DETERMINATION OF READY BIODEGRADABILITY

BY THE CARBON DIOXIDE EVOLUTION TEST METHOD- 5 -

STUDY INFORMATION- 6 -

TABLE OF CONTENTS

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

Good Laboratory Practice Co mpliance 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 S ubstance............................................................................................................ .... 10

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

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

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

Preparation of Test Chambers ................................................................................................. 12

Biodegradation Test Initiation ................................................................................................. 12

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

Test Termination............................................................................................................... ....... 12

Calculations ................................................................................................................... .......... 13

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

Carbon Analysis................................................................................................................ ....... 13

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

Conclusions .................................................................................................................... ...................... 14

Refere nces ..................................................................................................................... ....................... 15- 7 -

TABLE OF CONTENTS

- Continued -

TABLES

Table 1. Dissolved Organic Carbon (DOC) and pH of Test Solutions at Test Termination.............. 16

Table 2. Measured Inorganic Carbon Concentr ation of Trapping Solutions (mg C/L) ..................... 17

Table 3. Cumulative Milligrams of Carbon Dioxide Evolved ........................................................... 18

Table 4. Cumulative Percent of Theoretical Carbon Dioxide Evolved.............................................. 19

FIGURES

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved ........................................ 20

APPENDIX

Appendix I. Protocol and Protocol Deviation ................................................................................ 21- 8 -

SUMMARY

The ready biodegradability of the test substance was determined by the Carbon Dioxide

Evolution Test Method (OECD Guideline 301B). Tests of ready biodegradability are stringent tests

that provide limited opportunity for acclimation and biodegradation to occur. In the CO 2 test,

inoculated mineral medium was dosed with a known amount of test substance as the nominal sole

source of organic carbon and aerated with CO 2 -free air. The CO 2 produced from the mineralization

of organic carbon within the test chambers was displaced by the flow of CO 2 -free air and trapped as

K 2 CO 3 in KOH trapping solution. The amount of CO 2 produced by the test substance (corrected for

that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO 2

(TCO 2 ) that could have been produced if complete biodegradation of the test substance occurred. The

test contained a blank control group, a reference group and a treatment group. Each group contained

three replicate test chambers. The blank control was used to measure the background CO 2 production

of the inoculum and was not dosed with a carbon s ource. The reference chambers were dosed with

sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L.

The treatment group test chambers were used to eval uate the test substance at a nominal concentration

of 10 mg C/L. The results indicated that the ac tivated sludge inoculum was active, degrading the

reference substance 98.0%. The average cumulativ e percent biodegradation for the test substance

was 94.2%.

Test Substance Average

Cumulative Percent

Biodegradation Average

Final

pH Readily

Biodegradable 2- 9 -

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 ultimat e 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 additiona l testing may be needed.

This study was conducted by for at the

Ltd. biodegradation facility in Easton, Maryland. Original raw data generated

by . and the final re port are filed under Project Number 523E-101 in the

archives located on the site.

OBJECTIVE

The objective of the study was to measure the amount of carbon dioxide (CO 2 ) produced from

the biodegradation of a nonvolatile organic test substa nce. This value is expressed as a percentage of

the theoretical amount of CO 2 (TCO 2 ) that could have been produced if complete biodegradation of

the test substance occurred.

EXPERIMENTAL DESIGN

The test contained a blank control group, a re ference group and a treatment group. Each group

contained three replicate test chambers. The control group was used to measure the background CO 2

production of the inoculum and was not dosed with a carbon source. The reference group chambers

were used to check the viability of the inoculum and were dosed with sodi um benzoate, a substance

known to be biodegradable, at a nominal concentra tion of 10 mg C/L. The treatment group chambers

were used to evaluate the test substance at a nominal concentration of 10 mg C/L.

MATERIALS AND METHODS

This study was conducted according to the pr ocedures outlined in the protocol, “Test

Substance: Determination of Ready Biodegradability by the Carbon Dioxide Evolution Test Method”.

The protocol was based on the procedures specified in the OECD Guideline for Testing of Chemicals,

Method 301B (1), and Annex to Commission Directive 67/548/EEC.1992, Method C.4-C, Carbon

Dioxide (CO 2 ) Evolution (2).- 10 -

Test Substance

The test substance was received from . on April 19, 2005 and was assigned

. identification number 7161. The following is a description of the test

substance used in this study:

Lot Number: 8488/035

CAS Number: Not Given

Physical Description: Liquid

Purity: Not Given

Expiration Date: Not Given

Water Solubility: Not Given

Carbon Content: 72%

Storage Conditions: Ambient

.

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

addition. Direct weight addition of a test substance that is relatively insoluble in water is the most

appropriate route of administration. The amount of test substance used to dose the treatment group

test chambers were calculated based on the theoretical carbon content.

Reference Substance

The reference substance was received from Ma llinckrodt Chemicals on January 08, 2002 and

was assigned Identif ication number 5861. A stock solution of the

reference substance was prepared in NANOpure ® water at a nominal carbon concentration of 400 mg

C/L. The stock solution was analyzed for total organic carbon (TOC). The reference substance was

administered to the reference group test chambers by volumetric addition. Dosing amounts were

based on the measured carbon content of the referen ce substance solution. Following is a description

of the reference substance used in this study:

Name: Sodium benzoate

Manufacturer: Mallinckrodt Baker, Inc.

Paris, Kentucky

Lot Number: 0168 V38617

Physical Description: White powder

Chemical Abstract Number: 532-32-1

Expiration Date: January 8, 2007

Purity: 99.4%

Storage Conditions: Ambient- 12 -

Preparation of Test Chambers

The following were added to each chamber:

1) 2470 mL of NANOpure ® water

2) 3 mL calcium chloride solution (2.75%)

3 mL of ferric chloride solution (0.025%)

3 mL of magnesium sulfate solution (2.25%)

30 mL of phosphate buffer (pH 7.4)

3) 30 mL of the activated sludge inoculum

All chambers were aerated with CO 2 free air for approximately 24 hours at a rate of 50 to 100

mL per minute to purge the systems of CO 2 . After the aeration period, the flow of CO 2 free air was

stopped, and three CO 2 traps, each containing approximately 100 mL of 0.5 N KOH, were connected

to the exit air lines of each chamber. A volume of reference substance stock solution to achieve a

nominal concentration of 10 mg C/L was added to the reference group chambers. An amount of test

substance (approximately 0.0417 g) to achieve a nom inal concentration of 10 mg C/L was added to

the treatment group chambers. The final volume within all chambers was brought up to 3000 mL by

the addition of NANOpure ® water.

Biodegradation Test Initiation

The biodegradation test was started by bubbling CO 2 free air through each of the test chambers

at a rate of 50 to 100 mL per minute. The CO 2 produced from the degradation of organic carbon

sources within the test chamber was trapped as K 2 CO 3 in the KOH solution and the amount of

inorganic carbon in the trapping solution was measure d at various intervals during the study, using a

Shimadzu TOC-5000 carbon analyzer.

Sample Collection and Analysis

On days 1, 5, 9, 13, 15, 20, 22 and 26, the CO 2 trap nearest the test chamber was removed and

analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test

chamber and a new trap was placed on the end of the series.

Test Termination

On the 28th day of the test, an aliquot of the contents of each test chamber was removed and

the pH determined. The contents of all chambers were then acidified by the addition of 3 mL of- 13 -

concentrated hydrochloric acid to drive off inorga nic carbonate. All chambers were aerated overnight

and then a sample from each test chamber was rem oved for dissolved organic carbon (DOC) analysis

and then the trapping solutions closest to the t est chambers were analyzed for inorganic carbon.

Calculations

The results of the inorganic carbon analyses of the CO 2 traps were converted to mg CO 2

produced using the following equation:

mg CO 2 = result (mg C/L) X vol. of KOH (L) X 3.67 mg CO 2 /mg C

The cumulative mg of CO 2 for the test and reference substa nces were corrected for the amount

of CO 2 evolved by the control, using the following equation:

Cumulative mg CO 2 produced = Σ mg CO 2 test - mean Σ mg CO 2 control

The percentage of theoretical carbon dioxide (%TCO 2 ) evolved was calculated as follows:

% TCO 2 = 

### ) /mg Carbon mg CO

. t) x ( bon in tes (mg of car produced mg CO

2 2



### 67

3 X 100

RESULTS AND DISCUSSION

Carbon Analysis

The measured total organic carbon (TOC) concen tration of the reference substance stock

solution was 398.7 mg C/L. The volume of stock so lution used to dose the reference chambers was

adjusted based on the measured TOC value so that approximately 10 mg C/L was delivered.

Observations and Measurements

The temperature range recorded during the test was 18.6-26.3 oC. The mean result of the

standard plate count performed on the inoculum was 6.60 x 10 5 CFU/mL. The measured total

suspended solids concentration of the test solu tion was 3.467 mg/L and was within the acceptable

limit specified in the test guideline (1).- 14 -

The measured dissolved organic carbon (DOC) on Day 29 and pH values of the test chamber

contents on Day 28 are presented in Table 1. The measured concentrations of inorganic carbon in the

carbon dioxide trapping solutions are presented in Table 2. The cumulative amounts of CO 2 produced

over the test period are presented in Table 3. Th e cumulative percent of theoretical carbon dioxide

(% TCO 2 ) evolved is presented in tabular and graphical forms in Table 4 and Figure 1, respectively.

The control chambers evolved an average of 22.2 mg CO 2 over the test period. This value has

been corrected for the amount of CO 2 in the trapping solution since potassium hydroxide solution,

even when freshly prepared, contains carbonates. The amount of CO 2 evolved by the control

chambers did not exceed the 40 mg/L (120 mg to tal) value considered the acceptable limit for CO 2

evolution tests (1).

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

reference substance, sodium benzoate, from wh ich an average of 98.0% of theoretical CO 2 was

evolved. An average percent biodegradation of greater than 60% was achieved by Day 13, thereby

fulfilling the criteria for a valid test by reaching the pass level by Day 14 (1).

The final mean percent biodegradation for the test substance was 94.2%. The test Substance

may be considered readily biodegradable, since the pass level of 60% TCO 2 was achieved.

CONCLUSIONS

Evidence of ready biodegradability in a Ca rbon Dioxide Evolution Test is 60% TCO 2 within the

28-day test period (1,2). In addition, the pass leve l must be reached within 10 days of achieving 10%

TCO 2 (1,2). The final mean percent biodegradati on for the test substance was 94.2%. The test

substance may be considered readily biode gradable since the pass level of 60% TCO 2 was achieved.- 15 -

REFERENCES

1. Organisation for Economic Cooperation and Development . 1992. Guideline for the Testing

of Chemicals. Guideline 301B: Ready Biodegradability: CO 2 Evolution (Modified Sturm Test)

. Adopted by the Council on 17 July 1992.

2. Annex to Commission Directive 67/548/EEC. 1992. Carbon Dioxide (CO 2 ) Evolution . Official Journal of the European Communities No. L383. Method C.4-C,

3. Standard Methods for the Examination of Water and Wastewater . Seventeenth Edition.

Parts 2450 and 9215. American Public Health Association.- 16 -

Table 1

Dissolved Organic Carbon (DOC) and pH of Test Solutions at Test Termination

Test Chamber/Nominal Concentration DOC 1

(mg C/L) pH

Control Rep. A <1.0

Control Rep. B

Control Rep. C

Sodium Benzoate Rep. A (10 mg C/L)

Sodium Benzoate Rep. B (10 mg C/L)

Sodium Benzoate Rep. C (10 mg C/L) <1.0

<1.0

<1.0

<1.0

<1.0

Test Substance Rep. A (10 mg C/L)

Test Substance Rep. B (10 mg C/L)

Test Substance Rep. C (10 mg C/L) 1.0

1.1

0.9 7.2

7.2

7.2

7.3

7.2

7.3

7.1

7.2

7.2

1 Samples were filtered (0.45 μ m) and purged with CO 2 free air prior to analysis.- 17 -Table 2Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L)Date Day ControlRep. AControlRep. BControlRep. CReferenceRep. AReferenceRep. BReferenceRep. CDBATRep. ADBATRep. BDBATRep. CBlank16-Jun-0520-Jun-0524-Jun-0528-Jun-0530-Jun-055-Jul-057-Jul-0511-Jul-0514-Jul-051591315202226297.8219.5714.7915.9612.4213.137.357.9914.408.1415.5215.5012.839.559.637.318.2216.808.2517.4615.5513.619.0711.376.097.218.674.99151.0091.0657.1119.9731.1412.0516.1017.897.5762.4269.0143.2671.6862.757.3321.6554.687.80123.20111.7057.9316.7324.2913.5418.1820.9110.2990.69123.6057.1115.0223.3112.9412.8634.238.87105.0075.3273.8231.0141.8115.2518.6919.269.96110.90102.9057.4524.4839.0613.5216.7017.905.874.864.895.286.013.424.424.914.62- 18 -Table 3Cumulative Milligrams of Carbon Dioxide Evolved1,2Date Day ControlRep. AControlRep. BControlRep. CReferenceRep. AReferenceRep. BReferenceRep. CDBATRep. ADBATRep. BDBATRep. CControl3Rep. AControl3Rep. BControl3Rep. CBlankControlAverage16-Jun-0520-Jun-0524-Jun-0528-Jun-0530-Jun-055-Jul-057-Jul-0511-Jul-0514-Jul-051591315202226292.910.115.521.325.930.733.436.341.63.08.714.419.122.626.128.831.838.03.09.415.120.123.527.629.932.535.7-1.147.975.791.495.0102.2104.1107.2108.8-0.216.336.046.769.288.188.293.3108.5-0.138.774.190.192.597.299.7103.5106.30.827.767.483.284.989.391.593.4101.00.332.454.476.383.995.198.2102.1104.30.735.067.183.088.298.4100.8104.1105.70.76.19.713.716.019.620.721.825.40.84.78.611.412.715.016.117.321.70.95.59.412.513.616.517.118.019.42.23.95.77.79.911.112.814.616.23.09.415.020.224.028.230.733.638.41The results of the inorganic carbon analyses of the CO2traps were converted to mg CO2produced using the following equation:mg CO2= cumulative result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ.3Corrected for the CO2attributed to the inoculum and the KOHby subtracting the average amount of CO2evolved by the controls.- 19 -Table 4Cumulative Percent of Theoretical Carbon Dioxide Evolved1,2DateDayControlRep. AControlRep. BControlRep. CReferenceRep. AReferenceRep. BReferenceRep. CDBATRep. ADBATRep. BDBATRep. C16-Jun-0520-Jun-0524-Jun-0528-Jun-0530-Jun-055-Jul-057-Jul-0511-Jul-0514-Jul-051591315202226NANANANANANANANANANANANANANANANANANANANANANANANANANANA-1.043.568.783.186.392.894.697.398.9-0.214.832.742.462.980.080.184.798.5-0.135.167.381.984.088.390.594.096.50.725.161.275.677.181.183.184.891.80.329.449.469.376.286.489.192.894.80.631.861.075.480.189.391.594.596.0Cumulative Average (N=3)Std. Dev.NANA98.01.394.22.21The percentage of TCO2was calculated using the following equation:% TCO2=)/mg Carbonmg CO.t) x (bon in tes(mg of carproducedmg CO26732X 1002Calculations performed in Excel 2000 full precision mode. Manual calculations may differ.NA – Not Applicable- 20 -

-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

Time (days)Percent of Theoretical CO2 Production

Sodium Benzoate Rep.A Sodium Benzoate Rep.B Sodium Benzoate Rep.C

Test Substance Rep.A Test Substance Rep.B Test Substance Rep.C

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved- 21 -

APPENDIX I

Protocol and Protocol Deviation- 22 -- 23 -- 24 -- 25 -- 26 -- 27 -