GNEENWICH CHEMICAL CoNSULTING, Iruc.

3300 PGA BouLEVARD. Surrn 635

Pnru Bpecu GnnnExs, FL 33410

TELrpHONrr. 561.57 8.8 1 10

FecslHrrlp 561.578.8120

T\.} e

Grr

Muy 73,2075

Bv Federal Exutess

-

OPPT Document Control Office

EPA East Building

Room E6428

U.S. Environmental Ptotection Agency

7201 Constitution Ave., Nnfl

Washington, DC 20004

=

ffi

CONTAINS NO CONFID ENTEIL

BUSINESS INFORMATIUd{

CBIC Control hlumber

365025

for the Algae Study Re-conducted by

the TSCA Section 4 Test Rule on the

Volume Chemicals: Third Group of

ffi*:-r

__; ,l--.

t j ! l

.:-]:_,

r"* i t i

Attention:

Re-submission of the Final Test Repot

the PCBTF Testing Consortium undet

Testing of Certain lligh Prcduction

Chemicals

Benzene, 1-chloto-4- (uifluotome thyl)-

CAS RN 98-56.6

(p ar*Chlorobenzotdfluotide (PC BT F))

Docket ID Number: EPA-HQ-OPPT-2009-0112

40 cFR 799.5089

I) ocket I D lrJumb er: EPA-H Q-OPPT -2009 -01' 1.2

40 cFR 799.5089

Control Number 364790

Re:
c oNrAr Nrs N o{ o N5l"_ffi ,#

U.S. Environmental Protection Agency

Il'Iay 13,2015

Page 2

Deat Sit/Madam:

On behalf of the PCBTF Testing Consotdum, Gteenwich Chemical Consultirg, Inc.

(Greenwich Chemical Consulting) re-submits to the U.S. Envitonmental Protection Agency

FPA or the Agency) the final test report for the algae study re-conducted on the chemical

substance, benzene, 1,-chloto-4-(trifluotomethyl) [CAS RI\T 98-56-6] which is subject to a

Toxic Substances Control Act GSCA) Section 4 test rule.l

The final test report and cover letter have already been timely submitted to EPA both: (a)

through the Agency's Central Data Exchange (CD$ system on April 29, 2015 and ft) to the

TSCA Docket on a flash drive (including separate hatd copies of these trvo documents) on

April 23,2075. EPA notes, however, that it was not able to read the files on the flash drive

and requested re-submission of these files on CD. This CD is included hetein.z

The PCBTF Testing Consortium thanks the Agency fot its consideration rn this matter. Fot

all questions and cornments concerning this firattet, please use the contact infotmation given

below.

Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

Telephone: (561) 578-81 10

Facsimile: (561) 578-81 20

E-mail: iohn(Emcci.com

-

Sincerely,

John V. Massingale

Principal

1 76 Fed. Res. 65385; October 21.,2071..

2 Telephone conversations with Ms. Pam lv{osely FPA) on h{ay 11,2015 and IMay 13,201'5.

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GREENWICH CHEMICAL CONSULTING, INC.

33OO PGA BLVD

sunE 635

PALM BEACH GARDENS, FL 33410

SHIP TO: (202) 5fi{-8s30

OPPT Document Control ffiice

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Page I of 1

Frorn: (561) 578-8110

JOHN V. MASSINGALE

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https://3pl.freedomffship.com/viewlahel.php?airbill:78065 0655022&carrier:FedEx sltS/201s
CBIC Control Number

364790

GnEENWICH CHEMICAL CoNSULTING, I

3300 PGA Bour-EvnRD r Surrr, 635

Pnr-u Brecu GnnnErus, FL 33410

Te r-EpuouE 561.578.81 10

F,rr;srurr-r, 561.578.8120

NC;,-i'"

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Ap.iI 23,2015

Bv Federal Exuress

-

OPPT Document Control Oftice

EPA East Building

Room 86428

U.S. Environmental Ptotection Agency

1201 Constitution Ave., I\TW

Washington, DC 20004

Attention: DocketID I'Jumber: EPA-HQ-OPPT-2A09-0112

40 cF'R 799.5089

Dear Sir/Madam:

On behalf of the PCBTF Testing Consotdum,

(Greenwich Chemical Consulting) submits to the

CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

Gteenwich Chemical Consultiog, Inc.

U.S. Environmental Ptotection Agency

Re: Submission of the Final Test Report for the Atgae Study Re-conducted by the

PCBTF Testing Consortium under the TSCA Section 4 Test Rule on the

Tesrtng of Certain Eligh Pruduction Volume Chemicals: Thitd Group of

Chemicals

Benzene, 1-chloto-4-(trifluotomethyl)-

CAS RN 98.56-6

(1t aru-Chlotobenzotrifluotide (PCBT F))

Docket ID Numbet: EPA-HQ-OPPT-2009-0L12

40 cFR 799.5089
CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

U.S. Envrronmental Protection Agency

Ap.il 23,2015

Pa.ge 2

(EPA ot the Agency) the final test repoft fot the algae study te-conducted on the chernical

substance, benzene, 1-chloro-4-(trifluoromethyl)- [CAS RI\T 98-56-6] which is subject to a

Toxic Substances Conttol Act (TSCA) Section 4 test rule.1 Fot purposes of this letter, the

chemical substance, benzene, L-chloro-4-(trifluoromethyl)-, is herein referted to as, PCBTF.

The PCBTF Testing Consortium submits this test report in accordance with this test rule and

the tegulations set fotth at 40 CFR 799.5089.

I. General Background

Greenwich Chemical Consulti"g functions and operates as the panel manager for and

authodzed representative of the PCBTF Testing Consortium. Therefore, Greenwich Chemical

Consulting submits this letter and the enclosed final test report on behalf of the PCBTF

Testing Consortium's 15 member companies identified below:z

. Brenntag North America, Inc.

. Chemsynergy, Inc.

. China Specialty Chemical Company, Ltd.

. EMCO Chemical Distributors, Inc.

. Gteenchem Industries LLC

. I(owa American Colporation

. Makhteshim Agan of North America, Inc.

. MC International, LLC3

. Moris Technology Center, LLC

. Nexeo Solutions, LLC

. Silver Fetn Chemical, Inc.

. Special Matetials Company

. Superior Oil Company, Inc.

. Univat USA, Inc.

. Valspar Coqpotadon

This final test rule addresses 15 high ptoduction volume (HPV] chernicals that went

unsponsored under the Agency's voluntary HPV Chemical Challenge Ptogtam. One of the 15

chemicals identified in the rule is PCBTF. The final rule identifi.es the followlng trvo (2) studies

on PCBTF:

Aquatic Toxicity Testing: toxrcity to plants (algae))

Ready Biodegtadation

The PCBTF Testing Consortium conducted these tests and timely submitted final test reports

to the Agency in its letter dated April 18, 2013. Based on EPA's review of the algae test report,

t 76 Fed. Res. 65385; October 21,2011.

2 See Attachment A for the addresses of member companies.

3 MC International, LLC operates the entity, N{iami Chemical, Inc., as a DBA. For purposes of U.S. Customs

and TSCA, MC International, LLC is the importer.

a

I
coNrAIN'sNoJlR};'f-fffi ffi

U.S. Enr,rronmental Protection Agency

Aprit 23,2015

Page 3

however, th* Agency concluded that the study was inadequate fot purposes of the TSCA

Section 4 test nrle and must be re-conducted. The Agency's conclusions wete set fotth in its

letter of Septembet 26,2A1,3. The algae study was re-conducted as described below.

As required undet 40 CFR 790.50 and 799.5089, the PCBTF Testing Consortium timely

submitted to EPA the study plan for a flew algae test on the cornpound which was developed

by the gtoup's new contract lab, Harlan Laboratodes Ltd. (Hadan Laboratories). A. part of

the development of this study plan, the PCBTF Testing Consottium provided to Hadan

Laboratories the Agency's cotrunents of Septemb er 26,201,3. Hadan Laboratodes developed

this study plan taking into account and incorporating these cofiltnents and also addressing the

technical difficulries associated with testing PCBTF @, poor water solubility and high

volatiliry).

This study plan was subrnitted to the Agency onJanuary 9,2074 (ahead of the revisedJanuary

12,201,4 deadline). In its response dated March 6,201.4, EPA determined that the submitted

study plan was adequate with certain stipulations and modifications. In addiuon, EPA gtanted

the PCBTF Testing Consortium's request to agatrtuse OECD test guideline 207.4 Theteafter,

the PCBTF Testing Consortium worked with Hadan Laboratories to modify the study plan

to incorporate all of the Agency's stipulations and modifications. At the tequest of the PCBTF

Testing Consortium, additional guidance was subsequently requested ftom EPA on the use of

serial dilutions along with other minor matters. The Agency provided this guidance in its letter

dated October 2,2074.

The PCBTF Testing Consortium subsequently finalized the study plrn with Harlan

Laboratories, and the testing program began in }rTovember 2014. The final test report was

issued on April 21,,201.5 and is included herein ahead of the April 30,2015 deadline.

I[. Submission Deadlines for the Study Plan and Final Test Repoft

In its September26,2013 determination letter, EPA stated that a new study plan for the rlgae

test must be submitted to the Agency by Novemb er 12, 2013 and the final report submitted

by October 12, 2074. The Agency subsequendy gtanted the PCBTF Testing Consortium's

extension request dated November 6, 2013 fot a delay to these dates by which the study plan

would be submitted by January 12, 201,4 and final report submitted by Decemb er 12, 2014.

This request was granted by EPA in its response dated November 1.2,201,3.

Because of several technical issues, the PCBTF Testing Consortium requested an addirional

extension in its letter dated Novemb er 4,2014 fot the submission of the final test report. This

date was April 30,2015 which was granted by EPA in its response dated November 10, 2014.

a In the final TSCA Section 4 test de, EPA specified the use of an ASTM test guideline fot the algae study. For

both the first and second algae tests, the PCBTF Testing Consortium requested that the corresponding OECD

test gurdelines 201 be used in the conduct of the algae. The Agency granted this request in both cases.
CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

U.S. Envrronmental Protection Agency

April 23,2075

Page 4

III. List of Final Test Repot

Consistent with the fi.nal TSCA Section 4 test rule, the follo*irg final test report is included

herein.

Benryn4 I +hloml-(trzfluoromet@l) CAS RII ,8-56-5 (para-Chloroheniotrifluoride (PCBffl): Algal

Crowth Inhibition Test

lY. Submission of Robust Summaries thtouqh HPVIS

In the final test rule, EPA requests, but does not requite, that a robust summary also be

included for each test identified ur the rule consistent with the Agency's Draft Guidanm on

DeuelopingRobust Summaries. The Agency previously signaled to the PCBTF Testing Consortium

that the submission of these robust summaries was unnecessary. This is acceptable to the

group. If EPA decides at a later date that it wishes to receive such robust summades, the

PCBTF Testing Consoftium will provide them.

The PCBTF Testing Consotium thanks the Agency fot its considetation in this mattet. For

all questions and commeflts conceming this matter, please use the contact information given

below.

Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

Telephonc (561) 578-81 10

Facsimile (561) 578-8120

E-mail: iohn@.srcci.com

-

Sincetely,

John V. Massingale

Pdncipal

1

II
CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

U.S. Environmental Protection Agency

Ap.il 23,2015

Page 5

Mt. P. Lewis - U.S. EPA

Mr. M. Matteisen - U.S. EPA

Ms. H. Nio - U.S. EPA

Mt.J. Schaeffer - U.S. EPA

PCBTF Testing Consortium

Enclosute
Attachment A - Addresses of Member Comnanies

:Comnanv Addtess

Brenntag North America, Inc. 5083 Pottsville Pike

ReadinE. PA 19605

Chemsynergy, Inc. 1100 Burloak Drive

Suite 101

Burlington, ON L7L 6B.2

Canndz

China Specialty Chemical Company, Ltd. Unit A G.M 9) 3/tr

Cheong Sun Tower

116-118 Wing Lok Street

Sheung Wan

Honq Kong

EMCO Chemical Disributots, Inc. 8601 95th Street

Pleasant Prafuie, WI 53158

Gteenchem Industries, T J.6 222 Clernatis Street

West Palm Beach, FL 33401

Kowa American Co.p. 55 East 59th Street

19,h Floor

Irlew Yotk, NY 10022

Makhteshim Agan of North Amer{ca, Inc. 171 Madison Avenue

Room 1605

NIew York, NY 10016

MC International, LLC 2 NE 40th Street

Suite 50t

Miami, FL 33137

Moris Technology Center, IJ C 9 Ilene Court, Unit #2

Hillsboroush, Nf 08844

Nexeo Solutions, T.T.C Nexeo Solutions, Tf C

3 Watenray Square P1ace

Suite 1000

The Woodlands, TX 77380

Silver Fern Chemical Inc. 2226 Queen Anne Avenue North

Seattle. $fl4 98109

Special Materials Company 70 West 40th Street

2"d Floor

NewYork, NY 10018

Superiot Oil Company, Inc. 1,4A2 l.dorth Capitol Avenue

Suite 100

Indianapolis. IN 46202

Univar USA Inc. 1,7425 NE Union Hill Road

Redmond, $74 98052

Valspar Corporation PO Box 1,461

Minneapolis, MN 55440
harlqlt

REPORT r"

Benzene, l-chloro-4-(trifluoromethyt)- CAS RN 98-56-6

(para-Chlorobenzotrifluoride (PCBTF)):

Algal Growth Inhibition Test

Study Director:

Test f,'acility:

Sponsor:

Study Monitor:

Harlan Study Number:

Study Completion Date:

H Vryenhoef

Harlan Laboratories Ltd.

Shardlow Business Park

Shardlow

Derbyshire

DE72 2GD

UK

PCBTF Testing Consortium

c/o Greenwich Chemical Consulting, [nc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

41304731

21 April 2015

Page I of47
Harlan Study Number: 41304731 Report Page 2

3.4 Procedure............. ........ 13

3.4.1 Preliminary Media Preparation Trial.. ........ 13

3.4.2 Range-Finding Test....... .............. 13

3.4.3 Definitive Test .............. 14

3.4.4 Evaluations ........... ........ 15

3.4.5 Data Analysis ...-............ 16

Validation Criteria ....... l8

RESULTS............ ....... 19

4.1 Range-finding Test ...... 19

4.2 Definitive Test ............. 19

4.2.1 Verification of Test Concentrations............. ............. 19

4.2.2 Growth Data ................. 19

4.2.3 Validation Criteria ........20

4.2.4 Observations on Cultures ............21

4.2.5 Water Quality Criteria .................21

4.2.6 Observations on Test Item Solubility .........21

3.5
Harlan Study Number: 41304731 Report Page 3

LIST OF TABLES

Table I Cell Densities and Percentage Inhibition of Growth from the Rangefinding Test ....... ................24

Table 2 Cell Densities and pH Values in the Definitive Test .....25

Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive

Test........ .............26

Table 4 Inhibition of Growth Rate and Yield in the Definitive Test .........21

LIST OF FIGURES

Figure I Mean Cell Densities v Time for the Definitive Test........ .............28

Figure 2 Inhibition of Growth Rate Based on 0-Hour Measured Test

Concentrations ....... ...,.....,,29

Figure 3 Inhibition of Yield Based on O-Hour Measured Test Concentrations........................30

LIST OF APPENDICES

Appendix I Certificate of Analysis .......... ...... 3 I

Appendix 2 Positive Control ...........32

Appendix 3 Cultr:re Medium. ..........33

Appendix 4 Preliminary Media Preparation Trial .........34

Appendix 5 Analytical Investigations......... ..................35

Appendix 6 Monitoring Authority Statement of GLP Compliance.......... .................47
Harlan Study Number: 41304731 Report Page 4

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Harlan Study Number: 41304731

Study Title: Benzene, 1-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifl uoride (PCBTF)) :

Algal Growth Inhibition Test

This study was performed in compliance with UK GLP standards (Schedule l, Good Laboratory

Practice Regulations lg99 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice

(revised 1997, ENV/MC/CHEM(98)17); and a^re in accordance with, and implement, the

requirements of Directive s 2004 I 9 tEC and 2004 I lOlEC.

These principles axe compatible with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, the United States GPA and FDA),

and Japan (MHLW, MAFF and METI).

This report fully and accurately reflects the procedures used and data generated. There were no

circumstances considered to have affected the integrity of the study or the validity of the data.

Study Director: H Vryenhoef

/r{**,u,....

Da€ zl ApR zoll
Harlan Study Nurnber: 41304731 Report Page 5

QUALITY ASSURANCE STHTEMENT

Harlan Study Number: 41104731

Study Title: Benzene, l-chloro-4-(trifluoromethyl)- CAS RN 98-56-6 (paraChlorobenzotrifl uoride (PCBTF)) :

Algal Growth Inhibition Test

The general facilities and activities are inspected at least once a year and the results are reported

to the relevant responsible person and management.

Study-related procedures conducted at the test facility were audited and inspected. The details of

these audits and inspections are given below.

Dates and Types of QA Inspections

Reported to the

relevant Study

Director and Test

Facility

Management

Date of Inspection TWe of Inspection Phase Inspected Report Date

07 November 2014

26 January 2015

06 January 2015

06 January 2015

20 January 2015

12 March 2015

Study Plan

Verification

Process - based

Process - based

Process - based

Process - based

Report Audit

N/A

Test Item Preparation

Test System Preparation

and Exposure

Assessment of Response

Chemical Analysis

N/A

07 November 2014

26 January 2015

06 January 2015

06 January 2015

20 January 2015

12 March 2015

Verification of Study Plan Amendment for GLP compliance was performed during the course of

the study.

This statement conlirms that this report reflects the raw data and the procedures followed.

Quality Assurance: r-l lN\\tr_

Date: 2 0 ApR
Harlan Study Number: 41304731 Report Page 6

SIGNATURE OF CONTRIBUTING SCIENTISTS

Analytical Chemistry: D M Mullee

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire,DB7} 2GD, UK

2rmn20u
Harlan Study Number: 4 I 30473 I Report PageT

SUMMARY

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga

Pseudokirchneriella subcapitata. The rnethod followed that described in the OECD Guidelines

for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth

Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Information provided by the Sponsor indicated the water sotubility of the test item to be in the

range of 29 - 34 mglL. A preliminary media preparation trial indicated that a dissolved test item

concentration of approximately 57 mglL was obtained from a saturated solution method of

preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudohirchneriella subcapitafa was exposed to

solutions of the test item at nominal concentations of 1.0, 3.2, 10, 32 and 100% v/v saturated

solution (equivalent to O-Hour measured concentrations of 0.37, 0.84, 3.7 , 16 and 5l mg/L) (five

replicate flasks per concentration) for 72 horus, under constant illumination and shaking at a

temperafire of 24 + I oC. The test item solutions were prcpared by stirring an excess

(100 mg/L) of test item in culture medium using a magnetic stirrer at a rate such that a 504 vortex

was forrned for 24 hours. After the stirring period any undissolved test item was removed by

filtration (0.2 pm Sartorius Sartopore filter, first approximate 2 liters discarded in order to precondition the filter) to produce a 100% v/v saturated solution of the test item (equivalent to a

O-Hour measured concentration of 51 mg/I.). This saturated solution was then firrther diluted as

necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each

control and treatment group, using a Coulter@ Multisizer Particle Counter.

Due to the volatile nature of the test item, testing was conducted in completely filled (i.e., no

headspace), stoppered test vessels in order to minimize possible losses due to volatilization.

Following the recorrmendations of published data (Herman et al 1990) in order to prevent

inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate

was added to the culture medium to provide a source of carbon dioxide for algal growth.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations of 0.37, 0.84,

3.7,16 to 51 mg/L were obtained from the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test

preparations respectively. There was no significant change in the measured concentrations at 24,

48 and 72 hours (85% to 98% of the 0-Hour measured concentrations) and so the results are

based on 0-Hour measrued test concentrations only.
Harlan Study Number: 41304731

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on

the 0-Hour measured test concenhations:

Response

Yeriable EQ6 (mg/L) 95Yo Conlidence

Limits (mS/L)

No Observed Effect

Concentration (NOEC)

(mg/t)

Lowest Observed

Effect Concentration

(LOEC) (mg/L)

Growth Rate 6.2 4.8 8.1 0.84 J.t

Yield 2.2 1.8 2.6 0.84 5.t
Harlan Study Number: 41304731 Report Page 9

GENERAL INFORMATION

Schedule

Experimental Starting Date :

Experimental Completion Date:

29 Decemher 2014

23 January 2015

Ileviations from Study Plan

There were no deviations (unplanned changes) from the study plan.

Archiving

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific

supplement), all raw data (paper and electronic) and the final report will be retained in the Harlan

Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as

to furdrer retention or disposal. Funher retention or retum of the data will be chargeable to the

Sponsor.

No data will be discarded without contacting the Sponsor to obtain their written consent.
Harlan Study Number: 41304731 Report Page l0

1 INTRODUCTIONAND PURPOSE

This report contains a description of the methods used and results obtained dgring a study to

investigate the effect of the test item on the growth of the green alga Pse udohirchneriella

subcapitata.

Pseudolcirchneriella subcapitata is a freshwater unicellular alga, representative of primary

producers found in natural waters and can therefore be considered as an important non-target

organism in freshwater ecosystems.

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water

soluble test items, a modification of the standard method for the preparation of aqu.oui media

was performed. An approach endorsed by several important regulatory authorities in the EU and

elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of

the test item in cases where the test item is of high pudty and is poorly soluble in water and in

the permitted ar.r,xiliary solvents and surfactants. Using this approach, a saturated solution was

prepared hy stining an excess (100 mg/L) of test item in culture medium for a period of 24 hours

prior to removing any undissolved test item present by filnation (0.2 pm Sartorius Sartopore,

first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated

solution of the test item.

1.1 Guidelines / Regulations

This study was desigled to be compatible with the procedures indicated by the foltowing

internationally accepted guidelines and reconrmendations :

. OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and

Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission

Regulation (EC) No 761/2009

. OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances

and Mixtures. OECD Series on Testing and Assessment, No. 23. OECD Publishing.
Harlan Study Number: 413047i1 Report Page 1l

2 TEST ITEM

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is

given in Appendix 1.

Identification:

Physical state/Appe arance :

Batch:

Purity:

Exprry date:

Storage conditions:

Intended use/Application:

Benzene, l -chloro-4-(trifluoromethyl)- CAS RN 98-56-6

(para-Chlorobeneotrifluoride (PCBTF))

Clear colorless liquid

2014-02-08

99.77%

17 February 2016

Room temperature in the dark

Chemical intermediate
Harlan Study Number: 41304731 Report Page 12

3

3.1

Biomass:

EC*:

Growth:

Growth Rate (Average Specific

Growth Rate):

Yield:

LOEC (Lowest Observed

Eftect Concentrati on) :

NOEC (No Observed Effect

Concentration):

Treafinent:

Section-by-Section Growth

Rate:

MATERIALSANI} METHODS

Definitions and Abhreviations

Measurement variable (algal cell density) as surrogate measure

for algal biomass.

The calculated concenfration of test item that results in an xo/o

reduction of the respective growth parameter relative to the

conffol within a stated exposure period.

The increase in biomass over the test period.

The logarithmic increase in biomass during the exposure

period.

The value of the measurement variable at the end of the

expoflue period minus the measurement variable's value at the

start of the exposure period, calculated to express biomass

increase dwing the test.

The lowest test concenhation at which a statistically significant

inhibition of gowth is determined relative to the conffol.

The highest test concentration at which no statistically

significant inhibition of grourttr is determined relative to the

control.

Comprises test item treatments and control (test medium only).

Specific growth rate for each day during the course of the test

(days 0-1, l-2 and 2-3).

3.2 Test System

The test was carried out using Pseudokirchneriella subcapitala shain CCAP 278/4. Liquid

cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae

and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll,

Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of

culture medium (Section 3.3). The master cultures were maintained in the laboratory under

constant aeration and constant illumination at2l + I "C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL

volumes of culture media contained in conical flasks to give an initial cell density of

approximately 103 cells/ml. The flasks were plugged with polyilrethane foam stoppers and kept

r.mder constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 t I "C

until the algal cell density was approximately 104 - l0scells/ml.
Harlan Study Number: 41304731 Report Page 13

A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference

item. Details of the positive control are given in Appendix 2. The positive control was

conducted between 14 October 2014 and 3l October}}l4.

3.3 Test Water

The cultrue medium used for both the range-furding and defuritive tests was the silme as that

used to maintain the stock culture. For the pu{poses of testing an additional 500 m/L of sodium

bicarbonate was added to counteract the increase in pH due to algal growth in an enclosed

system (Herman et al 1990). At the request of the Sponsor the pH of the culture mediurn was

adjusted to pH 7.0 after the addition of the sodium bicarbonate.

The culture medium is defined in Appendix 3.

3.4 Procedure

3.4.1 Preliminary Media Preparation Trial

Information provided by the Sponsor indicated the water solubility of the test item to be in the

range of 29 - 34 mg/L based on the U.S. EPA's citation of a Merck Index of r:nknown reliability.

Based on this information the test item was categorized as being a 'difficult substance' as

defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances

and Mixtnres (OECD 2000). Therefore a media preparation trial was conducted in order to

determine the solubility of the test item under test conditions (see Appendix 4).

3,4,2 Range-Finding Test

The results obtained from the preliminary media preparation trial conducted indicated that a

dissolved test item concentration of approximately 57 mglL could be obtained using a saturated

solution method of preparation.

The test concentrations to be used in the definitive test were determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseerdokirchneriella subcapitata

cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution

(equivalent to 0-Hour measured concentrations of 0.067, 0.60, 5.0 and 50 mg/L) for a period of

72 hours.

A nominal amorurt of test item (1090 mg) was dispersed in 10.9 liters of culflre medium with the

aid of magnetic stirring at a rate such that a 50/o vortex was formed for 24 hours. After 24 hours

the stirring was stopped and any undissolved test item was removed by filtation through a

0.2 pm Sartorius Sartopore filter (frst approximate 2liters discarded in order to pre-condition

the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this
Harlan Study Number: 41304'13l Report Page 14

saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An

aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension

Q.7 mL) to give the required test concentrations of 0.10, 1.0, l0 and 100% v/v saturated solution

(equivalent to O-Hour meastued concenftations of 0.068, 0.60, 5.0 and 50 mgll).

The test was conducted in 250 mL glass conical flasks each completely filled (i.e., no headspace)

with test preparation and sealed with a ground glass stopper to reduce evaporation and minimize

losses due to the test items volatile nahue. Two replicate flasks were used for each control and

test concenffation.

The confrol group was maintained under identical conditions but not exposed to the test item.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multiuon@ Version 2

incubator) at 24 * I oC under continuous illumination (intensity approximately 7000 lux)

provided by warm white lighting (3S0 - 730 nm) and constantly shaken at approximately

150 rpm for 72 hours. Previous experience gained has shown that the use of ground glass

stoppers to seal test vessels is effective in attaining an airtight seal, thus preveting losses of test

item through volatility. This was demonstrated in this test by the analytical results obtained at 0

and72 hours.

After 72 hours the cell density of each flask was determined using a CoulteP Mdtisizer Paticle

Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to

determine the stability of the test item under test conditions.

3.4.3 Ilefinitive Test

Based on the results of the range-finding test the following test concentrations were assigned to

the definitive test: 1.0,3.2, 10, 32 and I 00% v/v saturated solution.

3.4.3.1 Experimental Preparation

A nominal amount of test item (1100 mS) was dispersed in ll liters of culture medium withthe

aid of magnetic stirring at a rate such that a 5oZ vortex was formed far 24 hours. After 24 horus

the stirring was stopped and any undissolved test item was removed by filtration through a

0.2 pm Sartorius Sartopore filter (first approximate 2liters discarded in order to pre-condition

the filter) to give a 100% v/v satuated solution. A series of dilutions was made from this

saturated solution to give stock solutions of 32, 10, 3,2 and I .\Yo vlv satwated solution. An

aliquot(2 liters)of eachof thestocksolutionswasseparatelyinoculatedwith 5.2mLof algal

suspension to give the required test concentrations of 1.0,3.2, 10,32 and 100% v/v saturated

solution (equivalent to 0-Hour measured concentrations of 0.37, 0.84, 3.7,16 and 5l mg/L).
Harlan Study Number: 4t304731 Report Page l5

The stock solutions and each of the prepared concentrations were inverted several times to

ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verifi.ed by chemical

analysis at0,24,48 and 72 hours (see Appendix 5).

3.4.3.2 Exposure Conditions

As in the range-finding test 250 mL glass conical flasks each completely frlled (i.r., no

headspace) with test solution were used. Of the five test replicates prepared at the start of the

test: (a) one replicate was kept sealed (with no prior sampling) until 24 hours, at which time

samples were taken for both cell density determination and chemical analysis; this replicate was

then discarded; (b) one test replicate was kept sealed (with no prior sampling) until 48 hours, at

which time samples were taken for both cell density determination and chemical analysis; this

replicate was then discarded; (c) three test replicates were kept sealed (with no prior sampling)

until 72 hor:rs, at which time samples were taken for both cell density determination and

chemical analysis. Therefore, each of the test replicates containing treated test solutions was

used only once for sampling before being discarded.

Of the nine control replicates prepared at the start of the test: (a) three replicates were kept sealed

(with no prior sampling) until 24 hours, at which time samples were taken for cell density

determination then discarded; (b) three replicates were kept sealed (with no prior sampling) until

48 hours, at which time samples were taken for cell density determination then discarded; and (c)

three replicates were kept sealed (with no prior sarnpling) until 72 hours, at which time samples

were taken for both cell density determination and chemical analysis.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditiorls gave an algal suspension in log phase growth characterized by a cell

density of l.16 x l0o cells per mL. Inoculation of 2 liters of test medium with 5.2 rn]- of this

algal suspension gave an initial nominal cell density of 3 x 103 cells per mL and had no

significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron@ Version 2

incubator) at 24 + I oC under continuous illumination (intensity approximately 7000 lux)

provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately

150 rpm fot 72 hours.

3.4,4 Evaluations

3.4.4.1 Test Organism Observations

The O-Hour cell density was determined by electronic particle counting (Coulter@ Multisizer

Particle Cotrnter) of the pre-culture and division by the dilution factor. Samples were taken at
Harlan Study Number: 41304731 Report Page 16

24, 48 and 72 hours and the cell densities determined using a Coultero Multisizer Particle

Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was

removed from each test and control culture (replicates pooled) at the end of the test. Thi shape

and size of the algal cells was inspected microscopically and any abnormalities recorded

3.4.4.2 ril/ater Qualrty Criteria

The pH of the control and each test preparation was determined at initiation of the test and after

72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The

temperature within the incubator was recorded daily.

3.4.4.3 Verification of Test Concentrations

Samples were taken from the control and each test group from the bulk test preparation at

0 hor:rs and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples

were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

A sample of the vessel sacrificed for cell density determination at 24 and48 hours was taken for

chemical analysis.

The method of analysis, recovery and test solution analyses are described in Appendix 5.

3.4.5 Data Analysis

3.4.5.1 Comparison of Growth Rates

The average specific growth rate for a specified period is calculated as the logarithmic increase

in biomass from the equation:

.. _ 

lnNo -lnN,

t, -t,

Where:

average specific growth rate from time t1 to t1

cell concentration at t1

cell concentration at tn

time of first measurement

time of ntr measurement

Fr:

N1 :

N"_

tr:

tn:
Harlan Study Number: 41304731 Report Page 17

The average specific growth rate over the test duration was calculated for each replicate control

and test item vessel using the nominally inoculated cell concentration as the starting value rather

than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1 ,l-Z and 2-3) was calculated for

the control cultues and the results examined in order to determine whether the growth rate

remained constant.

Percentage ffibition of growth rate for each replicate test item vessel was calculated using the

following equation:

I.-.tl"-Pt".x100

lro

Where:

I, : percentage inhibition of average specific grorarth rate

tl" = mean average specific growth rate for the control culfures

pt = average specific growth rate for the test culfure

3.4.5.2 Comparison of Yield

Yield is calculated as the increase

equation:

Y=No -No

Where:

in biomass over the exposure period using the following

Y:

No:

Nn:

yield

cell concentration at the start of the test

cell concentration at the end of the test

concentation and control the mean value for

calculated. Percentage inhibition of yield was

yield along with the standard

calculated using the following

For each test

deviation was

equation:

r - 

ff.- Y,) r.. x 100 ,Y,
Harlan Study Number: 41304731 Report Page l8

Iy:

Yc=

Y,:

Where:

percentage inhibition of yield

mean value for yield in the control group

mean value for yield for the treatment group

3.4.5.3 Determination of EC* Values

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic a1ris)

was plotted against test concentration (logarithmic axis) and a line fitted by computerizei

interpolation using the Xlfit software package (IDBS). EC* values were then determined from

the equation for the fiUed line.

Where appropriate 95% confidence limits for the ECso values were calculated, using the

simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

3.4.5.4 Statistical Analysis

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal

and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treafinents

with a control (Dunnett, 1955) was carried out on the growttr rate and yield data after 72 hours

for the control and all test concentrations to determine any statistically significant differences

between the test and conhol groups. All statistical analyses were performed using the SAS

computer software package (SAS, 1999 - 2001).

3.5 Validation Criteria

The results of the test are considered valid if the following performance criteria are met:

' The cell concentration of the control cultures must increase by a factor of at least

l6 over the test period.

' The mean of the coefficients of variation of the section by section daily growth rates in

the control cultr.ues during the course of the test (days 0-1, l-2 and 2-3) must not

exceed 35%.

The coefficient of variation of the average specific growth rate in replicate control

cultnres must not exceedTo/o.
Harlan Study Number: 41304731 Repot Page 19

4 RESULTS

4,1 Rang+finding Test

The cell densities and percentage inhibition of growth values from the exposure

Pseudohirchneriella subcapitata to the test item during the range-finding test are given

Table 1.

The results showed no effect on growth at the test concentrations of 0.10 and I.0% v/v saturated

solution. Howevet, growth was observed to be reduced at l0 and 100% v/v saturated solution.

Based on this information test concentrations of 1.0, 3 .2, 10,32 and 100% v/v saturated solution

were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours (see Appendix 5) showed measured test

concentrations to range from 0.067 to 50 mg/L whilst concentrations in the range of 0.059 to

48 mg/L were observed at72 hours (74%to 97Vo of the 0-Hour measured concentrations).

4.2 Definitive Test

4,2.1 Verification of Test Concentrations

Analysis of the test preparations at 0 hours (see Appendix 5) showed measured test

concentrations of 0.37, 0.84, 3.7,16 to 51 mg/L were obtained from the 1.0,3.2, 10, 32 and

100% v/v saturated solution test preparations respectively.. There was no significant change in

the measured concentrations at 24, 48 and 72 hours (S5% to 98% of the O-Hour measured

concentrations) and so the results are based on 0-Hour measured test concentrations only as

recommended in Section 39 of the OECD Test Guideline No.201.

4,2.2 Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given

in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Grourth rate

and yield values for the control and test cultures after 72 hows and percentage inhibition values

are given in Table 4.

The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage

inhibition values are plotted against test concentration in Figure 2 and Figure 3.

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of

Pseudokirchneriella subcapitara (CCAP 278/4) were affected by the presence of the test item

over the 72-Hour exposure period.

Accordingly the following results were determined frorn the data:

of

in
Harlan Study Number: 41304731 Report Page 20

4.2.2.1 Inhibition of Growth Rate

E.Cro (0 - 72 h) : 1.8 mg/L

E'Ceo (0 - 72 h) :2.9 mglL

ErCso (0 - 72 h) :6.2mglL;95% confidence limits 4.g - 

g.t mg/L

where ErC* is the test concenfration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test

concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity

of variance (Sokal and Rohlfl 19Sl) and Dunnett's multiple comparison procedure for comparing

several treatments with a conhol (Dru:nett, 1955). There were no statistically signifrcant

diffetences between the control, 0.37 and 0.84 mg/L test concentrations (P>0.05), however all

other test concentrations were significantly different (P<0.05) and, therefore the "No Observed

Effect Concentration" (NOEC) based on growth rate was 0.84 mg/L. Correspondingly the

"Lowest Obsenred Effect Concenfration" (LOEC) based on growth rate was 3.7 mglL.

4.2.2.2 Inhibition of Yield

&Cro (0 - 72 h) : 0.87 mg/L

&Czo (0 - 72 h) : l.Zms/L

&Cso (0 - 72 h) : 2.2 mglL;95Yo confidence limits 1.8 - 2.6 mglL

Where:

&C* is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no

statistically significant differences between the control, 0.37 and 0.84 mg/L test concentrations

(P>0.05), however all other test concentrations were significantly different (P<0.05) ffid,

therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.84 milL.

Correspondingly the "Lowest Observed Effect Concenhation" (LOEC) based on yield was

3.7 mglL.

4.2.3 Yalidation Criteria

The following data show that the cell concenhation of the control cultures increased by a factor

of 190 after 72 hours. This increase was in line with the OECD Guideline that states the

enhancement must be at least by a factor of 16 after ?2 hours.

Mean cell density of control at 0 hor.rs :

Mean cell density of control at72 hours :

3.00 x 103 cells per mL

5.96 x 10s cells per mL
Harlan Study Number: 41304731 Report Page 2l

The mean coefficient of variation for section by section specific $owth rate for the control

cultures was 22To and hence satisfied the validation criterion given in the OECD Guideline

which states the mean must not exceed 35%.

The coeffi.cient of variation for average specific growth rate for the control culfires over the test

period (0 - 72 h) was 2o/o and hence satisfied the validation criterion given in the OECD

Guideline which states that this must not excee d 7%.

4.2.4 Obseruations on Cultures

All test and contol cultures were inspected microscopically at72 horrs. After 72 horus there

were no abnormalities detected in the control or test cultures at0.37,0.84, 16 and 5l mg/L,

however some enlarged/swollen cells were observed to be present in the test cultr.res at

3.7 mgtL.

4,2.5 lVater Quality Criteria

The pH values of the control and each test preparation are given in Table 2. Temperaflue was

maintained at 24 + I "C throughout the test.

The pH value of the confrol cultues (see Table 2) was observed to increase from pH 7.9 at

0 honrs to pH 9.4 at 72 hours. The pH deviation in the control cultures was equal to 1.5 pH units

after 72 hours and therefore was within the limits given in the Test Guidelines.

4.2.6 Observations on Test Item Solubility

At the start of the test all control and test cultures \ryere observed to be clear colorless solutions.

After the 72-Hour test period all control, 0.37 and 0.84 mg/L test cultures were observed to be

green dispersions. The 3.7 mg/L test cultures were observed to be pale green dispersions whilst

the l6 and 51 mg/L test cultures were observed to be clear colorless solutions.
Harlan Study Number: 41304731 Report Page22

5 CONCLUSION

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been

investigated over a 72-Hour period and based on the O-Hour measured test concenhations gave

the following results:

Response

Yariable EC5j (mg/L) 959/o Confidence

Limits (mg/L)

No Observed Effect

Concentretion (NOEC)

(mg/L)

Lowest Observed

Effect Concentration

(L{}EC) (me/L)

Growth Rate 6.2 4.8 8.1 0.84 3.7

Yield )) t.8 2.6 0.84 3.7.
Harlan Study Number: 4 I 30473 I Report Page 23

6 REFERENCES

DI-INNETT, C.\tr/. (1955) A Multiple Comparison Procedure for Comparing Several Treatrnents

With a Control . J Am Stat Assoc S0, 1096-l l2l.

ENVIRONMENT DIRECTORATE, ORGANISATION FOR ECONOMIC CO-OPERATION

AND DEVELOPMENT (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of

Difficult Substances and Mixtures.

EUROPEAN CENTRE FOR ECOTOXICOLOGY AhID TOXICOLOGY OF CHEMICALS

(ECETOC) Monogaph No. 26 (1996) Aquatic Toxicity Testing of Sparingly Soluble, Volatile

and Unstable Substances.

HERMAN et al. (1990) Impact of volatile aromatic hydrocarbons, alone and in combination, on

growth of the freshwater alga Selenastrum capricornutum. Aquatic Toxicologt lS, 87 - 100

LITCHFIELD, J.T. and WILCOXON, F. (1949) A Simplified Method of Evaluating Dose-Effect

Experimerrts. J Pharmacol Exp Ther 96, gg-l13.

MAYER, P., NYHOLM, N., VERBRUGGEN, E. M. J., HERMEhls, J. L. M. and TOLLS, J-

(2000) Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials.

Environmental Toxicologt and Chemistry,l9, pp. 2SSI - ZSSG.

SAS/STATProprietary Software Release S.02 (1999 - 2001), SAS Institute Inc, Cary,NC, USA.

SOI(AL, R.R. and ROHLF, F.J. (1981) Biometry. New York: W H FREEMAN and Company.

Xlfit, ID Business Solutions Ltd.
Harlan Study Number: 41304731 Report Page 24

TABLES

Table I Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

' Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from

3 counts for each of the replicate flasks.

R1 and R2: Replicates I and 2

[Increase in growth compared to confiols ]

Nominal Concentration

(o/o vlv Saturated Solution)

Cell Densities* (cells per mL) Inhibition Yalues (7o)

0 Hours 72 Hours Growth Rate Yield

Conftol

R1 3.008+03 5.31E+05

R, 3.00E+03 5.94E+05

Mean 3.96f,+03 5.62E+05

0.10

R1 3.00E+03 6.40E+05

0 t2l R2 3.008+03 s.l2E{5

Mean 3.008+03 5.76E+05

1.0

R1 3.00E+o3 5.65E+05

R2 3.00E+03 4.638+05 3 I

Mean 3.00E+03 5.14E+05

l0

R1 3.00E+03 7.87E+04

R2 3.00E+03 6.20E+04 40 88

Mean 3.00E+03 7.038+04

100

R1 3.00E+03 3.63E+03

R2 3.00E+03 2.99E+03 99 100

Mean 3.00E+03 3.31E+03
Harlan Study Number: 4 130473 I Report Page 25

Table 2 Cell Densities and pH values in the Definitive Test

' Cell densities represert the mean number of cells per mL calculated from the mean of the cell corurts from

3 counts for each of the replicate flasks.

Rr -& : Replicates 1 to 3

Nominel Concentretion

(o/o vlv Saturated Solution)

pH Cell Densities* (cells per mL) pH

0h 0h 24h 48h 72h 72h

Control

R1

7.9

3.00E+03 t.3lE+04 l.l8E+05 5.72E+05

9.4

R2 3.00E{3 l.l6E+04 1.09E+05 5.50E+{5

R3 3.00E+03 l.3lE+04 l.l lE+05 6.67E{5

Mean 3.00E+{3 1.26E+04 1.13E+05 5.96E+05

1.0

R1

7.7

3.00E+03 1.228+04 1.23E+05 6.52E+05

9.1

R, 3.00E+03 6.10E+05

R3 3.00E+03 6.298+05

Mean 3.008+03 1.228+04 1.23E+05 6.30E+05

3.2

R1

7.6

3.00E+03 1.20E+04 8.41E+04 4.69E+05

9.2

R, 3.00E+03 6.29E+05

R3 3.00Er{3 4.96E+05

Mean 3.00E+03 1.20E+04 8.41E+04 5.32E+05

r0

R1

7.5

3.00E{3 6.13E+03 2.978+04 1.308+05

8.1

R2 3.00E+03 1.37E+05

& 3.00E+03 1.39E{5

Mean 3.00E+03 6.13E+03 2.97E+04 1.35E{{5

32

R1

7.5

3.00E+03 2.75E+03 5.42E+03 6.l9Er{3

7.7

R2 3.008+03 5.49E-+{3

R3 3.00E+03 6.318+03

Mean 3.008+03 2.75E+03 5.42E+03 5.99E+03

100

R1

7.4

3.00E+03 1.55E+03 3.74E+03 5.63E+03

7.5

R2 3.00E+03 4.63E+03

R3 3.00E+03 6.69E+03

Mean 3.008+03 1.55E+03 3.74E+03 5.658+03
Harlan Study Number: 41304731

Table 3 Daily Specific Growth Rates for the Control Cultures in the llefinitive Test

Daily Specific Growth Rate (cells/mlJhour)

Dey0-l DayI-2 Day2-3

Confrol

Rr 0.062 0.092 0.066

R2 0.056 0.093 0.067

& 0.062 0.089 0.075

Mean 0.060 0.091 0.069

Rr - Rr : Replicates I to 3
Harlan Study Number 41304731 Report Page27

Table 4 Inhibition of Growth Rate and Yield in the llelinitive Test

* In accordance with the OECD test guideline only the mean value for yietd for each test concenfration is

calculated

Rr - &: Replicates I to 3

SD : Standard Deviation

[Increase in growth compare to controls]

Nominal Concmtrrtion

("/o vlv Saturated Solution)

Growth Rate (cells/ml/hour) Yield (cells/ml)

0-72 h % Inhibition 0-72h 9/o Inhibition*

Control

Rl 0.073 5.69E+05

R2 o.072 5.47E+05

R, 0.075 6.64E+05

Mean 0.073 5.93E+05

SD 0.002 6.228+04

t.0

Rr 0.07s -3 6.49E+05

t6l

& 0.074 1 6.07E+0s

R3 0.074 I 6,26E+05

Mean 0.074 -l 6.27E+05

SD 0.001 2.12E+04

3.2

Rr 0.070 4 4.55E+05

1l

R2 0.074 I 6.26E+05

R3 0.071 J 4.93E+05

Mean 4.072 ) 5.29E+05

SD 0.002 8.59E+{4

10

Rl 0.052 29 1.27F+i5

78

& 0.053 27 1.34E+05

& 0.053 27 1.36E+05

Mean 0.053 27 1.32E+05

SD 0.001 4.67E+03

32

Rl 0.010 86 3.19E+03

99

R, 0.008 89 2.49E+03

R3 0.010 86 3.318+03

Mean 0.009 88 2.998+03

SD 0.001 4.ME+02

100

RI 0.009 88 2.63E+03

100

R2 0.006 92 1.63E+03

R3 0.01I 85 3.69E+03

Mean 0.009 88 2.65E+03

SD 0.003 1.03E+03
Harlan Study Number: 41304731 Report Page 28

FIGURES

Figure I Mean Cell Densities v Time for the Delinitive Test

Ft

E

tt

0) U

>.

a

E

fl

(l}

U

1.008+06

1.00E+05

1.00E+04

1.008+03

-€- 3.7mg/L

+- 16mg/L

-€- 51mg/L

Time(Ilours)
Harlan Study Number: 41304731 Report Page 29

Figure 2 Inhibition of Growth Rate Based on O-Hour Measured Test Concentrations

r00

90

80

70

60

50

40

30

20

l0

0

-10

0.1

Concentration (me/L)

- -L _ l_ _r_ _,-

--L-l-J-J-

-r___f--i-

-r---J--r-

-r---1"_Io

-o

10o\

l0 100
Harlan Study Number: 41304731 Report Page 30

Figure 3 Inhibition of Yield Based on 0-Hour Measured Test Concentrations

o

IJ

E

1€o\

100

90

80

70

60

50

40

30

20

IO

0

-10 L

0.1

ntt,rtir i i rr::i:i i i i/i liiii I i iiiiii i i t::il:i i j 

_ I lt:lll I I ; litij

_- _ _ _ _ _.- _ - -n_ __i__r_ _ i_r-.-r- I i iiiiiii i ifi iiiiii i i iiiiii

i i i itiiii t ', I i:iilt : i : lilli r I I i i iiii l, i I i : :lil i I i I I :ll

' -"- , i L i li I i i i I i : l

i '-i"'-'i"ii'1--i-i-i--'--'--/'i-'--j-'-'-i'--j--j'-i-l-i-j"---'---'-1'-- j ,t i Tii-i'i- I' i | | i :iii: I | : I I lll:t i I i I iiii

: i i l::iil I i : I tiiii: I I i ilt;i tt : I r :iilii I i I i ili:ii i : i ::l:i

i : I iii',ii/ I i I tll:lr : I : ::rrr i ; ; ;iiiiil i I i illlil I I i iil:l i i iiii'":.,{ i i iiiiiii i i iiiiii --------r-"---t-----i---i i-;--W;---- r -i,..-1 iii]_lI j--.---j----j.iiiii

t t/F t t

t}Ftttrl |

iii-:,-{tiiiiiiiiiiii iiiiiiii

l0 I00

Concentration (mg/L)
Harlan Study Number: 41304731 Report Page 3 I

APPBNDICES

Appendix I Certificate of Analysis

trrilFuprrty

CEnflFlED TRI.E AfE CoEffiicT

fmrumU trrhZJ|T/2014

He fraddon Etr:

GERTIFICATE OF AHALYSIS

^ .J(, ,0ftrr.00,srffir ffiffiffi

ProtrmilIfirnr; Parffi

Audtg SilC Smc. 000

Brtch Hulntrr: 2Ol4-O248

epefficrtbn

Acldtf, ppm {spe. hn probel

Alrlhlty, ppnl lls tgoH

ApprsrmaG

Actay. i6

Cobr(AP161

ilm Volilb Fhaidw. Wt. %

Sp.clfrc cmvity (20C/2SC)

Wgl*oofitcfll, ppm

S.0 PPlrl MN(IMUM

10.0 PFM riAxtirt H

Cl€tr llqdd. IrtG ot ru*pondrd

maElhl

90.00 16 MrHtMuM

?0 APHA tvl$(lMUM

0.0020 9t MNflHUM

r.3300To 1.3500G/CC

150 FFf,I Mru(IUUM

t.7

0.0

Glcff liq.dd, hlrr of :uipandtd

mrtedal

90.77

IO

0,0013

1,3S{0

78

hldDfr: lrnsrtl
Harlan Study Number: 41304731 Report Page 32

Appendix 2 Positive Control

A positive control (Harlan Study Number 41403365) used potassium dichromate as the reference

item at concentations of 0.050, 0.13, fr.32,0.80 and 2.0 mg/L.

Six flasks each completely filled with test preparation were used for the control, and three flasks

each completely filled with test preparation were used for each test concentration. The flasks

were sealed with ground glass stoppers and incubated at 24 + I oC r:nder continuous illumination

(intensity approximately 7000 lux) provided by wann white lighting (3S0 - 730 nm) and

constantly shaken at approximately 150 rpm for 72 hours. The culture medium used was

prepared with the addition of 500 mg/L NaHCOg.

Exposr:re of Pseudokirchneriella subcapitata (CCAP 27514) to the reference item gave the

following results:

E Cso (0 - 72 h) : I .2 melL;95% confidence limits 0.97 - 1.4 mg/L

&Cso (0 - 72 h) : 0.52 mglL;95% confidence limits 0.45 - 0.60 mgil,

No Observed Effect Concentration (NOEC) based on growth rate: 0.32 mglL

No Observed Effect Concentration (NOEC) based on yield: 0.32 mglL

Lowest Observed Efflect Concentration (LOEC) based on growth rate: 0.80 mg/L

Lowest Observed Effect Concentration (LOEC) based on yield: 0.80 mg/L

The results from the positive control with potassium dichromate were within the normal ranges

for this reference item.
Harlan Study Number: 41304731 Repot Page 33

Appendix 3 Culture Medium

Na]itO:

MgClz.6HzO

CaClz.ZHzO

MgSOa.THzO

KzHPO+

NaHCOT

HrBOI

MnCl2.4H2O

ZnClz

FeClr.6HzO

CoClz.6HzO

NazMoO+.ZHzO

CuClz.2HzO

NazEDTA.zthO

25.5 mglL

12.16 mg/L

4.41 mg/L

14.6 mglL

1.044 mgil,

15.0 mg/L

0.186 rnE/L

0,415 mg/L

0.00327 ffiglL

0.160 mg/L

0.00143 mg/L

0.00726 mg/L

0.000012 mg/L

0.30 mg/L

The culture rnedium was prepared using reverse osmosis purified deionized watert. For the

purposes of the definitive test, additional sodium bicarbonate (500 mg/L) was added to the

prepared culture medium prior to adjusting the pH to 7.0 with 0.1N NaOH or HCl.

'Etga Optima l5+ or Elga Purelab Option R-I5 Bp
Harlan Study Number: 41304731 Report Page 34

Appendix 4 Preliminary Media Preparation Trial

1. Saturated Solution Preparation

A nominal amount of test item (1090 mg) was dispersed, in duplicate, in 10.9 liters of culture

medium with the aid of magnetic stirring at a rate such that a vortex was formed which

represented approximately 5% of the overall media column height for periods of either 24 or

48 horus. After stirring samples were taken for chemical analysis after the following pretreatments:

Centrifugation at 10000 g for 30 minutes

Centrifugation at 40000 g for 30 minutes

Filtation through a 0.2 pm Sartorius Sartopore filter (approximately I liter discarded in

order to pre-condition the filter)

Filtration through a 0.2 pm Sanorius Sartopore filter (approximately 2 liters discarded in

order to pre-condition the filter)

Results

Stirring Period and Treatment Concentration Found (mg/L)

24 Hours Centifuged 10000 g 19.0

24 Hours Cenrifuged 40000 g I1.4

24 Hours Filtered - I liter discarded 57.2

24 Hours Filtered - 2 liters discarded 56.t

48 Hours Centrifuged 10000 g 18.5

48 Hours Centrifuged 40000 g 9.90

48 Hours Filtered - I liter discarded 54.2

48 Hours Filtered - 2 liters discarded 59.0

3. Iliscussion

The results obtained indicated that significantly higher dissolved test item concentrations were

obtained following removal of any undissolved test item by filtration rather than centrifugation.

This was considered to be due to losses of test item through volatility during the time taken to

remove the supernatant after centrifugation.

Based on this information the test item was prepared using a saturated solution method of

preparation at an initial loading rate of 100 mg[-, stirred for a period of 24 hours prior to the

removal of any undissolved test item by filtration through a 0.2 pm Sartorius Sartopore filter

(first approximate 2 liters discarded) to give a nominal test concentration of approximately

57 mg/L, the limit of water solubility of this Test item under test conditions.

a

I

t

2.
Harlan Study Number: 41304731 Report Page 35

Appendix! Analyticallnvestigations

1 MATERIALS AND IVIETHOI}S

1.1 Introduction

The test item concentration

using an external standard.

peak.

in the test samples was determined by gas chromatography (GC)

The test item gave a chromatographic profile consisting of a single

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd,

Shardlow, UK.

1.2 Test Item

The test item is described in the biological part of this study.

1.3 Analytical Standard

The test item described in the biological part of this study was also used as the analytical

standard.

1.4 Analytical Procedure

1,4.1 Storage

The samples were analyzed on the day of receip.

1.4.2 Reagents and Solvents

Test rnedium: as described in the biological part of this study.

Solvents: tetrahydrofuran,sigma-Aldrich,HPLCgrade.

hexane, Fisher Scientific, pesticide residue grade.
Harlan Study Number: 4130473I Report

Analytical Investigations

Page 36

1.4.3 Preparation of Calibration Standards

The test item (nominal 100 mg) was dissolved in hexane (100 mL) to prepare a stock solution

with a concenkation of 1000 mg/L. This stock solution was further diluted with hexane to

produce a solution of 50 mglL. Defined volumes of this solution were then diluted with hexane

to obtain calibration standards in the range of 0.1 to 1.0 mS/L. A second series of calibration

standards was similarly prepared in the range of 0.25 to 2.5 mg/L. These solutions were used to

determine the recovery and test sample concentrations.

1.4.4 Preparation of Linearity Standards

The test item (nominal 100 mg) was dissolved in hexane (100 mL) to prepaxe a stock solution

with a concenfration of 1000 mg/L. Defined volumes of this solution were then diluted with

hexane to obtain calibration standards in the range of 0.1 to 50 mg/L. A second series of

calibration standards was similarly prepared in the range of 0.25 to 25 mg/L. These solutions

were used to determine the test sample concentrations and to evaluate the linearity of the

analytical system.

1.4.5 Preparation of Spiked Recovery Samples

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked

ruith the test item and the recovery was assessed. The test item (nominal 100 mg) was initially

dissolved in tetrahydrofuran to prepare a stock solution with a concentration of 1000 mglL. This

stock solution was further diluted with tetralrydrofuran to produce a stock solution of 50 mg/L.

A defined volume of this stock solution was diluted with test medium to obtain spiked recovery

samples at a concentration of 0.050 mg/L. Five replicates were prepared and subjected to the

same freatment as the test samples. In addition, test medium without the addition of the test item

(synthetic control) was also analyzed.

1.4.6 Preparation of Test Samples

The test samples were analyzed on the day of receipt. The test samples were extracted directly

with hexane in the same bottle. An aliquot of the hexane layer was taken for analysis. See

Table I for more information on preparation volumes.
Harlan Study Number: 41304731

1.4.7 Instrumental Parameters

Gas chromatograph:

Detector:

Column:

Inlet liner:

Carrier gas:

Pressure:

Oven temperature

Program:

Injection mode:

Inlet purge time:

Injection volume

Inj ection temperature :

Detector temperature:

Retention time:

HP 5890 Series II, with autosampler

ECD

zB-624

(60 m x 0.32 mm i.d.;1.8 pm film thickness)

double gooseneck

niEogen

10 psi

oven: 40 oC, for 0 minutes

with I5 oC/minute to 220 oC, for 5 minutes

splitless

on at I minute

lpL

150 0c

250 0c

approximately 14.8 minutes

Page 37
Harlan Study Number: 41304731

1.4.8 Calculations

A calibration curve was prepared using a quadratic

The calibration data is presented in Table Z and

concentration "x" of the test item in an injected

equation:

Page 38

function of the form shown in equation (1).

Figure l. From the calibration curoe, the

sample was calculated using the follo*i.tg

where x:

v:

a, b, c:

The concentation

equation:

x

C =-rI{R/l 00

where c:

x:

F:

R:

The recovery rate

equation:

R - " .too

cfon

where R:

c:

(l)

cfortl

concentration of the test item in the injected sample [mg/L]

average peak area of the test item in the injected sample [counts]

constants

of the test itern in the test samples was calculated using the following

(2)

concentration of the test item in the test sample [mg/L]

concentration ofthe test item in the ir{ected sample solution [mg/L]

sample preparation factor

recovery rate [%] (If not correcting for recovery then R is taken as 100)

(R) of the spiked recovery samples was calculated using the following

(3)

recovery rate lYol

determined concentration ofthe test item in the spiked recovery sample

lme/Ll

fortified concentration of the test item in the spiked recovery sample

[*e/L]
Harlan Study Number: 41304731

Analytical Investigatiorls

Report Page 39

2 RESULTS

2.1 Range-Finding Test

The analytrcal methodology was not validated prior to the analysis of range-finding samples; full

validation was later conducted at a suitable concentration. The results for the range-finding test

samples axe summarized in Table 3.

2.2 Ilefinitive Test

2.2.1 Yalidation of the Analytical Method

Specificity

The biological control samples showed no significant interfering response at the retention time of

the test item. The standard solutions contained a peak specific for the test item whose area

changed accordingly with known concentration, hence the specificity of the method by retention

time was confirmed.

Linearity

The calibration data for the calibration standards of the tes item is given in Table 2 and Figure l.

The data was found to have a quadratic correlation within the calibration mnge of 0 to 52 mglL.

The R'? fit of the calibration curve to the data was 0.9997, and was considered to be acceptable.

Accuracy (Recovery) and Precision

A set of recovery samples accurately fortified at a relevant concentration of test item was

prepared five-fold and analyzed. The results obtained for the concentration of the test item in the

recovery samples are presented in Table 4.

The method was considered to be sufficiently accurate and precise for the purposes of this test.

The test sample results were not corrected for recovery.

Limit of Quantification

The limit of quantification (LOQ) was determined as ten times the standard deviation of five

synthetic controls. Using this method the LOQ was determined to be 0.0099 mgll.

2,2.2 Test Samples

The results obtained for the concentration of the test item found in the test samples are presented

in Table 5. Typical chromatograms are shown in Figures 2 to 5.
Harlan Study Number: 4 I 30473 1

Analytical Inveqtigations

Report Page 40

3 DISCUSSION

Jfe detection system was found to have acceptable correlation to the fit of a quadratic equation.

The analytical procedure was found to have acceptable recoveries of test item in test medium.

The method of analysis was validated and proven to be suitable for use.
Harlan Study Number: 4 I 30473 I

TABLES

Table I Preparation of Test Samples

Table 2 Linearity Data

Page 41

Nominal

Coucentration of

Test Item

lolo vlv Saturated

Solutionf

Sample

Volume

lmLI

F'inal Volume

lmLl

SampIe

Preparation

F'actor

F

Control 50 r0 0.2

1.0 50 10 0.2

3.2 50 t0 0.2

l0 50 10 0.2

32 50 100 2

100 50 100 2

Concentration of Test Item

lmg/Ll

Mean Area

Icountsl

0 1.584 x 104

0.104 l.l49x 105

0.264 2.352x 105

0.520 3.887 x l0s

1.04 6.535 x lOs

2.64 1.396 x 105

5.20 2.448 x 106

10.6 4.625 x 105

26.4 1.068 x 107

52.0 2.086 x 107
Harlan Study Number: 4 I 30473 1

Analytical Investigations

Report Page 42

Table 3 Results for Range-Finding Samples

Table 4 Results for Spiked Recovery Samples

For reporting purposes, x has been calculated from c rehospectively.

Time point

Ihours]

Nominal

Concentration

of

Test Item in

Range-Finding

Sample

Cno,

lYo vlv

Saturated

Solutionl

Measured

Concentration

of Test ltem in

Sample Vial

x

lmg/tl

Sample

Preparation

F'ector

F

Determined

Concentration

of Test Item in

Range-Finding

Sample

c

lmg/Ll

Erpressed as a

percentage of

the 0-Hour

Measured

Concentration

{o/ol

0 0. l0

1.0

l0

100

0.335

3.01

25.0

24.9

0.2

0.2

0.2

2.0

0.0659

0.602

5.00

49.8

NA

72 0.10

1.0

l0

100

0.293

2.22

20.3

24.1

0,2

0.2

0.2

2.0

0.0585

0.444

4.06

48.1

87

74

81

97

Nominal

Concentration

of

Test Item

Cnom

tmg/Ll

Fortilied

Concentration

of Test Item in

the Spiked

Sample

Cr .t

lms/tl

Measured

Concentration

of Test ltem in

the Sample

Vial

x

Ime/LI

Sample

Preparation

Factor

F

Determined

Concentration

of Test Item in

the Spiked

Sample

c

Ime/Ll

Mean

Analytical

Recovery

Rate

R

I%l

Precisiou

(Relative

Standard

Deviation of

Recovery)

t%t

0.050 0.0529

0.286

0.285

0.279

0.283

0.2'17

0.2

0.?

0.2

0.2

0.2

4.0572

0.0569

0.0557

0.0566

0.0554

107 1.4

Acceptance Target 80-120 <10
Harlan Study Number: 41304'13l

Analytical Inve stigations

Report Page 43

Table 5 Results for Test Samples

LOQ : Limit of Quantification

For reporting purposes, x has been calculated from c retrospectively.

Time point

Ihours]

Nominal

Concentration

of

Test Item in

Test Sample

cnom

lYovlv

Saturated

Solutionl

Measured

Concentration

of Test ltem in

Sample Vial

x

lmg/Ll

Sample

Preparation

Factor

F

Determined

Concentration

of Test ltem in

Test Sample

c

lmg/LI

0 Confrol

1.0

7, ''

l0

17

100

<LOQ

1.86

4.22

18.5

7.80

25.4

0.2

0.2

0.2

0.2

2

2

<LOQ

0.372

0.843

3.69

15.6

50.7

24 1.0

3.2

10

32

100

1.59

4.1s

17.5

7.55

24.8

0.2

0.2

0.2

,,

)

0.318

0.830

3.50

15.1

49.5

48 1.0

3.2

l0

32

100

1,.',l2

4.05

17.0

7.50

24.6

0.2

0.2

0.2

.)

)

0.344

0.809

3.39

15.0

49.2

72 Control

1.0

3.2

l0

3?

100

<LOQ

1.66

4.07

17.3

7.55

24.6

0.2

0.2

0.2

0.2

2

2

<LOQ

0.332

0.814

3.46

r5.l

49.2
Harlan Study Number: 41304?31

FIGURES

Figure I Linearity

6

'E 2soooooo

ert)

q

o

&a

& 20000000

J,(6

{) A

15000000

10000000

5000000

0

s0 60

Concentration (mdD

y = -259 .3502x2 + 4 10,505. 1239x+ 17g p,90.0943
Harlan Study Number: 41304731 Report

Analytical Investigations

Page 45

Figure 2 Chromatogram of Calibration Solution (S mg/L)

€oo

400

200

o

Figure 3 Chromatogram of Biological Control Sample.

600

400

20()

* 

Response seen determined to be less than the LOQ.
Harlan Study Number: 41304731

Figure 4 Chromatogram of Test Sample (1.0% v/v Saturated Solution)

Figure 5 Chromatogram of Test Sample (100% v/v Saturated Solution)

6t--^-- I

s

I

Elghrl{ i I i i

- 

ctiqeoer roo i : i

RstentiohTime i i i i

jilii

:i;ii
Harlan Study Number: 41304731 Report Page 47

Appendix 6 Monitoring Authority Statement of GLp Compliance

( Dil! Department \- ' of Health

THE DEPIIRTMENT OF HEALTH OF THE GOVERNTiENT

OF THE UNITED KINGDOM

GOOD LABORATORY PRACTICE

STATEi,IEi|T OF COMPLIANGE

IH ACCORDANCE WITH DIRECTIVE zOO4/S/EC

TEST FACILITY

Harlan Laboratorles Limlted

Shardlow Buclne*s Park

London Road

Shardlow

trorbyrhlrc

DE72 zGD

TEST TYPE(S)

AnalytlcellGl Inlcal Ghemletry

Envlronmental Fete

Environmsntal Toxicity

Phye.Ghern. Teetlng

Hutagenicity

Toxicology

DATE OF IHSPECTION 12 to 14 Harch 20t4

An inspection for compliance wlth the Princlples of Good Laboratory Prectice wes

carried out at the above test facility as part of the UK Good Laboratory Practlce

Compliance Monitoring Progremm€.

This statement confirms that, on the date of issue, the UK Good Laboratory

Practice Monitoring Authority were satisfled that the ebove test facility was

operating in compliance with the OECD Princlples of Good Laboratory Practice.

This staternent constitutes a Good Laboratory Practice lnstrument (as defined in the

UK Good Laboratory Practice Regulations 1999).

Dr. Andrew J. Gray

Head, UK GLP ltonitoring Authorlty
FedEx Ship Manager API Lahel Page

From: (56U 578-8110

JOHN V. MASSINGALE

Otigin lD:PBIA

GREENWCH CHEMICAT CONSULTING, INC.

33OO PGA BLVD

sutTE 635

PALM BEACH GARDENS, FL 33410

SHIP To: (202) 56+8S30

OPPT Document Control Office

U.S. Environmental Protection Agenc

Room E6428; EPA East Bldg.

1201 Constitution Ave., NW

Washington, DC 20004

Page I of 1

Ship Date: 23APR15

ActWgt 0.5 LB

CAD: 103160523 n SX12600

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