Bioaccumulation of Heavy Metals in Horseshoe Crabs (tachypleus Gigas) from Pekan, Pahang, Malaysia

ABSTRACT

An attempt was made to determine the various heavy metal accumulative concentrations in different body parts of horseshoe crab [Tachypleus gigas (Muller, 1785)]. Heavy metal accumulation levels were determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Highest mean concentration (μg g-1 wet weight) of iron (Fe), Zinc (Zn), Copper (Cu) and Cadmium (Cd) was observed in gill tissue (1336.73±2.08 ppm), apodeme (921.11±8.12 ppm), Gut (129.94±13.8 ppm) and apodeme (4.16±0.54 ppm) samples, respectively while lowest concentrations of metals were observed in gut (Fe = 556.61 ppm), mouth (Zn = 605.36 ppm), leg tissue (Cu = 60.85 ppm) and leg tissues (Cd = 2.12 ppm). Results clearly showed that bioaccumulation of essential metal concentration in all the analyzed body parts were higher than non-essential heavy metals with the flow of metals in Fe>Zn>Cu>Cd order. Statistical predictions revealed that bioaccumulation of metals were not significantly influenced by weight, total length and carapace width of the animal. The heavy metal accumulations in samples were higher than the national and international permissible limit range hence not suitable for human consumption.

INTRODUCTION

Horseshoe crabs are marine chelicerate arthropods remarkably retaining their genetic makeup and morphologically unchanged for more than 200 million years (Walls et al., 2002; Hurton and Berkson, 2004). Out of four known species of horseshoe crab, three species (Trachypleus gigas, T. tridentatus and Carcinoscorpius rotundicauda) inhabits Malaysian waters (Kassim et al., 2008; John et al., 2010) where they spawn throughout the year (Hajeb et al., 2005) with the peak spawning during May and June (John et al., 2010). Field observation showed that T. gigas and C. rotundicauda usually nest along the sandy and muddy beaches of Malaysia respectively while T. tridentatus nests only in East Malaysian coasts (Borneo). The utilization of horseshoe crab blood in pharmaceutical industries to detect the bacterial endotoxin level in biologicals including injectable drugs pose severe threats to its population. Earlier studies proved that the stress undergone by Limulus polyphemus due to blood extraction leads to 15-30% of mortality after being released in the ocean (Hurton and Berkson, 2004). However, It is also evident that the environmental contaminants has direct influence on limiting horseshoe crab population (Zaleha et al., 2010). A series of experiments by Botton (2000), Botton et al. (1998) and Itow et al. (l998a, b) have shown that horseshoe crab eggs are vulnerable to heavy metals, with mercury, organotin and cadmium which is being the most toxic. There were mortality and developmental defects were observed in developing embryos at the laboratory rearing at 39.5 mg L-1 (LC50) levels of cadmium and 3.2 mg L-1 (LC50) levels of mercury for continuous exposure. However, Itow et al. (l998a) did not found a high rate of abnormalities in the eggs or larvae of horseshoe crabs from delaware Bay. Further, horseshoe crab eggs were remarkably tolerant of heavy metal pollution in comparison to the tolerances of similar developmental stages of other marine crustacean. The relatively high tolerance of horseshoe crab eggs to heavy metals suggests that eggs might pose a problem for consumers (Botton et al., 1998).

Various studies had been carried out to determine the toxic heavy metal level in different parts of L. polyphemus (Botton, 2000; Burger et al., 2002). The studies on heavy metal bioaccumulation in T. gigas from Malaysian waters are still scanty except a finding on metal concentration in nesting grounds of horseshoe crabs (Zaleha et al., 2010). Hence present study was initiated to determine some heavy metal bioaccumulation in different parts of shore reaching matured horseshoe crab (T. gigas) from Pekan, Pahang, Malaysia.

MATERIALS AND METHODS

Sample collection and preparation: Matured horseshoe crab (T. gigas) samples were collected alive from Pekan coast (East Coast of peninsular Malaysia) nesting grounds (N 030 30' 0.00" E 1030 25' 1.20") on May 2010 during Full moon days and immediately transferred to INOCEM laboratory (Fig. 1). Data such as sex, total length (mm), carapace width (mm), weight of the samples (g) were noted after removing the symbionts attached to their body. Samples were killed by keeping them in -20oC for 2 h and washed thoroughly with running tap water prior to dissection. Clean and sterilized scissors were used to dissect open the animal from ventral region and body parts such as Gill tissue, legs, mouth parts, gut and internal flesh tissues (Apodeme) were exercised and washed with distilled water and kept separately in labeled Petri dishes and left in hot air oven at 60oC for 8 days. After the samples were completely dried, they were ground using mortar and pestle and kept in labeled falcon tubes prior to digestion.

Acid digestion method: An accurate sample dry weight, 0.5 g of different tissues was gently digested for 3 h in 10 mL concentrated HNO3 followed by 6 mL mix acid (3HCl:1H2O2:2H2SO4) and heated at 60oC until the digestion was completed. Then, 3 mL mixed acid (2HNO3:1H2O2) was added to the solution and then heated to dryness. After cooling, the solution was then transferred to a 50 mL polypropylene vial and completed with 5% HNO3 digestion. For each series of samples, three analytical blanks were prepared in a similar manner without samples to check the possible contamination. Finally, the samples were analyzed for Fe, Zn, Cu and Cd concentration by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Kamaruzzaman et al., 2010b). Analytical quality was assessed using standard reference materials, DORM (dogfish muscle: National Research Council Canada). Recoveries of all the elements ranged from 96 to 105% of the certified value.

Statistical prediction: Data were analyzed with non-parametric procedures to compare concentrations among metals and tissues. Kruskal Wallis correlation coefficients were used to compare the various metal concentrations (Corder and Foreman, 2009).

Fig. 1: Map showing location where horseshoe crab samples were collected (Source: Kamaruzzaman et al., 2010a)

Both arithmetic and geometric means are given to facilitate comparisons with other studies. Two way Anova test were used to check the influence of weight, sex, total length and carapace width over the bioaccumulation of different metals in different parts.

RESULTS

Overall there is no significant influence of parameters such as weight of the animal, total length, carapace width over the heavy metal accumulation in different body parts were observed (p>0.05)

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