Fine Particulate Matter-induced Toxic Effects in an Animal Model of Caenorhabditis elegans

Research has been focused on the health hazards of ambient PM2.5 related to humans. Many PM2.5 toxicity assessments using in vitro studies have focused on PM2.5-bounded hazardous pollutants. However, PM2.5 toxicity assessment by in vivo studies allow for better observation of the overall effects of PM2.5 exposure on entire organisms, making in vivo PM2.5 toxicity assessment relevant. The toxic effects of outdoor PM2.5, collected from National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS), Pingtung, Taiwan, on nematode Caenorhabditis elegans (C. elegans) were investigated. PM2.5 from NPUST and LJHS were found to be 4.5 and 2.5 μg Nm, respectively, which did not meet the standard. This levels of PM2.5 in Taiwan. For acute toxicity, no significant PM2.5 lethality on C. elegans was observed between NPUST and LJHS. PM2.5 from NPUST exhibited greater toxicity to lifespan (ageing), locomotion (head thrash), and reproduction (brood size) in the C. elegans animal models than that from LJHS; therefore, adverse effects could be correlated with PM2.5 concentrations. Prolonged exposure to PM2.5 led to more severe toxicity in nematodes as compared to acute exposure. In conclusion, this study suggests that the long-term adverse effects of ambient PM2.5 on environmental organisms should be carefully considered even when PM2.5 is at low levels. C. elegans is a sensitive animal model for the evaluation of PM2.5 ecotoxicity.


INTRODUCTION
Particulate matter 2.5 (PM 2.5 ) is a type of fine particles with an aerodynamic diameter of less than 2.5 µm. It is produced through processes including natural, indoor, and outdoor combustion processes, as well as other anthropogenic activities (Li et al., 2018a;Martins and da Graça, 2018). Some of the identified major sources of PM 2.5 emissions into the atmosphere are power plants (Mari et al., 2016;Dodla et al., 2017), waste incineration facilities (Yan et al., 2016), and industrial processes, such as the manufacture of steel stacks in the steelmaking industry (Gibson et al., 2013;Owoade et al., 2015). In addition, the large surface area of PM 2.5 facilitates easy adhesion of toxic compounds such as polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs). on its surface (Pandey et al., 2013;Chao et al., 2016;Lee et al., 2018). The World Health Organization (WHO) established guidelines on air quality that include limitations on exposure to PM 2.5 of not more than 25 µg m -3 day -1 and 10 µg m --3 year -1 (Hopke et al., 2018). In Taiwan, the allowable level of exposure to PM 2.5 per year is 35 µg m -3 , and for 24 hours, it is 15 µg m -3 , as regulated by the Taiwan Air Quality Monitoring Network (TAQMN) of the Environmental Protection Administration, Taiwan (TEPA).
Due to the extremely fine size of PM 2.5 particulates, they have been reported to be able to penetrate the blood brain barrier (Feng et al., 2016;Lin et al., 2017). Upon exposure, particulate matter induces overexpression of macrophages in the immune system and oxidative stress resulting in atherosclerotic plaques (Brook and Rajagopalan, 2010) and endothelial and vascular dysfunctions (Feng et al., 2016). PM 2.5 also affects the respiratory tract, particularly tiny sacs in the lungs called alveoli, where carbon dioxide and oxygen exchange takes place during respiration (Pinkerton et al., 2000). This can further result in the activation of a series of inflammatory responses in the lungs (Chao et al., 2018;Zhao et al., 2019). PM 2.5 is ubiquitous in the environment (Salako et al., 2012;Mohammadyan et al., 2017;Seneviratne et al., 2017), and it has been reported that people suffering from cardiopulmonary and respiratory diseases are at high risk because their exposure to fine particles can cause short-term health effects such as eye, nose, throat and lung irritation, coughing, sneezing, runny nose, and shortness of breath (Kim et al., 2018;Maciejczyk et al., 2018;Polezer et al., 2018). Several in vivo studies utilizing mice models have reported that exposure to airborne PM 2.5 prolongs lung tumour progression (Yang and Xiao, 2018), promotes abdominal aortic aneurysms (Jun et al., 2018), increases inflammatory cell infiltration , and causes lipid accumulation and hepatic function loss . In vitro studies have associated PM 2.5 exposure to induced cytotoxicity and autophagy in human endothelial cells (Zhou et al., 2018), as well as telomere lengthening (Miri et al., 2019). Most importantly, epidemiological studies have shown that both short-term and long-term exposure to PM increases the risk of developing neurological disorders such as stroke (Fu et al., 2019), Parkinson's disease , and congenital heart disease as a result of prenatal exposure , and generally stronger associations have been found with mortality and hospitalization (Karimi et al., 2019). Recently, Lo et al. (2017) reported that PM 2.5 was involved in deaths resulting from ischemic heart disease, stroke, lung cancer, and chronic obstructive pulmonary disease, wherein a fraction of 18.6% of the specified diseases was linked to PM 2.5 in Taiwan. Therefore, PM 2.5 is a major risk factor in Taiwan (Lung et al., 2016). Currently, there is no known established standard PM 2.5 exposure level that may be referenced to avoid its adverse health effects (Kiesewetter et al., 2015). Similarly, there is no singular in vivo animal model that can completely represent the human system. Therefore, there is a need for more research using other available model organisms to further understand the effects of PM 2.5 on human health (Feng et al., 2016).
Caenorhabditis elegans (C. elegans) was established by Sydney Brenner as a model organism in 1965 (Brenner, 1974). Compared with single-cell based in vitro assays, C. elegans-based assays allow for the evaluation of multiple toxicity endpoints (Kaletta and Hengartner, 2006). At the genetic level, more than half of the conserved signaling regions in humans are found in the nematode (Aoki and Mori, 2015). Numerous studies have reported the sensitivity of C. elegans in terms of pollution from different types of environmental media (Clavijo et al., 2016;Zuo et al., 2017;Rai et al., 2019). However, only a few studies have reported the sensitivity of C. elegans to air pollutants, specifically, to PM 2.5 (Zhao et al., 2014;Sun et al., 2015Sun et al., , 2016. In a study by Sun et al. (2015), exposure to coal combustion-related PM 2.5 induced lower response in all organism-level endpoints, specifically, prolonged exposure caused more severity than acute exposure to the nematodes. In addition, when Sun et al. (2016) determined the contribution of heavy metals found in coal combustionrelated PM 2.5 , he found that the combined exposure to specific metals caused greater toxicity than single exposure of said metals. Most importantly, transgenerational effects after exposure to diesel-and traffic-related PM 2.5 were discovered by Wang et al. (2019) and Zhao et al. (2014) wherein reproductive disability had been transferred to later generations of C. elegans.
C. elegans is an inexpensive, bioethical, and easy model animal to maintain, with attractive physiological characteristics that can be used to assess the toxicological effects of environmental pollutants (Leung et al., 2008). In this study, the toxicity of acute exposure to ambient outdoor PM 2.5 samples collected from Southern Taiwanese schools such as National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS) was assessed using the different C. elegans toxicological endpoints, which include survival (lethality and lifespan), reproduction (brood size), developmental (growth), and locomotion (head thrash and body bend) endpoints.

Chemical Reagents
The wild-type N2 strain of C. elegans was acquired from the Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University (Tainan, Taiwan) and was maintained in nematode growth medium (NGM) plates containing bacteriological agar and bactopeptone (Laboratories Conda, S.A., Spain) and NaCl (Honeywell Fluka™, New Jersey, USA). Additional NGM plate components such as CaCl 2 , K 2 HPO 4 , and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, USA), and MgSO 4 was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea). OP50 Escherichia coli (E. coli) cultures were acquired from the Bioresources Collection and Research Center (Hsinchu, Taiwan) and Luria-Bertani broth was obtained from Sigma-Aldrich (St. Louis, MO, USA). For the bleaching solution, NaOCl was obtained from J.T. Baker (Central Valley, PA), and KOH was obtained from Duksan Pure Chemicals (Gyeonggi-do, South Korea). KH 2 PO 4 used for the phosphate buffer was acquired from Avantor Performance Materials, LLC (Radnor, PA, USA), and Na 2 HPO 4 used for the M9 buffer was obtained from Honeywell Fluka™ (New Jersey, USA). All physiological observations were done under a dissecting microscope (Olympus, SZX10, Waltham MA, USA). For the K-medium ingredients, NaCl was obtained from Honeywell Fluka™ (New Jersey, USA), and KCl was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea). Dehydroethidium (DHE) for oxidative stress detection was purchased from Invitrogen™, Thermo Fisher Scientific (Waltham, MA USA).

Sample Collection
Outdoor ambient PM 2.5 samples were collected from two southern Taiwanese schools, NPUST and LJHS, located in Neipu, Pingtung County which is in the rural area. The air sampling site in NPUST was set up at the top floor of the College of Engineering in the NPUST campus. The other site in LJHS is nearby Linlou System Interchange of Formosa Highway. Each air sample was collected for 48 hours (2 days, 1 m 3 min -1 ) using a high-volume air sampler (SIBATA HV-1000R, Japan) following US EPA Reference Method TO9A. The PM 2.5 samples were collected onto quartz fibre filters that were pre-heated before sampling at 600°C for 2 hours. A balance with an accuracy of 0.1 mg was used to weigh the filter paper conditioned in an electronic desiccator before and after the sample collection for 24 h. The loaded filters were stored in a refrigerator at -20°C before extraction to limit the possible evaporation of volatile components.

Extraction, Purification, and Sample Preparation
After the collection of air samples, the mixture was extracted using a Soxhlet extractor with dichloromethane (DCM) for 24 h. The extracts were eluted with 15 mL DCM during the alumina column clean-up. The eluate was concentrated to approximately 1 mL and transferred to a vial. The concentrate was further concentrated to near dryness using a stream of nitrogen. For exposure concentrations, PM 2.5 was serially diluted 10X with 1% DMSO. The toxic effects in the study were limited in the organic fraction.

C. elegans Age Synchronization for Exposure Experiments
Wild-type N2 C. elegans were maintained on NGM plates seeded with E. coli as food and incubated at 22°C until the plates had a high density of eggs and gravid nematodes. These were then washed off the plates, placed into centrifuge tubes, and the gravid nematodes were lysed with a bleaching mixture leaving only the eggs to obtain age synchronized populations of L1 nematode larvae in preparation for the exposure experiments and toxicity assays.

Acute Exposure
Synchronized L1 larvae were seeded onto NGM plates and incubated until L3 or young L4 stage. The L3 and young L4 worms were rinsed from the plate with K-medium, gently washed, and centrifuged at 2500 x g for 4 minutes to remove the E. coli in the supernatant by aspirating without disturbing the nematode pellet. After washing, the pellet was re-suspended in K medium through continuous pipetting, and a seeding volume (worm per µL) was calculated by pipetting two 5 µL drops on a glass slide and manually counting the worms in each drop. Approximately 200 L3/young L4 worms were dispensed into each well of a 12-well plate each containing 1 mL of the different exposure concentrations for the two PM 2.5 samples (one set for NPUST and another for LJHS) diluted with K-medium. Acute (24-hour) exposure was performed at 22°C without the presence of food.

Lethality Assay
The lethality assay was performed by transferring 50 exposed nematodes (per exposure concentration) to NGM plates without food (OP50). These worms were gently prodded using the worm picker to assess viability. Nematodes that were non-responsive to stimulus were scored as dead. The assays were done in triplicates for each of the concentrations.

Lifespan Assay (Ageing Assay)
Fifty L3/young L4 worms from the acute exposure treatment were transferred to NGM plates with food (OP50). The worms were transferred to fresh NGM plates every day for the first 4 to 5 days (egg-laying period), and after that, the worms were no longer transferred. The plates were incubated at 22°C. Alive, censored (lost), and dead nematodes were recorded every 2 days within a span of 24 days (general lifespan of C. elegans). The survival plot or the Kaplan-Meier plot was constructed to evaluate the effects of the different concentrations of PM 2.5 on the lifespan or ageing of the nematodes. Triplicates were done for each of the exposure concentrations.

Reproductive or Brood Size Assay
L3/young L4 worms from the acute exposure treatment were assessed for 4 to 5 days of egg-laying. One L3/young L4 worm was transferred to each of the wells of a 12-well NGM plate with a fresh OP50 lawn. Each worm was transferred to a new plate every 2 days within the duration of egg-laying period. The old plates containing the eggs were hatched and incubated to L4 worms for easier counting of progeny. The total progeny for each of the worms was recorded. Twelve worms were evaluated for each of the exposure concentrations.

Growth Measurement Assay
L3/young L4 worms exposed to each of the PM 2.5 concentrations for 24 hours with prolonged exposure were measured for their length to assess the effects of PM 2.5 on the growth of the nematodes. Ten L3/young L4 nematodes exposed to each of the exposure concentrations were transferred to NGM plates with OP50 lawns and were incubated at 22°C for 48 hours until they reached the old L4 stage. The nematodes from each of the concentrations were exposed to a high temperature of 66°C in a dry bath for 5 minutes. This process straightens the body of the C. elegans, making it easier to measure the length. An optical microscope (Olympus) was used, and ImageJ software was utilized for the measurement of the worm length.

Locomotion Assay (Head Thrash and Body Bend Assay)
The endpoints of head thrash and body bending were used to evaluate the locomotion of the exposed L3/young L4 C. elegans by allowing the nematodes swim in K-medium. A head thrash was defined as a change in the direction of the mid body wherein the head and tail falls in the same direction as it moves. Body bending was regarded as a change in direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematode was traveling along the x axis. Head thrashing was counted for 1 minute, and body bending was counted for 20 seconds. Thirty nematodes were examined per treatment, and three replicates were performed.

Statistical Analysis
The locomotion, brood size, and length measurement data for the nematodes did not meet the normal distribution using a normality test such as the Shapiro-Wilk test. The Kaplan-Meier plots for the survival rates were used to evaluate the effects of the different PM 2.5 concentrations on the lifespan or ageing of the nematodes. The nonparametric Kruskal-Wallis H test was used to examine the differences between each the concentrations in comparison to the control. The survival plots or the Kaplan-Meier plot were derived for the lifespan data using GraphPad Prism 6 (San Diego, California, USA). The significance of each of the days were determined with the Kruskal-Wallis H test. Days with significance were chosen and tested further. Each of the concentrations from the chosen days was also compared to the control using the Mann-Whitney U test. The statistical analyses were tested using SPSS version 12 (International Business Machines Corp., New York, USA).

Lethality Effects of PM2.5 on C. elegans
Age-synchronized L4 worms were subjected to 24 hours of exposure to serially diluted samples of PM 2.5 , after which lethality was investigated. No lethality was observed in any of the PM 2.5 sample concentrations of NPUST (0.0014443-1444.3 mg L -1 ) and LJHS (0.0011518-1151.8 mg L -1 ) ( Fig. 1) collected from NPUST and LJHS. Similarly, in a study conducted by Sun et al. (2015), coal combustionrelated PM 2.5 with exposure concentrations ranging from 0.01 to 100 mg L -1 did not show any lethal effects on nematodes.

Life Span of C. elegans after PM2.5 Exposure
In this study, the toxicological endpoint lifespan was also evaluated since exposure of the nematodes to PM 2.5 concentrations greater than 1 µg L -1 was observed to cause toxicities. The concentrations of PM 2.5 determined from NPUST and LJHS were 4.5 and 2.5 µg Nm -3 respectively. The detected levels of PM 2.5 from both schools were lower than the allowable levels of outdoor PM 2.5 in Taiwan, as regulated by the TAQMN. However, prolonged exposure showed that the lifespan of C. elegans was significantly reduced for all sample concentrations from the NPUST sampling site. Specifically, the lowest and highest concentrations (1.4443 × 10 -3 mg L -1 and 1.4443 × 10 3 mg L -1 ) from NPUST caused the sharpest decrease in lifespan starting on post-exposure day 10 (Fig. 2). Additionally, a significant reduction in lifespan was observed on an earlier post-exposure day for LJHS for all concentrations. Concentrations of 1.5518 × 10 3 and 1.5518 × 10 2 mg L -1 caused a rapid decrease in the population of the nematodes. Lastly, C. elegans death occurred on post-exposure day 22, which is two days earlier than the usual lifespan of the nematode. In a similar report by Sun et al. (2015), coal combustion-related PM 2.5 at 100 mg L -1 significantly reduced the lifespan of C. elegans. However, there was no significant effect on the lifespan when the nematodes were exposed to concentrations 0.01-10 mg L -1 , which is inconsistent with the results of this study. Bases on the previous description of the Sun's study (Sun et al., 2015), it was little concern for their study deign. It was difficult to prevent from aggregation of PM 2.5 after air sampling, but they didn't show and explain how to maintain PM 2.5 -type particulate with diversely and homogenously widespread in the nematodes' medium. The finding of our studying is valuable due to our result indicating low levels of PM 2.5 exposure in nematodes was still significantly linked into lower lifespan compared with the control. According to the previous reports and the present study (Zhao et al., 2014;Sun et al., 2015Sun et al., , 2016Wang et al., 2017), PM 2.5 exposure decreased lifespan in the in-vivo and epidemiological studies including coal combustion in C. elegans (Sun et al., 2015(Sun et al., , 2016, trafficrelated pollutants in the C. elegans (Zhao et al., 2014), ambient air in Drosophila , ambient air in newborns (Martens et al., 2017), and ambient air in elderly (Li et al., 2018b). Therefore, the level of outdoor air PM 2.5 from the two schools, although lower than the national standard in Taiwan, was still shown to have a significant impact on microorganisms and ultimately may pose a threat on human health. To this end, additional endpoints (such as reproductive and locomotion) were investigated to further confirm this effect.

Growth Measurement Assay
No lethality was observed when the nematodes were subjected to acute exposure with the different concentrations of PM 2.5 from either sampling site (NPUST and LJHS). Also, specified concentrations of samples from both the NPUST (1.4443 × 10 -3 to 1.4443 × 10 3 mg L -1 ) and LJHS (1.5518 × 10 -3 to 1.5518 × 10 3 mg L -1 ) sampling sites did not lead to the alterations in body length (Fig. 3). Results showed that in all concentrations, the body length of the nematodes remained at 1 mm, the standard length of C. elegans (Leung et al., 2008). A similar study by Sun et al. (2015) reported that acute and prolonged exposure of C. elegans to PM 2.5 of concentrations 0.01 to 10 mg L -1 had no significant effect on the body lengths of the nematodes under study; however, acute exposure to PM 2.5 at a concentration of 100 mg L -1 and prolonged exposure to PM 2.5 at concentrations of 10 to 100 mg L -1 significantly reduced body length. Our result was inconsistent with that discussed in Sun's study (Sun et al., 2015).

Effect of Outdoor Air PM2.5 Levels on the Reproductive System of C. elegans
The secondary targeted organs of environmental pollutants include the neurons and the reproductive system (Leung et al., 2008;Aoki and Mori, 2015). In the C. elegans model, the reproductive system is one of the major targets of toxicants in the body, next to the neurosensory system (Aoki and Mori, 2015). In order to understand the effects of PM 2.5 on its translocation in C. elegans, the brood size was also assessed for each of the worms exposed to different PM 2.5 concentrations (Fig. 4). The total number of eggs, or brood size, was determined twice per concentration of samples from NPUST and LJHS. After acute exposure, the first measurement ( Fig. 4(A)) showed that brood size was significantly reduced as the concentration of samples from NPUST increased. Specifically, brood size at 1.4443 × 10 -3 mg L -1 was reduced to 18 eggs laid per worm, and concentrations from 1.4443 × 10 -2 to 1.4443 × 10 3 mg L -1 reduced the brood size to 21, 11, 10, 10, 10, and 3 eggs laid per worm, respectively. Similarly, the second assessment of brood size (Fig. 4(B)) showed that at concentrations ranging from 1.4443 × 10 -3 to 1.4443 × 10 3 mg L -1 , the eggs laid per worm decreased to 13, 6, 5, 2, 6, 3, and 1, respectively. For samples gathered from LJHS, the first and second assessment of brood size (Figs. 4(C) and 4(D)) showed no significantly different number of eggs laid per worm at concentrations ranging from 1.5518 × 10 -3 to 1.5518 × 10 3 mg L -1 . In this study, the results showed that exposure to high ambient PM 2.5 concentrations had a significant effect on the reduction of brood size of C. elegans. This result is analogous to the study conducted by Wang et al. (2019), wherein diesel particulate matter (DPM) caused reproduction deficits in C. elegans, as shown by a decrease in brood size during the first generation of offspring. Moreover, in the same study, it was observed that consecutive generation exposure to DPM led to severe limitations in brood size. Currently, studies have shown that the self-progeny of C. elegans is an effective parameter that can be used to determine the extent of reproductive disability due to the toxicity of various substances Moon et al., 2019). In general, the effect can be observed in a dosedependent manner . This was not the case for this study. The control group generated a relatively low brood size of 18. Dietary restriction (DR) can significantly lower the progeny number in C. elegans (El-Hajj and Newman, 2015). In addition, for the size, PM 2.5 is greater than E. coli OP50 (2.5 µm > 2 µm, general food source of C. elegans) (Khan et al., 2018). Therefore, toxicity of PM 2.5 is related to the size of the exposed organism.

Effects of Ambient PM2.5 on Locomotion Behavior of C. elegans
Locomotion assays used to analyze the head thrashing and body bending movements of the nematodes were conducted to assess the neurological toxicity brought about by their exposure to different concentrations of PM 2.5 (Fig. 5). A significant reduction in head thrashing movement and body bending were observed for nematodes exposed to PM 2.5 sampled from NPUST as compared to nematodes exposed to PM 2.5 sampled from LJHS. These results imply that levels of PM 2.5 may be correlated to the greatly disrupted functions of the reproductive and neurosensory systems in C. elegans. Additionally, prolonged exposure to PM 2.5 was observed to cause more sensitivity to C. elegans. Zhao et al. (2014) established that acute exposure to high concentrations of PM 2.5 causes adverse effects on both the reproduction and neurosensory functions of C. elegans. Moreover, prolonged exposure to low concentrations of PM 2.5 further significantly decreased the locomotion behavior of the nematodes. However, this was true for exposure to pollutant concentrations in the range of µg L -1 . This is because of the greater dispersion of PM 2.5 in DMSO, which increased the bioavailability of the particulate matter to the nematodes (Artifon et al., 2019). The main route of uptake of pollutants in C. elegans is through the alimentary system; the worms actively ingest particulates while feeding on bacteria. The presence of bacterial food causes the adsorption of a proportion of the particles and enhances the oral uptake, resulting in higher toxicity (Gonzalez-Moragas et al., 2015). Therefore, the dispersion of particulate matter in the media of choice is important in assessing their effects on in vivo systems. Several toxic endpoints were examined in the present study. Low PM 2.5 levels were found in our sampling areas. To make our amazement, the low levels of PM 2.5 -bounded pollutants induced several adverse effects including accelerated aging process, reduced reproduction, and delayed locomotion. The present study was limited based on the air sampling technique and pretreatment protocol. The standard methods of PM 2.5 air sampling and PM pretreatment were used in our study. This study excluded in the toxicity in inorganic fraction due to use of the Soxhlet extractors. The present study only considered the toxic effects of PM 2.5bounded hazardous pollutants after the high-volume PM 2.5 air samplers mainly due to occurrence of aggregation during the air sampling. Future work will be encouraged to gather airborne PM 2.5 -type particulate directly into nematodes' medium before aggregation.

CONCLUSIONS
Low PM 2.5 levels were obtained at NPUST and LJHS in this study. It was found that ambient PM 2.5 levels at NPUST were higher than the PM 2.5 level at LJHS. In addition, the in-vivo model of C. elegans was utilized to evaluate the possible health risks associated with differing PM 2.5 levels through the investigation of multiple toxic endpoints namely, lethality, lifespan, development, locomotion, and reproduction. The acute toxicity and growth measurement of C. elegans was unaffected by PM 2.5 collected from NPUST and LJHS, but its lifespan was significantly reduced in the case of airborne PM 2.5 at NPUST starting on post-exposure day 10. For the reproductive toxicity and locomotion, reduced brood size and the frequency of head thrashes for NPUST-PM 2.5 was observed, but there were no significant differences in lifespan, brood size, head thrashes, and body bends between the control and LJHS-PM 2.5 . Our significant findings were to indicate the potential toxic effects of long-term exposure to low PM 2.5 on C. elegans models, even though the measurements were lower than PM 2.5 standards. This study implored that the long-term adverse effects of ambient PM 2.5 on environmental organisms should be carefully considered even though PM 2.5 levels didn't meet the standards.