A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene

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 March 1999 to 29 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss authorities

Test material

Test material information

Constituent 1

Reference substance name:

3‐cyclohexyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

EC Number:

604-940-8

Cas Number:

154099-21-5

Molecular formula:

C39 H62 N4 O2

IUPAC Name:

3‐cyclohexyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 2

Reference substance name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-370-3

EC Name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

58890-25-8

Molecular formula:

C27 H36 N4 O2

IUPAC Name:

3‐cyclohexyl‐1‐[4‐({4‐[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 3

Reference substance name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-690-3

EC Name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

43136-14-7

Molecular formula:

C51 H88 N4 O2

IUPAC Name:

3‐octadecyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): MDI/CHA/ODA
- State of aggregation: solid

The data on the individual constituents CHA/MDI/CHA (EC 406-370-3) and ODA/MDI/ODA (EC 406-690-3) for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints are very similar to the data available for the different compositions of the multi-constituent or represent the outer boundaries of the data on the multi-constituent. Hence, they confirm that the different compositions are expected to have similar properties for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints.

Specific details on test material used for the study:

Solubility: insoluble

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

From the freshly prepared test medium and from the solvent control duplicate samples were taken just before the start of the test (without algae). Additional flasks with adequate volumes of the freshly prepared test medium and the solvent control were incubated during the test period under the same conditions as in the actual test but without algae. From these flasks duplicate samples were taken at the end of the test (after 72 hours exposure). Samples were stored deep frozen and protected from light until analysis was performed.

Test solutions

Vehicle:

yes

Remarks:

Tween 80

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the single test concentration of 100 mg/L was prepared by mixing 100 mg of the test item and 100 mg of Tween 80 with a small amount of test water by means of a mortar as homogenous as possible. Then the suspension was transferred quantitatively into the test water. The volume of the test medium was adjusted to the final volume of 1000 mL. The test medium was treated ultrasonically for 15 minutes and stirred intensively for 5 minutes at room temperature. The test medium was prepared just before inoculation of the algae (= start of the test).
- Controls: A solvent control (6 replicates with 100 mg/L Tween 80) and a control (3 replicates, test water without test item or emulsifier) were tested in parallel. Thus, the emulsifier concentration was 100 mg/L in the test medium and the solvent control.
- Vehicle: Pre-experiments have shown that it is not possible to obtain a stable suspension by mixing the test item into the test water. Solving or suspending the test item homogeneously in solvents like Acetone, Dimethylformamide (DMF) or Dimethylsulfoxide (DMSO) was not successful. Therefore, the test medium was prepared with the emulsifier Tween 80.
Concentration of vehicle in test medium: 100 mg/L
- Evidence of undissolved material: Yes. To secure that the maximum bioavailable concentration is maintained during the test period the test was performed with a suspension of the test item above the water solubility limit.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Source: Algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches lnstitut der Universitat Gottingen", D-37073 Gottingen. The algae had been grown in the laboratories of RCC under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): An exponentially growing pre-culture was set up 3 days prior to the test.

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

22 °C

pH:

- Start: 7.8-7.9
- End: 8.2-8.4

Nominal and measured concentrations:

- Nominal concentrations: 0 (control) and 100 mg/L.
- Measured concentrations: The mean concentrations found in the treatment samples were 102% (day 0) and 95% (day 3) of the nominal concentrations. The test substance was nearly stable during the performance of the biological test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks covered with glass dishes.
- Fill volume: 15 mL
- Initial cells density: 1E+04 cells/mL
- Blank control end cells density: ca. 72E+04 cells/mL
- Solvent control cells density: ca. 54E+04 cell/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-values were measured in samples from the single test concentration and the solvent control at the start and end of the test. The water temperature was measured at least daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not reported
- Photoperiod: continuous illumination
- Light intensity and quality: Illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the water bath in a distance of about 35 cm from the test flasks. The measured light intensity was about 6800 Lux (mean value), range: 6580 to 7150 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: After 24, 48 and 72 hours, algal cell densities in 1-2 mL samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample. After 72 hours the shape of the algal cells was microscopically examined.
- Other: The appearance of the test medium was recorded at least daily.
- Note: Since particles of the suspended test item in the test medium might have an influence on the algal counting by the electronic particle counter, additionally two replicates of the single test concentration and the solvent control were incubated with the test medium without addition of algae as "particle controls". These additional replicates were incubated and handled in the same manner as the replicates with algae and were used for possible corrections at the algal cell density determinations.

TEST CONCENTRATIONS
- Range finding study: Based on the results of a range-finding test a limit test was performed in accordance with the Commission Directive 92/69/EEC (non-GLP) to demonstrate that the test item has no toxic effect on the algae up to the concentration of 100 mg/L. Therefore, the maximum test item concentration of 100 mg/L was tested as the only test concentration.

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: To secure maximum bioavailability the test was performed with a supersaturated solution (i.e. suspension above the water solubility limit).

Details on results:

- Algal growth: The mean algal cell densities in the test medium were at all counting dates identical as or even slightly higher than in the parallel solvent control cultures. Thus, the test item had clearly no inhibitory effect on the growth of algae during the exposure period of 72 hours.
- Microscopic examinations: After the 72 hours test period no difference in cell shape was observed between the single test concentration of 100 mg/L and the algal cells in the control or solvent control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected. However, the algae in the test medium were partially aggregated with substance particle. Therefore, the samples at the end of the test were treated ultrasonically for one minute before determining the algal cell densities by means of the particle counter. The low algal cell densities in the solvent control after the incubation period of 48 hours could be due to an aggregation of the algal cells caused by the emulsifier Tween 80.
- Exponential growth in the control: Yes
- Effect concentrations exceeding solubility of substance in test medium: Yes. The test medium was turbid due to the suspended test item and remained turbid until the end of the test.

Reported statistics and error estimates:

Not applicable due to complete absence of toxic effects.

Any other information on results incl. tables

Table: Actual algal cell densities during the test period of 72 hours

Nominal test conc.	Flask No.	Density of algal cells (cell number x 10000/mL) * after
		24		48		72
Solvent control	1	4.55	4.26	15.54	15.36	76.55	77.86
	2	4.44	4.29	15.04	15.41	72.69	74.04
	3	4.73	4.21	14.55	14.78	63.83	66.23
	m	4.41		15.13		71.87
	s	0.05		0.38		6.22
	n	3		3		3
0 (control)	1	3.52	3.35	6.45	6.32	57.94	58.73
	2	4.21	3.95	4.79	4.86	53.13	52.87
	3	4.52	4.57	6.11	6.48	90.70	50.75
	4	5.17	4.74	4.47	4.06	53.08	51.85
	5	4.77	4.52	5.29	5.00	55.48	5.21
	6	3.96	3.63	5.69	5.71	- **	- **
	m	4.24		5.44 ***		54.07
	s	0.57		0.84		3.01
	n	6		6		5
100 mg/L	1	4.24	3.73	12.96	12.55	63.11	63.90
	2	4.87	4.30	14.11	13.33	69.35	68.58
	3	3.12	3.14	12.21	12.50	62.41	61.77
	m	3.90		12.94		64.85
	s	0.73		0.70		3.63
	n	3		3		3

* "particle controls" corresponding to the solvent control and the single test item concentration subtracted

** One of the six replicates was lost by accident before sampling after 72 hours. Therefore, only the   5 unaffected replicates were used for the calculation of the arithmetic mean and the standard deviation.

*** The low values of the algal cell densities in the solvent control after the incubation period of 48 hours might be due to an aggregation of the algal cells caused by the emulsifier Tween 80 and should therefore not be taken into account.

Table: Particle densities in the test medium and the solvent control (without algae)

Nominal test conc.	Flask No.	Particle density (particle number x 10000/ml)
		24		48		72
Solvent control	1	0.31	0.30	0.46	0.38	0.43	0.27
	2	0.19	0.18	0.68	0.46	0.72	0..40
	mean	0.25		0.50		0.46
100 mg/L	1	6.89	7.16	7.99	7.49	6.25	5.43
	2	8.60	7.51	7.64	7.52	7.87	6.66
	mean	7.54		7.66		6.55

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks"

Conclusions:

No toxic effects were observed in algae at a nominal test concentration of 100 mg/L. The 72-h ErC50 and 72-h ErC10 were determined to be >100 mg/L based on nominal concentrations (or > limit of solubility in test medium).

Executive summary:

The toxicity to algae was examined in a study according to OECD TG 201 and in compliance with GLP criteria. Cultures of green algae (S. subspicatus) at a starting cell concentration of ca. 1E+04 cells/mL were exposed to nominal concentrations of 0 (control) and 100 mg/L for 72 hours under static conditions and continuous illumination. No analytical determination of the concentrations of the dissolved test item could be performed due to the very low water solubility of the test item and the low sensitivity of the analytical method. For these reasons the test was not performed with filtrates of a stock suspension, but, in order to secure that the maximum bioavailable concentration is maintained during the test period, with a suspension of the test item above the water solubility limit. Therefore, the test medium was prepared with the emulsifier Tween 80. A solvent control was tested in parallel. For determination of the maintenance of the nominal test item concentration during the test period, quantification of the test medium was performed spectrophotometrically by VIS-detection at 250 nm at the start of the test and after 72 hours (in test solutions without algae). The mean recoveries found in the treatment samples were 102% at the start of the test, and 95% after 72 hours, exposure indicating that the test substance was nearly stable during the performance of the biological test. At the end of the test, algae in the test medium were partially aggregated with substance particle and therefore were treated ultrasonically before determining the algal cell densities. Possible disappearance of the test substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. Therefore, it was concluded that the concentration of the substance tested was satisfactorily maintained within ± 20 % of the nominal concentration, and the results are expressed as nominal.

The mean algal cell densities in the test medium were identical to, or even slightly higher than in the parallel solvent control cultures. Thus, the test item had clearly no inhibitory effect on the growth of algae during the exposure period of 72 hours. Based on these findings, both the 72 -h ErC50 and the 72-h ErC10 are determined to be >100 mg/L based on nominal concentrations (or > limit of solubility in test medium).