Marcela Havelková

Comparison of Mercury Distribution Between Liver and Muscle - A Biomonitoring of Fish from Lightly and Heavily Contaminated Localities

Tissue samples from 1,117 fish of 25 species were collected from 1991 through 1996 at 13 locations along the River Elbe. The principal indicator species were perch (Perca fluviatilis) (n=118), chub (Leuciscus cephalus L.) (n=113) and roach (Rutilus rutilus) (n=138). Mercury (Hg) concentrations in muscle and liver were determined by atomic absorption spectrometry. The liver/muscle index in three indicator species from heavily contaminated and lightly contaminated localities were significantly different. In fish from heavily contaminated localities, Hg was deposited preferentially in the liver (the depository for inorganic and organic forms of Hg), while in lightly contaminated areas, it was deposited preferentially in muscle.

Biochemical Markers for Assessing Aquatic Contamination

Biochemical markers, specifically enzymes of the first phase of xenobiotic transformation - cytochrome P450 and ethoxyresorufin-O-deethylase (EROD) - were used to determine the quantities of persistent organic pollutants (POPs) in fish muscle (PCB, HCB, HCH, OCS, DDT). Eight rivers were monitored (Orlice, Chrudimka, Cidlina, Jizera, Vltava, Ohře and Bílina; and the River Blanice was used as a control). The indicator species selected was the chub (Leuciscus cephalus L.). There were no significant differences in cytochrome P450 content between the locations monitored. The highest concentration of cytochrome P450 in fish liver was in the Vltava (0.241 nmol mg-1 protein), and the lowest was in the Orlice (0.120 nmol mg-1 protein). Analysis of EROD activity showed a significant difference between the Blanice and the Vltava (P< 0.05), and also between the Orlice and the Vltava (P< 0.01), the Orlice and the Bílina (P< 0.01), and the Orlice and the Ohře (P< 0.05). The highest EROD activity in fish liver was in the Vltava (576.4 pmol min-1 mg-1 protein), and the lowest was in the Orlice (63.05 pmol min-1 mg-1 protein). In individual locations, results of chemical monitoring and values of biochemical markers were compared. A significant correlation (P< 0.05) was found between biochemical markers and OCS, and PCB. Among the tributaries studied those that contaminated the Elbe most were the Vltava and the Bílina. These tributaries should not be considered the main sources of industrial contamination of the River Elbe, because the most important contamination sources were along the river Elbe itself.

Organic Pollutant Contamination of the River Tichá Orlice as Assessed by Biochemical Markers

·iroka Z. , J . Kri j t , T. Randak, Z. Svobodova, G. Pe‰kova, J . Fuksa, J . Haj‰ lova, J . Jarkovsk , M. Janska: Organic Pollutant Contamination of the River Elbe as Assessed by Biochemical Markers. Acta Vet Brno 2005, 74: 293-303. The aim of the study was to assess contamination of the River Elbe basin using selected biochemical markers. Biochemical markers selected were enzymes of the first stage of xenobiotic transformation, namely cytochrome P450 (CYP 450) and ethoxyresorufin-O-deethylase (EROD). The results were correlated with the most important inducers of the enzymes, i.e. polychlorinated biphenyls (PCB) concentrations in muscle tissue of fish, polyaromatic hydrocarbons (PAH) values in bottom sediments and 1-hydroxypyrene (1-OHPY) values in fish bile (terminal metabolite of PAH, or, rather, of one of them, i.e. pyrene), which were determined during the chemical monitoring of the River Elbe basin. The indicator species selected was chub (Leuciscus cephalus L.), which was captured at ten locations in the River Elbe basin. A comparison between the EROD activity and the CYP 450 content along the longitudinal profile of the Elbe showed a significant correlation at the level of significance of p < 0.05. The highest EROD activity levels in the liver were ascertained in Zelain (341 pmol·min-1·mg-1), Valy (263.2 pmol·min-1·mg-1) and Lysa nad Labem (179.17 pmol·min-1·mg-1). In Blanice (control location), EROD activity was significantly lower than in any of the other locations studied (p < 0.05). The study failed to produce an unambiguous proof of any correlations between detoxification enzyme activity (CYP 450 and EROD) in the liver and their two important inducers (PCB and PAHs). The possibility that other substances causing activation or inhibition of detoxification enzymes were in play is also discussed. Cytochrome P450, EROD, Leuciscus cephalus L., liver, PCB, PAH, 1-hydroxypyrene The River Elbe is one of the most important European rivers (total length 1103.5 km). Its extensive basin of a total of 148 268 km2 lies on the territory of two countries, i.e. the Czech Republic (51 336 km2) and Germany (96 932 km2). Intensive research of the Czech and German reaches of the Elbe started in 1991 under the Elbe I (1991 1994) project, and continued with the Elbe II (1995 1998) and Elbe III (1999 2002) projects. In those projects, large quantities of data regarding sources of pollution, chemical monitoring of hazardous substances in various components of the aquatic environment, water quality, etc. were collected and evaluated (Nesmurak 1994; BlaIkova et al. 1998; BlaIkova 2002). To enhance the relevance of results obtained by chemical monitoring, it is, however, also ACTA VET. BRNO 2005, 74: 293–303 Address for correspondence: ·iroka Zuzana Department of Public Veterinary Health and Toxicology University of Veterinary and Pharmaceutical Sciences Brno Palackeho 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 780 E-mail: sirokaz@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm necessary to assess the effects of anthropogenic pollution of the aquatic environment on fish. One of possible ways of assessing such effects is the use of biochemical markers of contamination. They are measurable biochemical parameters responding usually to substances with the same mechanism of toxic effect. That means they are not, with some exceptions, specific for individual xenobiotic substances. Their advantage lies in their ability to provide comprehensive information on the effects of pollution, i.e. to reflect synergic or antagonistic effects of individual components contributing to pollution. In 2003, the Elbe IV Project was started. For reasons mentioned above, chemical monitoring in fish was complemented with assay of biochemical contamination markers. Attention focused primarily on the enzymes of the first phase of xenobiotics conversion, i.e. cytochrome P450 and ethoxyresorufin-O-deethylase (EROD). Cytochrome P450 is an important biochemical marker of surface water contamination with some industrial and agricultural pollutants (Stegeman and Lech 1991). It is now believed that the most useful is the 1A family of cytochrome P450 (Machala et al. 1997; Schlenk and Di Giul io 2002). The most potent inducers for that isoform are substances from the groups of polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), nitrated polycyclic aromatic hydrocarbons (NPAH) and dioxins (e.g. 2, 3, 7, 8 TCDD) (White et al. 1997; Nilsen et al. 1998; Jung et al. 2001; Schlenk and Di Giul io 2002). On the other hand, chronic exposure to these contaminants can cause a lack of CYP1A induction response (Brammell et al. 2004), and also assessment of CYP1A at the time of spawning can influence its level, because estrogens can decrease CYP1A induction (Elskus 2004). Male fish seem to be more sensitive to PAH and PCB than female fish (McArdle et al. 2004). The induction of the CYP1A family is mediated by the aryl hydrocarbon receptor (AhR) (Bil l iard 2002). Following its interaction with xenobiotic substances, it is carried to the nucleus where it is the cause of enhanced expression of genes for CYP1A and, subsequently, of increased synthesis of cytochrome proteins. The potential toxicity of pollutants depends on their affinity to the AhR. The CYP1As are also responsible for the metabolic activation of most of the known promutagens and procarcinogens, and its elevated levels are responsible for such negative effects as cocarcinogenesis, immunotoxicity and reproduction disorders (Lewis et al. 2003; Carlson et al. 2004). The model CYP1A activity is the enzyme ethoxyresorufin-O-deethylase (EROD), with its ability to convert substrates to products demonstrating fluorescence, which can then be measured. This enzyme is an important biochemical marker of contamination. The aim of the study presented here was to use the assessment of biochemical markers cytochrome P450 and EROD in the livers of the indicator fish species (Leuciscus cephalus L.) to evaluate contamination levels in various locations within the River Elbe basin. Results of chemical monitoring relevant for the above contamination markers are also outlined and correlated in the paper. They were PCB concentrations in chub muscle tissues, concentrations of 1-hydroxypyrene (1-OHPY) in chub bile samples (i.e. the final metabolite of PAHs, or rather of pyrene), and PAH concentrations in bottom sediments in the locations studied. Materials and Methods Animals and Sampling The chub (Leuciscus cephalus L.) was selected as the most suitable indicator species. The chub is a common freshwater cyprinid species that inhabits both clean and polluted rivers (Baru‰ et al. 1995). The fish were captured with the use of a diesel-electric generator in 10 locations in the River Elbe basin. The locations studied were Podoli and Zelain at the River Vltava, a tributary to the Elbe, and Verdek, Numaice, Valy, Lysa nad Labem, Obfiistvi, Duain and Hfiensko along the River Elbe. The control location was upstream of the Husinec water reservoir at the river Blanice in the Vltava basin (Fig. 1). The fish were captured in July 2003 (average water temperature 21.4 °C). In the control location, fish were captured in September 2003 (water temperature 15 °C). In each location, eight chub (both males and females) were captured (except in Lysa nad Labem where only 3 chub were captured). The chub 294

Biochemical markers of contamination in fish toxicity tests

Biochemical markers of contamination in fish toxicity tests Markers of xenobiotic metabolization (cytochrome P450, ethoxyresorufin-O-deethylase, glutathione and glutathione-S-transferase) were investigated in the liver of the common carp Cyprinus carpio after 28-day exposure to different pesticide formulations. The fish exposed to herbicide Sencor 70 WG (metribuzin 700 g/kg) of 0.25 and 2.5 mg/l showed no change in cytochrome P450 and activity of ethoxyresorufin-O-deethylase when compared to control. Successor 600 (pethoxamid 600 g/l) of 0.06; 0.22 and 0.60 mg/l did not affect either cytochrome P450 or the activity of ethoxyresorufin-O-deethylase. However, in fish exposed to Successor 600 of 0.22 and 0.60 mg/l, there was a rise in glutathione and in the activity of glutathione-S-transferase (p<0.05), with Spearmans correlation r = 0.23 at p<0.05. Spartakus (prochloraz 450 g/l) of 0.36 and 1.08 mg/l induced cytochrome P450 and ethoxyresorufin-O-deethylase (p<0.05), with Spearmans correlation r = 0.49 at p<0.01. Glutathione increased in fish exposed to 1.08 mg/l (p<0.05), the activity of glutathione-Stransferase rose (p<0.05) in all concentrations tested (0.108; 0.36 and 1.08 mg/l). Spearmans correlation between glutathione and GST was r = 0.38; p<0.01). The obtained data contribute to a better understanding of detoxification of the selected xenobitics in fish. Although biomarkers of the first phase of metabolization are considered to be more sensitive, our results indicate higher sensitivity of the second phase biomarkers.

Biochemical markers for the assessment of aquatic environment contamination

Biochemical markers for the assessment of aquatic environment contamination The need for assessment of aquatic ecosystem contamination and of its impact on water dwelling organisms was developed in response to rising aquatic environmental pollution. In this field study, liver enzymes of phase I and phase II of xenobiotic transformation, namely cytochrome P450, ethoxyresorufin-O-deethylase, glutathione-S-transferase and tripeptide glutathione were used to assess the contamination of the aquatic environment at different rivers in the Czech Republic. The indicator species selected was the male chub (Leuciscus cephalus L.) and male brown trout (Salmo trutta fario). Chemical analyses included also the assessment of the most important inductors of previously mentioned biochemical markers. The major inductors of monitored biomarkers are industrial contaminants which belong to a large group of organic pollutants (PCB, PAH, PCDD/F, DDT, HCH, HCB and OCS), persistent in the environment. Four different groups of river basins were assessed: the River Tichá Orlice and its tributary the Kralický brook; important tributaries of the River Elbe (the rivers Orlice, Chrudimka, Cidlina, Jizera, Vltava, Ohře and Bílina); major rivers in the Czech Republic (the rivers Lužnice, Otava, Sázava, Berounka, Vltava, Labe, Ohře, Svratka, Dyje, Morava and Odra) and the River Vltava. The use of the biochemical markers together with chemical analyses seems to be an effective way to monitor the quality of aquatic environment.

Assessment of contamination of the Svitava and Svratka rivers in the Czech Republic using selected biochemical markers

The aim of the present study is to assess aquatic ecosystem contamination using selected biochemical markers: cytochrome P450, ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), tripeptide glutathione, vitellogenin, and 11-ketotestosterone in chub (Leuciscus cephalus L.). Seven locations on the Svitava and Svratka rivers (in the Brno conurbation, Czech Republic) were assessed. The results were compared with the levels of the most important inductors of these biomarkers: organic pollutants hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), DDT and its metabolites, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons in bottom sediment, fish muscle, and semipermeable membrane devices (SPMDs) and metals in sediment. The highest levels of pollutants were observed at sites situated downstream from Brno, especially at Modrice and Rajhradice. Significant positive correlations (p < 0.05) were found between EROD activity and HCH concentration in SPMDs, and also between GST and EROD activity with HCB concentration in muscle, after adjusting for age.