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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:
12 May 1999 to 23 June 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The study followed the same principles as the OECD 301 F guideline, however it did not include the abiotic and the toxicity checks, and pre-adapted inoculum was employed.
The ready biodegradability of the test material was investigated as part of a study in which a total of six substances was tested.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge: The inoculum used was the return activated sludge from the Mentor Waste Water Treatment plant (Mentor, OH), collected on the 12 May 1999. The inoculum was adapted to the test material for 14 days before the test initiation.
- Laboratory culture: Yes
- Method of cultivation: The sludge was aerated and stirred for 2-3 hours in a flask, homogenised in a Waring blender at low/medium speed for two minutes, and allowed to stand for to one hour. The supernatant was then pipetted out and used for inoculum pre-adaptation.
- Pre-adaptation of the inoculum: Sludge supernatant supplemented with ~25 mg/L vitamin free casamino acids and ~25 mg/L yeast extract. A two litre flask containing 100 mL of supplemented sludge supernatant and 900 mL test medium was prepared and the test material was added incrementally during the adaptation period, at concentrations approximately equivalent to 4, 8 and 8 mg carbon/L on days 0, 7, and 11 , respectively,
The mixture was aerated throughout the adaptation period by shaking at 150 rpm and 22 °C. On day 14, the adapted culture was homogenised in a blender for ~15 seconds and used as the inoculum. A composite culture was prepared by mixing equal amount of all homogenised cultures.
- Initial cell/biomass concentration: The bacterial count in the culture was estimated using Easicult®. The inoculum level was 10 % using the acclimated composite culture. The microbial level in the test mixture was 10³.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium was freshly prepared by using deionized water and the four stock solutions listed below. The 900 mL medium in each bioreactor contained 10 mL of stock solution A, and 1 mL each of the stock solutions B, C, and D and 887 mL of deionized water. One hundred mL of the acclimated inoculum was later added to each reactor to reach a final volume of one litre.
Stock solution A: 8.5 g/L KH2PO4, 21.75 g/L K2HPO4, 50.30 g/L Na2HPO4.7H2O and 0.50 g/LNH4Cl
Stock solution B: 36.40 g/L CaCl2.2H2O
Stock solution C: 22.50 g/L MgSO4.7H2O
Stock solution D: 0.25 g/L FeCl3.6H2O and 0.40 g/L Na2EDTA
- Test temperature: 23 ± 1 °C
- pH: The pH values of the initial medium in each bioreactor as well as the final pH values of the test mixtures were monitored to ensure biodegradation was not inhibited by extreme pHs. The pH of the test medium in each test vessel (reactor) was measured before the test/reference materials were added and then again at the end of the 28 day test period.
- Aeration of dilution water: The mixture was continuously stirred in a closed flask.
- Other: Magnetic stirring was provided for all test mixtures throughout the test period.

TEST SYSTEM
- Culturing apparatus: Reactor flasks, controlled by eight-channel respirometer reactor modules were used. Each reactor flask is equipped with an electrolytic cell assembly which generates oxygen to replace the amount consumed by the test mixture in the flask.
- Number of culture flasks/concentration: 2
- Measuring equipment: The consumption of oxygen was monitored using an electrolytic respirometer system. A BI-1000 electrolytic respirometer system was used; the oxygen uptake in all flasks was monitored continuously and recorded automatically every 4 hours throughout the test period using the BI Data Acquisition Software.

CONTROL AND BLANK SYSTEM
- Inoculum blank: The composite culture was used as the inoculum for procedure check and inoculum blank.
- Abiotic sterile control: No
- Toxicity control: No

STATISTICAL METHODS: No statistical analysis was performed
Reference substance:
benzoic acid, sodium salt
Key result
Value:
18.3
Sampling time:
28 d
Remarks on result:
other: The test material has been determined not to be readily biodegradable.
Details on results:
The test material was observed to biodegrade 18.3 % over the 28 day test period (average of two values, 11.0 and 25.5 %).


The BOD values of the test material are the values after the correction for background from the inoculum blank. The percent biodegradation level was the ratio of BOD and ThOD. The biodegradation rates of the duplicates agreed within 20 %.
The average oxygen uptake value of the two inoculum blanks was 30.7 mg and under the specified criteria of <60 mg.
The pH values were within the overall acceptable range of 6.0 to 8.5, indicating that the levels of biodegradation were not limited by extreme pHs.
All of the test validity criteria were met.
Results with reference substance:
The reference substance, sodium benzoate, reached a level of 94.3 % in the test period. Sodium benzoate degraded readily, reaching the pass level of 60 % within 3 days.

Table 1: Theoretical Oxygen Demand (ThOD)

Test Vessel

Substance

Specific ThOD (mg/mg)

Sample weight (mg)

ThOD
(mg)

BI 1 - 1

Sodium benzoate

1.825

105.1

191.8

BI 1 - 4

Test material

1.139

110.2

125.5

BI 2 - 1

Sodium benzoate

1.825

107.4

196.0

BI 2 - 4

Test material

1.139

107.2

122.1

Table 2: Accumulated Oxygen Demands and Final Biodegradation Levels (Day 28)

Test Vessel

Substance

Obs. Oxygen uptake
(mg)

BOD
(mg)

ThOD
(mg)

Biodeg.
(%)

BI 1 - 1

Sodium benzoate

209.2

173.0

191.8

90.2

BI 1 - 4

Test material

50.0

13.8

125.5

11.0

BI 2 - 1

Sodium benzoate

217.7

192.6

196.0

98.3

BI 2 - 4

Test material

56.2

31.1

122.1

25.5

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the conditions of the study, the test material has been determined not to be readily biodegradable.
Executive summary:

As part of an investigation to determine the ready biodegradability of a number of substances, the ready biodegradability of the test material was investigated in a respirometry test that followed the same principle as that in the standardised guideline OECD 301 F, except that it did not include the abiotic and toxicity checks and pre-adapted inoculum was employed. The study was conducted under GLP conditions.

The inoculum was adapted to the test material for 14 days before test initiation and the degradability was expressed as the ratio of the measured Biological Oxygen Demand (BOD) and the Theoretical Oxygen Demand (ThOD; calculated from the elemental analysis of the substance).

The initial test material concentration was 100 mg/L and the temperature throughout the 28 day test was 23 ± 1 °C. Oxygen consumption was measured with a BI-1000 electrolytic respirometer system; the oxygen uptake in all flasks were monitored continuously and recorded automatically every 4 hours throughout the test period. Sodium benzoate was employed as reference material.

The level of biodegradation at the end of the 28 day test period for the test material was 18.3 % (average of two replicates). The reference substance reached a biodegradation level of 94.3 % in the same test period.

Under the conditions of the study, the test material has been determined not to be readily biodegradable.

Description of key information

The test material has been determined not to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

As part of an investigation to determine the ready biodegradability of a number of substances, the ready biodegradability of the test material was investigated in a respirometry test that followed the same principle as that in the standardised guideline OECD 301 F, except that it did not include the abiotic and toxicity checks and pre-adapted inoculum was employed. The study was conducted under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality as set forth by Klimisch et al. (1997).

The inoculum was adapted to the test material for 14 days before test initiation and the degradability was expressed as the ratio of the measured Biological Oxygen Demand (BOD) and the Theoretical Oxygen Demand (ThOD; calculated from the elemental analysis of the substance).

The initial test material concentration was 100 mg/L and the temperature throughout the 28 day test was 23 ± 1 °C. Oxygen consumption was measured with a BI-1000 electrolytic respirometer system; the oxygen uptake in all flasks were monitored continuously and recorded automatically every 4 hours throughout the test period. Sodium benzoate was employed as reference material.

The level of biodegradation at the end of the 28 day test period for the test material was 18.3 % (average of two replicates). The reference substance reached a biodegradation level of 94.3 % in the same test period.

Under the conditions of the study, the test material has been determined not to be readily biodegradable.