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Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported; date of report is 11 October 1976. Test period was 96 hrs.
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
No mention is made of a relevant guideline or standard method used to conduct this toxicity test; however, study appears acceptable and meets basic scientific principles. Method to determine the oxygen levels was the "conventional" (according to report) Warburg Constant Volume Respirometer Method. Dilution water for incubations was according to a procedure described in "Standard Methods for Examination of Water and Wastewater" (13 ed., 1971).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The effects of 2,4,6-Tribromophenol on the biological oxidation rate of microorganisms were examined. Oxygen utilization in the presence of the test material at nine different concentration levels was determined manometrically using the Warburg Constant Volume Respirometer Method. This report describes the experimental procedures and presents the results obtained during 96 hours of incubation.
GLP compliance:
not specified

Test material

Details on test material:
Not available.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
Details on sampling:
Not available.

Test substrate

Details on preparation and application of test substrate:
"Due to the low water solubility of the chemical, the material had to be introduced to the flasks before addition of the bacterial suspension; appropriate quantities of test material dissolved in acetone were placed into the main compartment of the flask and the solvent allowed to evaporate completely. In the case of the 0.1, 1.0 and 10.0 percent samples, portions of 4, 40 and 400 mg of 2,4, 6-Tribromophenol, respectively, were weighed directly into the flask omitting the solvent."

Test organisms

Test organisms (inoculum):
other: The seed organisms were prepared by mixing equal portions of the supernatants from fresh domestic sewage and a fresh topsoil slurry. The fresh aerated sewage was obtained from North Shore Sanitary District, Highland Park, IL and settled for 24 hours at 2

Study design

Total exposure duration:
96 h

Test conditions

Test temperature:
Temperature ranged from 20ºC to 23.4ºC
NA - experiments were performed in solution (diluted supernatants from fresh domestic sewage and fresh topsoil slurry).
Details on test conditions:
- Test container: conventional Warburg instrument accomodating 12 respirometer units
- Amount of soil: not stated
- No. of replicates per concentration: 1
- No. of replicates per control: 1
- No. of replicates per vehicle control: no vehicle control required, since vehicle (acetone) was allowed to evaporate in test container before addition of seeded, diluted test water

RANGE-FINDING STUDY: none reported
Nominal and measured concentrations:
Nominal concentrations of the test material were 1, 10 and 100 ppb; 1, 10, and 100 ppm; and 0.1, 1.0, and 10%.
Reference substance (positive control):

Results and discussion

Details on results:
Reproduced from report. See below for tables.

IV. Results
"Changes in the level of the manometer fluid of the thermobarometer and of each reaction vessel were recorded at the specified time intervals
in mm deflections. The observed data were converted to actual O2 uptake..."

"Actual interval values expressed as oxygen uptake (mg 02) per liter are presented in Table II. The changes in the rate of O2 exchange during
incubation can be readily observed from these results. During the first 22 hours following inoculation, a general increase in oxygen uptake was
observed in all reaction flasks. The increases were highest in the lower concentrations (1, 10 and 100 ppb, and 1 ppm) with values slightly
exceeding those obtained in a control sample (seeded dilution water, no test material added). In the following intervals, the manometer readings
revealed a general decrease in O2 uptake in all samples. The low rate of oxygen exchange in the presence of the test material showed that
2,4,6-Tribromophenol did not provide a readily utilizable carbon source as did glucose. During the 24-48 hour interval, the rate of O2 uptake in
the test samples ranged from 2.86 to 9.54 mg per liter; a value of 6.69 mg O2 per liter was observed in the control vessel containing seeded dilution water only. Oxygen uptake in the media containing glucose (positive control) exceeded that of the test samples by a significant amount.

Cumulative oxygen uptake was also calculated; the values are presented in Table Ill. At the end of the incubation period (96 hours), total O2 uptake
amongst the test samples was highest in tests with concentrations ranging from 1.0 ppb to 10 ppm. At concentrations of 100 ppm, 0. 1, 1. 0 and
10 percent the total O2 uptake was less than when the test material was absent, indicating inhibited endogenous respiration due to the presence of
2,4,6 -Tribromophenol. Total O2 uptake in the positive control test containing glucose amounted to 75.9 mg O2 per liter media during 96 hours
of incubation."...
Results with reference substance (positive control):
Oxygen uptake in the positive control (glucose) exceed the rate of all test flasks by a significant amount, indicating utilization of a readily available carbon source.
Reported statistics and error estimates:
Not available.

Any other information on results incl. tables


TEST MATERIAL: 2,4,6-Tribromophenol

The Effect of 2,4,6-Tribromophenol on Oxygen Uptake by a Heterogeneous Seed Culture

Respirometer Unit Number Substrate Concentration Cumulative Oxygen Uptake (mg O2/l)
Time intervals (hours)
1 6 22 24 48 72 96
1 10% 2.20 9.12 19.40 8.92 13.47 14.45 11.29
2 1% 0.21 4.53 5.77 4.74 9.68 10.09 0.93
3 0.1% 0.50 9.43 19.11 11.66 18.36 18.61 15.88
4 100 ppm N.R. 5.54 15.08 4.31 13.85 18.77 13.85
5 10 ppm 0.25 11.71 13.17 25.85 33.65 35.6 23.16
6 1 ppm 1.02 17.05 31.46 28.21 33.08 36.43 30.54
7 100 ppb 1.25 10.73 28.87 23.10 28.67 33.21 25.59
8 10 ppb 1.26 10.73 31.16 21.94 29.67 34.40 23.68
9 1 ppb 2.00 3.98 26.02 22.27 25.13 34.04 29.63
10 Glucosea 0.81 6.60 25.37 23.77 48.73 65.31 75.90
11 Controlb 1.16 5.56 20.47 15.12 21.81 24.48 21.23

N.R. = No reading obtained.

a = 0.14% d-glucose seeded dilution water.

b = Seeded dilution water only (4 ml).

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
No guideline or standard method was specified as followed. However, study appears acceptable and meets basic scientific principles.
Overall, the study demonstrated that the test material did inhibit soil microorganism respiration at the higher test concentrations (>100 ppm) in a diluted aqueous solution of unidentified microorganisms seeded from sewage and soil supernatant. However, the approach did not allow for determination of effect concentrations.
Executive summary:

Reproduced from report:

II. Summary

The purpose of the study was to measure oxygen uptake by microorganisms in the presence of the test compound. Oxygen exchange was determined with the conventional Warburg respirometer. The biological seed culture was prepared from fresh sewage and topsoil. The test material was tested in 9 different concentrations (1, 10 and 100 ppb; l, 10, and 100 ppm and 0. l, 1.0 and 10 percent). Incubation of the samples was performed at ambient temperature (water bath at approximately 23° C) with constant shaking, and was continued for 96 hours. Pressure change readings were obtained at 1, 6 and 22 hours, and at 24 -hour intervals.

Oxygen exchange values were calculated as O2 uptake in milligrams per liter sample employing appropriate equations and flask constant values.

The test samples containing 2,4,6-Tribromophenol at concentrations of 100 ppm, 0.1, 1.0, and 10 percent exhibited slight inhibition of microbial respiration due to the presence of the test material. Following 96 hours of incubation, the rate of oxygen uptake in test samples with concentrations ranging from 1 ppb to 10 ppm amounted to 26.5 mg O2 per liter media (average). The amount of oxygen utilized in a control sarnple (seeded dilution water without test rnaterial) was 21.2 mg per liter. It is possible that the test compound in the lower concentrations (10 ppm and less) provided an additional carbon source. The highest value of absorbed oxygen was observed in a reaction flask containing 1 part 2,4,6 -Tribromophenol per million parts of microbial seed culture.

Oxygen uptake in the positive control (glucose) exceeded the rate of all test flasks by a significant amount, indicating utilization of a readily available carbon source.