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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-04-16 to 1996-04-19

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

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: Room temperature in a tightly sealed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of 240 ul of acetone/L dilution water was created at the maximum concentration of the test substance in the vehicle. This was then diluted down to the stated test concentrations of test substance.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
0 (water and solvent control), 90, 180, 360, 720, and 1440 ug test ingredient/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At test initiation, approximately 7.5 ml was collected from each concentration stock solution. At test termination (Day 3), an approximate 9-ml composite sample from the three replicates at each concentration was collected, Upon collection, samples were immediately submitted for analysis of the test substance. These analyses provided information on test concentrations over time.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Diluting various volumes of primary stock solution with algal nutrient medium
- Controls: Solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone suspended in water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 240 ul of acetone/L distilled water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms

Test organisms (species):

other: Selenastrum capricornutum

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection): Starr collection at the University of Texas.
- Age of inoculum (at test initiation): Several days old
- Method of cultivation: Algal nutrient medium slants

ACCLIMATION
- Acclimation period: SEveral days
- Culturing media and conditions (same as test or not): nutrient slant

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 d

Post exposure observation period:

The cells were dried and then observed less than <24 hours after the conclusion of the test due to an oven malfunction.

Test conditions

Hardness:

17.1 mg/L as CaCO3

Test temperature:

24.7-25.2

pH:

7.0-7.5

Conductivity:

116 umhos

Nominal and measured concentrations:

Nominal: 0 (water and solvent control), 90, 180, 360, 720, and 1440 ug test ingredient/L
Measured (average): 0 (water and solvent control), 62, 132, 340, 686, and 1540 ug test ingredient/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Borosilicate glass, 500 ml, 400 ml, 100 ml
- Aeration: No
- Initial cells density: 10,000 cells/ml
- Control end cells density: 242,000 cells/ml for the Water control and 249,000 cells/ml
- No. of organisms per vessel: 1,000,000 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, U.S. EPA medium (Miller et al. 1978)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized ultra-filtered water
- Alkalinity: <20 mg/L as CaCO3
- Conductivity: 116 umhos
- Culture medium different from test medium: No
- Intervals of water quality measurement: At test intiation and in eahc replicate at test termination.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: 4 Klux (80 uE/m^2/s) at the sureface of the test solution

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: compound microscope and hemocytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on a pilot study

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 d

Dose descriptor:

EC50

Effect conc.:

ca. 458 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

3 d

Dose descriptor:

EC50

Effect conc.:

ca. 344 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 3

Key result

Duration:

3 d

Dose descriptor:

NOEC

Effect conc.:

ca. 132 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Maximum specific growth rate, average specific growth rate, maximum cell count, terminal biomass, terminal cell count, and area under growth curve

Details on results:

- Exponential growth in the control (for algal test): no, but expected logarithmic growth occured
- Observation of abnormalities (for algal test): None

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

For each variable, a one-factor analysis of variance was performed on the test article concentrations. The statistical significance of linear treatment contrasts was tested at the 0.05 level in a sequential fashion (Tukey et al. 1985), to evaluate the no-observable-effect concentration levels. All concentration levels were compared against the solvent control for the evaluation of statistical significance. The specific growth rate(µ) was calculated in the following manner:
u = (ln (Xi/X(i-1)))/(ti-t(i-1))
where In= the natural logarithm, ti= number of days lapsed at time of sample the i-th sample, i = I, 2, 3; Xi= algal standing crop concentration (cells/ml) at day ti.

The maximum specific growth rate (u-max) was determined by the largest specific growth rate(µ) value for each replicate at each concentration during the test period.

The average specific algal growth rate during the linear-logarithmic phase was estimated by the slope from the simple linear regression of log cell counts on day number. For each test vessel, data from the linear-logarithmic phase of algal reproduction (Days O through 3) were fit to the following regression model:
In (Xi)= B0+B1*t;,
where B0 and B1 are the intercept and slope, respectively, of the regression. The estimated value of B1 was used as the measure of average specific algal growth rate for the corresponding test vessel and is denoted µ-reg.

The area under the growth curve (denoted AUC) is a measure of the growth of cell populations. The formula for the AUC can be reduced to:
AUC = X1+X2+(1/2)X3-(5/2)X0
where Xi is the algal standing crop concentration on the ith day.
The EC50 values were estimated based on the simple linear regressions of the percentage reductions in average specific growth rate and area under curve versus log concentration. The regression equations were solved for the concentration corresponding to one-half the solvent control value.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study found the 3 day EC50 and NOEC values of the test substance's toxicity to aquatic algae and cyanobacteria according to the specified guidelines.