Impact of pesticides lambda-cyhalothrin,emamectin benzoate,and thiophanate-methyl used alone and in mixtures on reproduction and oxidative stress markers in male Wistar rats

Abstract

Pesticide exposure is a growing public health concern, particularly in agricultural regions where workers and communities encounter multiple chemical compounds simultaneously. Although previous studies have investigated the reproductive toxicity of individual pesticides, the combined effects of commonly co-applied insecticide–fungicide mixtures remain poorly characterized. We hypothesized that the simultaneous administration of lambda-cyhalothrin, emamectin benzoate, and thiophanate-methyl, three pesticides routinely co-applied in eastern Algerian agriculture, would exert synergistic reproductive toxicity in male rats, exceeding the effects observed with each compound alone. Seventy-two adult male Wistar rats (n = 8 per group) were treated by oral gavage for 6 weeks with each pesticide at 1/20 LD₅₀ (high dose) or 1/60 LD₅₀ (low dose), or with a ternary mixture at the same dose levels; one group served as the control. Testicular and epididymal weights, plasma testosterone concentration, sperm parameters (concentration, motility, and speed), oxidative stress markers (GSH, GPx, and MDA), and tissue histopathology were evaluated. All three pesticides at the high dose significantly impaired reproductive parameters and induced oxidative stress; however, the mixture group (G8, 1/20 LD₅₀) exhibited the most pronounced alterations, including a highly significant decrease in testicular weight, severe testosterone suppression, marked sperm parameter decline, and extensive histopathological damage consistent with synergistic toxicity. These findings provide the first evidence that the combination of lambda-cyhalothrin, emamectin benzoate, and thiophanate-methyl produces synergistic reproductive toxicity in male rats, underscoring the need for mixture-based risk assessment in agricultural pesticide regulation.

Keywords

lambda-cyhalothrin emamectin benzoate thiophanate-methyl pesticide mixture reproductive toxicity oxidative stress synergism male Wistar rat

Introduction

Agriculture is a significant source of economic income, especially in developing countries, where farm workers frequently come into contact with potentially hazardous substances, the most common of which are pesticides (Mehrpour et al., 2014). In eastern Algeria, where agriculture is a key economic sector, the living environment is increasingly burdened by the excessive use of pesticides, leading to adverse biological effects on both target and non-target species (Akhter et al., 2024). Currently, food production relies on the intensive use of various pesticides (Costa et al., 2024), the application of which has increased significantly in recent decades (Napierkowska et al., 2024). According to the World Health Organization, pesticide poisoning represents a paramount public health issue, with approximately 3 million cases of pesticide toxicity occurring annually, resulting in 250,000–370,000 deaths (Mostafa et al., 2023). Consequently, pesticide exposure poses significant risks to the health of animals and humans, particularly to reproductive health, leading to adverse consequences including reproductive system dysfunction (Mbouh et al., 2022).

The impact of pesticides on the male reproductive system has been described through two principal hypotheses. The first posits that pesticide exposure induces oxidative stress (Adiguzel et al., 2024), which damages testicular cells and leads to decreased testosterone concentration (Mustafa and Jawad, 2023), histopathological changes in tissues, and subsequent impairment of spermatogenesis, manifesting as reduced sperm concentration, motility, and speed (Adiguzel et al., 2024) as well as diminished organ weight (Adam et al., 2019). The second hypothesis concerns the “mixture effect,” whereby combined pesticide exposure, compared with individual compounds, may produce synergistic or antagonistic toxicity in non-target organisms (Elnamaky et al., 2018).

These two mechanisms are not mutually exclusive: oxidative stress may serve as the convergent downstream pathway through which mixture interactions amplify reproductive damage. However, the extent to which specific pesticide combinations interact, and whether these interactions are additive, synergistic, or antagonistic, remains insufficiently characterized for many commonly co-applied compounds.

Lambda-cyhalothrin is a type II pyrethroid insecticide characterized by the presence of a nitrile group on the alpha carbon. This active ingredient, commercialized under numerous trade names, is widely used to control various insect pests in agriculture (Chakroborty et al., 2019). Pyrethroids act primarily by targeting voltage-dependent sodium channels in the nervous system, leading to disruption of nerve impulses (Field et al., 2017). Numerous studies have indicated that pyrethroid exposure exerts adverse effects on the male reproductive system, including hormonal disturbances (Adam et al., 2019), reduced sperm concentration and quality (Adam et al., 2019), induction of oxidative stress through the generation of reactive oxygen species (ROS) (Chakroborty et al., 2019), and damage to the anatomical structure of male reproductive organs (Ravula and Yenugu, 2021). Although Ahmed and Kandil (2023) reported that lambda-cyhalothrin bait exposure over four days resulted in adverse reproductive consequences in rats, their study was limited by the use of a single dose level (0.032%). The present study addressed this limitation by investigating the effects of multiple doses of lambda-cyhalothrin, both alone and in a mixture with other pesticides.

Emamectin benzoate is an insecticide belonging to the avermectin chemical family, commonly used to control pests in a variety of crop ecosystems (Guo et al., 2023). Avermectins act by blocking nerve impulse transmission through interference with the γ-aminobutyric acid (GABA) receptor, leading to irreversible paralysis and insect death (Kolianchuk et al., 2023). In non-target organisms, avermectins have been shown to disrupt reproductive hormone production (Ferri et al., 2013), induce oxidative stress (Dawoud et al., 2021) and cause testicular lesions with reduced sperm count and motility (EL-Shafey et al., 2011; Kumar, 2013; Tekeli, 2023). However, none of these studies assessed serum testosterone concentration in conjunction with oxidative stress markers and histopathological endpoints in an integrated manner.

In addition to insecticides, the fungicide class has also demonstrated notable side effects on non-target organisms. Thiophanate-methyl is a systemic fungicide belonging to the benzimidazole family, widely used to control fungal diseases in crops (Saleh and Al-Farttoosy, 2024). Benzimidazoles act by inhibiting spindle microtubule assembly, which can lead to chromatid or chromosome loss and non-disjunction in target cells (Bevilaqua et al., 2020). Benzimidazole fungicides have been reported to impair reproduction in rodents (Cédric et al., 2016) and induce testicular toxicity by disrupting seminiferous tubule cell assembly and function (Oh et al., 2014). Although Bououza et al. (2022) reported that thiophanate-methyl negatively affected testicular and epididymal weights, sperm quality, and histological structure in rats, that study did not address the impact of thiophanate-methyl on oxidative stress markers and testosterone concentration, parameters crucial for a comprehensive assessment of reproductive health.

A critical gap in the current literature concerns the combined toxicity of pesticide mixtures. In agricultural practice, risk assessments have traditionally been based on the toxicity of individual compounds (Wang et al., 2021). However, in real-world agricultural settings, organisms are exposed to complex mixtures of chemicals (Ernst et al., 2018) and combined toxicity studies provide more relevant information for predicting health risks (Chen et al., 2020; Dupraz et al., 2019). In eastern Algeria, combinations of insecticides and fungicides are routinely applied simultaneously to manage both insect pests and fungal diseases in orchards, reducing the number and cost of treatments (Wang et al., 2021). While previous studies have examined the reproductive toxicity of certain pesticide mixtures, such as beta-cypermethrin with emamectin benzoate (Zhang et al., 2020) and imidacloprid with lambda-cyhalothrin (Mbouh et al., 2022), no study to date has investigated the reproductive effects of the ternary combination of lambda-cyhalothrin, emamectin benzoate, and thiophanate-methyl, despite their frequent co-application in the field.

To address this gap, the present study aimed to evaluate the toxic effects of these three pesticides, administered either alone or as a ternary mixture at two dose levels (1/20 and 1/60 LD₅₀), on the male reproductive system of Wistar rats. Specifically, we tested the hypothesis that the ternary mixture would produce synergistic reproductive toxicity, exceeding the effects observed with each compound alone, through an integrated assessment of testicular and epididymal weights, plasma testosterone concentration, sperm parameters (concentration, motility, and speed), oxidative stress markers (GSH, GPx, and MDA), and testicular and epididymal histopathology.

Material and methods

Animals

Seventy-two adult male Wistar rats, aged 8 weeks (body weight 250 ± 20 g), were obtained from the Pasteur Institute, Algiers, Algeria. The animals were housed in polypropylene cages maintained at an ambient temperature of 22 ± 3°C under a natural photoperiod, with a relative humidity of 40%–60%. They were provided free access to water and were fed a diet consisting of 20 g croquettes per rat per day. The diet, purchased from the National Livestock Feed Office (ONAB, Mjasffa-Guelma, Algeria), contained corn, barley, phosphate, soy, cellulose, limestone, vitamins, and minerals.

Chemicals

The commercial pesticides used in this study were selected based on a field survey identifying the most commonly co-applied pesticides in agriculture in eastern Algeria. They were purchased from a pesticide dealer in the region.

• Lambda-cyhalothrin: C₂₃H₁₉ClF₃NO₃ (DECAPLUS® 5% EC, DEKACHIM, Algeria); CAS No. 91465-08-6.

• Emamectin benzoate: C₅₆H₈₁NO₁₅ (Palmer® 5% WP, DEKACHIM, Algeria); CAS No. 155569-91-8.

• Thiophanate-methyl: C₁₂H₁₄N₄O₄S₂ (MAPHYTO-PHANATE® 70% WP, SAPHYTO, France); CAS No. 23564-05-8.

The ternary mixture of these three pesticides was formulated based on farmers’ co-application practices to reflect real-world agricultural exposure scenarios. These pesticides are compatible in tank mixes, and combinations of insecticides with fungicides are commonly used to simultaneously treat insect pests and cryptogamic diseases in orchards.

Experimental design and dose justification

After an adaptation period of 15 days, the 72 rats were randomly assigned to nine groups of eight animals each (n = 8 per group) using a random number table. The group size of eight was selected based on guidelines for subchronic toxicity studies in rodents (OECD Test Guideline 407), which recommend a minimum of five animals per sex per dose group, and on prior toxicological studies using similar experimental designs (Ahmed and Kandil, 2023; Mostafa et al., 2023; Ravula and Yenugu, 2021). Treatments were administered orally by gavage for six consecutive weeks, as detailed in Table 1.

Table 1.

Experimental design: daily treatment protocol for nine groups of male Wistar rats (n = 8 per group) treated orally by gavage for 6 weeks.

Group	Daily treatment	Dose
G1	Control (water vehicle)	—
G2	Lambda-cyhalothrin (1/20 LD₅₀)	4.32 mg/kg bw
G3	Lambda-cyhalothrin (1/60 LD₅₀)	1.44 mg/kg bw
G4	Emamectin benzoate (1/20 LD₅₀)	3.35 mg/kg bw
G5	Emamectin benzoate (1/60 LD₅₀)	1.11 mg/kg bw
G6	Thiophanate-methyl (1/20 LD₅₀)	50 mg/kg bw
G7	Thiophanate-methyl (1/60 LD₅₀)	16.66 mg/kg bw
G8	Ternary mixture (1/20 LD₅₀)	LC + EB + TM combined
G9	Ternary mixture (1/60 LD₅₀)	LC + EB + TM combined

Note: bw, body weight; LC, lambda-cyhalothrin; EB, emamectin benzoate; TM, thiophanate-methyl. 1/20 and 1/60 LD₅₀ represent high and low dose levels, respectively.

The dose levels of 1/20 and 1/60 of the oral LD₅₀ were selected as high and low subchronic doses, respectively, based on an established toxicological logic. The 1/20 LD₅₀ fraction represents a commonly used high subchronic dose in reproductive toxicology studies, producing observable toxic effects without excessive lethality (Chukwudi et al., 2020; Mostafa et al., 2023), while the 1/60 LD₅₀ fraction represents a lower exposure level that more closely approximates chronic environmental exposure. These fractions are also consistent with the dose ranges used by farmers in the study region. The LD₅₀ values were obtained from recent studies: lambda-cyhalothrin, 86.40 mg/kg body weight (Kumar and Yadav, 2021); emamectin benzoate, 67 mg/kg body weight (Yasturb and Dontsova, 2021); and thiophanate-methyl, 1000 mg/kg body weight (Feki et al., 2017). All pesticides were prepared in water for gavage administration.

Blood and organ collection

At the end of the treatment period, rats were sacrificed by decapitation after overnight fasting. A volume of 3 ml of blood was collected in heparinized tubes and centrifuged at 3000 × g for 15 minutes using an Eppendorf 5804 R centrifuge equipped with a fixed-angle rotor to separate the plasma for the measurement of testosterone. Animals were then opened ventrally, and the testes and epididymis (left and right sides) were carefully dissected, cleaned of adipose tissue, and rinsed in 0.9% NaCl saline solution. The samples were weighed with a KERN PRS 320-3 precision balance (d = 0.1 mg) to obtain absolute weights. Some organs were immediately frozen at −20°C wrapped in Parafilm for the assessment of oxidative stress markers, while others were immediately fixed in a 10% formalin solution (Sigma, St. Louis, USA, CAS No. 50-00-0) for histological study.

Measurement of testosterone

Plasma testosterone concentration was measured using the enzyme-linked immunosorbent assay (ELISA) immunological method, according to the manufacturer’s instructions (ELISA EIA-1559, DRG Instruments GmbH, Germany). This competitive assay relies on the competition between an unknown amount of antigen in the sample and a fixed amount of enzyme-conjugated antigen for antibody binding sites immobilized in the wells. After incubation for 1–2 hours at room temperature or 37°C, the wells were washed to remove unbound material. Tetramethylbenzidine (TMB) solution (Sigma-Aldrich, Cat No: T0440, CAS No. 54827-17-7) was then added, and a blue color developed after 20 minutes of incubation. The addition of 1 N HCl (Sigma-Aldrich, Cat No: 320331, CAS No. 7647-01-0) stopped the reaction and transformed the blue color to yellow, which was measured using a spectrophotometer at 450 nm. The intra-assay coefficient of variation for the ELISA kit was <10%, as specified by the manufacturer. All samples were assayed in duplicate.

Semen collection

Semen analysis was performed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer, SCA®, Microptic, Barcelona, Spain). Following sacrifice, the epididymal fluid was collected, and 1 μl of sperm was diluted in 0.9% physiological NaCl solution. Then, 5 μl of the diluted sperm was placed in a GoldCyto® counting chamber using a micropipette and examined under a Nikon Eclipse E200-LED microscope at 40× magnification. Sperm concentration, motility, and speed (rapid, medium, slow, and immobile categories) were evaluated.

Measurement of oxidative stress markers

After thawing, oxidative stress parameters were measured in testicular and epididymal tissues as follows:

Reduced glutathione (GSH) was determined according to the method of Weckbecker and Cory (1988). This assay measures the optical absorbance of 2-nitro-5-mercapturic acid, formed by the reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) by the sulfhydryl groups (–SH) present in GSH. Tissue (100 mg) was cold-homogenized using an ultrasonic homogenizer with 4 ml of ethylenediaminetetraacetic acid (EDTA, 0.02 mol) solution (VWR International BVBA-EC, CAS No. 60-00-4). A volume of 0.8 ml of the homogenate was mixed with 0.2 ml of a sulfosalicylic acid solution (SSA, 0.25%) (BIOCHEM-Chemo-Pharma, France, CAS No. 5965-83-3), stirred, and left for 15 min in an ice bath, then centrifuged at 1000 × g for 5 min (Eppendorf 5810 R). A volume of 1 ml Tris(hydroxymethyl)aminomethane (0.4 mol) (BIOCHEM-Chemo-Pharma, CAS No. 77-86-1) and EDTA (pH 9.6) was added to 0.5 ml of the supernatant, mixed, then 0.025 ml of DTNB (0.01 mol) (Sigma, St. Louis, USA, CAS No. 120-78-5) was added. The mixture was left to react for 5 min, and absorbance was read at 412 nm.

Glutathione peroxidase (GPx) activity was determined using the method of Flohé and Günzler (1984). This method is based on the reduction of hydrogen peroxide (H₂O₂) in the presence of GSH, which is converted to oxidized glutathione (GSSG) by the action of GPx. Tissue (100 mg) was cold-homogenized with 1 ml of Tris-buffered saline (TBS: Tris 50 mM, NaCl 150 mM, pH 7.4). A volume of 0.2 ml of the homogenate was mixed with 0.4 ml of reduced glutathione (GSH, 0.1 mM) (Sigma, CAS No. 70-18-8) and 0.2 ml TBS buffer. After incubation in a water bath at 25°C for 5 min, 0.2 ml of H₂O₂ (1.3 mM) (Sigma, CAS No. 7722-84-1) was added to initiate the reaction (10 min), then 1 ml of trichloroacetic acid (TCA, 1%) (BIOCHEM-Chemo-Pharma, CAS No. 76-03-9) was added to terminate the reaction. After cooling for 30 min in an ice bath, the tubes were centrifuged at 3000 × g for 10 min (Eppendorf 5810 R), and the supernatant was collected. Then, 2.2 ml TBS buffer and 0.32 ml of DTNB (1 mM) (Sigma, CAS No. 69-78-3) were added to 0.48 ml of the reaction supernatant. After mixing and waiting 5 min, absorbance was recorded at 412 nm.

Malondialdehyde (MDA), a marker of lipid peroxidation, was quantified using the method of Ohkawa et al. (1979). The assay is based on the formation of a colored complex between MDA and thiobarbituric acid (TBA), absorbing at 530 nm, in a hot, acidic environment (100°C). A volume of 0.5 ml of tissue homogenate was mixed with 0.5 ml of TCA (20%) (Sigma, CAS No. 76-03-9), then 1 ml of TBA (0.67%) (Sigma, CAS No. 504-17-6) was added and incubated in a water bath at 100°C for 15 min. After cooling, 4 ml of n-butanol (Sigma, CAS No. 71-36-3) was added, and the mixture was centrifuged at 3000 × g for 15 min (Eppendorf 5810 R). The supernatant was collected and absorbance was read at 530 nm.

Total protein concentration was determined by the Bradford (1976) method using Coomassie Blue G-250 (Sigma, CAS No. 6104-58-1). A volume of 5 ml of Bradford’s reagent was added to 0.1 ml of the homogenate. After 5 minutes, absorbance was measured at 595 nm.

All biochemical assays were performed in duplicate, and mean values were used for statistical analysis.

Histopathological study

Histological examination of the testes and epididymis was performed following the method of Martoja and Martoja-Pierson (1967). Tissues were fixed in 10% formalin solution (Sigma, CAS No. 50-00-0) to preserve cellular morphology. They were then dehydrated through a graded ethanol series (70–100%) (Thermo Fisher Scientific, CAS No. 64-17-5), cleared in xylene (Sigma, CAS No. 1330-20-7), and embedded in paraffin. Sections of 3–5 μm were cut using a microtome, mounted on glass slides, and stained with hematoxylin and eosin (H&E). Samples were examined using a Euromex Micro Blue Microscope (MB.1051-LCD) equipped with a 7″ IPS LCD screen, at 100× and 400× magnification.

To provide standardized evaluation, a semi-quantitative scoring system was applied based on the severity of histopathological alterations in the seminiferous and epididymal tubules. Each specimen was assessed across multiple fields (minimum 10 fields per section at 400×) and graded as follows: Grade 0, no observable changes (normal architecture); Grade 1, minimal changes (<10% of tubules affected); Grade 2, mild changes (10–25% of tubules affected); Grade 3, moderate changes (25–50% of tubules affected); Grade 4, severe changes (>50% of tubules affected). Parameters scored included tubular degeneration, germ cell disorganization, spermatozoa depletion, interstitial hemorrhage, and epithelial hyperplasia/destruction. Histopathological examination was performed by an observer blinded to the treatment groups.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 9). Results are expressed as mean ± standard deviation (SD). Data normality was verified using the Shapiro–Wilk test, and homogeneity of variance was confirmed with the Brown–Forsythe test. Comparisons between groups were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. Differences were considered statistically significant at p < 0.05.

Results

Effect on absolute organ weights

As shown in Table 2, the high-dose groups exhibited significant reductions in absolute testicular weight compared with the control group: G2 (lambda-cyhalothrin, p < 0.05), G4 (emamectin benzoate, p < 0.05), and G8 (mixture, p < 0.01). Absolute epididymal weight was also significantly reduced in G2, G4, and G8 (all p < 0.05). The mixture group (G8) showed the most pronounced testicular weight reduction (2.364 ± 0.402 g compared with 3.42 ± 0.515 g in controls).

Table 2.

Absolute testicular and epididymal weights (g) across experimental groups (mean ± SD, n = 8 per group).

Parameters	Absolute testes weight (g)	Absolute epididymis weight (g)
Groups	Absolute testes weight (g)	Absolute epididymis weight (g)
G1	3.42 ± 0.515	1.481 ± 0.219
G2	2.635 ± 0.564*	1.025 ± 0.237*
G3	2.987 ± 0.530	1.127 ± 0.223
G4	2.624 ± 0.438*	0.9993 ± 0.358*
G5	3.095 ± 0.374	1.119 ± 0.142
G6	3.121 ± 0.593	1.162 ± 0.309
G7	3.311 ± 0.582	1.244 ± 0.229
G8	2.364 ± 0.402**	1.069 ± 0.291*
G9	3.314 ± 0.296	1.366 ± 0.226

Note: p: significance level; p > 0.05: no significant difference; *p < 0.05, **p < 0.01 compared with the control group (G1); Tukey’s post hoc test.

No significant changes in testicular or epididymal weights were observed in the low-dose groups (G3, G5, G7, G9) or in the high-dose thiophanate-methyl group (G6), compared with the control group (p > 0.05).

Effect on testosterone concentration

Plasma testosterone concentration was significantly reduced in the high-dose groups compared with the control group (G1): G6 (thiophanate-methyl, p < 0.05), G4 (emamectin benzoate, p < 0.01), and G8 (mixture, p < 0.001) (Figure 1). Notably, the low-dose mixture group (G9) also exhibited a significant reduction in testosterone concentration (p < 0.05), indicating a mixture effect even at the lower dose level. No significant changes were observed in G2 (high-dose lambda-cyhalothrin), G3, G5, or G7 (all low-dose individual pesticides).

Effect on sperm parameters

Semen parameter analysis using the CASA SCA® system demonstrated that exposure to these pesticides, whether administered alone or as a ternary mixture, significantly impaired both sperm quantity and quality. These detrimental effects on concentration, motility, and speed are illustrated in Figures 2 –4.

Sperm concentration (Figure 2) was significantly decreased in the high-dose groups: G2, G4, and G8 (all p < 0.01), and G6 (p < 0.05), compared with the control group (G1). The low-dose mixture group (G9) also showed a significant decrease (p < 0.05). No significant changes were observed in the low-dose individual pesticide groups (G3, G5, G7).

Sperm motility (Figure 3) was significantly decreased in G4 (high-dose emamectin benzoate, p < 0.001) and G8 (high-dose mixture, p < 0.001) compared with the control. No significant changes were observed in the other treatment groups.

Sperm speed (Figure 4) showed differential effects across speed categories. Medium sperm speed decreased significantly in G2 (p < 0.001), G3 (p < 0.01), G4 (p < 0.01), G8 (p < 0.001), and G9 (p < 0.05). Slow sperm speed increased significantly only in G8 (p < 0.001). The proportion of immobile sperm increased significantly in G2, G4, and G8 (all p < 0.001), G3 (p < 0.01), and G9 (p < 0.001). No significant changes in rapid sperm speed were observed across any treatment group.

Effect on oxidative stress markers

Testicular oxidative stress markers (Table 3): GSH was significantly decreased in the high-dose groups—G2, G4, and G6 (all p < 0.001) and G8 (p < 0.01)—compared with the control (G1). GPx activity was significantly reduced in all high-dose groups (G2, G4, G6, and G8; all P < 0.001). Testicular MDA was significantly elevated in G6 and G8 (both p < 0.001). No significant changes in GSH, GPx, or MDA were observed in the low-dose groups (G3, G5, G7, G9).

Table 3.

Oxidative stress markers in testicular tissue across experimental groups (mean ± SD, n = 8 per group).

Parameters	GSH nM/mg protein	GPx µM/GSH/min/mg protein	MDA nmol/mgTissue
Groups	GSH nM/mg protein	GPx µM/GSH/min/mg protein	MDA nmol/mgTissue
G1	0.107 ± 0.018	0.092 ± 0.012	39.39 ± 5.457
G2	0.037± 0.014***	0.045 ± 0.007 ***	44.26 ± 12.03
G3	0.098 ±0.021	0.072 ± 0.018	42.48 ± 9.745
G4	0.054 ± 0.002***	0.046 ± 0.004 ***	50.43 ± 9.045
G5	0.095 ± 0.007	0.080 ± 0.006	44.74 ± 10.58
G6	0.048 ± 0.014***	0.046 ± 0.007***	82.61 ± 5.338 ***
G7	0.122 ± 0.012	0.077 ±0.012	45.83 ± 10.53
G8	0.072 ± 0.012**	0.041 ± 0.002***	82.61 ± 16.34***
G9	0.085 ± 0.008	0.072 ± 0.009	56.52 ± 6.344

Note: GSH: reduced glutathione; MDA: malondialdehyde; GPx: glutathione peroxidase. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (G1).

Epididymal oxidative stress markers (Table 4): GSH was significantly decreased in G2, G6, and G8 (all p < 0.001), G4 (p < 0.01), and G9 (p < 0.01). GPx activity was significantly reduced in G2, G6, G8, and G9 (all p < 0.001). MDA was significantly elevated in G4 (p < 0.05), G6 (p < 0.01), and G8 (p < 0.001). No significant changes in epididymal GSH or GPx were observed in the low-dose individual pesticide groups (G3, G5, G7).

Table 4.

Oxidative stress markers in epididymal tissue across experimental groups (mean ± SD, n = 8 per group).

Parameters	GSH nM/mg protein	GPx µM/GSH/min/mg protein	MDA nmol/mgTissue
Groups	GSH nM/mg protein	GPx µM/GSH/min/mg protein	MDA nmol/mgTissue
G1	0.135 ± 0.025	0.1186 ± 0.013	75.39 ± 11.53
G2	0.074 ± 0.002***	0.057 ± 0.011***	98.04 ± 10.02
G3	0.115 ± 0.005	0.094 ± 0.007	77.96 ± 18.39
G4	0.100 ± 0.009**	0.081 ± 0.026**	98.85 ± 7.8*
G5	0.126 ± 0.012	0.116 ± 0.015	84.04 ± 7.50
G6	0.063 ± 0.009***	0.042 ± 0.015***	112.6 ± 6.88**
G7	0.137 ± 0.010	0.088 ± 0.015	86.65 ± 10.1
G8	0.071 ± 0.007***	0.030 ± 0.006***	123.3 ± 3.53***
G9	0.100 ± 0.016**	0.055 ± 0.009***	77.35 ± 16.7

Note: GSH: reduced glutathione; MDA: malondialdehyde; GPx: glutathione peroxidase. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (G1).

Histopathological findings

Testicular histopathology (Figure 5): The control group (G1) displayed normal architecture of adjacent seminiferous tubules surrounded by connective tissue, with intact germ cell layers (spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa) and visible interstitial Leydig cells (Grade 0).

The high-dose lambda-cyhalothrin group (G2) exhibited dilated and irregular seminiferous tubules, disorganized spermatogenic cells, absence of spermatozoa in certain tubular lumens, increased lumen diameter, and pycnotic nuclei with edematous interstitium (Grade 3). The low-dose lambda-cyhalothrin group (G3) showed germ cell hyperplasia with normal spermatozoa density in the tubular lumen (Grade 1).

The high-dose emamectin benzoate group (G4) displayed dilated and irregular tubules, degenerative changes with hyalinization, absence of spermatozoa in some tubules, and exfoliated spermatogenic cells in the lumen (Grade 3). The low-dose emamectin benzoate group (G5) retained normal architecture with adequate spermatozoa density (Grade 0).

The high-dose thiophanate-methyl group (G6) exhibited disorganized tubular epithelium, reduced spermatozoa density, and intertubular hemorrhage (Grade 2). The low-dose thiophanate-methyl group (G7) maintained organized spermatogenic cells with slight interstitial hemorrhage and pycnotic nuclei (Grade 1).

The high-dose mixture group (G8) showed the most severe changes: extensive germ cell degeneration, atrophy of most seminiferous tubules, a thin basal lamina, dilated and irregular tubules, increased lumen diameter, and intertubular hemorrhage (Grade 4). The low-dose mixture group (G9) displayed disrupted spermatogenic cell layers, luminal clogging with exfoliated necrotic cells, absence of spermatozoa in some tubules, and interstitial hemorrhage (Grade 2).

Epididymal histopathology (Figure 6): The control group (G1) showed normal epididymal tubule architecture, with intact epithelium surrounded by muscular tissue and numerous spermatozoa in the lumen (Grade 0).

In G2, many tubules lacked spermatozoa, and hyperplasia with destruction of some epithelia was observed (Grade 3). G3 displayed overall normal structure with reduced spermatozoa density (Grade 1).

G4 showed destruction of some germinal epithelial cells, enlarged interstitial space, and low sperm density (Grade 3). G5 exhibited normal architecture with low sperm density (Grade 1). G6 displayed destruction of some epididymal epithelia and low spermatozoa density (Grade 2). G7 demonstrated normal structure with moderate spermatozoa density (Grade 0–1).

G8 showed the most severe changes: disorganization, hyperplasia, and destruction of most epithelial cells, with most tubules devoid of spermatozoa (Grade 4). G9 exhibited partial epithelial destruction and reduced sperm density (Grade 2).

A summary of histopathological severity scores is presented in Table 5.

Table 5.

Semi-quantitative histopathological severity scores for testicular and epididymal tissues across experimental groups (Grades 0–4 scale).

Group	Treatment	Testicular score	Epididymal score
G1	Control	0	0
G2	Lambda-cyhalothrin 1/20 LD₅₀	3	3
G3	Lambda-cyhalothrin 1/60 LD₅₀	1	1
G4	Emamectin benzoate 1/20 LD₅₀	3	3
G5	Emamectin benzoate 1/60 LD₅₀	0	1
G6	Thiophanate-methyl 1/20 LD₅₀	2	2
G7	Thiophanate-methyl 1/60 LD₅₀	1	0–1
G8	Mixture 1/20 LD₅₀	4	4
G9	Mixture 1/60 LD₅₀	2	2

Discussion

Organ weight changes: Mechanisms and significance

Assessment of absolute organ weight is a key endpoint in toxicity studies, offering valuable insights into potential organ damage and overall health status (Adiguzel et al., 2024; Baali et al., 2023). In the present study, all three pesticides at the high dose significantly reduced testicular and/or epididymal weights, with the most pronounced effect observed in the ternary mixture group (G8). These findings are consistent with previous reports on lambda-cyhalothrin (Ahmed and Kandil, 2023; Oularbi et al., 2015; Pravallika et al., 2019), emamectin benzoate (Khaldoun et al., 2015), and thiophanate-methyl (Bououza et al., 2022).

The mechanisms underlying pesticide-induced reduction in reproductive organ weight are multifactorial. Decreased testosterone concentration, as observed in the present study, may directly impair the maintenance of testicular mass, as androgens are essential trophic factors for the seminiferous epithelium (Ahmed and Kandil, 2023). Furthermore, suppression of the hypothalamic–pituitary–gonadal (HPG) axis, resulting in decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion, may contribute to reduced gonadal weight (Alaa-Eldin et al., 2017; Mosbah et al., 2018). The histological evidence of seminiferous tubule shrinkage and germ cell depletion observed in the present study further supports the interpretation that reduced testicular weight reflects structural damage to the spermatogenic compartment (Abd-Elhakim et al., 2021). The reduced epididymal weight is likely a secondary consequence of diminished sperm production and reduced androgen-dependent secretory function (Chelbi et al., 2025).

The significantly greater testicular weight reduction observed in the mixture group G8 compared with the individual pesticide groups suggests a synergistic interaction. This is consistent with Zhang et al. (2020), who reported that inhalation exposure to a mixture of beta-cypermethrin and emamectin benzoate produced more pronounced changes in reproductive organ weight in male rats compared with individual exposures. The chemical reactivity, distinct toxicokinetics, and different mechanisms of action of the three pesticides in our mixture, a pyrethroid (sodium channel disruption), an avermectin (GABA receptor interference), and a benzimidazole (microtubule inhibition)—may converge on shared downstream pathways such as oxidative stress and HPG axis disruption, thereby amplifying the overall toxic effect (Amiour and Leghouchi, 2017; Hernández et al., 2013).

Testosterone suppression: Endocrine disruption pathways

The dose-dependent suppression of testosterone observed across treatment groups, with the most severe reduction in the mixture group G8 indicates endocrine disruption mediated through multiple pathways. One plausible mechanism is the down-regulation of steroidogenic acute regulatory protein (StAR) in Leydig cells, which is essential for cholesterol transport to the mitochondrial inner membrane and subsequent testosterone biosynthesis (Metwally et al., 2017). This process is regulated by the HPG axis: LH, secreted by the pituitary gland in response to gonadotropin-releasing hormone (GnRH), stimulates Leydig cell steroidogenesis, while FSH acts on Sertoli cells to support spermatogenesis (Ahmed and Kandil, 2023; Smith and Walker, 2014). Disruption of this axis at any level, hypothalamic, pituitary, or gonadal, may result in decreased testosterone output (Elnamaky et al., 2018).

Additionally, the oxidative damage to Leydig cells documented in the present study (elevated testicular MDA, depleted GSH) may directly impair steroidogenic capacity, as ROS-mediated damage to cellular membranes and mitochondria compromises the enzymatic machinery required for testosterone synthesis (Ahmed and Kandil, 2023). The histological observation of degenerative changes in Leydig cells in the high-dose groups supports this interpretation.

The finding that the low-dose mixture (G9) significantly reduced testosterone concentration, whereas low doses of individual pesticides did not, is particularly noteworthy, as it suggests that the mixture may lower the threshold for endocrine disruption. This could be attributed to increased stimulation of muscarinic receptors by acetylcholine in the testes, reducing steroidogenic activity in Leydig cells (Akande et al., 2024), or to the convergent disruption of the HPG axis by three distinct mechanisms simultaneously. This has important implications for human health, as decreased testosterone concentration in rodents correlates with reduced fertility and may predict reproductive risks in exposed human populations (Attia et al., 2012).

Impairment of sperm parameters: Dose-dependent and mixture effects

The significant reductions in sperm concentration, motility, and progressive speed observed in the high-dose groups, with consistently greater impairment in the mixture group, reflect the direct and indirect consequences of pesticide-induced testicular toxicity. The decrease in sperm concentration can be attributed to oxidative stress-mediated depletion of type A spermatogonia, which are essential for the proliferative phase of spermatogenesis (Mohamed et al., 2023). Pesticide-induced increases in ROS production damage testicular germ cells, leading to impaired spermatogenesis and reduced sperm output (Abedi et al., 2016; Lonare et al., 2016). Furthermore, oxidative stress induces DNA strand breaks, membrane lipid peroxidation, and apoptosis, all of which compromise overall sperm function (Lampiao et al., 2013).

The reduction in sperm motility observed particularly in the emamectin benzoate (G4) and G8 mixture group can be explained by several mechanisms. First, pesticide-induced cytotoxic effects may cause premature release of immature sperm from the germinal epithelium (Kenfack et al., 2015). Second, structural damage to the flagellum and midpiece—organelles essential for sperm propulsion, may directly impair motility (Kahalerras et al., 2022). Third, testosterone deficiency, as observed in our study, is known to compromise sperm function, since adequate intratesticular testosterone is required for normal spermatogenesis and sperm maturation (Abd-Elhakim et al., 2021; Mbouh et al., 2022).

The shift from progressive (medium-speed) to immobile sperm categories in the high-dose and mixture groups suggests that these pesticides disrupt mitochondrial function in the spermmid piece, inhibiting oxidative phosphorylation and glycolysis, which are the primary ATP-generating pathways powering sperm motility (Al-Ansary et al., 2016; Bouabdallah et al., 2022). The finding showed that even the low-dose mixture group (G9) exhibited significant increases in immobile sperm and decreased medium-speed sperm further supports the existence of a synergistic interaction at sub-toxic individual doses.

Oxidative stress: The convergent mechanism

The patterns of oxidative stress observed across tissues, decreased GSH and GPx activity accompanied by increased MDA, provide mechanistic insight into the reproductive toxicity of these pesticides. Pesticides are known to target mitochondria and disrupt cellular metabolism, leading to increased oxidative stress and impaired steroidogenesis, sperm quality, and reproductive organ function (Akande et al., 2024; Hussain et al., 2024).

The depletion of GSH observed in the present study likely reflects its increased consumption by antioxidant enzymes during the detoxification of pesticide-generated ROS. GSH serves as a critical substrate for GPx and other enzymes involved in xenobiotic detoxification (Zhang et al., 2020), and its depletion diminishes the cellular capacity to neutralize oxidative insults (Matuz et al., 2021). The concomitant decrease in GPx activity may be a direct consequence of GSH substrate limitation (Talbi et al., 2022), creating a vicious cycle: reduced GPx activity leads to accumulation of H₂O₂ and lipid peroxides, further depleting GSH and propagating oxidative damage (Ahmed and Kandil, 2023).

The elevation of MDA, a terminal product of lipid peroxidation, in the testicular and epididymal tissues of the high-dose groups, with the highest levels in the mixture group (G8), indicates extensive ROS-mediated membrane damage. Excessive ROS production reacts with membrane phospholipids, generating lipid peroxidation products such as 4-hydroxynonenal and MDA, which disrupt membrane integrity, alter receptor function, and compromise enzymatic activity (Su et al., 2019; Tekeli, 2023). The fact that the mixture group consistently showed among the highest MDA levels and lowest antioxidant defenses suggests that the convergence of three different pro-oxidant mechanisms overwhelmed the endogenous antioxidant system more effectively than any single pesticide.

Histopathological evidence of tissue damage

The histopathological alterations observed in the testes and epididymis corroborate the biochemical findings and provide visual evidence of the structural consequences of pesticide-induced toxicity. The severity of histopathological changes followed a clear dose-dependent pattern and was most pronounced in the high-dose mixture group (G8, Grade 4), which exhibited extensive germ cell degeneration, seminiferous tubule atrophy, and complete loss of spermatozoa in most lumens.

The degeneration of the seminiferous epithelium, characterized by disorganization and exfoliation of spermatogenic cells, reflects damage to the Sertoli cell–germ cell junctional complexes (Kobir et al., 2023). Sertoli cells provide structural and nutritional support essential for spermatogenesis (Pravallika et al., 2019), and their dysfunction leads to detachment of germ cells from the basement membrane (Said, 2019). Testosterone is required for maintaining the adhesion between Sertoli cells and germ cells within the seminiferous tubules; therefore, reduced intratesticular testosterone may directly contribute to germ cell exfoliation and subsequent apoptosis (Boukarine and Khelili, 2024; Metwally et al., 2017).

The epididymal alterations, epithelial destruction, reduced spermatozoa density, and interstitial changes, further reflect the downstream consequences of impaired spermatogenesis and altered androgen metabolism (Mohamed et al., 2023). The epididymis is androgen-dependent for its secretory and absorptive functions critical to sperm maturation and storage (Al Malahi et al., 2022), and pesticide-induced disruption of androgen receptor function may compromise these processes (Bal et al., 2012).

The consistent finding that the mixture groups (G8 and G9) exhibited more severe histopathological damage than the corresponding individual pesticide groups at the same dose level provides morphological evidence of synergistic toxicity. This is consistent with Oghenevwaire and Adeyemi (2025), who reported that simultaneous exposure to multiple pesticides led to more severe histopathological effects due to synergistic interactions. The fact that even the low-dose mixture group (G9, Grade 2) showed more extensive damage than some high-dose individual pesticide groups (e.g., G6 testes, Grade 2) further supports the mixture potentiation effect.

Our histopathological findings corroborate earlier reports of testicular damage in rats exposed to lambda-cyhalothrin (Al Malahi et al., 2022; Metwally et al., 2017; Mustafa and Jawad, 2023; Oularbi et al., 2015; Pravallika et al., 2019), emamectin benzoate (Khaldoun et al., 2015; Kumar, 2013), and thiophanate methyl (Bououza et al., 2022).

Novel contributions of the present study

The present study makes several distinct contributions to the existing literature on pesticide reproductive toxicology:

(1) First assessment of the ternary lambda-cyhalothrin/emamectin benzoate/thiophanate-methyl mixture: While previous studies have examined binary pesticide combinations, no prior study has investigated the reproductive toxicity of this specific three-compound mixture, despite their common co-application in eastern Algerian agriculture.

(2) Two-dose experimental design: Unlike earlier studies that relied on a single dose level (e.g., Ahmed and Kandil, 2023), the inclusion of two dose levels (1/20 and 1/60 LD₅₀) allowed dose-dependent trends to be examined and revealed mixture effects at the lower, individually sub-toxic dose; a broader range of doses would nonetheless be required to establish formal dose–response relationships.

(3) Integrated multi-parameter assessment: The simultaneous evaluation of organ weights, hormonal status, sperm parameters, oxidative stress markers, and histopathology within the same cohort provides a comprehensive and internally consistent picture of reproductive toxicity.

(4) Evidence of synergistic mixture toxicity: The mixture group exhibited the most severe impairment across all measured parameters, and importantly, the G9 low-dose mixture group produced significant effects on testosterone and sperm parameters that were not observed with any individual pesticide at the same low dose, providing evidence consistent with synergistic rather than merely additive interactions.

Study limitations

Several limitations should be acknowledged. First, the study did not measure serum FSH and LH levels, which would have provided direct evidence of HPG axis disruption. Second, the experimental design did not include binary mixture groups (two-pesticide combinations), which would have been informative for distinguishing pairwise interactions from ternary synergism. Third, the subchronic exposure duration of 6 weeks may not fully capture long-term or transgenerational reproductive effects. Fourth, the use of commercial formulations rather than pure active ingredients means that formulation adjuvants may have contributed to the observed toxicity. Finally, the semi-quantitative histopathological scoring, while providing standardized assessment, could be complemented by morphometric analysis and immunohistochemical markers (e.g., PCNA for proliferation, TUNEL for apoptosis) in future studies.

Future directions

Future studies should investigate the specific molecular pathways involved in the synergistic toxicity of this pesticide mixture, including gene expression analysis of steroidogenic enzymes (StAR, CYP11A1, 3β-HSD) and apoptotic markers (Bax, Bcl-2, caspase-3). Evaluation of HPG axis hormones (GnRH, FSH, LH) and inclusion of binary mixture groups would help elucidate the mechanistic basis of the observed synergism. Longer exposure durations and assessment of fertility outcomes (mating trials) would enhance the translational relevance. These data are essential for informing mixture-based risk assessment frameworks for agricultural pesticide regulation.

Conclusion

The present study demonstrated that 6 weeks of oral exposure to lambda-cyhalothrin, emamectin benzoate, and thiophanate-methyl, three pesticides commonly co-applied in eastern Algerian agriculture, produced significant dose-dependent reproductive toxicity in male Wistar rats. The ternary pesticide mixture at 1/20 LD₅₀ induced the most severe effects across all parameters: reduced testicular and epididymal weights, suppressed plasma testosterone, decreased sperm concentration, motility, and progressive speed, disrupted antioxidant defenses (depleted GSH and GPx, elevated MDA), and extensive testicular and epididymal histopathological damage. Critically, the low-dose mixture (1/60 LD₅₀) produced significant effects on testosterone concentration, sperm parameters, and oxidative stress markers that were not observed with any individual pesticide at the same dose, providing evidence consistent with synergistic toxicity. These findings highlight the inadequacy of single-compound risk assessment approaches and underscore the need for regulatory frameworks that account for the combined effects of pesticide mixtures to which agricultural workers and communities are routinely exposed.

Footnotes

Acknowledgment

The Animal Ecophysiology Research Laboratory supported this research. We would like to thank the head of the laboratory, Professor Cherif Abdennour, for his support.

ORCID iD

Narimene Chelbi

Ethical considerations

Animal experimentation was conducted in accordance with the guidelines approved by the Ethics Committee of the Directorate General of Scientific Research and Technological Development (DGRSDT), under the authority of the Algerian Minister of Higher Education in charge of scientific research, with approval number PNR/SF 08/201. The Animal Science Ethics Committee of the University Badji Mokhtar-Annaba (UBMA) also authorized the treatment of the animals. Animal treatment was performed in accordance with the International Guidelines for Laboratory Animal Care and Use (JO86/609/CEE) (Council of European Communities, 1986). This research study was conducted at the Animal Breeding Laboratory, Faculty of Sciences, Department of Biology, Badji Mokhtar Annaba University, Algeria.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors received financial support for laboratory fees from the Laboratory of Animal Ecophysiology, directed by Professor Cherif Abdennour, but not for authorship or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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