Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
GLP compliance:
yes
Remarks:
Deviation did not effect outcome of the study.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Purity of the sample: 97.4 weight% (using GC/FID)
Solubility in distilled deionized water: 0.40 ug/L at 22.5 °C
Lot/Batch No.: 0022268
Structure: confirmed using GC/MS

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
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 9 minutes, or until the dissolved oxygen (DO) level reached 0.30 mg/L.

Test solutions

Vehicle:
no
Details on test solutions:
The overnight sludge suspension was washed using the procedure described earlier except that the sludge solids were homogenized in a blender for 2 minutes after each wash. Homogenization breaks up the centrifuged pellet and ensures proper washing and a uniform cell suspension. The final sludge suspension was prepared in LDW at an MLSS of approximately 4 g/L. The final sludge concentration in the test beakers was 1.8 g/L. At time "0", 8 mL of each synthetic feed stock solution was placed in a 200-mL volumetric flask and brought to volume with distilled water. This mixture was placed in the Negative Control beaker. Next, 100 mL of distilled water was added to the beaker, and then 200 mL of sludge inoculum was added to give a final volume of 500 mL. The test solution was gently stirred using a stir plate and Teflon coated stir bar, and aerated at 0.5 to 1 L/minute using a pipette as an aeration device. At time "12 min" (12 minutes is an arbitrary, but convenient, interval) the above was repeated for the second beaker containing the lowest concentration of test substance. The process was repeated at 12-minute intervals to give a series of nominal concentrations of the test substance in the test vessels of 0.1, 0.3, 1.0, 3.2, and 10.0 mg/L. Due to low aqueous solubility, the test substance was added directly to the test vessels via small plastic weigh boats. The positive control (3,5-dichlorophenol) was tested on each batch of microbial inoculum in the same way, except that appropriate volumes of the positive control stock solution (each diluted with distilled water to 100 mL) were placed in the next three beakers instead of 100 mL of water. A total of three positive control solutions were tested at nominal concentrations of 31.25, 9.77, and 3.05 mg/L. Finally, a second Negative Control was prepared. Small plastic weigh boats were added to all of the Control vessels as the test substance was added via the weigh boats. 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 at test end.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge microorganisms were collected from a domestic wastewater treatment plant. Viability of the microorganisms was confirmed on receipt in the laboratory and activity checked by means of a positive control. In studies of this sort no attempt is made to identify the large variety of organisms present in the samples or the degree of absorption of the test and control substances.

Preparation of the Inoculum: 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 4 g/L. The sludge was washed three times with LDW using the following method: after centrifuging at approximately 7000 rpm for 15 minutes, the supernatant was decanted and the remaining sludge solids resuspended in LDW and mixed well. After the third spin, the wet solids were homogenized for 2 minutes in a blender. The 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 (19 hrs.) at 23 °C. The pH of the overnight suspension was 8.043.

Study design

Test type:
other: aquatic
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
Air temperature in the hood at test start was 22 °C.
pH:
No unusual variation in pH was noted in any of the test solutions at test start and at test end. The pH values ranged from 7.395 to 8.194.
Nominal and measured concentrations:
Nominal concentrations: 0.1, 0.3, 1.0, 3.2, and 10.0 mg/L
Details on test conditions:
Replicates: 1
Sludge per Replicate: 1.8 g/L
Exposure Vessels: 1-L glass beakers
Test Volume: 500 mL
Test Parameter: O2 consumption
Aeration: No
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol at nominal concentrations of 1.25, 9.77, and 3.05 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 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:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The respiration rates for the test values, starting from the lowest concentration level to the highest (0.1, 0.3, 1.0, 3.2, and 10.0 mg/L), were 42.09, 42.82, 41.40, 43.71, and 42.80 mg O2/L-hr resulting in inhibition values of -1.9%, -3.7%, -0.3%, -5.9%, and -3.7%, respectively (negative inhibition values represent enhanced respiration rates). These values are not considered significant due to the inherent experimental error of the test. Respiration rates for the negative controls were 42.92 and 39.66 mg O2/L-hr, resulting in a mean value of 41.3 mg O2/L-hr.
Results with reference substance (positive control):
Organisms in the positive control (31.25, 9.77, and 3.05 mg/L of 3,5-dichlorophenol) resulted in respiration rates of 20.13, 40.35, and 42.69 mg O2/L-hr, respectively. The resulting positive control EC50 value of 30.5 mg/L, was calculated using the graph of percent inhibition vs. concentration. While this value is slightly out of the accepted range of 5 - 30 mg/L, it is within the experimental error of the test. In addition, the sludge concentration in the beakers was slightly higher than the aim concentration of 1.6 g/L. The higher sludge concentration may have impacted the EC50 of the positive control. This deviation did not impact the outcome of the study, as sludge exposed to the test substance did not show inhibition.
Reported statistics and error estimates:
Microsoft Excel was used for data calculations and tabulations, and also to generate the graph showing percent inhibition vs. concentration. No attempt was made to determine potential adsorption of the test chemical to the sludge or potential biodegradation of the chemical by the microorganisms.

Any other information on results incl. tables

Beaker # Identification Concentration Respiration Rate (mg O2/L-hr) % Inhibition
1 Neg. Control 0 42.92 -
2 Test 0.1 42.09 -1.9
3 Test 0.3 42.82 -3.7
4 Test 1 41.4 -0.3
5 Test 3.2 43.71 -5.9
6 Test 10 42.8 -3.7
7 Pos. Control 13.25 20.13 51.2
8 Pos. Control 9.77 40.35 2.3
9 Pos. Control 3.05 42.69 -3.4
10 Neg. Control 0 39.66 -

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The negative control respiration rates were within 15% of each other. See additional remarks in the results section.
Conclusions:
DOTP is not expected to inhibit respiration of secondary waste treatment microorganisms.
Executive summary:

In a 3-hour toxicity study, sewage sludge microorganisms were exposed to DOTP at nominal concentrations of 0, 0.1, 0.3, 1.0, 3.2, and 10 mg/L under static conditions. There was no inhibition of respiration rate observed at any of the exposure concentrations during this study. The 3-hour EC50 value was >10 mg/L. The 3-hour NOEC value was >= 10 mg/L, the highest concentration tested. DOTP is not expected to inhibit respiration of secondary waste treatment microorganisms.