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

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:

20-23 May 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The test was conducted according to OECD Guideline 201 and in compliance with GLP. The stock solutions were prepared by agitation for about 24 hours and then filtered twice (porosity of 4-7 µm and then of 0.45 µm). The filtered solution was then further used to prepare the test solutions of the range-finding study. The measured concentrations of test substance in these solutions were below the limit of quantification (0.01 mg/L) at all measurement times. Based on the fact that the substance is highly insoluble in water (< 3 µg/L) and has a high adsorptive potential (Koc > 5.6), it can only be concluded that the EC50 > maximum solubility of the test substance in medium.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

see the field Overall remarks

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

see the field Overall remarks

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At t=0, a sample of 5 mL was taken from the limit test solution container (LS solution) and kept at -20°C until analysis. At t=24, 48 and 72, samples were taken from each limit test solution replicate, pooled (5 mL in total) and kept at -20°C until analysis. Further samples were also taken at t-24, 48 and 72 hours from the limit test solution which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test.

Vehicle:

no

Details on test solutions:

The stock solution, for the limit and range-finding tests, was prepared by dispersing the test item directly in LC reconstituted water.
A loading rate of I00 mg/L was used to prepare the stock solution with 23.5 hours agitation. As test item particles were still in suspension after agitation, the stock solution was filtered through a filter of porosity 4-7 µm and then a filter of porosity 0.45 µm to remove the suspended fraction. The filtered solution, corresponding to the limit of solubility of the test item under the experimental conditions (LS), was then used to prepare the test solutions. Test solutions were prepared by further dilution of this LS solution with LC reconstituted water to provide a geometric series of concentrations: 0 mg/L, LS/1000, LS/100, LS/10 for the range-finding study and LS for the limit test.
Glass test vessels containing algae and test solutions (or dilution water in the case of the controls) were filled directly from the test solution containers immediately after preparation and test solutions remained unchanged throughout the study.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Far Sawrey, Ambleside, Cumbria, LA22 OLP, UK. Cultured at CIT.
- Age of inoculum (at test initiation): the number of cells in the week-old culture was counted and the quantity of algal pre-culture to be added to the fresh test medium calculated to give a test solution concentration of 1 x 10^4 cells/mL.
- Method of cultivation: the algae are cultured under sterile conditions and maintained at exponential growth rate. Algae are cultivated for 7 days before harvesting for use in a new culture. New cultures are loaded at a concentration of 1 x 10^4 cells/mL. Each weekly culture is prepared under sterile conditions using autoclaved medium while week old cell cultures are checked for contamination before being transferred to fresh medium.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes, except that cultures are constantly aerated with air filtered using a 0.22 µm porosity filter to maximize the CO2 availability (and thereby the cell growth) while maintaining the sterility.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

34 mg CaCO3/L

Test temperature:

22.9-24.3 ºC

pH:

7.7-9.4

Dissolved oxygen:

Not determined

Salinity:

Not applicable

Nominal and measured concentrations:

LS = limit of solubility obtained from an initial loading rate of 100 mg/L for the limit test
Measured concentrations in the LS solution were below the limit of quantification (0.01 mg/L) throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks, stoppered with sterile cotton wool wrapped with lint
- Aeration: no, only constant agitation
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 145 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 with algae, one without algae for the limit test; 2 for the range-finding test
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: see below

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water:
Final concentrations
calcium nitrate (Ca(NO3)2.4H2O) ............................................................ 40 mg/L
potassium nitrate (KNO3) .......................................................................... 100 mg/L
magnesium sulphate (MgSO4.7H2O) ......................................................... 30 mg/L
monohydrogen potassium phosphate (K2HPO4)......................................40 mg/L
copper sulphate (CuSO4.5H2O) ................................................................ .15 µg/L
ammonium heptamolybdate [(NH4)6Mo7O24.4H2O] .............................30 µg/L
zinc sulphate (ZnSO4.7H2O) ..................................................................... .30 µg/L
cobalt chloride (CoC12.6H2O) ................................................................... .30 µg/L
manganese nitrate (Mn(NO3)2.4H2O) ....................................................... .30 µg/L
citric acid (C6H8O2.H2O) ........................................................................... .30 µg/L
boric acid (H3BO3) ..........................................................................................30 µg/L
iron citrate (ill) (C6H5FeO7) .......................................................................0.616 mg/L
iron sulphate (ll) (FeSO4.7H2O) ................................................................ .0.3125 mg/L
iron chloride (Ill) (FeC13.6H2O) ................................................................ ..0.3125 mg/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: A set of 15 and 30 W Osram Fluora® fluorescent tubes between "white" and "daylight" type (spectral range 400 to 700 rnm) were set approximately 40 cm above the cultures. These emit light measured using a lux meter equipped with a spherical collector at a level corresponding to half the height of the cultures in their conical flasks (corresponding to an irradiance of 252 mW/m2 for 15 W and 476 mW/m2 for 30 W tubes x no. of bulbs = 3724 mW/m2). The light intensity at each position to be occupied by a culture flask is measured regularly (6760-7580 lux). The color temperature of the fluorescent lamps is between 4400 K and 5000 K.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): number of cells at t=24, 48 and 72 hours
- Determination of cell concentrations: counting chamber (Malassez cell counter). An observation of less than 1 x 10^4 cells/mL is below the sensitivity of the Malassez cell counter. Possible observations of this type were considered as 1 x 10^4 cells/mL - i.e. the test item was taken to be algaestatic at this test concentration rather than algaecidal, as no further experiment was undertaken to determine whether algae exposed to algaestatic concentrations were capable of growth post-exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Range finding study: Test concentrations: LS/1000, LS/100, LS/10

Reference substance (positive control):

no

Key result

Remarks on result:

other: EC50 (72h): > maximum soluble concentration of test substance in test medium

Key result

Remarks on result:

other: NOEC (72h): > maximum soluble concentration of test substance in test medium

Details on results:

Inhibition of the growth rate at LS was 14%, 0% and 1.5% relative to the control at 24, 48 and 72 hours, respectively.
The report concluded that the 72h EC50 was > and the NOEC >= 100 mg/L (initial loading rate). The French Authorities reported the 72h EC50 as > and the NOEC >= 0.01 mg/L (measured initial).

Results with reference substance (positive control):

None

Reported statistics and error estimates:

None.

The validity criteria of OECD 201 (2006) were checked by the reviewer:

- The biomass was increased exponentially by a factor of > 16 within the 72h test period.

- The mean coefficient of variation for section-by-section specific growth rates (0-24h, 24-48 h, 48 -72h) in the controls was 21% (and thus < 35%).

- the coefficient of variation of average specific growth rates during the whole test period in replicate controls was 2% (and thus < 7%).

Validity criteria fulfilled:

yes

Conclusions:

The 72 -h ErC50 is higher than the limit of solubility in test medium at a nominal loading rate of 100 mg/L.
The 72 -h NOErC is higher or equal to the limit of solubility in test medium at a nominal loading rate of 100 mg/L.

Executive summary:

The substance was tested in the algae growth inhibition test according to OECD 201 and following GLP principles. Under the conditions of the test, no effects were observed in the limit test, conducted with the filtrate of a 100 mg/L loading rate. The measured concentration of test substance was < LOQ throughout the test. It is thus assessed that the EC50 and NOEC are above the maximum solubility limit of the test substance in test medium.

Description of key information

The 72-h ErC50 is higher than the limit of solubility in test medium at a nominal loading rate of 100 mg/L.

The 72-h NOErC is higher or equal to the limit of solubility in test medium at a nominal loading rate of 100 mg/L.

Additional information

The substance was tested in the algae growth inhibition test according to OECD 201 and following GLP principles. Under the conditions of the test, no effects were observed in the limit test, conducted with the filtrate of a 100 mg/L loading rate. The measured concentration of test substance was < LOQ throughout the test. It is thus assessed that the EC50 and NOEC are above the maximum solubility limit of the test substance in test medium.