N,N''-(methylenedi-4,1-phenylene)bis[N'-(4-methylphenyl)urea]

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:

8 November 1999 - 18 April 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

1989

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Stable in water for at least 96 hours

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=72
Volume: 10 mL
Storage: Samples were stored in a freezer until analysis.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and the end of the test period. Additionally, reserve samples of 10 mL were taken from all test solutions and stored in a freezer until possible analysis for a maximum of three months or until delivery of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: the test substance was not completely soluble in test medium (in an earlier study at NOTOX solubility in water was determined to be < 9 µg/L). Preparation of test solutions started with ultrasonic treatment of a stock solution at 100 mg/L. Magnetic stirring was applied for three days prior to filtration of the dispersion through a paper filter (5 µm). The lower concentrations tested in the range-finding test were prepared by subsequent dilutions of the filtrate. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
- Controls: test medium without test item or other additives (blank-control), treated in the same way as the filtrate (three days of stirring and subsequent filtration of the dispersion through a paper filter (5 µm).

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4
- Source: not indicated

CULTIVATION:
- Method: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 +/-2°C.
- Light intensity: 4000-9000 lux
- Medium different from test medium: not indicated

- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium different from test medium: no, M2

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3 mg/L

Test temperature:

23 °C

pH:

8.0 - 9.3 throughout the test period

Nominal and measured concentrations:

Nominal test concentrations: 0.1, 1.0, 10 and 100% of a filtered solution prepared at 100 mg/L
Measured test concentrations: 0.884 and 0.939 mg/L at t=0 for samples of 100 mg/L with and without algae respectively; 0.261 and 0.174 mg/L at t=72 for samples of 100 mg/L with and without algae respectively
Effect concentrations were based on geometric mean measured concentrations for samples of 100 mg/L with algae (0.48 mg/L).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 159.6 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6 + 1 replicate of test concentration without algae
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: not indicated

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps of 30 Watt yielding 3000-3300 lux, with a light intensity within the range of 46 to 50 µE.m^-2*s^-1.

EFFECT PARAMETERS MEASURED: growth rate and yield at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. temperature of the medium every day in a temperature control vessel.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Lambda spectrophotometer with a cuvette of 5 cm path length. Algal medium was used as blank.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (January 2000)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.
- Effect concentrations exceeding solubility of substance in test medium: yes

- Due to low solubility of the test substance in test medium, toxic levels for fresh water algae could not be reached. Mean growth rate reduction (0-72 h) was 1%, 0.4%, 2.1% and 3.6% for 0.1, 1, 10 and 100% of filtrates prepared at 100 mg/L. These were considered biologically not relevant. For details on results, see attached background information.
- Individual pH and temperature values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.4 mg/L, 95% confidence interval: 1.0 - 1.8 mg/L
- Other: the value for ErC50 exceeded the range given by the ISO-standard (0.6 - 1.03 mg/L), therefore it can be concluded that the algal stock used was somewhat less sensitive to toxic substances than the standard algal stock used (the lower limit of the 95% confidence interval was within the ISO-standard).

Reported statistics and error estimates:

Statistical analysis of the data was performed by the two sample T-test (TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).

Validity criteria fulfilled:

no

Remarks:

For details on validity criteria see 'overall remarks' section.

Conclusions:

72h-ErC50 for the test substance exceeded the water solubility at a concentration prepared at a loading rate of 100 mg/L (i.e. exceeded a mean measured concentration level of 0.48 mg/L). The NOEC was the highest tested concentration.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at the following concentrations 0 (blank-control), 0.1, 1.0, 10 and 100% of a filtered solution prepared at 100 mg/L. Results of samples taken from the 100 mg/L filtrate for analyis at t=0 and t=72 were 0.884 mg/L and 0.261 mg/L, respectively. The EC50 for growth rate reduction (72h-ErC50) exceeded the geometric mean measured concentration in the 100 mg/L filtrate (0.48 mg/L). Concluded was that due to low solubility of the test substance, no significant toxic levels (i.e. > 10%) for freshwater algal could be reached.  

Description of key information

A reliable study performed according to OECD guideline 201 and GLP principles resulted in an ErC50 of >0.48 mg/L and a NOEC of 0.48 mg/L, the geometric mean measured concentration in the 100 mg/L filtrate. Mean growth rate reduction (0-72 h) was 1%, 0.4%, 2.1% and 3.6% for 0.1, 1, 10 and 100% of filtrates prepared at 100 mg/L.

Additional information

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at the following concentrations 0 (blank-control), 0.1, 1.0, 10 and 100% of a filtered solution prepared at 100 mg/L. Results of samples taken from the 100 mg/L filtrate for analyis at t=0 and t=72 were 0.884 mg/L and 0.261 mg/L, respectively. The EC50 for growth rate reduction (72h-ErC50) exceeded the geometric mean measured concentration in the 100 mg/L filtrate (0.48 mg/L). Concluded was that due to low solubility of the test substance, no significant toxic levels (i.e. > 10%) for freshwater algal could be reached.