page 1 of 24

Final Report (2nd 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 03, 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 Gardens, FL 33410

United States of America

Test Facility

Institut für Biologische Analytik Project 78601163

und Consulting IBACON GmbH

Arheilger Weg 17

64380 Rossdorf

Germany
Final Report (2nd 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 

Gute Laborpnu:is/Good Laboratory Practice 

GLP-Bescbeinigung!Statemcnt of GLP Compliance 

{ymWaocordirlg to ~ 19b Ab$. I Chemll:altt•g=tz) 

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(Vnwrwtdl~e barc Be'Zcidlnung und Aclrcsse/UneqlllYQC:II ruune and adr=) 

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Final Report (2nd 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 Assurance 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 Result 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. Distribution 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 .......................................................................................22
Final Report (2nd 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 O2/L) in Test Flasks during the Test Period of 28 

Days.............................................................................................................................................22

Table 3. Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based 

on ThODNH4 .................................................................................................................................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 ThODNH4 .......................................................................................24
Final Report (2nd 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 (microorganisms from a domestic 

wastewater treatment plant) was supplied by the sewage 

works of Rossdorf, Germany.

Test Design: The test item Benzene, 1-chloro-4-(trifluoromethyl)- was 

investigated for its ready biodegradability in a manometric 

respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms 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 consumption of the aerobic activated sludge microorganisms 

after 28 days of incubation.

Test Item Loading Rate: 102.5 mg/L corresponding to an oxygen demand of about 

127.2 mg/L (ThODNH4) 

Reference Item: Sodium Benzoate

Reference Item Loading Rate: 101.6 mg/L corresponding to an oxygen demand of about 

169.3 mg/L (ThODNH4) 

Test Conditions: 22±1 °C, darkness

Results:

Biodegradation of Benzene, 1-chloro-4-

(trifluoromethyl)-:

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% 

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 (2nd 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 considered 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 degraded to 89% after 14 days and to 97% after 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 55% 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 (2nd 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 Gardens, 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

1st Study Plan Amendment: February 12, 2013

2nd Study Plan Amendment: March 06, 2013

3rd Study Plan Amendment: March 22, 2013

Experimental Starting Date: January 04, 2013

Experimental Completion Date: March 08, 2013

Study Completion Date: April 03, 2013
Final Report (2nd 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 recent 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 authorities 

throughout the European Community, the United States of 

America (FDA and EPA) and Japan (MHLW, MAFF and 

METI) 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 presented in the final report were audited in comparison to the raw 

data. A quality assurance statement, signed by the Quality 

Assurance Unit, is included in the final report.

2.3 Archiving The following documents and materials will be archived for 

the periods specified in the operative national GLP regulations of the Study Director:

− all raw data

− the study plan

− any study plan amendment

− the final report (1st original)

− any final report amendment or any revised final report 

(1st 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 Analytik 

und Consulting IBACON GmbH 

Germany

No raw data or material relating to the study will be discarded 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: 

lnstilut für BiologiSche Analytik und Consuiling I BACON Gmbli 

page 9 of 24 
Final 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 51 Study Plan Amendment 

2"d Study Plan Amendment 

3rd Study Plan Amendment 

Experimental Phase 

Dosing, Application 

Measurements 

Dosing, Application 

Draft Repmi 

Final Report 

~ 

lnslilul flir BiologiSche Analytik und 

Consulting !BACON GmbH 

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 2013 

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: 2013 

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 24 
Final 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 

Institut fOr Biolog;schc Analytik und 

Consuhing !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 24 
Final Report (2nd 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 biodegradability 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 days. The biodegradation was 

followed by the oxygen uptake of the microorganisms 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.

This type of study is recognized by the international test 

guidelines and should provide a rational basis to assess the 

ready biodegradation properties of the test item when incubated with aerobic activated sludge.

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Test Rule and the 

regulations set forth at 40 CFR 799.5089.

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

- 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
Final Report (2nd 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 Number: 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: C7H4ClF3 / 180.56

Theoretical Oxygen Demand: ThODNH4: 1.241 mg oxygen per mg test item (calculated)

Density: 1.334 g/cm3 (at 25°C) according to CoA

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

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: C7H5O2Na / 144.10 g/mol 

Theoretical Oxygen Demand: ThODNH4: 1.666 mg oxygen per mg sodium benzoate (calculated)

Storage: At room temperature (20 ± 5°C), under dark and dry conditions
Final Report (2nd 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, microorganisms 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 decanted. 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 was 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 

used for the experiment. 

6.3 Test Units

Type and Size: Manometric Test System with test flasks containing a volume 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 incubated 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 test flasks were closed gas-tight 

by a measuring head. Potassium hydroxide solution (45%)

was used for trapping the produced carbon dioxide. The 

amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

Identification: Each test unit was uniquely identified with the study number, 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 (measured 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 prepare the following stock solutions:

a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 
Final Report (2nd Original) IBACON Project 78601163 - Ready Biodegradability of Benzene, 1-

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

page 15 of 24

H2O, 0.5 g NH4Cl filled up with deionised water to 1000 

mL volume

b) 22.5 g MgSO4 x 7 H2O filled up with deionised water to 

1000 mL volume

c) 36.4 g CaCl2 x 2 H2O filled up with deionised water to 

1000 mL volume

d) 0.25 g FeCl3 x 6 H2O 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 concentrated HCl per litre was added.

50 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 

5000 mL with deionised water. The pH-value was 7.5 and 

therefore no adjustment was necessary.

6.6 Preparation of the Test Solutions

Table 1. Preparation of the Test Solutions

Treatment Flask Test 

Item

Reference 

Item1

HgCl2 Activated 

Sludge2

Test 

Water

Final 

Volume

mg /μl 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 Control5 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 HgCl2 (stock solution of 48.80 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 weighing 
Final Report (2nd 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: The 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: ThODNH4: Theoretical Oxygen Demand

is the total amount of oxygen required to oxidise a 

chemical completely. It is calculated from the molecular formula and expressed as mg oxygen required per mg test item.

BOD: Biochemical Oxygen Demand

is the amount of oxygen consumed by microorganisms 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 O2 per mg test item) exerted after 

each period was calculated as:

BOD =

mg O2 uptake of test item - mg O2 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 reference item concentration in the flask. 
Final Report (2nd 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 O2/mg test item or reference item) x 100

ThODNH4 (mg O2/mg test item or reference item)

6.10 Validity Criteria of the Study

Inoculum Control: The oxygen demand of the inoculum control (medium and 

inoculum) was 20 mg O2/L and thus not greater than 60 mg 

O2/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 therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item: The percentage degradation of the reference item should 

reach the level for ready biodegradability (>60%) within 14 

days as required by the test guideline.

The reference item sodium benzoate was degraded to more 

than 60% after 5 days of incubation.

Test Item: Since the biodegradation did not reach the pass criterion of 

60% degradation and no 10 day-window could be determined, 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% 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.

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 window and after 28 days.

Therefore, Benzene, 1-chloro-4-(trifluoromethyl)- is considered to be not readily biodegradable.
Final Report (2nd 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 degraded to 89% after 14 days and to 97% after 28 days of 

incubation. The results are represented in Tables 2 -3 and 

Figure 1.

Conclusion: The percentage biodegradation of the reference item confirms 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 reference 50% biodegradation was noted within 14 days and 

55% biodegradation was determined after 28 days of incubation. 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 not inhibitory on the aerobic activated sludge microorganisms 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 (2nd 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 Bekanntmachung 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 European Union No. L 50: 44 - 59

4. OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry 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 Development, 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 (2nd original)

IBACON: 1x (1st original)
Final Report (2nd Original) IBACON Project 78601163 - Ready Biodegradability of Benzene, 1-

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

page 20 of 24

Appendices
Final Report (2nd 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 KANGFENG CHEMICALS CO., LTD. Xueshh~ at industrinl z.one . Dongyang City, Zhpjiallg, China 

T c_o 1 ; 0006---:)79-86709687 r<nx : U08ß".,57!H36709618 Enlt.'li 1 : qhq666@hotlll3 i l. coro 

Certifi-cate of Aualys.is 

P-Cblorobcnzotrifluorlde 

Bateil Number.: ~111 02:8 

PO. NO.": P 1036861 . 

Qllantity~ 19SflOKQB , 

ExpirJ' dat~ .l013·t~l8 

--... ··----... ··-~-···· .......... -...... __________ ,.._", ___ .... _ ... _ ..... . ... ' -.. .,_.,' ". .... _. _____ .,....,. ..... _ ... ~ ·--·---.. --- Teilt items Speti6tationA Rts'tllts --.;-.............. _ .. ____ .. _.._.., ____ ... ____ ~ ..... -~ ----- --.. -...... .,.. .... _ ... _ ,.. __ .,. ________ ~ .. 

Appearance .. 

\ :·oe.-·.· . . 

-Assay 

Aeldity; ppui (spec. ion pro.bc) 

Colot- {APHA') 

Non-Vol~tile J:l,esiduc,. Wt% 

\Va-ter Ci)nten~ ppm 

· Öe~trLfquid,Frceof 

"Swpended Prü'tter · 

99%Mia.. 

3 ppm.M;tx. 

20APHAMax 

{).0020 % Max. 

1.330- 1.350 

lS~ ppro Mux. 

Conclusion: PASS 

Lub tecbniclan 

it~.l.it: .tt.t~ 

a,cck 

jlt~ . M:~J* 

C..'Ielr Liquid, Fffil .. M ·r: 

Suspwded Matter 

.. 0.8 

10 

·0..001 

tofr · 

. . • ·i· . •. 
Final Report (2nd 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 O2/L) in Test Flasks during 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 control
Final Report (2nd 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 ThODNH4

Time Percentage Biodegradation1

(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 ThODNH4 of Benzene, 1-chloro-4-(trifluoromethyl)-: 1.241 mg O2/mg test item 2 ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference item 
Final Report (2nd 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 28

Biodegradation (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 Reference Item Sodium Benzoate (Procedure Control) and of the Toxicity Control during the 

Exposure Period of 28 Days Related to ThODNH4