IRSPAS 2019
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/20453
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Item Novel solid phase micro extraction (SPME) Method for GC-MS analysis of Tributyltin.(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Bandara, K. R. V.; Chinthaka, S. D. M.; Manage, P.M.Tributyltin (TBT) is an organotin compound belongs to the group of Persistent Organic Pollutants (POPs) and one of the active ingredients in biocides used to control a broad spectrum of organisms. Antifouling paints which are used for boat hulls, docks, fishnets contain tributyltin to prevent the growth of aquatic fouling organisms. Increasing concentration of TBT in the environment, due to anthropogenic activities cause toxicological impact on target and non-target organisms having sexual disorders like sex changes at ultra-trace level called imposex. According to WHO, the No Observed Effective Level (NOEL) of TBT is below 1 ng L-1, thus a highly specific and sensitive analytical methods are needed to detect TBT in parts per trillion (ppt) level. In the present study, a new ultra-trace quantification method to detect TBT using Solid Phase Micro Extraction (SPME) followed by Gas Chromatography-Mass Spectrometry (GCMS) was optimized. TBT derivatization to tributyltinhydride was carried out using potassium borohydride (KBH4). Medium polar PDMS/DVB, fused silica (65 μm, 24 Ga) SPME fiber were found to be the most appropriate conditions for extraction and pre-concentration of derivatized TBT hydride compound. The best conditions for the extraction were optimized and analyses were done by using GC-MS under 270 ºC desorption temperature and 1 mL min-1 of helium gas flow rate. Field samples were collected from highly polluted and heavy boat traffic area along the coastal belt from Dikkovita to Mirissa, Sri Lanka. The optimal extraction conditions were found to be 10 minutes extraction time, pH 4.9 and the ambient temperature 30 ºC. The highest TBT concentration was detected in Colombo port (303±4.7 ng L-1) following the TBT was recorded in the fishery harbors at Beruwala (124±4.1 ng L-1), Galle (110±4.1 ng L-1), Dikkovita (97±4.3 ng L-1), Hikkaduwa (77±5.3 ng L-1) and Ambalangoda (57±5.2 ng L-1). TBT concentrations in Dehiwala, Wellawatta, Madu, Benthota and Gin ganga river mouths were recorded as 268±4.1 ng L-1, 240±3.4 ng L-1, 214±3.3 ng L-1, 145±2.7 ng L-1 and 50± 2.1 ng L-1 respectively. The recovery of the TBT extraction was 87±2.1 % for the artificial sea water while the Minimum Quantification Level (MQL) was 1 ng L-1. The Minimum Detection Level (MDL) of the method was calculated as0.3 ng L-1. The optimized method is a solvent free and the use of SPME is highly sensitive method to detect TBT in parts per trillion (ppt) levels.Item Bacterial degradation of tetracycline (TET) by TET resistant bacteria; A green solution for antibiotic pollution.(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Liyanage, G. Y.; Manage, P.M.Tetracyclines (TET) have been extensively used in aquaculture for chemotherapy against various fish diseases such as fin rot, skin ulcers. Overuse and misuse of antibiotics are widely regarded as two of the major factors promoting antibiotic resistance. Resistance to TET occurs via two primary mechanisms; ribosomal protection and enzyme inactivation. To minimize the development of resistance, excess amount of TET should be removed from the aquatic environment. Therefore, studies on the biodegradability of TET can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of TET by Enterobacter sp., Micrococcus luteus, Enterobacter ludwigii, Bacillus sp. and Streptomyces sp., which were isolated as TET resistance, non-pathogenic bacteria. In a previous study, TET resistant bacterial isolates were identified using the 16s rRNA sequencing. Overnight bacterial cultures grown in a medium without TET, were introduced into a medium containing TET at final concentration of 5 μg/ml. Triplicate samples were incubated at 28 °C with shaking at 100 rpm under dark conditions. Subsamples (0.5 ml) were removed at 2 days interval for a period of 14 days. Remaining TET in the subsamples was analyzed using High Performance Liquid Chromatography (HPLC). Complete degradation of TET by M. luteus and E. ludwigii was detected at the end of 14 days of incubation. Descending degradation percentages were followed by Enterobacter sp. (74%), Streptomyces sp. (72%) and Bacillus sp. (70%) at 14 days, respectively. Lowest half-life time of TET was shown by M. luteus and E. ludwigii (6 days), whereas half-life time of 8 days, 11 days and 12 days were recorded for Enterobacter sp, Streptomyces sp. and Bacillus sp., respectively. According to the degradation results obtained, it can be concluded that the selected bacterial strains can be used as potential candidates to be introduced into wastewater effluents to remove TET in effluent water, before reaching natural environmentsItem Cyanobacteria and 2-Methylisoborneol: the influence of Nitrogen and Phosphorous(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Ganegoda, S. S.; Chinthaka, S. D. M.; Manage, P.M.2-Methylisoborneol (2-MIB) is a key compound, which causes taste and odour (T&O) issues in water. Despite of no recorded health hazards reported associated with 2-MIB, water consumers reject the water with 2-MIB due to its unpleasant musty (moldy) T&O. The aim of this study was to study the correlation between cyanobacteria abundance, 2-MIB levels coupled with Nitrogen and Phosphorous levels. 2-MIB contamination level in sixteen raw water bodies, which are being used for drinking in six districts (Anuradhapura, Pollonnaruwa, Ampara, Batticaloa, Trincomalee and Hambanthota) were analyzed using Gas Chromatography–Mass Spectrometry coupled with Solid-phase micro extraction. Enumeration and identification of cyanobacteria was carried out using standard microscopic methods. Anabaena, Microcystis, Oscillatoria, and Cylindrospermopsis species were identified as the most abundant cyanobacteria. 2-MIB levels ranged from 5.3 ± 0.94 to 139.4 ± 0.21 ppt throughout the dry season of the sampling period, where the highest level was recorded in Kondawatuwana tank (139.4 ± 0.21 ppt) and the lowest was detected in Ridiyagama tank (5.3 ± 0.94). At wet season, 2-MIB levels ranged from 4.4 ± 0.78 to 73.8 ± 0.65 ppt, where the highest level was recorded in Jayanthi tank (73.8 ± 0.65 ppt), while the lowest was detected in Ridiyagama tank (5.3 ± 0.39 ppt). Recorded 2- MIB level was greater in dry season compared to the wet season. Seventy five percent (75%) of the sampling locations exceeded the human threshold levels of 2-MIB (5 ppt). Questionnaire survey showed that more than 95% end water consumers rejected drinking water contaminated with 2-MIB. Further it was found that the total cyanobacterial cell density (T.C.D) was positively correlated with 2-MIB and total phosphorous levels (p<0.05). Moreover, cell densities of Oscillatoria, Anabaena and Cylindrospermopsis species showed significant positive correlations (p<0.05) with 2-MIB contamination levels along with Pearson Correlation Coefficients (P.C.C) of 0.788, 0.682 and 0.731. However, no significant correlation was observed between Microcystis sp. and 2-MIB. Further, 2-MIB showed significant positive correlation (p<0.05) with total phosphorous (P.C.C 0.876), electrical conductivity (EC) (P.C.C, 0.771), and pH (P.C.C, 0.825). Increment of pH value leading to alkalinity is a known optimum condition for cyanobacteria growth while the current study shows alkalinity is favorable for 2-MIB. No significant correlation was obtained between total nitrogen (Nitrate –N, Nitrite –N, Ammonia –N) and 2-MIB nor T.C.D and total nitrogen. Thus, the results of the study indicate there is a direct positive correlation between 2-MIB, cyanobacteria and total phosphorous.Item Microcystin -LR contamination status of Nile tilapia (Oreochromis niloticus) and biomarker response(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Abeysiri, H. A. S. N.; Wanigasuriya, K.; Manage, P.M.Microcystin-LR (MC-LR) is a cyanotoxin derived from some cyanobacteria. Nile tilapia (Oreochromis niloticus) is the most popular freshwater fish among people in Sri Lanka. MC-LR accumulation in tilapia from thirteen reservoirs; Nallachchiya Wewa, Galkulama Wewa, Anakattiya Wewa, Padaviya Wewa, Nachchaduwa Wewa and Kalawewa in Anuradhapura District and from four reservoirs; Parakrama Samudraya, Halmilla Wewa, Kaudulla Wewa and Ambagas Wewa in Polonnaruwa District Muwapatigewela Wewa in Ampara District and Ulhitiya Wewa and Rathkinda Wewa in Badulla District was determined to evaluate the risk posed by the MC-LR contamination in fish on human health. Sample collection, transportation and analysis were followed according to the standard protocols and MC-LR was quantified by High Performance Liquid Chromatography. Fish skin, flesh and head were analyzed for MC-LR and Tolerable Daily Intake (TDI) values were calculated according to the WHO guidelines. MC-LR levels of fish in different reservoirs were compared with WHO standard of TDI (0.04 μg/kg/day). MC-LR level of fish skin (717.14 ± 0.82 μg/kg), flesh (105.11±0.08 μg/kg) and head (553.24±0.12 μg/kg) collected from Padaviya Wewa were recorded the highest mean concentrations of MC-LR. The mean concentrations of MC-LR in the skin and head of fish were significantly higher than that of flesh (p<0.05). The Average Daily Intake of MC-LR in the skin and head of all fish exceeded the provisional TDI set by WHO. Thus, the results of the present study revealed that consumption of head and skin of fish has a potential risk on accumulation of MC-LR in human body. Cyanobacterial toxins have been shown to affect aquatic organisms such as fish, resulting in oxidative stress. Among the antioxidant enzymes, glutathione peroxidase (GPx) plays an important role in the detoxification of MCs. Fish tissues such as head, flesh and skin were obtained for “GPx” gene expression analysis and results showed availability of detoxifying enzymes in fish skin and head collected from Parakrama Samudraya, Padaviya Wewa, Nallachchiya Wewa and Galkulama Wewa. MC-LR contamination in Galkulama Wewa showed a negative result. Therefore, previous exposure of fish for MC-LR shows indicator of the expression of gene of any detoxifying enzyme. Therefore, the results support the use of GPx in fish as a biomarker to assess the contamination by MC-LRItem Isolation of laccase producing fungi: Aspergillus niger from Sri Lankan textile wastewater effluents and its potential applicability on decolorization of an azo dye: CI Direct Blue 201(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Ekanayake, E. M. M. S.; Wijesekara, I.; Manage, P.M.The complex aromatic structural nature of synthetic dyes show resistance to natural oxidation processes and persist in the surface water and sediments for a long time. The existing physical and chemical treatment methods are costly and create secondary pollution. Therefore, the present study was focused on the degradation of an azo dye: CI Direct Blue 201 (DB 201), by myco-remediation. Aspergillus niger, a filamentous fungus, was isolated from textile wastewater effluent site in Sri Lanka and pure cultures were maintained on Potato Dextrose Agar (PDA) plates. Four cylinders (10 mm diameter in each) of actively growing A. niger cultures were cut and inoculated into mineral salt medium consisted of 50 mgL-1 DB 201 dye. All the experiments were carried out in triplicates, while controls were maintained without addition of the fungus. Flasks were incubated at 28 °C for seven days with shaking at 100 rpm. Three milliliters of sample aliquots were removed at 6 hrs intervals, centrifuged and the changes of the absorbance in the supernatant was analyzed through UV-Vis spectrophotometer at 570 nm. The laccase activity was determined by measuring the increase in the optical density at 420 nm. The reaction mixture for laccase assay contained 5 mM of 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) in 50 mM sodium acetate buffer (pH 4.5) and 50 μL of decolorized dye solution (ε420 = 36000 M−1 cm−1). Decolorized dye sample was analyzed through Fourier Transform Infrared (FTIR) Spectroscopy. A Bio-sorption test was carried out by providing the same incubation conditions using 3-day-old live and autoclaved fungi. The control without adding fungus, remained the same without showing any decolorization. The enzyme activity of laccase has increased during the decolorization processes from 18 Uml-1 to 254 Uml-1. The changes of the FTIR spectra relevant to the N=N Vibration (1723.3 cm-1), S=O Stretching (1227.3 cm-1) and N-O Stretching (742.88 cm-1) indicated the changes of the initial DB 201 dye structure after the treatment by A. niger. Furthermore, the bio-sorption assay by live (100%) and autoclaved fungi (12 ± 2%) confirmed the decolorization and the degradation of DB 201 dye would be based on the metabolic activity of the fungus rather than surface adsorption. Therefore, the present study emphasizes the potency of A. niger as an eco-friendly candidate for degradation of azo dyes. Further studies regarding the application of enzymes for real textile dye treatments are currently in progress.