Prevalence and Source Tracing of PFAS in Shallow Groundwater Used for Drinking Water in Wisconsin, USA

Samples from 450 homes with shallow private wells throughout the state of Wisconsin (USA) were collected and analyzed for 44 individual per- and polyfluoroalkyl substances (PFAS), general water quality parameters, and indicators of human waste as well as agricultural influence. At least one PFAS was detected in 71% of the study samples, and 22 of the 44 PFAS analytes were detected in one or more samples. Levels of PFOA and/or PFOS exceeded the proposed Maximum Contaminant Levels of 4 ng/L, put forward by the U.S. Environmental Protection Agency (EPA) in March 2023, in 17 of the 450 samples, with two additional samples containing PFHxS ≳ 9 ng/L (the EPA-proposed hazard index reference value). Those samples above the referenced PFAS levels tend to be associated with developed land and human waste indicators (artificial sweeteners and pharmaceuticals), which can be released to groundwater via septic systems. For a few samples with levels of PFOA, PFOS, and/or PFHxS > 40 ng/L, application of wastes to agricultural land is a possible source. Overall, the study suggests that human waste sources, septic systems in particular, are important sources of perfluoroalkyl acids, especially ones with ≤8 perfluorinated carbons, in shallow groundwater.

The range in grid cell areas (Figure 1 of the manuscript) is 316 to 326 km 2 (122-126 square miles).
Step 3 of sampling location selection consisted of several sub-steps and iterations.Initially, five WCRs were selected at random off of each list from Step 2. Invitation letters, offering the opportunity to participate in the study, were sent to, typically, three addresses from the five selected (based on determination of a useable mailing address).If more than one positive response to the mailed invitation was received for a grid cell, the participant selected for sampling was chosen randomly.If only one positive response was received, that location was selected for sampling.If no positive responses were received for a grid cell, letters were sent to additional randomly selected participants in the same grid cell (from the list produced in Step 2), in three additional mailing rounds until a positive response was received.Grid cells for the study and sampling locations are shown in Figure 1 of the manuscript.For nine out of the 450 grid cells, a second sampling location in a nearby grid cell was used, as no invited participant in the original (blue-shaded) grid cell accepted the study participation invitation.

Sampling procedure
Sampling was performed in teams of two trained samplers.Sampling kits were prepared before leaving for the field.After collection, samples were stored in battery/electrical-powered coolers capable of achieving cooling down to -20 °C, with the option for also setting cooling temperatures within the range 0°C to 6°C.An overview of sample bottles, field QC samples, preservation and holding times is provided in Table S1.
Due to potential for sample contamination, in particular for PFAS, the following precautions were taken: 1. Samplers typically wore cotton clothing that had been washed at least three times prior to sampling.

2.
No personal care products were applied after leaving the vehicle to begin sampling.

3.
Personal care products such as sunscreen and mosquito repellant were chosen in consideration of product information in PFAS Sampling Guidance from the Michigan Department of Environmental Quality 1 .These products were only applied in the morning and during breaks.

4.
No consumption of food or drink during sampling, with the exception of bottled water.

5.
Hands were washed thoroughly before filling sample bottles and care was taken to touch only parts of the bottles and caps that do not come in contact with sample water.
After arriving at a site, contact was made with the homeowner/resident if present and an information packet was left for them.Both samplers worked on measurement of field parameters.When it was time to fill sample bottles, sampling was performed following the "clean hands/dirty hands" technique.One member of the sampling team was designated prior to arrival at the site as "dirty hands" while the second member was designated as "clean hands".All operations involving contact with the sample bottle were handled by the individual designated as "clean hands", while the sampler designated as "dirty hands" performed other tasks (e.g., labeling, note taking, and turning the faucet on before sampling).
Steps performed were as follows: 1. Identification of the tap to be sampled.Typically, an outdoor tap was sampled.In some cases, an indoor tap near the pressure tank, before any installed treatment system, was sampled.
2. Run water, purging about 10 gallons through a hose directed to an area away from the home or other buildings.
3. Begin measuring field parameters using a flow-thru cell or, if not available, with probes at the bottom of a 5-gallon bucket.
4. Continue until stabilization of water temperature, specific conductance and pH, then those parameters plus dissolved oxygen were recorded.5. Sampler 1 (designated to keep clean hands) opened the PFAS sampling kit (LDPE bag with a loosely tied knot) and put on the powderless nitrile gloves.
6.Both PFAS sample bottles were filled so that they were about 90% full, allowing room for expansion when the sample freezes, then the field blank was collected by pouring lab-supplied blank water in one 250 mL PP bottle into another.7. Remaining sample bottles were filled in this order: 1000 mL amber glass bottle (human waste and herbicide indicators), 250 mL amber glass pre-filled with sulfuric acid (NOx, TAN and chloride), 250 mL HDPE (metallic and other elements), 60 mL HDPE (conductivity, pH, alkalinity).
8. The second sampler began labeling sample bottles with a fine-tip permanent marker after all sample bottles for PFAS, including the field blank, had been filled and capped.9. Sample bottles were placed in cold storage following Table S1.
Supporting Information -Page S6 of S47 were purchased from Honeywell and Sigma Aldrich, respectively.Table S3 shows additional information on analytical standards.For certain compounds, multiple names/abbreviations are shown in the tables due to inconsistencies in those identifiers between various relevant documentation (i.e., analytical standard product catalogues and other published methods cited in Table S4).The rationale for the choice of extracted internal standards is provided in Table S4.For PFECHS, the extracted internal standard in OTM-45 2 is 18 O2-PFHxS.During method development, PFECHS was quantitated with both 13 C8-PFOS and 13 C3-PFHxS.Recoveries using 13 C3-PFHxS were equal or better when compared to recoveries using 13 C8-PFOS.Since recoveries using 13 C3-PFHxS were equal or better than using 13 C8-PFOS and OTM-45 also uses labeled PFHxS (just a different isotope), 13 C3-PFHxS was chosen as the extracted internal standard.The extracted internal standard recovery was used to adjust target analyte concentrations.Internal standard mix was added to each quality control sample and field sample, in the bottle in which it was collected, and allowed to equilibrate for a minimum of fifteen minutes.AES lines were rinsed with water and methanol before each extraction.
SPE cartridges were pre-conditioned in three separate steps with 4 mL ammonium hydroxide in methanol, methanol, and water, rinsing at a flow rate of 5 mL min -1 for each solvent.Samples were then added to the SPE cartridges in ~4 mL increments at a flow rate of 3 mL min -1 .After the entire samples passed through the cartridges, 4 mL each of water and 25 mM ammonium acetate buffer were added successively at 3 mL min -1 .Cartridges were then blown to dryness with 4 mL of air flowing at 10 mL min -1 through each cartridge followed by nitrogen flow at approximately 10 L min -1 for 10 minutes.
At the elution step, each cartridge was rinsed with 4 mL methanol at a rate of 0.5 mL min -1 (the sample bottle and in-line filter are rinsed first with this methanol), followed by 4 mL 60mM ammonium hydroxide in methanol at 0.5 mL min -1 .Extracts were collected in 15-mL polypropylene centrifuge tubes.Extracts were further filtered through a graphitized carbon black (ENVI-Carb) cartridge immediately after the initial Supporting Information -Page S15 of S47 SPE step.The carbon SPE cartridge was rinsed with at least 6 mL 60mM ammonium hydroxide in methanol before samples were loaded.Sample extracts were added to rinsed cartridges at about 3-5 mL min -1 and collected in 15-mL polypropylene centrifuge tubes.
Filtered extracts were evaporated to between 0.5-1 mL under a gentle stream of nitrogen (~0.7 L min -1 ) in a heated water bath at 60-65°C.Sample volume was adjusted to exactly 1 mL with MeOH before extracts were filtered through a glass fiber filter or polypropylene syringe filter.S7).Ongoing quality control included analysis of method blanks (Milli-Q water, extracted by SPE), laboratory control spikes (Milli-Q water spiked with analytes, extracted by SPE), and field duplicates, each performed for every extraction batch.Calibration checks were performed every 10 samples.Isotope dilution was performed by adding spikes of extracted internal standards in the sample bottle.Table S7 also shows the method reporting limit (MRL) for each compound.The MRL is the minimum concentration reported as a quantitative value for a method analyte in a sample following analysis.This defined concentration is no lower than the concentration of the lowest calibration standard for that analyte and is only used if the recovery in the lowest standard is within 50 -150%.The extracted internal standard recovery was used to adjust target analyte concentrations.
Calibration by isotope dilution was utilized for those PFAS with an exact mass-labeled isotope (Table S3).
Calibration by internal standard was utilized for other compounds.A 450 mL aliquot of sample was treated with 50 mL of EDTA (80 g L -1 ), 300 µL sulfuric acid (1:1), and 125 µL benzoylecgonine-d3 (surrogate standard).Of this mixture, 100 mL was passed through a hydrophiliclipophilic balanced cartridge (Waters Oasis HLB 6 cc, 200 mg) using a Dionex Autotrace solid-phase extraction (SPE) unit.Prior to loading the sample, cartridges were conditioned with 5.0 mL of methanol then 5.0 mL ultra-pure water at 5.0 mL min -1 .After loading, cartridges were dried for 15 minutes under nitrogen gas then eluted with 5.0 mL methanol at 5.0 mL min -1 .Eluents were transferred to a Turbovap concentration station and dried with nitrogen gas at 50°C.After drying completely, 500 µL of methanol was used to rinse down the sides of the tubes, then extracts were brought to dryness again.Tubes were removed from the Turbovap and 50 µL of injection internal standard mix was added.This final extract was brought to a volume of 500 µL with 15 mM acetic acid in ultra-pure water and transferred to vials for analysis by LC/MS.

Aqueous extraction method for chloroacetanilide metabolites
This method is based upon USGS Open File Report 00-182 8 .A Dionex Autotrace Solid Phase Extraction (SPE) system fitted with a C18 cartridge (Waters SepPak C18 6cc, 500 mg).Cartridges were conditioned with 3 mL of each: methanol, ethyl acetate, methanol again, then ultra-pure water.After conditioning, the syringe was rinsed with 5 mL ethyl acetate, then 125 mL of sample was loaded.The cartridge was dried with nitrogen for 0.5 minutes, then eluted with ethyl acetate to remove the more non-polar compounds, followed by methanol to remove the more polar chloroacetanilide herbicide metabolites.
Both fractions were collected in 5 mL centrifuge tubes.The ethyl acetate fraction was discarded.The methanol fraction was transferred to a Turbovap concentration station and dried with nitrogen gas at 50°C.After drying completely, 500 µL of methanol was used to rinse down the sides of the tubes, then extracts were brought to dryness again.Tubes were removed from the Turbovap and 50 µL of Butachlor ESA injection internal standard mix was added.This final extract was brought to a volume of 500 µL with 15 mM acetic acid in ultra-pure water and transferred to vials for analysis by LC/MS.Ongoing quality control included analysis of laboratory reagent blanks (ultra-pure water extracted by SPE -for pharmaceuticals, personal products, and artificial sweeteners, the blank was prepared in the same manner as samples, with the addition of EDTA, sulfuric acid, and surrogate standard).Laboratory fortified blanks (ultra-pure water) and laboratory matrix spikes (study samples) were prepared similar to samples, with the addition of a target analyte mixture.Laboratory reagent blanks, fortified blanks, and matrix spikes were performed for every 20 samples.Field duplicates and blanks were also collected and analyzed.
Internal calibration was utilized.Calibration checks and blanks were performed every 10 samples.
1.5 Inorganics and total organic carbon Inorganics analysis was conducted at WEAL.The elemental forms of major cations, metals, sulfur and phosphorous were analyzed using EPA Method 200.7, rev.4.4.The elements analyzed were As, Ca, Cu, Fe, K, Pb, Mg, Mn, Na, P, S and Zn.Total ammonia/ammonium nitrogen (NHx), nitrate-nitrogen plus nitrite nitrogen (NOx) and chloride were analyzed using Standard Methods 9 4500-NH3 H, 4500-NO3 F and 4500-Cl G, respectively.Laboratory pH, conductivity and alkalinity were analyzed following Standard Methods 9 4500H+ B, 2510B and 2320B.Non-purgeable total organic carbon (TOC) was analyzed following Standard Methods 5310B-2000 TOC 9 .TOC was chosen, rather than dissolved organic carbon (i.e., TOC of filtered samples), because of the possibility of colloidal or particulate organic matter (which could transport PFAS) content in karst and fractured rock groundwater environments.Standards and reagents are shown in Table S13 and Table S14.

1 :
Wisconsin health advisory level of 20 ng L -1 applies to the sum of detected concentrations of PFOA, PFOS, PFOSA, NEtFOSA, NEtFOSAA and NEtFOSE.2) In the reference list at the end of the document: OTM 45 2 , EPA Draft Method 1633 3 , Barzen-Hanson and Field (2015) 4 , Meng et al. (2022) 5 ; EPA Method 533 6 1.3.2PFAS extraction Samples were extracted with a PromoChrom automated extraction system (AES).If samples contained light particulates, a PromoChrom in-line filter was used to prevent contamination and damage to AES lines and pumps.

1. 4 . 4 4 . 6
LC/MS/MS method for human waste indicators Analysis was performed on an Agilent 1200 series high performance liquid chromatograph coupled to an Agilent 6430 triple quadrupole mass spectrometer with an electrospray ionization source (ESI-LC/MS/MS) following a pre-programmed gradient (below) with an additional two minutes of post run time.The injection internal standard recovery was used to adjust target analyte concentrations.Analytical column: Zorbax Eclipse XDB-C18 column, 4.6 × 50 mm; 1.8 μ (Scheurer et.al., 2009) Supporting Information -Page S29 of S47 Guard column: Zorbax Eclipse Plus-C18, 2.1 x 12.5 mm; 5 μ Injection volume: 20 µL Flow Rate: 0.5 mL minute -1 Mobile phase: (A) 15 mM acetic acid in ultra-pure water; (B) 15 mM acetic acid in methanol Column temperature.5LC/MS/MS method for chloroacetanilide metabolites Analysis was performed on an Agilent 1200 series high performance liquid chromatograph coupled to an Agilent 6430 triple quadrupole mass spectrometer with an electrospray ionization source (ESI-LC/MS/MS) following a pre-programmed gradient (below) with an additional two minutes of post run time.The injection internal standard recovery was used to adjust target analyte concentrations.Analytical column: Thermo Betasil C-18, 150 x 2.1 mm; 3 µ Injection volume: 10 µL Flow Rate: 0.5 mL minute -1 Mobile phase: (A) 15 mM acetic acid in ultra-pure water; (B) 15 mM acetic acid in methanol Column temperatureQuality control -non-PFAS organics

Table S4 . PFAS analytes, WI public health values and extracted internal standards. Classes of PFAS within the analyte list are shown in italics on rows preceding each group.
13 C2-10:2FTUCA Exact isotope label Fluorosulfonamide/sulfonamido substances (PFBSA and PFHxSA are likely precursors in the environment of PFBS and PFHxS, respectively; all others are precursors of PFOS)