N,N-bis(2-hydroxypropyl)benzamide

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:

From July 2nd to July 5th, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after exactly 3 hours of incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode and was continously recorded for about 10 to 15 minutes. During measurement, the samples were continously stirred ona magnetic stirrer.

Vehicle:

no

Details on test solutions:

Based on the results of a pre-test, the test item was soluble at a concentration of at least 5 g/L in tap water.
Therefore, a stock solution of test item with a nominal concentration of 5g/L was prepared: 1002 mg of the substance was dissolved in 200 mL of tap water and thoroughly mixed by intense stirring for ten minutes. A clear solution was obtained. Adequate volumes of this stock solution were diluted with tap water to prepare test media at different concentrations. The exact amounts used are given in the specific table attached as background material.

For test media containing the reference item a stock solution of 3,5-dichlorophenol was prepared according to test guideline: 0.5 g dissolved in 10 mL 1M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was determined to be 7.5, and the final concentration amounted to 500 mg/L. Aliquots of this stock solution were mixed with synthetic wastewater and tap water to obtain desired concentrations (5, 16 and 50 mg/L) in the corresponding test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge froma wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During holding, the sludge was fed daily for three days with 50 mL synthetic wastewater per liter. On the fourth day of holding, immediately prior to use, the sludge was fed once again with 25 mL synthetic wastewater per liter. During the entire holding period, the sludge was kept at room temperature under continous aeration. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge was adjusted from 8.6 to 7.8 with a diluted sulfuric acd solution.

Synthetic wastewater:
16g peptone
11g meat extract
3g urea
0,7g NaCl
0,4g CaCl2 * 2H2O
0,2g MgSO4 * 7H2O
2,8g K2HPO4

Ingredient were filled up to a final volume of 1 liter with deionized water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not specified

Hardness:

Not specified

Test temperature:

22 °C at the start of the experiment
19 °C after 3 hours

pH:

7.8-8.0 at the start of the experiment
8.8 after 3 hours

Dissolved oxygen:

Not less than 2.5 mg/L during incubation.
6.5-6.6 mg/L at the start of the experiment.
6.5-8.1 after 3 hours

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentration of test item: 0 (control), 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

The test was performed in 1000 mL glass flasks.
At the start of the test 200 mL of activated sludge inoculum with a sludge concentration of 3.8 g dry weight/L was added. The sludge was added at time intervals of 15 minutes first to a control, then to the test solutions of reference substance, then to test item solutions, and finally to the second control.
During the incubation period of exactly 3 hours all test media and the controls were continously aerated with compressed air at a flow approximately one liter per minute.

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol

Key result

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

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No significant inhibitory effect of test item was recorded at any concentration tested (always <15% inhibition).
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 14%.
Guideline-recommended maximum variation is 15%, therefore this validity criteria was fulfilled.

Results with reference substance (positive control):

3h EC50 of 3,5-dichlorophenol = 16 mg/L.
This value is within the range recommended in the guideline (5-30 mg/L).
The result is valid

Validity criteria fulfilled:

yes

Conclusions:

The substance is not acutely toxic to microorganisms at doses up to 1000 mg/L.
The 3-hour EC50 is higher than 1000 mg/L.

Executive summary:

The toxicity of the substance to aerobic wastewater microorganisms of activated sludge was investigated in a 3 -hour respiration inhibition test according to OECD TG 209.

The nominal concentrations tested were 10, 32, 100, 320 and 1000 mg/L.

In addition, two controls and three different concentrations of the reference substance 3,5 dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

The test item did not show any significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of 3 hours, at any dose tested. Therefore, EC50 of the substance on microorganism was considered to be higher than 1000 mg/L

Description of key information

In study according to OECD TG 209, the substance did not show any inhibitory effect on the respiration rate of activated sludge after 3 hours at concentrations up to 1000 mg/L.

Therefore, 3-h EC50 of the substance to microorganisms was considered to be higher than 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L