3-methyl-1,1-diphenylurea

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10.04.-23.04.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

Principal investigator for analytical determination of the test substance concentrations was not Pavla Vlasáková, Dipl.Eng. In this study principal investigator was Jana Netušilová, Dipl.Eng., CETA, GLP Test Facility, Analytical Group I.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples of test solutions were prepared and delivered by Ecotoxicity group. The samples were analyzed on the day of delivery. The samples with nominal concentration 1 mg/L were analysed non-diluted. The samples with nominal concentration 100 mg/L were appropriate diluted (25 times) with UPW to the range of calibration curve. All samples were filtered through the syringe PTFE filter 0.45 μm before HPLC analyses.

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 preliminary and limit test. Because the test substance was poorly soluble for dilution was used ultrasound for 1 hours and stirring for 2 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM- Common name: Desmodesmus subspicatus - Source (laboratory, culture collection): Brinkmann 1953/SAG 86.81 obtained from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 3.6.2014. - 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. ACCLIMATION- Acclimation period: 3-4 days- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.5-24°C

pH:

7.6-7.8

Nominal and measured concentrations:

1, 5, 10, 50, 100 mg/L preliminary test100 mg/L limit test

Details on test conditions:

TEST SYSTEM- Test vessel: Erlenmayer flask with stoppers venting air- Material, size, headspace, fill volume: glass, 50 ml- Aeration: without- Initial cells density: 5000 cells in 1 ml- Control end cells density:-No. of vessels per concentration (replicates): 3OTHER TEST CONDITIONSTemperature: 21 - 24 °C controlled at ± 2 °CLighting: under continuous white light, 4 440 lux to 8 880 luxExposition time: 72 hoursVolume of tested mixture: 50 mLInitial concentration of algae culture: 5 000 cells in a 1 mL Without aeration.Agitation of algae culture by shaking.The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hour.The cell density was measured by direct counting of living cells in Burker´s counting chamber.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: NOECy

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

Results of reference testThe 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 30.3. to 2.4. 2015, are the following: 72 hour – ErC50 = 0.79 mg.L-1 (95% confidence limit: 0.70 – 0.89 mg.L-1)

Table No.: 1.         Individual cell densities– preliminary test

Nominal concentration mg·L^-1	Replicates	Cell density in 1 mL
		0 hours	24 hours	48 hours	72 hours
100	1	5000	6250	12500	206250
	2	5000	6250	12500	243750
	3	5000	6250	18750	231250
50	1	5000	6250	25000	231250
	2	5000	6250	31250	268750
	3	5000	6250	25000	243750
10	1	5000	6250	37500	300000
	2	5000	6250	43750	300000
	3	5000	6250	31250	368750
5	1	5000	6250	43750	350000
	2	5000	6250	50000	362500
	3	5000	12500	43750	362500
1	1	5000	12500	56250	375000
	2	5000	12500	62500	393750
	3	5000	12500	62500	375000
Control	1	5000	18750	87500	437500
	2	5000	18750	81250	443750
	3	5000	18750	75000	450000

Table No.: 2.         Growth rate (µ), yield (Y) – preliminary test

Nominal concentration mg·L^-1	Replicates	Growth ratefor section-by-section (m)			Growth rate (m) 0-72 h	Yield (Y)
		0-24 h	24-48 h	48-72 h
100	1	0.22	0.69	2.80	1.24	201250
	2	0.22	0.69	2.97	1.30	238750
	3	0.22	1.10	2.51	1.28	226250
50	1	0.22	1.39	2.22	1.28	226250
	2	0.22	1.61	2.15	1.33	263750
	3	0.22	1.39	2.28	1.30	238750
10	1	0.22	1.79	2.08	1.36	295000
	2	0.22	1.95	1.93	1.36	295000
	3	0.22	1.61	2.47	1.43	363750
5	1	0.22	1.95	2.08	1.42	345000
	2	0.22	2.08	1.98	1.43	357500
	3	0.92	1.25	2.11	1.43	357500
1	1	0.92	1.50	1.90	1.44	370000
	2	0.92	1.61	1.84	1.46	388750
	3	0.92	1.61	1.79	1.44	370000
Control	1	1.32	1.54	1.61	1.49	432500
	2	1.32	1.47	1.70	1.50	438750
	3	1.32	1.39	1.79	1.50	445000

The mean coefficient of variationfor section-by-section specific growth rates in the control cultures is3.5 %(CV_0-24h= 0 %, CV_24-48h= 5.3 % a CV_48-72h= 5.4 %).

The coefficient of variation of average specific growth ratesduring the whole test period in replicate control cultures is0.3 %.

Table No.: 7 Individual cell densiities-limit test

Nominal concentration mg·L^-1	Replicates	Cell density in 1 mL
		0 hours	24 hours	48 hours	72 hours
100	1	5000	12500	43750	200000
	2	5000	12500	50000	193750
	3	5000	12500	43750	193750
	4	5000	12500	56250	225000
	5	5000	6250	56250	287500
	6	5000	12500	43750	200000
Control	1	5000	18750	81250	425000
	2	5000	18750	75000	400000
	3	5000	12500	75000	418750
	4	5000	25000	81250	393750
	5	5000	18750	68750	418750
	6	5000	12500	75000	406250

Table No.: 8.         Growth rate (µ), yield (Y) – limit test

Nominal concentration mg·L^-1	Replicates	Growth rate for section-by-section (m)			Growth rate (m) 0-72 h	Yield (Y)
		0-24 h	24-48 h	48-72 h
100	1	0.92	1.25	1.52	1.23	195000
	2	0.92	1.39	1.35	1.22	188750
	3	0.92	1.25	1.49	1.22	188750
	4	0.92	1.50	1.39	1.27	220000
	5	0.22	2.20	1.63	1.35	282500
	6	0.92	1.25	1.52	1.23	195000
Control	1	1.32	1.47	1.65	1.48	420000
	2	1.32	1.39	1.67	1.46	395000
	3	0.92	1.79	1.72	1.48	413750
	4	1.61	1.18	1.58	1.46	388750
	5	1.32	1.30	1.81	1.48	413750
	6	0.92	1.79	1.69	1.47	401250

The mean coefficient of variation for section-by-section specific growth rates in the control cultures is 14.5% (CV_0-24h= 21.9 %, CV_24-48h= 17.2 % a CV_48-72h= 4.5 %).

 

The coefficient of variation of average specific growth rates during the whole test period
in replicate control cultures is 0.7 %.

Validity criteria fulfilled:

yes

Conclusions:

The highest inhibition of growth rate was 14.5 % and the highest inhibition of yield was47.8 % in the limit test.Therefore exact values of EC50 could not be calculated and the values of EC are givenin the form of a range.The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic). 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. So the nominal concentrations were used for all evaluations and results.

Executive summary:

The test substance,Akardit, 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 (EC) No. 761/2009, Annex IV.

 

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg·L^-1.

The analytical results showed, that the test substance Akardit was sufficiently stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L^-1.

The highest inhibition of growth rate was 14.9 % and the highest inhibition of yield was
49.4 % in the preliminary test.Based on no toxicity of the test substance found in preliminary test, the limit test was performed subsequently.

 

The concentration of 100 mg·L^-1was tested in the limit test.The highest inhibition of growth rate was 14.5 % and the highest inhibition of yield was 47.8 % in the limit test.

 

The nominal concentrations were used for all evaluations and results, because, 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, then the results can be based on nominal or measured initial values.

 

Test results:

72 hour – E_rC₅₀>100 mg·L^-1            (nominal concentration)

72 hour – E_yC₅₀>100 mg·L^-1           (nominal concentration)

72 hour – NOEC_r< 100 mg·L^-1       (nominal concentration)

 72 hour – NOEC_y< 100 mg·L^-1       (nominal concentration)

 

Description of key information

The test substance, Akardit, 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 (EC) No. 761/2009, Annex IV.

Only one study is available.

GLP study.

Klimish score 1.

The concentration of 100 mg·L^-1was tested in the limit test. The highest inhibition of growth rate was 14.5 % and the highest inhibition of yield was 47.8 % in the limit test.

72 hour – E_rC₅₀>100 mg·L^-1            (nominal concentration)

72 hour – E_yC₅₀>100 mg·L^-1           (nominal concentration)

72 hour – NOEC_r< 100 mg·L^-1       (nominal concentration)

72 hour – NOEC_y< 100 mg·L^-1       (nominal concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L