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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd to 23rd December 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
GLP compliance:
yes
Specific details on test material used for the study:
Off-white powder, 99.15% purity
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
dechlorinated tap water
Details on test solutions:
The results of a preliminary solubility trial showed that BMS 233101-01 was insufficiently soluble in water to allow the preparation of a suitable stock solution, so appropriate weights were added directly to test beakers. Dechlorinated tap water (284 ml) was added and the mixtures were treated with ultrasound for 10 minutes before the addition of remaining components of the mixture.
The prepared mixtures were aerated for three hours using a Pasteur pipette connected to a laboratory supply of oil-free compressed air

A concentrated solution of 3,5-DCP (500 mg/l) was prepared by dissolving 0.5 g in 10 ml of in sodium hydroxide and diluting to approximately 30 ml with ultrapure water. Sulphuric acid (IN) 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 organisms (species):
activated sludge of a predominantly domestic sewage
Details on 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 105oC 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/1) 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 g/1 by the addition of dechlorinated tap water. The pH of the sludge was also measured.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
18.7-21 C during the test
pH:
Before addition of sludge: 7.7 -7.8
After 3 hours of exposure: 8.2 - 8.4 (includes conditions for reference item)
Nominal and measured concentrations:
Nominal concentrations: 100mg
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass, 1 Litre, open bottles/vessels, fill volume: 500 mL
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the st
art was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the slu
dge flocs in suspension. Clean, oil-free air was used for air supply.
- No. of vessels per concentration (replicates): 5

Reference substance (positive control):
yes
Key result
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
Details on results:
Any observations that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):
Results with reference substance valid? yes

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 13.2 mg/l (95% confidence limits 10.4 -17.3 mg/l). The specific respiration rate of the control culture established at the end of the test (26.8 mg02/g/h)

was 104% of the rate of that established at the start (25.9 mg02/g/h). These results show that the test was valid and that the sample of activated sludge employed was

sensitive to inhibition. BMS 233101-01 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.

Validity criteria fulfilled:
yes
Conclusions:
Validity criteria fulfilled yes
1. Mean control oxygen uptake rate > 20 mg O2/g activated sludge in an hour (29 mg O2/g).
2. CV of oxygen uptake in controls < 30% (11%) 3. EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L for total respiration (estimated value: 12 mg/L).

BMS 233101-01 had no inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the test. The ECjo, ECso and EQo of the test substance could not, therefore, be calculated but these must be greater than 100 mg/1, the highest level tested.
The three-hour EC5o for 3,5-DCP (13.2 mg/1) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/1), and that relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.
Executive summary:

The effect of BMS 233101-01 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/1), fed with synthetic sewage, were exposed to the test substance at nominal concentrations of 1, 10 and 100 mg/1 for three hours. Single mixtures

were prepared at 1 and 10 mg/1 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/1, as a

positive control.

The specific respiration rate of the control culture established at the end of the test series (26.8 mgCVg/h) was 104% of the rate of that established at the start (25.9 mgCVg/h). The three-hour

50% effect concentration (ECso) for 3,5-DCP was calculated to be 13.2 mg/1 (95% confidence limits 10.4 - 17.3 mg/1). These results show that the test was valid and that the sample of activated sludge

employed was sensitive to inhibition. BMS 233101-01 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/1, the highest level tested

Description of key information

BMS 233101-01 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/1, the highest level tested

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information