Development and validation of a multi-substance method for routine analysis of pesticides in post-mortem samples by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Pesticides play an important role in forensic toxicology and are usually classified as a single class of chemicals. Despite their commonly perceived unity, pesticides encompass a spectrum of compounds, including organophosphates, carbamates, pyrethroids or organochlorines, among others, each with varying degrees of toxicity. Pesticide analysis in post-mortem samples can be difficult due to the complexity of the samples and to the high toxicity of these compounds. The aim of this study was to develop and validate an easy to use, sensitive, and robust method, using ultra-performance liquid chromatography-tandem mass spectrometry to be incorporated in the routine flow for pesticide analysis in post-mortem blood samples. Described herein is a streamlined, expeditious, yet highly efficient method facilitating the screening, qualitative assessment, and quantitative confirmation of 15 pesticides, including acetamiprid, azinphos-ethyl, bendiocarb, carbofuran, chlorfenvinphos, dimethoate, imidaclopride, malathion, methiocarb, methomyl, parathion, pirimicarb, strychnine, tetrachlorvinphos, and thiacloprid in post-mortem blood, recognizing the pivotal role blood plays in forensic investigations. The developed method was linear from 10 to 200 ng/mL; limits of detection were between 1 and 10 ng/mL, depending on the compound; it was successfully evaluated a dilution ratio of 1-2, 5 and 10; and 8 substances showed maximum stability for the time interval studied. This UHPLC-MS/MS method is useful and a powerful tool in a toxicology lab because it is fast, simple, effective, and trustworthy. The results of this validation highlight the robustness of the analytical method, providing a valuable tool for the accurate and sensitive detection of pesticides in post-mortem blood. Poised for routine implementation, this method has already found success in suspected intoxication cases, promising to elevate the standards of forensic pesticide analysis.

Optimization and uncertainty assessment of a gas chromatography coupled to Orbitrap mass spectrometry method to determine organic contaminants in blood: A case study of an endangered seabird.

Birds are excellent bioindicators of environmental pollution, and blood provides information on contaminant exposure, although its analysis is challenging because of the low volumes that can be sampled. The objective of the present study was to optimize and validate a miniaturized and functional extraction and analytical method based on gas chromatography coupled to Orbitrap mass spectrometry (GCOrbitrap-MS) for the trace analysis of contaminants in avian blood. Studied compounds included 25 organochlorine pesticides (OCPs), 6 polychlorinated biphenyls (PCBs), 8 polybrominated diphenyl ethers (PBDEs) and 15 polycyclic aromatic hydrocarbons (PAHs). Four extraction and clean-up conditions were optimized and compared in terms of efficiency, accuracy, and uncertainty assessment. Extraction with hexane:dichloromethane and miniaturized Florisil pipette clean-up was the most adequate considering precision and accuracy, time, and costs, and was thereafter used to analyse 20 blood samples of a pelagic seabird, namely the Bermuda petrel (Pterodroma cahow). This species, endemic to the Northwest Atlantic, is among the most endangered seabirds of the region that in the '60 faced a decrease in the breeding success likely linked to a consistent exposure to dichloro-diphenyl-trichloroethane (DDT). Indeed, p,p'-DDE, the main DDT metabolite, was detected in all samples and ranged bewteen 1.13 and 6.87 ng/g wet weight. Other ubiquitous compounds were PCBs (ranging from 0.13 to 6.76 ng/g ww), hexachlorobenzene, and mirex, while PAHs were sporadically detected at low concentrations, and PBDEs were not present. Overall, the extraction method herein proposed allowed analysing very small blood volumes (∼ 100 µL), thus respecting ethical principles prioritising the application of less-invasive sampling protocols, fundamental when studying threatened avian species.

Systematic investigation of organochlorine pesticides and polychlorinated biphenyls blood levels in Greek children from the Rhea birth cohort suggests historical exposure to DDT and through diet to DDE.

The blood levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) have been thoroughly investigated in Greek children from the Rhea birth cohort study. This investigation aimed to assess exposure levels, explore their possible relationship with children's age and sex, and indicate potential sources of exposure. Exposure patterns and common sources of PCBs and OCPs were analyzed using bivariate and multivariate statistics. A total of 947 blood samples from study participants were analyzed for OCP and PCB exposure, with 375 samples collected at 4 years old, 239 at 6.5 years old, and 333 at 11 years old. Elevated levels of DDE were observed in 6.5-year-old children compared to corresponding levels in other European countries. Higher levels of DDE were found in 4-year-old children, with the lowest concentrations in the 11-year-old group. The DDT/DDE ratio was consistently less than 1 among all the examined subjects. These results indicate exposure to DDT and DDE both in utero and through breastfeeding and dietary intake. For the entire cohort population, the highest concentration was determined for PCB 28, followed by PCBs 138, 153, and 180. The sum of the six indicator PCBs implied low exposure levels for the majority of the cohort population. Spearman correlations revealed strong associations between PCBs and OCPs, while principal component analysis identified two different groupings of exposure. DDE exhibited a correlation with a series of PCBs (153, 156, 163, 180), indicating a combined OCP-PCB source, and an anticorrelation with others (52, 28, 101), implying a separate and competing source.

Exposure of an endangered seabird species to persistent organic pollutants: Assessing levels in blood and link with reproductive parameters.

Ocean contamination, particularly from persistent organic pollutants (POPs), remains a significant threat to marine predators that occupy high trophic positions. Long-lived procellariform seabirds are apex predators in marine ecosystems and tend to accumulate contaminants. Prolonged exposure to pollutants negatively affects their fitness including reproductive success. Low breeding success may represent a hurdle for the restoration of small and endangered seabird populations, including several highly threatened gadfly petrels. Here we investigated the annual variation (2019 and 2022) in organochlorine pesticide (OCP), polychlorinated biphenyl ether (PCB), polybrominated diphenyl ether (PBDE), and polycyclic aromatic hydrocarbon (PAH) exposure in the endangered Bermuda petrel (Pterodroma cahow), and the relationship between female contaminant burden and breeding parameters. We found that petrels were exposed to a wide range of pollutants (33 out of 55 showed measurable levels) with PCBs dominating the blood contaminant profiles in both years. Only 9 compounds were detected in >50 % of the birds. Specifically, among OCPs, p, p'-DDE and hexaclorobenzene were the most frequently detected while fluorene and acenaphthene were the most common PAH. The concentrations of ∑5PCBs and ∑7POPs were higher in older birds. Furthermore, females with greater contaminant burdens laid eggs with a lower probability of hatching. However, female investment in egg production (size and volume) was unrelated to their blood contaminant load. Overall, this study highlights the presence of a wide range of contaminants in the petrel's food web, and it sheds light on the potential impact of chronic exposure to sub-lethal levels of PCBs on the breeding success of seabirds. We claim that toxicological testing should be a practice integrated in the management of seabirds, particularly of endangered species to monitor how past and present anthropogenic activities impact their conservation status.

Miniaturized flexible micro-tube plasma ionization source for the effective ionization of non-easily ionizable pesticides in food with liquid chromatography/mass spectrometry.

In this article, we have studied the potential of flexible microtube plasma (FµTP) as ionization source for the liquid chromatography high-resolution mass spectrometry detection of non-easily ionizable pesticides (viz. nonpolar and non-ionizable by acid/basic moieties). Phthalimide-related compounds such as dicofol, dinocap, o-phenylphenol, captan, captafol, folpet and their metabolites were studied. Dielectric barrier discharge ionization (DBDI) was examined using two electrode configurations, including the miniaturized one based on a single high-voltage (HV) electrode and a virtual ground electrode configuration (FµTP), and also the two-ring electrode DBDI configuration. Different ionization pathways were observed to ionize these challenging, non-easily ionizable nonpolar compounds, involving nucleophilic substitutions and proton abstraction, with subtle differences in the spectra obtained compared with APCI. An average sensitivity increase of 5-fold was attained compared with the standard APCI source. In addition, more tolerance with matrix effects was observed in both DBDI sources. The importance of the data reported is not just limited to the sensitivity enhancement compared to APCI, but, more notably, to the ability to effectively ionize nonpolar, late-eluting (in reverse-phase chromatography) non-ionizable compounds. Besides o-phenylphenol ([M - H]-), all the parent species were efficiently ionized through different mechanisms involving bond cleavages through the effect of plasma reagent species or its combination with thermal degradation and subsequent ionization. This tool can be used to figure out overlooked nonpolar compounds in different environmental samples of societal interest through non-target screening (NTS) strategies.

Analysis of serum levels of organochlorine pesticides and related factors in Parkinson's disease.

BACKGROUND: There is evidence that environmental factors contribute to the onset and progression of Parkinson's disease (PD). Pesticides are a class of environmental toxins that are linked to increased risk of developing PD. However, few studies have investigated the association between specific pesticides and PD, especially in China, which was one of the first countries to adopt the use of pesticides. METHODS: In this study, serum levels of 19 pesticides were measured in 90 patients with PD and 90 healthy spouse controls. We also analyzed the interaction between specific pesticides and PD. In addition, the association between pesticides and clinical features of PD was also investigated. Finally, we investigated the underlying mechanism of the association between pesticides and PD. RESULTS: Serum levels of organochlorine pesticides, which included α-hexachlorocyclohexane (HCH), ß-HCH, γ-HCH, δ-HCH, propanil, heptachlor, dieldrin, hexachlorobenzene, p,p'-dichlorodiphenyltrichloroethane and o,p'-dichloro-diphenyl-trichloroethane were higher in PD patients than controls. Moreover, α-HCH and propanil levels were associated with PD. Serum levels of dieldrin were associated with Hamilton Depression Scale and Montreal Cognitive Assessment scores in PD patients. In SH-SY5Y cells, α-HCH and propanil increased level of reactive oxygen species and decreased mitochondrial membrane potential. Furthermore, propanil, but not α-HCH, induced the aggregation of α-synuclein. CONCLUSIONS: This study revealed that elevated serum levels of α-HCH and propanil were associated with PD. Serum levels of dieldrin were associated with depression and cognitive function in PD patients. Moreover, propanil, but not α-HCH, induced the aggregation of α-synuclein. Further research is needed to fully elucidate the effects of pesticides on PD.

Exposure to organochlorine pesticides as a predictor to breast cancer: A case-control study among Ethiopian women.

Breast cancer (BC) is becoming one of the most prevalent non-infectious disease in low and middle income countries. The steady rise of BC incidence may be related to the different risk factors. Among many, rampant presence of environmental pollutants might be one of the risk factors. Therefore, the aim of this study is to investigate exposure to organochlorine pesticides as a risk factor to breast cancer. A case-control study design was employed among breast cancer patients and non-breast cancer individuals (controls). Blood samples were collected from 100 study participants (50 cases and 50 controls) followed by serum separation, extraction and cleanup using standard analytical procdures. The findings revealed that ten organochlorine pesticides were detected in the serum of the study participants. From the detected organochlorine pesticides, heptachlor was observed at higher concentration for breast cancer patients (6.90±4.37 µg/L) and controls (9.15±3.84 µg/L). Mean serum level of p,p'-DDE, p,p'-DDT, heptachlor, gamma-chlordane, endosulfan, and dibutyl-chlorendate were significantly higher in the serum of breast cancer patients than the controls. From the studied pesticides, p,p'-DDT and gamma-chlordane are significant predictors for BC, while, others are equivocal. A unit increment of the concentration of p,p'-DDT (AOR; 2.03, 95% CI: 1.041-3.969) increased the odds of developing breast cancer by two, while for gamma-chlordane (AOR;3.12, 95% CI; 1.186-8.203) by three. Our study results suggesting that, organochlorines are a risk factors for breast cancer in Ethiopia. Decreasing exposure to such organochlorines might have a significant public health relevance in reducing non-communicable chronic illnesses. Besides, continues monitoring of persistent organic pollutants using body biomarkers is important for disease prevention and device mitigation measures.

Utilization of rGO-PEI-supported AgNPs for sensitive recognition of deltamethrin in human plasma samples: A new platform for the biomedical analysis of pesticides in human biofluids.

In this study, the rGO-PEI-AgNPs sensor was designed as a new effective platform to sensitive monitoring of deltamethrin in human plasma samples. For this purpose, reduced graphene oxide (rGO)-supported polyethylenimine (PEI) was used as a suitable substrate for dispersion of silver nanoparticles (AgNPs) as amplification and catalytic element. Therefore, a novel interface (rGO-PEI-AgNPs) was prepared by the fully electrochemical method on the surface of glassy carbon electrodes. The engineered nano-sensor showed a wide dynamic range of 10 nM to 1 mM and low limit of quantification (LLOQ) as 10 nM in human plasma sample, which revealed excellent analytical performance for the recognition of deltamethrin with high sensitivity and reproducibility through differential pulse voltammetry and square wave voltammetry techniques. The results confirm that rGO-PEI-AgNPs as a novel biocompatible interface can provide appropriate, reliable, affordable, rapid, and user-friendly diagnostic tools in the detection of deltamethrin in human real samples.

Clinical utility of validated gas chromatography-ion trap mass spectrometry in patients with anticholinesterase pesticides poisoning.

Intentional or unintentional intake of anticholinesterase pesticides became common due to their extensive use in agricultural and domestic purposes, resulting in numerous poisoning cases. A simple, accurate, and sensitive gas chromatography-ion trap mass spectrometry-based method for the quantification of 12 anticholinesterase pesticides (monocrotophos, dimethoate, dichlorvos, azinphos-methyl, carbofuran, chlorpyrifos, dialifos, diazinon, malathion, parathion, methidathion, and terbufos) in serum was developed, and its utility in patients with alleged pesticides poisoning was assessed. The quantification was performed using liquid-liquid extraction by toluene/chloroform (4:1,v/v) with 500 µL of serum. On column limit of detection and limit of quantification were less than 50.00 µg/L. The recovery ranged from 97.54 to 103.23%. The calibration curves were linear (R2 > 0.9937). Accuracy was found to be between - 7.1 and 7.2%. Intra-day and inter-day reproducibility was less than 17% for the spiked quality control serum samples. The level of pesticide in serum quantified by the validated method correlated with clinical signs and symptoms, pseudo-cholinesterase activity, total atropine dose, length of hospital stay, and clinical outcome in 15 patients with alleged pesticide poisoning. The validated method may be used for monitoring and prognosis in patients with pesticide poisoning and diagnosis of poisoning in forensic toxicology.

Analysis of the Soil Fumigant, Dimethyl Disulfide, in Swine Blood by Dynamic Headspace Gas Chromatography-Mass Spectroscopy.

Plant parasites and soilborne pathogens directly reduce the overall yield of crops, vegetables, and fruits, negatively impacting the market demand for these products and their net profitability. While preplant soil fumigation helps maintain the consistent production quality of high-value cash crops, most soil fumigants are toxic to off-target species, including humans. Dimethyl disulfide (DMDS) has recently been introduced as a relatively low toxicity soil fumigant. Although DMDS exhibits low toxicity compared to other soil fumigants, it is volatile and exposure can cause eye, nasal, and upper respiratory tract irritation, skin irritation, nausea, dizziness, headache, and fatigue. While there is one analysis method available for DMDS from biological matrices, it has significant disadvantages. Hence, in this study, a dynamic headspace gas chromatography-mass spectroscopy (DHS-GC-MS) method was developed for the analysis of DMDS in swine whole blood. This method is highly sensitive and requires only three steps: 1) acid denaturation, 2) addition of internal standard, and 3) DHS-GC-MS analysis. The method produced a wide linear range from 0.1 - 200 µM with an excellent limit of detection of 30 nM. Intra- and interassay accuracy (100±14% and 100±11%, respectively) and precision (<5% and <6% relative standard deviation, respectively) were also excellent. The method worked well to quantify the DMDS levels in the blood of dimethyl trisulfide (DMTS)-treated swine (i.e., DMDS is a byproduct of DMTS treatment) with no interfering substances at or around the retention time of DMDS (i.e., 2.7 min). This simple, rapid, and extremely sensitive method can be used for the quantification of DMDS levels in blood to verify exposure to DMDS or to monitor levels of DMDS following DMTS treatment (e.g., for cyanide poisoning).

Multi-residue analysis of pesticides in blood plasma using hollow fiber solvent bar microextraction and gas chromatography with a flame ionization detector.

The challenges faced on pesticide extraction from biological samples are finding a method that allows a multi-residue extraction, pre-concentration, clean-up, and isolation of analytes in just one step. In this sense, the hollow fiber - liquid phase microextraction method (HF- LPME) in the "solvent bar" mode was used to optimize and validate a method for pesticide multi-residue analysis in blood plasma at trace levels, through gas chromatography coupled with a flame ionization detector (GC-FID). Hollow fiber solvent bar microextraction HF-SBME was carried out with octanol immobilized into the pores of hydrophobic polypropylene fiber and disposed within a matrix of blood plasma, spiked with a mixture of pesticides (monocrotophos, lindane, aldrin, methyl parathion, endosulfan, dieldrin, DDD, DDT, and endrin). The optimization parameters evaluated were: extraction temperature and time, stirring speed, and salt concentration. A principal component analysis was performed to visualize the analytes' behaviour based on their explained variance, and then, a Box-Behnken analysis was generated to identify the optimum parameters. According to the PCA, all pesticides showed similar responses to the extraction method and the response of dieldrin exhibit the lowest variance. Moreover, the stationary points selected from the Box-Behnken analysis were 25.5 °C for the extraction temperature, 870 rpm for stirring speed, 16 min for extraction time, and 8.3 % w/v of salt concentration. Moreover, the validation results proved that HF-SBME is an alternative technique for pesticide multi-residue extraction in blood plasma. The analytes were able to concentrate, reaching 46 fold enrichment. The solvent type, sample and solvent volume were narrowed down without changing the method's precision or accuracy. The relative standard deviation was under 10 %, and the recovery was between 55 % and 105 % for the different analytes excepting lindane, which had lower recovery (27 %). The detection limits were 0.02 until 0.13 µg mL-1 for most of the pesticides used. Finally, HF-SBME is a good alternative for pesticide multi-residue extraction in complex matrices like plasma.

Drugs, pesticides and metabolites in forensic post-mortem blood samples.

Forensic post-mortem toxicological data provide valuable information for the elucidation of cause of death. However, this is still not routine practice in Brazilian laboratories. This study investigated the presence of illicit and prescription drugs, pesticides and metabolites in 111 post-mortem blood samples from cases investigated by the Forensic Medical Institute of the Federal District, Brazil. Quantitative analysis was performed for 14 analytes using a validated programmed temperature vaporisation-large volume injection-gas chromatography-mass spectrometry method, which was also used as screening (qualitative analysis) for an additional 19 substances of forensic interest. At least one analyte was found in 61.2% of the samples, of which 34 were related to homicide, 15 to accidental death and 10 to suicide cases. The victims were 14-72 years old. The benzodiazepines diazepam, midazolam and 7-aminoflunitrazepan were detected in 46% of the positive samples (0.02-1.12 µg/mL; midazolam only qualitative). Cocaine was found in 34% (0.02-4.07 µg/mL), associated with substances commonly used as cocaine adulterants (e.g. caffeine, lidocaine and phenacetin). Three suicide cases involved the illegal rodenticide chumbinho, residues of which were found in the gastric content, and blood samples showed the presence of terbufos (0.03 and 0.04 µg/mL) and carbofuran (27.3 µg/mL). These results are discussed, along with autopsy and crime-scene information.

Magnetic Solid-Phase Extraction of Drugs and Pesticides from Human Plasma Using COOH-mMWCNTs.

Carbon nanotubes (CNTs) are useful for extracting chemical compounds due to their properties, such as surface area and the potential for chemical modification. Especially the formation of CNTs with carboxylic acid functional group makes them disperse in water-based samples and have strong interaction forces with cationizable analytes. Based on these features, carboxylic acid functionalized multi-walled CNTs (COOH-MWCNTs) have been used as extraction sorbents. CNT can also be gathered using an external magnet by forming complex with iron oxide (Fe3O4) magnetic nanoparticles (MNPs). In this study, COOH-MWCNTs with MNPs were subjected to magnetic solid-phase extraction (mSPE) in order to extract the targeted substances such as diphenhydramine, doxylamine, tramadol, escitalopram, zolpidem, diphenamid, paclobutrazol, hexaconazole, cyproconazole and mepronil from human plasma samples. The following five factors were optimized: (i) the ratio of COOH-MWCNTs to MNPs as a sorbent from 1:1 to 1:4; (ii) sorbent amount starting from 12.5 to 75%; (iii) sample pH tested pH 2 to pH 10 with 1 N hydrochloride and 1 N sodium hydroxide; (iv) agitating time from 0 to 4 min and (v) elution solvent. Limit of detection of 10 targeted substances in human plasma were in the range of 0.1-0.4 mg/L. The recovery of targeted substances (except diphenamid) in human plasma was 73.06-110.28% for intra-day and 83.00-107.70% for inter-day and the precision (relative standard deviation, %) in human plasma was 0.3-13.3% for intra-day and 2.9-15.6% for inter-day. The method was applied to nine authentic biological samples from overdose patients in the emergency room of Chungnam National University Hospital. The performance of mSPE was compared with the liquid-liquid extraction method using ethyl acetate. The results showed that the newly developed method in this study can be used for screening analysis in forensic and clinical toxicology.

Synthesis of sheet-like polypyrrole nanowires for the microextraction of trace residues of pyrethroid pesticides in human plasma and molecular dynamics-aided study of adsorption mechanism.

The synthesized sheet-like polypyrrole (ppy) nanowires were used as solid phase extraction materials, followed by gas chromatography-mass spectrometry (GC-MS) for the detection of traces residues of pyrethroid pesticides in human plasma. A multiresidue method was developed and verified for the determination of trace pyrethroid residues (transfluthrin, allethrin, resmethrin, fenpropathrin, etofenprox, fenvalerate) in human plasma. In this study, using the cationic surfactant cetyltrimethylammonium bromide (CTAB) as a soft template, ppy nanowires with regular morphology were prepared by oxidative polymerization. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and other techniques were employed for characterization. Molecular dynamics analyses were used to simulate the adsorption mechanism of each pyrethroid and ppy nanowires. Based on density analysis, molecular recognition analysis, and binding energy, the van der Waals force was considered as an important driving force for the adsorption of pyrethroids and ppy nanowires. The limits of detection (LOD) of six pyrethroids were 0.008-0051 ng mL-1, and the limits of quantification (LOQ) were 0.028-0.162 ng mL-1. The relative standard deviations of ppy nanowires were 2.12-5.09%, and the recoveries of six pyrethroids ranged from 76.9 to 110.4%. The enrichment factors were within the range of 47.09-51.30. The experimental results showed that the method could be an efficient detection method for trace residue analysis of pyrethroid pesticides in complex biological samples. It would be advantageous for clinical monitoring and toxicological studies of pyrethroids.

Simultaneous determination of polar pesticides in human blood serum by liquid chromatography coupled to triple quadrupole mass spectrometer.

In this study a simple and rapid method was used for the determination of 5 polar pesticides and 3 metabolites, ethephon, 2-hydroxyethyl phosphonic acid (HEPA), fosetyl-aluminum, phosphonic acid, glyphosate, chlorate, glufosinate and 3-methylphosphinicopropionic acid (MPPA) in human blood serum. Acidified water (1% formic acid) and acetonitrile (1% formic acid) were used as extraction solvents and n-hexane was utilized as a clean-up step for lipid removal. The analytical method was based on liquid chromatography coupled to triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The proposed method was validated, obtaining recoveries ranging from 70 to 110% and precision values lower than 17% for all analytes. Limit of quantification was 0.01 mg/L for ethephon, fosetyl-aluminum, MPPA and HEPA, 0.025 mg/L for glyphosate, phosphonic acid and chlorate and 0.05 mg/L for glufosinate. This is the first time that an analytical method was developed for the determination of a high number of polar pesticides in human blood serum, allowing the simultaneous monitoring of these compounds in a complex matrix as human blood serum.

Concentrations of persistent organic pollutants in maternal plasma and epigenome-wide placental DNA methylation.

BACKGROUND: Prenatal maternal plasma persistent organic pollutant (POP) concentrations have been associated with neonatal outcomes. However, the underlying mechanisms remain unknown. Placental epigenetic mechanisms may be involved, but no prior epigenome-wide studies have investigated the impact of maternal POPs on placental DNA methylation. We studied the association between maternal plasma POP concentration in early pregnancy and epigenome-wide placental DNA methylation among 260 pregnant women from the NICHD Fetal Growth Studies. RESULTS: Our analysis focused on POPs with more than 80% plasma concentrations above the limit of quantification, including 3 organochlorine pesticides (hexachlorobenzene, trans-nonachlor, p,p'-dichlorodiphenyldichloroethylene), 1 polybrominated diphenyl ether (PBDE 47), 3 polychlorinated biphenyls (138/158, 153, 180), and 6 poly- and perfluorinated alkyl substances (PFASs) (perfluorodecanoic acid, perfluorohexanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluoroundecanoic acid (PFUnDA)). Using 5% false discovery rate, POPs were associated with a total of 214 differentially methylated CpG sites (nominal p values ranging from 2.61 × 10-21 to 2.11 × 10-7). Out of the 214 CpG sites, 24 (11%) were significantly correlated with placental expression of 21 genes. Notably, higher PFUnDA was associated with increased methylation at 3 CpG sites (cg13996963, cg12089439, cg18145877) annotated to TUSC3, and increased methylation at those 3 CpG sites was correlated with decreased expression of TUSC3 in the placenta. Increased methylation at cg18145877 (TUSC3) and decreased expression of TUSC3 were correlated with shorter birth length. Out of the 214 CpG sites, methylation at 44 CpG sites was correlated (p value < 0.10) with at least one neonatal anthropometry measure (i.e., birth weight, birth length, and head circumference). Seven CpG sites mediated (p value < 0.05) the association between PBDE 47 and neonatal anthropometry measures. Genes annotating the top differentially methylated CpG sites were enriched in pathways related to differentiation of embryonic cells (PBDE 47) and in pathways related to brain size and brain morphology (PFASs). CONCLUSIONS: DNA methylation changes in the placenta were significantly associated with maternal plasma POPs concentration. The findings suggest that placental DNA methylation and gene expression mechanism may be involved in the prenatal toxicity of POPs and their association with neonatal anthropometry measures.

An epigenome-wide association study of ambient pyrethroid pesticide exposures in California's central valley.

BACKGROUND: Pyrethroid pesticide use is increasing worldwide, although the full extent of associated health effects is unknown. An epigenome-wide association study (EWAS) with exploratory pathway analysis may help identify potential pyrethroid-related health effects. METHODS: We performed an exploratory EWAS of chronic ambient pyrethroid exposure using control participants' blood in the Parkinson's Environment and Genes Study in the Central Valley of California (N = 237). We estimated associations of living and working near agricultural pyrethroid pesticide applications in the past 5 years (binary) with site-specific differential methylation, and used a false discovery rate (FDR) cut off of 0.05 for significance. We controlled for age, sex, education, cell count, and an ancestral marker for Hispanic ethnicity. We normalized methylation values for Type I/II probe bias using Beta-Mixture Quantile (BMIQ) normalization, filtered out cross-reactive probes, and evaluated for remaining bias with Surrogate Variable Analysis (SVA). We also evaluated the effects of controlling for cell count and normalizing for Type I/II probe bias by comparing changes in effect estimates and p-values for the top hits across BMIQ and GenomeStudio normalization methods, and controlling for cell count. To facilitate broader interpretation, we annotated genes to the CpG sites and performed gene set overrepresentation analysis, using genes annotated to CpG sites that were associated with pyrethroids at a raw p < 0.05, and controlling for background representation of CpG sites on the chip. We did this for both a biological process context (Gene Ontology terms) using missMethyl, and a disease set context using WebGestalt. For these gene set overrepresentation analyses we also used an FDR cut off of 0.05 for significance of gene sets. RESULTS: After controlling for cell count and applying BMIQ normalization, 4 CpG sites were differentially methylated in relation to pyrethroid exposures. When using GenomeStudio's Illumina normalization, 415 CpG sites were differentially methylated, including all four identified with the BMIQ method. In the gene set overrepresentation analyses, we identified 6 GO terms using BMIQ normalization, and 76 using Illumina normalization, including the 6 identified by BMIQ. For disease sets, we identified signals for Alzheimer's disease, leukemia and several other cancers, diabetes, birth defects, and other diseases, for both normalization methods. We identified minimal changes in effect estimates after controlling for cell count, and controlling for cell count generally weakened p-values. BMIQ normalization, however, resulted in different beta coefficients and weakened p-values. CONCLUSIONS: Chronic ambient pyrethroid exposure is associated with differential methylation at CpG sites that annotate to a wide variety of disease states and biological mechanisms that align with prior research. However, this EWAS also implicates several novel diseases for future investigation, and highlights the relative importance of different background normalization methods in identifying associations.

Salt-assisted acetonitrile extraction and HPLC-QTOF-MS/MS detection for residues of multiple classes of pesticides in human serum samples.

Detecting pesticide residues in human serum is a challenging process. In this study we developed and validated a method for the extraction and recovery of residues of multiple classes of pesticides from serum using one reagent. Salt-assisted acetonitrile extraction and high-performance liquid chromatography with quadrupole time of flight tandem mass spectrometry were used to quantitate 34 pesticides classified in nine groups of chemicals in human serum samples, which are frequently detected in food. The recoveries for 33 of analyzed pesticides ranged from 86 to 112% with relative standard deviations below 15%. The limits of quantitation and linearity of 31 of the pesticides were 1 µg/L and >0.990, respectively. The lower limit of quantitation has been reported in the literature particularly for multi-classes pesticide mixtures in human serum. The salt-acetonitrile reagent was allowed to achieve good recoveries and detection limits, which could be attributed to salt altering the solvent polarity, preferentially collecting the organic phase in the solution, and promoting the extraction. The developed method was applied for two organophosphate pesticide metabolites, diethylphosphate and 3,5,6-trichloro-2-pyridinol, in serum from rats that were fed a nonlethal quantity of chlorpyrifos. The concentrations of these two were 252.18 ± 15.47 and 0.63 ± 0.23 µg/L, respectively.

Rio Birth Cohort Study on Environmental Exposure and Childhood Development - PIPA Project.

Background/Objective: As a developing country, Brazil presents a wide range of environmental risks that can constitute hazards to child health. The country also presents different socio-economic-cultural conditions that could be responsible for determining different vulnerability and susceptibility levels for the population, which can potentiate the effects of the environmental pollutants. The Rio Birth Cohort Study (PIPA project) is a prospective maternal-infant health study, hosted in the city of Rio de Janeiro (Southeastern Brazil), designed to investigate separate and combined effects of environmental chemical pollutants, as well as the interactions between these exposures and sociocultural environment and epigenetic patterns. This paper presents the learned lessons and strategies to address the shortcomings detected from this pilot study. Methods: The study population will be all children born at the Federal University of Rio de Janeiro Maternity Hospital from July 1st, 2020 to June 30th, 2021. The estimated population is of 2,500 children. The study will collect social, demographic, and health information from pregnant women and their children up to four years of age. Biological samples from both mothers and newborns will be collected to assess metal, pesticide and plasticizer exposure. All newborns will have their landmarks of physical, neurological, psychological, and cognitive development recorded at specific ages. Findings: A pilot study was carried out between September 2017 and August 2018, totaling 142 enrolled pregnant women, leading to 135 (95%) births and the collection of umbilical cord (126-93%,) and mother (139-98%) blood samples, as well as both mother (142-100%) and newborn (54-40%) urine samples and newborn meconium samples (117-86.7%). Conclusions: The study proposes a comprehensive assessment of pre- and postnatal exposure to environmental chemicals at multiple time points in a population living in a highly urbanized developing country. As far as we know, this is the only birth cohort in Brazil specifically designed for this purpose.

Multiresidue analysis of 85 persistent organic pollutants in small human serum samples by modified QuEChERS preparation with different ionization sources in mass spectrometry.

In this study, a multiresidue analytical method was developed, validated, and applied for quantifying 85 persistent organic pollutants (POPs), including 38 polychlorinated biphenyls (PCBs), 23 polybrominated diphenyl ethers (PBDEs), and 24 organochlorine pesticides (OCPs) from 200 µL of human serum. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method was applied to minimize the required sample amount and optimize various conditions including the extraction solvent and the number of extractions. The extraction efficiency was optimized using double extraction with an ethyl acetate/hexane/acetone mixture. Gas chromatography coupled with triple-quadrupole mass spectrometry was used for analysis, and two different ionization sources, electron impact ionization (EI) and atmospheric pressure chemical ionization (APCI), were used to compare their sensitivity. The APCI source employed soft ionization at atmospheric pressure, producing abundant molecular ion formation with minimal fragmentation, in contrast to extensive fragmentation caused by EI. Of the 85 POPs analyzed, 59 target compounds (69.4%) showed lower limits of detection that were two- to fifty-fold lower in APCI than those determined using EI. The developed method was validated for its detection limit (0.5-10 pg/mL for PCBs, 2-20 pg/mL for PBDEs, and 2-40 pg/mL for OCPs), precision (0.8%-34.3% of coefficient of variation), recovery (49.6%-77.1%), matrix effect (46.7%-156.9%), and accuracy (81.2%-113.1% for PCBs, 85.8%-112.2% for PBDEs, and 55.2%-113.9% for OCPs). Its linearity was R2 > 0.99 for 84 compounds, and 96% average accuracy (for APCI) was obtained using the National Institute of Standards and Technology (NIST) standard reference materials (NIST 1957 and 1958). These ionization methods were compared by analyzing 25 real human serum samples. The observed species were 1.1-24.6 pg/mL of 28 PCBs, 2.5 pg/mL of BDE-47, and 6.5-195.1 pg/mL of 6 organochlorine pesticides (median concentration for each species), and only 11 compounds were detected with APCI owing to its enhanced sensitivity.