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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:

4 May 2017 - 26 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

SOURCE:
The source of the test organisms was activated sludge freshly obtained from a municipal sewage treatment plant, receiving predominantly domestic sewage. / `s-Hertogenbosch, The Netherlands
PREPARATION OF INOCULUM FOR EXPOSURE:
The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.9 g/L in the concentrated sludge.
Before use, the sludge was allowed to settle (37 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium

Duration of test (contact time):

>= 28 d

Initial conc.:

>= 19 mg/L

Based on:

test mat.

Initial conc.:

>= 12 mg/L

Based on:

other:

Remarks:

TOC/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Test vessels: 2 liter brown coloured glass bottles

- Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

- Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre,
pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water
and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and
made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and
made up to 1 litre.

- Mineral medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.

- Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

- Illumination: The test media were excluded from light

- Test temperature: from 22.2 to 23.3°C

- pH: Start of the test - 7.6 (adjusted with HCl from 7.8); End of the test - 7.5-7.8

- Aeration of the mixture (mineral components, Milli-RO water, inoculum): Yes, with synthetic air, overnight before the start of the test

- Suspended solids concentration: 3.9 g/L

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration:
The study consisted of six bottles:
- 2 inoculum blanks (no test substance),
- 2 test bottles with the test substance, at a test concentration 19 mg/L,
- 1 positive control (sodium acetate) and
- 1 toxicity control (Crosslinker CX-100 plus sodium acetate).
- Test performed in open system: Yes
- Details of trap for CO2: CO2 was trapped in barium hydroxide solution.
- Method used to create aerobic conditions / Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

SAMPLING
- Sampling frequency
Day: 1, 4, 6, 8, 11, 15, 18, 22, 25, 29 (3 times) for the test substance and the blank.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes
- Other: Positive control

- Details of trap for CO2 and volatile organics if used: Barium hydroxide 0.0125 M Ba(OH)2

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 1.1

Sampling time:

29 d

Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B^1)
1	0	0	0	0
4	1	1	1	0
6	2	2	2	0
8	2	4	3	2
11	2	5	4	3
15	2	7	5	5
18	2	7	5	5
22	2	8	5	6
25	2	9	6	7
29^2)	2	10	6	8
29^2)	2	11	7	9
29^2)	2	12	7	10
^1): Absolute difference in biodegradation between bottles A and B ^2): Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance was not readily biodegradable under the conditions of the modified Sturm test performed

Executive summary:

The ready biodegradability of the test substance was assessed using the carbon dioxide (CO2) evolution test according to OECD guideline 301B.

The test item was tested in duplicate at a concentration of 19 mg/L, corresponding to 12 mg TOC/L, during 28 days. Based on the obtained results, the test substance was classified as not readily biodegradable.

The present study is classified as reliable without restrictions since all acceptability criteria stated in the guideline were met.

Description of key information

The objective was the evaluation of the biodegradability in water of the test substance.

The ready biodegradability of the test substance was assessed using the carbon dioxide (CO2) evolution test according to OECD guideline 301B.

Based on the obtained results, the test substance was classified as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test substance was assessed using the carbon dioxide (CO2) evolution test (modified Sturm test) according to OECD guideline 301B. The source of test organism was activated sludge freshly obtained from a municipal sewage treatment plant. A single test concentration of 19 mg substance/L (corresponsing to 12 mg TOC/L) was tested during 28 days. The CO2 evolution was measured as parameter for biodegradation. Only 1.1% degradation was observed after 28 days. Based on the obtained results, the test substance was classified as not readily biodegradable.

The present study is classified as reliable without restrictions since all acceptability criteria stated in the guideline were met.