Registration Dossier

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: OECD guideline, GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2005-12-16

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
batch number: 173 JG-1
description: gel (at receipt), yellow hard wax (analytical certificate)
received: 2006-12-12
storage: at room temperature
purity 99.7%
hydrosolubility: 674 g/L (20°C)
expiry date: 2008-01-03

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
concentration: 100 mg/L

Test solutions

Vehicle:
no
Details on test solutions:
Test system
Groups of test and reference suspensions and controls were set up as follows:
• two control replicates containing inoculum at 1.6 g/L,
• five test concentrations containing the test item AQUAXYL at 10, 31.6, 100, 316 or
1000 mg/L and inoculum at 1.6 g/L, with one replicate per concentration,
• three reference concentrations containing the reference item 3,5-dichlorophenol at 4, 12 or
36 mg/L and inoculum at 1.6 g/L, with one replicate per concentration.
The two control replicates were used to determine the background respiration rate of the sludge
and were not exposed to the test or reference items.

Test and reference suspensions and controls were prepared in test flasks and then immediately
aerated for 3 hours before being transferred to an oxygen measuring apparatus.
Measurement of oxygen concentration of the above suspensions was determined using an
oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test
or reference suspension which was agitated with a magnetic stirrer. The oxygen probe was
connected to a meter equipped with a chart recorder.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Type: activated sludge collected from a water treatment works containing
effluent from a predominantly domestic origin.
Origin: the water treatment plant of Evreux (France).
Reason for this choice: activated sludge is recommended in OECD guideline for this test.
Preparation: After sampling, the inoculum was left to settle and the main part of the supernatant was rejected. It was washed once with dechlorinated
water and then filtered (to remove the biggest particles).
Four samples of 50 mL were taken to determine the content in suspended matter (dry weight/v). The concentration of sludge (5.46 g/L) was then adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h

Test conditions

Hardness:
280 +/- 20 mg/L
Test temperature:
between 21 and 22°C
pH:
7.68
Details on test conditions:
Temperature: between 21°C and 22°C.
Illumination: no special precautions were taken.
Dilution water: dechlorinated tap water was used, with a hardness of 280 ± 20 mg/L
as CaCO3.
Duration of test: 3 hours.
Aeration: air was bubbled through each suspension at the rate of 0.5 to 1 L/min
until the start of oxygen concentration measurements.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
The highest test item concentration without significant effect (difference ≤ 15%) on the respiration rate, relative to mean of the controls, was ≥ 1000 mg/L.
Results with reference substance (positive control):
As the validity criteria were met (the difference between the respiration rates of the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 13.6 mg/L i.e. between 5 and 30 mg/L), the test was considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under our experimental conditions, the 3-hour EC50 of the test item for activated sludge respiration inhibition was > 1000 mg/L.
The highest test item concentration without significant effect on the respiration was ≥ 1000 mg/L.
Executive summary:

The effect of the test item on the respiration of activated sewage sludge was evaluated using a 3-hour static test according to OECD guideline No. 209 (4th April 1984).

All concentrations in dechlorinated water experienced in this test were lower than the water solubility of this test item.

The inhibition potential of the test item on the respiration rate of an activated sludge at 1.6 g/L in dechlorinated water was investigated using the following suspensions:

• two control suspensions (activated sludge alone),

• five test item suspensions: activated sludge and test item at concentration of 10, 31.6, 100,

316 or 1000 mg/L,

• three reference item (3,5-dichlorophenol) suspensions: activated sludge and the reference item

at concentration of 4, 12 or 36 mg/L.

The five test item concentrations were prepared by dilution of a stock solution at 2000 mg/L (pH: 7.68).

The oxygen consumption (i.e. respiration rate) of the test item, reference item and control suspensions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours.

The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and that of the reference item were determined considering the mean oxygen consumption of the controls as 100%.

As the validity criteria were met (the difference between the respiration rates of the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 13.6 mg/L i.e. between 5 and 30 mg/L), the test was considered valid.

The respiration rate of the test item suspension at the highest concentration (1000 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the four other test item suspensions (10 to 316 mg/L) was not determined.

The highest test item concentration without significant effect (difference ≤ 15%) on the respiration rate, relative to mean of the controls, was ≥ 1000 mg/L.

Under our experimental conditions, the 3-hour EC50 of the test item for activated sludge respiration inhibition was > 1000 mg/L.

The highest test item concentration without significant effect on the respiration was ≥ 1000 mg/L.