PFAS Analysis: Overcome Challenges and Meet Evolving Requirements
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Last Updated: December 11, 2023
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Published: June 8, 2023
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Per-and polyfluoroalkyl substances (PFAS) are highly stable and resistant to degradation. They can persist in our environment for longer than any other artificial substance.
High contamination risks and the constant evolution of regulatory guidelines are just some of the challenges that researchers face in PFAS analysis for environmental monitoring. However, highly sensitive, robust solutions can aid researchers in overcoming such challenges and meeting regulatory compliance.
Download this whitepaper to learn more about solutions that can:
- Optimize the efficiency of PFAS analysis workflows
- Minimize sources of contamination
- Identify diverse compound types to confidently meet regulatory standards
ARTICLE
Overcoming Typical Challenges for
PFAS Analysis to Meet Evolving
Regulatory Requirements
Figure 1: Sources of PFAS compounds.
Introduction
Per- and polyfluoroalkyl substances (PFAS) represent a group of thousands of anthropogenic
compounds that have been produced and widely used in industrial applications and consumer
products since the 1950s. Some of the major industry sectors using PFAS include aerospace and
defense, automotive, aviation, food contact materials, textiles, leather and apparel, construction
and household products, electronics, firefighting, food processing, and medical supplies.
These compounds have unique physical and chemical characteristics: they all contain carbonfluorine bonds (among the strongest chemical bonds in organic chemistry), which means they are
highly stable and resistant to degradation and are known to persist in the environment longer than
any other artificial substance. This, along with their ubiquitous use, has led to the accumulation of
PFAS in the environment, with growing concern of human exposure to these chemicals.1-3
FIREFIGHTING
FOAMS
PERSONAL CARE
PRODUCTS
NON-STICK
COOKWARE PHOTOGRAPHIC
PROCESSES
COSMETICS
PESTICIDE AND
HERBICIDE PACKAGING
AND STORAGE
PAINTS
WATER RESISTANT
CLOTHING
MINING AND OIL WELL
SURFACTANTS
FLOOR POLISHES
AVIATION
HYDRAULIC FLUIDS
FAST FOOD
PACKAGING
STAIN RESISTANT
PRODUCTS
MICROWAVE
POPCORN BAGS
Identified
PFAS Sources
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
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Most PFAS are also easily transported in the environment,
covering long distances from the source of their release. Many
PFAS are found in human and animal blood and are present at
low levels in a variety of food products and in the environment
all across the globe. PFAS are found in soil and different water
resources, including drinking, surface, ground-, and wastewater.1-6
Among PFAS, perfluorooctanesulfonic acid (PFOS) and
perfluorooctanoic acid (PFOA) are the most prevalent in the
environment and so are included in many advisory guidelines.
This article gives a non-exhaustive overview of the most known
PFAS regulations, exploring the typical challenges encountered
in their LC/MS/MS analysis, with practical examples on how to
address them.
Mitigating the Adverse Impact of PFAS Exposure
With increasing awareness and scientific evidence of the
environmental and health impacts of PFAS, governmental agencies
have acknowledged the need for continuous monitoring of their
levels in a variety of samples. Regulations have been put in place to
surveil PFAS exposure in environmental matrices. Among them are:
U.S. EPA Method 537.1 for the determination of 18 PFAS in
drinking water. The LC/MS/MS method is based on isotopic
internal standards with reversed-phase solid-phase extraction
(SPE) sample preparation.9
U.S. EPA Method 8327, designed to measure a group of
24 PFAS compounds in ground-, surface, and wastewater
samples, uses LC/MS/MS with external calibration.10
U.S EPA Method 533 addresses some shorter chain and
more polar PFAS compounds. It contains a list of 25 PFAS
compounds (C4-C12) comprising the majority of those in
537.1, with the addition of some polar fluorotelomers and ether
carboxylic acids. The LC/MS/MS method uses isotopic dilution
and ion exchange SPE sample preparation.11
U.S. EPA Draft Method 1633 was introduced in 2021 and
is for use in the Clean Water Act (CWA). It is a composite
method for a broad range of PFAS in multiple matrices. The
method encompasses 40 targeted PFAS compounds in various
matrices, including aqueous, solids, biosolids, and tissues.12
Figure 2. PFOA and PFOS chemical structures.
ISO 25101:2009 is utilized for the determination of the PFOS
and PFOA in unfiltered samples of drinking, ground-, and
surface water by coupling SPE with LC/MS/MS.13
Directive (EU) 2020/2184 is the EU Drinking Water Directive,
which includes maximum limits for total PFAS of 0.5 µg/L.
For the sum of the 20 PFAS of most concern, the maximum
limit is 0.1 µg/L, which requires a limit of detection (LOD) of
30 ng/L for the sum and 1.5 ng/L for individual compounds.
Environmentally relevant PFAS should be determined not
only in clean drinking water but also in ground-, surface, and
wastewater, which means that interfering compounds and
matrices need to be separated.14,15
For a method showing sensitivity and robustness for the analysis
of the additional 20 PFAS compounds, refer to the application
note, “Analysis of PFAS in Drinking Water by Large Volume Direct
Injection Following the EU Drinking Water Directive 2020/2184.”
On December 30, 2022, the Chinese Ministry of Ecology
and Environment made public the List of New Pollutants for
Priority Management (2023), which was due to take effect
March 1, 2023. This list includes PFOS and PFOA.
The optimization of analytical methods for identification and
quantification of PFAS is essential for risk assessment. Because
of its high sensitivity, selectivity, and robustness, the most widely
used analytical method of PFAS detection is based on LC/MS/MS.
Liquid chromatography allows for the separation of the
various PFAS of interest. Triple quadrupole mass spectrometry
systems, with two quadrupoles and a collision cell, allow the
filtering of specific mass transitions for each analyte, adding
an extra layer of selectivity to the method. Mass spectrometers
provide exact masses and resolve peaks with small mass
differences, which makes them suitable for simultaneous
detection of thousands of substances.
While LC/MS/MS is a very sensitive detection method on its
own, there is still the need for sample preconcentration to
detect PFAS levels present in the environment. The choice of
sample preparation methods has implications for selectivity and
sensitivity and the possibilities of identifying the substances in
the sample. Sample handling and processing can significantly
alter the molecular composition of the samples and the results
of the analysis. Among sample preparation techniques, solvent
extraction and solid-phase extraction are widely used to extract
emerging contaminants from aqueous samples and have been
employed in EPA Method 537.1, as well as ISO 25101.
The examples of applications described below were
performed with the PerkinElmer QSight® LC/MS/MS System
(Figure 3).
Perfluorooctane Sulfonic Acid (PFOS)
Perfluorooctanoic Acid (PFOA)
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
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Typical Challenges for PFAS Analysis
Considering their ubiquity and large number of potential sources
of contamination, PFAS present some challenges for analytical
laboratories. These include:
The risk of contamination during the analytical workflow
The runtime needed to reach the detection and quantification
limits imposed by the regulations
Lab downtime caused by matrix-induced ion source
contamination
While analytical labs need to meet evolving regulatory
requirements for a list of compounds that is constantly expanding,
they also need to maintain their high level of operational efficiency.
Minimizing Sources of Contamination
The key challenge of measuring parts-per-trillion levels of PFAS is
that these compounds are ubiquitous throughout the environment
and accumulate everywhere, including laboratory equipment
and accessories. In fact, many of the components used in liquid
chromatographs, mass spectrometers, and solid-phase extraction
systems are made of polytetrafluoroethylene (PTFE) or PTFE
copolymers, which leach PFAS compounds and cause background
interference during sample measurement. Even the use of glass
sample containers can generate additional challenges, because
glass adsorbs PFAS compounds. Special care is required, and
alternate materials must be used throughout the laboratory to
reduce the absorption of these chemicals.
To reach accurate ultratrace levels, every step of the analytical
protocol must be free of PFAS, from sample collection to sample
preparation, analysis, and measurement. Table 1 summarizes the
necessary steps for reducing background contamination during
sample preparation and analysis.
Figure 3. PerkinElmer QSight 220 LC/MS/MS triple quadrupole system.
Source of Contamination Mitigation
Mobile Phases Purchase LC/MS-grade solvents
Use a delay column
PFAS Parts and Tubing in
HPLC Pump Use a delay column
PFAS Tubing in HPLC Autosampler Replace with PEEK tubing
Vials and PTFE Lined Caps Use only polyethylene vials and caps
PFAS Tubing in SPE Apparatus Replace with polyethylene tubing
Table 1. Steps to reduce PFAS contamination.
Mobile Phase and HPLC Pump
High-quality mobile phases and blank runs are important aspects
of PFOS analysis. Instead of using conventional glass vials
with PTFE-lined septa, polyethylene vials and caps can be used
to reduce the possibility of contamination. The HPLC pump,
autosampler, and SPE system all contain PFAS components that
require mitigation as well. The HPLC system pump has PTFE parts
that can leach PFAS compounds, and contamination is likely in all
but the highest grades of reagents.
To combat interference from these sources, a delay column can be
installed in the flow path between the pump and the autosampler.
The delay column captures PFAS contaminants coming from the
mobile phase, the solvent lines, or the pump before they reach the
autosampler. As a result, the captured compounds elute via the
gradient later than the analyte peak in the sample, enabling more
authentic measurements of PFAS in the sample.
Autosampler
In many cases, the HPLC autosampler contains fluoropolymer
tubing that introduces contamination on injection of the sample.
It is recommended to replace all tubing with high-performance
polyetheretherketone (PEEK) tubing to eliminate the possibility of
PFAS contamination during sample injection. For convenience,
PerkinElmer offers a kit specifically developed to replace the tubing
in their autosamplers for PFAS applications.
Solid-Phase Extraction (SPE) System
SPE extraction configurations normally include an abundance
of fluoropolymers. The tubing connecting sample bottles to
the SPE cartridges can be a significant source of PFAS
contamination. Replacement of all transfer tubing with linear
low-density polyethylene (LLDPE) or PEEK tubing is necessary
to avoid PFAS leaching. In addition, some of the valving on
the manifold may be constructed of PTFE; substitution with
polyethylene stopcocks is recommended. Finally, sample collection
during SPE extraction should employ polyethylene centrifuge tubes.
After proper mitigation of all possible PFAS sources, the
LC/MS/MS system will be ready to analyze PFAS at low parts
for trillion levels.
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
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For the detailed workflow description and the complete set of
results obtained with this approach please refer to the article,
“Analysis of Perfluoroalkyl and Polyfluoroalkyl Substances in
Drinking Water: Validation Studies of EPA Method 537.1 Using the
QSight 220 UHPLC/MS/MS.”
Optimizing the Runtime
There are thousands of PFAS, and the broad range of their
chemistries makes it difficult to establish a simple method for
testing them, even in drinking water. As regulations continue to
lower limits, it becomes more challenging to develop and validate
efficient analytical methods. The lowest-concentration minimum
reporting levels for current EPA methods are down to or below
single-digit parts per trillion.
In a recent study, PerkinElmer scientists optimized the EPA Method
533 and EPA Method 537.1 on the QSight 220 LC/MS/MS system.
EPA Method 537.1 describes a chromatographic run that
takes approximately 37 minutes to separate the 18 analytes,
surrogates, and internal standards. For the same method,
PerkinElmer scientists were able to achieve a runtime of about
10 minutes. This represents a significant time saving while
maintaining excellent chromatographic resolution and excellent
separation of the linear and branched isomers. An example of
their separation is shown in Figure 1.
In terms of instrument sensitivity, the limits of quantitation
(LOQ) and limits of detection (LOD) confirm that the QSight 220
LC/MS/MS system is highly capable of performing the method
successfully. With the 250-to-1 sample concentration from
the SPE extraction step, the limits were well below the current
requirements for all compounds – even those at extremely low
levels. The detailed extraction procedure, analytical method,
and results are presented in the application note, “Analysis of
Perfluoroalkyl and Polyfluoroalkyl Substances in Drinking Water:
Validation Studies of EPA Method 537.1 Using the QSight 220
UHPLC/MS/MS.”
Reducing Lab Downtime
Samples are often complex dirty matrices, which can impact the ion
source and increase the need for cleaning, causing lab downtime.
Matrix effect is the effect on an analytical method caused by all
other components of the sample except the specific compound
to be quantified. Several approaches have been investigated to
improve reproducibility and robustness of LC/MS/MS methods that
are subjected to matrix effect.
In LC/MS analysis, ions that are formed in an ion source are
normally sampled into a mass spectrometer through a small
aperture or capillary, followed by devices such as an ion funnel
or ion guide. Axial electric fields are often applied to transfer
ions through multiple pumping stages before reaching the
mass analyzer at high vacuum. During this transfer, ions,
neutrals, and solvated charged species can deposit on inner
surfaces, contaminating the path of the ions’ migration to high
vacuum, causing signal fluctuation, instability, and drift. In
addition, transporting ions under influence of the axial electric
field in a collision-rich environment causes ion scattering and
discrimination between high- and low-mass ions so that the mass
spectrometer requires tuning to set different lens parameters for
high and low masses.
Figure 1: Sources of PFAS compounds.
Analyte Peak # RT (min) IS# Ref
PFBS 1 3.54 2
PFHxA 2 4.15 1
HFPO-DA 4 4.34 1
PFHpA 6 4.78 1
PFHxS 7 4.77 2
ADONA 8 4.84 1
PFOA 9 5.30 1
PFOS 11 5.73 2
PFNA 13 5.74 1
9Cl-PF3ONS 14 5.93 2
PFDA 15 6.13 1
NMeFOSAA 17 6.31 3
PFUnA 19 6.45 1
NEtFOSAA 20 6.47 3
11Cl-PF3OUdS 22 6.56 2
PFDoA 23 6.72 1
PFTrDA 24 6.96 1
PDTA 25 7.16 1
13C2-PFHxA SS#1 3 4.15 1
13C3-HFPO-DA SS#2 5 4.34 1
13C2-PFDA SS#3 16 6.12 1
D5-NEtFOSAA SS#4 21 6.46 3
13C2-PFOA IS#1 10 5.29 -
13C4-PFOS IS#2 12 5.74 -
d3-NMeFOSAA IS#3 18 6.30 -
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
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To mitigate that, PerkinElmer has introduced QSight StayClean™
hot surface–induced desolvation (HSID™) technology, in which ions
and solvated charged species are entrained in a hot laminar flow
of sampling gas to be transported to a vacuum region. The laminar
flow gas shields ions and solvated species from striking the HSID
walls, acting as a constant cleaning agent. Additionally, solvated
species gain energy from the hot gas and become desolvated.
This enables enhanced uniform response across the entire mass
range with no lens optimization. Most mass spectrometers today
have an orthogonal sampling introduction to reduce contamination.
Because of its proximity to the ion source, the interface can still
be exposed to contamination as described above. Through the
combination of the coaxial flow ionization source and laminar flow
ion guide technologies, long-term signal stability and reproducibility
are achieved and frequent cleaning is no longer required.
Highlights
There are currently over 7,000 PFAS-related compounds, and
many more derivative compounds are expected to be created
in the future. Important developments dealing with new PFAS
challenges include:
Final Toxicity Assessments for GenX and Additional PFAS
The EPA has stated that the toxicity assessments for two PFAS,
hexafluoropropylene oxide dimer acid and its ammonium salt –
also referred to as GenX chemicals17 – will soon be published.
GenX chemicals are considered extremely persistent in drinking
water and have known impacts on human health, including
reproductive and immunological toxicities, and on the environment.
In addition to the assessments for GenX PFAS, the EPA Office
of Research and Development is also developing toxicity
assessments for five other PFAS, including PFBA, PFHxA, PFHxS,
PFNA, and PFDA.18
Identifying PFAS Categories
Part of the difficulty in gathering information on PFAS is that it
is such a large and diverse class of compounds. In response
to this complexity, the EPA is planning to reclassify PFAS
compounds into smaller categories based on parameters
such as chemical structure, physical and chemical properties,
and toxicological properties. The EPA has outlines two
approaches to categorizing PFAS:
Utilize toxicity and toxicokinetic data to develop PFAS
categories for further hazard assessment and to inform hazard
or risk-based decisions.
Develop PFAS categories based on removal technologies using
existing understanding of treatment, remediation, destruction,
disposal, control, and mitigation principles.
These approaches will help identify missing elements in the EPA’s
understanding of PFAS from hazard assessments and removal
technology perspectives, and further assist the EPA’s prioritization
for future actions.
The EU Drinking Water Directive establishes a new group-limit value
for PFAS of 0.5 µg/L, in addition to limits for 16 individual PFAS of
0.1 µg/L in drinking water. Such measures can be supported by
cost-effective and targeted monitoring of PFAS in the environment
to provide early warning of pollution.16
Limit Usage and Production
In June 2019, the European Council of Ministers highlighted the
widespread occurrence of PFAS in the environment, in products, and
in people and called for an action plan to eliminate all nonessential
uses of PFAS. The move toward zero pollution requires that product
lifecycles are made safer from the start, based on the concept of
“safe and circular by design.” This approach offers opportunities
to protect the health of European citizens and environments while
driving innovation for safer chemicals.
Innovations in Method Development Addressing
New PFAS Categories
As the future of PFAS testing moves toward expanding PFAS toxicity
assessments, optimizing methods, and identifying additional PFAS
categories, it will be even more critical that researchers develop
innovations within method development for enhanced
PFAS analysis.
Governmental agencies are planning to develop additional
targeted methods for detecting and measuring specific
PFAS and nontargeted methods for identifying known PFAS in
the environment.
Additional method development will be utilized for total PFAS
analyses that measure the amount of PFAS in environmental
samples without identifying specific PFAS.
Conclusions
Overcoming PFAS analytical challenges is critical for a
comprehensive understanding of PFAS toxicities and environmental
impacts. Properly developed and validated analytical methods
allow researchers to increase throughput and decrease sources
of contamination, while reducing runtimes. All these aspects are
crucial for an analytical laboratory to succeed.
The threat of PFAS contamination is a global concern, so it is
paramount that regulatory authorities, analytical technology
manufacturers, and water treatment authorities combine efforts
to develop the necessary legislation and innovations to facilitate
necessary PFAS research and mitigation.
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
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References
1. Nakayama, S. F. et al., Worldwide trends in tracing polyand perfluoroalkyl substances (PFAS) in the environment.
Trends in Analytical Chemistry [Accessed August 30th, 2022]
https://doi.org/10.1016/j.trac.2019.02.011.
2. Agency for Toxic Substances and Disease Registry, "Perand Polyfluoroalkyl Substances (PFAS) and Your Health",
last review July 6th, 2022 [Accessed August 30th, 2022]
https://www.atsdr.cdc.gov/pfas/index.html.
3. Lindstrom, A. B. et al., Polyfluorinated Compounds:
Past, Present, and Future, Environ. Sci. Technol. 2011,
45, 7954–7961.
4. Ciofi, L.; Renai, L.; Rossini, D.; Ancillotti, C.; Falai, A.; Fibbi,
D.; Bruzzoniti, M.; Santana-Rodriguez, J. J.; Orlandini, S.;
Del Bubba, M.; Applicability of the direct injection liquid
chromatographic tandem mass spectrometric analytical
approach to the sub-ng /L determination of perfluoro-alkyl
acids in waste, surface, ground and drinking water samples,
Talanta, 2018, 176, 412-421.
5. Schultz, M. M.; Barofsky, D. F.; Field, J. A.; Quantitative
determination of fluorinated alkyl substances by largevolumeinjection liquid chromatography tandem mass
spectrometry characterization of municipal wastewaters,
Environ. Sci. Technol. 2006, 40 (1), 289–295.
6. Sébastien Sauvé et al., Worldwide drinking water
occurrence and levels of newly-identified perfluoroalkyl
and polyfluoroalkyl substances, Science of The Total
Environment, 2018, 616–617, 1089-1100.
7. U.S. EPA: Drinking Water Health Advisories for PFOA and
PFOS, last review July 12th, 2022 [Accessed August 30th,
2022] Drinking Water Health Advisories for PFOA and
PFOS | US EPA.
8. ECHA: Perfluoroalkyl chemicals (PFAS) [Accessed
August 30th, 2022] Perfluoroalkyl chemicals (PFAS) -
ECHA (europa.eu).
9. Shoemaker, J. and Dan Tettenhorst. Method 537.1:
Determination of Selected Per- and Polyfluorinated Alkyl
Substances in Drinking Water by Solid Phase Extraction
and Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/ MS). U.S. Environmental Protection Agency,
Office of Research and Development, National Center for
Environmental Assessment, Washington, DC, 2018.
10. USA EPA Method 8327: Per-and Polyfluoroalkyl Substances
(PFAS) Using External Standard Calibration and Multiple
Reaction Monitoring (MRM) Liquid Chromatography/
Tandem Mass Spectrometry (LC/MS/MS) [Accessed
August 30th, 2022] Document Display | NEPIS | US EPA.
11. USA EPA Method 533 [Accessed August 30th, 2022] Method
533: Determination of Per- and Polyfluoroalkyl Substances
in Drinking Water by Isotope Dilution Anion Exchange Solid
Phase Extraction and Liquid Chromatography/Tandem Mass
Spectrometry (epa.gov).
12. USA EPA Draft Method 1633: Analysis of Per- and
Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid,
Biosolids, and Tissue Samples by LC-MS/MS [Accessed
August 30th, 2022] Document Display | NEPIS | US EPA.
13. ISO 25101:2009 Water quality — Determination of
perfluorooctanesulfonate (PFOS) and perfluorooctanoate
(PFOA) — Method for unfiltered samples using solid
phase extraction and liquid chromatography/mass
spectrometry [Accessed August 30th, 2022]
https://www.iso.org/standard/42742.html.
14. Directive (EU) 2020/2184 of the European Parliament
and of the Council of 16 December 2020 on the quality
of water intended for human consumption [Internet].
[cited 2022 Jan 26]. Available from:
https://eur-lex.europa.eu/eli/dir/2020/2184/oj.
15. PFAS pollution is widespread in Europe but risks are still
poorly understood [Internet]. [cited 2022 Dec 22]. Available
from: https://www.eea.europa.eu/highlights/pfas-pollution-iswidespread-in.
16. https://www.eea.europa.eu/publications/emerging-chemicalrisks-in-europe.
17. GenX is a registered trademark of the Chemours company.
https://www.chemours.com/en/about-chemours/genx.
18. Status of EPA Research and Development on PFAS
[Internet]. [cited 2022 Dec 22]. Available from:
https://www.epa.gov/chemical-research/status-epa-researchand-development-pfas.
Overcoming Typical Challenges for PFAS Analysis to Meet Evolving Regulatory Requirements
Additional Resources
PFAS Analysis Interactive Brochure
https://www.perkinelmer.com/libraries/bro-pfas-solutions-final
Case Study: PFAS Monitoring: How One Scientist is Thinking Ahead for the Environment
https://www.perkinelmer.com/libraries/cst-developing-methods-for-pfas-monitoring
Infographic: PFAS Health Concerns in Air, Water and Soil
https://www.perkinelmer.com/library/pfas-health-concerns-in-air-water-and-soil.html
Webinar: Microextraction Approaches for Environmental Monitoring of Emerging Contaminants
https://www.perkinelmer.com/library/microextraction-approaches-for-environmental-monitoring-of-emerging-contaminants.html
Webinar: Water Contaminants: Analysis of PFAS Using LC/MS/MS Technology
https://www.perkinelmer.com/library/water-contaminants-analysis-of-pfas-using-lc-ms-ms-technology.html
Webinar: What’s in your Water? Monitoring of PFAS in Drinking Water According to EPA Method 537.1
https://www.perkinelmer.com/library/what-is-in-your-water-monitoring-of-pfas-in-drinking-water.html
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