Friday, February 15, 2013 

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

ID Entry Date 1/10/2013 

## Project Title

Biodegradation Test

The OECD 301B method is designed to provide the screeni ng of chemicals for ready biodegradability in an aerobic aqueous medium. Samples are required to ac hieve a threshold of 60% degradation based on the maximum available carbon from a given sample formulatio n. 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 cr iteria 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 testing ( ). Neither test sample achieved the required level of degradation based on the total accumulated carbon dioxide over the 31 days of the test. Due the low rate of apparent respirati on within the test samples, chamber pH values were checked at day 17 and on the final day of the test and were within normal ranges. The microorganism concentrations were also checked on day 17 of the tes t, and were determined to exceed 1E7 CFU/ml per sample

chamber.

COD determination was conducted on each test samp le was performed by dichromate oxidation and spectrophotometric absorbance analysis. Lack of sample so lubility and agglomeration may affect the results of this analysis. Pro j ect Summar y



## Cover

Friday, February 15, 2013

### Sam p le List

Method Name

S ample # S ample Name S ample Notes

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution



### 1 Apply to silica gel as indicated in com.

2

3 Control

‐ Sodium Acetate

Chemical Oxygen Demand (COD) Determination



### 7

8



## Cover

Friday, February 15, 2013

## Title Biodegradation Test



## Project ID Test Start Date 1/10/2013



## Contact

Entry Date 1/10/2013 

# R e s u l t T a b l e

r.



# Result Table *

OECD 301 B ‐ Solution Biodegradation by CO2 Evolution 

### Test Method

1 Sample #



### Interval Result

Inoculum Mixed Environmental Or g anisms ()



### 3.7 31 final pH7.9, total sample carbon = 87.9% % ThCO2 day



### Image: Test Sample



## 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 95% (blue) and predicted 95% (red) boundary lines. Shading below the curve fit applies to the 10 day biodegradation window required for the determination of Ready Biodegradability.

2 Sample #



### Interval Result

Inoculum Mixed Environmental Or g anisms ()



### 0 31 final pH = 7.75; total sample carbon = 81.5% % ThCO2 day



### Image: Test Sample



## 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 95% (blue) and predicted 95% (red) boundary lines. Shading below the curve fit applies to the 10 day biodegradation window required for the determination of Ready Biodegradability.



## Result Table 1 of 4

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# Result Table *

3 Control
‐ Sodium Acetate Sample #



### Interval Result

Inoculum Mixed Environmental Or g anisms ()



### 60 14 final pH = 7.6; final plateau 75.5% +/

‐ 7% ThCO2 % ThCO2 day



### Image: Control Sample



## 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 95% (blue) and predicted 95% (red) boundary lines. Shading below the curve fit applies to the 10 day biodegradation window required for the determination of Ready Biodegradability.

Chemical Oxygen Demand (COD) Determination 

### Test Method

7 Sample #



### Interval Result

Inoculum None ()



### 2143 n / a dichromate oxidation ppm O2 consumed/mg



### 8 Sample #



### Interval Result

Inoculum None ()



### 981 n / a dichromate oxidation ppm O2 consumed/mg



## Result Table 2 of 4

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# Result Table *

OECD 301 B - Solution Biodegradation

Test conditions:

- inoculum: Surface water from water district. - proportion and nature of industrial waste water in sew age: unknown, discharge from waste treatment facility

within 1 mile. - test duration and temperature: 28 day s or as indicated, 22C +/- 2C

- bacterial inoculum ~1E5 cfu/ml

Legend Sample Analysis

TC - Total Carbon determined by catalyti c 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-vola tile) 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 wi th sample compositions that are linear organic (carbon containing) molecules lacking carbon to car bon double bonds. The total carbon (TC) provides an indication of the material composition, but does not provide information on chemic al structure or function. Inorganic carbon is typically low in most biodegradable materials, and incr eases 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 indicati on of nutrient abundance, but is not typically used as part of the test sample assessment.



## Result Table 3 of 4

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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.



## Result Table 4 of 4

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

Project ID Test Start Date 1/10/2013 

## Entry Date 1/10/2013



## Image Table

1 Sample #



### Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum



### Image: Test Sample



## Figure - Test chamber carbon dioxide (CO2) measurement as the percent of theoretical maximum (% ThCO2) derived fr om 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 st atistical confidence for 95% (blue) and predicted 95% (red) boundary lines. Shading below the curve fit applies to the 10 day biodegradation window required for the determination of Ready Biodegradability.



## Addendum 1 of 3

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## Image Table

2 Sample #



### Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum



### Image: Test Sample



## Figure - Test chamber carbon dioxide (CO2) measurement as the percent of theoretical maximum (% ThCO2) derived fr om 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 st atistical confidence for 95% (blue) and predicted 95% (red) boundary lines. Shading below the curve fit applies to the 10 day biodegradation window required for the determination of Ready Biodegradability.



## Addendum 2 of 3

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## Image Table

3 Control
‐ Sodium Acetate Sample #



### Test Method OECD 301 B ‐ Solution Biodegradation by CO2 Evolution

Mixed Environmental Organisms Inoculum



### Image: Control Sample



## Figure - Test chamber carbon dioxide (CO2) measurement as the percent of theoretical maximum (% ThCO2) derived fr om 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 st atistical confidence for 95% (blue) and predicted 95% (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

Friday, February 15, 2013 r.