1,1'-(methylenedi-4,1-phenylene)bis(3-butylurea)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-04 to 2005-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 201 (EU C3) Limit test at 100 mg/L

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Analytical purity: 99.18 % (HPLC/PDA)
Lot/batch No.: 23.05.2005

Analytical monitoring:

yes

Details on sampling:

Because of the poor water solubility, the water-accommodated fraction (WAF) of the concentration to be tested was prepared. This was done by weighing the nominal load of 125 mg/L, adding the appropriate amount of deionised water and shaking for 24 hours. After membrane filtration, the solution was used to prepare the treatment. Since the solution is diluted by algal pre-culture and nutrient medium, the nominal amount multiplied with 1.25 was used for the preparation of the WAF.

Vehicle:

no

Details on test solutions:

Because of the poor water solubility, the water-accommodated fraction (WAF) of the concentration to be tested was prepared. This was done by weighing the nominal load of 125 mg/L, adding the appropriate amount of deionised water and shaking for 24 hours. After membrane filtration, the solution was used to prepare the treatment. Since the solution is diluted by algal pre-culture and nutrient medium, the nominal amount multiplied with 1.25 was used for the preparation of the WAF.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test System
Unicellular freshwater green alga.

Genus, Species Desmodesmus subspicatus
Strain CHODAT
Family Chlorophyceae
Order Chlorococcales

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in June 2005 by the „Collection of Alga“, Institut für Pflanzenphysiologie of Universität Göttingen. The alga are kept as stock culture on solid agar at 8 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

NA

Hardness:

NA

Test temperature:

23 - 24 °C

pH:

8.2 - 9.1

Dissolved oxygen:

NA

Salinity:

NA

Nominal and measured concentrations:

Nominal load: 125 mg/L;
Preparation of the WAF: see sect. "Details on test solutions" as above.

Details on test conditions:

Experimental Conditions
Vessels: screw cap cuvettes d=6mm
Duration: 72 hours
Temperature: 23 +/- 2 °C
Lighting: 8000 ± 2000 Lux
Control: deionised water with nutrient medium and algae
Treatments: test solution with nutrient medium and algae
4.00 mL of the test solution was filled into each test vessel. Then the mixture of the adjusted pre-culture and the concentrated nutrient solution (0.50 mL each, resulting in 1.00 mL) was added to the test vessels. The test vessels were incubated open and stirred.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Absorption Values
The absorption values were determined through photometric measurement. The values are given in the following table: See Sect. "Remarks on results incl. tables and figures"
Temperature, Light Intensity, pH
The pH values in the control ranged from 8.2 to 9.1. The light intensity was 8000 lux. Temperature range was 23 – 24 °C. The details are given in the following table: See Sect. "Remarks on results incl. tables and figures"
Cell Concentration
The measured absorptions were corrected by the colour of the test solutions. With the corrected absorption values, the cell concentration was calculated. They are given in the following table: See Sect. "Remarks on results..."
Area under the Curve, Growth Rate, Yield
From the cell concentration, the Area under the Curve AUC, the Growth Rate µ and the Yield were calculated. The values with their corresponding means and standard deviations are given in the following table: See Sect. "Remarks on results..."

 Main Study

Absorption Values

The absorption values were determined through photometric measurement. The values are given in the following table:

Table1‑a: Absorption Values

Nominal Concentration in mg/L	Absorption values
	0 h	24 h	48 h	72 h
0	1	12	71	292
	1	13	75	297
	1	9	70	285
	1	6	65	256
	1	8	67	253
	1	8	66	244
100	1	6	70	289
	1	8	76	306
	1	7	72	303
	1	9	75	313
	1	8	85	309
	1	7	70	289

 

For t = 0 h, the absorption values were calculated from measurement of the pre-culture and the dilution factor.

Temperature, Light Intensity, pH

The pH values in the control ranged from 8.2 to 9.1. The light intensity was 8000 lux. Temperature range was 23 – 24 °C. The details are given in the following table:

Table1‑b: Temperature, Light Intensity and pH

	0 h	24 h	48 h	72 h
Temperature [°C]	23	23	24	23
Light intensity [lux]	8000	8000	8000	8000
pH (control)	8.2	-	-	9.1

 

 Cell Concentration

The measured absorptions were corrected by the colour of the test solutions. With the corrected absorption values, the cell concentration was calculated. They are given in the following table:

Table1‑c: Cell Concentration in mL^-1

Nominal Concentration in mg/L	Cell concentration in mL^-1
	0 h	24 h		48 h		72 h
			Mean (± SD)		Mean (± SD)		Mean (± SD)
0	6222	74664	58072 (± 16540)	441762	429318 (± 23281)	1816824	1687199 (± 141633)
	6222	80886		466650		1847934
	6222	55998		435540		1773270
	6222	37332		404430		1592832
	6222	49776		416874		1574166
	6222	49776		410652		1518168
100	6222	37332	46665 (± 6526)	435540	464576 (± 35124)	1798158	1875933 (± 63665)
	6222	49776		472872		1903932
	6222	43554		447984		1885266
	6222	55998		466650		1947486
	6222	49776		528870		1922598
	6222	43554		435540		1798158

SD = standard deviation

Area under the Curve, Growth Rate, Yield

From the cell concentration, the Area under the Curve AUC, the Growth Rate µ and the Yield were calculated. The values with their corresponding means and standard deviations are given in the following table:

Table1‑d: Growth Rate, AUC and Yield

Nominal Concentration in mg/L	Growth Rate [day^-1]		AUC [Cell Concentration in mL-1*day]		Yield [Cell Concentration in mL-1]
	Value	Mean (±SD)	Value	Mean (±SD)	Value	Mean (±SD)
0	1.89	1.87 (± 0.03)	1409283	1315435 (± 107506)	1810602	1680977 (± 141633)
	1.90		1455948		1841712
	1.88		1362618		1767048
	1.85		1222623		1586610
	1.84		1238178		1567944
	1.83		1203957		1511946
100	1.89	1.90 (± 0.01)	1356396	1433653 (± 66852)	1791936	1869711 (± 63665)
	1.91		1459059		1897710
	1.90		1418616		1879044
	1.92		1480836		1941264
	1.91		1524390		1916376
	1.89		1362618		1791936

SD = standard deviation

Inhibition

The following inhibition values were calculated for µ, AUC and Yield:

Table1‑e: Inhibition Values in %

Nominal Concentration in mg/L	Mean Inhibition in %
	µ	AUC	Yield
0	0	0	0
100	-2	-9	-11

Validity criteria fulfilled:

yes

Conclusions:

HAT ISO was treated in a static 72hr testing for toxicity to algae in a limit test with nominal concentration of 100 mg/L of test item. The NOEC 72 h was determined to be 100 mg/L, the LOEC was determined to be > 100 mg/L. No classification and labelling is necessary according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT ISO was treated in a static 72hr testing for toxicity to algae in a limit test with nominal concentration of 100 mg/L of test item.

For the treatment at a nominal concentration of 100 mg/L, it was tested whether the differences between treatment and control were significant. The values for the area under the curve, yield and the growth rate were used for the assessment of observed effect levels.

In the following table the values for the mean, variance and standard deviation are stated:

	Control			100 mg/L
	µ	AUC	Yield	µ	AUC	Yield
Mean	1.87	1315435	1680977	1.90	1433653	1869711
Standard Deviation	0.03	107506	141633	0.01	66852	63665
Variance	7.88E-4	1.16E+10	2.01E+10	1.30E-4	4.47E+09	4.05E+09

As the mean of the treatment is higher than the control for all three endpoints, no further statistical testing was necessary to determine the NOEC and the LOEC.

The nominal treatment concentration of 100 mg/L is stated as NOEC.

As treatment at a concentration of 100 mg/L was considered the NOEC, the LOEC is greater than 100 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993-11-24 to 1994-10-03

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Test material contaminated with solvent residue.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC 92/69

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

CAUTION

Analytical purity: >= 99.65 %;
- Purity test date: 1993-09-23;
- Lot/batch No.: 93.166;

Analytical monitoring:

yes

Details on sampling:

Each test medium will be sampled at the start and at the end of the test. Both the amount of test article incorporated into the algal biomass and the substance in solution will be taken into account. However, as determination of the test article concentration in the algal biomass may pose significant technical problems, concentration of the test article will be checked in the “non-inoculated treated” flask containing the highest test article concentration but without algae.
All samples will be immediately deep-frozen and analysed later to determine actual concentrations.

Vehicle:

yes

Details on test solutions:

Formulations of the test article were prepared as follows: Considering the low water solubility of the test article and to prevent impact of solid particles, an "extract" of the test article was prepared. A 1000 mg/L (preliminary study) or 2000 mg/L (main study) suspension of the test article was stirred for 24 hours. The test article in excess was then filtered. The saturated solution was the used as described in Sect. "Any other information on materials and methods incl. tables"

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Supplier. Aquarium de Trouville - France
TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: CHODAT 86.81 SAG;
- Source : Aquarium de Trouville, 17 rue de Paris, 14360 Trouville, France;
- Age of inoculum: about 72 hours old;
- Method of cultivation: axenic subcultures will be made at regular intervals in order to maintain the algae strain;


ACCLIMATION
- Acclimation period: 72 hours;
- Culturing media and conditions: same as test;
- Any deformed or abnormal cells observed: no;

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

NA

Hardness:

> 90 mg CaCO3/L

Test temperature:

19.9 - 21.5 °C (preliminary study)
19.8 - 22.3 °C (main study)

pH:

6.9 to 7.5 (preliminary study)
6.9 to 8.8 (main study)

Dissolved oxygen:

Not indicated

Salinity:

Not indicated

Nominal and measured concentrations:

Nominal concentrations as % extract of a 2 g/L water accomodated fraction: 1.4; 3; 7; 14; 32 (without algae);
Measured concentrations: No test article was detected in any of the study samples, the solubility being lower than the limit of detection of the analytical method (1 µg/mL).

Details on test conditions:

The algae culture were exposed to the test article for 72 hours.
Algae cell concentration was recorded about 24, 48 and 72 hours after the start of the test, by counting of living cells using the absorbance method.
Measurements of pH and temperature were performed at the beginning and at the end of the study.
Each test medium was sampled during the main study, at the start and at the end of the test, as well as a non-oculated test flask containing the highest test concentration but without algae. The samples were frozen immediately and analysed later to determine actual concentrations.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 18-24 %

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 54-108 %

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

ca. 6

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

ca. 14

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Inhibition rates:
See Sect. "Remarks on results incl. tables and figures"
Observation of test media:
All test media were limpid and colourless at the end of the study. The UV spectrum of the highest concentration showed no absorption of light between 400 and 685 nm.
Analysis of actual concentrations:
No test article could be detected, the actual concentrations being lower than the limit of detection of the analytical method (1 µg/mL).
Quality criteria:
The quality criteria were respected for the study:
- The cell density in the control cultures increased by a factor of at least 16 within three days.
- Despite no test article could be detected during analysis, any test article dissolved in the media was considered as stable because no other peak of possible degradation products appeared on the chromatograms.
For further details, see Sect. "Remarks on results incl. tables and figures.

Results with reference substance (positive control):

NA

- Inhibition rates:

 

Preliminary study

 

Nominal Concentrations ( % extract)	Inhibition rates
	growth curves IA	growth rates Iμ
1 %	44	19
10 %	67	32
50 %	76	49
70 %	67	36
100 %	74	39

 

Main study

 

Nominal Concentrations ( % extract)	Inhibition rates
	growth curves IA	growth rates Iμ
1.4 %	7	1
3 %	- 3	- 1
7 %	32	9
14 %	64	24
32 %	86	44

 

Growth curves: Comparison of areas under the growth curves (I_A)

Growth rates: Comparison of growth rates (Iμ)

CONCLUSION

 

From the results obtained under the experimental conditions employed, the conclusion can be expressed as follows:

 

EC50, for freshwater green algae (nominal concentrations*) :

 

From calculation using Probit method	Method
	Growth curves (IA)	growth rates (Iμ)
EC50 (% extract)	11 %	38 %
Confidence limits (95 %)	9 – 12 %	27 – 54 %

 

EC50, for freshwater green algae (nominal concentrations*) :

 

From experimental values:	Method
	Growth curves (IA)	growth rates (Iμ)
EC0** (% extract)	3 %	7 %

 

* : expressed as % extract of a 2 g/l water accomodated fraction.

** : an inhibition rate < 25 % was not considered as treatment-related.

Validity criteria fulfilled:

yes

Conclusions:

HAT ISO was tested in a 72 hours algae growth inhibition test. The test results showed that the EC50 of HAT ISO is below the solubility limit of HAT ISO. No classification and labelling could be performed according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT ISO was tested for toxicity to aquatic algae in a 72 h growth inhibition test. The test results showed that the EC50 value of HAT ISO is below the solubility limit of the substance. No inhibition effect which was caused by the test item could be detected. Due to contamination with solvent residues, the study was not evaluated for classification/labelling purposes.

Description of key information

NOEC was determined to be 100 mg/L, and LOEC to be >100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study

HAT ISO was treated in a static 72hr testing for toxicity to algae in a limit test with nominal concentration of 100 mg/L of test item.

For the treatment at a nominal concentration of 100 mg/L, it was tested whether the differences between treatment and control were significant. The values for the area under the curve, yield and the growth rate were used for the assessment of observed effect levels.

In the following table the values for the mean, variance and standard deviation are stated:

	Control			100 mg/L
	µ	AUC	Yield	µ	AUC	Yield
Mean	1.87	1315435	1680977	1.90	1433653	1869711
Standard Deviation	0.03	107506	141633	0.01	66852	63665
Variance	7.88E-4	1.16E+10	2.01E+10	1.30E-4	4.47E+09	4.05E+09

As the mean of the treatment is higher than the control for all three endpoints, no further statistical testing was necessary to determine the NOEC and the LOEC.

The nominal treatment concentration of 100 mg/L is stated as NOEC.

As treatment at a concentration of 100 mg/L was considered the NOEC, the LOEC is greater than 100 mg/L.

Supporting study

HAT ISO was tested for toxicity to aquatic algae in a 72 h growth inhibition test. The test results showed that the EC50 value of HAT ISO is below the solubility limit of the substance. No inhibition effect which was caused by the test item could be detected. Due to contamination with solvent residues, the study was not evaluated for classification/labelling purposes.