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: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 88%

Test solutions

Vehicle:
yes
Details on test solutions:
The test substance was dissolved in a stock solution of Barnstead DiamondTM water at 10000 mg/L. The concentration of the test substance in the stock solution was determined to be 9370 mg/L. The test substance was added to the test vessels at the following nominal concentrations: 1000, 320, 100, 32 and 10 mg/L.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Secondary activated sludge from Wilmington, DE USA Publically Owned Treatment Works (POTW) was used as the microbial inoculum. The activated sludge was kept aerated and fed with synthetic sewage feed. The amount of sludge to use as inoculum was determined by measuring its respiration rate at 50, 100, and 200 mL of sludge after diluting with 16 mL of synthetic sewage feed and sufficient dechlorinated water for a final volume of 500 mL. The respiration rate was measured after a minimum mixing time of 30 minutes. This pre-experiment to determine the amount of sludge to use as inoculum was not be performed in compliance with the GLP-Regulations. The raw data, however, were included in the study records and were archived under the project number of the study.

Study design

Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Details on test conditions:
All test vessels were 1000 mL glass flasks and contained a final volume of 500 mL. Test solutions were transferred to 300-mL Biological Oxygen Demand (BOD) glass bottles for Dissolved Oxygen (DO) determinations. Test solutions were aerated with compressed air at a flow rate of approximately 0.1 to 0.5 liter per minute at room temperature.

Prior to the start of the test, all components were added to the test vessels, less the volume of the inoculum. This volume was determined by a pre-test of the inoculum to find a concentration that gave an acceptable respiration rate. The test was initiated with the first positive control by adding the microbial inoculum and aeration started. After approximately 15 minutes the inoculum was added to the first reference substance. The procedure was repeated at approximately 15-minute intervals with the reference substance and then the test substance to give a series of vessels containing different concentrations of the reference and test substance. A negative control (synthetic sewage feed and test substance at the highest concentration, but without microbial inoculum) test was also evaluated. The final flask was a second positive control, prepared exactly as the first. The test was conducted in the following order so that the reference and test substances were bracketed by the two controls: first positive control; reference substance at 3 concentrations; test substance at 5 concentrations; negative (abiotic) control at highest concentration of test substance; second positive control.

For the measurement of the respiration rate a well-mixed sample of each treatment was transferred into a BOD bottle after 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode and recorded over a period of about 10 minutes or until the dissolved oxygen (DO) concentration fell below about 1 mg DO/L. During measurement, the samples were continuously stirred with the built-in stirrer. The rate of oxygen consumption (in mg O2/L/h) was determined from the most linear part of the respiration curve. Test sample temperature was measured concurrently with the DO measurements and recorded.

For the measurement of the respiration rate a well-mixed sample of each treatment was transferred into a BOD bottle after 3 hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode and recorded over a period of about 10 minutes or until the dissolved oxygen (DO) concentration fell below about 1 mg DO/L. During measurement, the samples were continuously stirred with the built-in stirrer. The rate of oxygen consumption (in mg O2/L/h) was determined from the most linear part of the respiration curve. Test sample temperature was measured concurrently with the DO measurements and recorded.

Concentrations of the test and reference substance stock solutions were estimated by determining the total dissolved organic carbon in one liter of water to which approximately 500 mg of test or reference substance had been added and mixed. Samples were analyzed for total carbon content via a Shimadzu TOC-V analyzer with an autosampler attachment. This system is based on the combustion/non-dispersive infrared gas analysis method widely employed for TOC measurement. Carbon dioxide-free carrier gas flows to the combustion tube, which was filled with an oxidation catalyst and heated to 680°C. The total carbon (TC) of a sample was burned in the combustion tube to form carbon dioxide. The carrier gas, containing the carbon dioxide and other combustion products, flowed from the combustion tube to an electronic dehumidifier, where it was cooled and dehydrated. The gas then passed through a halogen scrubber before going through the non-dispersive infrared (NDIR) gas analyzer, where the carbon dioxide was detected. The analog detection signal of the NDIR formed a peak, which was proportional to the TC concentration of the sample. A calibration curve equation that mathematically expresses the relationship between peak area and TC concentration was generated by analyzing various concentrations of a TC standard solution. The TC concentration in a sample was determined by analyzing the sample to obtain the peak area and then using the peak area in the calibration curve equation. Concentration was calculated using the following equation:

C = (TOC * MAWs)/(MWc * CA) where:

C = Concentration, mg/L
TOC = Total Dissolved Organic Carbon, mg/L
MWs= Molecular Weight of the substance, mg/mmole
MWc= Molecular Weight of Carbon, 12.01 mg/mmole
CA = Number of Carbon atoms in test substance

The respiration rate was calculated from the output as mg O2/L/h. This was done by graphing mg O2/L on the y-axis and time in hours on the x-axis, drawing the line of best fit, and then determining the slope of the line. The slope of the line was the extent to which the y-axis changes for 1 unit of change in the x-axis. Alternatively, the respiration rate can be calculated as follows:

b= [nΣxy – (Σx)( Σy)] / [nΣx2 – (Σx)2] where:

b = Respiration Rate
n = pairs of x and y values
x = time in hours
y = mg O2/L at time = x

The portion of the respiration curve over which the respiration rate is measured should be linear.

The inhibitory effects were determined by comparing the respiration rates at each concentration of either test or reference substance to the respiration rates in the controls. The results were expressed as percentage of the mean value of the respiration rates of the two controls according to:

[1-(2Rs/(Rc1 + Rc2))] * 100 = Percent inhibition, where:

Rs = oxygen consumption rate at tested concentration of test or reference substance
Rc1 = oxygen consumption rate of positive control 1
Rc2 = oxygen consumption rate of positive control 2

The 3-hour EC50 (Effective Concentration of the substance giving a calculated or interpolated inhibition of oxygen consumption of 50% compared with a blank control). If possible, the EC20 and the EC80 also were calculated and reported for the test substance.
The percent inhibition was calculated at each test concentration as above. For the test substance, the percent inhibition was plotted against concentration as a log-normal (or log-probability) graph and an EC50 value derived from the graph. For the reference substance, the percent inhibition was plotted against concentration as a normal-normal graph and an EC50 value derived directly from the graph.

The test results were valid if (1) the two positive control respiration rates (PCRR) were within 15 percent of each other and (2) the EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L , which was determined by graphing the percent inhibition on the y-axis and the concentration of the reference substance at which that level of inhibition was measured on the x-axis.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
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
Remarks on result:
other: 1000 mg/L was the maximum concentration tested.
Details on results:
Under the conditions of the test, there was no significant activated sludge respiration inhibition (inhibition less than 15%) at concentrations of the test substance as high as 1000 mg/L (1000 ppm) compared to the positive controls to which the test substance was not added.
Results with reference substance (positive control):
The Effective Concentration of the reference substance 3-5, dichlorophenol at which 50% inhibition occurred (EC50) was approximately 10 mg/L.
Reported statistics and error estimates:
The difference between the respiration rates of the two positive controls measured at the start and end of the test was less than 10%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, there was no significant activated sludge respiration inhibition at concentrations of the test substance as high as 1000 mg/L.
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was tested for toxicity towards activated sludge according to OECD Guideline 209 in the version dated 4-April-1984. For the determination of the toxic behavior of the test substance, activated sludge from the aeration tank of a municipal sewage treatment plant was exposed to the test substance at 10, 32, 100, 320, and 1000 mg/L nominal concentrations. For the reference substance 3,5-dichlorophenol, activated sludge was exposed at 3.2, 10, and 32 mg/L nominal concentrations. After a three hour incubation period, the inhibition of the respiration rate of the activated sludge was determined in comparison to a test solution without any test or reference substance.

Under the conditions of the test, there was no significant activated sludge respiration inhibition (inhibition less than 15%) at concentrations of the test substance as high as 1000 mg/L(1000 ppm) compared to the positive controls to which the test substance was not added.  The Effective Concentration of the reference substance 3-5, dichlorophenol at which 50% inhibition occurred (EC50) was approximately 10 mg/L. The difference between the respiration rates of the two positive controls measured at the start and end of the test was less than 10%. The Effective Concentrations of the test substance at which 20, 50, and 80% inhibition occurred (EC20, EC50, EC80, respectively) could not be determined because there was no inhibition at the highest test concentration of 1000 mg/L. The test is valid.