Fatty acid profile variations after exposure to textile industry effluents in Indian Major Carps

Indian major carps are the widely consumed fish species of Pakistan, being a cheap source of proteins and unsaturated fatty acids, they are good for cardiovascular health. Water pollution due to discharge of untreated industrial waste water into water bodies contaminates this precious source of nutrients. The present study therefore, was aimed to assess deterioration of fatty acid profile of three Indian major carp species due to different concentrations of industrial wastes. The water samples were collected from the river Chenab at the site where it receives industrial wastewater via Chakbandi drain. After exposure to 1.5%, 3.0%, and 4.5% dilutions of collected water in different aquaria it was observed that proportion of unsaturated fatty acids in selected fish species were decreased significantly as the intensity of the dose increased (P < 0.05). Conversely the level of saturated fatty acids increased with the increasing dose of treatment (P < 0.05). These findings suggest that untreated wastewater not only deteriorate the fatty acid profile of aquatic animals but also these toxic substances can reach human body through fish meat and pose further health hazards. Therefore, it is highly recommended that industrial effluents should be treated before they are dumped into water bodies.


Introduction
Indian major carp species, Labeo rohita (Ham.), Catla catla (Ham.), and Cirrhinus mrigala (Ham.) having great economic importance are the commercial fish species and important aquatic food item of Pakistan due to consumer prefer ability owing to their taste and quality of meat (Hussain et al., 2018).
In the recent past, fish culture has increased in Asian countries (Hussain et al., 2018). Fish has a highly nutritional Value like rich source of protein and omega 3 Fatty acid. Poly unsaturated omega-3 prevent cardio vascular effect like blood viscosity reduced, platelet inhibiting, Blood viscosity lower, inflammation suppression, Function of Abstract Indian major carps are the widely consumed fish species of Pakistan, being a cheap source of proteins and unsaturated fatty acids, they are good for cardiovascular health. Water pollution due to discharge of untreated industrial waste water into water bodies contaminates this precious source of nutrients. The present study therefore, was aimed to assess deterioration of fatty acid profile of three Indian major carp species due to different concentrations of industrial wastes. The water samples were collected from the river Chenab at the site where it receives industrial wastewater via Chakbandi drain. After exposure to 1.5%, 3.0%, and 4.5% dilutions of collected water in different aquaria it was observed that proportion of unsaturated fatty acids in selected fish species were decreased significantly as the intensity of the dose increased (P < 0.05). Conversely the level of saturated fatty acids increased with the increasing dose of treatment (P < 0.05). These findings suggest that untreated wastewater not only deteriorate the fatty acid profile of aquatic animals but also these toxic substances can reach human body through fish meat and pose further health hazards. Therefore, it is highly recommended that industrial effluents should be treated before they are dumped into water bodies.

Fatty acid profile variations after exposure to textile industry effluents in Indian Major Carps
tissues primarily involved (Lv et al., 2019). The higher concentrations of heavy metals in edible part of the fish are an additional threat to human health (Chaudhry and Jabeen, 2011).
The aim of present study was to assess perturbed fatty acid profiles in Indian major carps after treatment with different concentrations of industrial effluents as a potential bio-indicator of water pollution.

Study area
Water samples were collected from the River Chenab where it receives wastewater from the Chakbandi drain at latitude 31.570° and longitude 72.534°. The Chakbandi drain collects wastewater from different industries and pours these industrial wastes into River Chenab ( Figure 1). The water samples were analyzed for pH, total dissolved solids (TDS), salinity, alkalinity, electric conductivity (EC), hardness, dissolved oxygen (DO), CO 2 and Temperature (Table 1). Water samples from upstream of the site was used as control. Fish with average weight 10 grams, total length 9.0cm and fork length 7.0cm were collected from Satyana Road Fish Hatchery, Faisalabad and were acclimatized in cemented tanks 10x5 ft for 2 week. During acclimatization period fish were maintained on feeding regime of 25% protein feed 2 times a day. Effluent treatment in concentrations of 5%, 7.5% and 10% for Labeo rohita (Ham.), Catla catla (Ham.) and Cirrhinus mrigalae (Ham.) for 96 blood vessels improve and resistance of insulin. If fish intake increased so the risk of CVD may lead to decrease (Rhee et al., 2017).
Fish nutrients play a positive role in human health. The n-3 LC PUFA in fish have also a stabilized amino acid configuration, carry a more value of calcium minerals, taurine and vitamins D3, choline, B12, selenium, phosphorus and iodine. Moreover, fish as well contribute important proportion of iron, zinc and vitamin A to a population (Lund et al., 2013).
Municipal wastewater and unprocessed industrial direct discharge into drains, rivers on canals actually the aggravate of water pollution. Furthermore, big concentration of many pollutants may rise pH, total dissolved solids (TDS), biological oxygen demand (BOD), total suspended solids (TSS), electrical conductivity (EC), metals like as Cr, Cd, Ni and fecal coliform, Pb therefore making such water toxic for irrigation and domestic use (Tariq et al., 2020). This untreated water has proved to be detrimental for human consumption.
In Pakistan and other developing countries with minimal resources and infrastructure, industrial wastes are treated malfunctionally because of inadequacy of waste treatment machinery. It has been reported that only the smallest amount of waste (<8%) is treated efficiently while the majority of the industrial drainage goes untreated into the rivers, streams and canals, violating the directions of environmental protection agencies (Ensink et al., 2004).
Multiple researches have established a link between raised toxicant levels in water bodies and bioaccumulation in aquatic life forms, the fish gills, liver and muscle hours was used to determine LC 50 . After determination of LC 50 , sub-lethal concentrations i.e., 1.5% (T1), 3% (T2) and 4.5% (T3) were applied to three fish species polyculture system in 40L aquaria in triplicates and water samples from upstream sites was used as control. The experiment in 12 aquaria for 120 days (Jun-2020 to Sep-2020).

Preparation of fish samples
After treatment the muscle tissue specimens were collected and maintained at 0 °C until further analysis. After washing with distilled water, the sample were chopped (2-3cm) then oven dried at 65 °C until a constant weight was reached. Afterwards, they were ground and filtered with 1mm mesh size and stored in airtight containers, following the method described by Mahboob (2013).

Fatty acid profiling
After lipids extraction from pretreated samples with n-Hexane at 65°C using Soxhlet extraction, they were esterified using methanolic sulphuric acid (Tocher 2003). Then gas chromatography was performed; sample volume 0.2 ul, N 2 as carrier, H 2 , air 30, 40 and 500 mL/ min respectively (Column size 2m, stationary phase 10% Diethyl glycol succinate with diameter of 2 mm. Column temperature was set at 190°C, injection port temperature 200°C and detection port temperature 250°C. The resulting chromatograms were compared with those of obtained for the standard (Pure mixture of Fatty acid methyl esters (FAMEs)) (Hedayatifard and Jamali 2008).

Statistical analysis
The mean, standard error and analyses of variance (ANOVA) were calculated using SPSS-10 for PC. The means were compared by using Duncan's Multiple Range test. Probability values of P <0.05 were considered significant. Table 2 shows saturated fatty acid profile of Labeo rohita (Ham.) were significantly higher in treatments as compared to controls moreover maximum value of saturated fatty acid were observed in T3 as compared to T2 and T1 respectively. Concentration of unsaturated fatty acids decrease gradually as we increase the concentration of textile effluents for shop maximum value of unsaturated fatty acids present in T1 and gradually declined as we increase the effluent concentration in T2 an T3 similar observations were seen for polyunsaturated fatty acids like Omega 3 and Omega 6 fatty acids. Table 3 shows fatty acid profile of Cirrhinus mrigalae (Ham.) as can be seen in table to maximum concentration of saturated fatty acids were present in T3 and T2 and T1 and minimum concentration of saturated fatty acids can be seen in control for unsaturated fatty acids this trend was reversed and minimum concentration of unsaturated fatty acids was present in T3 and T2 and T1 while control samples showed maximum concentration of unsaturated fatty acids including Omega 3 and Omega 6 and other polyunsaturated fatty acids. Table 4 represents fatty acid profile of Catla catla (Ham.) similar trend was observed that is maximum concentration of saturated fatty acids was seen with increasing effluent treatment and minimum concentration of saturated fatty acids were present in controls and for unsaturated fatty acids minimum concentration of unsaturated fatty acids were present in T3 and T2 and T1 and maximum concentration was present in control samples.

Results and Discussions
The findings of present suggests that the values of PUFA decreased while that of SFA were increased in samples as compared to controls (Table 2-4).
The metal and other compounds are reported to cause oxidative stress that could be the cause of oxidation of PUFAs hence the decline in their concentrations (Gabryelak et al., 2000). Findings in brown trout (Samlo trutta) support this   Similar changes were observed in the study of Hussain et al. (2015), where fatty acid profiles of fish species collected from polluted sites of Chenab river were negatively affected as compared to farmed fish. The perturbed fatty acid profiles observed in present study are also in line with those found in Atlantic bluefin tuna after exposure by POPs. There was a positive correlation in levels of SFAs and PUFAs with the concentrations of dioxin-like polychlorinated biphenyls (DL-PCB). Moreover, the exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), also caused disturbance in normal fatty acid profiles of the exposed animal. Another study in marine fishes also reported a positive correlation between the levels of two essential FAs (EPA and DHA) and amounts of POPs (Geng et al., 2015).
The oxidative stress caused by pollutants can also interfere with the enzymatic activities of those enzymes which are involved in maintenance of a balance between SFAs, MUFAs and PUFAs. As the activity of fatty acid synthase (FAS) were reported to be elevated after triazol pesticide Paclobutrazol (PBZ) treatment. Thus there was an increased level of SFAs and MUFAs was observed in rockfish Sebastiscus marmoratus (Sun et al., 2013).

Conclusion
Presence of different pollutants in water bodies are accumulated in fish tissues and they deteriorate different physiological processes in fish body as we found in present study fatty acid profiles are vulnerable to heavy metal or textile industry effluent pollution, so fish can be used as a good bio indicator to assess environmental pollution specifically textile industry pollution in water bodies.