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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Nov 1993 - 20 apr 1994
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognized guideline, well documented study report; restriction due to lack of analytical monitoring of test solution (nominal concentrations used).

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:

Test material

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:
- Concentrations: 1, 10, 100, 1000, and 10,000 ppm

- Sampling method: The test material was not monitored in the test solutions

- Sample storage conditions before analysis: Not applicable

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)


-Test Solutions:

As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips. The nominal test material concentrations were 1, 10, 100, 1000, and 10,000 mg/L. Thus for 1mg/L, 0.0005 g was added to the test vessel; for 10 mg/L, 0.005 g; For 100 mg/L 0.05 g; for 1,000 mg/L 0.5 g; and for 10,000 mglL 5.0 g.

Reference Solutions:

3,5-dichlorophenol (97% purity) stock solution was prepared as follows:
1.5g of 3,5-dichlorophenol was dissolved in 10 mL of a 1N NaOH solution which was subsequently diluted to 30 mL with WCC laboratory reagent grade water, placed on a magnetic stirrer and stirred with a teflon-coated stir bar, followed by the addition of 8 mL of 1N H2SO4 (ACS grade) . The solution was then brought to a total volume of 1 L with WCZ laboratory reagent grade water for a nominal concentration of 500 ppm.
In the test, the nominal reference toxicant concentrations were diluted to 10, 20, and 40 ppm.

The control group was maintained under identical conditions but not exposed to the test material.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Activated sludge with associated aerobic organisms was obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool.

One gallon of activated sludge was collected by the plant operator, picked up on the day of collection by WCC personnel and transported to the testing laboratory. Immediately upon arrival, the sludge was aerated with low-pressure, oil-free air. The activated sludge organisms were fed a synthetic sewage feed at a rate of 50 ml per liter.

Triplicate 4-ml samples of the mixed sludge were dried at 100 oC in a Fisherbrand drying oven, until a constant weight was achieved. Based on these results, the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:

Test conditions

No data available
Test temperature:
20 deg. C
No data available
Dissolved oxygen:
No dissolved oxygen data was available, but changes in dissolved oxygen after various treatments were available and shown in the attached Table 2.
Not applicable. Study was performed in fresh water
Nominal and measured concentrations:
Test material: 1, 10, 100, 1000, and 10,000 ppm
Reference substance: 10, 20, and 40 ppm.
Details on test conditions:

-Test Vessels: the sludge/microbial inoculums were exposed to the test material in a 1-L glass flint glass bottle. Total volume within the test vessel for each test condition was 500 ml. Low pressure oil free air was delivered to each test vessles using a glass pipet. Air pressure was controlled by brass needle valves. All of the test vessels were held in a water-jacketed incubator in the dark during the 3 hour exposure period. The incubator was maintained at 20 deg. C.

-Number of Vessels and Concentrations (Range Finding Study): No data available.

-Number of Vessels and Concentrations (Definitive Study):

Controls – duplicate

Reference – one each at 10, 20 and 40 ppm 3,5-dichlorophenol

Test Material – one each at 1, 10, 100, 1000, and 10,000 ppm, nominal.


-Test Water: the dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confirmed with the DPD colorimetric method.


- Adjustment of pH: No data available.

- Photoperiod: tested in the dark.

- Light intensity: not applicable.

During testing, each mixture was aerated with oil-free compressed air. After the 3 hour contact time, 300 mL of the sludge mixture was removed from the vessels and transferred into a BOD bottle for measurement of dissolved oxygen.

-Measuring Vessel/Oxygen Meter: BOD bottles contained a teflon- coated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The test material, control or reference toxicity concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes until a linear trace covering a sufficient range of oxygen concentrations was obtained.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):

Change in mg/L Oxygen was recorded after 3 hours exposure.


- Range finding study: No data available.

- Results used to determine the conditions for the definitive study: No data available.

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
6 000.2 other: ppm
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Remarks on result:
other: 5573.6 - 6426.8 ppm
3 h
Dose descriptor:
Effect conc.:
1 000 other: ppm
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No data available.

- Effect concentrations exceeding solubility of substance in test medium: no effects observed for all tested concentrations.

Results with reference substance (positive control):
- Results with reference substance valid? Yes.

- Relevant effect levels: 3-Hour EC50 value was determined to be 15.5 mg/L.

Reported statistics and error estimates:
Analysis of Consumption Data:

Only the linear section on the chart paper representing the oxygen consumption was used to evaluate the results. The chart readings were transformed into mg of O2 per liter consumed per hour (mg O2/L*hr). To calculate the two control respiration rates for each test material concentrations:
1 ─ 2 *100* RS/(Rc1+Rc2),

RS = oxygen consumption rate of the inoculum exposed to the test material;

Rc1 = oxygen consumption rate, control 1;

Rc2 = oxygen consumption rate, control 2.

In addition to the assessment of the test material, the reference toxicant results were similarly evaluated. The reference toxicant data were subject to probit analysis to calculate the EC50.

To calculate the NOAEC, a t test was performed.

Consumption data were evaluated following the guidelines of USEPA(1989) in order to estimate the concentration which would produce a10%inhibition of oxygen consumption. The estimate is produced using a linear interpolation and requires that the inhibition increases monotonically with increasing exposure concentration. If this condition is not met, the data are smoothed to satisfy the condition.

Because the test concentrations were not replicated, the Jackknife statistical method was used to calculate a variance and the 95% confidence interval for the IC10. This method consists of calculating an IC10 using the first control value, then the second control value and finally the mean of the two control values. Additionally, each test concentration is dropped from the calculation in turn. This results in a number of IC10 estimates. The variance in these resultant IC10 estimates is used to produce a confidence interval with the degree of freedom based on the number of estimates.

Any other information on results incl. tables

Table 2: Oxygen consumption and Chemical-Induced Inhibition of Bacterial Respiration

Chemical Conc.

 (replicate #)




Percent Inhibition

Negative controls



Sample 1


Sample 2


Test material



1 ppm (1)



10 ppm (1)



100 ppm (1)



1000 ppm (1)



10,000 ppm (1)



Positive control



10 ppm



20 ppm



40 ppm



Statistical analysis of the results

The resultant respiration rates were respectively: 59.0, 50.7, 61.5, 66.4, and 47.2 mg O2/L*hr after treated with 1, 10, 100, 1000 and 10000 ppm test material. These data translate into percent inhibitions of -7.2, 7.9, -11.7, -20.6, and 14.3 %. Because none of the test concentrations produced an inhibition greater than 50%, an EC50could not be calculated. Each respiration rate was statistically compared as a single value versus the mean of the controls following the of Sokal & Rohlf (1981) using a single-sidedttest. The results of thettest revealed that the 10,000 ppm test concentration expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The discriminatory power of the test is low, however, due to having only one degree of freedom.

An IC10was calculated using a point estimate technique and Jackknifed to produce a variance from which a confidence could be derived. The result estimate for the IC10was 6000.2 ppm of the test material. The 95% confidence interval for this estimate is from 5573.6 to 6426.8 ppm.

Applicant's summary and conclusion

Validity criteria fulfilled:
The calculated no-observed-adverse-effect concentration (NOAEC) based on inhibition compared to the control was calculated to equal 1000 ppm. The EC50 could not be calculated. The IC10 was calculated to equal 6000.2 ppm with a 95 % confidence interval of 5573.6 to 6426.8 ppm.
Executive summary:

The potential impact of test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test" as prescribed by OECD (1984) and detailed in WCC Protocol OECD209 (Expanded Range Procedures). The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1000 and 10,000 ppm; a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5-dichlorophenol).

The activated sludge respiration test with test material passed the quality control criteria for an acceptable test. The EC50calculated for the reference toxicant was 15.5 rng/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15 % of each other, 54.7 and 55.4 mg O2/L*hr.

The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of test material were 59.0, 50.7, 61.5, 66.4 and 47.2 mg O2/L*hr respectively. The calculated NOAEC based on inhibition compared to the control calculated to equal 1000 ppm. The EC50 for this material regarding the inhibition of metabolism, as represented by respiration, was greater than 10,000 ppm, the IC10was calculated to equal 6000.2 ppm with a 95 % confidence interval of 5573.6 to 6426.8 ppm.