The impact of neonicotinoid pesticides on reproductive health

Abstract

Neonicotinoids are some of the most widely used insecticides in the world because they broadly target chewing and sucking insects. Neonicotinoids are used in commercial agricultural systems, sold for use in home gardens, and found in veterinary pharmaceuticals in the form of flea and tick preventatives for companion animals. They are also used as crop seed treatments and spread throughout crops as they mature. As a result, humans, wildlife, livestock, and pets are routinely exposed to neonicotinoids through the consumption of contaminated food and water as well as through the use of some veterinary pharmaceuticals. Although several studies indicate that neonicotinoid exposure causes genotoxicity, neurotoxicity, hepatotoxicity, and immunotoxicity in some non-target species, the impact of neonicotinoid pesticides on the male and female reproductive systems in mammals is largely understudied. This review summarizes current insights on the impact of common neonicotinoid pesticides such as acetamiprid, clothianidin, imidacloprid, and thiacloprid on male and female reproductive health in mammals. The review also summarizes the impacts of exposure to mixtures of neonicotinoids on reproductive endpoints. In addition, this review highlights where gaps in research on neonicotinoid pesticides and reproductive health exist so that future studies can be designed to fill current gaps in knowledge.

The history of pesticides is famously documented in “Silent Spring” by the environmental conservationist, Rachel Carson. Silent Spring thoroughly details the distribution of synthetic pesticides such as organochlorine pesticides (Carson 2002). Organochlorine pesticides were used as contact pesticides, which were topically applied to prevent the destruction of crops by unwanted pests. However, pesticides easily washed off by rain would runoff into groundwater and river systems or they would sit in the soil, lingering well past their initial application. Virtually all human beings and wildlife species have had contact with pesticides at some point in their lifetime. As Carson states, “They have entered and lodged in the bodies of fish, birds, reptiles, and domestic and wild animals so universally” that scientists find it hard to find organisms free from the touch of pesticides.

Synthetic pesticides are used globally to assist in food production. Pesticides are intended to be highly toxic for target species through mechanisms that should not impact the health of humans and other non-target species. However, pesticides exert toxicity when non-target species are unintentionally exposed to them (Mörtl et al. 2020). Some of the most memorable passages of Silent Spring describe the exposure of avian species to dichlorodiphenyltrichloroethane (DDT) and its metabolites. The bioaccumulation of DDT throughout the food chain negatively impacted the reproductive capabilities of birds, altering calcium metabolism in a way that resulted in thin eggshells. The thin eggshells fell apart and were unable to support the mass of the incubating bird they held. This led to a massive population loss of various avian species by the 1980s. Nesting colonies of grebes, an aquatic diving bird, dramatically decreased from more than 1,000 pairs to only 30 pairs after the application of DDT in Clear Lake, California (Carson 2002). In a similar fashion, we have recently witnessed the massive collapse of many bee colonies, though this is attributed to a different class of pesticides: neonicotinoids (Mörtl et al. 2020).

Neonicotinoid pesticides are nicotine derivatives that target nicotinic acetylcholine receptors (nAChRs) in the nervous system (Matsuda et al. 2020). This class of pesticides is marketed as a replacement for its predecessors. Neonicotinoid pesticides are used in large-scale agricultural systems, sold for private residential use, and are found in veterinary pharmaceuticals such as flea and tick preventatives. Neonicotinoids are highly water soluble compared with other pesticides. Neonicotinoids are readily absorbed throughout the plant, much unlike contact pesticides. When absorbed, the plant itself—from its nectar, pollen, leaves, stems, and fruit—contains the neonicotinoid pesticide. The neonicotinoids that are not absorbed by the plant remain in the soil and water. The prophylactic and often excessive use of neonicotinoids presents a tremendous potential for environmental accumulation and chronic exposure of non-target species, especially humans.

Neonicotinoids can act on nAChRs or act on interneuron synapses (Thany 2011). The binding affinity of neonicotinoids to invertebrate nACHRs is variable. For example, the American cockroach expresses 2 types of receptors that bind neonicotinoids: nAChR1 and nAChR2. Imidacloprid (IMI) binds to nAChR1, but not nAChR2, as an agonist. However, acetamiprid (ACE) and clothianidin (CLO) act as agonists of nAChR2 (Thany 2011; Bodereau-Dubois et al. 2012; Calas-List et al. 2013). Thiamethoxam (THM) is a poor agonist of nAChRs, but acts as a full agonist of cercal afferent/giant interneuron synapses (Thany 2011). Thiacloprid (THD) is a partial agonist of the Pameα7 receptor found in cockroaches (Cartereau et al. 2021). It is hypothesized that neonicotinoids act similarly in non-target species such as humans, however, the mechanisms of action in human cells are not fully understood.

Many studies demonstrate that neonicotinoid exposure causes adverse effects in mammals and other non-target species. Specifically, neonicotinoids have been shown to cause genotoxicity, neurotoxicity, hepatotoxicity, nephrotoxicity, and immunotoxicity (Wang et al. 2018; Zhao et al. 2020). However, the impact of neonicotinoid pesticides on male and female reproductive health is largely understudied. This review summarizes current insights on the impact of common neonicotinoid pesticides such as ACE, CLO, IMI, and THD on male and female reproductive health in mammals. In addition, this review highlights where gaps in research on neonicotinoid pesticides and reproductive health exist so that future studies can be designed to fill current gaps in knowledge.

Materials and methods

A PubMed search was conducted to identify relevant studies on the effects neonicotinoid pesticides on male and female reproductive health. Topics of interest included those related to reproductive outcomes in non-target mammalian species. Keywords searched included neonicotinoids, reproduction, infertility, puberty, pregnancy, ovary, testes, hypothalamus, sperm, and sex steroid hormones. Using the described criteria, an extensive inventory of studies was further refined and chosen based on reproductive outcomes being assessed in studies and relevance to known papers examining the impact of neonicotinoid exposure on reproductive outcomes. The evidence was then synthesized by neonicotinoid pesticide and summarized. Epidemiological studies presenting exposures to neonicotinoid mixtures were summarized in their own category.

Acetamiprid

ACE is used to eliminate pests on fruits, vegetables, cotton, and ornamental plants and flowers in the United States. Approximately 0.55 pounds of ACE are applied per acre per season. People and animals are commonly exposed to ACE via dietary exposure (US EPA 2002) (Table 1). ACE has a maximum acceptable daily intake (ADI) of 0.07 mg/kg bw (FAO 2017) (Table 1). The Joint FAO/WHO Meeting on Pesticide Residues (JMPR) reported the lowest observed adverse effect level (LOAEL) for ACE is 51.0 mg/kg bw/d in male rats and 60.1 mg/kg bw/d in female rats (US EPA 2002). ACE at the LOAEL decreased litter size and delayed preputial separation and vaginal opening in rats (US EPA 2002; FAO 2017). In 2012, the JMPR reconsidered acute dietary risks from maximum residue levels recommended for leafy vegetables (excluding spinach) and withdrew them. In 2015, additional maximum residue levels by the JMPR were evaluated; the meeting received information on supervised residue trials for cucumber and tomato from China and for sweet corn, mustard greens, and asparagus from the United States. Residue trials were also conducted on sweet corn in Canada. Ultimately, the JMPR concluded that short-term intake of ACE from the consumption of mustard greens may present a public health concern, but short-term intake of ACE for other commodities is unlikely (FAO 2017). The European Food Safety Authority recommended lowering the ADI for ACE multiple times. The ADI was lowered from 0.07 to 0.025 mg/kg following a recommendation released in 2013 (EFSA 2013) (Table 1). Another recommendation was recently published suggesting that the ADI should be further lowered from 0.025 to 0.005 mg/kg (EFSA et al. 2024) (Table 1).

Table 1.

Effects of select neonicotinoids reported by the Environmental Protection Agency and Food and Agriculture Organization of the United Nations.

Neonicotinoid	Exposure Window	Dose	Model	Sex	Reproductive endpoints	Conclusions	References
Acetamiprid	2-generation reproduction (∼ 5 mo)	NOAEL: 17.9/21.7 mg/kg bw per day, LOAEL: 51.0/60.1 mg/kg bw per day in male and female rats, respectively	Rat	Both	Abnormal reproductive outcomes, birth outcomes	Acetamiprid reduced litter weights, litter size, and delayed puberty in rats.	EPA 2002, FAO 2017
Clothianidin	2-generation study (∼5 mo)	NOAEL: 31.2/36.8 mg/kg bw per day, LOAEL: 163.4/188.8 mg/kg bw per day	Rat	Both	Abnormal reproductive outcomes, birth outcomes	Clothianidin delayed sexual maturation, decreased sperm motility, and caused abnormal sperm morphology in male rats. Clothianidin also caused ovarian interstitial gland hyperplasia in female rats and increased stillbirths among litters.	EPA 2003a
Imidacloprid	Gestational Days 6 to 18	NOAEL: 24 mg/kg bw per day, LOAEL: 72 mg/kg bw per day	Rabbit	Female	Abnormal reproductive outcomes, birth outcomes	Gestational exposure to imidacloprid caused post-implantation loss in dams.	Koshlukova 2006
Imidacloprid	Gestational Days 6 to 15	NOAEL: 30 mg/kg per day, LOAEL: 100 mg/kg per day	Rat	Female	Abnormal reproductive outcomes, birth outcomes	Imidacloprid increased the incidence of wavy ribs and high number of male fetuses.	Koshlukova 2006
Thiacloprid	Unspecified	NOAEL: 4.4 mg/kg bw/d, LOAEL: 25.6 mg/kg bw/d in dams (acute exposure)	Rat	Both	Abnormal reproductive outcomes, birth outcomes	Thiacloprid delayed sexual maturation in male offspring.	EPA 2003b
Thiacloprid	Unspecified	NOAEL: 1.6 mg/kg bw/d, LOAEL: 3.3 mg/kg bw/d (chronic exposure)	Rat	Female	Reproductive cancer	Thiacloprid increased the incidence of uterine tumors in rats.	EPA 2003b
Thiacloprid	Unspecified	NOAEL: 10.9 mg/kg bw/d, LOAEL: 475.3 mg/kg bw/d	Mouse	Female	Reproductive cancer	Thiacloprid increased the incidence of ovarian luteomas.	EPA 2003b

ACE has been shown to be toxic to the male reproductive system. Specifically, ACE interferes with sperm quality. In 1 study, male Sprague-Dawley rats were orally dosed with 12.5, 25, or 35 mg/kg of ACE for 3 mo (Arıcan et al. 2020). ACE at the 25 and 35 mg/kg doses significantly reduced sperm concentration and ACE at the 35 mg/kg dose significantly increased the amount of flattened-head sperm compared with control (Arıcan et al. 2020).

ACE exposure has also been shown to disrupt reproductive hormone levels in males. Specifically, ACE significantly reduced testosterone levels in a dose-dependent manner in male Sprague-Dawley rats compared with control (Arıcan et al. 2020) (Table 2). ACE at the 12.5 and 25 mg/kg doses significantly increased gonadotropin-releasing hormone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels compared with control (Arıcan et al. 2020). However, gonadotropin-releasing hormone can only be accurately measured in blood collected from the pituitary portal or the hypothalamus (Moenter and Evans 2022). The blood collected for analysis was taken from the orbital vein, which highlights a potential limitation of the study.

Table 2.

Effects of acetamiprid on the reproductive system.

Neonicotinoid	Exposure window	Dose	Model	Sex	Reproductive endpoints	Conclusions	References
Acetamiprid	Adult (3 mo)	12.5, 25, 35, mg/kg/d	Sprague-Dawley rats	Male	Apoptosis, gonad morphology, oxidative stress, sperm quality, steroidogenic hormone levels	Acetamiprid caused reproductive toxicity in the male reproductive system in the high-dose group. Mechanisms may be associated with oxidative stress, hormone disruption, and apoptosis.	Arıcan et al. 2020
Acetamiprid, Acetamiprid CF (commercial formulation)	In vitro (4 and 24 h)	0.1, 1, 10, 100 μM	HTR-8/SVneo Trophoblasts	Female	Apoptosis, cytotoxicity, oxidative stress	Acetamiprid and its commercial formulation are cytotoxic for human trophoblasts, and oxidative stress is the main mechanism of toxicity. Acetamiprid CF is more toxic than acetamiprid.	Gomez et al. 2020
Acetamiprid	Adult (1 mo)	10, 20 mg/kg bw	NMRI	Female	Follicle numbers, gonad morphology, oxidative stress, steroidogenic hormone levels	Acetamiprid caused inflammation in ovarian tissue of both groups, increased androgen-secreting cells and caused vascular hyperemia in the ovaries and area surrounding the follicle. Exposure to acetamaprid decreased the number of secondary and antral follicles.	Hassanzadeh et al. 2023

ACE also has been shown to affect apoptosis and proliferation in the testes of male Sprague-Dawley rats. Histological evaluation revealed that ACE at the 25 and 35 mg/kg doses significantly induced apoptosis compared with control and ACE at 35 mg/kg significantly reduced proliferation compared with control (Arıcan et al. 2020).

Although the exact mechanisms by which ACE causes male reproductive toxicity are unclear, oxidative stress may be a primary mechanism underlying ACE-induced toxicity in male Sprague-Dawley rats. During oxidative stress, reactive oxygen species likely form in cells that use molecular oxygen for steroid biosynthesis within the testes (Zaidi et al. 2013), leading to lipid peroxidation that could potentially decrease the amount of cholesterol available for steroidogenesis. This can directly decrease the amount of testosterone produced, resulting in low sperm concentration because testosterone is reported to have antioxidant and antiapoptotic functions in the testes, protecting sperm from potential damage (Vasconsuelo et al. 2011; Darbandi et al. 2018). Oxidative stress also impacts cell proliferation, apoptosis, and transcription within the testis (Li et al. 2016). Thus, ACE-induced low sperm concentration could be caused by ACE-induced oxidative stress. This possibility is supported by studies that show ACE exposure significantly reduced the levels of the antioxidative markers of glutathione (GSH) and total antioxidant status in plasma and testes tissues in a dose-dependent manner compared with control (Arıcan et al. 2020). Further, ACE exposure at 35 mg/kg significantly increased the levels of the oxidative markers of malondialdehyde and total oxidant status compared with control. Additionally, ACE caused lipid peroxidation in the plasma and testes of male Sprague-Dawley rats (Arıcan et al. 2020).

ACE has also been shown to be toxic to the female reproductive system. An in vitro study in human trophoblast cells demonstrated oxidative damage in ACE treatment groups. HTR-8/SVneo cells were treated with vehicle, ACE, or a commercial formulation of ACE (ACE CF) at 0.1, 1, 10, and 100 µM in vitro. The cells then were evaluated for changes in viability, apoptosis, and oxidative damage at 4 and 24 h (Gomez et al. 2020) (Table 1). ACE significantly reduced cell viability at 100 µM at 24 h compared with control. In contrast, ACE CF significantly reduced cell viability at 10 and 100 µM at 4 h, and at all concentrations at 24 h compared with control (Gomez et al. 2020). ACE at 1, 10, and 100 µM significantly increased the ratio of the pro-apoptotic factor Bcl-2 associated X protein to the anti-apoptotic factor B-cell lymphoma 2, whereas ACE CF at all concentrations significantly increased the Bcl-2 associated X protein and B-cell lymphoma 2 protein ratio compared with control (Gomez et al. 2020). ACE caused oxidative damage in cellular proteins, whereas ACE CF caused oxidative damage in cellular proteins, lipids, and DNA compared with control. Collectively, these data suggest that ACE CF is more cytotoxic than ACE in human trophoblast cells.

Although both the mechanism underlying the toxic effects of ACE and ACE CF on trophoblast cells and the long-term consequences of ACE and ACE CF toxicity on trophoblast cells are unknown, they may involve oxidative stress, leading to adverse pregnancy outcomes. This is because ACE causes oxidative stress in male rodents and oxidative stress in early pregnancy leads to preeclampsia (Matsubara et al. 2015), intrauterine growth restriction (Gurugubelli Krishna and Vishnu Bhat 2018), and preterm birth (Paules et al. 2019).

ACE exposure has been shown to cause inflammation and decrease ovarian follicles in mice. In 1 study, female NMRI mice were treated with 10 or 20 mg/kg ACE daily for 1 mo. ACE caused inflammation in the ovarian tissue of both groups compared with control. Additionally, ACE increased androgen-secreting cells and caused vascular hyperemia in the medulla area of the ovaries and peripheral area of the follicles compared with the control. Lastly, ACE significantly decreased the number of secondary and antral ovarian follicles in NMRI mice compared with the control (Hassanzadeh et al. 2023). This study contains a limitation regarding the exposure route of the mice. ACE was administered intraperitoneally, which does not reflect how humans would realistically be exposed to ACE.

Although ACE has a clear impact on some male and female reproductive outcomes, the available evidence is limited. More in vivo studies are needed to examine sex differences and the impact of neonicotinoid exposure during critical reproductive windows. Studies are also needed to examine which reproductive tissues contain target receptors for neonicotinoids. More in vitro studies are needed to define the reproductive toxicity of ACE in both sexes and its mechanisms of actions. For example, studies examining the impact of ACE exposure versus other neonicotinoids on ovarian follicles would be useful for comparing the degree of toxicity of neonicotinoids on the ovary. Similarly, in vitro studies could assess the direct effects of ACE on other cells from the reproductive system using uterine, testicular, or granulosa cell lines.

Clothianidin

CLO is registered for seed treatment use on corn and canola in the United States. The insecticide is applied between a range of 0.01 and 0.024 pounds of active ingredient per acre for canola, and 0.02 and 0.1 pounds per acre for corn. People and animals are commonly exposed to CLO via dietary exposure (US EPA 2003a) (Table 1). The maximum ADI for CLO is 0.1 mg/kg per body weight (FAO 2022) (Table 1). The EPA reported that the LOAEL for CLO is 163.4 mg/kg/d in male rats and 188.8 mg/kg/d in female rats. CLO at the LOAEL delayed sexual maturation, decreased sperm motility, and caused abnormal sperm morphology in male rats. Further, CLO at the LOAEL caused ovarian interstitial gland hyperplasia and increased stillbirths among litters in female rats (US EPA 2003a) (Table 1).

Limited additional information exists about the toxicity of CLO to the male reproductive system, but several additional studies indicate that CLO is toxic to the female reproductive system. In 1 study, CLO was metabolized maternally, transferred, and rapidly concentrated in breast milk in pregnant ICR mice. CLO and its metabolites were more concentrated in milk than in blood by 74.3%, at average concentrations of 2,230 and 1,656 ng/ml, respectively. This study indicates that the offspring experience acute exposure to CLO in comparison with the dams (Shoda et al. 2023) (Table 3).

Table 3.

Effects of clothianidin on the reproductive system.

Neonicotinoid	Exposure window	Dose	Model	Sex	Reproductive endpoints	Conclusions	References
Clothianidin	Prenatal, lactation (gestational day 1.5 to postnatal day 21)	65 mg per kg/bw/d	C57BL/6N	Female	Follicle viability, gonad morphology, oxidative stress, RNASeq, steroidogenic hormone levels	Clothianidin exposure in fetal and lactation stages manifested differently in adult animals.	Kitauchi et al. 2021
Clothianidin	Pregnancy (postnatal day 10 or 11 for a single dose)	6.5 mg/kg/d (1/10 of NOAEL)	ICR mice	Female	Lactational exposure	Clothianidin is metabolized maternally and transferred and concentrated in breast milk very rapidly in mice. Clothianidin is more concentrated in milk than blood.	Shoda et al. 2023

CLO induces morphological changes in the ovary. In 1 study, pregnant C57BL/6N mice were dosed with 65 mg/kg per body weight of CLO, the no-observed-adverse-effect level (NOAEL) in mice, daily until weaning. Female offspring were evaluated at 3 wks and 10 wks to assess differences of pre- and peri-natal exposure to CLO at prepuberty and adulthood, respectively (Kitauchi et al. 2021) (Table 3). CLO reduced ovarian weight and increased the intracellular space between granulosa cells in 3-wk-old female offspring compared with control. However, CLO did not impact the number of follicles and follicle quality in 3- or 10-wk old female offspring (Kitauchi et al. 2021). Kitauchi et al. hypothesized CLO exposure in prepubertal mice leads to an activation of the estrogen biosynthetic pathway, causing a decrease in ovarian weight and size (Kitauchi et al. 2021). These results are consistent with another study demonstrating that estrogen administration reduced ovarian weight without a change in the number of follicles (Wakabayashi 1962).

CLO also affects gene expression in ovaries. RNAseq analysis showed that CLO upregulated 62 genes and downregulated 37 genes in the ovaries of female C57BL/6N mice compared with control. The genes most affected by CLO had functions with annotations of “quantity of gonadal cells,” “quantity of ovarian follicles,” “quantity of germ cells,” and “quantity of gonads” (Kitauchi et al. 2021). Pathways impacted by exposure to CLO included the estrogen biosynthesis and gonadotropin-releasing hormone signaling relating to steroid hormone biosynthesis (Kitauchi et al. 2021). The RNAseq results were validated via qRT-PCR, showing that CLO significantly increased expression of inhibin subunit beta A, progesterone receptor, and aldo-keto reductase family 1 in 3-wk-old female offspring compared with control (Kitauchi et al. 2021).

CLO disrupts steroid hormone levels in female rodents. CLO exposure in utero and via lactation significantly reduced both corticosterone and 17OH-progesterone levels in 10-wk-old female offspring compared with control (Kitauchi et al. 2021). However, CLO did not affect the histology of the ovaries in 10-wk-old female offspring. This could mean CLO-induced effects on corticosterone and 17OH-progesterone may be due to CLO-induced effects on the adrenal glands because corticosterone is produced by the adrenal glands and 17OH-progesterone is produced both by adrenal glands and gonads (Kitauchi et al. 2021).

CLO may also induce oxidative stress in the ovary. In 1 study, the effects of CLO on glutathione peroxidase 4 (GPx4) and manganese-dependent superoxide dismutase (SOD) immunoreactivity were examined in the granulosa, theca, and lutein cells of female C57BL/6N mice. CLO reduced GPx4 reactivity in 3- and 10-wk-old female offspring compared with control. Conversely, CLO did not significantly impact the intensity of manganese-dependent SOD immunoreactivity in 3- and 10-wk-old female offspring compared with control (Kitauchi et al. 2021). Kitauchi et al. (2021) suggest that GSH is consumed by CLO exposure, leading to the intense immunoreactivity observed in the mouse ovary. GPx4 may use GSH and thus, if CLO exposure consumes GSH, this depletes the GSH needed for GPx4.

CLO has a clear impact on female reproductive health, but more studies are needed to evaluate the effects of CLO on other female reproductive outcomes in depth. For example, in vitro studies further examining how CLO may cause oxidative stress in the granulosa, theca, and luteal cells would be useful. Kitauchi suggests CLO may impact the adrenal glands (Kitauchi et al. 2021). Thus, in vivo studies examining the impact of CLO on the hypothalamic-pituitary-adrenal axis would further elucidate how CLO modulates levels of corticosterone and progesterone in non-target species. Furthermore, studies should investigate the impact of CLO on male reproductive health. Studies examining the impact of CLO on sperm morphology and the male gonads compared with other neonicotinoids are needed as are in vitro studies assessing the direct effects of CLO on testicular cells.

Imidacloprid

IMI is registered for use on agricultural and nursery crops for the control of insect pests (Koshlukova 2006) (Table 1). The maximum seasonal application rate ranges from 0.05 to 0.4 pounds per acre (USDA 2015) (Table 1). The ADI for IMI is 0.06 mg/kg per body weight (FAO 2018) (Table 1). The Department of Pesticide Regulation reported that the LOAEL for IMI is 72 and 100 mg/kg/d in rabbits and rats, respectively. IMI at the LOAEL caused post-implantation loss and decreased fetal weight in rabbits. In addition, IMI caused an increased incidence of wavy ribs and higher number of male fetuses in rats. IMI did not affect mating indices, fertility, gestation, litter size, pathology, or mortality at any dose level in Wistar rats (Koshlukova 2006). In contrast, the U.S. Department of Agriculture reported that IMI at the LOAEL caused birth defects, developmental toxicity, and reproductive impairment in pregnant rabbits and rats (USDA 2015). However, the Department of Agriculture report concluded that IMI did not affect reproductive variables or cause birth defects at doses that did not cause maternal toxicity and states that IMI may adversely affect reproduction and cause developmental delays as a result of maternal toxicity (USDA 2015).

In several additional studies, IMI has been shown to disrupt both the male and female reproductive systems. Specifically, some studies have shown that IMI induces morphological changes in male gonads. In 1 study, IMI exposure at 9 mg/kg per body weight daily for 1 mo increased testes weight in adult male Albino rats compared with control (Abdel-Razik et al. 2021) (Table 4). In contrast, IMI at 0.06, 0.8, and 2.25 mg/kg per body weight for 1 mo reduced testis weight in male Wistar rats. In another study, IMI significantly reduced testis weight in Wistar rats dosed with 2.25 mg/kg of IMI when compared with control and rats treated with 0.06 mg/kg of IMI (Tariba Lovaković et al. 2021) (Table 4). Histological evaluation revealed that IMI exposure caused vacuolated cytoplasm, seminiferous tubules with spermatogenic arrest, damaged germinal epithelium, hemorrhage, and increased intratubular space in the testis of Albino rats when compared with control. IMI also disrupted the basement membrane and caused homogenous eosinophilic materials between seminiferous tubules replacing the interstitial cells (Abdel-Razik et al. 2021). The discrepancy in testis weight observed between male Albino and Wistar rats is possibly attributed to the difference in amount of IMI used in both studies. The highest dose administered to the Wistar rats was 1/200 LD₅₀ (Tariba Lovaković et al. 2021), whereas Albino rats were exposed to a much higher dose of IMI at 9 mg/kg, approximately 1/50 LD₅₀ (Abdel-Razik et al. 2021).

Table 4.

Effects of imidacloprid on the reproductive system.

Neonicotinoid	Exposure window	Dose	Model	Sex	Reproductive endpoints	Conclusions	References
Imidacloprid	Adult (5 times a week for 1 mo)	9 mg/kg/d	Albino rats	Male	Gonad morphology, oxidative stress, sperm quality, steroidogenic hormone levels	Imidacloprid might alter reproductive functions.	Abdel-Razik et al. 2021
Imidacloprid	Adult (4 d)	0.2, 2, 20, 200 µg/ml	CD-1 mice	Female	Apoptosis, follicle viability, follicle morphology, steroidogenic hormone levels	Antral follicles may be the target of neonicotinoid toxicity, with the mechanism of toxicity varying between the parent compound and its metabolites.	Mourikes et al. 2023a
Imidacloprid	Adult (4 d)	0.2, 2, 20, 200 µg/ml	CD-1 Mice	Female	Follicle viability, steroidogenic hormone levels	Mouse ovarian follicles metabolize imidacloprid and induce ovarian Cyp expression over time.	Mourikes et al. 2023b
Imidacloprid	Adult (28 d)	Acceptable daily intake (ADI): 0.06 mg/kg Acceptable operator exposure level (AOEL): 0.8 mg/kg 1/200 of LD-50: 2.25 mg/kg	Wistar rats	Male	Gonad morphology, oxidative stress, essential elements	Low-dose exposure to imidacloprid negatively impacts aspects of male reproductivity in Wistar rats.	Tariba Lovaković et al. 2021

IMI exposure is detrimental to sperm quality because it affects the levels of essential elements within the testis and epididymis of male Wistar rats (Tariba Lovaković et al. 2021). Essential elements are necessary for spermatogenesis, sperm maturation, and overall sperm function (Mirnamniha et al. 2019). The essential elements sodium, potassium, and calcium are involved in the regulation of ion balance in spermatozoa, which is essential for motility and fertility (Syeda et al. 2020). IMI exposure at 2.25 mg/kg significantly increased the concentration of sodium in the testes compared with control (Tariba Lovaković et al. 2021). Further, IMI significantly reduced sperm concentration, motility, normality, and viability in male Albino rats compared with control (Abdel-Razik et al. 2021).

IMI disrupts reproductive hormone levels in male rodents. IMI significantly reduced serum testosterone levels and significantly elevated serum FSH levels compared with control in male Albino rats. However, IMI did not significantly affect serum LH levels compared with control (Abdel-Razik et al. 2021).

IMI exposure has also been shown to interfere with oxidative stress pathways in male reproductive organs. IMI at 0.06 mg/kg significantly increased GSH in the testes of male Wistar rats compared with control. IMI at 0.06, 0.8, and 2.25 mg/kg significantly increased glutathione peroxidase activity and SOD levels in the epididymis compared with control (Tariba Lovaković et al. 2021). Further, IMI at 9 mg/kg significantly reduced GSH and SOD and significantly increased CAT in the testis of male Albino rats compared with control. IMI also significantly induced lipid peroxidation and protein carbonyl content in the testes of male Albino rats compared with control (Abdel-Razik et al. 2021).

In addition to causing toxicity to the male reproductive system, IMI is a female reproductive toxicant. In 1 study, ovarian follicles from CD-1 mice were cultured in media containing vehicle, IMI, or the IMI metabolite known as desnitro-imidacloprid (DNI) (Mourikes et al. 2023a) (Table 4). IMI did not impact follicle growth or morphology, but DNI significantly reduced antral follicle growth at 200 µg/ml compared with control. Further, DNI, but not vehicle or IMI, caused follicle rupture at all concentrations (Mourikes et al. 2023a).

IMI also disrupts the ability of ovarian follicles to produce steroid hormones (Mourikes et al. 2023a, 2023b). IMI at 200 µg/ml significantly increased the levels of progesterone levels produced by CD-1 mouse ovarian follicles compared with control after 96 of culture. In contrast, DNI at 200 µg/ml significantly reduced progesterone, testosterone, and estradiol produced by ovarian follicles compared with control after 96 h (Mourikes et al. 2023a).

The mechanisms by which IMI and/or DNI disrupt ovarian steroidogenesis are not clear, but may involve the ability of IMI and DNI to impact the expression of steroidogenic regulators, hormone receptors, metabolic enzymes, and apoptosis regulators in female rodents. IMI and DNI at different concentrations significantly modulated gene expression in the steroidogenic hormone pathway over 96 h (Mourikes et al. 2023a). IMI and DNI impacted gene expression of estrogen receptor alpha and estrogen receptor beta in a nonmonotonic manner in cultured follicles (Mourikes et al. 2023a). IMI significantly induced the gene expression of cytochrome P450 family 2 subfamily E member 1 and cytochrome P450 family 4 subfamily f polypeptide 18 in cultured antral follicles (Mourikes et al. 2023b). Further, IMI and DNI significantly affected the gene expression of both Bcl-2 associated X and B-cell lymphoma 2 proteins at various times in vitro (Mourikes et al. 2023a).

Presently, information on the impact of IMI on reproductive outcomes is limited, with most studies being conducted in vitro. More studies are needed to understand the impact that IMI has on the hypothalamic-pituitary-gonadal axis in both the male and female reproductive systems. The impact of IMI on gonadal steroidogenesis also requires further investigation. Lastly, more studies are needed to fully understand the mechanisms of actions underlying IMI-induced reproductive toxicity.

Thiacloprid

THD is registered for the control of sucking insects on agricultural crops such as cotton and fruits. The maximum seasonal application rates are 0.50 pounds of active ingredient per acre of pome fruit, and 0.28 pounds of active ingredient per acre of cotton (US EPA 2003b) (Table 1). The ADI for THD is 0.01 mg/kg per body weight (FAO 2010) (Table 1).

THD induces morphological changes in the testis and prostate. In 1 study, pregnant outbred Swiss RjOrl mice were exposed to 0, 0.06, 0.6, and 6 mg/kg per body weight of THD at embryonic days E6.5—E15.5 and reproductive outcomes were assessed in the male offspring (Hartman et al. 2021; Dali et al. 2024) (Table 5). Embryonic exposure to THD increased relative testis weight in the first-generation of male offspring (Hartman et al. 2021) and reduced relative testis weight in the third-generation of male offspring compared with control (Dali et al. 2024). In another study, THD at 22.5 and 62.1 mg/kg reduced testis weight in Wistar rats compared with control (Mahmoud et al. 2024) (Table 5). Further, THD delayed sexual maturation in male offspring at a LOAEL of 25.6 mg/kg bw/d (US EPA 2003b).

Table 5.

Effects of thiacloprid on the reproductive system.

Neonicotinoid	Exposure window	Dose	Model	Sex	Reproductive endpoints	Conclusions	References
Thiacloprid	E6.5 to E15.5	0.06, 0.6, and 6 mg/kg/d	Swiss RjOrl mice	Male	DNA damage, gonad morphology, oxidative stress, sperm quality, hormone levels	Thiacloprid disrupts reproductive function and possibly induces epigenetic changes across 3 generations of mice.	Dali et al. 2024
Thiacloprid	E6.5 to E15.5	0.06, 0.6, and 6 mg/kg/d	Swiss RjOrl mice	Male	DNA damage, epigenetic changes, gonad morphology, sperm quality	Gestational exposure to thiacloprid affects epigenetic mechanisms controlling meiosis, which could negatively impact spermatogenesis.	Hartman et al. 2021
Thiacloprid	Adult (56 d)	22.5 (low), 62.1 (high) mg/kg/d	Wistar rats	Male	Gonad morphology, oxidative stress, sperm quality, hormone levels	Thiacloprid disrupts reproductive function, impairing fertility in Wistar rats.	Mahmoud et al. 2024
Thiacloprid	Puberty (28 d)	10, 50, 100 mg/kg/d	C57BL/6Jmice	Male	Sperm quality, hormone levels	Thiacloprid exposure at 50 and 100 mg/kg during puberty affects the male reproductive system.	Zou et al. 2023

THD exposure is also detrimental to sperm quality. THD at 0.6 and 6 mg/kg significantly reduced sperm concentration in male Swiss RjOrl mice compared with control (Hartman et al. 2021). THD also significantly reduced sperm concentration in a dose dependent manner in male Wistar rats (Mahmoud et al. 2024). In another study, 100 mg/kg of THD significantly reduced sperm concentration in C57BL/6J mice compared with control (Zou et al. 2023) (Table 4).

THD disrupts hormone levels as well as steroidogenic enzymes in males. Prenatal THD exposure significantly reduced testosterone levels in the third generation of male Swiss RjOrl offspring (Dali et al. 2024) as well as in male C57BL/6J mice and Wistar rats (Zou et al. 2023; Mahmoud et al. 2024). THD at 22.5 and 62.1 mg/kg significantly increased the levels of FSH and LH in male Wistar rats (Mahmoud et al. 2024). In contrast, THD at 50 and 100 mg/kg significantly increased FSH levels, whereas it slightly decreased LH levels in male C57BL/6J mice (Zou et al. 2023). THD at 22.5 and 62.1 mg/kg significantly reduced the testicular expression of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase compared with control in male Wistar rats (Mahmoud et al. 2024). THD at 100 mg/kg significantly reduced mRNA expression of steroidogenic acute regulatory protein and cytochrome P450 family 11 subfamily A member 1, and THD at 50 and 100 mg/kg significantly reduced protein expression of steroidogenic acute regulatory and cytochrome P450 family 11 subfamily A member 1 compared with control in male testis of C57BL/6J mice (Zou et al. 2023).

In addition to affecting the expression of steroidogenic regulators, THD impacts the expression of many other genes in the testes of Swiss RjOrl mice (Hartman et al. 2021; Dali et al. 2024). In 1 study, prenatal THD exposure led to 198 differentially expressed genes (DEGs) in first-generation male offspring and 2,277 DEGs in third-generation male offspring. Specifically, THD exposure downregulated genes associated with germ cell development and meiosis in first-generation male offspring and it downregulated genes associated with translation, protein folding, and mitochondrial electron transport, and upregulated genes associated with cholesterol metabolic process-related functions in third-generation male offspring (Dali et al. 2024).

THD exposure also induces DNA methylation changes in spermatozoa across multiple generations in male Swiss RjOrl mice, with 481 differentially methylated regions remaining consistent across all generations (Dali et al. 2024). Further, THD induced DNA damage in male germ cells of Swiss RjOrl mice (Hartman et al. 2021; Dali et al. 2024). THD also caused significant DNA fragmentation in testicular tissue samples from male Wistar rats (Mahmoud et al. 2024). THD may also induce oxidative stress because THD significantly increased malondialdehyde, a marker of lipid peroxidation, whereas it significantly decreased the antioxidants CAT, GSH, and SOD in male Wistar rats compared with control (Mahmoud et al. 2024).

Although several studies indicate that THD exposure causes male reproductive toxicity, the impact of THD on reproductive outcomes is descriptive and predominately limited to male reproductive outcomes. However, the EPA reported that THD increased the incidence of uterine tumors in rats and ovarian luteomas in mice (US EPA 2003b). Given the limited information on the effects of THD on both male and female reproduction, future studies should focus on the effects of THD on female reproductive endpoints and they should investigate the mechanisms underlying THD-induced toxicity in both male and female reproductive systems. Given that studies indicate that THD causes transgenerational effects on the male reproductive system, it is particularly important to determine whether THD causes transgenerational effects on the female reproductive system.

Epidemiological studies on pesticide mixtures containing neonicotinoids

Although the examination of individual neonicotinoids in animal models is highly informative for determining the toxicity of individual neonicotinoids, it is not an accurate reflection of modern human exposure because humans are exposed to mixtures of chemicals. Thus, the section below summarizes the current data from epidemiological studies examining the associations of exposure to a variety of neonicotinoid mixtures on reproductive outcomes in males and females.

One study evaluated the average estimated daily intake (EDI) of neonicotinoids through food for the Chinese general population. Composite dietary samples taken from the fifth (2009 to 2012) and sixth (2015 to 2018) Chinese total diet studies were used to evaluate average EDIs of the following neonicotinoid pesticides: ACE, CLO, dinotefuran (DIN), IMI, imidaclothiz, nitenpyram, THD, and THM. ACE, CLO, IMI, and THM had the highest average EDIs in both the fifth and sixth total diet studies, with the average EDI values of ACE (236.03 to 140.68 ng/kg bw/d) and IMI (96.23 to 33.67 ng/kg bw/d) decreasing over time and the EDI values of THM (27.11 to 43.11 ng/kg bw/d) and CLO (6.29 to 23.14 ng/kg bw/d) increasing over time. The average EDI for THD (0.15 to 0.56 ng/kg bw/d) slightly increased over time (Chen et al. 2020) (Table 6). The change in EDIs between the fifth and sixth total diet studies could indicate the replacement of older formulations of neonicotinoids (ACE and IMI) with newer neonicotinoids (THM and CLO) in agriculture. This highlights a cycle of replacement pesticides being approved for use without their adverse effects being thoroughly evaluated.

Table 6.

Effects of mixtures containing neonicotinoids on the reproductive system.

Neonicotinoid	Exposure	Exposure window	Model	Sex	Reproductive endpoints	Conclusions	References
Mixture (acetamiprid, clothianidin, dinotefuran, imidacloprid, imidaclothiz, nitenpyram, thiacloprid, thiamethoxam)	Average estimated daily intake in fifth and sixth Chinese total diet studies, respectively. Acetamiprid: 236.03 to 140.68 ng/kg bw/d Imidacloprid: 96.23 to 33.67 ng/kg bw/d Clothianidin: 6.29 to 23.14 ng/kg bw/d Thiamethoxam: 27.11 to 43.11 ng/kg bw/d Thiacloprid: 0.15 to 0.56 ng/kg bw/d	Fifth Chinese total diet study: 2009 to 2012 Sixth Chinese total diet study: 2015 to 2018	Human	Both	Not applicable	Humans are exposed to mixtures of neonicotinoids.	Chen et al. 2020
Mixture (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, thiamethoxam)	Imidacloprid and thiamethoxam were the most detected neonicotinoids in fruits and vegetables at 66% and 51%, respectively. This is in contrast with samples taken from the U.S. Department of Agriculture Pesticide Data Program where imicloprid was the most frequently detected neonictinoid at 7.3%.	Samples from U.S. Congressional Cafeteria and Hangzhou China studies were collected in 2015	Human	Both	Not applicable	Humans are exposed to mixtures of neonicotinoids.	Lu et al. 2018
Mixture (imidacloprid, imidacloprid-olefin, desnitro-imidacloprid, thiamethoxam, acetamiprid, clothinidin, desmethyl-clothinidin)	Average urinary concentration (ng/ml) of neonicotinoids and metabolites Imidacloprid: 0.07, Imidacloprid-olefin: 0.79 5-Hydroxy-imidacloprid: 1.60 Desnitro-imidacloprid: 0.20 Acetamiprid: 0.01 Desmethyl-acetamiprid: 2.08 Thiamethoxam: 0.41 Clothianidin: 0.21 Desmethyl-clothianidin: 0.26	Pregnancy (first trimester)	Human	Female	Gestational diabetes, oxidative stress	Exposure to individual neonicotinoids and mixtures of neonicotinoids were associated with gestational diabetes mellitus. Maternal age and pre-pregnancy weight may modify the association. Possible mechanism of underlying association may involve oxidative damage to nucleic acid, but more studies are needed.	Mahahi et al. 2023
Mixture [acetamiprid, imidacloprid, N-desmethyl-acetamiprid, 6-chloropyridine-3-carboxylic acid (6-CN), 2-chloro-1,3-thiazole-5-carboxylic acid (2CTCA)]	Urinary neonicotinoid concentration (µg/l) range Imidacloprid: 0.02 to 2.59 Acetamiprid: 0.03 to 1.01 6-CN: 0.25 to 90.29 N-desmethyl-acetamiprid: 0.18 to 8.85 2CTCA: 0.20–9.02	Prenatal (before delivery)	Human	Both	Birth outcomes, oxidative stress	Potential associations of prenatal exposure of neonicotinoids with head circumference.	Pan et al. 2022

Neonicotinoid	Exposure	Exposure window	Model	Sex	Reproductive endpoints	Conclusions	References
Mixture (flupyradifurone, clothianidin, acetamiprid, dinotefuran, nitenpyram, sulfoxaflor, thiacloprid, thiamethoxam)	Median serum neonicotinoid concentrations (ng/ml) in cases Acetamiprid: 0.016 Dinotefuran: 0.185 Clothianidin: <LOD Flupyradifurone: 0.029 Sulfoxaflor: 0.028 Thiacloprid: <LOD Imidacloprid: 0.078 Nitenpyram: 0.038 Thiamethoxam: 0.057	Prenatal (first trimester)	Human	Both	Birth outcomes	Maternal single and mixture neonicotinoids exposure were associated with fetal growth restriction.	Pan et al. 2023
Mixture (acetamiprid, clothianidin, thiacloprid, thiamethoxam, imidacloprid, dinotefuran, N-desmethyl-acetamiprid, 5-hydroxy-imidacloprid, olefin-imidacloprid)	Median serum neonicotnoid concentrations (ng/ml) in cases N-DM-Acetamiprid: 0.186 Thiacloprid: N/A Imidacloprid: 0.186 Thiamethoxam: 0.082	Prenatal (samples taken at birth)	Human	Both	Birth outcomes	Gestational exposure to neonicotinoids may be associated with an increased risk of septal defects, but more evidence is needed.	Qu et al. 2024
Mixture (extensive list of pesticides including neonicotinoids)	Exposure range: 0.03 to 0.05 mg/kg	Puberty (ages 7 to 8)	Human	Female	Pubertal timing	No relationship between agricultural pesticides and the development of precocious puberty. Larger sample sizes and controlled variables are needed to confirm the relationship.	Suh et al. 2020
Mixture (imidacloprid, acetamiprid, clothianidin, thiamethoxam, dinotefuran, thiacloprid, nitenpyram, flonicamid, sulfoxaflor, imidaclothiz, N-DM-Ace, Olefin-IMI, DM-CLO, DM-THM, 5-H-IMI)	Average neonicotinoid concentrations (ng/ml) in seminal plasma Desmethyl-acetamiprid: 0.044 Desmethyl-clothianidin: 0.005 Imidacloprid-olefin: 0.003	Adult (age 20 to 48)	Human	Male	Sperm quality	Inverse association between IMI-olefin concentration and semen quality.	Wang et al. 2022

Neonicotinoid	Exposure	Exposure window	Model	Sex	Reproductive endpoints	Conclusions	References
Mixture (pyrethroids, organophosphates, neonicotinoids (imidacloprid, acetamiprid, thiamethoxam, clothianidin, 5-h-imidacloprid, Olefin-IMI, DNI, DM-CLO, DM-ACE))	Average maternal urinary neonicotinoid concentration Imidacloprid: 0.04 Imidacloprid-olefin: 0.39 5-hydroxy-imidacloprid: 0.63 Desnitro-imidacloprid: 0.09 Desmethyl-acetamprid: 0.86 Thiamethoxam: 0.08 Clothianidin: 0.11 Desmethyl-clothianidin: 0.19	Pregnancy (first trimester)	Human	Female	Oxidative stress, pregnancy outcomes	Exposure to organophosphates, pyrethroids, neonicotinoids, their metabolites, and their mixture may increase oxidative damage to lipids, RNA, and DNA during pregnancy.	Wang et al. 2024
Mixture (imidacloprid, nitenpyram, acetamiprid, thiacloprid, imidaclothiz, thiamethoxam, clothianidin, dinotefuran, flonicamid, sulfoxaflor)	Average urinary neonicotinoid concentration (µg/g creatinine) Acetamiprid: 2.18 Imidacloprid: 0.45 Nitenpyram: 2.40 N-Acetamiprid: 18.17 Thiacloprid: 2.84 Imidaclothiz: 4.27 Thiamethoxam: 3.61 Clothianidin: 4.83 Dinotefuran: 2.70 Flonicamid: 1.53 Sulfoxaflor: 1.39	Puberty (ages 11 to 16)	Human	Both	Pubertal timing	Higher thiacloprid concentration was associated with delayed genitalia development in boys and early axillary hair development in girls. Neonicotinoid mixture was negatively associated with gentalia stage.	Yue et al. 2022

Another study evaluated the residue levels of 7 neonicotinoids in fruit and vegetable samples collected from cross-sectional studies conducted by the U.S. Congressional Cafeteria study and the Hangzhou China study. These samples were compared with data published by the U.S. Department of Agriculture Pesticide Data Program. ACE, CLO, DIN, IMI, nitenpyram, THD, and THM were evaluated in the study. IMI and THM were the most detected neonicotinoids in fruits and vegetables, with a 66% and 51% detection rate in the Hangzhou China study and a 52% and 53% detection rate in the U.S. Congressional Cafeteria study, respectively. In contrast, the overall frequency of detection for neonicotinoids in U.S. Department of Agriculture Pesticide Data Program samples was much lower than samples taken from the U.S. Congressional Cafeteria and Hangzhou China studies, with IMI being the most frequently detected at 7.3% (Lu et al. 2018) (Table 6).

The associations between neonicotinoid mixtures and abnormal birth outcomes has been examined in epidemiological studies (Pan et al. 2022, 2023; Qu et al. 2024) (Table 6). In 1 study, ACE, CLO, THD, THM, IMI, DIN, and their metabolites were measured in serum samples collected from pregnant women in a birth cohort study from Guangdong, China. IMI was the highest detected parent neonicotinoid in maternal and cord sera, with median concentrations of 0.79 ng/ml and 1.84 ng/ml, respectively. N-desmethyl-ACE was the most abundant neonicotinoid metabolite, with median concentrations of 0.08 ng/ml in maternal serum and 0.13 ng/ml in cord serum. Gestational exposure to neonicotinoid mixtures was not significantly associated with septal heart defects, but more studies are needed to confirm these data (Qu et al. 2024).

Another study evaluated the associations between gestational exposure to neonicotinoid mixtures and fetal growth restrictions (Pan et al. 2023). In the study, neonicotinoids (flupyradifurone, nitenpyram, sulfoxaflor, ACE, CLO, DIN, THD, and THM) were measured in maternal blood samples taken from pregnant women during the first trimester who were enrolled in the Guangxi Zhuang Birth Cohort Study (Pan et al. 2023). Subjects with pre-term birth were excluded from this study. The median concentrations of DIN, ACE, sulfoxaflor, and nitenpyram in study participants were 0.185, 0.016, 028, and 0.038 ng/ml, respectively. The concentration for THD was below the limit of detection in maternal serum. DIN (odds ratio (OR) [1.93; 1.69, 2.20]) and ACE (OR [1.31; 1.07, 1.59]) concentrations were significantly associated with higher odds of fetal growth restriction. THD (OR [0.23; 0.15, 0.34]), sulfoxaflor (OR [0.48; 0.41, 0.56]), and nitenpyram (OR [0.86; 0.80, 0.93]) were negatively associated with fetal growth restriction. These data suggest that exposure to individual and mixtures of neonicotinoids are associated with varying risk of fetal growth restrictions, and co-exposure to other pesticides may contribute to the observed results (Pan et al. 2023). The mechanisms by which neonicotinoid mixtures are associated with fetal growth restriction are unclear.

Another study evaluated the associations between neonicotinoid exposure during gestation and various birth outcomes including birth weight, birth length, ponderal index, head circumference, and gestational age in the Laizhou Wan Birth Cohort in China (Pan et al. 2022). ACE, IMI, and 6-chloropyridine-3-carboxylic acid had the highest detection frequencies at maximum urinary concentrations of 1.01, 2.59, and 90.29 ng/ml, respectively. Both ACE and IMI were significantly associated with a decrease in newborn head circumference. Maternal levels of 8-hydroxydeoxyguanosine, a marker of oxidative stress, demonstrated 38.5% to 65.5% mediating effects in negative association with IMI and ACE, with associations possibly differing between boys and girls (Pan et al. 2022).

Neonicotinoid mixtures have been correlated with adverse pregnancy outcomes. One nested case-control study of pregnant women enrolled in a birth cohort from Wuhan, China examined associations between gestational diabetes mellitus, maternal age, pre-pregnancy body mass index, and exposure to neonicotinoid mixtures (Mahai et al. 2023) (Table 6). Oxidative DNA damage, RNA damage, and oxidative stress were evaluated for mediating effects. Exposure to neonicotinoid mixtures was associated with increased odds of gestational diabetes mellitus. Maternal age and higher body mass index (overweight/obese) strengthened the effects of the association. Proportions mediated by DNA damage, RNA damage, and overall oxidative stress were 9.8%, 11.8%, and 14.5%, respectively (Mahai et al. 2023).

Another study examined the associations between exposures to pesticide mixtures featuring neonicotinoids and biomarkers of oxidative stress in pregnant women enrolled in a birth cohort from Wuhan, China between 2014 and 2017 (Wang et al. 2024) (Table 6). The concentrations of pyrethroids, organophosphates, neonicotinoids (ACE, CLO, IMI, and THM), and their metabolites were evaluated in maternal urine samples. Urinary desmethyl-CLO was associated with 8-hydroxydeoxyguanosine levels, suggesting that exposure to complex pesticide mixtures may increase oxidative damage to lipids, RNA, and DNA during pregnancy (Wang et al. 2024).

Neonicotinoid mixtures have been shown to have correlations with pubertal disruption. A cross-sectional study examined associations between pubertal development timing and neonicotinoid exposure by evaluating the concentration of neonicotinoids in urine samples. Urine samples were taken from a prospective puberty cohort established in urban areas of Chongqing, China in 2014. Higher urinary THD concentration (2.81 µg/g creatinine) was associated with delayed genitalia development in boys, whereas higher urinary THD (2.90 µg/g creatinine) was associated with early axillary hair development in girls. Overall, neonicotinoid mixtures were negatively associated with genitalia stage. The reasons for the associations between urinary THD and genitalia stage are unknown, but it is possible that neonicotinoids exert estrogenic and anti-androgenic effects, impacting the function of the hypothalamic-pituitary-gonadal axis and influencing the secretion of sex hormones that regulate genitalia stage (Yue et al. 2022) (Table 6). In contrast, an exploratory study examined associations between precocious puberty and agricultural pesticides by investigating clinical characteristics and analyzing urinary levels of 320 different agricultural pesticides (Suh et al. 2020) (Table 6). Samples were taken from female participants at Severance Children’s Hospital in Seoul, South Korea. Pesticides were detected in 1 of 30 participants with precocious puberty compared with 2 of 30 participants in the control group. Interestingly, DIN (0.0316 to 0.0524 mg/kg bw) was detected in all positive subjects (Suh et al. 2020). This study concluded exposures to agricultural pesticides were not associated with precocious puberty. However, a larger sample size as well as controlled variables are needed to properly assess the relationship between exposure to agricultural pesticides and precocious puberty.

Neonicotinoid mixtures have been correlated with changes in semen quality (Wang et al. 2022). Associations between semen quality and neonicotinoid exposure were examined by evaluating seminal plasma samples collected from men in Shijiazhuang, China from 2018 to 2019 (Wang et al. 2022) (Table 6). IMI-olefin, desmethyl-ACE, and desmethyl-CLO had the highest detection frequencies in all samples. IMI-olefin concentration was associated with decreased progressive motility of sperm (Wang et al. 2022).

Conclusions

In summary, exposures to various neonicotinoids such as ACE, CLO, IMI, and THD have been shown to adversely affect reproductive outcomes in both male and female mammals (Fig. 1). Further, exposures to mixtures of neonicotinoids have been associated with negative male and female reproductive outcomes in epidemiological studies. However, more research is needed to fully understand the extent to which neonicotinoids cause toxicity in the male and female reproductive system and to elucidate the underlying mechanisms of toxicity. Additionally, studies are needed to identify target receptors within reproductive tissues of non-target species. Further, given that most experimental studies have focused on the effects of individual neonicotinoids on male and female reproductive outcomes in animal models and that humans are exposed to mixtures of neonicotinoids, future research should focus on the effects of neonicotinoid mixtures on male and female reproduction. In addition, more research is needed to examine associations between neonicotinoid exposure, birth outcomes, pubertal timing, maternal health, and detrimental impact on male semen quality in epidemiological studies. Finally, a few studies suggest that neonicotinoids have the potential to induce epigenetic changes in offspring, leading to transgenerational effects of neonicotinoids, but this has not been investigated in detail. Thus, future studies should determine whether neonicotinoid exposures lead to transgenerational impacts on both male and female reproduction.

Fig. 1.

Schematic on the target tissues of neonicotinoid pesticides. The schematic shows that neonicotinoids can target the hypothalamic–pituitary–adrenal (HPA axis) and the hypothalamic-pituitary-gonadal (HPG) axis. The schematic also illustrates that when neonicotinoids target the HPA or HPG axis, they can cause endocrine disruption, which may be characterized by dysregulated steroidogenesis. Neonicotinoids can also cause reproductive toxicity, which includes abnormal gonad morphology, infertility, oxidative stress, and/or epigenetic DNA damage. Figure made with BioRender.

Funding

This work was supported by National Institutes of Health (NIH) R21 ES036520 and a Fellowship from the Graduate College at the University of Illinois Urbana-Champaign.

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