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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:
from 15 October 2002 to 22 November 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DV6850
- Substance type: powder
- Physical state: solid
- Analytical purity: 83.7%
- Lot/batch No.: R0332-52C
- Expiration date of the lot/batch: 2003/09/30
- Storage condition of test material: ca 4°C in the dark in dry conditions

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Oakley sewage treatment works, which treats predominantly domestic waste water
- Preparation of inoculum for exposure: Aliquots (25 mL) of a homogenised sample were filtered through dried (approximately 105 °C) and pre-weighed Whatman GFC filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Pretreatment: None
- Concentration of sludge: final suspended solids concentration 30 mg/L
- Initial cell/biomass concentration: no data
- Water filtered: no data
- Type and size of filter used, if any: no data
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
16.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
The mineral salts medium (MSM) for the final test was prepared by mixing 10 mL of stock solution 1 with 800 mL dilution water and then adding 1 mL of solutions 2 to 4 to each litre of water required for the test. The pH of each culture was determined in situ and adjusted to 7.4 ± 0.2 with 5N HCl.

Stock 1
Potassium dihydrogen phosphate: 8.50 g/L
di-Potassium hydrogen phosphate: 21.75 g/L
di-Sodium monohydrogen phosphate dihydrate: 33.40 g/L
Ammonium chloride: 0.50 g/L

Stock 2
Magnesium sulphate heptahydrate: 22.50 g/L

Stock 3
Calcium chloride dihydrate: 36.40 g/L

Stock 4
Iron {III) chloride hexahydrate: 0.25 g/L

- Additional substrate: not applied
- Test temperature: 20-24 °C
- pH: The pH of all test and control mixtures was 7.6 at the start of the test and ranged from 7.5 to 7.7 at the end.
- pH adjusted: the pH was adjusted to 7.4±0.2 at start of test with 5N HCl
- Suspended solids concentration: 30 mg solids/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: five-litre clear glass culture bottles that had been treated with a silylising agent (dimethyldichlorosilane) and wrapped in black polythene in order to exclude light and stoppered.
- Number of culture flasks/concentration:
1 flask with reference (inoculated mineral salts medium plus sodium benzoate (10 mgC/L))
2 flasks with test substance (10 mgC/L) plus inoculated mineral salts medium
- Method used to create aerobic conditions: The flasks were placed on electrically-operated magnetic stirrers and aerated overnight with a supply of air that had been treated to remove carbon dioxide by passing it through cylinders containing fused calcium chloride, Carbosorb AS and a trap containing 1M sodium hydroxide solution.
- Aeration: Air flow to all cultures ceased for approximately thirty minutes on Day 3 and ten minutes on Day 4 of the test. This short period of air failure is not thought to be significant nor to have affected the integrity of the study.
- Test performed in closed vessels: yes


SAMPLING
- Sampling frequency: samples were taken on day 2, 4, 5, 6, 7, 8, 11, 12, 13, 15, 21, 25, 28 and 29
- Sampling method: The vessels contents were continuously flushed for 29 days with treated air. The air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (100 mL). The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphthalein indicator. Following the removal of the first Dreschel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 flask with controls (mineral salts medium plus inoculum (30 mg solids/L))
- Toxicity control: 1 flask with sodium benzoate (10 mgC/L) plus test substance (10 mgC/L) plus inoculated mineral salts medium
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
4 d
Remarks on result:
other: DV6850
Key result
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
13 d
Remarks on result:
other: DV6850
Key result
Parameter:
% degradation (CO2 evolution)
Value:
77
Sampling time:
29 d
Remarks on result:
other: DV6850
Details on results:
Mean cumulative CO2 production by mixtures containing DV6850 was equivalent to approximately 10% of the theoretical value (TCO2, 110.1 mgCO2) after 4 days of incubation, 62% after 13 days and 77% by the end of the test on Day 29. A biodegradation plateau was not considered to have been achieved by the end of the test.
Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. DV6850 can, therefore, be considered to be readily biodegradable,

BOD5 / COD results

Results with reference substance:
Sodium benzoate had been biodegraded by 64% after 6 days and 78% after 29 days in the absence of DV6850, and by 61% after 6 days in its presence which confirmed that DV6850 was not inhibitory to the activity of the microbial inoculum.

Any other information on results incl. tables

Cumulative levels of CO2 production in the controls after 29 days (78.7 and 82.5 mg CO2) were within the acceptable range for this assay system (recommended maximum = 120 mg CO2 for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
DV6850 is readily biodegradable.
Executive summary:

The ready biodegradability of the test item DV685D was assessed in a valid GLP study according to the CO2 Evolution Test (Modified Sturm Test), Procedure C.4-C of the Annex to Directive 92/69/EEC, OECD Procedure 301B and US Environmental Protection Agency (EPA), Office of Prevention, Pesticides and Toxic Substances, (OPPTS) Method 835.3110 (m) (adopted January 1998).

A preliminary investigation to determine the carbon content of DV6850 was conducted using elemental (C,H,N) analysis. The measured carbon content (61.5%) was used to determine the weight of DV6850 required to achieve nominal test concentrations of 10 mg as Carbon/litre in culture vessels in the main biodegradability test.

DV6850 (nominally 48.9 mg) was added to two five-litre vessels containing three litres of mineral salts medium inoculated with activated sludge (30 mg solids/L). Two control vessels contained inoculated mineral salts medium alone and one contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mgC/L). An additional mixture containing sodium benzoate (10 mgC/L) and DV6850 (10 mgC/L) was established in order to assess the potential inhibitory effects of the test substance on the activity of the microbial inoculum.

Test, control and reference mixtures were aerated for 29 days with air that had been treated to remove carbon dioxide (CO2). The CO2, produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration.

The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. The pH of the test plus reference mixture was measured at the start of the test and on the day of its termination (Day 6).

Sodium benzoate had been biodegraded by 64% after 6 days and 78% after 29 days in the absence of DV6850, and by 61% after 6 days in its presence which confirmed that DV6850 was not inhibitory to the activity of the microbial inoculum. Cumulative levels of CO2 production in the controls after 29 days (78.7 and 82.5 mg CO2) were within the acceptable range for this assay system (recommended maximum = 120 mg CO2 for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.

Mean cumulative CO2 production by mixtures containing DV6850 was equivalent to approximately 10% of the theoretical value (TCO2, 110.1 mgCO2) after 4 days of incubation, 62% after 13 days and 77% by the end of the test on Day 29. A biodegradation plateau was not considered to have been achieved by the end of the test.

Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. As 62% degradation was achieved at the end of the 10 -day window, DV6850 can be considered to be readily biodegradable.