Evaluation of the toxic effect of a glyphosate-based herbicide on the health and reproduction of mammals: Case of the female rat (Rattus norvegicus) of the Wistar strain

 Corresponding author: Gnomoh Arnaud Pacôme Dolou. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Evaluation of the toxic effect of a glyphosate-based herbicide on the health and reproduction of mammals: Case of the female rat (Rattus norvegicus) of the Wistar strain Gnomoh Arnaud Pacôme Dolou 1, *, Yao Martin Siapo 1, Kassi Jean-Jacques Kablan 1, Koffi Roger Kouakou 2, and Yamousso Annick Tahiri 1 1 UFR Biosciences, Laboratory of Biology and Health, Félix Houphouët Boigny University, Ivory Coast. 2 UFR Sciences and Technology, Laboratory of Animal Biological Sciences, Alassane Ouattara University, Ivory Coast. World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 Publication history: Received on 21 May 2025; revised on 03 July 2025; accepted on 05 July 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.1.2553 Abstract Kalach 360 SL, a glyphosate-based herbicide used in agriculture in Côte d'Ivoire, is effective against weeds and improves crop yields. According to the UPL group, Kalach 360 SL is classified as low-hazard. However, evidence of the toxicity of the active ingredient (glyphosate) has been provided by some authors. This study aimed to evaluate the toxicity of Kalach 360 SL on mammalian health. Thirty-two rats, divided into four groups of eight, were used. The control group received distilled water. The other three groups received 0.1 mL, 0.2 mL, and 0.3 mL of Kalach 360 SL orally, corresponding to 250, 500, and 1000 mg/kg bw of glyphosate per day for 60 days. The results revealed a significant increase in relative liver mass. The relative mass of the kidney decreased significantly. The mass of the ovary decreased, unlike the mass of the uterine horn, which increased. The biochemical study revealed a significant increase in the serum concentration of ALAT, ASALT, Cholesterol, Creatinine and Urea. A decrease in the serum concentration of Total Protein was observed. Hormonal parameters revealed a decrease in the concentration of FSH and Progesterone. An increase in the concentration of Estradiol was observed. The histological study of the liver and kidney showed structural alterations within these organs. The histological study of the ovary and uterine horn revealed structural and functional alterations of these organs. These alterations are characterized by tissue lesions and necrosis. The daily use of this herbicide would present dangers to health and reproductive function. Keywords: Kalach 360 SL; Toxicity; Biochemical Parameters; Hormonal Parameters; Liver; Ovary 1. Introduction In recent years, labor for plantation maintenance has become very scarce in African countries. To fill this gap and improve yields, producers are most often turning to herbicides (Baldi et al., 2013) [1]. Herbicides are commonly used pesticides worldwide (Periquet et al., 2004) [2]. They belong to the large family of biocides (Periquet et al., 2004) [2]. Herbicides are used for crop protection, against herbaceous or woody plants. The associated agricultural practice is chemical weeding (Periquet et al., 2004) [2]. Depending on their mode of action, herbicides are classified into selective weedkillers, brush clearers, total weedkillers, products that destroy the aerial part of plants, anti-germs and Silicides more specifically target forest species or the natural regeneration process (Yahia, 2004) [3]. Among these different herbicides, total weedkillers are the most used in the agricultural sector (Yahia, 2004) [3]. However, the use of herbicides is problematic, because they do not only act in the desired location, but also disrupt the natural ecological balance (Camard, 2010) [4]. Indeed, the use of herbicides in agriculture can lead to the contamination of ecosystems and an impact on mammals (JardinSuisse, 2021) [5]. Kalach 360 SL, a Glyphosate-based herbicide distributed by Callivoire. It is used in Côte d'Ivoire in the agricultural sector. Kalach 360 SL is effective against weeds and improves World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 990 crop yields. Kalach 360 SL has an LD50 greater than 2000 mg/kg body weight. Therefore, Arysta LifeScience and UPL classify it as a low-hazard product. Although less hazardous, this substance may develop some adverse effects. The objective of this study is to evaluate the toxicity of the herbicide Kalach 360 SL (Glyphosate 360 mg/l) on mammalian health and reproduction. 2. Materials 2.1. Animal Materials The animal material consists of 32 adult male rats weighing between 120 g and 130 g. These animals all came from the ENS vivarium in Abidjan. They were fed corn, bread, and a soy + fish + corn-based formula. They were raised in polyethylene cages lined with wire-mesh wood shavings, with free access to water and food. The cages were cleaned every two days. 2.2. Chemical Equipment The chemical used was Kalach 360 SL (Glyphosate 360 mg/L). This herbicide is distributed in Côte d'Ivoire by Callivoire. Callivoire is a subsidiary of Unité Phosphorus Limited, a global player in the plant protection industry. In Côte d'Ivoire, Kalach 360 SL is used on a wide range of crops (tomatoes, carrots, cabbage, cucumbers, rice, oil palm, cotton). It is one of the most popular herbicides among Ivorian farmers due to its effectiveness against weeds and its non-toxicity on crops. 3. Methods 3.1. Preparation of the test solution According to Aymeric (2016) [6], the LD50 of glyphosate is greater than 8000 mg/kg of body weight. Three doses of glyphosate (Do) were defined according to the LD50: LD50/32, LD50/16, and LD50/8, i.e., 250 mg/kg, 500 mg/kg, and 1000 mg/kg of body weight. 3.2. Rat Treatment Four groups of 8 rats each were formed. The rats were treated daily by gavage for 60 days. For each group, the animals received 1 ml of the corresponding preparation individually and daily for the group for 60 days. Thus, • the control group received distilled water • group 1 received 250 mg/kg of body weight of glyphosate, i.e., 0.1 ml of Kalach 360 SL + 0.9 ml of distilled water; • Group 2 received 500 mg/kg body weight of Glyphosate, i.e. 0.2 ml of Kalach 360 SL + 0.8 ml of distilled water; • Group 3 received 1000 mg/kg body weight of Glyphosate, i.e. 0.3 ml of Kalach 360 SL + 0.7 ml of distilled water). 3.3. Sample Collection At the end of the test, the female rats were sacrificed after anesthesia. Blood was collected from the animals at the time of sacrifice. Blood was collected using tubes containing the anticoagulant EDTA (ethylenediamine tetraacetic acid) and dry tubes (without anticoagulant). The collected blood was used to measure biochemical parameters. Vital organs such as the liver and kidneys were removed after opening the abdominal cavity by dissection. They were rinsed with 0.9% sodium chloride (NaCl) and then weighed. After weighing, the organs were preserved in 10% formalin. The liver and kidney were used for histological study, which will reveal the effects of Kalach 360 SL on the internal structure of the kidney and liver. Reproductive organs such as the ovary and uterine horn were removed by dissection. They were rinsed with 0.9% sodium chloride (NaCl) and weighed. After weighing, the organs were preserved in 10% formalin. The testicle was used for the histological study. 3.4. Determination of Biochemical Parameters Biochemical assays were performed using a Mindray BS-200E automatic analyzer from China. The assay methods differ depending on the biochemical parameters being measured. The assay device is configured for each biochemical World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 991 parameter. Thus, AST, ALT, total protein, bilirubin, triglycerides, total cholesterol, urea nitrogen, and creatine are measured. 3.5. Determination of Hormonal Parameters The principle of assaying gonadal hormones (testosterone) combines the competitive method with final fluorescence detection (ELFA). To do this, the 60-test kit (VIDAS, BIOMERIEUX, France) containing all the immunological reaction reagents are used. The testosterone assay was performed using the HITACHI 902, Automatic analyzer, Japan. The sample to be tested is collected and then transferred to the well containing the testosterone derivative. A competition is carried out between the hormone present in the serum and the estradiol derivative of the conjugate. At the end of the test, the results are calculated by the machine compared to a stored calibration curve, then printed. The principle of assaying pituitary hormones (LH and FSH) combines the sandwich enzyme immunoassay method with a final fluorescence detection (ELFA). The assay steps are the same as for gonadal hormones except that the antigen (hormone) binds on the one hand to the immunoglobulins fixed on the cone and on the other hand to the conjugate, thus forming a "sandwich". 3.6. Histopathology of the Liver, Ovary, and Uterine Horn The liver, ovary, and uterine horn were removed and preserved in fixative (10% formalin + 9% NaCl) for 48 hours. Once removed from the fixative, these organs were rinsed and placed in perforated cassettes. The cassettes were labeled and successively placed in three alcohol baths of increasing strength (80° in 1 hour, 90° in 2 hours, and two baths of 100° in two hours each). After dehydration, the organs were placed in three successive toluene baths. The first bath lasted 1 hour, and each of the other baths lasted two hours. After passing through the toluene baths, the samples were directly immersed successively in two baths of molten paraffin. This process was carried out in an incubator (Memmert, Germany) between 58 and 60°C. The first bath lasts two (02) hours and the next one is done in three (03) hours. After impregnation, the cassettes are opened, the organs are removed and then placed in molds containing a thin quantity of paraffin. The mold was covered by the cassette inside which the liquid paraffin is poured until filling. Cooling, solidification of the molds and demolding reveals paraffin blocks in which the organ fixed to the back of the cassette is embedded. The formed blocks are roughened using the Leica RM2125 RTS brand microtome (Germany) and placed on ice for 30 min. After this step, the blocks are mounted on the microtome and then undergo 4 to 5 μm thick sections. 3.7. Statistical Data Analysis Graph Pad Prism7 software was used for statistical data analysis. Results are presented as mean plus or minus standard error (MSE). Data were separated using the one-way ANOVA method, followed by Tukey's multiple comparison test at the 5% significance level. Differences are considered: • Not significant if p > 0.05 • * Significant if p < 0.05 4. Results 4.1. Effect of Kalach 360 SL on the relative masses of vital and reproductive organs The relative liver masses of female rats treated with doses of 250, 500, and 1000 mg/kg body weight of glyphosate were 2.677 ± 0.060; 3.050 ± 0.092; 3.444 ± 0.130 g/100g versus 2.677 ± 0.060; 3.050 ± 0.092; 3.444 ± 0.130 g/100g for control female rats. The results reveal a significant increase (p < 0.05) in liver mass for female rats treated with the dose of 1000 mg/kg body mass of Glyphosate compared to control female rats (Table I). In contrast, in the kidneys, the relative mass is respectively 0.503 ± 0.023; 0.507 ± 0.020; 0.508 ± 0.025 g/100 g for the groups treated with doses of 250, 500 and 1000 mg/kg body weight of Glyphosate and 0.684 ± 0.058 g/100 g for the control group. The results show a significant decrease (p < 0.05) in female rats of the treated groups compared to the control rats (Table I). The values of the relative masses of the ovary of female rats treated with doses of 250, 500 and 1000 mg/kg body weight of Glyphosate are 0.035 ± 0.003; 0.035 ± 0.005 and 0.025 ± 0.003 g/100g body weight versus 0.051 ± 0.003 g/100g body weight for control female rats. The results showed a significant decrease (p < 0.01) in the relative mass of the ovaries in female rats treated with 1000 mg/kg bw doses of Glyphosate compared to the control (Table I). World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 992 For doses of 250, 500 and 1000 mg/kg bw of Glyphosate, the relative masses of the uterine horns were 0.314 ± 0.097; 0.312 ± 0.012; 0.406 ± 0.043 g/100 g body weight versus 0.176 ± 0.012 g/100 g body weight for the control group. Statistical analysis showed a significant increase in the relative mass of the uterine horns in all treated groups compared to the control group (Table I). Table 1 Relative mass of organs in female rats, control and treated with Kalach 360 SL at doses of 250, 500, 1000 mg/kg body weight of Glyphosate Glyphosate concentration (mg/kg m.c) Relative mass of organs (g/100g of body weight) Liver Kidney Ovary Uterine horn 0 2.677 ± 0.060 0.683 ± 0.058 0.051 ± 0.003 0.176 ± 0.012 250 2.677 ± 0.060 0.503 ± 0.023* 0.035 ± 0.003 0.314 ± 0.097* 500 3.050 ± 0.092 0.506 ± 0.020* 0.035 ± 0.005 0.312 ± 0.012* 1000 3.444 ± 0.130* 0.507 ± 0.025* 0.025 ± 0.003* 0.406 ± 0.043* Not significant if p > 0.05, Significant if p < 0.05 *, Vertical reading direction 4.2. Effects of Kalach 360 SL on Biochemical Parameters Liver parameters such as ALT, AST, and Total Protein were measured. The results of the data analysis from this assay are shown in Figures 1 and 2. Serum AST concentrations in the groups treated with doses of 250, 500, and 1000 mg/kg body weight were 182.300 ± 3.180 U/L; 213.000 ± 17.500 U/L; 236.700 ± 4.702 U/L; and 161.300 ± 7.667 U/L for the control group. The results showed a significant increase (p<0.05) in serum AST concentrations in rats treated with 500 and 1000 mg/kg doses compared to control female rats (Figure 1). For doses of 250, 500 and 1000 mg/kg body weight, serum ALT concentrations in treated female rats were 39.000 ± 1.528 U/L; 42.670 ± 3.333 U/L and 54.670 ± 1.333 U/L, respectively, compared to 33.330 ± 0.667 U/L for control female rats. Statistical analysis showed a significant increase (p<0.05) in serum ALAT concentration in female rats treated with doses of 500 and 1000 mg/kg body weight compared to control female rats (Figure 1). The serum Total Protein concentration in the groups treated with doses of 250, 500 and 1000 mg/kg body weight are 72.330 ± 0.667 U/l; 71.330 ± 0.333 U/l; 69.330 ± 0.333 U/l and 75.330 ± 0.333 U/l for the control group. The results showed a significant increase (p < 0.05) in the serum concentration of Total Protein in female rats treated with doses of 500 and 1000 mg/kg compared to control female rats (Figure 1). The serum concentration of Cholesterol in the groups treated with doses of 250, 500 and 1000 mg/kg of body mass are 0.630 ± 0.030 U/l; 0.773 ± 0.028 U/l; 0.400 ± 0.021 U/l compared to 0.436 ± 0.032 U/l for the control group. The results showed a significant increase in the serum concentration of Cholesterol in female rats treated with doses of 250, 500 and 1000 mg/kg compared to control female rats (Figure 1). For doses of 250, 500 and 1000 mg/kg body weight, serum Urea concentrations were respectively 0.450 ± 0.010 U/l; 0.496 ± 0.003 U/l; 0.570 ± 0.035 U/l versus 0.343 ± 0.023 U/l for the control group. The results also show a very significant increase (p<0.05) in serum urea concentration in female rats treated with the dose of 250, 500 and 1000 mg/kg body weight compared to the controls (Figure 1). The serum creatinine concentration in the groups treated with doses of 250, 500 and 1000 mg/kg body weight were 6.667 ± 0.333 U/l; 7.333 ± 0.333 U/l; 7.667 ± 0.667 U/l and 6.33 ± 0.667 U/l for the control group. The results showed a significant increase in the serum creatinine concentration of female rats treated with doses of 500 and 1000 mg/kg compared to the control female rats (Figure 1) World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 993 Not significant if p > 0.05, Significant if p < 0.05. Figure 1 Variation in serum concentration of biological markers in control and Kalach 360 SL-treated female rats at doses of 250, 500, 1000 mg/kg b.w. of Glyphosate 4.3. Effects of Kalach 360 SL on Hormonal Parameters The results revealed no significant changes (p>0.05) in serum LH concentrations in all treated groups compared to the control group (Figure 2). Groups treated with 500 and 1000 mg/kg bw doses of Glyphosate had low FSH levels compared to the control group. Statistical analysis showed a significant decrease (p<0.05) in serum FSH concentrations in female rats treated with 500 and 1000 mg/kg bw doses of Glyphosate compared to the control group of female rats (Figure 2). Groups treated with the respective doses of 250, 500, and 1000 mg/kg bw of Glyphosate all had elevated estradiol levels. Statistical analysis revealed a significant increase (p<0.05) in serum estradiol concentrations in the treated groups compared to the control group (Figure 2). Groups treated with the doses of 250, 500, and 1000 mg/kg bw of Glyphosate had low progesterone levels. The results showed a significant decrease (p<0.05) in serum progesterone concentrations in all groups of female rats treated with Kalach 360 SL compared to the control group (Figure 2). World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 994 A: Proportion of LH, B: Proportion of FSH, C: Proportion of Estradiol, D: Proportion of Progesterone; Not significant if p > 0.05, * Significant if p < 0.05 Figure 2 Proportions of Dosed Hormones 4.3. Impact of Kalach 360 SL on Liver Histology Microscopic observation of histological sections in control female rats shows normal architecture. Liver cells (hepatocytes) are arranged and arranged around the centrilobular vein (Figure 3 A). In treated female rats, the presence of hepatic necrosis, inflammatory cell infiltration, hepatocyte vacuolation, and congestion of the centrilobular veins is observed (Figures 3 B; 3 C; 3 D). These observations are absent in control female rats. World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 995 A: control; B: treated with 250 mg/kg bw; C: treated with 500 mg/kg bw; D: treated with 1000 mg/kg bw.1: Centrolobular vein; 2: sinusoidal capillaries; 3: hepatocytes; Vascular congestion; Hepatocyte vacuolation; N: Cell necrosis Figure 3 Histological sections of the liver of female rats treated with Kalach 360 SL (A) and control rats (B, C, D) 4.4. Effects of Kalach 360 SL on Kidney Histology Histology reveals normal kidney architecture in control female rats (Figure 4 A). Indeed, the observation shows no inflammation, necrosis, or tissue lesions. In female rats from the treated group, lesions within the tissue and necrosis in the glomerulus are observed (Figures 4 B; 4 C; 4 D). World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 996 G: Glomerulus; CB: Bowman's capsule; CG: Glomerular duct; TC: Convoluted tubule; NTG: Glomerular tissue necrosis; NT: Tissue necrosis; NCG: Glomerular duct necrosis; NTC: Convoluted tubule necrosis; R: Convoluted tubule diameter narrowing; NTG: Glomerular tissue necrosis; DG: Lomerulus destruction Figure 4 Histological sections of the kidney of control female rats (A) and female rats treated with Kalach 360 SL solution (B, C, D) at respective doses of 250, 500, 1000 mg/kg b.w. of Glyphosate 4.5. Impact of Kalach 360 on Ovarian Histology The histology of the ovaries in the control group (Figure 5 A) exhibits normal structural architecture. The presence of primary, secondary, and tertiary follicles, as well as the corpus luteum, is observed. These follicles develop correctly, with a high presence of follicular and egg cells. There is an absence of lesions and necrosis in the ovarian and follicular tissue. However, in the treated female rats (Figure 5 B, C, D), the ovarian structure exhibits abnormalities. The absence of eggs and corpus luteum, as well as a decrease in primary follicles, is noted. Ovarian tissue lesions and destruction of secondary and tertiary follicles were recorded. Follicular cell necrosis followed by cellular debris deposits were also observed. World Journal of Advanced Research and Reviews, 2025, 27(01), 989-1003 997 A: control; B: treated (250 mg/kg bw), C: treated (500 mg/kg bw), D: treated (1000 mg/kg bw); TI: theca interna; TE: theca externa; AN antrum; O: ovum; CJ: corpus luteum; CR: corona radiata; FT: tertiary follicle; FS: secondary follicle; FP: primary follicle; FTD: destroyed tertiary follicle; FSD: destroyed secondary follicle; DC: cellular debris; VS: blood vessel; SO: ovarian stroma Figure 5 Histological sections of the ovary of control (A) and treated (B, C, D) female rats with Kalach 360 SL 4.6. Effects of Kalach 360 SL on the Histology of the Uterine Horns Histological studies performed on the uterine horn of control female rats (Figure 6 A) reveal normal architecture. Indeed, we observe normal endometrial tissue and thickness. The myometrium also exhibits normal structure and thickness. A strong presence of uterine glands and epithelial cells is observed. In contrast, in treated female rats (Figure 6 B, C, D), some disorganization of the uterine tissue structure is observed. These abnormalities are characterized by a reduction in the lumen and thickness of the endometrium and myometrium. Necrosis is observed in epithelial cells and endometrial tissue. A significant reduction in uterine glands and the presence of tissue debris is observed.