Disodium 3-[[4-amino-9,10-dihydro-9,10-dioxo-3-[sulphonato-4-(1,1,3,3-tetramethylbutyl)phenoxy]-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

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:

November 02, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted April 4, 1984

GLP compliance:

no

Details on test solutions:

The test article was dissolved in distilled water at a concentration of 500 mg/I.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: aerobic activated sludge fron a domestic waste-water treatnent plant, ARA, Sissach/Switzerland.
- Pretreatment: the sludge was coarsely sieved, centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifuged. The procedure was repeated a further two times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (ml to its dry weight (g) detemined.
- Preparation: a volume of washed sludge suspension corresponding to 4.0 g dry material were made up with Soerensen buffer (pH 7) to 1 litre. To that mixture, 50 ml OECD recommended synthetic sewage feed were added.
- Incubation: overnight at 20 - 25 °C under aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21 °C

pH:

7.5 at the start of experiment

Nominal and measured concentrations:

Nominal 10 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: glass-flasks, more than 500 ml.
- Aeration: aeration with 0.2 l/minute.
- Sludge amount: 200 ml per vessel.
- Synthetic sewage amount: 16 ml per vessel.
- No. of vessels per concentration: 2 replicates.
- No. of vessels per control: 2 replicates.
- No. of vessels per positive control: 1 replicate per concentration.

EFFECT PARAMETERS MEASURED
Oxygen consumption was measured over a period of 10 rinutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The apparatus used for oxygen measurements was the WTW OXI 91(Wissenschaftlich-Technische Werkstaetten, Weilheim/Federal Republic of Germany).
The pH was determined with the pH 90 (WTW, Weilheim/FRG).

SAMPLING and ANALYSIS
After 180 minutes of aeration at 21 °C, the first sample was taken for oxygen measurement over a period of up to 10 minutes. Thereafter, in 15 minutes intervals, the rest of the samples were submitted to measurement of oxygen concentration.
Sampling was started with the control 1; thereafter, the measurements were continued with the positive controls and the test article samples. FinalIy, the oxygen consumption of control 2 was determined.

VALIOATION OF THE TESTS
The tests are validated by testing a known standard. i.e.3,5-dichlorophenol. The related IC50-value should be in the range of 5 - 30 mg/l.
Furthermore, the variation between the two control samples (start, end) should not be greater than 15 % of their mean.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorphenol

Duration:

3 h

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:

Due to aeration of the test mediun with test article, a foamy solution was obtained where mainly the foam was coloured. Under these conditlons, the respiration rate of the sludge was not inhibited (-2.4 %) at the hlghest concentrationts tested.

Results with reference substance (positive control):

At a concentration of the standard of 3.2 mg/l, the respiration rate was slightly inhibited to 16.7 %. The IC50 was found to be about 8.5 mg/l.
Almöst complete inhibition (92.9 %) was observed at 50 mg/l.

Influence of test item and positive reference oin oxygen consumption of activated sludge

Vessel	Oxygen consumption (mgO2/l min)	Inhibition (%)	Starting pH
Control	0.42	-	7.5
Control	0.42	-	7.5
Mean	0.42 ± 0 (< 0.01 %)	-
Test item 100 mg/l	0.43	-2.14	7.5
Test item 10 mg/l	0.43	-2.4	7.5
3 ,5-dichlorophenol 50 mg/l	0.03	92.9	7.5
3 ,5-dichlorophenol 10 mg/l	0.17	59.5	7.5
3,5-dichlorophenol 3.2 mg/l	0.35	16.47	7.5

Validity criteria fulfilled:

yes

Remarks:

IC50 of positive control was in the range of 5 - 30 mg/l and the variation between the two control samples was lower than 15 % of their mean

Conclusions:

IC50 (3h) > 100 mg/l (nominal)

Executive summary:

The inhibitory effect of test item on aerobic waste-water bacteria was investigated in a respiration test, according to the OECD guideline 209. Two concentrations were assayed, over a period of 3 hours: 10 and 100 mg/l, nominal. The test article showed no inhibition (-2.4 %) of the respiration rate up to a concentration of 100 mg/l. The IC50 for 3,5-dichlorophenol tested under the same conditions was about 8.5 mg/l.

Conclusion

IC50 (3h) > 100 mg/l (nominal)

Description of key information

IC50 (3h) > 100 mg/l (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

The inhibitory effect of Acid Violet 048 on aerobic waste-water bacteria was investigated in a respiration test, according to the OECD guideline 209. Two studies are available.

In the key study, two concentrations were assayed over the period of 3 hours: 10 and 100 mg/l, nominal. The test article showed no inhibition (-2.4 %) of the respiration rate up to a concentration of 100 mg/l.

In the second experiment, the nominal concentration for the definitive test was established on the basis of the range-finding study outcomes: the substance was tested at 1.0, 10, 100 and 1000 mg/l. 94 % of inhibition was recorded at the highested tested concentration and 78 % of inhibition was recorded at 100 mg/l. Thus, the limit test was conducted at 10 mg/l. During the definitive test, no inhibition occurred in the test vessels.

The effects recorded at 100 mg/l differ among the two experiments, thus the available data on Acid Violet 048 acid form has also been taken into consideration in order to better clarify the inhibitory effect potential.

The activated sludge respiration inhibition test on Acid Violet 048 acid form was conducted with a mixed population of activated sewage sludge microorganisms, over a period of 3 hours. Also in this case, the nominal concentration for the definitive test was established on the basis of the range-finding study outcomes: the substance was tested at 1.0, 10, 100 and 1000 mg/l. 95 % of inhibition was recorded at 1000 mg/l, 14 % of inhibition was recorded at 100 mg/l, 6 % of inhibition was recorded at 10 and 1 mg/l. Thus, the limit test was conducted at 100 mg/l.

The outcomes obtained into the experiment conducted with Acid Violet 048 acid form, agree with those obtained into the key experiment conducted with Acid Violet 048. It seems to be appropriate to conclude that the IC50 (3h) can be indicated as higher than 100 mg/l.