Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

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:

10 Oct 1989 to 20 Dec 1989

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Prior to use the activated sludge from the sewage plant (Frankfurt/M. -Niederrad) was washed twice with tap water to eliminate organic components and poisons from the sludge. After-resuspension the sludge was aerated or 4 hours. It was then homogenized in a waring blender at medium speed for 2 minutes and then settled for about 30 minutes. The supernatant was filtered through a coarse filter paper the first 200 mL being discarded. The filtrate was used as inoculum (1% of the final volume of the test solution).

Duration of test (contact time):

43 d

Initial conc.:

10 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Preparation of the test solutions: A stock solution with the test material could not be prepared in aqua bidest as indicated in the guideline. Also with special-measures such as ultrasonic treatment it was impossible to get a stable emulsion of the test material. For this reason 35.0 mg and 70.0 mg of the test material were weighed on small glass plates previously carefully cleaned by treatment with cromium sulphuric acid and aqua bidest. and dried at 104 °C. The glass plates were given into the test solutions at the starting point of the test. At the same time one Blank without any test substance but with inoculation and one reference (Na-Benzoate) with 20 mg C per litre were run in parallel. Before starting the test the mineral salt solutions with inoculation but without test substance were aerated with CO2- free air for 24 h in order to purge the system of CO2. Within this study the solutions with the test material were stirred by means of a magnetic stirrer in order to distribute the test substance in a way that it was dissolved at the maximal solubility.
- Composition of test solutions: The test solutions contained in a total volume of 3500 mL: 35 mL of the inoculum, 14 mL of the ferrichloride solution, 3.5 mL of the magnesium sulphate solution, 3.5 mL of the calcium chloride solution, 7 mL of the mixture solution and 3.5 mL of the ammonium sulphate solution according to the indicated guideline.
- Test temperature: 21.0 - 22.5 ˚C
- Aeration of dilution water: Yes. The aeration rate of the test system was controled at 4 L/h being assured by precise flowmeters.

TEST SYSTEM
- Details of trap for CO2 and volatile organics: CO2 generated by the test substance was trapped by 0.025 N Ba(OH)2 in a trap system described in the Guideline. Resting Ba(OH)2 was titrated with 0.05 N HCI. Each millilitre of HCl titrated corresponds to 1.1 mg of CO2 produced. Degradation was calculated as a percentage of the theoretical CO2 that should have been produced from the organic matter of the substance by complete combustion. The CO2 being generated was calculated to the nearest 0.01 mg and biodegradation values were rounded up to the nearest 0.1 percent. An additional plastic bottle with 700 mL 10N NaoH was introduced to the system for quantitative elimination of CO2 from the compressed air. As 16 test solutions were run in parallel the first NaOH-bottle was renewed each week, and the first two bottles at t29d in order to assure quantitative elimination of CO2 from the compressed air.

SAMPLING
- Sampling frequency: Determinations of CO2 were performed after following intervals: t4d, t8d, t14d, t17d, t22d, t27d, t30d (Blank only), t31d, t34d*, t36d, t38d, t41d*, and 43d.
*At t34d 1 mL conc. HCI was given into the test solution with 10 mg/L and at t41d 1mL conc. HCl was given into the test solution with 20 mg/L of the test substance in order to make the dissolved CO2 or inorganic carbon volatile and to purge the system of CO2. Final titration was made at t36d and at t43d respectively.

Reference substance:

other: Na-Benzoate

Key result

Parameter:

% degradation (CO2 evolution)

Value:

10.8

Sampling time:

14 d

Remarks on result:

other: test solution of 10 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

63.5

Sampling time:

31 d

Remarks on result:

other: test solution of 10 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

7.6

Sampling time:

31 d

Remarks on result:

other: test solution of 20 mg/L

Details on results:

An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.
After 4 days incubation, 1.3% of the tests substance was degraded in the 10 mg/L test solution group. The degradation increased to 10.8% after 14 days incubation. After that, 58.3, 63.5 and 78.3% biodegradation were found on day 27, day 31 and day 36. In the 20 mg/L test solution group, only 7.6% of the test substance was degraded after 31 days incubation.
The results suggested that the test substance was degraded at slower rates at 20 mg/L than it does at 10 mg/L. This behaviour may be due to many reasons. One reason for this behaviour may be the low water solubility of the test material though both test solutions were stirred by means of a magnetic stirrer in order to get a constant and water saturated solution of the test substance. It seems that the test substance is degraded in two steps being visible by two lag phases. This behaviour was visible in both test solutions.
From the data obtained in the test the test substance may be classified as "ultimately biodegradable" (regarding the 10 mg/L test solution) according to the definitions given by the OECD guidelines for testing "inherent biodegradability". Though only 24% biodegradation was found in the 20 mg/L test solution this classification may be right as a plateau of the biodegradation was not reached at t41d: biodegradation in this test solution seems to have been very slow but constant.

Results with reference substance:

The positive control Na-Benzoate was stopped after 141 h (~ 6d) because a degradation value of > 80% had been reached indicating that the inoculum used for the test was active enough to achieve the required conditions of the guideline. As about 10 to 15 % of the benzoate is normally used for generation of bacteria (results by own experience) a result of > 85% “ThCO2” in this test indicates a nearly complete degradation of the test material.

Table 1. CO2 results

ThCO2*: 2.483 mg CO2/ mg test substance

Theoretical amount of CO2 being generated if all test material is transformed to CO2:

10 mg/L - 86.905 mg CO2/ 3500 mL test solution

20 mg/L- 173.81 mg CO2/ 3500 mL test solution

Volume of the test solution: 3500 mL 

Time (td)	Test solution with 10 mg/L		Test solution with 20 mg/L
	mg CO2 generated in the test solution, cumulative	%TCO2** (=% biodegradation)	mg CO2 generated in the test solution, cumulative	%TCO2 (=% biodegradation)
4	0	0	0	0
8	1.17	1.3	1.17	0.7
14	9.36	10.8	4.02	2.3
17	22.35	25.7	6.28	3.6
22	39.85	45.9	9.64	5.5
27	50.68	58.3	13.03	7.5
31	55.18	63.5	13.2	7.6
34	62.17	71.5	20.52	11.8
36	68.03***	78.3	----	----
38	----	----	33.74	19.4
41	----	----	38.49	22.1
43	----	----	42.14***	24.1

*ThCO2:Theoretical amount of CO2 that can be generated by the test material

**TCO2: Percentage of total CO2 the test substance has generated in relation to

the ThCO2

*** The difference between this and the titration before is due to CO2 purged from the test solution after acidification.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Based on the findings, the test substance is "ultimately biodegradable" but not readily biodegradable under the conditions of the test.

Executive summary:

The ready biodegradability of the test item was investigated using aerobic activated sludge incubated at 25 °C for 14 days under continuous stirring. The test was conducted in accordance with OECD TG 301 B, and in compliance with GLP criteria. The tested sludge was sampled from the sewage plant (Frankfurt/M. -Niederrad) and was washed twice with tap water to eliminate organic components and poisons from the sludge. After-resuspension the sludge was aerated for 4 hours. It was then homogenized in a waring blender at medium speed for 2 minutes and then settled for about 30 minutes. The supernatant was filtered through a coarse filter paper the first 200 mL being discarded. The filtrate was used as inoculum (1% of the final volume of the test solution). The tested sludge was exposed to 10 mg/L, 20 mg/L of the test substance or reference substance, Na-Benzoate and was incubated at 21.0 to 22.5˚C for 43 days. The released CO2 from the test materials was trapped and analysed for indicating the biodegradation level.

This reference substance treated group was stopped after 141 h (~ 6d) because a degradation value of > 80% had been reached indicating that the inoculum used for the test was active enough to achieve the required conditions of the guideline. The test substance was degraded at slower rates at 20 mg/L than at 10 mg/L. After 4 days incubation, 1.3% of the tests substance was degraded in the 10 mg/L test solution group. The degradation increased to 10.8% after 14 days incubation. After that, 58.3, 63.5 and 78.3% biodegradation were found on day 27, day 31 and day 36. In the 20 mg/L test solution group, only 7.6% of the test substance was degraded after 31 days incubation.

Therefore, it was concluded that the test substance is "ultimately biodegradable" but not readily biodegradable under the conditions of the test.

Description of key information

Based on the findings, the test substance is "ultimately biodegradable" but not readily biodegradable under the conditions of the test.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was investigated using aerobic activated sludge incubated at 25 °C for 14 days under continuous stirring. The test was conducted in accordance with OECD TG 301 B, and in compliance with GLP criteria. The tested sludge was sampled from the sewage plant (Frankfurt/M. -Niederrad) and was washed twice with tap water to eliminate organic components and poisons from the sludge. After-resuspension the sludge was aerated or 4 hours. It was then homogenized in a waring blender at medium speed for 2 minutes and then settled for about 30 minutes. The supernatant was filtered through a coarse filter paper the first 200 mL being discarded. The filtrate was used as inoculum (1% of the final volume of the test solution). The tested sludge was exposed to 10 mg/L, 20 mg/L of the test substance or reference substance, Na-Benzoate and was incubated at 21.0 to 22.5˚C for 43 days. The released CO2 from the test materials was trapped and analysed for indicating the biodegradation level.

This reference substance treated group was stopped after 141 h (~ 6d) because a degradation value of > 80% had been reached indicating that the inoculum used for the test was active enough to achieve the required conditions of the guideline. The test substance was degraded at slower rates at 20 mg/L than it does at 10 mg/L. After 4 days incubation, 1.3% of the tests substance was degraded in the 10 mg/L test solution group. The degradation increased to 10.8% after 14 days incubation. After that, 58.3, 63.5 and 78.3% biodegradation were found on day 27, day 31 and day 36. In the 20 mg/L test solution group, only 7.6% of the test substance was degraded after 31 days incubation.

Therefore, it was concluded that the test substance is "ultimately biodegradable" but not readily biodegradable under the conditions of the test.