Researchers have looked at the impact of a large size range of NPs/MPs and tested the effects of various plastic types and shapes in a wide variety of animal species. As shown in Supplementary Tables S1, S2, it is clear that in most cases small MPs are more toxic than larger ones. For example, PS-MPs sized from 0.05 µm (NP) to 6 µm (MP) applied to marine medaka larvae led to decreased hatching rate, with the lowest values observed upon the smallest particle exposure. Paradoxically, this smallest sized NP induced higher expression level of the low choriolytic enzyme (LCE) (Chen et al., 2020a), a hatching enzyme, which could be a compensatory mechanism to counteract MP/NP induced reproductive inhibition. Similarly, in the pacific oyster, PS-NP (50 nm) reduced gamete fertilization, larval development, and embryo hatching, and this occurred regardless whether or not the PS-NPs were amino or carboxyl modified. On the contrary, PS-MPs of 2 µm had no effect on oyster reproduction (Tallec et al., 2018). A similar study using 50 nm amino-modified PS-NP showed no effect on reproduction in the oyster at lower NP concentrations, while higher concentrations of plastic particles reduced sperm motility due to sperm aggregation (Tallec et al., 2020), suggesting that concentration is a critical parameter in MP/NP-induced reproductive toxicity. In marine rotifer species, PS-NPs increased reproductive time and led to oxidative stress to a greater extent that PS-MPs did (Jeong et al., 2016). Exposure to smaller PE-MPs resulted in lower numbers of broods per female in the water flea (Daphnia magna), when compared to exposure to larger PE-MPs (Ogonowski et al., 2016). In C. elegans hermaphrodites 20 nm NPs caused greater transgenerational oxidative stress with greater induction of stress-responsive genes in the offspring of treated mothers than 100 nm NPs (Liu et al., 2021c), indicating that the smaller the NPs size is, the greater the observed reproductive defects are. Microplastic fibers were typically more toxic than beads. In the amphipod, Hyalelia azteca, and in the water flea a greater decrease in reproduction was observed with lower number of broods at lower MP concentrations upon fiber exposure than with plastic beads (Au et al., 2015; Ziajahromi et al., 2017). In the earthworm (Lumbricus terrestris) (Huerta-Lwanga et al., 2021) and springtail (Folsomia candida), microfibers decreased reproduction by inhibiting spermatogenesis (Jemec Kokalj et al., 2021). In the earthworm Eisenia andreii, PE-MP breakdown into NP induced sperm damage and decreased number of sperm bundles but did not cause damage to the oocyte in females, showing sexually dimorphic reproductive toxicity (Jeong et al., 2021). These data suggest that smaller particles are more toxic than larger ones, independent of the chemical composition of the MPs/NPs.

Smaller particles may be toxic because they might be preferentially ingested and thereby decrease ingestion of food. In the marine copepod, PS-MPs were preferred to food, and this led to decreased ingestion of food and increased time to egg hatching (Li et al., 2020b). However, MP ingestion does not preferentially occur if there is an excess of natural food, as can be seen with Daphnia magna (Aljaibachi and Callaghan, 2018), marine medaka (Cong et al., 2019), and marine rotifers (Xue et al., 2021). This suggests that active avoidance of plastic particles is possible, though this phenomenon appears to be reported in aquatic rather than in terrestrial species. When C. elegans was exposed to PS-MPs reproductive toxicity has been observed, even though the plastic particles were not detected in the reproductive tissues. However, reproductive toxicity were not due to styrene monomers leaching from the beads as their levels used in the study were far below toxicity and PS-MPs do not have to be ingested to have a toxic effect on the worms (Mueller et al., 2020). PS-MPs might indirectly affect reproduction in C. elegans, limiting food availability, as is suggested for copepods (Cole et al., 2015), since the inhibitory effects of PS increased with decreasing bacterial densities. In C. elegans, PS-MPs up to 3 µm could be ingested, but all sizes from 0.1 to 10 µm decreased the number of offspring. Indeed, the reproductive toxicity correlated with decreased food ingestion (Mueller et al., 2020), suggesting that the presence of PS-MPs interfered with feeding. In the pearl oyster and the planktonic doliolid, decreased feeding and lower ingestion of MPs have been observed instead (Gardon et al., 2018; Paffenhöfer and Köster, 2020); this led to gamete apoptosis to conserve energy for survival in the pearl oyster (Gardon et al., 2018). Smaller sizes of plastics may be more toxic due to longer periods of action, staying in the gut for a longer period of time (Mueller et al., 2020), or easier and preferential ingestion. Furthermore, when ingested, smaller particles could be taken up more easily by cells, using the cellular endocytic machinery or phagocytosis (Rejman et al., 2004; Xia et al., 2008; Ekkapongpisit et al., 2012; Monti et al., 2015). This could lead to cellular internalization and translocation of NPs from exposure site to distant tissues (Rubio et al., 2020).

Combining MPs with other pollutants could also alter the effect of MPs on reproduction due to change in particle size. For example, aggregation of PS-MPs caused by dibutyl phthalate (DBP) led to the formation of very large size particles which could not be ingested by the marine copepod. Hence, the presence of DBP decreased reproductive toxicity of PS-MP, measured as time to hatch, while PS-MP absorbed DBP and decreased DBP toxicity (Li et al., 2020b).

In C. elegans hermaphrodites, a study utilizing unmodified and amino-modified polystyrene NPs revealed that amino-modified NPs were more toxic to reproduction at both 10 μg/L and 100 μg/L concentrations across multiple (F0–F3) offspring generations. Amino modified NPs caused greater and dose dependent reduction in the number of germline cells, fertilized eggs and overall brood size, than pristine, unmodified NPs. The germline defects were due to an upregulation of the pro-apoptotic ced-3 and ced-4 genes and a concomitant decrease in the anti-apoptotic ced-9 gene expression (Sun et al., 2021). Overall, positively charged amino-modified nanoplastics were more toxic in C. elegans than neutral NPs, which however were more toxic than the negatively charged carboxylated NPs, possibly due to differential interaction of these compounds with membranes and organelles (Schultz et al., 2021). However, at short-term exposure (i.e., 24 h) and using polystyrene MPs rather than NPs, decreased number of progeny was seen independent of surface modification, although neutral PS particles had larger impact than amino- or carboxy-modified particles on another MP-altered pathway, purine metabolism (Kim et al., 2020). This reinforces the idea that in case of smaller sized particles, such as NPs, amino-modified plastic particles are the most toxic under chronic exposure.

In Daphnia magna, exposure to various doses (10–500 mg/L) of MPs (<60 µm) over 21 days revealed the greatest reduction in the number of offspring in the PVC MP treated group, when compared to polyurethane and polylactic acid particles (Zimmermann et al., 2020). However, in C. elegans and zebrafish, a comparison of PS, PVC, poly (p-phenylene oxide) (PPE), polyamide (PA) MPs at very low doses (0.001–10 mg/L) and sizes (0.5, 1, 10 μM) showed decreased growth and reproduction rate, independent of dose and chemical property of the particles applied. MP exposure caused intestinal damage and increased gluthathione-S-transferase (GST) levels in a particle size dependent manner (Lei et al., 2018a). PS-MPs also decreased sperm fertilization rate in the sea urchin to a greater extent than polymethyl-methylacrylate (PMMA) particles (Trifuoggi et al., 2019). In general, PS-MPs appear to be more reproductively toxic that other MPs in both aquatic species and in C. elegans, although this might be a consequence of most studies using PS particles and the general lack of comparative studies.

When investigating the impact of MPs/NPs on living organisms, we need to consider that bioactive compounds are almost always present on and readily released from plastic particles. MPs and NPs can carry various toxic pollutants, however, whether these act synergistically, additively or have no impact on effects of MPs/NPs is currently a controversial topic, due to the use of a diverse range of animal species, types of MPs/NPs and pollutants, as well as assessing various physiological or molecular readouts. Some reports indicate that MPs/NPs and their leached EDCs modify each other’s effects on animal survival, reproduction, stress or other signaling pathways, while others report the lack of these (Eder et al., 2021). For example, the pesticide deltamethrin caused delayed first brood production and decreased fertility in Daphnia magna, and similar impact was observed on the juvenile larvae number per surviving adult upon PE-MP exposure. The combined exposure to deltamethrin and PE-MPs led to a synergistic detrimental effect on brood number and survival in this species (Felten et al., 2020). However, addition of the insecticide and endocrine disruptor dichlorodiphenyltrichloroethane (DTT) (Kelce et al., 1995) to PE-MPs had neither additive nor synergistic effect on the larval yield of inland silversides (Menidia beryllina).

Sex-specific differences in sensitivity towards MP-pollutant mixtures have also been observed. For example, in the female Japanese medaka (Oryzias latipes), a decrease in estrogen receptor (ERα) expression, and expression of the egg proteins, vitellogenin and choriogenin was recorded after 2-months dietary exposure to virgin or marine-weathered PE-MPs (Rochman et al., 2014), indicating possible adverse effects on oogenesis (Murata et al., 1997). However, in the male Japanese medaka, only virgin PE-MPs influenced gene expression, and marine-weathered PE-MPs did not cause significant alterations in the measured outputs (Andrady, 2011; Rochman et al., 2014), suggesting sexual dimorphism in this response. When the medaka was exposed to UV-treated or marine-weathered MPs at larvae stage, it showed greater induction of vitellogenin expression, an in vivo biomarker of estrogen action linked to reproductive effects, than what was measured in larvae exposed to virgin MPs. This suggests that early developmental stages of marine species may be more sensitive to a combination of MPs and their leached EDCs, than to MPs alone, consistent with the long-lasting impact of EDCs alone in early development of animals (Patisaul and Adewale, 2009). This could indicate that timing and length of MP-pollutant treatment would be crucial in determining impacts of these relevant to environmental exposure. The synergistic effects of MPs/NPs and their pollutants might be the consequence of enhanced cellular uptake of the particles, as shown in mouse cell culture experiments performed with weathered MPs (Ramsperger et al., 2020). Interestingly, MPs and NPs could also act antagonistically with persistent organic pollutants, by absorbing and therefore decreasing bioavailability of EDCs. For instance, exposing the crustacean Gammarus roeseli to MPs mixed with the EDC phenanthrene led to less detrimental effects than observed by exposure to phenanthrene alone (Bartonitz et al., 2020).

It should be noted that there are a number of studies, mostly performed on aquatic species, where there were no negative effects observed on reproduction upon MP/NP exposure. In Daphnia pulex, NPs caused no difference in the offspring number/clutch or female and the number of clutches in exposure during F0 or F1 generations (Liu et al., 2020b). In another study, Daphnia magna exposure to a mix of NPs and MPs showed no reproductive effects despite uptake of these plastics (Rist et al., 2017). In quagga mussels (Dreissena rostriformis), MP had no effect on reproduction perhaps due to an acute and short (24 h) exposure (Pedersen et al., 2020). Similarly, exposure of MP in Danio rerio (zebrafish) for a short period (2 days) had no effect on egg fertilization (Pitt et al., 2018). In some cases, such as in the blackworm, ingested PE-MPs over a longer, 28-day exposure increased ROS and decreased aerobic energy production but did not alter reproduction (Silva et al., 2021). In two studies, PS-MP exposure in Java and Japanese medaka over 21 days (Assas et al., 2020) or in Daphnia magna over 100 days (Kelpsiene et al., 2020) caused no reproductive defects. It is possible that the used MP doses in these experiments, lying in the lower end of environmental concentrations (i.e., 10⁷ particles/l equivalent to 44 μg/L for the medaka and 0.32 mg/L for Daphnia magna), were too low to induce overt defects in the reproductive function of these animals. In one case, MPs could be used as a substrate for egg deposition which led to an increase in the numbers of water strider adults and juveniles in the North Pacific (Goldstein et al., 2012; Majer et al., 2012). It is therefore important to use a range of concentrations of plastics over both acute and chronic durations in a systematic way to test toxicity.

Sexual motivation is the first step of reproductive behavior and is part of the anticipatory component. As mating is key to species survival, animals are naturally motivated to perform this behavior, and this is intrinsically rewarded by the release of dopamine (Melis and Argiolas, 1995; Wise, 2004; Hull and Dominguez, 2007; Udupa and Chen, 2016). The dopaminergic meso (cortico)limbic system regulates the motivation for female sexual behavior and this circuit is regulated by estrogen signaling via the ER (Meisel and Mullins, 2006; Micevych and Meisel, 2017; Sanna et al., 2020).

When the effect of MPs on reproduction was examined after exposure to high levels of plastic particles, the planktonic crustacean Daphnia magna showed increased inter-brood periods and decreased average brood production, suggesting they had decreased motivation for reproduction (Ogonowski et al., 2016). In the zebra mussel (Dreissena polymorpha) exposure to different sizes of virgin PS-MP for 6 days increased dopamine levels (Magni et al., 2018), which could alter the motivation for reproduction. In echinoderms and bivalve molluscs, dopamine drives oogenesis (Khotimchenko, 1991). Since MP exposure causes dopaminergic neurotoxicity, a decrease in dopamine may influence oocyte quality in oysters (Hoelting et al., 2013). Similarly to what is observed in female mice, PS-MPs cause neurotoxicity in dopaminergic neurons and decreases dopamine levels in C. elegans. As dopamine also promotes egg-laying (Nagashima et al., 2016), PS-exposure leads to reduced egg-laying in the nematode model (Lei et al., 2018b). Supporting this, after exposure to and internalization of NPs, cultured human dopaminergic neurons developed neurospheres with increased oxidative stress (Hoelting et al., 2013). Given that MPs can alter the brain’s dopamine chemistry, possibly via ROS-induced apoptosis of dopaminergic neurons, the impact on reproductive motivation during and after exposure would be important to study.

Though the impact of MPs/NPs on consummatory components of sexual behavior has not yet been investigated, toxicity of EDCs that could leach from plastics have been widely studied, typically in rodents. For example, female rats and mice exposed to bisphenol A (BPA) as adults have increased plasma estrogen levels, which are linked to increased lordosis and reduced rejective behaviors during mating (Ribeiro et al., 2012; Wang et al., 2014). This may increase preference for less fit males (Patisaul and Adewale, 2009). In male rats, chronic adult exposure to BPA causes increased latencies to anticipatory and consummatory behaviors, including first mount, pelvic thrust, intromission, and ejaculation, and fewer intromissions when compared to the control animals (Picot et al., 2014). New, targeted studies assessing microplastic-induced alterations in nuclear-hormone-receptor signaling driving reproductive behaviors could shed light on their potential toxicity.

While C. elegans is a widely used model in investigating toxicology of MPs in eukaryotic multicellular organisms, our knowledge on the impact of MPs on reproductive behaviors even in this extensively studied species is limited. As in rodents, C. elegans male reproductive or sex behavior is a well-documented sequential mating behavior, with the males actively performing most of the sensory and motoric behaviors during the process (Figure 1) (Barr and Garcia, 2006). As in mammals, mating behaviors in C. elegans result from sexually dimorphic nervous systems. The hermaphrodite has 8 sex-specific neurons, whereas the male has 91 sex-specific motor, inter and sensory neurons, of which all but 4 are associated with the tail (Breedlove, 1986; Liu and Sternberg, 1995; Barr et al., 2018).

Experimental testing of reproductive capacity and mating behaviors so far has measured the time taken by the male to find the hermaphrodite, male spicule insertion, or measuring brood size to assess mating success (Barr, 2014). As in mammals, male mating effectivity in C. elegans decreases with age due to defects in mating execution rather than diminished sperm quality, suggesting that C. elegans infertility develops similarly to mammals (Chatterjee et al., 2013). Since there is a differential requirement for protein products across the mating sequence, the vulnerability of some behavioral mating stages to MPs/NPs may enable the identification of genes and pathways that are targeted by these pollutants. Due to the short lifecycle of C. elegans, which reaches reproductive stage in just 3 days post-hatching, along with the ease of obtaining replicates, and accuracy of behavior “scoring” parameters (Figure 1), C. elegans provide a cost-effective and rapid system for reproductive behavior testing when compared to rodents. Furthermore, C. elegans can provide insights into how early-life exposure to MPs/NPs might lead to deleterious consequences in later life. Due to its short lifespan, this nematode is ideal for studying the long term impacts of MP/NP exposure during the course of the whole lifetime (Litke et al., 2018). This is particularly relevant for the longer-lived human populations. Therefore, we propose that C. elegans is particularly suitable to investigate the impact of single environmental pollutants or combinations of these on the male mating behavior model (Figure 1) and resulting brood size in a longitudinal manner. In addition, investigating this will clarify if reproductive behavior and/or damage to the oocytes or sperms is the critical driver underlying decreased reproduction rate in animals upon exposure to plastics.

Dopamine signaling is well-conserved between vertebrates and C. elegans, and its function has been characterized in detail in the nematode model. C. elegans uses dopamine to react to environmental conditions, adjust its physiology and generate appropriate behaviors (McCloskey et al., 2017). The hermaphrodites have eight dopaminergic neurons that coordinates locomotion with egg-laying behavior (Cermak et al., 2020). The males have dopamine expression in the male ray sensory neurons, which enable them to respond to the presence of the hermaphrodite by moving towards and begin mating (Lints and Emmons, 1999), relevant to overall reproductive success of males. In C. elegans the expression of the dopaminergic neuron reporter dat-1pr::GFP shows decreased fluorescence upon exposure to polystyrene nanoplastics (PS-NP). This was also associated with decreased mitochondria function and increased oxidative stress (Liu et al., 2020c), suggesting dopaminergic specific toxicity upon PS-MP exposure. The exposure to UV-aged PS-MPs caused more severe dopaminergic defects than virgin MPs, probably due to the leaching of toxic materials (Chen et al., 2021a). Interestingly, PS-NP exposure in C. elegans causes significant increase in the expression level of intestinal dop-1, a gene encoding for a dopamine receptor (Qu et al., 2020a), further supporting the idea of MP-driven interference with the dopaminergic system in nematodes.

Changes of dopamine levels induced by MPs/NPs could affect a number of different dopamine-dependent behaviors that could be used to screen the toxic effects of MPs and their pollutants. For example, in C. elegans hermaphrodites touch response habituation is dependent on the availability of food. In the absence of food animals are habituated faster to the touch-triggered escape reflex than in the presence of food (Kindt et al., 2007). This response is regulated by dopamine (Kindt et al., 2007). Dopaminergic signaling is also required for the transition between locomotory gaits and slowing movement upon mechanosensation of food (Vidal-Gadea and Pierce-Shimomura, 2012; Sawin et al., 2000). Exposure to MPs decreased thrashing frequency when swimming in liquid and crawling speed on solid surface (Chen et al., 2021b) in a size and concentration dependent manner (Lei et al., 2018b). These behavioral assays could be used as high-throughput readouts upon exposure to plastic particles, prior to testing these in reproductive behavior assays (discussed in details in C. elegans is a Promising Model to Investigate Molecular Pathways Mediating MP/NP-Induced Reproductive Toxicity). Though reproductive motivation regulated by the dopamine system has not been studied in C. elegans, dopamine is involved in fine-tuning the activity of sensory-motor neurons and muscles during male copulation (Correa et al., 2012) and is a conserved candidate pathway. Hence, using dopamine synthesis or signaling worm mutants in these studies would be invaluable in understanding the contribution of dopaminergic neurons to reproductive behaviors as well as pinpointing possible action mechanisms for plastic pollutants.

One commonly identified signaling pathway regulated by MPs and NPs appears to be steroid hormone signaling, whereby altered expression levels of steroidogenic enzymes impact levels of steroid hormones, leading to possible changes in feedback of the hypothalamic-pituitary gonadal axis (Vadakkadath Meethal and Atwood, 2005) (Supplementary Table S1). This could potentially alter social behaviors, with detrimental consequences for reproduction.

As might be expected, cytochrome P450 enzymes involved in xenobiotic transformations are upregulated upon MP/NP exposure in some species. Long term PS-exposure in the marine medaka (Wang et al., 2021) and Daphnia pulex (Liu et al., 2020b) induced the expression of P450 enzymes. The P450 enzyme families also contain steroidogenic synthesis enzymes, some of which are affected by MPs or MP-EDC mixtures (Supplementary Table S1). In the medaka, exposure to PS-MPs decreased the number of mature eggs in the female and sperm in the male, and increased several steroidogenic enzymes including STaR, the rate limiting enzymes of steroid hormone synthesis, as well as the 11β-HSD and aromatase enzymes required for cortisol and estrogen synthesis, respectively. This leads to a higher estrogen/testerosterone ratio, which in some cases, could be due to MP carried EDCs. For example, co-exposure of male medaka to ethinylestradiol (EE) and MPs synergistically decreased the level of GnRH in the brain and increased Cyp19a in the testis, suggesting that increased estrogen/testosterone ratio led to lower sperm counts (Wang et al., 2022). Higher levels of estrogen could perhaps negatively feedback the level of the pituitary reproductive hormones, follicle stimulating hormone (FSH) and lutenizing hormone (LH) (Wang et al., 2021). Similarly, in the adult male Nile tilapia, irregular sized NPs decreased sperm numbers, and this was correlated with lower levels of LH and FSH (Ismail et al., 2021). In male rat (Amereh et al., 2020; Ijaz et al., 2021) or mouse (Xie et al., 2020), there was decreased testosterone and decreased LH/FSH (in rat only) upon MP exposure, supporting demasculinisation of the hypothalamo-pituitary-gonadal (HPG) axis. This was associated with altered morphology and viability of sperms, with evidence for increased DNA damage and tissue lesions (Amereh et al., 2020). In the adult male zebrafish, reduced aggression and increased shoaling behavior combined with increased vitellogenin synthesis and aromatase expression (Sarasamma et al., 2020) is observed upon MP exposure, suggesting possible demasculinisation of behavior, possibly due to decreased dopamine levels. In some cases, sexual dimorphism is seen in the hormone response to PS-MPs/NPs. For example, in the marine medaka, unlike in the previous examples, steroidogenic enzymes, estrogen, testosterone, LH and FSH increased in males but decreased in females (Wang et al., 2019b), though the mechanism that links MP/NPs to altered steroidogenesis remains unknown.

Studies covering a wide range of aquatic and terrestrial animal species indicated that oxidative stress, due to increased ROS production in cells and tissues, is a major molecular event triggered by MP/NP exposure (Supplementary Tables S1, S2). Increased ROS levels and/or altered expression of oxidative stress defense system were detected upon MP/NP exposure in the marine rotifer Brachionus species, copepods (Tigriopus japonicus) (Xue et al., 2021; Yoon et al., 2021), Daphnia species (Liu et al., 2021b), green mussel (Perna viridis) (Hariharan et al., 2021), marine medaka (Oryzias melastigma), zebrafish (Danio rero) (Qiang and Cheng, 2021), Nile tilapia (Oreochromis niloticus) (Ismail et al., 2021), as well as terrestrial organism including Eisenia sp., rat and mouse. In many instances, increased ROS content of cells and tissues was associated with reproductive defects, defined as decrease in viability, quality and number of oocytes or sperms, or decreased tissue/gonad integrity, as discussed above. ROS in turn induced apoptosis in the gonadal tissue leading to tissue damage, indicated by histopathological changes in reproductive tissues/gonads (Supplementary Table S1).

MPs/NPs could dysregulate the ROS scavenger system causing decreased gene expression or activity of these enzymes. Alternatively, higher ROS levels could deplete cellular ROS scavenging molecules, by increased use of these in battling oxidative stress. Increased oxidative stress in MP/NP exposed organisms would be expected to drive upregulation of the expression and/or activity of ROS-scavenging molecules and detoxification enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase, glutathione (GSH) and glutathione peroxidase, or glutathione-S-transferase. However, depending on the species, type of MP/NP used, exposure conditions and presence of pollutants on the plastics, a range of different responses were recorded. Studies found that expression levels or activity of SOD, CAT, and some components of the glutathione system decreased upon MP ingestion in worms (Huang et al., 2021), in zebrafish brain and liver (Umamaheswari et al., 2021), and the testes, ovaries and fertilized eggs of marine medaka (Wang et al., 2021), as well as in Daphnia pulex (Liu et al., 2020d), and Perna viridis (Hariharan et al., 2021). On the contrary, in the Nile tilapia nanoplastic particles of irregular shape caused increased serum levels of SOD and CAT, although these enzyme levels were not investigated in the reproductive organs of the fish, and the observed male reproductive deficit was most probably due to alterations in the serum luteinizing hormone and testosterone levels (Ismail et al., 2021). PS-MPs caused decreased levels of CAT and SOD in rats’ testes, which were associated with reduced sperm count, sperm motility and viability, probably due to the significant tissue damage seen in this tissue (Ijaz et al., 2021) (Supplementary Table S1). It has been suggested that MP-induced ROS could lead to DNA damage and defects in sperm cells, such as observed in earthworms (Huang et al., 2021), leading to decreased fertility. Interestingly, the availability of increased food supply in aquatic species could suppress the reproductive toxicity of PS-NPs (50 nm diameter spheres) (Yoon et al., 2021) or PE-MPs (10–22 μm) (Xue et al., 2021), affecting ROS levels or the function of endogenous ROS scavenging system altered by MPs and their pollutants. Therefore careful experimental design and replicable experimental conditions are vital for developing understanding of the real-life impact of MP/NP pollution on wild-life reproduction.

Increased ROS could be the consequence of enhanced ROS generation by mitochondria, as MP/NP exposure has been associated with altered mitochondria function. Decreased mitochondria membrane integrity was observed in MP treated rotifers (Jeong et al., 2016; Jeong et al., 2021), whereby increased oxidative stress and concurrent upregulation of the MAPK stress signaling pathway correlated with decreased fecundity (Jeong et al., 2016). Transcriptome analysis of PS-MP treated marine medaka also indicated the activation of MAPK pathways (Chen et al., 2020a) alongside reproductive deficits observed in the fish. Mice that ingested PS-MPs showed increased mitochondria membrane potential with increased ROS content and decreased GSH levels in oocytes, which were developing in inflamed ovaries, leading to overall decrease in reproduction (Liu et al., 2022b). A potential explanation for the observed mitochondrial dysfunction in various species upon MP/NP exposure could be lysosomal accumulation, and subsequent escape of the plastic particles to the cytosol via lysosomal rupture, which could lead to increased mitochondrial Ca²⁺ uptake and initiation of cell death, such as described in the murine RAW264.7 macrophage cells (Xia et al., 2008). Disruption of mitochondrial membrane potential was also observed in PS-MP-treated human epithelial colorectal adenocarcinoma cells (Caco-2) (Wu et al., 2019), suggesting a universal mechanism that could lead to increased ROS production and toxicity in animals upon plastic exposure. The resulting oxidative stress could cause damage to the DNA, lipids and proteins, ultimately leading to cell and tissue defects under sustained high ROS levels. Thus, increased ROS-induced cellular damage or mitochondria dysfunction-mediated cell death could be a probable explanation for reproductive tissue damage responsible to decreased fertility in animals.

It has been suggested that MP/NP-disruption of the blood-testis-barrier (BTB) leading to oxidative stress activates the p38/MAPK-Nrf2 pathway and induces apoptosis of spermatogenic cells, which could be responsible for the reduced reproductive capacity of PS-MP treated Wistar rats (Li et al., 2021c). PS-MP ingestion-induced elevated ROS in the testes of male Balb/c mice, which in turn activated the p38/MAPK stress signaling pathway, causing reproductive toxicity, seen by lower number and decreased motility of sperms, and increased rate of sperm deformity (Xie et al., 2020). Decreased BTB integrity following PS-MP feeding of male Balb/c mice was also linked to ROS-induced imbalance in mTORC1 and mTORC2 signaling, resulting in altered expression of actin cytoskeleton components, ultimately leading to spermatogenesis dysfunction (Wei et al., 2021). Transcriptome and protein expression data of PS-MP exposed mice also suggested an upregulation of the inflammatory signaling pathways, orchestrated by NF-κB. This was shown by increased expression of various inflammatory factors, along with downregulation of the phase II detoxification response regulator Nrf2 (Hou et al., 2021a; Jin et al., 2021), resulting in lower sperm quality in males. The ovaries of female rats fed with PS-MPs showed decreased GSH-Px, CAT, and SOD and increased MDA activities, while the number of growing follicles decreased with concurrent elevated levels of ovarian granulosa cell apoptosis and increased ovarian fibrosis (An et al., 2021b; Hou et al., 2021b). The latter process is thought to be enhanced by ROS initiated activation of Wnt/β-Catenin signaling pathway. Importantly, both ovarian apoptosis and fibrosis could contribute to the depletion of ovarian reserve capacity in female rats upon MP exposure.

Importantly, increased ROS content measured by in vivo dyes or fixative staining of NP/MP affected tissues/animals is broadly observed, even in studies which did not observe reproductive phenotypes (Silva et al., 2021). Furthermore, while reproductive deficits seen upon NP/MP exposure might require longer exposure times to plastic particles, cellular readouts of increased oxidative stress could be observed even after short (24 h) exposure time (Supplementary Tables S1, S2). Most studies reporting on reproductive deficits used polystyrene NPs (Supplementary Table S1), although increased ROS content, and consequently, upregulated oxidative stress responses seem to be the uniform response to NPs and MPs of various physicochemical properties. In support of the general role of ROS and subsequent activation of MAPK signaling pathway in mediating reproductive deficits of MPs/NPs, few studies have shown that supplementing the MP/NP-treated experimental models with antioxidants, such as N-acetylcysteine (NAC), or specific inhibitors of p38 MAPK, could lead to reversing or attenuating the deleterious effects of MPs/NPs on reproductive function (Xie et al., 2020; An et al., 2021b).

C. elegans was utilized widely to develop further understanding of the molecular events associated with MP/NP exposure (Supplementary Table S2). Taking advantage of tissue specific RNAi silencing of individual pathway components as well as utilizing readily available knockout mutants of virtually all genes of its genome, MPs and NPs have been shown to affect a range of signaling pathways in C. elegans. As shown by Shao et al. (Shao et al., 2019), PS-NP exposure (1 μg/L) caused downregulation of the insulin/IGF-1 signaling (IIS) pathway, decreasing expression levels of daf-2 (insulin receptor gene) and increasing the expression of daf-16, encoding for the FoxO orthologue in C. elegans. DAF-16 is a key transcription factor integrating signals from various pathways, including IIS, AMPK pathway, JNK pathway, germline and TOR signaling, to modulate aging and stress, via shuttling from cytoplasm to nucleus (Sun et al., 2017). Decreased insulin signaling leads to altered expression of DAF-16 target genes involved in detoxification response (Freedman et al., 1993; Honda and Honda, 1999). Other studies showed the activation on ERK/MAPK or p38/MAPK in the neurons (Yang et al., 2021) and changes to the JNK/MAPK and the insulin signaling in the intestinal cells of C. elegans upon PS-NP exposure (Qu et al., 2020b; Liu et al., 2021d). These pathways are all hallmarks of oxidative stress response. NP exposure therefore has been linked to changes in the expression level and activity of central transcriptional regulators in C. elegans with well conserved functions and orthologues in mammals, many of which are similarly involved in the MP/NP response. Most of these transcription factors (TFs) are key mediators of longevity and stress response pathways, that orchestrate the organismal response to environmental stimuli and metabolic status of the cells (Denzel et al., 2019). Although some of these have been associated with regulation of reproduction, currently there is no mechanistic link established between PS-NP exposure, changes in TF signaling and altered reproduction and fertility in C. elegans. A recent study using UV-aged PS-MPs in C. elegans provided evidence of increased germline DNA damage and consequent increased apoptosis of germ cells as probable cause for declining reproduction rate in treated animals (Chen et al., 2022). ROS content of worms indeed increased in MP/NP-exposed hermaphrodites across numerous studies (Yu et al., 2021). The incurred oxidative stress could lead to increased germline apoptosis via activation of the MAPK pathway (Salinas et al., 2006).

Altered oxidative stress response is an effect that is not only universally observed in the treated animal populations, but can also be transmitted to the offspring of MP/NP treated mothers along with reproductive deficits, even in the absence of NP/MP in the offspring generation (Zhang et al., 2019; Liu et al., 2020b; Chen et al., 2021b; Liu et al., 2021c; Sobhani et al., 2021; Wang et al., 2021). Long-lasting impact of MPs/NPs on the oxidative stress defense pathways could potentially contribute to neurotoxicity detected in the offspring of plastic treated C. elegans mothers (Chen et al., 2021b; Liu et al., 2021c). In C. elegans, supplementation of sulphate modified PS-NPs in the food led to decreased reproductive rate in four subsequent offspring generation, probably due to higher proportion of aberrant chromosomes formed in the oocytes (Yu et al., 2021), which might be the result of oxidative damage to the DNA of plastic exposed mothers. Increased germline apoptosis was observed in multiple offspring generations of PS-NP exposed C. elegans hermaphrodites, suggesting enhanced germline depletion as explanation for the decreased brood size in offspring (Sun et al., 2021; Yu et al., 2021).

As for the impact of MPs and NPs on NHR signaling and consequent defects in reproductive behavior in the C. elegans model organism, limited research is available in the literature. Even though typical vertebrate hormones that act by binding NHRs have not been identified in C. elegans, this nematode has 284 nuclear hormone receptors, considerably more than humans and mice (Sluder et al., 1999; Taubert et al., 2011; Zhang et al., 2004). Additionally, C. elegans expresses an estrogen receptor (ER) orthologue, NHR-14, and an androgen receptor (AR) orthologue, NHR-69. Molecular docking simulations performed with NHR-14 or NHR-69 have shown that the endogenous hormone ligands of the human ER and AR, 17β-estradiol and testosterone (respectively), have similar binding activity to NHR-14 and NHR-69 as to the human receptors (Jeong et al., 2019). Therefore, these NHRs might be relevant to study in regard of deficits in reproductive behaviors and fertility upon MP and NP treatment, as disturbance in the level of estrogen and androgen receptor ligands have been widely observed upon MP/NP ingestion in other species (as discussed in MPs/NPs Alter Nuclear Hormone Signaling and Biotransformation, Supplementary Table S1) (Wang et al., 2019b; Amereh et al., 2020; Xie et al., 2020; Ijaz et al., 2021; Jin et al., 2021; Wang et al., 2021; Wang et al., 2022). A nuclear hormone receptor that has been investigated in relation to MPs in C. elegans is the sterol-sensing NHR-8, which regulates fat metabolism and stress responses (Jones et al., 2013; Magner et al., 2013). PS-NP exposure significantly increased the expression of nhr-8 in wild-type worms, while loss-of-function nhr-8 mutation increased sensitivity towards PS-NP toxicity, decreased locomotion and increased ROS production (Huanliang et al., 2020). PS-NP exposure also increased the expressions of the intestinal linc-9 long non-coding RNA, which targets the nuclear hormone receptor NHR-77, linking nuclear hormone receptor signaling to MP/NP toxicity. linc-9 RNAi treated C. elegans showed increased susceptibility to PS-NP induced defects, which was diminished upon RNAi knockdown of nhr-77, indicating a functional role for NHR-77 in PS-NP toxicity (Zhao et al., 2021). This implies that MPs and NPs could potentially intersect NHR signaling pathways in worms, even without the additive effect of carried EDCs. Uncovering the MP/NP-driven disruption of NHR signaling in C. elegans may also give us clues as to what potential metabolic defects could be expected in animals due to increased plastic pollution in the environment and in the food chain, and what implication for reproductive behaviors these might have. This will allow us to generate new models of MP/NP action for behaviors and cellular endpoints that might be common to most organisms and for those that are specific for higher organisms.

Altogether, these studies have shown that 1) C. elegans responds to MP/NP toxic insult with altering a set of conserved signaling pathways, including oxidative stress-MAPK, IIS or JNK pathways, as observed in other animals; and 2) nuclear hormone signaling is probably one conserved target of MPs and NPs across species. This provides us with an excellent opportunity to further develop the C. elegans model for high-throughput screening to unravel the mechanistic links of MPs/NPs and reproductive toxicity.