Registration Dossier

Ecotoxicological information

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:
12.12.2016-10.02.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
The temperature was not in the prescribed range (20 ± 2 °C) during whole testing. The prescribed range was exceeded twice by about 0.4 and 0.1 °C in the preliminary test. This increase of the temperature takes only short period of time (2 hours).
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dimethyl-1,3-diphenylurea
EC Number:
210-283-4
EC Name:
1,3-dimethyl-1,3-diphenylurea
Cas Number:
611-92-7
Molecular formula:
C15H16N2O
IUPAC Name:
1,3-dimethyl-1,3-diphenylurea
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Preparation of Sample Solutions:
The test substance is poorly soluble in dilution water at concentrations needed for the test. For this reason the preliminary experiment was performed for finding suitable period for achieving the highest solubility.
For the preliminary experiment the solutions were prepared as follows: 100 mg of the test substance in 1000 mL of dilution water was stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter and the concentrations of the test substance in resulted solutions were determined.



Test solutions

Details on test solutions:
The stock solutions of the test substance were prepared in the dilution water. Due to lower solubility of the test substance were prepared two stock solutions for the preliminary and definitive test. 50 and 100 mg of the test substance was weighed into 1 000 mL of the dilution water for the preliminary test and 80 and 100 mg of the test substance was weighed into
1 000 mL of the dilution water for the definitive test No.2. The stock solutions were stirred for period 72 hours and subsequently filtered through 0.45 μm filter.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: own laboratory breeding.
- Age at study initiation: young Daphnia born in 24h
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no


ACCLIMATION
- Acclimation period: 48h
- Acclimation conditions: same as test
- Type and amount of food: a mixture of algae
- Health during acclimation: mortality control, healthy



Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
Ca+Mg ions
2.54 mmol/L preliminary study
2.52 mmol/L definitive test
Test temperature:
20±2°C
pH:
7.9
Dissolved oxygen:
8.5 mg//L preliminary study
8.6 mg/L definitive test
Conductivity:
2.26 µS.cm-1 preliminary study
0.88 µS.cm-1 definitive test
Nominal and measured concentrations:
100, 50, 10, 5, 1 mg/L preliminary test
100, 71, 51, 36, 26, 19 and 14mg/L definitive test
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers, 50 ml
- Type: open
- Aeration: without
Number of test animals: 20 for each concentration and control
Number of replicates (per concentration): 2
Number of test animals per replicate: 10
Volume of test substance solution: 50 mL per 10 individuals

Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1000 mL of deionized water.
A. 117.6 g CaCl2.2H20 in 1 L of deionized water
B. 49.3 g MgSO4.7H2O in 1 L of deionized water
C. 25.9 g NaHCO3 in 1 L of deionized water
D. 2.3g KCl in 1 L of deionized water
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
77.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
64.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
Reference test
The sensitivity of the test species and correctness of test performance is periodically verified
in six-month period by testing with the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 09.11. to 11.11.2016 are the following:

24 hour – EC50 = 1.34 mg·L-1 (95% confidence limit: 1.15 – 1.67 mg·L-1)
48 hour – EC50 = 0.74 mg·L-1 (95% confidence limit: 0.66 – 0.84 mg·L-1)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 per cent
of the nominal or measured initial concentration throughout the test, the results can be based
on nominal or measured initial values.
Because concentrations of the test substance have been satisfactorily maintained
within ± 20 percent of the measured initial concentration throughout the test, the nominal concentrations were used for all evaluations and results.

24 hour – EC50 = 77.3 mg·L-1 (nominal concentration)
(95% confidence limit: 63.4 – 105.2 mg·L-1)
48 hour – EC50 = 64.7 mg·L-1 (nominal concentration)
(95% confidence limit: 54.9 – 80.4 mg·L-1)

24 hour – EC0 = 19 mg·L-1 (nominal concentration)
48 hour – EC0 = 19 mg·L-1 (nominal concentration)

24 hour – EC100 ˃ 100 mg·L-1 (nominal concentration)
48 hour – EC100 ˃ 100 mg·L-1 (nominal concentration)

Executive summary:

The test substance, Methylcentralit, was tested in acute immobilisation test on Daphnia magna.

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

 

Since the test substance was poorly soluble in dilution water, the stock solutions were stirred for period 72 hours and subsequently filtered through 0.45 μm filter.

The preliminary test was performed in a range of the test substance nominal concentrations
1 – 100 mg·L-1.
Based on toxicityof the test substance found in the preliminary test, the definitive test was performed inappropriate concentration range.

The analytical results showed, that the test substance Methylcentralit was sufficiently stable in dilution water in the conditions of the test, therefore the definitive test was performed as static one.

 

The definitive test was performed in a range of the test substance nominal concentrations
14 – 100 mg·L-1. Samples for analytical determination were taken at the beginning and at the end of the test.

The definitive test was performed twice.In the first definitive test the result of analytical determinationof concentration of the test substance in the highest concentration (100 mg·L-1)was different from theanalytical determination of concentration of the test substancein the preliminary test and this concentration was not maintained within ± 20 percent of the nominal concentration.Therefore the definitive test was repeated (definitive test No.2).

The definitive test No.2 was used for all evaluation and results.

 

There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, the results can be based
on nominal or measured initial values.

The nominal concentrations were used for all evaluations and results.

 

Test results:

24 hour – EC50= 77.3 mg·L-1                            (nominal concentration)

(95% confidence limit: 63.4 – 105.2 mg·L-1)

48 hour – EC50= 64.7 mg·L-1                              (nominal concentration)

(95% confidence limit: 54.9 – 80.4 mg·L-1)

24 hour – EC0= 19mg·L-1                    (nominal concentration)

48 hour – EC0=19mg·L-1                    (nominal concentration)

24 hour – EC100˃ 100 mg·L-1                             (nominal concentration)

48 hour – EC100˃ 100 mg·L-1                             (nominal concentration)