1,3-dimethyl-1,3-diphenylurea

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:

12.12.2016-26.01.2017

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.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See Any other information....

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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:

The test sample of test solution were prepared and delivered by Ecotoxicity group. The samples were analyzed on the day of delivery. The delivered samples with nominal concentration 1 mg/L-100 mg/L were analysed.

Test solutions

Vehicle:

no

Details on test solutions:

The stock solution of the test substance was prepared in the test medium.
100 mg of the test substance was weighed into 1000 mL of the test medium for the preliminary and the limit test

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81
- Source: from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 27.5.2016
- Age of inoculum (at test initiation): Inoculum culture was kept 3-4 days under conditions at which the test was performed.
- Method of cultivation:
The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature.
Algae inoculum for the test was sampled from exponentially growing inoculum culture.

ACCLIMATION
- Acclimation period:3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Test temperature:

21.5-23°C

pH:

7,6-7,7

Conductivity:

preliminary test 2.26 µS·cm-1
full test 1.87 µS·cm-1

Nominal and measured concentrations:

preliminary test 100, 50, 10, 5 and 1 mg/L
full test 62, 41, 27, 18, 12, 8 and 5 mg/L

Details on test conditions:

Preliminary test
Range of nominal concentration: 1-100 mg/l
Preparation of the stock solutions
Stock solutions of the test substance: 99.91 mg·L-1 and 50.08 mg·L-1
Conductivity of deionized water: 1.28 µS·cm-1
pH of the test medium: 7.6

Preparation of the test concentrations
The test concentrations: 100, 50, 10, 5 and 1 mg·L-1
Conductivity of deionized water: 2.26 µS·cm-1
pH of the test medium: 7.6
Volume of inoculated algae culture: 1.50 mL in 200 mL of mixture
Lighting during the test: 8 350 – 8 360 lux
Temperature during the test: 21.5 – 22.5 °C

Full test
Range of nominal concentration:5-62 mg/L
Preparation of the stock solutions
Stock solutions of the test substance: 31.01 mg·500 mL-1 and 49.93 mg·L-1
Conductivity of deionized water: 2.26 µS·cm-1
pH of the test medium: 7.6

Preparation of the test concentrations
The test concentrations: 62, 41, 27, 18, 12, 8 and 5 mg·L-1
Conductivity of deionized water: 1.87 µS·cm-1
pH of the test medium: 7.7
Volume of inoculum algae culture: 1.6 mL in 250 mL mixture
Lighting during the test: 8 350 – 8 360 lux
Temperature during the test: 22.0 – 23.0 °C
Inicial density: 5000 cells per 1ml
Testing mixtures were incubated in Erlenmeyers’ flasks.
The test contained three parallel series of the test substance concentrations and three controls without the test substance.
The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours.
At the beginning and the end of the test the pH of the test mixtures was measured.
The light intensity and temperature were measured every 24 hours.
The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 18.04. to 21.04. 2016, are the following:

72 hour – ErC50 = 0.87 mg·L-1 (95% confidence limit: 0.64 – 1.17 mg·L-1)

Interlaboratory test data
From the results of the last interlaboratory test arranged by CSlab spol. s r.o. in 2015 we have obtained data for potassium dichromate 72h – ErC50 generated at 14 laboratories. The following reference range of values could be calculated from those data:

72 hour – ErC50 = 0.51 – 1.19 mg·L-1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

72 hour – ErC50 = 37.7 mg·L-1 (95% confidence limit: 35.3 – 40.0 mg·L-1)
(nominal concentration)

72 hour – EyC50 = 23.6 mg·L-1 (95% confidence limit: 20.1 – 27.5 mg·L-1)
(nominal concentration)

72 hour – ErC10 = 20.8 mg·L-1 (95% confidence limit: 19.9 – 21.9 mg·L-1)
(nominal concentration)

72 hour – EyC10 = 14.8 mg·L-1 (95% confidence limit: 12.9 – 16.9 mg·L-1)
(nominal concentration)


72 hour – NOECr ˂ 5 mg·L-1 (nominal concentration)
72 hour – NOECy ˂ 5 mg·L-1 (nominal concentration)

Executive summary:

The test substance, Methylcentralit, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according tomethod C.3. -Freshwater Algae and Cyanobacteria, Growth Inhibition Test,Commission Regulation (EU) No. 2016/266.

 

Since the test substance was poorly soluble in the test medium, 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.

The analytical results showed that the test substanceMethylcentralitwas stable in the test medium at conditions of the test (decrease of the test substance concentration is about 20 %).

Based on toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

The full test was performed in a range of the test substance nominal concentrations 5 – 62 mg·L^-1.

The samples for analytical determination were taken at the beginning, after 24 hours, 48 hours and at the end of the test.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in full test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the full test, then the results can be based on nominal or measured initial values, which is the case of this study.

The highest tested concentration (62 mg·L^-1) was not used for evaluation of results (see chapter 3.14 Deviation.). The other concentrations were maintained within ± 20 per cent of the nominal concentration during the test, therefore the nominal concentrations were used for all evaluations and results.

 

Test results:

72 hour – E_rC₅₀=37.7 mg·L^-1             (95% confidence limit: 35.3 – 40.0 mg·L^-1) (nominal concentration)

 

72 hour – E_yC₅₀= 23.6mg·L^-1            (95% confidence limit: 20.1 – 27.5 mg·L^-1) (nominal concentration)

 

72 hour – E_rC₁₀=20.8 mg·L^-1             (95% confidence limit: 19.9 – 21.9 mg·L^-1) (nominal concentration)

 

72 hour – E_yC₁₀=14.8 mg·L^-1            (95% confidence limit: 12.9 – 16.9 mg·L^-1) (nominal concentration)

72 hour – NOEC_r˂ 5 mg·L^-1              (nominal concentration)

72 hour – NOEC_y˂ 5 mg·L^-1              (nominal concentration)