Determination of nickel and chromium content in serum, emulsion, skin and viscera of Iranian tuna fish

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1 Indian Journal of Geo-Marine Sciences Vol. 44(9), September 2015, pp Determination of nickel and chromium content in serum, emulsion, skin and viscera of Iranian tuna fish Leila Zamani 1, Mohammad Hadi Givianrad 2*, Hamid Ezzatpanah 1 & Hossein Bakhoda 3 1 Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Marine Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran 3 Department of Agricultural Mechanization, Science and Research Branch, Islamic Azad University, Tehran, Iran * [ givianradh@yahoo.com] Received 28 May 2014 ; revised 21 June 2014 Contamination of heavy metals, namely, nickel and chromium was evaluated in serum, emulsion and tissues of Euthynnus affinis and Thunnus tonggol collected from Persian Gulf. The concentration of metals was determined by Graphite Furnace Atomic Absorption Spectrometry (GFAAS). Tissues analysis revealed that viscera in Euthynnus affinis accumulated the highest levels of Ni and Cr. Nickel concentration in the non-edible parts of the examined fish (viscera in both fish species and skin in Thunnus tonggol) were higher than the established values by World Health Organization (WHO), whilst concentration of chromium for all samples were higher than maximum permissible limits determined by the WHO, except for serum of Thunnus tonggol. [Keywords: Tuna fish, Nickel, Chromium, Graphite Furnace Atomic Absorption Spectrometry] Introduction Environmental pollution represents a main problem in both developed and undeveloped countries. Recent years have witnessed significant attention being paid to the problems of environmental contamination particularly chemical pollutants including the heavy metals 1. Thus, heavy metal is a common environmental problem facing many places world with the rapid development of industries 2. Heavy metals are emitted to the environment from different sources such as human activities, including agricultural, industrial and municipal wastes 3. Recent years, industry and agriculture in Persian Gulf area have been rapidly developed and human activities have increased. Metals such as iron, copper, zinc and manganese, nickel and chromium are essential nutritive elements, whereas mercury, lead and cadmium are non-essential metals, as they are toxic 4, even at low concentrations 5. The essential metals can also produce toxic effects at higher concentrations 6. The International Agency for Research on Cancer (IARC) has determined that some nickel compounds are carcinogenic to humans and that metallic nickel may possibly be carcinogenic to humans. Nickel is the 24 th most abundant element 7. Nickel can enter in body through drinking water or eating food contains nickel. Trivalent chromium is essential for human. Chromium (III) was classified by IARC (1990) into group 3 (agent is unclassifiable as to carcinogenicity in humans) and USEPA (1998a) placed it into group D (not classified as to its human carcinogenicity) 8. On the other hand, hexavalent form is considered to be toxic, because of its harmful effects on liver, lung and kidney 9. However, the classification of hexavalent chromium as a known human carcinogen raises some concern for the carcinogenic potential of trivalent chromium 8. Trace elements, which are present in seafood, are essential for human life at low concentrations but they may exert beneficial or harmful effects depending upon the concentration between Ni and Cr. Ni shows carcinogenic effect when consumed in high amount 10. The accumulation of heavy metals in liver, kidney, gonad and gill of the two species may result in harmful effects for the fish 11. The fish industry generates large quantities of by-products as fish viscera 12. Skin and viscera include as main source for feed. Thus, it is possible that heavy metal enter in body of animal, therefore the pollutants can be easily transferred to humans through ingestion 13. In the literature, few studies have been

2 1410 INDIAN J. MAR. SCI., VOL. 44, NO. 9 SEPTEMBER 2015 published on tuna species particularly Thunnus tonggol and Euthynnus affinis and were concerned mainly with muscle tissues. On the other hand, among the large groups of fish species, which have been consumed in Iran, E.affinis and T. tonggol are available and important species for local people in Persian Gulf. Being the complexity of heavy metal bioaccumulation of fishes, it was important to study the heavy metal accumulation in these two tuna fish. Processing steps may change the concentration of heavy metals in different parts fish before consumption. Defrosting might reduce the concentration of heavy metal in tuna fish due to possible separation of serum. Furthermore, it could be concluded that cooking might decline the concentration of heavy metal in tuna fish during processing 14. Therefore, samples of tuna fish during processing steps were collected from the output blood (serum) during defrosting and output water (emulsion) during cooking. In the literature, no study has been carried out on Ni and Cr accumulation in serum and emulsion. Heavy metals have been studied for their toxic effects, accumulation in organisms and bioaccumulation in food chains in the past decades 15. Several studies have shown that the accumulation of heavy metals in tissues is influenced by several factors such as seasonal variations, sex, size, age, molt of marine animals, water chemistry, feeding habit of fish, the duration of exposure of fish to contaminants in water, transportation, and storage. Fish have been found to be good bioindicator to monitor a variety of contaminants in the ecosystem. Fish is one of aquatic products humans consume 16 and also has long been recognized as an important source of high-quality protein and polyunsaturated fatty acids for many people. Fish are an excellent, low-fat source of omega-3 fatty acids, that contributes to low blood cholesterol and reduce the risk of heart disease, stroke 17. The main source of providing fish for canned tuna fish factories are the Persian Gulf where the possibility of heavy metal contamination is high because of heavy trafficking of oil 14. The aim of the present study was to determine the level of contamination heavy metal (nickel and chromium) in skin, viscera, serum and emulsion of two different fish species collected from the Persian Gulf. Materials and Methods Two fish species were examined in this study. These species were kawakawa tuna (Euthynnus affinis) and longtail tuna (Thunnus tonggol). Ten kawakawa tuna (E. affinis) with the average weight of 1.8 kg and longtail tuna (T. tonggol) with the average weight 3 kg were obtained from Rostami port in Boushehr province, Iran. Fish samples of each species were randomly selected and the size of fish for longtail tuna and kawakawa tuna was 65 cm and 50 cm, respectively. Samples were placed in polyethylene bags, sealed and kept in cold iced boxes and transported to the laboratory for the heavy metals determination. All samples were stored at - 20 C until analysis. All reagents were of analytical reagent grade, HNO 3, H 2 O 2. Deionized water (Millipore, 18.3 MΩ cm -1 resistivity) was use for all working and standard solutions. HNO 3 and H 2 O 2 were of suprapur quality (E. Merck). Special care was taken to prevent metal contamination of the samples by the hauling, and tissues were dissected by plastic knife. To minimize the risk of contamination, all the glasswares were cleaned by soaking in 10% nitric acid solution for 24 h and were rinsed with deionized water before use. A Varian Spectra AA-200 (Australia) atomic absorption spectrometer equipped with a GTA- 100 graphite furnace atomizer, deuterium lamp as a background corrector, a Varian programmable sample dispenser, were used for determination of Ni and Cr. For graphite furnace measurements, argon was used as inert gas. Solutions were injected into the graphite tube by means of autosampler that optimum conditions for GFAAS are given in Table 1. The samples were analyzed with GFAAS after microwave digestion. Milestone Ethos 1 closed vessel microwave digestion system (maximum pressure 1000 watt, maximum temperature 200 C, Italy) was used. Fish samples (0.5 g) were digested with 7 ml of HNO 3 (65%), 1 ml of H 2 O 2 (30%) in microwave digestion system for 25 minutes and diluted to a final volume of 25 ml with deionized water. Sample blanks were prepared in a similar manner to the field samples. Digestion conditions for microwave system were applied as follows: Step 1: 200 C for 20 min at 1000W; Step 2: 200 C for 30 min at 1000 W; then cooled to room temperature. 200 μl of phosphoric acid as a modifier was added to each flask. The clear solutions were transferred into lidded tubes in a

3 ZAMANI et al.: DETERMINATION OF NICKEL AND CHROMIUM CONTENT IN SERUM, EMULSION, SKIN AND VISCERA OF IRANIAN 1411 water bath of 60 C for 30 min. Advantages of microwave digestion against the classical methods Table 1 Operating condition for GFAAS Ni Wavelength/nm Slit/nm Hallow cathode lamps (HCL) current/ma 4 7 Atomization temperature/ C Injection volume/μl Table 2 Heavy metal content (μg/g wet weight) in various samples of T. tonggol (expressed as mean concentration ± SD, N = 10) Sample Nickel Chromium Serum 0.328±0.024 a 0.356±0.019 b Skin 0.700±0.023 c 0.360±0.029 b Viscera 0.553±0.020 b 0.322±0.025 b Emulsion 0.336±0.053 a 0.208±0.044 a Vertically, letters a, b and c show differences among samples. Means with the same letter are not statistically significant, p > 0.05 Cr Table 3 Heavy metal content (μg/g wet weight) in various samples of E. Affinis (expressed as mean concentration ± SD, N = 10) Sample Nickel Chromium Serum 0.300±0.046 a 0.173±0.008 a Skin 0.318±0.032 a 0.422±0.013 c Viscera 0.901±0.024 b 0.916±0.022 d Emulsion 0.292±0.011 a 0.244±0.014 b Vertically, letters a, b, c and d show differences among samples. Means with the same letter are not statistically significant, p > 0.05 Table 4 Maximum acceptable limits of Ni and Cr in guidelines Ni Cr Unit FAO μg/g wet wt National limit (China) mg/kg wet wt EEC(1979) μg/g dry wt Brazilian standard mg/kg wet wt are the shorter time, less utilization of acid and keeping volatile compounds in the solutions 18. Instrument was calibrated with standard solutions. Calibration standards of each metal were prepared by appropriate dilution of the stock solutions of 1000 mg/l. Calibration standard solutions were similarly analysed as the digested sample solution, and calibration curves constructed. All metal concentrations were determined on wet weight basis as μg/g. We used One-way analysis of variance (ANOVA) and Duncan s multiple range tests to identify significant differences between samples. Possibilities less than 0.05 (p < 0.05) were considered statistically significant. Data were presented as mean ± standard deviation of mean. The GFAAS determinations were carried out in triplicate to insure results reliability. All statistical calculations were performed with EXCEL and SPSS. Results and Discussion Ni and Cr levels in two tuna fish samples were determined and assessed by comparing element levels in these samples with maximum

4 1412 INDIAN J. MAR. SCI., VOL. 44, NO. 9 SEPTEMBER 2015 permissible limits set by the WHO. The mean metal concentrations in different parts of the sampled fish species, Thunnus tonggol and Euthynnus affinis from Persian Gulf are given in Tables 2 and 3. The mean range of Ni was μg/g in T. tonggol and μg/g in E. affinis. In T. tonggol, Ni content was highest in skin (0.700 μg/g) and lowest in serum and emulsion (0.328 and μg/g, respectively) (Table 2), whilst in E. affinis, viscera accumulated the highest amount of Ni (0.901 μg/g) (Table 3). Nickel concentrations obtained were in the range of , and μg/g for skin, emulsion and serum of fish, respectively. Results of this study were compared with those of other studies that specifically examined the viscera and skin tissues. Nickel levels in the literature have been reported as 0.68 μg/g wet weight for skin of fish 19, 0.05 μg/g dry weight for skin of fish 6, μg/g dry weight for intestine of fish 20 and μg/g wet weight 21. There is no information about maximum permissible nickel limits in fish tissues in Turkish standards 18. Mean Ni levels in serum and emulsion were below the maximum permissible value of 0.38 μg/g indicated by the WHO. On the other hand, mean nickel levels in viscera in examined fish species and for skin of Thunnus tonggol were found to be higher than permissible limits reported by WHO. Chromium was another trace element detected in our study. In Euthynnus affinis, chromium showed the highest and the lowest levels in viscera and serum, respectively (Table 3), whilst mean Cr concentration was lowest in emulsion of Thunnus tonggol (Table 2). The mean chromium concentration in the emulsion (0.244 μg/g) was higher than the average posted by the serum (0.173 μg/g) in Euthynnus affinis (Table 3), whilst the mean Cr concentration in the serum (0.356 μg/g) was higher than that in emulsion (0.208 μg/g) of Thunnus tonggol (Table 2). Present results indicate that lower concentrations of Cr were detected in serum of Euthynnus affinis when compared with the skin (Table 3). Chromium concentrations obtained were in the range of , and μg/g for skin, emulsion and serum of fish, respectively. Literature values for chromium were reported as mg/kg dry weight 16, 0.26 μg/g wet weight 19 and μg/g wet weight 21. There is no information about maximum permissible chromium concentrations in fish tissues in Turkish standards 18. However, the maximum Cr level permitted determined by the WHO is 0.2 μg/g, mean chromium levels of all parts in the examined species were higher than permissible limits proposed by the WHO, except for serum in Euthynnus affinis. Table 4 shows the metal level in international standards 1. In this study, the metal levels in the similar samples of the different species were statistically significant (p < 0.05), except Ni in serum and emulsion and Cr in emulsion (Fig. 1 and 2). The mean Cr accumulation (0.356 µg/g) in the serum of T. tonggol was higher than the data for E. affinis (0.173 µg/g) (Fig. 2). The amount of Ni in skin was higher in T. tonggol than in E. affinis (Fig. 1) but the skin of E. affinis accumulated higher level of Cr than that of T. tonggol (Fig. 2). The accumulation of metal in fish depended on the property of the examined fish part 3. It is very difficult to compare the metal accumulations even within the similar tissues of different species because of different feeding habits, the differences in the aquatic environments concerning the source and level of water pollution, growing rates of the species and types of tissues analyzed 21. Different from viscera and skin seemed to be independent on the species 22. On the other hand, viscera were accepted as storehouse of trace elements in various fish species 23. Statistical analysis showed significant differences (p < 0.05) in viscera between the two species for nickel and chromium (Fig. 1 and 2), which could be related to the bigger size of viscera in T. tonggol and lower accumulation of Ni and Cr. In longtail tuna, the concentration Cr in serum was higher than that in kawakawa tuna (Fig. 2), which could be related to the bigger size and higher initial concentration of longtail tuna and release of this metal due to separation of serum. Sensitivity was studied by means of the limit of detection (LOD), by using the following equation: 3 b LOD= m Where b is standard deviation of six successive measurements of blank; m is the slope of the standard calibration curve. According to this equation, the LOD of nickel and chromium were 3.7 μg/l and 2.1 μg/l, respectively 24. Precision, which is a measure of reproducibility, expressed as percent relative standard deviation (RSD) within the liner range, was defined as follows:

5 ZAMANI et al.: DETERMINATION OF NICKEL AND CHROMIUM CONTENT IN SERUM, EMULSION, SKIN AND VISCERA OF IRANIAN RSD x The repeatability of measurements, as the means of RSD was 1.3% for nickel and 1.5% for chromium for 6 successive individual measurements of the same sample. The accuracy of method has been investigated by means of spiking different amounts of nickel and chromium into samples 25. The recoveries were found within the range of 85% 87% for nickel and 87% 92% for chromium. Conclusion Present study provides primary information on the distribution of metal concentrations in tissues of T. tonggol and E. affinis from the Persian Gulf. In general, nickel recorded its lowest concentration in emulsion and serum. The results represent that heavy metal contents in fish parts varied according to concentrations in the environment and the type of fish tissue. Bioaccumulation of analyzed metals in this study can be a possible risk for the consumption Fig. 1 Comparison of Ni concentration in samples of T. tonggol with E. affinis of these species, if industry and recreational practices in the surroundings of the Persian Gulf increase unconsciously. Acknowledgements Authors are grateful to the Canned Tuna Fish Factory of Boushehr-Iran for providing the samples, Laboratory Complex of Islamic Azad University for valuable technical assistance. Fig. 2 Comparison of Cr concentration in samples of T. tonggol with E. Affinis References 1 Mendil, D., Ünal, O. F., Tüzen, M., Soylak, M., Determination of trace metals in different fish species and sediments from the River Yesilırmak in Tokat, Turkey, Food Chem. Toxicol., 48(2010): Givianrad, M. H., Rabani, M., Saber-Tehrani, M., Aberoomand-Azar, P., Hosseini-Sabzevari, M., Preparation and characterization of nanocomposite, silica aerogel, activated carbon and its adsorption properties for Cd(II) ions from aqueous solution, J. Saudi Chem. Soc., 17(2013): Atta, M. B., El-Sebaie, L. A., Noaman, M. A., Kassab, H. E., The effect of cooking on the content of heavy metals in fish (Tilapia nilotica), Food Chem., 58(1997): Bilandzic, N., Ðokic, M., Sedak, M., Metal content determination in four fish species from the Adriatic Sea, Food Chem., 124(2011): Tuzen, M., Determination of heavy metals in fish samples of the middle Black Sea (Turkey) by graphite furnace atomic absorption spectrometry, Food Chem., 80(2003): Dhaneesh, K. V., Gopi, M., Ganeshamurthy, R., Kumar, T. T. A., Balasubramanian, T., Bio-accumulation of metals on reef associated organisms of Lakshadweep Archipelago, Food Chem., 131(2012): Agency for Toxic Substances and Disease Registry (ATSDR), Toxicological profile for nickel, Department of Health and Human Services, Public Health Service, Atlanta, GA, U.S, USEPA, Integrated Risk Information System, US Environmental Protection Agency, Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH, 1998a, b. 9 Vercoutere, K., Comelis, R., Chromium speciation in enviromnental and biological samples, Qual. Assurance Environ. Anal., 17(1995): Malik, N., Biswas, A. K., Qureshi, T. A., Borana, K., Virha, R., Bioaccumulation of heavy metals in fish tissues of a freshwater lake of Bhopal, Environ. Monit. Assess., 160(2010):

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