External Peer Review of EPA’s Draft Aquatic Life Ambient Water Quality Criterion For Perfluorooctane Sulfonate (PFOS), Final Peer Review Report, August 26, 2021


Task Order 68HERH21F0090 under
Contract EP-C-17-017

External Peer Review of EPA's Draft
Aquatic Life Ambient Water Quality
Criterion for Perfluorooctane Sulfonate

(PFOS)

FINAL PEER REVIEW REPORT

August 26, 2021

Submitted to:

U.S. Environmental Protection Agency
Office of Water, Office of Science and Technology

1200 Pennsylvania Avenue, NW
Washington, DC 20460
Attn: James Justice
Justice.JamesR@epa.gov

Submitted by:
Eastern Research Group, Inc.

110 Hartwell Avenue
Lexington, MA 02421

%ERG

www.erg.com

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

CONTENTS

1.0 INTRODUCTION	1

1.1	Development of the Draft Documents	1

1.2	Peer Reviewers	2

2.0 SUMMARY OF REVIEWER COMMENTS ORGANIZED BY CHARGE QUESTION	2

2.1	Please comment on the overall clarity of the document as it relates to the derivation of

each criterion	2

2.2	Please comment on the approach used to derive the draft criterion for PFOS. Please

provide detailed comments	5

2.3	Please comment on the approach used to derive the draft acute estuarine/marine
benchmark for PFOS. Given the limited estuarine/marine test data available, a new
approach method was used to support the derivation of an acute estuarine/marine
benchmark to provide states and tribes with a protective value. Please provide detailed
comments	11

2.4	Please comment on the use of measured and unmeasured toxicity tests to derive the
respective criterion. In particular please comment on the supporting justification for

using unmeasured toxicity tests in Appendix 0	15

2.5	Please comment on the toxicity data used to derive the draft criteria	18

2.6	Please comment on the translation of the chronic water column criterion elements for
aquatic life to derive the tissue-based criterion elements, considering the

bioaccumulation of PFOA and PFOS. In particular, please comment on:	30

2.7	Please comment on the frequency and duration of the criterion elements, in particular

the tissue-based criterion elements	33

2.8	Please provide any additional technical comments that you believe should be considered	36

APPENDIX A CHARGE TO REVIEWERS	1

APPENDIX B INDIVIDUAL REVIEWER COMMENTS	1

REVIEWER 1	3

REVIEWER 2	 17

REVIEWER 3	25

REVIEWER 4	33

REVIEWER 5	47

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

1.0	INTRODUCTION

The U.S. Environmental Protective Agency (EPA) Office of Water (OW) is charged with protecting ecological
integrity and human health from adverse anthropogenic, water-mediated effects, under the purview of the
Clean Water Act (CWA). In support of this mission, EPA has developed draft water quality criteria to protect
aquatic life and aquatic-dependent wildlife from the presence of Perfluorooctanoic Acid (PFOA) and
Perfluorooctane Sulfonate (PFOS) in freshwater and saltwater environments. Derivation of these criteria is
described in two draft documents: Aquatic Life Ambient Water Quality Criteria for Perfluorooctanoic Acid
(PFOA) and Aquatic Life Ambient Water Quality Criteria for Perfluorooctane Sulfonate (PFOS).

This report documents the results of an independent letter peer review of the EPA's draft Aquatic Life Ambient
Water Quality Criteria for Perfluorooctane Sulfonate (PFOS). Eastern Research Group, Inc. (ERG), a contractor
to EPA, organized this external peer review for EPA OW and developed this report. Independent peer review of
the draft Aquatic Life Ambient Water Quality Criteria for Perfluorooctanoic Acid (PFOA) document is covered in
a separate report.

Section 2.0 of this report presents the individual reviewer comments organized by charge question. Appendix A
provides EPA's charge to reviewers and Appendix B presents the reviewer comments organized by reviewer.

1.1	Development of the Draft Documents

Toxicity studies used to derive the PFOA and PFOS criteria were carefully evaluated and thoroughly reviewed
to ensure studies were of sufficient data quality to use in criteria derivation. Scientists from EPA OW and Office
of Research and Development (ORD) conducted an extensive review of the PFOA and PFOS toxicity studies.
Additionally, EPA obtained replicate-level (or treatment-level, when replicates were unavailable)
concentration-response (C-R) data from publications, supplemental materials, or via contacting authors so that
EPA could independently fit C-R models to estimate acute LC5o and chronic ECio values that were used to derive
the criteria to ensure endpoints used were statistically sound. Individual C-R models and resultant point
estimates were also reviewed and discussed between OW and ORD to ensure the most statistically robust
models informed the derivation of the PFOA and PFOS criteria. In addition to contacting study authors for C-R
data (when not reported in the open literature), EPA also consulted primary authors for methods clarifications
in many instances during the data quality review phase to ensure that the studies used to derive criteria were
of high quality.

Overall, due to the paucity of measured freshwater toxicity data, EPA included a number of tests with
unmeasured treatments to derive criteria to ensure the dataset was representative of a range of taxa and
there were sufficient data to develop criteria. EPA also conducted meta-analyses to evaluate the relationship
between nominal and measured test concentrations using tests with measured treatment concentrations.
These meta-analyses (described in detail as Appendix L of the PFOA criteria document and Appendix O of the
PFOS criteria document) suggested measured concentrations were similar to nominal concentrations and that
the use of unmeasured tests, in light of data limitations, was appropriate. Additionally, estuarine/marine
toxicity data limitations did not allow for the direct derivation of acute or chronic estuarine/marine criteria for
PFOA or PFOS. Therefore, to develop recommendations that states and tribes could use in adopting protective
values for estuarine/marine waters, EPA developed acute PFOA and PFOS protective benchmarks using a New
Approach Methodology (detailed in Appendix K of the PFOA criteria document and Appendix L of the PFOS
criteria document).

Addressing data limitations to derive robust criteria/benchmarks, extensively reviewing studies, and
calculating point estimates meant that the derivation of the PFOA and PFOS aquatic life criteria were
developed via comprehensive, rigorous process that included collaborations across EPA scientists in OW and

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

Subsequently, EPA contracted with ERG to organize an independent external peer review of both draft
documents. Results of the PFOS review are described in this report. Results of the PFOA review are
documented in a separate report.

1.2 Peer Reviewers

ERG identified, screened, and selected the following five experts who met technical selection criteria provided
by EPA and had no conflict of interest in performing this review:

• Jason Conder, Ph.D.; Principal, Geosyntec Consultants

• Anu Kumar, Ph.D.; Principal Research Scientist, Environment Protection and Technologies,
Commonwealth Scientific and Industrial Research Organization (CSIRO)

• Ryan Prosser, Ph.D.; Associate Professor, University of Guelph

• Christopher J. Salice, Ph.D.; Director, Environmental Science and Studies Program, Towson University

• Jamie G. Suski, Ph.D.; Senior Scientist, EA Engineering, Science, and Technology, Inc.

ERG provided reviewers with instructions, the draft A qua tic Life Ambient Water Quality Criteria for
Perfluorooctane Sulfonate (PFOS), and the charge to reviewers (Appendix A of this report) prepared by EPA.
Reviewers worked individually to develop written comments in response to the charge questions. After
receiving reviewer comments, ERG compiled responses by charge question (see Section 2.0) and included the
responses organized by reviewer (Appendix B of this report).

2.0 SUMMARY OF REVIEWER COMMENTS ORGANIZED BY CHARGE QUESTION

This section organizes reviewer comments by charge question (see Appendix B for reviewer comments
organized by reviewer).

2.1 Please comment on the overall clarity of the document as it relates to the derivation of each
criterion.

2.1. Clarity of Document as it Relates to the Derivation of Each Criterion

Reviewer

Comments

Reviewer 1

Overall, the document is clear and the reader can follow the logic of criteria derivation, and
track the values used back to the cited research articles or values calculated by EPA.

Reviewer 2

1 thought that the document was well written and laid out. 1 thought that the document clearly
laid out the approach that the EPA used to derive each criterion. 1 thought it clearly outlined
the approach that the EPA chose in deciding which data to use in their derivation and how
these data would be used in derivation.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.1. Clarity of Document as it Relates to the Derivation of Each Criterion

Reviewer

Comments

The appendices are very useful in providing added detail and the data that were used in the
derivation of the criteria. The appendices allow for a high level of transparency around how
the criteria were generated.

In Table 3-1, the acronym "GMAV" was used as a heading in the table, but 1 could not locate
where this acronym was defined earlier in the document.

The captions of figures and tables are not sufficiently detailed. Figures and tables should be
able to stand on their own. Also, the use of acronyms in the caption and headings of tables and
figures decreases clarity, e.g., Figs 3-1, Tables 3-1, 3-2, 3-3, 3-4, 3-5. The use of acronyms in the
figure or table is valid to save space, as long as they are defined in the caption of the figure or
table.

Reviewer 3

Main Question: Perhaps this was missed in the draft document, but, is there guidance when
one criteria is exceeded and the other is not? For example, if the tissue based criteria are
exceeded yet, the CCC is not; perhaps this is an unlikely scenario as those receptors have been
accumulating PFOS for a duration likely under higher water concentrations (> 0.014mg/L).
Although if sediment concentrations remain elevated (but not water column concentrations)
this may also be a likely route of PFOS exposure to fish with sediment dwelling prey.

There are additional domestic criteria missing from the previously published criteria section;
please review those for Texas, Florida and California.

Are there two Sharpe et al.'s, 1 believe this is only one publication but flipping between Sharpe
et al. 2010, Sharpe et al. 2010a and Sharpe et al. 2010b throughout the document. If the goal is
to distinguish between supplemental vs the manuscript proper 1 suggest just clarifying in the
text instead of the reader looking for two pubs by Sharpe et al. 2010.

Page 238 - error: The study authors reported a 96-hour LC5o of 58.47 mg/L PFOS, based on the
results of the range finding test. The independently-calculated toxicity value was x.xx mg/L.

Page 296 - error: The independently-calculated toxicity value was x.xx mg/L.

Table 3-6 is not referenced/described in the text. Additionally, the title reads "Six" most
sensitive and lists "Seven".

Overall comment: ranking of sensitive genera flips back and forth between most and least
sensitive among tables, consistency would help the reader.

Reviewer 4

EPA has drafted the PFOS aquatic life criteria to be consistent with methods described in EPA's
"Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and Their Uses" (U.S. EPA 1985). 1 congratulate the EPA Team for a very thorough,
comprehensive analysis of the toxicological data to derive each criterion.

• The report is technically sound and is very clearly written.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.1. Clarity of Document as it Relates to the Derivation of Each Criterion

Reviewer

Comments

• The criteria have been derived using strong science-based evidence.

• Sub-sections on overview of PFAS, PFAS nomenclature, problem formulation, exposure
pathways, transformation and degradation of PFOS precursors in the aquatic
environment

• sources, concentration reported in environment and existing criteria (both national
and international) help to set the scene before toxicological data is presented and
assessed for developing various criterion.

• The freshwater acute water column-based criterion, the chronic water column-based
chronic criterion, the chronic fish whole-body tissue criterion, the chronic fish muscle
tissue criterion and the chronic invertebrate whole-body tissue criterion have been
developed and documented in this report are based on comprehensive assessment of
the toxicological data and consistent with the Guidelines.

• Acute and chronic MDRs for PFOS estuarine/marine criteria derivation were not met
due to fewer empirical PFOS toxicity data. To address this gap, the EPA Team
developed an acute aquatic life benchmark for estuarine/marine environments based
on Interspecies Correlation Estimation (ICE) model. Such predictive models should be
used when there is limited toxicity data.

• EPA Team has provided extensive background information on toxicity data assessment
and collated this information in various Appendices as additional line of evidence.

• Tables and Figures are very well laid out throughout the document and provide
additional information of the toxicity data used in developing Water Quality Criteria
for PFOS.

Reviewer 5

Overall, the document is clearly written and generally free of grammatical errors. 1 applaud the
scientists and EPA for compiling an impressive amount of work and communicating it in a
reasonably clean and coherent way. That said, there are a few instances of redundancy -
literally, the same sentences repeated. 1 have made note of these in the actual report and will
include that along with this document, if requested. Although these are easy enough to see
with careful review. The document is VERY LONG and very detailed so any efforts to shorten or
make more concise would be welcomed.

As for the clarity of technical elements of the document, 1 feel that many of the decisions to
use or not use data or endpoints could be more consistent and/or communicated better. For
example, in some cases the geometric mean of endpoints for a certain taxa is used for the
chronic value (pimephales) but for other taxa, this is not the case (e.g. daphnia; zebrafish). In
other cases, the decision to not use certain data seems as if it could be communicated more
clearly. So while the language of the document is pretty clear, the actual technical aspects are
less so.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.1. Clarity of Document as it Relates to the Derivation of Each Criterion

Reviewer

Comments

One major concern 1 have is with the overall approach of using the 4 most sensitive toxicity
values to then derive the final acute and final chronic values. Using this approach it would
seem the AWQC are then very sensitive to changes in any 1 of the 4 toxicity values. For
example, when EPA explored the impact of different toxicity values on the chronic freshwater
water column criteria, using higher toxicity values (e.g., pimephales in place of fatmucket Table
4-3) resulted in a lower chronic criteria. This is nonintuitive and suggests a possible flaw in the
approach. It's possible this is not the case and it makes sense both mathematically (steeper
slope) and perhaps even from a protection standpoint. Either way, EPA should explain why this
happens and what it means for the overall approach. 1 suspect the EPA is somewhat
constrained by the 1985 guidelines in developing the AWQC but 1 also see that New Approach
Methods (WEB-ICE) were used to derive criteria with limited data. 1 wonder if using a species
sensitivity distribution approach in which all the chronic or acute (freshwater)data are used
would result in more defensible criteria that are less impacted by changes in any one toxicity
value? At the very least, 1 think including a full SSD would be useful for comparison as part of
the characterization piece. In the PFOA document 1 mention revisiting and publishing and
updated 1985 guidelines...this is warranted when EPA has the bandwidth to do so. Having said
all this, 1 am aware that EPA likely has justification for their approach of using the 4 most
sensitive tox values but it would perhaps be good to mention this again as 1 suspect a lot of
people that may read the AWQC will not also read the 1985 guidelines.

Lastly, 1 had a hard time keeping track of all the decisions to use or not use data for each of the
tox values that supported the criteria. 1 think a more detailed table with all the tox values
considered (data shown in Figure 3-3) and including whether the data used were author-
reported, re-calculated by EPA, along with the lowest reported/calculated value that wasn't
used and why. This may be asking a lot and this information is throughout the document but
not in a single, easy to locate and read location.

2.2 Please comment on the approach used to derive the draft criterion for PFOS, Please provide
detailed comments.

• Is the technical approach used to derive the criterion elements logical?

• Does the science support the conclusions?

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

Reviewer 1

• Is the technical approach used to derive the criterion elements logical?

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

Yes, the technical approach used to derive the criteria elements is generally logical. 1 disagree
with some of the elements of the analyses, as noted in my detailed comments (see below,
responses to charge question 8)

• Does the science support the conclusions?

In general, the science is supportive of the general conclusions. As noted in my below detailed
responses to other charge questions, 1 believe the science is not supportive of the work in a
few key instances including:

1 believe the Criterion Continuous Concentration (CCC) should be potentially re-calculated
considering my comments provided in response to charge question 5a.

The science does not support the assumption of a 10-year recovery time for PFOS in aquatic
systems.

The generation of tissue criteria is weakly supported, and the uncertainty associated with
these criteria should be emphasized.

The NAM-generated marine Final Acute Value (FAV) and FAV/2 values (Appendix L) are highly
uncertain.

It is unclear if the EPA-calculated Effective Concentration 10% (EC10) values are supported;
additional details on the modeling and the variability and fit of each EC10 model need to be
provided.

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

The criteria derived are aimed at protecting aquatic life (e.g., fish, invertebrates) from the
direct acute and chronic toxicity of PFOS in water. Generally, the values applied are protective
and are generally similar to protective values derived by other regulatory organizations and
independent (i.e., academic, private sector) scientists. Although, as based on my comments, 1
believe there is room for improvement. The criteria derived for tissues attempt to provide
criteria that take into account bioaccumulation so that measurements in tissue can be
interpreted with respect to the potential for potential effects; however, the uncertainty with
the tissue criteria is high. The water and tissue criteria are not intended protective of
bioaccumulative effects that may affect higher trophic levels, such as wildlife that may
consume aquatic life.

Reviewer 2

Yes, the technical approach used by the EPA to derive the criterion is logical and defensible.
The approach is also clearly laid out in the document. Dividing the 5th centile of the acute GSD
by 2 is sufficiently conservative to ensure the protection of 95% of species, based on the data
currently available.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

Yes, 1 think the science supports the EPA's conclusions. However, there appears to be several
studies that were not considered by the EPA. 1 have listed these studies below.

Yes, 1 think the approach taken by the EPA is sufficiently conservative to be protective of
freshwater aquatic life from acute, chronic, and bioaccumulative effects based on the data that
was available at the time. It was a good idea to evaluate the influence on non-North American
species on the derivation of the criteria.

Reviewer 3

• Is the technical approach used to derive the criterion elements logical?

This is logical and follows the established GLRI guidance; however, both Canada and Australia
utilize a species sensitivity distributions to determine the 95th and 99th percentile of species
protection. Is there a defensible reason why EPA did not employ this approach or at the very
least present these distributions and analysis that would support the currently drafted criteria?

Additionally, thresholds from those SSDs (and others published) are lower than the draft
guidance here, this should be addressed:

Australia - 0.13 ng/L
Canada - 6.8 ng/L
Salice et al. 2018 - 1.12 ng/L
Conder et al. 2020 - 5.85 ng/L

*Giesy et al. 2010 - 5.1 ng/L (using CCC based on GLRI guidance)

• Does the science support the conclusions?

The GMCV for Zebrafish is 0.0165 mg/L, thus there are studies that result in chronic toxicity at
concentrations lower than this mean; however, this is very close to the CCC of 0.014mg/L. This
seems borderline protective when considering potential exposures to this species (and those
more sensitive).

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

1 have confusion over Tables 3-9 and 4-6 calculations. How is it that the inclusion of Lampsilis
with a higher GMAV results in a lower overall CMC (3.3 mg PFOS/L) compared to the CMC in
table 3-9 (3.6 mg PFOS/L)? Actually, looking more closely at this, the ln(GMAV)A2 are
inconsistent among the tables for Xenopus, this is likely are result of using table 3-6 as a
template for 4-6.

It is great to see the inclusion of the Burkhard et al. 2021 as this synthesis has been peer-
reviewed and published and is an exceptional overview of PFOS bioaccumulation;
unfortunately, there are not more current literature used within the draft document.

Reviewer 4

This EPA report provides a critical review of toxicity data identified in EPA's literature search
for PFOS, including the anionic form (CAS No. 45298-90-6), the acid form (CAS No. 1763-23-1),

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

potassium salt (CAS No. 2795-39-3), an ammonium salt (CAS No. 56773-42-3), sodium salt (CAS
No. 4021-47-0), and a lithium salt (CAS No. 29457-72-5). It quantifies the toxicity of PFOS to
aquatic life, and provides criteria intended to protect aquatic life from the acute and chronic
toxic effects of PFOS. The detailed assessment is as follows:

• These criteria have been derived using robust methods and the best available toxicity
data on aquatic life.

• The approach used to derive the draft criterion for PFOS is very logical and consistent
with the protection offered by acute and chronic aquatic life criteria derived using
empirical data, as prescribed in the 1985 Guidelines.

• Exclusion and inclusion criteria are appropriately discussed in the context of the
toxicological data reported in the literature and provide additional evidence on the
selection of toxicity data criteria development.

• With limited toxicity datasets to North American resident species, non-North American
resident species were included for criteria development. For example, inclusion of
non-resident species such as Planaria, Dugesia japonica and Japanese swamp shrimp,
Neocaridina denticulata for calculating acute water quality criteria and zebra fish,
Danio rerio for chronic criteria. The EPA team did not find any influence of excluding
non-North American resident species in criteria derivations and decided to retain the
full acute and chronic toxicity dataset. This was very rational decision and non-
Northern American species served as surrogate species for the broad range of the
thousands of untested species present in the freshwater environment in the U.S.

• The acute measures of effect on aquatic organisms selected included the lethal
concentration (LC50), effect concentration (EC50), or inhibitory concentration (IC50)
estimated to produce a specific effect in 50 percent of the test organisms as per the
Guidelines.

• The endpoint for chronic exposures incorporated the effect concentration estimated
to produce a chronic effect on survival, growth, or reproduction in 10 percent of the
test organisms (ECio). This approach has been also consistent with the harmonized
guidelines from OECD and the generally preferred effect level for countries such as
Canada, Australia, and New Zealand.

• Reported (No Observed Effect Concentrations) (NOECs) and (Lowest Observed Effect
Concentrations) (LOECs) were only used for the derivation of a chronic criterion when
a robust ECi0 could not be calculated for the genus.

• Furthermore, EPA independently calculated these toxicity values if sufficient raw data
were available for EPA to conduct statistical analyses. EPA's independently-calculated
toxicity values were used preferentially, where available.

• 1 agree with the authors' decision on not developing plant criteria based on their lesser
sensitivity to PFOS than in comparison to aquatic vertebrates and invertebrates. The

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

EPA team evaluated the toxicity data to plants as an additional line of evidence and
confirmed that the proposed PFOS freshwater acute and chronic criteria are expected
to be protective of freshwater plants.

• EPA developed protective tissue-based criteria through a bioaccumulation factor
approach. This was based on the application of evaluation criteria for screening
bioaccumulation factors (BAFs).

• Based on comprehensive toxicity data assessment, the EPA team has developed the
following criteria using the procedures described in the 1985 Guidelines. The
freshwater acute water column-based criterion magnitude is 3.6 mg/L and the chronic
water column-based criterion magnitude is 0.014 mg/L. The chronic freshwater
criterion also contains tissue-based criteria expressed as 43.0 mg/kg wet weight (ww)
for fish whole-body, 25.3 mg/L ww for fish muscle tissue, and 12.3 mg/kg ww for
invertebrate whole-body tissue.

• Acute and chronic MDRs for PFOS estuarine/marine criteria derivation were not met
and an estuarine/marine FAV could not be calculated to derive an estuarine/marine
acute criterion. Further benchmark was developed using predictive approach and
discussed in the follow-up question 3.

Reviewer 5

• Is the technical approach used to derive the criterion elements logical?

Overall, 1 think the technical approach is relatively sound although there are instances where it
was difficult to keep track of all the decisions with regard to data and whether these were
consistent and logical. Admittedly, 1 think this is a tough chemical and a tough dataset and EPA
did an excellent job with the background material and highlighted and used key studies (but
more have been published since and will, I'm sure be included). Unfortunately, the technical
approach to derive criterion elements is not universally logical. Moreover, as mentioned, using
only the 4 most sensitive toxicity endpoints followed by a regression (what type? Was this
specified?) seems less robust than using a full species sensitivity distribution with a more
"natural" distribution of sensitivity (s-shaped, for example). This last statement may not be
true so a reasonable compromise might be to include a full SSD as part of the characterization
piece related to "considering other toxicity values impact on the FCV, etc.". What really
confused me was that when EPA did what amounts to a sensitivity analysis of the FCV by
replacing toxicity values, the FCV DECREASED when higher toxicity values were used. While 1
suspect this happened because switching to higher toxicity values steepened the slope (or
something), it does not make intuitive sense to me and should be further explained.
Alternatively, an explanation and justification, even brief, would be helpful in supporting the 4
most sensitive toxicity value approach. 1 am aware that the 1985 guidelines may include this
but 1 suspect most users of the AWQC may not be familiar with the details of the guidelines.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.2. The Technical Approach Used to Derive the Draft Criterion for PFOS

Reviewer

Comments

because there were not enough tissue data from laboratory studies. 1 mention this because it
was confusing - there was a lot of explanation of using only dietary exposures and then one
sentence (basically) stating...EPA explored a BAF approach.

• Does the science support the conclusions?

Well, offhand, 1 think the final chronic value for freshwater organisms should likely be lower.
Importantly, several studies have been published in 2021 that should likely be included as
toxicity values and they may result in lower toxicity estimates. The fact that EPA's criteria are
higher than all other published criteria is worrisome. We are all using the same data and many
in the field are quite capable scientists.

These two papers come immediately to mind but 1 am sure there are others.

Sensitivity and Accumulation of Perfluorooctanesulfonate and Perfluorohexanesulfonic
Acid in Fathead Minnows (Pimephales promelas) Exposed over Critical Life Stages of
Reproduction and Development J.G. Suski, C.J. Salice, M.K. Chanov, J. Avers, J. Rewerts,
J. Field Environmental Toxicology and Chemistry, 2021, pp. 811-819.

Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture
Exposure Experiments with 6 Perfluoralkvl Substances Christopher J. McCarthy, Shaun
A. Roark, Demitria Wright, Kelly O'Neal, Brett Muckev, Mike Stanawav, Justin N.
Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J. Salice Environmental
Toxicology and Chemistry, 2021, pp. 2319-2333.

In my view, it is essential that EPA incorporate newly published toxicity data for PFOS (and
PFOA).

Furthermore, in several cases, EPA's decisions to use what look like higher estimates of toxicity
seem somewhat arbitrary and not internally consistent. 1 also noted above and mention here
again the sensitivity of the criteria development approach to changes in one of the 4 most
sensitive taxa/toxicity values.

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

1 believe the criteria are "NEARLY" protective of freshwater aquatic life for acute, chronic and
bioaccumulative effects of PFOS. 1 say "nearly" because it seems to me that the FCV, in
particular, could and maybe should likely be lower. Also, below 1 comment on the
appropriateness and utility of the frequency and duration elements of the criteria. Briefly, in
my opinion the frequency and criteria elements of the criteria certainly help the criteria
concentrations to be protective; it is unlikely that a 4-day exposure to the FCV would result in
adverse effects to any taxa for which there are data; however, these data are not commonly
reported (hourly or 4-day running average concentrations have never been reported to my
knowledge).

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.3 Please comment on the approach used to derive the draft aci larine/marine benchmark for
PFOS. Given the limited estuarine/marine test data available, a new approach method was used
to support the derivation of an acute estuarine/marine benchmark to provide states and tribes
with a protective value. Please provide detailed comments.

• Is the technical approach used to derive the benchmark logical?

• Does the science support the conclusions?

• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria
derived using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical
National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

2.3. The Technical Approach used to Derive the Draft Acute Estuarine/Marine Benchmark for PFOS

Reviewer

Comments

Reviewer 1

• Is the technical approach used to derive the benchmark logical?

The derivation of the acute marine benchmarks (FAV and Criterion Maximum Concentration
(CMC)) using the New Approach Method (NAM) is highly uncertain, and 1 would recommend
this analysis not be included as in this document. 1 do not feel that the analysis and subsequent
criteria have high confidence for use in a regulatory application. 1 understand that similar
analyses with other chemicals have about a 90% probability of the predicted effect value being
within a factor of 5 of the actual value (Raimondo et al., 2010 - cited in document). Given the
calculated CMC (0.43 mg/L), this implies the CMC has about a 90% probability of being within
0.086 to 2.2 mg/L. If the NAM approach stays in the document, this uncertainty and range of
values should be acknowledged in the discussion.

1 would rather see tentative or provisional acute criterion developed from the limited empirical
marine acute data highlighted in Appendix B and other recently published marine acute data.
This suggests a reasonable interim FAV of approximately 1 mg/L, which is similar to that
calculated using the NAM approach. 1 place higher confidence in the limited empirical data and
would suggest EPA emphasize it in addition to or in place of the values calculated by the NAM.

1 am hopeful that as new toxicity information on marine species are developed, these values
can be supplanted with a proper and robust criteria calculation. If such a future analysis is
possible, it should be noted.

• Does the science support the conclusions?

See above comment.

• Is it consistent with the protection offered by acute estuarine/marine aquatic life
criteria derived using empirical data, as prescribed in the 1985 Guidelines for
Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and Their Uses?

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2.3. The Technical Approach used to Derive the Draft Acute Estuarine/Marine Benchmark for PFOS

Reviewer

Comments

The approach seems to be consistent with the approach in the 1985 guidelines. As noted
above, the uncertainty with regards to the predictive capability of the interspecies correlations
should be acknowledged quantitatively.

Reviewer 2

The technical approach using Web-ICE to determine an acute benchmark for estuarine/marine
species is logical. The science has shown that Web-ICE can effectively be used to derive effect
measures for additional species using species for which data is available. 1 think the approach
taken by EPA has included sufficient conservatism to address the relatively large amount of
uncertainty around the acute toxicity of PFOS to estuarine and marine species. The proposed
acute benchmark for estuarine and marine species is an order of magnitude lower than the
acute benchmark for freshwater species, which 1 think underscores the conservatism used by
EPA in determining an acute benchmark for estuarine and marine species. That said, the
benchmark should be used cautiously due to the relatively large amount of uncertainty and
effort should be made to generate acute and chronic toxicity data for PFOS on estuarine and
marine species.

Reviewer 3

• Is the technical approach used to derive the benchmark logical?

After potential inclusion of the data mentioned below this approach may be appropriate. In
the current form with the limited data it may be misleading. Can this guidance be updated? 1
am aware of other researchers investigating PFAS on marine species (Ed Wirth, NOAA) and
maybe others that will be coming out soon.

• Does the science support the conclusions?

1 believe the data are incorrect for Fabbri et al. 2014. In table B.l the reported effect
concentration is recorded as >1 mg/L. However, looking at the paper, 1 read, "The PFCs PFOA
and PFOS induced a dose-dependent effect, with significant decreases in normal larval
development from 0.1 jig/L (17% and 27%, respectively; P 0.01). Maximal effects were
observed at 100 ng/L (about 40% and 50%, respectively; P 0.001) with no further decreases at
higher concentrations". There is a monotonic concentration-response curve. The associated
figure also supports an effect at O.lpig PFOS/L, see below. Furthermore, if the EC50 of the test
organisms is a needed endpoint (as noted in the PFOA justification, for which is lacking support
in the current form) looking at the figure below % of normal D-larvae for PFOS (although
incorrectly referred to in the legend as PFOAS) could be inferred at 0.1 mg/L. Furthermore, has
EPA considered calculating the MATC from this study?

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2.3. The Technical Approach used to Derive the Draft Acute Estuarine/Marine Benchmark for PFOS

Reviewer

Comments

Rs- 4. Effects of PFOA and PFOS (0.01-0.1 -1 -10-100-1000 tig/L) o
M. galloprovincialu normal larval development in 96-multhvell plates. Data are r«
ported as in Fig. 3a.

I did not see data included or the study evaluated for: Robertson JC (1986) Potential for
environmental impact of AFA-6 surfactant. Beak Consultants Ltd. Missassauga, Ontario,
Canada. EPA Docket AR226-1030a043.

There are data for saltwater spp in the ITRC from this citation.

• Is it consistent with the protection offered by acute estuarine/marine aquatic life
criteria derived using empirical data, as prescribed in the 1985 Guidelines for
Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and Their Uses?

No, this is a new approach; however, it follows the spirit of the 1985 guidelines.

Reviewer 4

• EPA applied the ICE model predictions to supplement the available test dataset to help
fill missing MDRs and allow the derivation of acute estuarine/marine benchmark
recommendations for aquatic life using procedures consistent with those in the 1985
Guidelines. A total of 3104 datapoints from 398 models were evaluated.

• ICE model has been recommended to predict the sensitivity of an untested taxon
(predicted taxa are represented by the y-axis) from the known, measured sensitivity of
a surrogate species (represented by the x-axis). The ICE model approach used is very
reasonable to predict toxicity of untested taxa.

• As documented in Section L.l, ICE-predicted models have been used by multiple
independent, international groups and further confirms that values developed from
ICE-generated SSDs will provide a level of protection that is consistent with using
measured laboratory data.

• In addition, prediction accuracy and robustness of the model is evaluated using robust
parameters (e.g., mean square error, R2), that fall within a defined range of
acceptability, and with close prediction confidence intervals that facilitate evaluating
the fit of the underlying data. This confirms the robustness of the model.

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2.3. The Technical Approach used to Derive the Draft Acute Estuarine/Marine Benchmark for PFOS

Reviewer

Comments

• ICE models predicted with acceptable accuracy for PFOS when invertebrates were used
to predict to invertebrate species and vertebrates were used to predict to vertebrate
species in these comparisons.

• The draft acute benchmark for estuarine/marine aquatic life developed using this
approach is 0.43 mg/L PFOS, it is lower than the recommended acute freshwater
criterion(3.6 mg/L), suggesting that estuarine/marine species may be more acutely
sensitive to PFOS. This is in line with Hayman et al., (2021), confirming marine species
have a higher sensitivity to PFOS than compared to the freshwater organisms.

• In this report, Mytilus galloprovincialis was not used in the FAV calculation because the
value was not definitive, and true sensitivity of this species is unknown. There are two
more studies published reporting the toxicity values for marine/estuarine species,
including Mytilus galloprovincialis.

o Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder,

Jennifer A Arblaster Aquatic toxicity evaluations of PFOS and PFOA for five
standard marine endpoints.

https://doi.Org/10.1016/i.chemosphere.2021.129699

It is recommended to assess the quality of the toxicity data on marine/estuarine species and
recalculate estuarine criteria based on this recently available information.

Reviewer 5

• Is the technical approach used to derive the benchmark logical?

Yes, given the lack of PFOS toxicity data for acute estuarine/marine species, 1 think applying
WEB-ICE is a REASONABLE APPROACH...perhaps the only approach that is defensible. Clearly,
more (or some) data would be a wonderful contribution. WEB-ICE, as mentioned, has been
reviewed and published quite a bit so 1 think, as an approach, it has merit and support of the
scientific community. EPA also did a good job presenting the approach and being clear about
the criteria being a draft. Overall, when data have been lacking, EPA has used state-of-the-art
approaches to developing criteria (my concerns are mostly when sufficient data are available).

• Does the science support the conclusions?

Yes, the science supports the conclusions. Interestingly, the acute criteria for estuarine/marine
species (o.43 mgPFOS/L) is almost an order of magnitude lower than the acute criteria for
freshwater organisms (3.6 mg/L). Whether estuarine/marine species are truly more sensitive
remains to be seen but, to me, it is more reasonable, given the lack of data, that the criteria
draft is lower.

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2.3. The Technical Approach used to Derive the Draft Acute Estuarine/Marine Benchmark for PFOS

Reviewer

Comments



• Is it consistent with the protection offered by acute estuarine/marine aquatic life
criteria derived using empirical data, as prescribed in the 1985 Guidelines for
Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and Their Uses?

Yes, and EPA justified this in the explanation of WEB-ICE that occurs in Appendix L and, overall,
the approach and resulting criteria are consistent with the protection of estuarine and marine
species.

2.4 Please comment on the use of measured and unmeasured toxicity tests to derive the respective
criterion. In particular please comment on the supporting justification for using unmeasured
toxicity tests in Appendix O.

2.4. The Use of Measured and Unmeasured Toxicity Tests to Derive Respective Criterion

Reviewer

Comments

Reviewer 1

The consideration of toxicity data from experiments in which PFOS measurements were not
made seems appropriate. The Appendix 0 analysis is supportive of the general observation
that actual concentrations in the toxicity test waters approximated nominal values for
freshwater. 1 agree that actual concentrations in the toxicity test waters for the marine test
may be lower than nominal values, thus, effect data originating from marine studies that only
report nominal concentrations may be biased high in some cases. Given the
tentative/temporary nature of the marine criteria developed in this study, this bias is
manageable until additional empirical data from experiment with measured concentrations in
water can be provided.

Reviewer 2

1 am concerned with the approach of using the agreement of measured and nominal
concentrations from studies that measured the concentration of PFOS in their tests to
determine whether to use toxicity data from studies that did not measure the concentration
PFOS in their tests. My concern stems from this approach having to assume that studies that
did not measure the concentration of PFOS in their experiments performed the dosing of PFOS
with the same care and skill as those studies that did measure the concentration of PFOS in
their experiments and measured concentrations within 20% of nominal. My concern is
compound by 58% and 65% of the freshwater and saltwater tests, respectively, only reporting
nominal test concentrations. The EPA's approach uses the agreement of measured and
nominal concentration in a minority of studies to determine whether to include the majority of
studies on their assessment.

1 am assuming that there wouldn't be sufficient data to determine a criterion without using
data from studies that did not measure the concentrations of PFOS in their experiment?

15

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2.4. The Use of Measured and Unmeasured Toxicity Tests to Derive Respective Criterion

Reviewer

Comments

1 think the approach that the EPA has used to determine the level of agreement between the
nominal and measured concentration of PFOS in the studies that measured the concentration
is logical and valid. It is encouraging that the agreement on average is high. Again, my largest
concern is assuming this agreement in a minority of studies is present in all studies.

Reviewer 3

This seems acceptable for the time being. Having worked in the laboratory with PFOS, 1 can
make a first-hand testament that mixing PFOS into exposures solutions does not guarantee a
homogenous mixture despite working at solutions well below the solubility limit. There are
nuances associated with achieving homogeneity of the exposure solution, we have developed
a PFAS mixing protocol to reduce chemical clumping and this increases uniformity of the
solutions. Furthermore, there is approximately 30% variability of PFOS quantitatively
(see...Rewerts et al. 2020); so, the best measurement still has significant variability.

Reviewer 4

PFOS is a highly stable compound, resistant to hydrolysis, photolysis, volatilization, and
biodegradation (as described in Section 1.1.1 of the Report) and, therefore, expected to vary
only minimally in the course of a toxicity test. To determine if nominal and measured PFOS
concentrations were typically in close agreement, pairs of nominal and corresponding
measured PFOS concentrations were compared to one another through (1) linear correlation
analysis and (2) an assessment of measured concentrations as a percent of its paired nominal
concentration. The authors reported, 22 freshwater studies with PFOS measured
concentrations, yielding 373 pairs of measured and nominal concentrations. In addition, there
were 7 estuarine/marine studies with measured concentrations, yielding 142 pairs of
measured and nominal concentrations. The data were grouped by classifications including
water type (salt/fresh) and experimental conditions (acute/chronic; solvent/no solvent;
fed/unfed, etc.). Data displayed a high degree of linear correlation and measured, and nominal
concentrations were in close agreement

The analysis conducted by EPA Team showed strong correlation (correlation = 0.9998) of the
326 pairs of nominal and measured concentrations from freshwater studies. In addition, the
experimental conditions did not influence the correlation between nominal and measured
concentrations. The detailed analyses of the data in Appendix 0 and the relevant Tables and
Figures provide very comprehensive analyses - this is very useful information and will assist
ecotoxioclogist in designing future experiments.

This confirms inclusion of unmeasured PFOS toxicity tests for quantitative use in criteria
derivation.

Personal experience on analyzing PFOS in ecotoxicological studies using freshwater species
have also exhibited strong correlation between nominal and measured concentrations.

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2.4. The Use of Measured and Unmeasured Toxicity Tests to Derive Respective Criterion

Reviewer

Comments

experimental conditions (acute/chronic and unfed/fed) could influence the observed
differences between measured and nominal concentrations. These results suggest that
measured and nominal concentrations from saltwater tests were not in close agreement, but
this analysis was based on limited set of data.

The measured concentrations in the recently published paper on marine/estuarine toxicity of
PFOS should also be included in this assessment:

Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder, Jennifer A Arblaster;
Aquatic toxicity evaluations of PFOS and PFOA for five standard marine endpoints.
https://doi.Org/10.1016/i.chemosphere.2021.129699

The second paper is on benthic organisms and PFOS is measured in overlying water, porewater
and sediment. This may provide further guidance on difference between PFOS measured and
nominal concentrations.

Stuart L. Simpson, Yawen Liu, David A. Spadaro, Xinhong Wang; Rai S. Kookana and Graeme E.
Batley; Chronic effects and thresholds for estuarine and marine benthic organism
exposure to perfluorooctane sulfonic acid (PFOS)-contaminated sediments: Influence of
organic carbon and exposure routes https://doi.Org/10.1016/i.scitotenv.2021.146008

Additional information for Appendix 0 based on a recently published paper:

According to Rewerts et al., 2021 additional handling steps, which are not typically reported
for ecotoxicological studies but may contribute to variability, include solution homogenization,
subsampling procedures, and the container materials selected for storage.
https://doi.org/10.1002/etc.4667

Reviewer 5

1 think the comparison of measured and nominal concentrations was an interesting read and a
useful contribution. That said, many toxicologists focused on PFAS have commented that
analytical confirmation is necessary for a high quality study - this was echoed (loudly) at the
SETAC Workshop on Risk of PFAS that occurred in summer, 2019. As well, in my own
experience there have been challenges in sometimes matching nominal and measured
concentrations for aquatic exposures. The paper by Rewerts et al. 2020 highlights some of the
challenges and provides recommendations for accurate solutions of PFAS. As a general rule, we
have erred on the side of reporting measured concentrations.

Two important thoughts. First, several very prominent analytical chemists that have made a
career of measuring PFAS have indicated to me that the analytical method is only about 30%
accurate - meaning that if the analytical measure was +/- 30% of nominal, they would be
considered "the same". EPA used 20% as a threshold (for deciding nominal and measured were
the same) and I'm not sure why this is. As far as 1 can tell, 30% is a more reasonable threshold.

Second, in the review and derivation of toxicity values for the MacDonald et al. 2014 paper,
EPA elected not to use the 20-day emergence rate endpoint, in part, because the nominal and
measured did not agree. This makes no sense to me. As long as the solutions were confirmed

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2.4. The Use of Measured and Unmeasured Toxicity Tests to Derive Respective Criterion

Reviewer

Comments

analytically and reported, that should be good enough and, in fact, preferred over nominal
alone.

Paper worth including in the section on nominal vs. measured PFOS concentrations:

Key Considerations for Accurate Exposures in Ecotoxicological Assessments of
Perfluorinated Carboxvlates and Sulfonates. Justin N. Rewerts, Emerson C. Christie, Alix
E. Robel, Todd A. Anderson, Christopher McCarthy, Christopher J. Salice, Jennifer A.
Field Environmental Toxicology and Chemistry, 2020

2.5 Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

• Are there relevant data that you are aware of that should be added to the analyses (note that
EPA is working on updating the toxicity data to reflect the data in ECOTOX between Sept, 2019
through the latest updati t, please provide references for consideration.

In particular, please comment on:

2.5.a.The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

2.5.a.i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data
from Yang et al. (2014) to suggest aquatic insects are relatively tolerant to acute
PFOS exposures. Specifically, Yang et al. (2014) conducted a 36-hour renewal,
measured PFOS acute test with the midge, Chironomus plumosus. This study was
not acceptable for quantitative use due to the potential problematic source of
the organisi : reported LC50 was 182 mg/t for PFOS indicating that insects
may not be one of the more sensitive taxonomic groups. Therefore, this test was
excluded from the acute criterion calculation, but used to waive the missing
insect MDR.

2.5.a„ii. the use of the quantitatively acceptable chronic toxicity value for mussel

(Lampsilis siliquoidea) from Hazelton et al. (2012), Specifically, Hazelton et al.
(2012) conducted a 36-day renewal, measured PFOS chronic test with fatmucket,
Lampsilis siliquoidea. The estimated EC10 was 0.05713 mg/t, which was
extrapolated from the author-reported data and the exposure response slope
from another PFOS toxicity study focused on another mussel species (Ellipto
complamata) as explained in Section 3.1.1.3.3, Therefore, this test was used in
the chronic criterion calculation.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5.a.iii. the use of the quantitatively acceptable chronic toxicity value for damselfly

(Enallagma cyathigerum) from Bots et al. (2010), Bots et al. (2010) conducted a
320-day renewal, unmeasured PFOS chronic test with blue damselfly nymphs,
Enallagma cyathigerum. The MATC was 0.03162 mg/L, which was calculated from
the author-reported value for nymph survival as explained in Section 3.1.1.3.2,
Therefore, this test was used in the chronic criterion calculation.

2.5.a.iv, the use of the quantitatively acceptable chronic toxicity value for midge

(Chironomus dilutus) from MacDonald et al. (2004), MacDonald et al. (2004)
conducted a 20-day renewal, measured PFOS chronic test with midge lava,
Chironomus dilutus. The EC10 was 0.05963 mg/L, which was an EPA-calculated
value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test was
used in the chronic criterion calculation.

2.5.b, EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as
well as the specific acute LC50 values (Appendix A.2) and chronic EC10 values (Appendix C.2)
that were estimated (for sensitive genera when C-R data were available) and used to derive
criteria.

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

Reviewer 1

• Were the data selected and/or excluded from the derivation of the criteria
derivation appropriately utilized?

In most cases, yes. Please see detailed comments on particular studies and interpretations in
response to other charge questions.

• Are there relevant data that you are aware of that should be added to the analyses
(note that EPA is working on updating the toxicity data to reflect the data in ECOTOX
between Sept. 2019 through the latest update)? If so, please provide references for
consideration.

Hayman, N.T., Rosen, G., Colvin, M.A., Conder, J., Arblaster, J.A. 2021. Aquatic toxicity
evaluations of PFOS and PFOA for five standard marine endpoints. Chemosphere
273:129699.

2.5.a.

2.5.a.i. 1 disagree with excluding this data point from the acute criteria calculations. 1 assume
this data has been removed under the assumption that these animals may have been pre-
exposed to PFOS and may have been more tolerant of PFOS exposures, which would result in
biased-high median lethal concentration (LC50) values. If so, this should be explicitly stated.
Assuming these Chironomus can develop tolerance to PFOS, it seems that they would have to
be exposed to rather high mg/L ranges of PFOS in water given the reported 96-hour LC50 of
182 mg/L. Based on published literature, 1 am unaware of natural ecosystems in China (where
the animals may have been originally harvested) with concentrations of PFOS that approach

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2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

this order of magnitude range (in which they could build up a tolerance). The animals were
obtained from a local market, so it is also possible that they were cultured for several
generations, presumably using uncontaminated water (which would further reduce the chance
that multiple generations were exposed at these levels). Overall, 1 think it is more reasonable
to assume that the animals used in the experiment have not built up an acute lethal tolerance
to PFOS, and the that LC50 result is unbiased. It does seem clearly show that insects may be
less sensitive to acute lethality effects of PFOS. As such, 1 think it should be included as a
quantitative endpoint.

Additionally, it seems inconsistent to exclude this Yang et al (2014) study, when chronic data
from an unpublished study by Funkhouser (2015) were included for quantitative consideration.
As noted on page C-25, the animals in the Funkhouser (2015) study were "purchased from a
private collector" and then kept for "several" generations prior to testing. The source of the
animals is just as uncertain as the Yang et al (2014) animals, and it is unclear (if PFOS tolerance
at lethal levels is possible) how many generations would be needed to shed adaptive tolerance
and how it would compare to "several." Simply put, if data from experiments like Funkhouser
(2015) are quantitatively included, those from Yang et al. (2014) should also be quantitatively
included (with some notes on the uncertainty of the animal sources).

2.5.a.ii. There were only three exposure levels in this experiment, including the control. One
PFOS dose (4.5 ng/L) indicated an absence of detectable effects on metamorphosis, the other
(69.5 ng/L) indicated an approximate 35% reduction relative to controls. This is not a definitive
test; there is little dose response information to fully confirm the effects/absence of effects
and predict an effective concentration (EC) value with a dose response model. Application of
another study's dose response curve to generate EC10 values for this study does not address
this fundamental shortcoming, and simply carries too much uncertainty. Although there are
only two PFOS doses, which is highly uncertain, use of a Maximum Acceptable Toxicant
Concentration (MATC) value may be a less uncertain path to including this study in quantitative
calculations. This would result in a more conservative chronic value for this study (0.018 mg/L
instead of 0.057 mg/L). Given the high uncertainty of using this result (due to only 2 PFOS
doses), 1 believe this value should be caveated in some way and re-evaluated for use or
excluded in future criteria derivation. For example, on page C-22, the Spachmo and Arukwe
(2012) value (which also featured a limited PFOS dose design), the document notes that the
limited doses "may limit its future use in the criteria derivation pending independent
verification of the toxicity values by EPA."

2.5.a.iii. 1 agree with the interpretation of the Bots et al. (2010) study and selection of the
MATC.

2.5.a.iv. First, on page 104, the document mentioned "an EC10 of 0.0586 mg/L for growth
following 10-days of exposure", but on page 115, the document noted "10-day growth with an
EC10 of 0.05963 mg/L". In Appendix C (page C-19), the document states "the independently-
calculated 10-day EC10 for growth was 0.0586 mg/L." There's some inconsistencies in the
value being obtained from EPA's EC10 modeling using this reference; please correct or clarify.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

One justification for using the 10-day EC10 growth result rather than other results is a lack of
being able to calculate EClOs. 1 do think the emergence results should not be discounted,
however. EPA notes "as for the emergence endpoint, there was a lack of a concentration-
response relationship and there were very similar levels of observed effects (which ranged
between 42.6 and 50.1%) despite the more than nine-fold increase in the mid-range treatment
concentrations (0.0023, 0.0144, 0.0217 mg/L, respectively)." The magnitude of the effect
(relative to controls), and the fact that there were statistically-detectable differences from
controls in some of these doses (0.0144, 0.0217 mg/L) seems to indicate an ecologically
meaningful adverse effect is occurring due to PFOS. This range of concentrations just might be
a portion of dose response curve that is relatively flat. There is a very clear adverse effect at
0.0949 mg/L. 1 think it would be reasonable to select the MATC for emergence (0.0071 mg/L
reported on page C-19) and treat it a second study point since it was a completely different
experiment from the 10-day experiment used to provide the EC10 of 0.05963 (or 0.0586) mg/L
value. A Species Mean Chronic Value using the 0.0071 and 0.0586 mg/L results would be 0.020
mg/L. This 0.020 mg/L value would seem to be protective while including the growth and
emergence data from these two experiments.

2.5.b. More details need to be provided on the dose response modeling using R. Appendix K
is helpful for providing the reader with details on the general approach, but where EClOs are
modeled by EPA, the model being used (out of the 22 available in the R software package)
needs to be specified. Providing some indication of variability (such as a 95% confidence
interval) for the model-generated EClOs is standard practice for dose response modeling, and
this information should be provided somewhere in the document. Showing the R package
output of the goodness of fit statistics (or equivalent) for the modeling in an Appendix would
be helpful; since this was used to select the model used in each instance of an EC10
calculation, it must be available, so 1 would recommend including it for full transparency and to
aid future efforts in understanding the aquatic toxicology of this chemical. Additionally, it
would be helpful to show the selected model fits for all calculated EClOs (as shown for the
most sensitive EClOs estimated). These steps would be helpful to ensure and demonstrate
quality of the model fits and reproducibility of the modeling work.

Additionally, somewhere in the document (Appendix K), the 22 dose response model
equations should be provided to the reader. Alternately, a reference could be made to a
document that clearly provides this information (ideally a peer-reviewed or EPA document)
containing all 22 models.

Reviewer 2

• Were the data selected and/or excluded from the derivation of the criteria
derivation appropriately utilized?

1 think the data used in the derivation of the criteria were appropriate. As mentioned above, 1
am a little concerned about the use of toxicity data from studies that did not measure the
concentration of PFOS in their experiments, especially considering the proportion of studies
that did not measure the concentrations. The confirmation of exposure concentrations is an
important principle of sound ecotoxicology.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments



• Are there relevant data that you are aware of that should be added to the analyses
(note that EPA is working on updating the toxicity data to reflect the data in ECOTOX
between Sept. 2019 through the latest update)? If so, please provide references for
consideration.

1 have listed a number of papers below that were published in 2020 and 2021 that the EPA may

want to consider in their assessment.

Hayman, N.T., Rosen, G., Colvin, M.A., Conder, J., Arblaster, J.A., 2021. Aquatic toxicity

evaluations of PFOS and PFOA for five standard marine endpoints. Chemosphere 273,
129699.. doi:10.1016/j.chemosphere.2021.129699

Logeshwaran, P., Sivaram, A.K., Surapaneni, A., Kannan, K., Naidu, R., Megharaj, M., 2021.

Exposure to perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at
ppb concentration induces chronic toxicity in Daphnia carinata. Science of The Total
Environment 769, 144577.. doi:10.1016/j.scitotenv.2020.144577

Simpson, S.L., Liu, Y., Spadaro, D.A., Wang, X., Kookana, R.S., Batley, G.E., 2021. Chronic effects
and thresholds for estuarine and marine benthic organism exposure to perfluorooctane
sulfonic acid (PFOS)-contaminated sediments: Influence of organic carbon and exposure
routes. Science of The Total Environment 776, 146008..
doi:10.1016/j.scitotenv.2021.146008

Li, R., Tang, T., Qiao, W., Huang, J., 2020. Toxic effect of perfluorooctane sulfonate on plants in
vertical-flow constructed wetlands. Journal of Environmental Sciences 92, 176-186..
doi:10.1016/j.jes. 2020.02.018

Aquilina-Beck, A.A., Reiner, J.L., Chung, K.W., Delise, M.J., Key, P.B., Delorenzo, M.E., 2020.
Uptake and Biological Effects of Perfluorooctane Sulfonate Exposure in the Adult
Eastern Oyster Crassostrea virginica. Archives of Environmental Contamination and
Toxicology 79, 333-342.. doi:10.1007/s00244-020-00765-4

Tornabene, B.J., Chislock, M.F., Gannon, M.E., Sepulveda, M.S., Hoverman, J.T., 2021. Relative
acute toxicity of three per- and polyfluoroalkyl substances on nine species of larval
amphibians. Integrated Environmental Assessment and Management 17, 684-690..
doi:10.1002/ieam.4391

Suski, J.G., Salice, C.J., Chanov, M.K., Ayers, J., Rewerts, J., Field, J., 2021. Sensitivity and

Accumulation of Perfluorooctanesulfonate and Perfluorohexanesulfonic Acid in Fathead
Minnows ( Pimephales promelas ) Exposed over Critical Life Stages of Reproduction and
Development. Environmental Toxicology and Chemistry 40, 811-819..
doi:10.1002/etc.4936

McCarthy, C.J., Roark, S.A., Wright, D., O'Neal, K., Muckey, B., Stanaway, M., Rewerts, J.N.,

Field, J.A., Anderson, T.A., Salice, C.J., 2021. Toxicological Response of Chironomus
dilutus in Single-Chemical and Binary Mixture Exposure Experiments with 6 Perfluoralkyl

22

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2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

Substances. Environmental Toxicology and Chemistry 40, 2319-2333..
doi:10.1002/etc.5066

2.5.a.

2.5.a.i. 1 think the EPA's decision that the data from Yang et al. (2014) was not acceptable for
quantitative use was appropriate. The source of the larvae is problematic. However, 1 don't
agree with the conclusion that insects may not be one of the most sensitive taxa. Chironomus
tentans is a relatively sensitive taxa to chronic exposure to PFOS (MacDonald et al. 2004). In
Table C.l, the EC10 for C. tentans is reported as 0.05963 mg/L. Chironomus tentans was also
the fourth most sensitive species used in calculating the chronic freshwater criterion (Table 3-
6). Also, another insect, Enallagma cyathigerum, another insect species, was the second most
sensitive species used in calculating the chronic freshwater criterion (Table 3-6).

2.5.a.ii. EPA used an EC10:EC35.4 from Drottar et al. (2000) for Elliptio complanata and applied
this ratio to derive an EC10 from the data reported in Hazelton et al. (2012) for Lampsilis
siliquoidea. The problem is that EPA have not clearly outlined in section 3.1.1.3.3 what
endpoint that Drottar et al. (2000) was measuring in Elliptio complanata (also note that the
genus and species are not spelled correctly in section 3.1.1.3.3). Is the endpoint measured in E.
complanata the same as the endpoint measure in L. siliquoidea? 1 tried to look up the endpoint
measure in Drottar et al. (2000) but 1 could not find the study and there was no reference
provided in the reference section for Drottar et al. (2000). This missing information makes it
difficult to comment on the validity of the approach that EPA has taken to derive an EC10 for L.
siliquoidea.

2.5.a.iii. 1 think the EPA's justification for the use of the survival data from Bots et al. (2010) is
valid. While control mortality reached 40% in the control, the plateau in control mortality after
60 days, the total duration of the test being 200 days, and 82.57% survival in the control from
day 60 to 200, justifies the inclusion of the MATC derived from Bots et al. (2010) for Enallagma
cyathigerum in the derivation of a chronic criterion.

2.5.a.iv. First, the wrong species is referenced in relation to the MacDonald et al. (2004) study.
MacDonald et al. (2004) reported the toxicity of PFOS to Chironomus tentans. The EPA's
derivation of a 10-d EC10 for Chironomus tentans using the data from MacDonald et al. (2004)
is not clear. In Appendix C, section C.2.4, the EPA writes, "EPA could not fit a curve to
independently verify the 10-day survival (due to a lack of a specific sample size for this
endpoint as the number of replicates was not stated in the paper; however, the number of
replicates was between 2 and 4 and EPA sought to obtain clarification and treatment level data
from the study authors)" It is not clear how EPA got the information necessary, e.g., number of
replicates, to fit a curve. It is also not clear what EPA means by "...and treatment level data
from the study authors."? Did EPA acquire the raw data for growth from the 10-day toxicity
test with C. tentans? If that is the case, they have not made that clear. If that is the case, it
would also strengthen their independently derived EC10 for growth in C. tentans. 1 think the
EPA needs to more clearly explain where they got the data necessary to derive the EC10 for C.
tentans used in the chronic criterion.

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2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

2.5.b. 1 think the approach that the EPA used to determine effect measure from
concentration-response data was appropriate. The use of the drc package in R to fit 22
different models to the empirical data and then using several criteria (e.g., AIC, residual
standard errors, confidence intervals) to evaluate the fit of the different models is robust. It
would have been useful if the EPA reported the 22 different models in Appendix K.

1 think the LC50 and EC10 values determined by the EPA using the approach mentioned in the
previous paragraph was appropriate. It is valid for these effect measures to be determined
when the concentration-response data has been provided by the authors of the study. The EPA
has also made is clear in Appendix A.2 and C.2 how they determined these effect measures
using the concentration-response data provide in the studies. This generates a high level of
transparency in the derivation of the criterion.

Reviewer 3

See collective responses below

• Were the data selected and/or excluded from the derivation of the criteria
derivation appropriately utilized?

Data selection and waiving of the MDR for insect family in the FAV seem reasonable.

The data selection for the derivation of the draft criteria are limited to published and/or
available studies from 2018 and prior. This significantly reduces the studies used in the
derivation as a number of publications have become available in recent years.

For example:

A newly published study is available for fathead minnows exposed to PFOS for chronic duration
and over the course of reproduction and development. Although, this study was static-
renewal, PFOS concentrations are measured ; importantly, this study resulted in a NOEC of
88ng/L based on reduced biomass seen in the second generation (Suski et al. 2020).
Importantly, follow-on work (in prep) indicates that this may be a maternal transfer impact as
PFOS exposures to juvenile fish alone do not share results.

Also, from the authors noted above is an ongoing full life-cycle fathead PFOS and PFAS mixture
exposure. This study is being conducted under flow through conditions and is expected to
reach termination in December 2021.

McCarthy et al. 2021 published data on chironomids (EC20 = 1.7ng/L), these are also not
included here.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

Bryan Brooks (Baylor) and Matt Simcik (UMN) also have acute data on the fathead minnow
with measured concentrations, these are not published just yet.

David Moore (Army Corps) is near completion of a full life-cycle fish study

In particular, SERDP has been funding this research for years and those data are published,
recently published or near final. EPA should reach out to SERDP Pis for data inquiries and
potential inclusion in these draft criteria.

2.5.a.

2.5.a.i. This seems appropriate, the flower market is most definitely an odd place to purchase
research organisms.

2.5.a.ii. From Section 3.1.1.3.3: "The in marsupia exposure was followed by a 24-hour free
glochidia exposure consisting of a factorial design, such that free glochidia from the control
group of the marsupia exposure were divided between a control and the two PFOS treatments
and the PFOS treatments were split into control and the same PFOS treatment group as the
marsupia exposure." - Comment: This is an exceptionally long and confusing sentence please
revise to help the reader understand this complex study and overall approach that EPA took.

The approach seems ok given the limited data availability at this time.

2.5.a.iii. Given the duration of the study the researchers likely hovered around the nominal
concentrations of PFOS. Inclusion seems appropriate.

2.5.a.iv. 1 am uncomfortable with this conclusion presented here, it may be more appropriate
to use MacDonald et al. data from the 20-day endpoint considering recent publication from
McCarthy et al. 2020 as noted above.

2.5.b. This seems like a reasonable and defensible approach if it is applied consistently across
genera.

Reviewer 4

The data selected to derive PFOS criteria are appropriate. Studies that did not fully meet the
data quality objectives outlined in the 1985 Guidelines were not considered for inclusion in the
criteria derivation, including some studies with other PFAS exposures, but were considered
qualitatively as supporting information. A brief summary of each study describing the
experimental conditions and summary tables providing all the relevant information such as
strengths and limitations of each study, end points selected for deriving criteria are well
documented by the EPA team and provides further confidence in data selection process.

The key acceptable exclusion/inclusion criteria used to derive draft criteria are listed below:

• Only single chemical toxicity tests with PFOS were considered for possible inclusion in
criteria derivation, studies that tested chemical mixtures, including mixtures with PFAS
compounds were excluded from criteria derivation.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

• Both controlled laboratory experiments and field observations/studies were included.

• PFOS toxicity tests were not excluded from quantitative use in criteria derivation on
the basis of unmeasured test concentrations alone.

• Due to lower sensitivity, insect MDR was excluded from the criterion calculation, but
were used to waive the missing insect MDR.

• Further supporting information on acceptable and unused studies for acute and
chronic endpoints and for freshwater and marine studies are documented and
summarized as appendices in this report.

Additional toxicity data published over the last six months is listed below:

Marine/estuarine

Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder, Jennifer A Arblaster
Aquatic toxicity evaluations of PFOS and PFOA for five standard marine endpoints.
https://doi. org/10.1016/i.chemosphere. 2021.129699

Stuart L. Simpson, Yawen Liu, David A. Spadaro, Xinhong Wang; Rai S. Kookana and Graeme E.
Batley Chronic effects and thresholds for estuarine and marine benthic organism
exposure to perfluorooctane sulfonic acid (PFOS)-contaminated sediments: Influence of
organic carbon and exposure routes https://doi.Org/10.1016/i.scitotenv.2021.146008

Fresh water

Christopher J. McCarthy, Shaun A. Roark, Demitria Wright, Kelly O'Neal, Brett Muckey, Mike
Stanaway, Justin N. Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J.

Salice, Toxicological Response of Chironomus dilutus in Single-Chemical and Binary
Mixture Exposure Experiments with 6 Perfluoralkyl Substances, Environmental
Toxicology and Chemistry, 10.1002/etc.5066, 40, 8, (2319-2333), (2021).
https://doi.org/10.1002/etc.5066

2.5.a.

2.5.a.i. Waiving an unfulfilled MDR when available data suggest it is not among the four most
sensitive genera is consistent with previous EPA criteria documents, including U.S. EPA (2016).
At this stage, 1 do not fully agree with the statement that midge larvae are tolerant to acute
exposures. The OECD protocol recommends 48h acute test for midge larvae and the 48h
exposure period is acceptable duration for assessing acute toxicity. The study by Olson (2017)
has limitations but this study can't be fully ruled out. The chronic toxicity data exhibits
sensitivity of insects to PFOS and this statement is also supported by the authors. In addition,
Stefani et al. (2014), Macdonald et al. (2004), and Marziali et al. (2019) conducted chronic
toxicity tests with Chironomus spp. and reported apical endpoints. Results of these studies,
taken together, also suggest that insects are among sensitive taxa to chronic PFOS exposures
(with adverse effects reports at low ug/L)

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

1 support the recommendation 'Additional insect toxicity data for PFOS would be very useful for
further examining the relative sensitivity of insects to PFOS exposures".

Unpublished work from our lab shows acute toxicity to midge larva, Chironomus tepperi at 1
mg/L PFOS (48 h EC50 value).

2.5.a.ii. The authors have provided detailed assessment of this study and explained the
approach used for the calculation of chronic toxicity value (section C.2.3-Third Sensitive
Freshwater Genus for Chronic Toxicity: Lampsilis siliquoidea (mussel). Hazelton et al. 2012 used
robust study design in spite of including only two concentration of PFOS in this study. The PFOS
exposure concentration was measured, and metamorphosis success was used as an endpoint
for inclusion in the criteria development. While viability of free glochidia at 24 hours post
removal from females was a less sensitive endpoint and did not meet the acceptability criteria.
The reduction in metamorphosis success at the 0.0695 mg/L was estimated to be 35.4% but
EC10 could not be calculated based on only two PFOS concentrations tested in this study. The
EPA team has calculated EC10 (0.05713 mg/L) using the exposure response slope from PFOS
toxicity study on another mussel species (Ellipto complamata). The explanation and logic
provided is reasonable to include the calculated EC10 value to derive the freshwater chronic
criterion and to better understand the effects of PFOS on aquatic insects.

2.5.a.iii. As a weight of evidence approach, EPA ran additional analyses with some of the other
toxicity values for E. cyathigerum to understand the influence of this study on the overall
chronic criterion. The 150-day MATC was more comparable to the other aquatic insect data
and more representative of life cycle effects than the 10-day MATC or NOEC at 60 and 320
days of exposure (Table 4.3 of the report). EPA has concluded that the 150-day MATC should
be used quantitatively to derive the chronic freshwater criterion toxicity. In addition, the
control survival of test organisms was determined to be acceptable at this time point in the
test. 1 am in agreement with this decision.

2.5.a.iv. The observed effects of PFOS on C. dilutus reported in the paper by the study authors
include survival and growth as weight (measured as mg of ash-free dry mass per individual) for
both the 10-day and 20-day exposure durations and emergence and reproduction over the 20-
day exposure duration. The author reported 10-day growth and survival EClOs for the study
were 0.0492 and 0.1079 mg/L, respectively. The study authors also reported NOECs of 0.0491
mg/L, LOECs of 0.0962 mg/L, and MATCs of 0.0687 mg/L for both endpoints. The author
reported 20-day ECi0s for growth, survival, and total emergence were 0.0882, 0.0864, and
0.0893 mg/L, respectively. And the study authors also reported NOECs of 0.0217 mg/L for
growth and survival and < 0.0023 mg/L for emergence, LOECs of 0.0949 mg/L for growth and
survival and 0.0217 mg/L for emergence, and MATCs of 0.0454 mg/L for growth and survival
and 0.0071 mg/L for emergence.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

a concern than the 10-day exposure, which had measured concentrations that were much
more in line with the expected nominal concentrations. The independently-calculated 10-day
ECio for growth was 0.0586 mg/L was used quantitatively to derive the chronic aquatic life
criterion.

The EPA team has reviewed publications by Stefani et al. (2014) and Marziali et al. (2019) as
additional supporting information. These authors conducted chronic toxicity tests with
Chironomus spp. and reported chronic apical endpoints (at low ug/l) but at only at one
concentration.

Use of the chronic toxicity data for PFOS in a recent publication should also be considered
to assess the reliability of 20-day endpoints (adverse effects reported at 2-3 ng/L).

Christopher J. McCarthy, Shaun A. Roark, Demitria Wright, Kelly O'Neal, Brett Muckey, Mike
Stanaway, Justin N. Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J. Salice,
Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture
Exposure Experiments with 6 Perfluoralkyl Substances, Environmental Toxicology and
Chemistry, 10.1002/etc.5066, 40, 8, (2319-2333), (2021).
https://doi.org/10.1002/etc.5066

2.5.b. This is an excellent approach utilized by the EPA Team. EPA's approach for fitting
concentration-response (C-R) data resulted in consistent approach across various
ecotoxicological studies. The R drc package was used to fit 22 different models to each
individual C-R dataset. A single model was then selected from the 22 models to serve as the
representative C-R model. The selected model represented the most statistically-robust model
available. In certain cases, this approach even improved and helped to select most sensitive
toxicological endpoint.

In depth analyses and associated dose-response graphs in Appendix A.2 and Appendix C.2
provides further in-depth information on the EPA's approach for fitting concentration-
response (C-R) data. As noted in Section 8 some of the values are missing.

Reviewer 5

• Were the data selected and/or excluded from the derivation of the criteria
derivation appropriately utilized?

As mentioned, 1 feel that there are some inconsistencies with how some data were included or
excluded. In the previous comment, for example, some data were excluded from the
MacDonald et al. 2014 paper because there was some disagreement between nominal and
measured. With regard to PFAS, 1 would say measured is almost always better than nominal
and the fact that these sometimes don't agree should not be too big of a deal as long as they
are not wildly different. EPA put substantial effort into sometimes justifying nominal - in all
cases, excluding studies that had analytical confirmation is less defensible than including
studies that only report nominal, in my opinion. This last statement is, of course, provided the
analytical methods are robust.

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2.5. The Toxicity Data to Derive the Draft Criterion

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Comments

Sensitivity and Accumulation of Perfluorooctanesulfonate and Perfluorohexanesulfonic
Acid in Fathead Minnows (Pimephales promelas) Exposed over Critical Life Stages of
Reproduction and Development. J.G. Suski, C.J. Salice, M.K. Chanov, J. Avers, J.

Rewerts, J. Field Environmental Toxicology and Chemistrv, 2021, pp. 811-819.

Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture
Exposure Experiments with 6 Perfluoralkvl Substances. Christopher J. McCarthy, Shaun
A. Roark, Demitria Wright, Kellv O'Neal, Brett Muckev, Mike Stanawav, Justin N.
Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J. Salice. Environmental
Toxicology and Chemistry, 2021, pp. 2319-2333.

2.5.a.

2.5.a.i. Given that insects are among the most sensitive organisms for the chronic exposures
to PFOS, it seems the Yang et al. 2014 paper is not very consistent with the prevailing data.
Additionally, McCarthy et al. 2021 reports toxicity to chironomids similar to that of MacDonald
et al. Additionally, while the Olson 2017 data for Aedes species was not acceptable (for valid
reasons), nonetheless the study shows very high sensitivity of another insect species to acute
exposures to PFOS. That said, given the EPA's stance and justification that "nominal generally
equal measured PFOS concentrations", I'm inclined to put more confidence in Olson's study.
Same for the 20-day data in the MacDonald et al. paper on chironomids. In that case, there
was a "relatively large difference between measured and nominal concentrations (p. 278)" and
so the data were not used. This seems odd to me - as long as there are measured data, that's
what 1 would suggest using. Regardless, the MacDonald et al. paper points to the sensitivity of
insects so, collectively, I'd be disinclined to sav that the Yang et al. paper shows insects are not
sensitive and the data requirement can be waived. 1 wonder if it's possible to somehow
estimate an acute toxicity value for aquatic insects based on chronic toxicity data? Basically, a
reverse of the Acute/Chronic ratio approach.

2.5.a.ii. Unfortunately, 1 cannot find the Drottar et al. (2000) paper which is the basis of
estimating the EC10 from the EC35 generated in the Hazelton et al. (2012) study. And..l think
the citation in the document is incorrect and this should be Drottar and Kreugar (2000g)... or
check to make sure the citations in text and references match. Moroever, the Drottar paper
appears to be an acute test which is VERY different than the Fatmucket study. This approach
seems like a "stretch" and, again, somewhat inconsistent with the approaches and decision
matrix EPA has used to utilize or discard other data and endpoints.

2.5.a.iii. Well, clearly it would have been better to be able to estimate an EC10 but this
appears appropriate. 1 note that for this study the exposure concentrations were an order of
magnitude apart; in other cases, EPA has used "too big of a difference between exposure
concentrations" to discard a study or two. Somewhere, it would be good to know at what point

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2.5. The Toxicity Data to Derive the Draft Criterion

Reviewer

Comments

there is too great a difference among exposure concentrations (lOx, 20x, ?) for the study to be
deemed acceptable for use quantitatively.

2.5.a.iv. 1 do not agree with the toxicity value used by EPA as obtained from the MacDonald et
al. study. The lowest toxicity value is the MATC for 20-day emergence of 0.0071 mg PFOS/L. It
is not clear why EPA did not use this value? Emergence is clearly extremely ecologically
relevant, and the value generated seems as defensible as most of the other endpoints EPA has
chosen to include?

Additionally, and as mentioned above, see the paper by McCarthy et al. (2021) that was just
published in Environmental Toxicology and Chemistry. Those data appear robust and should
meet acceptability criteria.

2.5.b. In general, the approach for fitting C-R data that EPA used is basically state-of-the-art.
The drc package is very powerful and provides a way to test many different curves to then
select the best fit model. Although EPA described some of this in the several sections related to
"fitting x data (K 1.2)", 1 think more details would be warranted. The description for the criteria
to select best fit models is rather vague. Perhaps a table of specific fit criteria would be
helpful? Perhaps this is not doable because every dataset is different.

When 1 teach modules on Akaike Information Criteria (AIC) 1 emphasize that the metric
"penalizes" fit for more parameters within a model. So, using AIC can yield the simplest, best
model that fits the data. This is because models with more parameters tend to yield a better fit
purely based on statistical properties and not the actual phenomena being studied. 1 am not
aware that AIC is a measure of the model fit to "true outcomes" which are only theoretical
constructs, 1 think. If we knew the "true outcomes" we would not really need the model.
Anyway, 1 would encourage the authors to review the AIC section and make edits if necessary
and certainly cite the source of the explanation.

For section K.2.2. are there actually any criteria (i.e., numbers) that are used to determine
when a model fit is appropriate? As a simple example, maybe one would consider an rA2 of 0.8
or better to be a "good model" for linear regression? Some statements to this effect and any
details regarding actual criteria used to select "good models" would be helpful. So, overall the
curve fitting approach is appropriate but more, specific details would be helpful.

2.6 Please comment on the translation of the chronic water column criterion elements for aquatic life
to derive the tissue-based criterion elements, considering the bioaccumulation of PFOA and PFOS.
In particular, please comment on:

2.6.a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the
chronic water column criterion elements into tissue-based criterion elements,

2.6.b, EPA's determination of appropriate BAFs and the tissue types that the tissue criterion
elements were based.

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.6. The Translation of the Chronic Water Column Criterion Elements for Aquatic Life to Derive the Tissue-
Based Criterion Elements Considering Bioaccumulation

Reviewer

Comments

Reviewer 1

The derivation of the tissue criteria in this manner is highly uncertain. To my knowledge this is
the first time EPA has applied ambient water quality criteria protective of aquatic life direct
toxicity with uptake factors (bioaccumulation factors (BAFs), bioconcentration factors (BCFs))
in this manner to calculate tissue criteria. References are made to the selenium tissue criteria,
but those are used in the reverse (i.e., criteria based on measured concentrations in tissue
used to calculate water criteria). The use of criteria for water with a assumed uptake factor
carries a large amount of uncertainty, and in general, the use of measured concentrations in
tissue linked to adverse effects is a more straightforward approach since it does not involve
uptake model predictions. This needs to be noted in the text. Also, are the predicted tissue
criteria meant to be a temporary stop-gap until tissue effect data become available? This
should be discussed and clarified.

2.6.a. The use of BAFs derived from field studies is inherently uncertain due to the wide
variety of techniques used in the compiled studies, their analytical data quality, the differences
in species and ecosystems, experimental designs, spatial uncertainties for mobile animals like
fish, etc. That being said, the use of a BAF value (or BCF) in criteria derivation is consistent with
other criteria developed by EPA. As noted above, the use of the tissue criteria needs to be
considered carefully, and 1 think empirical tissue data from toxicity experiments should form
the basis of a next iteration of a tissue criteria.

2.6.b. The development of BAFs for invertebrates, fish (whole body), and fish (muscle) seems
reasonable for the application in estimating a draft or interim tissue criteria until empirical
tissue data can be used to calculate tissue criteria directly.

Reviewer 2

2.6.a. 1 think the EPA has sufficiently addressed the uncertainty around the use of BAFs and
the chronic water column criterion in the derivation of tissue-based criterion. They have
indicated that tissue-based criterion should only be observed once in 10 years. The use of the
geometric mean of the reported BAFs incorporates the range of BAFs that may be present for
different invertebrate and fish species. The use of the chronic water column criterion also
builds in added conservatism to the tissue-based criterion.

Prosser et al. (2016) reported BAFs for PFOA in three freshwater species (two invertebrates
and one fish) (See Tables S29-31 in Supplementary Information), but it was not considered in
this assessment. It is not clear why it was not considered.

Prosser, R.S., Mahon, K., Sibley, P.K., Poirier, D., Watson-Leung, T., 2016. Bioaccumulation of
perfluorinated carboxylates and sulfonates and polychlorinated biphenyls in laboratory-
cultured Hexagenia spp., Lumbriculus variegatus and Pimephales promelas from field-collected
sediments. Science of The Total Environment 543, 715-726.
doi:10.1016/j.scitotenv.2015.11.062

2.6.b. The evaluation criteria for BAFs outline in Table 2-4 are appropriate and the decision to
only use high and medium quality BAFs is justified based on the criteria that would make a BAF
low quality. It was a good idea to use fish BAFs based on the concentration in muscle and

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2.6. The Translation of the Chronic Water Column Criterion Elements for Aquatic Life to Derive the Tissue-
Based Criterion Elements Considering Bioaccumulation

Reviewer

Comments

whole body (Table 3-12). Muscle tissue is usually exclusively sampled in large fish, especially as
part of fish consumption guidelines. The whole body is more appropriate for small fish and
invertebrate species, e.g., minnows, benthic macroinvertebrates.

Reviewer 3

Please clarify, the following sentence: "BAFs used in the derivation of the PFOS tissue criteria
consisted of > 2 water and organism samples each and were collected within one year and 2
km distance." It is unclear if the >2 samples refer to the tissue & water samples being
mismatched temporally or if there where >2 sets of water and tissue samples that were
collected in different years.

If the latter then this approach seems appropriate; if the former, EPA should discuss
differences in water chemistry between years to alleviate any concerns with matching tissue
concentration data to water samples that may have significant environmental temporal
variability.

A table summarizing the animal tissues used in deriving the BAFs would be helpful to assess
the range offish species and their dietary preferences.

Reviewer 4

2.6.a. The freshwater chronic PFOS toxicity data with measured tissue concentrations was
limited, with no quantitatively acceptable tissue-based tests. Therefore, there were insufficient
data to derive tissue-based criteria using a GSD approach from empirical tissue data from
toxicity studies.

Tissue criteria derived from the chronic water column concentration (CCC) with the use of
bioaccumulation factors were developed by EPA. The chronic freshwater criterion also
contains tissue-based criteria expressed as 43.0 mg/kg wet weight (ww) for fish whole-body,
25.3 mg/kg ww for fish muscle tissue, and 12.3 mg/kg ww for invertebrate whole-body tissue.

EPA developed protective tissue-based criteria through a bioaccumulation factor approach.
The authors reviewed PFOS BAF literature based on four criteria 1) number of water samples,
2) number of organism samples, 3) water and organism temporal coordination in sample
collection, and 4) water and organism spatial coordination in sample collection and developed
a ranking system. BAFs used in the derivation of the PFOS tissue-based criteria consisted of > 2
water and organism samples each and were collected within one year and 2 km distance. This
scheme assured selection of only BAFs of high and medium quality to derive the tissue criteria.

2.6.b. BAFs are different for muscle/fillet and whole-body tissues. Humans consume
muscle/fillets from fish and soft tissues from bivalves, therefore the water quality criteria
recommended by EPA used BAFs based on these tissues. In addition, muscle and whole-body
are the most commonly sampled tissue types in monitoring programs. These criteria were
developed based on the values reported for 50-60 samples (Table 3-12).

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External Peer Review Report for EPA's Draft PFOS Aquatic Life Water Quality Criteria

2.6. The Translation of the Chronic Water Column Criterion Elements for Aquatic Life to Derive the Tissue-
Based Criterion Elements Considering Bioaccumulation

Reviewer

Comments

representative tissue concentrations using tissue-specific bioaccumulation factors (BAFs).
Author's decision uses agreement on the use of female reproductive tissues due to its
relevance for potential maternal transfer to offspring. These additional tissue-based values
were calculated for comparative purposes and were not proposed as recommended criteria.

Reviewer 5

2.6.a. Using the BAF for PFOS to determine the tissue-based criterion elements is, 1 think, an
interesting and useful approach given the lack of tissue-based metrics associated with toxicity
data. The variability in observed bioaccumulation of PFOS is an active area of research but the
work by Burkhard 2021 (also an author on the AWQC) provides an excellent synthesis and
compendium of available BAFs for PFOS. That said, 1 noticed that the criteria for co-located
tissue and water samples for PFOS was that they were collected within a year of each other
and within 2 km distance (p. 134) - this likely contributes to significant variability in the BAFs.
Although there are few published datasets, there are some datasets that show considerable
temporal and spatial variability in PFAS water concentrations over the course of a few weeks
and over a spatial distance of less than 0.5 km. 1 wonder if the variability in BAFs would
decrease if the criteria were narrowed to co-collected samples measured at the same time?
Might be worth the exercise. Given the variability in PFOS BAFs, why not use something like
the 25% percentile BAF instead of the geometric mean? When developing a protective criteria
and there is a very noisy data set, it might be beneficial to err on the side of caution until
better data (many co-located samples in space and time) were available. So, in summary, 1
think the approach of using BAFs to estimate tissue-based criteria is reasonable but given the
variability in BAFs, 1 would encourage using a lower BAF instead of the geometric mean or
reconsidering the data that went into the BAFs used for criteria development.

2.6.b. Invertebrate whole body, fish whole body, and fish muscle are appropriate tissues for
the tissue-based criterion. These are the most commonly collected tissue types and are
relevant to monitoring efforts and are even useful for considerations offish advisories. That
said, the only other tissue worth considering would be for liver in fish since this tissue
accumulates considerably more PFOS than muscle - these are included in the appendices so
this is appropriate. Overall, tissues used for the tissue-based criteria are appropriate.

2.7 Please comment on t uency and duration of the criterion elements, in particular the tissue-
based criterion elements.

2.7. The Frequency and Duration of the Criterion Elements

Reviewer

Comments

Reviewer 1

The 4-day duration seems to be supported by the more sensitive chronic endpoints used to
derive the CCC.

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2.7. The Frequency and Duration of the Criterion Elements

Reviewer

Comments

For the tissue-based criterion (page 135), there is no clear support for assuming a 10-year
exceedance frequency. Given the uncertainty with the BAF-predicted tissue criteria, and how
little is known regarding the recalcitrance of PFOS in aquatic ecosystems and recovery time if
PFOS inputs in water were halted, the assignment of a 10-year exceedance frequency at this
stage seems completely arbitrary. We simply do not yet know the time frame over which
aquatic ecosystems recover from PFOS. It is not technically supported to cite recovery times
for selenium to support a 10-year recovery time for PFOS, these are completely different
toxicants that have their own unique fate and behavior. USEPA (1985) guidance suggests
assuming a 3-year frequency as a default, and the discussion on page 135-136 is not
scientifically convincing enough to modify it to 10 years.

Additionally, it should be noted that the exceedance frequency for another organic chemical,
Tributyltin (TBT) was set at 3 years by EPA in derivation of that criteria. TBT exhibits uptake
factors similar to PFOS (i.e., BCF of approximately 2,000 L/kg, wet weight for goldfish, as noted
in the EPA TBT criteria document, which is similar to the PFOS BAFs of 1,800-3,100 L/kg, wet
weight being used to calculate the fish tissue criteria). TBT is also persistent in aquatic
ecosystems, as noted by EPA. Given TBT is at least an organic chemical, it is a closer analog
than selenium, which is an element. As such, the exceedance frequency for the PFOS tissue
criterion should be set at the default of 3 years unless EPA can provide convincing technical
information specific to recovery times for PFOS.

Additionally, on page 136, the paragraph that begins with "Metals and other chemical
pollutants such as PFOS..." is not convincing as any quantitative support for EPA's 10-year
exceedance frequency for the chronic tissue-based criteria. The text as written may give the
reader the conclusion that PFOS recovery may be "on the order of decades", as EPA notes for
selenium. There is no support for the conjecture that PFOS recovery may be "relatively slow"
or require decades, as noted in my above comment.

Reviewer 2

As per Table 0-1, 1 think the chosen durations and frequencies for the acute and chronic
criteria are appropriate. They will ensure protection of aquatic life. The duration of the tissue-
based criterion is appropriate as the concentration will be measured when biota is collected.
The 10-year frequency is appropriate considering that for biota to reach the tissue-based
criteria, they would likely to have been exposed to concentrations at or above the chronic
criteria for an extended period of time.

Reviewer 3

This is a not an easy statement to comment on, as it may be unlikely that the aquatic receptors
will exceed or reach these tissue concentrations prior to exceedances from the CCC.

What 1 am not clear on is, if tissue concentrations exceed these proposed thresholds yet, PFOS
water concentrations do not exceed the CCC, what would be the proposed guidance?

Reviewer 4

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2.7. The Frequency and Duration of the Criterion Elements

Reviewer

Comments

point, or instantaneous, measurements that reflect integrative accumulation of PFOS over time
and space in population(s) at a given site. It was appropriate for EPA to inform the
recommended ten-year exceedance frequencies for the chronic tissue-based criteria given the
large variation in possible biological and physical variable influencing ecological recovery.

Reviewer 5

In my opinion, the frequency and duration of criterion elements is among the most uncertain
and potentially contentious elements of any type of protective criteria. The frequency and
duration for tissue-based criteria is that the tissue-based criteria cannot be exceeded more
than once in a 10 year period. This means that if the PFOS criterion for whole body in fish of 43
mg/kg bw is exceeded more than once in a 10 year period then the criteria is exceeded. This
also means the fish was likely exposed to the 0.014 mg/l concentration for longer than an
instantaneous exposure and likely longer than 4 days. So, to me, does this not mean that if 43
mg/kg bw was measured in a fish tissue, then the fish was likely exposed to 0.014 mg PFOS/L
for longer than 4 days, doesn't it? And, this also means that if fish whole body concentrations
were 42.5 mg/kg bw for 10 years, the criterion would not be exceeded. 1 would suspect that
long term PFOS exposures that consistently lead to 42.5 mg/kg bw in fish would likely translate
to adverse ecological impacts in some biota present in the same system. When 1 think of it this
way, these criteria do not seem appropriately protective. In my view, the water column
continuous exposure criteria should be adjusted downward which would then translate to a
lower tissue-based criteria which might be more reasonable. Although, as mentioned, another
protective approach would be to use something like the 25th percentile BAF or something
other than the mean. That said, at least in many cases fish tissue monitoring occurs on a yearly
basis so there is some potential for the criteria to be reasonably assessed against
environmental data. It is still possible that fish tissue concentrations could be exceeded every
year and this be missed by monitoring efforts. Nonetheless, because tissue concentrations are
an intergrative measure and because many monitoring programs probably do measure fish
every year, this is a better match than the water column criteria.

When we consider the acute and chronic water column criteria, the frequency and duration
elements are protective, in my opinion. The problem is that nobody knows if the criteria for
acute toxicity are exceeded for more than 1 hour or whether the chronic criteria was exceeded
for more than 4 days - this extent of temporal resolution (hourly concentrations or 4-day
running averages) just does not exist. So while 1 agree that conceptually, the frequency and
duration elements definitely would add to the protection of aquatic life...1 just don't see how
these can be implemented or regulated. Perhaps EPA is aware that my concern is not
warranted because in relevant circumstances, appropriately timed environmental data are
obtained.

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2.8 Please provide any additional technical comments that you believe should be considered.

2.8. Additional Technical Comments to Consider

Reviewer

Comments

Reviewer 1

1 have the additional detailed comments:

a) Please note that the comments provided in this file reflect a focus on of key portions
of the "Draft of the Aquatic Life Water Quality Criterion..." document as directed by
the above charge questions provided to me. Given time and resource constraints and
the scope of my review, it was not feasible to provide a detailed review of the entire
document and all of the supporting references and their associated results and
conclusions. As such, 1 reserve my right to supplement or amend my comments in
future, pending additional review or new information. Thank you for the opportunity
to assist EPA in its work on this very important matter, and 1 was honored to be
selected as a reviewer.

b) In general, the document needs some quality copy editing effort. 1 found many
typographical errors, issues with formatting, reference/citation issues, and in some
cases, poorly-worded text. 1 have noted a few of these instances below.

c) Page xv: "25.3 mg/L ww" is not correct units for a concentration in tissue.

d) Page 6: "The carbon chain can be fully fluorinated...". Please specify that this applies
to PFAS in general, not to PFOS.

e) Page 6: The reference to "Table 2-1"; should that be Table 1-2?

f) Page 9: Please note in Figure 2-1 that this is the linear isomer of PFOS. It would be
helpful to note that the PFOS data in this study are likely from experiments with water
spiked with the linear PFOS isomer. It is hypothesized that toxicity and
bioaccumulation may differ between branched and linear forms of PFCAs and PFASs.
Linear PFOS is thought to be more accumulative (as noted on Page 45) and potentially
more toxic to aquatic life when the dose is expressed as an external water
concentration. At some sites, a portion of the concentrations of PFOS in water (which
are reported as the sum of branched and linear PFOS) is branched PFOS, so criteria
derived from linear PFOS could be overly protective. Please include this uncertainty in
the discussion in the document.

g) Page 18: To my knowledge, FTSAs degrade to PFCAs, not PFSAs like PFOS. See Zhang
et al. (2016): Zhang, S., Lu, X., Wang, N., & Buck, R. C. (2016). Biotransformation
potential of 6:2 fluorotelomer sulfonate (6:2 FTSA) in aerobic and anaerobic sediment.
Chemosphere, 154, 224-230. doi:10.1016/j.chemosphere.2016.03.062

h) Page 62: Regarding "The importance of the sediment pathway for PFOS
bioaccumulation..." Larson et al. (2018) conducted some insightful food web modeling
on benthic and pelagic sources of PFOS. See: Larson, E.S., Conder, J.M., Arblaster, J.A.
2018. Modeling avian exposures to perfluoroalkyl substances in aquatic habitats

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2.8. Additional Technical Comments to Consider

Reviewer

Comments

impacted by historical aqueous film forming foam releases.
https://doi.Org/10.1016/j.chemosphere.2018.03.004 Chemosphere 201:335-341.

i) Page 66: Starting here on this page and in the rest of this section, most of the units
need to be specified for dry weight or wet weight for concentrations of PFOS in tissue.
There were other instances of this error in the document as well. For units of every
concentration of PFOS in tissue, please be sure to specify dry weight or wet weight.

j) Page 73-75: There are a few scientific names on these pages that are not italicized.
Also may occur in other portions of the document.

k) Page 78: USEPA (1998) is not cited in the references section; 1 fear there may be other
similar omissions.

1) Page 78: Where the 1985 guidelines are mentioned, please cite to USEPA (1985).

m) Page 78: Replace the "Stephan et al. (1985)" citation with USEPA (1985). Also, 1
believe the surname of senior author of USEPA (1985) is Stephen, not Stephan.

n) Page 80: At the start of Section 2.10.2, it would be good to discuss linear and
branched PFOS.

o) Page 86: The use of EC10 values instead of effective concentration 20% (EC20) values
for chronic values is inconsistent with EPA's general practice for developing aquatic
life values. The selection of EClOs for the selenium criteria (EPA, 2016) was associated
with the derivation of tissue guidelines. In the EPA (2016) document, EPA noted
"EC20s have historically been used in the derivation of EPA criteria applicable to the
water medium". As noted in the EPA (2016) selenium guidance EClOs were selected
over EC20s "given the nature of exposure and effects for this bioaccumulative
chemical." Additionally EPA (2016) selected EC10 for selenium because "it was found
that the dose-response curves for selenium across a broad range of fish genera are
very steep, such that a small change in selenium tissue concentration yielded a large
increase in observed adverse effect."

p) First, all the derivation of aquatic life criteria for "bioaccumulative chemicals" have
not followed the process used for selenium, and there is no quantitative discussion in
the current document that compares the bioaccumulation values for selenium to
those of PFOS in a manner than justifies the use of EClOs. For example, EPA in its
2016 aquatic life criteria for cadmium noted that cadmium "can bioaccumulate in
aquatic organisms", but EC20s (not EClOs) were used as chronic values in the
derivation of aquatic life criteria in that document. Fundamentally, there is a logical
disconnect between adding additional conservativism (i.e., using EClOs instead of
EC20s) simply because a chemical has a higher bioaccumulative potential than
another chemical or exceeds a BCF or BAF criteria used to determine a chemical has
"bioaccumulative" status by typical chemical registration guidelines. The use of
chronic exposure toxicology data generally assumes that concentrations in the
organisms have reached steady state and, and thus, any bioaccumulation that has

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2.8. Additional Technical Comments to Consider

Reviewer

Comments

occurred is accounted for and manifests in toxic action. Coincidentally, the general
assumption is that toxic responses have plateaued as well and that effective doses
(measured via external concentrations in water or concentrations in the organism)
will not change significantly with additional exposure time. The bioaccumulative
nature of the toxicant at that point is a moot point with regards to toxic effects in an
aquatic organism, so there seems no need to add additional conservatism in the
estimation of a threshold for potential ecologically-significant effects on aquatic life.
Adding additional conservatism to the aquatic life criteria to protect other trophic
levels (i.e. wildlife that consume aquatic life) or human consumers of aquatic life,
which does involve bioaccumulation of chemicals in aquatic organisms, is not justified.
Criteria to protect wildlife and humans exposed via exposure pathways involving
bioaccumulation of chemicals in aquatic life are handled via separate approaches, and
are completely disconnected from the acute and chronic toxicity data developed to
evaluate the risks to aquatic invertebrates and lower trophic level vertebrates like fish
and amphibians.

q) Second, EPA has not provided any analysis of the dose response curves that

demonstrates the need for EClOs versus EC20s (as was mentioned for selenium).
Additionally, justification of the use of EClOs by simply referencing the regulatory
policies of other countries seems to be insufficient as the basis for a US policy, and is
unsatisfying from a scientific perspective.

r) More discussion is needed to support the poorly-supported move from EC20s to
EClOs, or alternately, EC20s need to be used in throughout the document, as
consistent with past EPA practice in aquatic life criteria derivation. EClOs are more
conservative than EC20s, but there is often greater variability and uncertainty
associated with EC10 values given the typical 50% effect ranges that are generally
targeted in the experimental designs of typical toxicological studies. Additionally, as
noted in EPA's 2016 aquatic life criteria document for cadmium, EClOs are "rarely
statistically significantly different from the control treatment." A 20% effect has often
been discussed as a point of departure of ecologically-significant population- and
community-level effects (e.g., Suter, 2000: Suter, G.W., Efroymson, R.A., Sample, B.E.,
& Jones, D.S. (2000). Ecological Risk Assessment for Contaminated Sites. CRC Press.
April).

s) Overall, the adoption of a more conservative 10% effect level (i.e., EC10) for chronic
values used in criteria calculation carries large environmental management and policy
implications. As noted above, clarification and careful justification is needed. EPA
needs to clearly articulate (ideally with ample scientific support) why the additional
conservatism is needed. This important potential policy matter deserves an open and
earnest discourse among the scientific, stakeholder, and regulated communities.

t) Page 88: It appears that only studies in which organisms exposed via diet were

included for evaluation of tissue criteria. Is this correct? It is questionable to exclude
effect concentrations in tissue from experiments in which exposure of PFAS was only
via water. EPA (2016) took the "dietary exposure only" approach with selenium

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2.8. Additional Technical Comments to Consider

Reviewer

Comments

because the primary exposure route for selenium has been shown to be via the diet in
natural ecosystems. In contrast, for many aquatic animals (especially lower trophic
level fish and invertebrates), a significant portion of the exposure to PFOS is via non-
dietary pathways. Part of this is due to the fact that controlled studies (e.g., Martin et
al., 2003 studies cited in the document) have found that water-to-organism BCFs for
aquatic life such as fish are generally larger than diet-to-organism biomagnification
factors (BMFs). Additionally, there is no reason to expect dietary or non-dietary
exposure pathways would affect toxic responses given the relatively rapid internal
kinetics of PFAS in aquatic life (i.e., half-life of hours or days), especially for small
invertebrates and fish that are in relative equilibrium with their surrounding exposure
water.

Page 112: There's only one "Bots et al. 2010" in the references section. Multiple
instances of "Bots et al 2010b" are cited in this document. 1 believe there is only one
Bots et al. 2010 paper. Please clarify.

Page 125: The Aedes data point is missing from Figure 3-5. If the qualitative
Chironomus data point is included please include Aedes.

Page 132: "expected to protect P. primulas from chronic time-variable PFOA
exposures"... should that be "PFOS" instead?

Page 135: A reference for "Appendix Q" is made. Please provide Appendix Q.

The percentage effect for LOECs (relative to controls) needs to be clearly noted in the
Appendices, for example, in Table C.l and in the detailed summary text for the
reviews of each paper. This should be provided when LOECs or MATCs are used as
chronic values.

Page 173: "Reduction in superoxide dismutase" and "Changes in protein expression"
are atypical endpoints not well tied to ecologically significant effects. These should be
removed from the table and subsequent discussion, or presented separately as
qualitative analyses only.

aa]

Page 173: It is not appropriate to refer to the Gosner stage endpoint as "Length at
metamorphosis" in the table. Refer to it as "Gosner stage" if it is to be included.

bb) Page 176: Gosner stages are not a typical endpoint, and the use of the growth data
would be much more supportable. See comment below regarding Page C-42.

cc) Page C-2: When the MATC is used in tables in the Appendices, it would be helpful to
provide the percent effect level (relative to control) for the LOEC associated with the
MATC. Also, in cases in which the LOEC is provided as the chronic value, please
provide the percent effect level.

dd) Page C-3: How was the Species Mean Chronic Value (SMCV) for leopard frog

calculated? There is only one chronic value that is bolded in the data, and it does not

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2.8. Additional Technical Comments to Consider

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Comments

equal the SMCV. Please add text to clearly discuss which values are included (and how
the ">" values are used in subsequent calculations like geometric means).

ee) Page C-7: For Wang et al., since this value is the lowest used in the criterion

derivation, please share a table of the raw data graphed in the Figure on page C-7.

ff) Page C-16: Appears to be a missing figure.

gg) Page C-29: Typo "XX.XX mg/L". There are other typos like this in the document (search
for "XX").

hh) Page C-42: The amount of detail for the review of the Hoover et al. (2017) experiment
is insufficient. The selection of the Gosner stage as an endpoint requires additional
detail. The relationship between Gosner stage and more typical endpoints clearly
linked to ecological health (growth, reproduction, and survival is unclear). The effects
on Gosner stage in this study are subtle; all dosed animals indicated they had reached
tadpole stage (Gosner stages 25-41) at the 40-day endpoint noted. The maximum
difference in Gosner stages noted in the study was approximately 2 (control Gosner
stage result of ~30, 100 and 1000 ng/L Gosner stage results of ~28). A 1% difference in
Gosner stages (especially when both 28 and 30 values fall within a tadpole Gosner
stage development range) is difficult to translate to adverse ecological impact. As
shown in the Gosner stage chart for anurans (Virginia Herpetological Society,
http://www.virginiaherpetologicalsociety.com/amphibians/amphibian~
development/amphibian-development.htm) the difference between stage 28 and
stage 30 is the shape of the tail. It is unclear if this statistically detectable difference in
the tail shape that distinguishes Gosner stage 28 and 30 would result in an
ecologically significant decrease in the overall time period required to reach sexual
maturity or ultimately translate to a developmental malformation that would result in
an ecologically meaningful population-level effect (decrease in survival, decrease in
reproductive output, etc.). The uncertainty with this atypical endpoint is high, and
given the slight difference (~7%) between NOEC and LOEC exposures, 1 would
recommend this datum be removed from the quantitative analysis. Notably, Figure S2
of this paper presents results for a measurement of growth via the Snout Vent Length
(SVL) measurement endpoint. This endpoint provide more a continuous measurement
of growth and is more typical of endpoints used in criteria derivation.

ii) Page C-44: "In the later phases of the tests, (Bots et al. 2010a)" is repeated.

jj) Page D-3: Regarding the Han et al (2015) study, 1 disagree with the selection of the
less conservative growth endpoint. The reproductive effect does look to be valid and a
reasonable endpoint to consider. EPA's reasoning to exclude it is not compelling and is
unclear. The exposure duration was at least 10 days, which is likely sufficient for many
marine invertebrates with relatively short life cycles (i.e., mysids). Perhaps EPA could
reach out to the study authors to clarify the uncertainty around the exposure time (10
days or 20 days?). At any rate, 1 think the MATC should rely on the reproductive

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2.8. Additional Technical Comments to Consider

Reviewer Comments

endpoint, and given the good dose-response for the reproductive data, a robust EC10
or EC20 value could likely be calculated.

kk) Page G-3: Seems like the Olson (2017) snail experiment provides some useful chronic
(21-day exposure?) data for a relevant sublethal growth endpoints. Please explain
why this data was excluded from the chronic evaluation. Simply listing "Duration" in
this table does not provide enough detail.

II) Page H-l: Please explain the acceptable duration acceptable for the urchin test and
other tests. Simply listing "Duration too short" without noting the acceptable duration
that would be considered is not helpful. Perhaps a summary table for acceptable
durations for particular endpoints could be provided in this document.

mm) Page H-2: First use of "atypical duration" in the table. This entry is inconsistent
with other entries (e.g., "duration too short") and does not clearly describe why the
experiment is not considered. Please explain this table entry.

nn) Page P-12: The Hoover et al. (2017) paper is included twice. There may be more errors
like this in the document, it needs to be reviewed closely by a technical editor.

oo) Appendix L: The references cited in this section seem to be missing.

Reviewer 2 I think the EPA's criteria for PFOS are very defensible based on the science and data available. I
think they did a great job clearly laying out how they derived the criteria and providing all of
the data that was used in the derivation.

Reviewer 3 All technical comments have been previously mentioned

Reviewer 4 Additional suggestions are listed below:

1. The species listed in the table is Mytilus galloprovincialis not M. edulis

Table 0-1. The Three Most Sensitive Acute Estuariiiie/Marine Genera.
Ranked Below from Most to Least Sensitive.

Rank

Genus

Species

GMAV
(mg/L
PFOS)

Comments

Mytilus1

Mediterranean
mussel,
M. edulis
Mytilus

gallopro vin cialis

> 1

Not a resident species in North
America, but other species in this
genus are resident, commercially,
or ecologically important species

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2.8. Additional Technical Comments to Consider

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Comments

2. Page 115- second paragraph (values highlighted in red and underlined are not consistent)

The author reported 10-day growth and survival ECi0s for the study were 0.0492 and 0.1079
mg/L, respectively. The study authors also reported NOECs of 0.0491 mg/L, LOECs of 0.0962
mg/L, and MATCs of 0.0687 mg/L for both endpoints. And the author reported 20-day ECi0s for
growth, survival, and total emergence were 0.0882, 0.0864, and 0.0893 mg/L, respectively.
And the studv authors also reported NOECs of 0.0217 mg/L for growth and survival and <
0.0023 mg/L for emergence, LOECs of 0.0949 mg/L for growth and survival and 0.0217 mg/L
for emergence, and MATCs of 0.0454 mg/L for growth and survival and 0.0071 mg/L for
emergence. Also, it should be noted, the paper reported contrasting NOECs for 20-day survival.
The text in the paper stated that the NOEC was 0.0271 mg/L and Table 2 of the paper stated
0.0949 mg/L. EPA assumed the NOEC in Table 2 of the paper was not correct and that 0.0217
mg/L was the correct NOEC based on the data presented in Figure 3A of the paper. This
assumption was applied to the summary of the study results presented in this PFOS draft
criteria.

3. Page 138-middle of the paragraph The chronic freshwater criteria also contain tissue-
based criteria expressed as 43.0 mg/kg wet weight (ww) for fish whole-body, 25.3 mg/ —
ww for fish muscle tissue and 12.3 mg/kg ww for invertebrate whole-body tissue.

4. Page A-21 last paragraph The noted toxicity values provided in each study summary above
(ADD NUMBERS), comprising of both author-reported and independently-calculated LC50
values, were used to calculate the GMAV value (as the geometric mean of the three LC5o
values previously mentioned) of 22.48 mg/L, which was used to derive the freshwater
aquatic life criterion.

5. Page A-24- Fourth line from bottom-The study author reported LC50 was 22.2 ± 4.6 mg/L
for PFOS. The independently-calculated toxicity value was x.xx mg/L. The study author
reported value was used quantitatively to derive the draft acute water column criterion.

6. Page A-25- Fourth line from bottom The study author reported 96-hour LC5o was 50.51
mg/L PFOS. The independently-calculated toxicity value was x.xx mg/L. The study author
reported value was used quantitatively to derive the draft acute water column criterion.

7. Page A-27- Fourth line from bottom. The independently-calculated toxicity value was x.xx
mg/L. The study author reported value was used quantitatively to derive the draft acute
water column criterion.

8. Page A-29- First paragraph For comparison, the 7-day LC50 was 39.71 mg/L. The
independently-calculated toxicity value was x.xx mg/L. The 96-hour study author reported
value was used quantitatively to derive the draft acute water column criterion.

9. Page A-30- First paragraph The independently-calculated toxicity value was x.xx mg/L. The
study author reported value was used quantitatively to derive the draft acute water
column criterion.

10. Page A-36- 5th line from bottom in complete data x.xx mg/L.

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Comments

11. Also at A-37 in complete data x.xx mg/L.

Table 0-3. Summary of Assessment Endpoints and Measures of Effect Used in the Criteria

Assessment Endpoints for the

Aquatic Life: Survival, growth,
and reproduction of freshwater
and estuarine/marine aquatic
life (i.e., fish, amphibians,
aquatic invertebrates)

For effects from acute exposure:

1. LC5o concentrations in water, diet, and/or
tissue (e.g., )

2. NOEC and LOEC concentrations in water, diet,
and/or tissue (e.g., )

For effects from chronic exposure:

1. ECio concentrations in water, diet, and/or
tissue (e.g., )

2. NOEC and LOEC concentrations in water, diet,
and/or tissue (e.g., ); Only
used when an ECw could not be calculated for a
genus.

Note: only chronic exposures were considered for
derivation of the tissue-based criteria since PFOS is a
bioaccumulative chemical. These chronic tissue-based
criteria are expected to be protective of acute effects,
because acute effects were observed at much greater
concentrations than chronic effects.

Please review if the highlighted muscle, blood and egg would be relevant to this section in
terms of LC50, EC10, LOEC and NOEC endpoints .

12. 1.1.2 and page 3- Previously Published Chronic Water Criteria for Direct Aqueous
Exposure

The information on Australian guidelines to be updated based on NEMP2 published in 2020. I
will attach it as a PDF. https://www.environment.gov.au/svstem/files/resources/2fadflbc-
b0b6-44cb-al92-78c522d5ec3f/files/pfas-nemp-2.pdf

in Canada (ECCC 2018), and 0.000023 mg/L in Europe (RIVM 2010). Previously published
estuarine/marine chronic values were available for two geographic regions (Australia/New
Zealand and Europe). These publicly available values were 0.0000046 mg/L in Europe
(RIVM 2010) and

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2.8. Additional Technical Comments to Consider

Reviewer

Comments

The CRC marine guidelines are not valid as they are not based on the framework Freshwater
values are to be used on an interim basis

13. Page 4- Table 1.1 to be updated accordingly

Exposure
:enario

Freshwater

PFOS

0.00023
Hg/L

0.13
Hg/L

2 M-g/L

31 M-g/L

Exposure
scenario

99% species
protection -
high

conservation
value systems

95% species
protection -
slightly to
moderately
disturbed
systems

90% species
protection -
highly disturbed
systems

80% species
protection -
highly disturbed
systems

Comments and source

1

Australian and New Zealand Guidelines for
Fresh and Marine Water Quality - technical
draft default guideline values for PFOS and
PFOA.

Note 1: The 99% species protection level for
PFOS is close to the level of detection. Agencies
may wish to apply a 'detect' threshold in such
circumstances rather than a quantified
measurement.

Note 2: The draft guidelines do not account for
effects which result from the biomagnification
of toxicants in airbreathing animals or in
animals which prey on aquatic organisms.

Note 3: The WQGs advise3 that the 99% level of
protection be used for slightly to moderately
disturbed systems. This approach is generally
adopted for chemicals that bioaccumulate and
biomagnify in wildlife. Regulators may specify
or environmental legislation may prescribe the
level of species protection required, rather than
allowing for case by-case assessments.

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2.8. Additional Technical Comments to Consider

Reviewer

Comments

Exposure
scenario

PFOS

Exposure
scenario

Comments and source

Interim
marine

0.00023
Hg/L

99% species
protection

- high

conservation
value systems

As above.

Freshwater values are to be used on an interim
basis until final marine guideline values can be
set using the nationally-agreed process under
the Australian and New Zealand Guidelines for
Fresh and Marine Water Quality.

The WQG advise that in the case of estuaries,
the most stringent of freshwater and marine
criteria apply, taking account of any available
salinity correction.



0.13
Hg/L

95% species
protection

- slightly to
moderately
disturbed
systems



2 M-g/L

90% species
protection -
highly disturbed
systems

Marine guideline values developed by CRC
CARE are under consideration through the
nationally-agreed water quality guideline
development process.



31 M-g/L

80% species
protection -
highly disturbed
systems



ahttps://www.waterqualitv.gov.au/anz-guidelines/guideline-values/default/water-qualitv-
toxicants/local-conditions#bioaccumulation

14.	Table 1 2. Two Primary Categories of PFAS

Please refer to OECD 2021 to be consistent with PFAS terminology/nomenclature

OECD (2021), Reconciling Terminology of the Universe of Per- and Polyfluoroalkyl Substances:
Recommendations and Practical Guidance, OECD Series on Risk Management, No. 61, OECD
Publishing, Paris

15.	Table 1.3 Page 8

Please review Figure 9 OECD 2021 (also attached as PDF)

16.	Conceptual Model of PFOS in the Aquatic Environment and Effects

Figure 2.9 page 77- Growth as an endpoint missing in the endpoints - first pentagon

Reviewer 5

Specific comments to the various elements of the PFOS AWQC are above. Here, I want to
suggest that EPA revisit the 1985 Guidelines and publish either an updated version or an

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2.8. Additional Technical Comments to Consider

Reviewer

Comments



amendment. Basing critically important criteria on documents published in 1985 and then
using this to justify decisions seems like it would not pass muster in the scientific community.
I've had papers rejected because they did not include enough recent citations, for example.
Moreover, I've mentioned my concerns with the 4- most sensitive taxa + linear regression for
criteria derivation. No paper I've read on generating the 5th percentile most sensitive species
has used this approach. Granted, 1 may have missed them but my sense is that it is more
common to use a full SSD. It would be helpful, for example, if the revised Guidelines explored
this further or other means of criteria development (including new approach methods) and
published, used, and cited and updated guidelines document. I'd like to think we still generally
lead the world (more or less) in environmental protection so having an updated document
would be welcomed.

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APPENDIX A

CHARGE TO REVIEWERS

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Technical Charge to External Peer Reviewers
Contract No. EP-C-17-017
Task Order 68HERH21F0090 (ERG Task 49)

June 2021

External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

BACKGROUND

PFOA and PFOS toxicity studies have been conducted on a limited number of aquatic organisms, including
species of fish and invertebrates, indicating that exposure to elevated concentrations of PFOA and PFOS can
cause effects on survival, growth, and reproduction. In these draft criteria documents EPA is proposing two
separate water quality criteria to ensure the protection of aquatic life species from the exposure to PFOA
and PFOS individually.

Background on the PFOA and PFOS Aquatic Life Criteria Development Process:

Toxicity studies used to derive the PFOA and PFOS criteria were carefully evaluated and thoroughly reviewed
to ensure studies were of sufficient data quality to use in criteria derivation. Scientists from the Office of
Water (OW) and Office of Research and Development (ORD) conducted an extensive review of the PFOA and
PFOS toxicity studies. Additionally, EPA obtained replicate-level (or treatment-level, when replicates were
unavailable) concentration-response (C-R) data from publications, supplemental materials, or via contacting
authors so that EPA could independently fit C-R models to estimate acute LC50 and chronic EC10 values that
were used to derive the criteria to ensure endpoints used were statistically sound. Individual C-R models and
resultant point estimates were also reviewed and discussed between OW and ORD to ensure the most
statistically robust models informed the derivation of the PFOA and PFOS criteria. In addition to contacting
study authors for C-R data (when not reported in the open literature), EPA also consulted primary authors
for methods clarifications in many instances during the data quality review phase to ensure that the studies
used to derive criteria were of high quality.

Overall, due to the paucity of measured freshwater toxicity data, EPA included a number of tests with
unmeasured treatments to derive criteria to ensure the dataset was representative of a range of taxa and
there were sufficient data to develop criteria. EPA also conducted meta-analyses to evaluate the
relationship between nominal and measured test concentrations using tests with measured treatment
concentrations. These meta-analyses (described in detail as Appendix L of the PFOA criteria document and
Appendix O of the PFOS criteria document) suggested measured concentrations were similar to nominal
concentrations and that the use of unmeasured tests, in light of data limitations, was appropriate.
Additionally, estuarine/marine toxicity data limitations did not allow for the direct derivation of acute or
chronic estuarine/marine criteria for PFOA or PFOS. Therefore, to develop of recommendations that states
and tribes could use in adopting protective values for estuarine/marine waters, EPA developed acute PFOA
and PFOS protective benchmarks using a New Approach Methodology (detailed in Appendix K of the PFOA
criteria document and Appendix L of the PFOS criteria document).

Addressing data limitations to derive robust criteria/benchmarks, extensively reviewing studies, and
calculating point estimates meant that the derivation of the PFOA and PFOS aquatic life criteria were
developed via comprehensive, rigorous process that included collaborations across EPA scientists in OW and

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ORD. Beyond detailed discussions between OW and ORD, the PFOA and PFOS drafts also underwent two
rounds of review with the EPA Scoping Workgroup (consisting of additional scientists from both OW and
ORD) and one round of review with a group of internal EPA Reviewers that included representatives from
the Office of Water, Office of Research and Development, other EPA Program Offices, and EPA Regions.
In this peer review EPA is seeking to obtain a focused, objective evaluation of the two separate draft criteria
documents, one for PFOA and the other for PFOS. Generally, the charge questions below are the same for
EPA's PFOA and PFOS draft aquatic life water quality criteria. However, there are some unique questions
specific to the individual drafts and therefore, there are two separate sets of charge questions.

CHARGE QUESTIONS
PFOS

Charge Questions for the draft Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)
recommendations document

1) Please comment on the overall clarity of the document as it relates to the derivation of each
criterion.

2) Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed
comments.

• Is the technical approach used to derive the criterion elements logical?

• Does the science support the conclusions?

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

3) Please comment on the approach used to derive the draft acute estuarine/marine benchmark for
PFOS. Given the limited estuarine/marine test data available, a new approach method was used to
support the derivation of an acute estuarine/marine benchmark to provide states and tribes with a
protective value. Please provide detailed comments.

• Is the technical approach used to derive the benchmark logical?

• Does the science support the conclusions?

4) Please comment on the use of measured and unmeasured toxicity tests to derive the respective
criterion. In particular please comment on the supporting justification for using unmeasured toxicity
tests in Appendix O.

5) Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

• Are there relevant data that you are aware of that should be added to the analyses (note
that EPA is working on updating the toxicity data to reflect the data in ECOTOX between
Sept. 2019 through the latest update)? If so, please provide references for consideration.

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In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data
from Yang et al. (2014) to suggest aquatic insects are relatively tolerant to acute
PFOS exposures. Specifically, Yang et al. (2014) conducted a 96-hour renewal,
measured PFOS acute test with the midge, Chironomus plumosus. This study was
not acceptable for quantitative use due to the potential problematic source of the
organisms. The reported LC5o was 182 mg/L for PFOS indicating that insects may not
be one of the more sensitive taxonomic groups. Therefore, this test was excluded
from the acute criterion calculation, but used to waive the missing insect MDR.

ii. the use of the quantitatively acceptable chronic toxicity value for mussel (Lampsilis
siliquoidea) from Hazelton et al. (2012). Specifically, Hazelton et al. (2012)
conducted a 36-day renewal, measured PFOS chronic test with fatmucket, Lampsilis
siliquoidea. The estimated ECio was 0.05713 mg/L, which was extrapolated from the
author-reported data and the exposure response slope from another PFOS toxicity
study focused on another mussel species (Ellipto complamata) as explained in
Section 3.1.1.3.3. Therefore, this test was used in the chronic criterion calculation.

iii. the use of the quantitatively acceptable chronic toxicity value for damselfly
(Enallagma cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-
day renewal, unmeasured PFOS chronic test with blue damselfly nymphs, Enallagma
cyathigerum. The MATC was 0.03162 mg/L, which was calculated from the author-
reported value for nymph survival as explained in Section 3.1.1.3.2. Therefore, this
test was used in the chronic criterion calculation.

iv. the use of the quantitatively acceptable chronic toxicity value for midge

(Chironomus dilutus) from MacDonald et al. (2004). MacDonald et al. (2004)
conducted a 20-day renewal, measured PFOS chronic test with midge lava,
Chironomus dilutus. The ECio was 0.05963 mg/L, which was an EPA-calculated value
for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test was used in
the chronic criterion calculation.

5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as
well as the specific acute LC5o values (Appendix A.2) and chronic ECio values (Appendix C.2)
that were estimated (for sensitive genera when C-R data were available) and used to derive
criteria.

6) Please comment on the translation of the chronic water column criterion elements for aquatic life to
derive the tissue-based criterion elements, considering the bioaccumulation of PFOA and PFOS. In
particular, please comment on:

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic

water column criterion elements into tissue-based criterion elements.

6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion
elements were based.

7) Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

8) Please provide any additional technical comments that you believe should be considered.

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APPENDIX B
INDIVIDUAL REVIEWER COMMENTS

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COMMENTS SUBMITTED BY
REVIEWER 1

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External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

1. Please comment on the overall clarity of the document as it relates to the derivation of each criterion.

Overall, the document is clear and the reader can follow the logic of criteria derivation, and track the values
used back to the cited research articles or values calculated by EPA.

2. Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed
comments.

• Is the technical approach used to derive the criterion elements logical?

Yes, the technical approach used to derive the criteria elements is generally logical. I disagree with
some of the elements of the analyses, as noted in my detailed comments (see below, responses to
charge question 8).

• Does the science support the conclusions?

In general, the science is supportive of the general conclusions. As noted in my below detailed
responses to other charge questions, I believe the science is not supportive of the work in a few key
instances including:

1. I believe the Criterion Continuous Concentration (CCC) should be potentially re-calculated
considering my comments provided in response to charge question 5a.

2. The science does not support the assumption of a 10-year recovery time for PFOS in aquatic
systems.

3. The generation of tissue criteria is weakly supported, and the uncertainty associated with
these criteria should be emphasized.

4. The NAM-generated marine Final Acute Value (FAV) and FAV/2 values (Appendix L) are
highly uncertain.

5. It is unclear if the EPA-calculated Effective Concentration 10% (EC10) values are supported;
additional details on the modeling and the variability and fit of each EC10 model need to be
provided.

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

The criteria derived are aimed at protecting aquatic life (e.g., fish, invertebrates) from the direct
acute and chronic toxicity of PFOS in water. Generally, the values applied are protective and are
generally similar to protective values derived by other regulatory organizations and independent
(i.e., academic, private sector) scientists. Although, as based on my comments, I believe there is
room for improvement. The criteria derived for tissues attempt to provide criteria that take into
account bioaccumulation so that measurements in tissue can be interpreted with respect to the
potential for potential effects; however, the uncertainty with the tissue criteria is high. The water
and tissue criteria are not intended protective of bioaccumulative effects that may affect higher
trophic levels, such as wildlife that may consume aquatic life.

3. Please comment on the approach used to derive the draft acute estuarine/marine benchmark for
PFOS. Given the limited estuarine/marine test data available, a new approach method was used to

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support the derivation of an acute estuarine/marine benchmark to provide states and tribes with a
protective value. Please provide detailed comments.

• Is the technical approach used to derive the benchmark logical?

The derivation of the acute marine benchmarks (FAV and Criterion Maximum Concentration (CMC))
using the New Approach Method (NAM) is highly uncertain, and I would recommend this analysis
not be included as in this document. I do not feel that the analysis and subsequent criteria have high
confidence for use in a regulatory application. I understand that similar analyses with other
chemicals have about a 90% probability of the predicted effect value being within a factor of 5 of the
actual value (Raimondo et al., 2010 - cited in document). Given the calculated CMC (0.43 mg/L), this
implies the CMC has about a 90% probability of being within 0.086 to 2.2 mg/L. If the NAM approach
stays in the document, this uncertainty and range of values should be acknowledged in the
discussion.

I would rather see tentative or provisional acute criterion developed from the limited empirical
marine acute data highlighted in Appendix B and other recently published marine acute data. This
suggests a reasonable interim FAV of approximately 1 mg/L, which is similar to that calculated using
the NAM approach. I place higher confidence in the limited empirical data and would suggest EPA
emphasize it in addition to or in place of the values calculated by the NAM.

I am hopeful that as new toxicity information on marine species are developed, these values can be
supplanted with a proper and robust criteria calculation. If such a future analysis is possible, it
should be noted.

• Does the science support the conclusions?

See above comment.

• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria derived
using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical National Water
Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

The approach seems to be consistent with the approach in the 1985 guidelines. As noted above, the
uncertainty with regards to the predictive capability of the interspecies correlations should be
acknowledged quantitatively.

4. Please comment on the use of measured and unmeasured toxicity tests to derive the respective
criterion. In particular please comment on the supporting justification for using unmeasured toxicity
tests in Appendix O.

The consideration of toxicity data from experiments in which PFOS measurements were not made seems
appropriate. The Appendix O analysis is supportive of the general observation that actual concentrations in
the toxicity test waters approximated nominal values for freshwater. I agree that actual concentrations in
the toxicity test waters for the marine test may be lower than nominal values, thus, effect data originating
from marine studies that only report nominal concentrations may be biased high in some cases. Given the
tentative/temporary nature of the marine criteria developed in this study, this bias is manageable until
additional empirical data from experiment with measured concentrations in water can be provided.

5. Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

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In most cases, yes. Please see detailed comments on particular studies and interpretations in
response to other charge questions.

• Are there relevant data that you are aware of that should be added to the analyses (note that EPA
is working on updating the toxicity data to reflect the data in ECOTOX between Sept. 2019
through the latest update)? If so, please provide references for consideration.

Hayman, N.T., Rosen, G., Colvin, M.A., Conder, J., Arblaster, J.A. 2021. Aquatic toxicity evaluations of
PFOS and PFOA for five standard marine endpoints. Chemosphere 273:129699.

In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data from Yang et al.
(2014) to suggest aquatic insects are relatively tolerant to acute PFOS exposures. Specifically, Yang
et al. (2014) conducted a 96-hour renewal, measured PFOS acute test with the midge, Chironomus
plumosus. This study was not acceptable for quantitative use due to the potential problematic
source of the organisms. The reported LC50 was 182 mg/L for PFOS indicating that insects may not
be one of the more sensitive taxonomic groups. Therefore, this test was excluded from the acute
criterion calculation, but used to waive the missing insect MDR.

I disagree with excluding this data point from the acute criteria calculations. I assume this data has
been removed under the assumption that these animals may have been pre-exposed to PFOS and
may have been more tolerant of PFOS exposures, which would result in biased-high median lethal
concentration (LC50) values. If so, this should be explicitly stated. Assuming these Chironomus can
develop tolerance to PFOS, it seems that they would have to be exposed to rather high mg/L ranges
of PFOS in water given the reported 96-hour LC50 of 182 mg/L. Based on published literature, I am
unaware of natural ecosystems in China (where the animals may have been originally harvested)
with concentrations of PFOS that approach this order of magnitude range (in which they could build
up a tolerance). The animals were obtained from a local market, so it is also possible that they were
cultured for several generations, presumably using uncontaminated water (which would further
reduce the chance that multiple generations were exposed at these levels). Overall, I think it is more
reasonable to assume that the animals used in the experiment have not built up an acute lethal
tolerance to PFOS, and the that LC50 result is unbiased. It does seem clearly show that insects may
be less sensitive to acute lethality effects of PFOS. As such, I think it should be included as a
quantitative endpoint.

Additionally, it seems inconsistent to exclude this Yang et al (2014) study, when chronic data from
an unpublished study by Funkhouser (2015) were included for quantitative consideration. As noted
on page C-25, the animals in the Funkhouser (2015) study were "purchased from a private collector"
and then kept for "several" generations prior to testing. The source of the animals is just as
uncertain as the Yang et al (2014) animals, and it is unclear (if PFOS tolerance at lethal levels is
possible) how many generations would be needed to shed adaptive tolerance and how it would
compare to "several." Simply put, if data from experiments like Funkhouser (2015) are quantitatively
included, those from Yang et al. (2014) should also be quantitatively included (with some notes on
the uncertainty of the animal sources).

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0.05713 mg/L, which was extrapolated from the author-reported data and the exposure response
slope from another PFOS toxicity study focused on another mussel species (Ellipto complamata)
as explained in Section 3.1.1.3.3. Therefore, this test was used in the chronic criterion calculation.

There were only three exposure levels in this experiment, including the control. One PFOS dose (4.5
Hg/L) indicated an absence of detectable effects on metamorphosis, the other (69.5 ng/L) indicated
an approximate 35% reduction relative to controls. This is not a definitive test; there is little dose
response information to fully confirm the effects/absence of effects and predict an effective
concentration (EC) value with a dose response model. Application of another study's dose response
curve to generate EC10 values for this study does not address this fundamental shortcoming, and
simply carries too much uncertainty. Although there are only two PFOS doses, which is highly
uncertain, use of a Maximum Acceptable Toxicant Concentration (MATC) value may be a less
uncertain path to including this study in quantitative calculations. This would result in a more
conservative chronic value for this study (0.018 mg/L instead of 0.057 mg/L). Given the high
uncertainty of using this result (due to only 2 PFOS doses), I believe this value should be caveated in
some way and re-evaluated for use or excluded in future criteria derivation. For example, on page C-
22, the Spachmo and Arukwe (2012) value (which also featured a limited PFOS dose design), the
document notes that the limited doses "may limit its future use in the criteria derivation pending
independent verification of the toxicity values by EPA."

iii. the use of the quantitatively acceptable chronic toxicity value for damselfly (Enallagma
cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-day renewal, unmeasured
PFOS chronic test with blue damselfly nymphs, Enallagma cyathigerum. The MATC was 0.03162
mg/L, which was calculated from the author-reported value for nymph survival as explained in
Section 3.1.1.3.2. Therefore, this test was used in the chronic criterion calculation.

I agree with the interpretation of the Bots et al. (2010) study and selection of the MATC.

iv. the use of the quantitatively acceptable chronic toxicity value for midge (Chironomus dilutus) from
MacDonald et al. (2004). MacDonald et al. (2004) conducted a 20-day renewal, measured PFOS
chronic test with midge lava, Chironomus dilutus. The EC10 was 0.05963 mg/L, which was an EPA-
calculated value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test was used
in the chronic criterion calculation.

First, on page 104, the document mentioned "an EC10 of 0.0586 mg/L for growth following 10-days
of exposure", but on page 115, the document noted "10-day growth with an EC10 of 0.05963 mg/L".
In Appendix C (page C-19), the document states "the independently-calculated 10-day EC10 for
growth was 0.0586 mg/L." There's some inconsistencies in the value being obtained from EPA's
EC10 modeling using this reference; please correct or clarify.

One justification for using the 10-day EC10 growth result rather than other results is a lack of being
able to calculate EClOs. I do think the emergence results should not be discounted, however. EPA
notes "as for the emergence endpoint, there was a lack of a concentration-response relationship
and there were very similar levels of observed effects (which ranged between 42.6 and 50.1%)
despite the more than nine-fold increase in the mid-range treatment concentrations (0.0023,
0.0144, 0.0217 mg/L, respectively)." The magnitude of the effect (relative to controls), and the fact
that there were statistically-detectable differences from controls in some of these doses (0.0144,
0.0217 mg/L) seems to indicate an ecologically meaningful adverse effect is occurring due to PFOS.
This range of concentrations just might be a portion of dose response curve that is relatively flat.
There is a very clear adverse effect at 0.0949 mg/L. I think it would be reasonable to select the

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MATCfor emergence (0.0071 mg/L reported on page C-19) and treat it a second study point since it
was a completely different experiment from the 10-day experiment used to provide the EC10 of
0.05963 (or 0.0586) mg/L value. A Species Mean Chronic Value using the 0.0071 and 0.0586 mg/L
results would be 0.020 mg/L. This 0.020 mg/L value would seem to be protective while including the
growth and emergence data from these two experiments.

5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as well as the
specific acute LC50 values (Appendix A.2) and chronic EC10 values (Appendix C.2) that were
estimated (for sensitive genera when C-R data were available) and used to derive criteria.

More details need to be provided on the dose response modeling using R. Appendix K is helpful for providing
the reader with details on the general approach, but where EClOs are modeled by EPA, the model being
used (out of the 22 available in the R software package) needs to be specified. Providing some indication of
variability (such as a 95% confidence interval) for the model-generated EClOs is standard practice for dose
response modeling, and this information should be provided somewhere in the document. Showing the R
package output of the goodness of fit statistics (or equivalent) for the modeling in an Appendix would be
helpful; since this was used to select the model used in each instance of an EC10 calculation, it must be
available, so I would recommend including it for full transparency and to aid future efforts in understanding
the aquatic toxicology of this chemical. Additionally, it would be helpful to show the selected model fits for
all calculated EClOs (as shown for the most sensitive EClOs estimated). These steps would be helpful to
ensure and demonstrate quality of the model fits and reproducibility of the modeling work.

Additionally, somewhere in the document (Appendix K), the 22 dose response model equations should be
provided to the reader. Alternately, a reference could be made to a document that clearly provides this
information (ideally a peer-reviewed or EPA document) containing all 22 models.

6. Please comment on the translation of the chronic water column criterion elements for aquatic life to
derive the tissue-based criterion elements, considering the bioaccumulation of PFOA and PFOS.

The derivation of the tissue criteria in this manner is highly uncertain. To my knowledge this is the first time
EPA has applied ambient water quality criteria protective of aquatic life direct toxicity with uptake factors
(bioaccumulation factors (BAFs), bioconcentration factors (BCFs)) in this manner to calculate tissue criteria.
References are made to the selenium tissue criteria, but those are used in the reverse (i.e., criteria based on
measured concentrations in tissue used to calculate water criteria). The use of criteria for water with a
assumed uptake factor carries a large amount of uncertainty, and in general, the use of measured
concentrations in tissue linked to adverse effects is a more straightforward approach since it does not
involve uptake model predictions. This needs to be noted in the text. Also, are the predicted tissue criteria
meant to be a temporary stop-gap until tissue effect data become available? This should be discussed and
clarified.

In particular, please comment on:

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic water
column criterion elements into tissue-based criterion elements.

The use of BAFs derived from field studies is inherently uncertain due to the wide variety of techniques used
in the compiled studies, their analytical data quality, the differences in species and ecosystems,
experimental designs, spatial uncertainties for mobile animals like fish, etc. That being said, the use of a BAF
value (or BCF) in criteria derivation is consistent with other criteria developed by EPA. As noted above, the
use of the tissue criteria needs to be considered carefully, and I think empirical tissue data from toxicity
experiments should form the basis of a next iteration of a tissue criteria.

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6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion elements were
based.

The development of BAFs for invertebrates, fish (whole body), and fish (muscle) seems reasonable for the
application in estimating a draft or interim tissue criteria until empirical tissue data can be used to calculate
tissue criteria directly.

7. Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

The 4-day duration seems to be supported by the more sensitive chronic endpoints used to derive the CCC.

For the tissue-based criterion (page 135), there is no clear support for assuming a 10-year exceedance
frequency. Given the uncertainty with the BAF-predicted tissue criteria, and how little is known regarding
the recalcitrance of PFOS in aquatic ecosystems and recovery time if PFOS inputs in water were halted, the
assignment of a 10-year exceedance frequency at this stage seems completely arbitrary. We simply do not
yet know the time frame over which aquatic ecosystems recover from PFOS. It is not technically supported
to cite recovery times for selenium to support a 10-year recovery time for PFOS, these are completely
different toxicants that have their own unique fate and behavior. USEPA (1985) guidance suggests assuming
a 3-year frequency as a default, and the discussion on page 135-136 is not scientifically convincing enough to
modify it to 10 years.

Additionally, it should be noted that the exceedance frequency for another organic chemical, Tributyltin
(TBT) was set at 3 years by EPA in derivation of that criteria. TBT exhibits uptake factors similar to PFOS (i.e.,
BCF of approximately 2,000 L/kg, wet weight for goldfish, as noted in the EPA TBT criteria document, which
is similar to the PFOS BAFs of 1,800-3,100 L/kg, wet weight being used to calculate the fish tissue criteria).
TBT is also persistent in aquatic ecosystems, as noted by EPA. Given TBT is at least an organic chemical, it is a
closer analog than selenium, which is an element. As such, the exceedance frequency for the PFOS tissue
criterion should be set at the default of 3 years unless EPA can provide convincing technical information
specific to recovery times for PFOS.

Additionally, on page 136, the paragraph that begins with "Metals and other chemical pollutants such as
PFOS..." is not convincing as any quantitative support for EPA's 10-year exceedance frequency for the
chronic tissue-based criteria. The text as written may give the reader the conclusion that PFOS recovery may
be "on the order of decades", as EPA notes for selenium. There is no support for the conjecture that PFOS
recovery may be "relatively slow" or require decades, as noted in my above comment.

8. Please provide any additional technical comments that you believe should be considered.

I have the additional detailed comments:

a) Please note that the comments provided in this file reflect a focus on of key portions of the "Draft of
the Aquatic Life Water Quality Criterion..." document as directed by the above charge questions
provided to me. Given time and resource constraints and the scope of my review, it was not feasible
to provide a detailed review of the entire document and all of the supporting references and their
associated results and conclusions. As such, I reserve my right to supplement or amend my
comments in future, pending additional review or new information. Thank you for the opportunity
to assist EPA in its work on this very important matter, and I was honored to be selected as a
reviewer.

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b) In general, the document needs some quality copy editing effort. I found many typographical errors,
issues with formatting, reference/citation issues, and in some cases, poorly-worded text. I have
noted a few of these instances below.

c) Page xv: "25.3 mg/L ww" is not correct units for a concentration in tissue.

d) Page 6: "The carbon chain can be fully fluorinated...". Please specify that this applies to PFAS in
general, not to PFOS.

e) Page 6: The reference to "Table 2-1"; should that be Table 1-2?

f) Page 9: Please note in Figure 2-1 that this is the linear isomer of PFOS. It would be helpful to note
that the PFOS data in this study are likely from experiments with water spiked with the linear PFOS
isomer. It is hypothesized that toxicity and bioaccumulation may differ between branched and linear
forms of PFCAs and PFASs. Linear PFOS is thought to be more accumulative (as noted on Page 45)
and potentially more toxic to aquatic life when the dose is expressed as an external water
concentration. At some sites, a portion of the concentrations of PFOS in water (which are reported
as the sum of branched and linear PFOS) is branched PFOS, so criteria derived from linear PFOS
could be overly protective. Please include this uncertainty in the discussion in the document.

g) Page 18: To my knowledge, FTSAs degrade to PFCAs, not PFSAs like PFOS. See Zhang et al. (2016):
Zhang, S., Lu, X., Wang, N., & Buck, R. C. (2016). Biotransformation potential of 6:2 fluorotelomer
sulfonate (6:2 FTSA) in aerobic and anaerobic sediment. Chemosphere, 154, 224-230.
doi:10.1016/j.chemosphere.2016.03.062

h) Page 62: Regarding "The importance of the sediment pathway for PFOS bioaccumulation..." Larson
et al. (2018) conducted some insightful food web modeling on benthic and pelagic sources of PFOS.
See: Larson, E.S., Conder, J.M., Arblaster, J.A. 2018. Modeling avian exposures to perfluoroalkyl
substances in aquatic habitats impacted by historical aqueous film forming foam releases.
https://doi.Org/10.1016/j.chemosphere.2018.03.004 Chemosphere 201:335-341.

i) Page 66: Starting here on this page and in the rest of this section, most of the units need to be
specified for dry weight or wet weight for concentrations of PFOS in tissue. There were other
instances of this error in the document as well. For units of every concentration of PFOS in tissue,
please be sure to specify dry weight or wet weight.

j) Page 73-75: There are a few scientific names on these pages that are not italicized. Also may occur
in other portions of the document.

k) Page 78: USEPA (1998) is not cited in the references section; I fear there may be other similar
omissions.

I) Page 78: Where the 1985 guidelines are mentioned, please cite to USEPA (1985).

m) Page 78: Replace the "Stephan et al. (1985)" citation with USEPA (1985). Also, I believe the surname
of senior author of USEPA (1985) is Stephen, not Stephan.

n) Page 80: At the start of Section 2.10.2, it would be good to discuss linear and branched PFOS.

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criteria applicable to the water medium". As noted in the EPA (2016) selenium guidance EClOs were
selected over EC20s "given the nature of exposure and effects for this bioaccumulative chemical."
Additionally EPA (2016) selected EC10 for selenium because "it was found that the dose-response
curves for selenium across a broad range of fish genera are very steep, such that a small change in
selenium tissue concentration yielded a large increase in observed adverse effect."

p) First, all the derivation of aquatic life criteria for "bioaccumulative chemicals" have not followed the
process used for selenium, and there is no quantitative discussion in the current document that
compares the bioaccumulation values for selenium to those of PFOS in a manner than justifies the
use of EClOs. For example, EPA in its 2016 aquatic life criteria for cadmium noted that cadmium
"can bioaccumulate in aquatic organisms", but EC20s (not EClOs) were used as chronic values in the
derivation of aquatic life criteria in that document. Fundamentally, there is a logical disconnect
between adding additional conservativism (i.e., using EClOs instead of EC20s) simply because a
chemical has a higher bioaccumulative potential than another chemical or exceeds a BCF or BAF
criteria used to determine a chemical has "bioaccumulative" status by typical chemical registration
guidelines. The use of chronic exposure toxicology data generally assumes that concentrations in the
organisms have reached steady state and, and thus, any bioaccumulation that has occurred is
accounted for and manifests in toxic action. Coincidentally, the general assumption is that toxic
responses have plateaued as well and that effective doses (measured via external concentrations in
water or concentrations in the organism) will not change significantly with additional exposure time.
The bioaccumulative nature of the toxicant at that point is a moot point with regards to toxic effects
in an aquatic organism, so there seems no need to add additional conservatism in the estimation of
a threshold for potential ecologically-significant effects on aquatic life. Adding additional
conservatism to the aquatic life criteria to protect other trophic levels (i.e. wildlife that consume
aquatic life) or human consumers of aquatic life, which does involve bioaccumulation of chemicals in
aquatic organisms, is not justified. Criteria to protect wildlife and humans exposed via exposure
pathways involving bioaccumulation of chemicals in aquatic life are handled via separate
approaches, and are completely disconnected from the acute and chronic toxicity data developed to
evaluate the risks to aquatic invertebrates and lower trophic level vertebrates like fish and
amphibians.

q) Second, EPA has not provided any analysis of the dose response curves that demonstrates the need
for EClOs versus EC20s (as was mentioned for selenium). Additionally, justification of the use of
EClOs by simply referencing the regulatory policies of other countries seems to be insufficient as the
basis for a US policy, and is unsatisfying from a scientific perspective.

r) More discussion is needed to support the poorly-supported move from EC20s to EClOs, or
alternately, EC20s need to be used in throughout the document, as consistent with past EPA
practice in aquatic life criteria derivation. EClOs are more conservative than EC20s, but there is
often greater variability and uncertainty associated with EC10 values given the typical 50% effect
ranges that are generally targeted in the experimental designs of typical toxicological studies.
Additionally, as noted in EPA's 2016 aquatic life criteria document for cadmium, EClOs are "rarely
statistically significantly different from the control treatment." A 20% effect has often been
discussed as a point of departure of ecologically-significant population- and community-level effects
(e.g., Suter, 2000: Suter, G.W., Efroymson, R.A., Sample, B.E., & Jones, D.S. (2000). Ecological Risk
Assessment for Contaminated Sites. CRC Press. April).

s) Overall, the adoption of a more conservative 10% effect level (i.e., EC10) for chronic values used in
criteria calculation carries large environmental management and policy implications. As noted
above, clarification and careful justification is needed. EPA needs to clearly articulate (ideally with

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ample scientific support) why the additional conservatism is needed. This important potential policy
matter deserves an open and earnest discourse among the scientific, stakeholder, and regulated
communities.

t) Page 88: It appears that only studies in which organisms exposed via diet were included for
evaluation of tissue criteria. Is this correct? It is questionable to exclude effect concentrations in
tissue from experiments in which exposure of PFAS was only via water. EPA (2016) took the "dietary
exposure only" approach with selenium because the primary exposure route for selenium has been
shown to be via the diet in natural ecosystems. In contrast, for many aquatic animals (especially
lower trophic level fish and invertebrates), a significant portion of the exposure to PFOS is via non-
dietary pathways. Part of this is due to the fact that controlled studies (e.g., Martin et al., 2003
studies cited in the document) have found that water-to-organism BCFs for aquatic life such as fish
are generally larger than diet-to-organism biomagnification factors (BMFs). Additionally, there is no
reason to expect dietary or non-dietary exposure pathways would affect toxic responses given the
relatively rapid internal kinetics of PFAS in aquatic life (i.e., half-life of hours or days), especially for
small invertebrates and fish that are in relative equilibrium with their surrounding exposure water.

u) Page 112: There's only one "Bots et al. 2010" in the references section. Multiple instances of "Bots
et al 2010b" are cited in this document. I believe there is only one Bots et al. 2010 paper. Please
clarify.

v) Page 125: The Aedes data point is missing from Figure 3-5. If the qualitative Chironomus data point
is included please include Aedes.

w) Page 132: "expected to protect P. primulas from chronic time-variable PFOA exposures"... should
that be "PFOS" instead?

x) Page 135: A reference for "Appendix Q" is made. Please provide Appendix Q.

y) The percentage effect for LOECs (relative to controls) needs to be clearly noted in the Appendices,
for example, in Table C.l and in the detailed summary text for the reviews of each paper. This
should be provided when LOECs or MATCs are used as chronic values.

z) Page 173: "Reduction in superoxide dismutase" and "Changes in protein expression" are atypical
endpoints not well tied to ecologically significant effects. These should be removed from the table
and subsequent discussion, or presented separately as qualitative analyses only.

aa) Page 173: It is not appropriate to refer to the Gosner stage endpoint as "Length at metamorphosis"
in the table. Refer to it as "Gosner stage" if it is to be included.

bb) Page 176: Gosner stages are not a typical endpoint, and the use of the growth data would be much
more supportable. See comment below regarding Page C-42.

cc) Page C-2: When the MATC is used in tables in the Appendices, it would be helpful to provide the
percent effect level (relative to control) for the LOEC associated with the MATC. Also, in cases in
which the LOEC is provided as the chronic value, please provide the percent effect level.

dd) Page C-3: How was the Species Mean Chronic Value (SMCV) for leopard frog calculated? There is
only one chronic value that is bolded in the data, and it does not equal the SMCV. Please add text to
clearly discuss which values are included (and how the ">" values are used in subsequent
calculations like geometric means).

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ee) Page C-7: For Wang et al., since this value is the lowest used in the criterion derivation, please share
a table of the raw data graphed in the Figure on page C-7.

ff) Page C-16: Appears to be a missing figure.

gg) Page C-29: Typo "XX.XX mg/L". There are other typos like this in the document (search for "XX").

http://www.virginiaherpetologicalsociety.com/amphibians/amphibian~development/amphibian~
development.htirn) the difference between stage 28 and stage 30 is the shape of the tail. It is unclear
if this statistically detectable difference in the tail shape that distinguishes Gosner stage 28 and 30
would result in an ecologically significant decrease in the overall time period required to reach
sexual maturity or ultimately translate to a developmental malformation that would result in an
ecologically meaningful population-level effect (decrease in survival, decrease in reproductive
output, etc.). The uncertainty with this atypical endpoint is high, and given the slight difference
(~7%) between NOEC and LOEC exposures, I would recommend this datum be removed from the
quantitative analysis. Notably, Figure S2 of this paper presents results for a measurement of growth
via the Snout Vent Length (SVL) measurement endpoint. This endpoint provide more a continuous
measurement of growth and is more typical of endpoints used in criteria derivation.

ii) Page C-44: "In the later phases of the tests, (Bots et al. 2010a)" is repeated.

jj) Page D-3: Regarding the Han et al (2015) study, I disagree with the selection of the less conservative
growth endpoint. The reproductive effect does look to be valid and a reasonable endpoint to
consider. EPA's reasoning to exclude it is not compelling and is unclear. The exposure duration was
at least 10 days, which is likely sufficient for many marine invertebrates with relatively short life
cycles (i.e., mysids). Perhaps EPA could reach out to the study authors to clarify the uncertainty
around the exposure time (10 days or 20 days?). At any rate, I think the MATC should rely on the
reproductive endpoint, and given the good dose-response for the reproductive data, a robust EC10
or EC20 value could likely be calculated.

kk) Page G-3: Seems like the Olson (2017) snail experiment provides some useful chronic (21-day
exposure?) data for a relevant sublethal growth endpoints. Please explain why this data was
excluded from the chronic evaluation. Simply listing "Duration" in this table does not provide
enough detail.

II) Page H-l: Please explain the acceptable duration acceptable for the urchin test and other tests.

Simply listing "Duration too short" without noting the acceptable duration that would be considered
is not helpful. Perhaps a summary table for acceptable durations for particular endpoints could be
provided in this document.

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mm) Page H-2: First use of "atypical duration" in the table. This entry is inconsistent with other entries
(e.g., "duration too short") and does not clearly describe why the experiment is not considered.
Please explain this table entry.

nn) Page P-12: The Hoover et al. (2017) paper is included twice. There may be more errors like this in
the document, it needs to be reviewed closely by a technical editor.

oo) Appendix L: The references cited in this section seem to be missing.

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COMMENTS SUBMITTED BY
REVIEWER 2

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External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

1) Please comment on the overall clarity of the document as it relates to the derivation of each criterion.

I thought that the document was well written and laid out. I thought that the document clearly laid out the
approach that the EPA used to derive each criterion. I thought it clearly outlined the approach that the EPA
chose in deciding which data to use in their derivation and how these data would be used in derivation.

The appendices are very useful in providing added detail and the data that were used in the derivation of
the criteria. The appendices allow for a high level of transparency around how the criteria were generated.

In Table 3-1, the acronym "GMAV" was used as a heading in the table, but I could not locate where this
acronym was defined earlier in the document.

The captions of figures and tables are not sufficiently detailed. Figures and tables should be able to stand on
their own. Also, the use of acronyms in the caption and headings of tables and figures decreases clarity, e.g.,
Figs 3-1, Tables 3-1, 3-2, 3-3, 3-4, 3-5. The use of acronyms in the figure or table is valid to save space, as
long as they are defined in the caption of the figure or table.

2) Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed
comments.

• Is the technical approach used to derive the criterion elements logical?

• Does the science support the conclusions?

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

Yes, the technical approach used by the EPA to derive the criterion is logical and defensible. The approach is
also clearly laid out in the document. Dividing the 5th centile of the acute GSD by 2 is sufficiently
conservative to ensure the protection of 95% of species, based on the data currently available.

Yes, I think the science supports the EPA's conclusions. However, there appears to be several studies that
were not considered by the EPA. I have listed these studies below.

Yes, I think the approach taken by the EPA is sufficiently conservative to be protective of freshwater aquatic
life from acute, chronic, and bioaccumulative effects based on the data that was available at the time. It was
a good idea to evaluate the influence on non-North American species on the derivation of the criteria.

• Is the technical approach used to derive the benchmark logical?

• Does the science support the conclusions?

• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria derived
using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical National Water
Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

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species using species for which data is available. I think the approach taken by EPA has included sufficient
conservatism to address the relatively large amount of uncertainty around the acute toxicity of PFOS to
estuarine and marine species. The proposed acute benchmark for estuarine and marine species is an order
of magnitude lower than the acute benchmark for freshwater species, which I think underscores the
conservatism used by EPA in determining an acute benchmark for estuarine and marine species. That said,
the benchmark should be used cautiously due to the relatively large amount of uncertainty and effort should
be made to generate acute and chronic toxicity data for PFOS on estuarine and marine species.

I am concerned with the approach of using the agreement of measured and nominal concentrations from
studies that measured the concentration of PFOS in their tests to determine whether to use toxicity data
from studies that did not measure the concentration PFOS in their tests. My concern stems from this
approach having to assume that studies that did not measure the concentration of PFOS in their
experiments performed the dosing of PFOS with the same care and skill as those studies that did measure
the concentration of PFOS in their experiments and measured concentrations within 20% of nominal. My
concern is compound by 58% and 65% of the freshwater and saltwater tests, respectively, only reporting
nominal test concentrations. The EPA's approach uses the agreement of measured and nominal
concentration in a minority of studies to determine whether to include the majority of studies on their
assessment.

I am assuming that there wouldn't be sufficient data to determine a criterion without using data from
studies that did not measure the concentrations of PFOS in their experiment?

I think the approach that the EPA has used to determine the level of agreement between the nominal and
measured concentration of PFOS in the studies that measured the concentration is logical and valid. It is
encouraging that the agreement on average is high. Again, my largest concern is assuming this agreement in
a minority of studies is present in all studies.

5) Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

I think the data used in the derivation of the criteria were appropriate. As mentioned above, I am a little
concerned about the use of toxicity data from studies that did not measure the concentration of PFOS in
their experiments, especially considering the proportion of studies that did not measure the concentrations.
The confirmation of exposure concentrations is an important principle of sound ecotoxicology.

• Are there relevant data that you are aware of that should be added to the analyses (note that EPA
is working on updating the toxicity data to reflect the data in ECOTOX between Sept. 2019
through the latest update)? If so, please provide references for consideration.

I have listed a number of papers below that were published in 2020 and 2021 that the EPA may want to
consider in their assessment.

Hayman, N.T., Rosen, G., Colvin, M.A., Conder, J., Arblaster, J.A., 2021. Aquatic toxicity evaluations of PFOS
and PFOA for five standard marine endpoints. Chemosphere 273, 129699.
doi:10.1016/j.chemosphere.2021.129699

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Logeshwaran, P., Sivaram, A.K., Surapaneni, A., Kannan, K., Naidu, R., Megharaj, M., 2021. Exposure to
perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at ppb concentration
induces chronic toxicity in Daphnia carinata. Science of The Total Environment 769, 144577..
doi:10.1016/j.scitotenv. 2020.144577

Simpson, S.L., Liu, Y., Spadaro, D.A., Wang, X., Kookana, R.S., Batley, G.E., 2021. Chronic effects and

thresholds for estuarine and marine benthic organism exposure to perfluorooctane sulfonic acid
(PFOS)-contaminated sediments: Influence of organic carbon and exposure routes. Science of The
Total Environment 776, 146008.. doi:10.1016/j.scitotenv.2021.146008

Li, R., Tang, T., Qiao, W., Huang, J., 2020. Toxic effect of perfluorooctane sulfonate on plants in vertical-flow
constructed wetlands. Journal of Environmental Sciences 92, 176-186..
doi:10.1016/j.jes.2020.02.018

Aquilina-Beck, A.A., Reiner, J.L., Chung, K.W., Delise, M.J., Key, P.B., Delorenzo, M.E., 2020. Uptake and
Biological Effects of Perfluorooctane Sulfonate Exposure in the Adult Eastern Oyster Crassostrea
virginica. Archives of Environmental Contamination and Toxicology 79, 333-342.
doi:10.1007/s00244-020-00765-4

Tornabene, B.J., Chislock, M.F., Gannon, M.E., Sepulveda, M.S., Hoverman, J.T., 2021. Relative acute toxicity
of three per- and polyfluoroalkyl substances on nine species of larval amphibians. Integrated
Environmental Assessment and Management 17, 684-690. doi:10.1002/ieam.4391

Suski, J.G., Salice, C.J., Chanov, M.K., Ayers, J., Rewerts, J., Field, J., 2021. Sensitivity and Accumulation of
Perfluorooctanesulfonate and Perfluorohexanesulfonic Acid in Fathead Minnows ( Pimephales
promelas ) Exposed over Critical Life Stages of Reproduction and Development. Environmental
Toxicology and Chemistry 40, 811-819. doi:10.1002/etc.4936

McCarthy, C.J., Roark, S.A., Wright, D., O'Neal, K., Muckey, B., Stanaway, M., Rewerts, J.N., Field, J.A.,

Anderson, T.A., Salice, C.J., 2021. Toxicological Response of Chironomus dilutus in Single-Chemical
and Binary Mixture Exposure Experiments with 6 Perfluoralkyl Substances. Environmental
Toxicology and Chemistry 40, 2319-2333. doi:10.1002/etc.5066

In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data from Yang et al.
(2014) to suggest aquatic insects are relatively tolerant to acute PFOS exposures. Specifically, Yang
et al. (2014) conducted a 96-hour renewal, measured PFOS acute test with the midge, Chironomus
plumosus. This study was not acceptable for quantitative use due to the potential problematic
source of the organisms. The reported LC5o was 182 mg/L for PFOS indicating that insects may not
be one of the more sensitive taxonomic groups. Therefore, this test was excluded from the acute
criterion calculation, but used to waive the missing insect MDR.

I think the EPA's decision that the data from Yang et al. (2014) was not acceptable for quantitative use
was appropriate. The source of the larvae is problematic. However, I don't agree with the conclusion
that insects may not be one of the most sensitive taxa. Chironomus tentans is a relatively sensitive taxa
to chronic exposure to PFOS (MacDonald et al. 2004). In Table C.l, the EC10 for C. tentans is reported
as 0.05963 mg/L. Chironomus tentans was also the fourth most sensitive species used in calculating the
chronic freshwater criterion (Table 3-6). Also, another insect, Enallagma cyathigerum, another insect

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species, was the second most sensitive species used in calculating the chronic freshwater criterion
(Table 3-6).

ii. the use of the quantitatively acceptable chronic toxicity value for mussel (Lampsilis siliquoidea)
from Hazelton et al. (2012). Specifically, Hazelton et al. (2012) conducted a 36-day renewal,
measured PFOS chronic test with fatmucket, Lampsilis siliquoidea. The estimated ECi0 was
0.05713 mg/L, which was extrapolated from the author-reported data and the exposure response
slope from another PFOS toxicity study focused on another mussel species (Ellipto complamata) as
explained in Section 3.1.1.3.3. Therefore, this test was used in the chronic criterion calculation.

EPA used an EC10:EC35.4 from Drottar et al. (2000) for Elliptio complanata and applied this ratio to
derive an EC10 from the data reported in Hazelton et al. (2012) for Lampsilis siliquoidea. The problem is
that EPA have not clearly outlined in section 3.1.1.3.3 what endpoint that Drottar et al. (2000) was
measuring in Elliptio complanata (also note that the genus and species are not spelled correctly in
section 3.1.1.3.3). Is the endpoint measured in E. complanata the same as the endpoint measure in L.
siliquoidea? I tried to look up the endpoint measure in Drottar et al. (2000) but I could not find the study
and there was no reference provided in the reference section for Drottar et al. (2000). This missing
information makes it difficult to comment on the validity of the approach that EPA has taken to derive
an EC10 for L. siliquoidea.

iii. the use of the quantitatively acceptable chronic toxicity value for damselfly (Enallagma
cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-day renewal, unmeasured
PFOS chronic test with blue damselfly nymphs, Enallagma cyathigerum. The MATC was 0.03162
mg/L, which was calculated from the author-reported value for nymph survival as explained in
Section 3.1.1.3.2. Therefore, this test was used in the chronic criterion calculation.

I think the EPA's justification for the use of the survival data from Bots et al. (2010) is valid. While control
mortality reached 40% in the control, the plateau in control mortality after 60 days, the total duration of
the test being 200 days, and 82.57% survival in the control from day 60 to 200, justifies the inclusion of
the MATC derived from Bots et al. (2010) for Enallagma cyathigerum in the derivation of a chronic
criterion.

iv. the use of the quantitatively acceptable chronic toxicity value for midge (Chironomus dilutus)
from MacDonald et al. (2004). MacDonald et al. (2004) conducted a 20-day renewal, measured
PFOS chronic test with midge lava, Chironomus dilutus. The EC10 was 0.05963 mg/L, which was an
EPA-calculated value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test was
used in the chronic criterion calculation.

First, the wrong species is referenced in relation to the MacDonald et al. (2004) study. MacDonald et al.
(2004) reported the toxicity of PFOS to Chironomus tentans. The EPA's derivation of a 10-d EC10 for
Chironomus tentans using the data from MacDonald et al. (2004) is not clear. In Appendix C, section C.2.4,
the EPA writes, "EPA could not fit a curve to independently verify the 10-day survival (due to a lack of a
specific sample size for this endpoint as the number of replicates was not stated in the paper; however, the
number of replicates was between 2 and 4 and EPA sought to obtain clarification and treatment level data
from the study authors)" It is not clear how EPA got the information necessary, e.g., number of replicates, to
fit a curve. It is also not clear what EPA means by "...and treatment level data from the study authors."? Did
EPA acquire the raw data for growth from the 10-day toxicity test with C. tentans? If that is the case, they
have not made that clear. If that is the case, it would also strengthen their independently derived EC10 for
growth in C. tentans. I think the EPA needs to more clearly explain where they got the data necessary to
derive the EC10 for C. tentans used in the chronic criterion.

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5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as well as the
specific acute LC50 values (Appendix A.2) and chronic EC10 values (Appendix C.2) that were estimated
(for sensitive genera when C-R data were available) and used to derive criteria.

I think the approach that the EPA used to determine effect measure from concentration-response data was
appropriate. The use of the drc package in R to fit 22 different models to the empirical data and then using
several criteria (e.g., AIC, residual standard errors, confidence intervals) to evaluate the fit of the different
models is robust. It would have been useful if the EPA reported the 22 different models in Appendix K.

I think the LC50 and EC10 values determined by the EPA using the approach mentioned in the previous
paragraph was appropriate. It is valid for these effect measures to be determined when the concentration-
response data has been provided by the authors of the study. The EPA has also made is clear in Appendix
A.2 and C.2 how they determined these effect measures using the concentration-response data provide in
the studies. This generates a high level of transparency in the derivation of the criterion.

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic water
column criterion elements into tissue-based criterion elements.

I think the EPA has sufficiently addressed the uncertainty around the use of BAFs and the chronic water
column criterion in the derivation of tissue-based criterion. They have indicated that tissue-based criterion
should only be observed once in 10 years. The use of the geometric mean of the reported BAFs incorporates
the range of BAFs that may be present for different invertebrate and fish species. The use of the chronic
water column criterion also builds in added conservatism to the tissue-based criterion.

Prosser et al. (2016) reported BAFs for PFOA in three freshwater species (two invertebrates and one fish)

(See Tables S29-31 in Supplementary Information), but it was not considered in this assessment. It is
not clear why it was not considered.

Prosser, R.S., Mahon, K., Sibley, P.K., Poirier, D., Watson-Leung, T., 2016. Bioaccumulation of perfluorinated
carboxylates and sulfonates and polychlorinated biphenyls in laboratory-cultured Hexagenia spp.,
Lumbriculus variegatus and Pimephales promelas from field-collected sediments. Science of The
Total Environment 543, 715-726. doi:10.1016/j.scitotenv.2015.11.062

6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion elements were
based.

The evaluation criteria for BAFs outline in Table 2-4 are appropriate and the decision to only use high and
medium quality BAFs is justified based on the criteria that would make a BAF low quality. It was a good idea
to use fish BAFs based on the concentration in muscle and whole body (Table 3-12). Muscle tissue is usually
exclusively sampled in large fish, especially as part of fish consumption guidelines. The whole body is more
appropriate for small fish and invertebrate species, e.g., minnows, benthic macroinvertebrates.

7) Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

As per Table 0-1, I think the chosen durations and frequencies for the acute and chronic criteria are
appropriate. They will ensure protection of aquatic life. The duration of the tissue-based criterion is
appropriate as the concentration will be measured when biota is collected. The 10-year frequency is

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appropriate considering that for biota to reach the tissue-based criteria, they would likely to have been
exposed to concentrations at or above the chronic criteria for an extended period of time.

8) Please provide any additional technical comments that you believe should be considered.

I think the EPA's criteria for PFOS are very defensible based on the science and data available. I think they
did a great job clearly laying out how they derived the criteria and providing all of the data that was used in
the derivation.

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COMMENTS SUBMITTED BY
REVIEWER 3

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External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

1) Please comment on the overall clarity of the document as it relates to the derivation of each criterion.

o Main Question: Perhaps this was missed in the draft document, but, is there guidance when one
criteria is exceeded and the other is not? For example, if the tissue based criteria are exceeded yet,
the CCC is not; perhaps this is an unlikely scenario as those receptors have been accumulating PFOS
for a duration likely under higher water concentrations (> 0.014mg/L). Although if sediment
concentrations remain elevated (but not water column concentrations) this may also be a likely
route of PFOS exposure to fish with sediment dwelling prey.

o There are additional domestic criteria missing from the previously published criteria section; please
review those for Texas, Florida and California.

o Are there two Sharpe et al.'s, I believe this is only one publication but flipping between Sharpe et al.
2010, Sharpe et al. 2010a and Sharpe et al. 2010b throughout the document. If the goal is to
distinguish between supplemental vs the manuscript proper I suggest just clarifying in the text
instead of the reader looking for two pubs by Sharpe et al. 2010.

o Page 238 - error: The study authors reported a 96-hour LC5o of 58.47 mg/L PFOS, based on the
results of the range finding test. The independently-calculated toxicity value was x.xx mg/L.

o Page 296 - error: The independently-calculated toxicity value was x.xx mg/L.

o Table 3-6 is not referenced/described in the text. Additionally, the title reads "Six" most sensitive
and lists "Seven".

o Overall comment: ranking of sensitive genera flips back and forth between most and least sensitive
among tables, consistency would help the reader.

2) Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed

comments.

• Is the technical approach used to derive the criterion elements logical?

o This is logical and follows the established GLRI guidance; however, both Canada and Australia
utilize a species sensitivity distributions to determine the 95th and 99th percentile of species
protection. Is there a defensible reason why EPA did not employ this approach or at the very
least present these distributions and analysis that would support the currently drafted criteria?
o Additionally, thresholds from those SSDs (and others published) are lower than the draft
guidance here, this should be addressed:

¦ Australia - 0.13 ng/L

¦ Canada - 6.8 ng/L

¦ Salice et al. 2018 - 1.12 ng/L

¦ Conder et al. 2020 - 5.85 ng/L

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¦ *Giesy et al. 2010 - 5.1 ng/L (using CCC based on GLRI guidance)

• Does the science support the conclusions?

o The GMCV for Zebrafish is 0.0165 mg/L, thus there are studies that result in chronic toxicity at
concentrations lower than this mean; however, this is very close to the CCC of 0.014mg/L. This
seems borderline protective when considering potential exposures to this species (and those
more sensitive).

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

o I have confusion over Tables 3-9 and 4-6 calculations. How is it that the inclusion of Lampsilis
with a higher GMAV results in a lower overall CMC (3.3 mg PFOS/L) compared to the CMC in
table 3-9 (3.6 mg PFOS/L)? Actually, looking more closely at this, the ln(GMAV)A2 are
inconsistent among the tables for Xenopus, this is likely are result of using table 3-6 as a
template for 4-6.

o It is great to see the inclusion of the Burkhard et al. 2021 as this synthesis has been peer-

reviewed and published and is an exceptional overview of PFOS bioaccumulation; unfortunately,
there are not more current literature used within the draft document.

• Is the technical approach used to derive the benchmark logical?

o After potential inclusion of the data mentioned below this approach may be appropriate. In the
current form with the limited data it may be misleading. Can this guidance be updated? I am
aware of other researchers investigating PFAS on marine species (Ed Wirth, NOAA) and maybe
others that will be coming out soon.

• Does the science support the conclusions?

o I believe the data are incorrect for Fabbri et al. 2014. In table B.l the reported effect

concentration is recorded as >1 mg/L. However, looking at the paper, I read, "The PFCs PFOA
and PFOS induced a dose-dependent effect, with significant decreases in normal larval
development from 0.1 jig/L (17% and 27%, respectively; P 0.01). Maximal effects were observed
at 100 iug/L (about 40% and 50%, respectively; P 0.001) with no further decreases at higher
concentrations". There is a monotonic concentration-response curve. The associated figure also
supports an effect at O.lpig PFOS/L, see below. Furthermore, if the EC50 of the test organisms is
a needed endpoint (as noted in the PFOA justification, for which is lacking support in the current
form) looking at the figure below % of normal D-larvae for PFOS (although incorrectly referred
to in the legend as PFOAS) could be inferred at 0.1 mg/L. Furthermore, has EPA considered
calculating the MATC from this study?

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100

•PFOA

QJ
¦
>

ge 30

0,01 0,1

10 100 1000

PFCs (ng/L)

Fig. 4. Effects of PFOA and PFOS (0.01-0.1-1-10-100-1000 ng/L) on
M. galloprovincialis normal larval development in 96-multiwell plates. Data are re-
ported as in Fig. 3a.

o I did not see data included or the study evaluated for: Robertson JC (1986) Potential for

environmental impact of AFA-6 surfactant. Beak Consultants Ltd. Missassauga, Ontario, Canada.
EPA Docket AR226-1030a043.

¦ There are data for saltwater spp in the ITRC from this citation.

• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria derived
using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical National Water
Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

o No, this is a new approach; however, it follows the spirit of the 1985 guidelines.

4) Please comment on the use of measured and unmeasured toxicity tests to derive the respective

criterion. In particular please comment on the supporting justification for using unmeasured toxicity

tests in Appendix O.

o This seems acceptable for the time being. Having worked in the laboratory with PFOS, I can make a
first-hand testament that mixing PFOS into exposures solutions does not guarantee a homogenous
mixture despite working at solutions well below the solubility limit. There are nuances associated
with achieving homogeneity of the exposure solution, we have developed a PFAS mixing protocol to
reduce chemical clumping and this increases uniformity of the solutions. Furthermore, there is
approximately 30% variability of PFOS quantitatively (see...Rewerts et al. 2020); so, the best
measurement still has significant variability.

5) Please comment on the toxicity data used to derive the draft criteria.

o See collective responses below

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

o Data selection and waiving of the MDR for insect family in the FAV seem reasonable.

• Are there relevant data that you are aware of that should be added to the analyses (note that EPA
is working on updating the toxicity data to reflect the data in ECOTOX between Sept. 2019
through the latest update)? If so, please provide references for consideration.

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o The data selection for the derivation of the draft criteria are limited to published and/or
available studies from 2018 and prior. This significantly reduces the studies used in the
derivation as a number of publications have become available in recent years.

For example:

o A newly published study is available for fathead minnows exposed to PFOS for chronic duration
and over the course of reproduction and development. Although, this study was static-renewal,
PFOS concentrations are measured ; importantly, this study resulted in a NOEC of 88ng/L based
on reduced biomass seen in the second generation (Suski et al. 2020). Importantly, follow-on
work (in prep) indicates that this may be a maternal transfer impact as PFOS exposures to
juvenile fish alone do not share results.

o Also, from the authors noted above is an ongoing full life-cycle fathead PFOS and PFAS mixture
exposure. This study is being conducted under flow through conditions and is expected to reach
termination in December 2021.

o McCarthy et al. 2021 published data on chironomids (EC20 = 1.7ng/L), these are also not
included here.

o Bryan Brooks (Baylor) and Matt Simcik (UMN) also have acute data on the fathead minnow with
measured concentrations, these are not published just yet.

o David Moore (Army Corps) is near completion of a full life-cycle fish study

o In particular, SERDP has been funding this research for years and those data are published,
recently published or near final. EPA should reach out to SERDP Pis for data inquiries and
potential inclusion in these draft criteria.

In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data from Yang et al.
(2014) to suggest aquatic insects are relatively tolerant to acute PFOS exposures. Specifically, Yang
et al. (2014) conducted a 96-hour renewal, measured PFOS acute test with the midge, Chironomus
plumosus. This study was not acceptable for quantitative use due to the potential problematic
source of the organisms. The reported LC5o was 182 mg/L for PFOS indicating that insects may not
be one of the more sensitive taxonomic groups. Therefore, this test was excluded from the acute
criterion calculation, but used to waive the missing insect MDR.

o This seems appropriate, the flower market is most definitely an odd place to purchase research
organisms.

ii. the use of the quantitatively acceptable chronic toxicity value for mussel (Lampsilis siliquoidea)
from Hazelton et al. (2012). Specifically, Hazelton et al. (2012) conducted a 36-day renewal,
measured PFOS chronic test with fatmucket, Lampsilis siliquoidea. The estimated ECio was
0.05713 mg/L, which was extrapolated from the author-reported data and the exposure response
slope from another PFOS toxicity study focused on another mussel species (Ellipto complamata)
as explained in Section 3.1.1.3.3. Therefore, this test was used in the chronic criterion calculation.

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o From Section 3.1.1.3.3: "The in marsupia exposure was followed by a 24-hour free glochidia
exposure consisting of a factorial design, such that free glochidia from the control group of the
marsupia exposure were divided between a control and the two PFOS treatments and the PFOS
treatments were split into control and the same PFOS treatment group as the marsupia
exposure." - Comment: This is an exceptionally long and confusing sentence please revise to
help the reader understand this complex study and overall approach that EPA took,
o The approach seems ok given the limited data availability at this time.

iii. the use of the quantitatively acceptable chronic toxicity value for damselfly (Enallagma
cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-day renewal, unmeasured
PFOS chronic test with blue damselfly nymphs, Enallagma cyathigerum. The MATC was 0.03162
mg/L, which was calculated from the author-reported value for nymph survival as explained in
Section 3.1.1.3.2. Therefore, this test was used in the chronic criterion calculation.

o Given the duration of the study the researchers likely hovered around the nominal
concentrations of PFOS. Inclusion seems appropriate.

iv. the use of the quantitatively acceptable chronic toxicity value for midge (Chironomus dilutus) from
MacDonald et al. (2004). MacDonald et al. (2004) conducted a 20-day renewal, measured PFOS
chronic test with midge lava, Chironomus dilutus. The ECio was 0.05963 mg/L, which was an EPA-
calculated value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test was used
in the chronic criterion calculation.

o I am uncomfortable with this conclusion presented here, it may be more appropriate to use
MacDonald et al. data from the 20-day endpoint considering recent publication from McCarthy
et al. 2020 as noted above.

5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as well as the
specific acute LC50 values (Appendix A.2) and chronic ECio values (Appendix C.2) that were estimated
(for sensitive genera when C-R data were available) and used to derive criteria.

o This seems like a reasonable and defensible approach if it is applied consistently across genera.

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic water

column criterion elements into tissue-based criterion elements.

6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion elements
were based.

o Please clarify, the following sentence: "BAFs used in the derivation of the PFOS tissue criteria
consisted of > 2 water and organism samples each and were collected within one year and 2 km
distance." It is unclear if the >2 samples refer to the tissue & water samples being mismatched
temporally or if there where >2 sets of water and tissue samples that were collected in different
years.

¦ If the latter then this approach seems appropriate; if the former, EPA should discuss

differences in water chemistry between years to alleviate any concerns with matching tissue
concentration data to water samples that may have significant environmental temporal
variability.

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o A table summarizing the animal tissues used in deriving the BAFs would be helpful to assess the
range offish species and their dietary preferences.

7)	Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

o This is a not an easy statement to comment on, as it may be unlikely that the aquatic receptors will

exceed or reach these tissue concentrations prior to exceedances from the CCC.
o What I am not clear on is, if tissue concentrations exceed these proposed thresholds yet, PFOS
water concentrations do not exceed the CCC, what would be the proposed guidance?

8)	Please provide any additional technical comments that you believe should be considered.

o All technical comments have been previously mentioned

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COMMENTS SUBMITTED BY

1EW1E!

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External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

1) Please comment on the overall clarity of the document as it relates to the derivation of each criterion.

EPA has drafted the PFOS aquatic life criteria to be consistent with methods described in EPA's "Guidelines
for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their
Uses" (U.S. EPA 1985). I congratulate the EPA Team for a very thorough, comprehensive analysis of the
toxicological data to derive each criterion.

• The report is technically sound and is very clearly written.

• The criteria have been derived using strong science-based evidence.

• Sub-sections on overview of PFAS, PFAS nomenclature, problem formulation, exposure pathways,
transformation and degradation of PFOS precursors in the aquatic environment

sources, concentration reported in environment and existing criteria (both national and
international) help to set the scene before toxicological data is presented and assessed for
developing various criterion.

• The freshwater acute water column-based criterion, the chronic water column-based chronic
criterion, the chronic fish whole-body tissue criterion, the chronic fish muscle tissue criterion and
the chronic invertebrate whole-body tissue criterion have been developed and documented in this
report are based on comprehensive assessment of the toxicological data and consistent with the
Guidelines.

• Acute and chronic MDRs for PFOS estuarine/marine criteria derivation were not met due to fewer
empirical PFOS toxicity data. To address this gap, the EPA Team developed an acute aquatic life
benchmark for estuarine/marine environments based on Interspecies Correlation Estimation (ICE)
model. Such predictive models should be used when there is limited toxicity data.

• EPA Team has provided extensive background information on toxicity data assessment and collated
this information in various Appendices as additional line of evidence.

• Tables and Figures are very well laid out throughout the document and provide additional
information of the toxicity data used in developing Water Quality Criteria for PFOS.

2) Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed
comments.

• Is the technical approach used to derive the criterion elements logical?

• Does the science support the conclusions?

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

This EPA report provides a critical review of toxicity data identified in EPA's literature search for PFOS,
including the anionic form (CAS No. 45298-90-6), the acid form (CAS No. 1763-23-1), potassium salt (CAS No.
2795-39-3), an ammonium salt (CAS No. 56773-42-3), sodium salt (CAS No. 4021-47-0), and a lithium salt
(CAS No. 29457-72-5). It quantifies the toxicity of PFOS to aquatic life, and provides criteria intended to
protect aquatic life from the acute and chronic toxic effects of PFOS. The detailed assessment is as follows:

• These criteria have been derived using robust methods and the best available toxicity data on
aquatic life.

• The approach used to derive the draft criterion for PFOS is very logical and consistent with the
protection offered by acute and chronic aquatic life criteria derived using empirical data, as
prescribed in the 1985 Guidelines.

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• Exclusion and inclusion criteria are appropriately discussed in the context of the toxicological data
reported in the literature and provide additional evidence on the selection of toxicity data criteria
development.

• With limited toxicity datasets to North American resident species, non-North American resident
species were included for criteria development. For example, inclusion of non-resident species such
as Planaria, Dugesia japonica and Japanese swamp shrimp, Neocaridina denticulata for calculating
acute water quality criteria and zebra fish, Danio rerio for chronic criteria. The EPA team did not find
any influence of excluding non-North American resident species in criteria derivations and decided
to retain the full acute and chronic toxicity dataset. This was very rational decision and non-
Northern American species served as surrogate species for the broad range of the thousands of
untested species present in the freshwater environment in the U.S.

• The acute measures of effect on aquatic organisms selected included the lethal concentration
(LC50), effect concentration (EC50), or inhibitory concentration (IC50) estimated to produce a
specific effect in 50 percent of the test organisms as per the Guidelines.

• The endpoint for chronic exposures incorporated the effect concentration estimated to produce a
chronic effect on survival, growth, or reproduction in 10 percent of the test organisms (ECio). This
approach has been also consistent with the harmonized guidelines from OECD and the generally
preferred effect level for countries such as Canada, Australia, and New Zealand.

• Reported (No Observed Effect Concentrations) (NOECs) and (Lowest Observed Effect
Concentrations) (LOECs) were only used for the derivation of a chronic criterion when a robust ECio
could not be calculated for the genus.

• Furthermore, EPA independently calculated these toxicity values if sufficient raw data were available
for EPA to conduct statistical analyses. EPA's independently-calculated toxicity values were used
preferentially, where available.

• I agree with the authors' decision on not developing plant criteria based on their lesser sensitivity to
PFOS than in comparison to aquatic vertebrates and invertebrates. The EPA team evaluated the
toxicity data to plants as an additional line of evidence and confirmed that the proposed PFOS
freshwater acute and chronic criteria are expected to be protective of freshwater plants.

• EPA developed protective tissue-based criteria through a bioaccumulation factor approach. This was
based on the application of evaluation criteria for screening bioaccumulation factors (BAFs).

• Based on comprehensive toxicity data assessment, the EPA team has developed the following
criteria using the procedures described in the 1985 Guidelines. The freshwater acute water column-
based criterion magnitude is 3.6 mg/L and the chronic water column-based criterion magnitude is
0.014 mg/L. The chronic freshwater criterion also contains tissue-based criteria expressed as 43.0
mg/kg wet weight (ww) for fish whole-body, 25.3 mg/L ww for fish muscle tissue, and 12.3 mg/kg
ww for invertebrate whole-body tissue.

• Acute and chronic MDRs for PFOS estuarine/marine criteria derivation were not met and an
estuarine/marine FAV could not be calculated to derive an estuarine/marine acute criterion. Further
benchmark was developed using predictive approach and discussed in the follow-up question 3.

• Is the technical approach used to derive the benchmark logical?

• Does the science support the conclusions?

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• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria derived

using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical National Water

Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

• EPA applied the ICE model predictions to supplement the available test dataset to help fill
missing MDRs and allow the derivation of acute estuarine/marine benchmark recommendations
for aquatic life using procedures consistent with those in the 1985 Guidelines. A total of 3104
datapoints from 398 models were evaluated.

• ICE model has been recommended to predict the sensitivity of an untested taxon (predicted
taxa are represented by the y-axis) from the known, measured sensitivity of a surrogate species
(represented by the x-axis). The ICE model approach used is very reasonable to predict toxicity
of untested taxa.

• As documented in Section L.1, ICE-predicted models have been used by multiple independent,
international groups and further confirms that values developed from ICE-generated SSDs will
provide a level of protection that is consistent with using measured laboratory data.

• In addition, prediction accuracy and robustness of the model is evaluated using robust
parameters (e.g., mean square error, R2), that fall within a defined range of acceptability, and
with close prediction confidence intervals that facilitate evaluating the fit of the underlying data.
This confirms the robustness of the model.

• ICE models predicted with acceptable accuracy for PFOS when invertebrates were used to
predict to invertebrate species and vertebrates were used to predict to vertebrate species in
these comparisons.

• The draft acute benchmark for estuarine/marine aquatic life developed using this approach is
0.43 mg/L PFOS, it is lower than the recommended acute freshwater criterion(3.6 mg/L),
suggesting that estuarine/marine species may be more acutely sensitive to PFOS. This is in line
with Hayman et al., (2021), confirming marine species have a higher sensitivity to PFOS than
compared to the freshwater organisms.

• In this report, Mytilus galloprovincialis was not used in the FAV calculation because the value
was not definitive, and true sensitivity of this species is unknown. There are two more studies
published reporting the toxicity values for marine/estuarine species, including Mytilus
galloprovincialis.

o Stuart L. Simpson, Yawen Liu, David A. Spadaro, Xinhong Wang; Rai S. Kookana and
Graeme E. Batley Chronic effects and thresholds for estuarine and marine benthic
organism exposure to perfluorooctane sulfonic acid (PFOS)-contaminated sediments:
Influence of organic carbon and exposure routes
https://doi.Org/10.1016/i.scitotenv.2021.146008
o Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder, Jennifer A
Arblaster Aquatic toxicity evaluations of PFOS and PFOA for five standard marine
endpoints. https://doi.Org/10.1016/i.chemosphere.2021.129699

It is recommended to assess the quality of the toxicity data on marine/estuarine species and recalculate
estuarine criteria based on this recently available information.

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PFOS is a highly stable compound, resistant to hydrolysis, photolysis, volatilization, and biodegradation (as
described in Section 1.1.1 of the Report) and, therefore, expected to vary only minimally in the course of a
toxicity test. To determine if nominal and measured PFOS concentrations were typically in close agreement,
pairs of nominal and corresponding measured PFOS concentrations were compared to one another through
(1) linear correlation analysis and (2) an assessment of measured concentrations as a percent of its paired
nominal concentration. The authors reported, 22 freshwater studies with PFOS measured concentrations,
yielding 373 pairs of measured and nominal concentrations. In addition, there were 7 estuarine/marine
studies with measured concentrations, yielding 142 pairs of measured and nominal concentrations. The data
were grouped by classifications including water type (salt/fresh) and experimental conditions
(acute/chronic; solvent/no solvent; fed/unfed, etc.). Data displayed a high degree of linear correlation and
measured, and nominal concentrations were in close agreement

The analysis conducted by EPA Team showed strong correlation (correlation = 0.9998) of the 326 pairs of
nominal and measured concentrations from freshwater studies. In addition, the experimental conditions did
not influence the correlation between nominal and measured concentrations. The detailed analyses of the
data in Appendix O and the relevant Tables and Figures provide very comprehensive analyses - this is very
useful information and will assist ecotoxioclogist in designing future experiments.

This confirms inclusion of unmeasured PFOS toxicity tests for quantitative use in criteria derivation.

Personal experience on analyzing PFOS in ecotoxicological studies using freshwater species have also
exhibited strong correlation between nominal and measured concentrations.

The authors reported the strong correlation (0.8993) of the 142 pairs of nominal and measured
concentrations, the ratio of measured to nominal concentrations from the saltwater dataset showed bias
with a geometric mean value of 0.6178. Additionally, the median percent difference between measured and
nominal concentration was 30.82%. Furthermore, the saltwater comparison of nominal and measured
concentrations indicated that these experimental conditions (acute/chronic and unfed/fed) could influence
the observed differences between measured and nominal concentrations. These results suggest that
measured and nominal concentrations from saltwater tests were not in close agreement, but this analysis
was based on limited set of data.

The measured concentrations in the recently published paper on marine/estuarine toxicity of PFOS should
also be included in this assessment:

o Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder, Jennifer A Arblaster
Aquatic toxicity evaluations of PFOS and PFOA for five standard marine endpoints.
https://doi.Org/10.1016/i.chemosphere.2021.129699

The second paper is on benthic organisms and PFOS is measured in overlying water, porewater and
sediment. This may provide further guidance on difference between PFOS measured and nominal
concentrations.

Additional information for Appendix O based on a recently published paper:

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According to Rewerts et al., 2021 additional handling steps, which are not typically reported for
ecotoxicological studies but may contribute to variability, include solution homogenization, subsampling
procedures, and the container materials selected for storage. https://doi.org/10.10Q2/etc.4667

5) Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

• Are there relevant data that you are aware of that should be added to the analyses (note that EPA
is working on updating the toxicity data to reflect the data in ECOTOX between Sept. 2019
through the latest update)? If so, please provide references for consideration.

The data selected to derive PFOS criteria are appropriate. Studies that did not fully meet the data quality
objectives outlined in the 1985 Guidelines were not considered for inclusion in the criteria derivation,
including some studies with other PFAS exposures, but were considered qualitatively as supporting
information. A brief summary of each study describing the experimental conditions and summary tables
providing all the relevant information such as strengths and limitations of each study, end points selected
for deriving criteria are well documented by the EPA team and provides further confidence in data selection
process.

The key acceptable exclusion/inclusion criteria used to derive draft criteria are listed below:

• Only single chemical toxicity tests with PFOS were considered for possible inclusion in criteria
derivation, studies that tested chemical mixtures, including mixtures with PFAS compounds were
excluded from criteria derivation.

• Both controlled laboratory experiments and field observations/studies were included.

• PFOS toxicity tests were not excluded from quantitative use in criteria derivation on the basis of
unmeasured test concentrations alone.

• Due to lower sensitivity, insect MDR was excluded from the criterion calculation, but were used to
waive the missing insect MDR.

• Further supporting information on acceptable and unused studies for acute and chronic endpoints
and for freshwater and marine studies are documented and summarized as appendices in this
report.

Additional toxicity data published over the last six months is listed below:

Marine/estuarine

• Nicholas T Hayman , Gunther Rosen , Marienne A Colvin , Jason Conder, Jennifer A Arblaster Aquatic
toxicity evaluations of PFOS and PFOA for five standard marine endpoints.
https://doi.Org/10.1016/i.chemosphere.2021.129699

• Stuart L. Simpson, Yawen Liu, David A. Spadaro, Xinhong Wang; Rai S. Kookana and Graeme E. Batley
Chronic effects and thresholds for estuarine and marine benthic organism exposure to
perfluorooctane sulfonic acid (PFOS)-contaminated sediments: Influence of organic carbon and
exposure routes https://doi.Org/10.1016/i.scitotenv.2021.146008

Fresh water

• Christopher J. McCarthy, Shaun A. Roark, Demitria Wright, Kelly O'Neal, Brett Muckey, Mike
Stanaway, Justin N. Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J. Salice,

Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture Exposure
Experiments with 6 Perfluoralkyl Substances, Environmental Toxicology and Chemistry,
10.1002/etc.5066, 40, 8, (2319-2333), (2021). https://doi.org/10.1002/etc.5066

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In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

i. the use of the qualitatively acceptable acute midge (Chironomus plumosus) data from Yang et
al. (2014) to suggest aquatic insects are relatively tolerant to acute PFOS exposures. Specifically,
Yang et al. (2014) conducted a 96-hour renewal, measured PFOS acute test with the midge,
Chironomus plumosus. This study was not acceptable for quantitative use due to the potential
problematic source of the organisms. The reported LC50 was 182 mg/L for PFOS indicating that
insects may not be one of the more sensitive taxonomic groups. Therefore, this test was
excluded from the acute criterion calculation, but used to waive the missing insect MDR.

Waiving an unfulfilled MDR when available data suggest it is not among the four most sensitive genera is
consistent with previous EPA criteria documents, including U.S. EPA (2016). At this stage, I do not fully agree
with the statement that midge larvae are tolerant to acute exposures. The OECD protocol recommends 48h
acute test for midge larvae and the 48h exposure period is acceptable duration for assessing acute toxicity.
The study by Olson (2017) has limitations but this study can't be fully ruled out. The chronic toxicity data
exhibits sensitivity of insects to PFOS and this statement is also supported by the authors. In addition,

Stefani et al. (2014), Macdonald et al. (2004), and Marziali et al. (2019) conducted chronic toxicity tests with
Chironomus spp. and reported apical endpoints. Results of these studies, taken together, also suggest that
insects are among sensitive taxa to chronic PFOS exposures (with adverse effects reports at low ug/L)

I support the recommendation 'Additional insect toxicity data for PFOS would be very useful for further
examining the relative sensitivity of insects to PFOS exposures".

Unpublished work from our lab shows acute toxicity to midge larva, Chironomus tepperi at 1 mg/L PFOS (48
h EC50 value).

ii. the use of the quantitatively acceptable chronic toxicity value for mussel (Lampsilis siliquoidea)
from Hazelton et al. (2012). Specifically, Hazelton et al. (2012) conducted a 36-day renewal,
measured PFOS chronic test with fatmucket, Lampsilis siliquoidea. The estimated ECio was
0.05713 mg/L, which was extrapolated from the author-reported data and the exposure
response slope from another PFOS toxicity study focused on another mussel species (Ellipto
complamata) as explained in Section 3.1.1.3.3. Therefore, this test was used in the chronic
criterion calculation.

The authors have provided detailed assessment of this study and explained the approach used for the
calculation of chronic toxicity value (section C.2.3-Third Sensitive Freshwater Genus for Chronic Toxicity:
Lampsilis siliquoidea (mussel). Hazelton et al. 2012 used robust study design in spite of including only two
concentration of PFOS in this study. The PFOS exposure concentration was measured, and metamorphosis
success was used as an endpoint for inclusion in the criteria development. While viability of free glochidia at
24 hours post removal from females was a less sensitive endpoint and did not meet the acceptability
criteria. The reduction in metamorphosis success at the 0.0695 mg/L was estimated to be 35.4% but EC10
could not be calculated based on only two PFOS concentrations tested in this study. The EPA team has
calculated EC10 (0.05713 mg/L) using the exposure response slope from PFOS toxicity study on another
mussel species (Ellipto complamata). The explanation and logic provided is reasonable to include the
calculated EC10 value to derive the freshwater chronic criterion and to better understand the effects of
PFOS on aquatic insects.

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iii. the use of the quantitatively acceptable chronic toxicity value for damselfly (Enallagma
cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-day renewal,
unmeasured PFOS chronic test with blue damselfly nymphs, Enallagma cyathigerum. The MATC
was 0.03162 mg/L, which was calculated from the author-reported value for nymph survival as
explained in Section 3.1.1.3.2. Therefore, this test was used in the chronic criterion calculation.

As a weight of evidence approach, EPA ran additional analyses with some of the other toxicity values for E.
cyathigerum to understand the influence of this study on the overall chronic criterion. The 150-day MATC
was more comparable to the other aquatic insect data and more representative of life cycle effects than the
10-day MATC or NOEC at 60 and 320 days of exposure (Table 4.3 of the report). EPA has concluded that the
150-day MATC should be used quantitatively to derive the chronic freshwater criterion toxicity. In addition,
the control survival of test organisms was determined to be acceptable at this time point in the test. I am in
agreement with this decision.

iv. the use of the quantitatively acceptable chronic toxicity value for midge (Chironomus dilutus)
from MacDonald et al. (2004). MacDonald et al. (2004) conducted a 20-day renewal, measured
PFOS chronic test with midge lava, Chironomus dilutus. The EC10 was 0.05963 mg/L, which was
an EPA-calculated value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test
was used in the chronic criterion calculation.

The observed effects of PFOS on C. dilutus reported in the paper by the study authors include survival and
growth as weight (measured as mg of ash-free dry mass per individual) for both the 10-day and 20-day
exposure durations and emergence and reproduction over the 20-day exposure duration. The author
reported 10-day growth and survival EClOs for the study were 0.0492 and 0.1079 mg/L, respectively. The
study authors also reported NOECs of 0.0491 mg/L, LOECs of 0.0962 mg/L, and MATCs of 0.0687 mg/L for
both endpoints. The author reported 20-day ECi0s for growth, survival, and total emergence were 0.0882,
0.0864, and 0.0893 mg/L, respectively. And the study authors also reported NOECs of 0.0217 mg/L for
growth and survival and < 0.0023 mg/L for emergence, LOECs of 0.0949 mg/L for growth and survival and
0.0217 mg/L for emergence, and MATCs of 0.0454 mg/L for growth and survival and 0.0071 mg/L for
emergence.

Independent statistical analyses were conducted by EPA Team for both the 10-day and 20-day exposure
durations using data that were estimated The 20-day ECi0s for survival and emergence were not considered
to be reliable endpoints given the disparities in the calculated ECi0s and the level of data that was presented
in the paper, which made independent verification of the toxicity values less accurate. The dosing of the 20-
day exposure was more of a concern than the 10-day exposure, which had measured concentrations that
were much more in line with the expected nominal concentrations. The independently-calculated 10-day
ECio for growth was 0.0586 mg/L was used quantitatively to derive the chronic aquatic life criterion.

The EPA team has reviewed publications by Stefani et al. (2014) and Marziali et al. (2019) as additional
supporting information. These authors conducted chronic toxicity tests with Chironomus spp. and reported
chronic apical endpoints (at low ug/l) but at only at one concentration.

Use of the chronic toxicity data for PFOS in a recent publication should also be considered to assess the
reliability of 20-day endpoints (adverse effects reported at 2-3 ng/L).

Christopher J. McCarthy, Shaun A. Roark, Demitria Wright, Kelly O'Neal, Brett Muckey, Mike Stanaway,Justin
N. Rewerts, Jennifer A. Field, Todd A. Anderson, Christopher J. Sal ice, Toxicological Response of
Chironomus dilutus in Single-Chemical and Binary Mixture Exposure Experiments with 6 Perfluoralkyl
Substances, Environmental Toxicology and Chemistry, 10.1002/etc.5066, 40, 8, (2319-2333), (2021).
https://doi.org/10.1002/etc.5066

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5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as well as the
specific acute LC5o values (Appendix A.2) and chronic ECio values (Appendix C.2) that were estimated
(for sensitive genera when C-R data were available) and used to derive criteria.

This is an excellent approach utilized by the EPA Team. EPA's approach for fitting concentration-response (C-
R) data resulted in consistent approach across various ecotoxicological studies. The R drc package was used
to fit 22 different models to each individual C-R dataset. A single model was then selected from the 22
models to serve as the representative C-R model. The selected model represented the most statistically-
robust model available. In certain cases, this approach even improved and helped to select most sensitive
toxicological endpoint.

In depth analyses and associated dose-response graphs in Appendix A.2 and Appendix C.2 provides further
in-depth information on the EPA's approach for fitting concentration-response (C-R) data. As noted in
Section 8 some of the values are missing.

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic water
column criterion elements into tissue-based criterion elements.

The freshwater chronic PFOS toxicity data with measured tissue concentrations was limited, with no
quantitatively acceptable tissue-based tests. Therefore, there were insufficient data to derive tissue-based
criteria using a GSD approach from empirical tissue data from toxicity studies.

Tissue criteria derived from the chronic water column concentration (CCC) with the use of bioaccumulation
factors were developed by EPA. The chronic freshwater criterion also contains tissue-based criteria
expressed as 43.0 mg/kg wet weight (ww) for fish whole-body, 25.3 mg/kg ww for fish muscle tissue, and
12.3 mg/kg ww for invertebrate whole-body tissue.

EPA developed protective tissue-based criteria through a bioaccumulation factor approach. The authors
reviewed PFOS BAF literature based on four criteria 1) number of water samples, 2) number of organism
samples, 3) water and organism temporal coordination in sample collection, and 4) water and organism
spatial coordination in sample collection and developed a ranking system. BAFs used in the derivation of the
PFOS tissue-based criteria consisted of > 2 water and organism samples each and were collected within one
year and 2 km distance. This scheme assured selection of only BAFs of high and medium quality to derive the
tissue criteria.

6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion elements were
based.

BAFs are different for muscle/fillet and whole-body tissues. Humans consume muscle/fillets from fish and
soft tissues from bivalves, therefore the water quality criteria recommended by EPA used BAFs based on
these tissues. In addition, muscle and whole-body are the most commonly sampled tissue types in
monitoring programs. These criteria were developed based on the values reported for 50-60 samples (Table
3-12).

Within the body, PFOS tends to bioaccumulate within protein-rich tissues, such as the blood serum proteins
and liver. EPA Team calculated additional tissue values for liver, blood, and reproductive tissues by
transforming the freshwater chronic water column criterion into representative tissue concentrations using
tissue-specific bioaccumulation factors (BAFs). Author's decision uses agreement on the use of female

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reproductive tissues due to its relevance for potential maternal transfer to offspring. These additional tissue-
based values were calculated for comparative purposes and were not proposed as recommended criteria.

7) Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

PFOS concentrations in tissues are generally expected to change only gradually over time in response to
environmental fluctuations. The chronic tissue-based criteria averaging periods, or duration components,
were therefore specified as instantaneous, because tissue data provide point, or instantaneous,
measurements that reflect integrative accumulation of PFOS over time and space in population(s) at a given
site. It was appropriate for EPA to inform the recommended ten-year exceedance frequencies for the
chronic tissue-based criteria given the large variation in possible biological and physical variable influencing
ecological recovery.

8) Please provide any additional technical comments that you believe should be considered.

Additional suggestions are listed below:

1- The species listed in the table is Mytilus galloprovincialis not M. edulis
Table 0-1. The Three Most Sensitive Acute Estuarine/Marine Genera.

Ranked Below from Most to Least Sensitive.

Rank

Genus

Species

GMAV
(mg/L
PFOS)

Comments

Mytilus1

Mediterranean mussel,
M. edulis

Mytilus galloprovincialis

> 1

Not a resident species in North America, but other
species in this genus are resident, commercially, or
ecologically important species

2. Page 115- second paragraph (values highlighted in red and underlined are not consistent)

The author reported 10-day growth and survival ECi0s for the study were 0.0492 and 0.1079 mg/L,
respectively. The study authors also reported NOECs of 0.0491 mg/L, LOECs of 0.0962 mg/L, and MATCs of
0.0687 mg/L for both endpoints. And the author reported 20-day ECi0s for growth, survival, and total
emergence were 0.0882, 0.0864, and 0.0893 mg/L, respectively. And the study authors also reported NOECs
of 0.0217 mg/L for growth and survival and < 0.0023 mg/L for emergence, LOECs of 0.0949 mg/L for growth
and survival and 0.0217 mg/L for emergence, and MATCs of 0.0454 mg/L for growth and survival and 0.0071
mg/L for emergence. Also, it should be noted, the paper reported contrasting NOECs for 20-day survival. The
text in the paper stated that the NOEC was 0.0271 mg/L and Table 2 of the paper stated 0.0949 mg/L. EPA
assumed the NOEC in Table 2 of the paper was not correct and that 0.0217 mg/L was the correct NOEC
based on the data presented in Figure 3A of the paper. This assumption was applied to the summary of the
study results presented in this PFOS draft criteria.

3. Page 138-middle of the paragraph The chronic freshwater criteria also contain tissue-based criteria
expressed as 43.0 mg/kg wet weight (ww) for fish whole-body, 25.3 mg/ —ww for fish muscle tissue and
12.3 mg/kg ww for invertebrate whole-body tissue.

4. Page A-21 last paragraph The noted toxicity values provided in each study summary above (ADD
NUMBERS), comprising of both author-reported and independently-calculated LC5o values, were used to

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calculate the GMAV value (as the geometric mean of the three LC5o values previously mentioned) of
22.48 mg/L, which was used to derive the freshwater aquatic life criterion.

5. Page A-24- Fourth line from bottom-The study author reported LC50 was 22.2 ± 4.6 mg/L for PFOS. The
independently-calculated toxicity value was x.xx mg/L. The study author reported value was used
quantitatively to derive the draft acute water column criterion.

6. Page A-25- Fourth line from bottom The study author reported 96-hour LC5o was 50.51 mg/L PFOS. The
independently-calculated toxicity value was x.xx mg/L. The study author reported value was used
quantitatively to derive the draft acute water column criterion.

7. Page A-27- Fourth line from bottom. The independently-calculated toxicity value was x.xx mg/L. The
study author reported value was used quantitatively to derive the draft acute water column criterion.

8. Page A-29- First paragraph For comparison, the 7-day LC50 was 39.71 mg/L. The independently-
calculated toxicity value was x.xx mg/L. The 96-hour study author reported value was used
quantitatively to derive the draft acute water column criterion.

9. Page A-30- First paragraph The independently-calculated toxicity value was x.xx mg/L. The study author
reported value was used quantitatively to derive the draft acute water column criterion.

10. Page A-36- 5th line from bottom in complete data x.xx mg/L.

11. Also at A-37 in complete data x.xx mg/L.

Table 0-3. Summary of Assessment Endpoints and Measures of Effect Used in the Criteria Derivation for
PFOS

Assessment Endpoints for the
Aquatic Community

Measures of Effect

Aquatic Life: Survival, growth, and
reproduction of freshwater and
estuarine/marine aquatic life (i.e.,
fish, amphibians, aquatic
invertebrates)

For effects from acute exposure:

1. LC5o concentrations in water, diet, and/or tissue (e.g.,
muscle, blood, egg)

2. NOEC and LOEC concentrations in water, diet, and/or
tissue (e.g., muscle, blood, egg)

For effects from chronic exposure:

1. ECio concentrations in water, diet, and/or tissue (e.g.,
muscle, blood, egg)

2. NOEC and LOEC concentrations in water, diet, and/or
tissue (e.g., muscle, blood, egg); Only used when an ECio
could not be calculated for a genus.

Note: only chronic exposures were considered for derivation of the
tissue-based criteria since PFOS is a bioaccumulative chemical.
These chronic tissue-based criteria are expected to be protective of
acute effects, because acute effects were observed at much
greater concentrations than chronic effects.

Please review if the highlighted muscle, blood and egg would be relevant to this section in terms of LC50,
ECIO, LOEC and NOEC endpoints .

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12. 1.1.2 and page 3- Previously Published Chronic Water Criteria for Direct Aqueous Exposure

The information on Australian guidelines to be updated based on NEMP2 published in 2020. I will attach it as
a PDF. https://www.environment.gov.au/system/files/resources/2fadflbc-b0b6-44cb-al92-
78c522d5ec3f/files/pfas-nemp-2.pdf

"Previously published freshwater chronic values were available for two states (Minnesota and Michigan)
and three countries or geographic regions (Australia/New Zealand, Canada, and Europe). These publicly
available values for other jurisdictions were 0.019 mg/L and 0.14 mg/Lfor Minnesota (STS/MPCA 2007)
and Michigan (EGLE 2010), respectively, and were 0.00013 mg/L in Australia/New Zealand (CRC CARE
2017; EPAV2016), 0.00680 mg/L in Canada (ECCC 2018), and 0.000023 mg/L in Europe (RIVM 2010).
Previously published estuarine/marine chronic values were available for two geographic regions
(Australia/New Zealand and Europe). These publicly available values were 0.0000046 mg/L in Europe
(RIVM 2010) and 0.0078 mg/L in Australia/New Zealand (CRC CARE 2017; EPAV 2016)"

The CRC marine guidelines are not valid as they are not based on the framework Freshwater values are to be
used on an interim basis

13. Page 4- Table 1.1 to be updated accordingly

Exposure
scenario

PFOS

Exposure scenario

Comments and source

Freshwater

0.00023
Hg/L

99% species
protection - high
conservation value
systems

Australian and New Zealand Guidelines for
Fresh and Marine Water Quality - technical
draft default guideline values for PFOS and
PFOA.

0.13
Hg/L

95% species
protection - slightly to
moderately disturbed
systems

Note 1: The 99% species protection level
for PFOS is close to the level of detection.
Agencies may wish to apply a 'detect'
threshold in such circumstances rather
than a quantified measurement.

Note 2: The draft guidelines do not

2 Hg/L

90% species
protection - highly
disturbed systems

31 Hg/L

80% species
protection - highly
disturbed systems

account for effects which result from the
biomagnification of toxicants in
airbreathing animals or in animals which
prey on aquatic organisms.

Note 3: The WQGs advise3 that the 99%
level of protection be used for slightly to
moderately disturbed systems. This
approach is generally adopted for
chemicals that bioaccumulate and
biomagnify in wildlife. Regulators may
specify or environmental legislation may

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prescribe the level of species protection
required, rather than allowing for case by-
case assessments.

Exposure
scenario

PFOS

Exposure scenario

Comments and source

Interim
marine

0.00023
Hg/L

99% species
protection

- high conservation
value systems

As above.

Freshwater values are to be used on an
interim basis until final marine guideline
values can be set using the nationally-
agreed process under the Australian and
New Zealand Guidelines for Fresh and
Marine Water Quality.

The WQG advise that in the case of
estuaries, the most stringent of freshwater
and marine criteria apply, taking account
of any available salinity correction.

Marine guideline values developed by CRC
CARE are under consideration through the
nationally-agreed water quality guideline
development process.

0.13
Hg/L

95% species
protection

- slightly to
moderately disturbed
systems

2 M-g/L

90% species
protection - highly
disturbed systems

31 M-g/L

80% species
protection - highly
disturbed systems

ahttps://www.waterquality.gov.au/anz-guidelines/guideline-values/default/water-qualitv-toxicants/local-
conditions#bioaccumulation

14.	Table 1 2. Two Primary Categories of PFAS

Please refer to OECD 2021 to be consistent with PFAS terminology/nomenclature

OECD (2021), Reconciling Terminology of the Universe of Per- and Polyfluoroalkyl Substances:
Recommendations and Practical Guidance, OECD Series on Risk Management, No. 61, OECD Publishing, Paris

15.	Table 1.3 Page 8

Please review Figure 9 OECD 2021 (also attached as PDF)

16.	Conceptual Model of PFOS in the Aquatic Environment and Effects

Figure 2.9 page 77- Growth as an endpoint missing in the endpoints - first pentagon

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COMMENTS SUBMITTED BY
REVIEWER 5

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External Peer Review of EPA's Draft
Aquatic Life Water Quality Criterion for Perfluorooctane Sulfonate (PFOS)

1) Please comment on the overall clarity of the document as it relates to the derivation of each criterion.

RESPONSE: Overall, the document is clearly written and generally free of grammatical errors. I applaud the
scientists and EPA for compiling an impressive amount of work and communicating it in a reasonably clean
and coherent way. That said, there are a few instances of redundancy - literally, the same sentences
repeated. I have made note of these in the actual report and will include that along with this document, if
requested. Although these are easy enough to see with careful review. The document is VERY LONG and
very detailed so any efforts to shorten or make more concise would be welcomed.

As for the clarity of technical elements of the document, I feel that many of the decisions to use or not use
data or endpoints could be more consistent and/or communicated better. For example, in some cases the
geometric mean of endpoints for a certain taxa is used for the chronic value (pimephales) but for other taxa,
this is not the case (e.g. daphnia; zebrafish). In other cases, the decision to not use certain data seems as if it
could be communicated more clearly. So while the language of the document is pretty clear, the actual
technical aspects are less so.

One major concern I have is with the overall approach of using the 4 most sensitive toxicity values to then
derive the final acute and final chronic values. Using this approach it would seem the AWQC are then very
sensitive to changes in any 1 of the 4 toxicity values. For example, when EPA explored the impact of
different toxicity values on the chronic freshwater water column criteria, using higher toxicity values (e.g.,
pimephales in place of fatmucket Table 4-3) resulted in a lower chronic criteria. This is nonintuitive and
suggests a possible flaw in the approach. It's possible this is not the case and it makes sense both
mathematically (steeper slope) and perhaps even from a protection standpoint. Either way, EPA should
explain why this happens and what it means for the overall approach. I suspect the EPA is somewhat
constrained by the 1985 guidelines in developing the AWQC but I also see that New Approach Methods
(WEB-ICE) were used to derive criteria with limited data. I wonder if using a species sensitivity distribution
approach in which all the chronic or acute (freshwater)data are used would result in more defensible criteria
that are less impacted by changes in any one toxicity value? At the very least, I think including a full SSD
would be useful for comparison as part of the characterization piece. In the PFOA document I mention
revisiting and publishing and updated 1985 guidelines...this is warranted when EPA has the bandwidth to do
so. Having said all this, I am aware that EPA likely has justification for their approach of using the 4 most
sensitive tox values but it would perhaps be good to mention this again as I suspect a lot of people that may
read the AWQC will not also read the 1985 guidelines.

Lastly, I had a hard time keeping track of all the decisions to use or not use data for each of the tox values
that supported the criteria. I think a more detailed table with all the tox values considered (data shown in
Figure 3-3) and including whether the data used were author-reported, re-calculated by EPA, along with the
lowest reported/calculated value that wasn't used and why. This may be asking a lot and this information is
throughout the document but not in a single, easy to locate and read location.

2) Please comment on the approach used to derive the draft criterion for PFOS. Please provide detailed
comments.

• Is the technical approach used to derive the criterion elements logical?

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RESPONSE: Overall, I think the technical approach is relatively sound although there are instances where it
was difficult to keep track of all the decisions with regard to data and whether these were consistent and
logical. Admittedly, I think this is a tough chemical and a tough dataset and EPA did an excellent job with the
background material and highlighted and used key studies (but more have been published since and will, I'm
sure be included). Unfortunately, the technical approach to derive criterion elements is not universally
logical. Moreover, as mentioned, using only the 4 most sensitive toxicity endpoints followed by a regression
(what type? Was this specified?) seems less robust than using a full species sensitivity distribution with a
more "natural" distribution of sensitivity (s-shaped, for example). This last statement may not be true so a
reasonable compromise might be to include a full SSD as part of the characterization piece related to
"considering other toxicity values impact on the FCV, etc.". What really confused me was that when EPA did
what amounts to a sensitivity analysis of the FCV by replacing toxicity values, the FCV DECREASED when
higher toxicity values were used. While I suspect this happened because switching to higher toxicity values
steepened the slope (or something), it does not make intuitive sense to me and should be further explained.
Alternatively, an explanation and justification, even brief, would be helpful in supporting the 4 most
sensitive toxicity value approach. I am aware that the 1985 guidelines may include this but I suspect most
users of the AWQC may not be familiar with the details of the guidelines.

With regard to the tissue-based criteria, EPA mentions using "only PFOS studies in which organisms were
exposed in the diet" (or similar; p. 88) but then go on to say the BAF approach was used. I would edit this
section to start with mentioning that a BAF approach was used because there were not enough tissue data
from laboratory studies. I mention this because it was confusing - there was a lot of explanation of using
only dietary exposures and then one sentence (basically) stating...EPA explored a BAF approach.

• Does the science support the conclusions?

RESPONSE: Well, offhand, I think the final chronic value for freshwater organisms should likely be lower.
Importantly, several studies have been published in 2021 that should likely be included as toxicity values and
they may result in lower toxicity estimates. The fact that EPA's criteria are higher than all other published
criteria is worrisome. We are all using the same data and many in the field are quite capable scientists.

These two papers come immediately to mind but I am sure there are others.

Sensitivity and Accumulation of Perfluorooctanesulfonate and Perfluorohexanesulfonic Acid in
Fathead Minnows (Pimephales promelas) Exposed over Critical Life Stages of Reproduction and
Development J.G. Suski. C.J. Salice. M.K. Chanov. J. Avers. J. Rewerts. J. Field Environmental
Toxicology and Chemistry, 2021, pp. 811-819.

Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture Exposure
Experiments with 6 Perfluoralkyl Substances Christopher J. McCarthy, Shaun A. Roark, Demitria
Wright. Kelly O'Neal. Brett Muckey, Mike Stanaway, Justin N. Rewerts. Jennifer A. Field. Todd A.
Anderson. Christopher J. Salice Environmental Toxicology and Chemistry, 2021, pp. 2319-2333.

In my view, it is essential that EPA incorporate newly published toxicity data for PFOS (and PFOA).

Furthermore, in several cases, EPA's decisions to use what look like higher estimates of toxicity seem
somewhat arbitrary and not internally consistent. I also noted above and mention here again the sensitivity
of the criteria development approach to changes in one of the 4 most sensitive taxa/toxicity values.

• Is it consistent with the protection of freshwater aquatic life from acute, chronic, and
bioaccumulative effects?

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RESPONSE: I believe the criteria are "NEARLY" protective of freshwater aquatic life for acute, chronic and
bioaccumulative effects of PFOS. I say "nearly" because it seems to me that the FCV, in particular, could and
maybe should likely be lower. Also, below I comment on the appropriateness and utility of the frequency
and duration elements of the criteria. Briefly, in my opinion the frequency and criteria elements of the
criteria certainly help the criteria concentrations to be protective; it is unlikely that a 4-day exposure to the
FCV would result in adverse effects to any taxa for which there are data; however, these data are not
commonly reported (hourly or 4-day running average concentrations have never been reported to my
knowledge).

• Is the technical approach used to derive the benchmark logical?

RESPONSE: Yes, given the lack of PFOS toxicity data for acute estuarine/marine species, I think applying
WEB-ICE is a REASONABLE APPROACH...perhaps the only approach that is defensible. Clearly, more (or
some) data would be a wonderful contribution. WEB-ICE, as mentioned, has been reviewed and published
quite a bit so I think, as an approach, it has merit and support of the scientific community. EPA also did a
good job presenting the approach and being clear about the criteria being a draft. Overall, when data have
been lacking, EPA has used state-of-the-art approaches to developing criteria (my concerns are mostly when
sufficient data are available).

• Does the science support the conclusions?

RESPONSE: Yes, the science supports the conclusions. Interestingly, the acute criteria for estuarine/marine
species (o.43 mgPFOS/L) is almost an order of magnitude lower than the acute criteria for freshwater
organisms (3.6 mg/L). Whether estuarine/marine species are truly more sensitive remains to be seen but, to
me, it is more reasonable, given the lack of data, that the criteria draft is lower.

• Is it consistent with the protection offered by acute estuarine/marine aquatic life criteria derived
using empirical data, as prescribed in the 1985 Guidelines for Deriving Numerical National Water
Quality Criteria for the Protection of Aquatic Organisms and Their Uses?

RESPONSE: Yes, and EPA justified this in the explanation of WEB-ICE that occurs in Appendix L and, overall,
the approach and resulting criteria are consistent with the protection of estuarine and marine species.

RESPONSE: I think the comparison of measured and nominal concentrations was an interesting read and a
useful contribution. That said, many toxicologists focused on PFAS have commented that analytical
confirmation is necessary for a high quality study - this was echoed (loudly) at the SETAC Workshop on Risk
of PFAS that occurred in summer, 2019. As well, in my own experience there have been challenges in
sometimes matching nominal and measured concentrations for aquatic exposures. The paper by Rewerts et
al. 2020 highlights some of the challenges and provides recommendations for accurate solutions of PFAS. As
a general rule, we have erred on the side of reporting measured concentrations.

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Two important thoughts. First, several very prominent analytical chemists that have made a career of
measuring PFAS have indicated to me that the analytical method is only about 30% accurate - meaning that
if the analytical measure was +/- 30% of nominal, they would be considered "the same". EPA used 20% as a
threshold (for deciding nominal and measured were the same) and I'm not sure why this is. As far as I can
tell, 30% is a more reasonable threshold.

Second, in the review and derivation of toxicity values for the MacDonald et al. 2014 paper, EPA elected not
to use the 20-day emergence rate endpoint, in part, because the nominal and measured did not agree. This
makes no sense to me. As long as the solutions were confirmed analytically and reported, that should be
good enough and, in fact, preferred over nominal alone.

Paper worth including in the section on nominal vs. measured PFOS concentrations:

Key Considerations for Accurate Exposures in Ecotoxicological Assessments of Perfluorinated
Carboxylates and Sulfonates. Justin N. Rewerts, Emerson C. Christie. Alix E. Robel, Todd A. Anderson.
Christopher McCarthy, Christopher J. Salice, Jennifer A. Field Environmental Toxicology and
Chemistry, 2020

5) Please comment on the toxicity data used to derive the draft criteria.

• Were the data selected and/or excluded from the derivation of the criteria derivation
appropriately utilized?

RESPONSE: As mentioned, I feel that there are some inconsistencies with how some data were included or
excluded. In the previous comment, for example, some data were excluded from the MacDonald et al. 2014
paper because there was some disagreement between nominal and measured. With regard to PFAS, I would
say measured is almost always better than nominal and the fact that these sometimes don't agree should
not be too big of a deal as long as they are not wildly different. EPA put substantial effort into sometimes
justifying nominal - in all cases, excluding studies that had analytical confirmation is less defensible than
including studies that only report nominal, in my opinion. This last statement is, of course, provided the
analytical methods are robust.

• Are there relevant data that you are aware of that should be added to the analyses (note that EPA
is working on updating the toxicity data to reflect the data in ECOTOX between Sept. 2019
through the latest update)? If so, please provide references for consideration.

Sensitivity and Accumulation of Perfluorooctanesulfonate and Perfluorohexanesulfonic Acid in
Fathead Minnows (Pimephales promelas) Exposed over Critical Life Stages of Reproduction and
Development. J.G. Suski, C.J. Salice. M.K. Chanov, J. Avers, J. Rewerts. J. Field Environmental
Toxicology and Chemistry, 2021, pp. 811-819.

Toxicological Response of Chironomus dilutus in Single-Chemical and Binary Mixture Exposure
Experiments with 6 Perfluoralkyl Substances. Christopher J. McCarthy. Shaun A. Roark. Demitria
Wright. Kelly O'Neal. Brett Muckev. Mike Stanawav. Justin N. Rewerts. Jennifer A. Field. Todd A.
Anderson. Christopher J. Salice. Environmental Toxicology and Chemistry, 2021, pp. 2319-2333.

In particular, please comment on:

5a. The toxicity values used to derive the PFOS criteria, with a particular emphasis on:

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Chironomus plumosus. This study was not acceptable for quantitative use due to the potential
problematic source of the organisms. The reported LC5o was 182 mg/L for PFOS indicating that
insects may not be one of the more sensitive taxonomic groups. Therefore, this test was
excluded from the acute criterion calculation, but used to waive the missing insect MDR.

RESPONSE: Given that insects are among the most sensitive organisms for the chronic exposures to PFOS, it
seems the Yang et al. 2014 paper is not very consistent with the prevailing data. Additionally, McCarthy et
al. 2021 reports toxicity to chironomids similar to that of MacDonald et al. Additionally, while the Olson
2017 data for Aedes species was not acceptable (for valid reasons), nonetheless the study shows very high
sensitivity of another insect species to acute exposures to PFOS. That said, given the EPA's stance and
justification that "nominal generally equal measured PFOS concentrations", I'm inclined to put more
confidence in Olson's study. Same for the 20-day data in the MacDonald et al. paper on chironomids. In that
case, there was a "relatively large difference between measured and nominal concentrations (p. 278)" and
so the data were not used. This seems odd to me - as long as there are measured data, that's what I would
suggest using. Regardless, the MacDonald et al. paper points to the sensitivity of insects so, collectively, I'd
be disinclined to say that the Yang et al. paper shows insects are not sensitive and the data requirement can
be waived. I wonder if it's possible to somehow estimate an acute toxicity value for aquatic insects based on
chronic toxicity data? Basically, a reverse of the Acute/Chronic ratio approach.

ii. the use of the quantitatively acceptable chronic toxicity value for mussel (Lampsilis siliquoidea)
from Hazelton et al. (2012). Specifically, Hazelton et al. (2012) conducted a 36-day renewal,
measured PFOS chronic test with fatmucket, Lampsilis siliquoidea. The estimated ECio was
0.05713 mg/L, which was extrapolated from the author-reported data and the exposure
response slope from another PFOS toxicity study focused on another mussel species (Ellipto
complamata) as explained in Section 3.1.1.3.3. Therefore, this test was used in the chronic
criterion calculation.

RESPONSE: Unfortunately, I cannot find the Drottar et al. (2000) paper which is the basis of estimating the
EC10 from the EC35 generated in the Hazelton et al. (2012) study. And..I think the citation in the document
is incorrect and this should be Drottar and Kreugar (2000g)... or check to make sure the citations in text and
references match. Moroever, the Drottar paper appears to be an acute test which is VERY different than the
Fatmucket study. This approach seems like a "stretch" and, again, somewhat inconsistent with the
approaches and decision matrix EPA has used to utilize or discard other data and endpoints.

iii. the use of the quantitatively acceptable chronic toxicity value for damselfly (Enallagma
cyathigerum) from Bots et al. (2010). Bots et al. (2010) conducted a 320-day renewal,
unmeasured PFOS chronic test with blue damselfly nymphs, Enallagma cyathigerum. The MATC
was 0.03162 mg/L, which was calculated from the author-reported value for nymph survival as
explained in Section 3.1.1.3.2. Therefore, this test was used in the chronic criterion calculation.

RESPONSE: Well, clearly it would have been better to be able to estimate an EC10 but this appears
appropriate. I note that for this study the exposure concentrations were an order of magnitude apart; in
other cases, EPA has used "too big of a difference between exposure concentrations" to discard a study or
two. Somewhere, it would be good to know at what point there is too great a difference among exposure
concentrations (lOx, 20x, ?) for the study to be deemed acceptable for use quantitatively.

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an EPA-calculated value for 10-day growth as explained in Section 3.1.1.3.4. Therefore, this test
was used in the chronic criterion calculation.

RESPONSE: I do not agree with the toxicity value used by EPA as obtained from the MacDonald et al. study.
The lowest toxicity value is the MATC for 20-day emergence of 0.0071 mg PFOS/L. It is not clear why EPA did
not use this value? Emergence is clearly extremely ecologically relevant, and the value generated seems as
defensible as most of the other endpoints EPA has chosen to include?

Additionally, and as mentioned above, see the paper by McCarthy et al. (2021) that was just published in
Environmental Toxicology and Chemistry. Those data appear robust and should meet acceptability criteria.

5b. EPA's approach for fitting concentration-response (C-R) data (described in Appendix K) as well as the
specific acute LC5o values (Appendix A.2) and chronic ECio values (Appendix C.2) that were estimated
(for sensitive genera when C-R data were available) and used to derive criteria.

RESPONSE: In general, the approach for fitting C-R data that EPA used is basically state-of-the-art. The drc
package is very powerful and provides a way to test many different curves to then select the best fit model.
Although EPA described some of this in the several sections related to "fitting x data (K 1.2)", I think more
details would be warranted. The description for the criteria to select best fit models is rather vague. Perhaps
a table of specific fit criteria would be helpful? Perhaps this is not doable because every dataset is different.

When I teach modules on Akaike Information Criteria (AIC) I emphasize that the metric "penalizes" fit for
more parameters within a model. So, using AIC can yield the simplest, best model that fits the data. This is
because models with more parameters tend to yield a better fit purely based on statistical properties and
not the actual phenomena being studied. I am not aware that AIC is a measure of the model fit to "true
outcomes" which are only theoretical constructs, I think. If we knew the "true outcomes" we would not
really need the model. Anyway, I would encourage the authors to review the AIC section and make edits if
necessary and certainly cite the source of the explanation.

For section K.2.2. are there actually any criteria (i.e., numbers) that are used to determine when a model fit is
appropriate? As a simple example, maybe one would consider an rA2 of 0.8 or better to be a "good model"
for linear regression? Some statements to this effect and any details regarding actual criteria used to select
"good models" would be helpful. So, overall the curve fitting approach is appropriate but more, specific
details would be helpful.

6a. Uncertainty surrounding the bioaccumulation factors (BAFs) used to translate of the chronic water
column criterion elements into tissue-based criterion elements.

RESPONSE: Using the BAF for PFOS to determine the tissue-based criterion elements is, I think, an
interesting and useful approach given the lack of tissue-based metrics associated with toxicity data. The
variability in observed bioaccumulation of PFOS is an active area of research but the work by Burkhard 2021
(also an author on the AWQC) provides an excellent synthesis and compendium of available BAFs for PFOS.
That said, I noticed that the criteria for co-located tissue and water samples for PFOS was that they were
collected within a year of each other and within 2 km distance (p. 134) - this likely contributes to significant
variability in the BAFs. Although there are few published datasets, there are some datasets that show
considerable temporal and spatial variability in PFAS water concentrations over the course of a few weeks
and over a spatial distance of less than 0.5 km. I wonder if the variability in BAFs would decrease if the

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criteria were narrowed to co-collected samples measured at the same time? Might be worth the exercise.
Given the variability in PFOS BAFs, why not use something like the 25% percentile BAF instead of the
geometric mean? When developing a protective criteria and there is a very noisy data set, it might be
beneficial to err on the side of caution until better data (many co-located samples in space and time) were
available. So, in summary, I think the approach of using BAFs to estimate tissue-based criteria is reasonable
but given the variability in BAFs, I would encourage using a lower BAF instead of the geometric mean or
reconsidering the data that went into the BAFs used for criteria development.

6b. EPA's determination of appropriate BAFs and the tissue types that the tissue criterion elements were
based.

RESPONSE: Invertebrate whole body, fish whole body, and fish muscle are appropriate tissues for the tissue-
based criterion. These are the most commonly collected tissue types and are relevant to monitoring efforts
and are even useful for considerations of fish advisories. That said, the only other tissue worth considering
would be for liver in fish since this tissue accumulates considerably more PFOS than muscle - these are
included in the appendices so this is appropriate. Overall, tissues used for the tissue-based criteria are
appropriate.

7) Please comment on the frequency and duration of the criterion elements, in particular the tissue-
based criterion elements.

RESPONSE: In my opinion, the frequency and duration of criterion elements is among the most uncertain
and potentially contentious elements of any type of protective criteria. The frequency and duration for
tissue-based criteria is that the tissue-based criteria cannot be exceeded more than once in a 10 year period.
This means that if the PFOS criterion for whole body in fish of 43 mg/kg bw is exceeded more than once in a
10 year period then the criteria is exceeded. This also means the fish was likely exposed to the 0.014 mg/l
concentration for longer than an instantaneous exposure and likely longer than 4 days. So, to me, does this
not mean that if 43 mg/kg bw was measured in a fish tissue, then the fish was likely exposed to 0.014 mg
PFOS/L for longer than 4 days, doesn't it? And, this also means that if fish whole body concentrations were
42.5 mg/kg bw for 10 years, the criterion would not be exceeded. I would suspect that long term PFOS
exposures that consistently lead to 42.5 mg/kg bw in fish would likely translate to adverse ecological
impacts in some biota present in the same system. When I think of it this way, these criteria do not seem
appropriately protective. In my view, the water column continuous exposure criteria should be adjusted
downward which would then translate to a lower tissue-based criteria which might be more reasonable.
Although, as mentioned, another protective approach would be to use something like the 25th percentile
BAF or something other than the mean. That said, at least in many cases fish tissue monitoring occurs on a
yearly basis so there is some potential for the criteria to be reasonably assessed against environmental data.
It is still possible that fish tissue concentrations could be exceeded every year and this be missed by
monitoring efforts. Nonetheless, because tissue concentrations are an intergrative measure and because
many monitoring programs probably do measure fish every year, this is a better match than the water
column criteria.

When we consider the acute and chronic water column criteria, the frequency and duration elements are
protective, in my opinion. The problem is that nobody knows if the criteria for acute toxicity are exceeded
for more than 1 hour or whether the chronic criteria was exceeded for more than 4 days - this extent of
temporal resolution (hourly concentrations or 4-day running averages) just does not exist. So while I agree
that conceptually, the frequency and duration elements definitely would add to the protection of aquatic
life...I just don't see how these can be implemented or regulated. Perhaps EPA is aware that my concern is
not warranted because in relevant circumstances, appropriately timed environmental data are obtained.

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8) Please provide any additional technical comments that you believe should be considered.

RESPONSE: Specific comments to the various elements of the PFOS AWQC are above. Here, I want to
suggest that EPA revisit the 1985 Guidelines and publish either an updated version or an amendment. Basing
critically important criteria on documents published in 1985 and then using this to justify decisions seems
like it would not pass muster in the scientific community. I've had papers rejected because they did not
include enough recent citations, for example. Moreover, I've mentioned my concerns with the 4- most
sensitive taxa + linear regression for criteria derivation. No paper I've read on generating the 5th percentile
most sensitive species has used this approach. Granted, I may have missed them but my sense is that it is
more common to use a full SSD. It would be helpful, for example, if the revised Guidelines explored this
further or other means of criteria development (including new approach methods) and published, used, and
cited and updated guidelines document. I'd like to think we still generally lead the world (more or less) in
environmental protection so having an updated document would be welcomed.

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