SRC TR

- 15

- 051

HPV2 TEST RULE DATA ADEQUACY REVIEW ( FINAL ):

ACUTE TOXICITY TO FI SH, ACUTE TOXICITY T O DAPHNIA

,

AND TOXICITY TO ALGA E

Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1

Prepared by:

Megan Nelson, William Richards, Jennifer Rayner, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Cynthia Bowie, Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

August 27

, 2015Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 2524

- 04

- 1

i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1

B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 4

D

- 1. Acute Toxicity to Fish

................................ ................................ ................................ ....... 4

D

- 2. Acute Toxicity to Aquatic Invertebrates

................................ ................................ .......... 11

D

- 3. Toxicity to Aquatic Plants

................................ ................................ ............................... 18Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

- 04

- 1

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates aquatic toxicity tests of CASRN 2524

- 04

- 1 (phosphorochloridothioic acid, O,O

- diethyl ester; O,O

- diethyl phosphorochloridothioate [DEPCT]) submitted by Dow AgroSciences to EPA in July 2015 in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011;
referred to herein as HPV2; available online at http://www. regulations.gov/#!documentDetail;

D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of August 27

, 2015).

The HPV2 test rule requires the following toxicological testing for CASRN 2524

- 04

- 1: C1 (aquatic toxicity, consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia

, and toxicity to algae tests] if log K
ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae tests] if log K
ow ≥ 4.2), E1 (bacterial re verse mutation test), E2 (chromosomal aberrations or micronucleus test), and F2 (reproduction/developmental toxicity screening test). Based on an estimated log K
ow of 2.37 1 , Test Group 1 testing is required to determine the toxicity

of CASRN 2524

- 04

- 1 to aquatic organisms.

The July 2015 submission from Dow AgroSciences contained the following study reports:

 Marino, T.A., Hutchinson, K.L., Louch, D.W., Holzheuer, W.B., and Nelson, E.J. 2015a. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute to xicity study with the rainbow trout, Oncorhynchus mykiss

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

 Marino, T.A., Hutchinson, K.L., Louch, D.W., Holzheuer, W.B., and Nelson, E.J. 2015b. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute toxicity study with the freshwater Cladoceran, Daphnia magna

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

 Marino, T.A., Hutchinson, K.L., Holzheuer, W.B., and Nelson, E.J. 2015c. Phosphorochloridothioic acid, O,O

- diethyl ester: Growth inhibition test with t he freshwater green alga, Pseudokirchneriella subcapitata

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

B. EVALUATING DATA ADEQUACY

The submitted toxicological data are evaluated for adequacy in Section D. Each study was summarized according to a standard format and assigned a Klimi sch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were

1 U.S. EPA. 2012. Estimation Programs Interface SuiteTM [EPI Suite] for Microsoft ® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/ exposure/pubs/episuitedl.htm as of August 27 , 2015.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

- 04

- 1

2 adequate to satisfy test rule requirements, study details were compared to current standard test guidelines to establish whether ea ch study met current testing requirements

. In addition, each study was evaluated to determine whether recommendations provided in EPA’s study plan

reviews 1 were followed. Because CASRN 2524

- 04

- 1 has physicochemical properties (i.e., potential for hydroly sis 2 and volatilization 3 ) that necessitate special considerations during the aquatic toxicity testing process, EPA had previously provided recommendations aimed to minimize loss of the test substance during testing.

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Evaluation of Data Submitted for Aquatic Toxicity Testing Requirement for CASRN 2524

- 04

- 1: Data submitted by Dow AgroSciences and reviewed in this document are considered adequate to satisfy the aquatic toxicity (C1) testing requirement for CASRN 2524

- 04

- 1.

Other HPV2 Test Rule Toxicity Testing Requirements for CASRN 2524

- 04

- 1: Dow AgroSciences previously submitted data from a chromosomal aberrations test of CASRN

2524

- 04

- 1. 4 These data were reviewed in a separate report 5 and were determined to be adequate to satisfy the chromosomal aberrations (E2) testing requirement for CASRN 2524

- 04

- 1 promulgated in the HPV2 test rule.

Dow AgroSciences previously submitted data from a bacterial reverse mutation test of CASRN

2524

- 04

- 1

. 6 These data were reviewed in a separate report 7 and were determined to be not

1 “HPV2 Review of Toxicity Study Plan Dated August 9, 2014: Acute Toxicity to Fish, Acute Toxicity to Daphnia

, and Toxicity to Algae Tests, Phosphorochloridothioic acid, O,O

- d iethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #14

- 056, dated December 3, 2014 and “Review of Revised HPV2 Toxicity Study Plan Dated January 20, 2015: Acute Toxicity to Fish, Acute Toxicity to Daphnia , and Toxicity to Algae Tests, Phosphorochloridoth ioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 018, dated February 10, 2015.

2 West, R.J., and Hu, J. 2014. Phosphorochloridothioic acid, O,O

- diethyl ester: Determination of hydrolysis as a function of pH according to OECD Guide line 111. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

3 CASRN 2524

- 04

- 1 is considered volatile based on an estimated Henry’s Law Constant of 6.72 x 10

- 4 atm

- m 3 /mole

at 25°C (U.S. EPA. 2012. Estimation Programs Interface SuiteTM [EPI Suite] for Microsoft ® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/ exposure/pubs/episuitedl.htm as of August 27 , 2015).

4 Schisler, M.R., and McFadden, L.G. 2014. Evaluation of phosphorochloridothioic acid, O,O

- diethyl ester (DEPCT) in the mouse peripheral blood micronu cleus test. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0819 as of August 27 , 2015.

5 “HPV2 Test Rule Data Adequacy Review: Micronucleus Test, Phosphorochloridothioic acid, O,O

- diethyl ester,

CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 024, dated March 4, 2015. Available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0848 as of August 27 , 2015.

6 Tendulkar, K.E. 2014. Phosphorochloridothioic acid, O,O

- diethyl ester : Salmonella

- Escherichia coli mammalian

microsome reverse mutation assay pre

- incubation method with a confirmatory assay. Performing Laboratory: JAI Research Foundation, Gujarat, India. Sponsor: Dow AgroSciences LLC.

7 “HPV2 Test Rule Data Adequacy Review: Bacterial Reverse Mutation Test, Phosphorochloridothioic acid, O,O

diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 023, dated March 4, 2015. Available online at http://www.regul ations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0847 as of August 27 , 2015.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

- 04

- 1

3 adequate to satisfy the gene mutation (E1) testing requirement for CASRN 2524

- 04

- 1 promulgated in the HPV2 test rule.

Dow AgroSciences previously submitted data from a reprod uction/developmental toxicity screening test of CASRN 2524

- 04

- 1. 1 These data were reviewed in a separate report 2 and were determined to be adequate to satisfy the reproduction/developmental toxicity (F2) testing requirement for CASRN 2524

- 04

- 1 promulgated in the HPV2 test rule.

Summary of Data Adequacy for Toxicological Endpoints: Available data are adequate to satisfy the testing requirements for the acute toxicity to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to algae (component of C1), chromosomal aberrations (E2), reproductive toxicity (component of F2), and developmental toxicity (component of F2) endpoints. The gene mutation (E1) endpoint is considered to be a data gap and test ing is still required for this endpoint.

1 Andrus, A.K., Zablotny, C.L., Hannas, B.R., and Hukkanen, R.R. 2015. Phosphorochloridothioic acid, O,O

diethyl ester: Oral gavage reproduction/developmental toxicity screen ing test in Crl:CD(SD) rats. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

2 “HPV2 Test Rule Data Adequacy Review: Reproduction/Developmental Toxicity S creening Test, Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1”, SRC Technical Report #15

- 024, dated March 4, 2015.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

- 04

- 1

4 D . DATA FOR SIDS ENDPOINTS

D

- 1. Acute Toxicity to Fish

Summary of endpoint: A dequate

Study 1: Acute toxicity test in Oncorhynchus mykiss ( Marino et al.

, 2015

a)

Title: Phosphorochloridothioic a cid , O,O

- d iethyl e ster : An a cute t oxicity s tudy with the

r ainbow t rout

, Oncorhynchus mykiss

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Phosphorochloridothioic acid, O,O

- diethyl ester

, CASRN 2524

- 04

- 1 ; also referred to

as DEPCT.

Remarks: Purity 99.3%; Lot No. C032013081501

METHOD

Method/guideline followed: OECD TG 203 (1992) and ASTM E729

- 96 (2007)

Test type: Acute Toxicity to Fish

– Static Renewal

GLP compliant?: Yes

Year study was performed: 2015

Test species: Rainbow trout ( Oncorhynchus mykiss )

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested 1 :

Definitive Test:

Nominal DEPCT concentrations: 0 (control), 0.75, 1.5, 3.0, 6.0

, and 12 mg/L;

1 DEPTh ( phosphorothioic acid, O,O

- diethyl ester ) is the expected hydrolysis product of DEPCT. DHP ( O,O

diethyl hydrogen p hosphate ) is a potential oxidation product of DEPCT.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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5 Overall m ean 1 measured DEPCT conc entrations ( means of “fresh” and “spent” solutions):

< LLQ 2 (control), 0.327 , 0 .537

, 0.953, 1.73, and 3.91 mg DEPCT/L;

Overall m ean measured DEPTh conc entrations ( means of “fresh” and “spent” solutions): < LLQ 3
(control), 0. 280

, 0.554

, 1.07

, 1.81

, and 3.83 mg DEPTh/L;

Overall m ean measured DHP conc entrations ( means of “fresh” and “spent” solutions): < LLQ 4
(all test levels).

Test method/conditions remarks: Laboratory d ilution water (LDW) : Prior to use for culturing and testing, Lake Huron water from the City of Midland Water Treatment Plant was limed and flocculated with ferric chloride, sand

filtered, pH

- adjusted with gaseous CO 2 , carbon

- filtered

, and UV

- irradiated. Analysis of the

dilution water showed total organic carbon ( TOC ) was 0.95 mg/L, total suspended solids (TSS)

were < 3 mg/L, alkalinity was 24 mg CaCO 3 /L, hardness was 56 mg CaCO 3 /L

, and conduct ivity was 168 μS/cm. Concentrations of contamina nts were reported to be below levels that may

interf ere with the result of the study.

Test species : R ainbow trout ( Oncorhynchus mykiss ) were obtained as juveniles from Osage Catfisheries, Osage Beach, MO

. The fish were visually inspected and acclimated to dilution

water and test conditions for 28 days prior to testing

. “The mortality did not exceed 5% (0% actual) of the population in the 48

- hr period prior to test initiation. Feeding was withheld for a minimum of 24 hrs prior to test initiation (~32 hrs actual).” [ Marino et al. 2015a, p. 11] Weight and length

, measured for 10 of the fish used in the definitive test

, “were considered representative of the length and weight of all fish used in the study

. ” [ Marino et al. 2015a, p. 15 ]

M easured values were: 0.524 g (range: 0.400

– 0.672 g ) mean wet (blotted dry) weight and 4.1 cm (range: 4.0

– 4.3 cm) mean body length

.

Selection of test item concentrations: Target test item concentrations selected for the definitive test (0.75, 1.5, 3.0, 6.0, and 12 mg DEPCT/L) were based upon the results of two 96

- hour preliminary range

- finding tests that were briefly described in the current study report. In each prelimina ry test, fish mortality was 100% at target concentrations of 10 and 100 mg DEPCT/L and 0% at 1 mg DEPCT/L.

Preparation of test item concentrations: “ Aliquots of 5.64, 11.3, 22.5, 45, and 90 μL of test material (density = 1.2 g/cm 3 [per] Zhejiang Xinnon g Chemical Co., Ltd., 2007) were directly added to 9 L test water (LDW) to prepare each replicate exposure solution with a target (nominal) concentrations of 0.75, 1.5, 3.0, 6.0, and 12 mg DEPCT/L, respectively. Exposure solutions were vigorously stirred for approximately one to three mins with an overhead mixer. In addition, a replicate of lab dilution water (LDW) was prepared without the addition of the test

material and served as the water control. All exposure solutions ( ‘ fresh ’ and ‘ spent ’ ), including the water control, were clear and colorless over the course of the study. In the 6.0 and 12 mg/L test material exposure solutions ( ‘ fresh ’ and ‘ spent ’ ) a small amount of apparent undissolved substance (fine white precipitate) was noted on the bottom of the vessels

... All test solutions

1 Overall mean measured concentrations of DEPCT, DEPTh, and DHP were calculated by the reviewers.

2 The lower limit of quantitation (LLQ) for DEPCT was 0.313 mg/L.

3 The LLQ for DEPTh was 0.0750 mg/L.

4 The LLQ for DHP was 0.0750 mg/L.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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6 were utilized on the same day as preparation and on the same day as initial analytical confirmation; therefore, analysis of stability was [considered by the study authors to be] not necessary.” [ Marino et al. 20 15a, p. 14]

Exposure conditions: Due to potential for volatilization from water, exposure s to DEPCT were performed in closed vessels with minimized headspace using air

- impermeable closures (PTFE

lined plastic screw caps)

. A semi

- static test with daily (24

- hour) test medium renewal was performed with one replicate of seven fish per concentration in a glass jar of 9.73 L volume that

contained 9 L of control or test material exposure solution. Head space was approximately 730

mL. “ This minimal headspace was necessary to allow vigorous mixing of the test material into

the exposure medium, using an overhead stainless steel impeller mixer, while avoiding spillage. This remaining headspace in the test vessel also aided in maintaining dissolved oxygen

concent rations within required levels over each 24 hr. exposure renewal cycle. ” [ Marino et al. 2015a, p. 12 ] “... [F] ish were impartially added to each test vessel within 30 min of sampling for analytical verification of test solution concentrations. [Sampling wa s conducted during the mixing period of media preparation.] Fish were added to each replicate vessel, sequentially,

until all vessels contained seven fish. ” [ Marino et al. 2015a, p. 14] The biological loading rate was 0.4 g fish/L.

“ The test vessels were placed in a temperature

- controlled water trough according to a computer

generated randomization. The target test temperature was 13 ± 1°C. The photoperiod was set at 16 hrs light/8 hrs dark per day. Fish were not fed during the test.” [ Marino et al. 2015a, p. 15]

“The fish were observed at approximately 24, 48, 72, and 96 hrs of exposure for mortality (no response to touching of the caudal peduncle and no opercula movement) and sublethal effects.

Dead fish were recorded and removed when observed. Sublethal effects (behavioral and/or gross pathological findings) were also recorded when observed.” [ Marino et al. 2015a, p. 15]

“Dissolved oxygen, pH, and temperature data were measured daily from eac h test vessel at exposure initiation, a t each renewal period from the ‘freshly’ prepared and ‘spent’ solutions, and

at terminations. Water temperature was continuously monitored with a minimum/maximum thermometer placed in the surrogate vessel and the ran ge recorded daily. Light intensity was measured at each test vessel location on day 0. Water hardness, alkalinity, and conductivity were measured from the control water and the 12 mg/L [nominal] test level solution on day 0.”
[ Marino et al. 2015a, p. 15]

Analytical monitoring: “ S amples were collected from each test vessel at exposure initiatio n, at each renewal period from ‘freshly’ prepared and ‘spent’ solutions, and at termination. Analytical samples were collected during vigorous stirring of the test solution to generate a homogenous mixture of dissolved and any undissolved test material

- associated substances present. Samples (~200 mL) were collected from each test vessel and were extracted into toluene immediately to quench any further hydrolysi s of the parent test material. The organic and aqueous layers were allowed to separate in the extraction bottles, with the organic phase sampled for analysis of parent test material [DEPCT] via gas chromatography with electron capture detector (GC/ECD). The aqueous phase was sampled for the analysis of the expected degradation products [DEPTh and DHP] via high performance liquid chromatography with tandem mass spectrometryFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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- 1

7 (HPLC/MS/MS). Retainer samples were also collected at each sampling interval and a nalyzed, if necessary, or retained until results from the primary samples were verified. ” [ Marino et al. 2015a, pp. 15

- 16] LLQ values were 0.313, 0.0750, and 0.0750 mg/L for DEPCT, DEPTh, and DHP, respectively.

Statistical analysis : EPA’s Probit Program (v1.5) was used to calculate the 96

- hour LC 50 values

and 95% confidence intervals

. “ The NOEC was determined based on biological interpretation of the data and/or the highest concentration tested exhibiting no significant fish mortality or sublethal effects as determined by a Fisher’s Exact Test.” [ Marino et al. 2015a, p. 1 6 ]

RESULTS

Endpoint value s ( mg/L ):

The study authors provided the following LC 50 and NOEC values , based on nominal concentrations of DEPCT, measured concentrations of DEPCT in fresh solutions, or measured concentrations of degradation products (DEPTh or DHP) in spent solutions ; LOEC values were inferred by the reviewers as the next higher concentration above the NOEC, based on the mean

measured values provided by the study authors :

Nominal DEPCT Measured

DEPCT (fresh solutions) a Measured

DEPTh (spent solutions) b Measured DHP

(spent solutions) b

96

- hour LC 50 (95% confidence interval) 5.4 (3.6

- 8.1) 3.11

(2.10

- 4.76) 3.24

(2.33

- 4.53) N/A c

96

- hour NOEC 3.0 1.79 2.06 N/A

96

- hour LOEC 6.0 3.38 3.50 N/A

a Mea n of measured DEPCT concentrations in “fresh” solutions at 0, 24, 48, and 72 hours

. b Mea n s of mea sured DEPTh and DHP concentrations in “spent” solutions at 24, 48, 72, and 96 hours

.

c M easured DHP concentrations, in “spent” solutions at 24, 48, 72, and 96 hours, were all < LLQ.

The reviewers calculated the following endpoint values based on the mean measured concentrations of DEPCT in fresh and spent solutions combined:

Measured DEPCT (fresh and spent solutions)

96

- hour LC 50 1.65 1

96

- hour NOEC 0.953

96

- hour LOEC 1.73

1 The LC 50 was calculated using the Log

- Probit model in EPA’s Benchmark Dose Software.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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- 1

8 Results remarks: Analytical monitoring: For each nominal concentration of DEPCT that was tested in this study, the following table shows the m ean measured concentrations of DEPCT and its degradation products, DEPTh and DHP, that were determined in fresh and spent solutions over the duration of the study:

Mean Measured Concentrations (mg/L) a

DEPCT DEPTh DHP

Nominal

concentrations (mg/L) Fresh

solutions Spent

solutions Fresh

solutions Spent

solutions Fresh

solutions Spent

solutions

0 (Control) < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ

0.75 0.498 0.157 0.0375 0.522 < LLQ < LLQ

1.5 0.918 0.157 0.375 1.07 < LLQ < LLQ

3 1.75 0.157 0.0826 2.06 < LLQ < LLQ

6 3.23 0.235 0.124 3.50 < LLQ < LLQ

12 6.75 1.07 1.51 6.16 < LLQ < LLQ

a Means were calculated by the reviewer s as the mean of the measured concentrations in fresh or spent solutions over all sampling intervals

. If more than one measurement was available for a given time interval, these values were averaged before taking the average over all time intervals. (This differed from the method of averaging used by the study authors, who simply took the mean of all measurements.) For values < LLQ, one half of the LL Q was used in the calculation of the mean concentration. If all measurements for a given cell were < LLQ, then the mean is simply provided as < LLQ. (LLQs were 0.313 mg DEPCT/L, 0.075 mg DEPTh/L, and 0.075 mg DHP/L.)

The concentrations of DHP in all fre sh and spent samples were < LLQ.

The study authors calculated mass balance percentages from measured concentrations of

DEPCT

, DEPTh

, and DHP

. “ These mass balance (%) values were determined from the total moles of parent DEPCT plus total moles of degradation products (DEPTh, DHP) divided by nominal moles of parent, for each exposure solution analyzed at a given sampling interval. An average of th ese mass balance values was calculated for each nominal exposure concentration across all sampling intervals.” [Marino et al. 2015a, p. 3 5 ] “ For the 0.75, 1.5, 3.0, 6.0, and 12 mg DEPCT/L nominal test levels, the mass balance averaged (range in parenthes es) 65 (46

– 94), 60 (35

– 84), 62 (51

– 87), 57 (43

– 77), and 64 (50

– 93)%, respectively. These average mass balances indicate total measured test material (and reaction product) concentrations which fall near their nominal test concentrations, and ind icate that the test levels were dosed properly and that the test design and procedures were appropriate for maintaining the dosed test material and associated reaction products within the exposure vessels.” [ Marino et al. 2015a, pp. 16

- 17]

“ The apparent variability in mass balance values within and across the nominal exposure concentrations is likely reflective of both imprecision in the test material spiking procedure ( i.e

., dispensing of

- microliter volumes into large volumes of water) and of variabilit y in quantitative recovery of all soluble and insoluble test material species. It was impractical to extract the entire exposure solutions (9 L) and each exposure test vessel with toluene to give total recovery of all

dissolved and undissolved test materi al species. Although measures were taken to homogenize the exposure solutions while representative samples were removed for extraction, this sampling procedure may also introduce some imprecision in the test material mass balance

determinations.” [ Marino et al. 2015a, p. 17]Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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- 1

9 Exposure conditions: Over the course of the study, “[d]issolved oxygen levels ranged from 7.3

- 10.9 mg/L (70

- 106% air saturation), temperatures ranged from 12

- 14oC , and pH ranged from

6.8

- 7.7. Light intensity ranged from 539

- 874 lux f or all exposures.” [ Marino et al. 2015a, p. 17]

Biological observations : “ Following 24 hrs of exposure, sublethal effects (partial loss of equilibrium) was observed in 100% of the fish at the 12 mg DEPCT/L nominal exposure

concentration. Following 48 hrs of exposure, four of the seven fish (57 %) at the 12 mg

DEPCT/L nominal concentration were observed as dead. The remaining fish (3 of 7) at this test level continued to have partial loss of equilibrium. In addition, partial loss of equi librium was observed in 100 % of the fish at the 6.0 mg DEPCT/L nominal exposure concentration at the 48

- hr observation period. Following 72 hrs of exposure, mortality was observed in 29 % (2 of 7) and 71 % (5 of 7) of the fish at the 6.0 and 12 mg DEPCT/ L nominal test levels, respectively. Remaining fish at these test levels were exhibiting signs of complete or partial loss of equilibrium. In addition, one fish at the 1.5 mg DEPCT/L nominal concentration was observed

as immobile at the 72

- hr observation period. At exposure termination (96

- hr observation), mortality increased to 86 % (6 of 7) of the fish at the 6.0 and 12 mg DEPCT/L nominal test

levels. The remaining fish at these test levels had complete loss of equilibrium. One fish at the 1.5 mg DEP CT/L nominal concentration remained immobile at the 96

- hr observation period. No fish mortality or sublethal effects were observed at the 0.75 and 3.0 mg DEPCT/L nominal concentrations or in the water control during the conduct of this study.” [ Marino et al. 2015a, p. 18]

The following table summarizes the relationship between percent fish mortality at 96 hours and

overall mean measured concentrations (in fresh and spent solutions) of DEPCT, DEPTh, and

DHP over the duration of the study:

Overall Mean Measured Concentrations

(mg/L) a Percentage Mortality at

96 Hours (%) Nominal

concentrations (mg/L) DEPCT DEPTh DHP

0 (Control) < LLQ < LLQ < LLQ 0

0.75 0.327 0.280 < LLQ 0

1.5 0.537 0.554 < LLQ 0

3 0.953 1.07 < LLQ 0

6 1.73 1.81 < LLQ 86

12 3.91 3.83 < LLQ 86

a Overall means were calculated by the reviewer s as the mean of the measured concentrations in fresh and spent solutions. For values < LLQ, one half of the LLQ was used in the calculation of the mean concentration. If all measurements for a given cell were < LLQ, then the mean is simply provided as < LLQ. (LLQs were 0.313 mg DEPCT/L, 0.075 mg DEPTh/L, and 0.075 mg DHP/L.)Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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10 CONCLUSIONS

Based on nominal concentrations of DEPCT, the 96

- hour LC 50 was 5.4 mg DEPCT/L in Oncorhynchus mykiss

. The corresponding NOEC and LOEC were 3.0 and 6.0 mg/L, respectively.

Based on mean measured concentrations of DEPCT in “ fresh ” solutions , the 96

- hour LC 50 was 3.11 mg DEPCT/L in O ncorhynchus mykiss

. The corresponding NOEC and LOEC were 1.79 and 3.38 mg/L, respectively.

Based on mean measured concentrations of DEPCT in “fresh” and “spent” test solutions over the duration of the study, the 96

- hour LC 50 was 1.65 mg DEPCT/L in Oncorhynchus mykiss . The corresponding NOEC and LOEC were 0.953 and 1.73 mg/L, respectively.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. OECD TG 203 states that if measured concentrations of the test substance deviate by more than 20% from the nominal concentrations throughout the test, results

should be based on measured concentrations. Although t he study authors calculated an LC 50 value based on measured concentrations of DEPCT in fresh solutions, they did not provide an LC 50 value based on mean measured concentrations of DEPCT in fresh and spent solutions

combined. Th is missing value would be considered to be a more accurate estimate of the LC 50 of

DEPCT in rainbow trout than an LC 50 based on measured DEPCT concentrations in fresh solutions only. The reviewers calculated the LC 50 based on mean measured concentrations of DEPCT in fresh and spent solutions combined and found this value to be about 50 % less than the

LC 50 value based on measured concentrations of DEPCT in fresh solutions only , therefore providing a more conservative estimate of the toxicity of DEPCT to rainbow trout

.

REFERENCE

Marino, T.A., Hutchinson, K.L., Louch, D.W ., Holzheuer, W.B., and Nelson, E.J. 2015a. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute toxicity study with the rainbow trout, Oncorhynchus mykiss

. Performing Laboratory: The Dow Chemical Company (Toxicology and

Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

Text in quotes was taken directly from the report of Marino et al. (2015 a ).Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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11 D

- 2

. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Marino et al. , 2015b)

Title: Phosphorochloridothioic acid, O,O

- d iethyl ester: An a cute t oxicity s tudy with the

f reshwater Cladoceran, Daphnia magna

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1; also referred to

as DEPCT.

Remarks: Purity 99.3%; Lot No. C032013081501

METHOD

Method/guideline followed: OECD TG 202 (2004) and ASTM E729

- 96 (2007)

Test type: Acute toxicity to Daphnia

– Static Renewal

GLP compliant?: Yes

Year study was performed: 2015

Test species: Water flea ( Daphnia magna ), neonates (< 24 hours old), not first brood progeny,

from in

- house cultures.

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested 1 :

Definitive Test:

Nominal DEPCT concentrations: 0 (control), 0.313, 0.625, 1.25, 2.50

, and 5.00 mg/L;

Overall m ean 2 measured DEPCT concentrations ( means of “fresh” and “spent” solutions):

< LLQ 3 (control), 0.2 43 , 0. 554

, 0 .7 82

, 1

. 89, and 3

. 74 mg/L;

1 DEPTh (phosphorothioic acid, O,O

- diethyl ester) is the expected hydrolysis product of DEPCT. DHP (O,O

diethyl hydrogen phosphate) i s a potential oxidation product of DEPCT.

2 Overall mean measured concentrations of DEPCT, DEPTh, and DHP were calculated by the reviewers.

3 The lower limit of quantitation (LLQ) for DEPCT was 0.10 mg/L.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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12 Overall m ean measured DEPTh concentrations ( means of “fresh” and “spent” solutions): < LLQ 1
(control), 0. 085 , 0.1 05 , 0. 219 , 0. 344, and 0. 494 mg DEPTh/L;

Overall m ean measured DHP concentrations ( means of “fresh” and “spent” solutions): < LLQ 2
(all test levels).

Test method/conditio ns remarks: Laboratory dilution water (LDW): Prior to use for culturing and testing, Lake Huron water from the City of Midland Water Treatment Plant was limed and flocculated with ferric chloride, sand

filtered, pH

- adjusted with gaseous CO 2 , carbon

- filtered, and UV

- irradiated. Analysis of the dilution water showed total organic carbon (TOC) was 0.95 mg/L, total suspended solids (TSS)

were < 3 mg/L, alkalinity was 34 mg CaCO 3 /L, hardness was 48 mg CaCO 3 /L, and conduct ivity

was 200 μS/cm. Co ncentrations of contaminants were reported to be below levels that may interfere with the result of the study.

Test species: The study was performed with the freshwater daphnid, Daphnia magna , from in

house cultures initially obtained from Aquatic BioSystems, Colorado. Test organisms were

d aphnid neonates, which were less than 24 hours old and were only used from daphnids which

had had at least 3 broods. Daphnids were not fed during the test

.

Selection of test item concentrations: Target test item concentrations selected for the definitive test were based upon the results of two 48

- hour range

- finding tests, conducted under static

renewal conditions (with a renewal period of 24 hours), that were briefly described in the current study report. In the first range

- finding test, immobility at 48 hours was 67, 100, and 100% at target concentrations of 1, 10, and 100 mg DEPTC/L, respectively. I n the second range

- finding

test, “ [f] ollowing 48 hrs o f exposure, immobility was observed in 80, 80, 100, 100, and 100 % of the daphnids at the nominal 1.0, 3.0, 6.0, 12, 25, and 50 mg DEPCT/L exposure concentrations, respectively. ” [ Marino et al. 2015b, p. 13 ] It should be noted that page 13 of the study report indicates that six concentrations of DEPCT were tested in the second range

- finding test but reports only five immobilization values. The study report does not provide detailed information

for either ra nge

- finding test. Based on results of the range

- finding tests, concentrations selected

for the definitive test were 0.313, 0.625, 1.25, 2.50, and 5.00 mg DEPCT/L

.

Preparation of test item concentrations: “ Aliquots of 0.2, 0.4, 0.7, 1.5, and 2.9 μL of test material (density = 1.2 g/cm 3 ; Zhejiang Xinnong Chemical Co., Ltd., 2007) were directly added to 700 mL test water (LDW) to prepare each replicate exposure solution with target (nominal) concentrations of 0.313, 0.625, 1.25, 2.50, and 5.00 mg DEPCT/L, respectively. In addition, a replicate of lab dilution water (LDW) was prepared without the addition of the test material and

served as the water control. A surrogate replicate for each test level was prepared at exposure initiation and the renewal peri od [at 24 hours] for analytical verification and environmental parameter measurements ( e.g . pH, DO, temperature) of ‘ freshly ’ prepared solutions. Furthermore, another surrogate replicate for the control and highest test level was prepared at

exposure initiation and at the beginning of the ren e wal period for the measurement of water quality parameters ( e.g . hardness, alkalinity, conductivity) of ‘ freshly ’ prepared solutions. Exposure solutions were immediately sealed with air

- impermeable closures and v igorously

1 The LLQ for DEPTh was 0.050 mg/L.

2 The LLQ for DHP was 0.050 mg/L.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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13 mixed on a vortex shaker following addition of the test material. All dosed solutions ( ‘ fresh ’ and

‘ spent ’ ) were clear and colorless with a fine white precipitate becoming increasingly visible over the course of each 24 hr exposure [renewal] per iod.” [ Marino et al. 2015b, p. 14]

“ Test organisms were added within 30 min of initial addition of the test material to each test
vessel. ” [ Marino et al. 2015b, p. 1 4] “ All test solutions were utilized on the same day as preparation and on the same day as initial analytical confirmation; therefore, analysis of stability

was [considered by the study authors to be] not necessary. ” [ Marino et al. 2015b, p. 1 5 ]

Exposure conditions: “ Due to potential for volatilization from water, the exposure to DEPCT

was performed in closed vessels with minimized headspace using air

- impermeable closures
[PTFE

- lined polypropylene screw caps]

. ” [ Marino et al. 2015b, p. 1 5 ] A sem i

- static test with

24

- hour test medium renewal was performed with four replicates of five daphnids per

concentration in glass jars of 800 mL volume containing test solution. A cylindrical glass insert with NITEX screening (425 μm) was suspended within each test vessel to minimize the potential for physical effects due to contact with undissolved test material. “ The exposure solution and internal glass cylinder were determined to occupy approximately 700 mL, leaving approximately

100 mL of headspace in each test vessel. ” [ Marino et al. 2015b, p. 12 ] Loading was approximately one daphnid/140 mL test solution.

“ The replicate test vessels were placed in a temperature

- controll ed water bath according to a

computer

- generated randomization. The target test temperature was 20 ± 2 °C, not to vary by

> 1o C over the course of the exposure. The photoperiod was set at 16 hrs light/8 hrs dark per day.” [ Marino et al. 2015b, p. 1 5]

“ Daphnids were observed for immobility (inability to swim within approximately 15 sec after gentle agitation of the test vessel) at approximately 24 and 48 hrs of exposure. Immobile daphnids were recorded when observed, along with any noted sublethal effec ts (abnormal behavior or appearance). ” [ Marino et al. 2015b, p. 15]

“ Dissolved oxygen, pH, and temperature data were measured from each test level (surrogate vessels) at exposure initiati on, at the renewal period from ‘freshly’ prepared (surrogate vessel s) and ‘spent’ (replicate vessels) solutions, and at termination (replicate vessels). Water temperature was continuously monitored with a minimum/maximum thermometer placed in a surrogate vessel. Light intensity was measured at test initiation. Water hardness, alkalinity, and

c onductivity were measured from ‘freshly’ prepared (surrogate vessels) solutions of the control and the 5.00 mg/L [nominal] test level solution on day 0. ” [ Marino et al. 2015b, p. 15]

Analytical monitoring: “ S amples were collect ed from each test level (surrogate vessels) at exposure initiation, at the renewal period from ‘freshly’ prepared (surrogate vessels) and ‘spent’ (replicate vessels) solutions, and at termination (replicate vessels). Analytical samples were collected by e xtracting the whole test vessel with toluene. Samples were collected and extracted into toluene as soon as possible, and the organic and aqueous layers were allowed to separate in

the bottle. The organic phase was sampled for the analysis of parent test material [DEPCT] via gas chromatography with electron capture detector (GC/ECD). The aqueous phase was sampled for the analysis of the expected degradation products [DEPTh and DHP] via high performanceFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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14 liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). Retainer samples of each phase were also collected at each sampling interval and analyzed, if necessary, or retained until results from the primary samples were verified.” [ Marino et al. 2 015b, p. 16]

Statistical analysis: EPA’s Probit Program (v1.5) was used to calculate the 48

- hour EC 50 value s and 95% confidence intervals. “ The NOEC was determined based on biological interpretation of the data and/or the highest concentration tested exhibiting no significant daphnid immobility or sublethal effects as determined by a Fisher’s Exact Test. ” [ Marino et al. 2015b, p. 16]

RESULTS

Endpoint values (mg/L) :

The study authors provided the following EC 50 and NOEC values, based on nominal concentrations of DEPCT, measured concentrations of DEPCT in fresh solutions, or measured concentrations of degradation products (DEPTh or DHP) in spent solutions ; LOEC values were inferred by the reviewers as the next higher conc entration above the NOEC, based on the mean measured values provided by the study authors :

Nominal

DEPCT Measured

DEPCT (fresh solutions) a Measured

DEPTh (spent solutions) b Measured DHP

(spent solutions) b

48

- hr EC 50 (95% confidence interval) 3.14

(2.37

- 4.72) 2.84

(2.16

- 4.22) 0.623

(0.515

- 0.809) N/A c

48

- hr NOEC 1.25 1.07 0.333 N/A

48

- hr LOEC 2.50 2.36 0.583 N/A

a Mean of measured DEPCT concentrations in “fresh” solutions at 0 and 24 hours.

b Mean s of measured DEPTh and DHP concentratio ns in “spent” solutions at 24 and 48 hours.

c M easured DHP concentrations, in “spent” solutions at 24 and 48 hours, were all < LLQ.

The reviewers calculated the following endpoint values based on the mean measured concentrations of DEPCT in fresh and spent solutions combined:

Measured

DEPCT (fresh and spent solutions)

48

- hr EC 50 2.33 1

48

- hr NOEC 0.782

48

- hr LOEC 1.89

1 The E C 50 was calculated using the Log

- Probit model in EPA’s Benchmark Dose Software.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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15 Results remarks: Analytical monitoring: For each nominal concentration of DEPCT that was tested in this study, the following table shows the mean measured concentrations of DEPCT and its degradation products, DEPTh and DHP, that were determined in fresh and spent solutions over the duration of the study:

Mean Measured Concentrations (mg/L) a

DEPCT DEPTh DHP

Nominal

concentrations (mg/L) Fresh

solutions Spent

solutions Fresh

solutions Spent

solutions Fresh

solutions Spent

solutions

0 (Control) 0 0 0 0 < LLQ < LLQ

0.313 0.271 0.215 b 0.025 0.145 b < LLQ < LLQ

0.625 0.704 0.405 0.045 0.166 < LLQ < LLQ

1.25 1.07 0.500 0.104 0.334 < LLQ < LLQ

2.5 2.37 1.41 0.105 0.583 < LLQ < LLQ

5 4.40 3.08 0.172 0.816 < LLQ < LLQ

a Means were calculated by the reviewer s as the mean of the measured concentrations in fresh or spent solutions over all sampling intervals

. If more than one measurement was available for a given time interval, these values were averaged before taking the average over all time intervals

. (This differed from the method of averaging used by the study authors, who simply took the mean of all meas urements.) For values < LLQ, one half of the LLQ was used in the calculation of the mean concentration. If all measurements for a given cell were < LLQ, then the mean is simply provided as < LLQ. (LLQs were 0.10 mg DEPCT/L, 0.050 mg DEPTh/L, and 0.050 m g DHP/L.)

b These values exclude one replicate that had a calculated mass balance of 0%, since the study authors determined that this vessel may not have been dosed with the test material at exposure initiation.

The concentrations of DHP in all fresh and spent samples were < LLQ.

The study authors calculated mass balance percentages from measured concentrations of

DECPT

, DEPTh

, and DHP. “These mass balance (%) values were determined from the total moles of parent DEPCT plus total moles of degradation products (DEPTh, DHP) divided by nominal moles of parent, for each exposure solution analyzed at a given sampling interval. An average of these mass balance values was calculated for each nominal exposure concentration across all sampling intervals.” [Ma rino et al. 2015b, p. 3 3 ] “ For the 0.313, 0.625, 1.25, 2.50, and 5.00 mg DEPCT/L nominal test levels, the mass balance [percentages] averaged (range in parentheses) 101 (41

– 152), 93.8 (30

- 126), 68.5 (32

- 96), 81.6 (50

- 103), and 79.2 (63

- 92) %

of the nominal initial DEPCT concentrations, respectively. Analytical results of the ‘ spent ’ replicate solutions prepared on day 0 (24

- hr sampling period) yielded one replicate vessel at the 0.313 mg DEPCT/L (nominal) test level with a calculated mass balanc e of 0 %. The remaining replicates (n=3) at this test level had mass balance results ranging from 115 to 152 % of nominal. Thus, it was determined that this replicate test vessel may not have been dosed with the test material at exposure initiation (day 0). This potential experimental error had no adverse impact on results or interpretation of the study, as the NOEC was determined to be greater than this

concentration. The analytical results for this replicate vessel were excluded when calculating

mean measured concentrations, average % recovery, and the derived study endpoints. The average mass balances which fall near the nominal test concentrations indicate that the test levels were dosed properly and that the test design and procedures were appropri ate for maintaining the dosed test material and associated reaction products within the exposure vessels.” [ Marino et al. 2015b, p. 17]Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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16 “ The apparent variability in mass balance values within and across the nominal exposure concentrations is likely reflective of both imprecision in the test material spiking procedure ( i.e

., dispensing of sub

- microliter volumes) and of variability in quantitative recovery of all soluble and insoluble test material species. While the screened internal cylinders were a pplied to minimize physical toxicity of any undissolved substances present, they had to be quickly removed prior to addition of toluene extraction solvent and extraction of the remaining test

vessel contents. It is possible that these screens may have ent rained some undissolved/particulate test material species which were then not accounted for in the subsequent extraction and analyses.” [ Marino et al. 2015b, p. 18]

Exposure conditions: Over the course of the study, “[d]issolved oxygen levels ranged fr om 7.6

- 8.7 mg/L (85

- 100% air saturation), temperatures ranged from 20

- 21oC, and pH ranged from 7.3

- 7.7. Light intensity ranged from 502

- 832 lux.” [ Marino et al. 2015b, p. 18]

Biological observations: “ Following 24 hrs of exposure, immobility was observed in 20 % (4 of 20), and 5 % (1 of 20) of the daphnids at the 2.50, and 5.00 mg DEPCT/L nominal test levels, respectively. Lethargy was observed in 6 of 19 surviving daphnids at the 5.00 mg DEPCT/L

nomin al test level during the 24

- hr observations. At exposure termination (48

- hrs of exposure), immobility was observed in 15, 55, and 60%, of the daphnids at the 1.25, 2.50, and 5.00 mg DEPCT/L nominal test levels, respectively. One surviving daphnid at the 5.00 mg DEPCT/L nominal test level was lethargic at exposure termination. No immobility was observed at the

0.313

... [or] 0.625 mg DEPCT/L nominal test level [s] or in the water control over the course of the 48 hr exposures.” [ Marino et al. 2015b, p p

. 18

- 19]

The following table summarizes the relationship between percent daphnid immobilization at 48 hours and overall mean measured concentrations (in fresh and spent solutions) of DEPCT, DEPTh, and DHP over the duration of the study:

Overall Mean Measure d Concentrations

(mg/L) a Percentage

of

Immobilized Daphnids at

48 Hours (%) Nominal

concentrations (mg/L) DEPCT DEPTh DHP

0 (Control) < LLQ < LLQ < LLQ 0

0.313 0.243 0.085 < LLQ 0

0.625 0.554 0.105 < LLQ 0

1.25 0.782 0.219 < LLQ 15

2.5 1.89 0.344 < LLQ 55

5 3.74 0.494 < LLQ 60

a Overall means were calculated by the reviewer s as the mean of the measured concentrations in fresh and spent solutions. For values < LLQ, one half of the LLQ was used in the calculation of the mean concentration. If all measurements for a given cell were < LLQ, then the mean is simply provided as < LLQ. (LLQs were 0.10 mg DEPCT/L, 0.050 mg DEPTh/L, and 0.050 mg DHP/L.)Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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17 The reviewers conducted a Fisher’s Exact Test and confirmed that the percentage of immobilization (15%) observed at the nominal concentration of 1.25 mg DEPCT/L was not statistically significantly different from the control, confirming that this treatment level is the

NOEC.

CONCLUSIONS

Based on nominal concentrations of DEPCT, the 48

- hour EC 50 was 3.14 mg DEPCT/L in Daphni a magna

. The corresponding NOEC and LOEC were 1.25 and 2.5 mg/L, respectively.

Based on mean measured concentrations of the DEPCT in “fresh” solutions, the 48

- hour EC 50

was 2 .84 mg DEPCT/L in Daphnia magna

. The corresponding NOEC and LOEC were 1.07 and 2.36 mg/L, respectively.

Based on mean measured concentrations of DEPCT in “fresh” and “spent” test solutions over t he duration of the study, the 48

- hour E C 50 was 2.33 mg DEPCT/L in Daphnia magna

. The corresponding NOEC and LOEC were 0.782 and 1.89 mg/L, respectively.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. OECD TG 20 2 states that if measured concentrations of the test substance deviate by more than 20% from the nominal concentrations throughout the test, results

should be based on measured concentrations. Although the study authors calculated an EC 50 value based on me asured concentrations of DEPCT in fresh solutions, they did not provide an

EC 50 value based on mean measured concentrations of DEPCT in fresh and spent solutions combined. This missing value would be considered to be a more accurate estimate of the EC 50 o f DEPCT in daphnids than an EC 50 based on measured DEPCT concentrations in fresh solutions only. The reviewers calculated the EC 50 based on mean measured concentrations of DEPCT in fresh and spent solutions combined and found this value to be about 18 % less than the E C 50 value based on measured concentrations of DEPCT in fresh solutions only , therefore providing a more conservative estimate of the toxicity of DEPCT to daphnids

.

REFERENCE

Marino, T.A., Hutchinson, K.L., Louch, D.W., Holzheuer, W.B., and Nelson, E.J. 2015b. Phosphorochloridothioic acid, O,O

- diethyl ester: An acute toxicity study with the freshwater Cladoceran, Daphnia magna

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

Text in quotes was taken directly from the report of Marino et al. (2015b).Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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18 D

- 3. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1 : Toxicity to Pseudokirchneriella subcapitata (Marino et al., 2015c)

Title: Phosphorochloridothioic a cid, O,O

- d iethyl e ster: Growth i nhibition t est with the

f reshwater g reen a lga, Pseudokirchneriella subcapitata

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Phosphorochloridothioic acid, O,O

- diethyl ester, CASRN 2524

- 04

- 1; also referr ed to

as DEPCT.

Remarks: Purity 99.3%; Lot No. C032013081501

METHOD

Method/guideline followed: OECD TG 201 (2006) and ASTM E1218

- 04 (2012)

Test type: Toxicity to plants

– algae (static)

GLP compliant?: Yes

Year study was performed: 2015

Test species: Green algae ( Pseudokirchneriella subcapitata ); from in

- house cultures obtained from the University of Texas at Austin Culture Collection.

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested 1 :

Definitive Test:

Nominal DEPTC concentrations: 0 (control), 1.6, 4.6, 13, 37, and 106 mg/L ; Overall m ean 2 measured DEPCT concentrations ( means of concentrations measured at 0, 24, 48,

and 72 hours ): < LLQ 3 (control), 0

. 420 , 0. 355

, 1 .5 1

, 9

. 92 , and 3 4.8 mg/L ;

1 DEPTh (phosphorothioic acid, O,O

- diethyl ester) is the expected hydrolysis product of DEPCT. DHP (O,O

diethyl hydrogen phosphate) is a potential o xidation product of DEPCT.

2 Overall mean measured concentrations of DEPCT, DEPTh, and DHP were calculated by the reviewers.

3 The lower limit of quantitation (LLQ) for DEPCT was 0.313 mg/L.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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19 Overall m ean measured DEPTh concentrations ( means of concentrations measured at 0, 24, 48, and 72 hours) : < LLQ 1 (control), 1. 87 , 3. 04

, 8

. 24 , 1 5

. 9 , and 51

. 6 mg/L;

Overall m ean measured DHP concentrations ( means of concentrations measured at 0, 24, 48 , and 72 hours) : < LLQ 2 (control), 0. 0875

, 0.0517 , < LLQ, < LLQ, and <LLQ mg/L

Test method/conditions remarks: Culture medium: The Algal Assay Protocol (AAP) test medium for the range

- finding and definitive tests was prepared using sterile deionized water and reagent grade chemicals. “ The AAP used in the exposures was made with 20x the standard amount of NaHCO 3 to provide a carbon source for the algae in lieu of the atmospheric CO 2 which was unavailable to the sealed

vessels. The pH of the finished and augmented AAP medium was adjusted to ~ 7.5 prior to use.” [Marino et al. 2015c , p. 11 ]

Selection of test item concentrations: Target test item concentrations selected for the definitive test (1 .6, 4.6, 13, 37, and 106 mg DEPCT/L) were based upon the results of a 72

- hour preliminary range

- finding test that was briefly described in the current study report. This

p reliminary test used 6 inoculated replicates for the control (AAP medium ) and 2 inoculated replicates per treatment level (1, 10, 100 mg/L) under conditions similar to those of the definitive

test. Based on yield data at 72 hours, the EC 50 value in this r ange

- finding study was approximately 100 mg/L. To verify whether the study design could maintain a mass

- balance of the test material and degradation products, surrogate vessels 3 at concentrations of 1 and 100 mg/L were analytically monitored at 0, 24, 48

, and 72 hours; the results of this mass

- balance analysis were not reported.

Preparation of Test Solutions and Inoculation of Cultures: “ To minimize loss and ensure even distribution of test material, individual test solutions were prepared on day 0 in ea ch test vessel

via direct addition of the test material into the medium (AAP) and promptly crimp

- sealing with an aluminum seal containing a PTFE

- lined silicone septum after inoculation of algae. Two glass

beads were added to all test vessels. The vessels were filled at least halfway prior to the addition of test material by Hamilton Gas

- Tight syringe or pipette (when applicable). An aliquot of 0.2, 0.6, 1.7, 4.7, and 13.6 μL of test material (density = 1.2 g/cm 3 , per non

- GLP Zhejiang Zinnong

Chemical Co. , Ltd., 2007) was directly added to test medium (AAP; total volume = 154 mL) to prepare each replicate exposure solution with a target (nominal) concentration of 1.6, 4.6, 13, 37, and 106 mg DEPCT/L, respectively. Control replicates contained AAP without the addition of

test material. Test solutions were swirled to mix after the test material was added. Algal inoculum was then added to yield an approximate initial cell density of 5 x 10 3 cells/mL. Additional AAP was added to fill the vessel completely ( approximately 154

- mL total volume) and then the vessel was crimp sealed. Crimp sealed vessels were vortex mixed to ensure algal cells were evenly dispersed throughout the test solutions.” [Marino et al. 2015c , pp. 12

- 13 ]

1 The LLQ for DEPTh was 0.0750 mg/L.

2 The LLQ for DHP was 0.0750 mg/L.

3 “[V]essels containing no algae, prepared solely for the purpose of analytical measurements.” [Marino et al. 2015c

, p. 12 ]Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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20 “All test solutions were utilized on the same day as preparation and on the same day as initial analytical confirmation; therefore, analysis of stability was [considered by the study authors to be] not necessary.” [Marino et al. 2015c , p. 13 ]

Definitiv e Test Procedure: Prior to the final definitive test, t wo presumptive definitive tests were initiated but determined to

be invalid

. “ The first attempted exposure was determined not to be valid based on poor control growth incapable of satisfying the valid ity criteria for controls. This exposure was repeated and the inoculation density adjusted from 3,000 cells/mL to 5,000 cells/mL. The repeat exposure was terminated after 48 hours cell counts, when poor control growth indicated that all validity

criteria could not be met in that exposure.” [Marino et al. 2015c , p. 13 ] No explanation was provided for the poor control growth.

The final definitive test was conducted under static conditions with seven inoculated replicate

vessels ( initial algal cell density of 5 x 10 3 cells/mL) per test concentration and nine inoculated replicate vessels for the control (AAP medium). Additionally, “[f]our uninoculated replicate

vessels (counting blank) were prepared at each test concentration and control. The additi onal replicates at each test and control level were not inoculated with algae to serve as a counting blank (0 hr vessel [was used only] as a surrogate for pH and analytical [measurements] at test initiation). These blanks were used to correct the daily co unts [by electronic particle counting] for the interference of the test material and to monitor pH without the algal biomass.” [Marino et

al. 2015c , p. 13 ] E ac h test vessel was sealed as described above and was kept sealed (i.e., no prior sampling of test solutions) until the scheduled sampling time. The numbers of vessels sampled at each scheduled sampling time were:

0 hours 24 hours 48 hours 72 hours

Control 1 B 3 I

1 B 3 I

1 B 3 I

1 B

Treated 1 B 2 I

1 B 2 I

1 B 3 I

1 B B = Blank (uninoculated replicate) I = Inoculated replicate

At each scheduled sampling time, “ samples were taken for both analysis of cell counts and

determination of the concentration of test substance and degradation products. At 72 ho urs, samples were also... taken for the measurement of pH and morphological observations. The

vessels were terminated after cell counts, analytical sampling, measurement s of pH and/or observations [were] complete

... Cell counts were not assessed at 0 hours. Sampling [at 0 hours]

was [only] for confirmation of dosing and determination of pH.” [Marino et al. 2015c, p. 15]

“ At test initiation, samples were collected from surrogate vessels for the determination of pH. At test termination, aliquots were collected from inoculated replicates and pooled by test level for measurement of pH. At test termination, pH measurements were [also] taken from blank replicates at each test level. Temperature was continuously monito red with a minimum/maximum thermometer placed in a representative vessel on each shaker table. LightFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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21 intensity was measured at test initiation with a light intensity meter at each position where inoculated replicate vessels were placed during the in

- life phase.” [Marino et al. 2015c , p. 1 4 ]

Environmental Conditions: The sealed test vessels were incubated at 21

- 24°C under continuous lighting of 5200 ± 780 lux and constantly shaken for 24, 48, or 72 hours. The control group was

maintained under identical conditions but not exposed to the test item.

Evaluation of Cell Densities and Appearance: “Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea, California) fitted with a 100 μm aperture tube. Samples were taken by puncturing the septum with a syringe needle and withdrawing the necessary volume from vessels sacrificed at each time point... ; because remaining test solution was sacrificed for analytical analyses, additional test solution was set aside to re

- make samples for cell counts when

necessary. The total volume sampled each day was 5 mL (sample for cell count plus additional test solution) with the exception of blank vessels at test ter mination from which a total of 10 mL

was taken. Total cell counts were determined at approximately 24, 48, and 72 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equi valent spherical diameter of approximately 8.7 μm. Two cell count readings were made per replicate and averaged. The readings for the blank replicates were used to correct for background in daily calculations. The adjusted cell counts were converted to cells x 10,000/mL (cell density) for statistical analysis and reporting.” [Marino et al. 2015c , p. 14 ] Additionally, microscopic observations were conducted on the remaining inoculated replicates

for each test concentration at test termination.

Analytical Monitoring:

“ After samples for cell counts, measurements of pH, and/or observations were complete for selected test vessels... vessels were prepared for analytical analyses. Sample contents in the

bottles were transferred to a 250mL bottle. The sample vessel was rinsed with 25mL of toluene

and toluene was transferred to the 250mL bottle. The bottles were capped and mixed vigorously. The organic and aqueous layers were allowed to separate in the bottle. The organic phase was sampled for the ana lysis of parent test material [DEPCT] via gas chromatography with electron capture detector (GC/ECD). The aqueous phase was sampled for the analysis of the expected degradation products [DEPTh, DHP] via high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS).” [Marino et al. 2015c , p. 15 ]

Statistical Analys e s: For determination of the NOEC values based on 72

- hour yield and average specific growth rate

, t he data (untransformed, square root transformed for number of cells, and t he log of average specific growth rate) were initially checked for normality using the Shapiro

- Wilk’s Test (α = 0.01) and for homogeneity of variance using Levene’s Test (α = 0.01) . Based on the results of these tests, the data sets for yield and average specific growth rate passed the requirement for normality and homogeneity of variance; therefore, a one

- tailed Dunnett’s means comparison procedure (α = 0.05) was used to determine NOEC values

. EC 50 values and 95% confidence

i ntervals based on 72

- hour yield and average specific growth rate were calculated using two sigmoid

- shaped nonlinear models : the four

- parameter logistic model ( percent inhibition versus concentration) and the cumulative normal model ( response versus log concentrati on). TheFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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22 results from the model that best fit the data were reported. All statistical analyses were performed using the SAS version 9.2 software package.

RESULTS

Endpoint values (mg/L) :

The study authors provided the following EC 50 and NOEC values, based on nominal concentrations of DEPCT, measured concentrations of DEPCT at 0 hours , or mean measured concentrations of degradation products (DEPTh or DHP) at 24, 48, and 72 hours ; LOEC values

were inferred by the reviewers as the next higher co ncentration above the NOEC, based on the mean measured values provided by the study authors :

Yield Growth Rate

72

- hr EC 50 (95%

Confidence Interval) NOEC LOEC 72

- hr EC 50 (95%

Confidence Interval) NOEC LOEC

Nominal DEPCT 44.0

(32.5

- 59.6) 4.6 13 > 106 e 13 37

Measured DEPCT ( at 0 hours) a 34.8

(26.1

- 46.2) 1.21 d 5.57 > 74.8 e 5.57 26.1

Measured DEPTh ( at 24, 48, and 72 hours) b 23.4

(19.3

- 27.6) 3.25 10.1 > 60.0 e 10.1 19.5

Measured DHP ( at 24, 48, and 72 hours) b N/A c N/A N/A N/A N/A N/A

a DEPCT concentrations measured in surrogate vessels at test initiation.

b Mean s of measured DEPTh and DHP concentrations at 24, 48, and 72 hours.

c The study report indicated that the statistical analyses performed for DHP concentration data “were unreliable based on the overlapping measured concentrations that did not consistently increase across the test levels.” [Marino et al. 2015c, p. 25]

d The algal yield in the lowest two treatment levels was not significantly different from the control. Th e study authors selected the lower of these two treatment levels as the NOEC because it was the highest measured concentration exhibiting no effect

. ( T he measured concentration of DEPCT at time 0 was higher for the nominal concentration of 1.6 mg/L than i t was for the nominal concentration of 4.6 mg/L ; measured concentrations were 1.21 and 0.952 mg/L, respectively.)

e A 95% confidence interval was not determined.

The reviewers calculated 1 the following endpoint values based on the mean measured

concentrations of DEPCT across all sampling intervals (i.e., 0, 24, 48, and 72 hours) :

1 To determine t he E C 50 , the reviewers used the Hill model in EPA’s Benchmark Dose Software to d etermine the relationship between the biological response and test substance concentration; the resulting equation was used to

calculate the EC 50 by inserting a biological response value that was equivalent to 50% of the control value

.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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23 Yield Growth Rate

EC 50 NOEC LOEC EC 50 NOEC LOEC

Measured DEPCT ( at 0, 24, 48, and 72 hours) 12.7 0.355 1.51 > 34.8 1.51 9.92

Results remarks: Analytical monitoring: For each nominal concentration of DEPCT that was tested in this study, the following table shows the mean measured concentrations of DEPCT and its degradation products, DEPTh and DHP, that were determined at each scheduled sampling t ime (0, 24, 48, and 72 hours):

Mean Measured Concentrations (mg/L) at Each Scheduled Sampling Time a

DEPCT DEPTh DHP

Nominal

concentrations (mg/L) 0 Hours 24

Hours 48

Hours 72

Hours 0 Hours 24

Hours 48

Hours 72

Hours 0 Hours 24

Hours 48

Hours 72

Hours

0 (Control) < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ < LLQ

1.6 1.21 < LLQ < LLQ < LLQ 1.66 1.54 2.16 2.12 < LLQ 0.089 0.103 0.120

4.6 0.952 < LLQ < LLQ < LLQ 2.30 3.61 3.22 3.03 < LLQ < LLQ 0.094 < LLQ

13 5.57 < LLQ < LLQ < LLQ 2.05 11.3 11.0 8.64 < LLQ < LLQ < LLQ < LLQ

37 26.1 7.56 5.88 < LLQ 5.14 20.8 18.5 19.3 < LLQ < LLQ < LLQ < LLQ

106 74.8 44.5 13.5 6.45 31.9 43.9 59.5 71.0 < LLQ < LLQ < LLQ < LLQ

a Means were calculated by the reviewer s as the mean of the measured concentrations in fresh or spent solutions over all sampling intervals. If more than one measurement was available for a given time interval, these values were averaged before taking the average over all time intervals

. (This differed from the method of averaging used by the study authors , who simply took the mean of all measurements .) Only values from replicates containing algae were included in calculations of means (with the exception of data at 0 hours, for which concentr ations were only measured without algae). For values < LLQ, one half of the LLQ was used in the calculation of the mean concentration. If all measurements for a given cell were < LLQ, then the mean is simply provided as < LLQ. (LLQs were 0.313 mg DEPCT/ L, 0.075 mg DEPTh/L, and 0.075 mg DHP/L.)

The study authors calculated mass balance percentages from measured concentrations of

DEPCT

, DEPTh

, and DHP. “These mass balance (%) values were determined from the total moles of parent DEPCT plus total moles of degradation products (DEPTh, DHP) divided by nominal moles of parent, for each exposure solution analyzed at a given sampling interval. An average of these mass balance values was calculated for each nominal exposure concentration across all sampling int ervals.” [Marino et al. 2015b, p. 3 6 ] “ In the 1.6, 4.6, 13, 37, and 106 mg

DEPCT/L nominal test levels, the mass balance averaged (range in parentheses) 126 (84

– 172), 64.7 (52

– 79), 68.5 (44

– 112), 61.1 (20

– 83), and 73.0 (62

– 98)%, respectively. In the three lowest test levels (1.6, 4.6, and 13 mg DEPCT/L nominal), there was no measurable parent test material at 24 hours, indicating DEPCT was not stable under these test conditions. Average

mass balances near or above nominal test concentrations i ndicate that this test design was appropriate for maintaining the dosed test material and associate degradates in solution.” [Marino et al. 2015c , p. 17 ]

“ The apparent variability in mass balance values within and across the nominal exposure

concentratio ns is likely reflective of both imprecision in the test material spiking procedure ( i.e

., dispensing of sub

- microliter volumes) and of variability in quantitative recovery of all soluble and insoluble test material species. When preparing analytical samples, the entire bottle wasFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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24 extracted; thus, it was not possible to re

- prepare samples and no further determination of accuracy could be performed.” [Marino et al. 2015c , p. 17 ]

Exposure conditions: Over the course of the stu dy, “ [t]emperatures during the exposure period ranged from 23

– 24 oC. The pH ranged from 7.4

– 7.5 at test initiation and from 7.4

– 9.4 and

6.9

– 7.4 in replicates both with and without algae, respectively, at test termination. Light intensity ranged f

rom 4450

– 5650 lux from a continually lit source.” [Marino et al. 2015c , p. 17 ]

Biological observations : “ Mean yields at 0

- 72 hours were 59.62, 56.76, 59.42, 44.06, 36.78, and 8.363 (x10 4 ) cells/ml for the control, 1.6, 4.6, 13, 37, and 106 mg DEPCT/L n ominal test levels, respectively. Between 0 and 72 hours, the mean inhibition response relative to the control ranged from 0

– 86% inhibition of yield. At 0

- 72 hours, yields in 13, 37, and 106 mg DEPCT/L nominal test levels were significantly different f rom the control (means of 45.36 or smaller can be declared as significantly less than the control).” [Marino et al. 2015c , p. 18 ]

“Mean specific growth rates between 0 and 72 hours were 1.623, 1.578, 1.595, 1.496, 1.430, and 0.9379 (day

- 1 ) for the contro l, 1.6, 4.6, 13, 37, and 106 mg DEPCT/L nominal test levels, respectively. From 0 to 72 hours, mean inhibition response r elative to the control ranged from 2

– 42% of the mean specific growth rate. Mean specific growth rates for 37 and 106 mg

DEPCT/L nom inal concentration at 0

- 72 hours were significantly different from the control (means of 1.44 day

- 1 [or smaller] can be declared significantly less than the control).” [Marino et

al. 2015c , p. 19 ]

Morphological o bservations: “ Microscopic evaluation of cells at each test concentration and the control at test termination revealed no abnormal observations at any test level in the definitive exposure.” [Marino et al. 2015c , p. 19 ]

The following table summarizes the relationship between percent inhibition (of yield and growth rate) and overall mean concentrations of DEPCT, DEPTh, and DHP over the duration of the study (0

- 72 hours):

Overall Mean Measured Concentrations

(mg/L) a Percentage Inhibition at 72 Hours (%)

Nominal

conce ntrations (mg/L) DEPCT DEPTh DHP Yield Growth

Rate

0 (Control) < LLQ < LLQ < LLQ

- - 1.6 0.420 1.87 0.0875 5 3

4.6 0.355 3.04 0.0517 0 2

13 1.51 8.24 < LLQ 26 8

37 9.92 15.9 < LLQ 38 12

106 34.8 51.6 < LLQ 86 42

a Overall means were calculated by the reviewer s as the mean of the measured concentrations

from all sampling interval s (i.e., 0, 24, 48, and 72 hours). Only values from replicates containing algae were included in calculations of means (with the exception of data at 0 hours, for whichFinal HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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25 concentrations were only measured without algae). For values < LLQ, one half of the LLQ was used in the calculation of the mean concentration. If all measurements for a given cell were

< LLQ, then the mean is simply provide d as < LLQ. (LLQs were 0.313 mg DEPCT/L, 0.075 mg DEPTh/L, and 0.075 mg DHP/L.)

Acceptability Criteria: The definitive test met the following criteria for acceptability prescribed by the protocol: 1) “ The cell density in the control cultures increased exponentially by a factor of at least 16

within 72 hours. The 72

- hour mean cell density increased by approximately 120

- fold, thereby exceeding the minimal criteria. 2) The coefficient of variation average fo r specific growth rates throughout the exposure (days 0

– 3) in replicate control cultures was 3.1%, which is below the limit of 7% set by the guideline. 3) The mean coefficient of variation for section

- by

- section specific growth rates (days 0

- 1,

1

- 2 and 2

- 3, for 72

- hour tests) must be 35% or less. Because vessels were destructively sampled, it was necessary to average the daily values prior to determining the coefficient

of variation. The growth rate (per day) for the average of the control replicates was

c alculated for each time period and the coefficient of variation was calculated for each replicate based on each daily growth rate. 1 The mean coefficient of variation for the control replicates was 27%, which was below the maximum criterion.” [Marino et al.

2015c , pp. 1 9

- 20 ]

CONCLUSIONS

Based on nominal concentrations of DEPCT, the 72

- hour EC 50 for yield was 44.0 mg DEPCT/L

in P seudokirchneriella subcapitata

. The corresponding NOEC and LOEC values were 4.6 and

13 mg DEPCT/L

, respectively. The 72

- hour EC 50 for growth rate

, based on nominal

concentrations of DEPCT

, was > 106 mg DEPCT/L. The corresponding NOEC and LOEC

values were 13 and 37 mg DEPCT/L

, respectively.

Based on DEPCT concentrations measured from surrogate vessels at test initiation , the 72

- hour

EC 50 for yield was 34.8 mg DEPCT/L in Pseudokirchneriella subcapitata

. The corresponding

NOEC and LOEC values were 1.21 and 5.57 mg DEPCT/L, respectively. The 72

- hour EC 50 for growth rate

, based on DEPCT concentrations measured from surrogate vessels at test initiation

,

was > 74.8 mg DEPCT/L. The corresponding NOEC and LOEC values were 5.57 and 26.1 mg DEPCT/L, respectively.

Based on mean measured concentrations of DEPCT over the duration of the study (mean of 0, 24, 48, and 72 hours), the 72

- hour EC 50 for yield was 12.7 mg DEPCT/L in Pseudokirchneriella subcapitata

. The corresponding NOEC and LOEC values were 0.355 and 1.51 mg DEPCT/L, respectively. The 72

- hour EC 50 for growth rate

, based on mean measured concentrations of

DEPCT over the duration of the study (mean of 0, 24, 48, and 72 hours)

, was > 34.8 mg

1 It appears that th e phrase “for each replicate” should be removed from this sentence. Since control vessels were destructively sampled at each time period, it is not possible to calculate a coefficient of variation for section

- by

section growth rate for each replicate.Final HPV2 Test Rule Data Adequacy Review ( August 2015) CASRN 2524

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26 DEPCT/L. The corresponding NOEC and LOEC values were 1.51 and 9.92 mg DEPCT/L, respectively.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. OECD TG 20 1 states that if measured concentrations of the test substance deviate by more than 20% from the nominal concentrations throughout the test, results

should be based on mean measured concentrations. Although the study authors calculated E C 50 value s based on mea sured concentrations of DEPCT at test initiation , they did not provide E C 50 value s based on mean measured concentrations of DEPCT throughout the 72

- hour exposure period

. These EC 50 values would be considered to be more accurate estimate s of the E C 50 of

DEPCT in green algae than the E C 50 s based on measured DEPCT concentrations in solutions at
time 0 only. The reviewers calculated an E C 50 for yield, based on mean measured concentrations

of DEPCT in solutions throughout the exposure period (i.e., mean of 0, 24, 48, and 72 hours),

and found this value to be about 64 % less than the E C 50 for yield based on measured

concentrations of DEPCT in time 0 solutions, therefore providing a more conservative estimate of the toxicity of DEPCT to green algae

. (The EC 50 values for growth rate could not be compared because these values were greater than the highest concentration tested.)

REFERENCE

Marino, T.A., Hutchinson, K.L., Holzheuer, W.B., and Nelson, E.J. 2015c. Phosphorochloridothioic acid, O,O

- diethyl ester : Growth inhibition test with the freshwater green alga, Pseudokirchneriella subcapitata

. Performing Laboratory: The Dow Chemical Company (Toxicology and Environmental Research and Consulting), Midland, MI. Sponsor: Dow AgroSciences LLC.

Text in quotes was taken directly from the report of Marino et al. (2015c).