Benzoic acid, 5-amino-2-chloro-, 1,1-dimethyl-2-oxo-2-(2-propen-1-yloxy)ethyl 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

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-(5-amino-2-chlorobenzoyloxy)-2-methylpropanoate

EC Number:

696-318-8

Cas Number:

174489-43-1

Molecular formula:

C14H16ClNO4

IUPAC Name:

prop-2-en-1-yl 2-(5-amino-2-chlorobenzoyloxy)-2-methylpropanoate

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

The inoculum concentration in the test bottle was 25.3 mg suspended solids/L.

Duration of test (contact time):

ca. 29 d

Initial test substance concentration

Initial conc.:

ca. 56.48 other: %

Based on:

other: Organic carbon Content

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

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.

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 item, 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: 39.8 mg test item/1.5L = 15.0 mg ThOC/L
Replicate 2: 39.9 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 15.0 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 5 minutes 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 3, 6, 8, 10, 13, 16, 20, 24, 28 and 29 days.

The last measurement was performed after acidification of the test medium with 1ml of concentrated hydrochloric acid.

ANALYTICAL DEVICE:
At each sampling day the CO2 trapped in the scrubbers was measured as inorganic carbon with a carbon analyser (Shimadzu TOC-5050 A).

CALCULATIONS/STATISTICAL ANALYSIS:
The biodegradation was calculated on the basis of the theoretical carbon content of the test item and the quantities of inorganic carbon determined on the days of measurements in the absorbers

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

ca. 1

Sampling time:

29 d

Details on results:

The percentage biodegradation, based on CO2 evolution versus time for CA 2219 A and the reference substance (± CA2219 A) – Refer to Tables 1 and 2.
The theoretical calculated amount of CO2 in the test flasks with the test item alone were 82.4 and 82.4 mg/1.5L, equivalent to 22.5 and 22.5 mg TOC/1.5L. The actual found amount of TOC/1.5L at the end of exposure were -0.5 mg C (C measured in the test vessels minus mean C content measured in the blanks) equivalent to 4.0 and 0 mg/1.5L of CO2 in the test flasks with the test item alone.

The high rate of CO2 evolution in the toxicity control (reference + test item), indicates that the test item did not inhibite the biodegradation of the reference substance.
The biodegradation of the test item at the end of the test was 5 and -2 %, respectively (Tables 1 and 2). Therefore the degradation of the test item was:
1 % in 29 days.
Pass level (60 %): none
Time window: none

The mineralisation of CA 221 A under these test conditions was 0% in 29 days. Based on the results of this test alone and using the classification range given in Annex VI of directive 92/69/EEC C.4-C, the ecotoxicological classification is “not readily biodegradable”.

BOD5 / COD results

Results with reference substance:

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 60 mg/1.5.
These results show that the test system was working properly and no CO2 leaks were present.

The biodegradation 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, there the test is valid.

Any other information on results incl. tables

 

Table 1: CO2 evolution and biodegradation of the controls: together blank, reference item 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	15.6	58	53
6	5.8	5.6	5.7	23.9	21.0	22.5	22.5	75	75
8	6.4	6.4	6.4	26.7	25.5	26.1	24.8	87	81
10	7.0	6.8	6.9	27.7	27.0	27.3	25.9	91	84
13	7.8	7.6	7.7	29.4	29.4	29.4	28.2	96	91
16	8.5	8.2	8.3	31.1	31.0	31.1	30.5	101	99
20	9.0	8.8	8.9	32.3	32.1	32.2	32.0	104	103
24	9.9	9.3	9.6	33.2	33.0	33.1	33.1	105	105
28	10.2	9.8	10.0	33.8	33.7	33.8	34.4	105	108
29	10.3	10.2	10.2	34.3	34.3	34.3	34.5	107	108

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

 

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

 

CO2 evolution measured as mg C			Biodegradation % of test item
Day	1	2	1	2	mean
0 3 6 8 10 13 16 20 24 28 29	0.0 3.7 5.9 6.8 7.1 7.9 8.6 9.5 10.4 10.8 11.3	0.0 3.3 6.0 6.6 6.6 7.2 8.1 7.2 8.1 9.3 9.7	0 0 1 2 1 1 1 3 4 3 5	0 -2 1 1 -2 -2 -1 -8 -7 -3 -2	0 -1 1 1 0 -1 0 -2 -1 0 1

Negative degradation values, when obtained in a test, are a consequence of the comparison of values obtained in the blank (endogenous 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:

other: not readily biodegradable

Conclusions:

The mineralisation of CA 2219 A under these test conditions was 1% in 29 days.
Based on the results of this test alone and using the classification range given in Annex VI of directive 92/69/EEC C.4-C, the ecotoxicological classification is
“not readily biodegradable”.

Executive summary:

A measured volume (1.5 L) of inoculated mineral medium containing 39.8 mg resp. 39.9 mg test item

(22.5 resp. 22.5 mg TOC/1.5 L) as the nominal sole source of organic carbon is aerated by the passage of carbon dioxide-free air at a controlled rate in diffuse light. Degradation is followed over 29 days by determining the carbon dioxide produced, which is trapped in sodium hydroxide and which is measured as inorganic carbon by a carbon analyzer. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) is expressed as a percentage of theoretical carbon dioxide. 

A reference and a reference together with the test item as toxicity control were used as controls. 

The inoculum concentration in the test bottle was 25.3 mg suspended solids/L. 

 

The pH of the sludge after collection was 7.2. 

In the test with CA 2219 A tech. the temperature was maintained constant at 21 ± 1 °C. 

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

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

1 % in 29 days

Pass level (60 %): none

Time window: none

 

Conclusion:

The mineralisation of CA 2219 A under these test conditions was 1% in 29 days. 

Based on the results of this test alone and using the classification range given in Annex VI of directive 92/69/EEC C.4-C, the ecotoxicological classification is

“not readily biodegradable”.