Occupational use of agrochemicals results in inhibited cholinesterase activity and altered reproductive hormone levels in male farmers from Buea, Cameroon

Abstract

The efficiency of agro pesticides and fertilizers in eliminating pests and scaling up crop yield has motivated farmers to increase their use. Unfortunately, health hazards caused on farmers by these agrochemicals are of growing concern, though not well elucidated. In order to evaluate the effects of occupational exposure to agrochemicals on some key parameters of male farmers’ health in Buea Subdivision, Cameroon, a total of 101 men, including 62 farmers using the agrochemicals and a reference population of 39 men not involved in occupational utilization of the agrochemicals, were interviewed on use of protective equipment, exposure symptoms and reproductive health status. Thereafter, serum cholinesterase [acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)] activities, total antioxidant capacity and reproductive hormones [follicle-stimulating hormone (FSH), luteinizing hormone and testosterone] were assessed. Results revealed that farmers mainly used insecticides followed by fungicides, herbicides and fertilizers, but with inadequate protective measures. The use of agrochemicals resulted in several exposure symptoms including weakness, itches, burning sensation, headache, sneezing, coughing and vomiting, as well as decrease in serum AChE activity when compared to the reference population. The agrochemicals impacted negatively on the farmers’ reproductive health as evidenced by increased FSH levels. Taken altogether, these results suggested that exposure to agrochemicals adversely affects farmers’ health. Therefore, there is a need to further sensitize the farmers on the use of protective equipment to mitigate the exposure and resulting health hazards.

Introduction

Agro pesticides and fertilizers are deliberately introduced into the environment to scale up crop yield. Agro pesticides eliminate or control pests that interfere with crop production and storage of food grains and kill vectors involved in disease transmission [1], while fertilizers enhance soil characteristics and increase growth/productivity of plants [2]. The efficiency of these agrochemicals in scaling up agricultural production has motivated their continuous use to improve farmers’ likelihood particularly in Cameroon and globally [3, 4]. Unfortunately, exposure to these chemicals results into altered health status in non-target organisms including humans. Improper handling of agrochemicals can result in contamination of the environment with highly ubiquitous and persistent residues of agro pesticides such as organochlorines and organophosphates. Other agro pesticides such as carbamates, pyrethroids, triazines and chlorophenoxys, and recently generated neonicotinoids, though relatively less persistent within the environment, have also been shown to exhibit toxicity in humans and other nontargeted animals. The use of agro pesticides therefore represents a health hazard to agricultural workers, especially in their occupational milieu [3, 5, 6, 7]. Exposure to fertilizers has been associated with dermatitis, which was generally related to chemicals found in the fertilizers such as ammonium nitrates, phosphates and heavy metals [8]. Nitrate ions may be reduced into nitrite ions and thereafter converted into nitrous anhydride. The latter generate nitrosonium ions, which in turn react with secondary amine to give nitrosamines with high toxicity/carcinogenicity [9]. Moreover, phosphates and the heavy metals from fertilizers may induce endocrine disruption and alteration of reproductive hormone levels [10, 11, 12].

Farmers can be exposed to agrochemicals during transport, distribution, mixing, loading, spraying/application and cleaning of spraying equipment, especially when there is inadequate knowledge and training on handling of agrochemicals, inappropriate use of personal protecting equipment (PPE), use of faulty equipment or disregard of laws and rules governing the distribution and use of agrochemicals [13, 14]. Agrochemicals are generally taken up into the farmers’ body dermally, orally, optically and/or by inhalation [15, 16]. The agrochemical residues occur in food commodities and other environmental matrices. The general population is therefore exposed every day to these chemicals, not only from direct contact with product items but also through the food chain, water consumption and other indirect environmental sources [17]. However, exposure levels are more likely elevated among farm workers who might also uptake substantial quantities of the chemicals while manipulating them directly. Exposure to agrochemicals may be evidenced by detection of the chemicals and related residues in biological fluids including blood or urine [18–20]. Mechanistically, agro pesticides such as carbamates and organophosphates induce their deleterious effects by inhibiting cholinesterase enzymes, which break down the neurotransmitter acetylcholine into choline and acetate, preventing uncontrolled muscle spasms, tremors and convulsion [21–23]. Some agro pesticides can act as endocrine disruptors and impair the male reproductive system through alteration of the levels of the hormones of the hypothalamus–pituitary–testicular (HPT) axis including follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone [4, 24]. Pesticides may as well exhibit their deleterious effects on reproductive function through oxidative stress, which has been reported to alter sperm quality [25, 26]. Heavy metals from fertilizers, particularly lead, can alter sperm quality through oxidative stress and DNA damage [27]. Heavy metals may also alter serum levels of gonadotropins and suppress testosterone leading to impaired male reproductive function [10, 11, 28].

Recent studies in Buea, Cameroon, have reported impairment of liver and kidney function in agro pesticide users [29]. Diarrhoea, stomach ache, abdominal pain, nausea and vomiting associated to consumption of vegetables contaminated with fertilizers have been also reported in Buea [30]. Although agrochemicals have been increasingly used in Buea Subdivision in Cameroon [29, 31], exposure to these chemicals and related effects on male farmers’ health have not been thoroughly elucidated. Therefore, this study aimed at investigating the impact of agrochemical use on the biochemical markers of exposure (cholinesterase activities) as well as reproductive hormone levels of male farmers in Buea Subdivision.

Materials and Methods

Chemicals

S-Acetyl thiocholine iodide (ACTI), S-Butyryl thiocholine iodide (BCTI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), (2,4,6-Tris (2-pyridyl)-S-Triazine)₂ (TPTZ), NaH₂PO₄•H₂O, Na₂HPO₄, sodium acetate, glacial acetic acid and hydrochloric acid were obtained from Sigma-Aldrich (Germany); ELISA kits for FSH (catalogue N° FS232F, lot N° FSS5182), LH (catalogue N°LH231F, Lot N° LHS5021) and testosterone (TES5238 catalogue N^o Te187S, Lot N^o AB2259) were purchased from CALBIOTECH, USA. All reagents used in this study were of analytical grade.

Study area and population

Many inhabitants in Buea Subdivision regularly use agrochemicals to enable increased production of food and cash crops. Among these inhabitants, 101 apparently healthy men aged 23-50 years and resident in Maumu, Muea, Bolifamba, Molyko, Great Soppo and Wututu, and who freely signed an informed consent, were involved in the study. The participants included 62 farmers, who were using agrochemicals in their farming activities. A group of 39 men living in the same neighbourhood, with similar age but not involved in occupational use of agrochemicals, was involved as a reference population. Men who had been operated around the genital as well as those who were currently suffering from a given disease and/or under medication were excluded from the study. The study protocol was approved by the Institutional Review Board of the University of Buea (Ref: 2017/005/UB/SG/IRB/FHS), and the administrative authorization obtained from the Regional Delegation of Public Health, South-West Region, Cameroon (Ref: R11/MINSANTE/SWR/RDPH/PS/186/720).

Questionnaire

Participants who met the inclusion criteria and volunteered to be included in the study were interviewed using a structured questionnaire. The interview focused on reproductive history, lifestyle with regards to smoking, and consumption of coffee/tea and alcohol. The information on participants’ occupation, use of agrochemicals, names of the agrochemicals used, habits during and after agrochemical handling/application, use of PPE, frequency of agrochemical application, storage and disposal of agrochemical waste/leftover solutions; types of crops cultivated by the participants; as well as symptoms of agrochemical exposure (burning sensation, redness of the skin, itches, lacrimation, coughing, sneezing, headache, dizziness, vomiting and diarrhoea) were also investigated.

Collection of biological samples

Five millilitres (5 mL) of fasting blood was collected from each participant between 6:00 am and 8:00 am. The blood was collected aseptically into sterile vacutainer tubes without anticoagulant, and serum separated by centrifugation (839 × g, at 4°C for 15 min) and stored at −20°C for further assessment of biochemical markers of exposure and male reproductive hormones.

Assessment of biochemical parameters in serum samples

Serum acetylcholinesterase and butyrylcholinesterase activities

Serum acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were determined spectrophotometrically using a modified Ellman’s method adapted to a microplate reader, described by Jońca et al. [32], with slight modification. Briefly, 200X dilutions of serum samples were prepared in sodium phosphate buffer (0.1 M, pH 7.4) and 50 μL of the dilutions transferred into wells of 96 well plate. A DTNB solution (2 mM) prepared in the phosphate buffer was added into the wells (50 μL/well) and the mixture preincubated for 10 min. Thereafter, 100 μL of 10 mM substrate (either ACTI for AChE, or BCTI for BuChE) was added and the absorbencies of the mixtures were read at 405 nm at 1 minute interval for 20 min. Cholinesterase (AChE and BuChE) activities were expressed in units per litre (U/L) using the following formula:

Cholinesterase activity = where, TV, LP, SV, Abs15min, Abs5min and ε represent the total volume of the reaction medium in the well plate (200 μL), the light path (0.622 cm), serum volume in the well (0.25 μL), absorbance at 15 min, absorbance at 5 min and molar extinction coefficient of TNB (13.6 M⁻¹ cm⁻¹), respectively. It should be noted that the curve Δ Abs = f (t) remained linear under these experimental conditions for 20 min. The blank consisted of all the reagents except that serum was replaced with the buffer, and change of absorbance that was related to auto hydrolysis of DNTB was subtracted from the calculated cholinesterase activity of the samples.

Serum levels of male reproductive hormones

Serum concentration of testosterone, follicle-stimulating hormone and luteinizing hormone was analysed by competitive ELISA, as per instructions from the manufacturer.

For testosterone, 50 μL of either testosterone standard (0–18 ng/mL) or serum sample was pipetted into appropriate wells coated with streptavidin (in a 96 well plate). Testosterone-enzyme conjugate (100 μL) and antitestosterone biotin reagent (50 μL) were added to all wells. The plate was incubated for 60 min, the wells were washed 3 times, a solution of 3,3′,5,5′-Tetramethylbenzidine (TMB) was added into each well (100 μL/well) and the plate was incubated for 30 min. A stop solution was thereafter added into the wells (50 μL/well), absorbencies recorded at 450 nm and serum testosterone levels extrapolated from the standard curve. A similar protocol was applied for LH and FSH using FSH/LH standard and serum levels of each gonadotropin were extrapolated from the corresponding standard curve.

Total antioxidant capacity assay

Serum total antioxidant capacity (TAC) was determined by the ferric reduction antioxidant power (FRAP) method [33]. Summarily, 5 μL of either FeSO₄.7H₂O standard solution (100–1000 μM) or serum sample was pipetted into 96 well plates containing 65 μL of acetate buffer (300 mM, pH 3.6). One hundred microlitres (100 μL) of freshly prepared FRAP reagent (mixture of 25 mL of acetate buffer, 2.5 mL of 10 mM TPTZ prepared in 40 mM HCl and 2.5 mL of 20 mM FeCl₃.6 H₂O) was added to each well and absorbencies were read at 595 nm after 4 min. Serum TAC values were extrapolated from the FeSO₄.6H₂O standard curve and expressed in mM Fe (II) eq.

Statistical analyses

Descriptive statistics were performed on qualitative data extracted from the questionnaire, and results presented as relative or absolute frequencies. Further statistical analyses were carried out using MedCalc software version 14.8.1.0. The frequencies between the two groups were compared using Fisher’s exact test. Biochemical parameters were tested for normal distribution using Kolmogorov–Smirnov test, and averages compared between the reference group and agrochemical users using either t-test or Welch t-test, when appropriate. A P-value < 0.05 was considered statistically significant.

Results

General characteristics of the participants

A total of 101 individuals aged 23–50 years were included in the study. These participants comprised of 62 farmers who were using agrochemicals for farming activities and 39 men not involved in occupational use of the agrochemicals. The general characteristics of the study population are summarized in Table 1.

Table 1.

General characteristics of the study population

Characteristics		Agro pesticide users	Reference population	P-value
Age		41.5 6.0 years	40.1 8.2 years	0.086 (t-test)
Marital status		Married: 47 (75.81%) Single: 15 (24.19%)	Married: 25 (64.10%) Single: 14 (35.89%)	0.214 (Fisher’s exact test)
Consumption of stimulants	Tobacco/cigarettes	Yes: 14 (22.58%) No: 48 (77.42%)	Yes: 5 (12.82%) No: 34 (87.18%)	0.298 (Fisher’s exact test)
		6.9 ± 14.9 cigarettes/week	5.2 ± 15.2 cigarettes/week	0.591 (Welch-test)
	Alcoholic drinks	Yes: 49 (79.03%) No: 13 (20.97%)	Yes: 31 (79.49%) No: 08 (20.51%)	1.000 (Fisher’s exact test)
		6.24 ± 10.7 bottles/week	7.8 ± 11.3 bottles/week	0.495 (Welch-test)
	Coffee	Yes: 14 (22.58%) No: 48 (77.42%)	Yes: 19 (48.7%) No: 20 (51.3%)	*0.016 (Fisher’s exact test)
		0.68 ± 1.42 cups/week	1.2 ± 2.0 cups/week	0.195 (Welch-test)

The values in bold represent absolute (before the brackets) and relative (within the brackets, in %) frequencies. *Significant difference from the reference population at P < 0.05.

The average age and frequency of cigarette smoking, and alcohol consumption were similar between the two groups. However, there were more participants taking coffee/tea in the reference group than among farmers (P = 0.016, Fischer’s exact test), though average quantity (number of cups) of coffee/tea remained similar between the two study groups (P = 0.195). Likewise, the alcohol consumption and cigarettes/tobacco smoking were not significantly different between the agrochemical users and the reference population.

Crops cultivated by the farmers

The farmers generally grew cash and food crops as shown in Table 2. The most cultivated crops were tomatoes and cocoa, while the least cultivated crop was plantain.

Table 2.

Crops grown by farmers

Crops cultivated	Number of farmers growing the crops	Frequency (%)
Tomatoes	60	97%
Vegetables	24	39%
Pepper	26	42%
Cocoa	29	47%
Maize	22	35%
Plantain	19	31%

Agrochemicals used by farmers and related symptoms of exposure

Agrochemicals used by farmers in Buea Subdivision to improve on crop production and eliminate weeds are summarized in Table 3. The most represented groups of agrochemicals used were insecticides (32.7%) and fungicides (29.1%), while the least represented was insecticide–nematicides (1.9%).

Table 3.

Agrochemicals used by farmers in Buea subdivision to improve crop production and eliminate weeds

Agrochemical group	Active substance(s)	Agrochemical formulation	Frequency (%)
			% Relative to all active ingredients	% of farmers
Fungicide (29.1%)	Carbendazim	Banko plus	0.1	1.6
	Chlorothalonil	Banko 720 SC, Bravo 720	1.0	12.9
	Copper	Kocide 2000, Callomil super, Fungicao, Fungi off, Nordox 75 WG, Ok-mill unik, Ridomil, Metrostar, Golden blue, Fungioff 720 WP, Metalm 72 WP	8.7	50.0
	Mancozeb	Baobab, Coga, Cozeb 80 WP, Cleansed blue 80WP, Ivory, Mancobex 80WP, Mancomax, Mancostar, Mancozan, Monchamp, Penncozeb	6.6	56.5
	Maneb	Plantineb 80WP, Trimangol	4.6	56.5
	Mefenoxam (Metalaxyl-M)	Ridomil, Ok-mill	4.8	43.5
	Metalaxyl	Callomil plus, Monchamp, Metalm 72 WP, Fungi off 720 WP	2.8	38.7
	Methyl thiophanate	Metrostar	0.2	3.2
	Sulphur	Metrostar	0.2	3.2
Herbicide (19.1%)	2,4-Dichlorophenoxyacetic acid (2,4-D)	Decat-D 720, Amistar	0.3	5
	Glufosinate ammonium	Cyclone 200SL	0.1	2
	Glyphosate	Glyphader, Herbistar, Roundup, Kalach 360 SL, Labada 480 SL, Cleanfarm 360 SL, Glycot, Glyfomax, Finish	12.3	97
	Paraquat	Calloxone Super, Gramoxone	6.3	92
Insecticide (32.7%)	Abamectin	Acarius	0.9	13
	Acetamiprid	K-optimal, Pacha 25 EC, Supercot 35EC	2.1	29
	Alphamethrin	Alphacyga	0.2	3
	Chlorpyrifos	Nurelle D 20/200EC	0.1	2
	Chlorpyriphos-ethyl	Pyriforce, Dusban 4E	2.2	31
	Cypermethrin	Cypercot, Cypalm, Cypercal, Nurelle D 20/200EC, Somethrine 100 EC, Cigogne	7.7	73
	Dimethoate	Magic force	0.2	3
	Emamectin benzoate	Caiman B 50 WG, Emacot	1.2	18
	Fipronil	Capsidor 50 SC, Fixe 50EC	0.5	8
	Imidacloprid	Gamalin 80 EC, Iron 30 SC, Lamida Gold, Parastar, Mamira super 90EC, Dinacacao, Imida30 EC	8.0	71
	Lambda–Cyhalothrin	Supercot 35 EC, Lamida Gold, Mamira super 90EC, Gamalin 80 EC, K-optimal, Magic force, Pacha 25 EC, Parastar	9.4	79
	Temephos	Bastos 500 EC	0.1	2
	Thiamethoxam 240 g/L	Actara	0.1	2
Insecticide–nematicide (1.9%)	Oxamyl	Bastion Super, Vydate L	1.7	24.2
	Terbufos	Counter 15 FC	0.1	1.6
Nematicide (5.1%)	Ethoprophos	Mocap 15G	5.1	75.8
Fertilizers (12.2%)	Nitrogen, phosphorus, and potassium (NPK)	Agrovert (NPK 11:46:14 + OE), Fertivert (NPK 12:24:46 + OE), Cropmax,(OE) D. I. Grow (NPK 1.85:1.85:3.31 + OE), Codafol (NPK 14:6:5+ OE), Harvest more (NPK 10:4510 + OE; NPK 30:10:10 + OE; NPK 20:20:20 + OE), Solupotasse (NPK 0:0:100), Super Gro (OE), Bafoliar (NPK 12:24:46 + OE; NPK 11:46:14 + OE) NPK 20:10:10, NPK 30:10:10, and NPK 05:05:45	5.1	75.8
5	30	/	100	/

N = Nitrogen, P = Phosphorus, K = Potassium and OE = Oligo elements (trace elements or biostimulants).

Generally, the farmers had different experiences with regards to knowledge on agrochemicals, source of information on preparation and application of the agrochemicals, and habits during handling of agrochemicals (Table 4).

Table 4.

Knowledge on agrochemicals, source of information and eating/drinking habits during agrochemical application

Use of agrochemicals	Description	Frequency of agro pesticide users
Duration of agrochemical use	5 years	8 (12.90%)
	From 6 to 10 years	20 (32.26%)
	> 10 years	33 (53.2)
Knowledge on banned agrochemicals	/	28 (45.12%)
Purchase of agrochemicals	Road side vendors	49 (79.03%)
	Licensed retailers	34 (54.84%)
Source of training on agrochemical use	Seminars	19 (30.65%)
	Farming union	13 (20.97%)
	Neighbour’s advice	33 (53.23%)
	Personal knowledge	53 (85.48%)
Habits during application of pesticides	Eat, drink or smoke during application of agropesticides	16 (25.81%)

Values in parentheses are percentages (%) description of agro pesticide users.

Table 5.

Correlation between cholinesterase and reproductive hormones

		Hormone
Cholinesterase	Group	FSH	LH	Testosterone
AChE	Agrochemical users	r = −0.03235 P = 0.8029	r = −0.1011 P = 0.4342	r = 0.3095 *P = 0.0144
	Reference population	r = −0.04170 P = 0.8037	r = −0.1744 P = 0.2884	r = 0.01360 P = 0.9345
	All participants	r = −0.5162 P = 0.6100	r = −0.06801 P = 0.4992	r = 0.2111 *P = 0.0341
BuChE	Agrochemical users	r = 0.02989 P = 0.8170	r = −0.07309 P = 0.5724	r = 0.2437 P = 0.0563
	Reference population	r = 0.05274 P = 0.7532	r = −0.1172 P = 0.4772	r = 0.04437 P = 0.7889
	All participants	r = 0.01374 P = 0.6410	r = −0.04695 P = 0.6410	r = 0.2569 *P = 0.0095

AChE: acetylcholinesterase; BuChE: butyrylcholinesterase; r = correlation coefficient.

Correlation is significant at ^*P < 0.05 level (two-tailed).

Agrochemical users also had different experiences in terms of use of personal protective equipment (PPE). Twenty-four per cent (24%) of farmers reported that they did not use any PPE during application of agrochemicals. Also, 45% farmers used only one PPE: either eye glasses, gloves, hat, gas mask, raincoat or rubber boots but not a combination of the protective tools (Fig. 1A). The most represented protective equipment was rubber boots (72.6%), while the least represented were hat, raincoat and gloves (8.1–11.3%) (Fig. 1B).

Figure 1.

Use of personal protective equipment by farmers when applying agrochemicals on crops (A & B), and self-reported symptoms of exposure to the agrochemicals (C). PPE: Personal protective equipment.

The farmers also reported symptoms that generally occurred on them during or after agrochemical use/handling. As shown in Figure 1C, 73% farmers reported having felt burning sensations as a result of agrochemical use. Other exposure symptoms reported by 22–30% of farmers included dizziness, sneezing, coughing and itches, while vomiting and painful eyes were least represented (11.3%).

The outcome of interview reports on general and reproductive health status of the participants is summarized in Figure 2. The most represented reproductive difficulties were sexual weakness and fatigue, while the least was premature ejaculation. Agrochemical users generally reported more symptoms of reproductive dysfunction when compared to the reference population though this difference was not statistically significant (P = 0.44, chi-squared test).

Figure 2.

Self-reported symptoms of reproductive dysfunction among male farmers.

Biochemical parameters

Cholinesterase activities

The farmers had significantly lower activities of AChE (P = 0.048) when compared to the reference population (Fig. 3). The average BuChE activity was also reduced by 3% in the agrochemical users though not statistically significant (P = 0.620).

Figure 3.

Serum acetylcholinesterase (A) and butyrylcholinesterase (B) activities in agrochemical users and reference population. AChE: Acetylcholinesterase; BuChE: Butyrylcholinesterase; Ref. population: Reference population. Significant difference from the reference population at ^*P < 0.05 (Welch t-test).

Serum total antioxidant capacity

Assessment of serum TAC through FRAP assay did not reveal any significant difference between agrochemical users and the reference population, as shown in Figure 4.

Figure 4.

Serum total antioxidant capacity or FRAP value in a reference population and agrochemical users. FRAP: Ferric reduction antioxidant power; Ref. population: Reference population.

Serum levels of gonadotropins and testosterone levels

Concentrations of FSH, LH and testosterone were measured in serum of study participants and results presented in Figure 5. There was significant increase (P = 0.048) in serum FSH levels in agrochemical users when compared to the reference population, while LH and testosterone were not affected.

Figure 5.

Serum concentration of follicle-stimulating hormone (A), luteinizing hormone (B) and testosterone (C) in agrochemical users and reference population. FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; Ref. population: Reference population. Significant difference from the reference population at ^*P < 0.05 (Welch t-test).

Correlation between cholinesterase and reproductive hormones

Correlation analyses with data from all participants (Table 5) revealed a significant positive association (P < 0.05) between cholinesterase activities (AChE and BuChE) and testosterone. However, when data were dichotomized based on agrochemical use, testosterone correlated positively and significantly with AChE levels in agrochemical users only.

Discussion

This study was carried out to evaluate the effects of occupational exposure to agro pesticides and fertilizers on male farmers’ health in Buea Subdivision. It was a cross-sectional study involving farmers using agrochemicals and nonusers of agrochemicals (reference population), all living in the same neighbourhood. Both study groups were similar in terms of age and consumption of alcoholic drinks, whereas more participants from the reference population reported higher frequency of consumption of coffee. However, it is worth mentioning that findings related to the effect of coffee or caffeine intake on male reproductive function are inconsistent and inconclusive. While some authors did not report any significant effect of coffee/caffeine consumption on reproductive hormone levels (inhibin B and FSH) and sperm quality (motility and morphology) [34], others reported an increased risk of low sperm concentration with the increased quantity of coffee drank per day [35]. By contrast, other findings indicate that coffee consumption is associated with favourable profiles of male reproductive function biomarkers including increased testosterone levels and percentage of motile sperm [36, 37]. These inconsistencies make findings on adverse effects of coffee/tea consumption on male reproductive function inconclusive [38].

The farmers used 5 groups of agro pesticides containing 30 active ingredients, which were available in 85 different formulations (Table 3). Insecticides and fungicides were the most frequently used agro pesticides, suggesting that crops grown in Buea Subdivision were mostly affected by insect pests and fungal pathogens, thereby motivating intensive use of the insecticides and fungicides by farmers to increase their likelihood [29, 39].

The government of Cameroon provided technical assistance to farmers in the 1980s on the correct utilization of agrochemicals through the Marketing Board for Commodities [40, 41]. The economic crisis at the beginning of the 1990s led to the suppression of subventions allocated to the farmers and withdrawal of technical assistance with regards to purchase, distribution and application of agrochemicals. Consequently, the agricultural sector was liberalized [40], resulting into more involvement of private importers and distributors of agrochemicals. The liberalization of the agricultural sector in Cameroon thus makes agrochemicals more accessible to farmers and may justify the large number of agrochemicals reported being used in this study.

The farmers generally lack adequate knowledge and information on the use of agrochemicals as suggested by the use of banned/restricted agro pesticides. Despite government restriction on the use of agrochemicals containing the active principles such as metalaxyl and dimethoate [42], these active ingredients were still used by 38.7% and 3.0% of the farmers, respectively. Metalaxyl was used mainly in Callomil plus, by 32.3% farmers but also occurred in 3 other agro pesticide formulations including Monchamp, Metalm and Fungi-off (8.1–1.6% farmers) while dimethoate was used in the pesticide formulation “Magic force” only. Considering that restriction on agro pesticides is usually motivated by high toxicity associated with such products in nontarget species, the continuous use of the restricted agrochemicals observed in this study implies that the farmers lacked information or awareness on agro pesticide regulation. In fact, the vast majority of farmers (53–85%) either relied on neighbour’s knowledge or depended on their personal know-how for pesticide acquisition/choice and application, while only a minority (8–30%) either attended training and seminars on agro pesticide use and had documentation educating them on the use and handling of agrochemicals. In addition, the farmers generally poured leftover diluted agro pesticide solutions on the soil and disposed empty containers of agrochemicals in the farm. This poor handling of agrochemicals and related residues, as well as improper disposal of empty containers, was also consistent with farmers’ unawareness on agrochemical toxicity and may favour environmental contamination by the pollutants [43]. Contamination of the environmental matrices with residues of agrochemicals is of particular concern for highly environmentally persistent agro pesticides such as organochlorines and organophosphates. Other agro pesticides used by the farmers, such as carbamates (e.g., maneb, mancozeb) and pyrethroids, and recently generated neonicotinoids, though relatively less persistent within the environment, have also been toxic to humans and other animals. Therefore, the risk of exposure of humans and other nontargeted species to these toxic agro pesticide residues will unavoidably increase with their spillage into environmental matrices [5, 44]. Heavy metals in chemical fertilizers are highly resistant to physical, chemical and biotic degradation. These metals can be leached into water sources, absorbed by plants or biomagnified in animals hence contaminating food web [45].

Although appropriate use of PPEs is known to lower the risk of occupational exposure to agrochemicals, results from this study revealed that farmers did not adequately use the PPEs. Over 70% farmers used a maximum of 1 PPE, while 1/4 of them were not using any PPE when applying agrochemicals on crops. The efficiency of PPE in lowering exposure to agro pesticides is when the entire body is protected. Existing PPEs were generally not adequate with only some parts of the body being protected. This observation suggests that the farmers had contact with the agrochemicals (powder, mist or solutions) that could be absorbed through unprotected areas of the body resulting in occupational exposure to the chemicals. This was consistent with the self-reported cholinergic symptoms of exposure among farmers, such headache, dizziness and vomiting, which have been reported in previous studies [4, 29, 46]. Indeed, nonadherence to the use of PPEs during agro pesticides use has been shown to increase the risk of exposure to the agrochemicals among farm workers [14]. The symptoms of acute exposure are easily identified, while chronic exposure generally contributes to the development of chronic agrochemical-related illnesses and results in alteration of several biochemical parameters [12] such as inhibition of cholinesterase enzymes and reproductive hormones.

Inhibition of AChE and BuChE (also known as pseudocholinesterase) has been widely acknowledged as biomarkers of exposure to agro pesticides such as organophosphates and carbamates, which are known as inhibitors of the latter enzymes [14, 21, 22, 44]. The inhibition of AChE activity observed in the farmers using agrochemicals corroborated the studies of Miranda-Contreras et al. [47], who evaluated occupational exposure to organophosphates and carbamates in Venezuela farm workers, as well as with studies carried out by Tetteh et al. [48] and Thetkathuek et al. [49] in Ghana and Thailand, respectively. These organophosphate pesticides include chlorpyrifos, which was used by the farmers and whose cholinesterase-inhibiting effect has been reported [22]. Other pesticides applied by the farmers that have been shown to inhibit AChE are the herbicide active principles paraquat and glyphosate [50] and the pyrethroid insecticide cypermethrin [51]. It is worth mentioning that Miranda-Contreras et al. [47] reported a significant decrease of both AChE and BuChE among agro pesticide users compared to a control group, while the current study revealed significant decrease of AChE only (BuChE was not altered significantly; i.e. P = 0.61). This discrepancy might be related to variability in the efficiency of the enzymes involved in detoxification/metabolism of the cholinesterase-inhibiting pesticides, between the workers from Venezuela and farmers from Buea. In fact, previous studies showed that some individuals appear to have a higher susceptibility to xenobiotics due to the presence of genetic polymorphism that influences the metabolism of the agrochemicals [7, 12, 44, 52]. Difference in agrochemicals applied, type of PPE used and duration of pesticide application might also influence exposure levels among pesticide users between the two studies. The poor inhibitory activity of the agrochemicals towards BuChE could be explained by hydrophobic interactions, which are more prominent in BuChE when compared to AChE [53]. However, AChE inhibition is more sensitive than BuChE in the case of chronic exposure to organophosphates, whereas pseudocholinesterase inhibition does not reflect the biological effects of the organophosphates in the nervous system [23]. Therefore, the altered AChE activity in the farmers does not only suggest exposure to agrochemicals but also indicate that farmers may suffer from neurological disorders following exposure to the chemicals [6, 44]. Decrease in AChE activity has been reported in fish and amphibians exposed to industrial effluent and urea, respectively [54, 55]. The heavy metals manganese and cadmium have also been associated with decrease in AChE [50, 56].

The reproductive system in males is responsible for production, maturation and transfer of sperms into the female reproductive tract through a series of complex biochemical reactions controlled and regulated by enzymes and hormones. Pulsatile release of gonadotropin-releasing hormone from the hypothalamus stimulates the anterior pituitary gland, which secrets LH and FSH. LH stimulates the Leydig cells in the testes to carry out steroidogenesis including testosterone synthesis, while FSH stimulates testicular Sertoli cells to carry out spermatogenesis [1]. Sertoli cells also produce activin, inhibin B and follistatin, which through feedback mechanisms modulate FSH secretion by the anterior pituitary. Testosterone stimulates spermatogenesis and regulates the hypothalamus and the anterior pituitary gland on the release of GnRH and LH through a negative feedback mechanism [1, 24]. Exposure to agro pesticides has been shown to impair reproductive function through alteration of the levels of these reproductive hormones and/or induction of oxidative stress [4, 25, 26, 57]. In the current study, agrochemical users reported more frequent symptoms of reproductive dysfunction such as premature ejaculation, painful testes, sexual weakness and inability to erect. These symptoms were consistent with significant elevations in serum levels of FSH (P < 0.05) in the agro pesticide users. Likewise, elevated serum levels of FSH were reported in male farm workers occupationally exposed to agro pesticides in Venezuela and Egypt [47, 57]. The increased FSH level might be attributed to the active ingredients carbendazim, chlorpyrifos, cypermethrin, 2,4-dichlorophenoxyacetic acid and acetamiprid [58–62], used by the farmers and whose FSH-stimulatory effect has been reported. Moreover, fertilizers are well known as sources of heavy metals [63], such as lead, which may affect endocrine glands in the farmers, leading to elevated levels of gonadotropins [28, 64]. The observed elevated FSH levels in agrochemical users suggest deregulation of the hypothalamus–pituitary–testicular (HPT) axis in farmers and particularly impaired spermatogenesis, as correlation between elevated serum FSH levels and spermatogenesis impairment is well documented [57].

Serum LH and testosterone levels in farmers remained unaltered, a finding similar to that reported by Abdallah et al. [57]. However, the testosterone levels correlated positively with cholinesterase activities in the overall study population and/or among pesticide users. This correlation corroborated with previously reported increased cholinesterase activities in “levator ani” muscle of castrated rats under testosterone supplementation, probably attributed to direct anabolic action of testosterone on rat muscle [65]. By contrast, Durrant et al. [66] and Al-Aboudi et al. [53] observed an elevated activity of serum BuChE in orchidectomized mice and negative modulation of BuChE by testosterone in vitro, respectively. Despite these discrepancies, the significant association of testosterone with AChE activity, which persisted only in the farmers following dichotomization based on agrochemical use, suggests a differential modulation of the enzyme in the two study populations.

Exposure to agrochemicals has been reported to generate excess reactive oxygen species (ROS) [67]. Elevated ROS level alongside decreased antioxidant defence is known as oxidative stress and has been reported to induce oxidative damage to reproductive tissues/cells, which may result in decrease in male fertility [25, 26, 44]. However, assessment of the antioxidant status in this study through serum TAC did not reveal oxidative stress in the agrochemical users. An increase in the levels of this biomarker is usually associated with the depletion of the biological antioxidant potential [44]. Nonsignificant variation in the oxidative stress biomarker in this study carried out in men might be related to the gender, as findings from Costa et al. [6] suggested that women have a better response to oxidative damage from exposure to pesticides. The observation was similar to findings by Zepeda-Arce et al. [67], who evaluated oxidative stress and genetic damage among workers occupationally exposed to pesticides in Mexico.

Despite significant contribution of the data herein generated towards elucidation of agrochemical toxicity in farm workers, some shortcomings worth mentioning arose and may be addressed in subsequent investigations. The general population is exposed to agrochemicals through food, water, etc. [3, 17], and metabolism/detoxification of the xenobiotics is influenced by genetic polymorphism among individuals [7, 12, 44]. Therefore, genetic polymorphism assessment and determination of pesticide residues in the study population might enable better delineation of the effect of the agrochemicals among farmers.

Conclusion

Overall, results from this study suggested that farmers in Buea Subdivision were exposed to agrochemicals, which induced several symptoms and altered serum activity of AChE. Agrochemical exposure also resulted in alterations of serum FSH levels suggesting impaired reproductive function in the farmers. These results further stressed the endocrine disrupting potentials of agrochemicals, suggesting the need to sensitize the farmers on importance of PPEs in alleviation/mitigation of the exposure to the agrochemicals and related toxicity.

Conflicts of Interest

The authors declare no conflict of interest.