Short summary

In 2021 Danmark had the first PFAS contamination case situated i Korsør. Members of a Korsør Kogræsserforening had had cattle to graze on the contaminated area, and the cattle had elevated PFOS levels in their body. We send out an exposure questionnaire and offered the recipients PFAS bloodtests. We want to investigate if the recipients of the contaminated meat have have elevated serum PFAS and if yes, to examine the association between estimated amount of ingested meat and serum PFAS.

Rationale

Background Per- and polyfluorinated alkyl substances (PFAS) is a collective term for wide range of more than thousand chemical substances used on a large scale due to their chemical properties as water and grease repellent. Since the 1950's PFAS have been largely used in the manufacturing of clothes, carpets and other textiles, as impregnation of paper and cardboard, in connection with chrome-plating processes and in fire-extinguishing foam (Datasheet MST). Due to their detrimental environmental impact and negative health effects, PFAS' production and use have been subject to restrictions worldwide within the last decades. Since 2006, it has been forbidden to produce firefighting agents with PFOS and since 2011 all stored firefighting foam based on PFOS had to be destroyed. The European Chemicals Agency have for instance classified several PFAS as 'substances of very high concern (1) PFAS are persistent chemicals and some of the most used PFAS perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) have half-lives in the environment of 41 and 92 years respectively. Data thus indicate that no significant environmental degradation occurs as the substances are not biodegradable either in aerobic or non-aerobic conditions in water or soil (2)

PFAS bio accumulate in humans (3), for example PFOS concentrates in the lung, liver, kidneys and blood (bound to albumin) but is also found in several other human organs (CDC). PFOS' half-life is reported to be between 3.1 and 7.4 years (CDC). A Swedish cohort study with 106 participants has found that humans excrete about 20% of PFOS corresponding per year to a half-life of 3.4 years (4). PFAS is primarily excreted in urine and to a lesser extent in feces as well as in breast milk and during menstruation. Only free non-protein-bound PFAS is excreted in the urine (5). PFAS is reabsorbed in the intestine via the enterohepatic circulation (5) thus PFOS is excreted in the bile and reabsorbed in the small intestine in repeated cycles of excretion and absorption. Women eliminate PFAS faster than men, partly by blood loss during menstruation and partly during pregnancy and breastfeeding, where PFAS is transferred to the fetus/child. Other factors found to affect PFAS' half-life are age, kidney function and inflammation of the gut (4). Furthermore, a time-dependent PFAS elimination process is also suggested, with a more rapid elimination in the first year after end of exposure, and could be explained by a multi compartment mechanism of PFAS elimination with the serum compartments being replenished by from other compartments (i.e kidney and liver) at a slow rate (4). This hypothesis substantiates from animal studies, which, however, have had much higher PFAS concentrations than observed in exposed communities (2). There have been numerous cases worldwide where people have been extraordinarily exposed to PFAS either through work or through the environment by living near a source of pollution (6). For example, in 2013 in the Swedish city Ronneby the drinking water was discovered to be contaminated with PFAS by a nearby firefighting school, where large amounts of PFAS-containing firefighting foam had been used (7). Contamination of the drinking water was also found in the Veneto Region, Italy in 2013 caused by a nearby factory, which produced PFAS (8). A biomonitoring program in Flanders, Belgium found high levels of PFAS in the blood of people living around a PFAS-producing factory, especially those who consumed egg from their own free-range chicken (9).

PFAS has been associated with a wide range of health effects. Several reviews on epidemiological studies found an association between the most common PFAS and decreased antibody response to vaccine, decreased birth weight, increased cholesterol and increased risk of kidney cancer. Furthermore, effects on blood pressure during pregnancy, altered liver enzymes and thyroid function, increased risk of breast and testicle cancer and of ulcerative colitis have also been observed, but the evidence of a potential causal association is limited (2,10). According to PFOS and PFHxS, causal relationships have not been established. Yet, based on the available epidemiological studies, there is a significant suspicion that exposure to these compounds is associated with a risk of increased cholesterol in children and adults (11-15), a small reduction in birth weight (16,17), and reduced antibody response after vaccination among children and adults (18-21).

In the autumn 2020, high levels of PFOS and perfluorohexanesulfonic acid (PFHxS) were detected in a wastewater treatment plant in Korsoer, Denmark, and the source of contamination was a firefighting school (22). In February 2021 subsequent analyses of the meat from four calves, who had grazed in a field between the firefighting school and the lake Korsoer Nor in the previous summer revealed high levels of PFOS (156-230 nanogram/gram) and PFHxS (2.6-4.0 nanogram/gram) (22). The cattle had been the main source of beef intake for members of a local cow grazing association since 1999. The sum of these two PFOS and PFHxS values was 63 times higher than the tolerable intake of PFAS per person per week of 2.9 microgram/kg for the sum of four different PFAS (PFOS, PFOA, PFNA and PFHxS) according to EFSA (23). The members of the cow grazing association were advised to stop consumption of the meat immediately.

Knowledge about absorption, accumulation and steady-state plasma concentrations of PFAS over time has been gathered in animal models to extrapolate to humans. (5) Absorption from the gastrointestinal tract is simulated as the balance between first-order absorption in both animals and humans, but there are large differences in elimination rates between species which is expected to result in substantially different internal steady-state doses (i.e body burdens, serum concentrations) (5,24). Models to predict kinetics in animals and humans are developed (25) Data in the monkey models were based on a single-dose intravenous and oral exposure and repeated-dose oral exposure. The data used in evaluating the human model were serum PFAS in humans who had been exposed either from the environment or by working with fluorochemicals, but in these population studies PFOA and PFOS intakes and exposure durations were not known with certainty, and therefore these data cannot predict exact intake-plasma level relationships (25). Furthermore the biokinetic is complex and serum PFAS concentration in humans may be driven by other factors than just the current concentration of PFAS from a contaminated source (24). Our case is to our knowledge the first where humans been exposed to PFAS from contaminated beef from a PFAS "hotspot". We therefore want to investigate if the recipients of the contaminated meat have elevated serum PFAS and if yes, to examine the association between estimated amount of ingested meat and serum PFAS.

Description of the cohort

2.1Study population As our cohort comprises of members from the local cow grazing association who had eaten meat contaminated with only PFOS and PFHxS, this study solely focus on PFOS and perfluorohexanesulfonic acid (PFHxS), as it was the two types of PFAS found in contaminated cattle (22). The head of the Korsoer cow grazing association contacted all former and current members by phone or e-mail asking whether they wanted to participate in the study. The members of the association as well as those in their household and friends, who received meat from the association in a regular basis were asked to contact the Department of Occupational and Social Medicine in Holbaek if they wanted to participate in the study. They comprised the source population of this study. Shortly after, the participants received a questionnaire electronically or by mail regarding information on demographic, exposure, work, health, medication and other factors of relevance for PFOS elimination. To be included in the study population the participants must deliver the blood tests and complete the questionnaire with information on social security number and sex, period of membership in the years 1999-2020, amount of meat received per membership year in kg and number of persons in the household who have shared the meat. All of those who have taken the blood test will be offered medical consultation and individual risk counselling at the Department of Occupational and Social Medicine, Holbaek Hospital.

Data and biological material

2.2 The blood samples Blood samples is collected between May 2021 to December 2021. Blood levels of five common PFAS are available for each study participant. The specific chemical compounds are PFOS, PFHxS, PFOA, Perfluorodecanoic acid (PFDA) and perfluorononanoic acid (PFNA). Analyzes are carried out by the Laboratory Section of Clinical Pharmacology, Pharmacy and Environmental Medicine (KFFM), Institute for Health Services Research, University Hospital of Odense . As a part of the medical consultation and the risk counselling blood levels of potassium, sodium, creatinine, e-GFR, ALT, cholesterols, triglyceride, HbA1C, glucose and TSH are collected for each participant at the same time the PFAS samples were collected. These tests were chosen as biomarkers of possible health effects related to PFAS exposure. The results are registered in the electronic patient records.

2.3 The exposure questionnaire and exposure parameters Based on a questionnaire on exposure to PFAS developed by the CDC (Adult questionnaire - OMB 0923-0063, Multi-site Study Child Questionnaire - Short Form - OMB 0923-0063) we develop a questionnaire to describe the amount of potentially contaminated meat received per membership year - expressed in kg - in the period of 1999-2020 along with the number of persons in the household, who have shared the meat. We also retrieve demographic information (age and gender) as well as working activities in the period of 1999-2020 and medical history. In addition, information about possible additional exposure: intake of fish / shellfish caught from the nearby lake (Korsoer Nor), bathing in Korsoer Nor, residence as well as information about cultivation and intake of vegetables / berries in soil within a radius of 2 km from the fire school, as well as work history. General questions about diet, exercise and lifestyle, as well as health history, including factors such as blood transfusion and donation, and for women, questions about menstruation, contraception, pregnancy and childbirth, the baby's health and breastfeeding period.

2.4 Supplementary exposure information Quantity registrations from the cow grazing association with the exact amount of meat received per households in the period 2015-2020 were available for 49 households. Furthermore, the cow grazing association had registered that the calves grazed four months per year on the contaminated area nearby the firefighting school in the period 1999-2012. After 2013, the calves grazed two months on the contaminated site, and then two months on a non-PFAS contaminated area.

Collaborating researchers and departments

Klinisk Farmakologi, Farmaci og Miljømedicin, Institut for Sundhedstjenesteforskning Syddansk Universitet

• Flemming Nielsen, seniorforsker, Ph.d.
• Philippe Grandjean MD, DMSc, Professor
• Tina Kold Jensen MD, Professor

Korsør Kogræsser- og Naturplejeforening

• Kenneth Nielsen, formand for Korsør Kogræsser - og Naturplejeforening