LGC AXIO Proficiency Testing investigates what are per and polyfluoroalkyl substances (PFAS), what are the dangers of these compounds, and what are global bodies doing to regulate their contamination of the environment.
Emerging Pollutants of Environmental Concern Volume 2: Per and polyfluorinated alkyl substances (PFAS)
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LGC AXIO Proficiency Testing | Emerging Pollutants of Environmental Concern: PFAS
Introduction
The attention of analytical companies and legislators is moving from the ‘traditional’ pollutants to a new group of “emerging” environmental contaminants. They are increasingly of concern due to their widespread occurrence, their potential toxicity to mammals or other biota and their ability to persist in the environment, often resulting in bioaccumulation. Typically, emerging pollutants are introduced into the environment as a result of human interactions and processes. Over 350,000 chemicals and mixtures of chemicals have been registered for production and use, up to three times as many as previously estimated and with substantial differences across countries/regions. The chemicals bring many benefits to the society, but overuse is causing harm to the environment and consequently to our health. The determination of this wide range of compounds may require novel analytical techniques and/or monitoring strategies to determine concentrations in the µg/L concentration range or lower. To accurately determine the environmental fate of the emerging pollutants the sampling and analysis of water, soil, sediments, and biota must be carried out. In many cases the metabolites of pollutants may be as toxic as the parent compounds themselves or in some cases more toxic. The formation of metabolites, either by environmental processes such as microbial degradation, photolysis, and hydrolysis or by reaction with disinfectants in drinking water or during wastewater treatment. In this whitepaper we will explore the potential concerns surrounding Per and polyfluorinated alk yl substances (PFAS).
Definitions
" Emerging substances " can be defined as “substances that have been detected in the environment, not currently included in routine monitoring programmes and whose fate, behaviour and (eco)toxicological effects are not well understood”. [1] " Emerging pollutants " can be defined as “pollutants not currently included in routine monitoring programmes at the European level, but may be candidates for future regulation, dependant on: • Further research of their (eco)toxicity • The potential health effects • Public perception • Monitoring data regarding their occurrence in the various environmental compartments. [1]
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What are Per and polyfluorinated alkyl substances (PFAS)?
Per and polyfluorinated alkyl substances (PFAS) are a group of over 4,500 widely used, synthetic chemicals, that accumulate over time in the environment and in the human body. The most widely used, and most well-known PFAS, are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS). They are used in a wide variety of consumer products, some examples include; non-stick metal coatings for cooking pans, stain resistant coatings on carpets and textiles, paper food packaging, firefighting foams, lubricants, and personal care products such as eye makeup.
Figure 1 Perfluorooctanoic Acid (PFOA)
Perfluorooctane sulfonic acid (PFOS)
How to identify a PFAS? Per and polyfluoroalkyl substances (PFASs) are a family of synthetic chemicals that contain one or more Carbon atoms whereby all the Hydrogen substituents (present in the non- fluorinated analogues from which they are notionally derived) have been replaced by Fluoride atoms, in such a way that they contain the perfluoroalkyl moiety (CnF2n+1–). [4] The polyfluoroalkyl compounds have some H atoms left and not all are replaced with fluorine. There are also “ultra-short- chain” PFAS. [10]. Due to the high electronegativity of fluoride, the C-F structure is one of the strongest chemical bonds in organic chemistry. This means that compounds which contain this bond, do not easily degrade and are therefore extremely persistent and accumulative. This is one of the reasons that PFAS are sometimes referred to as “Forever Chemicals”. PFAS are also frequently described as “long-chain” or “short-chain”. The Organisation for Economic Co-operation and Development (OECD 2011, [5]) defines “long-chain” PFAS as: • Perfluoroalkyl carboxylic acids with eight carbons and greater (with seven or more perfluorinated carbons) • Perfluoroalkane sulfonates with six carbons and greater (with six or more perfluorinated carbons). In contrast, “Short-chain” PFAS refers to: • Perfluoroalkyl carboxylic acids with seven carbons and less (with six or less perfluorinated carbons) • Perfluoroalkane sulfonates with five carbons and less (with five or less perfluorinated carbons). 3
LGC AXIO Proficiency Testing | Emerging Pollutants of Environmental Concern: PFAS
How do they enter the environment?
Due to the wide range of contaminants, there are many mechanisms for entry into the environment. This includes; • Directly via pollution incidents • Over-spray during application • Disposal of waste materials • Indirectly via surface run off from agricultural land • Roads • Flood control systems Another significant route of entry is via the discharge from wastewater treatment as the effluents are a significant source for many of these
RISKS
~ 100 000 chemicals on the market
~ 500 chemicals extensively characterised for their hazards and exposures
EXPOSURES
HAZARDS
~ 10 000 chemicals fairly well characterised for a subset of their hazards and exposures ~ 20 000 chemicals with limited characterisation for their hazards and exposures
~ 22 600 chemicals with a use over 1 tonne per year
~ 4 700 chemicals with a use over 100 tonnes per year prioritised in hazard characterisation and evaluation
~ 70 000 chemicals with poor characterisation for their hazards and exposures
Figure 2 Chemical risks - The European environment - state and outlook 2020 report [17]
emerging contaminants. This is because of their extensive use in household products. After use, these chemicals are released into the wastewater system, many are not completely removed during wastewater treatment, thus, they enter rivers, and drinking water supplies. As shown in figure 2, it is estimated that robust information exists only for about 500 chemicals and according to the European Chemicals Agency (ECHA, 2019), 450 are sufficiently regulated. [17]
REACH is a regulation of the European Union that was adopted to assist the protection of the environment and human health from chemical hazards, while at the same time taking into account the needs of the chemicals industry. REACH stands for Registration, Evaluation, Authorisation and Restriction of Chemicals and it entered into force on 1 June 2007. [16] CAS REGISTRY®, the CAS substance collection includes 194 million organic and inorganic substances with a few thousand substances added every month. CAS REGISTRY officially became a self-supporting division of the American Chemical Society in 1956. It contains substances identified in the literature from 1957 to present, with some substances dating back to the early 1900s .
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PFAS Isomers
PFHxS 19F -NMR spectrum
PFNA 1 9F -NMR spectrum
During the last few years, the difference between isomeric forms of PFAS substances has emerged as an additional factor which affects the physicochemical properties of the compounds. This can have an effect in the adsorption on solid phases, the distribution in different environmental compartments etc. Some researchers suggest that linear PFAS are more readily adsorbed to soil and sediments, whereas branched isomers are more likely to remain in water compartments probably due to the differences in polarity between the two isomers. Studies in humans and animals have shown that the linear PFOS isomer is more likely to accumulate in animals whereas in humans, the branched isomer is more likely to accumulate. Above two 19F -NMR spectrums are shown. The one (PFNA) showing only linear chain with signal for the CF3 group at -80 ppm and the CF2 groups in the range of -115 to -125 ppm. On the other hand, the spectrum for PFHxS shows additional signals e.g. at -71 ppm indicating the presence of branched isomers in the product next to the signals for the linear chain.
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LGC AXIO Proficiency Testing | Emerging Pollutants of Environmental Concern: PFAS
The history of PFAS
The discovery of PFAS PFAS substances were actually discovered by accident in the 1930. In 1938, DuPont was conducting research to find new chemicals that could be used as refrigerants when its chemists stumbled upon an unusual coating in one of their test chambers. Testing revealed that the new substance, PTFE (polytetrafluoroethylene), was chemically very stable and had a remarkable ability to repel water and oil. This was the first PFAS ever invented, and it was soon put to good use in the Manhattan Project because it could resist corrosion from fluorine in the gaseous diffusion process used to enrich uranium. After World War II, Dupont marketed this substance in a very successful product it called Teflon that was used in non-stick cookware and water and stain resistant fabrics. The discovery of Teflon is often cited as an example of serendipity, or accidental discovery. The release of Scotchgard Shortly thereafter, 3M discovered its own PFAS chemical, PFOS (perfluorooctane sulfonate), when they were trying to develop a rubber which would not deteriorate from exposure to jet fuel. During their experiments, some of the new substance was dropped on a laboratory assistant’s tennis shoe, and when the assistant tried to clean the substance from her shoe, she discovered that it was impervious to water and alcohol. 3M marketed the new material in 1956 as Scotchgard. The US Navy Base fire Development of these chemicals increased in the late 1960s after a deadly fire aboard a U.S. Navy aircraft carrier, the USS Forrestal, in 1967. The fire resulted from the accidental launch of a rocket into armed planes and loaded fuel tanks. This blaze nearly destroyed the ship and killed more than 130 people. Soon after the tragic incident, manufacturers and scientists developed PFAS-containing aqueous film-forming foam (AFFF) — a foam mixture that rapidly extinguishes fire. The PFAS allow the mixture to spread, making it highly effective against petroleum fires and other flammable-liquid fires when mixed with water. PFAS-containing AFFF was subsequently installed on military and civilian ships, airplanes and airports. The DuPont plant scandal In 1999, Wilbur Tennant, a farmer who lived next to a DuPont plant in West Virginia, sued the company after his cows started acting deranged and dying. During the discovery process in the litigation, Mr. Tennant’s lawyers unearthed DuPont documents showing that the company’s Washington Works factory in Parkersburg, W.Va., had been dumping a type of PFAS into the Ohio River and that the chemicals had contaminated drinking water supplies for more than 100,000 people. Studies connected with this litigation advanced our knowledge of health hazards related to PFAS exposure. PFAS chemicals have been associated with kidney and testicular cancers, reduced immunity, thyroid problems, liver ailments, and developmental issues with fetuses and breastfed infants.
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United Nations Stockholm Conve n tion To eliminate the use of PFAS, at COP-4 in 2009, the Conference of the Parties decided to list perfluorooctane sulfonic acid (PFOS), its salts and perfluorooctane sulfonyl fluoride (PFOSF) the Stockholm Convention. Perfluorooctanoic acid (PFOA), its salts are also restricted. PFHxS and its salts were added to Stockholm convention in 2022. PFOS, its salts and derivatives as well as PFOA, its salts and PFOA-related compounds are banned under the EU Persistent Organic Pollutants (POPs) Regulation. POPs regulation will potentially be updated to include long-chain perfluorocarboxylic acids (PFCA's) (C9-C21), their salts and related compounds. [ 22 ] The European Commission and the member states have committed to phase out all PFAS, allowing their use only where alternatives cannot be used. PFOS and their derivatives are included as a priority hazardous substance under the EU Water Framework Directive (EU, 2013), with a much lower Environmental Quality Standard (AA-EQS) limit value of 0.65 ng/L (0.00065 µg/L) in inland surface waters and 0.13 ng/L in seawater. The majority of PFAS are industrial and consumer chemicals that are regulated under REACH regulation in the EU. According to a document by the Environment Agency, UK (2021) the UK has retained REACH in national legislation following withdrawal from the EU, but it will take several years for UK registration to be completed. [11] Drinking Water Directive The review of the Drinking Water Directive, which took effect in 2021, includes a limit of 0.5 µg/L for all PFAS and 0.1 µg/L for the sum of 20 individual PFAS. This is in line with a grouping approach for all PFASs. Several other PFASs are on the REACH Candidate List of substances of very high concern (SVHC). By January 2026, EU Member States shall take the measures necessary to ensure that water intended for human consumption complies with the parametric values set out in Part B of Annex I of the revised Drinking water directive, including Total PFAS (as defined in the directive) and the sum of PFAS. The Chemicals Strategy for Sustainability of the European Commission sets out a range of actions to regulate PFAS as a group [12]. In April 2024, EPA announced the final National Primary Drinking Water Regulation (NPDWR) for six PFAS. Public water systems must monitor for these PFAS and have three years to complete initial monitoring (by 2027), followed by ongoing compliance monitoring. Water systems must also provide the public with information on the levels of these PFAS in their drinking water beginning in 2027. [13] The ban of some commonly used long-chain PFAS has led scientists and the industry to find alternatives with shorter chain PFAS. Several of these alternatives are now under regulatory scrutiny in the REACH Regulation as they might still, pose a risk for the environment and health.
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In August 2024, the Drinking Water Inspectorate ( DWI ) published a guidance for PFAS in drinking water requiring water companies in England and Wales to monitor for PFAS with a guideline value of 0.1 micrograms per litre for the sum of 48 named PFAS (up from 47), which is equivalent to 0.1 parts per billion. [15] In Development The European Commission is in the process of developing a groundwater testing norm that includes a focus on 24 PFAS, with a proposed standard of 4.4 ng/L (as PFOA equivalents) for both surface and groundwater, as part of the Environmental Quality Standards Directive. [ 23 ] The Committees for Risk Assessment (RAC) and for Socio-Economic Analysis (SEAC) progressed their evaluation of the proposed restriction on PFAS under the EU REACH in March 2025. [ 24 ]
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LGC AXIO Proficiency Testing | Emerging Pollutants of Environmental Concern: PFAS
Why are PFAS a concern?
Within the past decade or so, several long-chain PFAS have been recognized as extremely persistent, bio accumulative and toxic. The distribution of individual PFAS between different environments, depends on the physicochemical properties of each chemical. Short-chain PFAS are more likely to be present in groundwater, whereas Long- chain substances have a higher potential to accumulate as they are more frequently detected in seabirds and other aquatic animals. [6]
Figure 3 PFAS effects on health
The European Environment Agency (EEA) warned that, due to the large number of PFAS, it is a difficult and time-consuming task to assess and manage risks for these substances individually. Although some efforts are being made. National Institute of Environmental Health Sciences (NIH) in the United States, is working with the Environment Protection Agency (EPA) and the Food and Drug Administration (FDA) on PFAS-related food safety, and the Centre for Disease Control and Prevention on monitoring PFAS exposure levels. Although it has been reported that over 95% of adults in the USA, have some amount of PFAS in their blood serum. [7], [8]. This is alarming, as exposure to such persistent and accumulative chemical compounds can be dangerous to our health, demonstrated by Figure 3 . [9] The European Human Biomonitoring Initiative, HBM4EU, was a Horizon 2020 project that identified 9 priority substance groups and work ed to harmonize procedures for human biomonitoring across the 28 participating countries. [2]. According to the HBM4EU Policy brief, around 14% of the teenagers tested across Europe, exceed the internal serum level of 6.9 µg/l PFASs, which equals to the European Food Safety Authority’s (EFSA) guideline value for a tolerable weekly intake of 4.4 ng/kg. Data related to PFAS compounds from 17 Human Biomonitoring-studies can be found on the online European HBM dashboard. [3] According to the “Cost of inaction” report by the Nordic Council of Ministers (2019) PFAS exposure is estimated to cause EUR 52-84 billion across Europe, for annual health related costs in all European Economic Area (EEA) countries. [10] Recent biomonitoring data has shown that people across the globe are substantially exposed to a range of PFAS. [2]
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Analysis of PFAS
Perfluorobutane sulfonic acid (PFBS) Perfluoropentane sulfonic acid (PFPS) Perfluorohexane sulfonic acid (PFHxS) Perfluoroheptane sulfonic acid (PFHpS) Perfluorooctane sulfonic acid (PFOS) Perfluorononane sulfonic acid (PFNS) Perfluorodecane sulfonic acid (PFDS) Perfluoroundecane sulfonic acid (PFUnDS) Perfluorododecane sulfonic acid (PFDoDS) Perfluorotridecane sulfonic acid (PFTrDS)
Perfluorobutanoic acid (PFBA) Perfluoro pen tanoic acid (PF P A) Perfluorohexanoic acid (PFHxA) Perfluorohe pt anoic acid (PFH p A) Perfluorooctanoic acid (PFOA) Perfluorononanoic acid (PFNA) Perfluorodecanoic acid (PFDA) Perfluoroundecanoic acid (PFUnDA) Perfluorododecanoic acid (PFDoDA) Perfluorotridecanoic acid (PFTrDA)
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12 13 14 15 16 17 18 19
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Table 1: List of 20 individual PFAS included in the revised Drinking Water Directive (EU) 2020/2184
The analysis of PFAS presents some considerable challenges for various reasons, one being the actual number of substances defined under PFAS, as well as the physicochemical properties and concentration of the substances in environmental samples. Accurate and robust analytical methods are essential for the detection and quantification of PFAS in water and will assist in the understanding on the prevalence and transportability of these compounds between different environments e.g., between soil and groundwater.
A report was prepared by the Environment Agency through NORMAN (February 2022) and collated information about Liquid Chromatography Mass Spectrometry (LC- MS), sum of PFAS, Total PFAS, Non-target screening (NTS) methods, Total Oxidizable Precursor (TOP) assays, Total Fluorine (TF) and Extractable organofluorine (EOF). [18] In figure 4, (NORMAN Network, 2022) the reported ranges for limits of detections of various PFAS are shown with drinking water, surface water and animals as examples. The Environmental Protection Agency (EPA) has developed, validated, and published three methods to support the analysis of 29 PFAS in drinking water, Method 533, 537 and 537.1. In addition, EPA’s Office of Water, in partnership with the Department of Defence (DoD) Research and Development Program, released Method 1633 in January 2024, which was then revised in December in 2024, reissued as 1633:A. This is a single laboratory validated method to test for 40 PFAS in wastewater, surface water, groundwater, soil, sediment, landfill leachate but also fish tissue. The European Commission has issued a Notice about Technical guidelines regarding methods of analysis for monitoring of PFAS in water intended for human consumption. [ 25 ]
Figure 4 Reported ranges for PFCAs, PFSAs and HFPO-DA limits of detection (LOD) in (a) drinking water (b) fresh surface water and in (c) animals. Red full and dotted lines represent the regulatory limits from the revised Drinking Water Directive of total PFAS (0.5 µg/L) and sum of 20 PFAS (0.1 µg/L). Blue lines represent the Water Framework Directive AA-EQS for PFOS in surface water and in fish [19]
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Non-targeted screening
The analysis of PFAS compounds typically requires the use of LC-MS/MS systems. However, as more PFAS compounds are discovered and more complex matrices are tested, both regulated and unregulated analytical methods will need to adapt. The non- targeted analysis allows the discovery of novel PFAS in the environment. EPA published Method 1621 in January 2024, which is a new screening for the Determination of Adsorbable Organic Fluorine (AOF) in Aqueous Matrices by Combustion Ion Chromatography (CIC) . This method provides an accumulative measurement of chemical substances that contain C-F bonds. The method measures organofluorine compounds f rom PFAS and non-PFAS fluorinated compounds that can be retained on at least 80 mg of granular activated carbon (GAC). The result is reported as the concentration of fluoride (F-) in the sample. EPA advises that this method may be used to estimate the aggregate contributions of the organofluorine compounds present in the sample. It advises that the numerical results generated by this method are not expected to be as accurate or precise as those from targeted methods for PFAS. [20, 21] In addition, Extractable Organic Fluorine (EOF) can be used. In both the cases of AOF and EOF, the inorganic fluorine must be removed.
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Conclusion
PFAS are a group of environmental contaminants which are of increasing concern in many countries. PFAS can be difficult to analyse due to the number of potential 'targets', which often exist in isotopic mixtures or groups of related compounds and which can exhibit different physical behaviour and interactions in the environment. Since PFAS are commonly used in the manufacture of consumer products and laboratory supplies, it is necessary to eliminate any potential sources of contamination during sample collection, transport, preparation, and analysis High quality analysis of PFAS contaminants therefore depends on laboratories having access to reliable and robust testing methodologies. LGC AXIO provides solutions to to support the quality control activities of laboratories undertaking work in this field. Through our range Dr Ehrenstorfer reference materials labs can use fully characterised, including isotopic composition, reference standards during method development, for spike recovery and as routine analytical calibrants. In addition, LGC AXIO supports laboratories by providing proficiency testing samples for the determination of PFAS in an environmental matrix, through the AQUACHECK scheme. Proficiency Testing is a independent assessment of a laboratories competence, testing the entire process from sample receipt to reporting of results and supporting the whole system of quality control.
The Authors
Dr. Matthew Whetton
Dr Matthew Whetton is the Head of Technical Operations for the Proficiency Testing group at LGC Standards and has almost 15 year’s experience in the field. In this role Matthew is responsible for the production, development and technical operation of over fifty proficiency testing schemes. Prior to joining LGC, Matthew has previously carried out a variety of roles in the fields of phytochemistry and analytical services, spending more than 10 years working in the field of analytical chemistry and specialising in the analysis of pesticides in food and environmental matrices.
Dr. Jens Seltmann
Jens is the Head of Product Development at LGC Labor GmbH (Augsburg/Germany) for Dr. Ehrenstorfer brand with almost 10 years’ experience in this field. He is mainly responsible for the development of new reference materials for residue analysis in the food, environmental and cannabis sector. He has a background in Organic Chemistry and Material Sciences.
Savvas Xystouris
Savvas is the Technical Development Manager for Chemistry LGC AXIO proficiency testing at LGC. He is mainly responsible for the development of new proficiency testing materials and technical management of PT schemes. He has a background in Chemistry and Food Science and Nutrition. Prior to joining LGC, he has worked at the State General Laboratory (SGL) of Cyprus as an Analytical Chemist. Savvas has been involved in the European Food Safety Authority's (EFSA) FoodEX II project, for the classification and re-coding of various food products as part of SGL’s EFSA Focal point projects. He is also member of the Institute of Food Science and Technology (IFST) since 2011.
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Bibliography
[1] http://www.norman-network.net/?q=node/19) [2] https://www.hbm4eu.eu/wp-content/uploads/2017/03/HBM4EU-in-brief.pdf [3] https://www.hbm4eu.eu/what-we-do/european-hbm-platform/eu-hbm-dashboard/ [4] https://setac.onlinelibrary.wiley.com/doi/10.1002/ieam.258 [5] https://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV-JM- MONO(2018)7&doclanguage=en [6] https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/ file/1012230/Poly-_and_perfluoroalkyl_substances_-sources_pathways_and_environmental_data_- _report.pdf [7] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483690/) [8] https://www.eea.europa.eu/publications/emerging-chemical-risks-in-europe [9] https://www.eea.europa.eu/publications/emerging-chemical-risks-in-europe/emerging-chemical- risks-in-europe [10] http://norden.diva-portal.org/smash/record.jsf?pid=diva2%3A1295959&dswid=8484 [11] https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/ file/1012230/Poly-_and_perfluoroalkyl_substances_-sources_pathways_and_environmental_data_- _report.pdf [12] https://ec.europa.eu/environment/pdf/chemicals/2020/10/SWD_PFAS.pdf [13] https://www.epa.gov/sdwa/and-polyfluoroalkyl-substances-pfas [14] https://www.epa.gov/superfund/proposed-designation-perfluorooctanoic-acid-pfoa-and- perfluorooctanesulfonic-acid-pfos [15] https://www.dwi.gov.uk/pfas-and-forever-chemicals/ [16] https://echa.europa.eu/regulations/reach/understanding-reach [17] https://www.eea.europa.eu/publications/soer-2020 [18] http://www.norman-network.net/sites/default/files/files/QA-QC%20Issues/2021%20NORMAN% 20network%20PFAS%20Analytical%20Exchange%20Final%20Report%2014022022.pdf [19] http://www.norman-network.net/sites/default/files/files/QA-QC%20Issues/2021%20NORMAN% 20network%20PFAS%20Analytical%20Exchange%20Final%20Report%2014022022.pdf [20] https://www.epa.gov/system/files/documents/2024-01/method-1621-for-web-posting.pdf [21] https://enveurope.springeropen.com/articles/10.1186/s12302-018-0135-3 [22] https://echa.europa.eu/view-article/-/journal_content/title/9109026-275 [23] https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex:52022PC0540 [24] https://echa.europa.eu/-/highlights-from-march-2025-rac-and-seac-meetings [25] https://eur-lex.europa.eu/eli/C/2024/4910/oj/eng
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