State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine
Abstract. Introduction. Objective is to identify hazard and asses reproductive toxicity risk of four generic lambda-cyhalothrin pesticides of different purity on male Wistar Han rats.
Methods. Lambda-cyhalothrin (LC1 — 97 %, LC2 — 96 %, LC3 — 97,1 %, LC4 — 96,7 % purity) was administered intragastrically on a daily basis, exceptfor Saturday and Sunday, at doses of 0,0; 0,3 and 3,0 mg/kg body weight for three groups of animals, 20 males in each, for 11 weeks. Control animals received an equivalent amount of solvent. In parallel with control and experimental animals, there were intact females, intended for mating. At the end of inoculation, functional parameters of the state of gonads and the ability of animals to reproduce offspring were studied. The state of reproductive function in intact females was taken into account at day 20 of pregnancy.
Results and discussion. The obtained data indicate that the exposure to four generic lambda-cyhalothrin substances at a dose of 3,0 mg/kg body weight for 11 weeks showed a general toxic effect (weight loss) in males under the effect of LC1. All four test substances have a reproductive toxicity at the maximum dose and have a pronounced antiandrogenic effect, which is manifested in changes in the morphological and functional parameters of the state of the gonads in the experimental groups of males. In addition, there was a negative effect on the fertility of males (LC2), which was concluded by the fertility index in intact females.
Conclusion. Based on the results obtained, it can be concluded that all studied lambda-cyhalothrin samples have reproductive toxicity at a dose of 3,0 mg/kg body weight. No observed effect level (NOEL) for all studies substances is the dose of 0,3 mg/kg body weight. In the range of studied doses, there is a dose-effect dependence.
Key words: pesticides, cyhalothrin, lambda-cyhalothrin, gonadotoxicity, reproductive toxicity.
Introduction. The widespread use of pesticides has led to an increase in health problems in humans and animals worldwide [1–7], because chemical plant protection products affect not only target objects but also non-targets, including the human body. Consequently, the problem of preventing the potentially dangerous effects of these substances on human health is one of the priority tasks of preventive toxicology. Study of potential reproductive toxicity of pesticides is an obligatory component during their toxicological and hygienic regulation.
Several decades ago, it was discovered that the reproductive toxicity of many pesticides is due to their ability to destructively affect endocrine regulation of the reproductive system of animals and humans, which leads to corresponding disorders of the reproductive function [6, 7].
As is known, endocrine disruptors include substances that penetrate production, storage, release, transport, binding, activity and elimination of natural circulating blood hormones, responsible for maintaining homeostasis and regulating the differentiation of vital processes [8]. The endocrine disparity properties of many pesticides are due to their structural similarity to natural hormones [9–11], penetrating the body and binding to nuclear receptors, they have a hormone-like effect that impairs homeostatic mechanisms of regulation of living organisms processes by endogenous hormones [12]. For example, pesticides of the class of pyrethroids, which include the pesticides studied by us and the use of which in recent years has increased several times [13,14,15], despite their neurotoxic properties, are endocrine disruptors.
Synthetic pyrethroids (SPs) is a unique group of chemicals that have pyrethrum-like structures with higher performance and make up over 30 % of the agents controlling harmful insects worldwide [16, 17]. Favourable properties of SPs, such as low-cost rates for crop processing and low volatility, have contributed to their widespread use in controlling harmful insects [18].
The functioning of the reproductive system is a unique, extremely complex, finely balanced process, which begins with the cushion, differentiation, growth and maturation of the germ cells and ends with reproduction of the offspring. Any negative effect on one of the links in this continuous chain of events can lead to impotence, menstrual disorders, fertility decline, spontaneous abortions, low birth weight and other developmental defects, including hereditary defects and various genetic diseases that affect the reproductive system and offspring [19]. Interaction and regulation of all reproductive processes are provided by the system, the centre of which is the neuroendocrine complex: hypothalamus-pituitary gland — gonads. Foetal reproductive system begins to form with the onset of embryogenesis, however, the structural and functional maturation does not end until the onset of puberty [20]. The effect of toxicants on the processes of the formation of the gonads and germinal cells in the early stages of embryonal development in the prenatal period can lead to irreversible changes in the genital organs and their functions at any time of the life cycle [21,22,23]. Malnutrition, low socioeconomic status, unhealthy lifestyle, stress, negative physical and chemical environmental factors in the postnatal period also lead to reproductive system abnormalities.
Mechanisms of toxic action of pesticides on mammal body are very diverse. As for pyrethroids, experimental studies have shown that one of the mechanisms of toxic effects of pyrethroids on gonads is the formation of a high level of free radicals in the process of metabolism, which cause oxidative stress in exposed animals [24, 25], which leads to a deterioration in the reproductive function of male rats. Spermatozoa are particularly susceptible to oxidative stress since their plasma membranes contain a high concentration of polyunsaturated fatty acids, and the cytoplasm contains low concentrations of antioxidant enzymes [26, 27]. These cells are naturally protected from such damage by the antioxidant properties of the seminal plasma, which contains a number of enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, etc.) and non-enzymatic antioxidants (taurine, hypotaurine, adrenaline, pyruvate, etc.), which can neutralise the potentially toxic effects of free radicals [28–30]. However, despite the well-developed antioxidant defence system, spermatozoa can be exposed to reactive oxygen species (ROC) through the action of toxic chemicals and environmental pollutants. In experiments of past years, the investigators described various structural changes in spermatozoa and their associated biochemical disorders in the testicles after the effects of SPs in vitro and in vivo such as cypermethrin, fenvalerate and lambda-cyhalothrin [31,32,33]. For example, the number of spermatozoa in testicles and appendages, as well as the daily production of sperm, were significantly reduced in male rats exposed to cypermethrin [34]. Yuan C. et al. showed that sperm motility was reduced in vitro 4 hours after incubation of Sprague-Dawley rat sperm with permethrin and cypermethrin [35]. These studies confirm the sensitivity of the plasma membrane of sperm, which is rich in polyunsaturated fatty acids, to the action of oxidative agents [36].
One of the widely used insecticides is lambda-cyhalothrin (LC) — type II SP [37,38,39]. By this time, the studies of the reproductive toxicity of the original LC molecule in a test of several generations have not been conducted. The only data we found was the study of the effect of lambda-cyhalothrin on the parameters of sperm in mice, which leads to damage to the morphology of semen [40, 41].
Evaluation of the risk of reproductive toxicity of LC was carried out on the basis of extrapolation of data obtained from an experiment in 3 generations of animals with cyhalothrin [40, 41]. The study of the effect of cyhalothrin was performed in rats in doses: 0.0; 0.67; 2.0 and 6.7 mg/kg body weight [42]. The reproductive toxicity in rats was not detected at a maximum dose tested. For the parent and offspring, no adverse effect level (NOAEL) of 2.0 mg/kg body weight has been established based on weight loss in adult animals and severe systemic toxicity for the young rats during lactation under exposure to the maximum dose (6.7 mg/kg body weight).
The objective of this work was to investigate the effect of four test compounds of LC (LC1, LC2, LC3 and LC4) on the reproductive function of male Wistar Han rats in a test system for studying gonadotoxicity, and also to compare the obtained data.
Materials and methods. The gonad and reproductive toxicity of four generic samples of synthetic pyrethroid lambda-cyhalothrin of different manufacturers was studied. The purity of the compounds studied was the following: LC1 – 97 %, LC2 – 96 %, LC3 – 97.1 %, LC4 – 96.7 %. The studies used male and female Wistar Han rats derived from the SPF nursery of the “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine, aged 5–6 weeks with a body weight of 80–100 g. Adaptation period was five days. During this time, daily observations were made of their physiological condition for the detection of signs of abnormalities. Rats were randomised by the body weight. In case of significant deviation from the average weight, animals were rejected. In parallel with control and experimental animals, there were intact females, intended for mating.
For each lambda-cyhalothrin test substance, the animals were divided into three groups of 20 male rats in each. Experimental animals were administered intragastrically a test substance ex tempore on a daily basis, except Saturday and Sunday, using a feeding tube, to three groups of animals at doses of 0.0; 0.3 and 3.0 mg/kg body weight for 11 weeks. The control group of males received distilled water with an emulsifier (OP-10) in equivalent amounts. The solution was administered at a rate of 0.5 mL per 100 grams of body weight of animals. To correct the volume of the administered solution in accordance with the increase in the body weight, animal were weekly weighed.
Animal studies have been conducted in accordance with the requirements and provisions of the Ethics Committee for Medical and Biological Studies of “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine and European Convention for the Protection of Animals used for Experimental and Other Scientific Purposes (Strasbourg, 18.03.1986) ETS No. 123, Guide for the Care and Use of Laboratory Animals (National Academies Press, USA, 2011) [43, 44].
Husbandry: the animals were placed in a conventional vivarium. The room was provided with forced ventilation (12 volumes per hour), which excluded air recirculation. The temperature and relative humidity of the air were recorded daily, temperature fluctuations ranged from 19 to 24 ºС, humidity — from 30 to 70 %. The illumination was natural.
Provision with water: the animals received deionised filtered drinking water disinfected with UV and purified by reverse osmosis ad libitum from plastic bottles in the volume of 0.5 L through metal tips.
Diet: throughout the experiment, rats received ad libitum balanced granular feed with low levels of natural phytoestrogens, Altromin (Germany).
Type of cages and number of animals in the cage: the animals were placed in cages of type M4 throughout the study period. The frame of the cage (40x30x15 cm) is made of a durable plastic material, top covered with metal demountable guards. The litter of sterilised, non-chlorinated paper was changed once a week.
Signs of general toxic action: all animals throughout the study period were examined in order to record any visible signs of reaction to the effect of the studied compound.
Body weight: experimental animals were weighed weekly throughout the exposure period. Intact females were weighed at day 0 and 20 of pregnancy.
Morphological and functional parameters of the condition of the testes: after the end of the exposure, 10 males, divided by weight, were selected from the experimental groups, and examined the morphological and functional condition of the gonads. The total spermatozoa count and the number of mobile semen, and the number of abnormal spermatozoa were determined. The calculation was carried out microscopically in 20 large squares of Goryaiev chamber.
Mating procedure: after the planned exposure period of LC, 10 experimental males from each group were coupled with intact females (in the ratio of 1 male to 2 females). Every morning during mating, vaginal smears were taken for each female and examined for the presence of spermatozoa. The day of finding sperm in the vaginal contents of the female was taken as day 0 of pregnancy. After establishing the fact of mating, the female was placed in a separate cage and taking smears was stopped. Duration of the mating period did not exceed 3 weeks.
Pre-mating interval: time elapsed from the moment the males were put to the females until the fertilisation was determined.
Parameters of reproductive ability: at day 20 of pregnancy in the control and intact females, coupled with experimental males, the following reproductive parameters were studied: total number of yellow bodies in the ovaries; number of implantation sites; the number of resorbed embryos and foetuses; the number of dead and living foetuses; presence of gross developmental abnormalities; average weight of foetuses; the total weight of the offspring.
Terminal studies: experimental males after the end of the mating period were sacrificed. All pregnant females were exposed to euthanasia at day 20 of pregnancy, and paired, but not pregnant females, at day 20 of expected pregnancy.
Females, in which during 3 weeks of mating the fact of conceiving has not been registered, were autopsied and examined in the absence of explicit signs of pregnancy. Euthanasia of animals was carried out using a CO2-chamber.
Gross examination: all autopsied experimental males underwent gross examination. Detected changes and deviations were registered. Testes and appendages from 10 experimental and control males were removed and weighed.
Mating ability and fertility: for each group and gender, the following indices were defined: mating, conception, fertility, pregnancy.
Statistical assessment: the statistical difference of between-group differences (P < 0.05) was evaluated according to Student’s t-test and calculated using two-way ANOVA.
Studies were performed in accordance with standard operating procedures of “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine, developed according to the recommendations and requirements of Good Laboratory Practice (GLP).
Results and discussion. The studied test substances of lambda-cyhalothrin in all studied doses did not affect the general physiological condition of the experimental males and did not cause their mortality.
In the group of males of the minimum dose of 0.3 mg/kg under the action of all four test substances, significant changes in body weight over time were not observed throughout the duration of the exposure. After exposure to a maximum dose of 3.0 mg/kg, LC2 and LC3 also did not show a general toxic effect. However, in the same group of males who received test substances LC1 and LC4, a probable decrease in the body weight of the experimental animals was noted in comparison with the control group: under the action of LC1 at week 10 and 11 of the experiment by 4.2 and 5.1 %, respectively, under LC4 at week 3 — 3.5 %. Also, a probable decrease in body weight at the exposure to LC4 at week 11 was observed between the doses of 0.3 mg/kg and 3.0 mg/kg — 4.5 %. Therefore, it has been established that LC1 has a general toxic effect under the action at the maximum dose tested (Fig. 1). Change in the body weight of the experimental males who received LC4 was found not to be related to the exposure.
No changes related to the effect of LC were found during gross examination of the internal organs in males. In all experimental groups of males in the study dose of 0.3 mg/kg, there were no significant changes in the weight and ratio of the weight of testes and appendages, the total number of spermatozoa, quantitative and percentage ratio of mobile spermatozoa, and pathological forms of semen, such as: abnormalities of the tail, head and middle (neck) of the spermatozoa.
Fig. 1. Changes of body weight of male rats over time in the period of lambda-cyhalothrin (ЛЦ) exposure (* – P ≤ 0.05; ** – P ≤ 0.01 – relative to animals in the control group)
During the study of morphological and functional parameters of the gonads in the experimental groups of males who received LC2, LC3 and LC4 at a dose of 3.0 mg/kg, a significant reduction in the total spermatozoa count by 12 %, 17 % and 12 %, respectively, has been established. Under the action of all studied substances, there was a significant decrease in the number of mobile spermatozoa: LC1 by 24 %, LC2 by 18 %, LC3 by 37 %, and LC4 by 22 %. At the same time, there was a probable decrease in the percentage of mobile spermatozoa by 16 % — LC1 and 24 % — LC3 (Fig. 2).
Gross examination of the testes and appendages did not found apparent abnormalities of these organs after the exposure to all samples of LC. However, in the group of males who received the maximum dose of LC1, a significant reduction in the weight of the testes by 4.9 % relative to the control group was noted. No significant changes in the weight of testes and appendages over time in this dose under the effects of other test compounds (LC2, LC3, and LC4) were observed (Fig. 3).
Fig. 2. General morphological and functional parameters of male semen (changes are expressed as a percentage of control) under the action of lambda-cyhalothrin (ЛЦ)
(* – P ≤ 0.05; ** – P ≤ 0.01; **** – Р ≤ 0.001 – relative to animals in the control group)
Fig. 3. General morphometric parameters of the testicles in males (* – P ≤ 0.05 – relative to the animals in the control group)
All LC samples at doses of 0.3 mg/kg and 3.0 mg/kg had no harmful effect in such parameters of the reproductive function of intact pregnant females, as the number of live foetuses in the experimental groups, the number of yellow bodies in the ovaries, the number and percentage of deaths before and after the implantation of the embryos and foetuses, as well as the average weight of foetuses and the total weight of offspring did not differ significantly from the value of the control group.
In the study of fertilising ability and fertility of males who received a dose of 3.0 mg/kg, which was evaluated according to the parameters derived from intact females, there was a tendency to decrease of conception and and fertility indexes, which reached the significance under the action of LC2: the conception index declined by 21 % and fertility by 25 %. In experimental groups of males at a dose of 0.3 mg/kg, fertilising ability and their fertility probably did not differ from the control (Fig. 4).
Fig. 4. The indices of mating, conception and fertility of intact females coupled to the experimental males (* – P ≤ 0.05 – relative to the animals in the control group).
The values of the pre-mating interval in the experimental groups did not significantly different from the control.
Conclusion
1. At intragastric administration of LC1 at a dose of 3.0 mg/kg body weight for 11 weeks, general toxic effect, namely, weight loss in males at week 10 and 11 is revealed.
2. All four test substances at a maximum dose have reproductive toxicity, which is expressed in the probable changes in the morphological and functional parameters of the state of gonads in the analysis of semen in the experimental groups of males, such as: decrease in the total number of spermatozoa (LC2, LC3, and LC4), a decrease in the number of mobile spermatozoa (LC1, LC2, LC3 and LC4), decrease in the percentage of mobile spermatozoa (LC1 and LC3), reduction in the absolute weight of the testes (LC1), and decrease in fertility and fertilising ability (LC2). The most toxic were LC1 and LC2 as per for gonad- and reproductive toxicity, and LC1 as per systemic toxicity. Therefore, there is no direct correlation between the severity of gonad- and systemic toxicity and the level of purity of the studied technical compounds. This fact allows us to assume that in this case, impurities, obviously, do not make a significant contribution to the induction of detected abnormal effects.
3. In the range of studied doses, there is a dose-effect dependence. It has been established that a dose level of 0.3 mg/kg body weight for all investigated compounds of LC does not exert a damaging effect on gonads and reproductive function (NOEL) of male Wistar Han rats.
4. The lack of changes in such parameters as the total number of the offspring, the number of live and dead foetuses, weight of the foetuses, the index of mating, conception, fertility, pregnancy in intact females, coupled with the experimental males (LC1, LC3, LC4) correlates with the results obtained in the study of cyhalothrin in the test system for 3 generations of animals, on the basis of which was concluded that there is no reproductive toxicity of cyhalothrin in the maximum dose of 6.7 mg/kg body weight [42]. However, this is the evidence of a lack of diagnostic sensitivity and informativeness of the test for 3 generations in the study of the reproductive toxicity of chemical compounds. The results of extensive studies in recent years suggest a high hazard of synthetic pyrethroids for reproductive toxicity that can break the processes of spermatogenesis and the endocrine function of the testes [24–37, 45, 46], which was very eloquently demonstrated by data we have obtained.
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