Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 15, No. (1) : 2013 : 5-8 © Global Science Publications ISSN-0972-3005 TRACE METALS IN THE SHELLS OF MUSSELS PERNA VIRIDIS TRANSPLANTED FROM POLLUTED TO RELATIVELY UNPOLLUTED SITES IN THE STRAITS OF JOHORE: SHELLS AS BIOMONITORING MATERIALS 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, 2Department of Environmental Sciences, Faculty of Environmental Studies, 3Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. (Received 2 August, 2012; Accepted 11 September, 2012) Key words : Depuration, Transplantation, Mussels, Trace metals. Abstract - In this paper, the shells of mussels Perna viridis were determined for Ag, As, Be, Co, Cr, Cs, Hg, Li, Mn, Se, Sr and V, based on transplanted mussels from a known polluted site at Kg Pasir Puteh (KPP) to two relatively unpolluted sites at Kg Sg Melayu (KSM) and Sg Belungkor (SB). It was found that significant (P< 0.05) decreased levels of Ag, Li and Se were found in mussels transplanted to KSM and SB after 2 and 6 weeks. However, significant (P< 0.05) increased levels of As, Mn, Sr and V were found in mussels transplanted to KSM. In comparison between KSM and SB, decreased levels of Ag, As, Li, Mn and Sr in SB were found to be more signficant (P< 0.05) in SB than KSM. Meanwhile there was no signficant (P> 0.05) changes for Cr, Co and Hg levels for both sites after the transplantation periods. When compared to the trace metals in the total soft tissues previously reported by Eugene Ng et al. (2012), only the increased level of As based on the shells was supported by the total soft tissue of mussels. All the differences of increased and decreased levels of trace metals between soft and hard (shells) tissues after the transplantation periods from KPP to KSM and SB, is thus difficult to explain. Further studies are neccesary in the future to understand the differences of metal accumulations between issues and shells of P. viridis. INTRODUCTION The Straits of Johore is a hotspot area for scientific studies due to anthropogenic activities in the surrounding. These activities can become major sources of trace metal pollution in the coastal area (Amin et al., 2009). Besides, the Straits of Johore is important for commercial mussel aquaculture because it is a high natural mussel spatfall area in Peninsular Malaysia besides Sebatu at Malacca coastal water (Al-Barwani et al., 2007). Mussel transplantation is employed in this study because of many advantages the sizes, spawning conditions and maturity status of transplanted mussels can be controlled (Alfonso et al., 2010; Giarratano et al., 2010; Hedouin et al., 2011). In Peninsular Malaysia, the Perna viridis is a marine mussel species known and has been proven to be a *Corresponding author’s email : yapckong@hotmail.com suitable and good biomonitor due to having low genetic variation (Yap et al., 2002a; Ong et al., 2009) and fulfilled most of the recommended criteria as a biomonitor (Yap et al., 2002b, 2003a, 2003b, 2004a, 2004b, 2006). In this study, mussel shell was focused upon because of positive characteristics of the shells to be used as a biomonitoring material of metal pollution (Yap et al., 2003). This included high precision and low variation in metal analysis, easy storage and positive relation with environment (Yap et al., 2003; 2004c). Three sites were focused in this study mussels, including Kg Pasir Puteh (KPP) where it had been reported as being metal-contaminated (Yap et al., 2003a, 2006); Kg. Sg. Melayu (KSM) and Sg. Belungkor (SB) where they are two relatively unpolluted sites in the Straits of Johore (Yap et al., EUGENE ET AL. 6 2006). Since information on metal levels in the mussel shells is lacking in the literature, 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 shells of P. viridis transplanted from KPP to KSM and SB. MATERIALS AND METHODS In this study, about 200 individuals of P. viridis were collected from KPP, in November 2009. After mussel collection from KPP, all mussels were rinsed using seawater for 3 times to get rid of any visible sediment on the mussel shells. A polyethylene cage (20 cm × 15 cm × 18 cm), with 40 individual mussels, was used in this study. At each site, four cages were used and they were left suspended in the water column at an average depth of 1.5m using a rope. Before transplantation, the collected mussels from KPP were analyzed for trace metals (week 0). Mussels were sampled at week 2 and week 6. The collected mussels were rinsed with seawater before being transported back to the laboratory in an ice compartment at 10°C. Shell lengths and shell growth rates of transplanted mussels in this study are presented in Table 1. The total shells (without byssus and soft tissues) of P. viridis were then dried in an oven for 72 hours at 105°C to constant dry weights. Shells 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 1 mL of H202 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 acidwashed pill box until metal analysis. After filtration, the samples were analyzed 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. Recoveries between measured and certified values for Ag, As, Co, Cr, Cs, Hg, Mn, Se and Sr were satisfactory being 80.3141.6%. However, the certified values for other metals are not available in the CRM. 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). RESULTS AND DISCUSSION Fom Table 2, significant (P< 0.05) decreased levels of Ag, Li and Se in the mussels shells were found in KSM and SB after tranplantation periods of 2 and 6 weeks. However, significant (P< 0.05) increased levels of As, Mn, Sr and V were found in mussel shells transplanted to KSM after 6 weeks. In comparison between KSM and SB, decreased levels of Ag, As, Li, Mn and Sr in SB were found to be more signficant (P< 0.05) in SB than KSM. Meanwhile there was no signficant (P> 0.05) changes for Cr, Be and Hg levels for both sites after the transplantation periods. The differences of trace metals in the mussel shells transplanted between KSM and SB were unlikely to be contributed by shell lengths and shell growth rates in P. viridis investigated in this study sinvce they not signficantly different (P> 0.05) (Table 1). Based on total soft tissues of P. viridis, Eugene Ng et Table 1. Shell lengths (mean ± SE, mm) and shell growth rate (cm/day) in the mussels transplanted from Kg Pasir Puteh to Kg. Sg. Melayu (KSM) and Sg. Belungkor (SB). Week KSM SB t-test KSM SB t-test 0 2 6 30.45 ± 2.64 53.17 ± 3.14 66.15 ± 4.57 34.46 ± 1.33 55.17 ± 2.37 67.49 ± 1.81 P> 0.05 P> 0.05 P> 0.05 NA 11.36 5.95 NA 10.36 5.51 P> 0.05 P> 0.05 Note: NA- not available Table 2. Trace metal concentrations (μg/kg dry weight) in shell of transplanted Perna viridis from Kg Pasir Puteh to Kg. Sg. Melayu (KSM) and Sg. Belungkor (SB). Cr As Weeks KSM SB t-test KSM SB t-test KSM SB t-test 0 2 6 24.47 + 5.07 19.82 + 0.00 5.92 + 0.39 24.47 + 5.07 7.61 + 2.29 1.18 + 0.98 NA p < 0.05 p < 0.05 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 NA p < 0.05 p < 0.05 69.93 + 3.33 68.16 + 5.65 73.45 + 13.66 69.93 + 0.66 63.40 + 0.00 53.05 + 0.00 NA p < 0.05 p < 0.05 Li Be Cs Weeks KSM SB t-test KSM SB t-test KSM SB t-test 0 2 6 217.40 + 16.61 203.46 + 1.20 211.92 + 0.39 217.40 + 16.61 115.25 + 16.61 1.18 + 0.98 NA p < 0.05 p < 0.05 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.20 + 0.00 0.59 + 0.39 NA p < 0.05 p < 0.05 1.19 + 0.20 1.00 + 0.10 0.37 + 0.29 1.19 + 0.20 2.25 + 0.70 3.45 + 0.28 NA p < 0.05 p < 0.05 Mn Se Weeks KSM SB t-test KSM 0 2 6 6370.92 + 704.72 7113.18 + 135.98 7067.61 + 185.02 6370.92 + 704.72 5838.29 + 479.26 5658.24 + 367.29 NA p < 0.05 p < 0.05 144.88 + 43.20 144.88 + 43.20 16.36 + 7.24 65.00 + 19.67 7.73 + 5.56 52.99 + 3.27 V SB Sr t-test KSM SB NA p < 0.05 p < 0.05 857852.71 + 22843.52 895239.11 + 630.47 898132.54 + 6683.41 857852.71+ 22843.52 NA 766232.83 + 44173.50 p < 0.05 722145.53 + 4365.40 p < 0.05 Co t-test Hg Weeks KSM SB t-test KSM SB t-test KSM SB t-test 0 2 6 17.23 + 1.18 25.97 + 0.20 87.94 + 5.06 17.23 + 1.18 40.22 + 1.59 74.47 + 2.05 NA p < 0.05 p < 0.05 135.81 + 1.98 142.04 + 1.47 137.95 + 6.88 135.81 + 1.98 162.89 + 10.65 162.32 + 4397 NA p < 0.05 p < 0.05 2580.82 + 3.81 2554.56 + 0.15 2547.93 + 6.30 2580.82 + 3.81 2561.73 + 8.00 2545.92 + 13.84 NA p < 0.05 p < 0.05 7 al. (2012) found that KSM and SB had decreased levels of Co, Cr, Hg, Sr and V if compared to KPP after the transplantation period of 6 weeks. However, Eugene Ng et al. (2012) also reported 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. Therefore, the present increased level of As was only supported by the total soft tissue data previously reported by Eugne Ng et al. (2012). All the differences of increased and decreased levels of trace metals between soft and hard (shells) tissues after the transplanation periods from KPP, is thus difficult to explain at the level our understanding. The question of which site is more contaminated by trace metals having higher bioavailabities is dependent of which tissues are analysed (whether soft tissues or hard tissues (shells of mussels). The answer would vary if it is based on the the total shells when compared total soft tissues of the mussels. 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