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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-10-2003 to 19-12-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2002 ; signature: December 2002

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): mixed population of sewage sludge micro-organisms was obtained from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: See below.
- Method of cultivation: See below.
- Storage conditions: Room temperature (at 21°C).
- Storage length: Used on day of collection (< 1 week).
- Preparation of inoculum for exposure: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration.
- Pretreatment: See above.
- Concentration of sludge: 1 drop per litre (is typically around 0.05 mL, to 5 ml/L per guideline).
- Initial cell/biomass concentration: Not reported.
- Water filtered: yes (water used to prepare culture medium).
- Type and size of filter used, if any: Aerated reverse osmosis purified and deionised water (Elga Optima 15+). The 'dilution water' was kept in a temperature controlled room, at approximately 21 °C, under gentle aeration overnight prior to use.

Duration of test (contact time):

28 d

Initial conc.:

1.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a: KH2PO4 8.50 g/L; K2HPO4 21.75 g/L; Na2HPO4.12H2O 33.40 g/L ; NH4Cl 0.50 g/L ; pH = 7.4 ; Solution c: MgSO4.7H2O 22.50 g/L ; Solution b: CaCl2 22.75 g/L ; Solution d: FeCl3.6H2O 0.25 g/L. 1 litre medium contains: 1 mL of solution (A to (D) and made up to 1 litre aerated reverse osmosis purified and deionised water (Elga Optima 15+). The concentration of dissolved oxygen was measured for control purposes. Test item concentration: In duplicate at 1.5 mg/L. Procedure control: Reference substance sodium benzoate, concentration 3 mg/L; Toxicity control: concentration 1.5 mg/L sodium benzoate plus 1.5 mg/L test item.
- Solubilising agent (type and concentration if used): None
- Test temperature: The BOD bottles were incubated in a temperature controlled water bath at 20 ± 0.8°C.
- pH: No.
- pH adjusted: No.
- Aeration of dilution water: Yes.
- Suspended solids concentration: Not reported.
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: 250-300 mL darkened BOD bottles with glass stoppers
- Number of culture flasks/concentration: In duplicate. The test media were transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test medium at each sampling occasion.
- Measuring equipment: Yellow Springs oxygen meter and BOD Probe.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: Not applicable.

SAMPLING
- Sampling frequency: In duplicate; on Days 0, 3, 6, 9, 12, 14, 18, 21,24 and 28
- Sampling method: See measuring equipment and sampling frequency above.
- Sterility check if applicable: Not reported.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive control - using reference item (Sodium Benzoate) only.

Reference substance:

benzoic acid, sodium salt

Remarks:

3 mg/L

Test performance:

1. Oxygen depletion did not exceed 1.5 mgO2/L after 28 days in inoculated controls
2. Oxygen depletion in test vessels was > 4.60 mgO2/L
3. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled on day 28.
4. Sodium Benzoate attained 68% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.
5. Oxygen concentrations were > 0.5 mg/L in all bottles during the test period
6. Inhibition of the degradation of a well-degradable compound, e.g. sodium Benzoate by the test item in the Closed Bottle test was not found, the toxicity control attained in excess of 25% degradation by day 14. After 28 days the toxicity control achieved 27% degradation.

Parameter:

% degradation (O2 consumption)

Value:

15

Sampling time:

28 d

Remarks on result:

other: mean % degradation; 1.5 mg/L test item concentration

Results with reference substance:

Sodium Benzoate attained 68% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Table 1. Dissolved oxygen measurements

Test Series	Replicate	Dissolved Oxygen (mg O2/L)
		Day
		0	3	6	9	12	15	18	21	24	28
a) Culture Medium with Inoculum	R1	8.90	8.50	8.50	8.45	8.25	8.20	8.20	8.00	7.95	7.85
	R2	8.90	8.40	8.45	8.45	8.35	8.00	8.15	8.15	7.90	7.90
b) Sodium Benzoate (3 mg/L) with Inoculum	R1	8.90	5.60	5.35	5.15	4.95	5.00	4.90	4.80	4.65	4.60
	R2	8.90	5.65	4.90	4.95	4.90	4.90	4.80	4.95	4.70	4.60
c) Test Item ( 1.5 mg/L) with Inoculum	R1	8.90	8.30	7.70	7.75	7.80	7.65	7.60	7.60	7.20	7.30
	R2	8.90	8.35	7.90	7.85	7.70	7.50	7.60	7.50	7.30	7.20
d) Test Item (1.5 mg/L) plus Sodium Benzoate (1.5 mg/L) with Inoculum	R1	8.90	7.05	6.60	6.60	6.50	6.45	6.40	6.30	6.20	6.00
	R2	8.90	7.00	6.60	6.55	6.45	6.20	6.30	6.35	6.10	6.10

 

Table 2. Oxygen Depletion and Mean Percentage Biodegradation Values

Test Series		Replicate	3	6	9	12	15	18	21	24	28
a ) Culture Medium with Inoculum	Mean O2 Depletion (mg O2/L)		0.450	0.425	0.450	0.600	0.800	0.725	0.825	0.975	1.025
b) Sodium Benzoate (3 mg/L) with Inoculum	O2 Depletion (mg O2/L)	R1	2.850	3.125	3.300	3.350	3.100	3.275	3.275	3.275	3.275
		R2	2.800	3.575	3.500	3.400	3.200	3.375	3.125	3.225	3.275
	% Degradation (mean)		57	67	68	68	63	66	64	65	65
c) Test Item ( 1.5 mg/L) with Inoculum	O2 Depletion (mg O2/L)	R1	0.150	0.775	0.700	0.500	0.450	0.575	0.475	0.725	0.575
		R2	0.100	0.575	0.600	0.600	0.600	0.575	0.575	0.625	0.675
	% Degradation (mean)		3	16	15	13	12	13	12	16	15
d) Test Item (1.5 mg/L) plus Sodium Benzoate (1.5 mg/L) with Inoculum	O2 Depletion (mg O2/L)	R1	1.400	1.875	1.850	1.800	1.650	1.775	1.775	1.725	1.875
		R2	1.450	1.875	1.900	1.850	1.900	1.875	1.725	1.825	1.775
	% Degradation (mean)		21	27	28	27	26	27	26	26	27

 

Other information:

- The oxygen depletion of the inoculated control did not exceed 1.5 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at > 4.20 mg O2/L or greater in all test vessels.

- The difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.

- On Day 14 the toxicity control had attained 25% degradation. After 28 days the toxicity control had attained 27% degradation.

- The positive control or standard reference item: sodium benzoate, attained 65% degradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mean biodegradation in duplicate was 15 % at day 28.

Executive summary:

The ready biodegradability test was carried out according to OECD 301D (Ready Biodegradability: Closed Bottle Test), EU Method C4-E and EPA OPPTS 835.3110 guidelines under GLP. The test item, at a concentration of 1.5 mg/l, was exposed to sewage treatment micro-organisms with culture medium in sealed culture vessels in the dark at 22°C ± 0.8°C for 28 days. The degradation of the test item was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the standard item, sodium benzoate, together with a toxicity control were used for validation purposes. Oxygen concentration measurements were in duplicate; on Days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28. In the toxicity control, the test substance was found not to inhibit microbial activity. All of the validity criteria for the test were met. Under the conditions of this study, the test item attained 15% degradation after 28 days and therefore cannot be considered as readily biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 15% (28 -days), OECD TG 301D, closed bottle test, 2004

Key value for chemical safety assessment

Type of water:

freshwater

Additional information

Key study : OECD TG 301D, 2004 : The ready biodegradability test was carried out according to OECD 301D (Ready Biodegradability: Closed Bottle Test), EU Method C4-E and EPA OPPTS 835.3110 guidelines under GLP. The test item, at a concentration of 1.5 mg/l, was exposed to sewage treatment micro-organisms with culture medium in sealed culture vessels in the dark at 22°C ± 0.8°C for 28 days. The degradation of the test item was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the standard item, sodium benzoate, together with a toxicity control were used for validation purposes. Oxygen concentration measurements were in duplicate; on Days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28. In the toxicity control, the test substance was found not to inhibit microbial activity. All of the validity criteria for the test were met. Under the conditions of this study, the test item attained 15% degradation after 28 days and therefore cannot be considered as readily biodegradable.