Underestimated health risks: polystyrene micro- and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis.

BACKGROUND: Micro- and nanoplastic pollution has become a global environmental problem. Nanoplastics in the environment are still hard to detect because of analysis technology limitations. It is believed that when microplastics are found in the environment, more undetected nanoplastics are around. The current "microplastic exposure" is in fact the mixture of micro- and nanoplastic exposures. Therefore, the biological interaction between organisms among different sizes of micro- and nanoplastics should not be neglected. RESULTS: We measured the biodistribution of three polystyrene (PS) particles (50 nm PS, PS50; 500 nm PS, PS500; 5000 nm PS, PS5000) under single and co-exposure conditions in mice. We explored the underlying mechanisms by investigating the effects on three major components of the intestinal barrier (the mucus layer, tight junctions and the epithelial cells) in four intestine segments (duodenum, jejunum, ileum and colon) of mice. We found that the amounts of both PS500 and PS5000 increased when they were co-exposed with PS50 for 24 h in the mice. These increased amounts were due primarily to the increased permeability in the mouse intestines. We also confirmed there was a combined toxicity of PS50 and PS500 in the mouse intestines. This manifested as the mixture of PS50 and PS500 causing more severe dysfunction of the intestinal barrier than that caused by PS50 or PS500 alone. We found that the combined toxicity of PS micro- and nanoplastics on intestinal barrier dysfunction was caused primarily by reactive oxygen species (ROS)-mediated epithelial cell apoptosis in the mice. These findings were further confirmed by an oxidants or antioxidants pretreatment study. In addition, the combined toxicity of PS micro- and nanoplastics was also found in the mice after a 28-day repeated dose exposure. CONCLUSIONS: There is a combined toxicity of PS50 and PS500 in the mouse intestines, which was caused primarily by ROS-mediated epithelial cell apoptosis in the mice. Considering that most recent studies on PS micro- and nanoplastics have been conducted using a single particle size, the health risks of exposure to PS micro- and nanoplastics on organisms may be underestimated.

Analysis of individual and combined estrogenic effects of bisphenol, nonylphenol and diethylstilbestrol in immature rats with mathematical models.

BACKGROUND: Traditional toxicological studies focus on individual compounds. However, this single-compound approach neglects the fact that the mixture exposed to human may act additively or synergistically to induce greater toxicity than the single compounds exposure due to their similarities in the mode of action and targets. Mixture effects can occur even when all mixture components are present at levels that individually do not produce observable effects. So the individual chemical effect thresholds do not necessarily protect against combination effects, an understanding of the rules governing the interactive effects in mixtures is needed. The aim of the study was to test and analyze the individual and combined estrogenic effects of a mixture of three endocrine disrupting chemicals (EDCs), bisphenol A (BPA), nonylphenol (NP) and diethylstilbestrol (DES) in immature rats with mathematical models. METHOD: In the present study, the data of individual estrogenic effects of BPA, NP and DES were obtained in uterotrophic bioassay respectively, the reference points for BPA, NP and DES were derived from the dose-response ralationship by using the traditional no observed adverse effect (NOAEL) or lowest observed adverse effect level (LOAEL) methods, and the benchmark dose (BMD) method. Then LOAEL values and the benchmark dose lower confidence limit (BMDL10) of single EDCs as the dose design basis for the study of the combined action pattern. Mixed prediction models, the 3 × 2 factorial design model and the concentration addition (CA) model, were employed to analyze the combined estrogenic effect of the three EDCs. RESULTS: From the dose-response relationship of estrogenic effects of BPA, NP and DES in the model of the prepuberty rats, the BMDL10(NOAEL) of the estrogenic effects of BPA, NP and DES were 90(120) mg/kg body weight, 6 mg/kg body weight and 0.10(0.25) µg/kg body weight, and the LOAEL of the the estrogenic effects of three EDCs were 240 mg/kg body weight, 15 mg/kg body weight and 0.50 µg/kg body weight, respectively. At BMDL10 doses based on the CA concept and the factorial analysis, the mode of combined effects of the three EDCs were dose addition. Mixtures in LOAEL doses, NP and DES combined effects on rat uterine/body weight ratio indicates antagonistic based on the CA concept but additive based on the factorial analysis. Combined effects of other mixtures are all additive by using the two models. CONCLUSION: Our results showed that CA model provide more accurate results than the factorial analysis, the mode of combined effects of the three EDCs were dose addition, except mixtures in LOAEL doses, NP and DES combined effects indicates antagonistic effects based on the CA model but additive based on the factorial analysis. In particular, BPA and NP produced combination effects that are larger than the effect of each mixture component applied separately at BMDL doses, which show that additivity is important in the assessment of chemicals with estrogenic effects. The use of BMDL as point of departure in risk assessment may lead to underestimation of risk, and a more balanced approach should be considered in risk assessment.

[Dietary intake and risk assessment of diethylhexyl phthalate in Chinese populations].

OBJECTIVE: To assess dietary exposure of diethylhexyl phthalate(DEHP) among Chinese population, including general population, children aged 2-6 years, adolescent aged 7-12, young people aged 13-17, adults aged 18-59 years old as well as older people aged 60 and above and its health risk. METHODS: A total of 6 650 food samples were collected during 2011 to 2013 from 140 local markets of 14 provinces in China, which covered major foods in China. Samples were detected by GC-MS and categorized into 22 food groups. Food consumption data were taken from China National Nutrition and Health Survey performed in 2002 including 68 959 subjects. Mean concentrations of DEHP in food were combined with individual food consumption data to estimate dietary exposure, and food contributors to dietary DEHP intake were also calculated. Then, the exposure was compared with the tolerable daily intake (TDI, 50 µg·kg(-1)-d(-1)) of DEHP. RESULTS: DEHP level in foods (n = 6 650) was in the range of not detected to 43.80 mg/kg. Mean dietary intakes of DEHP in general population was 2.07 (95% CI: 0.06-4.09) µg·kg(-1)·d(-1), accounting for 4.14 percent of TDI (50 µg·kg(-1)·d(-1)). Mean dietary intake for population aged 2-6, 7-12, 13-17, 18-59 as well as elderly aged 60 and above were 3.92 (95% CI: 0.83-7.01), 3.02 (95% CI: 0.69-5.36), 2.17 (95% CI: 0.54-3.81), 1.83 (95% CI: 0.46-3.21) and 1.66 (95% CI: 0.38-2.94) µg·kg(-1)·d(-1) respectively. The 97.5 percentile intakes in the general populations was 4.73 µg·kg(-1)·d(-1), accounting for 9.46% of TDI. Main food sources of DEHP were rice (28.4% (0.59/2.07)), melon solanaceous vegetables (14.7% (0.30/2.07)) and flour (13.2% (0.27/2.07)) for the general population. CONCLUSION: The results suggested that dietary exposure to DEHP among Chinese population was lower than tolerable daily intake of DEHP and there were no health concerns based on generally accepted exposure limits. Rice, melon solanaceous vegetables and flour were main food contributors of DEHP dietary intake for Chinese populations.

[Dietary intake and risk assessment of diisononyl phthalate (DINP) in Chinese population].

OBJECTIVE: To study DINP concentration level in the main food, evaluate DINP dietary intake level in Chinese population and its potential health risks. METHODS: Based on the deterministic assessment model, using concentration level of 25 kinds of food in 2011 - 2013 and Chinese national nutrition and health survey data in 2002, to calculate dietary intake of DINP in Chinese population. RESULTS: The average DINP concentration level in 24 kinds of foods was 0. 24 mg/kg, and the maximum value was 9. 55 mg/kg. In whole population, average dietary intake of DINP was 4. 39 µg/kg bw per day, only 2. 93% of TDI. The dietary intake of DINP in children aged 2 to 6 years old was highest, with an average of 8. 91 µg/kg bw per day, 5. 94% of the TDI. The dietary intake of DINP in children aged 7 to 12 years old was lower than 2 to 6 years old children, with an average of 6.53 µg/kg bw per day, 4. 35% of TDI. The dietary intake of DINP in high consumer(P97. 5) in all population was 8. 35 µg/kg bw per day, 5. 57% of the TDI. The range of dietary intake of DINP in high consumer (P97. 5) in each group (grouping by gender and age) was from 13. 84 to 5.44 µg/kg bw per day, which were all lower than the TDI. CONCLUSION: The dietary intake of DINP in different populations is considerably below the TDI and any health risk that would be expected to occur at this intake level is negligible.

The occurrence and exposure of microplastics in bivalves from Qingdao, China.

This study investigated the regional, seasonal, and species abundance and characteristics of microplastics (MPs) in bivalves from Qingdao, China and assessed the dietary exposure of MPs through bivalve consumption. The average abundance was 1.17 ± 1.07 items/individual or 0.17 ± 0.22 items/g wet weight. Fiber was the dominant shape (91.5 %). The average size of MPs was 995.63 ± 796.59 µm. Rayon, PE, and PET contributed mostly to the MPs composition. There were no significant regional or seasonal differences in MPs abundance (p > 0.05), while there were significant species differences (p < 0.05) when describing the abundance by wet weight. The estimated daily intakes of MPs through bivalve consumption by general population, consumer only population, and coastal residents in China were 3.32 × 10-3, 0.08, and 0.16 µg/kg BW, respectively. The exposure assessment method by converting the quantity of MPs into mass may facilitate the risk characterization in the future.

Identification of Active Components for Sports Supplements: Machine Learning-Driven Classification and Cell-Based Validation.

The identification of active components is critical for the development of sports supplements. However, high-throughput screening of active components remains a challenge. This study sought to construct prediction models to screen active components from herbal medicines via machine learning and validate the screening by using cell-based assays. The six constructed models had an accuracy of >0.88. Twelve randomly selected active components from the screening were tested for their active potency on C2C12 cells, and 11 components induced a significant increase in myotube diameters and protein synthesis. The effect and mechanism of luteolin among the 11 active components as potential sports supplements were then investigated by using immunofluorescence staining and high-content imaging analysis. It showed that luteolin increased the skeletal muscle performance via the activation of PGC-1α and MAPK signaling pathways. Thus, high-throughput prediction models can be effectively used to screen active components as sports supplements.

Macrophage polarization as a novel endpoint for assessing combined risk of phthalate esters.

Combined exposure to phthalate esters (PAEs) has garnered increasing attention due to potential synergistic effects on human health. This study aimed to develop an in vitro model using human macrophages to evaluate the combined toxicity of PAEs and explore the underlying mechanisms. A high-throughput screening system was engineered by expressing a PPRE-eGFP reporter in THP-1 monocytes to monitor macrophage polarization upon PAEs exposure. Individual PAEs exhibited varied inhibitory effects on M2 macrophage polarization, with mono(2-ethylhexyl) phthalate (MEHP) being the most potent. Isobologram analysis revealed additive interactions when MEHP was combined with other PAEs, resulting in more pronounced suppression of M2 markers compared to individual compounds. Mechanistic studies suggested PAEs may exert effects by modulating PPARγ activity to inhibit M2 polarization. Notably, an equimolar mixture of six PAEs showed additive inhibition of M2 markers. In vivo experiments corroborated the combined hepatotoxic effects, with mice exposed to a PAEs mixture exhibiting reduced liver weight, dyslipidemia, and decreased hepatic M2 macrophages compared to DEHP alone. Transcriptome analysis highlighted disruptions in PPAR signaling, and distinct pathway alterations on cholesterol metabolism in the mixture group. Collectively, these findings underscore the importance of evaluating mixture effects and provide a novel approach for hazard assessment of combined PAEs exposure with implications for environmental health risk assessment.

Characterization of Food-Additive Titanium Dioxide and Dietary Exposure to Titanium Dioxide Nanoparticles among the Chinese Population.

Titanium dioxide (TiO2) is a prevalent food additive, yet comprehensive data on particle size and dietary exposure are lacking in China. Transmission electron microscopy results revealed that the quantitative proportion of nanoparticles (NPs) in food-additive TiO2 was 37.7%, with a mass fraction of 9.89%. Laboratory test results showed that among the domestic products surveyed, candies excluding gum-based candies contained the highest content of TiO2. Using consumption data from the China Health and Nutrition Survey in 2018, the average dietary exposure for TiO2 and TiO2 NPs in the Chinese population were calculated at 34.84 and 3.44 µg/kg bw/day, respectively. The primary dietary sources were puffed food and powdered drinks. Exposure varied significantly across age and region, with children and Inner Mongolia residents having the highest intake. TiO2 NP exposure showed a negative correlation with age. Despite this, the dietary exposure risk of TiO2 NPs for the Chinese population remains deemed acceptable.

Natural Occurrence and Co-Occurrence of Beauvericin and Enniatins in Wheat Kernels from China.

A total of 769 wheat kernels collected from six provinces in China were analyzed for beauvericin (BEA) and four enniatins (ENNs), namely, ENA, ENA1, ENB and ENB1, using a solid phase extraction (SPE) technique with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results show that the predominant toxin was BEA, which had a maximum of 387.67 µg/kg and an average of 37.69 µg/kg. With regard to ENNs, the prevalence and average concentrations of ENB and ENB1 were higher than those of ENA and ENA1. The geographical distribution of BEA and ENNs varied. Hubei and Shandong exhibited the highest and lowest positive rates of BEA and ENNs (13.46% and 87.5%, respectively). However, no significant difference was observed among these six provinces. There was a co-occurrence of BEA and ENNs, and 42.26% of samples were simultaneously detected with two or more toxins. Moreover, a significant linear correlation in concentrations was observed between the four ENN analogs (r range: 0.75~0.96, p < 0.05). This survey reveals that the contamination and co-contamination of BEA and ENNs in Chinese wheat kernels were very common.

Concentration and health risk assessment of 16 rare earth elements in six types of tea in China.

Rare Earth Elements (REEs) have been implicated in potential health effects. However, the health risk of REE exposure among tea drinkers in China remains poorly understood. This study aimed to characterize the concentration of REEs in different tea categories and evaluate the associated health risks for tea consumers in China. By analyzing the content of 16 REEs in 4326 tea samples from China, the exposure level of REEs to the general population was estimated. The content of these 16 REEs was similar across six types of tea, with oolong tea exhibiting the highest levels. The concentration of light rare earth elements (LREEs) in six types of tea was higher than that of heavy rare earth elements (HREEs). The daily mean and 95th percentile (P95) exposure to REEs from tea for the general population in China were 0.0328 µg/kg BW and 0.1283 µg/kg BW, respectively, which are significantly lower than the temporary acceptable daily dose (tADI). Our findings suggest that REEs from tea do not pose a known health risk to Chinese consumers.

[Applicability of thresholds of toxicological concern in the chronic dietary risk assessment of transformation products of pesticide active substance].

OBJECTIVE: To establish the threshold of toxicological concern(TTC) approach and to apply it in the risk assessment of metabolites, degradation and reaction products of pirimicarb. METHODS: TTC decision tree approach based on Cramer classification was established and Lazar software was used to predict the genotoxicity of the seven transformation products of pirimicarb, namely, R34836, R34885, R35140, R31805, R34865, R16210 and R16192. Dietary exposure in general population as well as in six age population groups was estimated by using data from the Chinese National Nutrition and Health Survey and pirimicarb residue data from national chemical surveillance data in 2011. TTC decision tree approach was used for risk assessment and the exposure was compared with the corresponding TTC values. RESULTS: Of the seven transformations of pirimicarb active substance, namely, R34836, R34885, R35140, R31805, R34865, R16210 and R16192, the maximum dietary exposure of mean and large portion(P 97.5) were all belong to 2-6 age group. The mean exposures of the seven transformation products for 2-6 age group,were 0.0290, 0.0207, 0.0015, 0.0320, 0.0005, 0.6918 and 0.1274 µg/kg,respectively, and the corresponding P 97.5 exposures were 0.0817,0.0581,0.0042,0.0900,0.0014, 1.9459 and 0.3585 µg/kg. Besides, the mean and P 97.5 exposure of R16210 for 2-6 age group was the largest,which were 0.6918 and 1.9459 µg/kg, accounting for 46.12% and 129.73% of the TTC threshold,respectively. CONCLUSION: TTC decision tree approach is a useful tool for prior screening and primary risk assessment of the transformation products of pesticide active substance.

Trends of caffeine intake from food and beverage among Chinese adults: 2004-2018.

Caffeine is a kind of psychostimulant that naturally exist in foods. The benefits and risks of caffeine depend on the dose. Moreover, the intake of caffeine from dietary sources in China has seldom been assessed. We calculated the dietary caffeine intake of Chinese adult consumers from 2004 to 2018 and analyzed its consumption trends by using data from the China Health and Nutrition Survey and the National Food and Beverage Consumption Survey. Caffeine contents in different dietary items were determined by HPLC. Monte Carlo simulations were applied to estimate caffeine intake. Mann-Kendall trend test and linear regression were used to analyze the trend of caffeine consumption. Among 79,173 individuals, 3972 (5%) of the adult Chinese population consumed caffeine between 2004 and 2018. The average caffeine intake was 123 mg/day for male consumers and 116 mg/day for female consumers. The median and P75 caffeine intake raised over the 14 years. Traditional tea leaves, coffee and sodas are the main sources of caffeine intake. Our findings indicate that most Chinese adults consumed caffeine within the safe level (400 mg/day), but the caffeine consumption has shown an increasing trend in recent 14 years.

Advancing food safety risk assessment in China: development of new approach methodologies (NAMs).

Novel techniques and methodologies are being developed to advance food safety risk assessment into the next-generation. Considering the shortcomings of traditional animal testing, new approach methodologies (NAMs) will be the main tools for the next-generation risk assessment (NGRA), using non-animal methodologies such as in vitro and in silico approaches. The United States Environmental Protection Agency and the European Food Safety Authority have established work plans to encourage the development and application of NAMs in NGRA. Currently, NAMs are more commonly used in research than in regulatory risk assessment. China is also developing NAMs for NGRA but without a comprehensive review of the current work. This review summarizes major NAM-related research articles from China and highlights the China National Center for Food Safety Risk Assessment (CFSA) as the primary institution leading the implementation of NAMs in NGRA in China. The projects of CFSA on NAMs such as the Food Toxicology Program and the strategies for implementing NAMs in NGRA are outlined. Key issues and recommendations, such as discipline development and team building, are also presented to promote NAMs development in China and worldwide.

Impact of food matrices on the characteristics and cellular toxicities of ingested nanoplastics in a simulated digestive tract.

Microplastic and nanoplastic (MNP) pollution has become a major global food safety concern. MNPs can interact with food matrices, and their passage through the gastrointestinal tract can modify their properties. To explore whether and how food matrices influence MNP toxicity, we investigated the interactions between polystyrene nanoplastics (PS-NPs) and food matrices, using an in vitro gastrointestinal digestion model. Then, we tested cell viability, particle uptake and cellular toxicities induced by PS-NPs with food matrices in Caco-2 cells. The results showed that PS-NPs were aggregated, both with and without food matrices, after in vitro gastrointestinal digestion. Glyceryl trioleate exerted greater ability to stabilize digestas and to disperse PS-NPs than starch and bovine serum albumin. The protein corona's protein composition on PS-NPs varied when it interacted with different food matrices. Moreover, when combined with food matrices, the PS-NPs' uptake was enhanced, thus aggravating cellular inflammation, stress, and apoptosis levels. Finally, through co-exposure to a mixture of food matrices, we found a combined negative effect of PS-NPs and cadmium on cellular inflammation, stress, and apoptosis levels. This is the first study to compare the impact of various food matrices on the characteristics and cellular toxicities of ingested NPs in a simulated digestive tract.

Monosodium Glutamate Intake and Risk Assessment in China Nationwide, and a Comparative Analysis Worldwide.

The sixth Total Diet Study (TDS) of China included a countrywide study to assess the health effects of MSG (monosodium glutamate). MSG detection, consumption analysis, and risk assessment were conducted on 168 samples from seven food categories of the most typical Chinese daily diet. The highest value of MSG in the daily diet of the Chinese population was 8.63 g/kg. An MSG intake of 17.63 mg/kg bw/d for the general population of China was obtained from content measurements combined with food consumption, while the data from the apparent consumption survey alone gave 40.20 mg/kg bw/d. The apparent consumption did not consider the loss of MSG during food cooking, resulting in an overestimate. To offer a global perspective, MSG content, food category contributions, and ingestion levels across nations were summarized and thoroughly investigated. A realistic, logical, and precise risk assessment protocol for MSG daily intake was developed in this article.

Risk assessment of eighteen elements leaching from ceramic tableware in China.

Ceramic products are among the most frequently used food contact materials. Health risks associated with ceramic tableware usually arise from the migration of heavy metals. In this study, 767 pieces of ceramic tableware of different shapes and types were collected across China, and the migration levels of 18 elements were determined using inductively coupled plasma mass spectrometry. Migration tests were conducted according to the Chinese National Food Safety Standard - Ceramic Ware (GB 4806.4) with microwaveable and non-microwavable samples under different conditions. The food consumption of consumers via different shapes of ceramic tableware was obtained through a self-reported web-based survey, and the estimated dietary intakes of the studied elements were calculated accordingly. The exposure assessment showed that certain metals leached from the ceramic tableware at levels of concern. In addition, the applicability of the migration experiment conditions for microwaveable ceramic ware in GB 4806.4 needs to be further investigated.

Study of the evolution of 3-MCPDEs and GEs in the infant formula production chain employing a modified indirect method based on magnetic solid phase extraction.

Herein, a modified indirect method was established for the determination of 3-monochloropropane-1,2-diol esters (3-MCPDEs) and glycidyl esters (GEs), employing magnetic solid phase extraction by boronic acid-functionalized magnetic nanoparticles to replace the traditional clean-up procedure. Compared with routine methods, it has been proved to be more sensitive with limits of detection in the range of 0.02-1.5 µg/kg and less susceptible to contamination of phenylboronic acid derivatives and fatty acid methyl esters. The proposed method was applied to analyze 42 samples covering the entire infant formula (IF) production chain. Results revealed that homogenization process contributed 79-91 % of the total growth of the contaminants due to the vegetable oil addition, while the following evaporation and spray-drying processes contributed 9-21 % of the total growth owing to involved heat treatment. The GE levels in final IF products exceeded the maximum level set by EU regulation 2020/1322, indicating quality safety concerns in the production chain.

The occurrence and probabilistic risk assessment of 3-MCPDEs and GEs in infant formulas from Chinese market employing Monte Carlo simulation technique.

3-monochloropropane-1,2-diol esters (3-MCPDEs) and glycidyl esters (GEs) are food contaminants and have arisen continuous attention due to their toxicity, especially towards infants. Current risk assessment of these contaminants was mostly employing deterministic approaches, lacking quantitative characterization of the likelihood, incidence, or severity of the risks involved. Herein, 3-MCPDE and GE levels in 46 representative infant formulas (IFs) from Chinese market were determined by GC-MS/MS. Then, combining the occurrence data and consumption data from China National Food Consumption Survey, the Monte Carlo simulation-based probabilistic model for risk assessment of 3-MCPDEs and GEs in IFs from Chinese market was established. The results showed that all P90 (90th percentiles) hazard quotient values were below 1, demonstrating 3-MCPDEs didn't pose health risks to most populations aged 0-36 months old. However, for 0-12 months old groups, P10 (10th percentiles) margin of exposure values were all below 25000, indicating GEs may pose potential risks to 10% of this group. Uncertainty analysis revealed that the probabilistic model had considered uncertainties of model input and distribution, and realized refined assessment. This study is the first report on probabilistic assessment of 3-MCPDEs and GEs in IFs, which also provided references for the formulation of related regulatory limits in China.

Dietary Exposure to Glutamates of 2- to 5-Year-Old Toddlers in China Using the Duplicate Diet Method.

A duplicate diet collection method was used to estimate dietary exposure to glutamates in children aged 2-5 years in selected provinces of China. Daily duplicate diet samples were collected from 86 healthy toddlers over three consecutive days. Glutamates were analyzed using ultra-high-pressure liquid chromatography-MS/MS (UHPLC-MS/MS). Results showed that the highest glutamates content was found in mixed meals, at 5.12 mg/kg, followed by powdered formula (3.89 mg/kg), and milk and dairy products (2.29 mg/kg). The total mean daily dietary exposure for subjects was 0.20 mg/kg BW, and P95 daily dietary exposure was 0.44 mg/kg BW, both below the acceptable daily intake (ADI) (120 mg/kg BW) recommended by the Joint (FAO/WHO) Expert Committee on Food Additives (JECFA) and the ADI (30 mg/kg BW) set by the European Food Safety Authority (EFSA). Hence it can be considered that glutamates exposure would cause low risk in this group.

Investigation of Microplastics (≥10 µm) in Meconium by Fourier Transform Infrared Microspectroscopy.

Microplastics are prevalent emerging pollutants with widespread distribution in air, land and water. They have been detected in human stool, blood, lungs, and placentas. However, human fetal microplastic exposure remains largely under-studied. To assess fetal microplastic exposure, we investigated microplastics using 16 meconium samples. We used hydrogen peroxide (H2O2), nitric acid (HNO3) and a combination of Fenton's reagent and HNO3 pretreatment methods respectively to digest the meconium sample. We analyzed 16 pretreated meconium samples with an ultra-depth three-dimensional microscope and Fourier transform infrared microspectroscopy. The result showed that H2O2, HNO3 and Fenton's reagent combined with HNO3 pretreatment methods could not digest our meconium samples completely. Alternatively, we developed a novel approach with high digestion efficiency using petroleum ether and alcohol (4:1, v/v), HNO3 and H2O2. This pretreatment method had good recovery and non-destructive advantages. We found no microplastics (≥10 µm) in our meconium samples, indicating that microplastic pollution levels in the fetal living environment are miniscule. Different results between previous studies' and ours underscore that comprehensive and strict quality control are necessary for further studies on microplastic exposure using human bio-samples.