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Diss Factsheets

Administrative data

short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 December 1992 - 23 March 1993
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
The study has been performed according to OECD and/or EC guidelines and according to GLP principles. Limited information available to verify the composition of the used test substance.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
GLP compliance:

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
C23H24N4O2, C24H34N4O2
3-(2-methyl-3-{[(4-methylphenyl)carbamoyl]amino}phenyl)-1-(4-methylphenyl)urea; 3-(2-methyl-5-{[(4-methylphenyl)carbamoyl]amino}phenyl)-1-(4-methylphenyl)urea; 3-{2-methyl-3-[(octylcarbamoyl)amino]phenyl}-1-(4-methylphenyl)urea
Details on test material:
- Name of test material (as cited in study report): KY-MA
- Substance type: light yellow solid
- Physical state: solid
- Stability for at least 48 hours in water:Yes
- Storage condition of test material: RT in the dark
- Stable for at least 48h in water: Yes

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Duplicate samples were taken from all replicate test vessels, the Cremophor control and the supersaturated solutions.
- Sampling method: 10 mL from center of test vessels at t=0h, t=96h; ALSO, 10 mL taken from top and bottom of one vessel to determine homogeneity
- Sample storage conditions before analysis: t = 0 samples were stored at -20 degrees C until analysis. t = 96h samples were analysed on the day of sampling

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Cremophor addition to stock solutions (prepared at 1000 mg/L KY-MA) via 72h stirring to supersaturation
- Controls: Blank, Cremophor
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Cremophor RH40
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 g/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test solution was a suspension of test substance particles floating on the surface and deposited at the bottom of the vessels.

Test organisms

Test organisms (species):
Cyprinus carpio
Details on test organisms:
- Common name: carp
- Source: Zodiac, proefacc, "De Haar Vissen", "L.U., Wageningen, The Netherlands
- Length at study initiation (length definition, mean, range and SD): 2.0 (+/- 0.2) cm
- Weight at study initiation (mean and range, SD): 0.23 (+/- 0.03) g
- Feeding during test: NONE

- Acclimation period: 11d after delivery
- Acclimation conditions (same as test or not): Same except with feeding (no feeding during the test and 24h prior to the start of test)
- Type and amount of food: Touvit or Artemia
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): mortality <5% during 7d prior to test period

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h

Test conditions

250 mg CaCO3/L
Test temperature:
20.5-21.0 degrees C
Dissolved oxygen:
8.7-9.6 mg O2/L
Nominal and measured concentrations:
Nominal concentrations in the final test were set to 1000 mg/L. Aliquots from the start and end of the test were assessed via HPLC-UV and showed concentrations of 325-523 mg/L at the start of the test and 86-212 mg/L at the end of the test. The overall average concentration was 281 mg/L (measured). Tested concentrations were significantly above the solubility of KY-MA in water (0.006 mg/L).
Details on test conditions:
- Test vessel: 3L, glass
- Material, size, headspace, fill volume: glass, 3L
- Aeration: continuous
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.76 g/L

- Adjustment of pH: none
- Photoperiod: 16 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : motality and behavior (behavior could not be recorded due to opacity of test vessels)
Reference substance (positive control):

Results and discussion

Effect concentrations
96 h
Dose descriptor:
Effect conc.:
> 281 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Overall average value (note: there was variability in test concentrations between replicate vessels)
Details on results:
- Behavioural abnormalities: Could not be observed due to opacity of the test solutions
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: significant precipitation was seen in all test vessels. The test solutions were opaque suspensions.
- Effect concentrations exceeding solubility of substance in test medium: all test concentrations (1000 mg/L nominal) exceed the test substance solubility of KY-MA in water (0.006 mg/l)


- Measured test concentrations: t=0: 434/420 (A), 325/334 (B), 506/523 (C) mg/L; t=96h: 124/86 (A), 102/104 (B), 212/200 (C) mg/L
- Homogeneity test (replicate B, mean of duplicate samples taken at a height of 90%, 50% and 10%, respectively): t= 0: 340, 330 and 323 mg/L, t = 96h: 116, 103 and 99 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: 0 of 3 fish died at 0, 0.1 and 0.18 mg/L, 3 of 3 fish died at 0.32, 0.56 and 1.0 mg/L
- LC50: between 0.18 and 0.32 mg/L

Any other information on results incl. tables

Sublethal observations / clinical signs:

The measured test concentrations in medium were much higher than the maximum solubility in water (0.006 mg/L). No effects were observed at the tested concentration. Based on the overall average measured test concentration, the 96h-LC50 value was determined to be higher than 281 mg/L. Since this exceeds the maximum solubility of KY-MA in water (0.006 mg/L), it can be concluded that the 96h-LC50 value is above the maximum solubility in water of KY-MA.

Although there is variability of measured concentrations between the test vessels, the homogeneity test revealed that there is little variability between measured concentrations within a test vessel (at different heigths).

Applicant's summary and conclusion

Validity criteria fulfilled:
The measured 96-h LC50 of KY-MA was higher than 281 mg/L, which is higher than the maximum solubility of KY-MA in water (0.006 mg/L).

Executive summary:

A 96-h Acute toxicity test was performed in carp. The test substance was stirred for 72 hours to achieve maximum concentration possible in the test. Cremophore was used as a dispersant. There was no mortality observed in the time-frame of the test. The measured 96-h LC50 of KY-MA was higher than 281 mg/L, which is higher than the maximum solubility of KY-MA in water (0.006 mg/L).