BENZOPHENONE: READY BIODEGRADABILITY- CO2 

IN SEALED VESSELS (Headspace Test) 

WILDLIFE INTERNATIONAL PROJECT NO.: 264E-104 

Organisation for Economic Cooperation and Development 

 OECD Guideline 310 

AUTHORS: 

Edward C. Schaefer 

Molly E. Matthews 

STUDY INITIATION DATE: October 13, 2014 

STUDY COMPLETION DATE: March 10, 2015 

Submitted to: 

American Chemistry Council 

700 2nd Street NE 

Washington, D.C. 20002 

8598 Commerce Drive 

Easton, Maryland 21601 

(410) 822-8600 

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STUDY INFORMATION 

Study Initiation Date: October 13, 2014 

Experimental Start Date (OECD): October 16, 2014 

Experimental Start Date (EPA): October 22, 2014 

Experimental Termination Date: November 19, 2014 

Study Completion Date: March 5, 2015 

Study Director: Molly E. Matthews, Senior Biologist 

 Wildlife International 

Sponsor: American Chemistry Council 

 700 2nd Street NE 

 Washington, D.C. 20002 

 

Sponsor’s Representative: Bill Gulledge 

 

Study Personnel: Edward C. Schaefer, B.S., Director of Fate and Degradation 

Molly E. Matthews, B.A., Senior Biologist, Biodegradation

 Kyle Wheatley, B.S., Chemist, Biodegradation 
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TABLE OF CONTENTS 

Title/Cover Page ..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2 

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Study Information ................................................................................................................................... 5 

Table of Contents ................................................................................................................................... 6 

Summary ................................................................................................................................................ 8 

Introduction ............................................................................................................................................ 9 

Objective ................................................................................................................................................ 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods ........................................................................................................................... 9 

Test Substance ......................................................................................................................... 10 

Reference Substance ................................................................................................................ 10 

Test Medium ............................................................................................................................ 11 

Test Apparatus and Conditions ................................................................................................ 11 

Test Inoculum .......................................................................................................................... 11 

Preconditioning of Inoculum ................................................................................................... 11 

Inoculated Medium .................................................................................................................. 11 

Preparation of Test Chambers ................................................................................................ 11 

Sample Collection and Analysis .............................................................................................. 12 

Calculations ............................................................................................................................. 12 

Results and Discussion ....................................................................................................................... 13 

Observations and Measurements ............................................................................................. 13 

Conclusions .......................................................................................................................................... 13 

References ............................................................................................................................................ 14 
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TABLE OF CONTENTS 

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TABLES 

Table 1. Measured Concentrations of NaOH Solutions ..................................................................... 15 

Table 2. Measured Inorganic Carbon Concentration of Test Chambers (mg C/L) ............................ 16 

Table 3. Total Mass of Inorganic Carbon (mg) .................................................................................. 17 

Table 4. Percent of Theoretical Inorganic Carbon Evolved ............................................................... 18 

FIGURE 

Figure 1. Average Percentage of Theoretical Inorganic Carbon Evolved ........................................... 19 

APPENDICES 

Appendix I. Study Protocol ............................................................................................................ 20 

Appendix II. Certificate of Analysis ................................................................................................ 32 
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SUMMARY 

The ready biodegradability of Benzophenone was determined by following a protocol based 

upon OECD 310: Ready Biodegradability – CO2 in Sealed Vessels (Headspace Test). Tests of ready 

biodegradability are stringent tests that provide limited opportunity for acclimation and 

biodegradation to occur. In the Headspace Test, inoculated test medium was dosed with a known 

amount of test substance as the nominal sole source of organic carbon and sealed. CO2 evolution 

from the ultimate aerobic biodegradation of the test substance is determined by measuring the 

inorganic carbon (IC) produced in the test bottles over that produced in the blank control bottles. The 

amount of IC produced by the test substance (corrected for that evolved by the controls and the 

amount added by basification, where applicable) is expressed as a percentage of the theoretical 

amount of IC (ThIC) that could have been produced if complete biodegradation of the test substance 

occurred. The test contained a control group, a reference group and a treatment group. Each group 

contained twenty-seven replicate test chambers. The controls were used to measure the background 

IC production of the inoculum medium and were not dosed with a carbon source. The reference 

chambers were dosed with 1-octanol, a substance known to be biodegradable, at a nominal 

concentration of 20 mg C/L. The treatment group test chambers were used to evaluate Benzophenone 

at a nominal concentration of approximately 20 mg C/L. The results at the end of the 28-day study 

indicated that the activated sludge inoculum was active, degrading the reference substance 66.3%. 

The average cumulative percent biodegradation for Benzophenone was 65.0%. 

Test Substance 

Average 

Cumulative Percent 

Biodegradation 

Readily 

Biodegradable1

Benzophenone 65.0 Yes 

1 Meets or exceeds the OECD criteria for ready biodegradability (60% of ThIC within a 10-day window of reaching 

10% ThIC). 
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INTRODUCTION 

 Tests of ready biodegradability, by definition, provide limited opportunity for acclimation and 

biodegradation to occur. A positive result in a test of ready biodegradability is an indication that the test 

substance will undergo rapid and ultimate biodegradation in the environment. A negative result in a test 

of ready biodegradability does not necessarily mean that the test substance will not be biodegraded 

under relevant environmental conditions but that additional testing may be needed. 

This study was conducted by Wildlife International for American Chemistry Council at the 

Wildlife International biodegradation facility in Easton, Maryland. Original raw data generated by 

Wildlife International and the final report are filed under Project number 264E-104 in the archives 

located on the Wildlife International site. 

OBJECTIVE 

The objective of the study was to assess the biodegradability of Benzophenone in an aqueous 

medium by measuring inorganic carbon (IC) production. 

EXPERIMENTAL DESIGN 

The test contained one blank control, one reference and one treatment group. The blank control 

group was used to measure the background IC production of the inoculated medium and was not 

dosed with a carbon source. The reference group chambers were used to check the viability of the 

inoculated medium and were dosed with 1-octanol, a substance known to be biodegradable, at a 

nominal concentration of 20 mg C/L (2.9 mg/vessel). The treatment group chambers were used to 

evaluate Benzophenone at a nominal concentration of approximately 20 mg C/L (2.5 mg/vessel). 

MATERIALS AND METHODS 

 This study was conducted according to the procedures outlined in the protocol, 

“Benzophenone: Ready Biodegradability-CO2 in Sealed Vessels (Headspace Test)”. The protocol was 

based on the procedures specified in the OECD Guideline 310 (1) and ECHA Guidance (R7.9.4 and 

R7.9.5 –Evaluation of available information on degradation/biodegradation and Conclusions for 

degradation/biodegradation in Chapter R.7B –Endpoint Specific Guidance) (2). 
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Test Substance 

 The test substance was received from Chem Service on July 07, 2014 and was assigned 

Wildlife International identification number 11802. The following is a description of the test 

substance used in this study and a Certificate of Analysis can be found in Appendix II: 

 

 Identity: Benzophenone 

 CAS Number: 119-61-9 

 Lot Number: 2928800 

 Physical Description: Solid 

 Purity: 99.5% 

 Expiration Date: June 30, 2019 

 Storage Conditions: Ambient 

 Molecular Formula: C13H10O 

Carbon Content: 85.68% (Calculated by molecular formula) 

 The test substance was administered to the treatment group test chambers by direct weight 

addition. Direct Weight addition of a test substance that is relatively insoluble in water or of unknown 

solubility is the most appropriate route of administration. Dosing amounts were based on the 

calculated carbon content of the test substance. 

Reference Substance 

The reference substance was received from Sigma Aldrich on October 09, 2014 and was 

assigned Wildlife International Identification number 11950. The following is a description of the 

reference substance used in this study: 

 Name: 1-Octanol 

Batch Number: BCBL3176V 

Physical Description: Liquid 

Chemical Abstract Number: 111-87-5 

Expiration Date: May 2016 

Purity: 99.7% 

Storage Conditions: Ambient 

Molecular Formula: CH3(CH3)7OH 

Carbon Content: 73.83% (Calculated by molecular formula) 

The reference substance was administered to the treatment group test chambers by direct 

weight addition. Direct Weight addition of a test substance that is relatively insoluble in water or of 

unknown solubility is the most appropriate route of administration. Dosing amounts were based on 

the calculated carbon content of the reference substance. 
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Test Medium 

The test medium was a mineral salts media and was prepared using high quality water as 

described in the Protocol (Appendix I). All chemicals and reagents used in the preparation of the test 

medium were reagent grade or better. The constituents of the test medium are not known to contain 

any contaminants that are reasonably expected to be present or known to be capable of interfering 

with the study. 

Test Apparatus and Conditions 

The test chambers were glass serum bottles with a nominal volume of 160 mL. The chambers 

were sealed with butyl rubber septa and crimp caps. The test was conducted at 20±3oC. The test 

chambers were mixed at a rate sufficient to keep the bottle contents mixed and in suspension 

throughout the study by use of a shaker table set at 90 rpm. Test chambers were identified by project 

number, test substance ID, test concentration and replicate. 

Test Inoculum 

Activated sludge was collected from the Cambridge Wastewater Treatment Facility, 

Cambridge, Maryland on October 16, 2014. The Cambridge facility treats predominantly residential 

wastes. The sludge was sieved using a 2-mm screen and then aerated until its adjustment. 

Preconditioning of Inoculum 

The activated sludge was diluted in test medium to approximately 30 mg total suspended 

solids/L and aerated with CO2-free air for six days until its adjustment to 4 mg total suspended 

solids/L and use in inoculating the test medium. 

Inoculated Medium 

 The inoculated medium was prepared by diluting the preconditioned activated sludge in test 

medium to approximately 4 mg total suspended solids/L. After adjustment, the sludge was aerated 

for 30 minutes. Following aeration, the pH (7.6), dissolved organic carbon (DOC = 0 mg/L) and 

dissolved inorganic carbon (DIC = 0 mg/L) levels of the mixture were measured. 

Preparation of Test Chambers 

Aliquots of inoculated test medium were dispensed into 160 mL replicate bottles to give a 
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headspace to liquid ratio of approximately 1:2 (107 mL in a 160 mL bottle). The blank controls 

contained only inoculated medium and were not dosed with a carbon source. Reference group test 

chambers were dosed with sufficient reference substance necessary to deliver 20 mg C/L (2.9 mg). 

Treatment group test chambers were dosed with sufficient test substance necessary to deliver 20 mg 

C/L (2.5 mg). 

Sample Collection and Analysis 

 Three replicate chambers from each group were removed for IC analysis twice a week over the 

test period. 

 Samples for analysis were prepared by converting CO2 to carbonate. One milliliter of 7M 

NaOH was injected into the test chambers, which were subsequently returned to the shaker table 

where they had originally been incubating, and shaken at 90 prm for at least 1 hour at test 

temperature. After mixing, the chambers were removed from the shaker table, allowed to settle and 

then samples were removed for IC analysis. At each interval, the NaOH used was analyzed for its IC 

concentration. 

Calculations 

The total mass (mg) of inorganic carbon at each sampling interval was calculated using the 

following equation: 

 TIC = (mg IC/L in vessel – mg IC/L added from 7M NaOH Solution) x volume of liquid in vessel 

The percent degradation at each sampling interval was calculated using the following equation: 

Dt = Ct - Cb] /TOC x 100 

 

 Dt = % degradation at time t 

TICt = mg TIC in test chamber at time t 

TICb = mean mg TIC in blank at time t 

TOC = mg TOC added initially to the test chamber 
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RESULTS AND DISCUSSION 

Observations and Measurements 

 The temperature range recorded during the test was 19.2 to 21.4oC. The measured IC 

concentrations of the NaOH basification solutions used at each sampling interval are presented in 

Table 1. The measured concentrations of inorganic carbon in the test chambers are presented in Table 

2. The total mass of inorganic carbon measured in the test chambers are presented in Table 3. The 

percent degradation of the test vessels at each sampling interval is presented in tabular form in Table 

4. The average percent degradation at each sampling interval is presented in graphical form in Figure 

1. 

The control chambers evolved an average of 2.0 mg IC/L by Day 28 (Table 3). The amount of 

IC evolved by the control chambers was < 15% of the organic carbon added initially as the test 

substance (20 mg/L), which is considered the acceptable limit for this study. 

 The viability of the inoculum and validity of the test were supported by the results of the 

reference substance, 1-Octanol, from which an average of 66.3% of theoretical IC was evolved 

(Table 4). An average percent biodegradation of greater than 60% was achieved by Day 16, thereby 

fulfilling the criteria for a valid test by reaching the pass level by Day 28. The average cumulative 

percent biodegradation for Benzophenone was 65.0% (Table 4). Benzophenone may be considered 

readily biodegradable under aerobic conditions since the pass level of 60% ThIC was achieved within 

a 10-day window of reaching 10% ThIC. 

 

CONCLUSIONS 

 Biodegradation ≥ 60% ThIC within a 10-day window of reaching 10% ThIC in this test 

demonstrates that a test substance is readily biodegradable under aerobic conditions. The average 

cumulative percent biodegradation for Benzephenone was 65.0%. Benzophenone may be considered 

readily biodegradable under aerobic conditions since the pass level of 60% ThIC was achieved within 

a 10-day window of reaching 10% ThIC. 
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REFERENCES 

1. Organisation for Economic Cooperation and Development. 2006. Ready Biodegradability- 

 CO2 in sealed vessels (Headspace Test). OECD Guideline 310. 

2. ECHA Guidance (R7.9.4 and R7.9.5 –Evaluation of available information on 

 degradation/biodegradation and Conclusions for degradation/biodegradation in Chapter R.7B – 

 Endpoint Specific Guidance). 

 
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Table 1 

Measured Concentrations of NaOH Solutions 

Interval IC 

(Day) (mg IC/L) 

 

1 74.7 

5 70.8 

8 82.5 

12 83.4 

16 95.8 

19 84.8 

22 85.5 

26 111.1 

28 96.2 

 

 

 

 

 

 

 

 
Project 

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Table 2 

Measured Inorganic Carbon Concentration of Test Chambers (mg C/L) 

Date Day 

Control 

Replicate # Result 

1-Octanol 

Replicate # Result 

Treatment 

Replicate # R

23-Oct-14 23-Oct-14 23-Oct-14 27-Oct-14 27-Oct-14 27-Oct-14 30-Oct-14 30-Oct-14 30-Oct-14 

03-Nov-14 03-Nov-14 03-Nov-14 07-Nov-14 07-Nov-14 07-Nov-14 10-Nov-14 10-Nov-14 10-Nov-14 13-Nov-14 13-Nov-14 13-Nov-14 17-Nov-14 17-Nov-14 17-Nov-14 19-Nov-14 19-Nov-14 19-Nov-14 

1 1 1 5 5 5 8 8 8 

12 12 12 16 16 16 19 19 19 22 22 22 26 26 26 28 28 28 

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 

1.4 1.3 1.3 0.1 0.0 0.2 2.0 2.0 1.9 2.0 1.8 1.9 2.0 2.0 2.1 2.1 2.1 2.1 2.4 2.4 2.3 2.8 3.0 2.9 2.9 2.8 3.0 

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 

1.2 1.0 1.0 

10.8 

9.9 9.7 

11.2 13.0 14.0 14.2 15.3 10.3 14.3 15.1 14.6 15.8 17.0 10.4 16.3 17.1 16.7 16.7 19.5 17.9 17.2 13.8 17.4 

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 

 ”Result” values rounded for presentation purposes. 
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Table 3 

Total Mass of Inorganic Carbon (mg) 1,2

Date Day 

Control 

Replicate # Result 

1-Octanol 

Replicate # Result 

Treatment 

Replicate # Re

23-Oct-14 23-Oct-14 23-Oct-14 27-Oct-14 27-Oct-14 27-Oct-14 30-Oct-14 30-Oct-14 30-Oct-14 

03-Nov-14 03-Nov-14 03-Nov-14 07-Nov-14 07-Nov-14 07-Nov-14 10-Nov-14 10-Nov-14 10-Nov-14 13-Nov-14 13-Nov-14 13-Nov-14 17-Nov-14 17-Nov-14 17-Nov-14 19-Nov-14 19-Nov-14 19-Nov-14 

1 1 1 5 5 5 8 8 8 

12 12 12 16 16 16 19 19 19 22 22 22 26 26 26 28 28 28 

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 

0.1 0.1 0.1 

-0.1 -0.1 

0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 

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 

0.1 0.0 0.0 1.1 1.0 1.0 1.1 1.3 1.4 1.4 1.6 1.0 1.4 1.5 1.5 1.6 1.7 1.0 1.7 1.7 1.7 1.7 2.0 1.8 1.7 1.4 1.8 

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 

000

-0

00000000000111111111111

 1The total mass of IC was calculated using the following equation: 

 TIC = (mg IC/L in vessel – mg IC/L added from 7M NaOH) x volume of liquid in vessel 

 2Calculations performed in Excel 2010 full precision mode. Manual calculations may differ. 

 ”Result” values rounded for presentation purposes.
Project 

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Table 4 

Percent of Theoretical Inorganic Carbon Evolved 1,2

Date Day 

Control 

Replicate # Result 

1-Octanol 

Replicate # Result 

Treatment 

Replicate # Result 

23-Oct-14 23-Oct-14 23-Oct-14 27-Oct-14 27-Oct-14 27-Oct-14 30-Oct-14 30-Oct-14 30-Oct-14 

03-Nov-14 03-Nov-14 03-Nov-14 07-Nov-14 07-Nov-14 07-Nov-14 10-Nov-14 10-Nov-14 10-Nov-14 13-Nov-14 13-Nov-14 13-Nov-14 17-Nov-14 17-Nov-14 17-Nov-14 19-Nov-14 19-Nov-14 19-Nov-14 

1 1 1 5 5 5 8 8 8 

12 12 12 16 16 16 19 19 19 22 22 22 26 26 26 28 28 28 

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 

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

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 

-0.7 -1.5 -1.5 

53.5 49.0 47.9 46.2 55.0 

60.1 61.3 67.1 42.1 61.6 65.5 63.0 68.6 74.4 41.4 69.5 73.6 71.6 69.1 82.9 74.7 71.6 54.7 72.5 

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 

-0.5 -0.7 -0.1 -0.2 

0.7 1.4 0.0 

-1.4 -2.6 -1.8 -3.2 -3.4 -0.7 

5.5 6.8 

57.6 59.7 81.0 55.2 77.9 63.3 65.9 79.6 69.8 66.1 60.8 68.2 

Test Termination Average  (Day 28 Values)

NA 66.3 65.0 

1The percent degradation at each sampling interval was calculated using the following equation: Dt = [TICt – TICb] / TOC x 100 

TICt = mg TIC in test chamber at time t TICb = mean mg TIC in blank at time t TOC = mg TOC added initially to the test chamber 2Calculations performed in Excel 2010 full precision mode. Manual calculations may differ. NA – Not Applicable.
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-10

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Percent of Theoretical 

IC Production 

Time (Days)

Average Percent Degradation

Reference Benzophenone

Figure 1. Average Percentage of Theoretical Inorganic Carbon Evolved 
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Appendix I 

Study Protocol 
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Appendix II 

Certificate of Analysis 
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