Environment International 82 (2015) 28–34

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Elevated levels of PFOS and PFHxS in firefighters exposed to aqueous film forming foam (AFFF) Anna Rotander a,⁎, Leisa-Maree L. Toms b, Lesa Aylward a,c, Margaret Kay d, Jochen F. Mueller a a

National Research Centre for Environmental Toxicology (Entox), The University of Queensland, QLD 4108, Australia School of Public Health and Social Work and Institute of Health and Biomedical Innovation, Faculty of Health, Queensland University of Technology, QLD, 4001, Australia Summit Toxicology, LLP, Falls Church, VA 22044, USA d Discipline of General Practice, School of Medicine, The University of Queensland, Royal Brisbane and Women's Hospital QLD 4029, Australia b c

a r t i c l e

i n f o

Article history: Received 25 November 2014 Received in revised form 1 April 2015 Accepted 11 May 2015 Available online xxxx Keywords: Aqueous film-forming foam (AFFF) Biomarkers Firefighters Perfluoroalkyl acids (PFAA) Serum

a b s t r a c t Exposure to aqueous film forming foam (AFFF) was evaluated in 149 firefighters working at AFFF training facilities in Australia by analysis of PFOS and related compounds in serum. A questionnaire was designed to capture information about basic demographic factors, lifestyle factors and potential occupational exposure (such as work history and self-reported skin contact with foam). The results showed that a number of factors were associated with PFAA serum concentrations. Blood donation was found to be linked to low PFAA levels, and the concentrations of PFOS and PFHxS were found to be positively associated with years of jobs with AFFF contact. The highest levels of PFOS and PFHxS were one order of magnitude higher compared to the general population in Australia and Canada. Study participants who had worked ten years or less had levels of PFOS that were similar to or only slightly above those of the general population. This coincides with the phase out of 3M AFFF from all training facilities in 2003, and suggests that the exposures to PFOS and PFHxS in AFFF have declined in recent years. Selfreporting of skin contact and frequency of contact were used as an index of exposure. Using this index, there was no relationship between PFOS levels and skin exposure. This index of exposure is limited as it relies on self-report and it only considers skin exposure to AFFF, and does not capture other routes of potential exposure. Possible associations between serum PFAA concentrations and five biochemical outcomes were assessed. The outcomes were serum cholesterol, triglycerides, high-density lipoproteins, low density lipoproteins, and uric acid. No statistical associations between any of these endpoints and serum PFAA concentrations were observed. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Aqueous film-forming foams (AFFFs) are complex mixtures containing fluorinated- and hydrogenated surfactants used to extinguish fires involving highly flammable liquids. The older generation AFFF, produced by electrochemical fluorination (ECF) and based on perfluorooctanesulfonic acid (PFOS) was phased out around 2002 when 3M voluntarily discontinued its production of PFOS-based products (3M, 2000). Fluorinated surfactants are surfactants in which at least one hydrogen atom along the carbon backbone has been replaced with fluorine. The combination of the hydrophobic fluorocarbon chain and the hydrophilic head group gives repellency toward both water and oils (Moody and Field, 2000). The strength of the carbon–fluorine bond contributes to the physicochemical properties of fluorinated surfactants, such as strong chemical and thermal stability, making them both industrially attractive and environmentally persistent (Buck et al., 2012). ⁎ Corresponding author at: National Research Centre for Environmental Toxicology, Entox, The University of Queensland, QLD 4108, Australia. E-mail address: [email protected] (A. Rotander).

http://dx.doi.org/10.1016/j.envint.2015.05.005 0160-4120/© 2015 Elsevier Ltd. All rights reserved.

Perfluoroalkyl acids (PFAAs) such as PFOS are persistent in the environment once they have been released and are hence present in living organisms all over the world, including people in the general community (Kato et al., 2011; Giesy and Kannan, 2001; Kannan et al., 2004; Kärrman et al., 2007; Tao et al., 2006; Toms et al., 2014). Human exposure to PFAAs may occur through both direct and indirect exposures. Direct exposure implies that the PFAAs are present in the exposure source, and indirect exposure that a precursor compound undergoes environmental break-down processes or metabolizes in the body to PFAAs. The use of PFOS and other perfluoroalkyl acids (PFAAs) in AFFF formulations has been linked to environmental contamination related to handling, storage and usage (de Solla et al., 2012). Substantially elevated levels of PFOS have been reported in water and biological samples, such as molluscs, turtles, and wild mink downstream from airports with a history of firefighting training activities (de Solla et al., 2012; Kärrman et al., 2011; Persson et al., 2013). In Cologne, Germany, elevated levels of PFOS and perfluorohexanesulfonic acid (PFHxS) were found in five individuals who drank water from a private well contaminated with AFFF from a nearby airport (Weiss et al., 2012). Similarly, in Uppsala, Sweden, AFFF contaminated drinking water has been suggested

A. Rotander et al. / Environment International 82 (2015) 28–34

as one plausible factor behind increasing exposure to PFHxS in Uppsala residents (Glynn et al., 2012). In humans a large number of epidemiological studies have recorded associations between PFAAs and biomarkers of health and health outcomes, and several recent review papers summarize and discuss the epidemiologic evidence found between for example PFAAs and cancer incidence (Chang et al., 2014), blood lipids (Steenland et al., 2010), and developmental effects (Bach et al., 2015; Lam et al., 2014). High PFAA levels, up to 10 ppm, have been recorded in serum of 3M employees working at a fluorosurfactant and fluoropolymer manufacturing plant (Olsen et al., 2003). Firefighters belong to another occupational group with potentially elevated exposure to PFAAs through usage of AFFF. Although fire emergencies may be rare at a particular location (for example oil refineries, airports, and ships and oil platforms), firefighters have regular training which may include use of AFFF. PFOS based AFFF has commonly been used for training and operational purposes. However, in recent years the firefighting foam industry has moved toward telomer-based AFFF (Seow, 2013), made up of mixtures of predominantly 6:2 and 8:2 fluorotelomers (Wang et al., 2013). Over the last few years there has also been an increase in use of fluorine-free foams containing water-soluble non-fluorinatedpolymer additives and increased levels of hydrocarbon detergents (Seow, 2013). Little is known about the magnitude of exposure to PFAAs attributed to AFFF usage, which firefighters may have experienced. The objectives of the current study were to 1) Assess the concentration of PFAAs in blood sera of firefighters with past exposure to AFFF, 2) Compare findings with other Australian and international data, including data from groups with high occupational exposure, 3) Investigate potential associations between PFAA levels and different parameters targeted by a questionnaire (for example number of years in job with exposure to AFFF and frequency of blood donation), and 4) Investigate potential associations between levels of blood lipids and uric acid and measured serum levels of PFAAs on a cross-sectional basis. The firefighters had in the past been exposed to both PFOS-based (3M AFFF) and telomerbased AFFF (Ansul AFFF). 2. Materials and methods

29

low-density lipoprotein (LDL), high-density lipoprotein (HDL) levels, triglycerides, and uric acid. Total serum proteins (albumin and globulin) were also measured for inclusion in statistical analyses (See Supplementary Information for more detail).

2.4. Chemicals and extraction of PFAAs Details of chemicals and standard compounds used in the extraction are available in the Supplementary Information. Mass labeled internal standards were added to 0.2 mL serum. The analytes were then extracted with 1.5 mL 100% acetonitrile using ultrasonication followed by vortex extraction, centrifugation and evaporation to 0.2 mL under a gentle stream of nitrogen. Performance standards and 0.3 mL of 5 mM ammonium acetate in water were added prior to analysis.

2.5. Instrumental analysis PFAAs were determined by high-performance liquid chromatographytandem mass spectrometry (HPLC–MS/MS) using an API5500Q mass spectrometer (AB/Sciex, Concord, Ontario, Canada) equipped with an electrospray (TurboV) interface coupled to a Shimadzu Nexera HPLC system (Shimadzu Corp., Kyoto, Japan). Separation was achieved using a 4 μm 50 × 2.0 mm C18 Gemini column (Phenomenex, Torrance, CA) run at 45 °C, and a flow rate of 0.3 mL min−1. 2.6. Quality assurance The limit of detection (LOD) was calculated using an average of the lowest calibration point (n = 3) and 3*signal to noise (S/N). In the case of blank contamination the LOD was calculated using the average blank concentration (n = 6) plus three times the standard deviation. Details about method reproducibility and accuracy are provided in the Supplementary Information and method validation parameters, recovery rates and blank concentrations are presented in Tables S3, S4 and S5, respectively.

2.1. Study participants

2.7. Statistical analysis

Firefighters employed by a contractor providing firefighting capability in Australia were invited to participate in the study. Participation was voluntary and potential participants were provided with written information about the study and all documents necessary for participation at their work place. Participants provided informed consent, completed a written questionnaire, and provided a blood sample. Ethics approval for this study was granted by The University of Queensland Medical Research Ethics Committee (Number 2014000614).

An analysis of variance (ANOVA) was conducted using log10-transformed concentrations to examine factors that might influence blood serum levels of PFOS, PFHxS, and PFOA. Factors investigated included age, sex, serum protein levels, current smoking, blood donor (yes versus no), years of employment on jobs with foam contact, and indices of time-weighted intensity of foam contact before 2003 and after 2003 (see Supplementary Information). Potential associations between measured serum lipids, uric acid levels, and total serum protein levels were assessed to examine whether concentrations of these markers or the risk of out-of-range levels of these markers were influenced by serum PFAA concentrations. Additional demographic and lifestyle factors potentially influencing these markers were also considered, including age, sex, body mass index (BMI), exercise intensity, alcohol consumption, and current smoking status. Statistical analysis for lipids was conducted only including those participants whose blood samples were confirmed to be collected under fasting conditions. The measured concentrations of PFOS, PFHxS and PFOA in the firefighters were compared to available biomonitoring data sets from the general population in Australia and Canada. Data on PFAA concentrations in the Australian population are available from biomonitoring data conducted in Australia every 2 years (more information can be found in the Supplementary Information). The Canadian data is from the Canadian Health Survey 2010/2011 (Health Canada, 2013) and is based on plasma concentrations of approx. 1000 individuals.

2.2. Questionnaire The questionnaire was designed to capture information about basic demographic factors (age, gender), lifestyle factors (such as diet, alcohol consumption, exercise patterns), health conditions (including current medications) and potential occupational exposure (such as work history, self-reported skin contact with foam). The questionnaire has been attached to the Supplementary material. 2.3. Sample collection A blood sample was collected from each participant between April and December 2013 at a commercial pathology laboratory (a Sullivan Nicolaides (SNP) collection center or a SNP partner collection centre). All samples were couriered to the SNP Taringa pathology laboratory in Brisbane where the serum was analyzed for total serum cholesterol,

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A. Rotander et al. / Environment International 82 (2015) 28–34

3. Results

3.4. Comparison with PFAA levels in the general population

3.1. Participant demographics

The serum levels of PFOS were approximately six to ten times higher in firefighters compared to the levels found in the general population in Australia and in Canada (Fig. 1A). The median/mean level was 66/74 ng/mL in firefighters compared to 12 ng/mL (mean) and 6.8 (median) ng/mL in the general population in Australia and Canada, respectively. The serum levels of PFHxS in firefighters were approximately 10 to 15 times higher compared to the general population levels in Australia and Canada (Fig. 1B). The median/mean level was 25/33 ng/mL in firefighters compared to 3.2 ng/mL (mean) and 1.7 ng/mL (median) in the general population in Australia and Canada, respectively. However, the PFOA levels in firefighters were in the same range as the Australian and Canadian population (Fig. 1C).

Out of a total of 731 potentially eligible employees, a total of 149 firefighters participated in the study (20%). A summary of demographic, lifestyle and work exposure information is presented in Table S1 in the Supplementary Information. There was a higher percentage participating females compared to all eligible female employees, as well as a higher percentage of participants in the higher age groups between 50 and 64 years compared to all eligible employees (Table S2, Supplementary Information). The participants came from 18 training facilities throughout Australia.

3.2. Method performance 3.5. Comparison with PFAA levels in another occupationally exposed group Method validation parameters are summarized in Table S3 in the Supplementary Information. Mean recoveries for all labeled PFAAs ranged between 87% and 101%, LODs ranged between 0.02 and 0.06 ng/mL serum for all detected PFAAs. The reproducibility of PFOS, PFHxS and PFOA analysis, measured as the relative standard deviation (RSD) of multiple analysis (n = 7) of a pooled serum sample on different days, was below 10%. The accuracy of the method, calculated as the mean normalized difference (%) of a NIST reference serum sample (SRM 1957), were 23%, 22% and 33% for PFOS, PFHxS and PFOA, respectively.

The concentrations of PFOS found in the serum of the firefighters were compared to the data from 263 workers at the 3M Decatur plant in Alabama, USA (Fig. S3, Supplementary Information) who were exposed to PFAAs during the manufacturing of fluorosurfactant chemicals (Olsen et al., 2003). In this study, the geometric mean serum level of PFOS in firefighters was approximately 20 times lower than in the 3M workers, 51 ng/mL compared to 910 ng/mL, and the highest level in a firefighter was 391 ng/mL compared to 10,060 ng/mL in a Decatur employee (Olsen et al., 2003).

3.3. Levels of PFAAs

3.6. Evaluation of factors influencing serum levels of PFAAs

Multiple PFAAs were detected in the serum of all 149 firefighters. The three most prevalent PFAAs, detected in all samples, were PFOS, PFHxS and PFOA (Table 1). Other PFAAs were found at lower levels and more infrequently, and perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), and 6:2 fluorotelomer sulfonate (6:2 FTSA) were not detected in any of the samples. It can be noted that the detection limit for PFBA, PFPeA and 6:2 FTSA was close to or more than 0.1 ng/mL (Table S2, Supplementary Information). Serum concentrations of PFOS were ranged between 3 ng/mL and 391 ng/mL, between 1 and 277 ng/mL for PFHxS, and between 0.3 and 18 ng/mL for PFOA (Table 1). A strong correlation was found between PFOS and PFHxS concentrations (R2 = 0.93), which suggests that they have a common source of exposure (Fig. S1, Supplementary Information). A strong correlation between these two supports the hypothesis that serum levels of PFOS and PFHxS in firefighters have been influenced by direct or indirect contact with 3M AFFF. A weaker correlation (R2 = 0.43) was found between PFOS and PFOA, which suggests that the exposure scenario of these two chemicals varies more compared to PFOS and PFHxS (Fig. S2, Supplementary Information).

Various exposure and demographic factors were assessed as potential predictors of PFAA concentrations. The results of the ANOVA for 149 individuals with both measured PFAA concentrations and fully completed questionnaires regarding demographic and lifestyle factors are presented in Table 2. Levels of PFOS, PFHxS and PFOA were negatively associated with blood donation (lower levels in persons who report blood donation) (Fig. S4, Supplementary Information). While there were very few female participants (n = 5), levels of PFOS, PFHxS and PFOA in female participants were statistically significantly lower compared to males (Table 2). The concentrations of all three biomarkers were significantly positively associated with years of jobs with foam contact (Table 2, Fig. 2). Study participants who had worked ten years or less had levels of PFOS that were similar to or only slightly above those of the general population. This is consistent with the timeline of AFFF use, as the PFOS-containing 3M AFFF was phased out of use at all 18 training facilities in 2003. The time it took to phase out 3M AFFF from use has been estimated to approximately one year, in which all firefighters were still required to train with 3M AFFF once every 90 days (personal communication, see Supplementary Information). PFOS concentrations seemed to plateau with 20 years or more of AFFF use (Fig. 2). The concentrations were also independently associated with age. However, as expected, there was a strong and significant interaction between age and years of foam exposure that modifies the independent relationships of the two factors with blood levels of the compounds (Table 2, and see the Supplementary Information for more detail). These basic models accounted for approximately 56% of the variance in blood levels of PFOS and PFHxS, but only 17% of the variance in PFOA levels. There was no statistically significant difference in PFAA blood levels across training facilities (data not shown). Ninety participants completed questionnaire items related to frequency and extent of skin contact on each job assignment, allowing calculation of exposure intensity indices for the pre- and post-2003 time periods. Neither of the self-reported intensity indices was significantly associated with levels of these three PFAA compounds when they were included in the ANOVA regressions. p-Values for the skin exposure

Table 1 Serum levels (ng/mL serum) of eleven PFAAs found above LOD in 149 firefighters. Compound

% N LOD Mean (SD)

Median Range

Perfluorooctanesulfonic acid, PFOS Perfluorohexanesulfonic acid, PFHxS Perfluorooctanoic acid, PFOA Perfluoroheptanoic acid, PFHpA Perfluorononanoic acid, PFNA Perfluorodecanoic acid, PFDA Perfluoroundecanoic acid, PFUnDA Perfluorododecanoic acid, PFDoDA Perfluorotridecanoic acid, PFTrDA Perfluorobutanesulfonic acid, PFBS Perfluorodecanesulfonic acid, PFDS

100 100 100 50 100 99 88 6.6 7.9 2.6 3.3

66 25 4.2 0.07 0.69 0.27 0.14 b0.05 b0.06 b0.02 b0.03

NC = not calculated due to low detection rates.

74 (61) 33 (36) 4.6 (2.4) 0.10 (0.08) 0.76 (0.3) 0.29 (0.13) 0.16 (0.08) NC NC NC NC

3.4–391 0.7–277 0.3–18 b0.03–0.38 0.09–2.4 b0.04–0.99 b0.06–0.58 b0.05–0.12 b0.06–0.10 b0.02–0.09 b0.03–0.07

A. Rotander et al. / Environment International 82 (2015) 28–34

A

PFOS

200

B

100

40

20

50

0

0

AUS

C

PFHxS

80

60

ng/ml, ppb

ng/ml, ppb

150

31

CAN

THIS STUDY

AUS

CAN

THIS STUDY

PFOA

10

ng/ml, ppb

8

6

4

2

0 AUS

CAN

THIS STUDY

Fig. 1. Serum concentrations (ng/mL) of A) PFOS, B) PFHxS, and C) PFOA in 16 pooled samples from Queensland, Australia (AUS), from 2010/2011 (n = 1600), and individual plasma samples from a Canadian health survey from 2010 to 2011 (n = 1016), and in this study's 149 firefighters. The whiskers indicate the 95th percentile and the columns indicate median concentrations for THIS STUDY and CAN, and mean concentrations for AUS.

intensity indices in the regressions ranged from 0.3 to 0.99, indicating no predictive value for these indices on blood concentrations of PFOS, PFHxS, or PFOA. Thus, they were omitted from the final models. However, this finding needs to be interpreted cautiously because the index considered self-reported frequency and extent of direct skin exposure to AFFF and was likely to be a relatively crude measure of skin exposure.

3.7. Biochemical measures and PFAA levels We conducted an ANOVA to evaluate the influence of PFAA concentrations on the biochemical outcome measures (Table 3). Before beginning the regression exercise, we examined whether serum PFOA, PFOS, or PFHxS concentrations were associated with key covariates. No significant associations between these compounds and BMI or total serum protein (albumin plus globulin) were observed. The models for cholesterol, LDL, HDL, and triglycerides were limited to 38 participants who confirmed fasting at the time of blood sampling and did not report taking cholesterol-lowering medication. Results were similar when all participants with measured cholesterol and BMI were included in the analyses, regardless of fasting status. The model for uric acid omitted one participant who reported taking medication to treat gout. Only

individuals (in this case 93% of the participants) with reported height and weight measures (to allow calculation of BMI) were included in the models. Total serum cholesterol and LDL were not associated with any of the covariates except total serum protein levels. HDL levels were higher in females than males but the number of female participants with biochemical data was very small (n = 3). Serum triglycerides were not associated with any of the covariates examined, and no associations between cholesterol, HDL, and LDL, or triglycerides and PFOA, PFOS, or PFHxS were observed in the 38 participants included in the statistical analyses. None of the PFAA concentrations were significantly associated with serum total protein levels or uric acid.

4. Discussion 4.1. Method performance The comparison of the PFOS results of the analysis of the NIST reference sample, 14.1 (current method, using linear PFOS for quantification) compared to 17.7, ((Riddell et al., 2009) using a mixture of linear and branched isomers of PFOS for quantification), suggests that the results

Table 2 ANOVA for factors potentially influencing PFAA levels. ANOVA conducted on log10-transformed PFAA concentrations. log10PFOS

Intercept Female vs. male Foam exposure (yrs) Age (yrs) Blood donor (yes vs. no) Interaction term (age × yrs of foam exposure) Adj. R2 for model:

log10PFHxS

log10PFOA

Beta (SE)

p value

Beta (SE)

p value

Beta (SE)

p value

−0.185 (0.254) −0.292 (0.133) 0.094 (0.014) 0.032 (0.006) −0.198 (0.053) −0.001 (0.0003) 0.56

0.468 0.029 b0.001 b0.001 b0.001 b0.001

−1.009 (0.324) −0.350 (0.170) 0.115 (0.017) 0.037 (0.008) −0.266 (0.068) −0.002 (0.0003) 0.57

0.002 0.041 b0.001 b0.001 b0.001 b0.001

0.082 (0.20) −0.220 (0.105) 0.023 (0.011) 0.016 (0.005) −0.140 (0.042) −0.0005 (0.0002) 0.17

0.685 0.038 0.036 0.001 0.001 0.014

A. Rotander et al. / Environment International 82 (2015) 28–34

ng/ml, ppb

32

PFOS

400 350 300 250 200 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

0-10

11-20

21-30

31-42

Years of jobs with AFFF exposure Fig. 2. PFOS serum concentrations (ng/mL serum, y-axis) in 149 firefighters in relation to number of years of jobs with AFFF exposure (x-axis). The lines in the boxes indicate median concentrations, the outside of the boxes the 25th and 75th percentiles, and the whiskers min and max concentrations.

of total PFOS using the current method and the study by Riddell et al. are of equal magnitude. However, as all isomers of PFOS using the current method were quantified against linear PFOS (m/z 499 N 99), the PFOS results have an associated uncertainty factor of unknown magnitude due to that response factors for structural isomers differ in MS/MS (Riddell et al., 2009). Since the amount of branched isomers can vary considerably between individuals a 30–40% difference in calculated total PFOS concentrations between quantification methods, using the transition m/z 499 N 99 is not unusual (Berger et al., 2011). 4.2. PFAA exposure attributed to AFFF usage At the training facilities investigated in this study, 3M AFFF had been used until 2003 and Ansul AFFF between 2003 and 2010, and after 2010 a fluorine-free foam formulation had been used. Direct exposure (to the PFAAs themselves) and indirect exposure (to a precursor compound

that metabolizes in the body to a PFAA) to AFFF may have occurred through different routes and may have varied with many different factors, both in space and time. Two plausible exposure routes are skin contact with foam and inhalation of aerosolized foam present in air during foam training activities. Skin contact may have occurred with the actual foam as well as, for example, with protective gear impregnated with foam. Quantifying and documenting the exposure experienced by employees are always difficult. In this study we relied on retrospective self-reporting by participants of the direct exposure of the skin to these foams. Other exposure routes, such as inhalation of aerosolized foam during training, ingestion of foam or contact with safety equipment, were not possible to capture. The relatively high PFOS and PFHxS serum levels are likely attributable to exposure to 3M AFFF as perfluorinated sulfonates (PFSAs) have been identified as major components in 3M foam concentrates (Backe et al., 2013; Weiner et al., 2013), as well as in groundwater

Table 3 Results of ANOVA for possible associations between PFAA levels and biochemical outcomes. Models for cholesterol, HDL, LDL, and triglycerides (mmol/L) based on participants with measured BMI, confirmed fasting before blood sample, and those not taking cholesterol-lowering medications (n = 17). Model for uric acid (μmol/L) restricted to those with measured BMI and not taking gout medication (n = 1). p values b 0.05 are indicated in bold. Beta (SE) p value Parameter

Cholesterol (n = 38)

HDL (n = 38)

LDL (n = 38)

Triglycerides (n = 38)

Uric acid (n = 137)

Age (yrs)

0.038 (0.028) p = 0.187 0.197 (0.679) p = 0.774 0.055 (0.052) p = 0.300 −0.506 (1.046) p = 0.632 0.134 (0.054) p = 0.019 −0.874 (1.222) p = 0.480 1.201 (1.511) p = 0.433 −0.493 (1.074) p = 0.649 0.26

−0.001 (0.008) p = 0.871 0.67 (0.192) p = 0.002 −0.012 (0.015) p = 0.416 −0.334 (0.296) p = 0.269 0.003 (0.015) p = 0.824 −0.083 (0.346) p = 0.813 0.257 (0.428) p = 0.553 −0.135 (0.304) p = 0.660 0.28

0.04 (0.024) p = 0.111 −0.267 (0.586) p = 0.652 0.051 (0.045) p = 0.260 −0.221 (0.904) p = 0.808 0.106 (0.046) p = 0.030 −0.357 (1.055) p = 0.738 1.141 (1.305) p = 0.389 −0.775 (0.927) p = 0.410 0.28

−0.001 (0.019) p = 0.976 −0.435 (0.461) p = 0.353 0.038 (0.035) p = 0.286 −0.01 (0.71) p = 0.989 0.05 (0.037) p = 0.182 −0.862 (0.83) p = 0.308 −0.475 (1.026) p = 0.647 0.888 (0.729) p = 0.233 0.16

−0.0002 (0.0008) p = 0.773 −0.08 (0.03)p = 0.014

Sex (female vs. male) BMI (kg/m2) Current smoker (Y vs. N) Total serum protein (albumin + globulin) log10PFOA log10 PFOS log10 PFHxS Model adj. R2:

0.004 (0.0015) p = 0.011 −0.38 (0.032) p = 0.247 0.003 (0.001) p = 0.020 0.021 (0.032) p = 0.508 −0.045 (0.047) p = 0.342 0.040 (0.036) p = 273 0.14

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contaminated with AFFF (Backe et al., 2013; Houtz et al., 2013). 6:2 fluorotelomermercaptoalkylamido sulfonate (6:2 FTSAS) has been identified as the major fluorosurfactant in Ansul foams (Weiner et al., 2013). Other components in Ansul foam concentrates were identified as 8:2 fluorotelomer thioamido sulfonate (8:2 FTSAS), 8:2 fluorotelomer sulfonate (8:2 FTSA) and 6:2 FTSA (Weiner et al., 2013). 6:2 FTSA was measured in concentrations up to 14.6 mg/L in AFFF contaminated groundwater samples at U.S. military bases (Schultz et al., 2004), and it was suggested that these fluorotelomer sulfonates are biodegradation products of FTSAS as the ratio FTSA/FTSAS in groundwater far exceeded the ratio calculated in AFFF concentrate. Further degradation of 6:2 FTSA ultimately yields short-chain PFCAs (C ≤ 7) (Wang et al., 2011; Weiner et al., 2013). The training pad concrete at one of the training facilities in a previous study had been examined for PFAA distribution and potential future release (Baduel et al., 2015). The total mass (g) of individual PFAAs in the top 15 cm of the entire pad was estimated based on analysis of 15 individual samples of concrete dust extracted by drilling. The highest amounts (g) were estimated for PFBS N PFOS N PFHxA N PFHxS N PFBA N PFPeA N 6:2 FTSA, with amounts ranging between 600 g and 70 g. The amount of PFOA was merely 16 g in comparison. Much of the short-chain PFAAs at this site, such as PFHxA, could be break-down products of C6 based fluorotelomer surfactants in Ansul AFFF (Houtz et al., 2013; Seow, 2013). PFHxA has been found at similar or higher levels compared to 6:2 FTSA in AFFF contaminated groundwater (Backe et al., 2013). This relatively high presence of short-chain PFAAs is not reflected in the serum of the firefighters, which might be due the reduction of exposure to AFFF after 2010 and the relatively fast elimination of shorter perfluoro alkyl chain lengths (Chang et al., 2008; Olsen et al., 2009). In highly exposed humans the elimination half-life of PFHxA ranged between 14 and 49 days (Russell et al., 2013). 6:2 FTSA and 8:2 FTSA have rarely been detected in human sera and at relatively low levels with 8:2 FTSA being the most abundant of the two. In 50 sera collected from blood donors of both gender and varying age in the U.S. in 2009 the highest 6:2 FTSA and 8:2 FTSA level measured were 0.047 and 0.231 ng/mL, respectively (Lee and Mabury, 2011). Under appropriate conditions 8:2 FTSA can degrade to PFOA (Seow, 2013) and PFOA has been identified as an impurity in Ansul AFFF (Weiner et al., 2013) to which the firefighters had been potentially exposed between 2003 and 2010. However, in this study we did not see clear evidence for notably elevated PFOA exposure in the firefighters. 4.3. Evaluation of factors influencing serum levels of PFAAs That lower levels were associated with blood donation is consistent with that PFAAs bind to serum albumin (Jones et al., 2003; Han et al., 2003), and consequently removal of blood (through blood donation or disease treatment for hemochromatosis) can act as an elimination pathway and results in lower blood concentrations of these compounds (Lorber et al., 2015; Thompson et al., 2010). Similarly, although the number of female participants in this study is small, lower PFAA levels in female participants are consistent with previous data indicating that menstruation acts as an important elimination pathway (Kato et al., 2011; Toms et al., 2014; Wong et al., 2014). Compared to the highest levels in the manufacturing workers from Decatur, the highest PFOS levels in the firefighters were two orders of lower magnitude. However, serum from the Decatur workers was collected at a time when exposure was ongoing (i.e. they were working at the plant with fluorochemical manufacturing at the time of blood collection). Hence if assuming that 3M AFFF has been an important source for PFOS and PFHxS exposure to firefighters a direct comparison between these two groups is complicated by the fact that an important part of the PFOS exposure would have ceased around 2003 when 3M AFFF was replaced with Ansul AFFF. This assumption is supported by the result that the highest PFOS levels were, without exception, found in firefighters who were working when 3M AFFF was still in use. Previous studies have calculated the half-life of serum elimination as 5 years

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for PFOS and 8 years for PFHxS (Olsen et al., 2007). This suggests that levels measured in serum in the current study participants may have been approximately twice to four times as high prior to the phase-out of 3M AFFF in 2003. 4.4. Evaluation of biochemical measures and PFAA levels No clear associations between the biochemical markers and PFAA blood levels were apparent in the firefighters in this cross-sectional study. Previous cross-sectional studies have found relationships between some PFAA compounds and serum lipid and serum uric acid concentrations. In particular, in populations exposed to PFAAs, cholesterol (Lin et al., 2009; Nelson et al., 2010; Steenland et al., 2009), lowdensity lipoprotein (LDL) levels, and triglycerides (Steenland et al., 2009) have been positively associated with blood PFOA or PFOS concentrations. Because PFAAs bind to serum proteins (Butenhoff et al., 2012), a positive relationship between PFAA levels and serum protein levels would be expected. However, it is possible that the influence of other factors in this population was much greater than the influence of serum protein levels, making any association difficult to detect. 5. Conclusions Past employment with exposure to 3M AFFF was associated with significantly elevated levels of PFOS and PFHxS in Australian firefighters. Serum levels of PFOS and PFHxS reflected past exposures. Even ten years after the phase out of 3M AFFF, PFOS serum levels remained above 100 ng/mL and 200 ng/mL in 27% and 3% of the participating firefighters, respectively. Overall, no evidence of a measurable relationship between serum PFAA concentrations and the examined biochemical outcomes was found. Acknowledgments The participation of all firefighters is greatly acknowledged and appreciated. JFM is funded by ARC Future Fellowship (FF120100546). LMLT is funded by an ARC DECRA (DE120100161). Entox is jointly funded by The University of Queensland and Queensland Health. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.envint.2015.05.005. References 3M. Phase-out plan for PFOS-based products. in: Agency UEP, ed; 2000. Bach, C.C., Bech, B.H., Brix, N., Nohr, E.A., Bonde, J.P., Henriksen, T.B., 2015. Perfluoroalkyl and polyfluoroalkyl substances and human fetal growth: a systematic review. Crit. Rev. Toxicol. 45 (1), 53–67. Backe, W.J., Day, T.C., Field, J.A., 2013. Zwitterionic, cationic, and anionic fluorinated chemicals in aqueous film forming foam formulations and groundwater from U.S. military bases by nonaqueous large-volume injection HPLC–MS/MS. Environ. Sci. Technol. 47, 5226–5234. Baduel, C., Paxman, C., Mueller, J.F., 2015. Perfluoroalkyl substances in a firefighting training pad, distribution and potential future release. J. Hazard. Mater. 296, 46–53. Berger, U., Kaiser, M., Kärrman, A., Barber, J., van Leeuwen, S.J., 2011. Recent developments in trace analysis of poly- and perfluoroalkyl substances. Anal. Bioanal. Chem. 400, 1625–1635. Buck, R.C., Murphy, P., Pabon, M., 2012. Chemistry, properties, and uses of commercial fluorinated surfactants. In: Knepper, T.P., Lange, F.T. (Eds.), Polyfluorinated Chemicals and Transformation Products. Springer, Berlin Heidelberg. Butenhoff, J.L., Pieterman, E., Ehresman, D.J., Gorman, G.S., Olsen, G.W., Chang, S.C., Princen, H.M., 2012. Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions. Toxicol. Lett. 210, 360–365. Chang, S.C., Das, K., Ehresman, D.J., Ellefson, M.E., Gorman, G.S., Hart, J.A., Noker, P.E., Tan, Y.M., Lieder, P.H., Lau, C., Olsen, G.W., Butenhoff, J.L., 2008. Comparative pharmacokinetics of perfluorobutyrate in rats, mice, monkeys, and humans and relevance to human exposure via drinking water. Toxicol. Sci. 104, 40–53. Chang, E.T., Adami, H.-O., Boffetta, P., Cole, P., Starr, T.B., Mandel, J.S., 2014. A critical review of perfluorooctanoate and perfluorooctanesulfonate exposure and cancer risk in humans. Crit. Rev. Toxicol. 44 (S1), 1–81.

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