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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:
2012-06-15 to 2012-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes
Analytical monitoring:
yes
Test organisms (species):
other: Activated sludge from the aeration reactor of wastewater treatment plant
Details on inoculum:
Sampling site: Kurume central sewage treatment center (Kurume-shi, Fukuoka, Japan).
Sampling date: June 20, 2012
Concentration of suspended solid: 3050 mg/L
Cultivation: The activated sludge, which was cultivated for 27 hours at 20 +- 2°C after 150 mL of the synthetic sewage was added with 3 L of the activated sludge, was used.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
20+-2°C
pH:
no data
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
no data
Details on test conditions:
TEST SYSTEM
- Test vessel: About 300 mL glass vessel. Volume of test solution 250 mL
- Aeration: About 0.7 L/min with air pump (controllable flow rate at 0.5-1 L/min)
- Test temperature: 20+-2°C
- Dissolved oxygen analyzer: B-103Z (Iijima Electronics Corporation)
- Measurement bottle: 100 mL BOD bottles
- No. of vessels per concentration (replicates): 2
- No. of vessels per blank control (replicates): 2 (at the beginning and the end of the exposure Period in tests)
- No. of vessels per reference control (replicates): 1 at each concentration
- Preparation of synthetic sewage (reagent dissolved in 1L purified water):
pH: 7.2
Peptone: 16g
Meat extract: 11g
Urea: 3g
Sodium chloride: 0.7g
Calcium chloride dihydrate: 0.4g
Magnesium sulfate heptahydrate: 0.2g
Dipotassium hydrogen phosphate: 2.8g
- Preparation of activated sludge: Homogenization and washing of activated sludge were not performed
- Preparation of stock solution of test item: 100 mg of the test sample was accurately weighed by electronic analytical balance and dissolved in purified water in order to obtain 10.0 g/L stock solution of the test item.
- Preparation of stock solution for reference substance: 100 mg of 3,5-dichlorophenol was accurately weighed by electronic analytical balance and dissolved in purified water. The pH of this solution was adjusted with 1 mol/L sodium hydroxide solution. This solution was filled up by purified water, and obtained 1.00 g/L stock solution.
- Preparation of test solution: 25 mL of the 10.0 g/L stock solution of the test item, 8 mL of the synthetic sewage, the purified water (the volume subtracting the volume of the stock solution of the test item from 117 mL), and, 125 mL of the activated sludge were added to the test vessel
- Preparation of the reference control: Appropriate volume of the 1.00 mg/L stock solution of 3,5-dichlorophenol, 8 mL of the synthetic sewage, purified water (the volume subtracting the volume of the stock solution of 3,5-dichlorophenol from 117 mL), and, 125 mL of the activated sludge were added to the test vessel
- Preparation of the blank control: 8 mL of the synthetic sewage, 117 mL of the purified water, and, 125 ml of the activated sludge were added to the test vessel


EFFECT PARAMETERS MEASURED: After 3 hours of exposure, the test solution was promptly poured out softly and the measurement bottle was filled. Then, the concentration of dissolved oxygen in measurement bottle was measured continuously for about 15 minutes with stirring

TEST CONCENTRATIONS
- Test concentrations: 1000 mg/L
- Test concentration reference control: 4.0, 8.0, 16, and, 32 mg/L (Common ratio 2)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Results with reference substance (positive control):
EC50 = 13 mg/L

Oxygen consumption rate and percentage inhibition of respiration after 3 hours of exposure

 Test solution

 Concentration (mg/L)

 Oxygen consumption rate (mgO2/L/h)

 Percentage inhibition of respiration (%)

    Test vessel

 1000 63.3  0 (-1)*
 1000  65.5 0 (-4)*

          Reference control

 4.0  57.6
8.0   48.8  22
 16  17.8 72 
 32  10.1  84

    Blank control

 - 60.2   -
 65.5  -

Average oxygen consumption rate in blank control: 62.9 mgO2/L/h

* The percentage inhibition of respiration was regarded as 0% because the calculated value shown in parentheses was negative

Validity criteria fulfilled:
yes
Conclusions:
It is considered that the respiration inhibition of MMB is low because EC50 was >1000 mg/L in this test.

Description of key information

An OECD 209 Guideline "Activated Sludge, Respiration Inhibition Test" resulted in an EC50>1000 mg/L. 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

One reliable key study is available for testing the toxicity of MMB to microorganisms. A OECD 209 Guideline "Activated Sludge, Respiration Inhibition Test" resulted in an EC50>1000 mg/L. No respiration inhibition by MMB was observed in a 1000 mg/L limit test with a exposure duration of 3 hours. Based on this result a NOEC of >= 1000 mg/L was determined.