Coupling reaction products of 3,3’-dichlorobiphenyl-4,4’-bis(diazonium) dichloride with N-(2,4-dialkylphenyl)-3-oxobutanamide and potassium 4-[(3-oxobutanoyl)amino]substituted benzene and their basic aluminium and earth alkali metal salts

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar 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 (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): S-500
- Physical state: yellow powder
- Lot/batch No.: MF1456-2345
- Expiration date of the lot/batch: 18 August 2011
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable

Sampling and analysis

Analytical monitoring:

not required

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch of S-500 was a yellow powder that was not completely soluble in test medium at the concentration tested. The test concentrations were prepared separately in Milli-RO water. Initial loading rates of 200 mg/l were treated with ultrasonic waves for 5 minutes, followed by a stirring period of approximately 24 hours in the test bottles. Subsequently, synthetic sewage feed and sludge were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

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 7.8 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. The pH and temperature were determined.

Test conditions

Hardness:

Not applicable.

Test temperature:

18.5-18.9ºC

pH:

8.3

Dissolved oxygen:

measured and recorded for approx. 10 minutes; was between 7.5 and 8.0 mg O2/liter

Salinity:

Not applicable.

Nominal and measured concentrations:

a loading rate of 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass, approximately 300 ml oxygen bottles and 1 litre test bottles.
- 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: continuously during exposure with clean, oil-free air, no aeration during measurement.
- No. of organisms per vessel: 200 mL activated sludge (4.0 g/L suspended solids) in 500 mL final volume
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): two controls without test substance were tested in each test series, one at the start and one at the end.
- Biomass loading rate: 200 mL activated sludge (4.0 g/L suspended solids) in 500 mL final volume


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
- Test medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l


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.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Values of 1 and 8% inhibition at a loading rate of 100 mg/L
The EC50 of S-500 exceeded a loading rate of 100 mg/l (based on nominal concentrations).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50 for the reference substance was within the acceptable limits of 5-30 mg/L, with 36% inhibition at 5 mg/L and 76% inhibition at 32 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test, S-500 was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l. Hence, the EC50 of S-500 exceeded a loading rate of 100 mg/l (based on nominal concentrations).