Comparison of EOF concentrations of the Cohort 1 samples with other studies
A recent study from Tromsø, another Norwegian region, reported EOF in serum samples ranging from 12.6 to 45.3 ng F/mL in samples from 1986, 2007, and 2015. Specifically, in 2015, EOF concentrations ranged from 12.6 to 22.6, with a median of 18.5 ng F/mL. Miaz and co-workers reported a geometric mean of approximately 30 ng F/mL in pooled serum samples from first-time mothers in Sweden collected in 2015. Aro and co-workers reported detectable concentrations of EOF in human whole blood collected between 2017 and 2018 from Sweden in the range between 0.51–48.7 ng F/mL. The average EOF concentrations in plasma samples from German university students in Munster, collected between 1982 and 2009, ranged from 14.5 to 39.3 ng F/mL. In general, different methods have different extraction efficiency for different PFAS, thus a direct comparison of EOF concentrations may be challenging. The extraction methods and sample matrices (whole blood, plasma, and serum) differed between the present study with samples from the Oslo area (Cohort 1) and the one from Tromsø. However, the EOF concentrations in the samples from Cohort 1 (<7.5–32 ng F/mL, median: 14.6 ng F/mL) were similar to those observed in Tromsø (12.6 to 22.6 ng F/mL, median: 18.5 ng F/mL) collected around the same time.
Mass balance analysis of EOF
Similar to the results from other studies, our mass balance analysis of EOF revealed that quantifiable PFAS accounted for varying proportions of EOF in the samples (Figure 2). The current study measured up to 64 targeted PFAS in the samples aiming to close the mass balance of EOF. The majority of the EOF were mainly explained by legacy PFAS such as PFOA, PFNA, PFDA, PFHxS, PFOS which was similar to the results of other studies.,,, However, TFA stood out in the mass balance analysis; the median proportion of unknown fluorinated chemicals decreased from 62 to 29% (Figure 2) when TFA was excluded, as the concentrations of TFA exceeded other PFAS measured. It has been suggested in other studies that TFA might contribute to some proportions of the unrecognized EOF in the samples,, and the current study further confirmed the importance of measuring TFA to reduce the proportion of unrecognized EOF in the mass balance analysis.
TFA in human blood
As shown in the results from targeted analysis, concentrations of TFA were almost 2 times higher than those of PFOS in Cohort 1 (EuroMix study) suggesting significant human exposure to TFA. Since no recovery correction was carried out, real concentrations could be even higher. On the other hand, the determination of TFA only relied on a single transition in the multiple reaction monitoring (MRM) that may pose the possibility of reporting false positives. To ensure our TFA identification is valid, a subset of samples was analyzed with additional LC-MS/MS using the method reported by Covaci et al.
Holaday (1977) reported a half-life of TFA in human blood of approximately 16 hours, which is much shorter than other PFAS such as PFOS and PFOA. The detection of TFA in human samples at current concentrations may suggest exposure of significant amounts of TFA in daily life. TFA is known to be used in many industrial applications, consumer products and it is one of the final degradation products of chemicals used in cooling appliances potentially resulting in a persistent contamination of the environment and increasing human exposure. A study from Indiana, US showed that TFA was the only PFAA for which serum concentrations significantly correlated with both dust and water concentrations (Spearman correlation coefficients (r)=0.40, p<0.001 and r=0.28, p=0.01, respectively). Recent studies, reported TFA concentrations in drinking water in Sweden and Norway which ranged from 70 to 720 ng/L; specifically, the concentration in Oslo was 230 ng/L. These reported TFA concentrations in drinking water were below the guideline values for Germany (60 µg/L), Denmark (9 µg/L) and the Netherlands (2.2 µg/L). The German environmental protection agency (UBA) proposed to classify TFA as toxic to reproduction based on a recent study from Bayer on the reproductive toxicity of TFA in rabbits, which found severe fetal malformations. Further investigations are needed to understand human exposure to TFA and its impact on human health.
Previous studies also suggested that the unknown fraction may be caused by the metabolization of fluorinated pharmaceuticals carrying CF3-groups in their molecular structure. A recent study confirmed that fluorinated pharmaceuticals accounted for up to 63% of the EOF, and their contribution increased in recent years. We did not survey or measure any pharmaceuticals in the samples from Cohort 1 due to the lack of ethical clearance. However, interesting and important information can be obtained from the subjects in Cohort 2 that had taken fluoxetine, which is one of the CF3-containing drugs used to treat depression, obsessive-compulsive disorder, some eating disorders, and panic attacks. Significant higher concentrations of EOF were observed in the subjects who had taken fluoxetine compared to the control group, even though for two subjects from the control group who had elevated concentrations of target PFAS contributing to the EOF (Figure 1). It is known that fluoxetine will be metabolized into fluoxetine glucuronide, norfluoxetine, seproxetine, norfluoxetine glucuronide and other metabolites. In our current investigation, only seproxetine and fluoxetine were measured, thus part of the unrecognized EOF may be attributed to the transformation products of fluoxetine that we did not measure. Fluoxetine and norfluoxetine have long elimination half-lives, resulting in the drug remaining in the body for several weeks, even after discontinuation. The metabolism of fluoxetine is extensive, with approximately 2.5% of the administered dose excreted unchanged in the urine. Therefore, consistent use of the prescribed drug or even after stopping the use might have resulted in elevated concentrations of EOF. Further, we have no information on other fluorinated pharmaceuticals consumed by the subjects in Cohort 2 donors, which could further explain the variation in FOF and TFA in our study.
Temporal variations of EOF
Several studies have conducted temporal analyses of EOF. However, these analyses were conducted at intervals of one year or longer and were also cross-sectional.,, The current investigation is the first temporal analysis of EOF on a longitudinal basis (paired samples of the same individuals) and with a shorter interval between sampling occasions to also catch changes in fluorinated compounds with shorter half-lives such as TFA. As shown in Table 2, approximately 36% of the samples exhibited significant changes (25%), either an increase or decrease. The changes in EOF were not due to changes in legacy PFAS as these PFAS have long half-lives in humans and our results also showed no significant changes (25%) of these PFAS in the subjects. One hypothesis for the observed changes in EOF may be exposure to fast-eliminating compounds like TFA, which can cause fluctuations due to its short half-life (16 hours) and its significant contribution to the total PFAS. However, this assumption is based on the direct exposure to TFA, the parent compound, without taking into consideration the duration of transformation from unknown precursor compounds to TFA and some other unknown compounds. Nine individuals showed significant changes in TFA concentrations, but only 3 pairs showed significant changes in both EOF and TFA. Further characterization of unrecognized EOF present by using chemical oxidation may help interpret the results.