2021.11.30_CCO.p14_DEQ comments Eco Protocols

Appendix A: Comments on Eco (Aquatic) Protocols

Comments Responsiveness Table
DEQ comments to Chemours on 3/9/21 Chemours response to DEQ comments on
5/28/21
DEQ Comments 9/16/21 Outcome of 10/14/21 discussion
DEQ reviewed the following aquatic (eco)
toxicology master protocols received in
December 2020 from Chemours pursuant to
paragraph 14 of the Consent Order.
 Algae: 96-hour toxicity test with the
freshwater alga (Raphidocelis
subcapitata) OECD 201; EU Directive
92/69/EEC, Method C.3.; EPA OCSPP
Number 850.4500
 Daphnid acute: 48-hour static acute
toxicity test with the cladoceran
(Daphnia magna) OECD 202, EPA
OCSPP 850.1010
 Daphnid chronic: semi-static life-cycle
toxicity test with the cladoceran
(Daphnia magna) OECD 211, EPA
OCSPP 850.1300
 Fish acute: 96-hour static acute toxicity
test with the fathead minnow
(Pimephales promelas) OECD 203, EPA
OCSPP 850.1075
 Sediment 10-day not yet received.
Chemours Responses to DEQ Comments on
Aquatic Toxicology Protocols

Chemours submits the following responses to
DEQ’s March 9, 2021 comments on the aquatic
toxicology protocols. DEQ’s comments are
copied below, followed by Chemours’s
responses in orange font. Chemours has not
responded here to DEQ’s requests for protocols
for additional toxicology studies that are not
among those required by the Consent Order.

As noted in many of Chemours’s responses
below, in response to DEQ’s requests, Chemours
has engaged with the lab and the lab will use
the EPA-821-R-02-012 and EPA-821-R-02-013
guidelines. Chemours intends to submit updated
protocols to DEQ for approval, incorporating
these guidelines and other requested
modifications. Once DEQ approves the
protocols, it is important for the studies to move
forward on the schedules controlled by the lab.

Chemours submitted a master protocol on 7/14/21 for the
sediment method and then submitted revised master
protocols on 8/19/21 for Ceriodaphnia acute, Ceriodaphnia
chronic, and Fish acute methods. Comments on these
master protocols are provided below.

Chemours has not submitted a revised master protocol for
the algae method. Please see DEQ comments from 3/9/21
and below, and submit a revised master protocol that
addresses DEQ’s comments.
DEQ requested revised master protocols to
include the information throughout this
Comments Responsiveness Table.
1) Comments on toxicity study work plans: DEQ has previously provided comments on the toxicity study work plan in a letter dated 5/03/19 (RE: Comments on Draft Plan for Toxicity Studies) and an email dated
12/17/19 (RE: Toxicologist Meeting Minutes). These expectations still apply. Chemours has requested to submit components of the toxicity study work plan and supporting information separately. DEQ has agreed to
receive and review these items separately, but reminds Chemours that each of the following items are needed before DEQ can approve the final studies:
1(a). Revised master protocols must be provided
to DEQ with requested modifications and/or
additional supporting information. DEQ will
review and approve these protocols to enable
development of the compound specific
protocols.
Chemours’s Response: Chemours will resubmit
the protocols after revision with the additional
information.

Thank you

1(b). DEQ is requesting that Chemours submit
any previously existing toxicity information
available on these compounds. If toxicity data
for these compounds and these species do not
already exist, then DEQ requires that range-
finding toxicity tests be conducted.
Chemours’s Response: Chemours is not aware
of any substance-specific aquatic toxicity data
on these substances. Range finding studies are
part of the EPA-821-R- 02-012 and EPA-821-R-
02-013 guidelines.
After range-finding studies are conducted, Chemours should
provide DEQ with results from those studies along with the
proposed compound specific protocols.

Once the range-finding studies are complete, Chemours will
submit compound-specific protocols to DEQ for review and
approval. Please confirm that the dosing levels in the
compound-specific protocols will be as follows: the acute
studies must be designed to bracket EC50 values and chronic
studies must be designed to bracket LOEC and NOEC values.
State these targets in the compound-specific protocols as
well.
Eurofins will work with client to determine proper
dose ranges.

 Eurofins informed DEQ that there is a brief
time (2 weeks) between the Range Finding
experiments and when the main
experiments are started where they show
the clients the data and discuss the proper
concentrations; this time is also used to
complete the analytical method verifications
if needed.
 Eurofins confirmed DEQ could be privy to
that range finding data and be included in
the conversation with the understanding
that there is not an extensive amount of
time for review.
 DEQ emphasized that this is a priority and
will review as quickly as needed.

It has already been agreed upon that DEQ will
review and approve the compound specific
protocols along with range finding results prior to
initiation of the final studies. If Chemours
proceeds with the final studies without DEQ
approval of protocols and then Chemours runs
the risk of having to repeat the work. Chemours
should give DEQ at least 2 weeks’ notice prior to
sending range finding results and the compound-
specific protocols so that DEQ can block off time
to review in our schedules.
1(f). A work plan with key milestones and
approval status for each of these requested
items must be provided to DEQ for approval
prior to the commencement of the final studies.

Chemours’s Response: A work plan with key
milestones will be provided prior to the
commencement of the final studies but is not
appropriate to be subject to DEQ approval
because the lab controls the schedule. The lab
will only schedule a study once the protocol is
approved. Chemours will share the schedule as
soon as it is available after protocol approval.
Chemours will request “next available study
slot” following protocol approval.
Chemours should provide a work plan as one of the
supporting information documents.

Chemours had expressed concern about the timeline and the
impact it will have on DEQ’s ability to review and provide
feedback on the compound-specific protocols. However,
Chemours has not provided a study schedule for the EcoTox
studies, which was required by the Consent Order. Chemours
should define the study schedule and timeline in the work
plan, including points in the schedule when all protocols and
Eurofins committed to put together a timeframe
after the meeting where they will provide DEQ
time to evaluate the results of range finding study
before proceeding with the test if we agree on a
quick turnaround.

Eurofins provided the tentative range finding
schedule:
Algae, Fish and Cerio cute (Jan-Feb 2022);
Sediment (Mar 2022); Cerio Chronic (July 2022)

supporting information will be provided to DEQ for review and
approval.

If the lab is not able to break up steps in their work schedule
and report back on range-finding and analytical chemistry
development phases prior to starting the final toxicology
studies, then Chemours should ask the lab to provide a
written justification explaining this.

Chemours has asked to submit components separately, such
as the protocols and supporting information. DEQ has agreed
to receive and review these items separately, but reminds
Chemours that each of the items are needed for to DEQ
review before DEQ can approve the final compound-specific
protocols. Chemours may submit these ahead of time so that
DEQ can begin the review process. DEQ also recommends
that Chemours include a checklist in the work plan so that
DEQ and Chemours can track the progress of all requested
supporting information documents.

Eurofins’ range finding test concentrations will be
worked out with the manufacturer based on
weight/volume (test concentrations will be in
mg/L instead of by % concentration)

When the range finding test is complete, Eurofins
will discuss results with client while performing
analytical method validation. Eurofins will begin
definitive toxicity testing following completion of
analytical method validation (~3 weeks from start
of range finding studies)

Eurofins stated that the validation method report
can be a stand-alone study or can be embedded
in one of the studies. Eurofins can provide this
report a priori.
1(c). The final studies will be described by the
compound specific protocols, and these must be
reviewed and approved by DEQ. Compound
specific protocols will be the same in every way
as the master protocols except for compound
specific information like chemical name, CAS#,
and dose levels to be administered. Acute
studies must be designed to bracket EC50
values and chronic studies must be designed to
bracket LOEL and NOEL values. Additional work
plan comments were provided in the 5/03/19
letter from DEQ. Range finding results and other
data used to develop dose levels targeting these
response levels must be provided before DEQ
can approve the dose levels for the final studies.
Chemours’s Response: For acute aquatic work,
Chemours notes that it is not practical to have a
substantial delay between the range finder and
the main study. This is to minimize potential
changes in the test system apparatus (tanks,
tubing, animal batch, analytical equipment) that
could occur over time. The available time
between the range finder and the main study
will be defined by the contract laboratory in the
study schedule. Chemours will share the results
of the range finder study with DEQ as well as the
recommendations on main study doses from the
Study Director as soon as they are available.
Chemours welcomes input from DEQ on the
dose level selection, but the response must
occur within the window defined by the contract
laboratory’s study schedule.

Chemours notes that the scientific literature
suggests that per- and polyfluorinated materials
are not acutely toxic to these trophic levels, DEQ
has required an additional dose group over the
standard protocol to maximize the dose
response curve, and the performing laboratory is
well-versed in setting dose levels for these study
types. Because of these facts, Chemours
believes selecting main-study doses should be
straight-forward and easily completed in a short
time.
If Chemours proceeds without DEQ approval of compound
specific protocols, then they run the risk of having to repeat
any work that is not satisfactory to DEQ.

To expedite the review process, the following list of
individuals should be copied when submitting time sensitive
EcoTox documents to DEQ.
Frannie Nilsen [e-mail]
Amy Risen [e-mail]
Chris Ventaloro [e-mail]

Lorimar Henning [e-mail]
Julie Woosley [e-mail]
1(d). Analytical chemistry methods and reporting
limits for each media to be analyzed for the
toxicity studies must be reviewed and approved
by DEQ for this work. DEQ is outlining
requirements for describing modified methods
in a separate document and will provide this
shortly. Information provided should be
consistent with guidelines in Appendix 1.
Chemours’s Response: Analytical methods will
be developed for these assays only after
protocol approval. In general, the workflow for
contracting a study starts with agreement on the
protocol. Once agreement is reached, the
contract lab will schedule all phases of the
study. This includes method development
(including the
development of the analytical method), range
finder studies, and the main study. The
analytical methods are a separate report that
Chemours will supply for review and approval
prior to the study start.
Chemours should refer to the document called “Guidelines
for Submittal of Reference Materials and Modified Methods”,
which was submitted via email on 4/07/21 and provide all
requested information about the methods that will be used.
Even if Chemours has provided parts of this information in
the past for other projects, the full set of requested
information should be provided again for the EcoTox studies.

DEQ encouraged Chemours and Eurofins to
develop all analytical chemistry supporting
information on this page as soon as possible and
to provide to DEQ before the end of the range-
finding studies.
Chemours should prepare a list of all supporting
information documents and begin providing them
to DEQ ASAP. The analytical work may require
additional review and feedback to Chemours, and
it is not likely that there will be sufficient time to
review these items between the range-finding
and final studies.

Chemours is reminded that if they proceed with
the final studies without DEQ approval of
protocols and all supporting information then
Chemours runs the risk of having to repeat the
work.
1(e). PFAS reference materials used for these
toxicity studies are not commercially available
and additional information must be provided to
DEQ:

1(e)(i). Describe production and handling of all
batches of reference material to be used.
Samples from each batch must be made
available to DEQ for independent analysis.
Chemours’s Response: None of the five
compounds being studied are commercially
available in quantities sufficient for the studies.
As such, Chemours synthesized four of the
compounds using starting compound material
taken from the appropriate commercial
process(es). The fifth, PFMOAA, was custom
synthesized for Chemours by ExFluoro Research
Corp. of Round Rock, Texas. The substances
may be stored in a controlled environment at
room temperature. Samples can be made
available to NCDEQ.
Chemours should provide a supporting information document
for the reference materials. This should include a list of all
batches of material that will be used for these studies, a
description of their production and handling, preparation and
expiration dates, unique identification numbers for lots or
batches, and CoAs for each.
Chemours chemist was not present at the
meeting but will address this in the protocols.
Chemours’ Consent Order Progress Report for First Quarter
2021 stated that “One substance required additional
purification; that purification is currently on‐going and
expected to finish in the second quarter of 2021”. Please
identify which substance this is and provide documentation
on the additional purification.
The compound that required additional
purification was Nafion By-product 2. There was a
high concentration of an unnamed contaminant,
Chemours’ chemists are working to removing it.

DEQ requests the documentation on the
additional purification when the chemist
completes the work.
1(e)(ii). A third-party laboratory must
characterize the reference materials as
described below and provide information to DEQ
and laboratories contracted for the toxicity
studies.
Chemours’s Response: Chemours has
characterized these samples by a combination
of multinuclear NMR spectroscopy, IR
spectroscopy and GC & GC/MS spectroscopy (of
the volatile methyl esters, when necessary). Of
CoAs should be provided to DEQ with the supporting
information document for the reference materials. Chemours
should provide all of the information requested here and in
the “Guidelines for Submittal of Reference Materials and
Modified Methods”.
Shawn confirmed that the LC/MS/MS method is
primary, and the NMR is secondary. Chemours
chemist was not present at the meeting but will
address this in the protocols.

these techniques, multinuclear NMR is the most
valuable in the indubitable structural
identification. Chemours can provide samples to
NCDEQ for confirmation.

Characterization of the material should include LC/MS/MS
analysis, which is a useful and more sensitive method of
determining purity than NMR.

Available NMR data should be submitted as well. NMR is
useful for qualitative determination of a substance as
mentioned in Chemours response. NMR is also useful in
purity determination, but as a second confirmatory technique
with LC/MS/MS as the primary technique.
1(e)(ii)(1). Certificate of analysis including but
not limited to: identity (including isomer
structure), purity, and composition. Information
provided should be consistent with guidelines in
Appendix 1.
Chemours’s Response: COAs of the test
substances will include a test substance name
which will specifically identify the isomer. Also
included will be sample composition and
concentration of constituents.

1(e)(ii)(2). Homogeneity and stability tests must
be conducted for each compound in all solvents
and stock solutions that will be used in the
toxicity studies. Work by other investigators
showing potential for instability in certain
solvents should be reviewed (Liberatore et al.
2020).
Chemours’s Response: Homogeneity and
stability tests will be run as part of method
development for each specific study. It is not
feasible to do stability and homogeneity tests in
all solvents and stock solutions as independent
experiments.

Knowledge of long-term stability is crucial. If it is not practical
to conduct homogeneity and stability tests for all solutions,
then itemize them and explain cases where this is
unnecessary.

Work by other investigators shows the potential for instability
in certain solvents. Chemours should provide this research
paper to all contract labs conducting this work and ask them
to review it (Liberatore et al. 2020).
Chemours does not believe that this information
is necessary, because it is built-in with each
study.
DEQ asked that all analytical chemistry
information be provided as soon as possible as it
may require additional review and feedback from
DEQ.

Chemours agreed to provide the Liberatore study
to staff at all contract labs contributing to this
work.

1(e)(iii). DEQ is outlining requirements for
characterizing reference materials in a separate
document and will provide this shortly.

(No response provided) As further discussed in other comments, Chemours should
refer to the document called “Guidelines for Submittal of
Reference Materials and Modified Methods”, which was
submitted via email on 4/07/21.

2) Comments on protocols
2(a). General comments on protocols proposed:
2(a)(i). As specified in EPA-821-R-02-012,
Subsection 4.7.2 "... the testing laboratory must
perform at least one acceptable reference
toxicant test per month for each toxicity test
method conducted in that month (Subsection
4.15). If a test method is conducted only
monthly, or less frequently, a reference toxicant
test must be performed concurrently with each
effluent toxicity test." DEQ requests submission
of the lab’s in-house reference toxicant testing
for all the test protocols and also submittal of
the reference toxicity test information that is
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline. Chemours
will ask the lab if it is able to share this
information.

When submitting revised protocols, Chemours should confirm
in the protocol what their plans are for reporting the
following: reference toxicant testing during the main study,
historical in-house reference toxicant testing for all the test
protocols, and reference toxicant testing information that is
provided by the biological supplier from which all test species
are purchased.

The only species they expect to obtain from a
vendor is Lumbriculus. Fish, Ceriodaphnia and
algae are maintained in-house.

Fish are maintained as brood stock and
Ceriodaphnia are maintained as mass cultures.
For this testing Fish will be transferred to
spawning chambers and Ceriodaphnia culture
boards will be started.

RTTs are only done routinely for Daphnia magna
as that comprises the bulk of their testing. Fish

provided by the biological supplier from which all
test species are purchased.
and Ceriodaphnia RTTs are only completed when
testing occur.

No historical RTT for any of the species DEQ is
requesting. Eurofins will be performing RTT with
each test performed.

DWR has asked Eurofins to develop a CuSum
chart for Ceriodaphnia dubia with a minimum of
5 tests (three RTTs will be performed prior to the
range finding test, one with the range finding test
and the 5th with the final test).
Eurofins has committed to look at their schedule
to see if they can accommodate the request of 5
RTTs.

It was not determined in this discussion what
historical data would be made available for
Lumbriculus worms. Eurofins should propose
additional historical RTT data to provide in the
revised protocols.
2(a)(ii). In the different protocols, the word
“indiscriminately” is being used instead of
randomly. EPA-821-R-02-013, Subsection
9.4.4.1 states that “Statistical independence
among observations is a critical assumption in
all statistical analysis of toxicity data. One of the
best ways to ensure independence is to properly
follow rigorous randomization procedures.
Randomization techniques should be employed
at the start of the test, including the
randomization of the placement of test
organisms in the test chambers and
randomization of the test chamber location
within the array of chambers...” Examples of
randomization procedures are using a template
for randomization or by using a table of random
numbers as stated in Subsection 11.3.4.5.1.
DEQ is asking for a more in-depth explanation of
what “indiscriminately” means.
Chemours’s Response: EPA-821-R-02-013 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Protocols were updated by replacing the word
“indiscriminate” with the word “random”. Please update the
protocols to provide more description of what this means.
One option would be to describe the randomization
procedures used by the lab. Another option would be to state
the importance of using randomization techniques and to
reference guidelines.

Please add that weak looking organisms are removed and
replaced with more fit individuals before test start.

Eurofins explained that indiscriminate means
pooling organisms in a dish vs randomization.
They will start randomizing cups as per EPA
method.

Eurofins stated dead and weak organisms will be
removed.

2(a)(iii). Each proposed protocol states that
changes to protocols and final reports will be
made in the form of an amendment, signed by
the Study Director, and approved by the
Sponsor. Chemours must provide such
amendments to DEQ for review and approval.
Changes to the protocols are to be approved by
DEQ before starting the experiments and
changes to the reports are to be approved by
DEQ prior to completing the writing of the final
report.

Chemours’s Response: Seeking DEQ approval is
not practical in all cases for the reasons
discussed below. However, all protocol
amendments that are generated during a study
are included in the final report so there is a clear
record of any changes made during a study.

Protocol amendments that impact the design of
a study rarely occur between protocol approval
and initiation of the study. In the unlikely event
this happens, Chemours will seek approval of
the change from DEQ. Chemours will inform DEQ
of protocol amendments that do not alter the
study design (for example, correcting of
typographical errors and similar) as they occur.

Some protocol amendments address situations
that require an immediate decision, particularly
where vertebrate animals are involved. In these
cases, the Study Monitor (Chemours) will
consider the advice from the Study Director
(subject matter expert at the contact laboratory)
and act. Chemours will inform DEQ of the
amendment but may not be able to wait for
approval. All amendments are included in the
final report so there is a clear record of any
changes that occurred during the study.

Changes to a final report are also rare. Should
the need to amend a final report occur,
Chemours will share that amendment with DEQ
for approval prior to re-finalization.

Chemours asks DEQ to identify a single point of
contact who has the authority to receive
amendments and approve them if needed.
If Chemours proceeds with any changes to protocols without
DEQ approval, then they run the risk of having to repeat any
work that is not satisfactory to DEQ.
To expedite the review process, the following list of
individuals should be copied when submitting time sensitive
EcoTox documents to DEQ.
Frannie Nilsen [e-mail]
Amy Risen [e-mail]
Chris Ventaloro [e-mail]
Lorimar Henning [e-mail]
Julie Woosley [e-mail]

2(a)(iv). DEQ has a preference for following
guidelines within EPA 821-R-02-012 and EPA
821-R-02-013 where feasible in order to be
consistent with NC Whole Effluent Testing
guidelines.
Chemours’s Response: In response to DEQ’s
request, Chemours has engaged with the lab
and the lab has confirmed that it can use these
guidelines instead.

Thank you

2(a)(v). New comment. Data Collection & Analysis Chemours should update all the master protocols to state
that the following will be conducted:

The biological data collection section should be updated to
state that “General observations of appearance and
behavior, such as erratic swimming, loss of reflex,
discoloration, excessive mucus production, hyperventilation,
opaque eyes, curved spine, hemorrhaging, molting, and
cannibalism, should also be noted in the daily record.” as
stated in the EPA methods.

Provide a tabular indexed summary of all biological
measurement metrics (i.e. general observations in the daily
record and study endpoints).

Provide descriptive statistics (mean, range, standard
deviation) for each parameter and treatment variable of each
protocol (i.e. temperature, water quality, etc.).

Provide the NOEC, LOEC, EC10, EC25, and EC50 for each
endpoint (where the effect concentrations (ECs) may include
lethality (LC) and inhibition (IC) as applicable).

Provide details regarding the dose-response curve including
confidence intervals and graphs of the fitted model used for
their calculation, the slopes of the dose-response curves and
their standard error.

The data analysis section should describe methods that will
be used to develop each value. Describe any deviation from
the suggested OECD/EPA statistical methods and rationale
for their change in each protocol if applicable.

Eurofins stated they can add the statement to the
protocol although it mostly applies to the fish.

Eurofins confirmed it will be included in final
reports

Eurofins confirmed it will be included in final
reports

Eurofins stated they will comply with this
expectation, but will depend on the response
data

Noted and complied; will be in the final reports
and can be provided a priori if needed.

Eurofins positively confirmed.
2(a)(vi). New comment. Sample handling Provide a discussion of sample collection, storage, and
processing to be consistent with the special needs for this
class of chemicals. Describe the special needs that will be
addressed for this class of chemicals and reference sources
of information. Include specifications about the materials
that are allowable for sample containers as well as
instructions around which media may be sub-sampled from
the sample container when preparing samples for analysis.
Chemours/Eurofins positively confirmed.

2(b). Algae: 96-hour toxicity test with the freshwater alga (Raphidocelis subcapitata) OECD 201; EU Directive 92/69/EEC, Method C.3.; EPA OCSPP Number 850.4500

2(b)(i). The Raphido protocol states "Test
chambers will be held at 24 ± 2°C under
continuous fluorescent lighting..." The EPA-821-
R-02-013, Subsection 14.10.3.1 states that
"Test flasks are incubated under continuous
illumination at 86 ± 8.6 μE/m2/s (400 ± 40 ft-
c), at 25 ± 1°C, and should be shaken
continuously at 100 cpm on a mechanical
shaker or twice daily by hand...” DEQ is
requesting the protocol be updated to be
consistent with EPA recommendations or that an
explanation be provided for why the alternate
temperature range is proposed.
Chemours’s Response: EPA-821-R-02-013 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Chemours provided a revised master protocol on 9/21/21,
but the EPA-821-R-02-013 method has not been referenced.
Please update the method with the new reference.

The revised method does includes the change from 24 ± 2°C
to 25 ± 1°C. However, not all requested revisions were
made. See additional comments below in this section and
the general comments section 2(a).

Chemours/Eurofins positively confirmed.

2(b)(ii). The EPA-821-R-02-013, Subsection
14.10.3.1 also states that the “Flask positions
in the incubator should be randomly rotated
each day to minimize possible spatial
differences in illumination and temperature on
growth rate. If it can be verified that test
specifications are met at all positions, this need
not be done." DEQ is requesting an explanation
of how the protocol ensures that all flasks are
exposed to the desired light intensity and
temperature ranges.
Chemours’s Response: EPA-821-R-02-013 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

The EPA 821 Methods do not specifically state how to ensure
the incubators have the right light intensity and temperature
range.

Chemours should ask the lab to provide a written explanation
about the lab procedures established to ensure these
parameters are within the desired levels. This written
explanation should be provided to DEQ for review along with
the revised protocol.
Chemours/Eurofins positively confirmed.
2(c). Daphnid acute: 48-hour static acute toxicity test with the cladoceran (Daphnia magna) OECD 202, EPA OCSPP 850.1010
2(c)(i). DEQ is recommending the use of
Ceriodaphnia dubia instead of Daphnia magna
because C. dubia is a more sensitive species of
the two. EPA-821-R02-012 (Subsection 2.1)
states that “D. magna distribution appears to be
absent from the eastern United States (except
for Northern New England) and Alaska”. DEQ
has a preference to use C. dubia, but also has a
preference for using test species that the lab
has expertise with. Please reevaluate species
choice and provide explanation for species
selected.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline. As DEQ
requests, the lab will use C. dubia.

The revised master protocol was received, and additional
comments are below.

2(c)(ii). The Daphnia acute and chronic protocols
are using the well water with a hardness of
~140mg/L as a dilution test water. EPA typically
recommends a hardness of 160-180mg/L as
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
The revised protocol continues to target hardness of 140
mg/L and alkalinity of 180 mg/L. This is considerably higher
than the ranges targeted by NCDEQ. NCDEQ is also
concerned about using alkalinity and hardness levels that are
The 140-180mg/L is the range used as per OECD
method. Eurofins will be using reconstituted
water at 80-100mg/L for the Ceriodaphnia and
fish test. Eurofins is also planning on culturing

CaCO3 for D. magna culturing (EPA-821-R- 02-
012, Subsection 4.2.1), but based on the
language in EPA-821-R-02-012, Subsection
7.1.1.2, it may be acceptable as long the lab is
culturing the organisms in the dilution water
before testing, which the lab is intending to do.
However, if a decision is made to switch to C.
dubia, then the desired range is 80-100mg/L for
reconstituted water and 30-50mg/l for surface
water as stated in the NC rule 15A NCAC 02H
.1111(a)(13). DEQ is recommending that the lab
match species with the recommended hardness
levels in these EPA guidelines. DEQ is
recommending use of C. dubia in order to be
more consistent with what we require for the NC
Whole Effluent Toxicity Program.
and the lab will use that guideline. As DEQ
requests, the lab will use C. dubia.

consistent with how organisms are cultured in this lab.
Please ask the lab to provide a written explanation of their
rationale behind choosing these values. The lab should
include a description of their experience using C. dubia and
the hardness and alkalinity levels that they target routinely
for culturing and testing. This written explanation should be
provided to DEQ for review along with the revised protocol.

Additionally, as per EPA-821-R-02-013, the quality of the
water is extremely important for culturing and testing of
Cladoceran organisms. Therefore, at a minimum of once per
year the water used for culture/testing should be analyzed
for metals and pesticides. The EPA manual provides
guidelines on the acceptable levels of the different analytes
in Subsection 4.4.1. When submitting revised protocols,
Chemours should provide a copy of the metals and pesticide
analysis of the water that will be used for this study.

Ceriodaphnia weeks prior to tests to acclimate to
this water.

Eurofins will send these reports a priori.

2(c)(iii). The daphnia acute protocol states that
“loading in each test chamber during test will be
at least 20 mL of test solution per daphnid”. In
Table 13 of the EPA-821-R-02-012 manual, the
test conditions listed for the acute testing with
D. magna includes the minimum recommended
test solution volume of 25ml. Please provide
comment and explanation for this test solution
volume.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Updates made. Thank you.
2(c)(iv). The daphnia acute protocol states
"Adults will be fed during the 24-hour period
prior to test initiation, but neonates will not be
fed during the test." DEQ is requesting that the
protocol be updated to be consistent with EPA-
821-R-02-012 (Table 13); newly released young
should have food available a minimum of 2 hrs
prior to use in a test".
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Updates made. Thank you.
2(c)(v). The daphnia acute protocol states "Prior
to test initiation, neonate daphnids<24 hours
old will be collected from cultures and
indiscriminately distributed one or two at a time
to transfer containers until each contains its
complement of five daphnids." DEQ is
Chemours’s Response: Chemours will ask the
lab for this information and will provide the
information from the lab to DEQ when the
updated protocols are submitted for approval.

DEQ is requesting additional information about the
randomization procedure for all protocols. Please see
comment 2(a)(ii).

Chemours should also ask the lab to provide a written
description of the measures taken to avoid cross-
Eurofins explained that indiscriminate means
pooling organisms in a dish vs randomization.
Eurofins will randomize cups as per EPA method.

requesting more information regarding the
methodology of obtaining organisms from the
culture. Is the lab doing the pooling or blocking
method? What are the measures taken to avoid
cross-contamination when populating the tests
chambers?
contamination when populating the test chambers. This
written explanation should be provided to DEQ for review
along with the revised protocol.

Organisms are transferred to a K cup and from
there they populate each treatment. A clean
pipette is used in each treatment.

2(c)(vi). The daphnia acute protocol states "The
dissolved oxygen concentration will be ≥3 mg/L
throughout the test." EPA-821-R-02-012
(Subsection 9.14.1) states that "the DO in the
test solution should not be permitted to fall
below 4.0 mg/L for warm water species and 6.0
mg/L for cold water species." This validation
criterion should be set at ≥4 mg/L DO, not 3
mg/L.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Updates made. Thank you.

2(c)(vii). New comment Page 7 discusses the frequency of taking measurements and
says that the temperature, DO, and pH will be measured at
the beginning and at the end of the test. As per EPA 821-R-
02-012, Subsection 10.2.1 the DO, pH, and temperature are
to be measured in the control and all test concentrations at
the beginning of the test, daily thereafter, and at test
termination. Please update the protocol to state that these
measurements will also be taken at 24 hours.

Similarly, the % immobility should be calculated at each
treatment level for both 24 and 48 hours rather than the
‘cumulative % immobility observed in the treatment groups’;
please update the protocol to reflect this.

Please provide additional details regarding the dose-
response curve including confidence intervals and graphs of
the fitted model used for their calculation, the slopes of the
dose-response curves and their standard error. For additional
information on data collection and analysis, see item 2(a)(v).
Chemours/Eurofins positively confirmed.

Chemours/Eurofins positively confirmed.

Eurofins positively confirmed; will be in the final
reports and can be provided a priori.

2(d). Daphnid chronic: semi-static life-cycle toxicity test with the cladoceran (Daphnia magna) OECD 211, EPA OCSPP 850.1300
2(d)(i). DEQ is recommending the use of
Ceriodaphnia dubia instead of Daphnia magna
because C. dubia is a more sensitive species of
the two. EPA-821- R02-012 (Subsection 2.1)
states that “D. magna distribution appears to be
absent from the eastern United States (except
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline. As DEQ
requests, the lab will use C. dubia.

The revised master protocol was received, and additional
comments are below.

for Northern New England) and Alaska". Please
use C. dubia or provide explanation of why using
D. magna instead of C. dubia.
2(d)(ii). Proposed protocol specifies that test will
be initiated with neonates <24 hours old. EPA
821-R-02-012 specifies <24 hours and born
within 8 hours of each other for C. dubia. If C.
dubia is used, add to the chronic protocol that
neonates used will have been born within 8
hours of each other.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline. As DEQ
requests, the lab will use C. dubia.

Page 4 states that “The test will be initiated with neonate C.
dubia < 24 hours old.” Please update the method to state
that neonates will be within 8 hours of each other.
Chemours/Eurofins positively confirmed.
2(d)(iii). The Daphnia acute and chronic
protocols are using the well water with a
hardness of ~140mg/L as a dilution test water.
EPA typically recommends a hardness of 160-
180mg/L as CaCO3 for D. magna culturing (EPA-
821-R-02-012, Subsection 4.2.1) but based on
the language in EPA-821-R-02-012, Subsection
7.1.1.2, it may be acceptable as long the lab is
culturing the organisms in the dilution water
before testing, which the lab is intending to do.
However, if a decision is made to switch to C.
dubia, then the desired range is 80-100mg/L for
reconstituted water and 30-50mg/L for surface
water as stated in the NC Statute 15A NCAC
02H .1111(a)(13). DEQ is recommending that
the lab match species with the recommended
hardness levels in these EPA guidelines. DEQ is
recommending use of C. dubia in order to be
more consistent with what we require for the NC
Whole Effluent Toxicity Program.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline. As DEQ
requests, the lab will use C. dubia.

The revised protocol continues to target hardness of 140
mg/L and alkalinity of 180 mg/L. This is considerably higher
than the ranges targeted by NCDEQ. NCDEQ is also
concerned about using alkalinity and hardness levels that are
consistent with how organisms are cultured in this lab.
Chemours should ask the lab to provide a written explanation
of their rationale behind choosing these values. The lab
should include a description of their experience using C.
dubia and the hardness and alkalinity levels that they target
routinely for culturing and testing. This written explanation
should be provided to DEQ for review along with the revised
protocol.

Additionally, as per EPA-821-R-02-013, the quality of the
water is extremely important for culturing and testing of
Cladoceran organisms. Therefore, at a minimum of once per
year the water used for culture/testing should be analyzed
for metals and pesticides. The EPA manual provides
guidelines on the acceptable levels of the different analytes
in Subsection 4.4.1. When submitting revised protocols,
Chemours should provide a copy of the metals and pesticide
analysis of the water that will be used for this study.

The 140-180mg/L is the range used as per OECD
method. Eurofins will be using reconstituted
water at 80-100mg/L for the Ceriodaphnia and
fish test. Eurofins is also planning on culturing
Ceriodaphnia weeks prior to tests in order to
acclimate to this water.

Eurofins will send this report a priori.
2(d)(iv). New comment Changes were made to the approach for statistical analysis
with specific statistical details removed and replaced with a
citation to the OECD and EPA methods. Please reference the
specific Figures/Sections of the EPA methods that describes
the choice of statistical analysis and rationale for each
parameter (survival and reproduction) if using the citation is
preferred.

Chemours/Eurofins positively confirmed that
these details will be included in the revised
method.

Please add the IC25 and IC50 which are specific to
reproductive toxicity are relevant to this protocol.

See general comments on data collection and analysis, item
2(a)(v).
Chemours/Eurofins positively confirmed.
2(d)(v). New comment Page 5 discusses the test organism but does not account for
all stressors that should be checked for. There is a sentence
starting with “A culture will not be used to provide neonates
for the test if …..” This sentence should be updated to
include ephippia.
Chemours/Eurofins positively confirmed.
2(e). Fish acute: 96-hour acute toxicity test with the fathead minnow (Pimephales promelas) OECD 203, EPA OCSPP 850.1075.
2(e)(i). The fathead minnow acute protocol
states that organisms will be purchased from a
supplier. DEQ notes that hardness of the
shipping water may be lower than the hardness
in the well water (~140mg/L) used as dilution
water. What is the acclimation process and
period used to change the fish from the shipping
water to the lab dilution water?
Chemours’s Response: Chemours will ask the
lab for this information and will provide the
information from the lab to DEQ when the
updated protocols are submitted for approval.
Additional information was not provided with the revised
protocol to explain this. Page 4 continues to state that the
water hardness will be 140 mg/L. DEQ typically recommends
that labs use a hardness of 30-50mg/L for surface waters or
80-100mg/L for synthetic lab water as stated in NC Statute
15A NCAC 02H .1111(a)(13). However, if the supplier
provides fish cultures that have been acclimated to
hardwater conditions, higher values are sometimes used,
and their acclimatization information should be obtained
from the supplier and detailed in the study notes.

Chemours should ask the lab to provide a written explanation
of their rationale behind choosing this hardness value. The
lab should include a description of their experience using and
culturing P. promelas, acclimatization information obtained
from the supplier, and the hardness levels that they target
routinely for culturing and testing. This written explanation
should be provided to DEQ for review along with the revised
protocol.

The 140-180mg/L is the range used as per OECD
method. Eurofins will be using reconstituted
water at 80-100mg/L for the Ceriodaphnia and
fish test. Eurofins is also planning on culturing
Ceriodaphnia weeks prior to tests to acclimate to
this water.

2(e)(ii). The fathead minnow acute protocol
states "Fathead minnows will be held for at least
14 days prior to the test in water from the same
source and at approximately the same
temperature as used during the test." DEQ is
concerned that waiting until the fish are older
(>14 days) the test may miss the more sensitive
life stage. Additionally, keeping fish age at 1-14
days old would be more consistent with what
DEQ requires for the NC Whole Effluent Toxicity
Program. DEQ is requesting that protocols be
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

The protocol was revised and now the lab will be culturing the
fish to obtain larval fathead minnows for testing. Chemours
should ask the lab to provide written explanation of their
culturing process and culturing system. This written
explanation should be provided to DEQ for review along with
the revised protocol.
Eurofins will provide a description of their
culturing facilities and a summary of how larval
fish will be reared from their current brood stock.
This will include a description of breeding tank
set up with mixed sex fish, how eggs will be
obtained, and larval fish will be maintained.

updated to be consistent with EPA guidance as
per EPA-821-R-02-012 (Table 14); the age of
test organisms required for the acute toxicity
test is 1-14 days old with less than or equal to
24-h range in age.
2(e)(iii). The fathead minnow acute protocol
states "Fish will not be fed for at least one day
prior to the test or during the test." DEQ is
requesting that protocols be updated to be
consistent with EPA-821-R-02-012 (Table 14,
and Subsection 9.11.1), which states that "food
is made available to test organisms while
holding before they are placed in the test
chambers."
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Updates made. Thank you.
2(e)(iv). The fathead minnow acute protocol
states "Specifications for acceptable levels of
contaminants in fish diets have not been
established." The EPA-821-R-02-012,
Subsection 4.8.3 provides guidance of the
allowable levels of contaminants in fish food, "If
the concentration of total organochlorine
pesticides exceeds 0.15 μg/g wet weight, or the
concentration of the total organochlorine
pesticides plus PCBs exceeds 0.30 μg/g wet
weight, or toxic metals (Al, As, Cr, Co, Cu, Pb, Ni,
Zn, expressed as total metal) exceed 20 μg/g
wet weight, the food should not be used...”.
Every batch of fish food should be analyzed to
meet these guidelines before using for toxicity
tests.
Chemours’s Response: Chemours will ask the
laboratory for additional information on this
point and will provide the information from the
lab to DEQ when the updated protocols are
submitted for approval.

The updated protocol did not include the required
information regarding acceptable levels of contaminants in
fish food and still say that the ‘acceptable levels have not
been established’. Please modify protocol to include this
information that has been provided by the EPA method
referenced in this protocol – details in first column.
Eurofins will send these reports a priori.

2(e)(v). The fathead minnow acute protocol
states, "The test will be conducted at a target
water temperature of 22 ± 1°C." DEQ is
requesting more information about the culturing
conditions and other factors contributing to this
decision. EPA-821-R-02-012 (Table 14)
recommends the use of either 20±1°C or
25±1°C. A 25±1°C temperature would be more
consistent with what DEQ requires for the NC
Whole Effluent Toxicity Program.
Chemours’s Response: EPA-821-R-02-012 was
not specified initially, but in response to DEQ’s
request, Chemours has engaged with the lab
and the lab will use that guideline.

Updates made. Thank you

2(e)(vi). The fathead minnow acute protocol
states "Prior to test initiation, juvenile fathead
minnows will be collected from holding tanks. To
initiate the test, the fish will be indiscriminately
distributed one or two at a time to the test
chambers until each contains its complement of
ten fish." DEQ is requesting more details on the
measures taken to avoid cross-contamination
when populating the tests chambers, and to
minimize test dilution with water from fish
population. When populating test organisms into
test chambers it is recommended to rinse the
transfer apparatus between the different test
concentrations and minimize the amount of
water introduced into each test chamber while
transferring the tests organisms.
Chemours’s Response: Chemours will ask the
laboratory for additional information and will
provide the information from the lab to DEQ
when the updated protocols are submitted for
approval.

Chemours should ask the lab to provide a written explanation
of the measures taken to avoid cross-contamination when
populating the tests chambers, and to minimize test dilution
with water from fish population. This written explanation
should be provided to DEQ for review along with the revised
protocol.
Organisms are transferred to a K cup and from
there they populate each treatment. A clean
pipette is used in each treatment.

2(e)(vii). Protocol describes several potential
test chambers options, one of which is Teflon-
lined. DEQ recommends not using Teflon due to
potential for contributing PFAS to the test
system.
Chemours’s Response: Chemours will ask the
lab not to use Teflon to the extent other
materials are available.

Please describe the test chambers without a Teflon lining in
the protocol.
Eurofins will use glass beakers or chambers.
2(f). Sediment 10-day:
2(f). Sediment 10-day: Chemours asked DEQ to
identify the specific OECD guideline. Please use
Sediment-Water Lumbriculus Toxicity Test Using
Spiked Sediment toxicity study – OECD 225 and
refer to previous communications about this
method (See 3/27/19 Chemours letter Re:
Work Plan for Toxicity Studies Pursuant to
Consent Order Paragraph 14. See 5/03/19 DEQ
Comments on Chemours’ Proposed
Toxicity Study Work Plan Pursuant to Paragraph
14 of the Consent Order, dated 3/27/2019).
Chemours’s Response: Noted. Thank you.

On 7/14/21 Chemours submitted a proposed master
protocol for the 28-day sediment toxicity study to DEQ for
review.

After reviewing the proposed master protocol, DEQ is
concerned that the methods are not specific enough to be
replicated in the laboratory- which was a requirement of the
master protocol. As such, the following comments aim to
elucidate the missing information. If a more specific bench
protocol exists, please submit that with the revised proposed
master protocol for review.
Details provided below.
2(f)(i). New comment. Experimental Design Please provide an experimental design diagram to improve comprehension of the intricate
experiment proposed, including the number of chambers per treatment, number of
organisms per chamber and per treatment, dose concentrations, and anticipated sample
sizes for each statistical comparison (see details below; 2(f)(v) Statistical Analysis).
DEQ rescinded this request as the explanation of
the experimental details and documents Eurofins
committed to sharing with DEQ both a priori and
in the final report have satisfied the questions
DEQ had about the experimental quality and
design.

The proposed master protocol states that “To control bias, oligochaetes will be impartially
assigned to exposure chambers at test initiation.” and “After preparation, the test chambers
will be impartially positioned in an environmental chamber or water bath designed to
maintain the target test temperature.” NCDEQ requests clarification on the exact meaning
of “impartial”. As per OCED 225(41 and 43) and EPA/600/R-99/064 (Subsection 16.2.2.6)
assignment and positioning of organisms should be randomized. See general comment
2(a)(ii).

Feeding:
The protocol discusses the addition of food (YCT) to the test organisms during the holding
time and the addition of wheat grass to the sediments before adding the overlying water. As
per EPA/600/R-99/064 (Subsection 9.9) NCDEQ is recommending performing compound
analyses on the food to be added during this test.

Eurofins has committed to providing this report to
DEQ a priori.

2(f)(ii). New comment. Materials and Methods – chamber set-up 2(f)(ii)(a). Preparation of Test Sediment:
The OECD 218 method is referenced. Chemours should ask the lab to provide a written
explanation of why the sediment preparation in the chironomid test (OECD 218) was
selected instead of OECD 225.

The method for mixing chemicals into the sediment can have a large impact on the outcome
of the test. The general approach to mixing was provided but it is vague. The revised
protocol should provide more clarification on how the test sediment will be prepared, the
exact equipment to be used, the amount of time mixing will occur, and provide references
for the procedure. In addition, clearly describe how the dose concentrations will be
maintained/verified through overlying water renewal.
Eurofins stated that both are the same method of
preparation and will ensure that the same
method is referenced consistently in such cases.

OECD 225 method does not do overlying water
renewal unless there is a spike in ammonia,
which is rare. For water quality analysis purposes
removal of water is required which is replaced
with RO.

The dose concentrations into the sediments will
be mixed in batches and split into replicates.
Eurofins will describe detailed process in the
revised protocol.

2(f)(ii)(b). Preparation of Test Chambers:
The proposed protocol stated that ”Approximately 600 mL of overlying water will be slowly
added to each test chamber in such a manner as to minimize disturbance of the sediment.”
Please update the revised protocol with more information regarding the practices used to
prevent disturbance of sediments and what is the Corrective Action Plan in place if a
sediment disturbance does occur.

The proposed protocol stated that “…sediment/water mixtures will be allowed to equilibrate
for approximately 48 hours to 7 days prior to adding the test organisms.” However, as stated
on the OECD 255(29) and EPA/600/R-99/064 (Subsection 8.3.2.2.3), this equilibrium time
can be sediment and chemical specific. Since there is a paucity of information regarding
these PFAS, how is the length of time determined to be adequate? Please update the
revised protocol with this information.
Eurofins has agreed to describe detailed process
in protocol.
Briefly, a baffle will be used to pour water to
avoid disturbing sediments. If there is any
disturbance, there will be a 48hr settling time
prior to continuing the experiment.

Eurofins confirmed that there is a spike test
(28days) to check for equilibration.

2(f)(iii). New comment. Materials and Methods – organism details 2(f)(iii)(a). Provide details about the % recovery of organisms that will yield a successful
experiment and provide a reference for the established protocol that the % recovery is
based on.
Eurofins indicated that % recovery is not an
appropriate indicator as the organisms are
expected to multiply and that the better measure
is to look for the required increase mentioned
above.
(c)Test Procedures and Biological Measurement:
The protocol stated that.“All surviving oligochaetes in each replicate at the end of the test
will then be combined by replicate and measured for dry weight.” The dry weight protocol
can introduce bias due to gut content and the Ash-Free Dry Weight (AFDW) procedure is
recommended by OECD 255 (51) and EPA/600/R-99/064 (Subsection 13.3.7). NCDEQ is
requesting that the lab review the AFDW procedure and provide a written explanation for the
procedure they choose.

Eurofins will follow the AFDW.

(d). Conditions for the Validity of the Test:
The protocol states “The average number of living worms per replicate in the controls should
have increased by a factor of at least 1.8 at the end of exposure compared to the number of
worms per replicate at the start of exposure”. Please explain this expectation of 1.8. Is there
an expectation of reproduction and how much mortality is expected?

The proposed study is using organisms from an outside supplier; therefore, NCDEQ is
requesting the lab to perform a 96hr water-only Reference Toxicant Test (RTT) associated
with each batch of organisms received from the supplier to “satisfactorily demonstrate the
sensitivity and condition of the test animals” as stated in the OECD 225 (12) and
EPA/600/R-99/064 (Subsection 9.8.1). Both the 96-hr water-only RTT conducted by the
contract lab and an RTT report from the supplier should be provided to DEQ along with the
results from the final toxicology studies. See comment 2(a)(i) for additional information.

The EPA manual indicates a 90% control survival test acceptability for the 96hr RTT (water-
only test; Tables 9.1, 9.2), is this the guideline that will be followed? Provide rationale and
references if a different guideline will be followed.
Eurofins confirmed that the factor of 1.8 comes
from reproduction. At least 18 worms at the end
of the exposure divided by the total of worms
exposed = 1.8

The only species they expect to obtain from a
vendor is Lumbriculus. Fish, Ceriodaphnia and
algae are maintained in-house.
 It was not determined in this discussion
what historical data would be made
available for Lumbriculus worms.
Eurofins should propose additional
historical RTT data to provide in the
revised protocols.
 DWR has asked Eurofins to develop a
CuSum chart for Ceriodaphnia dubia
with a minimum of 5 tests (three RTTs
will be performed prior to the range
finding test, one with the range finding
test and the 5th with the final test).
 Eurofins has committed to look at their
schedule to see if they can fit this
request.

Eurofins indicated that this is not applicable as
the organisms are expected to multiply which will
skew % recovery calculations.
2(f)(iv). New comment. Materials & Methods – Statistical Analysis (a) Add sublethal endpoints to the statistical design. For example, the protocol stated that
“Test chambers will be observed daily and visual assessments of any abnormal behavior
(e.g. sediment avoidance/leaving sediment, fecal pellets visible or not visible on the surface
of the sediment) will be recorded.” NCDEQ is requesting that these behavioral sublethal
effects be evaluated as endpoints and not just recorded.

(b) The protocol states “The no-observed-effect-concentration (NOEC) and lowest-observed-
effect-concentration (LOEC) will be determined by visual interpretation of the dose-response
pattern and statistical analyses of the data.” Please use a more specific approach and
describe in detail.

Eurofins will evaluate sublethal endpoints
whenever possible. They stated that most
sublethal responses are difficult to observe as
the organisms remain mostly in the sediment.

(c) The goal of the statistical tests needs to be clearly stated, as that would determine which
primary and post-hoc tests should be used. The protocol should be revised to state each
goal (endpoint and type of effect concentration) to be determined and clearly explain the
data that will be compared (including how many data sets will be assessed in each
evaluation) and the associated statistical methods that will be used for each step. The
experimental design figure should be referenced in explanations.

(d) See general comments on data collection and analysis, item 2(a)(v).
3). Additional protocols requested
• CRITICAL NEED - Bioaccumulation in Fish:
Aqueous and Dietary Exposure – EPA (OCSPP)
850.1730; This is study critical for DEQ to
establish water supply and fish consumption
surface water standards. PFAS do not
accumulate using the same mechanisms as
other organic chemicals, so modeling is not the
preferred approach. Paragraph 14 of the
Consent Order states that this testing should be
“… sufficient to aid in development of surface
water and groundwater regulatory standards…”
Chemours has not responded here to DEQ’s
requests for protocols for additional toxicology
studies that are not among those required by
the Consent Order.
These studies are being requested by DEQ because they are
needed for development of health-based standards for
surface water.

DEQ asks that Chemours makes reference material available
to DEQ and other researchers to conduct additional work to
support regulatory development.

DEQ reserves the right to ask for additional studies as we
review and identify important endpoints to investigate and
obtain feedback from the public.
Chemours toxicologist (Shawn) has confirmed
that Chemours can provide reference materials in
the scope of grams and not kilograms. DEQ will
confirm with external partners regarding weight
of reference material needed and contact Shawn
to continue the conversation and obtain the
reference material.
• Fish Early Life-Stage Test – EPA (OCSPP)
850.1400; Using Pimephales promelas; 28-day
study focused on embryonic through early
juvenile life-stage effects.
• Fish Sexual Development Test – OECD 234;
60-day study focused on endocrine disrupting
effects.
• Amphibian Metamorphosis Assay – OECD
231; 21-day exposure study provides
information on development in tadpoles. This is
important to consider as amphibians are often
underrepresented in ecotoxicity studies.
• Empirically derived chemical characteristics
including: half-life in water, octanol:water
coefficient (logP)
4. References
DEQ letter to Chemours 5/03/19. “Comments on Draft Plan for Toxicity Studies” Accessed via
https://files.nc.gov/ncdeq/GenX/consentorder/paragraph14/05032019-Holman-to-Long-Letter-Re-Comments-on-Draft-Toxicity-Study-Paragraph-14-of-CO.pdf

Liberatore, H., S. Jackson, M. Strynar, and J. McCord. 2020. Solvent Suitability for HFPO-DA (GenX” Parent Acid) in Toxicological Studies. Environ Sci Technol Lett.
18;7(7):477-481

USEPA. 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. U.S.
Environmental Protection Agency, Washington, DC 20460. EPA-821-R-02-012.

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. U.S.
Environmental Protection Agency, Washington, DC 20460. EPA-821-R-02-013.

USEPA 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Second Edition. U.S.
Environmental Protection Agency, Washington, DC 20460. EPA-600-R-99-064.

Appendix B: Example Workflow Diagram, Timeline, and Checklist

Appendix C: Documents that Eurofins committed to providing to DEQ a priori

1. Analytical method validation report
2. Details regarding the dose-response curve including confidence intervals and graphs of the fitted model used for their
calculation, the slopes of the dose-response curves and their standard error.
3. Results of annual culture/testing analysis for metals and pesticides.
4. Reports regarding acceptable levels of contaminants in fish food used at Eurofins.
5. Report detailed the compound analyses on the YCT sediment organism food