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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April - 24 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

A stock solution with a nominal concentration of 3200 mg/L was prepared with direct addition of the powdered test item, mixed into deionised water using ultrasonic bath for approximately 6 minutes. The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary
Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.75. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
21.0 – 21.9 °C
pH:
7.56 - 8.19
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Preliminary test: 10, 100, 1000 mg/L (nominal)
Definitive test: 59.60, 95.37, 152.59, 244.14, 390.63, 625.0, 1000.0 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
- Aeration: With compressed air (about 1 L/min)
- No. of vessels per concentration (replicates): 5
- No. of vessels per reference control (replicates): 1
- No. of vessels per untreated control (replicates): 8
- No. of vessels per abiotic control (replicates): Not required as no oxygen uptake in the preliminary test.
- Sludge concentration (weight of dry solids per volume): 3g sludge innoculum/L of suspended solids
- Nutrients provided for bacteria: Synthetic Sewage Feed : Peptone 16.0 g; Meat extract 11.0 g; Urea 3.0 g; NaCl 0.7 g; CaCl2 x 2H2O 0.4 g; MgSO4 x 7H2O 0.2 g; K2HPO4 2.8 g; Deionised water add. 1000.0 mL
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Inhibition of respiration rate.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: 10 mg/L: 3.49 % inhibition; 100 mg/L: 36.73 % inhibition; 1000 mg/L: 67.51 % inhibition
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol at nominal test concentrations of 1.0; 3.2; 10 and 32 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
99.54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- The average specific total respiration rate of the untreated (blank) controls was 30.07 mg O2/L/h/g solid, so the validity criteria was met.
- The variation coefficient of oxygen uptake rate in above untreated replicates was 17.94 % and thus, the validity criteria was met.
Results with reference substance (positive control):
- Results with reference substance valid? Yes. The EC50 values of the positive control substance 3,5-Dichlorophenol (3,5-DCP) were in the recommended range for total respiration using activated sludge derived from domestic sewage.
- Relevant effect levels: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 12.88 mg/L with 95% confidence limits of 10.51 – 15.79 mg/L.
Reported statistics and error estimates:
The 3-hour EC20, EC50 and EC80 values of the test item and the 3-hour EC50 values of the reference item with their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
With respect to the inhibitory effect of the test item on the total respiration rate, the 3-hour EC50 value of the test item was determined to be in the range of 10 mg/L to 100 mg/L (99.54 mg/L was calculated, with 95 % confidence limits of 65.74 – 150.71 mg/L).
Executive summary:

The effect of the substance on the respiration rate of activated sludge microorganisms was investigated by the method as described in the OECD Guidelines for Testing of Chemicals, No.: 209 Adopted: 22 July 2010.

Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test, a further definitive test was conducted with the following concentrations: 59.60, 95.37, 152.59, 244.14, 390.63, 625.0 and 1000.0 mg/L.

The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

The purpose of the test was only to assess the effects on total oxygen uptake, the inhibition effect in respect of nitrification process was not determined.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

With respect to the inhibitory effect of the test item on the total respiration rate, the 3-hour EC50 value of Amphosol CDB special Dried was determined to be in the range of 10 to 100 mg/L (99.54 mg/L was calculated, with 95 % confidence limits of 65.74 – 150.71 mg/L).

Description of key information

3 -h EC50 = 99.54 mg/L (OECD TG 209).

Key value for chemical safety assessment

EC50 for microorganisms:
99.54 mg/L

Additional information

The effect of the substance on the respiration rate of activated sludge microorganisms was investigated by the method as described in OECD TG 209 Adopted: 22 July 2010.

Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test, a further definitive test was conducted with the following concentrations: 59.60, 95.37, 152.59, 244.14, 390.63, 625.0 and 1000.0 mg/L.

The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

The purpose of the test was only to assess the effects on total oxygen uptake, the inhibition effect in respect of nitrification process was not determined.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

With respect to the inhibitory effect of the test item on the total respiration rate, the 3-hour EC50 value of Amphosol CDB special Dried was determined to be in the range of 10 to 100 mg/L (99.54 mg/L was calculated, with 95 % confidence limits of 65.74 – 150.71 mg/L).