Dodeca (lithium, sodium)-2-({4-[4-(4-{bis[alkyl-(sulfonatoalkoxy)-4-({-sulfonato-[(substituted-phenyl)diazenyl]phenyl} diazenyl)anilino]-triazin-yl}-6-[alkyl-(sulfonatoalkoxy)-4-(sulfonato--[(substituted-phenyl)diazenyl]phenyl)diazenyl)anilino]piperazin-yl)-triazin-ylamino]-alkyl-5-(sulfonatoalkoxy)phenyl}diazenyl)-5-[(sulfonatophenyl)diazenyl]benzensulfonate

Toxicity to microorganisms

Administrative data

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:

other: The study was performed according to OECD guidelines and follwoing GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): E-BW102
- Substance type: reddish brown powder
- Physical state: solid
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: 23/12/2015
- Storage condition of test material: at room temperature, in the dark

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The batch of E-BW102 tested was a reddish brown powder with a purity of 93.0% and indicated to be light sensitive. Preparation was as much as possible performed under low light conditions.

A stock solution of 0.5 g/l was prepared by adding 125.04 mg test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (5 minutes) was used to accelerate dissolution and to ensure homogeneity. Volumes of the dark red stock solution corresponding to the test concentration were then added to the test media.
A concentration of 100 mg/l was tested in duplicate.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (4.0 g/l of sludge, as used for the test). The pH was 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Oxygen consumption was measured and recorded for approximately 10 minutes.

Test conditions

Test temperature:

19.7 - 20.8 C

pH:

7.7 - 7.9

Dissolved oxygen:

6.2 - 7.4 mg/L

Nominal and measured concentrations:

A nominal concentration of 100 mg/L was tested in duplicate

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and 1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate:4 g/l of sludge as used for the test
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/l/hr, was
calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.
TEST CONCENTRATIONS
- Test concentrations: limit study at a loading rate of 100 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

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

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

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:

3 and 10% inhibition of respiration rate of the sludge was recorded at 100 mg E-BW102 per litre in the duplicate measurements, which was not significant. Therefore, no further testing was needed. Hence, the EC50 of E-BW102 exceeded 100 mg/l (based on nominal concentrations).

Results with reference substance (positive control):

Results with reference substance valid: yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing
concentration , ranging from a 38 % inhibition at 5.0 mg/l to 83% at 30 mg/l. Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 7.0 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test, E-BW102 was slightly inhibitory to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/l (3 and 10% inhibition of respiration rate of the sludge).
The 3-hour EC50 of E-BW102 exceeded a nominal concentration of 100 mg/l.

Executive summary:

The influence of E-BW102 on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 

The study procedures described in this report were based onthe OECD guideline No. 209, 1984.In addition, the procedures were designed to meet the test methods of theCouncilRegulation (EC) No. 440/2008 of, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

 

The batch of E-BW102 tested was a reddish brown powder with a purity of 93.0% and indicated to be light sensitive. Preparation was as much as possible performed under low light conditions.

 

A stock solution of 0.5 g/l was prepared by adding 125.04 mg test substance to 250 ml of Milli-RO water. Thorough mixing (5 minutes) was used to accelerate dissolution and to ensure homogeneity. An amount of synthetic sewage feed (16 ml), activated sludge (200 ml) and an adequate amount of the dark red stock solution were mixed and made up to 500 ml with Milli-RO water in a 1-litre bottle. A concentration of 100 mg/l was tested in duplicate. Two controls without test substance were tested in each test series. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.

 

No significant inhibition of respiration rate of the sludge was recorded at 100 mg E-BW102 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC₅₀of E-BW102 exceeded 100 mg/l (based on nominal concentrations).

The batch of activated sludge was checked for sensitivity by testing the reference substance

3,5-dichlorophenol.