Is Nontarget Analysis Ready for Regulatory Application? Influence of Peak-Picking Algorithms on Data Analysis.

The use of peak-picking algorithms is an essential step in all nontarget analysis (NTA) workflows. However, algorithm choice may influence reliability and reproducibility of results. Using a real-world data set, the aim of this study was to investigate how different peak-picking algorithms influence NTA results when exploring temporal and/or spatial trends. For this, drinking water catchment monitoring data, using passive samplers collected twice per year across Southeast Queensland, Australia (n = 18 sites) between 2014 and 2019, was investigated. Data were acquired using liquid chromatography coupled to high-resolution mass spectrometry. Peak picking was performed using five different programs/algorithms (SCIEX OS, MSDial, self-adjusting-feature-detection, two algorithms within MarkerView), keeping parameters identical whenever possible. The resulting feature lists revealed low overlap: 7.2% of features were picked by >3 algorithms, while 74% of features were only picked by a single algorithm. Trend evaluation of the data, using principal component analysis, showed significant variability between the approaches, with only one temporal and no spatial trend being identified by all algorithms. Manual evaluation of features of interest (p-value <0.01, log fold change >2) for one sampling site revealed high rates of incorrectly picked peaks (>70%) for three algorithms. Lower rates (<30%) were observed for the other algorithms, but with the caveat of not successfully picking all internal standards used as quality control. The choice is therefore currently between comprehensive and strict peak picking, either resulting in increased noise or missed peaks, respectively. Reproducibility of NTA results remains challenging when applied for regulatory frameworks.

Interlaboratory Comparison of Extractable Organofluorine Measurements in Groundwater and Eel (Anguilla rostrata): Recommendations for Methods Standardization.

Research on per- and polyfluoroalkyl substances (PFAS) frequently incorporates organofluorine measurements, particularly because they could support a class-based approach to regulation. However, standardized methods for organofluorine analysis in a broad suite of matrices are currently unavailable, including a method for extractable organofluorine (EOF) measured using combustion ion chromatography (CIC). Here, we report the results of an international interlaboratory comparison. Seven laboratories representing academia, government, and the private sector measured paired EOF and PFAS concentrations in groundwater and eel (Anguilla rostrata) from a site contaminated by aqueous film-forming foam. Among all laboratories, targeted PFAS could not explain all EOF in groundwater but accounted for most EOF in eel. EOF results from all laboratories for at least one replicate extract fell within one standard deviation of the interlaboratory mean for groundwater and five out of seven laboratories for eel. PFAS spike mixture recoveries for EOF measurements in groundwater and eel were close to the criterion (±30%) for standardized targeted PFAS methods. Instrumental operation of the CIC such as replicate sample injections was a major source of measurement uncertainty. Blank contamination and incomplete inorganic fluorine removal may introduce additional uncertainties. To elucidate the presence of unknown organofluorine using paired EOF and PFAS measurements, we recommend that analysts carefully consider confounding methodological uncertainties such as differences in precision between measurements, data processing steps such as blank subtraction and replicate analyses, and the relative recoveries of PFAS and other fluorine compounds.

Impact of Microplastics on the Ocular Surface.

Plastics are synthetic materials made from organic polymers that are ubiquitous in daily living and are especially important in the healthcare setting. However, recent advances have revealed the pervasive nature of microplastics, which are formed by degradation of existing plastic products. Although the impact on human health has yet to be fully characterised, there is increasing evidence that microplastics can trigger inflammatory damage, microbial dysbiosis, and oxidative stress in humans. Although there are limited studies investigating their effect on the ocular surface, studies of microplastics on other organs provide some insights. The prevalence of plastic waste has also triggered public outcry, culminating in the development of legislation aimed at reducing microplastics in commercial products. We present a review outlining the possible sources of microplastics leading to ocular exposure, and analyse the possible mechanisms of ocular surface damage. Finally, we examine the utility and consequences of current legislation surrounding microplastic regulation.

Serum measures of hexabromocyclododecane (HBCDD) and polybrominated diphenyl ethers (PBDEs) in reproductive-aged women in the United Kingdom.

We investigated the serum concentrations of two brominated flame retardants (BFRs) - polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCDD) -in 59 women aged between 23 and 42 from the United Kingdom. We also collected demographic data, including age, bodyweight and height in order to test for associations with BFR levels. Temporal and global differences were also assessed using previously published data. HBCDD was detected in 68% of samples with a mean concentration of 2.2 ng/g lipid (range = <0.3-13 ng/g lipid). The dominant stereoisomer was α-HBCDD with an average contribution of 82% (0-100%) towards ΣHBCDD, was followed by γ-HBCDD (average contribution = 17%). PBDEs were detected in 95% of samples with a mean ∑PBDE (sum of BDEs -28, -47, -99, -100, -153, -154 and -183) concentration of 2.4 ng/g lipid (range = <0.4-15 ng/g lipid). BDEs -153 and -47 were the dominant congeners, contributing an average of 40% and 37% respectively, to the average ΣPBDE congener profile. Data from this study suggests that HBCDD levels decrease with age, it also suggests a positive association between bodyweight and HBCDD levels, which likewise requires a large-scale study to confirm this. The data also show that 10 years after their European ban, PBDE body burden has begun to decrease in the UK. Whilst it is too early to draw any firm conclusions for HBCDDs, they appear to be following a similar pattern to PBDEs, with levels decreasing by a factor of >2.5 since 2010. Whilst the human body burden appear to be decreasing, both PBDEs and HBCDD are still consistently detected in human serum, despite legislative action limiting their production and use. This highlights the need to continuously assess human exposure and the effectiveness of policy aimed at reducing exposure.

Influence of extraction windows for data-independent acquisition on feature annotation during suspect screening.

Non-target analysis (NTA) using high-resolution mass spectrometry is becoming a useful approach to screen for suspect and unknown chemicals. For comprehensive analyses, data-independent acquisition (DIA), like Sequential Windowed Acquisition of all THeoretical Mass Spectra (SWATH-MS) on Sciex instruments, is necessary, usually followed by library matching for feature annotation. The choice of parameters, such as acquisition window number and size, may influence the comprehensiveness of the suspect features detected. The goal of this study was to assess how mass spectrometric DIA settings may influence the ability to obtain confident annotations and identifications of features in environmental (river water, passive sample extract (PSE)), wastewater (unpreserved and acidified) and biological (urine) sample matrices. Each matrix was analysed using 11 different MS methods, with 5-15 variable size acquisition windows. True positive (TP) annotation (i.e., matching experimental and library spectra) rates were constant for PSE (40%) and highest for urine (18%), wastewater (34% and 36%, unpreserved and acidified, respectively) and river water (8%) when using higher numbers of windows (15). The number of annotated features was highest for PSE (12%) and urine (8.5%) when using more acquisition windows (9 and 14, respectively). Less complex matrices (based on average total ion chromatogram intensities) like river water, unpreserved and acidified wastewater have higher annotation rates (7.5%, 8% and 13.2%, respectively) when using less acquisition windows (5-6), indicating matrix dependency of optimum settings. Library scores varied widely for correct (scores between 6 and 100) as well as incorrect annotations (scores between 2 and 100), making it hard to define specific ideal cut-off values. Results highlight the need for properly curated libraries and careful optimization of SWATH-MS and other DIA methods for each individual matrix, finding the best ratio of total annotations to true positive, (i.e., correct) annotations to achieve best NTA results.

Identification of novel polyfluoroalkyl substances in surface water runoff from a chemical stockpile fire.

In 2018, over 30,000 L of fluorine-free firefighting foam was used to extinguish an industrial warehouse fire of uncharacterized chemical and industrial waste. Contaminated firewater and runoff were discharged to an adjacent freshwater creek in Melbourne, Australia. In this study, we applied nontarget analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) to 15 surface water samples to investigate the presence of legacy, novel and emerging per-and polyfluoroalkyl substances (PFAS). We identified six novel and emerging fluorotelomer-based fluorosurfactants in the Australian environment for the first time, including: fluorotelomer sulfonamido betaines (FTABs or FTSA-PrB), fluorotelomer thioether amido sulfonic acids (FTSASs), and fluorotelomer sulfonyl amido sulfonic acids (FTSAS-So). Legacy PFAS including C6-C8 perfluoroalkyl sulfonic acids, C4-C10 perfluoroalkyl carboxylic acids, and perfluoro-4-ethylcyclohexanesulfonate were also detected in surface water. Of note, we report the first environmental detection of ethyl 2-ethenyl-2-fluoro-1-(trifluoromethyl) cyclopropane-1-carboxylate. Analysis of several Class B certified fluorine-free foam formulations allowed for use in Australia revealed that there was no detectable PFAS. Patterns in the homologue profiles of fluorotelomers detected in surface water are consistent with environments impacted by fluorinated aqueous film-forming foams. These results provide strong evidence that firewater runoff of stockpiled fluorinated firefighting foam was the dominant source of detectable PFAS to the surrounding environment.

Tracing Environmental Exposure from Neurodevelopment to Neurodegeneration.

Interplay between genetic and environmental factors during critical time windows can have effects that span from neurodevelopment to neurodegeneration. We present the concept of the 'neuroexposome', emphasizing the brain's distinctive response to environmental exposure, and how current 'omics' sciences can inform on both disease pathogenesis and future public health policies.

Urinary metabolites of organophosphate esters: Concentrations and age trends in Australian children.

There is growing concern around the use of organophosphate esters (OPEs) due to their suspected reproductive toxicity, carcinogenicity, and neurotoxicity. OPEs are used as flame retardants and plasticizers, and due to their extensive application in consumer products, are found globally in the indoor environment. Early life exposure to OPEs is an important risk factor for children's health, but poorly understood. To study age and sex trends of OPE exposures in infants and young children, we collected, pooled, and analysed urine samples from children aged 0-5years from Queensland, Australia for 9 parent OPEs and 11 metabolites. Individual urine samples (n=400) were stratified by age and sex, and combined into 20 pools. Three individual breast milk samples were also analysed to provide a preliminary estimate on the contribution of breast milk to the intake of OPEs. Bis(1-chloroisopropyl) phosphate (BCIPP), 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP), bis(1,3-dichloroisopropyl) phosphate (BDCIPP), dibutyl phosphate (DBP), diphenyl phosphate (DPHP), bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate (3OH-TBOEP), and bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were detected in all urine samples, followed by bis(methylphenyl) phosphate (80%), and bis(2-ethylhexyl) phosphate (BEHP, 20%), and bis(2-chloroethyl) phosphate (BCEP, 15%). Concentrations of tris(2-chloroethyl) phosphate (TCEP), BCEP, tris(2-ethylhexyl) phosphate (TEHP), and DBP decreased with age, while bis(methylphenyl) phosphate (BMPP) increased with age. Significantly higher concentrations of DPHP (p=0.039), and significantly lower concentrations of TEHP (p=0.006) were found in female samples compared to males. The estimated daily intakes (EDIs) via breastfeeding, were 4.6, 26 and 76ng/kg/day for TCEP, TBP and TEHP, respectively, and were higher than that via air and dust, suggesting higher exposure through consumption of breast milk.

Rapid screening and identification of chemical hazards in surface and drinking water using high resolution mass spectrometry and a case-control filter.

Access to clean, safe drinking water poses a serious challenge to regulators, and requires analytical strategies capable of rapid screening and identification of potentially hazardous chemicals, specifically in situations when threats to water quality or security require rapid investigations and potential response. This study describes a fast and efficient chemical hazard screening strategy for characterising trace levels of polar organic contaminants in water matrices, based on liquid chromatography high resolution mass spectrometry with post-acquisition 'case-control' data processing. This method allowed for a rapid response time of less than 24 h for the screening of target, suspect and non-target unknown chemicals via direct injection analysis, and a second, more sensitive analysis option requiring sample pre-concentration. The method was validated by fortifying samples with a range of pesticides, pharmaceuticals and personal care products (n = 46); with >90% of target compounds positively screened in samples at 1 ng mL-1, and 46% at 0.1 ng mL-1 when analysed via direct injection. To simulate a contamination event samples were fortified with compounds not present in the commercial library (designated 'non-target compounds'; fipronil and fenitrothion), tentatively identified at 0.2 and 1 ng mL-1, respectively; and a compound not included in any known commercial library or public database (designated 'unknown' compounds; 8Cl- perfluorooctanesulfonic acid), at 0.8 ng mL-1. The method was applied to two 'real-case' scenarios: (1) the assessment of drinking water safety during a high-profile event in Brisbane, Australia; and (2) to screen treated, re-circulated drinking water and pre-treated (raw) water. The validated workflow was effective for rapid prioritisation and screening of suspect and non-target potential hazards at trace levels, and could be applied to a wide range of matrices and investigations where comparison of organic contaminants between an affected and control site and or timeframe is warranted.

Non-targeted, high resolution mass spectrometry strategy for simultaneous monitoring of xenobiotics and endogenous compounds in green sea turtles on the Great Barrier Reef.

Chemical contamination poses a threat to ecosystem, biota and human health, and identifying these hazards is a complex challenge. Traditional hazard identification relies on a priori-defined targets of limited chemical scope, and is generally inappropriate for exploratory studies such as explaining toxicological effects in environmental systems. Here we present a non-target high resolution mass spectrometry environmental monitoring study with multivariate statistical analysis to simultaneously detect biomarkers of exposure (e.g. xenobiotics) and biomarkers of effect in whole turtle blood. Borrowing the concept from clinical chemistry, a case-control sampling approach was used to investigate the potential influence of xenobiotics of anthropogenic origin on free-ranging green sea turtles (Chelonia mydas) from a remote, offshore 'control' site; and two coastal 'case' sites influenced by urban/industrial and agricultural activities, respectively, on the Great Barrier Reef in North Queensland, Australia. Multiple biomarkers of exposure, including sulfonic acids (n=9), a carbamate insecticide metabolite, and other industrial chemicals; and five biomarkers of effect (lipid peroxidation products), were detected in case sites. Additionally, two endogenous biomarkers of neuroinflammation and oxidative stress were identified, and showed moderate-to-strong correlations with clinical measures of inflammation and liver dysfunction. Our data filtering strategy overcomes limitations of traditional a priori selection of target compounds, and adds to the limited environmental xenobiotic metabolomics literature. To our knowledge this is the first case-control study of xenobiotics in marine megafauna, and demonstrates the utility of green sea turtles to link internal and external exposure, to explain potential toxicological effects in environmental systems.

The Neurobiology and Age-Related Prevalence of the ε4 Allele of Apolipoprotein E in Alzheimer's Disease Cohorts.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterised by amyloid beta (Aß) plaques and tau neurofibrillary tangles in the brain. Human apolipoprotein E (ApoE) is a lipid transport protein coded by the polymorphic APOE gene, with three major alleles: ε2, ε3 and ε4. After age, the ε4 allele is the greatest risk factor for developing sporadic AD, conferring an increased risk of 3-4 and 8-12 times for one or two copies of the allele, respectively. This risk is reported to vary by demographic factors including sex, ethnicity and geography. In order to understand the risk of ApoE ε4 in relation to age, the primary risk factor for developing AD, we need to understand how the prevalence of APOE genotypes changes with age. Here, we present the first data on age-related prevalence of APOE ε4 in AD in three AD cohorts in Australia and the USA. There is a significant association between age and ε4 prevalence, particularly for ε4 homozygotes, such that as age increases the prevalence of ε4 decreases. Further studies on a random, population-based sample of the population are needed to provide more generalizable data, particularly in the >90-year-old age group.

Monitoring exposure to polycyclic aromatic hydrocarbons in an Australian population using pooled urine samples.

Integrated exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs). The aim of this study was to provide the first assessment of exposure to PAHs in a large sample of the population in Queensland, Australia including exposure to infant (0-4years). De-identified urine specimens, obtained from a pathology laboratory, were stratified by age and sex, and pooled (n=24 pools of 100) and OH-PAHs were measured by gas chromatography-isotope dilution-tandem mass spectrometry. Geometric mean (GM) concentrations ranged from 30ng/L (4-hydroxyphenanthrene) to 9221ng/L (1-naphthol). GM of 1-hydroxypyrene, the most commonly used PAH exposure biomarker, was 142ng/L. The concentrations of OH-PAHs found in this study are consistent with those in developed countries and lower than those in developing countries. We observed no association between sex and OH-PAH concentrations. However, we observed lower urinary concentrations of all OH-PAHs in samples from infants (0-4years), children (5-14years) and the elderly (>60year old) compared with samples from other age groups (15-29, 30-44 and 45-59years) which may be attributed to age-dependent behaviour-specific exposure sources.

Age as a determinant of phosphate flame retardant exposure of the Australian population and identification of novel urinary PFR metabolites.

The demand for alternative flame retardant materials such as phosphate flame retardants and plasticizers (PFRs) is increasing, although little is known of their possible effects on human health and development. To date, no information on the exposure of children or general Australian population to PFRs is available. The objectives of this study were to characterize the average levels and age-related patterns of PFR metabolites in urine in the general Australian population and to identify novel hydroxylated PFR metabolites in urine. Surplus pathology urine samples from Queensland, Australia were stratified and pooled by age and sex (3224 individuals aged 0 to 75years into 95 pools) according to two different pooling strategies at two different time periods. Samples were analyzed by solid phase extraction and liquid chromatography-tandem mass spectrometry following enzymatic treatment. Nine PFR metabolites were measured in the Australian population, including the first report of a hydroxylated metabolite of TCIPP (BCIPHIPP). Diphenyl phosphate (DPHP), BCIPHIPP and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) were detected in >95% of samples. DPHP, a metabolite of aryl-PFRs, was found in several samples at levels which were one order of magnitude higher than previously reported (up to 730ng/mL). Weighted linear regression revealed a significant negative association between log-normalized BDCIPP and DPHP levels and age (p<0.001). Significantly greater levels of BDCIPP and DPHP were found in children's urine compared with adults, suggesting higher exposure to PFRs in young children. BCIPHIPP was identified for inclusion in future PFR biomonitoring studies.

Pooled biological specimens for human biomonitoring of environmental chemicals: opportunities and limitations.

Biomonitoring has become the "gold standard" in assessing chemical exposures, and has an important role in risk assessment. The pooling of biological specimens-combining multiple individual specimens into a single sample-can be used in biomonitoring studies to monitor levels of exposure and identify exposure trends or to identify susceptible populations in a cost-effective manner. Pooled samples provide an estimate of central tendency and may also reveal information about variation within the population. The development of a pooling strategy requires careful consideration of the type and number of samples collected, the number of pools required and the number of specimens to combine per pool in order to maximise the type and robustness of the data. Creative pooling strategies can be used to explore exposure-outcome associations, and extrapolation from other larger studies can be useful in identifying elevated exposures in specific individuals. The use of pooled specimens is advantageous as it saves significantly on analytical costs, may reduce the time and resources required for recruitment and, in certain circumstances, allows quantification of samples approaching the limit of detection. In addition, the use of pooled samples can provide population estimates while avoiding ethical difficulties that may be associated with reporting individual results.

Short term variability in urinary bisphenol A in Australian children.

Used frequently in food contact materials, bisphenol A (BPA) has been studied extensively in recent years, and ubiquitous exposure in the general population has been demonstrated worldwide. Characterizing within- and between-individual variability of BPA concentrations is important for characterizing exposure in biomonitoring studies, and this has been investigated previously in adults, but not in children. The aim of this study was to characterize the short-term variability of BPA in spot urine samples in young children. Children aged ≥2-<4 years (n=25) were recruited from an existing cohort in Queensland, Australia, and donated four spot urine samples each over a two day period. Samples were analysed for total BPA using isotope dilution online solid phase extraction-liquid chromatography-tandem mass spectrometry, and concentrations ranged from 0.53 to 74.5 ng/ml, with geometric mean and standard deviation of 2.70 ng/ml and 2.94 ng/ml, respectively. Sex and time of sample collection were not significant predictors of BPA concentration. The between-individual variability was approximately equal to the within-individual variability (ICC=0.51), and this ICC is somewhat higher than previously reported literature values. This may be the result of physiological or behavioural differences between children and adults or of the relatively short exposure window assessed. Using a bootstrapping methodology, a single sample resulted in correct tertile classification approximately 70% of the time. This study suggests that single spot samples obtained from young children provide a reliable characterization of absolute and relative exposure over the short time window studied, but this may not hold true over longer timeframes.