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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-11-24 to 1994-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

EC 92/69

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Analytical purity: >= 99.65 %
Lot/batch No.: 93.166

Analytical monitoring:

yes

Details on sampling:

Stock solutions of the desired strength are prepared by dissolving the test article in deionized water.The chosen test concentrations will be prepared by dilution of the stock solution. If high concentrations are tested, the test article is dissolved in the dilution water directly.
Test medium samples were taken at the start of the test (before introduction of the test animals) and at the end of the test. Samples were immediately deep-frozen and analysed later to determine actual concentrations.
Samples were thawed and filtered through Acrodisc LC 13 PVDF 0.45 µm syringe filter to remove any undissolved test article.

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 suspension of the test article with the auxiliary substance (DMSO - 50 mg/L) was stirred for 4 hours. The test article in excess was then filtered. The saturated solution obtained was used at the following concentrations:
- preliminary study: 25, 50, 75, 90 and 100 %;
- main study: 10, 50, 75 and 100 %;
100 % extract without auxiliary substance was also tested in the main study.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species, strain: Freshwater microcrustacea, Daphnia magna;
Supplier: Société d'Elevage Piscicole Contrôlé - La Grande Rivière - Saint Forgeux - France;
Age at initiation of treatment. less than 24 hours old;

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

NA

Hardness:

180 mg CaCO3/L

Test temperature:

19.3 - 20.9 °C

pH:

7.5 - 8.0

Dissolved oxygen:

8.4 - 8.6 mg/L

Salinity:

NA

Nominal and measured concentrations:

A 1000 mg/L suspension of the test article with the auxiliary substance (DMSO - 50 mg/L) was stirred for 4 hours. The test article in excess was then filtered. The saturated solution obtained was used at the following concentrations:
- preliminary study: 25, 50, 75, 90 and 100 %;
- main study: 10, 50, 75 and 100 %;
100 % extract without auxiliary substance was also tested in the main study.

Details on test conditions:

For each study, twenty daphnies were used per treated group, in comparison with an untreated control group and an auxiliary substance control group. Each group was constituted of four tubes, each containing 5 Daphnia for 10 mL of test medium (one tube contained 6 Daphnia).
Immobilisations were recorded about 24 and 48 hours after the start of the test.
Measurements of pH, dissolved oxygen and temperature were performed at the beginning and at the end of the test for each test medium.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobilisations:
The number of immobilisations noted was not proportional to the test article concentration. Only 10 % immobilisation was noted in the high concentration treated group without auxiliary substance (100 % extract).
Observation of the test media:
The extracts tested were colourless and limpid throughout the study.
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.
Further details: See Sect. "Remarks on results including tables and figures"

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

NA

Quality criteria:

The quality criteria were respected for the study:

- No immobilisations were noted in the control and auxiliary control groups.

- No control Daphnia was trapped at the surface of water.

- The oxygen concentration was higher than 2 mg/L.

- 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.

PRELIMININARY STUDY

 

Number of Daphnia per group at the start of study: 20

(except 100 % concentration: 21 daphnies).

 

Nominal Concentration (% extract)	Cumulated immobilizations
	24 hours		48 hours
	nb	%	nb	%
0 (Control)	0	0	0	0
Auxiliary control	0	0	0	0
25 %	0	0	15	75
50 %	0	0	19	95
75 %	1	5	14	70
90 %	1	5	6	30
100 %	1	5	3	14.3

nb : number

MAIN STUDY

Number of Daphnia per group at the start of study: 20

(except 100 % concentration: 21 daphnies).

 

Nominal Concentration (% extract)	Cumulated immobilizations
	24 hours		48 hours
	nb	%	nb	%
0 (Control)	0	0	0	0
Auxiliary control	0	0	0	0
10 %	1	5	1	5
50 %	0	0	17	85
75 %	0	0	2	10
100 %	0	0	3	14.3
100 * %	0	0	2	10

* : Without auxiliary substance.

CONCLUSION

 The immobilizations observed were not proportional to the test article concentration.

 

Despite the use of auxiliary solvent is recommended in the Guidelines to improve the solubility of the test article, it may not help the interpretation of the results. DMSO is not toxic to Daphnia at the concentration used, as proved by the auxiliary control group, but it may have contributed to obtain erratic results.

This is the reason why a group of Daphnia was treated without auxiliary solvent, at the maximum concentration of test article. 10 % immobilizations were obtained.

 

Therefore the results can be expressed as follows:

 

EC50, for Daphnia magna (nominal concentrations):

 

Time of exposure (hours)	EC50	Nominal concentration (% extract of a 1 g/l preparation)
24	higher than limit of solubility	up to 100 %
48	higher than limit of solubility	up to 100 %

 

EC0 (no observed effect concentration after 48 hours) : not determined

EC100 (lowest concentration causing 100 % immobilization after 48 hours) : not determined

 

Validity criteria fulfilled:

yes

Conclusions:

HAT ISO was tested for acute toxicity to Daphnia magna in a 48 hours static test. The immobilisations observed were not proportional to the test concentration. Thus the EC50/48 hours for Daphnia magna was higher than the limit of solubility. No classification and labelling is required according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT ISO was tested for acute toxicity to Daphnia magna in a 48 hours static test.

Immobilisations:

The number of immobilisations noted was not proportional to the test article concentration. Only 10 % immobilisation was noted in the high concentration treated group without auxiliary substance ( 100 % extract).

Observation of test media:

The extracts tested were colourless and limpid throughout the study.

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:

- No immobilisations were noted in the control and auxiliary control groups.

- No control Daphnia was trapped at the surface of water.

- The oxygen concentration was higher than 2 mg/L.

- 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.

CONCLUSION

 The immobilizations observed were not proportional to the test article concentration.

 

Despite the use of auxiliary solvent is recommended in the Guidelines to improve the solubility of the test article, it may not help the interpretation of the results. DMSO is not toxic to Daphnia at the concentration used, as proved by the auxiliary control group, but it may have contributed to obtain erratic results.

This is the reason why a group of Daphnia was treated without auxiliary solvent, at the maximum concentration of test article. 10 % immobilizations were obtained.

 

Therefore the results can be expressed as follows:

 

EC50, for Daphnia magna (nominal concentrations):

 

Time of exposure (hours)	EC50	Nominal concentration (% extract of a 1 g/l preparation)
24	higher than limit of solubility	up to 100 %
48	higher than limit of solubility	up to 100 %

 

EC0(no observed effect concentration after 48 hours) : not determined

EC100(lowest concentration causing 100 % immobilization after 48 hours) : not determined

Description of key information

EC50 was determined to be higher than the water solubility limit.

Additional information

HAT ISO was tested for acute toxicity to Daphnia magna in a 48 hours static test.

Immobilisations:

The number of immobilisations noted was not proportional to the test article concentration. Only 10 % immobilisation was noted in the high concentration treated group without auxiliary substance ( 100 % extract).

Observation of test media:

The extracts tested were colourless and limpid throughout the study.

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:

- No immobilisations were noted in the control and auxiliary control groups.

- No control Daphnia was trapped at the surface of water.

- The oxygen concentration was higher than 2 mg/L.

- 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.

CONCLUSION

The immobilizations observed were not proportional to the test article concentration.

 

Despite the use of auxiliary solvent is recommended in the Guidelines to improve the solubility of the test article, it may not help the interpretation of the results. DMSO is not toxic to Daphnia at the concentration used, as proved by the auxiliary control group, but it may have contributed to obtain erratic results.

This is the reason why a group of Daphnia was treated without auxiliary solvent, at the maximum concentration of test article. 10 % immobilizations were obtained.

 

Therefore, the results can be expressed as follows:

 

EC50, for Daphnia magna (nominal concentrations):

 

Time of exposure (hours)	EC50	Nominal concentration (% extract of a 1 g/L preparation)
24	higher than limit of solubility	up to 100 %
48	higher than limit of solubility	up to 100 %

 

EC0 (no observed effect concentration after 48 hours) : not determined

EC100 (lowest concentration causing 100 % immobilization after 48 hours) : not determined