Occurrence of perfluoroalkyl substances in cow’s, goat’s and sheep’s milk – dietary intake and risk assessment

Altogether, 73 milk samples from cows (n = 38), goats (n = 20) and sheep (n = 15) were collected from various regions of Poland by the Veterinary Inspectorate (Fig. 1). Sampling was carried out according to Commission Implementing Regulation 2022/1428 of 24 August 2022 laying down methods of sampling and analysis for the control of perfluoroalkyl substances in certain foodstuffs. Samples were analysed by the National Reference Laboratory for halogenated POPs at the Radiobiology Department of the National Veterinary Research Institute, Puławy, Poland.

Fig. 1.

Fourteen PFASs were investigated: perfluorobutanesulfonic acid (PFBS), perfluoropentanesulfonic acid (PFPeS), PFHxS, perfluoroheptanesulfonic acid (PFHpS), linear and branched PFOS (l-PFOS and br-PFOS), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, PFNA, perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoA), perfluoro-tridecanoic acid (PFTrDA), and perfluorotetradecanoic acid (PFTeDA).

Isotopically labelled analogues were used: sodium perfluoro-1-(2,3,4-¹³C₃) butanesulfonate, sodium perfluoro-1-hexane(¹⁸O₂)sulfonate, sodium perfluoro-1-(1,2,3,4-¹³C₄) octanesulfonate, perfuoro-n-1,2,3,4,6-¹³C₄) hexanoic acid, perfluoro-n-(1,2,3,4-¹³C₄) heptanoic acid, perfluoro-n-(1,2,3,4-¹³C₈) octanoic acid, perfluoro-n-(1,2,3,4,5-¹³C₅) nonanoic acid, perfluoro-n- (1,2-¹³C₂) decanoic acid, perfluoro-n-(1,2,3,4,5,6,7-¹³C₇) undecanoic acid, perfluoro-n-(1,2-¹³C₂) dodecanoic acid, and perfluoro-n-(1,2-¹³C₂) tetradecanoic acid. Perfluoro-(1,2,3,4,5,6,7,8-¹³C₁₂) octanesulfonate was used as a recovery standard. All standards were purchased from Wellington Laboratories Inc. (Guelph, ON, Canada).

Oasis WAX solid-phase extraction (SPE) cartridges (150 mg, 6 mL) were used from Waters Corp. (Milford, MA, USA) and ENVI-Carb SPE sorbent (500 mg, 6 mL) was selected from Supelco (Bellefonte, PA, USA). Methanol was obtained from LGC Standards (Wesel, Germany), sodium hydroxide pellets (>98%) were purchased from Honeywell Fluka (Seelze, Germany), ammonium acetate and ammonium solution 32% were ordered from Merck (Darmstadt, Germany), ultrapure water (18.2 MΩ/cm) was sourced from Supelco and acetic acid was procured from J.T. Baker (Phillipsburg, NJ, USA).

Raw milk samples (250 mL) were freeze dried and 2 g masses of lyophilised sample were fortified with isotopically labelled standards. Extraction was carried out using 10 mL of 0.01M methanol/ potassium hydroxide. Samples were shaken for 1 min and left for 20 h. Purification was performed on an Oasis WAX (150 mg, 6 mL) SPE cartridge and ENVI-Carb SPE sorbent (500 mg, 6 mL). Recovery standards were added before liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

Concentrations of analytes were determined using LC-MS/MS on a Triple Quad 7500 system (Sciex, Framingham, MA, USA) operated in negative electrospray ionisation (ESI–) mode. For chromatographic separation, a Gemini C18 column (3 μm, 50 × 2.0 mm) equipped with a guard column (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of 20 mM of ammonium acetate aqueous solution and methanol. The limits of quantification (LOQ) were between 0.001 μg/kg and 0.004 μg/kg and met the criteria set down in Commission Recommendation (EU) 2022/1431 of 24 August 2022 on the monitoring of perfluoroalkyl substances in food. Based on this recommendation, the LOQ for milk analysis should be at or below 0.010 μg/kg for PFOS and PFOA, 0.020 μg/kg for PFNA and 0.040 μg/kg for PFHxS. Acquisition and processing were performed using the Sciex OS software.

The SPE cartridges and all solvents were checked for purity before routine analysis. Blank samples and IRMM-427 Pike-perch certified reference material purchased from the European Commission Joint Research Centre Institute for Reference Materials and Measurements (JRC, Geel, Belgium) were analysed with each series of samples to achieve internal quality assurance and control (QA/QC). Successful participation in proficiency testing (PT) served as external QC organised by the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (EURL, Freiburg, Germany).

The Shapiro–Wilk test was used to verify the normal distribution of the data. Differences between individual experimental groups were checked using the Mann–Whitney and Kruskal–Wallis tests (at p ≤ 0.05). To find associations between occurrences of PFASs and fat content, Spearman’s rank correlation coefficient was used.

Concentrations of individual PFASs and the sum concentration are expressed as μg/kg of milk. Sum concentrations are given as lower-bound (LB) concentration (concentrations below the LOQ were replaced with the value of 0) and as upper-bound (UB) concentration (concentrations below the LOQ were replaced with the value of the LOQ).

Dietary intake was calculated for the statistically average monthly milk consumption in Poland of 3.5 L (43) and for the recommended daily milk consumption of 0.5 L. To characterise the potential health risk associated with ∑4 PFAS intake, doses ingested with milk were compared to the TWI (4.4 ng/kg b.w. per week). Body weight was assumed to be 70 kg for an adult and 23.1 kg for a 3–10-year-old child (9).

The LB ∑4 PFAS concentrations declined from their highest in sheep’s milk to lower ones in goats’ milk and the lowest in cow’s milk (Fig. 2). Statistically significant differences were observed both between sheep’s milk and cow’s milk (p = 0.002) concentrations and between goat’s milk and cow’s milk (p = 0.027) concentrations, but the goat’s milk and sheep’s milk LB ∑4 PFAS concentrations did not differ significantly (p = 0.401). The LB ∑14 PFAS concentrations descended in order from sheep’s milk to goat’s milk and lastly to cow’s milk (Fig. 2). The cow’s milk LB ∑14 PFAS concentration did not differ significantly from that of goat’s milk (p = 0.057) but was statistically significantly lower than that of sheep’s milk (p = 0.005). No differences between voivodeships in LB ∑4 PFAS levels in cow’s, goat’s or sheep’s milk (p = 0.878, p = 0.984 and p = 0.771, respectively) were found.

Fig. 2.

The LB ∑14 PFAS concentrations also did not differ significantly between voivodeships (at p = 0.982 for cow’s milk, p = 0.912 for goat’s milk and p = 0.999 for sheep’s milk). The Lubelskie and Świętokrzyskie voivodeships were not included in statistical analysis respectively for goat’s and cow’s milk because the number of samples was insufficient.

Summarised data on four PFAS concentrations in the analysed milk samples from the three species are presented in Table 1. None of the samples exceeded the indicative values established by Commission Recommendation (EU) 2022/1431, and even the maximum detected concentrations were an order of magnitude lower. Only in one, which was the most contaminated goat’s milk sample, was the sum br-PFOS and l-PFOS (∑PFOS) concentration result (0.0174 μg/kg) close to the indicative value of 0.020 μg/kg, but the mean concentration was below 24% of the IL. The mean PFOA concentrations were almost equal in the three types of milk and were below 17% of the IL. For PFNA, this share in relation to the limit was smaller at <4%. In the only sample in which PFHxS was detected, the concentration-to-limit ratio was <5%.

The most frequently detected substance was l-PFOS, which was found in 33%, 76% and 93% of cow’s, goat’s and sheep’s milk samples, respectively. Branched PFOS was much less frequently found in cow’s (5%) and goat’s milk (35%) but was detected in similar abundance to l-PFOS in sheep’s milk samples (87%). The highest mean levels of l-PFOS and ∑PFOSs transpired to be in goat’s milk at 0.0033 μg/kg and 0.0048 μg/kg, respectively, but no statistical significance (p = 0.197, p = 0.414) was found between species (Fig. 2) in these concentration differences. Perfluorooctanoic acid was the second most frequently occurring compound and was detected in 13% of cow’s milk, 35% of goat’s milk and 47% of sheep’s milk samples. The mean concentrations (0.0016–0.0017 μg/kg) did not differ significantly between species (p = 0.981). The similarity in concentrations between PFOS and PFOA despite the greater transmission to milk of the former may suggest that cows were exposed to much higher levels of PFOA than PFOS.

Perfluorononanoic acid was detected to a lesser extent in cow’s milk, found only in 5% of samples, and in goat’s milk, noted in 12% of samples, than in sheep’s milk. This perfluoroalkyl substance was identified in 53% of sheep’s milk samples. The mean levels in all three milk types showed similarity (0.0014–0.0016 μg/kg).

Perfluorohexanesulfonic acid contaminated only a single sample of goat’s milk, where its concentration was 0.003 μg/kg.

Excluding one PFHxS detection result in goat’s milk, the percentage shares in the LB ∑4 PFASs indicate the dominant role of PFOS: 53% and 40% in goat’s and sheep’s milk, respectively. Contrastingly, in cow’s milk the proportions of PFOS, PFOA and PFNA were almost equal.

The occurrence of the rest of the analysed PFASs in milk samples from the three species is presented in Table 2. Generally, the levels found for most of the analysed PFASs in milk were low and below or only slightly above the LOQ. This is in line with European data, in which most PFASs were not detected in milk, and the detected compounds were noted with a frequency of below 4% (8). Regardless of the species, PFPeS and PFHpS were not detected in any samples. Differences between species in some compounds’ occurrences were noticed. Perfluorohexanoic acid was not found in any cow’s or goat’s milk, while it was detected in 20% of sheep’s milk samples, concentrated at 0.0055 μg/kg (mean).

No sample of cow’s milk contained PFHpA or PFTrDA, whilst goat’s and sheep’s milk samples yielded both compounds. The frequencies of PFHpA detection were 6% and 13% and for PFTrDA 12% and 27% in goat’s and sheep’s milk, respectively. The compound which transpired to be the most abundant was PFTeDA, being in 13% of cow’s milk, 29% of goat’s milk and 33% of sheep’s milk samples. This compound also occurred in the highest concentrations of all 14 investigated PFASs, peaking in sheep’s milk at a mean 0.0073 μg/kg. Interspecies differences in its concentration were not significant (p = 0.704).

Based on mean UB ∑4 PFAS concentrations and average full milk consumption, the estimated intakes of PFASs were 0.153–0.266 ng/kg b.w. for children and 0.050–0.88 ng/kg b.w. for adults (Table 3), which indicates that exposure is very low and is merely <7% of the TWI for children and <2% of the TWI for adults (Fig. 3).

Fig. 3.

When 95^th percentile concentrations are considered, exposure increases by approximately 30% from cow’s milk consumption and approximately 80–100% from goat’s and sheep’s milk. However, relating these values to the TWI raises the fraction of the intake to <12%. Calculating exposure based on LB concentrations, the intake would be over 80% lower for cow’s milk, 27% lower for sheep’s milk and 37% lower for goat’s milk.

Since the recommended portion of milk is over four times larger than the average portion, exposure based on mean UB ∑4 PFAS concentration from ingestion of the recommended portion would increase by a factor of four. Intake would be <26% and <9% of the TWI for children and adults, respectively, and based on the 95^th percentile concentration would be <48% and <16% of the TWI (Fig. 3). Goat’s and sheep’s milk are less popular and consumed in lesser amounts, especially by children, but cow’s milk should not be of concern despite being consumed more often. Cow’s milk is safe, since the estimated intake via the recommended weekly consumption of 3.5 L was <20% of the TWI for children and <7% for adults (based on the 95^th percentile UB concentration). The calculation based on the upper-bound concept is an overestimation, and exposure based on the LB 95th percentile concentration would be <12% of the TWI for children and <4% for adults.

To reach the TWI dose, an adult would have to drink 70 L and a child over 23 L of cow’s milk contaminated at the average UB level in a week. This suggests that neither the statistical average nor the recommended consumption of cow’s, goat’s or sheep’s milk analysed in this study contributes significantly to the overall PFAS intake, even when the milk is the most contaminated.