4-mesyl-2-nitrotoluene

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 April 1999 to 21 May 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, (non-OECD) guideline study. No analytical monitoring, no effects observed at the highest concentration tested.

Qualifier:

according to guideline

Guideline:

other: Water Quality: Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test). ISO 10712 (1995)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal stock solution of 40 mg/L was prepared by adding 0.0100 g of the test substance to 250 mL of deionised water, stirring and sonificating until fully dissolved. Test concentrations achieved by adding appropriate volumes of stock directly into flasks.
- Eluate: Not applicable
- Controls: Two blank controls and a positive control (3,5-dichlorophenol)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Not applicable
- Laboratory culture: Freeze-dried culture from the National Collections of Industrial and Marine Bacteria Ltd, 23 St. Machar Drive Aberdeen, UK. Stored at 4°C until used.
- Method of cultivation: Freeze-dried culture rehydrated in 0.5 mL nutrient broth, streaked onto nutrient agar in a universal bottle and incubated at 25°C for 24 hours then stored at room temperature.
- Preparation of inoculum for exposure: 18-20 hours prior to test start 4 mL of growth medium added to 46 mL deionied water. Loop of stock culture added to this anth then incubated overnight at 25°C in an orbital shaker at 150 rpm. After this cells were diluted by growth medium to give optical density absorbance of 0.80 ± 0.05 at 600 nm (4 cm cells).
- Initial biomass concentration: Not reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

6 h

Hardness:

Not reported

Test temperature:

25.0 ± 0.5°C

pH:

Not reported

Dissolved oxygen:

Not reported

Nominal and measured concentrations:

Nominal concentrations of 0.40, 2.2, 4.0, 22 and 32 mg/L in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical Flasks
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 50 mL test medium
- Aeration: None
-Shaking: Continual 150 rpm.
- Type of flow-through (e.g. peristaltic or proportional diluter): Static, none used
- Renewal rate of test solution (frequency/flow rate): Static test
- No. of vessels per concentration (replicates): One
- No. of vessels per control (replicates): Three blank controls under identical conditions but without test material. Three positive control at 18 mg/L. Five test substance blanks at the test concentrations but no innoculum, one deionised water only.
- Biomass loading rate: not reported.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: After 6 hours incubation time,opictcal density at 600 nm (Uvikon 930 spectrophotometer)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No consistent factor

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

6 h

Dose descriptor:

EC50

Effect conc.:

> 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: Highest concentration tested

Duration:

6 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: Highest concentration tested

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 6 h inhibition of growth 96% (18 mg/L 3,5-dichlorophenol)

Reported statistics and error estimates:

Not applicable as no effects seen at highest test concentration

Table 1: Influence of the test substance on growth of Pseudomonas putida in a 6-hour respiration inhibition test

Test Concentration mg/L	Mean optical density	Blank corrected optical denisty	Mean % inhibition
Control	0.367	0.361	-
32	0.396	0.395	<5
22	0.455	0.455	<5
4	0.394	0.395	<5
2.2	0.421	0.425	<5
0.4	0.425	0.428	<5
Positive control	0.013	0.013	96

Validity criteria fulfilled:

yes

Conclusions:

Up to and including the highest test concentration of 32 mg/L, the test item had no significant inhibitory effect on the growth of Pseudomonas putida after the incubation period of 6 hours.

Executive summary:

Pseudomonas putida were incubated for 6 hours in the presence of different concentrations of the test substance. The nominal test concentrations were 0.40, 2.2, 4.0, 22 and 32 mg/L. Up to and including the highest test concentration of 32 mg/L, the test item had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 6 hours. The 6-hour EC50 could not be calculated but was clearly higher than 32 mg/L.

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Only a single concentration tested. Not a dedicated study to investigate microorganism toxicity.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Information on microorganism toxicity can be extracted from the toxicity-control for the biodegradability test.

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

activated sludge

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Waste water treatment plant, 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

224 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Functional inhibition

In the toxicity control, containing the test substance and diethylene glycol (each corresponding to 50% of total DOC) and activated sludge (inoculum), the initial DOC concentration of 208 mg/L measured on Day 0 rapidly decreased by 53% within the first 14 days of exposure. Thus, according to the test guidelines, the test item can be assumed not to be inhibitory to activated sludge at the tested concentration of 224mg/L because the extent of biodegradation was clearly above 35% within 14 days of incubation.

Validity criteria fulfilled:

not applicable

Conclusions:

The substance is concluded to be not inhibitory to activated sludge at the test concentration of 224 mg/L. The toxicity control concentration is considered the equivalent of a NOEC for risk assessment purposes.

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 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.

Description of key information

EC50>32 mg/L, NOEC=32 mg/L, Pseudomonas putida, 6hour, ISO 10712, Magor 1999
Not inhibitory, 224 mg/L, Zahn-Wellens toxicity control, OECD 302B, Bätscher 2005

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

224 mg/L

Additional information

Pseudomonas putida were incubated for 6 hours in the presence of different concentrations of the test material (Magor 1999). The nominal test concentrations were 0.40, 2.2, 4.0, 22 and 32 mg/L. Up to and including the highest test concentration of 32 mg/L, the test item had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 6 hours, giving a NOEC=32 mg/L. The 6-hour EC50 could not be calculated but was clearly higher than 32 mg/L, as was potentially the true NOEC. While this study is reliable, use of this NOEC for risk assessment purposes would result in an overly conservative PNEC.

Further information on microorganism toxicity can be extracted from the biodegradability toxicity-control test (See CSR Chapter 4.1.2). The inherent biodegradability of the test substance was investigated using a Zahn-Wellens test over 28-days (Bätscher 2005). In the toxicity control the DOC decreased by 53% within 14 days of incubation, thus indicating that the test substance was not inhibitory to activated sludge at the control test concentration of 224 mg/L. The toxicity control concentration is considered the equivalent of a NOEC for risk assessment purposes.

The use of the biodegradation toxicity-control test concentration as a NOEC is considered to be a reasonable approach to obtaining a realistic value for risk assessment purposes (Guidance IRCSA, Chapter R.10, R.10.4.2, May 2008). Because the biodegradation toxicity-control test is not a dedicated microorganism toxicity test, an assessment factor should be applied to derive the PNECSTP.