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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 10, 2003 to October 27, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The sample of 3-chloro-o-xylene (T.R. Wilbury Laboratories sample number 1763) used for the study was delivered on March 10, 2003. It was contained in four 50 mL amber bottles that were shipped in a 20 gallon plastic bucket at ambient temperature. The label attached to the bottles included the following information: "3-chloro-1,2-dimethyl benzene, CAS 608-23-1, Clariant, Lot 26.11.02, 3-cl-1,2-dimethyl benzene, CAS# 608-23-1, 25 g, 98%, combustible. The substance (a colorless liquid) was supplied by GE Global Research, 1 Research Circle, Niskayuna, New York. Prior to use the test substance was stored at room temperature in the dark. The stability of 3-chloro-o-xylene under exposure conditions was not determined during this study. Triplicate analyses of an aliquot of 3-chloro-o-xylene for chemical oxygen demand (COD) by Galbraith Laboratories, Inc., Knoxville, Tennessee, resulted in a mean COD value of 1.327 mg O2/mg test substance.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Fresh activated sludge was obtained from the municipal waste treatment facility at Newburyport, Massachusetts on July 10, 2003. This facility treats predominantly domestic waste. Viability of the microorganisms was confirmed after inoculation of the test solutions and activity was checked by means of the positive control. The sludge was used on the day it was collected and it was aerated for approximately 4 hours prior to use. A subsample of the activated sludge (approximately 1000 mL) was homogenized for 2 minutes with a mechanical blender and then centrifuged for 30 minutes. The supernatant was decanted, pooled, and 0.35 mL was added to each mixing vessel, except the media blank. The inoculum contained 50 mg/L total suspended solids.
Duration of test (contact time):
28 d
Initial conc.:
5 mg/L
Based on:
test mat.
Initial conc.:
1.327 other: mg O2/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral Medium (MM), acc. to the guideline
- Preparation of medium: The mineral media was aerated vigorously for 20 minutes and allowed to stand for 20 hours at room temperature prior to use. The dissolved oxygen concentration of the mineral media immediately prior to use was 8.9 mg/L, the temperature was 21.0°C, and the pH was 7.4 ± 0.2.
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: test start: 20 ± 1°C
- pH: 7.4 ± 0.2
- pH adjusted: no
- Aeration of minera medium: yes
- Suspended solids concentration: 50 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: clear glass BOD bottles, 300 mL
- Number of culture flasks/concentration: 10
- Method used to create aerobic conditions: sludge was aerated for approximately 4 hours prior to use
- Measuring equipment: YSI Model 57 meter (instrument number 18)
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: closed

SAMPLING
- Sampling frequency: days 5, 14, 21, and 28
- Sampling method: Duplicate BOD bottles from each series were removed on days 5, 14, 21, and 28 and the dissolved oxygen concentration was determined.
- Sterility check if applicable: no
- Sample storage before analysis: not idicated

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (no test substance, 0.35 mL of secondary effluent)
- Positive control: yes (2 mg/L sodium benzoate)
- Media blank (abiotic control): yes (mineral media without secondary effluen)

CALCULATION METHODS:
- Percentage biodegradation was calculated acc. to the guideline; see also any other information on matrials and methods
Reference substance:
benzoic acid, sodium salt
Preliminary study:
A definitive biodegradability test was attempted from June 26 to July 1, 2003. The test was terminated after 5 days due to unacceptable oxygen consumption in the media blanks and inoculum blanks, and the test was repeated.
Test performance:
Test concentrations of the final definitive biodegradability test were prepared by siphoning approx. 200 mL of mineral media into each of four 3.5 liter clear glass mixing vessels. Test substance was added to one of the vessels at a nominal concentration of 5 mg/L by weighing 0.0175 g of test substance onto a plastic weigh pan and adding the weigh pan with test substance to the mixing vessel. The solution was inoculated with 0.35 mL of the secondary effluent (0.1 mL/L) and the total volume was brought up to 3.5 liters with mineral media. The inoculum blank, which contained no test substance, received only 0.35 mL of secondary effluent brought up to a total volume of 3.5 liters with mineral media. The positive control, 2 mg/L sodium benzoate, was prepared by adding 7.0 mg to 200 mL of mineral media containing 0.35 mL of secondary effluent that was brought up to 3.5 liters with mineral media. The media blank was prepared by adding 3.5 liters of mineral media without secondary effluent to the mixing vessel. The solutions were gently mixed using a magnetic stir plate, avoiding the formation of a vortex. The test was started by gently siphoning the contents from the lower quarter of the mixing vessel into 10 replicate iodine washed clear glass BOD bottles with a volume of approx. 300 mL. The bottles were filled to overflowing, stoppered, and placed into an incubator with a photoperiod of 0 hour light and 24 hours dark (on day 6 of the definitive test, all remaining test vessels were covered with foil to exclude light due to the initiation of a 16 hour light/8 hour dark photoperiod in the incubator). The dissolved oxygen concentration in duplicate test vessels from each series was immediately measured. The remaining test vessels were maintained in the dark at 20 ± 1°C for up to 28 days (the temp. was recorded daily during the test period). Duplicate BOD bottles from each series were removed on days 5, 14, 21, and 28 and the dissolved oxygen concentration was determined.
Key result
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
28 d
Details on results:
The loss of oxygen in test vessels containing 3-chloro-o-xylene indicated that there was 2% biodegradation. The degradation lag phase, the 10-day window of degradation, the slope of the degradation line, and the degradation phase could not be determined because of this low degradation. Because degradation was less than 60% after 28 days, these results indicate that 3-chloro-o-xylene was not readily biodegradable under test conditions.
Results with reference substance:
The positive control, sodium benzoate, yielded 84% of the theoretical degradation during the test, demonstrating the adequacy of the inoculum. Temperature remained within the acceptable range of 20 ± 1°C throughout the test.

Dissolved oxygen data from the ready biodegradability (biotic degradation) of 3-chloro-o-xylene using the closed bottle test.

Dissolved Oxygen Concentration (mg/L)
Day Inoculum Blank  Positive Control
Rep. 1  Rep. 2  Mean Rep. 1   Rep. 1 BOD* Rep. 2 Rep. 2 BOD* Mean Percent Biodeg
0 8.7 8.6 8.7 8.6 - 8.6 - -
5 8.3 8.6 8.5 6.5 1 6.4 1 60
14 8.6 8.6 8.6 6.1 1.2 6.3 1.1 72
21 8.4 8.5 8.5 5.9 1.3 5.9 1.3 78
28 8.5 8.6 8.6 5.8 1.4 5.7 1.4 84

* mg O2/mg test/reference substance

Dissolved Oxygen Concentration (mg/L)
Day Media Blank  3-chloro-o-xylene
Rep. 1  Rep. 2  Mean Rep. 1   Rep. 1 BOD*  Rep. 2 Rep. 2 BOD*  Mean Percent Biodeg
0 8.8 8.7 8.8 8.7 - 8.6 - -
5 8.5 8.4 8.5 8.5 0 8.3 0.02 1
14 8.4 8.7 8.6 8.6 0 8.5 0 0
21 8.5 8.6 8.6 8.5 0 8.5 0 0
28 8.7 8.4 8.6 8.4 0.04 8.6 0 2

* mg O2/mg test/reference substance

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under test conditions the test item was not readily biodegradable, i.e. observed biodegradation was 2% after 28 days.
Executive summary:

The test on ready biodegradation of the test item was conducted according to OECD 301D (Closed Bottle Test) and in compliance with GLP. Fresh activated sludge, obtained from the municipal waste treatment facility at Newburyport, Massachusetts, and was used as the source of unacclimated microorganisms for the test. Biodegradability was determined by measuring the amount of oxygen taken up by the microbial population over the 28 day test period. The degradation was defined as the amount of oxygen taken up, expressed as a percentage of the biochemical oxygen demand (BOD), relative to the chemical oxygen demand (COD) or theoretical oxygen demand (ThOD) as was used for the positive control. The test was performed with a single concentration of test substance, i.e. 5 mg/L whole test substance. The chemical oxygen demand for the sample of 3-chloro-o-xylene was 1.327 mg O2/mg test substance. A series of 10 replicate test vessels was established for the test concentration, an inoculum blank, a media blank, and a positive control (2 mg/L sodium benzoate). After 28 days, the oxygen consumption for the test substance at a concentration of 5 mg/L resulted in 2% biodegradation. Because degradation was less than 60%, these results indicate that 3-chloro-o-xylene was not readily biodegradable under test conditions.

The study was fulfilled the validity criteria of the test guideline OECD 301D and was considered reliable and adequate for the environmental hazard assessment.

Description of key information

The test item is not readily biodegradable, i.e. observed biodegradation was 2% after 28 days (OECD 201D, 2003)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable test on ready biodegradation of the test item was conducted according to OECD 301D (Closed Bottle Test) and in compliance with GLP (RL1, 2003). Fresh activated sludge, obtained from the municipal waste treatment facility at Newburyport, Massachusetts, and was used as the source of unacclimated microorganisms for the test. Biodegradability was determined by measuring the amount of oxygen taken up by the microbial population over the 28 day test period. The test was performed with a single concentration of test substance, i.e. 5 mg/L whole test substance. A series of 10 replicate test vessels was established for the test concentration, an inoculum blank, a media blank, and a positive control. After 28 days, the oxygen consumption for the test substance at a concentration of 5 mg/L resulted in 2% biodegradation. As degradation was less than 60%, the results indicate that 3-chloro-o-xylene was not readily biodegradable under test conditions. The study fulfilled the validity criteria of the test guideline OECD 301D and was considered reliable and adequate for the environmental hazard assessment.