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## Bioassay and Safety Assessment Lab SHANGHAI ACADEMY OF PUBLIC MEASUREMENT
FINAL REPORT
Ready Biodegradability by the Manometric Respirometry Test ofCode: Study Number of DOW: Study director:

SPONSOR: The Dow Chemical Company Midland, MI 48674 USABioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 2 of24 

### Contents
Title
.............................................................................................................................1 Contents

.....................................................................................................................2 Statement ofGLP Compliance

.................................................................................3 QualityAssurance 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 ofTest Substance

..........................................................................9 6 Date
......................................................................................................................9 7 Objective

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

...........................................................................................................10 9 Principle oftheTest..
........................................................................................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 Departurefrom Procotol
...............................................................................16 19 References

.....................................................................................................16 20 Tables and Figures
........................................................................................17 Appendix
..................................................................................................................23 Information on Test Substance (provided bythe sponsor)

.................................23 Certificate ofAnalysis ofTest Substance (provided bythe sponsor)

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

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### Statement of GLP Compliance



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## 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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## IJ chemical testing goodlaboratorypractices (HJIT 155-2004). The report fully and accurately reflects the procedures used and data generated.**~()..8· )-~**  Date 

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### QualityAssurance Statement

The study conduct, raw data and report were inspected by the QualityAssurance 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 ofthe 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 SafetyAssessment Lab, APM 

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### Laboratory Statement

1. This test report would be invalid without the seal ofthe lab and the signatures of Study director, QA and Manager ofTesting facility. 2. Any unauthorized alteration ofthe test report would make the report invalid. 3. The test report only reflects the test substance received from sponsor. 4. The ownerofthe test report shall be responsible for any consequences caused by using incomplete information from the test report.Bioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 6 of24



### Signature Page

Submitted by: Bioassay and SafetyAssessmentLab, APM

Prepared by:

Approved by: 1500 Zhangheng Road, Zhangjiang Hi-tech Park, Shanghai, 201203, P.R. China

Bioassay and SafetyAssessment Lab, APM


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### Key Study Personnel

The following personnel were responsible forthe conduct ofthe 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 testBioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 8 of24


### 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 domesticwaste watertreatment plant. The tested concentration of the test substance was 21.20 mg/L 

### (Le. 50.67 mg ThOOIL). 

### The concentration ofsludge 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 ofthe 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 ofthe test substance at the end ofthe test was less than 20%. The results of the inhibition control showed that the test substance met the criteria for not being inhibitoryto 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 ofthe 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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# L Bioassay and SafetyAssessment 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 SafetyAssessment 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 ofTest Substance Name: Code: Receipt date:**2012~02-13** Conditions ofstorage: Store in dark at room temperature L.,j Full details ofthe test substance are given in AppendixA.



### , , 

### 6 Date Study initiation date 2012-04-09 Experimental starting date 2012-04-27 Experimental completion date 2012-05-25Bioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 10 of24 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 ofthe 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 ofthe total pressure in the airtighttestflasks. The pressure drop is detected and converted into an electrical signal by means ofan electrode type manometer. 10 Materials and Reagents 10.1 Mineral medium (MM) The preparation ofthe mineral medium is described in detail in 20.1. 10.2 Reference substance Name: sodium (Shanghai Linfeng Chemical Reagent Co. Ltd); Code: REF01 0-2010-01; CAS number: 532-32-1;
Purity: ;:,,99.5%; Lot number: 20100310. 11 Apparatus BOD respirometer 0NTW, OxiTop 11OC); 

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### Mould incubator (YIHENG TEC, MJ-2501) pH meter

## ryvTW Multi 34301); Moisture meter (MettlerToledo, HG63); Balance (MettlerToledo, XS205). 12 Species Page 11 of24



### Inoculum: Activated sludge, from a domesticsewage 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 ofthe 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 dryweight 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 liquorsuspended solids level of 3 

### gIL. The final concentration ofthe activated sludge in the test medium was 

### 30 mg/L. 13 Test Methods 13.1 

### Preparation oftest systemBioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 12 of24 13.1.1 Preparation ofTest Substance Incubates Test substance is not miscible with water according to the information supplied bytlJe sponsor. Therefore, the test substancewas 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. 1.00081g 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 itfrom being opened. The above samplewas placed on a roller mixing device to mixfor overnight (roller rate was 30 rpm). A TOe (total organic carbon) analyzerwas used to verifythat 5 replicates ofthe 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 ofsodium benzoate was dissolved in 500 ml mineral medium and the concentration ofstock 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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### 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 

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No. (ml) Inoculum Substance. Substance. 1 Test Substance (TS1) 250 21.20 

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# - 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.Bioassay and SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 14 of24 4) Insertthe rubber sleeves into the flasks. 5) Screwthe OxiTop® measuring head tightly. 6) Startthe measurement on the OxiTop® head. 7) Place the measuring flask on the inductive stirring system, then in the incubator for 28 days at22 ± 1°C, in the dark. 8) Auto-read the results once per 112min during 28 days. 9) Determine pH atthe beginning and the end ofthe 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 ofthe reference substance was 1.67 mg 02/mg. 100 mg/L ofreference substance was 167.00 mg ThOD NH3/L. chemical formula: C25H2404 (MW:388.46), purity (99.90%). ThODNH30ftest 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. 13.3.2Biodegradability Percent Calculated by BOD Raw data is exported byAchat OCv2.03 into Excel forms. Biodegradct>ilitypercent%=( a - bm ) x 100% Co xThODNH3
a - BOD ofTS, PC orTC 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 n I I i J 

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### 14 Method Validation The mean BOD ofthe 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 ofthe solution in test substance flasks were in the range of7.457 - 7.719, which metthe requirement of pH 6 - 8.5 ofthe test substance solution. The difference between replicate values ofthe removal ofthe test substance at the the end ofthe testwas 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 metthe 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 forthe 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 ofthe test substance and the ThODNH3 ofthe 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. 

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### 16 Conclusions The test substance was not readily biodegradable under the presenttest conditions. 

### 17 Archives The signed copies ofthe 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 ofthe 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 ofPublic Measurementforfive years, and returned to the sponsor at designated address on agreed date. 

### 18 Departure from Procotol None. 

### 19 References OECD, Guidelinesforthe Testing ofChemicals, 2006, Section 3 Introduction: Revised Introductions ofthe OECD Guidelines for Testing ofChemicals, Section 3 OECD, Series on the Test Guidelines Programme, Number2, 1998, Detailed Review Paper on BiodegradabilityTesting. 

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2HP04: 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 purewater. (C) Magnesium Sulphate Solution MgS04'7H2 0 : Dissolve in and make up to 1000 ml with ultra pure water. (D) Ferric Chloride Solution FeCI3'6H2 0: Dissolve in and make up to 1000 ml with ultra pure water. Add one drop ofconcentrated HCI per litre. 8.50 

### 9 21.75g 26.64 9

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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 ifa precipitate forms. Test Medium: BSM is prepared before use in deionised water and is prepared as follows: Mix 10 ml ofsolution A with 800 ml ultra pure water, and then add 1 ml each ofsolutions B, C, and D. Bring volume to 1 L with deionised water. 20.2 pH Readings atthe Beginning and End ofthe Experimental Phase ofthe Study Inhibition I Test Substance Inoculum Blank Reference Control Toxicity Oay(d) Control Flask 1 Flask 2 Flask 3 Flask4 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 8.178Bioassayand SafetyAssessment Lab, APM Code: Ready biodegradability bythe manometric respirometer of Page 18of24 

### 20.3 Records of Biochemical Oxygen Demand (mg/L) Inoculum Reference Inhibition TestSubstance Blank Control Control Oay(d) Flask 1 Flask 2 Flask 3 Flask4 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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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 SafetyAssessment Lab, APM Code: Ready biodegradability by the manometric respirometer of Page 19 of24 20.4 Calculation of Biodegradation ofTest Substance Test Inoculum Blank: Substance: Corrected uptake of % Biodegradation ofTest 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 ofTest 3&4 In Flask 1 In Flask2 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 ofthe 02 uptake from Flask 3 and 4). "% Biodegradation undergone by Test Substance under experimental conditions: Calculated by taking the corrected uptake of02 related to the test substance in the individual testflask and dividing bythe total ThOD in the flask (i.e. the ThOD ofthe 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 SafetyAssessment Lab, APM Code: Ready biodegradability bythe manometric respirometer of Page 20 of24 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 of02 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 ofthe 02 uptake from Flask 3 and 4). ** % Biodegradation undergone by Reference Substance underexperimental 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 ofthe Reference substance multiplied by the Reference substance concentration in the flask). :

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### the Inhibition Control Inoculum Blank: Inhibition Control: Corrected uptake % Biodegradation of Day 02 Uptake (mg/L) 02 Uptake (mg/L) of02 by Inhibition Inhibition Control" Flask 3 Flask4 Mean Flask Flask 7 Flask 7 - Mean % Biodegdn. In Flask 1 3.40 4.00 3.70 36.60 32.90 15.13 

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20.7 BOD and Biodegradation Curve A. BOD ofVarious Test Permutations Studies

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Other name

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