Mixture of hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-5-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate and hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-7-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to EC, OECD and ISO guidelines and following GLP criteria.

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

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): E-BK105
- Substance type: black powder
- Physical state: solid
- Lot/batch No.: MB-1
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Stable
- Expiration date of the lot/batch: 23 December 2015

Specific details on test material used for the study:

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

Sampling and analysis

Analytical monitoring:

not required

Details on sampling:

not applicable

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 0.5 g/l was prepared by adding 124.39 mg test substance to 250 ml of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the dark blue
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:

- Preparation of inoculum for exposure:
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.

- Initial biomass concentration: 4.0 g/l of sludge

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

none

Test conditions

Hardness:

no data

Test temperature:

19.9 and 20.8°C.

pH:

7.7-7.8

Dissolved oxygen:

7.0 - 8.0 mg/l

Nominal and measured concentrations:

nominal: 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass, approximately 300 ml oxygen bottles and 1-litre test bottles.
- Type: closed
- Aeration: yes
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): procedure was repeated for the duplicate concentration. Two controls without test substance were tested in each test series, one at the start and one at the end.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tapwater purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)


OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Oxygen consumption was measured and recorded for approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH and temperature were determined in the remaining part of the reaction mixture.


TEST CONCENTRATIONS
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:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

No significant inhibition of respiration rate of the sludge was recorded at 100 mg E-BK105 per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of E-BK105 exceeded 100 mg/l (based on nominal concentrations).

Results with reference substance (positive control):

EC50 = 7.0 mg/l

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test, E-BK105 was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/l (EC50 > 100 mg/L; NOEC = 100 mg/L).