Stepping-up accurate quantification of chlorinated paraffins: Successful certification of the first matrix reference material.

BACKGROUND: Chlorinated paraffins (CPs) are industrial chemicals categorised as persistent organic pollutants because of their toxicity, persistency and tendency to long-range transport, bioaccumulation and biomagnification. Despite having been the subject of environmental attention for decades, analytical methods for CPs still struggle reaching a sufficient degree of accuracy. Among the issues negatively impacting the quantification of CPs, the unavailability of well-characterised standards, both as pure substances and as matrix (certified) reference materials (CRMs), has played a major role. The focus of this study was to provide a matrix CRM as quality control tool to improve the comparability of CPs measurement results. RESULTS: We present the process of certification of ERM®-CE100, the first fish reference material assigned with certified values for the mass fraction of short-chain and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively). The certification was performed in accordance with ISO 17034:2016 and ISO Guide 35:2017, with the value assignment step carried out via an intercomparison of laboratories of demonstrated competence in CPs analysis and applying procedures based on different analytical principles. After confirmation of the homogeneity and stability of the CRM, two certified values were assigned for SCCPs, depending on the calibrants used: 31 ± 9 µg kg-1 and 23 ± 7 µg kg-1. The MCCPs certified value was established as 44 ± 17 µg kg-1. All assigned values are relative to wet weight in the CRM that was produced as a fish paste to enhance similarity to routine biota samples. SIGNIFICANCE AND NOVELTY: The fish tissue ERM-CE100 is the first matrix CRM commercially available for the analysis of CPs, enabling analytical laboratories to improve the accuracy and the metrological traceability of their measurements. The certified CPs values are based on results obtained by both gas and liquid chromatography coupled with various mass spectrometric techniques, offering thus a broad validity to laboratories employing different analytical methods and equipment.

Calibration and Application of PUF Disk Passive Air Samplers To Assess Chlorinated Paraffins in Ambient Air in Australia, China, and Vietnam.

Ambient air samples were collected in Brisbane (Australia), Dalian (China), and Hanoi (Vietnam) during Mar 2013-Feb 2018 using polyurethane foam based passive air samplers. A sampling rate calibration experiment was conducted for chlorinated paraffins (CPs, i.e., short-chain, medium-chain, and long-chain CPs), where the sampling rates were 4.5 ± 0.7, 4.8 ± 0.3, and 4.8 ± 2.1 m3 day-1 for SCCPs, MCCPs, and LCCPs, respectively. The atmospheric concentration of CPs was then calculated and the medians of ∑CPs were 0.079, 1.0, and 0.89 ng m-3 in Brisbane, Dalian, and Hanoi, respectively. The concentration of CPs in Brisbane's air remained at low levels, with no significant differences observed between the city background site and the city center site, indicating limited usage and production of CPs in this city. The highest concentration of MCCPs was detected in Dalian, while the highest concentration of SCCPs was detected in Hanoi. A decrease of SCCP concentration and an increase of MCCPs' were found in Brisbane's air from 2016 to 2018, while increasing trends for both SCCPs and MCCPs were observed in Dalian. These results indicated impacts from different sources of CPs in the investigated cities.

Nuclear magnetic resonance as a tool to determine chlorine percentage of chlorinated paraffin mixtures.

A new simple method for chlorine percentage calculations (method C), from proton nuclear magnetic resonance (1H NMR) spectroscopy, has been established and applied to an industrial chlorinated paraffin (CP) mixture and 13 single-chain CPs of known carbon chain lengths. Two modified methods (method A and B), originating from the work of Sprengel et al., have been utilized on the same single-chain mixtures. All samples were analysed by 1H NMR and two-dimensional heteronuclear quantum coherence (HSQC) for this purpose. All three methods worked well for medium chlorinated (45-55% Cl) single-chain mixtures of known carbon chain lengths. Method A yielded the best result for mixtures of lower chlorine content (<45% Cl), method C gave better estimations for higher chlorine contents (>55% Cl). Compared to Mohr's titration, method A showed a deviation of 0.7-7.8% (3.6% average), method B 4.1-11.3% (7.0% average) and method C 0.6-11.6% (5.2% average), for all 13 single-chain mixtures. The new method C is the only method that could be applied for determining the chlorine percentage of industrial mixtures of multiple, unknown chain lengths.

Semiquantitative Characterization of Bromo-chloro Paraffins and Olefins in the Australian Environment.

A semiquantitative high-resolution mass spectrometry method was developed and applied to assess the occurrence of bromo-/chloro paraffins (BCPs) and olefins (BCOs) in the environment. More than 400 possible BCPs and BCO congener groups were detected in dust, air, and sewage sludge samples collected from Australia. Median chain analytes with the number of halogen atoms <7 (CnHmClxBry, 14 ≤ n ≤ 17, x + y < 7) prevailed in the dust and sludge samples, while short chain analytes (CnHmClxBry, 10 ≤ n ≤ 13, x + y < 7) predominated the air samples. The estimated concentrations of ∑BCPs and ∑BCOs in dust and sludge were approximately 20% that of the chlorinated paraffins (CPs) present, with the median concentrations of 5.4 µg/g (dust) and 0.18 µg/g (sludge) for ∑BCPs and 22 µg/g (in dust) and 0.50 µg/g (sludge) for BCOs. In the air samples, the concentrations of BCPs (0.020 pg/m3) and BCOs (0.032 pg/m3) were 3-4 orders of magnitudes lower than the concentrations of CPs (790 pg/m3). Significant correlations (P < 0.001) were found between the concentration of CPs, BCPs, and BCOs in all the matrices.

Determination of chlorinated paraffins (CPs): Analytical conundrums and the pressing need for reliable and relevant standards.

The determination of chlorinated paraffins (CPs) has posed an intractable challenge in analytical chemistry for over three decades. The combination of an as yet unspecifiable number (tens - hundreds of thousands) of individual congeners in mass produced commercial CP mixtures and the steric interactions between them, contrive to defy efforts to characterise their residual occurrences in environmental compartments, food and human tissues. However, recent advances in instrumentation (mass spectrometric detectors and nuclear magnetic resonance), combined with interlaboratory studies, have allowed a better insight into the nature of the conundrums. These include the variability of results, even between experienced laboratories when there is insufficient matching between analytical standards and occurrence profiles, the poor (or no) response of some instrumentation to some CP congener configurations (multiple terminal chlorines or < four chlorines) and the occurrence of chlorinated olefins in commercial mixtures. The findings illustrate some limitations in the existing set of commercially available standards. These include cross-contamination of some standards (complex CP mixtures), an insufficient number of single chain standards (existing ones do not fully reflect food/biota occurrences), lack of homologue group standards and unsuitability of some configurationally defined CP congeners/labelled standards (poor instrument response and a smaller likelihood of occurrence in commercial mixtures). They also indicate an underestimation in reported occurrences arising from those CPs that are unresponsive during measurement. A more extensive set of standards is suggested and while this might not be a panacea for accurate CP determination, it would reduce the layers of complexity inherent in the analysis.

Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram.

This study presents, for the first time, the successful application of analyzing a whole gas chromatography (GC) chromatogram by nuclear magnetic resonance (NMR) spectroscopy using a continuous repeatable and stable (n = 280) high-resolution (HR) GC fractionation platform with a 96-well plate. Typically with GC- or liquid chromatography-mass spectrometry analysis, (isomer) standards and/or additional NMR analysis are needed to confirm the identification and/or structure of the analyte of interest. In the case of complex substances (e.g., UVCBs), isomer standards are often unavailable and NMR spectra too complex to achieve this. This proof of concept study shows that a HR GC fractionation collection platform was successfully applied to separate, purify, and enrich isomers in complex substances from a whole GC chromatogram, which would facilitate NMR analysis. As a model substance, a chlorinated paraffin (CP) mixture (>8,000 isomers) was chosen. NMR spectra were obtained from all 96 collected fractions, which provides important information for unravelling their full structure. As a proof of concept, a spectral interpretation of a few NMR spectra was made to assign sub-structures. More research is ongoing for the full characterization of CP isomers using multivariate statistical analysis. For the first time, up to only a few CP isomers per fraction were isolated from a highly complex mixture. These may be further purified and certified as standards, which are urgently needed, and can also be used for persistency, bioaccumulation, or toxicity studies.

Optimization of a low flow sampler for improved assessment of gas and particle bound exposure to chlorinated paraffins.

An optimized low volume sampler was developed to determine both gas- and particle bound concentrations of short and medium-chain chlorinated paraffins (S/MCCPs). Background contamination was limited by the sampler design, providing method quantification limits (MQLs) at least two orders of magnitude lower than other studies within the gas (MQL: 500 pg (ΣSCCPs), 1.86 ng (ΣMCCPs)) and particle (MQL: 500 pg (ΣSCCPs), 1.72 ng (ΣMCCPs) phases. Good repeatability was observed between parallel indoor measurements (RSD ≤ 9.3% (gas), RSD ≤ 14% (particle)) with no breakthrough/saturation observed after a week of continuous sampling. For indoor air sampling, SCCPs were dominant within the gas phase (17 ± 4.9 ng/m3) compared to MCCPs (2.7 ± 0.8 ng/m3) while the opposite was observed in the particle bound fraction (0.28 ± 0.11 ng/m3 (ΣSCCPs) vs. 2.7 ± 1.0 ng/m3 (ΣMCCPs)). Only SCCPs in the gas phase could be detected reliably during outdoor sampling and were considerably lower compared to indoor concentrations (0.27 ± 0.10 ng/m3). Separation of the gas and particle bound phase was found to be crucial in applying the appropriate response factors for quantification based on the deconvoluted S/MCCP sample profile, thus avoiding over- (gas phase) or underestimation (particle phase) of reported concentrations. Very short chain chlorinated paraffins (vSCCPs, C5-C9) were also detected at equal or higher abundance compared to SCCP congener groups (C10-C13) congener groups, indicating an additional human indoor inhalation risk.

In vitro biotransformation and evaluation of potential transformation products of chlorinated paraffins by high resolution accurate mass spectrometry.

Chlorinated paraffins (CPs) are high production chemicals, which leads to their ubiquitous presence in the environment. To date, few studies have measured CPs in humans and typically at relatively low concentrations, despite indications that exposure may be high compared to various persistent organic pollutants. The aim of this study is to investigate the in vitro biotransformation of CPs by human liver fractions. We determined the changes of the CP concentrations after the enzymatic transformation with human liver microsomes using a two-tiered in vitro approach. CP concentrations decreased with human liver microsomes, with the decreases of 33-94% after incubating with different groups of enzymes for 2 h. The profiles of CP rapidly shifted after the incubation with human liver microsomes. In addition, the concentrations of CPs and the biotransformation products were tentatively measured using high-resolution mass spectrometric analysis, including very short CP (carbon chain length <10), alcohols, ketones, and carboxylic acids. C‒C bond cleavage is a potential transformation pathway for CPs, and ketones are potential products of CP biotransformation, especially for long-chain CPs (C>17). The ketone products may be investigated as CP exposure biomarker in biomonitoring studies.

Spatial variation of short- and medium-chain chlorinated paraffins in ambient air across Australia.

Atmospheric levels of chlorinated paraffins (CPs) at five remote, six rural and four urban sites in Australia were measured using XAD-2 passive air samplers (XAD-PAS). While long-chain CP (LCCP, C>17) levels were below method detection limits (MDLs), short-chain CPs (SCCPs, C10-13) and, for the first time, medium-chain CPs (MCCPs, C14-17) and CPs with a carbon chain length of nine (CP-C9) were found at many sites (88%, 81% and 88%, respectively) across the Australian continent, representing a range of environmental conditions. Applying preliminary sampling rates of the XAD-PAS for CPs, gaseous CP levels in Australian air were

Evaluating age and temporal trends of chlorinated paraffins in pooled serum collected from males in Australia between 2004 and 2015.

Chlorinated paraffins (CPs) are high production volume chemicals of which some show resistance to environmental degradation, long-rang transport, bioaccumulation and toxicity potential. Information regarding their presence in humans is limited, including their human bioaccumulation potential. The present study aimed to evaluate CP levels in human serum from Australia in order to better understand their exposure and current pollution status as well as trends associated with age and time between 2004 and 2015. For this, we selected a male sub-group of the Australian population under 60 years old (n = 16 pools, total 1600 serum samples). While long-chain CP (C18-20) and most short-chain CP (C10-13, SCCPs) levels were below method detection limits (MDL), medium-chain CPs (C14-17, MCCPs) were found in most serum samples (detection frequency 94%) as well as CPs with a carbon chain length of nine (detection frequency 76%). The levels of ΣSCCPs and ΣMCCPs ranged from

First detection of short-chain chlorinated paraffins (SCCPs) in humpback whales (Megaptera novaeangliae) foraging in Antarctic waters.

Short-chain chlorinated paraffins (SCCPs) are particularly prone to environmental dispersal through long range atmospheric transport. Consequently, they have been detected in biota and environmental matrices at both the North Pole and South Pole. This study shows the first detection of SCCPs in southern hemisphere humpback whales feeding in Antarctic waters. Blubber of specimens stranded along the Australian coastline was analysed and SCCPs were detected in 7 out of 9 individuals. Levels of SCCPs detected in this study were generally low with concentrations up to only 46 ng/g lw. These results were significantly lower than those detected in Northern Hemisphere odontocetes from previous studies, although no reported burdens in northern hemisphere baleen whales are available for comparison. Both the highest level and lowest ( C13. Further investigation is needed in order to evaluate the presence and distribution of SCCPs in the remote Antarctica ecosystem, and delineate longer term environmental consequences of recent inclusion of SCCPs under Annex A of the Stockholm Convention, securing their phase out in ratifying nations.

Chlorinated paraffins in indoor dust from Australia: Levels, congener patterns and preliminary assessment of human exposure.

Chlorinated paraffins (CPs) are a group of polychlorinated n-alkanes with high production volumes. Until now, there are only limited data on the levels of CPs in the environment, especially in the indoor environment. In this study, dust samples were collected from 44 indoor environments, including 27 private houses, 10 offices, and 7 vehicles. Short-, medium-, and long-chain CPs were detected in all dust samples. The median concentration of ∑CPs (C10-C21) was 57, 160 and 290 µg/g, in houses, offices, and vehicles, respectively. Medium-chain CPs were the dominant group, on average accounting for 86% of ∑CPs. Cl6 and Cl8 groups had the highest contributions to ∑CPs across all the different microenvironments, while C13 and C14 were the predominant groups of SCCPs and MCCPs. Median exposure to ∑CPs via indoor dust were estimated at 80 ng/kg/day and 620 µg/kg/day for Australian adults and toddlers respectively. The daily intake of CPs via dust, in the worse scenario, was still 2-3 orders of magnitudes lower than the reference doses based on neoplastic effects.

Medium-Chain Chlorinated Paraffins (CPs) Dominate in Australian Sewage Sludge.

To simultaneously quantify and profile the complex mixture of short-, median-, and long-chain CPs (SCCPs, MCCPs, and LCCPs) in Australian sewage sludge, we applied and further validated a recently developed novel instrumental technique, using quadrupole time-of-flight high resolution mass spectrometry running in the negative atmospheric pressure chemical ionization mode (APCI-qTOF-HRMS). Without using an analytical column the cleaned extracts were directly injected into the qTOF-HRMS followed by quantification of the CPs by a mathematical algorithm. The recoveries of the four SCCP, MCCP and LCCP-spiked sewage sludge samples ranged from 86 to 123%. This APCI-qTOF-HRMS method is a fast and promising technique for routinely measuring SCCPs, MCCPs, and LCCPs in sewage sludge. Australian sewage sludge was dominated by MCCPs with concentrations ranging from 542 to 3645 ng/g dry weight (dw). Lower SCCPs concentrations (<57-1421 ng/g dw) were detected in the Australian sewage sludge, which were comparable with the LCCPs concentrations (116-960 ng/g dw). This is the first time that CPs were reported in Australian sewage sludge. The results of this study gives a first impression on the distribution of the SCCPs, MCCPs, and LCCPs in Australia wastewater treatment plants (WWTPs).

Chlorinated paraffins in the environment: A review on their production, fate, levels and trends between 2010 and 2015.

This review provides an update on information regarding the production volumes, regulations, as well as the environmental levels, trends, fate and human exposure to chlorinated paraffin mixtures (CPs). CPs encompas thousands congeners with varying properties and environmental fate. Based on their carbon chain lengths, CPs are divided into short- (SCCPs; C10-13), medium- (MCCPs; C14-17) and long- (LCCPs; C ≥ 18) chained groups. They are high production volume and persistent chemicals, and their cumulative global production already surpasses that of other persistent anthropogenic chemicals (e.g. PCBs). However, international regulations are still curbed by insufficient information on their levels and fate, including bioaccumulation and toxicity potential. An increasing number of studies since 2010 demonstrate that CPs are detected in almost every compartment in the environment, including remote areas. Consensus on the long range transport and high bioaccumulation potential (BCF > 5000 & TMF > 1) has recently been reached for SCCPs, fulfilling criteria under the Stockholm Convention for designation as a persistent organic pollutant; information on their levels is, however, still sparse for many countries. M/LCCPs have received comparatively little attention in the past, but as replacement chemicals for SCCPs, MCCPs are now considered in an increasing number of studies. The limited data to date suggests MCCPs are widely used. Although data on their bioaccumulation and toxicity are still inconclusive, MCCPs and LCCPs with C<20 may also have a bioaccumulation potential. Considering this and their high production volumes, use, and ubiquitous occurrence in the environment, a better understanding on the levels and fate of all CPs is needed.

Recent developments in capabilities for analysing chlorinated paraffins in environmental matrices: A review.

Concerns about the high production volumes, persistency, bioaccumulation potential and toxicity of chlorinated paraffin (CP) mixtures, especially short-chain CPs (SCCPs), are rising. However, information on their levels and fate in the environment is still insufficient, impeding international classifications and regulations. This knowledge gap is mainly due to the difficulties that arise with CP analysis, in particular the chromatographic separation within CPs and between CPs and other compounds. No fully validated routine analytical method is available yet and only semi-quantitative analysis is possible, although the number of studies reporting new and improved methods have rapidly increased since 2010. Better cleanup procedures that remove interfering compounds, and new instrumental techniques, which distinguish between medium-chain CPs (MCCPs) and SCCPs, have been developed. While gas chromatography coupled to an electron capture negative ionisation mass spectrometry (GC/ECNI-MS) remains the most commonly applied technique, novel and promising use of high resolution time of flight MS (TOF-MS) has also been reported. We expect that recent developments in high resolution TOF-MS and Orbitrap technologies will further improve the detection of CPs, including long-chain CPs (LCCPs), and the group separation and quantification of CP homologues. Also, new CP quantification methods have emerged, including the use of mathematical algorithms, multiple linear regression and principal component analysis. These quantification advancements are also reflected in considerably improved interlaboratory agreements since 2010. Analysis of lower chlorinated paraffins (

Exceptionally strong sorption of infochemicals to activated carbon reduces their bioavailability to fish.

The addition of activated carbon (AC) to sediments is a relatively new approach to remediate contaminated sites. Activated carbon strongly sorbs hydrophobic organic contaminants, thereby reducing their bioavailability and uptake in organisms. Because of its high sorption capacity, AC might, however, also sorb other chemicals that are not contaminants but instead have ecological functions. Examples of such compounds are infochemicals or pheromones (i.e., compounds serving as chemical inter- and intraspecies information vectors). The present study investigated the sorption of 2 known infochemicals, hypoxanthine-3-N-oxide (H3NO) and pyridine-N-oxide (PNO), to 5 different powdered ACs. Sorption isotherms of these low-molecular-weight, polar fish kairomone substances appeared highly nonlinear, with logarithmic Freundlich sorption coefficients of up to 7.6. At physiologically relevant concentrations, sorption was up to 7 to 9 orders of magnitude stronger than expected on the basis of hydrophobic forces only (i.e., the compounds' log octanol-water partition coefficient, being approximately -1), indicating exceptionally strong binding to specific sites. This binding effectively reduced the bioavailability of H3NO to Sarasa goldfish, as was shown in a behavioral assay. The present study demonstrates the previously unrecognized potential of AC to sorb ecologically relevant chemicals. Whether this potential may lead to subtle, unwanted ecological effects in the field will have to be investigated in more detail during future research.

Applicability of passive sampling to bioanalytical screening of bioaccumulative chemicals in marine wildlife.

Quantification of bioaccumulative contaminants in biota is time and cost-intensive and the required extensive cleanup steps make it selective toward targeted chemical groups. Therefore tissue extracts prepared for chemical analysis are not amenable to assess the combined effects of unresolved complex mixtures. Passive equilibrium sampling with polydimethylsiloxane (PDMS) has the potential for unbiased sampling of mixtures, and the PDMS extracts can be directly dosed into cell-based bioassays. The passive sampling approach was tested by exposing PDMS to lipid-rich tissue (dugong blubber; 85% lipid) spiked with a known mixture of hydrophobic contaminants (five congeners of tetra- to octachloro-dibenzo-p-dioxins). The equilibrium was attained within 24 h. Lipid-PDMS partition coefficients (Klip-PDMS) ranged from 20 to 38, were independent of hydrophobicity, and within the range of those previously measured for organochlorine compounds. To test if passive sampling can be combined with bioanalysis without the need for chemical cleanup, spiked blubber-PDMS extracts were dosed into the CAFLUX bioassay, which specifically targets dioxin-like chemicals. Small quantities of lipids coextracted by the PDMS were found to affect the kinetics in the regularly applied 24-h bioassay; however, this effect was eliminated by a longer exposure period (72 h). The validated method was applied to 11 unspiked dugong blubber samples with known (native) dioxin concentrations. These results provide the first proof of concept for linking passive sampling of lipid-rich tissue with cell-based bioassays, and could be further extended to other lipid rich species and a wider range of bioanalytical end points.