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Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

Page 1 of 24 

Bioassay and Safety Assessment Lab 

SHANGHAI ACADEMY OF PUBLIC MEASUREMENT 

FINAL REPORT 

Ready Biodegradability by the Manometric Respirometry Test 

of

Code: 

Study Number of DOW: 

Study director: 

SPONSOR: 

The Dow Chemical Company 

Midland, MI 48674 USA 

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Page 2 of 24 

Contents 

Title ............................................................................................................................. 1 

Contents ..................................................................................................................... 2 

Statement of GLP Compliance ................................................................................. 3 

Quality Assurance Statement .................................................................................. ·4 

Laboratory Statement ............................................................................................... 5 

Signature Page .......................................................................................................... 6 

Key Study Personnel ................................................................................................ 7 

1 Abstract ............................................................................................................... 8 

2 Study Sponsor .................................................................................................... 8 

3 Study Monitor ...................................................................................................... 9 

4 Study Director ..................................................................................................... 9 

5 Information of Test Substance .......................................................................... 9 

6 Date ...................................................................................................................... 9 

7 Objective ............................................................................................................ 10 

8 Guideline ........................................................................................................... 10 

9 Principle of the Test.. ........................................................................................ 10 

10 Materials and Reagents ................................................................................. 10 

11 Apparatus ....................................................................................................... 10 

12 Species ........................................................................................................... 11 

13 Test Methods .................................................................................................. 11 

14 Method Validation .......................................................................................... 15 

15 Results and Discussions .............................................................................. 15 

16 Conclusions ................................................................................................... 16 

17 Archives .......................................................................................................... 16 

18 Departure from Procotol ............................................................................... 16 

19 References ..................................................................................................... 16 

20 Tables and Figures ........................................................................................ 17 

Appendix .................................................................................................................. 23 

Information on Test Substance (provided by the sponsor) ................................. 23 

Certificate of Analysis of Test Substance (provided by the sponsor) ................ 24 

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Bioassay and Safety Assessment Lab, APM Code: 

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Page 3 of 24 

Statement of GLP Compliance 

r ; L The work to which this study refers was performed in compliance with the OECD 

guideline of Good Laboratory Practices (ENVIMCICHEM (98)17), requirements of 

lJ conduct for good laboratory practice (GBIT 22275.1-2008) and the guidelines of 

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chemical testing good laboratory practices (HJIT 155-2004). 

The report fully and accurately reflects the procedures used and data generated. 

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Quality Assurance Statement 

The study conduct, raw data and report were inspected by the Quality Assurance Unit 

(QAU) at Bioassay and Safety Assessment Laboratory, APM to determine adherence 

with the study protocol and laboratory standard operating procedures. This report is 

an accurate representation of the raw data. Type and date of study inspections, and 

date of report to the Study Director and to Facility Management are listed below: 

Inspecting Type Inspection Date Report to study directorl 

Facility Management 

Study plan 2012.3.26 2012.3.26/2012.3.26 

Report 2012.6.18/19 2012.6.19/2012.6.19 

Sample and test systems 2012.4.27 2012.5.4/2012.6.19 preparation 

Determinations and 2012.4.27 2012.5.4/2012.6.19 

data record 2012.5.4/11/25 2012.5.25/2012.6.19 

Date 

(signed in original- 3t~5f) 

Bioassay and Safety Assessment Lab, APM 

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Page 5 of 24 

Laboratory Statement 

1. This test report would be invalid without the seal of the lab and the signatures of 

Study director, QA and Manager of Testing facility. 

2. Any unauthorized alteration of the test report would make the report invalid. 

3. The test report only reflects the test substance received from sponsor. 

4. The owner of the test report shall be responsible for any consequences caused by 

using incomplete information from the test report. 

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Bioassay and Safety Assessment Lab, APM Code: 

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Page 6 of 24 

Signature Page 

Submitted by: Bioassay and Safety AssessmentLab, APM 

Prepared by: 

Approved by: 

1500 Zhangheng Road, Zhangjiang Hi-tech Park, 

Shanghai, 201203, P.R. China 

Bioassay and Safety Assessment Lab, APM 

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Date 

(signed in original- JllUj§- X) 

Bioassay and Safety Assessment Lab, APM 

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Bioassay and Safety Assessment Lab, APM Code: 

. Ready biodegradability by the manometric respirometer of

Page 7 of 24 

Key Study Personnel 

The following personnel were responsible for the conduct of the work and reporting of 

the study results 

Yang Jing Ecotoxicologist & Study Director 

Chen Xiaoqian Ecotoxicologist & Report Writer 

Zhang Jingji Ecotoxicologist & Biodegradation test 

Zhang Kun Ecotoxicologist & Biodegradation test 

Zhang Ying Ecotoxicologist & Biodegradation test 

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Page 8 of 24 

1 Abstract 

The study was conducted according to the guidelines of "OECD, Guidelines for the 

Testing of Chemicals, 1992, Ready Biodegradability 301 F: Manometric Respirometry 

Test". 

Under specific experimental conditions, the ready biodegradability of 

( was determined in a 28-day dissolved oxygen 

depletion test using activated sludge from a domestic waste water treatment plant. 

The tested concentration of the test substance was 21.20 mg/L (Le. 50.67 mg 

ThO OIL). The concentration of sludge inoculum in the test system was 30 mg/L, and 

the tested concentration of sodium used as reference substance was 100 

mg/L (Le.167.00 mg ThOD/L). An inhibition control was also included. 

During the test, the temperature was maintained at 21.5-23.0°C. The mean total O2 

uptake in the inoculum blanks at the end of the test was less than 60 mg 02/L in 28 

days. Biodegradation of the reference substance (sodium reached an 

average of 78.53% by Day 14, meeting the pass level of >60% within 14 days. The 

difference between the maximum and minimum replicate values of the amount of 

removal of the test substance at the end of the test was less than 20%. The results of 

the inhibition control showed that the test substance met the criteria for not being 

inhibitory to the microbial inoculum. Thus, the test is valid. 

During the 28-day test period, the biochemical oxygen demand (BOD) of the test 

substance didn't show a significant increase. The mean biodegradation percentage of 

the test substance at Day 28 was 11.15%, which didn't meet the 10-d window 

criteria within 28-d period of the test. 

Therefore, the test substance cannot be considered to be readily biodegradable under 

the experimental conditions according to this test method. 

2 Study Sponsor 

Name: The Dow Chemical Company 

Address: Midland, M148674, USA 

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Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

Page 90f24 

Contact person: Lorraine Francourt 

Tel: +49 7227912761 

Email: LFrancourt@dow.com 

3 Study Monitor 

Contact person: Summer Shen 

Address: No.936, Zhangheng Road, Zhangjiang Hi-Tech Park, Shanghai 201203, 

P.R.C . 

Tel: +86-21-38513187 Fax: +86-21-20249979 

Email: smshen2@dow.com 

4 Study Director 

Name:

Address: Room 209, Bioassay and Safety Assessment Laboratory, 1500 Zhangheng 

Road, Zhangjiang Hi-tech Park, Shanghai, 201203, P.R. China 

Tel: 86-21-50798123 Fax: 86-21-50798125 Email: yangj@apm.sh.cn 

5 Information of Test Substance 

Name: 

Code: 

Receipt date: 2012~02-13 

Conditions of storage: Store in dark at room temperature 

L.,j Full details of the test substance are given in Appendix A. 

, , 

6 Date 

Study initiation date 2012-04-09 

Experimental starting date 2012-04-27 

Experimental completion date 2012-05-25 

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Page 10 of 24 

Study completion date 

7 Objective 

The ready biodegradability of the test substance was investigated using the 

Manometric Respirometry Test. 

8 Guideline 

OECD, Guidelines for the Testing of Chemicals, 1992, Ready Biodegradability 301 F: 

Manometric Respirometry test. 

9 Principle of the Test 

The biodegradation process consumes the dissolved oxygen in the test medium and 

generates C02. The CO2 is absorbed by sodium hydroxide, which results in a 

decrease of the total pressure in the airtight test flasks. The pressure drop is detected 

and converted into an electrical signal by means of an electrode type manometer. 

10 Materials and Reagents 

10.1 Mineral medium (MM) 

The preparation of the mineral medium is described in detail in 20.1. 

10.2 Reference substance 

Name: sodium (Shanghai Linfeng Chemical Reagent Co. Ltd); 

Code: REF01 0-201 0-01; 

CAS number: 532-32-1; 

Purity: ;:,,99.5%; 

Lot number: 20100310. 

11 Apparatus 

BOD respirometer 0NTW, OxiTop 11 OC); 

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Mould incubator (YIHENG TEC, MJ-2501) 

pH meter ryvTW Multi 34301); 

Moisture meter (Mettler Toledo, HG63); 

Balance (Mettler Toledo, XS205). 

12 Species 

Page 11 of 24 

Inoculum: Activated sludge, from a domestic sewage treatment plant, is normally used 

as the microbial inoculum based on "OECD, Guidelines for the Testing of Chemicals. 

Ready Biodegradability 301F: Manometric respirometry test, 1992". The activated 

sludge is suitable for the ready biodegradability test because of its higher cell 

densities, no pre-adaption to the test chemical, and its consistent behavior in the test 

system. 

Sludge collection: A fresh sample of activated sludge was collected on Apr 2th

, 

2012 from the aeration tank of the Longhua Wastewater Treatment Plant in Shanghai 

which treats predominantly domestic sewage by membrane bioreactor (MBR). The 

activated sludge was kept aerobic until used. 

Sludge treatment: Beofer use, the activated sludge was passed through a 0.5 mm 

sieve to remove coarse particles. The sludge was washed with mineral medium by 

settling the sludge in a graduated cylinder, decanting the supernatant liquid, replacing 

with fresh mineral medium, aerating, and settling. The above washing process was 

repeated 3 times, then the sludge was centrifuged one time (1500 rpm, 4°C, 

centrifuge 10 minutes). A small amount (about 0.4 g) of the concentrated sludge is 

weighed, and thEm dried at 105°C for 1h to calculate the moisture. The dry weight of 

sludge was 4.48%. 

Sludge suspension: Based on the dry weight, the amount of wet sludge was 

calculated and suspended in mineral medium to give a mixed liquor suspended solids 

level of 3 gIL. The final concentration of the activated sludge in the test medium was 

30 mg/L. 

13 Test Methods 

13.1 Preparation of test system 

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Page 12 of 24 

13.1.1 Preparation of Test Substance Incubates 

Test substance is not miscible with water according to the information supplied by tlJe 

sponsor. Therefore, the test substance was coated on silica gel to introduce it into test 

system. 

An adequate amount of the silica gel (35-60 Mesh) was dried in a muffle furnace at 

500-550oe for 2 hours. The silica gel was removed from muffle furnace, cooled to 

room temperature, and kept dry by storing the silica gel in a desicator. 

A 9 g portion of dried silica gel was weighed into the glass vial. 1.00081 g test 

substance also weighed into the glass vial which containing the weighed silica gel. 

The vial containing the test substance-silica gel mix was capped and wrap tape was 

placed around the cap to prevent it from being opened. The above sample was placed 

on a roller mixing device to mix for overnight (roller rate was 30 rpm). 

A TOe (total organic carbon) analyzer was used to verify that 5 replicates of the mixed 

sample contained the same amount of carbon. The results were 69.48, 68.98, 72.04, 

66.39,66.22 g/kg (68.62 ± 2.41 g/kg; average ± 1 standard deviation); Based on these 

results, the test substance was considered to be distributed uniformly in the silica gel. 

53.00 mg test substance-silica gel was weighed separately and added directly into 

the respirometer flask filled with 250 ml mineral medium containing inoculum. The 

concentration of the test substance was 21.20 mg IL (i.e. 50.67 mg ThOD/L). Test 

substance incubates were prepared in duplicate (Flask No.1). 

53.01 mg test substance-silica gel was weighed separately and added directly into 

respirometer flask filled with 250 ml mineral medium containing inoculum. The 

concentration of the test substance was 21.20 mg/L (i.e. 50.67 mg ThOD/L). Test 

substance incubates were prepared in duplicate (Flask No.2). 

13.1.2 Preparation of Reference Substance Incubates 

To prepare the stock solution of reference substance, 499.9 mg of sodium benzoate 

was dissolved in 500 ml mineral medium and the concentration of stock solution was 

1000 mg/L. 

To 147.6 ml of inoculum mineral medium, 16.4 ml of reference stock solution and 

31.09 mg silica gel were added to give a final test solution concentration of 100 mg/L 

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Bioassay and Safety Assessment Lab, APM Code: 

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Page 13 of 24 

(Le. 167.00 mg ThOD IL). Reference substance incubates were prepared in duplicate 

(Flask NO.5 and 6). 

13.1.3 Inoculum Blank Incubates 

Inoculum blank incubates containing only 365 ml mineral medium and 69.64 mg/69.65 

mg silica gel with inoculum were prepared in duplicate (Flask NO.3 and 4). 

13.1.4 Toxicity Control Incubates 

A single toxicity control incubates was prepared (Flask No.7). 34.64 mg test L substance-silica gel and 16.4 ml of reference stock solution were added into 147.6 ml 

of inoculum mineral medium to give a final concentration of the test substance of L 21.10 mg IL (Le. 50.43 mg ThOD/L) and a final concentration of reference substance 

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of 100 mg/L (i .. e 16700 mg ThOD/L) 

Incubate Contents (mg/L) Flask 

Description Volume Test Reference. No. 

(ml) Inoculum 

Substance. Substance. 

1 Test Substance (TS1) 250 21.20 - 30 

2 Test Substance (TS2) 250 21.20 - 30 

3,4 Inoculum Blanks (IB) 365 - - 30 

Reference Substance 

5,6 (Procedure Control PC) 

164 - 100 30 

7 Toxicity Control (TC) 164 21.10 100 30 

13.2 Respirometer Operation 

When the test system was prepared completely, Respirometer 0NTW, OxiTop 110C, 

Weilheim, Germany) Operational procedure as follows: 

1) The estimation and option of measuring span, test system preparation as above 

table (see 13.1) 

2) Insert a magnetic stirrer bar into the flasks. 

3) Place 2 sodium hydroxide pellets in the rubber sleeve. 

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Page 14 of 24 

4) Insert the rubber sleeves into the flasks. 

5) Screw the OxiTop® measuring head tightly. 

6) Start the measurement on the OxiTop® head. 

7) Place the measuring flask on the inductive stirring system, then in the incubator 

for 28 days at 22 ± 1°C, in the dark. 

8) Auto-read the results once per 112min during 28 days. 

9) Determine pH at the beginning and the end of the study (see table 18.2). 

13.3 Data Analysis Method 

13.3.1 Theoretical Oxygen Demand (ThOD) 

ThODNH3 was theoretical oxygen demand without nitrification. ThODNo3 was 

theoretical oxygen demand with nitrification. 

The ThOD NH3 of the reference substance was 1.67 mg 02/mg. 

100 mg/L of reference substance was 167.00 mg ThOD NH3/L. 

chemical formula: C25H2404 (MW:388.46), purity (99.90%). 

ThODNH30f test substance = 2.39 mg 02/mg. 

21.20 mg/L test substance was 50.67 mg ThOD/L; 

21.10 mg/L test substance was 50.43 mg ThOD/L. 

13.3.2 Biodegradability Percent Calculated by BOD 

Raw data is exported by Achat OCv2.03 into Excel forms. 

Biodegradct>ilitypercent%= ( a - bm ) x 100% 

Co x ThODNH3 

a - BOD ofTS, PC or TC flask, mg/L; 

bm- BOD of IB flask, mg/L; 

Co- test or reference substance concentration, mg/L; 

ThODNH3- Theoretical oxygen demand, mg 02/mgo 

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Page 15 of 24 

14 Method Validation 

The mean BOD of the inoculum blanks was 27.45 mg 02/L in 28 days, which was less 

than 60 mg/L, meeting the guideline requrement. The pH values of the solution in test 

substance flasks were in the range of 7.457 - 7.719, which met the requirement of pH 

6 - 8.5 of the test substance solution. The difference between replicate values of the 

removal of the test substance at the the end of the test was less than 20%. 

The mean biodegradation of the reference substance (Sodium benzoate) was 

78.53% by Day 14, meeting the pass level for the ready biodegradation test (> 60% 

within 14 days). 

The biodegradation percent of the toxicity control was 62.39% by day 14, ;::25 % 

ThOD, which met the guideline requirement. 

Thus, the test is considered valid. 

15 Results and Discussions 

15.1 The Results Presented in 20.3 and 20.4. 

During the 28-day test period, the biochemical oxygen demand (BOD) of the test 

substance didn't show a significant increase. The mean biodegradation percentage of 

the test substance at at Day 28 was 11.15%, which didn't meet the 10-d window 

criteria within 28-d period of the test.Therefore, the test substance cannot be 

considered to be readily biodegradable under the experimental conditions according 

to this test method. 

15.2 The biodegradation results for the toxicity control are presented in 20.3 and 20.6. 

The percent of biodegradation in the toxicity control, containing both the test 

substance and reference substance, was calculated based on the sum of the 

ThODNH3 of the test substance and the ThODNH3 of the reference substance. 

In the toxicity control, within 14 days of exposure, biodegradation reached 62.39%. 

Since biodegradation in the toxicity control was greater than 25% within 14 days, the 

test substance was considered not to have a toxic effect on the activity of the 

microbial inoculum. 

15.3 pH values presented in 20.2. 

r1 LJ The pH of the solutions in all flasks at the start and end of the test were measured. 

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The pH values were in the range of 7.457 to 8.314. 

16 Conclusions 

The test substance was not readily biodegradable under the 

present test conditions. 

17 Archives 

The signed copies of the final reports (English and Chinese) and notarized copies of 

all raw data generated and recorded during the study will be sent to the sponsor at 

designated address after issuance of the final reports. All original raw data generated 

and recorded during the study and signed copies of the final reports (English and 

Chinese) will be stored in the Bioassay and Safety Assessment Laboratory of 

Shanghai Academy of Public Measurement for five years, and returned to the sponsor 

at designated address on agreed date. 

18 Departure from Procotol 

None. 

19 References 

OECD, Guidelines for the Testing of Chemicals, 2006, Section 3 Introduction: Revised 

Introductions of the OECD Guidelines for Testing of Chemicals, Section 3 

OECD, Series on the Test Guidelines Programme, Number2, 1998, Detailed Review 

Paper on Biodegradability Testing. 

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Page 17 of 24 

I , 20 Tables and Figures ~ 

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20.1 Composition of Basal Salts Medium (BSM) and Stock Solutions 

Stock Solutions: 

(A) Phosphate Solution 

KH2P04: 

K2HP04: 

Na2HP04 : 

NH4CI: 

Dissolve in and make up to 1000 ml with ultra pure water. 

The pH should be 7.4 ± 0.2. 

(B) Calcium Chloride Solution 

CaCI2: 

Dissolve in and make up to 1000 ml with ultra pure water. 

(C) Magnesium Sulphate Solution 

MgS04'7H20 : 

Dissolve in and make up to 1000 ml with ultra pure water. 

(D) Ferric Chloride Solution 

FeCI3'6H20: 

Dissolve in and make up to 1000 ml with ultra pure water. 

Add one drop of concentrated HCI per litre. 

8.50 9 

21.75g 

26.64 9 

0.50 9 

27.50 9 

22.50 9 

0.25g 

Stock Solution Preparation: Stock solutions A-D are filtered through 0.45,um pore 

cellulose nitrate membrane. The sterilized filtrates are refrigerated at 4°C. Prepare 

fresh stock solution if a precipitate forms. 

Test Medium: BSM is prepared before use in deionised water and is prepared as 

follows: Mix 10 ml of solution A with 800 ml ultra pure water, and then add 1 ml each 

of solutions B, C, and D. Bring volume to 1 L with deionised water. 

20.2 pH Readings at the Beginning and End of the Experimental Phase of the Study 

Inhibition I 

Test Substance Inoculum Blank Reference Control Toxicity Oay(d) Control 

Flask 1 Flask 2 Flask 3 Flask 4 Flask 5 Flask 6 Flask 7 

0 7.719 7.719 7.733 7.733 7.716 7.716 7.703 

28 7.457 7.486 7.499 7.498 8.314 8.217 8.178 

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20.3 Records of Biochemical Oxygen Demand (mg/L) 

Inoculum Reference Inhibition 

Test Substance 

Oay(d) Blank Control Control 

Flask 1 Flask 2 Flask 3 Flask 4 Flask 5 Flask 6 Flask 7 

1 1.40 2.80 3.40 4.00 28.10 30.90 36.60 

2 7.00 8.50 7.90 8.50 70.30 76.00 76.00 

3 9.90 12.70 10.70 11.30 87.20 101.00 107.00 

4 12.70 14.10 13.00 13.60 101.00 118.00 121.00 

5 15.50 16.90 14.70 15.30 115.00 127.00 129.00 

6 15.50 16.90 15.80 16.40 124.00 132.00 135.00 

7 16.90 18.30 16.40 17.00 129.00 135.00 141.00 

8 18.30 19.70 17.00 18.10 132.00 138.00 143.00 

9 19.70 21.10 18.10 19.20 135.00 141.00 146.00 

10 19.70 22.50 18.70 19.80 138.00 141.00 149.00 

11 19.70 22.50 19.20 20.90 141.00 143.00 149.00 

12 21.10 24.00 20.30 21.50 146.00 149.00 155.00 

13 24.00 25.40 21.50 23.20 152.00 152.00 158.00 

14 24.00 25.40 21.50 23.20 155.00 152.00 158.00 

15 33.80 36.60 26.00 27.70 172.00 172.00 174.00 

16 25.40 26.80 22.60 24.30 158.00 155.00 160.00 

17 26.80 28.20 23.20 24.90 158.00 158.00 163.00 

18 26.80 28.20 23.20 25.40 158.00 158.00 163.00 

19 26.80 28.20 23.20 25.40 160.00 158.00 163.00 

20 26.80 29.60 23.70 25.40 160.00 158.00 166.00 

21 26.80 29.60 24.30 26.00 160.00 158.00 166.00 

22 28.20 31.00 24.30 26.60 163.00 160.00 169.00 

23 29.60 32.40 24.90 27.10 163.00 163.00 172.00 

24 29.60 32.40 25.40 27.10 166.00 163.00 169.00 

25 31.00 32.40 26.00 27.10 163.00 163.00 172.00 

26 31.00 32.40 25.40 27.70 166.00 163.00 172.00 

27 32.40 33.80 26.00 27.70 166.00 163.00 172.00 

28 32.40 33.80 26.60 28.30 166.00 166.00 172.00 

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Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

Page 19 of 24 

20.4 Calculation of Biodegradation of Test Substance 

Test 

Inoculum Blank: Substance: 

Corrected uptake of 

% Biodegradation of Test Substance 

O2 Uptake (mg/L) O2 Uptake 

O2 by Test ... (mg/L) 

Substance ": 

Mean 

Flask 1 - Flask 2- Mean 

Flask Flask Flask % % Flask Mean Mean Biodegdn. Flask Biodegdn. Biodegdn. 3 4 1 2 Flask Flask of Test 3&4 In Flask 1 In Flask 2 3&4 3&4 Substance 

3.40 4.00 3.70 1.40 2.80 -2.30 -0.90 -4.54 -1.78 -3.16 

7.90 8.50 8.20 7.00 8.50 . -1.20 0.30 -2.37 0.59 -0.89 

10.70 11.30 11.00 9.90 12.70 -1.10 1.70 -2.17 3.36 0.60 

13.00 13.60 13.30 12.70 14.10 -0.60 0.80 -1.18 1.58 0.20 

14.70 15.30 15.00 15.50 16.90 0.50 1.90 0.99 3.75 2.37 

15.80 16.40 16.10 15.50 16.90 -0.60 0.80 -1.18 1.58 0.20 

16.40 17.00 16.70 16.90 18.30 0.20 1.60 0.39 3.16 1.78 

17.00 18.10 17.55 18.30 19.70 0.75 2.15 1.48 4.24 2.86 

18.10 19.20 18.65 19.70 21.10 1.05 2.45 2.07 4.84 3.46 

18.70 19.80 19.25 19.70 22.50 0.45 3.25 0.89 6.41 3.65 

19.20 20.90 20.05 19.70 22.50 -0.35 2.45 -0.69 4.84 2.08 

20.30 21.50 20.90 21.10 24.00 0.20 3.10 0.39 6.12 3.26 

21.50 23.20 22.35 24.00 25.40 1.65 3.05 3.26 6.02 4.64 

21.50 23.20 22.35 24.00 25.40 1.65 3.05 3.26 6.02 4.64 

26.00 27.70 26.85 33.80 36.60 6.95 9.75 13.72 19.24 16.48 

22.60 24.30 23.45 25.40 26.80 1.95 3.35 3.85 6.61 5.23 

23.20 24.90 24.05 26.80 28.20 2.75 4.15 5.43 8.19 6.81 

23.20 25.40 24.30 26.80 28.20 2.50 3.90 4.93 7.70 6.32 

23.20 25.40 24.30 26.80 28.20 2.50 3.90 4.93 7.70 6.32 

23.70 25.40 24.55 26.80 29.60 2.25 5.05 4.44 9.97 7.21 

24.30 26.00 25.15 26.80 29.60 1.65 4.45 3.26 8.78 6.02 

24.30 26.60 25.45 28.20 31.00 2.75 5.55 5.43 10.95 8.19 

24.90 27.10 26.00 29.60 32.40 3.60 6.40 7.11 12.63 9.87 

25.40 27.10 26.25 29.60 32.40 3.35 6.15 6.61 12.14 9.38 

26.00 27.10 26.55 31.00 32.40 4.45 5.85 8.78 11.55 10.17 

25.40 27.70 26.55 31.00 32.40 4.45 5.85 8.78 11.55 10.17 

26.00 27.70 26.85 32.40 33.80 5.55 6.95 10.95 13.72 12.34 

26.60 28.30 27.45 32.40 33.80 4.95 6.35 9.77 12.53 11.15 

Related Calculations: 

"Corrected uptake of 02 by Test Substance: the oxygen uptake recorded in the individual flask (Flask 1 and 2 

minus the mean oxygen uptake related to the Inoculum (i.e. the mean of the 02 uptake from Flask 3 and 4). 

"% Biodegradation undergone by Test Substance under experimental conditions: Calculated by taking the 

corrected uptake of 02 related to the test substance in the individual test flask and dividing by the total ThOD in the 

flask (i.e. the ThOD of the test substance multiplied by the test substance concentration in the flask). 

- 
Day 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

27 

28 

Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

Page 20 of 24 

20.5 Calculation of Biodegradation of Reference Substance 

Reference Corrected uptake of Inoculum Blank: Substance: % Biodegradation of Reference 

O2 Uptake (mg/L) O2 Uptake O2 by Reference Substance **: 

(m ILl Substance *: 

Mean Flask 5 Flask 6 % % Mean 

Flask Flask Flask Flask Flask - Mean - Mean Biodegdn. Biodegdn. Biodegdn. of 

3 4 5 6 Flask Flask Reference 3&4 3&4 3&4 In Flask 5 In Flask 6 Substance 

3.40 4.00 3.70 28.10 30.90 24.40 27.20 14.61 16.29 15.45 

7.90 8.50 8.20 70.30 76.00 62.10 67.80 37.19 40.60 38.90 

10.70 11.30 11.00 87.20 101.00 76.20 90.00 45.63 53.89 49.76 

13.00 13.60 13.30 101.00 118.00 87.70 104.70 52.51 62.69 57.60 

14.70 15.30 15.00 115.00 127.00 100.00 112.00 59.88 67.07 63.48 

15.80 16.40 16.10 124.00 132.00 107.90 115.90 64.61 69.40 67.01 

16.40 17.00 16.70 129.00 135.00 112.30 118.30 67.25 70.84 69.05 

17.00 18.10 17.55 132.00 138.00 114.45 120.45 68.53 72.13 70.33 

18.10 19.20 18.65 135.00 141.00 116.35 122.35 69.67 73.26 71.47 

18.70 19.80 19.25 138.00 141.00 118.75 121.75 71.11 72.90 72.01 

19.20 20.90 20.05 141.00 143.00 120.95 122.95 72.43 73.62 73.03 

20.30 21.50 20.90 146.00 149.00 125.10 128.10 74.91 76.71 75.81 

21.50 23.20 22.35 152.00 152.00 129.65 129.65 77.63 77.63 77.63 

21.50 23.20 22.35 155.00 152.00 132.65 129.65 79.43 77.63 78.53 

26.00 27.70 26.85 172.00 172.00 145.15 145.15 86.92 . 86.92 86.92 

22.60 24.30 23.45 158.00 155.00 134.55 131.55 80.57 78.77 79.67 

23.20 24.90 24.05 158.00 158.00 133.95 133.95 80.21 80.21 80.21 

23.20 25.40 24.30 158.00 158.00 133.70 133.70 80.06 80.06 80.06 

23.20 25.40 24.30 160.00 158.00 135.70 133.70 81.26 80.06 80.66 

23.70 25.40 24.55 160.00 158.00 135.45 133.45 81.11 79.91 80.51 

24.30 26.00 25.15 160.00 158.00 134.85 132.85 80.75 79.55 80.15 

24.30 26.60 25.45 163.00 160.00 137.55 134.55 82.37 80.57 81.47 

24.90 27.10 26.00 163.00 163.00 137.00 137.00 82.04 82.04 82.04 

25.40 27.10 26.25 166.00 163.00 139.75 136.75 83.68 81.89 82.79 

26.00 27.10 26.55 163.00 163.00 136.45 136.45 81.71 81.71 81.71 

25.40 27.70 26.55 166.00 163.00 139.45 136.45 83.50 81.71 82.61 

26.00 27.70 26.85 166.00 163.00 139.15 136.15 83.32 81.53 82.43 

26.60 28.30 27.45 166.00 166.00 138.55 138.55 82.96 82.96 82.96 

Related Calculations: 

* Corrected uptake of 02 by Reference Substance: the oxygen uptake recorded in the individual flask (Flask 5 and 

6) minus the mean oxygen uptake related to the Inoculum (i.e. the mean of the 02 uptake from Flask 3 and 4). 

** % Biodegradation undergone by Reference Substance under experimental conditions: Calculated by taking the 

corrected uptake of 02 related to the Reference substance in the individual test flask and dividing by the total 

ThOD in the flask (i.e. the ThOD of the Reference substance multiplied by the Reference substance concentration 

in the flask). 

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Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

Page 21 of 24 

20.6 Calculation of Biodegradation of Test and Reference Substances in the 

Inhibition Control 

Inoculum Blank: Inhibition Control: Corrected uptake % Biodegradation of 

Day 02 Uptake (mg/L) 02 Uptake (mg/L) of 02 by Inhibition Inhibition Control" 

Flask 3 Flask 4 Mean Flask Flask 7 Flask 7 - Mean % Biodegdn. In Flask 

1 3.40 4.00 3.70 36.60 32.90 15.13 

2 7.90 8.50 8.20 76.00 67.80 31.18 

3 10.70 11.30 11.00 107.00 96.00 44.15 

4 13.00 13.60 13.30 121.00 107.70 49.53 

5 14.70 15.30 15.00 129.00 114.00 52.43 

6 15.80 16.40 16.10 135.00 118.90 54.68 

7 16.40 17.00 16.70 141.00 124.30 57.17 

8 17.00 18.10 17.55 143.00 125.45 57.70 

9 18.10 19.20 18.65 146.00 127.35 58.57 

10 18.70 19.80 19.25 149.00 129.75 59.67 

11 19.20 20.90 20.05 149.00 128.95 59.31 

12 20.30 21.50 20.90 155.00 134.10 61.68 

13 21.50 23.20 22.35 158.00 135.65 62.39 

14 21.50 23.20 22.35 158.00 135.65 62.39 

15 26.00 27.70 26.85 174.00 147.15 67.68 

16 22.60 24.30 23.45 160.00 136.55 62.80 

17 23.20 24.90 24.05 163.00 138.95 63.91 

18 23.20 25.40 24.30 163.00 138.70 63.79 

19 23.20 25.40 24.30 163.00 138.70 63.79 

20 23.70 25.40 24.55 166.00 141.45 65.06 

21 24.30 26.00 25.15 166.00 140.85 64.78 

22 24.30 26.60 25.45 169.00 143.55 66.02 

23 24.90 27.10 26.00 172.00 146.00 67.15 

24 25.40 27.10 26.25 169.00 142.75 65.65 

25 26.00 27.10 26.55 172.00 145.45 66.90 

26 25.40 27.70 26.55 172.00 145.45 66.90 

27 26.00 27.70 26.85 172.00 145.15 66.76 

28 26.60 28.30 27.45 172.00 144.55 66.48 

Related Calculations: 

* Corrected uptake of 02 by Inhibition Control: the oxygen uptake recorded in Flask 7minus the mean oxygen 

uptake related to the Inoculum (i.e. the mean of the 02 uptake from Flask 3 and 4). 

**% Biodegradation undergone by Test Substance in Inhibition Control under experimental conditions: Calculated 

by taking the corrected uptake of 02 related to the Inhibition Control and dividing by the total test substance ThOD 

in the flask (I.e. the ThOD of the Test substance multiplied by the test substance concentration and reference 

substance concentration in the inhibition I toxicity flask) 

- 
Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of

20.7 BOD and Biodegradation Curve 

A. BOD of Various Test Permutations Studies 

230.00 

180.00 

_ 130.00 

t 

§ 

80.00 

30,00 

·20,00 

BOD of Various Test Permutations Studies 

1 2 3 4 ~ 0 1 8 P 10 11121314 1~ 1@ 11 18 1~ 20 ~1 2223 24 2~ 2@ ~r 2~ 

D.VB. Biodegradation of 

Biodegradation Curve of Catechol Dlbenzoate 

100,00 

po.oo 

80,00 

70,00 

.. 

Page 22 of 24 

-TS1(ll 

-T~Z(Z) 

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p ... lovol 

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10.00 

0.00 ~ ~:e~?r=~F~~~;~~~~~~~~~~~;;~_;;;_:;;~, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

~10.00 Days 

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Bioassay and Safety Assessment Lab, APM Code: 

Ready biodegradability by the manometric respirometer of 

Page 23 of 24 

Appendix Information on test substance (provided by the sponsor) 

Chemical name 

Other name 

Molecular formula 

CAS No 

Batch No 

Appearance Off-white solid 

Purity 

Odour Very slight 

Density n.a. 

Solubility in water 3.69 x10·6 gIL 

Melting point n.a. 

Boiling point (760mm Hg) n.a. 

Freezing point n.a. 

Vapour pressure 9.4 x 10-7 Pa (25°C) 

Absorption n.a. 

Flash point n.a. 

Ignition temperature n.a. 

Self-ignition temperature n.a. 

Oxidizing properties Predicted negative 

Explosion Predicted negative 

Log Pow 6.18 

pH n.a. 

Conditions of storage Store in dark at room temperature 

Stability Stable in water 

Expiry date 2014-02-01 

i ' n.a.: no applicable , 

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