DEPURATION OF TRACE METALS IN TRANSPLANTED PERNA VIRIDIS FROM POLLUTED 1 Jr. of Industrial Pollution Control 29(1)(2013) pp 1-6 © EM International Printed in India. All rights reserved www.envirobiotechjournals.com DEPURATION OF TRACE METALS IN TRANSPLANTED PERNA VIRIDIS FROM POLLUTED SITE AT KG PASIR PUTEH TO RELATIVELY UNPOLLUTED SITES AT KG SG MELAYU AND SG BELUNGKOR IN THE STRAITS OF JOHORE Y.J. EUGENE NG 1, C.K. YAP *1, M.P. ZAKARIA 2, A.Z. ARIS 2 AND S.G. TAN 3 1 Department of Biology, Faculty of Science, 2 Department of Environmental Sciences, Faculty of Environmental Studies, 3 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. Key words: Accumulation, Transplantation, Mussels. (Received 31 July, 2012; accepted 1 September, 2012) ABSTRACT Green-lipped mussels Perna viridis were transplanted from a known polluted site at Kg Pasir Puteh (KPP) to two relatively unpolluted sites at Kg Sg Melayu (KSM) and Sg Belungkor (SB). Total soft tissues of mussels were determined for Ag, As, Be, Co, Cr, Cs, Hg, Li, Mn, Se, Sr and V. After 6 weeks of transplantation, KPP had higher levels of Co, Cr, Hg, Sr and V if compared to KSM and SB because of the negative value in rate of depuration. As expected, the coastal waters at KSM and SB can be used for depuration of trace metals Co, Cr, Hg, Li, Sr and V since their levels were lower when compared to KPP population after transplantation. However, it was found that after weeks 2 and 6 of transplantation from KPP, KSM and SB had higher levels of Ag, As, Be and Se if compared to KPP. Present finding revealed an unknown source of trace metal pollution at KSM and SB and future studies are much needed to understand the sources for such elevation of trace metals in the active mussel aquacultural site especially at KSM. INTRODUCTION Anthropogenic activities are major sources of trace metal pollution in the coastal area (Amin et al., 2009). Transplantation is widely used nowadays for a more accuracy of metal pollution in the coastal waters. In particular, marine mussels are suitable for transplantation experiments because of several advantages. Firstly, monitoring sites may be chosen *Corresponding author’s email: yapckong@hotmail.com independently without considering the presence of natural populations (Hedouin et al., 2011). Secondly, the transplanted mussels can be located to those coastal waters in which the accessibility of bioaccumulation status is hard-to-reach (Hunt and Slone, 2010; Giarratano et al., 2010). Thirdly, the intrinsic factors inherent in marine mussels including age, size, stage of sexual maturity of mussels can be controlled (Alfonso et al., 2010) besides the exposure 2 EUGENE ET AL. times and depth of transplantation. Fourthly, the all marine mussels investigated have a low genetic variation and in the same phase of the reproductive cycle since they are abundant and are of the same source (Phillips and Segar, 1986). In addition, the green-lipped mussel Perna viridis populations focused in this study has been reported to have low genetic variation and thus the mussel species is suitable to be employed as a valid biomonitor in Peninsular Malaysia (Yap et al., 2002a; Ong et al., 2009). Therefore, by conducting transplantation experiments, the influence of external and internal factors such as seasonal variation, size or age which are susceptible to induce bias in data comparison is minimized (Regoli and Orlando, 1994 and Hedouin et al., 2011). Hence, the trace metals accumulated in the transplanted P. viridis in this study can provide more accurate information on the trace metal contamination in the study area. In this study, mussels P. viridis were collected from Kg Pasir Puteh (KPP) was used for transplantation purpose because this site had been reported as being contaminated by Cd, Pb, Cu and Zn (Yap et al., 2002b, 2003a, 2003b, 2004a, 2004b, 2006). Meanwhile, Kg. Sg. Melayu (KSM) and Sg. Belungkor (SB) are two relatively unpolluted sites (Yap et al., 2006) and the mussels collected from KPP were placed in the coastal waters of KSM and SB for two and six weeks for depuration purpose. All the above three sites are located in the Straits of Johore. The Straits of Johore is a high mussel spatfall area in Peninsular Malaysia besides Sebatu at Malacca coastal water (Al-Barwani et al., 2007). Therefore, the objective of this paper was to determine the levels of 12 trace metals (Ag, As, Be, Co, Cr, Cs, Hg, Li, Mn, Se, Sr and V) in the total soft tissues (TST) of transplanted P. viridis from KPP to KSM and SB. MATERIALS AND METHODS About 200 individuals of P. viridis were collected from KPP in the Straits of Johore (Figure 1), on 28 November 2009. Mussel transplantation was conducted on the same day in which those mussels collected from KPP were transplanted in the coastal waters at KSM and SB, both located in the Straits of Johore (Figure 1). Samplings of mussels were conducted after 2 and 6 weeks from KSM and SB. Prior to transplantation, after the mussels were collected from KPP, all mussels were rinsed using seawater for 3 times to get rid of any visible sediment on the mussel shells. The mussels were then divided randomly into sub-groups of 40 individuals and each sub-group was placed in a polyethylene cage of 20 × 15 × 18 cm which permitted water circulation through it. Four cages were used per site and left suspended in the water column at an average depth of 1.5m using a rope which was modified from Faverney et al. (2010). The collected mussels were rinsed with seawater before being transported back to the laboratory in an ice compartment at 10°C. After the byssus of P. viridis were discarded, the total soft tissues were then dried in an oven for 72 hours at 105°C to constant weight. TST were homogenized and about half gram of sample was digested using the CEM Microwave Sample Preparation System following essentially the methods of Zhou and Liu (1997). Seven mL of HNO3 and 1mL of H2O2 were mixed with the dried samples in closed Teflon vessels. The vessels were then sealed and placed in a microwave oven at 220ºC for 30 minutes. The resulting digests were then transferred to 100mL volumetric flasks and diluted to 100mL using double distilled water. The sample was then filtered through Whatman No. 1 filter paper and the filtrate was stored in an acid-washed pill box until metal analysis. After filtration, the samples were analysed for Ag, As, Be, Co, Cr, Cs, Hg, Li, Mn, Se, Sr and V using an Inductively Coupled Plasma-mass Spectrometer with Dynamic Reaction CellTM (ICP-MS DRCplus) (Perkin Elmer ELAN DRCplus). The data are presented in µg/ kg dry weight. To avoid possible contamination, all glassware and equipment used were acid-washed and the accuracy of the analysis was checked against blanks. For data validation, Certified Reference Materials (CRM) for Mussel Tissue, no. 2976 (National Institute of Standard and Technology (NIST), USA) were also analyzed and checked for recoveries as shown in Table 1. t-test was conducted to see check if the metal levels after transplantation periods (Weeks 2 and 6) are significantly different from the initial week 0 metal levels. The statistical analysis was done by using STATISTICA StatSoft Inc. version 8.0 for Windows. The rate of metal depuration was calculated according to the following formula as suggested by Yap et al. (2004a): Rate of metals depuration = Metal level end of metal exposure - Metal level end of metal depuration Day(s) of metal depuration DEPURATION OF TRACE METALS IN TRANSPLANTED PERNA VIRIDIS FROM POLLUTED RESULTS The concentrations of all 12 metals in the total soft tissues of transplanted P. viridis from KPP to KSM and SB are presented in Table 2. The concentrations of Co, Cr, Hg, Li, Sr and V for the transplanted mussel from KPP to KSM and SB were decreased after 2 and 6 weeks of transplantation. Thus, this is within our expectation that the coastal waters at KSM and SB can be used for depuration of trace metals Co, Cr, Hg, Li, Sr and V. 3 On the other hand, the concentrations of Ag, As, Be and Se were increased during the transplantation period in both KSM and SB. For Cs and Mn, the concentrations were increased at KSM, while the concentrations were decreased in SB in both week 2 and week 6. Generally, all the trace metal levels in weeks 2 and 6 were higher in KSM than in SB. This indicated SB is a cleaner site for metal depuration purpose when compared to KSM. The depuration rate for the transplanted P. viridis was shown in Table 3. The negative value in the Table Table 1. A comparison of trace metal concentrations (µg/kg dry weight) between measured values and certified values in the Certified Reference Materials (CRM) for Mussel Tissue, no. 2976 (National Institute of Standard and Technology (NIST), USA). Metals Certified values (C) Measured value (M) Percentage of recovery [(M/C) x 100] Ag As Cr Hg Mn Se Sr 11.00 13,300.00 500.00 61.00 33,000.00 1,800.00 93,000.00 10.90 13,420.00 707.80 49.00 28,009.70 1,781.00 89,310.40 99.09 100.90 141.56 80.33 84.88 98.94 96.03 Note: Certified values for other trace metals that are not indicated are not available in the CRM. Fig. 1 Transplantation map for Perna viridis. Note: A- Kampung Pasir Puteh; B- Sungai Melayu; C- Sg Belungkor. EUGENE ET AL. 4 Table 2. Trace metal concentrations (mean±SE, µg/kg dry weight) in total soft tissue (TST) of transplanted Perna viridis from Kg Pasir Puteh (week 0) to Kg. Sg. Melayu and Sg. Belungkor after weeks 0 and 6. Metal Weeks Sg Melayu Ag 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 76.338 210.534 213.614 4757.637 5464.833 5956.471 12.160 56.035 106.011 563.091 430.736 419.616 8026.202 7388.831 5745.234 78.636 274.735 513.898 122.597 10.697 4.955 13012.720 12694.295 9877.933 104257.215 124748.984 132477.894 1221.951 1485.530 1891.323 130747.688 123261.262 102359.888 4962.260 3987.353 3033.800 As Be Co Cr Cs Hg Li Mn Se Sr V Belungkor ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 15.060 1.611 30.083 32.088 292.442 1572.625 0.584 4.696 34.233 6.204 55.467 59.654 95.464 184.677 218.017 9.974 34.424 223.440 9.223 0.309 1.611 32.880 399.852 358.737 948.782 9070.285 393.230 51.103 29.364 89.730 432.922 14.407 426.971 50.484 178.249 161.181 3 is due to the accumulation of Ag, As, Be, Cs, Mn and Se, instead of depuration, in the transplanted mussels after weeks 2 and 6. DISCUSSION From this study, the higher levels of Ag, As, Be and Se in KSM and SB after transplantation periods when compared to week 0 (original KPP population) could be due to higher bio availabilities of these metals which are related to anthropogenic inputs at KSM and SB. The only direct observable activities found in KSM are fish and mussel aquaculture and rubber plantation (Yap et al., 2006) meanwhile only a jetty and 76.338 105.958 157.000 4757.637 5003.482 5936.400 12.160 20.330 26.900 563.091 422.699 344.300 8026.202 7083.293 5418.700 78.636 58.584 47.300 122.597 5.597 4.900 13012.720 11701.484 10340.000 104257.215 81915.097 70312.700 1221.951 1421.951 1533.000 130747.688 111003.234 95873.600 4962.260 3522.050 2893.400 T-Test ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 15.060 19.759 15.600 32.088 119.119 43.617 0.584 17.499 0.656 6.204 27.145 2.432 95.464 200.758 264.800 9.974 20.272 2.202 9.223 0.656 1.000 32.880 56.891 66.437 948.782 264.586 234.468 51.103 13.510 41.956 432.922 179.034 358.524 50.484 46.838 117.836 p< 0.05 p< 0.05 p< 0.05 p>0.05 p< 0.05 p< 0.05 p>0.05 p< 0.05 p>0.05 p>0.05 p< 0.05 p< 0.05 p< 0.05 p>0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p< 0.05 p>0.05 agricultural site was found in SB. Thus, both sites are considered as relatively unpolluted sites in the Straits of Johore. Present finding indicates a contamination by Ag, As, Be and Se at KSM and SB. The presence of higher Ag level in KSM and SB could be due to the source from residential area nearby because Ag was used as ornaments in people’s daily life and erosion of silver ornaments in commercial areas (Han et al., 2006). On the other hand, high level of As found in this two sites could be due to the toxicity in arsenic which can be used in wood preservative to kill insects, bacteria and fungi (Rahman et al., 2004) especially widely used in our rubber plantations and As was also used as various agricultural insecticides DEPURATION OF TRACE METALS IN TRANSPLANTED PERNA VIRIDIS FROM POLLUTED Table 3. Rates of depuration for transplanted Perna viridis from Kg Pasir Puteh to Kg. Sg. Melayu and Sg. Belungkor. Rate of depuration Metal weeks Sg Melayu Belungkor Ag 2 6 2 6 2 6 2 6 2 6 2 6 2 6 2 6 2 6 2 6 2 6 2 6 -9.585 -3.268 -50.514 -28.544 -3.134 -2.235 9.454 3.416 45.526 54.309 -14.007 -10.363 7.993 0.205 22.745 74.638 -1463.698 -671.921 -18.827 -15.937 534.745 675.900 69.636 45.916 -2.116 -1.921 -17.560 -28.066 -0.584 -0.351 10.028 5.209 67.351 62.083 1.432 0.746 8.357 0.025 93.660 63.636 1595.866 808.203 -14.286 -7.406 1410.318 830.335 102.872 49.259 As Be Co Cr Cs Hg Li Mn Se Sr V Note: negative rates of depuration indicate higher metal accumulations instead of depuration. (Sabina et al., 2005). In addition, Se which was used in glass production and rubber industry could be the reason why Se level was high in KSM and SB. However, whether all the above activities are the major sources of tarce metal pollution at KSM and SB, only future studies should be conducted to confirm this. As expected, lower levels of some trace metals including Co, Cr, Hg, Li, Sr and V were found in KSM and SB after transplantation periods. The higher levels of the above six metals at KPP could be related to large shipyard repair and construction facilities, fossil fuel fired electrical power plants and shipping dock activities (Yap et al., 2003a; 2003b; 2006). Therefore, KSM and SB are suitable sites for depuration of Co, Cr, Hg, Li, Sr and V from the TST of contaminated polluted P. viridis. CONCLUSION Present findings indicated that the coastal waters at KSM and SB can be used for depuration of trace metals 5 Co, Cr, Hg, Li, Sr and V since their levels were lower when compared to KPP population after 2 and 6 weeks transplantation. However, mussels transplanted to KSM and SB had higher levels of Ag, As, Be and Se if compared to KPP, after the transplantation periods. This revealed an unknown source of trace metal pollution at KSM and SB. 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