N-(cyclohexylthio)phthalimide

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:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(cyclohexylthio)phthalimide

EC Number:

241-774-1

EC Name:

N-(cyclohexylthio)phthalimide

Cas Number:

17796-82-6

Molecular formula:

C14H15NO2S

IUPAC Name:

N-(cyclohexylthio)phthalimide

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the wastewater treatment plant of Pardubice processing predominantly municipal sewage.

The fresh collected wastewater was filtered through paper filter. COD was determined and wastewater was aerated till using.
The mineral medium was strongly aerated for 20 min. and let standing for 20 h at the test temperature. The volume of wastewater for inoculation of mineral medium was chosen 1.6 mL per 1 L of medium ( COD of waste water: 121 mg/L)
30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 48 mL of modified wastewater. The pH value of medium: 7.4.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

3.46 mg/L

Based on:

COD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS

- Solutions and chemicals: The deionised water with conductivity less then 5 uS/cm was used for the preparation of all solutions. The same bath of water was used for each set of the experiments. Analytical grade reagents were used.

Stoc solution for the preparation of mineral medium:
solution a) KH2PO4 8.50 g, K2HPO4 21.75 g, Na2HPO4.2H2O 33.4 g, NH4Cl 0.50 g, water up to the volume of 1000 mL.
- pH:7.4
- pH adjusted: yes
solution b) MgSO4.7H2O 22.5 g in 1000 mL of water
solution c) CaCl2 27.5 g in 1000 mL of water
solution d) FeCl3.6H2O 0.25 g in 1000 mL of water
- Composition of medium: preparation of 1 L of mineral medium. 1 mL of solution a), b), c) and d) were mixed with apprex. 500 mL of water, the mixture was replenished with water up to 1000 mL. The solution was prepared from aerated water and it was left to stay one day at laboratory temperature.

- Test temperature: 20 ± 1.0°C
- Continuous darkness: yes
- Exposition time: 28 days

TEST SYSTEM
-oxygen vesel with glass stopper, volume of approx. 280 mL
-large glass bottles of 5 and 10 L volume for the preparation and inoculation of medium
-closed thermostat
-Ph meter WTW inoLab Level 2
-equipment for the determination of dillolved oxygen
-equipment for the determination of nitrite
-equipment for the determination of nitrate
-volumetric flasks, flasks, beakers, pipettes...

Series of bottles for the test:
- 2x9 bottles with test substance and inoculated medium
- 9 bottles with test substance and inoculated medium for determination of oxidized nitrogen forms
- 2x9 bottles with test substance and inoculated medium for the check of inoculum activity
- 2x5 bottles with test and reference substance mixture and inoculated medium for the toxicity test
- 2x9 bottles with inoculated medium only for the blank determination
The bottles were lined up and placed into thermosta.

SAMPLING
- Sampling frequency:0 -3rd-7th-10th-14th-17th-21st-24th-28th day
- Sampling method: appropriate bottles from each series were taken off and they were analysed for dissolved oxygen.

- Sample storage before analysis: enclosed in a full closed bottles, in the dark, at constant temperature
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: The inoculated mineral medium without test substance was used for the blank determination.
- reference substance: The stock solution of sodium benzoate was prepared in concentration 1.0000 g/L of water. From this solution the 12.6 mL (2.1 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L.
The solution prepared in this way contained 2.10 mg/L of sodium benzoate, and had COD 3.45 mg/L. The pH value of solution: 7.4.
- Toxicity control:4.7 mL of dispersion of test substance were dosen into individual bottles and completed with inoculated solution of sodium benzoate of concentration 2.10 mg/L. mixtures prepared in this way contained COD of test substance 3.46 mg/L and COD of reference substance 3.40 mg/L. The pH value of mixtures: 7.4.
- Other:

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

Measurement
The zero-time bottles were analysed immediately for dissolved oxygen by the Winkler method.
At the 3rd, 7th, 10th, 14th, 17th, 21st, 24th, 28th day of test the appropriate bottles from each series were taken off and they were analysed for dissolved oxygen.
The determination of nitrates and nitrites was caried out in parallel bottles in these time intervals by means of spectrometric methods.
Evaluation
The oxygen consumption and percentage of the biodegradation of test substance for each test interval and each bottle were calculated. In time intervals when the increase of oxidized nitrogen forms was detected, the correction of oxygen consumption for nitrification was carried out.
The evaluation of the reference substance and of the test and reference substance substance mixture was done by the same way, but withouth the correction of nitrification. All the calculations were related to the initial values of COD of the test and reference substance.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

16

Sampling time:

28 d

Any other information on results incl. tables

Determination of dissolved oxygen concentration in the main test

	Conc. C		Disolved oxygen (mg/L) after period (days)
			0	3	7	10	14	17	21	24	28
Test substance	Cz	1	8.57	8.55	8.39	8.53	8.49	8.45	8.12	8.07	7.96
		2	8.57	8.58	8.39	8.56	8.49	8.43	8.12	8.10	8.02
		average	8.57	8.57	8.39	8.55	8.49	8.44	8.12	8.09	7.99
Reference substance	Cr	1	8.55	6.72	6.47	6.59	6.33	6.25	6.07	6.02	5.92
		2	8.52	6.75	6.50	6.53	6.33	6.25	6.02	6.02	5.97
		average	8.54	6.74	6.49	6.56	6.33	6.25	6.05	6.02	5.95
Blank experiment	Co	1	8.58	8.57	8.42	8.65	8.59	8.51	8.55	8.58	8.49
		2	8.55	8.57	8.42	8.65	8.59	8.54	8.55	8.56	8.50
		average	8.57	8.57	8.42	8.65	8.59	8.53	8.55	8.57	8.50

 

Biodegradation of test and reference substance in the main test

		Biodegradation (%) after period (days)
		0	3	7	10	14	17	21	24	28
Test substance	1	0	1.7	2.0	4.6	4.0	3.5	13.6	14.5	16.8
	2	0	0.9	2.0	3.8	4.0	4.0	13.6	13.6	15.0
	average	0	1.3	2.0	4.2	4.0	3.8	13.6	14.1	15.6
Reference substance	1	0	53.0	55.9	59.1	64.9	65.5	71.3	73.3	74.2
	2	0	51.3	54.2	60.0	64.1	64.6	71.9	72.5	71.9
	average	0	52.2	55.1	59.6	64.5	65.1	71.6	72.9	73.1

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

other: not ready biodegradable