Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

Perfluoroalkyl acids (PFAAs) are a family of synthetic substances used in a wide variety of consumer and industrial applications, including non-stick and stain-resistant products. PFAAs, specifically perfluorinated sulfonates and carboxylates, are chemically stable and virtually non-biodegradable in the environment. In recent years, PFAAs have been detected in tissues and blood of humans and wildlife. Furthermore, PFAAs have a tendency to bioaccumulate and biomagnify in biota. Perfluorooctane sulfonate and perfluorooctanoate are known to be toxic when animals are exposed to environmentally-relevant levels, but scientists and regulators are challenged with determining and predicting their modes of action. There is some evidence to suggest that PFAAs can impact the thyroid hormone (TH) pathway and neurodevelopment. The studies presented in this thesis investigated the developmental effects and potential modes of action of newer PFAAs that are being introduced into the global market place. Egg injection experiments were performed in domestic chicken (Gallus gallus domesticus) embryos to assess the in ovo toxicity of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) during development. Real-time RT-PCR was used to measure the transcription of candidate genes in the liver and cerebral hemisphere of day 21-22 embryos. Candidate genes were selected based on their responsiveness to PFAA exposure in an in vitro screening assay conducted previously. In ovo exposure to PFHxS decreased embryo pipping success and overall growth at 38,000 ng/g; several orders of magnitude higher than concentrations reported in wild bird eggs. The expression of TH-responsive genes, including type II and III 5'-deiodinase, neurogranin, and octamer motif binding factor 1, were induced. In addition, PFHxS diminished free thyroxine (T4) levels in plasma. PFHxA had no affect on pipping success, gene expression or T4 levels in chicken embryos at the doses assessed. The transcriptional profiles in the cerebral hemisphere of chicken embryos exposed to 890 and 38,000 ng/g PFHxS were compared to a solvent control using microarray technology. The expression of 78 different genes were significantly altered (fold change > 1.5, p < 0.001) by PFHxS. Functional analysis showed that PFHxS affected genes involved in tissue development and morphology and cellular assembly and organization. Pathway and interactome analysis suggested that gene expression may be affected through integrin receptors and signaling pathways via TH–dependent and –independent modes of action. It is expected that the findings presented in this thesis will be of general relevance and importance to regulatory agencies and of interest to research scientists and risk assessors.

<div> <p>In recent years, the presence of per- and polyfluoroalkyl substances (PFAS) in aquatic systems has led to research on their fate, effects and treatability. PFAS have been found in various environmental matrices including wastewater effluents, surface, ground, and drinking water. Perfluoroalkyl acids (PFAAs) are the class of PFAS most commonly tested due to their ability to migrate rapidly through groundwater and include perfluoroalkyl sulfonic acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs). Of the globally distributed and persistent PFAAs, PFSAs are the most resistant to biological and oxidative chemical attack. This doctoral study focused on a reductive treatment approach with zero valent metals/bimetals nanoparticles (NPs) synthesized onto a carbon material to reduce NP aggregation. Initial work focused on exploring reactivity of different combinations of nano (n) Ni, nFe⁰ and activated carbon (AC) at 22 ^oC to 60 ^oC for transforming perfluorooctanesulfonate (PFOS) from which nNiFe⁰-AC at 60 ^oC led to transformation of both linear (L-) and branched (Br-) PFOS isomers. The remaining research focused on work with nNiFe⁰-AC at 60 ^oC in batch reactors including optimizing nNiFe⁰-AC preparation, quantifying PFOS transformation kinetics and evaluating the effects of PFAA chain length (C4, C6 and C8) and polar head group (PFSA versus PFCA) as well a groundwater matrix on transformation magnitude. Optimization of analytical methods to provide multiple lines of evidence of transformation including fluoride, sulfite and organic product generation was an ongoing throughout the research.</p> <p>nNiFe⁰-AC prepared with a 3-h synthesis stirring time led to the highest PFOS transformation of 51.1 ± 2.1% with generation of ~ 1 mole of sulfite (measured as sulfate) and 12 moles of fluoride. Several poly/per-fluorinated intermediates with single and double bonds were identified using quadrupole time-of-flight mass spectrometry (QToF-MS) in negative electrospray ionization (ESI-) mode with MS/MS fragmentation confirmation as well as one and later two desulfonated products with QToF negative atmospheric pressure chemical ionization (APCI-). All organic transformation products were found in only particle extracts as well as most of the sulfite generated. PFOS transformation kinetics showed that generated fluoride concentrations increased for the first day whereas sulfate concentrations continued to increase during the 5-d reaction. The transformation products identified showed defluorination of single- and double-bond structures, formation of C8 to C4 PFCAs and paraffins from cleavage of the C-S bond.</p> <p>The length of the perfluoroalkyl chain affected the length of time to achieve peak removal, but overall magnitude of transformation when reactions appeared complete were similar for both PFSAs and PFCAs. Like PFOS, PFOA transformation maxed in 1 d whereas shorter chains required more time to reach their peak removal, which is hypothesized to be due to lower sorption of the shorter chain PFAAs to the reactive surfaces. Measured F mass balance was higher for PFOS and PFOA (>90% F) compared to shorter chain PFAAs (~50-70% F). The Perfluorohexanesulfonate (PFHxS) and perfluorobutanesulfonate (PFBS) degradation products include single bond polyfluoroalkyl sulfonates and shorter-chain perfluoroalkyl carboxylates. For example, PFHxS transformation resulted in perfluorohexane carboxylic acid (PFHxA) and perfluorobutane carboxylic acid (PFBA). PFCA transformation products included per- & polyfluoroalkyl carboxylates with single bonds and alcohols with single and double bonds. The effect of inorganic matrix on transformation with nNiFe⁰-AC at 60 ^oC was explored using a contaminated groundwater collected at a former fire-training area in Massachusetts. Transformation appeared ‘generally’ lower than in the single-solute clean water systems, which may have been due to the presence of PFAS precursors that degraded to PFAAs and competitive adsorption between anionic PFAAs and inorganic ions onto the NP surface.</p><p>The research presented here demonstrates that nNiFe⁰-AC at 60 ^oC can mineralize PFAAs even in a typical groundwater matrix. Additional lab and pilot scale studies are needed to clarify the mechanisms leading to transformation as well as why transformation reactions plateau prior to all the parent compounds being transformed. The latter may be due to a poisoning phenomenon that can occur in closed systems, which may not occur in a flowing system more characteristic of an environmental scenario, as well as surface area and reactive site constraints or particle passivation.</p></div>

The persistent and toxic perfluoroalkyl acids (PFAAs) are ubiquitously present in the environment and reach humans predominantly via food and drinking water. The aim of the present study was to investigate the effect of low-grade (&lt;10 ng L-1 of single PFAAs) contaminated PFAAs drinking water on serum PFAA concentrations in a representative Swedish adolescent population, and to examine the influence of potential determinants on the variation of the PFAAs serum concentrations. This was done by using multivariate regression analysis on the possible determinants of blood serum PFAA concentrations in 479 Swedish adolescents, 10 to 21 years of age, who had left complete dietary and life style information in 2016-17 in the nation-wide food consumption survey Riksmaten Ungdom. Raw and drinking water samples (DW) from water treatment plants (WTPs) that delivered DW to participants schools were sampled in 2018, analysed for PFAAs, and used for assessing the participants DW PFAA exposure. Maternal education level and maternal birth country, consumption of fish, as well as age and sex were significantly associated with the participants PFAAs serum concentrations. DW concentrations as low as &lt;1 ng L for PFOA and PFHxS, &lt;0.45 ng L-1 for PFNA and &lt;4 ng L-1 for PFOS were significantly associated with increased adolescent serum concentrations of the PFAAs in question, which suggests that low-grade contaminated drinking water is an important exposure route for Swedish adolescents. For risk assessment purposes, it was investigated whether parts of the adolescent population exceeded the serum PFOS and PFOA concentrations corresponding to the current health-based reference intakes as assessed by the European Food Safety Authority (EFSA) and the U.S. Agency for Toxic Substances and Disease Registry (ATSDR). Around 1.7% and 2.7% of participants had PFOS serum concentrations exceeding serum levels used to derive the tolerable daily intake (TDI) (EFSA) and the minimum risk level (MRL) (ATSDR), respectively and a cause for concern was consequently identified. However, the high serum concentrations of participants exceeding the TDI and MRL serum concentrations belong to participants suspected to have been previously exposed to highly contaminated drinking water and not from consuming foods and beverages containing background concentrations of PFAAs.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22058号 / 工博第4639号 / 新制||工||1723(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 清水 芳久, 教授 高野 裕久 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Atmospheric deposition has been suggested to be a major input pathway of perfluoroalkyl acids (PFAAs) to the Baltic Sea catchment area and to the Baltic Sea itself. However, the sources of PFAAs to the atmosphere are not well characterized. In this study we investigated if waste incineration plants in Sweden could be a source of PFAAs to the atmosphere and to the environment in general. Samples of the end products from waste incineration were collected at four different incineration plants. The plants differed in size and technical advancement and were considered to be representative for the majority of waste incineration plants in Sweden. The collected samples were slag from the furnaces, fly ash from the flue gases, “bambergkaka” (a mix of fly ash and sludge from wastewater treatment) as well as condensate and wastewater from the cleaning process of the flue gases. Two methods were developed, one for analysis of PFAAs in solid samples and one for water samples. Method validation showed good performance for both methods in terms of precision and accuracy, despite low recoveries obtained for the method for solid samples. The results from sample analysis revealed that PFAAs were present in all solid samples at concentrations in the low to sub ng/g range and in all but one condensate and wastewater samples at concentrations in the low to sub ng/L range. The quantified concentrations were used to estimate the potential annual discharges of PFAAs from waste incineration plants to the environment. Emission scenarios via landfills, via wastewater treatment plants and to the atmosphere were considered. The main conclusion of this study is that waste incineration in Sweden is not a significant source of PFAAs to the atmosphere or to the environment in general.

Per- and polyfluoroalkyl substances (PFASs) are persistent organic pollutants used in industrial applications and are globally distributed in the environment. A group of PFASs that are difficult to measure with today’s method are perfluoroalkyl acid precursors (PFAA precursors) that, when degraded, serves as indirect sources of PFAAs. This study has optimized a previously developed method for quantification of PFAA precursors in soil; through total oxidizable precursor assay (TOP assay) under alkaline conditions, to be applicable on sewage sludge. To achieve and maintain an alkaline environment during the entire oxidative treatment, several parameters were tested: concentrations of NaOH, persulfate and sample; additional clean-up with graphitized non-porous carbon and reaction time. Solid phase extraction-weak anion exchange (SPE-WAX) was used for clean-up and separation of analytes, and LC-MS/MS was used for quantification. The optimal conditions with the highest levels of PFAAs detected was obtained with 1.33 M NaOH, 60 mM persulfate, 3.57 g/L sludge with a reaction time of 6 hours. The use of graphitized non-porous carbon reduced matrix effects on oxidative conversion resulting in a higher pH as well as a higher degree of oxidation, but with some analyte loss.

Background In some cross-sectional epidemiologic studies the shape of the association between serum concentrations of perfluoroalkyl acids (PFAAs) and lipids suggests departures from linearity. Objectives We used statistical approaches allowing for non-linearity to determine associations of prenatal exposures of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) with lipid concentrations. Methods PFAAs were measured in serum from pregnant women collected in 1991–1992 at enrollment in the Avon Longitudinal Study of Parents and Children and lipids in serum from their daughters at ages 7 (n = 111) and 15 (n = 88). The associations of PFAAs with lipids were first explored by cubic splines, followed by piecewise linear regressions by tertiles to obtain regression coefficients (β) and their 95% confidence limits (95% CL) (in mg/dL per 1 ng/mL). Results At age 7, total cholesterol was positively associated with prenatal PFOA concentrations in the lower tertile (β = 15.01; 95% CL = 2.34, 27.69) but not with PFOA concentrations in the middle (β = − 3.63; 95% CL = − 17.43, 10.16) and upper (β = − 1.58; 95% CL = − 4.58, 1.42) tertiles. At age 15, a similar pattern was noted as well. Positive associations between LDL-C and prenatal PFOA concentration in the lower tertile were observed in daughters at ages 7 (β = 14.91; 95% CL = 3.53, 28.12) and 15 (β = 13.93; 95% CL = 0.60, 27.26). LDL-C was not associated with PFOA concentrations in the middle or upper tertile at any age. Neither HDL-C nor triglycerides was associated with prenatal PFOA exposure. Non-linear patterns of association of total cholesterol and LDL-C with prenatal PFOS were less consistently noted. Conclusion Exposure to low levels of PFOA during prenatal development may alter lipid metabolism later in life. Given the small sample size further replication of the association in large independent cohorts is important.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.