4-mesyl-2-nitrotoluene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jun 2005 - 05 Jul 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Waste water treatment plant, predominantly domestic, ARA Ergolz II, Füllinsdorf, Switzerland.
- Preparation of inoculum for exposure: Sludge was washed twice with tap water by centrifugation and decanting of supernatant liquid phase.

Duration of test (contact time):

28 d

Initial conc.:

224 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: 300 mg (± 10%) dry suldge material per litre water (water prepared as per test guidelines)
- Test temperature: 21 - 24°C
- pH: 6.7 - 7.5 (before adjustment)
- pH adjusted: Yes, by sodium hydroxide solution to 7.5 ± 0.5, prior to test start and before each sampling
- Suspended solids concentration: 300 mg dry material / L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 2000-mL Erlenmeyer flasks, loosely covered with aluminium foil
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The covering was such to allow exchange of air between the flask and the atmosphere
- Measuring equipment: TOC analyzer equipped with an autosampler (Shimadzu TOC-5000A)
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: Loose lids
- Other: test water prepared by dissolving analytical grade salt in purified water according to test guidelines. Water evapouration losses were compensated for by the addition of purified water prior to sampling.

SAMPLING
- Sampling frequency: At 0 hours, 3.0 ± 0.5 hours, 1, 3, 7, 10, 14, 21 and 28 days.
- Sampling method: Deposits on the test vessels were scraped off and resuspended in the test vessel.
- Sample storage before analysis: Samples were analysed on the day of sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes (sample poisoned with 10 mg/L mercury dichloride)
- Toxicity control: Yes
- Other: Yes, Procedure control, adsoption control

Reference substance:

diethylene glycol

Preliminary study:

Not reported

Test performance:

Not reported

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

3 h

Remarks on result:

other: Mean of test substance replicates

Parameter:

% degradation (DOC removal)

Value:

9

Sampling time:

3 d

Remarks on result:

other: Mean of test substance replicates

Parameter:

% degradation (DOC removal)

Value:

3

Sampling time:

7 d

Remarks on result:

other: Mean of test substance replicates

Parameter:

% degradation (DOC removal)

Value:

9

Sampling time:

14 d

Remarks on result:

other: Mean of test substance replicates

Parameter:

% degradation (DOC removal)

Value:

5

Sampling time:

28 d

Remarks on result:

other: Mean of test substance replicates

Details on results:

A full set of results at all sampling occasions are shown in Table 1.
The measured DOC in the test item replicates did not vary significantly from the initial mean DOC.
In the abiotic control no degradation of the test item occured
In the adsorption control no DOC-removal was observed in the first 24 hours of exposure indicating that the test item did not adsorb on activated sludge.

Results with reference substance:

In the procedure controls, containing the reference item diethylene glycol and activated sludge, the reference item was completely biodegraded within 7 days, thus confirming suitability of the activated sludge.

Table 1: Degradation Measurements

Percentage Biodegradation^1
Time (days)	Test substance			Procedure Control			Abiotic Control	Toxicity Control
	Replicate number		Mean	Replicate number		Mean
	1	2		1	2
0^2	0	0	0	0	0	0	0	0
1	4	3	3	5	5	5	-1	2
3	9	8	9	29	28	28	9	11
7	3	2	3	99	99	99	3	40
10	8	6	7	99	100	99	8	51
14	8	9	9	99	100	100	10	53
21	1	0	0	99	99	99	3	49
28	5	5	5	99	100	100	0	48

¹Corrected for the innoculum control (except for the abiotic control)

²0 days = 3 ± 0.5 hours after the addition of the test substance

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material was found not to biodegrade under the conditions of the test.

Executive summary:

The inherent biodegradability of the substance was investigated using a Zahn-Wellens/EMPA test over 28-days. The mean concentration of DOC was constant during the exposure period indicating that the test substance is not biodegradable under the test conditions. In the toxicity control the DOC decreased by 53% within 14 days of incubation, thus the test material is concluded to be not inhibitory to activated sludge at the test concentration of 224 mg/L.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Apr 2007 to 17 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods for the Examination of Waters and Associated Materials (1995). The Instrumental Determination of Total Organic Carbon and Related Determinants. HMSO, London

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge obtained form Buckland Sewage Treatment works, Newton Abbot, Devon, UK treating sewage of predominantly domestic origin.
- Storage conditions: Sludge kept aerated at approximately 20°C and pH 7.
- Storage length: Sludge obtained 12 April 1999, test start 19 April 1999.
- Preparation of inoculum for exposure: Five days prior to test sludge centrifuged, washed and resuspended in test medium, then diluted in medium, added to test bottles and stirred until use.
- Concentration of sludge: Concentration equivalent to 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: Test water was prepared according to guidelines, with nutrients dissolved in deionised water to obtain stock solutions.
- Test temperature: 22 ± 2°C.
- pH: 7.07 - 7.27
- Continuous darkness: Yes, test flasks were incubated in a dark room

TEST SYSTEM
- Culturing apparatus: 500 mL glass bottle with magnetic stirrer.
- Number of culture flasks/concentration: 6 replicates
- Measuring equipment: Hach Manometric biochemical oxygen demand apparatus. At end of test DNPOC analysed (Methods for the Examination of Waters and Associated Meterials (1995). The Instrumental Determination of Total Organic Carbon and Related Determinands. HMSO, London)
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Hach Manometric biochemical oxygen demand apparatus adsorbs CO2.

SAMPLING
- Sampling frequency: Days 5, 10, 15, 20 and 28.
- Sampling method: One sample of about 10 mL was taken from each test flask per sampling date.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes - poisoned with HgCl2
- Toxicity control: No
- Other: Procedure control used (Sodium acetate).

STATISTICAL METHODS:
Percentage degradation = (BOD/COD) * 100%

Reference substance:

acetic acid, sodium salt

Test performance:

No unusual observations or other information that may have affected results.

Parameter:

% degradation (CO2 evolution)

Value:

< 5

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

< 5

Sampling time:

28 d

Details on results:

See Table 1.

No degradation of the test item was observed in the poisoned samples.

Parameter:

BOD5*100/COD

Value:

< 5 other: %

Parameter:

BOD5

Value:

< 0.05 g O2/g test mat.

Parameter:

COD

Value:

1.3 g O2/g test mat.

Results with reference substance:

In the procedure controls, containing the reference item sodium acetate and activated sludge, sodium acetate was 64% biodegraded within 10 days, thus confirming suitability of the activated sludge.

Table 1. Biodegradation of the test substance and the reference item in a over 28 days. Values corrected for the inoculum controls.

	Percentage Biodegradation^1
Time	Test item	Procedure control
(days)
5	<5	61%
10	<5	64%
15	<5	64%
20	<5	66%
28	<5	66%

¹Mean values of 6 per sample (5 for 28 day procedure control

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material was found not to be biodegradable under the conditions of the test.

Executive summary:

The ready biodegradability of the substance was determined in an OECD 301F test by observing the evolution of CO2 over 28 days. An inoculum control, procedure control (sodium acetate) and a sterile control (samples poisoned with HgCl2) were also incubated for 28 days. Aerobic activated sludge was used as the inoculum. The test material was found not to be biodegradable under the conditions of the test within 28 days.

Description of key information

Not readily biodegradable, Manometric Respirometry, OECD 301F, Magor 1999
Not inherently biodegradable, Zahn-Wellens, OECD 302B, Bätscher 2005

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The ready biodegradability of the substance was determined in an OECD 301F test by observing the evolution of CO2 over 28 days. An inoculum control, procedure control (sodium acetate) and a sterile control (samples poisoned with HgCl2) were also incubated for 28 days. Aerobic activated sludge was used as the inoculum. The test material was found not to be biodegradable under the conditions of the test within 28 days.

The inherent biodegradability of the substance was investigated using a Zahn-Wellens/EMPA test over 28-days. The mean concentration of DOC was constant during the exposure period indicating that it is not biodegradable under the test conditions. In the toxicity control the DOC decreased by 53% within 14 days of incubation, thus the substance is concluded to be not inhibitory to activated sludge at the test concentration of 224 mg/L.

Both guideline studies are considered to be relevant, reliable and adequate for risk assessment, classification and labelling. Neither showed significant biodegradability under the conditions of the tests, and this is the result carried forward for risk assessment purposes.