Propanoic acid, 2-hydroxy-2-methyl-, 2-propen-1-yl ester

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Only one CO2 scrubber used

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

EC Number:

700-908-3

Cas Number:

19444-21-4

Molecular formula:

C7H12O3

IUPAC Name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge collected from the sewage treatment plant of CH-4153 Reinach on August 23, 1999. The pH after collection was 7.2. The preparation was carried out according to the method described in the guideline. The concentration of the inoculum in the test was 25.3 mg sludge/L.

Duration of test (contact time):

ca. 29 d

Initial test substance concentration

Initial conc.:

ca. 58.35 other: %

Based on:

other: Organic Carbon Content

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:



VESSELS:
2 L flasks (dark brown glass) equipped with gas inlet and magnetic stirrer.
RECONSTITUTED WATER:
The reconstituted water was prepared according to the method described in the guideline.
TEMPERATURE:
20 ± 2°C
DURATION:
29 days
AIR:
≡ 25 ml/min purified from carbon dioxide.
CO2 –SCRUBBERS:
Approx. 250 ml scrubbers with gas inlet tubes equipped with sintered glass tips containing 250 ml 0.05N sodium hydroxide.
DEVIATION:
Only one CO2 scrubber was used per test bottle. Theoretically, a solution of 0.05N NaOH is sufficient to trap at least two times more CO2 than the maximum ThCO2 which can evolve in each test bottle (including CO2 possibly evolved from the bacteria e.g., endogenous respiration). Moreover, experimentally it was confirmed that no measurable CO2 carry over has ever occurred with the scrubbers used.

FLASKS:
Flasks 1 and 2: test medium and inoculum (inoculum blank)
Flask 3 and 4: reference substance and inoculum (procedure control)
Flask 5 and 6: test item and inoculum
Flask 7: reference substance, test item and inoculum (control of toxicity and inhibition of the bacteria’s activity by the tests substance)
Flask 8: dist. Water


TEST CONCENTRATION:
Test item: Replicate 1: 38.4 mg test item/1.5L = 14.9 mg ThOC/L
Replicate 2: 38.6 mg test item/1.5L = 15.0 mg ThOC/L
Reference substance: 15 mg DOC/L
For the toxicity control, where the test item and reference were applied together, the reference amount was 15.0mg DOC/L and the test item was 15.2 mg ThOC/L.
Before application, the inoculum was pre-acclimated to the test medium over-night. The aeration was performed inside 1.2L water, containing the amount of mineral salts for 1.5L.
The final volume was made up to 1.5L with 300 ml mixture made from the test item (the above mentioned amount) in bidistilled water. This mixture was homogenized by ultrasonification for 1 minute and added to the test vessel, which was immediately connected to the CO2 scrubber.

DURATION AND SAMPLING:
The test duration was 29 days. During this time the evolved CO2 trapped by the 0.05 N NaOH solution of the blank and test item was measured at 0, 3, 6, 8, 10, 13, 16, 20, 24, 28 and 29 days.
The last measurement was performed after acidification of the test medium wit 1 ml of concentrated hydrochloric acid.


ANALYTICAL DEVICE:
At each samplingday the CO2 trapped in the scrubbers was measurred as inorganic carbon with a carbon analyzer. (Shimadzu TOC-5050 A)

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

The cumulative amount of CO2 in the blank flasks were 37.8 and 37.4 mg/1.5L, equivalent to 10.3 and 10.2 mg TOC/1.5L.
The total CO2 evolution in the inoculum blank at the end of the test did not exceed 60mg/1.5L.
These results show that the test system was working properly and no CO2 leaks were present.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

ca. 78

BOD5 / COD results

Results with reference substance:

The bidegradation of the reference substance reached the level for ready biodegradablility in a 10-day window. After 10 days on incubation 91 % of the reference compound sodium benzoate, had degraded, therefore the test is valid. 

Any other information on results incl. tables

Table 1: CO2 evolution and biodegradation of the controls; together Blank, reference substance and reference + test item. 

CO2 evolution measured as mg C							Biodegradation
	Blank			Reference			Reference + test Substance	Reference	Reference + Test Substance
	1	2	mean	1	2	mean
Day	mg	mg	mg	mg	mg	mg	mg	%	% *
0	0.00	0.0	0.0	0.0	0.0	0.0	0.0	0	0
3	3.7	3.7	3.7	16.8	16.6	16.7	19.8	58	72
6	5.8	5.6	5.7	23.9	21.0	22.5	34.5	75	128
8	6.4	6.4	6.4	26.7	25.5	26.1	41.9	87	158
10	7.0	6.8	6.9	27.7	27.0	27.3	45.0	91	169
13	7.8	7.6	7.7	29.4	29.4	29.4	48.7	96	182
16	8.5	8.2	8.3	31.1	31.0	31.1	51.1	101	190
20	9.1	8.8	8.9	32.3	32.1	32.2	53.2	104	197
24	9.9	9.3	9.6	33.2	33.0	33.1	53.7	105	196
28	10.2	9.8	10.0	33.8	33.7	33.8	54.0	105	195
29	10.3	10.2	10.2	34.3	34.3	34.3	55.4	107	201

 

*The biodegradation of the ‘reference + test item’ control is calculated with the applied amount of the reference alone, ie., 15 mg DOC/L

 

 

 

 

Table 2: CO2 evolution and biodegradation of the test item.

Day	CO2 evolution Measured as mg C		Biodegradation Of test substance
	1	2	1	2	mean
0	0.0	0.0	0	0	0
3	3.4	3.5	-1	-1	-1
6	10.2	11.1	20	24	22
8	13.4	13.9	31	33	32
10	15.1	15.9	37	40	38
13	20.6	20.9	57	59	58
16	23.5	23.6	68	68	68
20	25.3	20.9	73	53	63
24	26.8	23.6	77	62	70
28	27.4	25.8	78	70	74
29	28.1	27.5	80	77	78

 

Negative degradation values, when obtained in a test, are a consequence of the comparison of values obtained in the blank (edogenous respiration leading to a carbon dioxide evolution) and the low values obtained in the presence of the test item. 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mineralisation of CA 2215 A under these test conditions was 78% in 29 days. Based on the results of this test alone and using the classification range given in Annex VI of directive 67/548/EEC, the ecotoxicological classification is “readily biodegradable”.

Executive summary:

The pH of the sludge after collection was 7.2. 

In the test with CA 2215 A tech. the temperature was maintained constant at 21 ± 1°C. 

The biodegradablilty of the reference substance after 10 days was 91%. 

The biodegradation calculated as percentage of measured amount of inorganic carbon over the theory was:

78% in 29 days

Pass level (60%): 16 days

Time window; 10 days

The mineralisation of CA 2215 A under these test conditions was 78% in 29 days. Based on the results of this test alone and using the classification range given in Annex VI of directive 67/548/EEC, the ecotoxicological classification is “readily biodegradable”.