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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 April 2010 to 6 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-conform study according to OECD 209 guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
In the reference test, the respiration rates at the two highest treatment levels were assessed between 7.69 O2/L and 9.12 O2/L, which is outside the range of 6.5 mg O2/L to 2.5 mg O2/L that is recommended in the guideline.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples from test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
At start of exposure, samples were taken from each test vessel after preparation of the test solutions before adding the microbial inoculum.
At the end of exposure, samples were taken after the oxygen measurements. Appropriate volumes of the test solutions taken at the end of the exposure period were filtered using folded paper filters (pore size: 4 to 7 µm) to remove the microbial inoculum. The filtrates, and the homogenised solutions from the start of exposure were sent to the analytical test site for determination of the test concentrations. The retained solids were stored in the freezer as a reserve until potential further processing.
Vehicle:
no
Details on test solutions:
The test solutions were prepared without using a stock solution.
The test item concentration levels were prepared by stirring corresponding amounts of the test item in 284 mL of deionised water at room temperature in the dark for seven days before adding the synthetic sewage feed and the microbial inoculum. A magnetic stirrer was used which was set to 300 rpm in order to avoid settling of particles.
The controls were prepared by combining 284 mL of temperature adapted dilution medium (deionised water) with 16 mL of the synthetic sewage feed and adding the microbial inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was obtained from the sewage treatment works in frankfurt/Main Niederrad, Germany, treating predominantly domestic sewage. Activated sludge was washed with reconstituted water (according to OECD guideline) and stored for 4 days at 20 ± 2 °C. During storage 50 mL synthetic sewage feed were added per litre activated sludge and per day. The activated sludge was stirred and aerated during storage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
257.2 mg CaCO3/L
Test temperature:
20.3-20.6°C
pH:
6.2
Nominal and measured concentrations:
nominal added test item concentrations: 0-4.1-12.3-41-123-411 mg/L
nominal added bismuth concentrations: 0-3.0-9.0-30-90-300 mg V/L
measured dissolved bismuth concentrations: <0.3 mg/L, except for highest dose: 14.1 mg Bi/L.
Details on test conditions:
Storage time of the activated sludge before use: 3 days
Test vessels: 1000 mL glass beakers without cover
Amount of test mixture per test vessel: 500 mL
Volume of microbial inoculum per test mixture: 200 mL
Volume of synthetic sewage feed per test mixture: 16 mL
Mixed liquor suspended solids level in the microbial inoculum: 3.78 g/L
Mixed liquor suspended solids level in the final test mixtures: 1.51 g/L
Number of test concentrations: 5 plus control
Number of replicates per test item concentration: 2 plus one for chemical analysis
Number of replicates in the control: 2 plus one for chemical analysis
Aeration of test vessels: 40 to 50 litre of air per hour (ambient air, oil-free air-compressor)
Effect parameter measured: inhibition of respiration.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 300 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Bi
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: with inhibition around 17%
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
175.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Bi
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 127.3-209.1 mg Bi/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
341.2 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Bi
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 292.4-434.1 mg Bi/L
Details on results:
A concentration-related effect was observed only in the highest test item concentration level, where respiration compared to the controls was inhibited by approximately 17%. Since this inhibition was not statistically significant at p ≤ 0.05, EC50 values were not calculated.
3h-EC10 for respiration rate in activated sludge inoculum: 175.4 mg Bi/L (95%CL: 127.3-209.1)
3h-EC20 for respiration rate in activated sludge inoculum: 341.2 mg V/L (95%CL: 292.4-434.1)

The measured test concentrations throughout the test were outside the range of ± 20% (average recoveries of the test concentrations <10%) of the nominal test concentrations. Therefore the calculations of the biological effect concentrations were expressed based on the average recovery of the largest dose tested (4.7%):
3h-NOEC for respiration rate in activated sludge inoculum: ≥14.1 mg Bi/L
3h-EC10 for respiration rate in activated sludge inoculum: 8.2 mg Bi/L (95%CL: 6.0-9.8)
3h-EC20 for respiration rate in activated sludge inoculum: 16.0 mg V/L (95%CL: 13.7-20.4)
Results with reference substance (positive control):
In the reference test 4 concentration levels (1.8, 5.9, 18.8, 60 mg/L) were tested. The reference test was performed according to ECT's SOP A 16.1. A 3h-EC50 value of 8.5 mg/L was obtained. The EC50 value is within the accepted range of 5 to 30 mg/L as required by the test guideline.
Reported statistics and error estimates:
Welch-t test for inhomogeneous variances with Bonferroni-Holm adjustment was used to detect significant differences from the control (p ≤ 0.05)
Validity criteria fulfilled:
yes
Conclusions:
GLP-conform study according to OECD 209 guideline. A 3h-EC10 value for activated sludge respiration inhibition of 175.4 mg Bi/L is obtained and selected for the effects assessment.
Executive summary:

GLP study concucted according to OECD 209 guideline. A 3h-EC10 value for activated sludge respiration inhibition of 175.4 mg Bi/L is obtained and selected for the effects assessment.

Description of key information

The 3h-EC10 value for activated sludge respiration inhibition is 175.4 mg Bi/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
175.4 mg/L

Additional information

A GLP study was conducted according to OECD 209 guideline with bismuth subnitrate.

A 3h-EC10 value for activated sludge respiration inhibition of 175.4 mg Bi/L is obtained and selected for the effects assessment.