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HEAVY METALS IN FISH AS A BIOINDICATOR FOR MONITORING OF METAL RECALCITRANTS IN MALAYSIAN RIVERS

Maimon Abdullah1, Salem Alzahrani2 and Abdullah Samat3

1,2,3 School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, National University of (UKM), Bangi 43600 , Malaysia

The Langat River is one of the main sources of water supply for the Selangor state in Malaysia and heavy metals are among the most persistent types of pollutants that can pose a threat to the aquatic ecosystem and human health. This study was conducted to determine the concentrations of heavy metals, namely aluminum (Al), cadmium (Cd), copper (Cu), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb) and zinc (Zn) in the bones, gills, guts, fins, head, muscles and skin of Tilapia, which is a widespread species of freshwater fish that can be used as a potentially useful bioindicator for monitoring metal pollution in water resources. A total of 33 fishes were collected from three stations located in selected parts of the Langat River during two sampling occasions, in January and February 2009, respectively. The metal contents in dried fish samples were extracted by the acid digestion method and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Among the metals analyzed, Fe and Al were the most abundant in the different body parts, while Cd and Ni were the least abundant and were not detectable in the edible body parts. The mean concentrations of metals in Tilapia in descending order are as follows: Fe (4583±1.9); Al (2882±2.07); Zn (62.09±5.15); Mn (100.9±0.017); Cu (20.17±0.011); Pb (10.28±0.039); Cr (2.74±0.04); Ni (1.39±0.13) and Cd (20.17±0.011). Macro and trace element accumulation in fish can pose a health risk to consumers in a contaminated water catchment. However, the results of this study show that the metal contents in Tilapia sampled from the Langat River are still within the normal range except for Fe and Mn, which have exceeded the allowable limit set by the Malaysian Food Act (1983) and Food Regulations (1985). The Langat River is within the safety limit for potable water usage and the Tilapia fish harvested from the river is considered safe for human consumption.

Keywords: heavy metals; Tilapia; biological indicator; Langat River

Introduction As Malaysia undertakes to realize its vision 2020 through its industrialization and urban expansion policies (Muyibi et al. 2008), heavy metals pollution of rivers have become a matter of grave concern as reflected in several studies conducted locally, such as for Kelang River (Law and Singh 1986), Selangor River (Mat and Maah 1994), Linggi River (Khan and Lim 1994), Sepang River (Ismail and Ramli 1997) and Langat River (Sukiman 1989). The Klang-Langat River basin is undergoing a very intensive urbanization process due to rapid population growth and socioeconomic development, while the Langat River is one of the main sources of water supply for the Selangor state. Thus, the river status and its water quality should be continually monitored, particularly with respect to the heavy metals, which are among the most persistent types of pollutants that can pose a threat to the aquatic ecosystem and human health. The impacts of human activities on water bodies require appropriate monitoring tools to facilitate detection and characterization of the causes and sources of chemical, physical and biological impairment of the aquatic habitats. Among these tools, aquatic biota (fish, frogs, insects, benthos and plants) are identified as potential bioindicators to detect pollutant loads in water (Gibson et al. 1996). In tracking long-term changes ID: 326 of a specific water body such as a river system, fish (such as Tilapia, Oreochromis mossambicus) are known to be useful and reliable indicators of long-term effects and broad habitat conditions as highlighted by many investigators over the years (Brinley 1942, Araújo et al. 2000; Vidal, 2008). Tilapia is the common name for around 70 species of perch-like fishes (family Cichlidae) native to the fresh waters of tropical Africa (Bhassu et al. 2004). This study has been carried out to determine the metal concentrations in Tilapia and to screen for its potential as a bioindicator of water pollution by recalcitrants such as heavy metals in the Langat River and other water bodies in Malaysia.

Material and Methods

Study Area The Langat River Basin (area 2938 km2) lies in the mid western part of Peninsular Malaysia and flows through parts of the industrial area of the Klang Valley in two states, namely Selangor and Negeri Sembilan. The Langat River is about 120 km long and the main tributaries are the River and the Labu River (Sukiman 1989). Fish samplings were conducted at three stations in selected parts of the Langat River tributary system. First station is located at Engineering Lake (EL) within the main campus of Universiti Kebangsaan Malaysia in Bangi. The second station is situated in the Langat River (LR) near area and the third station is located at Cempaka Lake (CL), also in Bandar Baru Bangi (Figure 1).

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Figure 1 Three sampling stations in Langat River tributary system, Selangor

Sample Collection A total of 33 fish samples were collected by using a cast net and scoop net with a mesh size of about 2.5 cm. Fresh fish samples were stored in cool boxes, each labeled with dates and location. Likewise, GPS and land use data were recorded for each sampling station. Morphological data of fish recorded are the total length (tip of caudal fin to head), standard length (front of caudal fin to head), and body weight, physical appearance and color. Fresh specimens were immediately taken to the ID: 326 laboratory and all samples were kept frozen in the refrigerator at -20 °C for further preparation and analysis works.

Preparation of the Fish Samples Fish samples were each was separated into seven parts: head, muscles, gills, skin, gut, fin and long bone, using a stainless steel knife to prevent metal contamination. The fresh tissues were weighed, then oven-dried at 80 °C to constant weight. The dried samples were pounded into powder form using a mortar and pestle.

Acid Digestion Method for Fish Heavy metals in fish body parts were extracted by the acid digestion method, using the Kjeldatherm digester model Gerhardt and following the AOAC standard method (1996). Powdered dried sample of fish part (0.5 g) was soaked in 2 ml of concentrated nitric acid in a PTFE beaker, and then fully digested in the Kjeldatherm digester at 100 °C (for about 2 hours); until the dark fumes turned white to indicate that all the nitric acid had evaporated. The digested samples were air-cooled, and then 2 ml of hydrochloric acid added, and further heated for one hour to complete the digestion, resulting in a clear yellowish solution. The cooled solution was filtered through an acid-resistant filter paper (0.90 µm pore size). The filtrate was further diluted with 25ml of distilled water and then analyzed for metals content using the inductively coupled plasma mass spectrometry (ICP-MS, model Perkin-Elmer OPTIMA 4300).

Results and Discussion Sampling efforts using the cast nets were hampered by the presence of floating garbage and debris that were discarded in the upper and midstream parts of the Langat River. The body length of fish samples ranged from 11-22 cm, and the body weight ranged from 40-167 gm.

Heavy Metals in Tilapia

Aluminium (Al) Results showed that overall mean Al level for all the fish body parts (dry weight) was 250±97.54 µg/g (Table 1), with highest mean value from station 3 CL (428±241 µg/g) and the lowest, from station 2 LR (43.71±6.28 µg/g). Among the body parts, the highest mean Al concentration was in the guts (947.86±1174.34 µg/g), and the lowest, in the muscles (3.06±2.926 µg/g). Kruskal-Wallis H test shows that there are statistically significant differences in the Al contents of the various body parts, with the guts having the highest value of Al (Chi-Square=32.66, df;6, P<0.001). The effects of Al on fish behavior have not been reported much. In reviews of dietary studies in fish, Poston (1991) reported no adverse effects on growth, survival, or feed conversion in the Atlantic salmon fed up to 2000 μg/g dietary aluminum. Poston (1991) also noted that in fact, trace amounts of Al had some nutritional benefits to the Atlantic salmon, thus suggesting that Al may be an essential inorganic element and most likely metabolically regulated. However, other studies showed that under acid rain conditions (i.e., low pH), certain Al complexes can be toxic to fish even at low levels (Haines 1981).

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Table 1 Mean Al levels in the fish body parts sampled from the Langat River

Al S1 S2 S3 Total Mean

Skin 2.07±1.92 9.85±2.15 5.966±2.037 5.966±2.037

Bone 15.51±3.27 20.29±5.61 16.43±3.27 17.41±4.05

Fins 8.13±5.34 3.30±1.004 10.61±3.84 7.35±3.42

Muscles - 9.18±8.78 - 3.06±2.926

Gills 31.47±1.78 75.33±6.30 249.51±30.86 118.77±1298

Head 7.45±4.13 7.51±4.29 19.58±3.86 11.52±4.10

Gut 1882±296 2882±1664 - 1588±653.5 Total 277±44.36 428±241 43.71±6.28 250±97.54 Mean

Nickel (Ni) Results showed that Ni concentrations for all the fish body parts ranged from 0.13±0.11 µg/g to 1.39 ±0.08 µg/g. Ni content was the lowest among nine heavy metals tested in the various fish body parts and sampling stations (Table3). The guts contained the highest mean levels of Ni (0.39±0.29 µg/g). Nickel was also detectable in the fins (0.46±0.47 µg/g), but was not detected in the skin, bone, gills, heads and muscles of Tilapia. Nickel level was highest for fish collected from station 1 EL (0.218±0.027 µg/g) and lowest for station 2 LR (0.124±0.014 µg/g). However, Kruskal-Wallis H test shows no significant difference in Ni content between the various body parts of Tilapia. (Chi-Square=53.086, df; 6, P<0.001). Ni content in the body parts of Tilapia is comparatively lower than other metals, ranging from 1.39 – 0.13 µg/g, and well within the safety limit. This indicates that Ni pollution is not of main concern in the Langat River.

Table 2 Mean Ni levels in the fish body parts sampled from the Langat River Ni S1 S2 S3 Total Mean

Skin - - - -

Bone - - - -

Fins 1.397±0.0806 - - 0.465±0.28

Muscle - - - -

Gills - - - - ID: 326

Head - - - -

Gut 0.134±0.111 0.745±0.08 0.285±0.04 0.388±0.08 Total 0.218±0.027 0.124±0.01 0.047±0.007 0.121±0.05 Mean

Copper The guts contained the highest mean Cu contents (10.68±7.38 µg/g), followed by gills (1.21±0.45 µg/g), skin (0.33±0.19 µg/g), head (0.21±0.14 µg/g) and muscles (0.17±0.07 µg/g), respectively. However, Cu was not detectable in the bone and fins of Tilapia. The mean Cu contents in the body parts of Tilapia ranged from 0.011±0.119 µg/g to 20.17±3.26 µg/g (Table 3) and were below the allowable limit of 30 µg/g set by the Malaysian Food Act 1983 and Food Regulations 1985. Fish from station 2 LR had the highest Cu contents whereas those from station 3 LC had the lowest levels. There are significant differences in the Cu content between the different body parts (Chi-Square=54.92, df;6, P<0.001). Tthe Cu levels were higher in the guts compared to the gills, likely due to ingestion of Cu in the sediment as well as the food, while Cu uptake in the gills was likely facilitated by the large surface area available for adsorption and the large volume of water passing over the gills.

Table 3. Mean Cu levels (µg/g) in the fish body parts sampled from the Langat River Cu S1 S2 S3 Total Mean

Skin 0.231±0.042 0.571±0.072 0.18±0.027 0.327±0.047

Bone 0.011±0.119 0.088±0.064 - 0.03±0.061

Fins - - - -

Muscles 0.094±0.028 0.203±0.058 0.198±0.043 0.165±0.043

Gills 0.638±0.129 1.35±0.035 1.63±0.048 1.21±0.070

Head 0.047±0.020 0.371±0.036 0.204±0.02 0.207±0.028

Gut 4.98±1.19 20.17±3.26 6.88±0.124 10.68±1.52 Total 0.857±0.218 3.25±0.503 1.29±0.038 1.80±0.252 Mean

Zinc (Zn) Results showed that the mean Zn contents for all the fish body parts in this study ranged from 5.15±0.261 µg/g to 62.09±3.81 µg/g (Table 4). The guts contain the highest mean value (49.51±14.75 µg/g), whilst the muscles recorded the lowest (5.78±0.76 µg/g), and the values were significantly different (Chi-Square=51.008, df;6, P<0.001). Fish in station 2 LR had the highest Zn levels, followed by those from stations 1 EL and 3 LC, respectively. The Zn levels were higher in the fins and guts compared to the head, bone, gills, muscles and skin, but did not exceed the maximum allowable limit of 100 µg/g set by the Malaysian Food Act 1983 and Food ID: 326

Regulations 1985. The Zn levels were also lower compared to values reported from the Sepang coast (3.79–95.4 µg/g) by Ismail and Saed (2000).

Table 4 Mean Zn levels in the fish body parts sampled from the Langat River Total Zn S1 S2 S3 Mean

Skin 26.33±1.79 22.17±1.61 8.73±0.631 19.07±1.34

Bone 16.20±0.98 21.13±0.41 14.94± 17.42± 1.91

Fins 29.97±1.61 36.63±1.17 27.45±3.05 31.35±1.94

Muscles 5.15±0.261 6.72±0.210 5.46±0.0343 5.77±0.271

Gills 14.72±0.29 16.36± 0.15 15.08±0.64 15.39±0.36

Head 12.84±0.380 14.09±0.773 14.26±6.04 13.73±2.40

Gut 54.91±8.11 62.09±3.81 31.53±4.95 49.51±5.62 Total 22.87±1.91 25.59±1.16 16.77±2.36 21.74±1.82 Mean

Cadmium (Cd) Results showed that the mean Cd contents for all the fish body parts ranged from 5.15±0.261 µg/g to 62.09±3.81 µg/g (Table 9) the guts contain the highest mean Cd contents (0.19±0.25 µg/g), whilst the skin recorded the lowest (0.031±0.021 µg/g). The fish from station 2 LR had the highest mean level of Cd, whereas those from station 1 EL had the lowest mean Cd level. Cd was not detected in the head, gills and bone. There are significant differences in the Cd content between the different body parts of Tilapia (Chi-Square=29.34, df;6, P<0.001). Cadmium was not detected in bone, gills and head of Tilapia, while detectable levels were found in muscles, skin, fins and gut. However the overall Cd levels in Tilapia are still below the permissible limit of 1.00 mg/g set by the Malaysian Food Act and Food Regulation 1985. The main sources of Cd in the Langat River are likely to be from the use of phosphate fertilizers, land application of municipal sewage sludge and atmospheric deposition. However, fish generally contain Cd levels in the range of 5-10 µg/kg fresh weight, which are representative for these food classes. (OECD 1994).

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Table 5 Mean Cd levels in the fish body parts sampled from the Langat River Cd S1 S2 S3 Total Mean

Skin 0.094±0.063 - - 0.031±0.021

Bone - - - -

Fins 0.142±0.082 - - 0.047±0.027

Muscles 0.023±0.013 - - 0.007±0.004

Gills - - - -

Head - - - -

Gut 0.002±0.001 0.501±0.158 0.501±0.15 0.194±0.059 Total 0.037±0.022 0.071±0.022 0.011±0.002 0.04±0.015 Mean

Lead (Pb) Results showed that the mean Pb contents in Tilapia ranged from 0.039±0.029 µg/g to 10.28±1.713 µg/g (Table 6), indicating that the river was moderately polluted. Among the different body parts, the skin recorded the lowest (0.045±0.026 µg/g). Fish from station 3 LC accumulated the highest mean level of Pb (2.045±0.454 µg/g) compared to the other two stations, while fish from station 2 LR had the lowest value (0.649±0.083 µg/g). Statistical analysis (Kruskal-Wallis H test) shows that there are significant difference in the Pb contents between the different body parts. Although the values did not exceed the permissible limit of 2.0 µg/g set by the Food Act 1983, however, Pb in the guts (5.72±3.60µg/g) was found to have exceeded the allowable limit. Sources of Pb pollution could be derived from surface runoffs, emissions from mobile sources (combustion of leaded fuels by motorized vehicles), as well as from industrial processes (WHO 1989).

Table 6 Mean Pb levels in the fish body parts sampled from the Langat River Pb S1 S2 S3 Total Mean

Skin 0.096±0.050 0.039±0.029 0.045±0.026 0.045±0.026

Bone 0.222±0.370 - 1.103±0.540 0.441±0.303

Fins 1.560±0.164 0.163±0.094 1.206±0.261 0.976±0.173

Muscles - 0.072±0.041 - 0.024±0.013

Gills 2.11±0.121 0.110±0.099 1.18±0.332 1.13±0.184

Head 0.089±0.021 - 0.552±0.335 0.213±0.118

Gut 2.71±0.518 4.16±0.323 10.28±1.713 5.720±0.851 Total 0.969±0.177 0.649±0.083 2.045±0.454 1.221±0.238 Mean ID: 326

Iron (Fe) Results showed that the mean Fe contents for all the fish body parts in this study ranged from 4583±403 µg/g to 1.90±1.10 µg/g (Table 7). The guts contain the highest mean Fe contents (3298.28±1481.10 µg/g); whilst the muscles recorded the lowest (2.77±4.05 µg/g). Fish from station 1 EL accumulated the highest mean level of Fe while fish from station 2 LR had the lowest mean Fe level. In general, Fe was accumulated in the highest levels in all the fish body parts compared to the other metals. Statistical analysis (Kruskal-Wallis H test) shows there are significant differences in the Fe content between the various body parts of Tilapia (Chi- Square=52.456, df;6, P<0.001), and Fe levels have exceeded the maximum permissible limit of 0.5 µg/g set by the Malaysian Food Act 1983 and Food Regulations 1985.

Table 7 Mean Fe levels in the fish body parts sampled from the Langat River Fe S1 S2 S3 Total Mean

Skin 21.76±1.40 22.77±5.43 16.66±0.510 20.40±2.450

Bone 35.01±6.63 15.82±3.23 9.82±6.90 20.22±5.59

Fins 12.39±1.086 6.66±5.218 8.77±3.438 9.27±3.247

Muscles - 6.42±5.17 1.90±1.10 2.777±2.091

Gills 97.46±11.72 62.27±3.59 77.78±9.70 79.17±8.33

Head 23.71±2.355 12.39±2.318 12.72±7.150 16.28±3.941

Gut 4583±403 3777±1016 1533±294 3298±571 Total 681±60.88 557±148 237±46.11 492±85.23 Mean

Manganese (Mn) The mean Mn levels in all the fish body parts ranged from 0.017±0.016 µg/g to 100.9±7.08 µg/g (Table 8). The guts contain the highest mean Mn contents (69.63±24.66 µg/g); whilst the muscles recorded the lowest (0.415±0.623 µg/g). Fish from station 2 LR accumulated the highest mean level of Mn (19.27±1.91 µg/g), while fish from station 3 LC had the lowest mean Mn level (13.34±2.019 µg/g). Statistical analysis (Kruskal-Wallis H test) of Mn in different organs of Tilapia shows there are significant differences (Chi-Square=53.06, df; 6, P<0.001). The Mn concentrations in the different body parts of Tilapia have exceeded the allowable limit of 0.3 µg/g for edible parts according to Malaysian Food Act 1983 and Food Regulations 1985. The highest mean content of Mn was recorded in Tilapia from Station 2 LR. This may be due to its location, which is surrounded by many factories and is also considered to be a favorite recreational site for the local anglers. There are also two busy roads adjacent to station 2 that can contribute metal pollutants from non-point sources. Discharges of effluents may play a significant role in increasing the concentrations of Mn and other metals in fish.

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Table 8 Mean Mn levels in the fish body parts sampled from the Langat River Mn S1 S2 S3 Total Mean

Skin 0.318±0.052 1.83±1.45 0.619±0.097 0.923±0.534

Bone 4.33±1.017 9.91±1.57 9.80±1.16 8.01±1.25

Fins 5.78±1.17 10.83±0.829 13.03±1.78 9.88±1.26

Muscles 0.017±0.016 1.12±1.77 0.104±0.081 0.415±0.623

Gills 4.15±0.133 6.07±0.244 8.34±0.271 6.192±0.216

Head 2.27±0.101 4.27±0.431 6.77±1.46 4.43±0.665

Gut 53.15±8.91 100.9±7.08 54.73±9.29 69.62±8.34 Total 10.002±1.62 19.27±1.91 13.34±2.019 14.21±0.758 Mean

Chromium Table 9 shows that Cr was only detected in the bones, gut, head and fins of Tilapia, with the highest mean level in the guts (1.684±1.054 µg/g) and the lowest value in the head (0.0148±0.008 µg/g). In contrast, only low levels of Cr were found in the edible parts such as muscles and skin, while none was detected in the skin, gills, heads and muscles. Fish collected from station 2 LR had the highest mean level of Cr (0.397±0.256 µg/g), while those from station 3 LC had the lowest value (0.122±0.07 µg/g). There are significant differences in the Cr content between the different body parts of Tilapia (Chi-Square=29.9, df; 6, P<0.001). Cr could be sourced from wastewater effluents from the nearby industrial area in Bangi. Studies by Tacon and Beveridge (1982) using tracer trivalent Cr in the Rainbow trout did not show any significant accumulation of Cr in the carcass, fins, bones, skin, gills, muscle, or intestines, even at the highest concentration tested (8.2 μg/g).

Table 9 Mean Cr levels in the fish body parts sampled from the Langat River Mn S1 S2 S3 Total Mean

Skin - - - -

Bone 0.050±0.106 - - 0.016±0.035

Fins 0.054±0.001 0.040±0.023 - 0.031±0.008

Muscles - - - -

Gills - - - -

Head 0.044±0.025 - - 0.0148±0.008

Gut 1.45±0.891 2.740±1.774 0.859±0.496 1.684±1.054 Total 0.221±0.14 0.397±0.256 0.122±0.07 0.249±0.157 Mean ID: 326

Conclusion Among the metals analyzed, Fe and Al were the most abundant in the different body parts, while Cd and Ni were the least abundant and none being detectable in some tissues. The metal concentrations (in µg/g) in fish samples in descending order are as follows: Fe > Al > Zn > Mn > Cu > Pb > Cr > Ni > Cd, respectively. Potentially toxic metals are Pb, Cd, Cr and Cu, and their content in fish samplings have not exceeded the allowable levels set by the Malaysian Food Act (1983) and Food Regulations (1985). The metal concentrations in Tilapia are still within the normal range except for Fe (0.3µg/g) and Mn (0.5 µg/g), which exceeded the allowable limit, particularly for the edible body parts. Although the levels of heavy metals accumulated in Tilapia are not particularly high, there is a potential trend of increasing pollution in the Langat River in the future, derived from domestic sewage, industrial wastes and agricultural activities in the Langat basin in tandem with its population growth. Rather high levels of recalcitrants are found in the guts and gills of the Tilapia, while the muscles and bone have higher resilience against metals pollution, therefore, Tilapia harvested from the Langat River are currently quite safe for human consumption. However, as a precautionary measure, regular monitoring of fish as well as the water quality of the river is highly recommended.

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