CWA Analytical Methods for Per- and Polyfluorinated Alkyl Substances (PFAS)

8.2.1 Note

Note:   In the absence of source-specific information (e.g., historical data) on the levels of PFAS or project-specific requirements, collect at least three aliquots of all aqueous samples to allow sufficient volume for an original whole-volume analysis, a re-extraction and second analysis, and for the determination of percent solids and for pre-screening analysis. …

Note:   In the absence of source-specific information (e.g., historical data) on the levels of PFAS or project-specific requirements, collect at least three aliquots of all aqueous samples to allow sufficient volume for an original whole-volume analysis, a re-extraction and second analysis, and for the determination of total suspended solids and for pre-screening analysis. …

10.1.1

During the development of this method, instrumental parameters were optimized for the precursor and product ions of the linear isomers of the target analytes listed on Table 9.  If a qualitative or quantitative standard containing an isomeric mixture (branched and linear isomers) of an analyte is commercially available for an analyte, the quantification ion used must be the quantification ion identified in Table 9, unless interferences render the product ion unusable as the quantification ion. …

During the development of this method, instrumental parameters were optimized for the precursor and product ions of the linear isomers of the target analytes listed on Table 10.  If a qualitative or quantitative standard containing an isomeric mixture (branched and linear isomers) of an analyte is commercially available for an analyte, the quantification ion used must be the quantification ion identified in Table 10, unless interferences render the product ion unusable as the quantification ion. …

10.2.2.5

… The laboratory must demonstrate that the analytical conditions provide a separation of at least 1 minute between the bile salts and the retention time window of PFOS, as described in Section 7.3.3. …

… The laboratory must demonstrate that the analytical conditions provide a separation of at least 1 minute between the bile salts and the retention time window of PFOS, as described in Section 10.3.5. …

10.3.3.2

… The response ratio (RR) for each native compound calibrated by isotope dilution is calculated according to the equation below, separately for each of the calibration standards, using the areas of the quantification ions (Q1) with the m/z shown in Table 9. …

… The response ratio (RR) for each native compound calibrated by isotope dilution is calculated according to the equation below, separately for each of the calibration standards, using the areas of the quantification ions (Q1) with the m/z shown in Table 10. …

11.0

For aqueous samples that contain particles and solid samples, percent solids are determined using the procedures in Section 11.1. …

For aqueous samples that contain particles and solid samples, total suspended solids are determined using the procedures in Section 11.1. …

11.2.5

… If necessary, adjust pH with 50% formic acid (Section 7.1.13.4) or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous ammonium hydroxide [Section 7.1.6.2]). …

… If necessary, adjust pH with 50% formic acid (Section 7.1.13.4) or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous ammonium hydroxide [Section 7.1.6]). …

11.4.9

… Check that the pH = 6.5 ± 0.5 and adjust as needed with 50% formic acid (Section 7.1.13.4) or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous ammonium hydroxide [Section 7.1.6.2]). …

… Check that the pH = 6.5 ± 0.5 and adjust as needed with 50% formic acid (Section 7.1.13.4) or ammonium hydroxide (or with 5% formic acid [Section 7.1.13.3] and 3% aqueous ammonium hydroxide [Section 7.1.6]). …

13.3

… The volume injected for samples and QC samples must be identical to the volume used for calibration (Section 10.2.3). …

… The volume injected for samples and QC samples must be identical to the volume used for calibration (Section 10.3.2). …

14.3.5

Using the data from the CV standard, compute the ion abundance ratio (IAR) for each target analyte listed with a confirmation ion mass in Table 9, using the equation below. …

Using the data from the CV standard, compute the ion abundance ratio (IAR) for each target analyte listed with a confirmation ion mass in Table 10, using the equation below. …

Note below 14.3.5

Note:    Some of the target analytes in Table 9 do not produce confirmation ions or produce confirmation ions with very low relative abundances; therefore, for those analytes, the IAR does not apply.

Note:    Some of the target analytes in Table 10 do not produce confirmation ions or produce confirmation ions with very low relative abundances; therefore, for those analytes, the IAR does not apply.

14.5.1

After completing the first 6 steps in the analytical sequence described in Section 13.3, analyze the extracts of the low-level OPR (LLOPR) and the mid-level OPR) (Sections 11.3.3, 11.3.2, and 11.4.2) prior to analysis of samples from the same batch to ensure the analytical process is under control.

After completing the first 6 steps in the analytical sequence described in Section 13.3, analyze the extracts of the low-level OPR (LLOPR) and the mid-level OPR) (Sections 11.2.3, 11.3.2, and 11.4.2) prior to analysis of samples from the same batch to ensure the analytical process is under control.

14.5.2

Compute the percent recovery of the native compounds by the appropriate quantification method depending on the compound (Section 10.3).  Compute the percent recovery of each EIS compound by the non-extracted internal standard method (Sections 1.2 and 10.3).

Compute the percent recovery of the native compounds by the appropriate quantification method depending on the compound (Section 10.3).  Compute the percent recovery of each EIS compound. by the non-extracted internal standard method (Sections 1.2 and 10.3).

15.2

… The EIS is quantitated with respect to a non-extracted internal standard (NIS), as shown in Table 9, using the response ratios or response factors from the most recent multi-level initial calibration (Section 10.3). …

… The EIS is quantitated with respect to a non-extracted internal standard (NIS), as shown in Table 10, using the response ratios or response factors from the most recent multi-level initial calibration (Section 10.3). …

8.5.1

The method does not call out extracting samples ASAP for NFDHA in aqueous samples, as it does for solid and tissue samples.  However, Section 8.5.5 makes it sound like this caveat is required for all matrices. 

The single-laboratory validation study included a holding time study that covered both samples and extracts.  The observed behavior of some of the ether sulfonates in the extracts led to the shorter, 28-day extract holding time for those analytes in soils, sediments, and tissues, versus 90 days for the extracts for all other analytes.  The caveat at the end of Section 8.5.5 is incorrect in that section.  In the revised method, EPA will delete that sentence from Section 8.5.5, as shown below:

Store sample extracts in the dark at or below 6 ºC until analyzed.  If stored in the dark at or below 0 6 ºC, sample extracts may be stored for up to 90 days, with the caveat that issues were observed for some ether sulfonates after 28 days (see Reference 10).  These issues may elevate the observed concentrations of the ether sulfonates in the extract over time.  Samples may need to be extracted as soon as possible if NFDHA is an important analyte.

10.1.7

The mass calibration must be verified prior to the analysis of any standards and samples and after each subsequent mass calibration. Each laboratory must follow the instructions for their instrument software to confirm the mass calibration, mass resolution, and peak relative response. In addition to the mass calibration verification performed using a standard specified by the manufacturer, the mass calibration must also be verified with respect to the ion masses monitored by this method.

Some readers were misinterpreting the section to mean that a mass calibration verification was needed with every sample batch, which is not true.  As part of this clarification, the text will be revised to read:

The mass calibration must be verified prior to the analysis of any standards and samples and after each subsequent mass calibration, at the frequency indicated in Table 11.  The laboratory must follow the instructions for their instrument software to confirm the mass calibration, mass resolution, and peak relative response. If the manufacturer’s instructions include options for evaluation of mass resolution, the tightest resolution requirements (typically called unit resolution) must be met.  In addition to the mass calibration verification performed using a standard specified by the manufacturer, the mass calibration must also be verified with respect to the ion masses monitored by this method.

10.1.7.1 and 10.1.7.2

These sections will be deleted in their entirety.  Commenters noted the discrepancy between calling out “unit resolution” (e.g., 1 Da) and then having a QC specification of 0.2 Da. Different manufacturers have different mass shift criteria.  Some are wider than 0.2 Da. Therefore, the text in Section 10.1.7 will be revised as described above to resolve that discrepancy as well as addressing the frequency issue.

11.1.1.5 vs. 11.2.4

Section 11.1.1.5 states the following: Subject to project-specific approval, laboratories may utilize other documented strategies for minimizing the disruptions due to SPE clogging and slow extractions, including centrifuging the sample before adding the EIS and treating the supernatant aqueous phase and the solid phase as separate samples.

Section 11.2.4 states the following: If centrifugation is used to prevent samples with high TSS from clogging the SPE, the EIS compounds must be spiked into the original sample container prior to centrifugation.

The presumed conflict is due to the example provided in 11.1.1.5.  That use of centrifugation is subject to approval by the client, and the example involves two closely related steps: centrifuging the sample into two “fractions” and then treating the fractions as separate samples from that point on, including two separate extractions.

11.2.4 is the more general case where centrifugation is used, but the solids are added to the top of the SPE column and only one extraction is performed.  That use of centrifugation would not require project-specific approval.  The EPA will revise Section 11.2.4 and move some of the text to a new Note in that section, as shown below.

Spike an aliquot of EIS solution (Section 7.3.1) directly into the sample in the original bottle (or subsampled bottle) as well as to the bottles prepared for the QC samples.  Mix by swirling the sample container.  If centrifugation is used to prevent samples with high TSS from clogging the SPE, the EIS compounds must be spiked into the original sample container prior to centrifugation.

Note:   Except as described in Section 11.1.1.5, where centrifugation is used to create two separate samples, when centrifugation is used to prevent samples with high TSS from clogging the SPE but the solids are extracted along with the supernatant liquid, the EIS compounds must be spiked into the original sample container prior to centrifugation.

15.1.3

Please clarify why the ratio requirement does not apply to PFPeA as Table 10 includes a confirmation ion for this PFAS.

The last sentence in the second paragraph of Section 15.1.3 reads “The ratio requirement does not apply for PFBA, PFPeA, NMeFOSE, NEtFOSE, PFMPA, and PFMBA because suitable (not detectable or inadequate S/N) secondary transitions (Q2) are not available.”

As noted in the parenthetical phrase in the sentence above, those six target PFAS do not have “suitable secondary transitions.”  In some cases, there is no detectable transition ion, and in other cases, such as PFPeA, the signal-to-noise ratio for the ion at m/z 68.9 is too low to be used reliably.  The EPA will revise the last sentence in Section 15.1.3 as follows:

The ratio requirement does not apply to PFBA, PFPeA, NMeFOSE, NEtFOSE, PFMPA, or PFMBA because secondary transitions (Q2) for these analytes are either not detectable or have inadequate S/N to be reliably used in an ion ratio calculation.

15.2 Equation

Recommend adding the percent solids to the equation for solid samples.

The final term in the equation, W_s is described as the sample volume in liters for an aqueous sample, or the weight in grams of a solid sample.  The solid sample weight should be expressed in terms of dry weight until such time as the EPA revises the method, at which time, we will add an additional term for the percent solids that only will apply to soil, sediment, and biosolid samples.

15.3.1 and 15.3.2

Section 15.3.1 states that a diluted extract result can be used if: “… the responses for any EIS in the diluted extract that is associated with one of those analytes meet the S/N and retention time requirements in Sections 15.1.1 and 15.1.2, and the EIS recoveries from the analysis of the diluted extract are greater than 5% ...”

The problem with this statement is that the EIS percent recovery should not change significantly when the extract is diluted.  The EIS recovery is calculated using the NIS response, which will also be diluted proportionately.  Thus, the EIS recovery should not change when the extract is diluted. 

The EPA will revise Sections 15.3.1 and 15.3.2 as follows:

15.3.1   If the Q1 area for any compound exceeds the calibration range of the system, dilute a subsample of the sample extract with the methanolic ammonium hydroxide and acetic acid solution in Section 7.1.9 and analyze the diluted extract for the analyte(s) that exceeded the calibration range. If the responses for any EIS in the diluted extract that is associated with one of those analytes meet the S/N and retention time requirements in Sections 15.1.1 and 15.1.2, and the EIS recoveries from the analysis of the diluted extract meet the requirements in Tables 6 or 8greater than 5%, then the compounds associated with those EIS compounds may be quantified using the EIS response. Adjust the compound concentrations, detection limits, minimum levels, and LOQs to account for the dilution.

If the EIS responses in the diluted extract do not meet those S/N and retention time requirements, then the compound cannot be measured reliably by isotope dilution in the diluted extract. In such cases, the laboratory must take a smaller aliquot of any affected aqueous sample and dilute it to 500 mL with reagent water and prepare and analyze the diluted aqueous sample, or prepare and analyze a smaller aliquot of soil, biosolid, sediment, or tissue sample. Adjust the calibration ranges, detection limits, and LOQs to account for the tested sample mass/volume and any dilution factors.

If a dilution results in an EIS recovery less than 5% for the analyte that required the dilution, then the laboratory must prepare and analyze a diluted aqueous sample or a smaller aliquot of a solid sample.

Note: Target analyte results with a corresponding EIS that has less than 5% EIS recovery cannot be used for monitoring compliance with an NPDES permit. If the EIS recovery does not meet the requirements of Tables 6 or 8,is less than 5% but the EIS response has a S/N greater than or equal to 20:1, the result may have some utility as an estimated value. Some non-NPDES projects may accept such an estimated value if the result is so high that it is well above the project criteria or action level, especially if additional sampling is planned for that site.

15.3.2   If the recovery of any EIS in a sample is outside of the acceptance limits in Tables 6 or 8, then a diluted sample extract must be analyzed, or a smaller sample mass/volume must be extracted (Section 15.3.1). If the recovery of any EIS in the diluted sample extract is below 5%, the method does not apply to the sample being analyzed and the result may not be reported or used for permitting or regulatory compliance purposes. If all cleanup procedures in this method are used and EIS recovery remains outside of the acceptance range, extraction and/or cleanup procedures that are beyond this scope of this method may be needed to analyze the sample.