4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 209, EEC Directive 87/302 No. L 133/118 "Respiration Inhibition Test" and in accordance with the Principles of Good Laboratory Practices (GLP)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302 No. L 133/118 "Respiration Inhibition Test"

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

yes

Details on test solutions:

5 g of MORTRACE SB CONC. was dissolved in 100 ml of acetone, resulting in a dark-brown stock solution. Aliquots of 1000, 500, 320, 200 and 100 µl were withdrawn from this stock solution under stirring, and the acetone was evaporated at room temperature in a stream of nitrogen. Then, 284 ml of bidistilled water was added to each residue. These mixtures were treated with an ultrasonic processor (Vibra-Cell VCX-600, Sonics and Materials Inc.) under cooling with ice for 2 x 5 minutes with a 2 to 3-minute break between sonication, resulting in lightly yellowish solutions with a brown precipitate.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The test was performed with aerobic activated sludge from a domestic waste water treatment plant (ARA Ergolz II, Fullinsdorf / Switzerland).
The sludge was centrifuged and the supernatant liquid phase decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre, were made up with Soerensen buffer (pH 7). To this mixture, 50 ml OECD recommended synthetic sewage feed per litre was added daily and the sludge was aerated until use. Before use, the pH was adjusted to 7.9 with diluted H2S04.
Synthetic sewage feed: 16g peptone, 11g meat extract, 3g urea, 0.7g NaCI, 0.4g CaCl2.2H2O, 0.2g MgSO4.7H2O and 2.8g K2HPO4 - filled up to a final volume of 1 litre with bidistilled water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Post exposure observation period:

not applicable

Hardness:

no information available

Test temperature:

The test was performed at 21 °C.

pH:

8.1 - 8.2

Dissolved oxygen:

oxygen consumption ranged between 0.329 - 0.392 mg/l

Salinity:

not applicable

Nominal and measured concentrations:

not applicable

Details on test conditions:

The test was carried out in glass flasks (>500 ml) aerated with an air-flow of about 0.2 I/minute instead of 0.5 I/minute to prevent possible foam development.
The flasks were filled according to the table provided. Samples were prepared in about 7-minute intervals. Thereafter, the pH of the samples was noted, and aeration started using compressed air at an air-flow rate of approximately 0.2 l per minute.
After 30 minutes of aeration at 21 °C, the first sample was taken for oxygen measurement over a period of up to 10 minutes. Thereafter, in 10-minute intervals, the rest of the samples was submitted to measurement of oxygen concentration.
Sampling was started with the control flask 1. Thereafter, the measurements were continued with the reference compound (flasks 2 -6), control flask 7 and the test article samples (flasks 8- 12). Finally, the oxygen consumption of control flask 13 was determined.
Oxygen consumption was measured aver a period at about 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The apparatus used for oxygen measurements was the WTW CXI 539 (WissenschaftlichTechnische Werkstaetten, Weilheim/Germany).
The pH was determined with the pH 90 (WTW Weilheim/Germany).

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The respiration rate of aerobic waste water bacteria was not inhibited (-2.2 %) when exposed to the lowest test concentration of 10 mg/I. At 20 mg/i, 32 mg/I, 50 mg/i and 100 mg/I, the respiration rate was slightly inhibited by 7.4%, 5.4%, 4.6 % and 10.4 %, respectively. Based on these results the EC5O (30 minutes) can be defined as >100 mg/I.

Results with reference substance (positive control):

The respiration rate of the activated sludge was practically not inhibited (2.5 %) at the lowest concentration of 1.0 mg/I. At 3.2 mg/I and 10 mg/I, the respiration rate was slightly inhibited by 7.0 % and 24.6 %, respectively. At 32 mg/I and 50 mg/I, the respiration rate was inhibited by 64.5 % and 76.8 %, respectively.
The EC50 (30 minutes) determined by the Logit model was calculated to be 21.1 mg/I with the 95 % confidence limits of 20.4 - 21.7 mg/I and the 95 % confidence limits for the slope were: 1.339 - 1.340 mg/I

Reported statistics and error estimates:

standard statistical methods were employed

None

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the EC50 (30 minutes) can be defined as >100 mg/I.

Executive summary:

The inhibitory effect of MORTRACE SB CONC. on aerobic waste water bacteria of activated sludge was investigated in a respiration test. This study examined nominal test concentrations in the range from 10 mg/l to 100 mg/l. The respiration rate of aerobic waste water bacteria was not inhibited (-2.2 %) when exposed to the lowest test concentration of 10 mg/l. At 20 mg/l, 32 mg/l, 50 mg/l and 100 mg/l, the respiration rate was slightly inhibited by 7.4 %, 5.4 %, 4.6 % and 10.4 %, respectively. Nevertheless, the EC50 (30 minutes) was determined to be >100 mg/l. The EC50 (30 minutes) for the reference compound 3,5 -dichlorophenol, which was tested under the same conditions as the test article, was found to be at 21 .1 mg/l and therefore lies within the range of 5 - 30 mg/I recommended by the test guidelines.

Description of key information

Based on an activated sludge, respiration inhibition test, the 30-minute EC50 for Mortrace SB Conc was determined to be > 100 mg/L, based on initial nominal loading rates of the test material. 

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

One microorganism study with Mortrace SB Conc was available for review. Based on the available data which indicated that the study was conducted under GLP and met the test validity criteria, a Klimisch score of 1 was assigned to this study. A 30-minute EC50 value of > 100 mg/L was reported for Mortrace SB Conc based on only slight levels of respiration inhibition (10.4%) at the highest tested concentration of 100 mg/L (initial nominal loading rate). Additionally, the respiration rate of aerobic waste water bacteria was not inhibited when exposed to the lowest test concentration of 10 mg/L, so 10 mg/L is the NOEC in this study.