Organochlorine pesticides residue affinity in fish muscle and their public health risks in North West Ethiopia

Abstract Pesticides are the parent compounds, their metabolites, and associated impurities of agricultural and health chemical inputs. If they are found at concentration levels higher than the standard limits, they have potential negative impacts on the ecosystem in general and on fish and humans in particular. This study investigates organochlorine pesticides (OCPs) residue occurrences in fish muscle and assesses their public health potential risks, in North West Ethiopia. The concentration of OCPs residue under gas chromatography with electron capture detector (GC‐ECD) was detected in 37.84% of fish muscle samples. The mean amounts detected were Endosalfan I, 341.50 ± 32.19 μg/kg; Endosalfan II, 36.01 ± 2.3 μg/kg; Endosalfan sulfate, 5.43 ± 4.06 μg/kg; 4, 4, DDE (4,4‐dichlorodiphenyldichloroethylene), 64.01 ± 9.08 μg /kg; 4,4, DDD (4,4‐dichlorodiphenyldichloroethane), 5.65 ± 3.12 μg/kg; and 4, 4, DDT (4,4‐dichlorodiphenyltrichloroethane), 1.58 ± 0.30 μg/kg. The mean concentration of Endosalfan I tested in fish muscle samples was higher than that of the permissible limit of different international standards. However, due to the low per capita consumption rate of fish origin food in Ethiopia, the health risk index (HRI) ranges from 0.002 to 0.1275, which shows there is no public health risk. This study highlights the possibility of chemical residue occurrence in fish food products, and hence pesticide use regulations and monitoring concentration levels should be implemented regularly to avoid human and environmental health risks.

The risks associated with consumption of animal-origin food should be taken into account in the agricultural sector by reasonable and achievable good practices in the agricultural industry. The maximum levels of OCPs residues in foodstuffs of animal origin should be set at the strictest possible level due to the lipophilic nature of these chemicals (Fair et al., 2018;USEPA, 2009). According to the WHO/ FAO (2009), humans, animals, and fishes are considered at risk when they consume OCPs residues at levels higher than the maximum residue limits and acceptable daily intake.
Exposure to OCPs compounds is especially dangerous during prenatal development and infancy, as it causes irreversible changes in the central nervous system. OCPs have been shown in studies to have a high potential to cross placental barriers and cause serious neonatal damage even at low concentrations (Jayara et al., 2016; Thompson et al., 2017). Long-term health consequences include an increased risk of reproductive disorders, kidney and liver dysfunction, nervous system and birth defects, endocrine disruption, immune system dysfunction, and carcinogenesis (Okoffo et al., 2016;Yazgan & Tanik, 2010). Besides, it causes cardiovascular diseases, disrupts heme biosynthesis and vitamin D metabolism, and disrupts mineral metabolism (Jayara et al., 2016).
The problem of OCPs stems not only from their toxic properties but also from low levels of exposure to these chemicals. Their effects can be observed only at the physiological or biochemical level.
The acute effect of OCPs is the intoxication of fish, humans, and other animals by direct contact or through the food chain (Jayara et al., 2016; Thompson et al., 2017). Numerous studies on both humans and animals provide strong evidence of the toxic potential of OCPs (Ravindran et al., 2016). According to the study conducted by Yazgan and Tanik (2010), the acute toxicity effect of OCPs causes environmental risks that may cause ecological damage, including to the flora and fauna of the entire ecosystem.
According to various studies, the level of OCPs in developing countries is increasing as it is still used for agriculture and public health purposes, while it is declining in developed countries (Sadasivaiah et al., 2007;Thompson et al., 2017;WHO, 2010).
Ethiopia has a relatively well-developed pesticide legislation on registration and control of pesticides intended to address their environmental and health effects (Negatu et al., 2016). In addition, Ethiopia is a signatory to the Stockholm Convention and agreed to support research on persistent organic pollutants. But there are gaps between policy and practical enforcement of prohibiting the import and application of banned OCPs including DDT and Endosulfan (Negatu et al., 2016). However, limited data are available about the occurrence and level of pesticides in the aquatic environment, specifically in fish tissue. In Ethiopia, studies in rift valley water bodies have revealed the contamination of the environment (sediment) and fish species (Oreochromis niloticus, Clarias gariepinus, Tilapia zilli, and Carrassiusspp.) by pesticides (Yohannes et al., 2014). The study conducted by Deribe et al. (2014) (Thompson et al., 2017).
The first version of this study indicates that there is still an increasing trend of OCPs applications for improving agricultural production in the catchments of Lake Tana (Agmas & Adugna, 2020).
Accordingly, high concentrations of OCPs can be found in the environment and fish muscle of our study area. In Ethiopia, the studies conducted to assess the contamination of OCPs in the environment are scarce; none has been conducted in Lake Tana. Dried fish products are being exported to neighboring countries, especially to the Republic of Sudan and Eritrea recently. Hence, the research result will help to amend fish product export policies and associated environmental safety considerations regionally and locally. Therefore, the aim of this study was to assess OCPs accumulation in the muscle of fish and estimate its potential human health risks in fish of Lake Tana, North West Ethiopia.

| Study area
The study was conducted in Lake Tana, the headwaters of the Blue Nile River. It is located in the province of North West Ethiopia. It is located approximately 580 km north west of the city of Addis Ababa. The lake is one of the largest (3600 km 2 ) and best fishing sites in Ethiopia. Geographically, Lake Tana is found at latitude 12°1'35.75"N and longitude 37°18'12.54"E. Mean annual temperatures range from 13 to 22°C. The annual average rainfall of Lake Tana is 1248 mm per year (Stave et al., 2017).
The lake has three main commercially important fish groups, such as: Labeobarbus intemedius, Clarias gariepinus, and Oreochromis niloticus. They are consumed by larger part of the community and traded widely in the region, even to neighboring Sudan in dry form.

| Study design and sampling method
The cross-sectional study design was carried out from January 2018 to December 2020. Three commercially important fish species of the lake, including Labeobarbus intermedius, Clarias gariepinus, and Oreochromis niloticus muscle tissue, were sampled for this study. A total of 137 fish were sampled, with an average size of 446 g for O.
niloticus, 675 g for C. garipinus, and 378 g for L. intermedius. Muscle tissue was taken from the dorsolateral sides of each sampled fish.
The samples were wrapped in aluminum foil, packed in clean polyethylene bags, labeled and sealed, and then transported in a thermoinsulated container with ice packs.

| Sample extraction and clean ups
Gas chromatography (GC) was used for the pesticide residue analysis.
The gas chromatography with electron capture detector (GC-ECD) processing and analysis were conducted in the laboratories of JIJE analytical and testing service laboratory, Nifas silk, Lafto Sub-city, Addis Ababa, Ethiopia. All glassware and containers were washed with detergent, rinsed with purified water and acetone, and kept in an oven at 180°C for 2 h for processing (Anastassiades et al., 2003).
The extractions were carried out according to the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method described by Anastassiades et al. (2003), with some necessary modifications.
Lyophilized dry fish muscle sample was chopped on a chopping board with a sharp knife and ground using a mini-chopper, then 10 g of the chopped sample was transferred to a 50 ml Teflon centrifuge tube. Then, 10 ml of acetonitrile was added to the centrifuge tube and agitated well for proper mixing, followed by the addition of 7.5 g of anhydrous MgSO 4 and 1 g of NaCl, and the centrifuge tube was vigorously shaken for 1 min. Later, it was centrifuged at 5000 rpm for 5 min. After centrifugation, 2 ml of the supernatant was transferred to an Eppendorf tube containing 100 mg of primary secondary amine (PSA), 150 mg of MgSO 4 , and 100 mg of charcoal for cleanup, followed by vigorous shaking for 2 min. Again, the prepared sample extract was centrifuged at 10,000 rpm for 5 min. Afterward, the sample extract was filtered through a 0.45μm filter using a syringe and transferred to auto-sampler vials for further gas chromatography (GC) analysis.

| GC analysis
The OCPs residues were analyzed by a Shimadzu GC-2010 with an electron capture detector (ECD), an auto-injector (AOC-20i; Shimadzu, Kyoto Prefecture, Japan), and GC solution software. The capillary column used in ECD was Rtx-CL, 30.0 m length/0.25 mm in diameter 0.32μm film thickness. The GC was run under the following conditions: injector temperature: 250°C; detector temperature 330°C; oven temperature: 260°C starting from 0 to 180°C for 0.3 min and continued at 5°C/min to 220°C, held for 12 min, and continued at 5°C/min to 260°C; injected sample volume: A 1-2μl mode of injection: split; carrier gas: N 2 with a 77.8 kPa flow rate; runtime: 28 min.
Standard peaks were detected by inserting a high concentration of the standard (1 ppm), and the retention time for OCPs was estimated.

| GC calibration standards
The GC quantification for individual OCPs was performed using external standard calibration because of the simplicity and sensitivity of the ECD. The lowest concentration calibration standard analyzed during initial calibration was established (the lowest detection limit was 0.001 μg/kg). Commercially prepared stock standard solutions of OCPs (98%-99% purity) that are certified were used (purchased from Thermo Fisher Scientific, USA). Thus, individual standard stock solutions (100 mg/L) were kept in a brown bottle ready for use.

Calibration factor
Sample peak areas (or peak heights) are compared to the peak areas (or heights) of the standards. Our external standard calibration was employed by calculating the calibration factors for each analyte at each concentration, the mean calibration factor, and the relative standard deviation (RSD) of the calibration factors, using the formulas below.
Calculate the calibration factor for each analyte at each concentration as: Calculate the mean calibration factor for each analyte as:

CF =
Peak Area (or Height) of the Compound in the Standard Mass of the Compound Injected (in nanograms) CFi n F I G U R E 1 Map of the study area (Source: Modified map from spatial data in DIVA-GIS 2015).
Where n is the number of standards analyzed.
Calculate the standard deviation (SD) and the RSD of the calibration factors for each analyte as: If the RSD for each analyte was <20%, then the response of the instrument was considered linear fitted model and the mean calibration factor may be used to quantify sample results. Each analyte in each subsequent standard run during the 12-h period (analytical shift) must fall within its respective retention time window.
The amount of sample for each chromatogram component peak that corresponds to the compounds used for calibration was calculated as follows. The appropriate selection of a baseline from which the peak area or height was necessary to determine for proper quantification: Where, A x , Area (or height) of the peak for the analyte in the sample; V t , Total volume of the concentrated extract (μl); D, Dilution factor, however we did not make dilution, D = 1; CF, Mean calibration factor from the initial calibration (area/ng); V i , Volume of the extract injected (μl). The injection volume for samples and calibration standards was the same; W s = Weight of the sample extracted (g).
The dry weight was used. Since we use kilograms, we multiply our results by 1000.
Using the units given here for these terms resulted in a concentration in units of ng/g, which is equivalent to μg/kg. For quality control purposes, we analyzed standards after every 20 samples to prevent the number of samples being re-injected when the standards fail the quality control limits.

| Public health risk calculation
Comparison of observed concentrations of pollutants in the environment with established maximum permissible levels (MPLs) is the basic approach used to assess the potential risk posed to ecosys-

| Data analysis
The data were entered into an excel spreadsheet, cleaned by EPI-Info V.3.5.3, and exported via state transfer to be cleaned, edited, and analyzed by SPSS version 20. Data cleaning was performed to check for accuracy, consistencies, and missed values. A multiple logistic regression analysis model was used to assess the association between pesticide residue occurrence in fish meat and risk factor variables. The adjusted odds ratio (OR) at a 95% confidence interval (CI) value, not including 1, was considered statistically significant.

| Occurrence of pesticide residues in fish meat
Overall, gas chromatography (GC) laboratory analysis of the 137 fish muscle samples revealed 37.84% were positive for OCPs. This analysis study indicated that six pesticide residues, including Endosalfan I, Endosalfan II, Endosalfan sulfate, 4, 4, DDE, 4, 4, DDD, and 4, 4, DDT residues, were detected (Table 1). This study's finding was different from that of Gustav Gbeddy et al. (2012), who found OCPs in 100% of the tested samples, which is higher than that of the present study. Another higher frequency of occurrences of OCPs detected by Bedigama and Gabadage (2018) in Sri Lanka was 80%.
The detection levels were from not detected for Dimethotes (detection limit = 0.001 μg/kg), to 4041.73 μg/kg dry weight for Endosalfan I recorded in the present study (Table 1).  (Bedigama & Gabadage, 2018). Lower than our findings were detected in a study done by Kasozi et al. (2006), that DDTs in fish samples from Lake Victoria, Uganda were 0.80-0.86 μg/kg.
This high occurrence in our study area might be due to the long-term use of organochlorine pesticides to improve agricultural productivity in the catchments of the lake. The high lipophilicity, bioaccumulation nature, long half-life, and potential for long-range leaching nature of the chemicals result in the accumulation of persistent toxic substances in soil, water, and air (Agrawal et al., 2010;Ravindran et al., 2016;USEPA, 2009) According to our findings, the detected level of 4, 4, DDT was lower than its metabolites (4, 4, DDE and 4, 4, DDD), and the Endosalfan sulfate concentration in fish muscle was lower than those of its metabolites (Endosalfan I and Endosalfan II) ( Table 1).
The lower concentration of the parent compounds may reflect the current limited use of those persistent OCPs in the Lake Tana catchments. The detected concentration may be due to a previous historic event with a long half-life (2-15 years) (Jayaraj et al., 2016). However, a non-negligible concentration of parent compounds of these persistent pollutants was detected, which indicates the continued use of these OCPs in the water shed of Lake Tana.
This study highlights that more pesticide was detected from African catfish (Clarias gariepinus) species than from other main commercially important fish groups (Labeobarbus intermedius and O. niloticus). Among all the covariances which were imported into multivariable logistic regression analysis, fish species and sampling sites of the study lake were independent variables significantly associated with the occurrence of pesticide ( Figure 2). influence OCP pollutant levels in fish muscle (Bonito et al., 2016;Borga et al., 2004;Cardona, 2015;USEPA, 2009).
Another study by Hasan et al. (2022) in Bangladesh detects less than 1 HRI organochlorine pesticide in fish. In contrast to our finding, the study done by Yohannes et al. (2014) on a Rift Valley Lake-Lake in Ziway, Ethiopia, indicated a potential cancer risk from consumption of the fish from the calculated hazard ratio (HR). Another study in Sri Lanka by Bedigama and Gabadage (2018) found HRI values > 1.
As a limitation of this study, it is necessary to conduct further studies that assess the detection of OCPs residues in human serum to examine the burden of these OCPs and integrating biomarkers of exposure and health risk effects, and modeling exposure routes via fish consumption.

| CON CLUS ION AND RECOMMENDATIONS
The present research finding showed that high concentrations of Endosalfan I residues in fish muscle were detected in fish samples, which are higher than the standard limit level. However, as a result of the low consumption rate of fish meat in the meantime, it will have little or no significant adverse public health effects on consumers.
So, applying a more conservative threshold limit (WHO/FAO standard) implementation is a necessary action by considering health risks of local consumers, other sources of exposure, and vulnerable populations (children and pregnant women). The study highlights the presence of a non-negligible level of OCPs in the muscle of fish that indicates there is still use of these pesticides in the catchments of Lake Tana, North West Ethiopia. As a result, one health intervention is recommended to protect against adverse effects on flora and fauna of the entire eco-system as well as public health hazards.
Collaborative work on awareness creation about the possible occurrence of OPCs residues should also be done among environmental health, agricultural, and public health offices. In addition, further study of the OCP residue in other agricultural food items and its effects on the ecology of the aquatic environment is recommended.

ACK N OWLED G M ENTS
We wish to extend our sincere appreciation to the Ethiopian Institute

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed this study are available from the corresponding author on reasonable request. The data are not publicly available due to privacy or ethical restrictions.

E TH I C A L A PPROVA L
The study was granted an exemption from requiring ethics approval from the College of Agriculture and Environmental Science, Research Ethics and Review Board.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.