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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:
from 2009-03-09 to 2009-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008/440/EC
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 1984
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no details given.
Analytical monitoring:
no
Details on sampling:
no analytical measurement.
Vehicle:
yes
Details on test solutions:
Defined amounts of test item were directly weighed into the designated test flasks to reach the planned nominal concentrations. The nominal test item concentrations were prepared by mechanical dispersion (using orbital shaker). These test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.41. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no post exposure period
Hardness:
no data
Test temperature:
19.8 – 20.3 °C (during the incubation) and
18.8 – 20.6 °C (during oxygen measurement)
pH:
The pH of the activated sludge inoculum was determined to be pH 7.41.
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 6.6 mg O2/L.
Salinity:
no data
Nominal and measured concentrations:
Before the start of the test defined amounts of the test item were directly weighed into the test flasks and mechanically dispersed using orbital shaker.
The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
Details on test conditions:
One test solution with a final volume of 330 mL was tested per treatment in a glass flask. 10.56 mL synthetic sewage and an adequate amount of the test item or an adequate volume of the stock solution of the reference item was filled up with deionised water to 198 mL before the start of the test. At the start of the test 132 mL activated sludge inoculum with a sludge concentration of 4 g/L (dry weight) was added, first to first control (C1), then in time intervals of 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item and the test item and finally to a second control (C2).
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after exactly 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with a stirring O2 electrode and was recorded for about ten minutes. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all treatments. The temperature was measured in the climate chamber with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all BOD bottles.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration of range of 10-31 mg potassium cyanate /L. Slight stimulation of respiration rate was observed (-1.8 %) at the concentrations of 10 mg/L. The respiration rate was inhibited between 1.8 % and 17.5 % in the examined nominal test concentration range. Concentrations exceeding 1000 mg/L nominal were not tested.
Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L.
The NOEC was determined to be 1000 mg/L.
The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were not inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (17.5 %) at the highest examined concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.470 (at 1000 mg/L) was in the historical control data range (0.514 +/- 0.077), and the deviation from the control was slightly higher than 15 %, but still it can be considered as a biological variability of the test system.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be 13.58 mg/L and thus the EC50 was in the acceptable the range of 5 to 30 mg/L.
Reported statistics and error estimates:
Standard deviation and EC50 calculation
Validity criteria fulfilled:
yes
Conclusions:
Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L.
Executive summary:

In a 3 -h activated sludge test, an inoculum (domestic) were exposed to potassium cyanate at nominal concentrations of 10, 31, 100, 313 and 1000 mg/L under static conditions. 

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration of range of 10-31 mg potassium cyanate /L. Slight stimulation of respiration rate was observed (-1.8 %) at the concentrations of 10 mg/L. The respiration rate was inhibited between 1.8 % and 17.5 % in the examined nominal test concentration range. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L.

The NOEC was determined to be 1000 mg/L.

Description of key information

No studies are available for sodium cyanate regarding activated sludge tests. Read across from potassium cyanate was performed instead. In aqueous solution cyanate salts dissociate very quickly to cyanate ion and the respective alkali metal ion. It is not expected that Na+ or K+ contribute to the toxic potential.
Based on measured inhibition rates for potassium cyanate it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In a 3 -h activated sludge test, an inoculum (domestic) were exposed to potassium cyanate at nominal concentrations of 10, 31, 100, 313 and 1000 mg/L under static conditions. 

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration of range of 10-31 mg potassium cyanate /L. Slight stimulation of respiration rate was observed (-1.8 %) at the concentrations of 10 mg/L. The respiration rate was inhibited between 1.8 % and 17.5 % in the examined nominal test concentration range. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L.

The NOEC was determined to be 1000 mg/L.