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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2011 - April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP issued by the People's Republic of China, according the OECD guideline meeting all validity criteria.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
no
Remarks:
Study performed under GLP issued by the People's Republic of China, according the OECD guideline meeting all validity criteria.
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge microorganisms were obtained from a chengdong waste water treatment plant in Nanjing city. On arrival at the laboratory, a small amount of the fresh sludge was weighed, oven-dried, and reweighed. A calculation was made from these results to determine the amount of wet sludge that must be suspended in laboratory dilution water (LDW) in order to obtain an activated sludge with a mixed-liquor suspended solids (MLSS) level of approximately 3 g/L. The sludge was washed three times with LDW using the following method: after centrifuging at approximately 10000 rpm for 10 minutes, the supernatant was decanted and the remaining sludge solids re-suspended in LDW and mixed well. After the third spin, the wet solids were homogenized for 2 minutes in a blender. The 90 g wet solids were suspended in 3 L of LDW and 75 mL of each of the two synthetic feed stock solutions. The sludge suspension was aerated overnight (24 h) at (20±2)oC. The pH of the overnight suspension was 7.5. Viability of the microorganisms was checked by means of a reference control. In studies of this sort no attempt was made to identify the large variety of organisms present in the samples or the degree of absorption of the test and control substances.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature of the reaction mixtures after the incubation period ranged from 20+/-2°C.
pH:
The pH of the reaction mixtures after the incubation was 7.5.
Dissolved oxygen:
aerobic during test
Nominal and measured concentrations:
The nominal concentrations of 3,5-Dichlorophenol tested were: 3, 10 and 30 mg/L
The nominal concentrations of test substance (range finding) were: 10, 100 and 1000 (highest concentrations in triplicate).
Details on test conditions:
A reference control stock solution of 3, 5-dichlorophenol, a known inhibitor of microbial respiration, was prepared as follows: Dissolved 1.000 g of 3, 5-dichlorophenol in 20 mL of 1 moL/L NaOH; Diluted to approximately 60 mL with 40 mL distilled water; Added, while stirring, 0.5 moL/L H2SO4 to the point of incipient precipitation-approximately 16 mL of 0.5 moL/L H2SO4 was required; Finally, diluted the mixture to one liter with laboratory water. The pH should then be in the range 7 to 8 and adjusted with 0.5 moL/L H2SO4.
The test substance (meta-Diisopropanolbenzene) was directly added to test bottles after weighting appropriate amounts of meta-Diisopropanolbenzene.

At time “0”, 16 mL of the synthetic sewage feed and 250 mL of microbial inoculum were added to a first vessel (first blank control FB1), then fixed final volume of 500 mL with deionised water. Whereafter aerate the mixture at 0.8 litre per minute using a Pasteur-pipette by an aeration device.
At time “15 min” the above was repeated, except that appropriate amount of the test substance was added to the 16 mL of synthetic sewage. Then 250 mL microbial inoculum was added to make a total volume of 500 mL. This mixture was then be poured into a second vessel and aerated as above. This process was repeated at 15-minute intervals with different amounts of the test substance to give a series of vessels containing widely-spaced concentrations of the test substance. Finally, a second blank control (FB2) was prepared.
In the range-finding test, the test solutions of the test substance with nominal concentration 10, 100, 1000 mg/L were established, with no replicates for each concentration, but triplicates at the highest tested concentration 1000 mg/L.
The reference control was tested on the microbial inoculums in the same way. Appropriate volumes stock solution of reference substance was added to test bottle with exposure solutions at 3 mg/L, 10 mg/L, and 30 mg/L.
Finally, a second Negative Control was prepared. Oxygen consumption was measured and recorded after an aeration time of three hours. Air temperature was recorded at test start. The pH of all test solutions were measured at test start and test end.
After three hours the respiration rate was measured directly in the vessel over a period of up to 10 minutes.
This determination was repeated on the contents of each vessel at 15-minute intervals, in such a way that the contact time in each vessel was three hours.
During the test, the following conditions were maintained:
-Duration time: 3 hours
-Water: deionised water
-Air supply: Clean, oil-free air. 0.8 liter/minute
-Temperature: (20±2)°C

Measurements
Air temperature in the hood at test start was (20±2) oC. No unusual variation in pH was noted in any of the test solutions at test start and at test end. The pH value was 7.5. After three hours, a standard BOD measuring bottle was filled from the contents of the first beaker and the respiration rate was measured over a period of 15 min, or until the dissolved oxygen level reached 0.30 mg/L. The dissolved oxygen meter downloaded the readings directly to a computer. This determination was repeated on the contents of each vessel at 15 min interval so that the exposure time in each beaker was three hours.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Test validity
1) Two negative control respiration rates were within 30% of each other.
2) The 3-hour EC50 of 3, 5-dichlorophenol was in the range 2 mg/L ~25 mg/L.

Test Results
The test results of range-finding test were provided in Table 1. The 3 h-EC50 value of 3,5-dichlorophenol was 19.0 mg/L and the 95% confidence limit ranged from 15.0 mg/L to 24.2 mg/L. The 3 h-EC50 value of the test substance (meta-Diisopropanolbenzene) was >1000 mg/L. EC50 values were calculated by regression analysis.
According to “The guidelines for the hazard evaluation of new chemical substances (HJ/T 154-2004)”, the test substance belongs to low toxic chemical.
Results with reference substance (positive control):
The EC50 of the reference compound (3,5-dichlorophenol) was 19 mg/L which is within the prescribed range of 2 to 25 mg/L
Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
The test is valid as shown by the EC50 of the reference compound 3,5-dichlorophenol (19 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (7.5%) The average oxygen uptake rate for the control replicates was lower than prescribed (15 mg O2/g dry weight/hour) but this does not affect the sensitivity of the activated sludge as shown by the result of the reference substance nor the reproducibility of the oxygen measurements as shown by the coefficient of variation of the replicates of the control. The lower oxygen uptake rate does therefore not invalidate the test.
The toxicity of the test substance to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance.
The EC50 of meta-Diisopropanolbenzene after 3 hours contact time is > 1 g/L.
meta-Diisopropanolbenzene is therefore considered not harmful to activated sludge
Executive summary:

A 3-hour activated sludge respiration inhibition test was performed using activated sludge from a domestic waste treatment plant according to “The guidelines for the testing of chemical (HJ/T 153-2004)” and with reference to OECD TG 209 etc.The sludge microorganisms were exposed to three concentrations of the test substance (meta-Diisopropanolbenzene) at nominal concentration of 10 mg/L, 100 mg/L, and 1000 mg/L. The respiration rate, expressed as oxygen consumption by the microbes in mg O2per liter per hour, was measured under defined conditions following the 3-hour exposure period. Inhibition values were calculated by comparing test respiration rates to negative control rates. EC50values were calculated by regression analysis. The result indicated that under valid test conditions the 3 h-EC50 value of 3, 5-dichlorophenol was 19.0 mg/L and 95% confidence limit ranged from 15.0 mg/L to24.2 mg/L. The 3 h-EC50 value of the test substance (meta-Diisopropanolbenzene) was >1000 mg/L.According to “The guidelines for the hazard evaluation of new chemical substances (HJ/T 154-2004)”, the test substance (meta-Diisopropanolbenzene) belongs to low toxic chemical.

Description of key information

The 3 h-EC50value of the test substance (meta-Diisopropanolbenzene) was >1000 mg/L.

Key value for chemical safety assessment

Additional information