Phenolphthalein

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:

2010-02-16 to 2010-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge from a domestic waste water treatment plant

- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. The sediment was re-suspended in test water and aerated for two days. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

104 mg/L

Based on:

test mat.

Initial conc.:

103 mg/L

Based on:

ThOD

Remarks:

NH4

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water
- Test temperature: 22 ± 1 °C
- pH: 7.5 (measured at the start of the test), 7.1 - 7.5 (measured at the end of the test)
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks, volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45%)

SAMPLING
- Sampling frequency: every day of the exposure period

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Abiotic sterile control: 1 replicate
- Toxicity control: 1 replicate

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Results with reference substance:

The reference item was sufficiently degraded to 88% after 14 days and to 96% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Validity Criteria

Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 20 mg O₂/L and thus, not greater than 60 mg O₂/L within 28 days as required by the test guideline.

pH-Value: The pH-value of the test item flasks at the end of the test was within the range of pH 7.4 to 7.5 as required by the test guideline.

Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.

Test Item: The difference of duplicate values for the degradation of the test item at the plateau and at the end of the test were than 20%. The validity criterion was fulfilled.

Biodegradation of test Item

The mean percentage biodegradation at the end of the 28-day exposure period was 76% (ThOD_NH4). The degradation rate of the test item did not reach 60% within the 10-day window but after 28 days of incubation. Therefore, the test item is considered to be readily biodegradable, but failing 10 day window.

Biodegradation of Reference Item

The reference item was sufficiently degraded to 88% after 14 days and to 96% after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Biodegradation in the Toxicity Control: In the toxicity control containing both the test item and reference 37% biodegradation was noted within 14 days and 64% biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Oxygen Demand in Abiotic Control

The oxygen demand in the abiotic control was from 0 to 5 mg/L. No correction of the test item degradation rates had to be done, because the correction will not affect the outcome of the study.

Table 1: Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThOD_NH4

Time (Days)	Percentage Biodégradation ^1
	Test Item^1		Sodium Benzoate ^2	Toxicity Control ^1, 2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	0	0	29	14
2	0	2	44	19
3	0	0	47	24
4	0	0	61	27
5	2	2	64	28
6	0	0	64	28
7	0	2	67	29
8	2	2	70	30
9	2	2	73	30
10	4	4	79	33
11	6	6	83	34
12	7	7	85	34
13	9	11	88	35
14	11	13	88	37
15	13	16	90	38
16	15	18	90	38
17	20	22	93	40
18	24	27	93	44
19	29	29	96	47
20	35	36	96	51
21	40	40	96	53
22	46	45	96	56
23	51	49	96	57
24	55	54	96	59
25	60	58	96	61
26	64	65	96	63
27	68	72	96	63
28	75	76	96	64

¹ ThOD_NH4 of test item: 2.161 mg/mg

² ThOD_NH4 of sodium benzoate: 1.666 mg/mg

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test item was found to be readily biodegradable, but failing 10-day window.

Executive summary:

The ready Biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 104 mg/L in duplicate, corresponding to an oxygen demand of about 225 mg/L (ThOD_NH4) and the reference item was tested at a concentration of 103 mg/L corresponding to an oxygen demand of about 171 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. Since the test item contains no nitrogen, the evaluation of biodegradation has to be based ThOD_NH4. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 88% after 14 days and to 96% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 37% biodegradation was noted within 14 days and 64% biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms. The mean biodegradation after 28 days of the test item was 76% (ThOD_NH4); the 10 day window criterion was not passed. Therefore, the test item is considered to be readily biodegradable, but failing 10 day window.

Description of key information

The test item was found to be readily biodegradable, but failing 10-day window (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

The ready Biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 104 mg/L in duplicate, corresponding to an oxygen demand of about 225 mg/L (ThOD_NH4) and the reference item was tested at a concentration of 103 mg/L corresponding to an oxygen demand of about 171 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. Since the test item contains no nitrogen, the evaluation of biodegradation has to be based ThOD_NH4. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 88 % after 14 days and to 96 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 37 % biodegradation was noted within 14 days and 64 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms. The mean biodegradation after 28 days of the test item was 76 % (ThOD_NH4); the 10 day window criterion was not passed. Therefore, the test item is considered to be readily biodegradable, but failing 10 day window.