Mustafa Canli

The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species

Heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) concentrations in the muscle, gill and liver of six fish species (Sparus auratus, Atherina hepsetus, Mugil cephalus, Trigla cuculus, Sardina pilchardus and Scomberesox saurus) from the northeast Mediterranean Sea were measured and the relationships between fish size (length and weight) and metal concentrations in the tissues were investigated by linear regression analysis. Metal concentrations (as microg/g d.w.) were highest in the liver, except for iron in the gill of Scomberesox sauris and lowest in the muscle of all the fish species. Highest concentrations of Cd (4.50), Cr (17.1) and Pb (41.2) were measured in liver tissues of T. cuculus, Sardina pilchardus and A. hepsetus, respectively. The liver of M. cephalus showed strikingly high Cu concentrations (202.8). The gill of Scomberesox saurus was the only tissue that showed highest (885.5) iron concentrations. Results of linear regression analysis showed that, except in a few cases, significant relationships between metal concentrations and fish size were negative. Highly significant (P<0.001) negative relationships were found between fish length and Cr concentrations in the liver of A. hepsetus and M. cephalus, and Cr concentrations in the gill of T. cuculus. Cr and Pb concentrations in the liver and Cu concentrations in all the tissues of Scomberesox saurus also showed very significant (P<0.001) negative relationships. Negative relationships found here were discussed.

Response of antioxidant system of freshwater fish Oreochromis niloticus to acute and chronic metal (Cd, Cu, Cr, Zn, Fe) exposures.

Antioxidant systems are known to be sensitive to metal exposures and are suggested to use in predicting sublethal metal toxicity. In this study, several antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPX) and glutathione reductase (GR) were measured in the liver and kidney of Oreochromis niloticus exposed to sublethal concentrations of metals (Cd, Cu, Cr, Zn and Fe), using an acute (20 μM, 48 h) or subchronic (10 μM, 20 d) protocol. Beside the several increases of antioxidant enzyme activities, general inhibition was recorded after acute and chronic metal exposures. Results indicated that there were variations in responses of the enzymes to metal exposures, depending upon tissues, metals and exposure types. This study emphasized that the antioxidant enzymes are very sensitive to metals as their activities altered significantly, suggesting they could be helpful in predicting sublethal metal toxicity and useful as an early warning tool in natural monitoring studies.

Changes in serum biochemical parameters of freshwater fish Oreochromis niloticus following prolonged metal (Ag, Cd, Cr, Cu, Zn) exposures.

Fish serum may reflect status of many biochemical processes in the metabolism. Heavy metals, as environmental stressors, may alter serum biochemical parameters in fishes. Thus, freshwater fish, Oreochromis niloticus, were exposed to low levels (0.05 mg/L) of metals (silver [Ag], cadmium [Cd], copper [Cu], chromium [Cr], zinc [Zn]) to investigate responses of serum biochemical parameters over different exposure periods (0, 5, 10, 20, 30 d). Fish mortality occurred only in Ag exposure, as all fish died between days 12 to 16. Activities of alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase (AST) were altered only in Cu- and Cd-exposed fish. Both Cd and Cu exposures decreased the activity of ALP, although they increased the activities of ALT and AST. Glucose concentrations increased in Ag-, Cd-, and Cu-exposed fish, with a sharp increase occurring in Ag-exposed fish before mortality began. Total protein and triglyceride concentrations increased in Ag-exposed fish, although they decreased in Cu-exposed ones. However, all metal exposures increased cholesterol concentration in the serum. Concentration of blood urea nitrogen increased in Ag-, Cd-, and Cu-exposed fish, although it decreased in Cr-exposed ones. Calcium level decreased only in Cu-exposed fish, and Cl(-) level decreased in Ag-exposed fish. Silver and Cu exposures also decreased Na(+) level in the serum. Cadmium and Cu exposures increased serum K(+) levels. The present study, investigating the effects of environmentally realistic metal exposures on serum biochemical parameters, demonstrated that fish serum could sensitively reflect environmental metal stress. Thus, it suggests that serum biochemical parameters could be used as important and sensitive biomarkers in ecotoxicological studies concerning the effects of metal contamination and fish health.

Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures

In this study, levels of reduced glutathione (GSH) and metallothionein (MT) which are known to be biomarker of metal exposures were measured in a freshwater fish Oreochromis niloticus following exposure to 0, 5, 10 and 20μM concentrations of Cu, Zn, Cd and Pb for 14 days. Metals and GSH were measured in the liver, gill, intestine, muscle and blood, and MT in the liver. Copper accumulation occurred only in the gill, while Zn accumulation occurred only in the muscle. Lead accumulated in the liver and gill, whereas Cd accumulated in all the tissues. Metal exposures did not alter GSH levels in the blood, muscle and gill, but its levels increased in the liver following Cd, Zn and Cu exposures. MT levels in the liver increased only in Cd-exposed fish. The results showed that there was no significant change in tissue GSH levels following metal exposures, except in the liver. The levels of liver GSH increased significantly by all the metals, except lead. Data indicated that only the liver may be suitable indicator tissue to determine the response of GSH and MTs to metal exposure in environmental monitoring studies.

Investigations on the osmoregulation of freshwater fish (Oreochromis niloticus) following exposures to metals (Cd, Cu) in differing hardness.

Hardness is one of the most important factors in water chemistry as it affects fish physiology and metal toxicity. The aim of this study was to investigate osmoregulatory responses in the Nile tilapia Oreochromis niloticus exposed to copper and cadmium (1.0μg/mL) in soft water (SW) (hardness 80mg CaCO3/L and conductivity 1.77mS/cm) and hard water (HW) (hardness 320mg CaCO3/L and conductivity 5.80mS/cm) for 0, 1, 7 and 14 days. Following the exposures, Na(+)/K(+)-ATPase activity, ion and Cu levels in the gill, kidney and intestine were measured. There was no fish mortality within 14 days, except Cu exposure in SW which killed all fish between 8 and 12 days. Generally, Na(+)/K(+)-ATPase activity was altered by both metal exposures in the gill and kidney as it increased in HW condition, but decreased in SW condition. There were also alterations in Na(+)/K(+)-ATPase activity in the intestine as its activity generally decreased. Data, in general, showed that Cd was more effective on Na(+)/K(+)-ATPase activity comparing to Cu. However, ion levels altered mainly in the kidney and intestine. Tissue metal accumulation was higher in fish tissues from SW condition comparing to HW condition. Data represented here showed that the effects of metals differed in differing water hardness. This suggests that special attention should be paid to the water chemistry when natural monitoring studies are carried out. This study also suggests that the response of osmoregulation system of fish may be a sensitive indicator under stressful conditions in different natural waters.

Alterations in the serum biomarkers belonging to different metabolic systems of fish (Oreochromis niloticus) after Cd and Pb exposures.

The serum of vertebrates including human gives sufficient data about the current health status of organism in concern. Biomarkers have gained importance in evaluation of data from biological monitoring studies. In this study, freshwater fish Oreochromis niloticus were individually exposed Cd or Pb using acute (10μM, 2 d) and chronic (20μM, 20 d) exposure protocols to investigate the alterations in serum parameters (ALP, ALT, AST, LDH, lipase, glucose, protein, cholesterol and triglyceride) and response of the endocrine system functioning in different axis, namely HPI (cortisol), HPT (TSH, T3, T4), gonadal (LH, FSH) and prolactin. Data showed that except LH, the levels of all hormones decreased significantly following exposure to Cd and Pb both in acute and chronic exposures. In acute exposures, the activity of ALP and levels of cholesterol and triglyceride decreased significantly following both Cd and Pb exposures, while glucose levels increased only after Cd exposure. In chronic exposures, both metal exposures caused significant decreases in ALP activity and levels of cholesterol and triglyceride, though there were increases in glucose level after Cd exposure and AST, ALT and LDH levels after Pb exposure. Data emphasized the importance of biomarker selection and multiparameter usage of relevant systems in ecotoxicological research to achieve proper evaluation of environmental data.

The effects of increased freshwater salinity in the biodisponibility of metals (Cr, Pb) and effects on antioxidant systems of Oreochromis niloticus

Anthropogenic activities can increase the salinity of freshwaters and this may cause stress for fish and affect metal bioavailability. Oxidative stress biomarkers are of great interest due to their responses to environmental stressors which provide valuable data for biological monitoring of aquatic pollution. Thus, the individual and combined effects of salinity and metals (Cr, Pb) were investigated in the liver of freshwater fish Oreochromis niloticus in the present study. Fish were exposed to salinity (2 and 8 ppt) alone and salinity+metal (1 μg/mL Pb and Cr) combination exposures for 0, 1, 7 and 14 days and subsequently antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPX; glutathione reductase, GR and glutathione S-transferase, GST) activities and glutathione (GSH) levels in the liver were measured. Data showed that all the parameters varied in relation to metal species, exposure durations and salinity levels. Profound alterations on the measured parameters were detected at the lower salinity compared to the higher one. Salinity increase effectively stimulated the antioxidant parameters. The effects of salinity and metals on the measured parameters increased as the exposure duration prolonged. SOD was the most affected antioxidant parameter from both salinity and metals. Because metal and salinity stresses affect fish antioxidant system, this work suggests that the chemistry of freshwaters should be taken into account in natural monitoring for metal contamination in the field.

The effects of temperature and metal exposures on the profiles of metallothionein-like proteins in Oreochromis niloticus

Freshwater fish Oreochromis niloticus were exposed individually to 1mg/L concentrations of cadmium and zinc for 14 days at 20, 25 and 30°C. Heat-treated (80°C) liver tissue homogenates were run on a column packed with Sephadex G-75 to obtain eluats for determination of metallothionein-like proteins, sulfhydryl groups and metal concentrations. Elution profiles of the liver showed that there were three heat-resistant protein peaks detected at 254 and 280nm in all samples. Approximate molecular weights of proteins obtained from the first, second and third peaks were 70,000, 43,000 and 6500Da, respectively. Metallothionein-like proteins (6500) of control fish contained sulfhydryl groups and zinc, but not cadmium. Concentrations of zinc and cadmium in MT-like proteins increased in metal exposed fish and these increases were temperature dependent. However, zinc concentration in control fish decreased in relation to temperature increase. Data indicated that temperature plays significant role on metals bound on metallothioneins. This suggests that season of sampling should be taken into account when natural monitoring studies are carried out with metallothioneins.

Low water conductivity increases the effects of copper on the serum parameters in fish (Oreochromis niloticus).

The conductivity is largely determined by ion levels in water, predominant ion being Ca(2+) in the freshwaters. For this reason, the effects of copper were evaluated as a matter of conductivity of exposure media in the present study. Thus, freshwater fish Oreochromis niloticus were exposed to copper in differing conductivities (77, 163 and 330 μS/cm), using acute (0.3 μM, 3 d) and chronic (0.03 μM, 30 d) exposure protocols. Following the exposure serum parameters of fish were measured. Data showed that there was no significant alteration (P>0.05) in serum parameters of control fish. However, activities of ALP, ALT and AST decreased significantly at the lower conductivities in chronic copper exposure, but not in acute ones. Protein levels did not differ significantly in any of the exposure conditions. However, Cu exposure at the lowest conductivity sharply increased the levels of glucose in the acute exposure, while there was no significant difference in the chronic exposure. Cholesterol levels decreased only at the lower conductivities in chronic exposure, but increased in acute exposure. Similarly, triglyceride levels increased in acute exposures and decreased in chronic exposures at the lowest conductivity. There was no change in Na(+) levels, while there was an increase in K(+) levels and a decrease in Ca(2+) level at the lowest conductivity of acute exposures. However, Cl(-) levels generally decreased at the higher conductivities of chronic exposures. There was a strong negative relationship between significant altered serum parameters and water conductivity. In conclusion, this study showed that copper exposure of fish at lower conductivities caused more toxicities, indicating the protective effect of calcium ions against copper toxicity. Data suggest that conductivity of water may be used in the evaluation of metal data from different waters with different chemical characteristics.

Characterization of antioxidant system parameters in four freshwater fish species.

The potential use of antioxidant system parameters has gained considerable interest due to their pivotal role of detoxification mechanisms in environmental studies and culture fish point of view. Fish with different ecological needs may have different antioxidant capacity and response to environmental contaminants. Thus, the optimal working conditions and specific enzyme activities (Vmax and Km) of antioxidant system parameters (Superoxide dismutase, SOD; Catalase, CAT; Glutathione peroxidase, GPX; Glutathione reductase, GR and Glutathione S-transferase, GST) and glutathione (GSH) were determined in four commonly cultured freshwater fish species (tilapia; Oreochromis niloticus, carp; Cyprinus carpio, trout; Onchorhynchus mykiss and catfish; Clarias garipienus). Data showed that optimal concentrations of different buffers, pH and specific chemicals for each enzyme and GSH were similar in most cases for all fish species, except a few differences. The highest Vmax and Km values were found in carp for GPX and GST, though these values were the highest in tilapia, catfish and trout for CAT, SOD and GR, respectively. As a conclusion, optimization assays of these parameters in different bioindicator organisms based on their physiological and ecological differences may be useful for the aquatic ecosystem biomonitoring studies and also present fundamental data for utilization in aquaculture.