5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-300-1

EC Name:

-

Cas Number:

138564-59-7

Molecular formula:

C12H9N3O2S

IUPAC Name:

5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

97.7%
Item code QA405K
Lilly lot no: 356MO1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 356M01
- Purity test date: 1995-11-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature in the dark.
- Solubility and stability of the test substance in the solvent/vehicle: Soluble up to 384 ml per 500 ml of test solution
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluting a saturated aqueous solution to the desired test level with water and the concentration was obtained via HPLC.
- Final dilution of a dissolved solid, stock liquid or gel: The diluted aqueous solution was mixed with the inoculum and excess water.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
30.67, 76.68, 191.69, 479.23, and 1198.08 µg test material/L for the test material

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 30.67, 76.68, 191.69, 479.23, and 1198.08 µg test material/L
- Sampling method: Samples were taken in triplicate and then immediately assayed. Temperature was measured continuously using a continuous-monitoring temperature recorder. Additionally, dissolved oxygen measurements were made using an oxygen electrode and an accessory overflow funnel with built-in stir bar. The electrode was connected to a digital ph meter to allow for direct measurement at 10 second intervals.
- Sample storage conditions before analysis: None

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dissolving the test substance in methanol, coating the walls of the flask in the solution, and then drying to evenly deposit the test substance on the walls of the volumetric flask. Then water was added and the test substance dissolved in it to its maximum solubility.
- Controls: A solvent control was used
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 384 ml water/484 ml of test solution
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None mentioned.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Greenfield municipal sewage treatment plant that treats predominately domestic sewage.
- Storage conditions: Aerated continuously at room temperature.
- Storage length: <24 h
- Preparation of inoculum for exposure: The amount of suspended solids was measured and the solution was homogenized.
- Pretreatment: Homoginized with a Brinkman polytron for approximately 1 minute.
- Concentration of sludge: 1598 mg dry weight equivalent sludge/L in the incubation flasks.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Test conditions

Hardness:

Not specified

Test temperature:

20-24 degrees Celsius over the duration of the test.

pH:

7-8

Dissolved oxygen:

Test parameter

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal Concentrations were not specified
Measured Concetrations:
30.67, 76.68, 191.69, 479.23, and 1198.08 µg test material/L for the test concentrations
0 ug test material/L for the controls

Details on test conditions:

TEST SYSTEM
- Test vessel: Volumetric Flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, 1 L, 516 ml, 484 ml
- Aeration: Yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 3
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1598 mg dry weight equivalent sludge/L in the incubation flasks.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Conditioned drinking water that was purged with oil-free air for a minimum of 20 hours at room temperature before use.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Not specified
- Light intensity: Not specified
- Details on termination of incubation: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Dissolved O2 concentrations and test substance concentrations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on geometric spacing to the solubility limit with the requisitie number of tested concentrations to meet the specified guideline
- No limit test was conducted

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

other: Inhibitory Effect

Remarks:

Not a respiatory inhibitor

Effect conc.:

>= 1 198.08 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other:

Remarks:

no effects seen at the highest dose of 1198.08 ug

Details on results:

The test substance showed no inhibitory effect on microbial respiration at any of the concentrations tested.

Results with reference substance (positive control):

The positive reference compound, at concentrations of 5000; 14,999; and 30,006 µg/L, inhibited respiration by 4.51 %, 59.86%, and 75.95%, respectively, demonstrating susceptibility of the inoculum to a known inhibitor of respiration.

Reported statistics and error estimates:

STATISTICAL METHODS:
Respiration rates (oxygen consumption rates) were calculated as the slope of a least-squares linear regression analysis of concentration of dissolved oxygen (mg Oz/L) versus elapsed time (seconds). A 10-minute window was selected to determine regression statistics. The period chosen was between the 13th and 73rd observation for each series of measurements, which for readings every 10 seconds over 10 minutes provided 60 data points. This window was selected to provide uniformity in
the data analysis and avoid bias.

Calculation of Percent Inhibition
Percent inhibition of the positive reference and test substances were calculated as a percentage of the mean of the control respiration rates according to the following equation:
(1-((2*Rs)/(Rc1+Rc2))*100 = percent inhibition

where:
Rs=oxygen consumption rate at tested concentration of test or positive reference substance
Re1=oxygen consumption rate, control before test or positive reference substance
Rc2=oxygen consumption rate, control after test or positive reference substance

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This test validly assessed the toxicity to microorganisms of the test substance according to the specified guideline