Bioaccumulation of Heavy Metals in Horseshoe Crabs (Tachypleus gigas) from Pekan, Pahang, Malaysia
B. Akbar John,
M.H. Aqilah Megat
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.
Received: February 22, 2011;
Accepted: May 20, 2011;
Published: July 16, 2011
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 -20°C 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 60°C 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 60°C 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).
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.
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) except animal sex which had meager influence.
Female crabs accumulate considerabely higher amount of metals such as Fe, Zn
and Cu in their body parts (except gill tissue). Two fold increase in bioaccumulation
of Fe in male crab gill tissues were detected besides Zn and Cu which were also
higher accumulated in comparison to female crabs (Fig. 2).
Cadmium level were lower in male crab parts compared to female crabs (except
in apodeme). It was also observed that both male and female crabs accumualates
same level of cadmium content in their gill tissues (Fig. 3).
Average concentration of heavy metals in different parts of T. gigas
(regardless of sex) indicated that gill tissue accumulate more amount of Fe
(1336.73 ppm) followed by mouth (965.6 ppm), Apodeme (942.83 ppm), legs (814.45
ppm) and gut (566.61 ppm). Zinc accumulation in different body parts flowed
in Apetome (921.11 ppm) > Gill (912.68 ppm) > Leg (615.01 ppm) > Gut
(606.44 ppm) > Mouth (605.36 ppm) order. It was evident from the present
study that the accumulation Fe and Zn in comparison to Cu level was 3-13, 3-10
times higher in different body parts respectively. The flow of Cu concentration
in horseshoe crabs showed Gut (183.42 ppm) > Apodeme (129.95 ppm) > Gill
(102.22 ppm) > Mouth (97.10 ppm) > Leg (60.85 ppm) order.
||Comparison of mean bioaccumulation level of Iron (Fe), Zinc
(Zn) and Copper (Cu) in different parts of male and female T. gigas.
X-axis represents different body parts of Male (M) and Female (F) horseshoe
crabs and Y-axis represents the metal bioaccumulation level (ppm). Overall
data represented as Mean±SE
||Comparison of mean bioaccumulation level of Cadmium (Cd) in
different parts of male and female T. gigas. X-axis represents different
body parts of Male (M) and Female (F) horseshoe crabs and Y-axis represents
the metal bioaccumulation level (ppm). Overall data represented as Mean±SE
|| Overall means of metals in different tissues of horseshoe
crab (Tachypleus gigas) collected from Pekan, Pahang in 2010
Cadmium accumulated in least concentration compared to other metals studied
and bioaccumulative flow was observed in apotome (4.16 ppm) > Gut (3.80 ppm)
> Gill (3.70 ppm) > Mouth (2.59 ppm) > Leg (2.12 ppm) order (Table
1). Overall metal bioaccumulation in different tissues of T. gigas collected
from Pekan coastal area, Pahang was in Fe > Zn > Cu > Cd order.
Comparative study of detected bioaccumulation in present study with earlier
studies (Burger et al., 2003) and national and
international Maximum Permissible standard Limits (MPL) clearly showed the exceeded
level of metals in T. gigas from Pekan coast (Fe = 817.59±279.35
ppm; Zn = 638.22±168.74 ppm; Cu=103.19±45.6 ppm; Cd = 2.87±0.86
ppm) with reference to MPL while the conspecifics of T. gigas in Atlantic
coast (L. polyphemus) accumulate 5-8 fold increase (13-23 ppm) in cadmium
concentration (Table 2).
||Guidelines on heavy metals concentration (μg g-1
dry weight) for food safety set by local and international body and comparison
of results with previous studies on Atlantic horseshoe crab (L. polyphemus)
|MPL: Maximum permissible limit, WHO: World health organization,
FDA: Food and drug administration and MRF: Malaysian regulation on Food.
Data represented in Mean±SD
The knowledge on metal bioaccumulation in native species is very important
for their management, utilization of these species for human consumption and
to determine the useful bioindicator species through various biomonitoring programs.
It was suggested that interpreting the levels of metals in invertebrates is
difficult because both toxicity and susceptibility differ due to their detoxification
ability (Burger et al., 2002). Considerable amount
of studies were carried out on Atlantic horseshoe crab (L. polyphemus)
due to their higher population density, their utilization in bite fishery and
biomedical industries besides their importance in balancing migratory shorebirds
food chain along the Mexican coasts (Botton et al.,
1994). It was also found out that heavy metal pollution is a major factor
responsible for the declining horseshoe crab population in Delaware bay (Botton,
2000). The environmental and toxicological studies on other three conspecific
species are still scanty. It is evident from present study that the essential
metals bioaccumulate in higher quantity than the nonessential metals. The elevated
concentration of Fe, Zn and Cu in horseshoe crab might be due to the major role
played by these elements in maintaining the proper physiological functions of
the organism through various enzymatic activities. This observation was well
corresponded with the previous study by Kanakaraju et
al. (2008) who postulated that these metals play an important role as
an essential element in all living systems from invertebrates to human, hence
the organisms tend to accumulate high concentration of Fe from the surrounding
environment. This observation might also be due to the organisms capacity
to regulate and accumulate elevated concentration of these metals (Kamaruzzaman
et al., 2010c). The observed high concentration of metals in female
crabs might be due to (1) their long term exposure to the bottom sediments compared
to male (2) their feeding behavioral pattern, physiologic condition and metabolic
activity of the animal.
Comparison with earlier studies on conspecific species from Mexican coast (L.
polyphemus), T. gigas accumulate 5-8 times less amount of cadmium.
This may relate partly to the lack of industry near the nesting beaches along
the Malaysian coasts and to the relative distance from ocean which is a source
of some naturally occurring metals (mercury, cadmium) (Burger
et al., 2002). Comparison with the recent report suggested that the
source of Cd in Pekan coast was primarily through the natural sources and virtually
no anthropogenic activities through various industrial processes were observed
along this coast. Hence the minimal enrichment concentration of Cd in nesting
ground of T. gigas would have been reflected in low accumulated concentration
of Cd in different body parts of horseshoe crab (Zaleha
et al., 2010) whereas industrialization and various anthropogenic
activities would influenced the higher accumulation of Cd in horseshoe crab
samples from New Jersey and Delaware bay (Burger et al.,
The detected concentration of heavy metals in different parts of horseshoe
crabs was more than Maximum Permissible Limit (MPL) and hence T. gigas
from Pekan site should not be used for human consumption. These heavy metal
contaminated horseshoe crabs might result in bioaccumulation of toxic metals
in the human system via food chain and may lead to adverse health effects (Bergback
et al., 1992; Koller et al., 2004).
Unlike in the Mexican coast, horseshoe crab in Malaysian coast neither being used for bait fishery nor their eggs are being prayed upon by the shore birds due to low population density of horseshoe crabs and less number of landing during mating season due to site preference. Hence, only limiting source of their population would be the predatory activity or environmental pollution. Thus the knowledge on various contaminants in the horseshoe crabs and their surrounding environment would pave the way in understanding the healthiness of the environment and enacting various managemental laws to protect the horseshoe crabs in Malaysian coasts.
Authors express their sincere gratitude to the International Islamic University Malaysia for providing infrastructure facilities.
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