Toxicity of waters from two streams to early life stages of brown trout (Salmo trutta f. fario L.), tested under semi-field conditions.

The development of brown trout (Salmo trutta f. fario L.) in water of two differently polluted streams and in a control situation was monitored in order to get insights into the impact of anthropogenic chemical stressors on the reproductive success of this fish species indigenous to both streams. The test streams, situated in the south of Stuttgart, Germany, were the complexly polluted K?rsch stream and the less polluted Kr?henbach stream. Bypass systems connected to the streams and a laboratory control system were used for continuous exposure of early brown trout stages shortly after fertilisation up to the end of the embryonic development. Temperature and oxygen conditions were standardised in all test series in order to minimise unspecific effects. The examined endpoints were: (1) mortality, (2) developmental rate, (3) time course of hatching, (4) malformations, and (5) growth. A retarded development, reduced growth rates and higher mortality rates of K?rsch stream water exposed embryos indicated an embryotoxic potential for the more polluted stream. High infection-related mortality rates of embryos suggested the presence of confounding factors also in the less polluted Kr?henbach stream. In parallel to the exposure experiment, physicochemical and limnochemical parameters as well as concentrations of organic contaminants and heavy metals were monitored. Analytical data confirm the different degrees of pollution of both streams.     

Environmental monitoring by gene expression biomarkers in Barbus graellsii: laboratory and field studies

Barbel (Barbus graellsii) is a freshwater fish used as a sentinel species in environmental monitoring programmes and ecotoxicological studies in northern Spain, particularly in the Ebro River basin, the largest freshwater resource in Spain. We developed specific primers for the quantification of CYP1A and metallothionein (MT)-1 and -2 gene expression by QRT-PCR in barbel in order to assess their suitability in biological effect monitoring of dioxin-like compounds and metals. All three genes responded as expected in laboratory tests, using model inducers. Hepatic CYP1A mRNA levels showed a twofold induction in fish injected intraperitoneally with beta-naphthoflavone, whereas MT-1 and MT-2 gene expression was strongly induced by cadmium (15- and 13-fold, respectively) and mercury (five- and eightfold). Barbel populations from different sites on the Ebro River basin showed a good correlation between the historical records of organochlorine pollution, CYP1A expression levels and EROD activity. Nevertheless, although metallothionein protein levels in the liver of wild fish correlated with hepatic levels of mercury, MT-1 and MT-2 gene expression did not correlate with the mercury content or with the concentration of metals in sediments from the sites inhabited by the fish. These results demonstrate the utility of barbel CYP1A-mRNA expression, but not that of MT-1 or MT-2, as a biomarker in field studies. The tools and protocols developed here are likely to apply to other species of the genus Barbus, with some 700 species distributed throughout most of the Old World.     

EROD and CYP1A protein in channel catfish (Ictalurus punctatus) from an urban estuary relative to that in benzo[a]pyrene-exposed hatchery specimens

Bottom-feeding fish such as flounder and killifish have been widely used in monitoring hepatic monooxygenase induction in polluted water bodies. While channel catfish are often utilized in tissue monitoring of fresh and estuarine water bodies, few data are available on their use in environmental monitoring of hepatic monooxygenase activity. In this project, the presence of CYP1A protein was verified in channel catfish through recognition by Mab 1-12-3, an antibody which recognizes the CYP1A homologue in a variety of teleost species. CYP1A protein levels and 7-ethoxyresorufin-o-deethylase (EROD) activity in laboratory control and benzo-a-pyrene (BaP)-challenged channel catfish were compared to those in feral channel catfish from Back River, an urban estuarine tributary to Chesapeake Bay. Though more variable, mean CYP1A protein levels in the field-collected fish were similar to those of the BaP-induced laboratory fish. However, EROD activity of the Back River fish was less than one half that observed in the BaP-induced laboratory fish. When normalized to CYP1A protein levels, EROD activity was slightly lower in the Back River fish than either the laboratory control or BaP-treated fish. This finding may indicate possible inhibition or inactivation of the CYP1A protein in the feral fish.     

Survival of brown trout during spring flood in DOC-rich streams in northern Sweden: the effect of present acid deposition and modelled pre-industrial water quality

Mortality and physiological responses in brown trout (Salmo trutta) were studied during spring snow melt in six streams in northern Sweden that differed in concentrations of dissolved organic carbon (DOC) and pH declines. Data from these streams were used to create an empirical model for predicting fish responses (mortality and physiological disturbances) in DOC-rich streams using readily accessible water chemistry parameters. The results suggest that fish in these systems can tolerate higher acidity and inorganic aluminium levels than fish in low DOC streams. But even with the relatively low contemporary deposition load, anthropogenic deposition can cause fish mortality in the most acid-sensitive surface waters in northern Sweden during spring flood. However, the results suggests that it is only in streams with high levels of organically complexed aluminium in combination with a natural pH decline to below 5.0 during the spring where current sulphur deposition can cause irreversible damage to brown trout in the region. This study support earlier studies suggesting that DOC has an ameliorating effect on physiological disturbances in humic waters but the study also shows that surviving fish recover physiologically when the water quality returns to less toxic conditions following a toxic high flow period. The physiological response under natural, pre-industrial conditions was also estimated.     

Biological effect monitoring in dab (<Emphasis Type="Italic">Limanda limanda</Emphasis>) using gene transcript of CYP1A1 or EROD—a comparison

BACKGROUND, AIM, AND SCOPE: Gene expression analyses with real-time (RT)-polymerase chain reaction (PCR) gains importance in marine monitoring. This new technique has to be compared to the classical approaches like the well known biomarker ethoxyresorufin-O-deethylase (EROD) to test their suitability for monitoring programmes. The goal of the present study is to compare EROD activity and CYP1A1 mRNA expression in the important monitoring fish species dab (Limanda limanda) and to answer the question of whether these parameters reflect the polycyclic aromatic hydrocarbon (PAH) contamination of the fish. Further on, glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was investigated as a potential housekeeping gene. MATERIALS AND METHODS: Female dab were caught in the summer of 2004 in the North Sea and in the Baltic. EROD activity was determined in liver samples by a kinetic fluorimetric assay according to a standard protocol. The gene expression of CYP1A (cytochrome P450 1A) and GAPDH were determined by means of RT-PCR. Results were compared to gonado somatic index and to the concentration of PAH metabolite 1OHPyr (1-hydroxypyrene) analysed in the bile fluids of the fish, respectively. RESULTS: Dab from all stations showed a considerable individual variation in the levels of both CYP1A mRNA and EROD. Highest mean values for CYP1A mRNA and EROD were detected in the northern part of the sampling area. In contrast, the PAH metabolite 1OHPyr was found at the highest concentration in fish caught near the German coast. CYP1A mRNA and EROD showed only a minor but significant correlation (r = 0.32, p < 0.05, n = 123). 1OHPyr in bile correlated significantly (p < 0.05) with the amount of GAPDH mRNA content in the liver. DISCUSSION: The significant but low correlation of CYP1A mRNA and EROD activity on an individual basis illustrates that these two parameters are apparently not closely linked. However, maximum EROD values correspond with maximum CYP1A mRNA concentrations when station means are regarded. Because EROD and CYP1A mRNA in dab follow different physiological principles, their application will lead to related but not identical monitoring results. This should be taken into account when future marine monitoring programmes are designed. The results also indicate that PAH are not the crucial factor for CYP1A and EROD levels in dab from the off-shore areas in the North Sea. This is remarkable because the PAH metabolism is known to be CYP1A-dependent and the widely used biomarker EROD has been recommended for monitoring PAH-related effects in fish from the North Sea. Due to a correlation between GAPDH and 1OHPyr, GAPDH was not suitable as housekeeping gene for dab. CONCLUSIONS: Neither the results from EROD nor from CYP1A1 mRNA measurements in dab reflected their exposure to PAH as measured by the PAH metabolite 1OHPyr. Thus, the question arises of whether EROD or CYP1A mRNA is a suitable biomarker at all to indicate PAH exposure in dab from the open North Sea. RECOMMENDATIONS AND PERSPECTIVES: For future biological effect monitoring, it is advisable to measure more and predominately independent parameters by RT-PCR and to incorporate more components of the detoxification system.     

Structure dependent induction of CYP1A by polychlorinated biphenyls in hepatocytes of male castrated pigs

Hepatocytes cultures prepared from castrated pig hepatocytes (Great Yorkshire x Dutch Landrace), as a model for human liver, were used to study the effect of twenty polychlorinated biphenyls (PCBs) on CYP1A activity, measured as the dealkylation of either ethoxyresorufin or methoxyresorufin. The selection of the PCBs was based on their differences in physico-chemical properties. The non-ortho and mono-ortho substituted PCBs were the most potent CYP1A inducers in pig hepatocytes. In addition, several multiple-ortho substituted congeners, with five or more chlorine atoms, were inducers of CYP1A activity as well. Their relative effect potencies (REP) were proximately 10,000 times lower than the most potent congener, 3,3',4,4',5 PeCB (PCB#126). Using partial least-squares (PLS) modeling, predictions of CYP1A activity could be made for all tetra to hepta substituted congeners. Several multiple-ortho substituted PCBs, which are highly abundant in the biotic and abiotic environment, have been found to induce CYP1A activity in pig hepatocytes. Because induction of CYP1A activity is used as biomarker for Ah-receptor mediated responses, it is suggested to include these congeners in future risk assessment.     

Water chemistry and fish community responses to episodic stream acidification in Pennsylvania, USA

Five streams were studied on the Northern Appalachian Plateau of Pennsylvania from October 1988 through June 1989 to determine chemical changes that occur during episodic storm run-off and the responses of fish to these events. These second-order streams flowed through undisturbed, wooded, sandstone bedrock catchments with surface areas ranging from 500 to 1000 hectares. Median pH of precipitations was about 4.2, and among streams it ranged from 5.0 to 6.2. During storm events, pH declined by as much as 1.2 units and peak concentrations of total monomeric Al ranged from < 0.01 to 0.75 mg litre(-1). Organically bound A1 was generally a minor component of total monomeric A1. Wild brook trout (Salvelinus fontinalis) were found in all streams, although only a remnant population existed in the most acidic stream. Sculpins (Cottus bairdi or C. cognatus) were collected only in the two streams with the least severe episodes. Mortality of brook trout and sculpins in in situ bioassays ranged from 0 to about 80% among streams during acidic episodes and was positively related to concentrations of total dissolved Al. Radio-tagged brook trout moved downstream during episodes when Al reached toxic concentrations. Some displaced trout were found near groundwater seeps, where pH was higher and dissolved Al was lower than in the main channel.     

‘Fishing’ for Alternatives to Mountaintop Mining in Southern West Virginia

Mountaintop removal mining (MTR) is a major industry in southern West Virginia with many detrimental effects for small to mid-sized streams, and interest in alternative, sustainable industries is on the rise. As a first step in a larger effort to assess the value of sport fisheries in southern West Virginia, we estimate the potential abundances of two popular sport fishes—smallmouth bass (Micropterus dolomieu) and brook trout (Salvelinus fontinalis)—in the Coal River Basin (CRB). A self-thinning model that incorporates net primary production and terrestrial insect subsidies is first used to predict potential densities of adult (age 1+) smallmouth bass and brook trout. Predicted densities (fish ha^?1) are then multiplied by the surface area of the CRB stream network (ha) to estimate regional abundance. Median predicted abundances of bass and trout are 38 806 and 118 094 fish (total abundances with the CRB), respectively. However, when streams that intersect permitted MTR areas in the CRB are removed from the dataset, predicted abundances of bass and trout decrease by ~12–14 %. We conclude that significant potential exists in the CRB to capitalize on sport fisheries, but MTR may be undermining this potential.     

Tissue distribution and effects on hepatic xenobiotic metabolising enzymes of 2,3,3',4,4'-pentachlorobiphenyl (PCB-105) in COD (Gadus morhua) and rainbow trout (Oncorhynchus mykiss)

2,3,3',4,4'-Pentachlorobiphenyl, PCB-105 (IUPAC no. 105) was orally administered twice with a 4-day interval between dosings (total dose 10 mg kg(-1) body weight) to gonadally immature cod and rainbow trout of both sexes. The fish were killed 9 and 17 days after the first treatment, and the effects of PCB-105 on hepatic xenobiotic metabolising enzymes were determined by examining the cytochrome-P450-dependent ethoxyresorufin-O-deethylase (EROD) and aldrin epoxidase activities, and the EROD-catalysing P450 1A1 protein by indirect enzyme-linked immunosorbent assay (ELISA). Glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene was included. The hepatic levels of the compound were determined. In addition, the distribution patterns of radio-labelled PCB-105 were studied by whole-body autoradiography. In exposed rainbow trout EROD activity and P450 1A1 by enzyme linked immunosorbent assay (ELISA) were significant induced, while GST activity was significant reduced. Exposed cod did not show significantly different enzyme values from controls, but percentage fat in the liver was significantly reduced. The whole cod liver contained about 1000 times more PCB-105 than the corresponding trout liver, and on a fat-weight basis the PCB level was five to six times higher in cod liver than in the rainbow trout liver. The autoradiographical investigation revealed high concentrations of radiolabelled compound in the liver and the brain of cod, while in rainbow trout the radiolabel was mainly confined to extrahepatic fat depots. These results demonstrate that the mono-ortho chlorinated coplanar analogue, PCB-105, has a different distribution pattern in the two fish species and that the potential for induction of the hepatic xenobiotic metabolising enzyme system seems to be lower in the cod than in the rainbow trout.     

Hepatic monooxygenase (CYP1A and CYP3A) and UDPGT enzymatic activities as biomarkers for long-term carbofuran exposure in tench (Tinca tinca L)

The effect of a long-term exposure of tenchs to different concentrations (10 and 100 μ g/L) of the pesticide carbofuran has been evaluated. Microsomal hepatic cytochrome P450 subfamily 1A (CYP1A) and 3A (CYP3A) activities, as well as the phase II enzyme uridine diphospho-glucuronosyltransferase (UDPGT) activity were evaluated as adequate biomarkers of fish exposure to environmentally relevant concentrations of the pesticide carbofuran in freshwater ecosystems. A clear time-dependent inhibition of both CYP1A and UDPGT activities was observed in fish exposed to the highest dose of carbofuran with respect to controls, whereas in the case of CYP3A activity, values of exposed animals did not show a clear pattern of alteration during the experiment. The results of the present study demonstrated that hepatic CYP1A and UDPGT activities from tench could be considered as sensitive biomarkers for carbamate pesticides in polluted water, thus allowing future and ecologically relevant biomonitoring studies with this species.     

Hepatic monooxygenase (CYP1A and CYP3A) and UDPGT enzymatic activities as biomarkers for long-term carbofuran exposure in tench (Tinca tinca L)

The effect of a long-term exposure of tenchs to different concentrations (10 and 100 micro g/L) of the pesticide carbofuran has been evaluated. Microsomal hepatic cytochrome P450 subfamily 1A (CYP1A) and 3A (CYP3A) activities, as well as the phase II enzyme uridine diphospho-glucuronosyltransferase (UDPGT) activity were evaluated as adequate biomarkers of fish exposure to environmentally relevant concentrations of the pesticide carbofuran in freshwater ecosystems. A clear time-dependent inhibition of both CYP1A and UDPGT activities was observed in fish exposed to the highest dose of carbofuran with respect to controls, whereas in the case of CYP3A activity, values of exposed animals did not show a clear pattern of alteration during the experiment. The results of the present study demonstrated that hepatic CYP1A and UDPGT activities from tench could be considered as sensitive biomarkers for carbamate pesticides in polluted water, thus allowing future and ecologically relevant biomonitoring studies with this species.     

Uptake of 17β-estradiol and biomarker responses in brown trout (Salmo trutta) exposed to pulses

In streams, chemicals such as 17β-estradiol (E2) are likely to occur in pulses. We investigated uptake and biomarker responses in juvenile brown trout (Salmo trutta) of 3- or 6-h pulses of concentrations up to 370 ng E2 L⁻¹. Uptake by the fish was estimated from disappearance of E2 from tank water. A single 6-h pulse of 370 ng E2 L⁻¹ increased the plasma vitellogenin concentration, liver Erα- and vitellogenin-mRNA. Exposure to 150-160 ng E2 L⁻¹ for 6 h increased vitellogenin in one experiment but not in another. Two 6-h pulses had a larger effect one pulse. Brown trout in the size range 24-74 g took up E2 linearly with time and exposure concentration with a concentration ratio rate of 20.2 h⁻¹. In conclusion, the threshold for induction of estrogenic effects in juvenile brown trout at short term pulse exposure appears to be in the range 150-200 ng E2 L⁻¹.     

Use of blood parameters in fish to assess acidic stress and chloride pollution in French running waters

The study investigated whether plasmatic parameters in fish such as Cl- content could serve as physiological indicators to evaluate water quality. The variations of plasma Cl- content in two fish species caught in a wide range of rivers representative of the hydrographic system of Lorraine (N-E France) were investigated. First, we studied autochthonous populations of chub (Leuciscus cephalus) which is a widespread species in the rivers of Lorraine. Organisms living in highly mineralised rivers (>1500 microScm(-1))--either naturally or due to salt mine contamination--showed plasma Cl- content significantly greater than organisms living in river with a lower mineralisation. Second, we investigated plasma Cl- content of brown trout (Salmo trutta fario) in poorly mineralised streams (<80 microS cm(-1)) with different degrees of acidification, both on autochthonous and transferred organisms. While indigenous trout maintained their Cl- content even in the acidic streams, transferred trout exhibited an important decrease of Cl- content after 48 h of exposure under acidic conditions.     

The response of stream-dwelling fish to liming

The fish fauna in 22 limed and seven unlimed small streams was monitored using yearly electrofishing to assess the effects of liming on species occurrence and abundance. The liming techniques were divided into three main methods, lake liming, doser liming and wetland liming, to evaluate whether different strategies had different effects on the fish fauna. The predominant species at the investigated stations were salmonids, mainly brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). Other species generally occurred in low numbers. The average number of fish species did not increase significantly after liming, which was probably due to recolonization difficulties caused by the frequent occurrence of migration obstacles in the streams. After liming the density of salmonids increased significantly, irrespective of the liming method. Other fish species showed no general increase, but in individual streams significant increases of European minnow (Phoxinus phoxinus) and bullhead (Cottus gobio) occurred. Some unlimed streams had acid spates with a pH below 6, which immediately lowered the numbers of salmonid parr. Other unlimed streams lost fish species progressively due to increasing acidification. pH, and probably increased levels of metals, were the major factors regulating the fish fauna. Few examples of biotic interactions were observed, but with an increase of Atlantic salmon parr after liming, brown trout abundances decreased in three streams.     

Altitudinal and thermal gradients of hepatic Cyp1A gene expression in natural populations of Salmo trutta from high mountain lakes and their correlation with organohalogen loads

The biomarker of xenobiotic exposure cytochrome p450A1 (Cyp1A) was used to analyze the biological response to chemical pollution in Salmo trutta (brown trout) from nine high mountain European lakes in Norway, Tatras, Tyrol, and central Pyrenees. Hepatic Cyp1A mRNA levels correlated both with the reciprocal of absolute annual average air temperatures of the sampled lakes and with muscle concentrations of several hydrophobic organohalogen compounds (OC), including chlorinated polychlorobiphenyls (PCB), DDE, and DDT. The correlation between Cyp1A expression and OC content was observed across the whole temperature range (between −0.7 °C and +6.2 °C), but also in the absence of any thermal gradient. We concluded that airborne pollutants accumulate in high mountain lake fish at concentrations high enough to increase Cyp1A expression, among other possible effects. As geographical distribution of semi-volatile OC is strongly influenced by air temperatures, future climate modifications will potentially enhance their physiological effects in lake ecosystems.     

PCB153 and p,p′-DDE disorder thyroid hormones via thyroglobulin,deiodinase 2, transthyretin,hepatic enzymes and receptors

Polychlorinated biphenyls (PCBs) and DDT are widespread environmental persistent organic pollutants that have various adverse effects on reproduction, development and endocrine function. In order to elucidate effects of PCBs and DDT on thyroid hormone homeostasis, Sprague–Dawley rats were dosed with PCB153 and p,p′-DDE intraperitoneally (ip) for five consecutive days and sacrificed within 24 h after the last dose. Results indicated that after combined exposure to PCB153 and p,p′-DDE, total thyroxine , free thyroxine, total triiodothyronine, and thyroid-stimulating hormone in serum were decreased, whereas free triiodothyronine and thyrotropin-releasing hormone were not affected. Thyroglobulin and transthyretin levels in serum were significantly reduced. mRNA expression of deiodinases 2 (D2) was also suppressed, while D1 and D3 levels were not significantly influenced after combined exposure. PCB153 and p,p′-DDE induced hepatic enzymes, UDPGTs, CYP1A1, CYP2B1, and CYP3A1 mRNA expressions being significantly elevated. Moreover, TRα1, TRβ1, and TRHr expressions in the hypothalamus displayed increasing trends after combined exposure to PCB153 and p,p′-DDE. Taken together, observed results indicate that PCB153 and p,p′-DDE could disorder thyroid hormone homeostasis via thyroglobulin, deiodinase 2, transthyretin, hepatic enzymes, and hormone receptors.     

Cytochrome P4501A induction and testosterone hydroxylation in cultured hepatocytes of four fish species

Primary hepatocytes of the rainbow trout (Oncorhynchus mykiss), flounder (Platychthis flesus), dab (Limanda limanda) and lemon sole (Microstomus kitt) were exposed to 3,3'4,4'5 pentachlorobiphenyl (PCB 126) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for two days. This resulted in a dose-dependent induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD), or methoxyresorufin O-deethylase (MROD) activity. In all species, a linear relationship was observed between EROD and MROD activities, suggesting that the same CYP1A enzyme metabolizes the two alkoxy-resorufin substrates. Exposures of hepatocytes of flounder or dab to TCDD, resulted in a 59-fold and 8.2-fold induction of EROD activity, respectively. This did not concur with a change in the in vitro testosterone hydroxylation profiles of both species. These and other in vitro data indicate that TCDD exposure does not influence monooxygenase activities involved in testosterone hydroxylation. Furthermore, CYP1A is of minor importance for testosterone hydroxylation in these fish species.