Influence of Sublethal Lead Concentrations on Glucose, Serum Enzymes and Ion Levels in Tilapia (Oreochromis mossambicus) Hasan Kaya1, Mehmet Akbulut2, Sevdan Yılmaz3 1 Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department of Basic Sciences, Çanakkale-Turkey, e-mail: hasankaya@comu.edu.tr 2 Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department of Basic Sciences, Çanakkale-Turkey, e-mail: mehakbulut@comu.edu.tr 3 Çanakkale Onsekiz Mart University, Marine Sciences and Technology Faculty, Department of Aquaculture, Çanakkale-Turkey, e-mail: sevdanyilmaz@comu.edu.tr Abstract. In this study, alterations in glucose, blood enzymes (alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate aminotransferase (AST)) and serum ion (P+++, Mg+, Cl-, Ca++, Fe++) levels were investigated in Tilapia (Oreochromis mossambicus), which were semi-statically exposed to different lead concentrations in vivo. The fish were exposed to low (0.5 mg/L), medium (2.5 mg/L) and high (5 mg/L) concentrations of lead during 14 days. At the end of the experiment, biochemical blood parameters such as glucose, ALP, LDH, AST, chloride and magnesium increased (p<0.05). While, LDL and calcium levels decreased (p<0.05); ALT, cholesterol, albumin, iron and phosphor were fluctuated (p<0.05). Consequently, it was found that exposure of O. mossambicus to lead concentrations affected serum biochemical parameters negatively. Keywords: lead, toxicity, glucose, serum enzymes, ion levels, Oreochromis mossambicus 1 Introduction Lead is a persistent contaminant in the natural environment that can enter the water column through geologic weathering and volcanic action, or by various anthropogenic activities including mining and smelting of lead-ores, burning of coal, effluents from storage battery industries, automobile exhausts, metal coating and finishing operations, fertilizers, pesticides and from additives in pigments and gasoline (WHO, 1995). Contamination of water through anthropogenic practices is the primary cause of lead poisoning in fish (Sorensen, 1991). Due to its nondegredable nature, it’s get into the environment and eventually enters to fish and human body system. When it can enter to the body lead can accumulate to soft tissues such as liver, kidney, nervous system and brain of fish (Berman, 1980). It is well documented that lead can impair 858 the health of humans and other organisms by neurotoxicity, renal toxicity, and deleterious effects on the hematological and cardiovascular systems (ATSDR, 2006). In studies examining the toxicity of lead on fish, it was determined that lead inhibited to Na+, K+-ATPase enzyme activity and caused to oxidative stress in tilapia (Kaya and Akbulut, 2015), it damage Ca2+ and Na+ homeostasis in trout fish at concentrations found in ecosystems (Rogers et al. 2003; Patel et al. 2006), and caused hematological (Kaya et al. 2013) and neurological (Davies et al. 1976) effects in fish under chronic conditions. However, no studies evaluating the effects of sublethal lead concentrations on the biochemical parameters of fish could be found. The present study aimed to investigate the effects of water-borne lead on fish with special reference to the blood glucose, serum enzymes and ions. 2 Material and Methods 2.1 Experimental design Tilapia fish used in this study (n=144) were obtained from Çanakkale Onsekiz Mart University (Marine Sciences and Technology Faculty, Aquaculture Department), Çanakkale, Turkey, and were adapted to ambient conditions in 12 stock aquariums, each with dimensions of 45x28x80 cm and containing 80 L of rested¸ Çanakkale city tap water, for 4 weeks. Fish weighting 45.2±5 g (mean±SD) were divided into 12 experimental aquariums, each containing 12 fish, and an experimental design with three replicates was established. Feeding was interrupted 24 h before the start of the experiments to help maintain water quality. During the experiment, the fish were fed twice a day with feed at about 2% of their body weight and their behavior was observed during each feeding. Care was also taken to ensure that all of the feed added to the tanks was eaten and that fecal waste was quickly removed from the tanks at every water change. In the experiment, fish were exposed to the following sublethal concentrations of lead: low, 0.5 mg/L; medium, 2.5 mg/L; and high, 5 mg/L. The control group was maintained in freshwater only. Concentrations were determined by considering in Ay et al. (1999). The experiment had a semi-static regime, and water was changed every day: a 75% change in the morning and a 25% change in the evening (modified from Smith et al. 2007). After each water parameters were as follows: temperature, 25.4±0.3◦C (mean±SE); dissolved oxygen, 6.31±0.11 mg/L; pH, 7.15±0.04; hardness, 125.0±6.2 mg/L CaCO3; total ammonia, 0.151±0.02 mg/L. The electrolyte composition of the dechlorinated Çanakkale tap water was measured as 0.310±0.005, 0.049±0.001, 0.534±0.001, and 0.828±0.006 mmol/L for Na+, K+, Mg+, and Ca2+, respectively. Fish were randomly sampled on days 0, 7, and 14 for blood biochemistry analysis. The experiments were performed in accordance with the guidelines for fish research established by the Animal Ethics Committee at Çanakkale Onsekiz Mart University. 859 2.2 Preparation of the Pb(NO3)2 Solution and Application The heavy metal salt, Pb(NO3)2 (99.5% purity; Sigma-Aldrich, Steinheim, Germany), was used in the experiment. To obtain the needed concentrations, the main stock solution was prepared in ultra-distilled water and appropriate dilutions made from it. 2.3 Blood Sampling In the experiment, total 12 fish on the first day (from the stock aquarium), 6 fish from each aquarium on the 7th and 14th day were used for blood analysis. For blood sampling, fish were anaesthetized with MS222 (Smith et al. 2007). They were well wiped and cleaned in order to avoid mucus mixing into the blood, and then, blood was taken from the fish through the caudal vein by a 5 ml plastic syringe, without harming the fish (Val et al. 1998). Then, a sample of blood was transferred to EDTA tubes, BD Microtainer®, UK for hematological analysis. Plastic biochemistry tubes (Kima-vacutest®, Italy) were used for biochemical analysis. Blood serum was isolated by centrifugation (4000xg, 10 min) and it was stored below -20°C. 2.4 Biochemical Analysis For biochemical analysis, the blood collected was centrifuged at 4000 rpm for 10 minutes and blood serum was separated (Bricknell et al. 1999). Then, the serum extracted was analyzed on the spectrophotometer (T80+UV/VIS) using an analyzer (Bioanalytic Diagnostic Industry, Co). The biochemical parameters that were detected during the test included glucose (GLU), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), calcium (Ca2+), magnesium (Mg+), iron (Fe++), phosphorus (P+++) and chlorine (Cl-). 2.5 Statistical analysis ANOVA with Dunnett post-test (one-way ANOVA for comparison between exposure groups and control group) was used. The statistical analysis was made by using SPSS 17.0, and the significance level was considered to be 0.05 (Logan, 2010). 3 Results Glucose and serum enzymes obtained from the study were given in Table 1.While the glucose levels registered an increase in medium and high concentrations on 7th and 14th days compared to the control group, they did not show any differences with the control group in low concentrations. While the ALP increased across all groups on day 7 compared to the control group, on day 14, the low and medium 860 concentrations registered an increase. The enzyme AST was revealed to be high across all groups on day 7 and on day 14, it was found out to be higher in low and medium concentrations compared to control group. While the ALT activity was revealed to be lower on day 7 compared to control group, on day 14, a decrease in low and medium dose was determined compared to the control group as an increase in high group was experienced. While the enzyme LDH showed similarities with control group in every group, (p>0.05), it increased across all groups on day 14. Serum electrolytes during the study are given in Table 1. While the Ca2+, one of the serum ions of the study decreased on days 7 and 14 across all groups compared to control group, the Mg+ increased in medium and high concentrations on day 7. On the other hand, while Fe++ showed a decrease in low and high doses on day 7 compared to control group, the medium dose increased. On day 14; a decrease compared to the control group in low and medium doses was determined while an increase was registered in high doses. The Cl- electrolyte showed an increase on days 7 and 14 across all groups compared to the control group. P+++ on the other hand, showed an increase in low and high concentrations on day 7 and showed a decrease on day 14 in high group compared to the control group. 861 Table 1. Effects of different concentrations of lead on glucose, serum enzymes and some serum minerals. Exposure groups are represented as follows control: 0 (Control), low (0.5 mg/L), medium (2.5 mg/L), high (5 mg/L); ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase, Ca2+, calcium; Mg+, magnesium; Cl-, chloride; Fe++, iron; P+++, phosphor. The differences among the times shown with the small letters for each ion are significant (p<0.05). GLU ALP AST ALT LDH Ca2+ Mg+ Fe++ Cl- P+++ (mg/dL) (U/L) (U/L) (U/L) (U/L) (mmol/L) (mmol/L) (µg/dL) (mmol/L) (mmol/L) Init 172.044±0 16.71±2.0 58.22±2.9 10.621±0. 3.026±0.0 79.681±0. 162.035±4.6 5.281±0.4 5.13±0.26 6.33±0.87 ial .78 1 3 50 5 8 7 3 Cont 167.199±4 5.73±0.77 6.52±0.44 16.58±0.6 61.17±4.1 10.599±0. 3.006±0.1 77.134±1. 163.107±6.6 5.371±0.2 rol .10c d c 6a 9 39a 8b 58b 2c 0b 182.484±4 9.52±0.47 6.56±0.12 13.05±0.7 73.17±2.7 9.149±0.3 3.130±0.0 61.087±1. 345.095±4.6 7.607±0.5 Low 7th .98c c c 7ab 0 7b 3b 36c 8b 2a Medi day 215.884±7 18.76±1.7 18.21±0.9 13.26±1.3 74.38±3.0 7.391±0.1 3.583±0.1 129.717±2 379.362±2.5 6.044±0.1 um .68b a 9b 9ab 2 6c 1a .8a 8a 9b 271.954±2 13.41±0.8 38.15±1.6 9.34±0.75 85.29±2.7 8.186±0.2 3.707±0.0 59.148±1. 390.79±6.63 8.366±0.4 High .80a 7b a b 7 7bc 6a 31c a 3a Cont 172.900±2 5.89±0.72 6.80±0.60 17.88±1.4 54.27±3.0 10.373±0. 3.258±0.0 78.374±2. 170.763±8.0 5.642±0.5 rol .60c b d 6b 3b 28a 7ab 66b 4c 8a 182.424±5 10.19±1.1 21.81±2.8 2.12±0.15 145.03±4. 9.616±0.2 3.473±0.0 58.289±0. 161.338±4.6 6.459±0.3 Low 14th .91c 2a 6b c 21a 0a 5a 86c 8c 7a Medi day 206.338±7 8.90±0.77 14.86±1.0 7.50±0.61 163.30±5. 6.080±0.0 2.962±0.1 61.463±0. 392.107±6.6 5.874±0.1 um .67b a 5c c 60a 07c 2b 94c 8a 3a 229.235±5 3.13±0.19 85.48±2.7 27.77±1.6 142.41±3. 7.638±0.0 2.924±0.0 97.392±3. 346.97±1.26 3.961±0.1 High .94a b 8a 1a 24a 07b 2b 31a b 0b 862 4 Discussion The glucose is the primal source of the energy that is required for the vital actions and its level in serum is regulated through the endocrine system (Dange, 1986). In fish, in addition to the stress induce such as hunger, dense stocking etc; the pollutants such as metals also increase the secretion of the cortisol, epinephrine and glucocorticoid thus leading to the changes in carbohydrate metabolism (Sastry and Subhadra, 1985). Under the influence of lead metal and the environmental concentrations, it was revealed that the glucose levels in serum increased compared to the control group. It is thought that such an increase forms hyperglycemia and in addition, leads to damages in liver and hormonal irregularities (insulin deficiency). The enzymes of dehydrogenize and phosphatase are important and critical enzymes in terms of biological processes and thus they are responsible for the detoxification and biosynthesis of macro molecules (Yousef et al. 2007). In tilapia; increases in LDH and ALP activities (both are blood serum enzymes) indicate that liver damage due to the presence of lead metal. The increases are thought to be occurring due to the fact that as the result of the liver damage, the liver cytoplasm leaks into the blood stream (Wang and Zhai, 1988). Rahman et al. (2000) reported that in fish that were exposed to the pollutants, the increase results from the LDH enzyme, mixing into blood due to the necrosis in liver. The transaminase enzymes such as ALT and AST play an important role in the metabolism of protein and amino acids. In this study, while the AST activity was found out to be increasing in all concentrations, time and concentration varied increases and decreases were registered in ALT enzyme activity. It is thought that the increases in such enzyme activities are resulted after the enzymes are introduced to the circulatory system due to the damages in liver tissue. ALT, AST, ALP and LDH serum enzymes that were assessed within the scope of this study are prone to be used as sensitive biomarkers in ecotoxicological studies because of their characteristic of being an early warning mechanism against the heavy metal based pollution in aquatic ecosystems (Vaglio and Landriscina, 1999). The aquatic organisms have to preserve the osmotic pressure of the plasma in order to survive the ever changing environmental conditions and to maintain the water and ion homeostasis. In bony fish, there are advanced structures to provide the aforementioned regulations and such structures keep the inorganic ion concentrations of the fish in close levels. The changes that may occur in electrolyte levels may induce stress and thus may lead to the damages (Sjöbeck and Larsson, 1979). Calcium is an ion that has various roles in ion regulation, membrane permeability, muscle and neuron cell functions and skeletal bone metabolism and in the blood clotting. The most important serum electrolyte for the lead toxicity is calcium. In this study, the calcium levels of tilapia fish that were subjected to lead concentrations were observed to be lower compared to the control group. Such decreases are found out to be consistent with the literature and the decreases in blood calcium levels (hypocalcaemia) was observed. In this study, unlike the calcium ions, increases were revealed in magnesium electrolyte under the impact of lead. The previous study 863 indicates an inverse relationship between calcium and magnesium ions (Marshall, 2002). Chloride plays an important role in osmotic pressure and ion balance as well as in acid–base equilibrium. In this study, under the effect of water-borne lead concentrations, CI- ion levels of experimental groups registered increases compared to the control group. Na+ and Cl- levels in fish are responsible for the osmolarity. Changes in such ions may cause increase in gill permeability and damages to osmoregulation. It is known that in some studies, under the effect of the pollutants, the inhibitions that occur in the gill Na+, K+-ATPase enzyme activity lead to the disarray in ion regulation (Haux and Larsson, 1979). The changes in parameters that were examined within the scope of this study such as glucose, LDH, ALP, ALT and AST showed that sublethal lead concentrations inflict damage to the liver as the changes in magnesium; calcium and chloride indicate gill damages. Acknowledgments. This study was supported by the Çanakkale Onsekiz Mart University Scientific Foundation (BAP) (project no: 2010/26). References 1. ATSDR, 2006. Agenyc for Toxic Substances and Disease Registry, CERCLA Priority List of Hazardous Substances. Online: 05 October 2011 from http://www.atsdr.cdc.gov/cercla/05list.html. 2. Ay, Ö., Kalay, M and Canlı, M. (1999). Copper and lead accumulation in tissues of freshwater fish Tilapia zillii and its effects on the branchial Na+, K+-ATPase activity. The Bulletin of Environmental Contamination and Toxicology, 62, p.160-168. 3. Berman, E. (1980). Lead in ‘’Toxic Metals and Their Analysis’’. Heyden and Son LTD., London, p.117-132. 4. Bricknell I.R., Bowden T.J., Bruno D.W., Maclachlan P., Johntone, R and Ellis, A.E. (1999). Susceptibility of Atlantic Halibut, Hippoglossus hippoplossus to infection with typical and atypical Aeromonas salmonicida. Aquaculture, 175, p.1-13. 5. Dange, D.A. (1986). Changes in carbohydrate metabolism in tilapia, Oreochromis mossambicus, during short-term exposure to different types of pollutants environmental pollution series A. Ecological and Biological, 41(2), p.165-177. 6. Davies, P. H., J. P. Goettl., Sinley, J R and Smith, N. F. (1976). Acute and chronic toxicity of lead to rainbow trout Salmo gairdneri, in hard and soft water. Water Research, 10, p.199 –206. 7. Haux, C., Larsson, A., Lithner, G and Sjobeck, M.L. (1986). A field study of physiological effects on fish in lead-contaminated lakes. Environmental Toxicology and Chemistry, 5, p.283-288. 864 8. Kaya, H., Akbulut, M., Çelik, EŞ and Yılmaz, S. (2013). Impacts of sublethal lead exposure on the hemato-immunological parameters in tilapia (Oreochromis mossambicus). Toxicological and Environmental Chemistry, 95(9), p.1554-1564. 9. Kaya, H. and Akbulut, M. (2015). Effects of waterborne lead exposure in Mozambique tilapia: oxidative stress, osmoregulatory responses, and tissue accumulation. Journal of Aquatic Animal Health, DOI: 10.1080/08997659.2014.1001533, 0, p.1–11. 10. Logan, M. 2010. Biostatistical design and analysis using r: a practical guide. Wiley- Blackwell, London. p.546. 11. Marshall, W. (2002). Na, Cl, Ca2 and Zn2 transport by fish gills: retrospective review and prospective synthesis. The Journal of Experimental Biology, 293, p.264-283. 12. Patel, M., Rogers, J. T., Pane, EF and Wood ,C. M. (2006). Renal responses to acute lead waterborne exposure in the freshwater Rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology, 80, p.362 –371. 13. Rahman, M.F., Siddiqui, MK and Jamil, K. (2000). Acid and alkaline phosphatase activities in a novel phosphorothionate (rpr-11) treated male and female rats. Evidence of dose and time-dependent response. Drug and Chemical Toxicology, 23, p.497-509. 14. Rogers, J.T., Richards, J.G. and Wood, C.M. (2003). Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology, 64, p.215-234. 15. Sastry, K.V. and Subhadra, K.M. (1985). In vivo effects of cadmium on some enzyme activities in tissues of the freshwater catfish, Heteropneustes fossilis. Environmental Research, 36, p.32-45. 16. Sjöbeck, M.L.J. and Larsson, A. (1979). Effects of inorganic lead on delta- aminolevulinic acid dehydratase activity and hematological variables in the rainbow trout, Salmo gairdnerii. Archives of Environmental Contamination and Toxicology, 8, p.419-431. 17. Smith, C., Shaw, B and Handy, R. D. (2007). Toxicity of single walled carbon nanotubes to Rainbow trout, (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects. Aquatic Toxicology, 82(2), p.94 – 109. 18. Vaglio, A. and Landriscina, C. (1999). Changes in liver enzyme activity in the teleost Sparus aurata in response to cadmium intoxication. Ecotoxicology and Environmental Safety, 43B, p.111-116. 19. Val, A.L., De Menezes, GC and Wood, C.M. (1998). Red blood cell adrenergic responses in amozonion teleost. Journal of Fish Biology, 52, p.83-93. 20. Wang, X. and Zhai, W. (1988). Cellular and biochemical factors in bronchoalveolar lavage fluids of rats exposed to fenvalerate. Chinese Journal of Pharmacology and Toxicology, 2, p.271-276. 21. World Health Organization (1995). Environmental Health Criteria 165. Geneva: International Programme on Chemical Safety. 865 22. Yousef, M.I., Awad, T.I., Elhag, FA and Khaled, F.A. (2007). Study of the effect of ascorbic acid against the toxicity of stannous chloride on oxidative damage, antioxidant enzymes and biochemical parameters in rabbits. Toxicology, 235(3), p.194-202. 866