1,3'-Bipyridinium, 5'-[[4-[[4-[(1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo[1,3'-bipyridinium]-5'-yl)azo]benzoyl]amino]phenyl]azo]-1',2'-dihydro-6'-hydroxy-3,4'-dimethyl-2'-oxo-, salt with 2-hydroxypropanoic acid (1:2)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The test substance is the acid form of the substance under registration; justification for Read Across is detailed in the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted on April 4th, 1984

GLP compliance:

no

Details on test solutions:

The test article (0.5 g) was suspended up to 1000 ml in bidistilled water and sonicated for about 10 minutes (red suspension with precipitation).

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source: the tests were run with aerobic activated sludge from a domestic waste-water treatment plant (ARA, Sissach/Switzerand).
- Preparation of inoculum for exposure: the sludge was coarsely sieved, centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifuged. This 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) determined. Based on this ratio, 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. Thereafter, the sludge was aerated at 20 - 25 °C until use. The pH measured was 7.7.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

22 °C

Nominal and measured concentrations:

10 and 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass-flasks, > 500 ml.
- Aeration: aerated with an air-flow of about 0.2 l/minute instead of 0.5 l/minute to prevent possible foam development. Aeration performed with a "Pasteur" pipette.
- No. of vessels per concentration: test item + sewage + sludge + water, one replicate per concentration.
- No. of vessels per control: sewage + sludge + water, two replicates.
- No. of vessels per positive control: 3,5-dichlorophenol + sewage + sludge + water, one replicate per concentration.
- Sludge amount: 200 ml.
- Nutrients provided for bacteria: 16 ml synthetic sawage.
- Preparation of samples: samples were prepared in 15 minutes intervals. Thereafter, the pH of the samples was noted.

EFFECT PARAMETERS MEASURED
Oxygen consumption was measured over a period of 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 UXI 91 (Wissen- schaftlich-Technische Werkstaetten, Weilheim/Federal Republic of Germany). The pH was determined with the pH 90 (WTW, Weilheim/FRG).
The inhibitory effect of the test article on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean of the two control respiration rates.

VALIDITY OF THE TEST
The tests are validated by testing a known standard, i.e. 3,5-dichlorophenol. Its IC50-value should be in the range of 5 - 30 mg/l.
Furthermore, the variation between the two control samples should not be greater than 15 %.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (tested concentrations: 3.2, 10 and 50 mg/l)

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

Results with reference substance (positive control):

At a concentration of the standard of 3.2 mg/l, no inhibition of the respiration rate was observed (-7.6 %). Almost complete inhibition (89.2 %) was observed at 50 mg/l. The IC50 was found to be about 13.7 mg/l.

Influence of 3,5 -dichlorophenol sodium salt on oxygen consumption of active sludge

Flask	Conc. of test chemical	Oxygen consumption (mg O2/l/min)	Inhibition %	Startinh pH
Blank control	--	0.97	--	7.8
Blank control	--	1.01	--	7.8
Mean		0.99
Deviation		3.30 %
3,5-dichlorophenol	50	0.11	89.2	7.8
3,5-dichlorophenol	10	1.07	-7.6	7.8
3,5-dichlorophenol	3.2	1.02	-2.8	7.8

Validity criteria fulfilled:

yes

Remarks:

the variance between the control samples was 3.3 % and the inhibitory effect or 3,5-dichiorophenol was with 13.7 mg/l within the range of the IC50-value of 5 to 30 mg/l

Conclusions:

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

Executive summary:

The inhibitory effect of test item on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions. The exposure time of the test substance on the activated sludge was 3 hours.

The nominal concentrations of test item were 10 and 100 mg/l.

No chemical analysis of test concentrations was conducted. For the calculation of the effective concentration leading to 20, 50, and 80 % respiration inhibition (EC20, EC50, EC80) the nominal content of the test substance was used, assuming the test compound to be stable in water over 3 h.

At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

Conclusion

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

Description of key information

Not harmful/toxic to microorganisms.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

There are two old internal reports on Basic Orange 064 potential for short-term toxicity to fish. The test were performed according to ETAD 103 method. In both the cases, the IC50 values were reported to be higher then 100 mg/l. Unfortunately, only a sheet of the test results is available in both the cases, thus a reliability cannot be confirmed due to the absence of the testing procadures and results details.

In order to confirm and to assess the substance behaviour, the available data on the structural analogous Similar Substance 01 have been taken into consideration. Similar Substance 01 is the acid form of the Basic Orange 064; the read across approach can be considered as adequate and suitable to assess the toxicity potential to microorganisms of the substance under registration (details on read across approach are given in attachment in the IUCLID section 13).

The inhibitory effect of Similar Substance 01 on the oxygen consumption of activated sludge microorganisms was investigated under aerobic static conditions. The exposure time of the test substance on the activated sludge was 3 hours and the nominal concentrations assayed were 10 and 100 mg/l. For the calculation of the effective concentration leading to 20, 50, and 80 % respiration inhibition (EC20, EC50, EC80) the nominal content of the test substance was used, assuming the test compound to be stable in water over 3 hours. At the applied test concentrations, the test substance showed no significant toxic effects to the activated sludge.