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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the OECD 209 Guideline and following GLP principles. All validity criteria of the Guideline 209 (1984) were accepted.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

The test solutions at 31.6, 100, 316 and 1000 mg/L were prepared by direct addition of the test item in the test vessels and vigorous agitation of these solutions for a few minutes before the beginning of the test (i.e. just before addition of the inoculum).
The test solution at 10 mg/L was prepared by means of a suspension at 20 mg/L in dechlorinated water because the test item quantity necessary for direct addition to the test flask was too low to be accurately weighed(< 10 mg). The suspension was agitated for 1 hour and 40 minutes in order to solubilize as much of the test item as possible and obtain an homogeneous suspension. The test solution was prepared immediately by further dilution of this suspension, maintained under agitation, with dechlorinated water.
A reference stock solution was prepared by dissolving 0.5 g of the reference item, 3,5-dichlorophenol, in 10 mL of NaOH at 1 N. The obtained preparation was then diluted to 30 mL with purified water. Aliquots of H2S04 at 1 N were added while stirring until the point of incipient precipitation was obtained (approximately 8 mL of H2S04 at 1 N were required). The solution was made up to 1000 mL using purified water. The reference stock solution was agitated for 18 hours. Reference solutions were prepared by further dilution of the stock solution with dechlorinated water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: the water treatment plant of Evreux (France)
- Preparation of inoculum for exposure: the inoculum was left to settle and the supernatant rejected. It was then filtered (to remove the biggest particles) and washed with dechlorinated water. Four samples of 50 mL were taken to determine the content in suspended matter (dry weight). The concentration of sludge (2.6 g/L) was then adjusted by allowing it to form floccules in the stock flask (by stopping aeration) and then rapidly decanting off a portion of the supernatant until a predetermined volume was reached. The quantity of suspended matter was 4g/L± 10%.
- Pretreatment: The inoculum was maintained under agitation for 1 day before the test. As the sludge was not used immediately, 50 mL of sewage feed were added, per liter, just before agitation. Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.
- Initial biomass concentration: 1.6 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

280 +/- 20 mg/L as CaCO3

Test temperature:

21 -22 ºC

pH:

The pH of the test solution at 1000 mg/L was 7.7.
The pH of the stock solution at 20 mg/L was 7.9 after agitation.
The pH of the reference stock solution was 7.7 after agitation.

Dissolved oxygen:

Dissolved oxygen concentrations are not reported, only respiratory uptake in mg O2/L h.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations of 10, 31.6, 100, 316 and 1000 mg/L
All concentrations of KY-UN in this test were higher than the water solubility of this item and should therefore be considered as loading rates (solutions including the dissolved and undissolved fractions of the item).

Details on test conditions:

TEST SYSTEM
- Test vessel: opaque BOD flask
- Aeration: air was bubbled through each solution at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechloriniated water

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of inoculum was adjusted, if necessary, to 6.0- 8.0 using NaHC03 or HCI.
- Photoperiod: not stated
- Light intensity: no special precautions were taken

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Measurement of oxygen concentration of the above solutions was determined using an oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test or reference solution which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter equipped with a chart recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

See attached Table 1.

Results with reference substance (positive control):

The 3h-EC50 was determined to be 11 mg/L (95% CI 9.24-13 mg/L)

Validity criteria fulfilled:

yes

Remarks:

see "Overall remarks'.

Conclusions:

The substance is not toxic to STP microorganisms up to 1000 mg/L.

Executive summary:

The effect of the test substance on the respiration rate of microorganisms was assessed according to the OECD 209 and following GLP principles. Under the experimental conditions, the 3-hour EC50 of the test item KY -UN is > 1000 mg/L (solution including the dissolved and undissolved fractions of the item) for activated sludge. The test item should be considered as non-toxic for the micro-organisms of a water treatment plant.

Description of key information

Based on the results of an activated sludge respiration inhibition study, the substance is not toxic to STP microorganisms up to 1000 mg/L.

Additional information

The effect of the test substance on the respiration rate of microorganisms was assessed according to the OECD 209 guideline and following GLP principles. Under the experimental conditions, the 3-hour EC50 of the test item KY -UN is > 1000 mg/L (solution including the dissolved and undissolved fractions of the item) for activated sludge. The test item should be considered as non-toxic for the micro-organisms of a water treatment plant (as at 1000 mg/L only 9% inhibition was observed).