Assessment of Heavy Metals (Arsenic, Cadmium, Lead and Total Mercury) in Tilapia sp. in Sri Lanka B.K.K.K.Jinadasa1*, E.M.R.K.B.

Edirisinghe1 Post harvest Technology Division, National Aquatic Resources Research and Development Agency (NARA), Colombo-15, Sri Lanka. E-mail- kkamal@nara.ac.lk

Abstract In a 2008, samples of the muscles, liver and gills of Tilapaia sp. (Oreochromis sp.) in five size classes (<18, 18-19.9, 20-21.9, 22-23.9 and >24 cm) were obtained from a eighteenth (18) Reservoirs in 3 province and four district which are Anuradapura and Polonnaruwa in North Central province, Ampara in Eastern province and Hambanthota in Southern province in Sri Lanka. The elements Arsenic (As), Cadmium (Cd), Lead (Pb) and Total Mercury (Hg) were detected in the sample by atomic absorption spectroscopy. In Tilapia sp the mean Cd, Pb, Hg and As concentration in muscle 0.0340, 0.0571, 0.2624 and 0.2585, gills 0.0225, 0.4374, 0.3640 and 0.2506 and liver 0.2966, 2.0651, 0.5247 and 0.3552 mg/Kg in dry weight basis. That result revealed that the levels of heavy metal concentration in Tilapia sp is not exceed the maximum permissible level for human consumption according to the EU, WHO and Food Standards Australia and New Zealand guidelines. Introduction: Sri Lanka is well known for its dams construction, and is reputed to have one of the highest densities of reservoirs in world (McCully 1996; De Silva 1989). Most of the reservoirs of Sri Lanka are located in the dry zone. At all reservoirs are used for freshwater aquaculture. Common features of this fishery is the dominance in the landing of the exotic Cichlid species introduced in 1952, the Mossambicus Tilapia (De Silva 1985; Fernando & Indrasena 1969). Reservoir fish are the main source of animal protein of the rural people living in dry zone of the country (G. Allinson etal 2008, Amarasinghe and De Silva, 1999; Chandrasiri, 1986).

Metals are found throughout the earth, in rock, soil and sediments primarily trapped in some stable from bedrock by weathering and erosion and allowed to circulate in water and air. This is essential because many biochemical processes require a given amount of some metals. But some heavy metals introduced to environment by anthropogenic activities may adversely affect the system. Heavy metals can be uptake in difference ways by the aquatic organisms. Include passive diffusion, facilated diffusion, active transport and endocytosis. Fish can uptake metals through gills, through digestive tract and via skin. Bioaccumulation is the uptake and sequestration of contaminants by organisms from their ambient environment, in certain cases, the uptake of contaminants by biota directly from the ambient medium by this air, soil, sediments or water (Phillips, 1993 and Senadheera and Pathiratne 2003). Of all the heavy metals, lead probably has the longest history of environmental contamination and toxicity to humans. Environment lead contamination arise s source including agriculture and manufacture process, atmosphere emission from motor vehicles and incineration of municipal solid waste. The primary sources of lead intake in the general population are food including water, accidentally, via paint dust even or soil (Sonia Lucia Blanco etal; 2007). Cadmium is a high toxic element. Even minute amounts of cd are sufficient to cause poisoning. Cd is widely distributed at low levels in the environment; the sources for human exposure air, water food and tobacco, with food representing the major route of the uptake for general public (Anon; 1993). Mercury is one of the ancient metals and it occurs naturally in the environment. Inorganic mercury enters the environment from industry, mining and pollution and natural geochemical processes; its accumulation in sediment generally transformed by microorganisms in to organic mercury, resulting high toxic molecules, such as mono methyl mercury, which bioaccumulates in fish. Arsenic also one of the most toxic metals can be released to the environment by natural and industrial sources; fish can be accumulated high levels and contributes significantly to daily intake of the elements. In many cases Tilapia was used as a bioassay organism in toxicological studies in which it was substantiated with the highest sensitivity to toxic effect (Rashed M.N. 2001). Chronic renal failure (CRF) was reached in high levels in the North Central province area in last few years (Bandara

J.M.R.S. etal 2008). Heavy metals such as As, Cd, Pb and Hg are a threat to public health, even at low concentrations, since long term, chronic exposure may result in the accumulation of toxic level. Low to intermediate levels exposure is associated with chronic toxicity, so detection of food is related from a toxicological point of view. The maximum permissible levels of export fish As, Cd, Pb and Hg have been gazette by government of Sri Lanka (No 1528/7). The level of above metal levels varies according to species. These study focuses on the distribution of As, Cd, Pb and Hg level in Tilapia sp. in North Central province and compare with same sp. in Southern and Eastern province. Materials and methods: The study areas were four district, Anuradapura and Polonnaruwa (North Central province), Hambanthota (Southern province) and Ampara (Eastern province) in Sri Lanka. Number of reservoir selected from above district for ease of access and also to cover the fully area of district. Six reservoirs from Anuradapura district (Kalawewa, Koobichchiyankulama, Thuruwila, Padaviya, Rajanganaya and Wilachchiya), five reservoirs form Polonnaruwa district (Minneriya, Girithale, Maduruoya, Parakrama Samudraya, and Kaudulla), 3 reservoirs form Ampara district (Senanayake Samudraya, Ambalanoya and Nawagiriyawa) and four reservoirs form Hambantota district (Udawalawe, Lunugamwehere, Udukiriwila and mahagalwewa). Fish samples were obtained from commercial landings and brought to the laboratory in ice. In the laboratory the total length and body weight were determined. Fish were divided in to five length classes (<18, 18-19.9, 20-21.9, 22-23.9 and >24 cm) and pooled within categories under the each reservoirs name. Then fish were gutted and the liver and gills removed. Individual fish were filleted and from the middle of the right fillet and above 50 g of block of muscle devoided. All samples were oven dried at 800C to a constant weight and stored in air tight bag until further analysis. Fish samples were digested using Mars CEM XP-1500 (model No 927065) microwave digester. Sample was weighted (0.5 g) in four decimal places and 5 mL of C. HNO3 (supra pure) was added allowed 15 min in fume hood for pre digestion. Then digested were filter (Whatman No 542) in to one mark grade A 100 mL volumetric flask and volume up with double distilled water. The CRM (FAPAS/canned fish/T0774) samples and blank samples were run with each batch.

Spectra AA Varian atomic absorption spectrometer (AAS-240 FS) from Varian with graphite tube atomizer (Varian GTA 120) was used for lead and cadmium determinations. Vapor generation accessory (Varian VGA 77) with close end cell use for mercury determination and open end cell were used for determination arsenic. The following analytical wavelengths were monitored: As, 193.7; Cd, 228.8; Pb, 283.3 and Hg, 253.7 nm, respectively. The limit of detection (LOD) was based on mean sample blank value + 3s. All statistical analysis was conducted using Microsoft Excel 2009 version and SPSS version 16.0 for windows. Results and Discussion: Analysis of the certified reference material ((FAPAS/canned fish/T0774/except lead) found that analyzed trace elements to between excepted values (75%-125% recovery). The concentration and the ranges of the four metals of the muscles, gills and liver tissues of Tilapia sp. in the dry weight in each tank and mean value of each district are presented in table 1 and figure 1 and the analyzed metal concentration of each length classes are presented in table 2. 374 Tilapia samples were collected from 18 reservoirs in four districts. The mean length 21.1 4.0 cm and the mean length was 216.6 162.1 g. The number of samples in each length classes (A; <18, B; 18-19.9, C; 20-21.9, D; 22-23.9 and E; >24 cm) 75, 86, 83, 61 and 69 respectively. Results indicate that the mean level of the cadmium in the muscle tissues of the Tilapia sp was 0.0340 0.03 mg/Kg, dry wt basis (n = 56). The highest value (0.1003 mg/Kg in dry wt) was recorded from Mahagalwewa reservoir in Hambanthota district. The mean level of lead, total mercury and arsenic in the muscle tissues 0.0571 0.13, 0.26240.23 and 0.25850.15 mg/Kg in dry wt basis (n = 56). The highest value of lead (0.6591 mg/Kg dry wt), total mercury (1.1173 mg/Kg dry wt) and arsenic (0.6039 mg/Kg dry wt) were recorded from Mahagalwewa tank, Senanayake Samudraya in Ampara district and Wilachchiya reservoir in Anuradapura district. The mean level of the cadmium, lead, total mercury and arsenic in the gills (n = 56) of Tilapia sp. was 0.0225 0.04, 0.4374 0.83, 0.3640 0.47 and 0.2506 0.17 mg/Kg in dry wt basis. The highest value recorded from Rajanganaya tank in Polonnaruwa district, Wilachchiya tank in Anuradapura district, Ambalanoya tank in Ampara district and Rajanganaya tank respectively. As

same cadmium, lead, total mercury and arsenic in the liver sample (n =55) of Tilapia sp was 0.2966 0.35, 2.0651 2.23, 0.5247 0.93 and 0.3552 0.38 mg/Kg in dry wt basis. The highest value was recorded in Rajanganaya tank, Udukiriwila tank in Hambantota district, Senanayake samudraya and Thuruwila tank in Anuradapuraya district. The higher concentrations of heavy metals were recorded in the gills and liver than in muscle tissues of fish sample. Those levels are greater than in maximum permissible level in some sampling places. There is no statistically significant correlations (p<0.05) between Tilapia length classes and metals concentration muscles, gills and liver tissues and between four district and metals concentration mussels, gills and liver samples. That is excepted results, because freshwater fish maintain constant internal metal concentration; that metal concentration do not increase with age (Allinson etal, 2003). As best of our knowledge unable to found the maximum permitted levels for metals in fish in Sri Lanka, but there is government of Sri Lanka published gazette notification (No.1528/7-17th Dec. 2007) to heavy metals in fish for the fisheries export purpose. According to that the maximum level of Pb, Cd and Hg is 0.20, 0.05 and 0.50 mg/Kg in wet weight basis, but there is not mention about the As. Guidance value of the WHO/FAO committee on food additives (JECFA) and food standard Australia and New Zealand (FSANZ) have established the value with permissible level. FAO Codex level for metals in fish are 0.5 mg/Kg and 1.0 mg/Kg wet weight basis in non predatory and predatory fish (Allison etal, 2008). FSANZ general accepted level of Hg, Pb 1mg/day and 0.5mg/day, JECFA also established the As and Pb level as 0.12 mg/day and 3.6 mg/day. As well as EU regulation specifies the maximum heavy metals limits in fish meat as 0.05 mg/Kg Cd and 0.2 mg/Kg for Pb and 0.50 mg/Kg for Hg (Anon 2005). Sri Lankan consumes an average of 12.3 Kg fish per person per year (Anon 2007). After calculating these value, observed that the daily intake of the metals after consumption of Tilapia is below the standard levels. Though the metals concentration of gills and liver samples of Tilapia is comparatively higher than the muscles tissues, are not arise problem, because when the preparing Tilapia for cooking removes those parts normally.

The heavy metals content of Tilapia fish has variously been reported in previous studies were given in table no 03. The heavy metals levels in Tilapia in Sri Lanka are slightly lower than with the compare with other countries. Conclusion Present study showed that the total Hg, As, Cd and Pb level in Tilapia muscle was below the maximum permissible level. Acknowledgement: Authors wish to thanks to the National Aquatic Resources Research and development Agency to the financial assistant. Follow-up action
1. Finding the Bio Transfer Factor (BTF) of heavy metals

2. Calculate Provisional Tolerable Weekly Intake (PTWI) amount of Tilapia

3. Assess the heavy metal concentration of other indigenous sp and aquatic plant.

References:

Allinson G., Salzman S.A., Turoczy N., Nisikawa M., Amarasinghe U.S., Nirbadha K.G.S., De Silva S.S. (2008). Observation on metal concentrations in two species of Tilapia (Oreochromis mossambicus and Oreochromis niloticus) in cascades of reservoirs of Sri Lanka. Submitted paper of Environmental Contamination & toxicology. Amarasinghe, U.S. and De Silva SS (1999). Sri Lankan reservoir fishery; a case for introduction of a co-management strategy. Fish Mange. Ecol.6, pp 387-399. Anon (1993). Guidance document for cadmium in shellfish. Washington (DC); center for food safety and applied nutrition, US FDA. Anon (2005). Commission regulation EC No 78/2005 of 16th January 2005 amending regulation EC No 466/2001 as regards heavy metals. Official journal L 16/43.20/01/2005 pp 43-45. Annon (2007). National Aquatic Resources research and Development Agency, Sri Lanka fisheries year book, 2007. http://www.nara.ac.lk/Year%20Book-2007/chapter7.html#ch7n4. Arain M.B., Kazi T.G., Jamali M.K., Jalbani N., Afrid H.I., Shah A. (2008). Total dissolved and bioavailable elements in water and sediments samples and their accumulation in Oreochromis mossambicus of Manchar lake. Chemosphere 70; pp1845-1856. Bandara J.M.R.S., Senevirathne D.M.A.N., Dasanayake D.M.R.S.B., Herath V., Bandara J.M.R.P., Abeysekara T., Rajapaksha K.H. (2008). Chronic renal failure among farm families in cascade irrigation system in Sri Lanka associated with elevated dietary cadmium level in rice and freshwater fish (Tilapia) Environ Geochem Health 2008. Chandrasiri J.M.K.D. (1986). Socio economic conditions of inland fisherman in Sri Lanka. Agrarian research institute, Colombo. De Silva S.S. (1985). The Mahaweli baisn, Inland fisheries in multipurpose river basin planning development in tropical Asian counties; three case studies. FAO fisheries technical paper, 265. pp 91-166. De Silva S.S. (1989). Reservoirs of Sri Lanka and their fisheries. FAO fisheries technical paper 298. pp 128.

Fernando C.H., Indrasena H.H.A. (1969). The freshwater fisheries of Ceylon. Bull fisheries Stat Ceylon 26: pp83-95. Khallaf E.A., Galal M. Authman m. (2003). The biology of Oreochromis niloticus in polluted canal. Ecotoxicolgy 12; pp 405-416. McCully, P. (1996). Silenced Rivers. The ecology and politics of large Dams. Zed books, London. Phillips D.J.H (1993). Bioaccumulation; In handbook of ecotoxicology pp 378-396. Blackwell Scientific Publication, Oxford. Rashid M.N. (2001). Monitoring of environmental heavy metals in fish from Nasser lake. Environ Int 27: pp 27-33. Senadheera S.P.S.D. and Pathirathne K.A.S. (2003). Bioaccumulation potential of three toxic heavy metals is shrimp, Penaeus monodon from different fractions of the culture environment. Sri Lanka Journal of Aquatic Science Vol 8, 2003. Sonia Lucia Blanco, Jose Carlos Gonzalez and Juan M. Vieites (2008). Mercury, cadmium and lead levels in samples of the main traded fish and shellfish species in Galicia, Spain. Food additives and contaminants 2008, 1-7. iFirst.

Table 1: Level of Cd, Pb, Hg, As in Tilapia Muscles, Gills and Liver in four distric1 and 18 reservoirs in Sri Lanka (value in mg/Kg dry weight basis)
District name1 Muscle Gill Liver

Reservoir name Anuradapuraya Kalawewa Koobichchiyankulama Padawiya Rajanganaya Thuruwila Wilachchiya Polonnaruwa1 Minneriya Girithale Parakramasamudraya Maduruoya Kaudulla Hambanthota1 Udawalawa Mahagalwewa Udukiriwila Lunugamwehera Ampara1 Senanayake Samudraya Amabalanoya Nawagiriyawa
1

Cd 0.0463 0.0958 0.0016 0.0563 0.0539 0.0215 0.0348 0.0205 0.0321 0.0010 0.0298 0.0108 0.0117 0.0404 0.0310 0.0858 0.0248 0.0328 0.0198 0.0095 0.0350 0.0201

Pb 0.0245 0.0871 0.0000 0.0000 0.0484 0.0000 0.0173 0.0218 0.0000 0.0160 0.0151 0.0317 0.0445 0.1248 0.0697 0.5541 0.0000 0.0000 0.0768 0.0205 0.1459 0.0922

Hg 0.1436 0.1393 0.0534 0.0771 0.1016 0.0735 0.5763 0.2393 0.0717 0.3676 0.1654 0.2809 0.4176 0.3377 0.2421 0.3184 0.3788 0.3725 0.4592 0.6709 0.2049 0.3961

As 0.2655 0.1630 0.0940 0.1183 0.3083 0.5249 0.3020 0.2899 0.3767 0.1737 0.2738 0.2114 0.3073 0.2041 0.1648 0.1287 0.2643 0.2147 0.2852 0.3609 0.2153 0.2415

Cd 0.0406 0.0091 0.0029 0.0630 0.0945 0.0338 0.0139 0.0133 0.0290 0.0000 0.0193 0.0001 0.0046 0.0173 0.0269 0.0450 0.0087 0.0000 0.0086 0.0028 0.0258 0.0000

Pb 0.9753 0.3241 0.3567 0.5263 1.4583 1.2337 2.2274 0.1323 0.2465 0.6154 0.1144 0.0000 0.0145 0.3132 0.0312 0.4472 0.5263 0.1580 0.0173 0.0000 0.0604 0.0000

Hg 0.0495 0.0937 0.0442 0.0343 0.0461 0.0424 0.0379 0.3969 0.0341 0.2915 0.4941 0.5504 0.5737 0.5853 0.5344 0.7441 0.4915 0.6215 0.6233 0.6020 1.0695 0.2090

As 0.2770 0.1437 0.0854 0.0548 0.4349 0.4645 0.5008 0.2816 0.3575 0.1767 0.2416 0.1983 0.3343 0.1665 0.1695 0.1900 0.2155 0.0852 0.2925 0.3621 0.2427 0.2380

Cd 0.2511 0.1528 0.0002 0.0960 0.8060 0.1985 0.2323 0.2821 0.2017 0.4198 0.4345 0.1640 0.2347 0.3892 0.5671 0.2408 0.5252 0.1971 0.2466 0.2826 0.2004 0.2007

Pb 2.2207 3.7090 0.2449 2.0246 3.6630 1.2944 2.0457 0.9007 1.3018 1.4574 1.0224 0.1736 0.6023 3.3022 0.9159 2.7917 6.1908 1.8640 1.5092 0.3996 0.3059 4.1978

Hg 0.1662 0.1176 0.1909 0.1863 0.0865 0.1417 0.3424 0.6164 0.0533 0.2794 0.2543 0.9546 1.4568 0.5182 0.6863 0.5848 0.4417 0.4380 1.2639 2.5996 0.2704 0.2624

As 0.3880 0.2761 0.0869 0.1836 0.2795 0.9396 0.3253 0.4564 0.5204 0.1575 0.3472 0.5011 0.5538 0.2438 0.3319 0.1515 0.3832 0.0726 0.2931 0.2068 0.2928 0.3822

Table 2: Level of Cd, Pb, Hg, As in Tilapia in each length classes (A; <18, B; 18-19.9, C; 20-21.9, D; 22-23.9 and E; >24 cm) in four districts (value in mg/Kg dry weight basis)
District Length Flesh Gills Liver

Class A B Anuradapuraya C D E A B Polonnaruwa C D E C Ampara D E A B Hambanthota C D E

Cd 0.232 3 0.000 0 0.072 4 0.117 2 0.0591 0.140 0 0.397 7 0.095 3 0.264 5 0.254 8 0.118 7 0.000 0 0.176 4 0.197 1 0.289 6 0.136 7 0.2562 0.094 0

Pb 2.045 7 0.000 0 0.0759 3.444 1 0.722 0 0.670 6 1.1605 0.363 4 0.642 8 0.6518 4.084 2 0.000 0 1.694 6 1.864 0 4.834 1 3.792 7 3.396 3 1.5625

Hg 0.342 4 0.000 0 0.143 2 0.100 1 0.108 6 0.866 1 0.516 4 0.268 4 0.766 3 0.845 8 0.388 0 0.000 0 0.414 5 0.438 0 0.2582 0.2150 0.488 3 0.2859

As 0.325 3 0.000 0 0.116 2 0.310 5 0.2659 0.5929 0.5253 0.120 0 0.323 1 0.670 1 0.510 4 0.000 0 0.385 4 0.072 6 0.183 8 0.194 7 0.270 4 0.000 0

Cd 0.129 7 0.000 0 0.008 3 0.004 5 0.064 9 0.016 4 0.028 6 0.011 0 0.010 9 0.027 6 0.200 7 0.019 0 0.021 4 0.033 6 0.028 6 0.026 9 0.030 6 0.020 6

Pb 0.000 0 0.000 0 0.372 8 0.000 0 0.020 0 0.000 0 0.006 7 0.000 0 0.020 0 0.037 0 4.197 8 0.110 9 0.0551 0.000 0 0.000 0 0.201 9 0.316 6 0.000 0

Hg 0.091 8 0.000 0 0.0550 0.067 9 0.039 5 0.250 3 0.119 2 0.276 8 0.1558 0.266 8 0.262 4 0.429 3 0.5675 0.353 1 0.372 2 0.153 8 0.101 2 0.464 2

As 0.000 9 0.000 0 0.116 0 0.531 3 0.221 3 0.247 7 0.333 4 0.111 0 0.309 9 0.241 2 0.382 2 0.286 9 0.272 9 0.267 1 0.193 6 0.0561 0.104 6 0.231 4

Cd 0.1965 0.000 0 0.127 9 0.001 9 0.041 5 0.030 4 0.024 6 0.001 6 0.0156 0.008 1 0.000 0 0.008 1 0.0115 0.000 0 0.006 2 0.0285 0.017 4 0.000 0

Pb 1.614 0 0.000 0 3.5550 0.401 5 2.999 2 0.204 6 1.201 8 0.018 8 0.083 9 0.261 8 0.000 0 0.033 2 0.007 1 0.021 9 0.3505 0.388 7 0.232 7 0.629 8

Hg 0.034 7 0.000 0 0.108 8 0.019 9 0.040 0 0.260 1 0.106 7 0.353 0 0.457 6 0.305 2 0.000 0 0.866 8 0.5072 0.631 8 0.5465 0.604 0 0.2151 1.278 2

As 0.0221 0.0000 0.1176 0.4542 0.3178 0.3176 0.3632 0.3224 0.2295 0.2702 0.0000 0.3692 0.2193 0.0577 0.3058 0.1224 0.0800 0.1275

Table 3: Investigated heavy metal level in Tilapia sp in previous studies.

Metals level in muscle(mg/Kg wet wt) As Cd Hg Pb _ 0.02 0.45 0.15 0.65 0.09 0.07 0.29 0.59 0.35 _ 0.6 0.26 0.05 0.02 _ <0.90 <0.15 0.01 _ 0.03* 0.06* 0.26* 0.26*

Fish Sp. O. niloticus O. mossambicus O. mossambicus O. mossambicus O. niloticus Oreochromis sp.

Country Egypt China Pakisthan Sri Lanka Sri Lanka Sri Lanka

Reference Khallaf etal Allinson etal Allinson etal Allinson etal Allinson etal This study

* Value in mg/Kg dry weight basis

Figure 1: Mean concentration of Total Hg, As, Cd and Pb (mg/Kg dry wt.) in the muscle, gill and liver tissues of Tilapia sp. in four districts.

3.00 2.50 2.00 1.50 1.00 0.50 0.00

concentartion (mg/Kg)

3.50

Anuradapuraya Polonnaruwa Ampara Hambanthota

Cd

Pb

Hg

As

Cd

Pb

Hg

As

Cd

Pb

Hg

As

Flesh

Gills Sample type and metals

Liver