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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
July, 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
yes
Remarks:
stock solutions were prepared with chlorine-free tap water
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacity of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. The Inoculum was not adapted. The activated sludge was kept aerated until use at test temperature. Dry solid of the activated sludge was determined as 3.8 g/L (range-finder test) and 5.3 g/L (full test) by weight measurements before and after drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
N/A
Post exposure observation period:
N/A
Hardness:
2.19 mmol/L
Test temperature:
18.3-21.8 °C
pH:
7.3-7.4
Dissolved oxygen:
N/A
Salinity:
N/A
Conductivity:
N/A
Nominal and measured concentrations:
Range-finder test with three test concentrations: 10, 100 and 1000 mg/L
Full test with six concentrations (nominal): 62.5, 125, 250, 500, 1000, 2000 mg/L
Details on test conditions:
The different vessels were started separately with an interval of exactly 15 min in order to be able to measure the respiration rates one by one while making sure that the aeration time is 3 h in every vessel. At time "0", 16 ml synthetic sewage and the test item or reference compound were added to the test vessel and made up to 250 mL with chlorine-free tap water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids. For the controls the same procedure was made as with the test vessels, however without test
or reference substance. All vessels were stirred and aerated throughout the contact time with room air to assure an oxygen saturation of 60-70% using a membrane pump. Before starting the test the oxygen saturation rate was measured.

After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.

This determination was repeated for each vessel at 15-min intervals, ensuring that the contact time in each vessel was 3 h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol 97% at concentrations 1, 10 and 30 mg/L (both in range finder and main test)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
359.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
62.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Range-finder
A range-finder test with the concentrations of 10, 100 and 1000 mg/L was performed. The pH in the test vessels was 7.3, an adjustment was not necessary.
An inhibition between 49 and 58% was observed in the test vessels with 1000 mg/L test concentration. In the test vessels with the test concentration of 100 mg/L the observed inhibition was between 17 and 27%.
The temperature range of the range-finder test within the test duration was between 20.7 and 22.0°C.

Full test
The inhibiting effect of the test item on the total respiration of activated sludge was observed with six concentrations (62.5, 125, 250, 500, 1000, 2000 mg/L).
At the lowest test concentration 62.5 mg/L no significant different respiration rate than in the blank controls was observed. In the test vessels with 125.0 mg/L test concentration an inhibition of 21% (mean of five replicates), with 2000.0 mg/L test concentration an inhibition of 86% (mean of five replicates) was observed.
The pH in all test vessels at the beginning of the test vessels was between 7.3 and 7.4.

Blanks
The mean respiration rate of six blanks was 47.9 mg/(L*h) per one gram activated sludge. The coefficient of variation (CV) was 25% and therefore less than 30%. The pH in the blank vessels was 7.3.
Results with reference substance (positive control):
In the full test the EC50 of 3,5-dichlorphenol was calculated as 5.8 mg/l and was therefore within the acceptable range of 2 to 25 mg/l. The pH in the reference vessels was 7.3.
Reported statistics and error estimates:
EC50, LOEC and NOEC were given by ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH

EC50 = 359.4 mg/L (95%-CL: 157.201 – 840.753 mg/L)
EC10 = 62.6 mg/L (95%-CL: 30.085 – 130.182 mg/L)
LOEC = 250.0 mg/L
NOEC = 125.0 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The results for the inhibition of the total respiration of activated sludge were:
EC50 = 359.4 mg/L (95%-CL: 157.201 – 840.753 mg/L)
EC10 = 62.6 mg/L (95%-CL: 30.085 – 130.182 mg/L)
LOEC = 250.0 mg/L
NOEC = 125.0 mg/L

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
359.4 mg/L
EC10 or NOEC for microorganisms:
62.6 mg/L

Additional information

NOEC >= 125 mg/L

EC10 = 62.6 mg/L