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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-03-27 to 1996-03-29

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.2 (Acute Toxicity for Daphnia)

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 room temperature in a tightly secured container.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in acetone to act as the carrier in the stock solution to disperse the test substance in water.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
0 (water and acetone controls), 125, 250, 500, 800, and 1500 µg test ingredient/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Actual intennediate 290437 concentrations were determined at test initiation and termination. At test initiation, 7 .5 ml was collected from each solution prior to distribution to the replicates. At test termination, a composite sample of 12 ml (3 ml from each of the four vessels containing organisms) was collected from the four replicates containing animals at each concentration. All samples were collected into appropriately labeled containers, diluted with methanol (to a final methanol concentration of 25%), and immediately submitted for analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dissolution of the test substance into a stock solution of acetone, and then diluting this in water to the proper test concentrations.
- Controls: Two vehicle controls (acetone/water mixture and just water) were run simultaneously with the main test
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone acting as a dispersant and water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 250 ul acetone/L of dilution water

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Daphnia magna
- Source: Lilly Environmental Toxicology Laboratory
- Age of parental stock (mean and range, SD): All were 24 hours old or younger
- Feeding during test: Daphnids were not fed during the test, but were prior to testing.


ACCLIMATION
- Acclimation period: Less than or equal to 24 hours
- Acclimation conditions (same as test or not): same as test
- Food type: unicellular green algae and cerophyl
- Frequency: daily
- Health during acclimation (any mortality observed): none

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
A plastic pipet (4-mm I.D.) was used to place five first-instar daphnids into each of the 4 replicates/treatment.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

120 mg/L as CaCO3

Test temperature:

19.7 +-0.2 degrees C

pH:

8.1-8.2

Dissolved oxygen:

9.4 +- 0.3 mg/L

Salinity:

121 mg/L as CaCO3

Conductivity:

211 umhos

Nominal and measured concentrations:

The average assayed intermediate 290437 concentrations for the study were 108, 228, 448, 730, and 1312 ug test ingredient/L at nominal concentrations of 125, 250, 500, 800, and 1500 ug test ingredient/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Borosilicate glass, 250 ml, 50 ml, 200 ml
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Fresh condition well water 7 times/week
- Alkalinity: 115 mg/L as CaCO3
- Conductivity: 244 umhos
- Culture medium different from test medium: Same as test medium
- Intervals of water quality measurement: 7 times/week, at test intiation, and 24/48 hours into the test.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to 8.1
- Photoperiod: 16 light / 8 dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Physical condition of each organism at intiation, 24 h, and 48 h

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

>= 274 - <= 370 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 108 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

- Behavioural abnormalities:In the water control, on organism was hypoactive at 24 hours; additionally, at 48 hours one organism was hypoactive, one was prostrate, and one was immobile. In the solvent control a single organism was hypoactive at 24 hours and prostrate at 48 hours. No treatment-related immobilization or other physical signs of toxicity were observed in Daphnia magna exposed to an average assayed test substance concentration of 108 ug/L. At average assayed test substance concentrations >227 ug/L, hypoactivity, prostration, and immobilization were observed.
- Mortality of control: None
- Other adverse effects control: In the water control, on organism was hypoactive at 24 hours; additionally, at 48 hours one organism was hypoactive, one was prostrate, and one was immobile. In the solvent control a single organism was hypoactive at 24 hours and prostrate at 48 hours.

Reported statistics and error estimates:

Statistical Evaluation
The EC50 value was calculated by probit analysis (SAS, 1990). All values expressed as "x ± y" represent means± standard deviations and were calculated using SAS (1990) or a hand calculator.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study validly assessed the test substance's short-term toxicity to aquatic invertebrates according to the specified guidelines.