Chickens’ eggs and the livers of farm animals as sources of perfluoroalkyl substances

Sampling of chickens’ eggs (n = 25), cows’ livers (n = 10), chickens’ livers (n = 7) and horses’ livers (n = 3) was carried out according to the 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 (10). Eggs were collected from both free-range and cage production. Samples were collected by the Veterinary Inspectorate in various regions of Poland. The liver samples were taken from the Podlaskie, Świętokrzyskie, Mazowieckie, Wielkopolskie and Pomorskie voivodeships. Eggs were sampled from free-range production (n = 7) in the Lubuskie, Opolskie, Wielkopolskie and Lubelskie voivodeships and from cage production (n = 18) in the Małopolskie, Podkarpackie, Wielkopolskie, Dolnośląskie, Śląskie and Kujawsko-Pomorskie voivodeships.

The compounds which were investigated and their limits of quantification (LOQ) and measurement uncertainty (MU) are listed in Table 1. Commission Recommendation (EU) 2022/1431 sets requirements regarding the LOQ which are 0.10 μg/kg for PFS, PFOA, PFNA and PFHxS in the meat of terrestrial animals and 0.30 μg/kg for these substances in eggs (11). The LOQ requirements were met for all compounds for both matrices.

The isotope dilution technique which was applied called for the use of the following isotopically labelled analogues: sodium perfluoro-1-(2,3,4-¹³C₁₂)butanesulphonate, sodium perfluoro-1-hexane(¹⁸O₂)sulphonate, sodium perfluoro-1-(1,2,3,4-¹³C₁₂)octanesulphonate, perfluoro-n-(1,2,3,4,6-¹³C₁₂)hexanoic acid, perfluoro-n-(1,2,3,4-¹³C₁₂)heptanoic acid, perfluoro-n-(1,2,3,4-¹³C₁₂)octanoic acid, perfluoro-n-(1,2,3,4,5-¹³C₁₂)nonanoic acid, perfluoro-n-(1,2-¹³C₁₂)decanoic acid, perfluoro-n-(1,2,3,4,5,6,7-¹³C₁₂)undecanoic acid and perfluoro-n-(1,2-¹³C₁₂)dodecanoic acid. Perfluoro-(1,2,3,4,5,6,7,8-¹³C₁₂)octanesulphonate was used as a recovery standard. All standards were supplied by Wellington Laboratories Inc. (Guelph, ON, Canada).

Samples were freeze dried and a 2 g mass of lyophilised sample was spiked with an isotopically labelled standard. Extraction was carried out using 10 mL of 0.01M methanol (LGC Standards, Wesel, Germany) and potassium hydroxide (Honeywell Fluka, Seelze, Germany). After being shaken for 1 min, samples were left for 20 h. Next, two steps of purification were performed. In the first cleanup step, Oasis WAX solid-phase extraction cartridges (6 mL, 150 mg) were used (Waters Corp., Milford, MA, USA), and in the second step ENVI-Carb Solid Phase sorbent (6 mL, 500 mg) (Supelco, Bellefonte, PA, USA) was applied. Before detection, recovery standard was added. Liquid chromatography–tandem mass spectrometry was selected for detection (Triple Quad 7500 system; Sciex, Framingham, MA, USA). A Gemini C18 column (3 μm, 50 × 2.0 mm) equipped with a guard column (Phenomenex, Torrance, CA, USA) was used for chromatographic separation. Methanol and 20 mM of ammonium acetate aqueous solution (Merck, Darmstadt, Germany) were used as a mobile phase.

A blank sample and the certified reference material IRMM-427 Pike-perch, supplied from the European Commission Joint Research Centre Institute for Reference Materials and Measurements (JRC, Geel, Belgium), were analysed with each batch of samples. All reagents and chemicals used were verified to be PFAS free before routine analysis. The method was accredited in accordance with the ISO 17025:2018-02 standard. Performance of the method meeting the necessary standard was confirmed by successful participation in proficiency testing organised by the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (EURL, Freiburg, Germany).

The normality of the distribution of the data was checked by the Shapiro–Wilk test. Differences between individual experimental groups were checked using the Mann–Whitney and Kruskal– Wallis tests.

Concentrations are expressed as μg/kg wet weight. Sum concentrations are given as lower-bound (LB) concentration (concentrations below the LOQ were replaced with the value of 0).

There is no data on average liver consumption in Poland, which is why one portion of 50 g and one of 100 g consumed per week were used for PFAS intake calculation. In terms of eggs, the average Polish weekly consumption of three eggs (38) and double this average were applied. Each egg was taken to weigh 50 g. The consumer’s body weight was assumed to be 23.1 kg for a 3–10-year-old child and 70 kg for an adult, according to EFSA guidelines (14). To characterise the potential health risk associated with ∑4 PFAS intake, doses ingested with milk and liver were compared to the TWI (4.4 ng/kg b.w. per week).

Fig 1.

The mean LB ∑4 PFAS concentration was the highest in cows’ livers (0.52 μg/kg), the next highest in chicken’ livers (0.17 μg/kg), lower in horses’ livers (0.13 μg/kg) and the lowest in chickens’ eggs (0.096 μg/kg). None of the samples exceeded the maximum levels for ∑4 PFASs set by Commission Regulation (EU) 2023/915 (Table 2). The concentration-to-limit ratio for ∑4 PFASs was <7% for liver and <6% for eggs. A 64% proportion of all analysed compounds was ∑4 PFASs in cows’ and chickens’ livers, a 68% proportion of all compounds was ∑4 PFASs in horses’ livers and 100% was ∑4 PFASs in eggs. Livers were significantly more contaminated than eggs (P-value < 0.03). None of the compounds were detected in eggs from cage production.

The compound detected most frequently was linear PFOS (L-PFOS) (8% in eggs and 48% in all livers). In cows’ livers it was detected in 80% of samples. Besides having the highest detection frequency, this compound also reached the highest concentration in cows’ and chickens’ livers and in eggs (Table 2). The highest level of L-PFOS was in cows’ livers (0.24 μg/kg), where it accounted for 46% of the ∑4 PFASs; together with branched PFOS (br-PFOS) the share of ∑4 PFASs increased to 63%. Branched PFOS was much more frequently detected in cows’ (90%) and horses’ (80%) livers than in chickens’ livers (14%), while no samples of eggs contained this contaminant. The ratio of br-PFOS to ∑PFOSs was the highest in cows’ livers (mean 28%) and markedly lower in horses’ and chickens’ livers (15–18%). The total PFOS concentration-to-limit ratio was <8% for eggs, 6% for cows’ livers and <2% for chickens’ and horses’ livers.

Perfluorononanoic acid was the compound reaching the second highest levels (0.21 μg/kg in cows’ livers). Perfluorooctanoic acid was measured at the highest concentration in chickens’ livers but was found only in one sample, where compared to its concentration in other analysed liver samples and eggs it was at a high concentration of 0.17 μg/kg; however, in relation to the maximum level this contaminant’s concentration was <25% (Table 2).

For PFNA the mean concentration-to-limit ratio was <53% for cows’ livers, and was lower for chickens’ livers at 25%, for horses’ livers at <7% and for eggs at <5%. One sample of cow’s liver exceeded the maximum level for PFNA (0.40 μg/kg) set by Commission Regulation (EU) 2023/915 (Table 2). Perfluorohexanesulphonic acid was only detected in one chicken liver sample (0.047 μg/kg) and had a concentration of around 10% of the maximum level.

None of the analysed samples contained quantifiable concentrations of PFHxA, PFHpA or PFPeS. Chickens’ eggs contained only one of the remaining analysed PFASs (PFUnDA), which was found only in one sample of free range eggs in a concentration slightly over the LOQ (0.030 μg/kg). Perfluorodecanoic acid and PFUnDA were found in all cow’s liver samples, PFDoA in over 40% of them and PFHpS in 33%, but PFBS was seen in only one sample. The following compounds were detected in the livers of horses: PFDA, PFuDA, PFDoA, PFBS and PFHpS.

The estimated intakes of ∑4 PFASs were the highest when cows’ livers were eaten and were 0.37 ng/kg b.w. for a 50 g portion and 0.74 ng/kg b.w. for a 100 g portion for adults and 1.13 ng/kg b.w. and 2.25 ng/kg b.w. for the same portions for children (Fig. 2). This corresponds to exposure of up to 17% of the TWI for adults and over 50% of it for children. A much lower intake was calculated for consumption of chickens’ and horses’ livers by adults (0.09–0.24 ng/kg b.w. from the smaller portion of the less contaminated of the two to the larger portion of the more contaminated) and children (0.28–0.74 ng/kg b.w. analogously) (Fig 2.). Referring these values to the TWI, exposure would be <6% for adults and <17% for children (Fig. 3). Dietary intake for children via the average portion of three eggs would be 0.62 ng/kg b.w., which corresponds to exposure of <15% of the TWI, and intake for adults would be 0.21 ng/kg b.w., amounting to <5% of the TWI. A six-egg portion would double the exposure but still leave it far below 100% of the TWI (Fig. 3).

Fig 2.

Fig 3.

To reach the TWI dose based on mean LB concentrations, a child would have to consume around 200 g of cow’s liver, 600 g of chickens’ livers, almost 800 g of a horse’s liver, or 22 chickens’ eggs. The same simulation for adults indicates that they would have to consume 600 g of cow’s liver, 1.8 kg of chickens’ livers, almost 2.4 kg of a horse’s liver, or 64 chickens’ eggs.