Firefighters are routinely exposed to per- and polyfluoroalkyl substances (PFAS) through the use of Aqueous Film-Forming Foams (AFFFs) for the suppression of Class B fire, which derive from flammable and combustible liquids, such as gasoline and alcohol. The addition of surfactants and PFAS in the AFFFs allows them to form an aqueous film that can extinguish the fire, while also coating the fuel. As such, AFFFs are often used for fire extinction in airports and military bases. Exposure to PFAS in the general population may arise from ingestion of contaminated food or water, usage of consumer products containing PFAS, such as non-stick cookware or stainresistant carpets and textiles, and inhalation of PFAS-containing particulate matter.1 Detection of increased serum PFAS concentrations has been linked to an elevated risk for kidney cancer in humans,2 and firefighters are known to have increased serum concentrations of certain PFAS after attending training exercises.3 In the same study it was also observed that the average urinary excretions of 2-butoxyacetic acid (2-BAA) a surfactant often added in AFFFs exceeded the reference limit of the occupationally unexposed population, ranging from 0.5 to 1.4 mmol/mol creatinine. Furthermore, an increased risk of mortality from kidney cancer has been observed in firefighters compared to the U.S. population.4 The detrimental health effects of PFAS are exacerbated by their increased half-lives in humans. A recently published study examined the half-lives of short- and long- chained PFAS in the serum of 26 airport employees and observed a wide range of half-lives which was dependent on the length and chemical structure of each substance that was examined. Indicatively, the shortest half-life was described for perfluorobutanesulfonic acid (PFBS), while the linear isomer of perfluorooctanesulfonic acid (PFOS) had the longest half-life (average of 44 days and 2.93 years, respectively), findings which are in agreement with other sources in the literature.5 One aspect of this phenomenon could be attributed to renal reabsorption, as humans actively transport PFAS in the proximal tubules.6,7 A recently published scoping review of 74 epidemiologic, pharmacokinetic, and toxicological studies examined the relationship between PFAS exposure and kidney-related health outcomes. It was observed that exposure to PFAS was associated with lower kidney function, including chronic kidney disease (CKD), and histological abnormalities in the kidneys, as well as alterations in key mechanistic pathways, that can induce oxidative stress, and metabolic changes leading to kidney disease.8 The alarming number of studies showcasing the harmful health effects pertaining to PFAS exposure has led to the banning of the production of AFFFs containing highly toxic, long chain PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) since 2015.9 However, this regulation is gradually being implemented across states and little is known about the toxicity of the next generation AFFFs. Based on the above, in the present study we evaluate cellular proliferation and toxicity in kidney-derived cells (HEK-293) that were exposed to three widely used AFFFs.
Human embryonic kidney 293 (HEK-293) cells were purchased from ATCC (Manassas, VA) and were grown in Dulbecco's Modified Essential Medium (DMEM) with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Glutamine supplement (100× PSG) supplement. Three Class B AFFFs currently used by U.S. fire departments were studied. They all contain various solvents, salts, and surfactant agents. The following components are identified as potentially toxic agents: Foam A, an Alcohol Resistant—Aqueous Film Forming Foam (AR-AFFF) 3% × 6%, contains methylene chloride, 1,3-dichloropropene, and perfluorooctanoic acid (PFOA); while Foam B, an AFFF 3% Fire Fighting Agent and Foam C, an AR-AFFF 3% × 3%, both produced by the same company, and contain a similar proprietary foamer blend with the addition of C6 fluorosurfactant in Foam C (Table 1).
TABLE 1 -
Detailed Information of the Composition of Foams A, B, and C According to Their Individual Safety Data Sheet (SDS) as Provided by the Respective Manufacturing Companies
| Lauryl Imino Propionate, Sodium Salt
| Perfluorooctanoic Acid
| Methylene Chloride
| Sodium octyl sulfate
| Proprietary foamer blend (water, amphoteric copolymer, amphoteric polymer, amphoteric surfactant, acrylic copolymer, propylene glycol, ethanol)
| Sodium decyl sulfate
| Sodium octyl sulfate
| Proprietary foamer blend (water, amphoteric copolymer, amphoteric polymer, C6 fluorosurfactant, acrylic copolymer, propylene glycol, ethanol)
| Sodium decyl sulfate
Cell Proliferation Assay
HEK-293 cells were seeded on 96-well plates at confluency of 1000 cells/mL and incubated at 37°C in a humidified, 5% CO2 atmosphere for 24 hours. Three foam products were added into the wells at several concentrations ranging from 0% to 100% dilution of stock foam concentration. All dilutions were performed using Dubelcco's PBS (Thermo Fisher Scientific, Waltham, MA) and while accounting for the dilution factor due to the presence of culture media. The 0% stock concentration was pure DPBS (ratio of DPBS to cell media; 1:10) and regarded as the negative control, while the 100% stock concentration was pure foam and regarded as the positive control of toxicity. For any given well cell culture media accounted for 90% of the volume added and the remaining 10% was either DPBS, foam, or mixture of the two for the dilutions. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) proliferation assays were conducted over the course of 72 hours and evaluated using CellTiter 96® AQueous One solution MTS assay (Promega Corporation, Madison, WI). The absorbance values were read in a Clariostar luminometer plate reader at 490 nm (CLARIOstar, BMG Labtech). Baseline absorbance of each foam concentration was measured prior to the execution of the proliferation assays and all analyses were performed on the normalized values.
Cell Viability Assay
Similar to the cell proliferation assay, HEK-293 cells were seeded on 96-well plates at confluency of 1000 cells/mL and incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. The AFFFs were added into the wells at different concentrations ranging from 0% to 100% dilution of stock foam concentration. A cell viability assay was performed at 72 hours post plating and evaluated using the LIVE/DEAD® Viability/Cytotoxicity Kit for Mammalian Cells (Invitrogen Corporation, Calsbad, CA). In particular, calcein AM was added to the AFFF-treated cells, which is a cell-permeant, non-fluorescent dye that is enzymatically converted into the intensely green, fluorescent dye, calcein, in live cells with intracellular esterase activity. Fluorescence imaging was conducted on the fluorescence microscope Keyence BZ-X710, and quantification of captured images was performed on ImageJ (now renamed as FIJI).
All experiments were performed in triplicates. ImageJ, now renamed as FIJI was used for the quantitation of fluorescence and all data analysis was performed on GraphPad Prism. Data are expressed as the mean ± SD of measures used. Differences between treatment groups were analyzed either by Student's t tests (two-tailed) or by one-way analysis of variance (ANOVA) tests, followed by Dunnett's or Tukey's multiple comparisons post-hoc test. Test results with P-values < 0.05 are considered statistically significant, and the asterisks throughout the manuscript depict levels of statistical significance, that is, ∗indicates P < 0.05, “indicates P < 0.01, etc.
The effect of AFFFs on HEK-293 cells was evaluated using established methods of cellular toxicity assessment. Each foam was added in triplicates in the cells in concentrations ranging from 100% to 0% of the working stock concentration. All foams exhibited significant cytotoxic effects when cells were treated with any foam concentration >3%, which is the working concentration used by firefighters (Fig. 1A-C). Interestingly, we observed that for foams A and C treatment even with 0.3% foam led to significant decrease of the proliferation rate by 21.9% and 12.5%, respectively (P < 0.0001, P < 0.05, respectively, Supplementary Table S1, https://links.lww.com/JOM/B77). This cytotoxic effect was also observed after incubating AFFF-treated cells with a non-fluorescent dye which is activated only in live and enzymatically active cells (Fig. 2A and B). All foams had significantly lower fluorescent emission both at 3% and 0.3% stock when compared to control Supplementary Table S2, https://links.lww.com/JOM/B78). Foam A emitted the weakest fluorescent signal potentially suggesting that Foam A might have the highest toxicity, out of all tested products, even at 0.3% stock dilution, results that agree with our MTS assay findings (Fig. 2B).
This is the first study to examine the cytotoxicity of firefighting foams in a human kidney cell line. We investigated the effect of three commonly used AFFFs used by two large U.S. Fire Departments on kidney-derived cells and documented that cells treated with AFFFs for Class B fires had significant decrease in cell proliferation, even in concentrations 10-fold lower than the working concentration used for fire suppression. AFFFs themselves are comprised of several chemical components including fluorinated compounds, which are thermally stable and can encapsulate burning fuel, as well as surfactants to improve the speed with which the foams can cover the fuel. Surfactants themselves are known to have deleterious effects on cell membranes, which contribute to cytotoxicity observed in the present study. AFFFs also contain harmful PFAS, whose toxic health effects have led to increased concern about their usage in the firefighting service and the community environment. Our findings are in agreement with previous research on the effect of PFAS on kidney cells where it was observed that exposure to PFAS could lead to DNA damage and reduced cellular proliferation in Xenopus laevis A6 kidney cells10 and caused cell apoptosis and increased transcription of inflammatory cytokines in a rat renal proximal tubular epithelial cell line (NRK-52E).11 Similarly, studies on different cell systems have shown that PFAS exhibit direct neurotoxicant action that affects neural cell differentiation12 while also having detrimental immunological effects via suppression of cytokine secretion by immune cells.13 The toxic effects of an undisclosed legacy AFFF was recently studied in Zebrafish embryos, and it was observed that larvae exposed to the PFOS/ PFHxS mixture identified in the AFFF formulation had developmental challenges, such as a significantly shorter body length, without however affecting pancreas development or resulting into engorged liver, as could potentially be expected to occur, as result of the developmental exposure to AFFFs.14 Regarding toxicity on HEK-293 cells, we observed across the three different foams we examined, Foam B exhibited the least toxicity, an observation that could potentially be attributed to the presence of surfactants and other components of this formulation (Table 1). Foam A exhibited the greatest toxicity which might be due to the addition of three highly toxic agents in the formulation: methylene chloride, 1,3- dichloropropene, and perfluorooctanoic acid (PFOA). The presence of PFOA, a long chain PFAS in Foam A, indicates that the recall of the legacy AFFFs and the switch to next-generation short chain PFAS-containing foams, has yet to be fully enforced. Our findings indicate that Foam C followed in toxicity. Although not much is known about the detailed composition of this foam (Table 1), we hypothesize that the presence of a C6 fluorosurfactant in the proprietary foam blend might have contributed to the observed decrease of cell viability and proliferation. This finding points to a potential toxicity deriving from a short chain fluorosurfactant which supports two recent FDA-led studies regarding the toxicity of 6:2 fluorotelomer alcohol (FTOH), a short chain PFAS often used in food packaging and stain- and water-resistant textiles. In particular, the findings indicated the accumulation of 6:2 FTOH in the fat, liver, and plasma of rodents and adverse histopathological lesions in the animals’ kidneys.15,16 All of the above highlight the necessity for proper regulation of AFFF composition. The routine and persistent nature of firefighters’ exposure to PFAS is evident, as fluorine particles can be detected on turn-out gear and dust particles collected from fire stations that no longer use PFAS-containing AFFFs.17 What is even more alarming is that despite the increased concern among the scientific community regarding PFAS exposure, there is relatively low awareness among firefighters regarding the presence of PFAS in flame-suppressing foams, as observed in a recent survey-based study conducted by our group on more than a hundred firefighters employed in 67 Florida fire stations.18 In particular, out of the 142 firefighters that participated in the study, only 25 (17.6%) respondents knew whether the AFFFs used in their fire station was a legacy or newer formulation and only 4.6% of participants were aware whether PFOA or PFOS were one of the main components of the foams used by their department. The widespread use of AFFFs in the firefighting service is a matter of national importance, as a growing body of evidence demonstrates PFAS threaten human health, however our understanding of their mechanistic action remains unknown. For that reason, the industry has been moving towards substituting legacy foams with fluorine- free foams that do not contain long chain PFAS. Nonetheless, it should be highlighted that rigorous regulation and enforcement of this switch is necessary, along with robust assessment and monitoring potential health effects of the next-generation AFFFs within the fire service.
The authors would like to thank the fire departments who provided samples of the Aqueous Film-Forming Foams to be evaluated in this study.
1. ATSDR U. Toxicological Profile for Perfluoroalkyls 2021.
2. Shearer JJ, Callahan CL, Calafat AM, et al. Serum concentrations of per- and polyfluoroalkyl substances and risk of renal cell carcinoma. J Natl Cancer Inst
3. Laitinen JA, Koponen J, Koikkalainen J, Kiviranta H. Firefighters’ exposure to perfluoroalkyl acids and 2-butoxyethanol present in firefighting foams. Toxicol Lett
4. Pinkerton L, Bertke SJ, Yiin J, et al. Mortality in a cohort of US firefighters from San Francisco, Chicago and Philadelphia: an update. Occup Environ Med
5. Xu Y, Fletcher T, Pineda D, et al. Serum half-lives for short- and long-chain perfluoroalkyl acids after ceasing exposure from drinking water contaminated by firefighting foam. Environ Health Perspect
6. Olsen GW, Burris JM, Ehresman DJ, et al. Half-life of serum elimination of perfluorooctanesulfonate,perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ Health Perspect
7. Yang CH, Glover KP, Han X. Characterization of cellular uptake of perfluorooctanoate via organic anion-transporting polypeptide 1A2, organic anion transporter 4, and urate transporter 1 for their potential roles in mediating human renal reabsorption of perfluorocarboxylates. Toxicol Sci
8. Stanifer JW, Stapleton HM, Souma T, Wittmer A, Zhao X, Boulware LE. Perfluorinated chemicals as emerging environmental threats to kidney health: a scoping review. Clin J Am Soc Nephrol
9. R Sontake A, M Wagh S. The phase-out of perfluorooctane sulfonate (PFOS) and the global future of aqueous film forming foam (AFFF), INNOVATIONS IN FIRE FIGHTING FOAM. Chem Eng Sci
10. Gorrochategui E, Lacorte S, Tauler R, Martin FL. Perfluoroalkylated substance effects in Xenopus laevis A6 kidney epithelial cells determined by ATR-FTIR spectroscopy and chemometric analysis. Chem Res Toxicol
11. Wen LL, Lin CY, Chou HC, Chang CC, Lo HY, Juan SH. Perfluorooctanesulfonate mediates renal tubular cell apoptosis through PPARgamma inactivation. PLoS One
12. Slotkin TA, MacKillop EA, Melnick RL, Thayer KA, Seidler FJ. Developmental neurotoxicity of perfluorinated chemicals modeled in vitro. Environ Health Perspect
13. Corsini E, Sangiovanni E, Avogadro A, et al. In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs). Toxicol Appl Pharmacol
14. Annunziato KM, Doherty J, Lee J, et al. Chemical characterization of a legacy aqueous film-forming foam sample and developmental toxicity
in Zebrafish (Danio rerio). Environ Health Perspect
15. Rice PA, Aungst J, Cooper J, Bandele O, Kabadi SV. Comparative analysis of the toxicological databases for 6:2 fluorotelomer alcohol (6:2 FTOH) and perfluorohexanoic acid (PFHxA). Food Chem Toxicol
2020; 138: doi: 10.1016/j.fct.2020.111210.
16. Kabadi SV, Fisher JW, Doerge DR, Mehta D, Aungst J, Rice P. Characterizing biopersistence potential of the metabolite 5:3 fluorotelomer carboxylic acid after repeated oral exposure to the 6:2 fluorotelomer alcohol. Toxicol Appl Pharmacol
2020; 388: doi: 10.1016/j.taap.2020.114878.
17. Young AS, Sparer-Fine EH, Pickard HM, Sunderland EM, Peaslee GF, Allen JG. Per- and polyfluoroalkyl substances (PFAS) and total fluorine in fire station dust. J Expo Sci Environ Epidemiol
18. Caban-Martinez AJ, Solle NS, Feliciano PL, et al. Use of aqueous filmforming foams and knowledge of perfluorinated compounds among Florida firefighters. J Occup Environ Med