Organochlorine Pesticide Residues in Freshwater Fish Species from Sri Lanka: A Human Health Risk Assessment

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1 American Journal of Chemistry and Applications 2018; 5(3): ISSN: (Print); ISSN: (Online) Organochlorine Pesticide Residues in Freshwater Fish Species from Sri Lanka: A Human Health Risk Assessment Bedigama Kankanamge Kolita Kamal Jinadasa *, Gabadage Dona Thilini Madurangika Jayasinghe Analytical Chemistry Laboratory (ACL), National Aquatic Resources Research & Development Agency (NARA), Colombo, Sri Lanka address * Corresponding author To cite this article Bedigama Kankanamge Kolita Kamal Jinadasa, Gabadage Dona Thilini Madurangika Jayasinghe. Organochlorine Pesticide Residues in Freshwater Fish Species from Sri Lanka: A Human Health Risk Assessment. American Journal of Chemistry and Applications. Vol. 5, No. 3, 2018, pp Received: May 15, 2018; Accepted: June 24, 2018; Published: July 26, 2018 Abstract Twenty organochlorine pesticide residues (OCP) were analysed in the muscle tissue of the seven selected fish species (n42) known as Tilapia (Oreochromis sp.), Striped snakehead (Channa striata), Eel (Anguilla sp.), Pearl spot (Etroplus suratensis), Long whiskers catfish (Mystus gulio), Stinging catfish (Heteropneustes fossilis), Zig-zag eel (Mastacembelus armatus) with the aim of assessing the health risk to adult Sri Lankan. The fish were collected from the landing site located at the freshwater reservoir in Anuradhapura district from Nov-Dec The QuEChERS method was used to extracted and purified of the samples and gas chromatography (GC) coupled with Electron Capture Detector (ECD) was used to OCP analyse. The γ-bhc was the most commonly detected compounds in the fish samples and it was accounted as 71% of the analysed samples respectively. Out of the 20 OCP considered in this study, 10 were present in the various concentrations. The Eel was the most contaminated fish species found through this study (2, ng/g, ww basis) and followed by Tilapia (1, ng/g, ww). The results raise concern the possible non-carcinogenic risk and there was no carcinogenic health risk the results of exposure of the consumption of fish selected fish species. Keywords Organochlorine Pesticide Residues (OCP), Freshwater Fish, Anuradhapura, Contamination 1. Introduction Pesticides are synthesised toxic chemical agents used as a pests killer for killing the insects, rodents, fungi and unwanted plants [1]. They are broadly divided into several classes of which the most vital is an organochlorine pesticide (OCP) and its residues in food have been concern for several years [2]. The OCPs are broad spectrum pesticide which is widely used in many countries including Sri Lanka for the agricultural purposes due to low cost and versatility [3]. The OCPs are highly stable and lipid soluble compounds. As a result, it has the potential to bioaccumulation and biomagnification through the food chain, especially in breast milk, blood and fatty tissues [4, 5]. Fish are identified as biomagnifies OCPs from the surrounding environment and transfer it to humans when consumed [6]. Due to consumption of OCPs contaminated food, highly affected to the human health; include Inuit infants, immune system malfunction, endocrine disruption and cancer [7, 8]. Well known OCP s are namely α-bhc, aldrin, and β-endosulfan which are associated with the development of chronic kidney diseases unknown aetiology, CKDU [9]. The number of CKDU patients were recorded in the sampling area and exposure of pesticides identified as a one of the risk factor [10]. The long-range environmental transport and high hydrophobicity of OCP s cause to accumulate the aquatic water bodies especially sediments where they may be transferred through food chain [11]. Bioaccumulation of the OCPs into the fish tissues and making them to the useful

2 American Journal of Chemistry and Applications 2018; 5(3): biological indicator in environmental assessment and have a tendency to the transfer higher trophic levels [12]. The human exposure for OCP s are mainly through the diet (fish, meat, dairy products etc) [13], breastfeeding and placental transfer [14]. Sri Lanka ratified the Stockholm Convention on POPs in December of 2005 and has taken several steps to banned persistent pesticides found in the country under the control of pesticides act 1980, its amendment act 1994 and Malathion control act 1984 [15]. The capture and culture reservoir fisheries are most important fisheries in Sri Lanka, especially in Anuradhapura district. As a result, published by the ministry of fisheries and aquatic resources development, Sri Lanka, Anuradhapura district catch-up highest contribution (17.7%) for total inland fish production in the year 2015 [16]. But the cadmium (Cd), fluoride and cyanotoxin have been reported as serious environmental health problems in the most reservoir in North Central Province area including Anuradhapura district [17]. However, the level of OCP s in edible freshwater fish species has not been well documented. Hence, through this study hopes to determine the concentration of OCPs in the fish species collected in the Anuradhapura district, Sri Lanka. 2. Material & Methods 2.1. Sample Collection and Preparation Forty-two (42) fish samples in seven fish species, namely Tilapia, (Oreochromis sp.), Striped snakehead (Channa striata), Eel (Anguilla sp.), Pearl spot (Etroplus suratensis), Long whiskers catfish (Mystus gulio), Stinging catfish (Heteropneustes fossilis), Zig-zag eel (Mastacembelus armatus) were collected from commercial fishermen in the landing sites of Anuradhapura reservoirs (Rajanganaya, Abaya wewa, Nuwara wewa, Thisa wewa, Wilachchiya, Mahakanadarawa) on November to December After collection, the entire sample put on the ice in insulated box and transferred immediately to the analytical chemistry laboratory. Then the fish were dissected to obtain a bone and skinless dorsal axial muscle tissues. The muscle tissues were homogenized and packed in sealed bags and stored refrigerator (-20 C) until further analysis Extraction Procedure The extraction procedure was based on AOAC official method The fish sample was defrosted and chopped, then weighted 15±0.01 g accurately into a 50 ml centrifuge tube. Acetonitrile (15 ml, with 1% acetic acid) and internal standard (IS) were added to each sample. To achieve phase separation between water and acetonitrile, the tube containing 6 g of anhydrous MgSO 4 and 1.5 g of anhydrous CH 3 COONa, QuEChERS salt packet (Agilent bond elute-5982) was added into it and vortex vigorously for 1 min. After vortexing, the samples were centrifuged for 1 min at 1500 rpm. Then, dispersive solid phase extraction (dspe) with the resin used to removes fatty acids. The 6 ml of sample was taken in a tube containing 150 mg of Primary and secondary amine exchange material (PSA) and 1 g of MgSO 4 (Agilent dispersive kit-5982), vortex for 2 min and centrifuged for 1 min at 1500 rpm to separate the solid materials. Approximately 1 ml of the extract was transferred to a gas chromatography (GC) vial through filtering by using 0.22 µm syringe filter and injected to the GC MS (gas chromatography coupled with electron capture detector) Gas Chromatography Condition In total 20 OCPs, α-bhc, β-bhc, δ-bhc (Lindane), γ- BHC, Heptachlor, Aldrin, Heptachlor exo-epoxide-isomer B (HCEI-B), γ-chlordane, α-chlordane, α-endosulfan, 4,4- DDE, Dieldrin, Endrin, β-endosulfan, 4,4-DDD, Endrin aldehyde, Endosulfan sulfate, 4,4-DDT, Endrin ketone and Methoxychlor were analysed in fish sample. The analysis was performed on Shimadzu GC 2014 (Shimadzu, Japan) equipped with an Electron Capture Detector (ECD). Agilent J&W DB-5ms Ultra Inert (30 m 0.25 mm 0.25 µm) column employed for the separation of analytes and high purity nitrogen gas used as a carrier gas. The analytical conditions are as follow, 825 C to 175 C (15 C/min), 10 C/min to 260 C (1 min) Quality Assurance of Analysis Blank and IS samples were run with every 6 samples. FAPAS T05113QC (oily fish) sample was run with each batch to assess instrument response and extraction procedure accuracy Health Risk Assessment The estimated daily intake (EDI) for the OCP residues in each fish species were calculated based on the following equation. Where C; average measured concentration of OCP (ng/g, ww), CR; estimated daily fish consumption rate (g/day) and BW; body weight of a person (kg) in here set as 50.5 kg. The daily fish consumption rate per person (43.2 g/day) obtained from the fisheries statistic data sheet published by the ministry of fisheries aquatic resources development, Sri Lanka [16]. Risk characterization was calculated for two ways (i) for non-carcinogenic risk (ii) for carcinogenic risk To evaluate the potential non-carcinogenic health risk due to consumption of OCP contaminated fish was assessed by calculating the hazard index (HI). Where, EDI; Estimated Daily Intake and ADI; Acceptable Daily Intake. Buah-Kwofie, Humphries [12] highlighted for preliminary risk assessment study, if HI 0.2 that there are negligible adverse health effects as a result of exposure while HI>0.2, it is required to further detailed risk assessment and (1) (2)

3 75 Bedigama Kankanamge Kolita Kamal Jinadasa and Gabadage Dona Thilini Madurangika Jayasinghe: Organochlorine Pesticide Residues in Freshwater Fish Species from Sri Lanka: A Human Health Risk Assessment risk management measures to be undertaken. For carcinogenic effects evaluation, hazard ratio (HR) was calculated using the following equation Where CBC is the cancer benchmark concentration which was calculated using the following equation Where, RL; maximum acceptable risk level (1x10-6, dimensionless), OSF; oral slope factor (mg/kg, day) BW; body weight (50.5 kg) and CR; consumption rate (43.2 g/day). The OSF values for OCP were obtained from the United States Environmental Protection Agency (USEPA) Integrated Risk Information System (IRIS) for each contaminant ( An HR<1 indicates (3) (4) that there are no adverse health effects are expected as a result of exposure while, HR<1 indicates that adverse health effects are possible [13]. 3. Results and Discussion The GC chromatogram of 100 ng/ml OCPs mix standards are given in figure 1. The detection limit was calculated according to the EURACHEM guideline-2014 (The Fitness for Purpose of Analytical Methods). FAPAS T05113QC quality control material contained only Dieldrin and its recovery was 33.4±4.8 ng/g (n8). The IS recovery was maintained between % and RSD<15% throughout the analysis. This is proved that the analytical protocol of OCP analysis of fish samples complied with the US-EPA requirement. The OCP concentrations in fish samples not corrected for recoveries. Figure 1. GC chromatogram of OCP mixture (100 ng/ml), (1) α-bhc (2) β-bhc (3) δ-bhc (Lindane) (4) γ-bhc (5) Heptachlor (6) Aldrin (7) Heptachlor exo-epoxide-isomer B (8) γ-chlordane (9) α-chlordane (10) α-endosulfan (11) 4,4-DDE (12) Dieldrin (13) Endrin (14) β-endosulfan (15) 4,4-DDD (16) Endrin aldehyde (17) Endosulfan sulfate (18) 4,4-DDT (19) Endrin ketone (20) Methoxychlor. Varies concentrations of OCP were detected in muscle tissues of all studied fish species. Ten OCP including α-bhc, β-bhc, δ-bhc, γ-bhc, Aldrin, Heptachlor exo-epoxideisomer B, Endrin, β -Endosulfan, 4,4-DDD and 4,4-DDT were detected in varying concentration (Table 1). γ-bhc was the most commonly detected OCP among selected fish species and it was accounted for 71%. The general pattern of OCP contaminations were in the order of γ-bhc > Aldrin> 4,4-DDT> Endrin> β-bhc, γ-bhc > Heptachlor exoepoxide-isomer B> α-bhc> δ-bhc> β-endosulfan> 4,4- DDD. The 8 number of OCPs were detected in Oreochromis sp. out of 10. Table 1. Mean concentration of OCPs (SD) in muscle tissues of selected fish species (ng/g). Concentrations are compared to maximum residue limits (MRLs) set by the European Commission (source: Pesticides EC MRL (ng/g Oreochromis sp. C. striata E. suratensis M. gulio soluble fat) H. fossilis Anguilla sp M. armatus α-bhc (171.09) <23.01 <23.01 <23.01 < (258.33) <23.01 γ- BHC 10 <25.90 <25.90 <25.90 <25.90 < (298.33) <25.90 β-bhc (290.67) <28.00 <28.00 <28.00 < (383.21) <28.00 δ-bhc (234.81) (328.57) (23.33) (289.38) (53.33) <21.90 <21.90 Aldrin (182.24) <13.01 <13.01 <13.01 < (197.97) <13.01 Heptachlor exoepoxide-isomer B (100.87) (72.12) < (36.57) <31.50 < (187.13) Endrin (447.52) < (51.78) (40.54) (16.67) <50.00 <50.00 β-endosulfan 50* (169.01) <70.10 <70.10 <70.10 <70.10 <70.10 < ,4-DDD (143.70) <40.40 <40.40 <40.40 < ( ) < ,4-DDT 1000** (192.37) <20.00 *ΣDDT and **ΣEndosulfan, the bold numbers were exceeded EU, MRLs (239.02) <20.00 < (628.37) (403.48)

4 American Journal of Chemistry and Applications 2018; 5(3): The ΣOCP were ranged from ng/kg in ww, while the highest ΣOCP were found in Anguilla sp. This is a common concept because most of the EU countries have been used yellow Eel (Anguilla anguilla) as a time-trend monitoring of OCP [18]. The other possible reasons for the recorded high amount of ΣOCP are mentioned as the Eel are relatively insensitive to poor water quality and considered the high amount of fat content [19]. Jürgens, Chaemfa [20] reported even after two decades of banned or server restriction into the United Kingdom, comparatively high amount of ΣDDT ( ng/g) into the European Eel (Anguilla anguilla) from Thames river. There was a considerable difference observed within the species (Figure 2). Figure 2. Variation of OCP concentration (ng/g, ww) detected in studied fish species The metabolites, 4,4-DDD observed in high amount in Oreochromis sp and Anguilla sp. This was accounted for 32% and 76% of ΣDDT concentration respectively. The predominance of 4,4-DDD was observed in many other previous studies also [12]. It may be due to the longer halflife of the 4,4-DD in the environment (7 years). BHC profiles were common compared with the most studied species and γ-bhc was the dominant OCPs found in the fish. It was only detected residues in C. striata (Figure 3). Commercially two types of BHC namely technical BHC (α- BHC and γ-bhc) and δ-bhc (Lindane) were introduced to the environment worldwide. The ratio between βbhc/σbhc<0.5 indicates that recently use of BHC while and α-bhc/γ-bhc<1 suggest that use of Lindane [21]. In here βhch/σbhc value was 0.37 and α-bhc/γ-bhc value was Hence, selected fish species were contaminated by recently used BHC from the reservoir catchment area in Anuradhapura district. Figure 3. The relative contribution of individual residues in muscle tissues of studied fish species.

5 77 Bedigama Kankanamge Kolita Kamal Jinadasa and Gabadage Dona Thilini Madurangika Jayasinghe: Organochlorine Pesticide Residues in Freshwater Fish Species from Sri Lanka: A Human Health Risk Assessment To evaluate the non-carcinogenic health risk, the EDI and HI were calculated for the fish species (Table 2). The highest EDI value was observed in 4,4-DDD in Anguilla sp ( ng/kg, day). However, HI value was not calculated due to unavailability of oral reference dose (RfD). The 3 HI values (Aldrin, HCEI-B and Endrin) were exceeded the recommended value while the highest HI value was recorded Pesticides Aldrin for M. gulio (52.97). All studied species, exceeded HI values more than 1 for at least 1 OCP. The exposure levels are an age-dependent parameter, researchers published the infants and younger have higher food consumption ratio per body weight [22], hence they have the highest possibility to non-carcinogenic health risk compare with the adults. Table 2. EDI and HI values for studied fish species; the bolded HI values are exceeded 1. Oreochromis sp. C. striata E. suratensis M. gulio H. fossilis Anguilla sp M. armatus EDI HI EDI HI EDI HI EDI HI EDI HI EDI HI EDI HI α-bhc NA _ NA _ NA _ NA _ NA NA _ NA γ-bhc _ β-bhc NA _ NA _ NA _ NA _ NA NA _ NA δ-bhc NA NA NA NA NA _ NA _ NA Aldrin _ 3.98 _ _ _ HCEI-B Endrin β-endosulfan ,4-DDD NA _ NA _ NA _ NA _ NA NA 4,4-DDT HCEI-B - Heptachlor exo-epoxide-isomer B The carcinogenic effect was assessed for the OCP residues detected in the selected fish species and hazard ratio (HR) were calculated (Table 3). The relevant oral reference dose (RfD) and slope factor were obtained from USEPA s Integrated Risk Information System (IRIS). Table 3. Hazards ratio of OCPs residues in selected fish species for adults. Pesticides OSF Oreochromis sp. C. striata E. suratensis M. gulio H. fossilis Anguilla sp M. armatus Hazard Ratio (HR) α-bhc _ β-bhc _ γ-bhc 1.3 _ 0.22 _ Aldrin _ HCEI-B _ ,4-DDT _ The hazard ratio (HR>1) obtained for Heptachlor exoepoxide-isomer B for Anguilla sp. while all other calculated values below 1. Those values (except HR>1 scenario) are suggesting that the lifetime cancer risk associated with studied fish species is greater than one in a one million. Meanwhile, HR was almost close to 1 for α-bhc for Anguilla sp. (Table 3). However, this study has number limitations such as (i) not consider the potential exposure of age (ii) not studied most common cultured fish sp. like Indian and Chinese carp from sampling area except Oreochromis (iii) possible interaction between elements and number of other elements also not studied. 4. Conclusion Even with the limitations, the overall conclusion of the study revealed that the fish species from Anuradhapura district, Sri Lanka contaminated for OCPs and they were associated with non-cancer risk while almost all species were not associated with cancer risk. Acknowledgements The work was supported by National Aquatic Resources Research & Development Agency (NARA) and the authors are thankful to Dr. EMRKB Edirisinghe, Mrs. JM Chandrika and Mrs. UK Kusumalatha who assisted in field and laboratory. References [1] WHO. Pesticide residues in food [2] Stephen, W. C. C. and L. S. C. Benedict, Determination of organochlorine pesticide residues in fatty foods: A critical review on the analytical methods and their testing capabilities. Journal of chromatography A, : p [3] Guruge, K. S. and S. Tanabe, Contamination by persistant organochlorine and butyl tin compounds in the west cost of Sri Lanka. Marine pollution bulletin, (3): p

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