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C u s t o m e r   R e p o r t

ID Entry Date 3/17/2013

Project Title

Biodegradation Test

The OECD 301B method is designed to provide the screening of chemicals for ready 

biodegradability in an aerobic aqueous medium. Samples are required to achieve a threshold 

of 60% degradation based on the maximum available carbon from a given sample formulation. 

Total carbon is determined analytically for each sample and used as the reference for the 

determination of the percentage of carbon dioxide (% ThCO2) produced by microbiological 

degradation. 

For the purpose of determining biological degradation, two criteria can be achieved. Ready 

Biodegradability can be achieved by obtaining the 60% threshold within a 10 day window within 

a total of 28 days of testing. The second criteria, Ultimate Biodegradation, can be achieved if 

the amount of biodegradation meets or exceeds the 60% threshold at a time point determined 

in the test (e.g. when the rate of degradation reaches a plateau). 

Two test samples were submitted for OECD 301B biodegradation re- testing (

) using a customer supplied industrial inoculum. Following arrival of the inoculum, 

the material was coarse filtered to remove particulate material. OECD 301B buffer salts were 

added to the inoculum filtrate and then incubated with constant agitation and normalized for 7 

days at 24C prior to setup of the test chambers. Each test sample (and control) was introduced 

into the test chambers by homogenizing the sample material with silica gel in a manner 

described by the customer supplied method. 

In this test series, neither test sample achieved the required level of degradation based on the 

total accumulated carbon dioxide over the 35 days of the test. Due the low rate of apparent 

respiration within the test samples, chamber pH values were checked at day 20 and on the final 

Project Summary

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Cover

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day of the test and were within normal ranges. The microorganism concentrations were also 

checked on day 0 and day 20 of the test, and were determined to exceed 1E7 CFU/ml per 

sample chamber. Control sample performed normally for each of the test parameters achieving 

Ready Biodegradation by Day 17 of the test.

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Sample List

Method Name

Sample # Sample Name Sample Notes

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

1 Apply to silica gel as indicated in com.

2

3 Control ‐ Sodium Acetate

4 Silica gel blank

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Title Biodegradation Test

Project ID Test Start Date 1/10/2013

Contact

Entry Date 3/17/2013

R e s u l t   T a b l e

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Result Table *

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Sample # 1

Interval Result

Inoculum Mixed Environmental Organisms  ()

final pH = 7.4; total sample carbon = 88%; ‐ no net CO2 production 35 day 0 % ThCO2

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines.

Sample # 2

Interval Result

Inoculum Mixed Environmental Organisms  ()

final pH 7.4; TOC = 82%;  ‐ no net CO2 production 35 day 0 % ThCO2

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines. 

Result Table 1 of 3

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Result Table *

Sample # 3 Control ‐ Sodium Acetate

Interval Result

Inoculum Mixed Environmental Organisms  ()

17 day 60 % ThCO2

35 day 72 % ThCO2

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent 

of theoretical maximum (% ThCO2) derived from the test sample. 

Average values are plotted with the standard deviation (+/- SD) for the 

time course of the test. Curve fit is applied to calculate the statistical 

confidence for 90% (blue line) and predicted 90% (red) boundary lines. 

Shading below the curve fit applies to the 10 day biodegradation window 

required for the determination of Ready Biodegradability.

Sample # 4 Silica gel blank

Interval Result

Inoculum Mixed Environmental Organisms  ()

35 day 0 % ThCO2

Result Table 2 of 3

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Result Table *

*   This report is governed by and incorporates by reference, the conditions of testing as posted on the date of issuance and is intended for your exclusive use.  

Any Copying or replication of this report to or for any other person or entity, or use of our company name or Service Mark is permitted only with our prior written

consent.  This report sets forth our findings solely with respect to test samples identified herein.  The results set forth in this report are not indicative or

representative of the quality or characteristics of the lot from which a test sample was taken or any similar identical product unless specifically and expressly

noted.  Our report includes all tests requested and the results there of based upon the information provided.  You have 60 days from the date of issuance of this

report to notify us of any material error or omission caused by our handling of the samples, provided however that such notice shall be in writing and shall

specifically address the issue you wish to raise.  A failure to raise such issue within the prescribed time shall constitute your unqualified acceptance of the

completeness of this report, the test conducted and the correctness of the report contents.

OECD 301 B - Solution Biodegradation

Test conditions:

- inoculum: Surface water from water district.

- proportion and nature of industrial waste water in sewage: unknown, discharge from waste treatment facility 

within 1 mile.

- test duration and temperature: 28 days or as indicated, 22C +/- 2C

- bacterial inoculum ~1E5 cfu/ml 

Legend

Sample Analysis

TC - Total Carbon determined by catalytic oxidation of the test sample.

IC - Inorganic Carbon 

TOC - Total Organic Carbon - determined by the subtraction of TC from IC.

TN - Total Nitrogen determined by chemical luminescence.

%S - Percent Solids- is the dry (non-volatile) percent of the test sample. 

For the sample analysis, percent solids is determined when estimating the weight of material to test. For 

biodegradable materials, the best degradability will be obtained with sample compositions that are linear organic 

(carbon containing) molecules lacking carbon to carbon double bonds. The total carbon (TC) provides an 

indication of the material composition, but does not provide information on chemical structure or function. Inorganic 

carbon is typically low in most biodegradable materials, and increases over the course of the test due to the action 

of the microorganisms in creating waste, or biological compounds that are generated from the consumption of the 

carbon based test sample. Total nitrogen can be an indication of nutrient abundance, but is not typically used as 

part of the test sample assessment. 

Result Table 3 of 3

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R e p o r t   A d d e n d u m

Project ID Entry Date 3/17/2013 Test Start Date 1/10/2013

Image Table

Sample # 1

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines.

Addendum 1 of 3

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Image Table

Sample # 2

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines. 

Addendum 2 of 3

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Image Table

Sample # 3 Control ‐ Sodium Acetate

Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Inoculum Mixed Environmental Organisms

Image: Chart

Figure - Test chamber carbon dioxide (CO2) measurement as the percent of 

theoretical maximum (% ThCO2) derived from the test sample. Average values 

are plotted with the standard deviation (+/- SD) for the time course of the test. 

Curve fit is applied to calculate the statistical confidence for 90% (blue line) and 

predicted 90% (red) boundary lines. Shading below the curve fit applies to the 

10 day biodegradation window required for the determination of Ready 

Biodegradability.

Addendum 3 of 3

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