish from Paddy Fields in the Middle Reach of the Yangtze River

Metal content was determined in two fish species (Misgurnus anguillicaudatus and Monopterus albus) inhabiting rice paddies along the Middle Reach of the Yangtze River. Both are species important food fish in East Asia. The levels of eight metals (Fe, Mn, Zn, Cu, Cr, As, Cd, Pb) in skin, muscle and intestinal tissues were measured and correlated with fish size. Metal distribution among the tissues was examined and the potential risk of fish consumption was evaluated. Correlations between metal concentration and fish size were not significant. Concentrations of essential metals varied significantly among tissues, but the differences were not significant for non-essential metals. Correlations among metals in skin tissue were stronger than in intestinal and muscle tissues. Metal concentrations in the edible tissues of both fish met permissible levels for consumption, except Pb in loach skin and Cr loach in muscle. The potential risk caused by Pb from skin and Cr from muscle of loach consumption should be considered in future risk assessmeents.     

Bioaccessibility and content of Se in fish and shellfish widely consumed in Mediterranean countries: influence of proteins,fat and heavy metals

Twenty samples of fish and seafood products were selected for determination of total and bioaccessible Se (i.e. soluble and dialyzable). The Se contents ranged between 0.638–0.052?µg/g. The samples of fatty fish such as mackerel and tuna showed higher contents than the species of lean fish, and mainly the fresh water fish, such as salmon, trout and panga showed the lowest contents. The fraction of soluble Se ranged between 43–84% while that the fraction of dialyzable Se did not exceed 14%. No statistically significant correlation was observed between protein content and total and bioaccessible Se content. In contrast, a negative interaction was observed between bioaccessible Se and fat content, and between bioaccessible Se and presence of heavy metals like Cd.     

Relative sensitivity of bull trout (Salvelinus confluentus) and rainbow trout (Oncorhynchus mykiss) to acute copper toxicity

Bull trout (Salvelinus confluentus) were recently listed as threatened in the United States under the federal Endangered Species Act. Past and present habitat for this species includes waterways contaminated with heavy metals released from mining activities. Because the sensitivity of this species to copper was previously unknown, we conducted acute copper toxicity tests with bull and rainbow trout (Oncorhynchus mykiss) in side-by-side comparison tests. Bioassays were conducted using water at two temperatures (8 degrees C and 16 degrees C) and two hardness levels (100 and 220 mg/L as CaCO3). At a water hardness of 100 mg/L, both species were less sensitive to copper when tested at 16 degrees C compared to 8 degrees C. The two species had similar sensitivity to copper in 100-mg/ L hardness water, but bull trout were 2.5 to 4 times less sensitive than rainbow trout in 220-mg/L hardness water. However, when our results were viewed in the context of the broader literature on rainbow trout sensitivity to copper, the sensitivities of the two species appeared similar. This suggests that adoption of toxicity thresholds that are protective of rainbow trout would be protective of bull trout; however, an additional safety factor may be warranted because of the additional level of protection necessary for this federally threatened species.     

Sensitivity of mottled sculpins (Cottus bairdi) and rainbow trout (Onchorhynchus mykiss) to acute and chronic toxicity of cadmium, copper, and zinc

Studies of fish communities of streams draining mining areas suggest that sculpins (Cottus spp.) may be more sensitive than salmonids to adverse effects of metals. We compared the toxicity of zinc, copper, and cadmium to mottled sculpin (C. bairdi) and rainbow trout (Onchorhynchus mykiss) in laboratory toxicity tests. Acute (96-h) and early life-stage chronic (21- or 28-d) toxicity tests were conducted with rainbow trout and with mottled sculpins from populations in Minnesota and Missouri, USA, in diluted well water (hardness = 100 mg/L as CaCO3). Acute and chronic toxicity of metals to newly hatched and swim-up stages of mottled sculpins differed between the two source populations. Differences between populations were greatest for copper, with chronic toxicity values (ChV = geometric mean of lowest-observed-effect concentration and no-observed-effect concentration) of 4.4 microg/L for Missouri sculpins and 37 microg/L for Minnesota sculpins. Cadmium toxicity followed a similar trend, but differences between sculpin populations were less marked, with ChVs of 1.1 microg/L (Missouri) and 1.9 microg/L (Minnesota). Conversely, zinc was more toxic to Minnesota sculpins (ChV = 75 microg/L) than Missouri sculpins (chronic ChV = 219 microg/L). Species-average acute and chronic toxicity values for mottled sculpins were similar to or lower than those for rainbow trout and indicated that mottled sculpins were among the most sensitive aquatic species to toxicity of all three metals. Our results indicate that current acute and chronic water quality criteria for cadmium, copper, and zinc adequately protect rainbow trout but may not adequately protect some populations of mottled sculpins. Proposed water quality criteria for copper based on the biotic ligand model would be protective of both sculpin populations tested.     

Oxidative stress biomarkers in Senegal sole, Solea senegalensis, to assess the impact of heavy metal pollution in a Huelva estuary (SW Spain): seasonal and spatial variation

The response of wild fish to heavy metals was studied in sole (Solea senegalensis) collected in 2004, 2005 and 2006 at three sampling sites from Huelva estuary (SW Spain), in the vicinity of a petrochemical and mining industry. Heavy metals As, Cd, Cu, Fe, Pb and Zn were analyzed in samples collected from sediment, water and tissue (liver) to examine their bioconcentration and effects in fish such as lipid peroxidation (LPO), catalase (CAT; EC 1.11.1.6), glutathione peroxidase (GPx; EC 1.8.1.7), glutathione S-transferase (GST; EC 2.5.1.18) and glutathione reductase (GR; EC 1.11.1.6) were also analyzed in the fish liver. The results showed different effects in sole from diverse locations with varying degrees of pollution. Significant differences in LPO, CAT and GR activities between control fish and fish from sampling sites were observed as well as seasonal differences for biomarkers. Significant correlations were established between some biomarkers and heavy metals concentrations in liver, sediment and water. This study indicates the usefulness of integrating a set of biomarkers to assess the effects of pollutants in aquatic environments under complex mix of pollutants and chronic pollution situation.     

Effects of natural organic matter source on reducing metal toxicity to rainbow trout (Oncorhynchus mykiss) and on metal binding to their gills

Juvenile rainbow trout (Oncorhynchus mykiss, 3 g) were exposed for 74 h in ion-poor (soft) water to a mixed-metal solution in the presence of 4, 6, and 10 mg C/L natural organic matter (NOM). The metals were 0.2 microM Pb, 0.1 microM Hg, 0.1 microM Cd, 1.3 microM Cu, 0.05 microM Ag, and 3.5 microM Co, and the natural organic matter was isolated by reverse osmosis from three sources in southern Ontario, Canada. The six-metal solution alone was extremely toxic to the fish. Increasing concentrations of each NOM increased trout survival, but the NOM having the most allochthonous properties (from Luther Marsh) increased fish survival most, while the NOM having the most autochthonous properties (from Sanctuary Pond, Point Pelee) increased fish survival least. This pattern was reflected in the degree of reduction of Pb and Cu accumulation by the gills. Relatively simple chemical characterization of NOM, such as protein-to-carbohydrate ratios, or optical characterization, such as absorbance-to-fluorescence ratios (e.g., representing aromaticity), may adequately reflect these biologically relevant differences in organic matter quality.     

Bioaccumulation of heavy metals in mullet (Mugil cephalus) and oyster (Crassostrea madrasensis) from Pulicat lake, south east coast of India

The accumulation of six heavy metals (Cr, Cd, Cu, Zn, Pb and Ni) in sediment, water and in tissue parts of Mugil cephalus and Crassostrea madrasensis was studied in two locations of Pulicat lake, Southeast coast of India, which receives considerable quantity of effluents from industries located in North Chennai coastal region. The results reveal that the metal concentration in water is decreasing in the following order of Zn > Ni > Cu > Cr > Pb > Cd both in lake and barmouth and highest concentration was observed for Zn (32.5 μg L(-1) in lake and 25.2 μg L(-1) in bar mouth). Metals were highly concentrated in sediments when compared to water and biota. Metals abundance in sediments has following sequential order of Cr > Ni > Zn > Cu > Pb > Cd and the accumulation pattern in barmouth showed minor variation indicating the following pattern of Zn > Ni > Cr > Cu > Pb > Cd. The geoaccumulation index (I(geo)) for Pulicat lake sediments indicate that the sediments are extremely contaminated with Cd and moderately contaminated with Cu and Ni. Bioaccumulation of heavy metals in Mugil cephalus and Crassostrea madrasensis showed marked differences in the accumulation patterns. It is observed that Zn, Cu and Pb are accumulated in elevated concentrations in various parts of the fish and oyster when compared with other metals.     

Liver Histopathology in Brown Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> f. fario) from the Tinhela River,Subjected to Mine Drainage from the Abandoned Jales Mine (Portugal)

The objective of this study was to compare the occurrence of toxicopathic liver lesions in brown trout (Salmo trutta fario) from Tinhela River near the Jales Mine, both before implementation (2002) and after completion of the governmental mitigation program (2006). Fish were caught in April 2002 and May 2006, using an electrofishing system at four sites: S0, reference station; S1, S2 and S3 as contaminated stations. In 2002, the hepatosomatic index (HSI) was significantly higher for trout captured at the contaminated sites S2 and S3 than in S0. After the rehabilitation program, the HSI of fish sampled at the contaminated sites did not differ from the reference group. The liver of trout caught at S0 exhibited the normal parenchymal and stromal architecture described for the species and there were no pathological abnormalities. In contrast, fish sampled at S3 and S2 sites had diverse toxicopathic alterations. Specifically, livers from the two contaminated sites showed bile duct hyperplasia, often with mild epithelial dysplasia and fibrotic adventitial sleeve, foci of smaller and more basophilic hepatocytes and foci of hepatocellular necrosis; the latter conditions were frequently associated. Compared with the reference animals, increased hepatocellular vacuolization was found in livers from the polluted sites. Histopathological examination revealed differences among sampling sites in the severity and diversity of hepatic lesions clearly related to the proximity of the tailings. No pathological alterations were observed in the livers of brown trout caught in the same four areas of the Tinhela River after the mitigation program in 2006. In conclusion, our results supported that drainage from the abandoned Jales Mine had deleterious toxicological effects in brown trout. Our data suggested that the governmental mitigation program may have reduced the impact of Jales tailings.     

Oil shale processing as a source of aquatic pollution: monitoring of the biologic effects in caged and feral freshwater fish.

The biologic effects of the oil shale industry on caged rainbow trout (Oncorhynchus mykiss) as well as on feral perch (Perca fluviatilis) and roach (Rutilus rutilus) were studied in the River Narva in northeast Estonia. The River Narva passes the oil shale mining and processing area and thus receives elevated amounts of polycyclic aromatic hydrocarbons (PAHs), heavy metals, and sulfates. The effects of the chemical load were monitored by measuring cytochrome P4501A (CYP1A)-dependent monooxygenase (MO) activities [7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase (AHH)] as well as conjugation enzyme activities [glutathione S-transferase (GST) and UDP-glucuronosyltransferase] in the liver of fish. CYP1A induction was further studied by detecting the amount and occurrence of the CYP1A protein. Histopathology of tissues (liver, kidney, spleen, and intestine) and the percentage of micronuclei in fish erythrocytes were also determined. Selected PAHs and heavy metals (Cd, Cu, Hg, and Pb) were measured from fish muscle and liver. In spite of the significant accumulation of PAHs, there was no induction of MO activities in any studied fish species. When compared to reference samples, AHH activities were even decreased in feral fish at some of the exposed sites. Detection of CYP1A protein content and the distribution of the CYP1A enzyme by immunohistochemistry also did not show extensive CYP1A induction. Instead, GST activities were significantly increased at exposed sites. Detection of histopathology did not reveal major changes in the morphology of tissues. The micronucleus test also did not show any evidence of genotoxicity. Thus, from the parameters studied, GST activity was most affected. The lack of catalytic CYP1A induction in spite of the heavy loading of PAHs was not studied but has been attributed to the elevated content of other compounds such as heavy metals, some of which can act as inhibitors for MOs. Another possible explanation of this lack of induction is that through adaptation processes the fish could have lost some of their sensitivity to PAHs. Either complex pollution caused by oil shale processing masked part of the harmful effects measured in this study, or oil shale industry did not have any severe effects on fish in the River Narva. Our study illustrates the difficulties in estimating risk in cases where there are numerous various contaminants affecting the biota.     

Persistent organic pollutants (POPs) in fish collected from the urban tract of the river Tiber in Rome (Italy)

European eel and chub samples were analyzed to determine the levels of non-dioxin-like polychlorobiphenyls (NDL-PCBs), polychlorodibenzodioxins (PCDDs) and polychlorodibenzofurans (PCDFs), dioxin-like PCBs (DL-PCBs), and brominated polybromodiphenyl ethers (PBDEs) in order to evaluate the extent of contamination of the river Tiber along the urban tract through the city of Rome (Italy). All samples presented detectable levels of the chemicals analyzed, and exhibited species-specific differences in terms of congener composition and total concentrations. On average the European eel presented the highest values. In this species the dioxin-like compound sums (WHO-TEQs) exceeded the pertinent maximum levels (MLs). Non-ortho PCBs constituted approximately 80% of WHO-TEQ toxicological potential whereas NDL-PCB and PBDE concentrations appeared to match values determined in other polluted aquatic ecosystems where non-point contamination sources were present. The contamination patterns determined in fish tissues seemed to reflect the impact of generic contamination source(s).