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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 and 2 September 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not specified in the study report.
Analytical monitoring:
no
Details on sampling:
Appropriate concentrated solutions of the test substance were prepared in THF and aliquots (2 ml) were added to empty test vessels (1-litre glass beakers).
Vehicle:
yes
Details on test solutions:
The results of a preliminary solubility trial showed that ADK STAB HP-10 was insufficiently soluble in water to allow the preparation of an aqueous stock solution, but was soluble in tetrahydrofuran (THF; Fisher Scientific UK, Code no. T/0706, Batch no. 9916283169). Appropriate concentrated solutions of the test substance were prepared in THF and aliquots (2 ml) were added to empty test vessels (1-litre glass beakers). The THF was then evaporated using a gentle stream of nitrogen to leave the test substance deposited on the walls of the beakers, which were left to stand for one hour and then reflushed with nitrogen to remove any residual THF. Dechlorinated tap water (284 ml) was added and the mixtures were treated with ultrasound for 10 minutes.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
PREPARATION OF THE MICROBIAL INOCULUM: A sample of activated sludge was obtained the day before the start of the test from Oakley Sewage Treatment Works, which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required.
The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, aliquots (25 ml) of the activated sludge were filtered through dried and preweighed Whatman GFC filter papers which were 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 and adjusted to 4gll by the removal of supernatant. Synthetic sewage (50 ml/l) was added and the mixture aerated overnight.
On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 μ/l by the removal of supernatant. The pH of the sludge was also measured.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
200 - 250 mg/1 as CaC03
Test temperature:
19.0 – 21.1°C
pH:
7.4 - 8.2
Dissolved oxygen:
Not applicable - respiration rate study
Salinity:
Not applicable - freshwater study
Nominal and measured concentrations:
Nominal concentrations
Details on test conditions:
PREPARATION OF SOLUTIONS OF THE REFERENCE SUBSTANCE (3,5-DICHLOROPHENOL): A concentrated solution of 3,5-DCP (500 mg/l) was prepared by dissolving 0.5 g in 10 ml of 1N sodium hydroxide and diluting to approximately 30 ml with ultrapure water. Sulphuric acid (1N) was added to the point of incipient precipitation and the solution made up to a final volume of one litre with ultrapure water. The pH of this solution was then measured.
Nominal concentrations of 3.0, 10.0 and 32.0 mg/l were prepared by dilution of this concentrated solution.

TEST METHODS: The results of a preliminary solubility trial showed that ADK STAB HP-10 was insufficiently soluble in water to allow the preparation of an aqueous stock solution, but was soluble in tetrahydrofuran (THF; Fisher Scientific UK, Code no. T/0706, Batch no. 9916283169). Appropriate concentrated solutions of the test substance were prepared in THF and aliquots (2 ml) were added to empty test vessels (1-litre glass beakers). The THF was then evaporated using a gentle stream of nitrogen to leave the test substance deposited on the walls of the beakers, which were left to stand for one hour and then reflushed with nitrogen to remove any residual THF. Dechlorinated tap water (284 ml) was added and the mixtures were treated with ultrasound for 10 minutes.
The prepared mixtures were aerated for three hours using a Pasteur pipette connected to a laboratory supply of oil-free compressed air.
Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (capacity; 270 ml) and its rate of oxygen consumption over a period of ten minutes was measured using a Yellow Springs Instruments (YSI) dissolved oxygen meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder.
The pH and temperature of the samples were measured at the start and end of the test.
Reference substance (positive control):
yes
Remarks:
3,5-DICHLOROPHENOL
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The specific respiration rate of the control culture established at the end of the test (38.5 mgO2/g/h) was 90% of the rate of that established at the start (42.9 mgO2/g/h).
These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition.
ADK STAB HP-10 had no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test.
Results with reference substance (positive control):
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 by the Moving Average method to be 10.4 mg/l (95% confidence limits 7.1- 15.5 mg/l).
Validity criteria fulfilled:
yes
Conclusions:
The EC20, EC50 and EC80 of the test substance could not, be calculated but these must be greater than 100 mg/l, the highest level tested.
Executive summary:

The effect of ADK STAB HP-10 on the respiration rate of activated sludge was assessed by the methods detailed in EC Directive 88/302, 'Biodegradation - Activated Sludge Respiration Inhibition test' and OECD Test Guideline 209, 'Activated Sludge, Respiration Inhibition test'.

Samples of activated sludge (suspended solids 1.6 g/l), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 1, 10 and 100 mg/l for three hours. Single mixtures were prepared at 1 and 10 mg/l and the highest level was prepared in triplicate. Their rates of oxygen consumption were determined and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of the culture series.

The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed at 3.0, 10.0 and 32.0 mg/l, as a positive control.

The specific respiration rate of the control culture established at the end of the test series

(38.5 mgO2/g/h) was 90% of the rate of that established at the start (42.9 mgO2/g/h). The three-hour 50% effect concentration (EC50) for 3,5 -DCP was calculated to be 10.4 mg/1 (95% confidence limits 7.1 - 15.5 mg/l). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition.

ADK STAB HP-10 had no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/l, the highest level tested.

Description of key information

Key value determined using EU Method C11 and OECD guideline 209.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

The effect of ADK STAB HP-10 on the respiration rate of activated sludge was assessed.

Samples of activated sludge (suspended solids 1.6 g/l), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 1, 10 and 100 mg/l for three hours. Single mixtures were prepared at 1 and 10 mg/l and the highest level was prepared in triplicate. Their rates of oxygen consumption were determined and compared with those of controls, containing activated sludge and synthetic sewage alone, which were established at the beginning and end of the culture series.

The reference inhibitor 3,5-dichlorophenol (3,5-DCP) was employed at 3.0, 10.0 and 32.0 mg/l, as a positive control.

The specific respiration rate of the control culture established at the end of the test series

(38.5 mgO2/g/h) was 90% of the rate of that established at the start (42.9 mgO2/g/h). The three-hour 50% effect concentration (EC50) for 3,5 -DCP was calculated to be 10.4 mg/1 (95% confidence limits 7.1 - 15.5 mg/l). These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition.

ADK STAB HP-10 had no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The EC20, EC50 and EC80 of the test substance could not, therefore, be calculated but these must be greater than 100 mg/l, the highest level tested.