Abstract: Muscle and feather in tissue of 40 juveniles and 40 adult green-lipped mussel Perna veridis (L.) collected from Muar Estuary, Johor were analyzed for copper (Cu), cadmium (Cd), lead (Pb) and zinc (Zn) concentration using a fast and sensitive Inductively Coupled Plasma Mass Spectrometer (ICP-MS). In this study, the average concentration of Cu was 8.96 μg g-1 dry weights, Cd with 0.58 μg g-1 dry weight, Pb averaging 2.28 μg g-1 dry weights and Zn averaged to 86.73 μg g-1 dry weight. The highest accumulation of metal studied was found in feather sample compared to the muscle. The positive relationship of Cu, Cd, Pb and Zn with P. virdis length suggesting that the accumulation of these metals were formed in the mussel. In all cases, metal levels found were lower than the guideline of international standards of reference and the examined bivalve were not associated with enhanced metal content in their tissues and were safe within the limits for human consumption.
INTRODUCTION
The wide diversity of human activities that introduce pollutants into the environment, as well as their magnitudes make the assessment of environmental impact a subject of utmost interest. Contamination of aquatic environments with potentially harmful substances, in particular non-degradable heavy metals and its subsequent impact on organism, is more dramatic within estuaries and semi-closed coastal zones, especially when they are near highly populated or industrial areas. Heavy metals may enter an estuary from different natural and anthropogenic sources, including industrial or domestic sewage, storm runoff, leaching from landfills, shipping activities and atmospheric deposits (Goodwin et al., 2003; Saifullah et al., 2002).
The green-lipped mussel Perna veridis (L.) is widely distributed in the coastal waters of the Asia-Pacific region (Tanabe, 2000) and therefore its use as a biomonitoring agent has been interesting for scientific purposes. The marine mussels can act as a quantitative indicator to reflect the heavy metal contamination in the coastal areas (Phillips, 1995; Yap et al., 2003). Another reason that mussels are often chosen for biomonitoring studies, are they are sedentary organisms, long-lived, easily identified and sampled, reasonably abundant and available throughout the year, tolerant of natural environmental fluctuations and pollution. Besides, they have good net accumulation capacities and they are important ecologically (Yap et al., 2004).
Some metals such as Cu, Cd, Pb and Zn have long been known to accumulate within the aquatic food chain. These metals are widely distributed in the coastal environment, both from natural source and anthropogenic activities, thus the metals are readily accumulated in the soft tissue of P. veridis. Mussels are well known to accumulate a wide range of contaminant in their tissues and have been suggested as a potential biomonitoring agent of heavy metals in many countries (Phillips, 1995; Yap et al., 2003). In recent years, the study area especially for the first km along the Muar River has been heavily impacted by discharges from municipal and industrial outflows. This was due to the rapid development of the area via expansion of the industrialization area as well as the increase in population. The aim of this study is to obtain the background data on levels of heavy metals (Cu, Cd, Pb and Zn) in muscle and feather of P. virdis that were found abundant in the study areas.
MATERIALS AND METHODS
Description of the study area: Muar estuary located in southern of peninsular Malaysia, encompasses an estimated wide of 150 to 300 m. It flows through from north to southwest of Johor and entering the Malacca Straight (Fig. 1). The indigenous people inhabiting Muar River are mostly fisherman, factory worker and grocer. This river is intensively exploited by the surrounding community not only as a route for fisherman but also as a port for shipyard, route of industrial wastes and also an active area for sand mining activities. Nevertheless, this river has a diverse ecosystem and also highly utilizable natural resources such as oyster, crab, prawn and some mangrove species. In recent years, the study area especially for the first km along the Muar River has been heavily impacted by discharges from municipal and industrial outflows especially near Muar City and Tanjung Agas areas. This was due to the rapid development of the area via expansion of the industrialization area as well as the increase in population. Electronics and petro-chemicals are the main industry in the area and is the catalyst for other supportive industries to develop around the same area. Bivalve species that were dominantly caught in 11th September 2006 were selected; green-lipped mussel Perna veridis (L.).
Analytical procedures: Forty juveniles and 40 adults P. veridis were collected randomly with a sufficient number of individuals along Muar estuary, especially near the fish culture areas. The samples were transported to the laboratory and stored at -20 °C prior to analysis. Then the samples were thawed at room temperature and their length was recorded using the callipers and 3 decimal points of measuring weight, respectively. They were dissected using stainless steel scalpels and Teflon forceps using a laminar flow bench. A part of the mussel (muscle and feather) were removed and transferred in polypropylene vials. Before acid digestion, a porcelain mortar was employed to grind and to homogenise the dry tissue samples.
Briefly, the digestion method involved the heating of 1.0 g of sample
in a teflon beaker with mixed concentrated acids of HNO3 and
H2SO4 in the ratio of 1:1. The teflon beakers were
kept at 100 °C for 2-3 h. After cooling, a H2O2
solution was added in order to break down any recalcitrant lipid material
in the tissue and a clear solution with no residue should be obtained
at this stage. Deionized water was added and allowed to evaporate a few
times to dryness and finally the digested tissues were transferred into
a 50 mL volumetric flask. An inductively coupled plasma mass spectrometer
(ICP-MS) was used, for the quick and precise determination of Cu, Cd,
Pb and Zn in the tissue samples. The precision assessed by replicate analyses
was within 3%. The accuracy was also examined by analyzing a blank and
a material standard from the National Research Council of Canada standard
(NBS DORM 2) and the results coincided with the certified values within
a difference of ± 3%.
| Fig. 1: | The study area and sampling locations at Muar River |
Statistical analysis: Statistics were performed using a two factor analysis of variance. This method was based on the procedure of general linear models, where samples were examined for potential influence on Cu, Cd, Pb and Zn. Differences between level means per factor were treated using Tukey`s multiple comparisons of means. When ANOVA assumptions such as sample normality and homoscedasticity were not respected, multiple (n>2), sample comparisons were performed by nonparametric Kruskal-Wallis tests. An ANOVA paired tests were then used for two-sample comparisons. A p ≤ 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
A total of 80 P. veridis (40 juveniles and 40 adults) were analyzed
for Cu, Cd, Pb and Zn concentration with a mean length size from 38 to
129 mm. Figure 2 shows the mean values of the measured
heavy metals (Cu, Cd, Pb and Zn) in muscle and feather of P. veridis.
There are significant differences between the samples within the same
area. The concentration of Cu in muscle from the juvenile (38-80 mm) to
the adult size (81-130 mm) of P. veridis was varied within the
average of 6.12 and 8.16 μg g-1 dry weight, respectively.
Feather indicates the concentration of Cu was relatively high compared
to the muscle concentration which was 11.20 and 10.36 μg g-1
dry weight, respectively. For Cd, the concentration in adult P. veridis
muscle was 0.56 μg g-1 dry weight and juvenile was 0.42
μg g-1 dry weight. The Cd concentration in P. veridis
feather was higher in juvenile mussel (0.85 μg g-1 dry
weight) compared to the adult mussel (0.50 μg g-1 dry
weight). Meanwhile the concentration of Pb in muscle of juvenile and adult
mussel was 1.39 μg g-1 dry weight and 1.72 μg g-1
dry weight, respectively. Similar with other metals, the Pb concentration
in adult mussel feather was lower (2.32 μg g-1 dry weight)
compared to the juvenile mussel (3.70 μg g-1 dry weight).
Zn was found higher in the adult size of P. veridis mussel which
indicating the average concentration of 85.49 μg g-1 dry
weight followed by the juvenile size which is 42.13 μg g-1
dry weight. However, the concentration Zn is even higher in the feather
of juvenile P. veridis which as high as 118.94 and 100.36 μg
g-1 dry weight for adult mussel feather.
| Fig. 2: | The concentration of heavy metal in the muscle and feather content of P. veridis from Muar River, Johor, (a) Muscle and (b) Feather |
| Fig. 3: | Relationship of Cu, Cd, Pb and Zn concentrations in P. veridis versus mussel length |
Generally, the concentration of all study metal in mussel feather was slightly higher compared to the muscle in both juvenile and adult mussel. Meanwhile, the metal concentration in juvenile mussel feather was higher than the adult mussel. This may due to the P. veridis has the greatest growth rate of the mussels. Maximum growth of the green mussel occurs 2 m below the surface because of the increased productivity of the water at that depth and a narrow area of temperature and salinity fluctuation (Yap et al., 2003).
The well relationship formed between the study metal concentration and P. veridis suggesting that metals bioaccumulation has occurred and many results for different species in other studies indicating the occurrence of bioaccumulation. In this study, a statistically significant correlation was also observed between metal concentrations in P. veridis with the length of the mussel (Fig. 3). The correlation of the Cu, Cd, Pb and Zn concentration in P. veridis were fairly significantly with r = 0.8099, r = 0.6619, r = 0.7170 and r = 0.6769, respectively.
Present study shows that the level of Cu in the P. veridis collected
from Muar estuary were lower than other studies done in Malaysia but higher
than Kuala Dinding, Perak and Bagan Lalang, Selangor. Meanwhile, the concentration
of Cd and Pb of present study were lower than other study by Yap et
al. (2004). For Zn, P. veridis collected from Sebatu, Malacca
has the lower concentration compared to this study (Table
1). Finally, when this study compared with metal levels found in P.
veridis from other area of this region, our concentration level are
lower and comparable to the study by other scientist (Table
2). The metal levels were also lower than the recommended guidelines
for Cu, Cd, Pb and Zn set by different countries (Table
3). Thus, the average concentration of heavy metal of P. veridis
at Muar estuary was considered safe to be consume but however there was
still an obvious bioaccumulation of pollutant in the muscle content and
feather content.
| Table 1: | A comparison of mean concentration (μg g-1 dry weight) of heavy metals in Perna veridis from other studies done in Malaysia with present study |
| Table 2: | A comparison of mean concentration (μg g-1 dry weight) of heavy metals in Perna veridis from regional studies with present study |
| Table 3: | Guidelines on heavy metals concentration (μg g-1 dry weight) for food safety set by different countries (Yap et al., 2004) |
CONCLUSION
The concentration of Cu, Cd, Pb and Zn in the P. veridis of the Muar estuary was found not to be serious. In this study, P. veridis accumulates these metals in their soft tissues and constitutes one of the important food-chains in the coastal environment. From the data obtained, these metal concentrations could be contributed to natural and anthropogenic metal sources that affecting their habitats. From the human public health point of view, these results seem to show no possibility of acute toxicities of Cu, Cd, Pb and Zn if the edible mussels are consumed by human.
ACKNOWLEDGMENTS
This research was conducted with funding from the Malaysia Ministry of Science, Technology and Innovation (MOSTI), under the Intensified Research for Priority Areas (IRPA) project number 55007. The authors wish to express their gratitude to Oceanography Laboratory teams for their invaluable assistance and hospitality throughout the sampling period.