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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-04-08 to 1996-04-12

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.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

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: 1995-11-15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in a tightly sealed condition
- Solubility and stability of the test substance in the solvent/vehicle:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 0.9g of test substance was dissolved in acetone to prepare a 6 mg test substance/ml stock solution
- Final dilution of a dissolved solid, stock liquid or gel: The final set of concentrations were prepared by diluting the stock solution with acetone/chilled conditioned well water to a final volume of approximately 134 L

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Nominal test concentrations were 0 (water and acetone control), 70, 800, 1200, 1500, and 1800 µg/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (water and acetone control), 70, 800, 1200, 1500, and 1800 µg/L
- Sampling method: Samples for analysis were collected at test initiation and termination. In addition, samples of aged and newly prepared test solutions were collected at each renewal period. Each collected sample was 7.5 ml and was diluted with 2.5 ml of methanol. Samples were collected prior to the daily renewal
- Sample storage conditions before analysis: Samples were immediately submitted for analysis.

Test solutions

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Water and acetone control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone/conditioned distileed well water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 298.5 µI acetone/L of dilution water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Juvenile rainbow trout
- Strain: Onchorhynchus mykiss
- Source: Hatched at the Eli Lilly Environmental Toxicology Laboratory on December 8, 1995, from eyed eggs supplied by Trout Lodge in McMillin, WA
- Age at study initiation (mean and range, SD): 134 days
- Length at study initiation (length definition, mean, range and SD): Averaged 64 ± 4.8 mm and ranged between 55 and 69 mm, total length
- Weight at study initiation (mean and range) : Averaged 2.20 g and ranged from 1.29 to 2.79 g.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Same as test except for the addition of aeration
- Type of food during acclimation: Dry pelleted formulation daily (Trout feed, Formula No.1, manufactured by Zeigler Brothers, Gardners, PA).
- Feeding frequency during acclimation: Daily until 48 hours before the test
- Health during acclimation (any mortality observed): <0.1%

FEEDING DURING TEST
- The fish were not fed during the test

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 d

Post exposure observation period:

Fish were immediately euthanized following the conclusion of the test.

Test conditions

Hardness:

137mg/l as CaCO3

Test temperature:

10.4-11.6 degrees Celsius over the exposure period

pH:

7.3-7.7 over the exposure period of the test

Dissolved oxygen:

10.3-11.9 mg/L over the exposure period

Conductivity:

256 umhos

Nominal and measured concentrations:

Nominal: 0 (water and acetone control), 70, 800, 1200, 1500, and 1800 µg/L
Measured: Averages were 0, 77, 885, 1364, 1703, and 2076 µg/L respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Large aquarium
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, >134 L, 134L
- Aeration: None
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: Unspecified
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.02 g/L approximately

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water
- Alkalinity: 122 mg/L as CaCO3

OTHER TEST CONDITIONS
- Adjustment of pH: The dilution water was conditioned
- Photoperiod: 16/8 light/dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Fish behavior on a scale was measured at test intiation and after approximately 2, 24, 48, 72, and 96 hours of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Determined by standard procedure
- Range finding study: None

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

other: Median lethal concentration

Effect conc.:

1 279 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

77 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

- Behavioural abnormalities: At concenttations of >=885 µg/L, the trout exhibited exposure-related signs of toxicity including hypoactivity, impaired swimming, prostration, and death.
- Mortality of control: None

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

The LC50 value was calculated using the probit procedure (SAS 1990). All values expressed as "x ± y" represent means± standard deviations and were calculated using either SAS (1990) or a hand calculator.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study validly assessed the short term aquatic toxicity of the test substance according to the specified guideline. The 96-hour median lethal concentration of the test substance to rainbow trout was 1279 µg/L with 95% confidence limits of 1073 to 1458 µg/L. The slope of the concentration-mortality curve at 96 hours was 9.5. The no-observed effect concentration to rainbow trout was an average assayed concentration of 77 µg/L.

Executive summary:

The study was carried out in accordance with EU method C.1. The 96 hour median lethal concentration was 1279ug/l and the NOEC was 77ug/l.