2019.04.10_CCO.p14_CommentsToxicityTestProtocolsFINAL

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Comments on Chemours’ Proposed Toxicity Study Work Plan Pursuant to Paragraph 14 of the Consent
Order, dated 3/27/2019
Prepared by: Sandra L Mort, PhD, DEQ Environmental Toxicologist
April 10, 2019

General Work Plan comments
1. The objective of this effort is to define a range of statistically significant sensitive adverse effect
responses for each chemical and assay, i.e. dose-response or concentration-response
characterization. No-effect levels or lowest effect levels (NOEAC, LOAEC) alone will not meet the
objectives of the prescribed toxicity testing program.
1.1. NOAEC, LOAEC, 50% response levels and multiple other effect concentrations (i.e., EC10,
EC20, EC80), as specified by DEQ, are to be reported.
1.2. Response statistics are to be performed using appropriate software. Concentrations –
response plots are to be included for each reported endpoint. Concentrations–response
plots manually plotted on graphical paper are not an acceptable data reporting method.
1.3. Ideally, the highest test concentration should result in 100% effect response and the lowest
concentration no observable effect (or less than 20% effect response). Not less than 5 test
concentrations are recommended for all assays, where 5 or less treatments are specified in
the method.
2. All test organism cultures, toxicity assays and associated procedures (e.g., culture and test
environmental conditions, analyses of test solutions, statistical analyses and criteria) are to
satisfy the minimum specifications, performance criteria, quality assurance and quality control
requirements as specified in the referenced OECD, OPPT, USEPA or other method references, as
well as meeting established GLP protocols.
2.1. This includes not less than the minimum number of exposure (treatment) concentrations
and replicates per treatment concentrations and controls selected with the objective to
provide a range of effect levels suitable for dose/concentration-response modeling
(benchmark dose modeling).
3. Data that does not provide a dose/exposure-response suitable for modeling may require re-
testing.
4. Due to the unique physicochemical properties of the test chemicals (PFAS) the concentration of
the test substance should be measured, at a minimum, at the highest and lowest test
concentration, at the beginning and end of the test. Control solutions are to be verified as PFAS-
free for at the beginning and end of the test. PFAS measurements are to be completed using
DEQ-approved analytical methods and obtain DEQ-approved PFAS sensitivity criteria. Less than
±20% variation is required.
5. Confirmation that all test apparatus is PFAS-free is required.
6. Test data reporting is to meet at a minimum the specifications in each method, with individual
treatment or replicate data recording sheets made available at DEQ’s request.
7. Assays reported to DEQ are to include the most recent quality control testing of the test
organism cultures, culture media and test media.
8. For mortality endpoint tests (acute) an additional observation at 6-8 hours after organisms are
exposed to the test and control solutions (i.e., at the end of the work day on the day of test
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initiation) is recommended. At all observation periods report any observed mortalities or other
observations of abnormal behavior or effects.

Comments on Specific Assay Specifications noted by Chemours
A. Mammalian Studies – no additional comments
B. Ecological Studies –
a. Algal Acute Assay –
i. Limit tests are acceptable only at the solubility limit of the specific PFAS in the
test-specific medium, derived using appropriate methods approved by DEQ. The
test solutions must be verified by analysis at the end of the exposure period. A
minimum of 6 replicates are required for each treatment and controls.
ii. Recommended test organism – Freshwater algae Pseudokirchneriella
subcapitata, (formerly known as Selenastrum capricornutum)
b. Daphnia Acute Immobilization Assay –
i. Reported effect-levels (immobilization) at 24 and 48 hours are to include EC10,
EC20, EC50 and EC80.
ii. Lethal effect concentrations are to be reported for the same response levels and
exposure periods as indicated for immobilization.
iii. Behaviors, adverse effects or visible abnormalities in addition to immobilization
are to be reported for all treatments/replicates at the prescribed exposure
durations.
iv. Limit tests must meet the specifications in the referenced test method
(including minimum number of test organisms) and be conducted at the
solubility limit of the specific PFAS in the test-specific medium. The solubility
limit must be derived using appropriate methods approved by DEQ.
c. Fish Acute Assay
i. Limit tests must meet the specifications in the referenced test method
(including minimum number of test organisms) and be conducted at the
solubility limit of the specific PFAS in the test-specific medium. The solubility
limit must be derived using appropriate methods approved by DEQ.
ii. Recommended test species is Oncorhynchus mykiss (rainbow trout) or
Pimephales promelas (fathead minnow). All assays for all PFAS are to be
completed using a single species throughout these investigations, unless
otherwise specified by DEQ.
iii. A minimum of 10 fish per treatment and controls is recommended to provide
additional statistical power.
d. Daphnia magna Reproduction Assay
i. Control and any one treatment accidental mortality of the parent animal shall
not exceed 20%.
ii. The concentration of the test substance should be measured, at a minimum, at
the highest and lowest test concentration and controls, at the beginning and
end of the test and at each media change-over. Less than ±20% variation is
required.
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e. Sediment-Water Lumbriculus variegatus Toxicity Test Using Spiked Sediment
i. The concentration of the test substance in the lowest and highest test sediment
concentrations is to be measured after sediment spiking at the beginning of the
test.
ii. The test substance concentration in the lowest and highest concentration
sediment and water phases individually are to be determined at the end of the
equilibration phase and at the end of the 28-day exposure, or at the end of the
exposure period as dictated by organism mortality.
iii. The most recent reference toxicity test data is to be reported with the PFAS
assay data to document the ability of the testing lab to generate data of high
quality and a consistent response of test organisms.
iv. The laboratory must document the suitability of the un-spiked sediment
substrate to provide a suitable environment for the test organism culture
survival and reproduction.
v. 6 replicates are recommended for the control.
C. Dose Selection
a. DEQ agrees that development and pooling of the full mass of test substance needed to
complete the specified assays is a logical approach to eliminate lot-to-lot variations and
artificial response variability.
b. DEQ does not agree with the proposed modified 2-test concentration plus controls test
design. This test design will not provide the desired dose/effect-response data needed
to adequately characterize the toxicity of the individual PFAS to the individual test
organisms and provide the data desired for regulatory development. As specified above,
the multi-test treatment design designated in each of the individual assays is to be
followed, with not less than 5 treatment concentrations, or the minimal number
specified in each assay if that minimum number of treatments (not including the
controls) is more than 5.
c. DEQ urges Chemours pursue contract services for the development of PFAS test
material if this will generate the needed volume of test material more quickly that the 1-
year period anticipated for Chemours to generate the material.
D. Work Plan Schedule
a. DEQ urges that test protocol design and submittal to DEQ for approval be initiated as
soon as possible to facilitate initiation of the individual PFAS assays as soon as adequate
testing material is available for the Tier 1 assays. Protocol design should not be delayed
until the test material has been generated.
E. Mammalian Studies
a. See comment above regarding acquiring assay protocol design approval from DEQ in
the interim period during which the PFAS test materials are being generated, so that the
assays can be initiated soon (1-3 months) after the pooled material is received.
F. Ecotoxicology Studies
a. Six months to complete the 72-hour algae and fish acute assays seems excessive. Three
months would seem to be more than adequate time to complete this work, with an
additional 3-4 months for the Daphnia reproduction assays.
G. Estimated Timeline
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a. As noted above, DEQ urges Chemours or their contractor to proceed with test design
and submittal to DEQ for approval as soon as is feasible to allow for initiation of the
toxicity assays to begin as soon as possible (within 1-3 months) after the pooled
individual PFAS test material is available.
b. DEQ would urge Chemours to complete reporting of all rodent study data to DEQ no
more than 24 months after receiving the test material.