## 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 

### FinalTest 

## 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) 

### thefinal 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 4test 

### rule.l 

### Thefinaltest 

### 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) tothe 

### TSCA Docket on aflash 

### 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 theflash drive and requested re-submission 

### of these files 

### on 

### CD.This CD is 

### included hetein.z



### The 

### PCBTFTesting Consortium thanks the 

### Agency

### fot its consideration rnthis 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

176 Fed. Res. 65385; October 21.,2071.. 2Telephone conversations with Ms. Pam lv{osely FPA) on h{ay 11,2015 and IMay 13,201'5.



## /\

I
I

# i,'

il 

## ilr ,l 

# li 

# !l 4 j

### ,FedEx Ship Manager 

### API Label Page

GREENWICH CHEMICAL CONSULTING, INC. 33OO PGA BLVD sunE 635 PALM BEACH GARDENS, FL 33410
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### Control

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### Environmental Protection Agenc Room E6428; EPA East Bldg. 1201

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## I of 1

Frorn: (561) 578-8110 JOHN V. MASSINGALE Origin lD: PBIA 

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### https://3pl.freedomffship.com/viewlahel.php?airbill:78065 0655022&carrier:FedEx 

### sltS/201sCBICControl 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'" t r l-. l,'t

. 1l_ : l-iji'i



### 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 

### ofthe Final Test Reportfor theAtgae Study Re-conducted 

### by the 

### PCBTFTesting Consortium under 

### the TSCA Section 

## 4Test 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 799.5089CONTAINS 

### 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

### finaltest 

### reporton 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 

### undertheAgency'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 algaetest 

### report,
t 76 Fed. Res. 65385; October 21,2011. 2 SeeAttachment Afor the addresses of member companies. 3MC International, LLC operatesthe 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 

### thatthe study was inadequate 

### fot purposes

### of the TSCA Section 

### 4test 

### nrle and 

### must be 

### re-conducted.The Agency's conclusions 

### wete set

### fotth in its 

### letter 

### of Septembet 26,2A1,3.

### The algaestudy 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 

### cornpoundwhich 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 

## @, poorwater solubility and high volatiliry). This study plan was subrnitted 

### to the Agency 

### onJanuary 9,2074 (ahead

### of the revisedJanuary 12,201,4deadline). 

### In its response dated 

### March 6,201.4, 

### EPA determinedthat 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 theAgency's 

### stipulations and 

### modifications. 

### At thetequest 

### ofthe 

### PCBTF Testing Consortium, additional guidancewas subsequently requested

### ftom EPA on the use

### of serial 

### dilutions along

### with other 

### minor matters. 

### TheAgency 

### provided this guidance 

### in its 

### letter dated 

### October2,2074. 

## The PCBTF Testing Consortium subsequently 

### finalized 

## the study

## plrn with Harlan Laboratories, 

### andthe 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 

### forthe Study Plan and 

### Final Test Repoft 

### In its September26,2013 determination letter, 

### EPA stated

### that anew 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

### whichthe 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 severaltechnical 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.

aIn the final TSCA Section 4 test

### de, EPA specified the use of anASTM 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 

### 4test 

### 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 thefinal test 

### rule, 

### EPA requests, 

### but does 

### not requite,that a robust summary also be 

### included

### for each 

### test 

### identified 

### urthe rule consistent

### with the Agency's 

### Draft Guidanm on DeuelopingRobust Summaries. The Agency 

### previously signaled to the 

### PCBTF Testing 

### Consortium 

### thatthe 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

### IICONTAINS 

### 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 SunTower 116-118Wing 

### Lok Street SheungWan Honq Kong 

### EMCO Chemical 

### Disributots, Inc. 8601 95th Street Pleasant Prafuie,

### WI 53158 Gteenchem Industries, TJ.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 3Watenray Square P1ace Suite 1000 The Woodlands, 

### TX 77380 Silver Fern Chemical Inc. 2226 Queen Anne Avenue 

### North Seattle. 

### $fl4 98109 Special Materials Company 70West 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 r" REPORT
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 of47Harlan 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 onTest Item

# Solubility .........21 3.5Harlan 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 inthe 

### 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 on0-Hour Measured Test

Concentrations 

# ....... ...,.....,,29 Figure

# 3 Inhibition ofYield Based on O-Hour Measured Test Concentrations........................30 

## LIST OFAPPENDICES 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 MonitoringAuthority Statement 

### of GLP

## Compliance.......... .................47Harlan Study Number: 41304731 Report Page 4



## STUDY 

## DIRECTOR STATEMENT OF 

## GLP

## COMPLIANCE Harlan Laboratories 

### Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE722GD, 

### 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 SI2004/0994)). 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 s2004I9tEC and2004IlOlEC. 

### These 

### principles axe compatible 

### with Good Laboratory Practice regulations specified by regulatory authoritiesthroughout the European Community, the 

### United States 

### GPA and FDA), and Japan 

### (MHLW, MAFF and 

### METI).
This report

### fully andaccurately reflectsthe procedures used anddatagenerated. 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

### zollHarlan 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 andactivities are inspected at least once ayear andthe results are reported tothe 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 

### ofQA 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 January2015 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 January2015
06 January 2015 20 January 2015 12 March 2015 

### Verification 

### of Study Plan Amendment 

### for GLP compliance was performedduring the course 

### of the study. This statement conlirms thatthis report reflects the raw data and the proceduresfollowed. Quality Assurance: 

## r-l

# lN\\tr_ Date: 2

## 0 ApRHarlan 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



# 2rmn20uHarlan Study Number: 4 I30473 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 indicatedthewater 

### sotubility 

### of the test item 

### to be 

### in the range 

### of29 

### - 34 

### mglL. A preliminary media preparation 

### trial indicated that adissolved 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 ofthis item undertest conditions. 

### Following a preliminary range-finding test, Pseudohirchneriella subcapitafa 

### was exposed to solutions 

### of thetest 

### 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 504vortex wasforrned 

### for 24 

### hours. After the stirring period any undissolvedtest 

### itemwas removed by 

### filtration (0.2 pm Sartorius Sartopore 

### filter, first approximate 

### 2 liters discarded 

### in order 

### to pre

### condition 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 

### tothe restriction 

### of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide asource of carbondioxide 

### for algal growth.


### Results Analysis

### ofthe test preparations at 

### 0 hours showed measured test concentrations 

### of 0.37, 0.84, 

### 3.7,16to 51 

### mg/L were obtained 

### fromthe 1.0,3.2, 10, 32 and 100%

### v/v saturatedsolution 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 sothe 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.tHarlan 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) andthefinal report

### will be retained in the Harlan Laboratories 

### Ltd, Shardlow, 

### UK archives 

### forfive 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 discardedwithout contacting the Sponsor to obtain theirwritten 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 onthe growth 

### of the green 

### alga Pseudohirchneriella 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 exposeorganisms 

### to a saturated solution 

### of thetest 

### 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 aperiod

### of 24 hours 

### priorto removing any undissolved test 

### item present 

### byfilnation (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 onAquatic 

### Toxicity Testing 

### of 

### Difficult Substances and Mixtures. OECD Series onTesting and Assessment, No. 23. OECD Publishing.Harlan Study Number: 413047i1 Report Page 1l



# 2 TEST 

# ITEM Information as provided 

### bythe Sponsor. 

### A Certificate 

### of Analysis supplied 

### bythe 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 intermediateHarlan 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 

### oftest 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

### atthe end

## of the expoflue period minus the measurement variable's value atthe 

### start 

### of the exposure 

### period, calculated 

### to express biomass increase 

### dwing the test. The lowest test concenhation atwhich astatistically significant 

### inhibition of

### gowth isdetermined 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 

### ofthe test (days0-1, 

### 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 subcapitatawere 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 wasapproximately 104

### - 

### l0scells/ml.Harlan Study Number: 41304731 Report Page 13



### A positive control (Harlan Study Number 41403365) used potassium dichromate asthe reference 

## item. Details 

### of the positive control are given 

### in Appendix

## 2. The positive control was conducted between 14October 2014 and

### 3l October}}l4. 

# 3.3 Test

## Water The cultrue medium used 

### for both the range-furding and 

### defuritive tests was

### the silme asthat 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 Sponsorthe 

### pH 

### of the culture mediurn was adjustedto 

### pH 7.0 after the addition of the sodium bicarbonate. The culture medium is defined inAppendix 3. 

# 3.4 Procedure 

# 3.4.1 Preliminary Media Preparation 

### Trial Information provided 

### by the Sponsor indicatedthewater 

### 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 

### AquaticToxicity 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 undertest 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 testwere determined by a preliminary range

### finding 

### 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 saturatedsolution (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 magneticstirring at a rate such that a 50/ovortex wasformed for 24 

### hours. After 24 hours 

### the stirringwas 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 thisHarlan 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 eachof the stock solutionswas separately inoculated

### with algal suspension 

### Q.7 mL) to givethe 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 conicalflasks each completely 

### filled (i.e., no headspace) 

### with test preparation and sealed

### with a ground glass stopper to reduce evaporation and minimize losses due

### tothetest 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. Thiswas 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 orderto determine the stability of the test item undertest conditions. 

## 3.4.3 Ilefinitive Test Basedon the results 

### of the range-finding testthe

### 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 magneticstirring at a rate such that a 5oZ vortex was formed far 24 

### hours. After 24 horus 

### thestirring 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 eachofthestocksolutionswasseparatelyinoculatedwith 

### 5.2mLof algal suspension 

### to givethe 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 andstability 

### 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 atthe start 

### of the 

### test: (a) one replicate was 

### kept sealed

### (with no 

### prior sampling) 

### until 24 hours, 

### atwhich 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 atthe startof 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, atwhich time samples were taken for cell density determination then discarded; and (c) three replicates were kept sealed 

### (with no 

### prior sarnpling) 

### until 72 hours, atwhich 

### time samples were taken for both cell density determination and chemical analysis. The control group was maintained under identical conditions but not exposed to thetest 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 

### of2 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 thefinal test concentration. The flaskswere 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 warmwhite lighting (380

## - 730 

### nm) and constantly shaken

### at approximately 150 

### rpmfot 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 bythe 

### dilution 

### factor. Samples were taken atHarlan 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 thetest item on the appearance 

### of algal cells, a sample was removed

### from each test andcontrol culture (replicates pooled) atthe end 

### of the

### test. 

### Thi shape and size 

### of the algal cells was inspected microscopically and anyabnormalities 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 

### bulktest 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 

### forfurther 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 testsolution 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 asthe 

### logarithmic increase in biomass from the equation:

..

### _ 

### lnNo 

## -lnN, 

## t,

# -t, Where: average specific growth rate from time t1to t1

cell concentration at t1 cell concentration at tn time 

### offirst 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 asthe starting value rather than the measured starting value in orderto increase the precision of the calculation. 

### In addition the section 

### by section specific growth rate (days 0-1

## ,l-Z and2-3) 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 vesselwas 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 

### ofYield 

### Yield 

### is calculated as

### the increase equation: 

## Y=No -No Where: 

### in biomass 

### overthe exposure 

### period using

### the following



# Y: No: Nn: yield

cell concentration atthe start of the test cell concentration atthe end of the test 

### concentation and 

### controlthe mean 

### value for 

### calculated. Percentage 

### inhibition 

### of yield was 

### yield along 

### with the standard calculated 

### using

### thefollowing 

### 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 

### foryield in the control group mean value for yield for thetreatment group 

### 3.4.5.3 Determination of EC* Values 

### For each 

### individual testvessel (mean values 

### for yield), percentage 

### inhibition (arithmetic a1ris) 

### was 

### plotted against 

### test concentration 

### (logarithmic 

### axis) and

### a line fitted 

### by computerizei interpolation usingthe

### Xlfit software package 

### (IDBS). EC* values werethen 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 severaltreafinents 

### 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 

### thetest 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 consideredvalid 

### if the

### following performance criteria are met: 

## ' The 

### cell concentration 

### of the control cultures 

### must increase 

### by afactor

### of at least 

### l6 over the test period. 

## ' The mean

### of the coefficients 

### ofvariation of the section by section 

### daily growth rates 

### in the control cultr.ues

### during the course

### of thetest (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 atthe 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 saturatedsolution 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/Lwhilst concentrations 

### in the range 

### of 0.059to 48 

### mg/L were observed at72 hours 

### (74%to 97Voof 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 39of the OECD Test Guideline 

### No.201. 

## 4,2.2 Growth Data Cell density values determined at each sampling time and 

### pH values at0 and 72 hours are given 

### in Table 

### 2. Daily specific growth rates for the control cultures aregiven in Table

### 3. Grourth rate and

### yield values 

### for the control and test cultures after 72 hows and percentage 

### inhibition values aregiven 

### in Table 4. The mean 

### cell densities versus 

### timeforthe

### definitive test are presented 

### in Figure 

### 1. Percentage 

### inhibition values are plotted againsttest 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 inHarlan 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* isthe test concenfration that reduced growth rate byx%. 

### 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 ratewas3.7 

### mglL. 

### 4.2.2.2 Inhibition ofYield &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 theyield 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 

### ofthe control cultures increased 

### by afactor 

## of 190 after 

### 72

### hours. This increase 

### was 

### in line 

### with the OECD Guideline

### that states the enhancement must be at least by afactor 

### of 16after ?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 

### mLHarlan 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 hencesatisfied 

### the validation criterion given 

### in the OECD Guideline which states the mean must not exceed 35%. The coeffi.cient 

### ofvariation 

### for average specific growth rate

### for the control 

### culfires over thetest 

### 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 

### cellswere observed

## to be present 

## in thetest 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 andtest cultures \ryereobservedto be clear colorless solutions. 

### After the 72-Hour test period 

### all control, 0.37 and 0.84 

### mg/L test cultureswere observed

### to be green dispersions. The 3.7 

### mg/L test cultures were observedto 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 thetest item 

### on the growth

## of Pseudokirchneriella 

### subcapitata 

### has been investigated over a 72-Hour period and based onthe 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 I30473 I Report Page23



# 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) 

### AquaticToxicity Testing

### of Sparingly Soluble,

### Volatile and Unstable Substances. 

### HERMAN et

### al. (1990) Impact

### ofvolatile aromatic hydrocarbons, alone and 

### in combination, on growth 

### of thefreshwater 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 countsfrom 3 countsfor eachof the replicate flasks. R1and R2: Replicates 

### I and 2 [Increase in growth comparedto confiols ] Nominal Concentration (o/ovlv 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 3 

### I

R2 3.00E+03 4.638+05 Mean 3.00E+03 5.14E+05 l0 R1 3.00E+03 7.87E+04 40 88

R2 3.00E+03 6.20E+04 Mean 3.00E+03 7.038+04

100 R1 3.00E+03 3.63E+03 99 100

R2 3.00E+03 2.99E+03 Mean 3.00E+03 3.31E+03Harlan 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 countsfor each of the replicate flasks. Rr

## -& : Replicates 1to 3 Nominel Concentretion (o/ovlv 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+03Harlan 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 3Harlan 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 foryietdfor each test concenfration is calculated Rr

### -

## &: Replicates I to 3 SD

## : Standard Deviation [Increase in growth compare to controls] Nominal Concmtrrtion ("/ovlv 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+03Harlan Study Number: 41304731 Report Page 28



## FIGURES Figure 

## I Mean Cell Densities 

### vTime

### 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)Page 29 Harlan Study Number: 41304731 Report


### 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
10 o\

100 l0Harlan 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 Li 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 i Tii-i'i-

,t 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

I00 l0 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 llstgoH 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€trllqdd. 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: lrnsrtlHarlan Study Number: 41304731 Report Page 32



### Appendix 

## 2 Positive 

### Control A positive control (Harlan Study Number 41403365) used potassium dichromate asthe reference item atconcentations 

### of 0.050, 0.13, fr.32,0.80 and 2.0 mg/L. Six flasks each completely

### filledwith test preparation were used 

### for the control, andthree flasks each completely 

### filled with test preparation were used

### for each test 

### concentration. The flasks were sealed

### with ground glass stoppers and incubated at24

### + 

## 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 pHto 7.0 

### with 0.1N NaOH or HCl.

'Etga Optima l5+ or Elga Purelab Option R-I5 BpHarlan 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 

### weretaken

### for chemical analysis 

### after 

### thefollowing 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) Filtrationthrough a 0.2 pm Sanorius Sartopore

### filter (approximately 2 liters discarded 

### in orderto pre-condition the

### filter)
Results Stirring Period andTreatment 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 indicatedthat 

### significantly higher dissolved test 

### item concentrations were obtained

### following removal 

### of any undissolved test item 

### byfiltration 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 

### onthis information 

### thetest 

### itemwas 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 MATERIALSAND IVIETHOI}S 

# 1.1 Introduction Thetest 

### 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 Thetest item isdescribed inthe biological part ofthis 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 dayof 

### receip. 

## 1.4.2 Reagents 

### and Solvents
Test 

### rnedium: as described in the biological part ofthis 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

### ofthis 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 0.25to 2.5

### mg/L. These solutions were usedto 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 

### dilutedwith 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 thetest 

### item andthe recoverywas assessed. The test item 

### (nominal 100 mg) was 

### initially dissolved in tetrahydrofuran to prepare a stock solution

### with aconcentration 

### of 1000 

### mglL. This stock solution wasfurther diluted 

### with tetralrydrofuran

### to produce a stock solution 

### of 50 mg/L. 

### A definedvolume 

### of this stock solution was diluted 

### with test medium

### to obtain spiked recovery samples 

### ata concentration

### of 0.050

### mg/L. Five replicates were prepared and subjected 

### to the samefreatment asthetestsamples. 

### 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 sampleswere analyzed on the day

### of receipt. The test samples were extracted directly 

### with hexane

### in the same 

### bottle. An aliquot 

### of the hexane 

### layer wastaken 

### 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

onat 

### I minute 

## lpL
150 0c 250 0c approximately 14.8 minutes Page 37Harlan 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 ofthe test item inthe injected sample 

### [mg/L]

average peak areaofthetest item in the injected sample [counts] constants 

### of thetest 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 inthe ir{ected sample solution [mg/L] sample preparation factor recovery rate 

### [%] 

### (If not correcting for recovery then R istaken as 100) 

### (R) 

### of the spiked recovery samples 

### was calculated 

### using 

### the following
(3)
recovery rate lYol determined concentration ofthe test item inthe spiked recovery sample 

### lme/Ll fortified concentration 

### of the test item inthe 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 ata suitable concentration. The results 

### for the range-finding test samples axe summarized in Table 3. 

# 2.2 Ilefinitive Test 

# 2.2.1 Yalidation ofthe

### Analytical Method Specificity The biological control samples showed no significant interfering response atthe retention time

### of the test

### item. The standard solutions contained a peak specific 

### for thetest item whose area changed accordingly 

### with known concentration, hence the specificity 

### of the method 

### by retention time wasconfirmed. 

### 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 curveto 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 methodwas considered 

### to be 

### sufficiently accurate and precise 

### for the purposes 

### of this test. Thetest sample results were not corrected for recovery. 

### Limit of

### Quantification The 

### limitof quantification 

### (LOQ) was determined asten 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 thetest item found in thetest samples are presented in Table

### 5. Typical chromatograms areshown 

### in Figures 2to 5.Harlan Study Number: 4 I30473 1 Analytical Inveqtigations Report Page 40 

# 3 DISCUSSION Jfe detection systemwas foundto have acceptable correlationtothe

### fit of a quadratic equation. The analytical procedure was found 

### to have acceptable recoveries 

### of test 

### item 

### in test medium. The method of analysis wasvalidated and proven to be suitable for use.Harlan Study Number: 4 I30473 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 107Harlan Study Number: 4 I30473 1 Analytical Investigations Report Page42



### 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, lYovlv 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 the0-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 <10Harlan Study Number: 41304'13l Analytical Inve stigations Report Page43



### 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 ofTest 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 49.2Harlan Study Number: 41304?31



## FIGURES Figure

# I Linearity

6 'E 2soooooo 

### e rt) q o & a & 20000000 J,( 6 {) A 15000000

10000000

5000000

0 

### s0 60 Concentration (mdD y= -259 .3502x2 + 4 10,505. 1239x+ 17gp,90.0943Harlan 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 ofTest Sample (1.0% 

### v/v Saturated 

### Solution)

Figure

# 5 Chromatogram ofTest 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;iiHarlan Study Number: 41304731 Report Page47



### 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 12to 14 Harch 20t4 An inspection for compliance wlth the Princlples of Good Laboratory Prectice wes carried out atthe abovetest facility as part ofthe UK Good Laboratory Practlce Compliance Monitoring Progremm€. This statement confirmsthat, on the date 

### of issue,the UK Good Laboratory Practice Monitoring Authority were satisfled thatthe ebove testfacility was operating in compliance with the OECD Princlples of Good Laboratory Practice. This staternent constitutes a Good Laboratory Practice lnstrument (as defined inthe UK Good Laboratory Practice Regulations 1999).

Dr. Andrew J. Gray Head, UK GLP ltonitoring Authorlty

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