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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 2012 to 19 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance was poorly soluble in the dilution medium. Therefore at test initiation weights of the test substance were added to the test bottles o give final, nominal concentrations of 10, 100 and 1000 mg/L.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: yes
- Method of cultivation: Synthetic sewage feed for activated sludge was prepared by dissolving the following per one litre of RO water: g/L
Peptone 16.0
Meat extract 11.0
Urea 3.0
Sodium chloride 0.7
Calcium chloride dihydrate 0.4
Magnesium sulphate heptahydrate 0.2
di-Potassium hydrogen phosphate 2.8
The pH of this solution was 7.5 ± 0.5.

- Preparation of inoculum for exposure: On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.


Test type:
static
Water media type:
other: Reverse osmosis water
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2ºC.
pH:
Synthetic sewage 7.39
3,5-DCP stock solution 7.24
Activated sludge 6.99
Nominal and measured concentrations:
Nominal: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: bottle
- Type: closed
- Material, size, headspace, fill volume: 500 mL bottles fitted with an oxygen probe
- No. of vessels per concentration: 5
- No. of vessels per control: 5
- No. of vessels per positive control: 1
- Suspended solids: 1.6 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: 10, 100 and 1000 mg/L
- Positive control concentrations: 3, 10 and 32 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 16.7 mg/L.
- The mean specific respiration rate of the control cultures established was 26.8 mgO2/gh with a coefficient of variation of 17.2%.
- These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.
- RIKABINOL HB did not significantly inhibit the respiration rates of the samples of activated sludge. The mean respirations rates of the sludge exposed to RIKABINOL HB was decreased, at most, by 2% at 1000 mg/L. Consequently the EC50 for inhibition could not be calculated but must be >1000 mg/L, the highest concentration tested in this study.
The no observed effect concentration (NOEC) for RIKABINOL HB was 1000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The three-hour EC50 for 3,5-DCP (16.7 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L). The validity criterion relating to the respiration rates in the control (specific respiration rate ≥20 mgO2/gh and a coefficient of variation of ≤30%) was also satisfied.


Reported statistics and error estimates:
Where possible, the EC20, EC50 and EC80 (and 95% confidence intervals) and no observed effect concentration (NOEC) of the test substance were calculated using SAFEstat programme SAS Proc NLIN (SAS Institute, 2002).
Validity criteria fulfilled:
yes
Conclusions:
RIKABINOL HB did not significantly inhibit the respiration rates of the samples of activated sludge. Consequently the EC50 for inhibition could not be calculated but must be >1000 mg/L, the highest concentration tested in this study.
The no observed effect concentration (NOEC) for RIKABINOL HB was 1000 mg/L.

Description of key information

3 Hour ASRIT:

EC50 > 1000 mg/L

NOEC 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

An Activated Sludge Respiration Inhibition Test (ASRIT) was conducted (Huntingdon Life Sciences Ltd, 2012, Study MOG0016) to determine the effect of HBPA on microorganisms. The test was conducted according to OECD test guideline 209, and in compliance with GLP.

Activated sludge used in the test was taken from a sewage treatment plant predominantly treating domestic sewage. Cultures were exposed to HBPA at concentrations 10, 100 and 1000 mg/L for 3 hours.

HBPA did not significantly inhibit the respiration rates of the samples of activated sludge. The mean respiration rates of activated sludge exposed to HBPA was decreased, at most, by 2% at 1000 mg/L. The effect of HBPA on the respiration of activated sewage sludge micro organisms gave a 3 Hour EC50 > 1000 mg/L whereas the NOEC after 3 hours exposure was 1000 mg/L.