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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2003 - October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6,8-trimethylnonan-4-ol
EC Number:
204-606-8
EC Name:
2,6,8-trimethylnonan-4-ol
Cas Number:
123-17-1
Molecular formula:
C12H26O
IUPAC Name:
2,6,8-trimethylnonan-4-ol
Details on test material:
- Name of test material (as cited in study report): Trimethylnonanol
- Molecular weight (if other than submission substance): 186.34
- Physical state: Transparent pale yellow tinted liquid
- Analytical purity: 95.7% (gas chromatography)
- Lot/batch No.: RC1355T3D4(2)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
The microbial inoculum consisted of secondary effluent, collected from the City of Midland Municipal Wastewater Treatment Plant (Midland, Michigan). This facility treats an excess of 3.0 x 10(6) gallons of wastewater daily, of which > 90% is from domestic sources. The secondary effluent was collected one day prior to initiation of the test and filtered through Whatman 114V filter paper prior to use. The Mineral Medium was inoculated with the filtered effluent at a concentration of 4 mL per liter of mineral medium. The inoculated mineral medium was aerated for at least 30 minutes, and allowed to stand at test temperature for at least 20 hours prior to adding the test and reference chemicals. The pH of the inoculated mineral medium was 7.4 and did not require adjustment. The concentration of dissolved oxygen in the equilibrated, inoculated mineral medium was 8.7 mg O2/L prior to initiating the test.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2.14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
A total of 16 liters of inoculated mineral medium was prepared. The Inoculum Blanks were prepared by dispensing the inoculated mineral medium into 300 mL glass BOD bottles. Procedure Controls were prepared by volumetrically removing a 2 liter aliquot of the inoculated mineral medium, fortifying it with 5.0 mL of the 1608 mg/L sodium benzoate stock solution (4.02 mg/L), and dispensing into 300 mL glass BOD Bottles. The Test Suspensions were prepared by combining a 2L aliquot of fortified inoculated mineral media (described above) with 6 liters of inoculated mineral medium (2.14 mg/L) and dispensing into 18 BOD Bottles. To prepare Toxicity Controls, a final 2 liter aliquot of the test chemical amended mineral medium was fortified with 5.0 mL of the 1608 mg/L sodium benzoate (2.14 mg/L test material and 4.02 mg/L benzoate) and dispensed into 300 mL glass BOD Bottles. Each BOD bottle was filled to capacity, and sealed with ground glass stoppers and plastic covers to prevent entrapment of air bubbles and evaporation of the aqueous media.

A total of 18 inoculum blanks, containing only the inoculated mineral medium, were prepared to allow for at least duplicate measurements over at least nine? sampling intervals. These blanks were used to determine mean values for oxygen consumption due to endogenous respiration of the inoculum. Similarly, a total of 18 test suspensions, containing 2.14 mg/L of the test material were prepared. A total of six Procedure Control mixtures were prepared to allow biodegradation of sodium benzoate (4.02 mg/L) to be determined in duplicate after 0, 3, 7, and 14 days incubation. Similarly, six replicate Toxicity Control mixtures, containing both the test material and reference substance, were prepared to examine the potential inhibition of the inoculum at the same sampling intervals.

Incubation
The reaction mixtures were statically incubated in a Forma Scientific walk- in incubator room, which was maintained under complete darkness except during dissolved oxygen measurements. The air temperature within this incubator was automatically recorded at three hour intervals over the course of the test. In addition, the local air temperature at the bench top where the reaction mixtures were kept was periodically recorded using an NIST-traceable ASTM thermometer.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (O2 consumption)
Value:
2.6
St. dev.:
0.1
Sampling time:
3 d
Parameter:
% degradation (O2 consumption)
Value:
1.6
St. dev.:
0.4
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
2.4
St. dev.:
0.3
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
0.8
St. dev.:
2.7
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
5.1
St. dev.:
0.7
Sampling time:
28 d
Details on results:
Very limited biodegradation of trimethylnonanol was observed under the stringent test conditions used in the OECD Closed Bottle Test. The OECD Guideline 301D criterion for “readily biodegradable” classification requires achieving 60% biodegradation within a 10-day window following onset (10% degradation), within a total test duration not exceeding 28 days. Biodegradation of trimethylnonanol reached only 5% after 28 days. Therefore, trimethylnonanol cannot be considered as ”readily biodegradable” according the current OECD criteria for the Closed Bottle Test.

The results of this test met each of the criteria established by OECD for determining validity of its ready biodegradability tests. For example, a test is considered valid if the difference of extremes in replicate values of percent biodegradation of the test chemical is less than 20% at the plateau, end the test, and end of the 10 day window. In this test, the maximum difference in percent biodegradation among replicates was 5.1% occurring at day 28. In addition, percent biodegradation of the reference compound (sodium benzoate) must exceed the pass level (60%) by day 14. In this
test, the mean percent biodegradation (+/- 1 standard deviation) of benzoate was 81.9 +/- 0% at day 14. For Toxicity Controls, containing both the test compound and the reference substance, if less than 25% biodegradation occurrs within 14 days, the test substance can be judged to be inhibitory to the inoculum. In this test, 42.8 +/- 0.3% biodegradation was observed at day 14. Lastly, the maximum total oxygen consumption in the inoculum blanks must not exceed 1.5 mg/L over the 28 day test, and dissolved oxygen levels in the test suspensions must not fall below 0.5 mg/L. In this test, the maximum oxygen consumption in the inoculum blanks after 28 days was 0.91 mg/L, while the minimum dissolved oxygen level observed in the test suspensions was 7.4 mg/L. Therefore, the results of this test can be considered as fully valid according to the current OECD criteria.

BOD5 / COD results

Results with reference substance:
In this test, the mean percent biodegradation (+/- 1 standard deviation) of benzoate was 81.9 +/- 0% at day 14.

Any other information on results incl. tables

Complete dissolution of the test material was confirmed by analysis of dissolved organic carbon in the dosing stock solution. Of the calculated 6.6 mg carbon/L added to the stock solution, 89% was measured as DOC. The incubation temperature ranged from 19.8 to 20.4oC,and averaged 20.1 + 0.1oC during the course of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: cannot be considered as “readily biodegradable” according the current OECD criteria for the Closed Bottle Test
Conclusions:
Based on the study results, the maximum dissolved oxygen level observed in the test suspensions was 7.4mg/L. Therefore, the results of this test can be considered as fully valid according to the current OECD criteria.
Executive summary:

The ready biodegradation of trimethylnonanol was evaluated according to the OECD Guideline 301D: Closed Bottle Test. This test examined biodegradation of the test chemical (2.14 mg/L) in a defined mineral medium after inoculation with secondary effluent (4 mL/L) from the City of Midland, Michigan Wastewater Treatment Plant. Consumption of dissolved oxygen in replicate inoculum blanks (no test substance added) and test chemical suspensions was determined after 3, 5, 7, 11, 14, 17, 21, and 28 days incubation at a temperature of 20.1 + 0.1oC. In order for the test material to be considered as “readily biodegradable” by the test guideline, average net oxygen consumption in the test suspensions, corrected for oxygen consumed in the inoculum blanks, must have exceeded 60% of the theoretical oxygen demand (ThOD), within a 10 day window from onset (10% degradation). Biodegradation of trimethylnonanol reached only 5.1% after 28 days. Therefore, trimethylnonanol cannot be considered as “readily biodegradable” according the current OECD criteria for the Closed Bottle Test.

The reference compound, sodium benzoate (4.02 mg/L), achieved > 60% biodegradation (i.e., 81.9%) after 14 days. Consumption of dissolved oxygen in the inoculum blanks did not exceed 1.5 mg/L over the 28 day test. The difference between extremes in percent biodegradation among replicate test suspensions did not exceed 2.7% at any of the sampling intervals. Toxicity controls (benzoate + trimethylnonanol) exceeded the 25% biodegradaion threshold at 14 days (i.e., 42.8), confirming the test material was not inhibitory to the inoculum under these conditions. Therefore, the results of this test met each of the validation criteria established for the OECD Closed Bottle Test.