Chemicals
The present study was conducted from the period from October 2017 to October 2020 at Fisheries College and Research Institute, Thoothukudi.
Standard solutions of organochlorine pesticides (99% purity) containing α, β, γ, δ-BHC, heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin, endrinalde-hyde, endosulfan I, endosulfan II, endosulfan sulfate, p,p’-DDE, p,p’- DDT, p,p’-DDD, methoxychlor (M-8270-14-ASL) was obtained from AccuStandard, Inc. USA. Chemicals of QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) kit such as primary secondary amines (PSA), carbon-18 (C18), graphitized carbon black (GCB), magnesium sulfate (MgSO
4) and sodium acetate (NaOAc) were individually purchased from Sigma-Aldrich (Bangalore, Karnataka, India). All other solvents such as ethyl acetate, dichloromethane, acetone nitrile, n-hexane, methanol, acetic acid and petroleum ether used for sample analysis were of HPLC grade obtained from Sigma-Aldrich and Rankem Chemicals (Gurugram, Haryana, India). The storage vials of 2 ml in capacity were purchased from Agilent Technologies, Germany.
Study area
Five sampling sites
viz. Papanasam (Site 1), Cheranmadevi (Site 2), Vannarapettai (Site 3), Srivaikuntam (Site 4) and Punnakayal (Site 5) were chosen from the source to the mouth of the river and their locations are given in Plate 1 and 2.
A global position system (GPS, Leica G20, USA) was used to locate the sampling locations more precisely during the sampling. Fish samples were collected from the respective sites and brought to the laboratory in iced condition and held in deep-freezer at -20°C until analysis. Three freshwater fish
viz catla (
Catla catla), rohu (
Labeo rohita) and tilapia (
Oreochromis mossambicus) were collected from the first four sampling sites. Four marine and estuarine fishes such as Indian mackerel (
Rastrelliger kanagurta), Milk fish (
Chanos chanos), Indian oil sardine (
Sardinella longiceps) and Mullet (
Mugil cephalus). were collected from the station 5, which is an estuary region receiving influx of marine and freshwater.
Extraction and cleanup
Pesticides from fish muscle and their organs were extracted as per AOAC 2007.01 using QuEChERS method
(Anastassiades et al., 2003a,b; Norli et al., 2011). For the analysis, 5g of each homogenized sample was mixed separately with 15 ml of 1% of acetic acid in acetonitrile in a centrifuge tube and vortexed well for 1min to maintain the pH between 5 to 7. From that, 4-5 ml of supernatant was taken in another centrifuge tube and kept at -20°C for 15 min to enable fat deposition. From which, 1.5 ml of upper layer was taken and 150 mg of MgSO
4 and 100 mg of CaCl
2 were added, vortexed well for 1 min and centrifuged again at 5000 rpm for 5 min to increase the solvent partitioning. Finally, 1 ml of supernatant was taken and added to QuEChERS clean up kit. The kit contained PSA - 25 mg (to remove the sugars, fatty acids and organic acids), C18 - 25 mg (to remove the long chain fatty compounds, sterols and non-polar interference), GCB- 7 mg (to remove the pigments, polyphenols and other polar compounds without loss of planar pesticides like HCB and Terbufos and MgSO
4 -150 mg (to separate the solvent layer and improve the recovery of pesticides). The mixture was shaken vigorously and centrifuged at 9000 rpm for 5 min. After the clean-up process, 1 ml of supernatant was taken for GC-ECD analysis.