Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE
The test substance was soluble in water therefore a stock solution of 502.1 mg/l was prepared in Milli-Q water. A volume of 100 ml of prepared stock solution in 500 ml final volume corresponding to an actual concentration of 100 g/l being the maximum concentratio to be tested

CONTROL
A solution of 3,5-Dichlorophenolwas prepared by dissolving 128.80 mg in 10 ml 1N NaOH, diluted to approximately 30 ml with Milli-Q water and adding under stirring 1N H2SO4 to thje point of incipient precipitation. Finally the solution was diluted to one litre with Milli-Q water. The pH was 7.69.

Test organisms (species):

activated sludge

Details on inoculum:

-Source: municipal sewage treatment plant: 'Waterschap de Dommel 's-Hertogenbosch, the Netherlands.
-Number of micro-organisms: determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium
-Preparation: the sludge was coarsely sieved and washed with tap-water. A small amount of the sludge (250 ml) was weighed and dried at 100-105°C. From this result the amount suspended solids was calculated: the level was 4.0 g/l of sludge. The batch of sludge was used on subsequent days (maximum four days), therefore 50 ml of synthetic sewage feed was added to each litre of activated sludge at the end of each working day. The sludge was kept aerated at 20 ± 2 °C until use. Before use the pH was checked.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Test temperature:

20 ± 2 °C

pH:

7.5 - 7.6

Nominal and measured concentrations:

-test subatance: 100.4 mg/l
-control: 51.5, 33.0, 10.3, 3.3 and 1.0 mg/l

Details on test conditions:

TEST SYSTEM
-Test vessel: all glass, 500 ml beakers and 300 ml oxygen bottles.
-Material, size, fill volume: 184 - 284 ml of Milli-Q water
-Aeration: yes, Clean, oil-free air, Airflow 0.5-1 l/min


OTHER TEST CONDITIONS
-Oxygen meter: Oxygen electrode (Tri Ox EO 200, WTW, FRG) supplied with a recorder (Kipp BD40).
-Sodium bicarbonate solution: 70 g NaHCOs dissolved per litre Milli-Q water
-Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H20
0.2 g MgS04 .7H2O
2.8 g K2HPO4
Dissolved in 1 l Milli-Q water, filtered and adjusted to pH 7.5 ± 0.5. The nutrient solution could be used for one week if stored at 4 °C. Before use, it shall be mixed thoroughly and the pH adjusted.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

30 min

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No significant inhibition of the respiration rate of the sludge (<10 %) was recorded at 100.4 mg substance per litre (8.4%).
Acceptability of the test:
-The mean respiration rate of control 1 and 2 was 48.4 mg O2/l/hr. The difference between the controls is 4 %.
-The mean respiration rate of control 2 and 3 is 50.8 mg O2/l/hr. The difference between the controls is  5 %.
-The EC50 of 3,5 -Dichlorophenol was 11.37 mg/l  (95 % fiducial limits: 7.79 - 16.78  mg/l).
-Since tha substance did not inhibit the respiration of active sludge at the maximum concentration to be tested  (100 mg/l),  no further testing was required.

Results with reference substance (positive control):

At a concentration of the reference substance of 1.0 mg/l the respiration rate was inhibited to 10.9 %. The EC50 was 11.37 mg/l (95% fiducial limits: 7.79 - 16.78 mg/l). At 51.5 mg/l, the maximum concentration test the inhibition was 82.4%.

Validity criteria fulfilled:

yes

Conclusions:

No significant inhibition of the respiration rate of the sludge (<10%) was recorded at 100.4 mg per litre (8.4%)

Executive summary:

The inhibitory effect of the substance on aerobic waste-water bacteria was investigated in a respiration inhibition test, according to EEC Directive 67/548 amended November 18, 1987 (87/302), Part C: Methods for the determination of ecotoxicity, Publication No. L133, "Biodegradation: Activated sludge respiration inhibition test" (May 30, 1988). The oxygen concentration was measured after a contact time of 30 minutes with activated sludge fed with a standard amount of synthetic sewage feed. The test substance showed no significant inhibition (8.4 %) of the respiration rate at a concentration of 100 mg/l. Thus, the IC 50 (30 min) was >100 mg/l.

In conclusion, as the validity conditions were respected, the substance was found not to be toxic to waste-water bacteria.

Description of key information

IC50 (30 min) > 100 mg/l

Additional information

The inhibitory effect of the substance on aerobic waste-water bacteria was investigated in a respiration inhibition test, according to EEC Directive 67/548 amended November 18, 1987 (87/302), Part C: Methods for the determination of ecotoxicity, Publication No. L133, "Biodegradation: Activated sludge respiration inhibition test" (May 30, 1988). The oxygen concentration was measured after a contact time of 30 minutes with activated sludge fed with a standard amount of synthetic sewage feed.

The IC 50 (30 min) was found to be >100 mg/l. As the validity conditions were respected, the substance was found not to be toxic to waste-water bacteria.