The widespread use of glyphosate in agriculture and aquatic environments has raised concerns about its potential impact on non-target organisms, including fish [1]. Glyphosate has been shown to affect fish physiology, behavior, and ecology, leading to changes in population dynamics and community structure [2]. Moreover, glyphosate can accumulate in fish tissues, including muscle, liver, and kidneys, which can have negative impacts on their growth, reproduction, and overall health [3].

The proximate composition of fish muscle, including moisture, protein, fat, ash, and fiber content, is an important indicator of its nutritional quality [4]. Changes in the proximate composition of fish muscle can affect its texture, flavor, and overall acceptability to consumers [5]. Furthermore, the nutritional quality of fish can have significant impacts on human health, particularly for consumers who rely heavily on fish as a source of protein [6].

Clarias gariepinus, a catfish species, is an important food fish in many parts of the world, particularly in Africa and Asia [7]. The species is widely cultivated in aquaculture systems, where it is often exposed to pesticides, including glyphosate [2]. Therefore, it is essential to investigate the impact of glyphosate on the proximate composition of Clarias gariepinus muscle.

Several studies have investigated the effects of glyphosate on fish physiology and ecology [2,8]. However, few studies have examined the impact of glyphosate on the proximate composition of fish muscle [3]. Moreover, the majority of these studies have focused on acute exposures to high concentrations of glyphosate, rather than chronic exposures to sub-lethal concentrations [1].

This study aimed to evaluate the effect of sub-lethal concentrations of glyphosate on the proximate composition of muscle and accumulation in the muscle of Clarias gariepinus. The results of this study will contribute to our understanding of the potential impacts of glyphosate on fish health and nutritional quality and provide valuable information for policymakers, aquaculture practitioners, and consumers.

Study Area

The experiment was conducted in the wet laboratory in the Department of Fisheries, College of Natural Resources and Environmental Management, Michael Okpara University of Agriculture Umudike, Abia State, in South Eastern Nigeria. The institution is situated in the tropical rainforest zone on Latitude 05°26¹–5°25¹N and Longitude 07°34¹–7°36¹E with total annual rainfall ranging from 1800 to 2100 mm and an altitude of 122 m above sea level [9].

Ethical Approval

Approval was granted by the Michael Okpara University of Agriculture Umudike Ethical Committee at the College of Veterinary Medicine to conduct research on pesticides using an animal model (MOUAU/CVM/REC/202418). All procedures involving test fishes were carried out with utmost care, adhering to the established ethical principles for animal use in scientific research, as outlined in the EU directive 2010/63/EU.

Fish Sample Collection

One hundred and twenty (120) fingerlings of Clarias gariepinus of mixed uniform sizes, with an average weight of 2.9±1.90 g, and measuring 6.08±0.07 cm in total length and 5.40±0.20 cm in standard length, were procured from a reputable fish farm and transported to the experimental site.

Fish were acclimated in the experimental tank of 500 liters for a period of two (2) weeks in continuously aerated tap water in a static system. Fish was fed on a commercial diet (35% crude protein) twice daily during the period of acclimation.

Glyphosate Source

Glyphosate turbo was sourced as a commercially available herbicide from an agro-based shop in Umuahia, Abia State, Nigeria.

Preparation of Test Solution

The preparation of the test solution was according to the dilution method of Reish and Oshida [10]. From the results that were obtained from the acute toxicity, sub-lethal concentrations of 0.00 mg/l, 0.02 mg/l, 0.03 mg/l, and 0.04 mg/l were used for the study. The sub-lethal concentrations were calculated from one-fifth (1/5), one-tenth (1/10), and one-twentieth (1/20) of LC₅₀ as recommended by Oladimeji and Ologunmeta [11] in a static experiment. A fresh solution was prepared after 72 hours (3 days) to maintain the concentration level.

Experimental Design and Treatment

The experiment was designed as four (4) treatments of graded concentrations of various levels of glyphosate including control with zero glyphosate. Each of these treatments was replicated three (3) times to provide a total of twelve (12) experimental treatments.

Two distinct experimental periods were used for this study: the acute toxicity period, which lasted for four (4) days (96 h), and the sub-lethal toxicity period, which lasted for ninety (90) days.

Records of fish weight were taken with an electronic sensitive weighing balance to ascertain the weight of fish before the commencement of the study.

Water Quality Monitoring

Water quality parameters, including pH, temperature, dissolved oxygen, and ammonia, were monitored daily using a water quality analyzer (YSI, USA).

Determining Glyphosate Turbo Accumulation in Fish Muscle

Glyphosate accumulation in the muscle tissue of control and exposed fish groups was determined using gas chromatography, following the protocol of AOAC [12]. 10 g of homogenized fish muscle was mixed with 60 g of anhydrous sodium sulphate to remove moisture. The mixture was then extracted with 300 ml of n-hexane for 24 hours. The resulting crude extract was evaporated using a rotary vacuum evaporator at 40°C until dryness. A 1 ml aliquot of the filtered residue was dissolved in 50 ml of chloroform and transferred to a 100 ml volumetric flask, where it was diluted to the mark. After evaporating the chloroform at room temperature, 1 ml of a reagent mixture (20% benzene and 55% methanol) was added. The sample was then sealed, heated to 40°C, and subsequently cooled. Following the addition of 1 ml each of hexane and water, the sample was centrifuged. The organic layer was then injected into a gas chromatography column using a Buck 530 Gas Chromatograph (CA, USA).

Determining Proximate Composition of Experimental Fish Muscle

The proximate composition of muscle tissue from both control and exposed groups was analyzed using the method of AOAC [12]. The parameters evaluated included moisture content, crude fiber, crude protein, crude fat, carbohydrates, and ash content.

Moisture Content

Moisture content was determined by drying 2 g of sample in a petri dish at 100°C for one hour. The weight of the petri dish was calculated before and after drying. The moisture content was expressed as a percentage (%).

Where

W1 = Weight of petri dish and sample before drying

W2 = Weight of petri dish and sample after drying

Ash Content

Ash content was determined by incinerating 2 g of sample in a platinum crucible at 500°C for three hours. The weight of the ash was measured, and the ash content was calculated as a percentage.

Where

W1 = Weight of empty platinum crucible

W2 = Weight of platinum crucible and sample before burning

W3 = Weight of platinum and ash

Crude Fiber

Crude fiber was determined by defatting 2 g of sample with petroleum ether and then boiling it with 200 ml of solution containing 1.25 g of H₂SO₄ per 100 ml of solution. The residue was filtered, dried, and weighed, and the crude fiber content was calculated as a percentage.

Crude Fat

Crude fat was determined using the Soxhlet fat extraction method. A 1g sample was extracted with petroleum ether for two hours, and the weight of the extracted fat was measured.

Crude Protein

Crude protein was determined using the Kjeldahl method. A 0.5g sample was digested with sulfuric acid and catalyst, and the ammonia was distilled and titrated with hydrochloric acid. The protein content was calculated as a percentage.

Carbohydrate

Carbohydrate content was determined using the differential method, where the carbohydrate content was calculated as the difference between 100% and the sum of the percentages of protein, moisture, ash, fat, and fiber.

Statistical Analysis

Data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test to determine significant differences between treatment groups. A significance level of (p < 0.05) was used.

Mean Physicochemical Parameters of the Experimental Water during the Study

The result of the physicochemical analysis of water is presented in Table 1. After exposure to sub-lethal concentrations, the temperature of the study ranged between 26.2±0.06 and 26.1±0.17, indicating that the temperature range was under a close check of range for the research. The pH and DO reduced down the treatment with an increase in glyphosate concentration, while the TDS and EC increased down the treatments.

Table 1:   Mean Physico-Chemical Parameters of Water during Exposure of Fish to Glyphosate.

Values with different Alphabets varies significantly at (P < 0.05) in the column.

T (°C) - Temperature, pH - potential Hydrogen, DO (mg/l) - Dissolved Oxygen, TDS - Total Dissolved Solids, EC (µs) - Electrical Conductivity.

Effects of Glyphosate Turbo on the Proximate Composition of Muscles of Clarias gariepinus

The result of table 2 showed that moisture content was found to be highest in the control group with a mean (72.2±0.40) and lowest value (23.5±0.48) with glyphosate concentration of 0.04 mg/l. Moisture content of specimens decreased with increase in doses of glyphosate turbo. The value of crude protein also decreased with an increase in glyphosate concentrations, with the highest means (17.2±0.62) in the control group (0.0 mg/l) and the lowest means (16.3±0.30) in the 0.04 mg/l concentration. The value of lipid content decreased significantly (P < 0.05) as the concentration of glyphosate increased, ranging from 5.21 ± 0.34 (control) to 3.20 ± 0.15 (0.03 mg/l) of glyphosate. Crude ash content increased slightly, but not significantly (P > 0.05), ranging from 3.59% (control) to 3.93% (0.04 mg/L glyphosate). The value of crude fiber and carbohydrate content decreased significantly (P < 0.05) as the concentration of glyphosate increased.

Table 2: Proximate Composition of Clarias Gariepinus Muscle Exposed to Varying Levels of Glyphosate Turbo.

Means with the same superscript along the columns are not significantly different (P>0.05).

Glyphosate Turbo Accumulation in the Muscle Tissue of Clarias Gariepinus

Glyphosate accumulation in the muscle of test organisms were found to be highest (7.02±0.26) in the muscle of clarias gariepinus exposed to a higher toxicant (0.04 mg/l) followed by (0.03 mg/l) concentration which had 4.52±0.00 while the lowest values were recorded in the control (0.35±0.01).

Table 3: Glyphosate Turbo Accumulation In Muscle Tissue Of Clarias Gariepinus Exposed To Different Levels Of Glyphosate.

Means with the same superscript along the columns are not significantly different (P>0.05).

The introduction of agrochemicals like glyphosate turbo into aquatic systems can lead to a decline in dissolved oxygen levels, ultimately causing mortality among organisms [13]. This study's findings revealed significant decreases in physicochemical parameters, specifically dissolved oxygen and pH, as the concentration of toxicants increased. This decline likely contributed to the observed mortality rates. Similarly, Akinsoroton [14] found that increased concentrations and toxicity of glyphosate-based herbicides correlated with decreased dissolved oxygen and pH levels in culture water for Clarias gariepinus fingerlings.

Previous studies carried out by researchers documented that proximate composition of fish differs greatly, and the variation could be due to age, feed intake, sex and sexual changes connected with spawning, the environment, and season [15]. Proximate composition serves as a good pointer of physiology needed for routine analysis of fisheries [16]. In the present study, proximate compositions of the control group were found to be significantly different when compared with exposed groups, which suggests that glyphosate turbo affected the proximate composition of the carcass of exposed groups.

The value of protein content recorded in control was within the limit reported by Huidobro [5], Osibona [17], and Nwali et al. [18] but disagrees with Obaroah et al. [19], Fagbenro et al. [20], and Laurat [21], who reported 28%, 66.6%, and 54–62%, respectively.

Moisture content recorded was in line with results from Ibhadon et al. [22], Osibona [17], and Nwali [18], who reported 73%, 76%, 71%, and 72.75%, respectively, but not in consonance with Laurat [21], who reported moisture content in the carcass of Clarias gariepinus juvenile to be between 8.21% and 11.86%. Normally, moisture and lipid contents in fish fillets are inversely related, and their sum is approximately 80% [23]. The result of carbohydrate content in this study disagrees with Nwani et al. [24], who reported zero detection of carbohydrate in the proximate composition of wild-farmed Clarias. The result of lipid content in the control was higher than that recorded by Osibona [17] (1.15%) and Nwali et al. [18] (1.06%) but lower than the values from Obaroah et al. [19], Fagbenro et al. [20], and Laurat [21], who reported 18.22%, 18.86%, and 9-11%, respectively. Fish and fish products are highly nutritious with protein content of 15 to 20% and are particularly efficient in supplementing the cereal and tuber diets widely consumed in Africa [20].

Glyphosate accumulation in the muscle of exposed fish species was found to be dose dependent, with higher glyphosate turbo accumulation in 0.04 mg/l. Aquatic animals in polluted waters tend to accumulate many chemicals in high concentrations, which is a potentially hazardous situation for the entire food chain [25,26]. From the result of glyphosate accumulation in the present study, it can be concluded that the increase in levels of glyphosate residues in the muscle tissue of Clarias gariepinus may pose environmental and health concerns, particularly for humans consuming contaminated fish.

This study investigated the impact of sub-lethal concentrations of glyphosate on the proximate composition and accumulation in the muscle tissue of Clarias gariepinus. The results showed significant changes in water quality parameters and proximate composition of fish muscle, including decreases in moisture, protein, and lipid content. Additionally, glyphosate accumulation in the muscle tissue was found to be dose-dependent. These findings suggest that glyphosate exposure can affect the nutritional quality of Clarias gariepinus and pose environmental and health concerns. The study's results contribute to our understanding of the potential impacts of glyphosate on fish health and nutritional quality, suggesting the need for responsible use and regulation of glyphosate in aquatic environments.

Declaration of Competing Interest

The authors declare no competing interest.

Acknowledgements

We thank all the staff of Fisheries and Aquatic Resources Management, Michael Okpara University of Agriculture Umudike, Umuahia, Abia State, Nigeria, for their effort in making sure that this research was successful and completed.

1. He H, Yu J, Chen G, Li W, He J, Li H. Acute toxicity of Butachlor and Atrazine to freshwater green alga Scenedesmus obliquus and cladoceran Daphnia carinata. Ecotoxicol Environ Saf. 2012; 80: 91-96.
2. Perez GL, Vera MS, Miranda L. Effects of herbicide glyphosate on the biodiversity of zooplankton communities in a freshwater ecosystem. Ecotoxicology. 2011; 20: 597-607.
3. Lushchak OI, Kubrak OI, Storey JM, Storey KB. Low oxygen and high temperatures induce oxidative stress in gills and liver of goldfish. Journal of Experimental Biology. 2009; 212: 233-242.
4. Sanches-Silva A, Costa D, Albuquerque TG, Costa HS. Nutritional and chemical composition of fish species: A review. Journal of Food Science. 2014; 89: S1448-S1456.
5. Huidobro A, Tejada M, Borderias AJ. Quality index method developed for raw gilthead seabream (Sparus aurata). J Food Sci 2001; 66: 1298-1304.
6. Williams GM, Kroes R, Munro IC. Safety evaluation and risk assessment of the herbicide Roundup and Its Active Ingredient, Glyphosate, for Humans. 2000; 31: 117-165.
7. De Graaf GJ, Galemoni F, Banzoussi B. Market orientation and fish supply chain in West Africa: The case of the catfish market in Cotonou, Benin. Food Policy. 2005; 30: 578-586.
8. Folmar LC, Sanders HO, Julin AM. Toxicity of the herbicide glyphosate and its decomposition products to fish and aquatic invertebrates. Bull Environ Contam Toxicol. 1979; 22: 67-74.
9. National Root Crops Research Institute 2010. Annual Report. North Carolina Cooperative Extension Service (NCCS). Soil facts; poultry manure as a fertilizer source. Publication AG-439-5.
10. Reish DL, Oshida PS. Short term bioassay. In: Manuals of Methods in Aquatic Environmental Research, part 6. FAO. Fish Technical Paper 247. 1987; 1-62.
11. Oladimeji AA, Ologunmeta RT. Chronic Toxicology of water borne lead to Tilapia nilotica (L). Nigeria Journal of Applied Fish and Hydrobiology. 1987; 117: 19-24.
12. Official Methods of Analysis. Association of Official Analytical Chemists. 2005.
13. Duke SO, Powles SB. Glyphosate-resistant weeds and crops. Pest Manag Sci. 2008; 64: 317-327.
14. Akinsoroton AM. Toxicity of dizensate (Glyphosate herbicide) on Clarias gariepinus fingerlings, Adv Res Biol Sci. 2014; 2.
15. Silva JJ, Chamul RS. Composition of marine and freshwater finfish and shell fish species and their products. In: marine and freshwater products hand book, Technomic publishing company Inc. 2000; 31-46.
16. Cui Y, Wootton R. Effects of ration, temperature and body size on the body composition, energy content and condition of the Minnow, Phoxinus phoxinus. J Fish Biol. 2011; 32: 749-764.
17. Osibona AO. Comparative study of proximate composition, amino and fatty acids of some economically important fish species in Lagos, Nigeria. Academic journals.com. 2011; 5: 581-588.
18. Nwali BU, Egesimba GI, Okechukwu-Ugwu PC, Ogbanshi ME. Assessment of the nutritional value of wild and farmed Clarias gariepinus. International Journal of Current Microbiolological Applied Scinces. 2015; 4: 179-182.
19. Obaroh IO, Haruna MA, Ojibo A. Comparative study on proximate and mineral element composition of Clarias gariepinus from the cultured and wild sources. European Journal of Basic and Applied Sciences. 2015; 2.
20. Fagbenro OA, Akinbulumo OM, Adeparusi OE, Raji AA. Flesh yield, waste yield, proximate and mineral composition of four commercial West African freshwater food fishes. Journal of Animal and Veterinary Advances. 2005; 4: 848-851.
21. Laurat T, Maimunatu A, Vandi P. Page Nutritive Value of the Carcass of African Catfish Clarias gariepinus (Burchell, 1822) Fingerlings fed at Different Frequencies. Journal of Agriculture and Veterinary Science. 2017; 83-87.
22. Ibhadon S, Abdulsalam MS, Emere MC, Yilwa V. Comparative Study of Proximate, Fatty and Amino Acids Composition of Wild and Farm-Raised African Catfish, Clarias gariepinus in Kaduna, Nigeria. Pakistan Journal of Nutrition. 2015; 14: 56-61.
23. Food and Agriculture Organisation. World production of fish, crustaceans and mollusks by major fishing areas. Fisheries Information Data and Statistics Unit (FIDI), Fisheries Department, FAO Rome. 1999; 33.
24. Nwani CD, Lakra WS, Nagpure NS, Kumar R, Kushwaha B, Srivastava SK. Toxicity of the herbicide Atrazine: Effects on lipid peroxidation and activities of antioxidant enzymes in the freshwater fish Channa punctatus (Bloch). International Journal of Environmental Research. 2010; 7: 3298-3312.
25. Ikeogu CF, Nsofor CI, Igwilo IO, Ngene AA. The effects of Crude Oil on the Blood Parameters and Serum Enzymes of the African catfish Clarias gariepinus. Journal of Pharmcrutical Sciences and Bioscientific Resesearch. 2017; 7: 341-345.
26. Solomon KR, Takacs P. Probabilistic risk assessment of pesticides. In R. M. Harrison & R. E. Hester (Eds.), Environmental and Health Impact of Pesticides. Royal Society of Chemistry. 2002; 147-164.