Human health risk assessment of screened pesticides residues in two edible sh species of river Ganga, India

Study was carried out to determine the concentration and bioaccumulation of pesticide residues in edible sh from river Ganga, India, to assess human health risk via consumption of sh. Two commonly edible sh species bagrid catsh and common carp were collected. The n-hexane extract of the muscle tissues were characterized by gas chromatography coupled to mass spectrometry and quantied by electron capture detector for pesticide residues. Bioaccumulation factors (BAFs) in common carp of pesticides were found to be higher than those in bagrid sh. Daily exposures of pesticides for consumers via sh consumption were determined by calculating and comparing estimated daily intake (EDI) with ADI values. The EDI results in our study were insignicantly high from ADI values. Non-carcinogenic and carcinogenic risks were evaluated by Target hazard quotient (THQ) and risk ratio (R), respectively. Hazard quotients (THQ) were found to be lower than the set 1.0, inferring non-carcinogenic risk by consumption of sh from the river. Regard to contaminants carcinogenic affects the total risk ratio (R) value of pesticides were found lower than threshold of tolerable risk except of heptachlor indicating carcinogenic risk via consumption of sh. The results demonstrate that due to increased pollution in the ecosystem required more attention.


Introduction
Agrochemical pollution being a global concern in past few decades due to their persistence in the environment, bioaccumulation in organisms and having a toxic impact on humans and other organisms.
Bioaccumulation of residues has a wide range of acute and chronic adverse impacts on reproduction, physiology and biochemical parameters (Shah and Parveen, 2020).
Among all aquatic ecosystem organisms sh is considered as suitable bio-indicator of monitoring environmental contamination pollution. Fish uptakes pollutants directly through the water via gills, integuments and indirectly from the food web. The pollutants present in the sh not only indicate environmental persistence but are also transferred to other organisms through the trophic web. Fish as nutrition is an important source of not only proteins but also omega-3-polyunsaturated fatty acids which are a cure for cardiovascular diseases (Jabeen and Chaudhry, 2011) and used in the pharmaceutical and cosmetic preparations (Nengroo and Rauf 2019). Consumption of sh from the contaminated environment causes accumulation of pollutants in the human body.
River Ganga is important not only for irrigation purposes but also provides basic nutrition for common people (Sharma et al., 2014). With the increase in population India has been undergoing rapid industrialization and economic development. Use of pesticides for agricultural purposes had increased to hundred times to sustain more population. Enormous quantities of pesticides are being applied along with Ganga river basin agricultural elds.
These residues nally nd their way into the river by ash oods, drainage and surface runoff. Large numbers of reports are available that ground waters in India are highly polluted with pesticides. Kaushik et al., 2008 reported organochlorine pesticides in Ghaggar river of Haryana. Sankararamakrishnan et al., 2005 reported organochlorine and organophosphate pesticides in river Ganga at Kanpur station. In the present study we choose river Ganga at Narora as sampling site. These pesticides residues are accumulated in aquatic organisms and pose a potential carcinogenic risk for humans.
Therefore the objective of the present study is to clarify the concentration and accumulation of pesticides in water and edible sh of river Ganga. Further the assessment of daily exposure and human health risk via consumption of sh from the river. The study provides a broader overview of pesticide status in edible sh of river so that effective measures to be taken to reduce the potential human health risk.

Materials And Methods
2.1. Study site and the Sampling: Figure 1 displays the details of sampling site, thirty sh and twenty-ve water samples were collected at Narora site of the river Ganga in August 2019. The sh samples included 10 bagrid cat sh and 10 common carp, belonging to bagridae and cyprinidae families, commonly consumed shes of India (Gupta, 2015, Nongbri andSyiem, 2012). The sh and water samples were analyzed for pesticide residues. All of the collected samples were stored in an ice-box and are immediately transported to the laboratory.
In the laboratory, shes were dissected and dorsal muscle tissues of the sh were taken and analyzed for pesticide residues. Collected muscle samples were freeze-dried, grounded to ne powder, and stored at − 20 °C before the process of extraction begins. The impurity particulates in the collected water samples were separated by ltration through 0.45-µm hydrophilic lters.

Sample extraction and clean up:
Pesticides in water samples were described by the method of Muir and Sverko (2006). 2,4,5,6-tetrachlorom-xylene (TCmX) as recovery surrogate was added in 1 litre of ltered water sample. Liquid-liquid extraction with dichloromethane (35 ml) was performed. Na 2 So 4 column was used to remove water in the organic phase further n-hexane was used as an organic solvent. The column was packed from bottom to top with neutral silica, neutral alumina, and anhydrous sodium sulfate, to remove impurities in the extract.
The extract was nally blown to dryness by purity nitrogen and the residues were redissolved with 20 µl of n-hexane.
Matrix-solid phase dispersion method (Villaverde- de-Saa et al., 2013) was done to extract pesticides in sh muscle. Samples (0.5 g) was grounded to ne powder with known amount of surrogate (TcmX) and 0.5 g octadecylsilane bonded silica (C18) as dispersion sorbent in a mortar and pestle for 5 min to get a homogeneous mixture. The homogeneous mixture was transferred into (0.22 µm) membrane lter polyethylene (10 ml) syringe. The column was packed with neutral silica, acidic silica, orisil acidic alumina and sample mixture from bottom to top with other membrane lter placed on top. 15 ml of dichloromethane was used to elute the packed column at a ow rate of 2 ml min − 1 . High purity nitrogen at the gentle stream was used to dry eluent, followed by dry residues redissolution with 200 µl of nhexane and addition of internal standard (pentachloronitrobenzene). Qualitative as well as quantitative analysis of analytes were done with GC-MS and GC-ECD.

Lipid:
Determination of lipid content was done gravimetrically (Smedes, 1999). Two grams of ne grounded muscle powder was dissolved in twenty millilitres of water, cyclohexane and 16 ml isopropyl mixture. Ultrasonic extraction was done, mixture reached statically separated equilibrium, and the organic phase was collected. Extraction was repeated with18 ml of cyclohexane and 3 ml isopropyl alcohol and combined with earlier then dried under a gentle nitrogen stream. The residue was weighed.

Gas Chromatography and Mass Spectrometry:
3 µl of Sample were injected into a gas chromatography (Agilent 7890A) instrument equipped with an electron capture detector (GC-ECD) (Agilent Technologies, USA) the analytical capillary column was DB-5 (30 m × 0.25 mm i.d. × 0.25-µm lm thickness, Agilent). Nitrogen was used as carrier gas with the ow rate of (1 ml min − 1 ). Injector and detector temperatures were adjusted at 250 and 300 °C. Started at 80 °C with 1 min hold, and the oven temperature was raised to 150 °C at 20 °C min − 1 rate and nally to 300 °C (5 min hold) at the rate of 5 °C min − 1 .
The instruments were calibrated with calibration standards during analysis. Each sample was analyzed in duplicate. The recoveries of TCmX (surrogate standard) were 75 ± 6% in water samples and 68 ± 6% in sh samples. The recoveries of pesticides ranged from 73 to 100% in water samples and from 66 to 84% in sh samples. The method detection limits (MDLs) concentration of analytes were con rmed whose signal-to-noise (S/N) ratio was three and ranged from 0.05 to 100 µg L − 1 in water samples and from 0.01-100 µg g − 1 in sh samples. Concentration detected less than MDLs in samples were treated as not detected (nd).

Data analysis:
Biota-water accumulation factor (BAF) is calculated by the following equation: Where C l is the pollutant concentration in the sh (µg g −1 ) normalized by lipid content of sh and c is concentration of pollutant in water (µg l −1 ).
In order to determine potential human health risk of tested shes, the estimate daily intake (EDI), THQ and R were calculated. The calculation formulas of EDI, THQ and R are listed as follows: Where, C = Concentration of pollutant in food (μg g −1 ), W F = Average daily sh consumption in India is 55 g day −1 person −1 , and W AB = Average adult body weight (70 kg).
The target hazard quotients (THQs), and carcinogenic risk ratio (R) were used in risk assessment. The THQ>1 denotes that the daily exposure seems to cause human health hazard effects. Where,

Results And Discussion
3.1. Concentration of pesticides in water samples: Table 1 depicts the mean value (µg/l) concentration of pesticide residues detected in analyzed water samples. In India, 60,000 MT of pesticides are being annually used of which maximum consumption occurs along river Ganga basin (Kumar et al., 2013). Pesticides used in the agricultural elds could easily concentrate in the water through surface runoff streams and tributaries. Besides the continuous agricultural activities done along the Ganga basin, the dry beds of the river are used to grow vegetables and fruits, also add pesticides to the river during monsoon season. In the present study eight pesticides were detected, chlordane were found in higher quantity with 0.104 µg/l and heptachlor were found in a lower quantity of 0.006 µg/l. Chlordane, dimethoate and malathion were detected in all of the analyzed samples while atrazine, heptachlor were detected in 8 of the 25 water samples. No obvious change in the trend of detection and concentration were found for malathion, heptachlor and chlordane which shows their historical use and persistence from the past. Dimethoate, atrazine, dichlorvos, azinphosmethyl and cypermethrin were found in the present study, they are newly introduced pesticides and are being used in increasing trend along the basin (Indiastat, 2018 Pesticide concentration detected in sh samples is given in Table 2. Mean concentrations (wet weight, ww) ranged from 0.167 µg g − 1 for heptachlor to 0.045 µg g − 1 for azinphosmethyl in bagrid cat sh.
Mean concentration in common carp ranged from 0.182 µg g − 1 for dichlorvos to 0.017 µg g − 1 for malathion. Aktar et al., 2009 reported 13 pesticides from muscle tissues of sh from river Ganga with 0.1 µg g − 1 of dimethoate and 5.4 µg g − 1 of malathion. Dimethoate and malathion in the present study were reported lower than the early ndings. All the examined samples contain residues except for azinphosmethyl, only 4 samples reported concentration in bagrid cat sh. In common carp, only 6 out of 15 examined samples show concentrations of malathion and dimethoate residues. Heptachlor and chlordane were reported at below permissible limit by (Samanta, 2006) in sh from the river at West Bengal site. Due to continuous exposure, contaminants accumulate and get concentrated in the muscle tissues compared to water. Accumulation of pesticides depends upon different physiological, environmental and feeding habits. The diverse feeding habit of shes prefers more accumulation of pesticides. Table 2 The concentration of pesticide residues in surface water (ug/l) and sh tisuues (µg/g ww) from river Ganga, India.

Bioaccumulation factors:
The bioaccumulation factor is the ratio of chemical contaminant found inside the tissue of the sh to that found in the surrounding water. Pesticides being lipophllic, the concentration inside the tissue is normalized with lipid content. In the present study, the BAF of heptachlor was found higher in both shes than the other pesticides. Higher BAF of heptachlor might be accredited to poor water solubility and relative high log K ow ( Table 2). BAF of common carp were found to be higher than that found in bagrid cat sh. From the studies conducted it is known that bioaccumulation of chemical contaminant in the sh is due to the cumulative effect of many physiological and environmental conditions such as trophic level, sh age, total lipid percentage and environmental concentration of the contaminant. In this study the lipid content of bagrid cat sh is much higher than common carp and BAFs were also higher than found in later. Some earlier studies reported that no relationship between lipid percentages found in the sh and concentration of lipophllic chemicals in the sh Gobas, 1999. 3.4. Risk assessment of pesticides in the sh: Fish consumption is known as the most common way for contaminants into human body Hoekstra et al., 2013. EDIs were calculated to assess potential pesticide exposure to humans with the concentration (mean values) of determined pollutants in the sh. Table 3 compares our calculated results with the ADI value issued by USEPA 2009. From the results the daily exposure to pesticides via consumption of sh from the river, potential risks are present as the calculated results are insigni cantly high. Table 3 shows the health risk assessment gures for the two shes. The THQs values for both the shes were less than 1.0. The HI values of pesticides to humans via consumption of sh indicated lower health risk from the study area. The calculated R values of pesticides related to consumption of sh were lower than 1 × 10 − 4 indicating their cancer risks were negligible (USEPA 2009). Nevertheless, the calculated R value of heptachlor were found of high value than 1 × 10 − 4 in both two shes, therefore which suggested the cancer risk of heptachlor associated with consumption of sh cannot be ignored.

Conclusion
Bagrid and common carp two commonly consumed sh from river Ganga were collected from Narora site and were analyzed for pesticide residues. Residues found in the muscle tissues of sh show a slight change in concentration than found in water samples. More concentration of detected pesticides was found in Common carp than bagrid. The EDIs in our study were all insigni cant and slightly higher than the ADI value. The HI of the detected pesticides was lower than 1.0, which implying lower human noncarcinogenic risk via consumption of sh from the river. Carcinogenic risks of detected pesticides to human exposure were negligible, but for heptachlor exposure to carcinogenic risk were high. Finally consumption of sh from the study river cannot be ignored with carcinogenic risk because of enormous use of pesticides in the agricultural basin.