{"paper_id":"344ded0b-082a-4f0f-9f22-7c7ac8ddb144","body_text":"Microplastic-mediated delivery of di-butyl phthalate alters C. elegans lifespan and reproductive fidelity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Microplastic-mediated delivery of di-butyl phthalate alters C. elegans lifespan and reproductive fidelity Chiara Angelyn O. Maldonado, David M. Mares, Paola C. Garcia, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6629868/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Nov, 2025 Read the published version in Microplastics → Version 1 posted You are reading this latest preprint version Abstract Microplastics harbor chemical additives and absorb pollutants from the environment. Microplastics pose a human health threat and have been found in nearly all human tissues. The toxicological pathways and physiological effects of microplastic-mediated chemical exposure following ingestion remain unknown. Here we use C. elegans to investigate the effects of di-butyl phthalate and polystyrene microplastic mixtures on fertility and lifespan. Our studies demonstrate that 1 µm microplastics at 1 mg/L exposure levels result in decreased brood size, whereas 1000 times fewer microplastics (1 µg/L) did not affect the number of eggs laid. While there was no change in brood size at 1 µg/L microplastic exposure levels, there was an increase in embryonic lethality. Microplastics-mediated delivery of di-butyl phthalate to C. elegans significantly reduced brood size and increased embryonic lethality compared to exposure to microplastics alone. This reproductive toxicity is potentially due to a stress response via DAF-16, as observed with microplastics and di-butyl phthalate co-exposure. Furthermore, chronic exposure to microplastics shortened the lifespan of C. elegans , which was further reduced with di-butyl phthalate co-exposure. The exacerbated defects observed with co-exposure to phthalate-containing microplastics underscore the risks associated with microplastics releasing the additives and/or chemicals that they have absorbed from the environment. Biological sciences/Biological techniques/Experimental organisms Biological sciences/Biological techniques/Experimental organisms/Model invertebrates/Caenorhabditis elegans Biological sciences/Developmental biology Biological sciences/Physiology Earth and environmental sciences/Environmental sciences Earth and environmental sciences/Natural hazards Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Plastic and chemical pollutants are ubiquitous in the environment, posing an emerging human health concern. Microplastic (MP) particles have been identified in nearly all human tissues, from blood 1 to the brain 2 , and most recently in human reproductive organs 3 , 4 . Microplastic exposure includes plastics from various environmental sources and exists in a wide range of chemical types, mixtures, and conditions. Primary microplastic particles are manufactured and used as ingredients in biomedical products and various health and beauty items (i.e., scrubs, make-up, cleansers, and toothpaste) 5 , 6 . Whereas secondary microplastics result from the breakdown of larger plastic items. Human exposure to microplastics occurs through direct use of microplastic-containing products, exposure to secondary microplastics released into food, or environmental pollutants. Both primary and secondary microplastics undergo chemical and physical changes in their structure due to environmental exposure. Factors such as UV light, physical breakdown, and exposure to environmental pollutants can lead to alterations in the size, surface structure, and chemical composition of microplastics. Despite efforts to reduce plastic use, it remains prevalent, with much already having entered the ecosystem 7 . Dating back to the 1950s, approximately 400 million tons of plastic waste accumulate each year 8 . Microplastics (MPs, plastic particles < 5 mm 9 ) and nanoplastics (NPs, plastic particles ≤ 1 µm 9 ) are detected in the air, water, and our food sources 10 – 14 . Meanwhile, microplastics have been found in tissue exhibiting the ability to enter mammalian cells 15 . The smallest particles, nanoplastics, invade endosomes, lysosomes, lymph and circulatory systems, and the lungs 16 – 18 , leading to deleterious effects on the cellular level 17 , 18 . The direct health risks of human ingestion of microplastics have not been quantified. Moreover, these risks amplify as plastics already contain additives, and they can absorb, accumulate, and transfer chemicals from the surrounding environment into organisms. Plastic and resulting microplastic particles can be composed of homogenous or heterogeneous polymer mixtures, contain additives from the manufacturing process (plasticizers, by-products, and monomers), and may absorb chemical pollutants 19 – 21 . These additives and pollutants can later leach into the environment 22 . Persistent organic pollutants have been shown to be transferred from plastic particles to fish with adverse effects in environmentally relevant concentrations 23 and led to a transfer of chemicals from microplastics into the guts of lung worms ( Arenicola marina ), and a reduction of biological functions 24 . In mice, it was demonstrated that phthalate esters were released from microplastics leading to intestinal permeability and inflammation 25 . A study using C. elegans showed that 19 chemicals (including phthalates and agrochemicals) increased DNA damage and physiological dysregulation when compared to Bisphenol A (BPA) 26 . BPA has been widely recognized as a threat to human health, and its use has been highly restricted, with a total ban on its use in infant-related products 27 , 28 . Due to these concerns, most manufacturers have voluntarily removed BPA from consumer products. With these regulations and demands from consumers, manufacturers must rely on other plasticizers to aid in the performance and functionality of plastic products. Di-butyl phthalate (DBP), a widely used plasticizer, can compose 20–80% of some plastic products 21 and its use is regulated by both US and European agencies 29 . DBP is classified as a phthalate esters, which as a group, are considered endocrine-disrupting chemicals and pose cytotoxic and estrogenic effects 28 , 30 – 32 . DBP is most commonly used as a plasticizer, primarily in polyvinyl acetate emulsion adhesives, as a solvent for oil-based dyes, and insecticides 33 . It can be found in consumer products including nail polish, paints, adhesives, and perfume oils. Prior to 2008, many products for children contained high levels of DBP, butyl benzyl phthalate (BBP), and di (2-ethyexyl) phthalate (DEHP). The United States and European federal restrictions now limit the use of some phthalates, including DBP, in children’s products. Despite bans on some products, DBP and other phthalate esters are widely used in everyday household items or manufacturing processes. Many household products contain these chemical additives, which, when disposed of, will likely end up in municipal landfills 33 . Thus, DBP could leach into the environment due to the improper disposal and breakdown of products containing it. Once in the environment, DBP absorbs onto suspended particles and is less likely to degrade than dissolved DBP 33 . DBP has a high likelihood of being absorbed into MPs in the environment as it is readily absorbed by plastic particles, possesses properties that make it absorptive, and it is chemically related to compounds that are easily absorbed into plastics 34 . Despite the risks of microplastic exposure to humans and the danger of plastic particles carrying chemical pollutants, microplastics remain under-investigated. It is crucial to determine how microplastic-chemical mixtures alter the development and health of humans and other land animals. We use the C. elegans model system to examine the cellular and physiological effects of nanoplastic mixtures on whole-organism health and development. In this study, we investigate whether 1 µm polystyrene microplastics can transport absorbed chemicals into an animal after ingestion and lead to physiological responses. The 1 µM polystyrene size regime is ideally situated at the interface of micro- and nano-particles, and because polystyrene is commonly used in packing foams, food containers, and disposable cutlery, it is highly relevant to human oral microplastic exposure risks 21 . The additive, DBP, was chosen as a representative chemical due to its wide use as an industrial additive. Studies of additives commonly found in plastics show that the many common plasticizers with relatively low molecular weight will transfer from the plastic material into foods 21 . We hypothesized that microplastics can contain a chemical pollutant and mediate physiological defects that are greater than either pollutant alone. We find that microplastic exposure reduces brood size and leads to embryonic lethality in a concentration-dependent manner. The microplastic-mediated delivery further decreased reproduction compared to microplastics or DBP alone. Reproductive defects due to microplastic -DBP co-exposure appear to cause stress via DAF-16 and reduce C. elegans lifespan. Results and Discussion Polystyrene microplastic surface structure is unchanged after exposure to di-butyl phthalate. To verify the diameter and surface structure characteristics of polystyrene microplastics (PS MP, hereon referred to as microplastics), scanning electron microscopy (SEM) was used to image microplastics with and without exposure for 24 hours to 0.1 M di-butyl phthalate (DPB) or the solvent (DMSO). SEM analysis revealed no change to the surface morphology of microplastics exposed to DBP for 24 hours. A small but significant increase in the average size of microplastic particles occurred when soaked in DMSO, but not 0.1 M DBP (Fig. 1 , p ≤ 0.0001). It was anticipated that exposure to the plasticizer DBP may change the size or surface structure of microplastics. Environmental exposure of microplastics to heat, UV and mechanical stress can change their characteristics 20 , 35 , and this was not observed after 24 hour exposure of microplastics to DBP or DMSO (Fig. 1 ). DBP has high rates of sorption into microplastics, with highest rates seen in polystyrene (PS) microplastic when compared to polyvinyl chloride (PVC) and polyethylene (PE) 34 . Sorption of compounds on or into microplastics is size dependent, with smaller particles containing more readily available chemicals, due to the increase in surface to size ratio that occurs with the reduced particle size 20 , 36 , 37 . Microplastic and DBP mixtures were prepared by soaking microplastics in DBP for 24 hours, followed by dilution into C. elegans food, E. coli OP50. In each dilution step, microplastics were sonicated to aid in resuspension. This process would disrupt any DBP that would be adsorbed onto the microplastic surface, and thus any DBP present in the microplastic is assumed to be absorbed within the microplastic particles. The slightly larger but non-significant increase in mean size of the 0.1 M DBP soaked microplastics and significantly larger diameter of microplastics after DMSO exposure may be due to swelling of the microplastic particles after absorption. Polystyrene microplastics are ingested by C. elegans and act as a vehicle for chemical exposure. C. elegans exposed to 1 µg/L and 1 mg/L Dragon-Green microplastic show the presence of microplastics in their gut tube (Fig. 2 a). Nematodes were initially exposed to two different doses (1 µg/L and 1 mg/L), which were within the range of other studies and that mimic concentrations determined from human samples such as blood 1 , 38 – 41 . Microplastics were present at all larval life stages, and the percentage of total worms containing microplastics increased with life stage and exposure time. By adulthood, nearly all worms contained microplastics (Fig. 2 c). These results are consistent with other studies demonstrating that microplastics (> 1 µm) accumulate in the gut, while smaller nanoplastic (< 1 µm) particles can cross cell membranes 15 , 40 , 42 – 44 . When examining the eggs of adult nematodes exposed to Dragon-Green microplastic, no microplastics were observed (Fig. 2 b). Smaller microplastics, less than 1 µm in size, in the nanometer range, have been observed to cross into the body cavity in C. elegans 42 . Microplastics contain chemical additives or may absorb pollutants in the environment that can then be transferred into living organisms, leading to physiological effects 21 , 34 . Phthalates, including DBP, are classified as endocrine disruptors that can be long-lived in the environment 21 , 36 . DBP is most commonly used as a plasticizer in PVC followed by PET 21 . While DBP is not commonly used in PS products, we choose to study it as a potential environmental chemical that could be absorbed in microplastics and transferred into an organism after ingestion. To visualize this, we soaked PS microplastics in the lipophilic dye, Nile Red, to ask if microplastics could mediate the delivery of a chemical into an organism. Nematodes exposed to microplastics that have been soaked in Nile Red do not show puncta, indicating the location of each microplastic, but rather there is diffuse staining in the worm (Fig. 3 a). This indicates that the Nile Red dye, which had been absorbed into the PS microplastic, transfers into the worm’s body after ingestion. This transfer is present in more than 90% of the worms examined (Fig. 3 b). When nematodes are exposed to an equivalent amount of Nile Red without microplastics, the Nile Red is not present in the worm’s body. Thus, the microplastics mediate the delivery of Nile Red, which is then absorbed. This mimics what one would expect if microplastics can act as a vector to deliver chemicals into an organism after ingestion. Exposure to polystyrene microplastics and polystyrene microplastic DBP mixtures results in reproductive toxicity. To determine if microplastics and chemical pollutants cause reproductive toxicity, C. elegans were exposed to different concentrations of microplastics and DBP for varying times. When L1 life stage, nematodes were exposed to 1 µm MP (at 1 µg/L concentration) for 48 hours, until the young adult stage, or continuously, there was no observed significance in decreased total eggs laid or hatched (Fig. 4 a, S1a). Despite no change to the number of eggs laid, chronic microplastic exposure to nematodes significantly increased embryonic lethality (p = 0.04) and the total number of eggs unhatched per nematode (p = 0.007) (Fig. 4 b, S1b). Embryonic lethality is determined by taking the total number of eggs that are unhatched and dividing by the total number of eggs laid per worm. These results are in range with other studies that observed continuous microplastics of the same or smaller size leading to minimal or no change in brood size, with no embryonic lethality 44 . While some studies noted a decrease in brood size with exposure to microplastics, this difference may be due to a different size or composition of microplastics or varying exposure conditions 45 . When we exposed C. elegans to a 1000x higher concentration of microplastics (1 mg/L), we observed a significant reduction in the total number of eggs laid (p = 0.016), but this did not lead to an increase in embryonic lethality (Fig. 4 a,b). C. elegans exposure to microplastics (1 µg/L) harboring 0.1 M DBP, further reduced the number of eggs laid per worm compared to exposure to microplastics alone (p = 0.017, Fig. 4 c). This combined exposure did not lead to an increase in embryonic lethality (Fig. 4 d). Examination of microplastics loaded with either 100 µM or 3.7 mM DBP did not affect brood size, egg hatching, or embryonic lethality (Fig. 4 e,f and S2 a-f). Typical concentrations of DBP used as a plasticizer can range from 10–35%, or higher in some products 21 . Our study employed 0.1 M DBP, a concentration equivalent to less than 10% w/w, which is in the range of DBP found in everyday products. While 0.1 M DBP led to defects in fertility, these were not seen with lower concentrations (100 µM and 3.7 mM). Comparable levels of chemical pollutants could reach organisms through the ingestion of microplastics that have absorbed chemicals from the environment or by the leaching of additives from microplastics that initially contained higher chemical amounts. In particular, it is worth noting that recycled plastics can contain additives from recycled feedstocks 46 . Impaired reproduction with exposure to microplastics alone or in combination with DBP could be caused by various factors. DBP is an endocrine disrupting chemical and leads to cryptogenic effects 32 . Exposure to DBP leads to defects in reproduction and development 29 . Specifically, DBP exposure reduced human sperm function and in rodent studies defects occur in both development and reproduction 47 – 50 . In C. elegans , exposure to 100 µM DBP, in the absence of MP, led to increased embryonic lethality and DNA damage 26 . C. elegans exposure to DBP alone, at a lower dose (500 µM) than used in this study, leads to a reduction in the number of eggs laid and increased embryonic lethality 26 . These defects were attributed to defects in early embryogenesis, with elevated levels of DNA double-strand breaks, activation of a DNA damage checkpoint, and impaired embryogenesis 26 . Errors in cell division could reduce the ability of eggs to hatch and could be caused by a multitude of sources 51 . In humans and other organisms, these errors may lead to aneuploidy, spontaneous abortions, and birth defects 26 , 51 , 52 . Additionally, a reduction in the total number of eggs laid could be attributed to a nutrient deficiency of the parent caused by intestinal damage from polystyrene microplastic exposure 45 , 53 – 56 . Another study suggested that the associated defects with polystyrene microplastic exposure could be due to a reduction in ATP levels and a reduced energy budget toward reproduction 57 . The exacerbated defects we observed with co-exposure to MPs containing 0.1 M DBP demonstrate that microplastics can mediate the delivery of a chemical into an organism after ingestion. This microplastic-mediated delivery of DBP underscores the risks of co-exposure when microplastics can release the additives they already contain and/or chemicals that they have absorbed from the environment. Exposure to polystyrene microplastics and polystyrene microplastic DBP mixtures elicits a stress response. A possible mechanism of polystyrene microplastic toxicity resulting in a reduction in fertility is the generation of reactive oxygen species (ROS). Polystyrene microplastic exposure leads to higher levels of reactive oxygen species 16 , 39 , 53 and increased expression of GST-4 (glutathione S-transferase 4, an enzyme that is involved in clearing ROS) in nematodes 44 , 55 . Additionally, DBP and other phthalate esters cause DNA damage and chromosomal abnormalities 26 , 52 , 58 that can be caused by ROS generation 52 . In response to stress, C. elegans activate a DAF-16 stress response that leads to the activation of genes encoding proteins involved in response to oxidative stress 59 . Therefore, to investigate if stress may be an underlying mechanism of reduced fertility, we asked if microplastic-mediated DBP exposure in C. elegans would initiate a DAF-16 stress response. The C. elegans strain TJ356 contains a reporter GFP fused to DAF-16 that is driven by the daf-16 promoter 60 . This GFP reporter is cytoplasmic under normal conditions and relocates to the nucleus to activate genes involved in an anti-oxidative response, under stress. Therefore, the localization of this DAF-16 GFP reporter can be used as a readout of a DAF-16-mediated stress response 39 , 44 , 61 . Under control conditions, when C. elegans were not exposed to microplastics, the majority of worms examined had cytoplasmic DAF-16 localization (Fig. 5 a,b). Some worms showed an intermediate localization where less than 30% of the nuclei showed nuclear expression. This same expression pattern is present with C. elegans exposure to 1 µg/L microplastics (Fig. 5 b). These results are consistent with what others see with similar PS microplastic exposure 44 . In contrast, microplastic-mediated DBP exposure lead to a significant activation of the DAF-16 stress response, with 40% of the total worms showing an intermediate expression pattern and nearly 20% nuclear localization (Fig. 5 b). This indicates that the DAF-16 transcription factor is located in the nucleus where it will activate genes in response to oxidative stress. Importantly, exposure to microplastics containing DMSO, the solvent for DBP, or an equivalent amount of DBP or DMSO alone, did not affect DAF-16 localization (Fig. 5 b). Expression of DAF-16 in these controls, showed no significant change when compared to the negative control ( E. coli OP50 only) and microplastic exposure alone (Fig. 5 b). In our initial reproductive toxicity studies, we observed that higher concentrations of MPs (1 mg/L) induced a reduction in total eggs laid with no effect on embryonic lethality (Fig. 4 a,b). Therefore, we asked if microplastics alone, at a higher concentration, would elicit a stress response via DAF-16. Continuous exposure of C. elegans to 1 mg/L microplastics increased the percentage of worms with intermediate and nuclear expression of DAF-16 (Fig. 5 b). This indicated that the concentration of microplastic exposure can mitigate the effect. In contrast to our results, Leon et al observed a stress response with C. elegans exposure to 100 nm PS microplastics at a concentration of 10 mg/L 44 . This may be due to differences in the size of the tested plastic particles. The stress response observed in Leon et al was with microplastics of the same composition, polystyrene, but with a smaller diameter (100 nm) and at 10,000 x greater concentration than in this study. Indeed, we observed that 1 µm microplastics at our higher, 1 mg/L, concentration elicited a DAF-16 stress response. This indicates that a stress response via DAF-16 may be concentration-dependent. While we did not see a DAF-16-mediated stress response with our primary level of microplastic exposure (1 µM/L) alone, a significant response was elicited when DBP delivery was mediated with this same concentration of microplastics. Interestingly, Leon et al observed that with the removal of worms from microplastic exposure, the microplastics left the worm’s body, and the DAF-16 stress response reduced. We did not test for the effect of DAF-16 localization with the removal of microplastic exposure from C. elegans , but it would be interesting to ask if the microplastic-mediated DBP stress response via DAF-16 would be maintained, as the microplastics might clear the worm body, while the DBP is absorbed. Exposure to polystyrene microplastics and microplastic DBP mixtures reduces life span. In some cases, the activation of a DAF-16 stress response is associated with an extended life span 59 , 62 – 64 , and this response can be tissue-specific, with greater extension of lifespan associated with intestinal-specific DAF-16 activation 65 . Increased longevity induced by stress events is associated with the insulin/insulin-like growth factor (IGF-1) receptor signaling pathway, or diet, amongst other pathways 64 . In particular, due to the presence of microplastic accumulation in the gut of the worm (Fig. 2 a), and studies indicating intestine-specific stress due to microplastic exposure 56 , we wondered if the presence of microplastics may reduce nutrient intake and increase lifespan. We next addressed whether microplastics or microplastic-mediated DBP exposure would alter C. elegans lifespan. Short-term exposure (48 hours) of C. elegans to 1 µm microplastic (1 µg/L) did not change the lifespan of animals compared to the control (Fig. 6 a). Interestingly, when nematodes were exposed to the same conditions, but without removal after 48 hours, there was a significant reduction in lifespan (p < 0.0001) (Fig. 6 a, S3a,b). This reduction in lifespan was equal to what was observed with continuous exposure to 1000 x more concentrated microplastics (1 mg/L) with continuous exposure (Fig. 6 a). The lifespan of animals was further reduced when exposed to microplastics that mediated the delivery of 0.1 M DBP (p = 0.00357) (Fig. 6 b, S3c). This reduction in lifespan was not observed under the control conditions of microplastics containing DMSO, or with exposure to DBP or DMSO alone (Fig. 6 b, S3c). Additionally, the reduction in lifespan with microplastic-mediated DBP exposure appears to be dependent on the concentration of DBP, as the lowest concentration of DBP (100 µM) used in this study did not lead to a further reduction in lifespan as compared to microplastic exposure alone (Fig. S3c). There are conflicting studies on the effect of microplastic exposure on C. elegans lifespan. In one study, polystyrene microplastics initiated a DAF-16 stress response that was not associated with a change in lifespan in C. elegans , and this was speculated to be due to a counteracting mechanism 44 . Other reports were consistent with our results, showing reduced lifespan with exposure to polystyrene microplastics 39 . Furthermore, our work shows that microplastic-mediated delivery of DBP further reduced lifespan. Additional reports examined the effects of leachates from plastic 66 , phthalates 67 , 68 , and other types of MP exposure on life span, all leading to reduced longevity. This work shows that microplastic-mediated delivery of plastic-containing compounds, such as DBP, enhances the physiological defects observed with microplastic exposure alone. The exposure level of microplastics, the time of exposure, and the concentration of the absorbed chemicals influence the defects observed. Overall, our results show that chronic microplastic exposure has detrimental effects on reproduction and reduces lifespan. These defects are further exacerbated by the co-exposure of microplastics and DBP. Microplastics mediate the delivery of DBP in C. elegans , further decreasing fertility and lifespan as well as leading to a stress response via DAF-16 activation. Further studies are needed to identify other mechanisms involved in the toxicity due to microplastic-mediated exposure to DBP. The worsened physiological defects seen with co-exposure to phthalate-containing microplastics demonstrate that microplastics can leach contents into an organism after ingestion. This highlights the risks of microplastics releasing the additives they already contain and/or chemicals that they have absorbed from the environment. Methods Characterization of microplastics Polystyrene microplastic (Poly Sciences, 19518-500) size and surface were characterized by Scanning Electron Microscopy (JEOL JSM-6010 PLUS/LA). Samples were sputter-coated with 2 nm gold particles prior to imaging (Quorum Technologies EMS150T ES). Microplastic diameter was measured using the measuring tool in the JOEL associated software. Preparation of microplastic and chemical mixtures Microplastics were suspended in E. coli ( strain OP50), seeded on Nematode Growth Media (NGM) plates (Teknova, N1105) and allowed to dry overnight before adding nematodes to the plates for exposure. To prepare microplastic E. coli suspensions, microplastics were suspended in a 1% solution with M9 buffer, then diluted into E. coli OP50. At each dilution step, the solution was sonicated for 15 minutes (Emerson Branson 3800). Wide-boar tips were used to transfer liquid. Dry 1 µM polystyrene microspheres (Poly Sciences, 19518-500) were used for all experiments unless otherwise noted. Di-butyl phthalate (DBP, Sigma, 524980) was diluted into 100% DMSO (Sigma, D8418) at 0.1 M concentration. Additional dilutions were made from the 0.1 M stock into water. DBP polystyrene microplastic mixtures were prepared by adding DBP (either 0.1 M, 3.7 mM, or 100 µM concentration) to dry polystyrene microplastics, to create a 1% solution, which was incubated overnight, on a rotating rack, in the dark, at room temperature (19–22°C). DMSO polystyrene microplastic controls were prepared by adding 100% DMSO, 3.7% DMSO, or 0.0001% DMSO to dry polystyrene microplastics at 1%. Polystyrene microplastics were diluted into E. coli OP50 to a final concentration of either 1 µg/L or 1 mg/L. Maintenance of C. elegans Caenorhabditis elegans (C. elegans) stains, N2 (wild type) and TJ356 (zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]) that were used in this study were provided by the Caenorhabditis Genome Center. Worms were grown on Nematode Growth Media Agarose (NGM) plates seeded with E. coli OP50 at 20°C. Exposure design Bleach-synchronized L1 larvae were exposed to E. coli OP50 containing 1 µm polystyrene microplastics at either 1 µg/L or 1 mg/L, or polystyrene microplastics soaked in DBP (100 µM, 3.7 mM or 0.1 M) or DMSO (0.1%, 3.7%, or 100%) for 24 hours, or DBP or DMSO added directly to the OP50 E. coli alone on NGM plates made within 48 hours. DBP and DMSO only control concentrations were determined by the amount of solution carried into the final E. coli OP50, after dilution from the prepared 1% polystyrene microplastics. From a 1% polystyrene microplastic in 0.1 M DBP solution that was diluted 1:10,000 into E. coli OP50 for a final concentration of 1 µg/L microplastic, would have an associated 10 µM DBP control in E. coli OP50. L1s were exposed for 48 hours and then moved to E. coli OP50 plates for short-term exposure and maintained on prepared plates until the end points for experimental conditions. At least 2 biological replicates were performed for each assay. Visualizing microplastics in C. elegans To visualize microplastic distribution and location, L1 bleach-synchronized worms were exposed to 1 µm internally dyed Dragon Green polystyrene microspheres (Bangs Laboratories Inc., FS03F). L1 synchronized larvae were exposed to E coli OP50 and 1 µM/L and 1 mM/L Dragon Green microplastics suspended in E. coli OP50 and seeded on NGM plates. Worms were paralyzed in levamisole on an agarose pad before imaging at each life stage. Visualizing Nile Red in C. elegans To assess if chemical compounds absorbed by microplastic beads could transfer into the worm’s body after ingesting the chemically soaked beads, we fed L1 synchronized C. elegans microplastic beads soaked in 1 mg/L Nile Red in methanol (Sigma Aldrich, 19123). The beads were rocked for 1 hour and left to dry overnight, before being suspended in a 1% solution in M9 buffer. The 1% M9 solutions were diluted to either 1 µg/L or 1 mg/L into OP50 E. coli before seeding on NGM plates and left to dry overnight. Lifespan Assay Lifespan assays were performed at 20°C as previously described. Approximately 50 synchronized L1 larvae were placed onto corresponding treatment conditions for 48 hours and then moved to new plates containing the same treatment, with 100 µM 5’-fluorordeoxyuridine. Worms were counted as dead or alive every day until the number of live worms reached zero. Evaluation of reproductive toxicity To assay the total number of eggs laid per worm and the percentage hatched, bleach-synchronized L1 larvae were seeded onto each condition. 48 hours after seeding, L4 larval worms were then singled out onto respective plates. The following day, L4 singling is repeated from the previous day’s plate, and both plates are kept. After 24 hours, L1s and embryos are counted from the first day of singling, then repeated every day, counting the L4-free plate, until the worm stops laying (around 5–6 days). Imaging and microscopy Fluorescent and bright field images were all taken using an EVOS M5000 (Invitrogen). For live imaging (Dragon-Green polystyrene microplastic localization, Nile Red soaked polystyrene microplastics transfer, and DAF-16-GFP localization), worms were transferred to an agarose pad on a slide and immobilized with levamisole. All image processing and analysis was done with ImageJ software. Activation of stress response To determine if microplastic and chemical exposure lead to stress activation, the strain TJ356, expressing DAF-16 fused to GFP was exposed to polystyrene microplastics alone, and with DBP or DMSO, and DBP and DMSO alone. L1 synchronized larvae were placed on each condition and after 72 hours of exposure, imaged live. Live imaging was completed within 5 minutes of paralysis for each strain. Each nematode was classified by the localization of DAF-16::GFP expression (cytoplasmic, intermediate or nuclear) 61 , 62 . Statistical Analysis Statistical analysis and figure design were done with GraphPad Prism 8 (GraphPad Software, CA, USA). Data were checked for normality between treatments and statistical analysis was done using Kruskal-Wallis followed by Dunn’s post hoc test (microplastic diameter, brood size, polystyrene microplastic exposure). A t-test (two-tailed) was done to analyze control vs. Nile Red data. Experiments were all carried out with a minimum of 2–3 biological replicates (N) and number of total worms (n) examined that are used in statistical analysis as described. Declarations Acknowledgements We thank V. Culotta for valuable discussions and advice on the development and design of experiments. Many undergraduate student researchers have played a part in moving this project forward, and we thank them for their time and valuable input. We also thank St. Mary’s University for its continued support, including funds from the Department of Biological Sciences, the Benjamin F. Biaggini Endowment, and San Antonio Area Foundation. C. elegans strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Research funding was provided by the National Institutes of Health, NIGMS (R16GM150406). C. Maldonado, P. Garcia, and M. Flores were funded by NIH grants (T34GM00873 and T34GM149455). Alyssa was funded by an NIH grant (R25GM102783). Author contributions C.M. planned and evaluated results on brood size and longevity, and wrote the paper. D.M. provided invaluable technical help throughout, performed experiments, and evaluated results. P.G. planned and carried out experiments on brood size and longevity. M.G. planned and performed fluorescent analysis. M.F. planned experiments and performed chemical transfer assays. A.F. and R.L.P. planned and carried out experiments to validate plastic particles using SEM. J.C.H. conceptualized, planned, trained students, executed some of the experiments, evaluated all results, and wrote the paper. All authors provided feedback on the manuscript. Competing interests We have no competing interests to disclose. Materials & Correspondence All requests and correspondence should be addressed to Jennifer C. Harr, PhD. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Leslie, H. A. et al. Discovery and quantification of plastic particle pollution in human blood. Environ Int 163 , 107199 (2022). https://doi.org/10.1016/j.envint.2022.107199 Nihart, A. J. et al. Bioaccumulation of microplastics in decedent human brains. Nat Med (2025). https://doi.org/10.1038/s41591-024-03453-1 Hu, C. J. et al. Response to Comment on: \"Microplastic presence in dog and human testis and its potential association with sperm count and weights of testis and epididymis\". Toxicol Sci (2024). https://doi.org/10.1093/toxsci/kfae137 Montano, L. et al. First evidence of microplastics in human ovarian follicular fluid: An emerging threat to female fertility. Ecotoxicol Environ Saf 291 , 117868 (2025). https://doi.org/10.1016/j.ecoenv.2025.117868 GESAMP. GESAMP Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection. Sources, Fate and Effects of Microplastics in the Marine Environment: Part 2 of a Global Assessment. 93 , 220 (2016). Protection), G. I. F. U.-I. U. W. I. U. U. U. J. G. o. E. o. t. S. A. o. M. E. Report of the forty-second session of GESAMP. GESAMP Reports & Studies Series 92 , 57 (2016). Yates, J. et al. Plastics matter in the food system. Commun Earth Environ 6 , 176 (2025). https://doi.org/10.1038/s43247-025-02105-7 Geyer, R., Jambeck, J. R. & Law, K. L. Production, use, and fate of all plastics ever made. Science Advances 3 (2017-07). https://doi.org/10.1126/sciadv.1700782 Frias, J. P. G. L. & Nash, R. Microplastics: Finding a consensus on the definition. Marine Pollution Bulletin 138 (2019/01/01). https://doi.org/10.1016/j.marpolbul.2018.11.022 Danopoulos, E., Twiddy, M. & Rotchell, J. M. Microplastic contamination of drinking water: A systematic review. PLOS ONE 15 (Jul 31, 2020). https://doi.org/10.1371/journal.pone.0236838 De-la-Torre, G. E. & De-la-Torre, G. E. Microplastics: an emerging threat to food security and human health. Journal of Food Science and Technology 2019 57:5 57 (2019-10-19). https://doi.org/10.1007/s13197-019-04138-1 Qian, N. et al. Rapid single-particle chemical imaging of nanoplastics by SRS microscopy. Proc Natl Acad Sci U S A 121 , e2300582121 (2024). https://doi.org/10.1073/pnas.2300582121 Yates, J. et al. A systematic scoping review of environmental, food security and health impacts of food system plastics. Nat Food 2 , 80-87 (2021). https://doi.org/10.1038/s43016-021-00221-z Chen, Q. et al. Long-range atmospheric transport of microplastics across the southern hemisphere. Nat Commun 14 , 7898 (2023). https://doi.org/10.1038/s41467-023-43695-0 Lehner, R., Weder, C., Petri-Fink, A. & Rothen-Rutishauser, B. Emergence of Nanoplastic in the Environment and Possible Impact on Human Health. Environmental Science & Technology 53 (January 10, 2019). https://doi.org/10.1021/acs.est.8b05512 Hussain, N., Jaitley, V. & Florence, A. T. Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. Advanced Drug Delivery Reviews 50 (2001/08/23). https://doi.org/10.1016/S0169-409X(01)00152-1 Berntsen, P. et al. Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells. Journal of The Royal Society Interface 7 (2010-6-6). https://doi.org/10.1098/rsif.2010.0068.focus Fröhlich, E. et al. Cytotoxicity of nanoparticles independent from oxidative stress. The Journal of Toxicological Sciences 34 (2009/08/01). https://doi.org/10.2131/jts.34.363 Teuten, E. L. et al. Transport and release of chemicals from plastics to the environment and to wildlife. Philos Trans R Soc Lond B Biol Sci 364 , 2027-2045 (2009). https://doi.org/10.1098/rstb.2008.0284 Alijagic, A. et al. The triple exposure nexus of microplastic particles, plastic-associated chemicals, and environmental pollutants from a human health perspective. Environment International 188 (2024). https://doi.org/ARTN 108736 10.1016/j.envint.2024.108736 Hahladakis, J. N., Velis, C. A., Weber, R., Iacovidou, E. & Purnell, P. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. Journal of Hazardous Materials 344 , 179-199 (2018). https://doi.org/10.1016/j.jhazmat.2017.10.014 Yu, Y. et al. Various additive release from microplastics and their toxicity in aquatic environments. Environmental Pollution 343 (2024/02/15). https://doi.org/10.1016/j.envpol.2023.123219 Rochman, C. M., Hoh, E., Kurobe, T. & Teh, S. J. Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress. Sci Rep 3 , 3263 (2013). https://doi.org/10.1038/srep03263 Browne, M. A., Niven, S. J., Galloway, T. S., Rowland, S. J. & Thompson, R. C. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. Curr Biol 23 , 2388-2392 (2013). https://doi.org/10.1016/j.cub.2013.10.012 Deng, Y. et al. Microplastics release phthalate esters and cause aggravated adverse effects in the mouse gut. Environ Int 143 , 105916 (2020). https://doi.org/10.1016/j.envint.2020.105916 Shin, N., Cuenca, L., Karthikraj, R., Kannan, K. & Colaiacovo, M. P. Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans. PLoS Genet 15 , e1007975 (2019). https://doi.org/10.1371/journal.pgen.1007975 FDA. Bisphenol A (BPA) , <https://www.fda.gov/food/food-packaging-other-substances-come-contact-food-information-consumers/bisphenol-bpa> (2023). Rochester, J. R. Bisphenol A and human health: a review of the literature. Reprod Toxicol 42 , 132-155 (2013). https://doi.org/10.1016/j.reprotox.2013.08.008 Monti, M., Fasano, M., Palandri, L. & Righi, E. A review of European and international phthalates regulation: focus on daily use products. Eur J Public Health 32 (2022). Siracusa, J. S., Yin, L., Measel, E., Liang, S. & Yu, X. Effects of bisphenol A and its analogs on reproductive health: A mini review. Reprod Toxicol 79 , 96-123 (2018). https://doi.org/10.1016/j.reprotox.2018.06.005 Sicinska, P., Mokra, K., Wozniak, K., Michalowicz, J. & Bukowska, B. Genotoxic risk assessment and mechanism of DNA damage induced by phthalates and their metabolites in human peripheral blood mononuclear cells. Sci Rep 11 , 1658 (2021). https://doi.org/10.1038/s41598-020-79932-5 Mankidy, R., Wiseman, S., Ma, H. & Giesy, J. P. Biological impact of phthalates. Toxicol Lett 217 , 50-58 (2013). https://doi.org/10.1016/j.toxlet.2012.11.025 TOXICOLOGICAL PROFILE FOR DI-n-BUTYL PHTHALATE. Agency for Toxic Substances and Disease Registry , 225 (2001). Liu, F.-f., Liu, G.-z., Zhu, Z.-l., Wang, S.-c. & Zhao, F.-f. Interactions between microplastics and phthalate esters as affected by microplastics characteristics and solution chemistry. Chemosphere 214 (2019/01/01). https://doi.org/10.1016/j.chemosphere.2018.09.174 Andrady, A. L. et al. Oxidation and fragmentation of plastics in a changing environment; from UV-radiation to biological degradation. Sci Total Environ 851 , 158022 (2022). https://doi.org/10.1016/j.scitotenv.2022.158022 Yan, Y. et al. Dibutyl phthalate release from polyvinyl chloride microplastics: Influence of plastic properties and environmental factors. Water Res 204 , 117597 (2021). https://doi.org/10.1016/j.watres.2021.117597 Mao, S. & He, C. Effect of particle size and environmental conditions on the release of di(2-ethylhexyl) phthalate from microplastics. Chemosphere 345 , 140474 (2023). https://doi.org/10.1016/j.chemosphere.2023.140474 Lenz, R., Enders, K. & Nielsen, T. G. Microplastic exposure studies should be environmentally realistic. Proc Natl Acad Sci U S A 113 , E4121-4122 (2016). https://doi.org/10.1073/pnas.1606615113 Qiu, Y., Liu, Y., Li, Y., Li, G. & Wang, D. Effect of chronic exposure to nanopolystyrene on nematode Caenorhabditis elegans. Chemosphere 256 , 127172 (2020). https://doi.org/10.1016/j.chemosphere.2020.127172 Peng, M., Felix, R. C., Canario, A. V. M. & Power, D. M. The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts. Sci Total Environ 914 , 169979 (2024). https://doi.org/10.1016/j.scitotenv.2024.169979 Liu, Z. et al. Effects of microplastics on the innate immunity and intestinal microflora of juvenile Eriocheir sinensis. Sci Total Environ 685 , 836-846 (2019). https://doi.org/10.1016/j.scitotenv.2019.06.265 Jeong, A., Park, S. J., Lee, E. J. & Kim, K. W. Nanoplastics exacerbate Parkinson's disease symptoms in C. elegans and human cells. Journal of Hazardous Materials 465 (2024/03/05). https://doi.org/10.1016/j.jhazmat.2023.133289 Mueller, M. T. et al. Surface-Related Toxicity of Polystyrene Beads to Nematodes and the Role of Food Availability. Environ Sci Technol 54 , 1790-1798 (2020). https://doi.org/10.1021/acs.est.9b06583 Errazuriz Leon, R. et al. Photoaged polystyrene nanoplastics exposure results in reproductive toxicity due to oxidative damage in Caenorhabditis elegans. Environ Pollut 348 , 123816 (2024). https://doi.org/10.1016/j.envpol.2024.123816 Lei, L. et al. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. Sci Total Environ 619-620 , 1-8 (2018). https://doi.org/10.1016/j.scitotenv.2017.11.103 Pivnenko, K., Eriksen, M. K., Martin-Fernandez, J. A., Eriksson, E. & Astrup, T. F. Recycling of plastic waste: Presence of phthalates in plastics from households and industry. Waste Manag 54 , 44-52 (2016). https://doi.org/10.1016/j.wasman.2016.05.014 Gray, L. E., Jr., Laskey, J. & Ostby, J. Chronic di-n-butyl phthalate exposure in rats reduces fertility and alters ovarian function during pregnancy in female Long Evans hooded rats. Toxicol Sci 93 , 189-195 (2006). https://doi.org/10.1093/toxsci/kfl035 Sen, N., Liu, X. & Craig, Z. R. Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reprod Toxicol 53 , 15-22 (2015). https://doi.org/10.1016/j.reprotox.2015.02.012 Alam, M. S. et al. Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate. Reproduction 139 , 427-437 (2010). https://doi.org/10.1530/REP-09-0226 Okayama, Y. et al. In Utero Exposure to Di( n-butyl)phthalate Induces Morphological and Biochemical Changes in Rats Postpuberty. Toxicol Pathol 45 , 526-535 (2017). https://doi.org/10.1177/0192623317709091 Schneider, I. & Ellenberg, J. Mysteries in embryonic development: How can errors arise so frequently at the beginning of mammalian life? PLoS Biol 17 , e3000173 (2019). https://doi.org/10.1371/journal.pbio.3000173 Hornos Carneiro, M. F. et al. Antioxidant CoQ10 Restores Fertility by Rescuing Bisphenol A-Induced Oxidative DNA Damage in the Caenorhabditis elegans Germline. Genetics 214 , 381-395 (2020). https://doi.org/10.1534/genetics.119.302939 Liang, B. et al. Underestimated health risks: polystyrene micro- and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis. Part Fibre Toxicol 18 , 20 (2021). https://doi.org/10.1186/s12989-021-00414-1 Qu, M. et al. Nanopolystyrene at predicted environmental concentration enhances microcystin-LR toxicity by inducing intestinal damage in Caenorhabditis elegans. Ecotoxicol Environ Saf 183 , 109568 (2019). https://doi.org/10.1016/j.ecoenv.2019.109568 Wu, Y., Tan, X., Shi, X., Han, P. & Liu, H. Combined Effects of Micro- and Nanoplastics at the Predicted Environmental Concentration on Functional State of Intestinal Barrier in Caenorhabditis elegans. Toxics 11 (2023). https://doi.org/10.3390/toxics11080653 Yu, Y. et al. Polystyrene microplastics (PS-MPs) toxicity induced oxidative stress and intestinal injury in nematode Caenorhabditis elegans. Sci Total Environ 726 , 138679 (2020). https://doi.org/10.1016/j.scitotenv.2020.138679 Huang, C. W., Yen, P. L., Kuo, Y. H., Chang, C. H. & Liao, V. H. Nanoplastic exposure in soil compromises the energy budget of the soil nematode C. elegans and decreases reproductive fitness. Environ Pollut 312 , 120071 (2022). https://doi.org/10.1016/j.envpol.2022.120071 Tseng, I. L., Yang, Y. F., Yu, C. W., Li, W. H. & Liao, V. H. Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans. PLoS One 8 , e82657 (2013). https://doi.org/10.1371/journal.pone.0082657 Senchuk, M. M. et al. Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans. PLoS Genet 14 , e1007268 (2018). https://doi.org/10.1371/journal.pgen.1007268 Lee, R. Y., Hench, J. & Ruvkun, G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol 11 , 1950-1957 (2001). https://doi.org/10.1016/s0960-9822(01)00595-4 Ke, T. et al. N,N' bis-(2-mercaptoethyl) isophthalamide induces developmental delay in Caenorhabditis elegans by promoting DAF-16 nuclear localization. Toxicol Rep 7 , 930-937 (2020). https://doi.org/10.1016/j.toxrep.2020.07.012 Leite, N. R. et al. Baru Pulp (Dipteryx alata Vogel): Fruit from the Brazilian Savanna Protects against Oxidative Stress and Increases the Life Expectancy of Caenorhabditis elegans via SOD-3 and DAF-16. Biomolecules 10 (2020). https://doi.org/10.3390/biom10081106 Lin, K., Hsin, H., Libina, N. & Kenyon, C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat Genet 28 , 139-145 (2001). https://doi.org/10.1038/88850 Zhou, K. I., Pincus, Z. & Slack, F. J. Longevity and stress in Caenorhabditis elegans. Aging (Albany NY) 3 , 733-753 (2011). https://doi.org/10.18632/aging.100367 Libina, N., Berman, J. R. & Kenyon, C. Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. Cell 115 , 489-502 (2003). https://doi.org/10.1016/s0092-8674(03)00889-4 Reyes, M. S. S. & Medina, P. M. B. Leachates from plastics and bioplastics reduce lifespan, decrease locomotion, and induce neurotoxicity in Caenorhabditis elegans. Environ Pollut 357 , 124428 (2024). https://doi.org/10.1016/j.envpol.2024.124428 Zongur, A. Evaluation of the Effects of Di-(2-ethylhexyl) phthalate (DEHP) on Caenorhabditis elegans Survival and Fertility. Appl Biochem Biotechnol 196 , 8998-9009 (2024). https://doi.org/10.1007/s12010-024-05032-z Pradhan, A., Olsson, P. E. & Jass, J. Di(2-ethylhexyl) phthalate and diethyl phthalate disrupt lipid metabolism, reduce fecundity and shortens lifespan of Caenorhabditis elegans. Chemosphere 190 , 375-382 (2018). https://doi.org/10.1016/j.chemosphere.2017.09.123 Additional Declarations No competing interests reported. Supplementary Files SupplementalFiguresMaldonado.docx Cite Share Download PDF Status: Published Journal Publication published 30 Nov, 2025 Read the published version in Microplastics → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6629868\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":454407185,\"identity\":\"940ff2a9-8c46-4252-870a-7329513d6d30\",\"order_by\":0,\"name\":\"Chiara Angelyn O. 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Representative SEM images of 1 µM PS MPs to verify their size and surface structure before and after exposure to 0.1 M DBP and 100 % DMSO. Error bar is 1 µM. Quantification of PS MP size before and after 24-hour exposure to 0.1 M DBP and 100 % DMSO. n= 31, 104, 81, ***p ≤ 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/d55006b418d28c80dbdcb498.png\"},{\"id\":83202658,\"identity\":\"dbca43f1-763b-4c9f-acb0-61d126b1d2c2\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:39:53\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":208562,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e1 µm Dragon Green Polystyrene (PS) Microplastics (MPs) are ingested by \\u003cem\\u003eC. elegans\\u003c/em\\u003e. MPs are ingested by \\u003cem\\u003eC. elegans\\u003c/em\\u003e at all larval stages. a) Representative images of L1-Adult \\u003cem\\u003eC. elegans\\u003c/em\\u003e after continuous exposure to 1 µg/L Dragon Green 1 µm PS MP particles. Scale bar=100 µm. b) Representative image of \\u003cem\\u003eC. elegans\\u003c/em\\u003e eggs from an adult worm that was continuously exposed to Dragon Green PS MPs. Scale bar=10 µm. c) Quantification of the number of worms containing MPs at each larval stage (L1- Adult). YA, young adult. N=3, n=173, 247, 199, 111, 111, 105.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/11bfb864ef5663a6707aa1ff.png\"},{\"id\":83202662,\"identity\":\"f187bb2f-80df-4a59-a85b-76881eb90f2e\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:39:53\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":134850,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMicroplastics soaked in Nile Red act as a carrier to deliver Nile Red into \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eC. elegans\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e after ingestion. \\u003c/strong\\u003ea)\\u003cstrong\\u003e \\u003c/strong\\u003eRepresentative images of adult \\u003cem\\u003eC. elegans\\u003c/em\\u003e fed \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 with and without Nile Red-stained microplastics (MPs). Control = \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 only\\u003cem\\u003e. \\u003c/em\\u003eScale bar = 100 µm.\\u003cem\\u003e \\u003c/em\\u003eb) Quantification of the presence of Nile Red inside \\u003cem\\u003eC. elegans\\u003c/em\\u003e after ingestion of Nile Red-stained MPs, N = 3, n = 50, 50, 30. ***p\\u0026lt;0.00001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/8f3649f9f98a28d1f21d0ebb.png\"},{\"id\":83202661,\"identity\":\"a1809a9f-587a-43dc-b205-5b9dbc7eba1b\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:39:53\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":278657,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTotal eggs laid, number of fertilized and unfertilized eggs, and embryonic lethality of \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposed to\\u003cstrong\\u003e \\u003c/strong\\u003epolystyrene (PS) microplastics (MP) and/or containing DBP. a) Number of eggs laid per nematode with \\u003cem\\u003eE. coli \\u003c/em\\u003eOP50 only, 48-hour exposure to PS MP or continuous PS MP exposure. N=8, n=43, 46, 52. b) Percent embryonic lethality. c) Total number of eggs with exposure to continuous PS MP with and without 0.1 M DBP. N= 4, n= 28, 28, 28, 25, 29. d) Percent embryonic lethality. e) Total number of eggs with exposure to continuous PS MP with and without 3.7 mM DBP. N=4, n=27, 28, 27, 28, 28. f) Total number of eggs with exposure to continuous PS MP with and without 100 µM DBP. N=3, n=27, 27, 25, 27, 26. Violin plots show the mean with the standard deviation. Kruskal-Wallis was used for statistical analysis, using Dunn's as a post hoc test (*p ≤ 0.05; **p ≤ 0.01).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/3d190223cc3776b7be053d46.png\"},{\"id\":83202882,\"identity\":\"72c7b209-1751-4d6b-890b-20bd37acd9be\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:47:53\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":115740,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStress response via DAF-16 in \\u003cem\\u003eC. elegans\\u003c/em\\u003e after exposure to polystyrene (PS) microplastic (MPs) and DBP. a) Representative images of DAF-16-GFP expression in adult \\u003cem\\u003eC. elegans\\u003c/em\\u003e, showing cytoplasmic (left), intermediary (center), and nuclear localization (right). Scale bar = 100 µm. b) Quantification of DAF-16-GFP localization in \\u003cem\\u003eC. elegans\\u003c/em\\u003e with exposure to their regular E. coli diet only, with 1 µg/L PS MP, 1 µg/L PS MP and DBP, 1 µg/L PS MP and DMSO (vehicle control), DBP alone, and DMSO alone. N=2, n=53, 65, 170, 149, 140, 129, 43.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/bd6435ced9050e212c9511fd.png\"},{\"id\":83202883,\"identity\":\"d588491e-3ad1-4c9f-ab45-3b98f5b6c98c\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:47:53\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":173389,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLifespan of \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposed to\\u003cstrong\\u003e \\u003c/strong\\u003epolystyrene (PS) microplastic (MPs) containing different concentrations of MPs or MPs that had been soaked in different concentrations of DBP (0.1 M or 100 µM). a) Survival assay for nematodes exposed to control (no MPs), 1 µg/L PS-MPs for 48 hours, 1 µg/L or 1 mg/L PS-MPs continuously. n=47, 49, 33, 41. p\\u0026lt;0.0001. b) Survival assay for N2 nematodes continuously exposed to 1 µg/L PS MPs, with and without being soaked in 0.1 M DBP or vehicle alone (100 % DMSO) for 24 hours. n=48, 36, 52, 47, 45. p=0.00357. c) Survival assay for N2 nematodes continuously exposed to 1 µg/L PS-MPs, with and without being soaked in 100 µM DBP or vehicle alone (0.1 % DMSO) for 24 hours. n=33, 44, 42, 49, 44. Not significant, p=0.4. Log-rank (Mantel-Cox).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/293b1f2facb6d3a2d76998a0.png\"},{\"id\":97804415,\"identity\":\"bd8cdc24-759a-488c-8b03-666fe9f6f7f0\",\"added_by\":\"auto\",\"created_at\":\"2025-12-09 14:25:46\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2003089,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/af43ca39-b328-4fa1-85c4-e5db0ffdb3d5.pdf\"},{\"id\":83202664,\"identity\":\"73181e65-e522-4243-a7dc-e19d2b0c62f9\",\"added_by\":\"auto\",\"created_at\":\"2025-05-21 06:39:53\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1226929,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalFiguresMaldonado.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6629868/v1/68a16b636b62937ca3d289d9.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003eMicroplastic-mediated delivery of di-butyl phthalate alters \\u003cem\\u003eC. elegans\\u003c/em\\u003e lifespan and reproductive fidelity\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePlastic and chemical pollutants are ubiquitous in the environment, posing an emerging human health concern. Microplastic (MP) particles have been identified in nearly all human tissues, from blood\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e to the brain\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e, and most recently in human reproductive organs\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Microplastic exposure includes plastics from various environmental sources and exists in a wide range of chemical types, mixtures, and conditions. Primary microplastic particles are manufactured and used as ingredients in biomedical products and various health and beauty items (i.e., scrubs, make-up, cleansers, and toothpaste)\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Whereas secondary microplastics result from the breakdown of larger plastic items. Human exposure to microplastics occurs through direct use of microplastic-containing products, exposure to secondary microplastics released into food, or environmental pollutants.\\u003c/p\\u003e \\u003cp\\u003eBoth primary and secondary microplastics undergo chemical and physical changes in their structure due to environmental exposure. Factors such as UV light, physical breakdown, and exposure to environmental pollutants can lead to alterations in the size, surface structure, and chemical composition of microplastics. Despite efforts to reduce plastic use, it remains prevalent, with much already having entered the ecosystem\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Dating back to the 1950s, approximately 400\\u0026nbsp;million tons of plastic waste accumulate each year\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Microplastics (MPs, plastic particles\\u0026thinsp;\\u0026lt;\\u0026thinsp;5 mm\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e) and nanoplastics (NPs, plastic particles\\u0026thinsp;\\u0026le;\\u0026thinsp;1 \\u0026micro;m\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e) are detected in the air, water, and our food sources\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR11 CR12 CR13\\\" citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Meanwhile, microplastics have been found in tissue exhibiting the ability to enter mammalian cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e. The smallest particles, nanoplastics, invade endosomes, lysosomes, lymph and circulatory systems, and the lungs\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR17\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e, leading to deleterious effects on the cellular level\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. The direct health risks of human ingestion of microplastics have not been quantified. Moreover, these risks amplify as plastics already contain additives, and they can absorb, accumulate, and transfer chemicals from the surrounding environment into organisms.\\u003c/p\\u003e \\u003cp\\u003ePlastic and resulting microplastic particles can be composed of homogenous or heterogeneous polymer mixtures, contain additives from the manufacturing process (plasticizers, by-products, and monomers), and may absorb chemical pollutants\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR20\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. These additives and pollutants can later leach into the environment\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. Persistent organic pollutants have been shown to be transferred from plastic particles to fish with adverse effects in environmentally relevant concentrations\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e and led to a transfer of chemicals from microplastics into the guts of lung worms (\\u003cem\\u003eArenicola marina\\u003c/em\\u003e), and a reduction of biological functions\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. In mice, it was demonstrated that phthalate esters were released from microplastics leading to intestinal permeability and inflammation\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. A study using \\u003cem\\u003eC. elegans\\u003c/em\\u003e showed that 19 chemicals (including phthalates and agrochemicals) increased DNA damage and physiological dysregulation when compared to Bisphenol A (BPA)\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. BPA has been widely recognized as a threat to human health, and its use has been highly restricted, with a total ban on its use in infant-related products\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. Due to these concerns, most manufacturers have voluntarily removed BPA from consumer products. With these regulations and demands from consumers, manufacturers must rely on other plasticizers to aid in the performance and functionality of plastic products.\\u003c/p\\u003e \\u003cp\\u003eDi-butyl phthalate (DBP), a widely used plasticizer, can compose 20\\u0026ndash;80% of some plastic products\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e and its use is regulated by both US and European agencies\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. DBP is classified as a phthalate esters, which as a group, are considered endocrine-disrupting chemicals and pose cytotoxic and estrogenic effects\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR31\\\" citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. DBP is most commonly used as a plasticizer, primarily in polyvinyl acetate emulsion adhesives, as a solvent for oil-based dyes, and insecticides\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. It can be found in consumer products including nail polish, paints, adhesives, and perfume oils. Prior to 2008, many products for children contained high levels of DBP, butyl benzyl phthalate (BBP), and di (2-ethyexyl) phthalate (DEHP). The United States and European federal restrictions now limit the use of some phthalates, including DBP, in children\\u0026rsquo;s products. Despite bans on some products, DBP and other phthalate esters are widely used in everyday household items or manufacturing processes. Many household products contain these chemical additives, which, when disposed of, will likely end up in municipal landfills\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. Thus, DBP could leach into the environment due to the improper disposal and breakdown of products containing it. Once in the environment, DBP absorbs onto suspended particles and is less likely to degrade than dissolved DBP\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. DBP has a high likelihood of being absorbed into MPs in the environment as it is readily absorbed by plastic particles, possesses properties that make it absorptive, and it is chemically related to compounds that are easily absorbed into plastics\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eDespite the risks of microplastic exposure to humans and the danger of plastic particles carrying chemical pollutants, microplastics remain under-investigated. It is crucial to determine how microplastic-chemical mixtures alter the development and health of humans and other land animals. We use the \\u003cem\\u003eC. elegans\\u003c/em\\u003e model system to examine the cellular and physiological effects of nanoplastic mixtures on whole-organism health and development. In this study, we investigate whether 1 \\u0026micro;m polystyrene microplastics can transport absorbed chemicals into an animal after ingestion and lead to physiological responses. The 1 \\u0026micro;M polystyrene size regime is ideally situated at the interface of micro- and nano-particles, and because polystyrene is commonly used in packing foams, food containers, and disposable cutlery, it is highly relevant to human oral microplastic exposure risks\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. The additive, DBP, was chosen as a representative chemical due to its wide use as an industrial additive. Studies of additives commonly found in plastics show that the many common plasticizers with relatively low molecular weight will transfer from the plastic material into foods\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. We hypothesized that microplastics can contain a chemical pollutant and mediate physiological defects that are greater than either pollutant alone. We find that microplastic exposure reduces brood size and leads to embryonic lethality in a concentration-dependent manner. The microplastic-mediated delivery further decreased reproduction compared to microplastics or DBP alone. Reproductive defects due to microplastic -DBP co-exposure appear to cause stress via DAF-16 and reduce \\u003cem\\u003eC. elegans\\u003c/em\\u003e lifespan.\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003ePolystyrene microplastic surface structure is unchanged after exposure to di-butyl phthalate.\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo verify the diameter and surface structure characteristics of polystyrene microplastics (PS MP, hereon referred to as microplastics), scanning electron microscopy (SEM) was used to image microplastics with and without exposure for 24 hours to 0.1 M di-butyl phthalate (DPB) or the solvent (DMSO). SEM analysis revealed no change to the surface morphology of microplastics exposed to DBP for 24 hours. A small but significant increase in the average size of microplastic particles occurred when soaked in DMSO, but not 0.1 M DBP (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, p ≤ 0.0001). It was anticipated that exposure to the plasticizer DBP may change the size or surface structure of microplastics. Environmental exposure of microplastics to heat, UV and mechanical stress can change their characteristics\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e, and this was not observed after 24 hour exposure of microplastics to DBP or DMSO (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). DBP has high rates of sorption into microplastics, with highest rates seen in polystyrene (PS) microplastic when compared to polyvinyl chloride (PVC) and polyethylene (PE)\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Sorption of compounds on or into microplastics is size dependent, with smaller particles containing more readily available chemicals, due to the increase in surface to size ratio that occurs with the reduced particle size\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. Microplastic and DBP mixtures were prepared by soaking microplastics in DBP for 24 hours, followed by dilution into \\u003cem\\u003eC. elegans\\u003c/em\\u003e food, \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50. In each dilution step, microplastics were sonicated to aid in resuspension. This process would disrupt any DBP that would be adsorbed onto the microplastic surface, and thus any DBP present in the microplastic is assumed to be absorbed within the microplastic particles. The slightly larger but non-significant increase in mean size of the 0.1 M DBP soaked microplastics and significantly larger diameter of microplastics after DMSO exposure may be due to swelling of the microplastic particles after absorption.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003ePolystyrene microplastics are ingested by C. elegans and act as a vehicle for chemical exposure.\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposed to 1 µg/L and 1 mg/L Dragon-Green microplastic show the presence of microplastics in their gut tube (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). Nematodes were initially exposed to two different doses (1 µg/L and 1 mg/L), which were within the range of other studies and that mimic concentrations determined from human samples such as blood\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR39 CR40\\\" citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. Microplastics were present at all larval life stages, and the percentage of total worms containing microplastics increased with life stage and exposure time. By adulthood, nearly all worms contained microplastics (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec). These results are consistent with other studies demonstrating that microplastics (\\u0026gt; 1 µm) accumulate in the gut, while smaller nanoplastic (\\u0026lt; 1 µm) particles can cross cell membranes\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR43\\\" citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. When examining the eggs of adult nematodes exposed to Dragon-Green microplastic, no microplastics were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb). Smaller microplastics, less than 1 µm in size, in the nanometer range, have been observed to cross into the body cavity in \\u003cem\\u003eC. elegans\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eMicroplastics contain chemical additives or may absorb pollutants in the environment that can then be transferred into living organisms, leading to physiological effects\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Phthalates, including DBP, are classified as endocrine disruptors that can be long-lived in the environment\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. DBP is most commonly used as a plasticizer in PVC followed by PET\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. While DBP is not commonly used in PS products, we choose to study it as a potential environmental chemical that could be absorbed in microplastics and transferred into an organism after ingestion. To visualize this, we soaked PS microplastics in the lipophilic dye, Nile Red, to ask if microplastics could mediate the delivery of a chemical into an organism. Nematodes exposed to microplastics that have been soaked in Nile Red do not show puncta, indicating the location of each microplastic, but rather there is diffuse staining in the worm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea). This indicates that the Nile Red dye, which had been absorbed into the PS microplastic, transfers into the worm’s body after ingestion. This transfer is present in more than 90% of the worms examined (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). When nematodes are exposed to an equivalent amount of Nile Red without microplastics, the Nile Red is not present in the worm’s body. Thus, the microplastics mediate the delivery of Nile Red, which is then absorbed. This mimics what one would expect if microplastics can act as a vector to deliver chemicals into an organism after ingestion.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eExposure to polystyrene microplastics and polystyrene microplastic DBP mixtures results in reproductive toxicity.\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo determine if microplastics and chemical pollutants cause reproductive toxicity, \\u003cem\\u003eC. elegans\\u003c/em\\u003e were exposed to different concentrations of microplastics and DBP for varying times. When L1 life stage, nematodes were exposed to 1 µm MP (at 1 µg/L concentration) for 48 hours, until the young adult stage, or continuously, there was no observed significance in decreased total eggs laid or hatched (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, S1a). Despite no change to the number of eggs laid, chronic microplastic exposure to nematodes significantly increased embryonic lethality (p = 0.04) and the total number of eggs unhatched per nematode (p = 0.007) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, S1b). Embryonic lethality is determined by taking the total number of eggs that are unhatched and dividing by the total number of eggs laid per worm. These results are in range with other studies that observed continuous microplastics of the same or smaller size leading to minimal or no change in brood size, with no embryonic lethality\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. While some studies noted a decrease in brood size with exposure to microplastics, this difference may be due to a different size or composition of microplastics or varying exposure conditions\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. When we exposed \\u003cem\\u003eC. elegans\\u003c/em\\u003e to a 1000x higher concentration of microplastics (1 mg/L), we observed a significant reduction in the total number of eggs laid (p = 0.016), but this did not lead to an increase in embryonic lethality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea,b).\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposure to microplastics (1 µg/L) harboring 0.1 M DBP, further reduced the number of eggs laid per worm compared to exposure to microplastics alone (p = 0.017, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec). This combined exposure did not lead to an increase in embryonic lethality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). Examination of microplastics loaded with either 100 µM or 3.7 mM DBP did not affect brood size, egg hatching, or embryonic lethality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee,f and S2 a-f). Typical concentrations of DBP used as a plasticizer can range from 10–35%, or higher in some products\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Our study employed 0.1 M DBP, a concentration equivalent to less than 10% w/w, which is in the range of DBP found in everyday products. While 0.1 M DBP led to defects in fertility, these were not seen with lower concentrations (100 µM and 3.7 mM). Comparable levels of chemical pollutants could reach organisms through the ingestion of microplastics that have absorbed chemicals from the environment or by the leaching of additives from microplastics that initially contained higher chemical amounts. In particular, it is worth noting that recycled plastics can contain additives from recycled feedstocks\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eImpaired reproduction with exposure to microplastics alone or in combination with DBP could be caused by various factors. DBP is an endocrine disrupting chemical and leads to cryptogenic effects\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. Exposure to DBP leads to defects in reproduction and development\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. Specifically, DBP exposure reduced human sperm function and in rodent studies defects occur in both development and reproduction\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR48 CR49\\\" citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. In \\u003cem\\u003eC. elegans\\u003c/em\\u003e, exposure to 100 µM DBP, in the absence of MP, led to increased embryonic lethality and DNA damage\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposure to DBP alone, at a lower dose (500 µM) than used in this study, leads to a reduction in the number of eggs laid and increased embryonic lethality\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. These defects were attributed to defects in early embryogenesis, with elevated levels of DNA double-strand breaks, activation of a DNA damage checkpoint, and impaired embryogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. Errors in cell division could reduce the ability of eggs to hatch and could be caused by a multitude of sources\\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e. In humans and other organisms, these errors may lead to aneuploidy, spontaneous abortions, and birth defects\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e. Additionally, a reduction in the total number of eggs laid could be attributed to a nutrient deficiency of the parent caused by intestinal damage from polystyrene microplastic exposure\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR54 CR55\\\" citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u003c/sup\\u003e. Another study suggested that the associated defects with polystyrene microplastic exposure could be due to a reduction in ATP levels and a reduced energy budget toward reproduction\\u003csup\\u003e\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe exacerbated defects we observed with co-exposure to MPs containing 0.1 M DBP demonstrate that microplastics can mediate the delivery of a chemical into an organism after ingestion. This microplastic-mediated delivery of DBP underscores the risks of co-exposure when microplastics can release the additives they already contain and/or chemicals that they have absorbed from the environment.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eExposure to polystyrene microplastics and polystyrene microplastic DBP mixtures elicits a stress response.\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eA possible mechanism of polystyrene microplastic toxicity resulting in a reduction in fertility is the generation of reactive oxygen species (ROS). Polystyrene microplastic exposure leads to higher levels of reactive oxygen species\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e and increased expression of GST-4 (glutathione S-transferase 4, an enzyme that is involved in clearing ROS) in nematodes\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e. Additionally, DBP and other phthalate esters cause DNA damage and chromosomal abnormalities\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e\\u003c/sup\\u003e that can be caused by ROS generation\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e. In response to stress, \\u003cem\\u003eC. elegans\\u003c/em\\u003e activate a DAF-16 stress response that leads to the activation of genes encoding proteins involved in response to oxidative stress\\u003csup\\u003e\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, to investigate if stress may be an underlying mechanism of reduced fertility, we asked if microplastic-mediated DBP exposure in \\u003cem\\u003eC. elegans\\u003c/em\\u003e would initiate a DAF-16 stress response. The \\u003cem\\u003eC. elegans\\u003c/em\\u003e strain TJ356 contains a reporter GFP fused to DAF-16 that is driven by the \\u003cem\\u003edaf-16\\u003c/em\\u003e promoter\\u003csup\\u003e\\u003cem\\u003e\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e. This GFP reporter is cytoplasmic under normal conditions and relocates to the nucleus to activate genes involved in an anti-oxidative response, under stress. Therefore, the localization of this DAF-16 GFP reporter can be used as a readout of a DAF-16-mediated stress response\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eUnder control conditions, when \\u003cem\\u003eC. elegans\\u003c/em\\u003e were not exposed to microplastics, the majority of worms examined had cytoplasmic DAF-16 localization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea,b). Some worms showed an intermediate localization where less than 30% of the nuclei showed nuclear expression. This same expression pattern is present with \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposure to 1 µg/L microplastics (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). These results are consistent with what others see with similar PS microplastic exposure\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. In contrast, microplastic-mediated DBP exposure lead to a significant activation of the DAF-16 stress response, with 40% of the total worms showing an intermediate expression pattern and nearly 20% nuclear localization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). This indicates that the DAF-16 transcription factor is located in the nucleus where it will activate genes in response to oxidative stress. Importantly, exposure to microplastics containing DMSO, the solvent for DBP, or an equivalent amount of DBP or DMSO alone, did not affect DAF-16 localization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). Expression of DAF-16 in these controls, showed no significant change when compared to the negative control (\\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 only) and microplastic exposure alone (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). In our initial reproductive toxicity studies, we observed that higher concentrations of MPs (1 mg/L) induced a reduction in total eggs laid with no effect on embryonic lethality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea,b). Therefore, we asked if microplastics alone, at a higher concentration, would elicit a stress response via DAF-16. Continuous exposure of \\u003cem\\u003eC. elegans\\u003c/em\\u003e to 1 mg/L microplastics increased the percentage of worms with intermediate and nuclear expression of DAF-16 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). This indicated that the concentration of microplastic exposure can mitigate the effect. In contrast to our results, \\u003cem\\u003eLeon et al\\u003c/em\\u003e observed a stress response with \\u003cem\\u003eC. elegans\\u003c/em\\u003e exposure to 100 nm PS microplastics at a concentration of 10 mg/L\\u003csup\\u003e44\\u003c/sup\\u003e. This may be due to differences in the size of the tested plastic particles. The stress response observed in \\u003cem\\u003eLeon et al\\u003c/em\\u003e was with microplastics of the same composition, polystyrene, but with a smaller diameter (100 nm) and at 10,000 x greater concentration than in this study. Indeed, we observed that 1 µm microplastics at our higher, 1 mg/L, concentration elicited a DAF-16 stress response. This indicates that a stress response via DAF-16 may be concentration-dependent. While we did not see a DAF-16-mediated stress response with our primary level of microplastic exposure (1 µM/L) alone, a significant response was elicited when DBP delivery was mediated with this same concentration of microplastics. Interestingly, \\u003cem\\u003eLeon et al\\u003c/em\\u003e observed that with the removal of worms from microplastic exposure, the microplastics left the worm’s body, and the DAF-16 stress response reduced. We did not test for the effect of DAF-16 localization with the removal of microplastic exposure from \\u003cem\\u003eC. elegans\\u003c/em\\u003e, but it would be interesting to ask if the microplastic-mediated DBP stress response via DAF-16 would be maintained, as the microplastics might clear the worm body, while the DBP is absorbed.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eExposure to polystyrene microplastics and microplastic DBP mixtures reduces life span.\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn some cases, the activation of a DAF-16 stress response is associated with an extended life span\\u003csup\\u003e\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR63\\\" citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e, and this response can be tissue-specific, with greater extension of lifespan associated with intestinal-specific DAF-16 activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e\\u003c/sup\\u003e. Increased longevity induced by stress events is associated with the insulin/insulin-like growth factor (IGF-1) receptor signaling pathway, or diet, amongst other pathways\\u003csup\\u003e\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e. In particular, due to the presence of microplastic accumulation in the gut of the worm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea), and studies indicating intestine-specific stress due to microplastic exposure\\u003csup\\u003e\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u003c/sup\\u003e, we wondered if the presence of microplastics may reduce nutrient intake and increase lifespan.\\u003c/p\\u003e \\u003cp\\u003eWe next addressed whether microplastics or microplastic-mediated DBP exposure would alter \\u003cem\\u003eC. elegans\\u003c/em\\u003e lifespan. Short-term exposure (48 hours) of \\u003cem\\u003eC. elegans\\u003c/em\\u003e to 1 µm microplastic (1 µg/L) did not change the lifespan of animals compared to the control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). Interestingly, when nematodes were exposed to the same conditions, but without removal after 48 hours, there was a significant reduction in lifespan (p \\u0026lt; 0.0001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea, S3a,b). This reduction in lifespan was equal to what was observed with continuous exposure to 1000 x more concentrated microplastics (1 mg/L) with continuous exposure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). The lifespan of animals was further reduced when exposed to microplastics that mediated the delivery of 0.1 M DBP (p = 0.00357) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb, S3c). This reduction in lifespan was not observed under the control conditions of microplastics containing DMSO, or with exposure to DBP or DMSO alone (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb, S3c). Additionally, the reduction in lifespan with microplastic-mediated DBP exposure appears to be dependent on the concentration of DBP, as the lowest concentration of DBP (100 µM) used in this study did not lead to a further reduction in lifespan as compared to microplastic exposure alone (Fig. S3c).\\u003c/p\\u003e \\u003cp\\u003eThere are conflicting studies on the effect of microplastic exposure on \\u003cem\\u003eC. elegans\\u003c/em\\u003e lifespan. In one study, polystyrene microplastics initiated a DAF-16 stress response that was not associated with a change in lifespan in \\u003cem\\u003eC. elegans\\u003c/em\\u003e, and this was speculated to be due to a counteracting mechanism\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. Other reports were consistent with our results, showing reduced lifespan with exposure to polystyrene microplastics\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Furthermore, our work shows that microplastic-mediated delivery of DBP further reduced lifespan. Additional reports examined the effects of leachates from plastic\\u003csup\\u003e\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e\\u003c/sup\\u003e, phthalates\\u003csup\\u003e\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e\\u003c/sup\\u003e, and other types of MP exposure on life span, all leading to reduced longevity.\\u003c/p\\u003e \\u003cp\\u003eThis work shows that microplastic-mediated delivery of plastic-containing compounds, such as DBP, enhances the physiological defects observed with microplastic exposure alone. The exposure level of microplastics, the time of exposure, and the concentration of the absorbed chemicals influence the defects observed. Overall, our results show that chronic microplastic exposure has detrimental effects on reproduction and reduces lifespan. These defects are further exacerbated by the co-exposure of microplastics and DBP. Microplastics mediate the delivery of DBP in \\u003cem\\u003eC. elegans\\u003c/em\\u003e, further decreasing fertility and lifespan as well as leading to a stress response via DAF-16 activation. Further studies are needed to identify other mechanisms involved in the toxicity due to microplastic-mediated exposure to DBP. The worsened physiological defects seen with co-exposure to phthalate-containing microplastics demonstrate that microplastics can leach contents into an organism after ingestion. This highlights the risks of microplastics releasing the additives they already contain and/or chemicals that they have absorbed from the environment.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\n\\n\\n\\n\\n \\n\\n\\n\\n \"},{\"header\":\"Methods\",\"content\":\"\\u003ch2\\u003eCharacterization of microplastics\\u003c/h2\\u003e\\u003cp\\u003ePolystyrene microplastic (Poly Sciences, 19518-500) size and surface were characterized by Scanning Electron Microscopy (JEOL JSM-6010 PLUS/LA). Samples were sputter-coated with 2 nm gold particles prior to imaging (Quorum Technologies EMS150T ES). Microplastic diameter was measured using the measuring tool in the JOEL associated software.\\u003c/p\\u003e\\u003ch3\\u003ePreparation of microplastic and chemical mixtures\\u003c/h3\\u003e\\u003cp\\u003eMicroplastics were suspended in \\u003cem\\u003eE. coli (\\u003c/em\\u003estrain OP50), seeded on Nematode Growth Media (NGM) plates (Teknova, N1105) and allowed to dry overnight before adding nematodes to the plates for exposure. To prepare microplastic \\u003cem\\u003eE. coli\\u003c/em\\u003e suspensions, microplastics were suspended in a 1% solution with M9 buffer, then diluted into \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50. At each dilution step, the solution was sonicated for 15 minutes (Emerson Branson 3800). Wide-boar tips were used to transfer liquid. Dry 1 µM polystyrene microspheres (Poly Sciences, 19518-500) were used for all experiments unless otherwise noted. Di-butyl phthalate (DBP, Sigma, 524980) was diluted into 100% DMSO (Sigma, D8418) at 0.1 M concentration. Additional dilutions were made from the 0.1 M stock into water. DBP polystyrene microplastic mixtures were prepared by adding DBP (either 0.1 M, 3.7 mM, or 100 µM concentration) to dry polystyrene microplastics, to create a 1% solution, which was incubated overnight, on a rotating rack, in the dark, at room temperature (19–22°C). DMSO polystyrene microplastic controls were prepared by adding 100% DMSO, 3.7% DMSO, or 0.0001% DMSO to dry polystyrene microplastics at 1%. Polystyrene microplastics were diluted into \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 to a final concentration of either 1 µg/L or 1 mg/L.\\u003c/p\\u003e\\u003ch3\\u003eMaintenance of C. elegans\\u003c/h3\\u003e\\u003cp\\u003e \\u003cem\\u003eCaenorhabditis elegans (C. elegans)\\u003c/em\\u003e stains, N2 (wild type) and TJ356 (zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]) that were used in this study were provided by the Caenorhabditis Genome Center. Worms were grown on Nematode Growth Media Agarose (NGM) plates seeded with \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 at 20°C.\\u003c/p\\u003e\\u003ch3\\u003eExposure design\\u003c/h3\\u003e\\u003cp\\u003eBleach-synchronized L1 larvae were exposed to \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 containing 1 µm polystyrene microplastics at either 1 µg/L or 1 mg/L, or polystyrene microplastics soaked in DBP (100 µM, 3.7 mM or 0.1 M) or DMSO (0.1%, 3.7%, or 100%) for 24 hours, or DBP or DMSO added directly to the OP50 \\u003cem\\u003eE. coli\\u003c/em\\u003e alone on NGM plates made within 48 hours. DBP and DMSO only control concentrations were determined by the amount of solution carried into the final \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50, after dilution from the prepared 1% polystyrene microplastics. From a 1% polystyrene microplastic in 0.1 M DBP solution that was diluted 1:10,000 into \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 for a final concentration of 1 µg/L microplastic, would have an associated 10 µM DBP control in \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50. L1s were exposed for 48 hours and then moved to \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 plates for short-term exposure and maintained on prepared plates until the end points for experimental conditions. At least 2 biological replicates were performed for each assay.\\u003c/p\\u003e\\u003ch2\\u003eVisualizing microplastics in C. elegans\\u003c/h2\\u003e\\u003cp\\u003eTo visualize microplastic distribution and location, L1 bleach-synchronized worms were exposed to 1 µm internally dyed Dragon Green polystyrene microspheres (Bangs Laboratories Inc., FS03F). L1 synchronized larvae were exposed to \\u003cem\\u003eE coli\\u003c/em\\u003e OP50 and 1 µM/L and 1 mM/L Dragon Green microplastics suspended in \\u003cem\\u003eE. coli\\u003c/em\\u003e OP50 and seeded on NGM plates. Worms were paralyzed in levamisole on an agarose pad before imaging at each life stage.\\u003c/p\\u003e\\u003ch3\\u003eVisualizing Nile Red in C. elegans\\u003c/h3\\u003e\\u003cp\\u003eTo assess if chemical compounds absorbed by microplastic beads could transfer into the worm’s body after ingesting the chemically soaked beads, we fed L1 synchronized \\u003cem\\u003eC. elegans\\u003c/em\\u003e microplastic beads soaked in 1 mg/L Nile Red in methanol (Sigma Aldrich, 19123). The beads were rocked for 1 hour and left to dry overnight, before being suspended in a 1% solution in M9 buffer. The 1% M9 solutions were diluted to either 1 µg/L or 1 mg/L into OP50 \\u003cem\\u003eE. coli\\u003c/em\\u003e before seeding on NGM plates and left to dry overnight.\\u003c/p\\u003e\\u003ch3\\u003eLifespan Assay\\u003c/h3\\u003e\\u003cp\\u003eLifespan assays were performed at 20°C as previously described. Approximately 50 synchronized L1 larvae were placed onto corresponding treatment conditions for 48 hours and then moved to new plates containing the same treatment, with 100 µM 5’-fluorordeoxyuridine. Worms were counted as dead or alive every day until the number of live worms reached zero.\\u003c/p\\u003e\\u003ch2\\u003eEvaluation of reproductive toxicity\\u003c/h2\\u003e\\u003cp\\u003eTo assay the total number of eggs laid per worm and the percentage hatched, bleach-synchronized L1 larvae were seeded onto each condition. 48 hours after seeding, L4 larval worms were then singled out onto respective plates. The following day, L4 singling is repeated from the previous day’s plate, and both plates are kept. After 24 hours, L1s and embryos are counted from the first day of singling, then repeated every day, counting the L4-free plate, until the worm stops laying (around 5–6 days).\\u003c/p\\u003e\\u003ch2\\u003eImaging and microscopy\\u003c/h2\\u003e\\u003cp\\u003eFluorescent and bright field images were all taken using an EVOS M5000 (Invitrogen). For live imaging (Dragon-Green polystyrene microplastic localization, Nile Red soaked polystyrene microplastics transfer, and DAF-16-GFP localization), worms were transferred to an agarose pad on a slide and immobilized with levamisole. All image processing and analysis was done with ImageJ software.\\u003c/p\\u003e\\u003ch2\\u003eActivation of stress response\\u003c/h2\\u003e\\u003cp\\u003eTo determine if microplastic and chemical exposure lead to stress activation, the strain TJ356, expressing DAF-16 fused to GFP was exposed to polystyrene microplastics alone, and with DBP or DMSO, and DBP and DMSO alone. L1 synchronized larvae were placed on each condition and after 72 hours of exposure, imaged live. Live imaging was completed within 5 minutes of paralysis for each strain. Each nematode was classified by the localization of DAF-16::GFP expression (cytoplasmic, intermediate or nuclear)\\u003csup\\u003e\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e\\u003cp\\u003eStatistical analysis and figure design were done with GraphPad Prism 8 (GraphPad Software, CA, USA). Data were checked for normality between treatments and statistical analysis was done using Kruskal-Wallis followed by Dunn’s post hoc test (microplastic diameter, brood size, polystyrene microplastic exposure). A t-test (two-tailed) was done to analyze control vs. Nile Red data. Experiments were all carried out with a minimum of 2–3 biological replicates (N) and number of total worms (n) examined that are used in statistical analysis as described.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank V. Culotta for valuable discussions and advice on the development and design of experiments. Many undergraduate student researchers have played a part in moving this project forward, and we thank them for their time and valuable input. We also thank St. Mary\\u0026rsquo;s University for its continued support, including funds from the Department of Biological Sciences, the Benjamin F. Biaggini Endowment,\\u0026nbsp;and San Antonio Area Foundation. \\u003cem\\u003eC. elegans\\u003c/em\\u003e strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Research funding was provided by the National Institutes of Health, NIGMS (R16GM150406). C. Maldonado, P. Garcia, and M. Flores were funded by NIH grants (T34GM00873 and T34GM149455). Alyssa was funded by an NIH grant (R25GM102783).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eC.M. planned and evaluated results on brood size and longevity, and wrote the paper. D.M. provided invaluable technical help throughout, performed experiments, and evaluated results. P.G. planned and carried out experiments on brood size and longevity. M.G. planned and performed fluorescent analysis. M.F. planned experiments and performed chemical transfer assays. A.F. and R.L.P. planned and carried out experiments to validate plastic particles using SEM. J.C.H. conceptualized, planned, trained students, executed some of the experiments, evaluated all results, and wrote the paper. All authors provided feedback on the manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe have no competing interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMaterials \\u0026amp; Correspondence\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll requests and correspondence should be addressed to Jennifer C. Harr, PhD.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eLeslie, H. A.\\u003cem\\u003e et al.\\u003c/em\\u003e Discovery and quantification of plastic particle pollution in human blood. \\u003cem\\u003eEnviron Int\\u003c/em\\u003e \\u003cstrong\\u003e163\\u003c/strong\\u003e, 107199 (2022). https://doi.org/10.1016/j.envint.2022.107199\\u003c/li\\u003e\\n\\u003cli\\u003eNihart, A. J.\\u003cem\\u003e et al.\\u003c/em\\u003e Bioaccumulation of microplastics in decedent human brains. \\u003cem\\u003eNat Med\\u003c/em\\u003e (2025). https://doi.org/10.1038/s41591-024-03453-1\\u003c/li\\u003e\\n\\u003cli\\u003eHu, C. J.\\u003cem\\u003e et al.\\u003c/em\\u003e Response to Comment on: \\u0026quot;Microplastic presence in dog and human testis and its potential association with sperm count and weights of testis and epididymis\\u0026quot;. \\u003cem\\u003eToxicol Sci\\u003c/em\\u003e (2024). https://doi.org/10.1093/toxsci/kfae137\\u003c/li\\u003e\\n\\u003cli\\u003eMontano, L.\\u003cem\\u003e et al.\\u003c/em\\u003e First evidence of microplastics in human ovarian follicular fluid: An emerging threat to female fertility. \\u003cem\\u003eEcotoxicol Environ Saf\\u003c/em\\u003e \\u003cstrong\\u003e291\\u003c/strong\\u003e, 117868 (2025). https://doi.org/10.1016/j.ecoenv.2025.117868\\u003c/li\\u003e\\n\\u003cli\\u003eGESAMP. GESAMP Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection. Sources, Fate and Effects of Microplastics in the Marine Environment: Part 2 of a Global Assessment. \\u003cstrong\\u003e93\\u003c/strong\\u003e, 220 (2016). \\u003c/li\\u003e\\n\\u003cli\\u003eProtection), G. I. F. U.-I. U. W. I. U. U. U. J. G. o. E. o. t. S. A. o. M. E. Report of the forty-second session of GESAMP. \\u003cem\\u003eGESAMP Reports \\u0026amp; Studies Series\\u003c/em\\u003e \\u003cstrong\\u003e92\\u003c/strong\\u003e, 57 (2016). \\u003c/li\\u003e\\n\\u003cli\\u003eYates, J.\\u003cem\\u003e et al.\\u003c/em\\u003e Plastics matter in the food system. \\u003cem\\u003eCommun Earth Environ\\u003c/em\\u003e \\u003cstrong\\u003e6\\u003c/strong\\u003e, 176 (2025). https://doi.org/10.1038/s43247-025-02105-7\\u003c/li\\u003e\\n\\u003cli\\u003eGeyer, R., Jambeck, J. R. \\u0026amp; Law, K. L. Production, use, and fate of all plastics ever made. \\u003cem\\u003eScience Advances\\u003c/em\\u003e \\u003cstrong\\u003e3\\u003c/strong\\u003e (2017-07). https://doi.org/10.1126/sciadv.1700782\\u003c/li\\u003e\\n\\u003cli\\u003eFrias, J. P. G. L. \\u0026amp; Nash, R. Microplastics: Finding a consensus on the definition. \\u003cem\\u003eMarine Pollution Bulletin\\u003c/em\\u003e \\u003cstrong\\u003e138\\u003c/strong\\u003e (2019/01/01). https://doi.org/10.1016/j.marpolbul.2018.11.022\\u003c/li\\u003e\\n\\u003cli\\u003eDanopoulos, E., Twiddy, M. \\u0026amp; Rotchell, J. M. Microplastic contamination of drinking water: A systematic review. \\u003cem\\u003ePLOS ONE\\u003c/em\\u003e \\u003cstrong\\u003e15\\u003c/strong\\u003e (Jul 31, 2020). https://doi.org/10.1371/journal.pone.0236838\\u003c/li\\u003e\\n\\u003cli\\u003eDe-la-Torre, G. E. \\u0026amp; De-la-Torre, G. E. Microplastics: an emerging threat to food security and human health. \\u003cem\\u003eJournal of Food Science and Technology 2019 57:5\\u003c/em\\u003e \\u003cstrong\\u003e57\\u003c/strong\\u003e (2019-10-19). https://doi.org/10.1007/s13197-019-04138-1\\u003c/li\\u003e\\n\\u003cli\\u003eQian, N.\\u003cem\\u003e et al.\\u003c/em\\u003e Rapid single-particle chemical imaging of nanoplastics by SRS microscopy. \\u003cem\\u003eProc Natl Acad Sci U S A\\u003c/em\\u003e \\u003cstrong\\u003e121\\u003c/strong\\u003e, e2300582121 (2024). https://doi.org/10.1073/pnas.2300582121\\u003c/li\\u003e\\n\\u003cli\\u003eYates, J.\\u003cem\\u003e et al.\\u003c/em\\u003e A systematic scoping review of environmental, food security and health impacts of food system plastics. \\u003cem\\u003eNat Food\\u003c/em\\u003e \\u003cstrong\\u003e2\\u003c/strong\\u003e, 80-87 (2021). https://doi.org/10.1038/s43016-021-00221-z\\u003c/li\\u003e\\n\\u003cli\\u003eChen, Q.\\u003cem\\u003e et al.\\u003c/em\\u003e Long-range atmospheric transport of microplastics across the southern hemisphere. \\u003cem\\u003eNat Commun\\u003c/em\\u003e \\u003cstrong\\u003e14\\u003c/strong\\u003e, 7898 (2023). https://doi.org/10.1038/s41467-023-43695-0\\u003c/li\\u003e\\n\\u003cli\\u003eLehner, R., Weder, C., Petri-Fink, A. \\u0026amp; Rothen-Rutishauser, B. Emergence of Nanoplastic in the Environment and Possible Impact on Human Health. \\u003cem\\u003eEnvironmental Science \\u0026amp; Technology\\u003c/em\\u003e \\u003cstrong\\u003e53\\u003c/strong\\u003e (January 10, 2019). https://doi.org/10.1021/acs.est.8b05512\\u003c/li\\u003e\\n\\u003cli\\u003eHussain, N., Jaitley, V. \\u0026amp; Florence, A. T. Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. \\u003cem\\u003eAdvanced Drug Delivery Reviews\\u003c/em\\u003e \\u003cstrong\\u003e50\\u003c/strong\\u003e (2001/08/23). https://doi.org/10.1016/S0169-409X(01)00152-1\\u003c/li\\u003e\\n\\u003cli\\u003eBerntsen, P.\\u003cem\\u003e et al.\\u003c/em\\u003e Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells. \\u003cem\\u003eJournal of The Royal Society Interface\\u003c/em\\u003e \\u003cstrong\\u003e7\\u003c/strong\\u003e (2010-6-6). https://doi.org/10.1098/rsif.2010.0068.focus\\u003c/li\\u003e\\n\\u003cli\\u003eFr\\u0026ouml;hlich, E.\\u003cem\\u003e et al.\\u003c/em\\u003e Cytotoxicity of nanoparticles independent from oxidative stress. \\u003cem\\u003eThe Journal of Toxicological Sciences\\u003c/em\\u003e \\u003cstrong\\u003e34\\u003c/strong\\u003e (2009/08/01). https://doi.org/10.2131/jts.34.363\\u003c/li\\u003e\\n\\u003cli\\u003eTeuten, E. L.\\u003cem\\u003e et al.\\u003c/em\\u003e Transport and release of chemicals from plastics to the environment and to wildlife. \\u003cem\\u003ePhilos Trans R Soc Lond B Biol Sci\\u003c/em\\u003e \\u003cstrong\\u003e364\\u003c/strong\\u003e, 2027-2045 (2009). https://doi.org/10.1098/rstb.2008.0284\\u003c/li\\u003e\\n\\u003cli\\u003eAlijagic, A.\\u003cem\\u003e et al.\\u003c/em\\u003e The triple exposure nexus of microplastic particles, plastic-associated chemicals, and environmental pollutants from a human health perspective. \\u003cem\\u003eEnvironment International\\u003c/em\\u003e \\u003cstrong\\u003e188\\u003c/strong\\u003e (2024). https://doi.org/ARTN 108736 10.1016/j.envint.2024.108736\\u003c/li\\u003e\\n\\u003cli\\u003eHahladakis, J. N., Velis, C. A., Weber, R., Iacovidou, E. \\u0026amp; Purnell, P. An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. \\u003cem\\u003eJournal of Hazardous Materials\\u003c/em\\u003e \\u003cstrong\\u003e344\\u003c/strong\\u003e, 179-199 (2018). https://doi.org/10.1016/j.jhazmat.2017.10.014\\u003c/li\\u003e\\n\\u003cli\\u003eYu, Y.\\u003cem\\u003e et al.\\u003c/em\\u003e Various additive release from microplastics and their toxicity in aquatic environments. \\u003cem\\u003eEnvironmental Pollution\\u003c/em\\u003e \\u003cstrong\\u003e343\\u003c/strong\\u003e (2024/02/15). https://doi.org/10.1016/j.envpol.2023.123219\\u003c/li\\u003e\\n\\u003cli\\u003eRochman, C. M., Hoh, E., Kurobe, T. \\u0026amp; Teh, S. J. Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress. \\u003cem\\u003eSci Rep\\u003c/em\\u003e \\u003cstrong\\u003e3\\u003c/strong\\u003e, 3263 (2013). https://doi.org/10.1038/srep03263\\u003c/li\\u003e\\n\\u003cli\\u003eBrowne, M. A., Niven, S. J., Galloway, T. S., Rowland, S. J. \\u0026amp; Thompson, R. C. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. \\u003cem\\u003eCurr Biol\\u003c/em\\u003e \\u003cstrong\\u003e23\\u003c/strong\\u003e, 2388-2392 (2013). https://doi.org/10.1016/j.cub.2013.10.012\\u003c/li\\u003e\\n\\u003cli\\u003eDeng, Y.\\u003cem\\u003e et al.\\u003c/em\\u003e Microplastics release phthalate esters and cause aggravated adverse effects in the mouse gut. \\u003cem\\u003eEnviron Int\\u003c/em\\u003e \\u003cstrong\\u003e143\\u003c/strong\\u003e, 105916 (2020). https://doi.org/10.1016/j.envint.2020.105916\\u003c/li\\u003e\\n\\u003cli\\u003eShin, N., Cuenca, L., Karthikraj, R., Kannan, K. \\u0026amp; Colaiacovo, M. P. Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans. \\u003cem\\u003ePLoS Genet\\u003c/em\\u003e \\u003cstrong\\u003e15\\u003c/strong\\u003e, e1007975 (2019). https://doi.org/10.1371/journal.pgen.1007975\\u003c/li\\u003e\\n\\u003cli\\u003eFDA. \\u003cem\\u003eBisphenol A (BPA)\\u003c/em\\u003e, \\u0026lt;https://www.fda.gov/food/food-packaging-other-substances-come-contact-food-information-consumers/bisphenol-bpa\\u0026gt; (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eRochester, J. R. Bisphenol A and human health: a review of the literature. \\u003cem\\u003eReprod Toxicol\\u003c/em\\u003e \\u003cstrong\\u003e42\\u003c/strong\\u003e, 132-155 (2013). https://doi.org/10.1016/j.reprotox.2013.08.008\\u003c/li\\u003e\\n\\u003cli\\u003eMonti, M., Fasano, M., Palandri, L. \\u0026amp; Righi, E. A review of European and international phthalates regulation: focus on daily use products. \\u003cem\\u003eEur J Public Health\\u003c/em\\u003e \\u003cstrong\\u003e32\\u003c/strong\\u003e (2022). \\u003c/li\\u003e\\n\\u003cli\\u003eSiracusa, J. S., Yin, L., Measel, E., Liang, S. \\u0026amp; Yu, X. Effects of bisphenol A and its analogs on reproductive health: A mini review. \\u003cem\\u003eReprod Toxicol\\u003c/em\\u003e \\u003cstrong\\u003e79\\u003c/strong\\u003e, 96-123 (2018). https://doi.org/10.1016/j.reprotox.2018.06.005\\u003c/li\\u003e\\n\\u003cli\\u003eSicinska, P., Mokra, K., Wozniak, K., Michalowicz, J. \\u0026amp; Bukowska, B. Genotoxic risk assessment and mechanism of DNA damage induced by phthalates and their metabolites in human peripheral blood mononuclear cells. \\u003cem\\u003eSci Rep\\u003c/em\\u003e \\u003cstrong\\u003e11\\u003c/strong\\u003e, 1658 (2021). https://doi.org/10.1038/s41598-020-79932-5\\u003c/li\\u003e\\n\\u003cli\\u003eMankidy, R., Wiseman, S., Ma, H. \\u0026amp; Giesy, J. P. Biological impact of phthalates. \\u003cem\\u003eToxicol Lett\\u003c/em\\u003e \\u003cstrong\\u003e217\\u003c/strong\\u003e, 50-58 (2013). https://doi.org/10.1016/j.toxlet.2012.11.025\\u003c/li\\u003e\\n\\u003cli\\u003eTOXICOLOGICAL PROFILE FOR DI-n-BUTYL PHTHALATE. \\u003cem\\u003eAgency for Toxic Substances and Disease Registry\\u003c/em\\u003e, 225 (2001). \\u003c/li\\u003e\\n\\u003cli\\u003eLiu, F.-f., Liu, G.-z., Zhu, Z.-l., Wang, S.-c. \\u0026amp; Zhao, F.-f. Interactions between microplastics and phthalate esters as affected by microplastics characteristics and solution chemistry. \\u003cem\\u003eChemosphere\\u003c/em\\u003e \\u003cstrong\\u003e214\\u003c/strong\\u003e (2019/01/01). https://doi.org/10.1016/j.chemosphere.2018.09.174\\u003c/li\\u003e\\n\\u003cli\\u003eAndrady, A. L.\\u003cem\\u003e et al.\\u003c/em\\u003e Oxidation and fragmentation of plastics in a changing environment; from UV-radiation to biological degradation. \\u003cem\\u003eSci Total Environ\\u003c/em\\u003e \\u003cstrong\\u003e851\\u003c/strong\\u003e, 158022 (2022). https://doi.org/10.1016/j.scitotenv.2022.158022\\u003c/li\\u003e\\n\\u003cli\\u003eYan, Y.\\u003cem\\u003e et al.\\u003c/em\\u003e Dibutyl phthalate release from polyvinyl chloride microplastics: Influence of plastic properties and environmental factors. \\u003cem\\u003eWater Res\\u003c/em\\u003e \\u003cstrong\\u003e204\\u003c/strong\\u003e, 117597 (2021). https://doi.org/10.1016/j.watres.2021.117597\\u003c/li\\u003e\\n\\u003cli\\u003eMao, S. \\u0026amp; He, C. Effect of particle size and environmental conditions on the release of di(2-ethylhexyl) phthalate from microplastics. \\u003cem\\u003eChemosphere\\u003c/em\\u003e \\u003cstrong\\u003e345\\u003c/strong\\u003e, 140474 (2023). https://doi.org/10.1016/j.chemosphere.2023.140474\\u003c/li\\u003e\\n\\u003cli\\u003eLenz, R., Enders, K. \\u0026amp; Nielsen, T. G. Microplastic exposure studies should be environmentally realistic. \\u003cem\\u003eProc Natl Acad Sci U S A\\u003c/em\\u003e \\u003cstrong\\u003e113\\u003c/strong\\u003e, E4121-4122 (2016). https://doi.org/10.1073/pnas.1606615113\\u003c/li\\u003e\\n\\u003cli\\u003eQiu, Y., Liu, Y., Li, Y., Li, G. \\u0026amp; Wang, D. Effect of chronic exposure to nanopolystyrene on nematode Caenorhabditis elegans. \\u003cem\\u003eChemosphere\\u003c/em\\u003e \\u003cstrong\\u003e256\\u003c/strong\\u003e, 127172 (2020). https://doi.org/10.1016/j.chemosphere.2020.127172\\u003c/li\\u003e\\n\\u003cli\\u003ePeng, M., Felix, R. C., Canario, A. V. M. \\u0026amp; Power, D. M. The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts. \\u003cem\\u003eSci Total Environ\\u003c/em\\u003e \\u003cstrong\\u003e914\\u003c/strong\\u003e, 169979 (2024). https://doi.org/10.1016/j.scitotenv.2024.169979\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Z.\\u003cem\\u003e et al.\\u003c/em\\u003e Effects of microplastics on the innate immunity and intestinal microflora of juvenile Eriocheir sinensis. \\u003cem\\u003eSci Total Environ\\u003c/em\\u003e \\u003cstrong\\u003e685\\u003c/strong\\u003e, 836-846 (2019). https://doi.org/10.1016/j.scitotenv.2019.06.265\\u003c/li\\u003e\\n\\u003cli\\u003eJeong, A., Park, S. J., Lee, E. J. \\u0026amp; Kim, K. W. Nanoplastics exacerbate Parkinson\\u0026apos;s disease symptoms in C. elegans and human cells. \\u003cem\\u003eJournal of Hazardous Materials\\u003c/em\\u003e \\u003cstrong\\u003e465\\u003c/strong\\u003e (2024/03/05). https://doi.org/10.1016/j.jhazmat.2023.133289\\u003c/li\\u003e\\n\\u003cli\\u003eMueller, M. T.\\u003cem\\u003e et al.\\u003c/em\\u003e Surface-Related Toxicity of Polystyrene Beads to Nematodes and the Role of Food Availability. \\u003cem\\u003eEnviron Sci Technol\\u003c/em\\u003e \\u003cstrong\\u003e54\\u003c/strong\\u003e, 1790-1798 (2020). https://doi.org/10.1021/acs.est.9b06583\\u003c/li\\u003e\\n\\u003cli\\u003eErrazuriz Leon, R.\\u003cem\\u003e et al.\\u003c/em\\u003e Photoaged polystyrene nanoplastics exposure results in reproductive toxicity due to oxidative damage in Caenorhabditis elegans. \\u003cem\\u003eEnviron Pollut\\u003c/em\\u003e \\u003cstrong\\u003e348\\u003c/strong\\u003e, 123816 (2024). https://doi.org/10.1016/j.envpol.2024.123816\\u003c/li\\u003e\\n\\u003cli\\u003eLei, L.\\u003cem\\u003e et al.\\u003c/em\\u003e Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. \\u003cem\\u003eSci Total Environ\\u003c/em\\u003e \\u003cstrong\\u003e619-620\\u003c/strong\\u003e, 1-8 (2018). https://doi.org/10.1016/j.scitotenv.2017.11.103\\u003c/li\\u003e\\n\\u003cli\\u003ePivnenko, K., Eriksen, M. K., Martin-Fernandez, J. A., Eriksson, E. \\u0026amp; Astrup, T. F. Recycling of plastic waste: Presence of phthalates in plastics from households and industry. \\u003cem\\u003eWaste Manag\\u003c/em\\u003e \\u003cstrong\\u003e54\\u003c/strong\\u003e, 44-52 (2016). https://doi.org/10.1016/j.wasman.2016.05.014\\u003c/li\\u003e\\n\\u003cli\\u003eGray, L. E., Jr., Laskey, J. \\u0026amp; Ostby, J. Chronic di-n-butyl phthalate exposure in rats reduces fertility and alters ovarian function during pregnancy in female Long Evans hooded rats. \\u003cem\\u003eToxicol Sci\\u003c/em\\u003e \\u003cstrong\\u003e93\\u003c/strong\\u003e, 189-195 (2006). https://doi.org/10.1093/toxsci/kfl035\\u003c/li\\u003e\\n\\u003cli\\u003eSen, N., Liu, X. \\u0026amp; Craig, Z. R. Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. \\u003cem\\u003eReprod Toxicol\\u003c/em\\u003e \\u003cstrong\\u003e53\\u003c/strong\\u003e, 15-22 (2015). https://doi.org/10.1016/j.reprotox.2015.02.012\\u003c/li\\u003e\\n\\u003cli\\u003eAlam, M. S.\\u003cem\\u003e et al.\\u003c/em\\u003e Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate. \\u003cem\\u003eReproduction\\u003c/em\\u003e \\u003cstrong\\u003e139\\u003c/strong\\u003e, 427-437 (2010). https://doi.org/10.1530/REP-09-0226\\u003c/li\\u003e\\n\\u003cli\\u003eOkayama, Y.\\u003cem\\u003e et al.\\u003c/em\\u003e In Utero Exposure to Di( n-butyl)phthalate Induces Morphological and Biochemical Changes in Rats Postpuberty. \\u003cem\\u003eToxicol Pathol\\u003c/em\\u003e \\u003cstrong\\u003e45\\u003c/strong\\u003e, 526-535 (2017). https://doi.org/10.1177/0192623317709091\\u003c/li\\u003e\\n\\u003cli\\u003eSchneider, I. \\u0026amp; Ellenberg, J. Mysteries in embryonic development: How can errors arise so frequently at the beginning of mammalian life? \\u003cem\\u003ePLoS Biol\\u003c/em\\u003e \\u003cstrong\\u003e17\\u003c/strong\\u003e, e3000173 (2019). https://doi.org/10.1371/journal.pbio.3000173\\u003c/li\\u003e\\n\\u003cli\\u003eHornos Carneiro, M. F.\\u003cem\\u003e et al.\\u003c/em\\u003e Antioxidant CoQ10 Restores Fertility by Rescuing Bisphenol A-Induced Oxidative DNA Damage in the Caenorhabditis elegans Germline. \\u003cem\\u003eGenetics\\u003c/em\\u003e \\u003cstrong\\u003e214\\u003c/strong\\u003e, 381-395 (2020). https://doi.org/10.1534/genetics.119.302939\\u003c/li\\u003e\\n\\u003cli\\u003eLiang, B.\\u003cem\\u003e et al.\\u003c/em\\u003e Underestimated health risks: polystyrene micro- and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis. \\u003cem\\u003ePart Fibre Toxicol\\u003c/em\\u003e \\u003cstrong\\u003e18\\u003c/strong\\u003e, 20 (2021). https://doi.org/10.1186/s12989-021-00414-1\\u003c/li\\u003e\\n\\u003cli\\u003eQu, M.\\u003cem\\u003e et al.\\u003c/em\\u003e Nanopolystyrene at predicted environmental concentration enhances microcystin-LR toxicity by inducing intestinal damage in Caenorhabditis elegans. \\u003cem\\u003eEcotoxicol Environ Saf\\u003c/em\\u003e \\u003cstrong\\u003e183\\u003c/strong\\u003e, 109568 (2019). https://doi.org/10.1016/j.ecoenv.2019.109568\\u003c/li\\u003e\\n\\u003cli\\u003eWu, Y., Tan, X., Shi, X., Han, P. \\u0026amp; Liu, H. Combined Effects of Micro- and Nanoplastics at the Predicted Environmental Concentration on Functional State of Intestinal Barrier in Caenorhabditis elegans. \\u003cem\\u003eToxics\\u003c/em\\u003e \\u003cstrong\\u003e11\\u003c/strong\\u003e (2023). https://doi.org/10.3390/toxics11080653\\u003c/li\\u003e\\n\\u003cli\\u003eYu, Y.\\u003cem\\u003e et al.\\u003c/em\\u003e Polystyrene microplastics (PS-MPs) toxicity induced oxidative stress and intestinal injury in nematode Caenorhabditis elegans. \\u003cem\\u003eSci Total Environ\\u003c/em\\u003e \\u003cstrong\\u003e726\\u003c/strong\\u003e, 138679 (2020). https://doi.org/10.1016/j.scitotenv.2020.138679\\u003c/li\\u003e\\n\\u003cli\\u003eHuang, C. W., Yen, P. L., Kuo, Y. H., Chang, C. H. \\u0026amp; Liao, V. H. Nanoplastic exposure in soil compromises the energy budget of the soil nematode C. elegans and decreases reproductive fitness. \\u003cem\\u003eEnviron Pollut\\u003c/em\\u003e \\u003cstrong\\u003e312\\u003c/strong\\u003e, 120071 (2022). https://doi.org/10.1016/j.envpol.2022.120071\\u003c/li\\u003e\\n\\u003cli\\u003eTseng, I. L., Yang, Y. F., Yu, C. W., Li, W. H. \\u0026amp; Liao, V. H. Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans. \\u003cem\\u003ePLoS One\\u003c/em\\u003e \\u003cstrong\\u003e8\\u003c/strong\\u003e, e82657 (2013). https://doi.org/10.1371/journal.pone.0082657\\u003c/li\\u003e\\n\\u003cli\\u003eSenchuk, M. M.\\u003cem\\u003e et al.\\u003c/em\\u003e Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans. \\u003cem\\u003ePLoS Genet\\u003c/em\\u003e \\u003cstrong\\u003e14\\u003c/strong\\u003e, e1007268 (2018). https://doi.org/10.1371/journal.pgen.1007268\\u003c/li\\u003e\\n\\u003cli\\u003eLee, R. Y., Hench, J. \\u0026amp; Ruvkun, G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. \\u003cem\\u003eCurr Biol\\u003c/em\\u003e \\u003cstrong\\u003e11\\u003c/strong\\u003e, 1950-1957 (2001). https://doi.org/10.1016/s0960-9822(01)00595-4\\u003c/li\\u003e\\n\\u003cli\\u003eKe, T.\\u003cem\\u003e et al.\\u003c/em\\u003e N,N\\u0026apos; bis-(2-mercaptoethyl) isophthalamide induces developmental delay in Caenorhabditis elegans by promoting DAF-16 nuclear localization. \\u003cem\\u003eToxicol Rep\\u003c/em\\u003e \\u003cstrong\\u003e7\\u003c/strong\\u003e, 930-937 (2020). https://doi.org/10.1016/j.toxrep.2020.07.012\\u003c/li\\u003e\\n\\u003cli\\u003eLeite, N. R.\\u003cem\\u003e et al.\\u003c/em\\u003e Baru Pulp (Dipteryx alata Vogel): Fruit from the Brazilian Savanna Protects against Oxidative Stress and Increases the Life Expectancy of Caenorhabditis elegans via SOD-3 and DAF-16. \\u003cem\\u003eBiomolecules\\u003c/em\\u003e \\u003cstrong\\u003e10\\u003c/strong\\u003e (2020). https://doi.org/10.3390/biom10081106\\u003c/li\\u003e\\n\\u003cli\\u003eLin, K., Hsin, H., Libina, N. \\u0026amp; Kenyon, C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. \\u003cem\\u003eNat Genet\\u003c/em\\u003e \\u003cstrong\\u003e28\\u003c/strong\\u003e, 139-145 (2001). https://doi.org/10.1038/88850\\u003c/li\\u003e\\n\\u003cli\\u003eZhou, K. I., Pincus, Z. \\u0026amp; Slack, F. J. Longevity and stress in Caenorhabditis elegans. \\u003cem\\u003eAging (Albany NY)\\u003c/em\\u003e \\u003cstrong\\u003e3\\u003c/strong\\u003e, 733-753 (2011). https://doi.org/10.18632/aging.100367\\u003c/li\\u003e\\n\\u003cli\\u003eLibina, N., Berman, J. R. \\u0026amp; Kenyon, C. Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan. \\u003cem\\u003eCell\\u003c/em\\u003e \\u003cstrong\\u003e115\\u003c/strong\\u003e, 489-502 (2003). https://doi.org/10.1016/s0092-8674(03)00889-4\\u003c/li\\u003e\\n\\u003cli\\u003eReyes, M. S. S. \\u0026amp; Medina, P. M. B. Leachates from plastics and bioplastics reduce lifespan, decrease locomotion, and induce neurotoxicity in Caenorhabditis elegans. \\u003cem\\u003eEnviron Pollut\\u003c/em\\u003e \\u003cstrong\\u003e357\\u003c/strong\\u003e, 124428 (2024). https://doi.org/10.1016/j.envpol.2024.124428\\u003c/li\\u003e\\n\\u003cli\\u003eZongur, A. Evaluation of the Effects of Di-(2-ethylhexyl) phthalate (DEHP) on Caenorhabditis elegans Survival and Fertility. \\u003cem\\u003eAppl Biochem Biotechnol\\u003c/em\\u003e \\u003cstrong\\u003e196\\u003c/strong\\u003e, 8998-9009 (2024). https://doi.org/10.1007/s12010-024-05032-z\\u003c/li\\u003e\\n\\u003cli\\u003ePradhan, A., Olsson, P. E. \\u0026amp; Jass, J. Di(2-ethylhexyl) phthalate and diethyl phthalate disrupt lipid metabolism, reduce fecundity and shortens lifespan of Caenorhabditis elegans. \\u003cem\\u003eChemosphere\\u003c/em\\u003e \\u003cstrong\\u003e190\\u003c/strong\\u003e, 375-382 (2018). https://doi.org/10.1016/j.chemosphere.2017.09.123\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6629868/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6629868/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eMicroplastics harbor chemical additives and absorb pollutants from the environment. Microplastics pose a human health threat and have been found in nearly all human tissues. The toxicological pathways and physiological effects of microplastic-mediated chemical exposure following ingestion remain unknown. Here we use \\u003cem\\u003eC. elegans\\u003c/em\\u003e to investigate the effects of di-butyl phthalate and polystyrene microplastic mixtures on fertility and lifespan. Our studies demonstrate that 1 \\u0026micro;m microplastics at 1 mg/L exposure levels result in decreased brood size, whereas 1000 times fewer microplastics (1 \\u0026micro;g/L) did not affect the number of eggs laid. While there was no change in brood size at 1 \\u0026micro;g/L microplastic exposure levels, there was an increase in embryonic lethality. Microplastics-mediated delivery of di-butyl phthalate to \\u003cem\\u003eC. elegans\\u003c/em\\u003e significantly reduced brood size and increased embryonic lethality compared to exposure to microplastics alone. This reproductive toxicity is potentially due to a stress response via DAF-16, as observed with microplastics and di-butyl phthalate co-exposure. Furthermore, chronic exposure to microplastics shortened the lifespan of \\u003cem\\u003eC. elegans\\u003c/em\\u003e, which was further reduced with di-butyl phthalate co-exposure. The exacerbated defects observed with co-exposure to phthalate-containing microplastics underscore the risks associated with microplastics releasing the additives and/or chemicals that they have absorbed from the environment.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Microplastic-mediated delivery of di-butyl phthalate alters C. elegans lifespan and reproductive fidelity\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-21 06:39:48\",\"doi\":\"10.21203/rs.3.rs-6629868/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"34fc33e7-3c5b-495c-96f4-c1a0eac8dd46\",\"owner\":[],\"postedDate\":\"May 21st, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":48831080,\"name\":\"Biological sciences/Biological techniques/Experimental organisms\"},{\"id\":48831081,\"name\":\"Biological sciences/Biological techniques/Experimental organisms/Model invertebrates/Caenorhabditis elegans\"},{\"id\":48831082,\"name\":\"Biological sciences/Developmental biology\"},{\"id\":48831083,\"name\":\"Biological sciences/Physiology\"},{\"id\":48831084,\"name\":\"Earth and environmental sciences/Environmental sciences\"},{\"id\":48831085,\"name\":\"Earth and environmental sciences/Natural hazards\"}],\"tags\":[],\"updatedAt\":\"2025-12-09T14:25:41+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6629868\",\"link\":\"https://doi.org/10.3390/microplastics4040096\",\"journal\":{\"identity\":\"microplastics\",\"isVorOnly\":true,\"title\":\"Microplastics\"},\"publishedOn\":\"2025-12-01 00:00:00\",\"publishedOnDateReadable\":\"December 1st, 2025\"},\"versionCreatedAt\":\"2025-05-21 06:39:48\",\"video\":\"\",\"vorDoi\":\"10.3390/microplastics4040096\",\"vorDoiUrl\":\"https://doi.org/10.3390/microplastics4040096\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6629868\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6629868\",\"identity\":\"rs-6629868\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}