page 1 of 24 

## Final Report ( 2 nd 

## Original)



# Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

(GLP compliant study based on the Commission Regulation 440/2008/EC, C.4

- D, 2008 and the OECD No. 301 F, 1992)

Author: Dr. Ute Hammesfahr

Study Completion Date: April 0 3 , 2013

CiToxLAB study code: 12/ 056

- 914AN (previous study code 12/056

- 322AN)

Sponsor
PCBTF Testing Consortium c/o Greenwich Chemical Consulting, Inc. 3300 PGA Blvd Suite 635 Palm Beach Garden s, FL 33410

United States of America

Test Facility
Institut für Biologische Analytik Project 78601163 und Consulting IBACON GmbH Arheilger Weg 17 64380 Rossdorf GermanyFinal Report ( 2 nd Original) IBACON Project 78601163 - Ready Biodegradability of Benzene, 1

- chloro -4- (trifluoromethyl) - in a Manometric Respirometry Test

page 2 of 24 Copy of GLP Certificate 

# IBAC N

lnsutut für Biologische Analytik und**Consu~ing** !BACON GmbH

G ute Labo rpnu :is/Good Labora t ory Practice

GL P-B esc b e ini g un g!S tat emcnt o f GLP Co mplian ce {ymWaocordirlg to**~** 19b Ab$

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- Obcrwadwng.sbellorde/Namc and oddren oftlre GL.P M 0t1itoring AuthorityFinal Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 3 of 24 

### Contents
Copy of GLP Certificate

................................ ................................ ................................ ................................ 2 1. Summary

................................ ................................ ................................ ................................ ................... 5 2. Survey of the Study

................................ ................................ ................................ ................................ ... 7

2.1 General Information

................................ ................................ ................................ ............................ 7 2.2 Good Laboratory Practice

................................ ................................ ................................ ................... 8 2.3 Archiving

................................ ................................ ................................ ................................ ............. 8 2.4 Signatures

................................ ................................ ................................ ................................ ............ 9 3. Quality Ass urance Unit Statement

................................ ................................ ................................ .......... 10 4. Statement of Compliance

................................ ................................ ................................ ........................ 11 5. Objectives of the Study

................................ ................................ ................................ ........................... 12

5.1 Title

................................ ................................ ................................ ................................ ................... 12 5.2 Purpose

................................ ................................ ................................ ................................ .............. 12

5.3 Guidelines/Recommendations

................................ ................................ ................................ ........... 12

6. Material and Methods

................................ ................................ ................................ .............................. 13

6.1 Test Item and Reference Item

................................ ................................ ................................ ........... 13 6.2 Test System

................................ ................................ ................................ ................................ ....... 14

6.3 Test Units

................................ ................................ ................................ ................................ .......... 14

6.4 Test Conditions

................................ ................................ ................................ ................................ . 14

6.5 Test Water

................................ ................................ ................................ ................................ ......... 14 6.6 Preparation of the Test Solutions

................................ ................................ ................................ ...... 15

6.7 Course of the Test

................................ ................................ ................................ ............................. 15

6.8 Test Parameters

................................ ................................ ................................ ................................ . 16

6.9 Resu lt Evaluation

................................ ................................ ................................ .............................. 16 6.10 Validity Criteria of the Study

................................ ................................ ................................ .......... 17 6.11 Deviations to the Study Plan

................................ ................................ ................................ ........... 17

7. Results and Discussion

................................ ................................ ................................ ............................ 17

7. 1 Biodegradation of Test Item

................................ ................................ ................................ .............. 17 7.2 Biodegradation of Reference Item Sodium Benzoate

................................ ................................ ....... 18 7.3 Biodegradation in the Toxicity Control

................................ ................................ ............................ 18

7.4 Abiotic Control

................................ ................................ ................................ ................................ .. 18

8. References

................................ ................................ ................................ ................................ ............... 19
9. Distributio n of the Final Report

................................ ................................ ................................ .............. 19 Appendices

................................ ................................ ................................ ................................ .................. 20 Appendix I Certificate of Analysis of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- ................................ 21 Appendix II Additional Tables and Figures

................................ ................................ ....................... 22Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 4 of 24 List of Tables

Table 1. Preparation of the Test Solutions

................................ ................................ ................................ .. 15 Table 2. Cumulative Biochemical Oxygen Demand (mg O 2 /L) in Test Flasks during the Test Period of 28 Days

................................ ................................ ................................ ................................ ............. 22

Table 3. Percenta ge Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD NH4
................................ ................................ ................................ ................................ . 23 Table 4. pH

- Values at the End of the Test

................................ ................................ ................................ .. 24

List of Figures

Figure 1. Biodegradation of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- (Flasks 1 and 2), of the Reference Item Sodium Benzoate (Procedure Control) and of the Toxicity Control during the Exposure Period of 28 Days Related to ThOD NH4
................................ ................................ ....................... 24Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 5 of 24 1. Summary

Title: Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

Guidelines:

− Commission Regulation 440/2008/EC, Method C.4

- D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)

− OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992

Material and Methods:

Test Item: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- ; Batch No.:
20111028

Test Species: Aerobic activated sludge (micro organisms from a domestic

waste water treatment plant) was supplied by the sewage works o f Rossdorf

, Germany.
Test Design: The test item Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- was investigated for its ready biodegra dability in a manometric respirometry test over a period of 28 days. The biodegrad ation was followed by the ox ygen uptake of the micro orga nisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
Endpoints: Degradation rate of test item calculated by the oxygen co nsumption of the aerobic activated sludge microorganisms after 28 days of incubation.
Test Item Loading Rate: 10 2.5 mg/L corresponding to an oxygen demand of about
127.2 mg/L (ThOD NH4 )

Reference Item: Sodium Benzoate

Reference Item Loading Rate: 10 1.6 mg/L corresponding to an oxygen dem and of about
1 69.3 mg/L (ThOD NH4 )

Test Conditions: 22 ±1 °C, darkness

Results:
Biodegradation of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- : T he criterion for ready biodegradability under the cond itions of a manometric respirometry test is the 10

- day wi ndow, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The mean biodegradation after 28 days of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- was 4 %; the 10 day window criterion was not passed.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 6 of 24 Therefore, Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- is consi dered to be not readily biodegradable. The results are represented in Tables 2

- 3 and Figure 1

.

Biodegradation of Sodium Benzoate: The reference item sodium benzoate was sufficiently d egraded to 8 9 % after 14 days and to 9 7 % a fter 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. The results are represented in Tables 2

- 3 and Figure 1

. Biodegradation of the Toxicity Control: In the toxicity control containing both, the test item and the reference item sodium benzoate, 50 % biodegradation was noted within 14 days and 5 5% biodegradation after 28 days of incubation. According to the test guidelines the test item
can be assumed to be not inhibitory to the aerobic activated sludge microorganisms. The results are represented in Tables 2

- 3 and Figure 1.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 7 of 24 2. Survey of the Study

2.1 General Information

Sponsor: PCBTF Testing Consortium c/o Greenwich Chemical Consulting, Inc.
3300 PGA Blvd Suite 635 Palm Beach Garden s, FL 33410

United States of America

Monitoring: David Esdaile CiToxLAB Hungary Ltd. 8200 Veszprém

– Szabadságpuszta Hungary

Test Item: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

-

Test Facility: Institut für Biologische Analytik und Consulting IBACON GmbH Arheilger Weg 17 64380 Rossdorf Germany

IBACON

- Project: 78601163

Project Staff:
Test Facility Management: Dr. Ralf Petto
Study Director: Dr. Ute Hammesfahr
Head of Quality Assurance Unit (QAU): Christiane Rutschmann

- Fröhlich (Biologist)

Schedule:
Study Initiation Date: December 17, 2012

1 st Study Plan Amendment: February 12, 2013

2 nd Study Plan Amendment: March 06, 2013

3 rd Study Plan Amendment: March 22, 2013
Experimental Starting Date: January 04, 2013

Experimental Completion Date: March 08 , 2013
Study Completion Date: April 0 3 , 2013Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 8 of 24 2.2 Good Laboratory Practice This study was performed in compliance with the most r ecent edition of:

− The OECD Principles of Good Laboratory Practice

− German Chemicals Act (ChemG), Annex 1

− Directive 2004/10/EC These requirements are accepted by regulatory author i ties throughout the European Community, the United States of America (FDA and EPA) and Japan (MHLW, MAFF and
ME TI) on the basis of intergovernmental agreements. This study was assessed in compliance with the study plan and the IBACON Standard Operating Procedures. This study and procedures were periodically inspected by the Quality Assurance Unit (QAU) and the dates and the phases of the inspections are included in the final report. The data presen ted in the final report were audi t ed in comparison to the raw data. A quality assurance statement, signed by the Quality Assurance Unit, is i n cluded in the final report.

2.3 Archiving The following documents and materials will be archived for the periods specified in the operative national GLP regul ations of the Study Director:

− all raw data

− the study plan

− any study plan amendment

− the final report (1 st original)

− any final report amendment or any revised final r e port (1 st original)

− one sample of the test item and of any reference item

following the date on which the final report is audited by the Quality Assurance Unit at: Institut für Biologische Analy tik und Consulting IBACON GmbH Germany No raw data or material relating to the study will be di s car ded without the sponsor's prior written consent.Final Report (2"d Original) ISAGON Project 78601163

- Ready Siedegradability of Benzene, 1- chloro-4-(trifluoromethyl)- in a Manometrie Respirometry Test

2.4 Signatores

Study Director: Dr. Ute Rammesfahr

Date: 

# cßtrit**~ ~()'j:J** 

Test Facility Management: Dr. Ralf Petto

Date: l nst i

l ut für BiologiSche Analytik und Consu il ing I BACON G m bli

page 9 of 24Final Report (2 " d Original) IBACON Project 78601163- Ready Siedegradability of Benzene

, 1

- chloro-4-(trifluoromethyl)- in a Manometrie Respirometry Test 

# IBAC

3. Quality Assurance Unit Statement

Test Facility:

IBACON Project:

Title ofthe Study:

Test Item:

Study Director :

Study Based Inspections

Phases inspected

Study Plan

I 5 1 Study Plan Amendment

2"d Study Plan Amendment

3rd Study Plan Amendment

Experimental Phase

Dosing, Application

Measurements

Dosing, Application

Draft Repmi

Final Report**~** l nsli l ul flir BiologiSche Analytik und Consu lt ing ! BACON G m bH

Institut flir Biologische Analytik
und Consulting !BACON GmbH Arheilger Weg 17
64380 Rossdorf Germany

78601163

Ready Biodegradability of Benzene, 1-chloro-4- (trifluoromethyl)- in a Manometrie Respirometry Test

Benzene

, 1-chloro-4-(trifluoromethyl)-

Dr. Ute Hammesfahr

Dates ofQAU Inspections

December 17

, 2012

February 12, 2013

March 05, 2013

March 21, 2013

January 04, 2013

February 01

, 2013

February 08

, 2013

March 12

, 2013

April 02,2013



### APR 0 3 2 0 13 Dates of Reports to Study
Director and to Test Facility Management

December 17,2012

February 12

, 2013

March 05,2013

March 21,2013

January 04

, 2013

February 01, 2013

February 08, 2013

March 12, 2013

April 02

, 2013



### APR 0 3 : 2 01 3

This statement confirms that the final report accurately reflects the raw data.

Quality Assurance Unit:

Date: Food Analyst Kirsten Laura Schönfeld

# ~"&f'<v<.

APR 0 3

. 2013

page 10 of 24Final Report (2"d Original) IBACON Project 78601163

- Ready Siedegradability of Benzene, 1- chloro-4-(trifluoromethyl)- in a Manometrie Respirometry Test 

# IBAC

4. Statement of Compliance

IBACON Projeet:

Title of the Study:

Test Item :
Study Direetor:

GLP-Regulations:

Integrity ofthe Study:

Study Direetor: . 78601163 I nst i t ut fOr Biolog;schc Analytik und Consu h ing ! BACON GmbH

Ready Biodegradability of Benzene, 1-ehloro-4- (trifluoromethyl)- in a Manometrie Respirometry Test

Benzene, 1-ehloro-4-(trifluoromethyl)-

Dr. Ute Rammesfahr

- The OECD Prineiples of Good Laboratmy Praetiee

- German Chemieals Aet (ChemG), Annex 1

- Direetive 2004/1 0/EC

These requirements are aeeepted by regulatory authorities throughout the European Community, the United States of
Ameriea (FDA and EPA) and Japan (MHLW, MAFF and METI) on the basis of intergovernmental agreements.

This study, performed in the test faeility of !BACON, was eondueted in eomplianee with the Good Labaratory Praetiee regulations

. There were no eireumstanees that may have
affeeted the quality or integrity ofthe study.

Dr. Ute Rammesfahr

page 11 of 24Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 12 of 24 5. Objectives of the Study

5.1 Title Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

5.2 Purpose The purpose of this study was to determine the ready bi odegradability of the test item Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- . The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 da ys. The biodegrad a tion was followed by the oxygen uptake of the micro o r ganisms du ring exposure. As a reference item sodium benzoate was tes ted simultaneously under the same co n ditions as the test item, and functioned as a procedure control. This type of st udy is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when inc ubated with aerobic activated sludge. This study is being performed based on the U.S. Toxic Su bstances Control Act (TSCA) Section 4 Test Rule and the regulations set forth at 40 CFR 799.508 9

.

5.3 Guidelines/Recommendations This study was designed to comply with the following met hods:

- Commission Regulation 440/2008/EC, Method C.4

- D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)

- OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirom e try Test", adopted July 17, 1992Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 13 of 24

6. Material and Methods

6.1 Test Item and Reference Item

Test Item The test item and the information concerning the test item were provided by the sponsor:

Test Substance Name: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

-
Chemical Name: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- Synonym: P

- Chlorobenzotrifluoride

Supplier: Zhejiang Kangfeng Chemicals Co., Ltd. (via EMCO Chemical Distributors, Inc. )

Batch /Lot N umber : 20111028

CAS Number: 98

- 56

- 6
Purity: 99.69%

Appearance : Clear colourless liquid
Report Date of CoA : October 28, 2011
Expiry (Retest) Date: October 28, 2013

Empirical Formula / Molecular Weight: C 7 H 4 ClF 3 / 180.56
Theoretical Oxygen Demand: ThOD NH4 : 1.241 mg oxygen per mg test item (calculated)

Density: 1.334 g/cm 3 (at 25°C) according to CoA

Storage: At room temperature (20 ± 5°C), under dark and dry cond itions

Reference Item The information concerning the reference item was provided by the manufacturer:

Name: Sodium Benzoate

Batch No.: 110M1219V

Retest Date: November 2014
Purity: 100.4 %
Quality Release Date: December 09, 2010
Empirical Formula / Molecular Weight: C 7 H 5 O 2 Na / 144.10 g/mol

Theoretical Oxygen Demand: ThOD NH4 : 1.666 mg oxygen per mg sodium benzoate (ca lculated)

Storage: At room temperature (20 ± 5°C), under dark and dry cond itionsFinal Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 14 of 24 6.2 Test System
Species/Origin: Aerobic activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the sewage plant Rossdorf , Germany.
Conditioning: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was d ecanted. The solid material was re

- suspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension wa s weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water (see 6.5). This suspension was

use d for the experiment.

6.3 Test Units
Type and Size: Manometric Test System with test flasks containing a vo lume of approximately 500 mL (see 6. 6 ).

Apparatus: BSB/BOD

- Sensor

- System, Aqualytic Dortmund, Germany
Principle: The test flasks prepared according paragraph 6. 6 were inc ubated at 22 ± 1 °C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD

- Sensor

- System, Aqualytic Dortmund, Germany

. The tes t flasks were closed gas

- tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O 2 consumed by the activated sludge was calc ulated from the decrease of pressure in the reaction vessel.

Identification: Each test unit was uniquely identified with the study nu mber, treatment and replicate number.

6.4 Test Conditions
Surrounding Type: Climatised room

Temperature: 22 ±1 °C
Light Conditions: Darkness

pH

- Value of Test Solutions: 7.5 (measur ed at the start of the test)
7. 5

– 7.6 ( measured at the end of the test)

6.5 Test Water

Reconstituted Test Water: Analytical grade salts were added to deionised water to pr epare the following stock solutions:

a) 8.5 g KH 2 PO 4 , 21.75 g K 2 HPO 4 , 33.4 g Na 2 HPO 4 x 2Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 15 of 24 H 2 O, 0.5 g NH 4 Cl filled up with deionised water to 1000 mL volume b) 22.5 g MgSO 4 x 7 H 2 O filled up with deionised water to 1000 mL volume
c) 36.4 g CaCl 2 x 2 H 2 O filled up with deionised water to
1000 mL volume
d) 0.25 g FeCl 3 x 6 H 2 O filled up with deionised water to
1000 mL volume In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of conce ntrated HCl per litre was added.

5 0 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of
5 000 mL with deionised water. The pH

- value wa s 7. 5 a nd therefore no adjustment was necessary.

6.6 Preparation of the Test Solutions

Table 1 . Preparation of the Test Solutions

Treatment Flask Test
Item Reference
Item 1 HgCl 2 Activated Sludge 2 Test
Water Final
Volume

m g / μ l mg mL mL mL mL

Benzene, 1

- chloro

- 4

-

(trifluoromethyl)

- 1 25. 0 / 18.7

— — 5 239 244

2 25.0 / 18.7

— — 5 239 244

Inoculum Control 3 3

— — — 5 239 244

4

— — — 5 239 244

Procedure Control 4 5

— 24.8

— 5 239 244

Abiotic Control 5 6 25. 0 / 18.7

— 5

— 239 244

Toxicity Control 7 25. 0 / 18.7 25. 4

— 5 239 244

1 Reference item: sodium benzoate

2 Final sludge concentration in test flasks : 30.7 mg sludge/L ( Stock suspension of 1

. 5 g/L on dry matter base )

3 The inoculum control was also used for other projects which ran in parallel.

4 The procedure control was also used for other projects which ran in parallel.

5 Poisoned with HgCl 2 (stock solut ion of 48.8 0 mg/mL)

— : Not applicable

6.7 Course of the Test
Preparation of Test Flasks: The amounts of test item and reference item were directly weighed / pipetted into the test flasks. The test flasks were capped immediately after the different pipetting or weig hingFinal Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 16 of 24 steps to prevent loss of the test item due to volatilisation. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of
the test item.

Incubation: The closed test flasks were incubated in a climatised room under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.

Test Duration: 28 days

6.8 Test Parameters
Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD

- Sensor

- System, Aqualytic Dortmund, Germany) each day.

Temperature: T he temperature was recorded by means of the automated software RMT Wincontrol ©

. pH

- Value: pH

- values were measured in procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of
the test using a pH

- electrode WTW pH 340i.

6.9 Result Evaluation

Definitions: ThOD NH4 : Theoretical Oxygen Demand is the total amount of oxygen required to oxidise a chemical completely. It is calculated from the m olecular formula and expressed as mg oxygen r equired per mg test item.

BOD: Biochemical Oxygen Demand

is the amo unt of oxygen consumed by micro orga nisms when metabolising a test item; also expressed as mg oxygen uptake per mg test item.

10

- day window: the 10 days immediately following the attainment of 10% biodegradation

Calculation of BOD: The biodegradability (% BOD = mg O 2 per mg test item) exerted after each period was calculated as:

BOD = mg O 2 uptake of test item

- mg O 2 uptake of inoculum control

mg test item in flask
For the toxicity control, the BOD of the treatment is the sum of test item
and reference item and is calculated as the sum of test item and refe rence item concentration in the flask.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 17 of 24 The percentage biodegradation of the test item and of the reference item sodium benzoate was calculated as:

% degradation = BOD (mg O 2 /mg test item or reference item) x 100 ThOD NH4 (mg O 2 /mg test item or reference item)

6.10 Validity Criteria of the Study

Inoculum Control: The oxygen demand of the inoculum control (medium and
inoc ulum) was 2 0 mg O 2 /L and thus not greater than 60 mg
O 2 /L within 28 days as required by the test guideline.

pH

- Value: The pH

- value of the test item flasks at the end of the test was
7. 5 and t herefore within the range of pH 6.0 to 8. 5 as r equired by the test guideline.

Reference Item: The percentage degradation of the reference item should reach the level for ready biodeg radability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 6 0% after 5 da ys of incubation.

Test Item: Since the biodegradation did not reach the pass criterion of 60% degradation and no 10 day

- window could be dete rmined, the validity criterion was not applicable. At the end of the experiment, the difference in replicate variation was
0 %.

6.11 Deviations to the Study Plan
There were no deviations to the study plan.

7. Results and Discussion

7.1 Biodegradation of Test Item
Percentage Biodegradation: The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10

- day window, describing the period between reaching at least 10% degr adation and 60% degradation. This period should not exceed 10 days. The mean biodegradation after 28 days of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- wa s 4 %; the 10 day window criterion was not passed. The results are represented in Tables 2

- 3 and Figure 1

. Conclusion: The degradation rate of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- did not reach 60% within the 10

- day wi ndow and after 28 days.
Therefore, Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- is consi dered to be not readily biodegradable.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 18 of 24 7.2 Biodegradation of Reference Item Sodium Benzoate
Percentage Biodegradation: The reference item sodium benzoate was sufficiently d egraded to 8 9 % after 14 days and to 9 7 % after 28 days of incubation. The results are represented in Tables 2

- 3 and Figure 1.

Conclusion: The percentage biodegradation of the reference item co nfirms the suitability of the used aerobic activated sludge
inoculum.

7.3 Biodegradation in the Toxicity Control
Percentage Biodegradation: In the toxicity control containing both the test item and re ference 50 % biodegradation was noted within 14 days and
5 5 % biodegradation was determined after 28 days of incub ation. The results are represented in Tables 2

- 3 and Figure 1.

Conclusion: According to the test guidelines the test item can be assumed

to be no t inhibitory on the aerobic activated sludge mi cro o rganisms because degradation was > 25% within 14 days.

7.4 Abiotic Control
Oxygen Demand: The oxygen demand in the abiotic control was zero (see Table 2). No correction of the test item degradation rates had to be done.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 19 of 24 8. References

1. Chemikaliengesetz der Bundesrepublik Deutschland (ChemG), Anhang 1, in der Fassung der B ekanntmachung vom 2. Juli 2008 (BGBl. I S. 1146), zuletzt geändert durch Art. 5 Abs. 39 G v. 24. Februar 2012 (BGBl. I S. 212)

2. Commission Regulation 440 /2008/EC, Method C.4

- D of May 30 , 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)

3. Directive 2004/10/EC of 11 February 2004 amending Commission Directive 87/18/EEC, Official Journal of the Euro pean Union No. L 50: 44

- 59

4. OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Resp irometry Test", adopted July 17, 1992

5. OECD Principles of Good Laboratory Practice, adopted by Commission on 26th November 1997 [C(97)186 /Final], Environment Directorate, Organisation for Economic Co

- operation and Develo pment, ENV/MC/CHEM(98)17, Paris 1998

6. Simple determination of biodegradability in accordance with OECD 301 F. Melliand reprint 10/1996, E 148

- E 150

7. U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the regulations set forth at 40 CFR 799.5089

9. Distribution of the Final Report

Sponsor: 1x (2 nd original)

IBACON: 1x (1 st original)Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 20 of 24 

## Appendi cesFinal Report ( 2 nd Original) IBACON Project 78601163 - Ready Biodegradability of Benzene, 1

- chloro -4- (trifluoromethyl) - in a Manometric Respirometry Test

page 21 of 24 Appendix I Certificate of Analysis of Benzene, 1

- chloro -4- (trifluoromethyl) -

· , .

:· · 

# IBAC

Institut für Biologische Analytik und Consulting IBACON GmbH



# ZHEJIANG KANGFEN G CHEMICALS C O., LTD.**Xueshh~ at**  industrinl z.one

. Dongyang City, Z hpjiallg, Chi n a T

c_o 1 ; 0006---:)79-86709687 r<nx : U08ß "., 57!H36709618 E nlt.'li 1 : qh q666@hotlll3 i l. coro



# Certifi-cate of Aualys.is

P-Cblorobcnzotrifluorlde

Bateil Number.:**~111** 02:8 PO

. NO ." : P 10 36 861

.**Qllantity~** 19SflOKQB

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.Final Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 22 of 24 Appendix II Additional Tables and Figures

Table 2 . Cumulative Biochemical Oxygen Demand (mg O 2 /L) in Test Flasks d uring the Test Period of 28 Days

Time Flask No.
(days) 1 2 3 4 5 6 7

1 0 0 0 0 50 0 50

2 0 0 5 0 70 0 70

3 5 0 5 5 95 0 95

4 5 5 10 10 110 0 120

5 10 10 10 10 125 0 130

6 10 10 10 10 135 0 140

7 15 15 15 10 140 0 145

8 15 15 15 15 145 0 150

9 15 15 15 15 150 0 155

10 15 15 15 15 150 0 155

11 20 15 15 15 155 0 160

12 20 15 15 15 160 0 160

13 20 15 15 15 160 0 165

14 20 15 15 15 165 0 165

15 20 20 15 15 165 0 165

16 20 20 15 15 170 0 165

17 25 20 20 20 170 0 170

18 25 20 20 20 170 0 170

19 25 25 20 20 170 0 170

20 25 25 20 20 170 0 175

21 25 25 20 20 175 0 175

22 25 25 20 20 175 0 175

23 25 25 20 20 175 0 175

24 25 25 20 20 180 0 180

25 25 25 20 20 180 0 180

26 25 25 20 20 180 0 180

27 25 25 20 20 185 0 185

28 25 25 20 20 185 0 185

Flasks 1 and 2: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- Flasks 3 and 4: inoculum control Flask 5: reference (procedure control)
Flask 6: abiotic control Flask 7: toxicity controlFinal Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 23 of 24 Table 3 . Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD NH4

Time Percentage Biodegradation 1

(Days) Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- 1 Sodium Benzoate 2 Toxicity
control 1, 2

Flask 1 [%] Flask 2 [%] Flask 5 [%] Flask 7 [%]

1 0 0 30 17

2 0 0 40 22

3 0 0 53 30

4 0 0 59 37

5 0 0 68 40

6 0 0 74 43

7 2 2 75 44

8 0 0 77 45

9 0 0 80 47

10 0 0 80 47

11 4 0 83 48

12 4 0 86 48

13 4 0 86 50

14 4 0 89 50

15 4 4 89 50

16 4 4 92 50

17 4 0 89 50

18 4 0 89 50

19 4 4 89 50

20 4 4 89 52

21 4 4 92 52

22 4 4 92 52

23 4 4 92 52

24 4 4 95 53

25 4 4 95 53

26 4 4 95 53

27 4 4 97 55

28 4 4 97 55

1 ThOD NH4 of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- : 1.241 mg O 2 /mg test item

2 ThOD NH4 of sodium benzoate: 1.666 mg O 2 /mg reference itemFinal Report ( 2 nd Original) IBACON Project 78601163

- Ready Biodegradability of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- in a Manometric Respirometry Test

page 24 of 24 Table 4 . pH

- Values at the End of the Test

Flask No. Treatment pH

- value

1 Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- 7. 5

2 Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- 7. 5

3 Inoculum control 7. 5

4 Inoculum control 7. 5

5 Procedure control 7. 6

6 Abiotic control 6.7

7 Toxicity control 7.8

-10

10 30 50 70 90 110

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Biodegradation (ThODNH4)

Days

Test Item Flask 1 Test Item Flask 2 Procedure Control Toxicity Control

Figure 1 . Biodegradation of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- (Flasks 1 and 2), of the Refe rence Item Sodium Benzoate (Procedure Control) and of the Toxicity Control d uring the Exposure Period of 28 Days Related to ThOD N H4