## BENZOPHENONE: READY BIODEGRADABILITY- CO 2 

## IN SEALED VESSELS (Headspace Test)

WILDLIFE INTERNATIONAL PROJECT NO.: 264E-104

Organisation for Economic C ooperation 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 2 nd 

## Street NE

Washington, D.C. 20002

8598 Commerce Drive

Easton, Maryland 21601

(410) 822-8600

Page 1 of 33

## Project Number 264E-104

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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 2 nd 

## 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 .............................................................................................. 1 2

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 Concentrati ons of NaOH Solutions ..................................................................... 15

Table 2. Measured Inorganic Carbon Concentr ation 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 Theo retical 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 Seal ed 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 so le source of organic carbon and sealed. CO 2 

## evolution

from the ultimate aerobic biodegradation of the te st 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 me dium and were not dosed with a carbon source. The reference

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

concentration of 20 mg C/L. The treatment group te st 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

Biodegradable 1



## Benzophenone 65.0 Yes

1 

### Meets or exceeds the OECD criteria for ready biodegradabil ity (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 ultima te biodegradation in the environmen t. 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 additiona l testing may be needed.

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

Wildlife International biodegradation facility in East on, 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 refere nce and one treatment group. The blank control

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

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

inoculated medium and were dosed with 1-octa nol, 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 th e procedures outlined in the protocol,

“Benzophenone: Ready Biodegradability-CO 2 

## in Sealed Vessels (Headspace Test)”. The protocol was

based on the procedures specified in the OECD Gu ideline 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 –Endpoin t 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 Certificat e 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: C 13 

## H 10 

## O

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 relativel y 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 numbe r 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: CH 3 

## (CH 3 

## ) 7 

## OH

Carbon Content: 73.83% (Calculated by molecular formula)

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

weight addition. Direct Weight addition of a test s ubstance 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 constituen ts 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 ca ps. The test was conducte d at 20±3oC. The test

chambers were mixed at a rate sufficient to k eep 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 collect ed from the Cambridge Wastewater Treatment Facility,

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

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

Preconditioning of Inoculum

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

solids/L and aerated with CO 2 

## -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 dilu ting the preconditioned activated sludge in test

medium to approximately 4 mg total suspended so lids/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 subs tance necessary to deliver 20 mg C/L (2.9 mg).

Treatment group test chambers were dosed with suffi cient 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 CO 2 

## 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 sh aken at 90 prm for at least 1 hour at test

temperature. After mixing, the chambers were remo ved 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:

D

t



## =  C

t



## -  C

b 

## ] /TOC x 100

D

t 

## = % degradation at time t

TIC

t 

## = mg TIC in test chamber at time t

TIC b 

## = 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 av erage 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 subs tance 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-

CO 2 

## in sealed vessels (Headspace T est). 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 96.2Project Number 264E-104- 16 -Table 2Measured Inorganic Carbon Concentration of Test Chambers (mg C/L)DateDayControlReplicate #Result1-OctanolReplicate #ResultTreatmentReplicate #Result23-Oct-1423-Oct-1423-Oct-1427-Oct-1427-Oct-1427-Oct-1430-Oct-1430-Oct-1430-Oct-1403-Nov-1403-Nov-1403-Nov-1407-Nov-1407-Nov-1407-Nov-1410-Nov-1410-Nov-1410-Nov-1413-Nov-1413-Nov-1413-Nov-1417-Nov-1417-Nov-1417-Nov-1419-Nov-1419-Nov-1419-Nov-141115558881212121616161919192222222626262828281234567891011121314151617181920212223242526271.41.31.30.10.00.22.02.01.92.01.81.92.02.02.12.12.12.12.42.42.32.83.02.92.92.83.01234567891011121314151617181920212223242526271.21.01.010.89.99.711.213.014.014.215.310.314.315.114.615.817.010.416.317.116.716.719.517.917.213.817.41234567891011121314151617181920212223242526271.21.21.30.10.20.42.01.71.41.51.31.21.93.13.413.614.018.313.417.915.016.118.816.916.115.116.6”Result” values rounded for presentation purposes.Project Number 264E-104- 17 -Table 3Total Mass of Inorganic Carbon (mg)1,2DateDayControlReplicate #Result1-OctanolReplicate #ResultTreatmentReplicate #Result23-Oct-1423-Oct-1423-Oct-1427-Oct-1427-Oct-1427-Oct-1430-Oct-1430-Oct-1430-Oct-1403-Nov-1403-Nov-1403-Nov-1407-Nov-1407-Nov-1407-Nov-1410-Nov-1410-Nov-1410-Nov-1413-Nov-1413-Nov-1413-Nov-1417-Nov-1417-Nov-1417-Nov-1419-Nov-1419-Nov-1419-Nov-141115558881212121616161919192222222626262828281234567891011121314151617181920212223242526270.10.10.1-0.1-0.10.00.10.10.10.10.10.10.10.10.10.10.10.10.20.20.20.20.20.20.20.20.21234567891011121314151617181920212223242526270.10.00.01.11.01.01.11.31.41.41.61.01.41.51.51.61.71.01.71.71.71.72.01.81.71.41.81234567891011121314151617181920212223242526270.10.10.1-0.10.00.00.10.10.10.10.10.00.10.20.31.41.41.91.31.81.51.61.91.71.61.51.71The 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 vessel2Calculations performed in Excel 2010 full precision mode. Manual calculations may differ.”Result” values rounded for presentation purposes.Project Number 264E-104- 18 -Table 4Percent of Theoretical Inorganic Carbon Evolved1,2DateDayControlReplicate #Result1-OctanolReplicate #ResultTreatmentReplicate #Result23-Oct-1423-Oct-1423-Oct-1427-Oct-1427-Oct-1427-Oct-1430-Oct-1430-Oct-1430-Oct-1403-Nov-1403-Nov-1403-Nov-1407-Nov-1407-Nov-1407-Nov-1410-Nov-1410-Nov-1410-Nov-1413-Nov-1413-Nov-1413-Nov-1417-Nov-1417-Nov-1417-Nov-1419-Nov-1419-Nov-1419-Nov-14111555888121212161616191919222222262626282828123456789101112131415161718192021222324252627NANANANANANANANANANANANANANANANANANANANANANANANANANANA123456789101112131415161718192021222324252627-0.7-1.5-1.553.549.047.946.255.060.161.367.142.161.665.563.068.674.441.469.573.671.669.182.974.771.654.772.5123456789101112131415161718192021222324252627-0.5-0.7-0.1-0.20.71.40.0-1.4-2.6-1.8-3.2-3.4-0.75.56.857.659.781.055.277.963.365.979.669.866.160.868.2Test Termination Average(Day 28 Values)NA 66.3 65.01The percent degradation at each sampling interval was calculated using the following equation: Dt= [TICt– TICb] / TOC x 100TICt= 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 chamber2Calculations 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 30Percent of TheoreticalIC 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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