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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
n.a.
Vehicle:
no
Details on test solutions:
Appropriate amounts of the test substance were added directly to the different samples at the beginning of the incubation.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
For the preparation of the microbial inoculum activated sludge was used. The sludge was collected from one of the return sludge channels of the sewage treatment plant in Baden near Vienna. Baden is a spa with only a small amount of industry and the waste water of the sewage treatment plant is predominantly domestic.
A sample of activated sludge was collected on the day of the test. The container for transportation was only about 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (about one hour after sample collection) the sludge sample was aerated by means of compressed air.
The concentration of suspended solids was determined by filtering 5 mL of the sample through a pre-dried and pre-weighed glass microfibre filter. After drying and re-weighing the amount of solids was calculated. On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L by diluting the sludge sample with tap water. This suspension was used as inoculum for the samples. The inoculum was aerated until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None.
Hardness:
N.a.
Test temperature:
The temperature inside the samples was determined: 20.5 °C.
pH:
pH of the standardised activated sludge: 7.6, determined with a pH-meter.
pH at the end of the incubation: 8.0 to 8.2.
Dissolved oxygen:
The oxygen concentration was determined with a DO meter OXI 537 and an oxygen electrode.
Salinity:
n.a.
Nominal and measured concentrations:
Five concentrations of the test substance (nominal 19, 38, 75, 150 and 300 mg/L) were tested versus two negative controls (tap water).
The actual concentrations used in these test were 18.6, 39.0, 76.6, 153.4 and 301.4 mg/L.
As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (nominal 5, 15 and 45 mg/L).
Details on test conditions:
All incubation mixtures were prepared by mixing 200 mL of microbial inoculum and 200 mL of synthetic sewage solution with 100 mL of the appropriate dilution of the control substance (or 100 mL water and the appropriate amount of test substance) in glass beakers with a nominal volume of 1000 mL.
After mixing, the samples were aerated at a flow rate between 0.5 and 1.0 L/min using oil free compressed air and a Pasteur-pipette as aeration device. The incubation of the individual samples was started at intervals of 12-14 min and each sample was aerated for 3 hours at 20.5 °C. At the end of the incubation time aliquots of the samples were transferred to measuring bottles and oxygen consumption was recorded.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: 3,5-dichlorophenol
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The respiration rates and %-inhibition for the samples are found in the attachment.
The test substance did not inhibit the respiration in an activated sludge. The highest inhibition obtained was 7.2 % that is within the limit of 15 % which is allowed for the variation of control samples.
The EC20, EC50 and EC80-values for 7-AMCA are therefore greater than 300 mg/L. Also the EC10 is >300 mg/L because the highest inhibtion was 7.15 %.
No 95 % confidence limits can be calculated.
Results with reference substance (positive control):
EC20 = 4.1 mg/L.
EC50 = 11.2 mg/L.
EC80 = 30.7 mg/L.

Validity of the test

• The respiration rates of the two control samples were within 15 % of each other, the actual individual values were 96.4 and 103.6% of their calculated mean value.

• The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L: EC20 = 4.1 mg/L EC50 = 11.2 mg/L EC80 = 30.7 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The EC10 and EC50 - values for 7-AMCA are greater than 300 mg/L.
Executive summary:

The study was performed to estimate possible effects of 7-AMCA on aerobic microbial sewage treatment plants. The test was performed according to the OECD Guideline for Testing of Chemicals 209 "Activated Sludge, Respiration Inhibition Test" and the EU method in 87/302, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Five concentrations of the test substance (nominal 19, 38, 75, 150 and 300 mg/L) were tested versus two negative controls (tap water). As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (nominal 5, 15 and 45 mg/L). The microbial inoculum was a preparation of activated sludge collected on the day of test. The concentration of the microbial inoculum was adjusted to 4 g of dry weight per litre which gives a final amount of 1.6 g dry weight per litre in the test medium. The inoculum was kept aerated before the begin of the test. After mixing the inoculum with synthetic sewage solution and appropriate amounts of control or test substance solutions the samples were aerated for a contact time of 3 hours at 20.5 °C.

Negative controls were run as first and as last sample. After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negative controls as 100 %.

 

Results

The test results fulfilled the criteria for validity.

The test substance did not inhibit the respiration in an activated sludge. The highest inhibition obtained was 7.2 % that is within the limit of 15 % which is allowed for the variation of control samples.

The EC10 and EC50 - values for 7-AMCA are greater than 300 mg/L.

The pH of the incubation mixtures after the 3 hour incubation was between 8.0 and 8.2.

Description of key information

The EC10 and EC50 for respiration inhibition of microorganisms in activated sludge

is >300 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
300 mg/L
EC10 or NOEC for microorganisms:
300 mg/L

Additional information

The EC10 and EC50 are not 300 mg/L, as one is forced to enter in the field above, but the EC10/EC50 are >300 mg/L.