FTOH Biotransformation Research Articles

Assessing aerobic biotransformation of 8:2 fluorotelomer alcohol in aqueous film-forming foam (AFFF)-impacted soils: Pathways and microbial community dynamics

Production of 8:2 fluorotelomer alcohol (8:2 FTOH) for industrial and consumer products, including aqueous film-forming foams (AFFFs) used for firefighting, has resulted in its widespread occurrence in the environment. However, the fate of 8:2 FTOH at AFFF-impacted sites remains largely unknown. Using AFFF-impacted soils from two United States Air Force Bases, microcosm experiments evaluated the aerobic biotransformation of 8:2 FTOH (extent and byproduct formation) and the dose-response on microbial communities due to 8:2 FTOH exposure. Despite different microbial communities, rapid transformation of 8:2 FTOH was observed during a 90-day incubation in the two soils, and 7:2 secondary fluorotelomer alcohol (7:2 sFTOH) and perfluorooctanoic acid (PFOA) were detected as major transformation products. Novel transformation products, including perfluoroalkane-like compounds (1H-perfluoroheptane, 1H-perfluorohexane, and perfluoroheptanal) were identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and used to develop aerobic 8:2 FTOH biotransformation pathways. Microbial community analysis suggests that species from genus Sphingomonas are potential 8:2 FTOH degraders based on increased abundance in both soils after exposure, and the genus Afipia may be more tolerant to and/or involved in the transformation of 8:2 FTOH at elevated concentrations. These findings demonstrate the potential role of biological processes on PFAS fate at AFFF-impacted sites through fluorotelomer biotransformation.

Biotransformation of 8:2 Fluorotelomer Alcohol in Soil from Aqueous Film-Forming Foams (AFFFs)-Impacted Sites under Nitrate-, Sulfate-, and Iron-Reducing Conditions

The environmental fate of per- and polyfluoroalkyl substances (PFAS) in aqueous film-forming foams (AFFFs) remains largely unknown, especially under the conditions representative of natural subsurface systems. In this study, the biotransformation of 8:2 fluorotelomer alcohol (8:2 FTOH), a component of new-generation AFFF formulations and a byproduct in fluorotelomer-based AFFFs, was investigated under nitrate-, iron-, and sulfate-reducing conditions in microcosms prepared with AFFF-impacted soils. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (HRMS) were employed to identify biotransformation products. The biotransformation was much slower under sulfate- and iron-reducing conditions with >60 mol % of initial 8:2 FTOH remaining after ∼400 days compared to a half-life ranging from 12.5 to 36.5 days under nitrate-reducing conditions. Transformation products 8:2 fluorotelomer saturated and unsaturated carboxylic acids (8:2 FTCA and 8:2 FTUA) were detected under all redox conditions, while 7:2 secondary fluorotelomer alcohol (7:2 sFTOH) and perfluorooctanoic acid (PFOA) were only observed as transformation products under nitrate-reducing conditions. In addition, 1H-perfluoroheptane (F(CF2)6CF2H) and 3-F-7:3 acid (F(CF2)7CFHCH2COOH) were identified for the first time during 8:2 FTOH biotransformation. Comprehensive biotransformation pathways for 8:2 FTOH are presented, which highlight the importance of accounting for redox condition and the related microbial community in the assessment of PFAS transformations in natural environments.

Biotransformation and tissue bioaccumulation of 8:2 fluorotelomer alcohol in broiler by oral exposure

In order to examine whether 8:2 FTOH exposure would lead to a contamination risk of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in broiler derived food, the biotransformation, and tissue distribution and accumulation of 8:2 FTOH following oral exposure in male broilers were investigated. The main metabolites of 8:2 FTOH in plasma and six tissues (muscle, liver, kidney, fat, heart, and lungs) identified by LC-Q-TOF were 2-perfluorooctyl ethanoic acid (8:2 FTCA), 8:2 fluorotelomer unsaturated carboxylic acid (8:2 FTUCA), 3-perfluoroheptyl propanoic acid (7:3 FTCA), perfluoropentanoic acid (PFPeA), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), 8:2 FTOH glucuronide conjugate, and 8:2 FTOH sulfate conjugate. The tissue distribution and bioaccumulation of 8:2 FTOH and its unconjugated metabolites were determinated by LC-MS/MS. 8:2 FTOH was quickly depleted in plasma and all six tested tissues, while PFOA, PFNA, and 7:3 FTCA showed strong accumulation in blood and all six examined tissues and were eliminated more slowly than the other metabolites. The tissues with the highest accumulation levels for 8:2 FTOH and its metabolites were heart, kidneys and liver, and the tissue with the lowest accumulation levels was muscle. The elimination half-lifes of PFNA in kidney and 7:3 FTCA in lung were longer compared to those of other metabolites in all six determined tissues. Thus, PFNA and 7:3 FTCA can be selected as potential biomonitoring markers after 8:2 FTOH exposure. This study has improved our understanding of 8:2 FTOH biotransformation and tissue bioaccumulation in broilers, which will help us monitor human exposure risk via food derived from broilers polluted by 8:2 FTOH.

Fungal biotransformation of 6:2 fluorotelomer alcohol

AbstractFungal degradation of 6:2 fluorotelomer alcohol (6:2 FTOH, C6F13CH2CH2OH) by two wood‐decaying fungal strains and six fungal isolates from a site contaminated with per‐ and polyfluoroalkyl substances (PFASs) was investigated. 6:2 FTOH is increasingly being used in FTOH‐based products, and previous reports on the microbial fate of 6:2 FTOH have focused on bacteria and environmental microbial consortia. Prior to this study, one report demonstrated that the 6:2 FTOH biotransformation by the wood‐decaying fungus, Phanerochaete chrysosporium, generated more polyfluoroalkyl substances, such as 5:3 acid (F(CF2)5CH2CH2COOH), and diverted away from producing the highly stable perfluorocarboxylic acids (PFCAs). Most of the fungi (Gloeophyllum trabeum and isolates TW4‐2, TW4‐1, B79, and B76) examined in this study showed similar degradation patterns, further demonstrating that fungi yield more 5:3 acid (up to 51 mol% of initial 6:2 FTOH dosed) relative to other metabolites (up to 12 mol% total PFCAs). However, medium amendments can potentially improve 6:2 FTOH biotransformation rates and product profiles. The six fungal isolates tolerated up to 100 or 1,000 milligrams per liter of perfluorooctanoic acid and perfluorooctane sulfonic acid, and some isolates experienced increased growth with increasing concentrations. This study proposes that fungal pathways must be considered for the biotransformation of potential PFAS precursors, such as 6:2 FTOH, and suggests the basis for selecting proper microorganisms for remediation of fluoroalkyl‐contaminated sites.

Impact of 6:2 fluorotelomer alcohol aerobic biotransformation on a sediment microbial community

Sediment microbial communities are responsible for many chemical biotransformation processes in the aquatic environment and play a critical role in various ecosystems and biogeochemical cycling. However, the impact of polyfluoroalkyl substances on sediment microbial communities remains unclear. These substances are increasingly being used in consumer and industrial products to replace environmentally persistent perfluoroalkyl substances. In this study, we investigated the effects of low (5mg/L) and high (15mg/L) doses of 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] on the structure of a sediment microbial community. 6:2 FTOH biotransformation was rapid in the sediment mixture with a half-life <3days, regardless of the initial doses. After 28days, major products produced in the high dose condition included 28mol% 5:2 sFTOH [F(CF2)5CH(OH)CH3], 9.6mol% 5:3 Acid [F(CF2)5CH2CH2COOH] and 11mol% PFHxA [F(CF2)5COOH], while 73mol% 5:2 sFTOH, 23mol% 5:3 Acid and 26mol% PFHxA were observed in the low dose condition. In the original (control) sediment without 6:2 FTOH dosing, Proteobacteria was the predominant microorganism (18%), followed by Chloroflexi (14%), Verrucomicrobia (13%), Firmicutes (3.4%), Bacterioidetes (2.4%), Actinobacteria (1.7%) and Planctomycetes (1.3%). The presence of 6:2 FTOH and the accumulation of transient transformation products in the sediment exerted selection pressure on the microbial taxonomic distribution and diversity. Our observations indicate that potential 6:2 FTOH degraders and tolerant strains, such as Dokdonella spp., Thauera spp., Albidovulum spp. and Caldanaerovirga spp., existed in the sediment mixture and began to dominate over time. This suggests that these genera might have higher tolerance towards elevated 6:2 FTOH and its transformation products. These findings on the characterization of sediment microbial community stability and dynamics will help predict changes in response to perfluoroalkyl and polyfluoroalkyl substances and also help identify robust microbial strains to degrade polyfluoroalkyl substances in the environment.

Engineering artificial communities for enhanced FTOH degradation

Fluorotelomer alcohols (FTOHs, [F(CF2)nCH2CH2OH]) are concerned environmental pollutants with perfluorinated carbon chains. FTOHs can be biotransformed; however, the extent, the pace of the defluorination, and types of metabolites produced vary depending on degradative microorganisms under different environment. In this study, we examined ways to increase the effectiveness of the FTOH defluorination process to less persistent major metabolites. Defined mixed cultures and bioaugmented microbial cultures were engineered to study their ability to biotransform 6:2 fluorotelomer alcohol [F(CF2)6CH2CH2OH]. The effects of carbon sources and the concentration of carbon sources were also examined. All experiments resulted in 5:2 sFTOH [F(CF2)5CH(OH)CH3] as the primary metabolite at the end point. The carbon sources resulted in different amounts of pathway utilization as well as overall changes in effectiveness. The best overall effectiveness was observed when cosubstrate carbon was kept at low concentrations. Pathway II was best utilized by the P. butanovora+P. fluorescens mixed culture, with lactate having a slight negative impact on pathway II utilization. Additional carbon to augmented activated sludge resulted in decreased 6:2 FTOH biotransformation by 60%. Enrichment cultures showed that shorter chain FTOHs are easier to degrade, with the n-octane enriched culture transforming 20% of 8:2 FTOH, 60% of 6:2 FTOH and 70% of 4:2 FTOH. The microbial communities of the enrichment cultures and the alkane hydroxylase gene were also examined to help understand FTOH biotransformation mechanisms.

6:2 Fluorotelomer iodide in vitro metabolism by rat liver microsomes: Comparison with [1,2-14C] 6:2 fluorotelomer alcohol

6:2 Fluorotelomer iodide [6:2 FTI, F(CF2)6CH2CH2I] is the industrial raw material used to manufacture 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] and 6:2 FTOH-based products. During its manufacture and industrial use, workers may be exposed to via oral, dermal or inhalation of 6:2 FTI. Therefore it is useful to understand how 6:2 FTI may be metabolized and into what transformation products. 6:2 FTI in vitro rat liver microsomal metabolism was explored for the first time to compare its biotransformation potential with that of [1,2-14C] 6:2 FTOH [F(CF2)614CH214CH2OH]. 6:2 FTI and 6:2 FTOH metabolite yields were determined in closed-bottle systems using Sprague Dawley and Wistar Han rat microsomes after incubation at 37°C for up to 6h with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-addition and NADPH-regenerating systems, respectively. 5:3 acid [F(CF2)5CH2CH2COOH] was the most abundant metabolite for 6:2 FTI (3.3–6.3mol%) and 6:2 FTOH (9–12mol%). Perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), and perfluorohexanoic acid (PFHxA) in sum accounted for 1.3–2.2mol% from 6:2 FTI and 2.7–4.4mol% from 6:2 FTOH biotransformation. Perfluoroheptanoic acid (PFHpA) accounted for 0.14–0.36mol% from 6:2 FTI but only 0.01–0.06mol% from 6:2 FTOH biotransformation. These results suggest that mammalian systems exposed to 6:2 FTI or 6:2 FTOH would form 5:3 acid, PFBA, PFPeA, PFHxA as the primary stable metabolites, whereas more PFHpA would be expected from 6:2 FTI biotransformation.

Biotransformation of 6:2 Fluorotelomer Alcohol (6:2 FTOH) by a Wood-Rotting Fungus

Biotransformation of 6:2 FTOH [F(CF2)6CH2CH2OH] by the white-rot fungus, Phanerochaete chrysosporium, was investigated in laboratory studies. 6:2 FTOH is a raw material increasingly being used to replace products that can lead to long-chain perfluoroalkyl carboxylic acids (PFCAs, ≥ 8 carbons). During a product's life cycle and after final disposal, 6:2 FTOH-derived compounds may be released into the environment and potentially biotransformed. In this study, P. chrysosporium transformed 6:2 FTOH to perfluorocarboxylic acids (PFCAs), polyfluorocarboxylic acids, and transient intermediates within 28 days. 5:3 Acid [F(CF2)5CH2CH2COOH] was the most abundant transformation product, accounting for 32-43 mol % of initially applied 6:2 FTOH in cultures supplemented with lignocellulosic powder, yeast extract, cellulose, and glucose. PFCAs, including perfluoropentanoic (PFPeA) and perfluorohexanoic (PFHxA) acids, accounted for 5.9 mol % after 28-day incubation. Furthermore, four new transformation products as 6:2 FTOH conjugates or 5:3 acid analogues were structurally confirmed. These results demonstrate that P. chrysosporium has the necessary biochemical mechanisms to drive 6:2 FTOH biotransformation pathways toward more degradable polyfluoroalkylcarboxylic acids, such as 5:3 acid, with lower PFCA yields compared to aerobic soil, sludge, and microbial consortia. Since bacteria and fungi appear to contribute differently toward the environmental loading of FTOH-derived PFCAs and polyfluorocarboxylic acids, wood-rotting fungi should be evaluated as potential candidates for the bioremediation of wastewater and groundwater contaminated with fluoroalkyl substances.

6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions

Factors affecting microbial aerobic biodegradation of 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF₂)₆CH₂CH₂OH] were investigated using three alkane-degrading bacteria (Mycobacterium vaccae JOB5, Pseudomonas oleovorans, and Pseudomonas butanovora) and one fluoroacetate-degrading bacterium (Pseudomonas fluorescens DSM 8341). In the presence of formate (an external reducing energy source), P. fluorescens DSM 8341 produced perfluorobutanoic acid by removing three -CF₂- groups from 6:2 FTOH. Only P. fluorescens DSM 8341 transformed 5:3 acid to 4:3 acid and perfluoropentanoic acid. However, formate showed no effects on the degradation rates, patterns, or transformation products of 6:2 FTOH by M. vaccae JOB5. When dicyclopropylketone (an alkane hydroxylase inducer) or formate was added, P. oleovorans rapidly degraded 6:2 FTOH and produced PFPeA. In the presence of lactate, P. butanovora degraded 6:2 FTOH slowly but produced diverse metabolites. Our results demonstrate that the extent and mechanisms of 6:2 FTOH biotransformation are affected by strain types, enzyme inducers, and levels of reducing energy.

6:2 Fluorotelomer alcohol aerobic biotransformation in activated sludge from two domestic wastewater treatment plants

6:2 Fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] is a major basic chemical being used to manufacture FTOH-based products. After the end of use, 6:2 FTOH-based products may be released to domestic wastewater treatment plants (WWTPs) as a first major environmental entry point. Activated sludge collected from two WWTPs was dosed with 6:2 FTOH to investigate its biotransformation rate and to identify major transformation products. The volatile 5:2 sFTOH [F(CF2)5CH(OH)CH3] is the most abundant transformation product and accounted for an average of 40mol% of initially dosed 6:2 FTOH after two months of incubation with activated sludge, with 30mol% detected in the headspace. PFPeA [F(CF2)4COOH] averaged 4.4mol% after two months, 2.4–7 times lower than that in sediment and soils. The much lower level of PFPeA formed in activated sludge compared with soil indicates that microbial populations in activated sludge may lack enzymes or suitable environment conditions to promote rapid 5:2 sFTOH decarboxylation to form PFPeA, resulting in more 5:2 sFTOH partitioned to the headspace. PFHxA [F(CF2)5COOH] and 5:3 [F(CF2)5CH2CH2COOH] acid are major non-volatile transformation products in activated sludge. For example, PFHxA averaged 11mol% after two months, which is about 30% higher compared with sediment and soils, suggesting that microbes in WWTPs may utilize similar pathways as that in sediment and soils to convert 5:2 sFTOH to PFHxA. 5:3 Acid averaged 14mol% after two months, comparable to that in soils and slightly lower than in sediment, further confirming that 5:3 acid is a unique product of 6:2 FTOH biotransformation in the environment.

6:2 Fluorotelomer alcohol biotransformation in an aerobic river sediment system

The 6:2 FTOH [F(CF2)6CH2CH2OH] is a major raw material being used to replace 8:2 FTOH [F(CF2)8CH2CH2OH] to make FTOH-based products for industrial and consumer applications. A novel aerobic sediment experimental system containing 20g wet sediment and 30mL aqueous solution was developed to study 6:2 FTOH biotransformation in river sediment. 6:2 FTOH was dosed into the sediment to follow its biotransformation and to analyze transformation products over 100d. The primary 6:2 FTOH biotransformation in the aerobic sediment system was rapid (T1/2<2d). 5:3 acid [F(CF2)5CH2CH2COOH] was observed as the predominant polyfluorinated acid on day 100 (22.4mol%), higher than the sum of perfluoropentanoic acid (10.4mol%), perfluorohexanoic acid (8.4mol%), and perfluorobutanoic acid (1.5mol%). Perfluoroheptanoic acid was not observed during 6:2 FTOH biotransformation. The 5:3 acid can be further degraded to 4:3 acid [F(CF2)4CH2CH2COOH, 2.7mol%]. This suggests that microbes in the river sediment selectively degraded 6:2 FTOH more toward 5:3 and 4:3 acids compared with soil. Most of the observed 5:3 acid formed bound residues with sediment organic components and can only be quantitatively recovered by post-treatment with NaOH and ENVI-Carb™ carbon. The 6:2 FTCA [F(CF2)6CH2COOH], 6:2 FTUCA [F(CF2)5CF=CHCOOH], 5:2 ketone [F(CF2)5C(O)CH3], and 5:2 sFTOH [F(CF2)5CH(OH)CH3] were major transient intermediates during 6:2 FTOH biotransformation in the sediment system. These results suggest that if 6:2 FTOH or 6:2 FTOH-based materials were released to the river or marine sediment, poly- and per-fluorinated carboxylates could be produced.

6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants

The aerobic biotransformation of 6:2 FTS salt [F(CF 2) 6CH 2CH 2 SO 3 - K +] was determined in closed bottles for 90 d in diluted activated sludge from three waste water treatment plants (WWTPs) to compare its biotransformation potential with that of 6:2 FTOH [F(CF 2) 6CH 2CH 2OH]. The 6:2 FTS biotransformation was relatively slow, with 63.7% remaining at day 90 and all observed transformation products together accounting for 6.3% of the initial 6:2 FTS applied. The overall mass balance (6:2 FTS plus observed transformation products) at day 90 in live and sterile treatments averaged 70% and 94%, respectively. At day 90, the stable transformation products observed were 5:3 acid [F(CF 2) 5CH 2CH 2COOH, 0.12%], PFBA [F(CF 2) 3COOH, 0.14%], PFPeA [F(CF 2) 4COOH, 1.5%], and PFHxA [F(CF 2) 5COOH 1.1%]. In addition, 5:2 ketone [F(CF 2) 5C(O)CH 3] and 5:2 sFTOH [F(CF 2) 5CH(OH)CH 3] together accounted for 3.4% at day 90. The yield of all the stable transformation products noted above (2.9%) was 19 times lower than that of 6:2 FTOH in aerobic soil. Thus 6:2 FTS is not likely to be a major source of PFCAs and polyfluorinated acids in WWTPs. 6:2 FTOH, 6:2 FTA [F(CF 2) 6CH 2COOH], and PFHpA [F(CF 2) 6COOH] were not observed during the 90-d incubation. 6:2 FTS primary biotransformation bypassed 6:2 FTOH to form 6:2 FTUA [F(CF 2) 5CF = CHCOOH], which was subsequently degraded via pathways similar to 6:2 FTOH biotransformation. A substantial fraction of initially dosed 6:2 FTS (24%) may be irreversibly bound to diluted activated sludge catalyzed by microbial enzymes. The relatively slow 6:2 FTS degradation in activated sludge may be due to microbial aerobic de-sulfonation of 6:2 FTS, required for 6:2 FTS further biotransformation, being a rate-limiting step in microorganisms of activated sludge in WWTPs.

Elucidating the Pathways of Poly- and Perfluorinated Acid Formation in Rainbow Trout

Several studies have shown that fluorotelomer-based compounds can be metabolized to poly- and perfluorinated carboxylates, such as perfluorooctanoate (PFOA). Research has predominately focused on the 8:2 fluorotelomer alcohol (8:2 FTOH), however, the biotransformation pathway is not well understood. Specifically, there is uncertainty regarding the biological fate of the 8:2 fluorotelomer unsaturated carboxylate (FTUCA) and 7:3 fluorotelomer saturated carboxylate (FTCA). The objective of this study was to further elucidate the pathway for 8:2 FTOH biotransformation through dosing rainbow trout with three 8:2 FTOH metabolism intermediates: the 7:3 FTCA (CF(3)(CF(2))(6)CH(2)CH(2)COO(-)), 8:2 FTCA (CF(3)(CF(2))(7)CH(2)COO(-)), and 8:2 FTUCA (CF(3)(CF(2))(6)CF horizontal lineCHCOO(-)). This study represents the first investigation of these three labile intermediate metabolites in an in vivo system. The parent compounds were dosed via the diet and the parent compounds and intermediates were monitored in the blood and liver during the 7-day uptake phase and 10-day elimination phase. Exposure to the 7:3 FTCA did not result in the formation and accumulation of PFOA, but resulted in low levels of the 7:3 FTUCA and perfluoroheptanoate, a novel finding. PFOA was formed in the 8:2 FTCA and 8:2 FTUCA dosing. In addition, the 7:3 FTCA was formed during exposure to both the 8:2 FTCA and 8:2 FTUCA. Elimination half-lives were 5.1 d (95% confidence interval: 3.1-14 d) for 7:3 FTCA, 1.2 d (1.1-1.3 d) for 8:2 FTCA, and 0.39 d (0.31-0.53 d) for 8:2 FTUCA. The observed differences in the elimination half-life may be the result of differences in either the depuration or metabolism rate. Based on the findings of this study, and reported analogous literature pathways, we proposed a "beta-like-oxidation" pathway for PFOA formation proceeding from the 8:2 FTUCA > 7:3 beta-keto acid > 7:2 ketone > PFOA. Alternatively PFOA could be formed directly through the beta-oxidation of the 7:3 beta-keto acid.

Bioactivation of fluorotelomer alcohols in isolated rat hepatocytes

Fluorotelomer alcohols (FTOHs; C x F 2 x+1 C 2H 4OH) are intermediates in the production of specialty surfactants and stain-repellent polymers. The magnitude and pathways of human exposure to FTOHs are not understood, but FTOHs are present in ambient air and house dust, and FTOH-derivatives are used in food-contact applications. Previously, electrophilic FTOH biotransformation products were detected in rat hepatocytes, and liver lesions were found in FTOH exposed rodents. To begin elucidating the mechanism(s) of action, freshly isolated rat hepatocytes were incubated with FTOHs, or FTOH biotransformation products, and toxicity was followed in the presence or absence of carbonyl scavengers and metabolic enzyme modulators. The LC 50 depended on perfluorinated chain length, with the shortest (4:2 FTOH; x = 4) and longest (8:2 FTOH; x = 8) FTOHs tested being more toxic than the medium chain length FTOH (6:2 FTOH; x = 6); a structure–toxicity relationship that is consistent with that for 2-alkenals. For hepatocytes treated with 8:2 FTOH, cytotoxicity corresponded to depletion of glutathione (GSH), increased protein carbonylation, and lipid peroxidation. Aminobenzotriazole, a P450 inhibitor, diminished cytotoxicity for all FTOHs tested, and decreased protein carbonylation and lipid peroxidation for 8:2 FTOH, indicating that a biotransformation product was responsible for FTOH cytotoxicity. Preincubation of hepatocytes with hydralazine or aminoguanidine decreased the cytotoxicity of 8:2 FTOH, suggesting that reactive aldehyde intermediates contributed to the cytotoxicity. A GSH-reactive α/β-unsaturated acid metabolite was also more toxic than the corresponding FTOH, and may have contributed to the observed effects. Overall, these results suggested that FTOH toxicity was related to electrophilic aldehydes or acids through GSH depletion and protein carbonylation. Further research into the nature of protein modification is warranted for these current-use fluorochemicals.