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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
according to guideline
other: Real Decreto 363/1995 (BOE núm. 133 de Junio de 1995)
GLP compliance:
Analytical monitoring:
Details on test solutions:
- Test concentrations: 10 mg/L, 32 mg/L, 100 mg/L, 320 mg/L and 1000 mg/L, and two controls.
- Preparation of the stock and test solutions: A stock solution of a concentration of 1 g/L of active ingredient of the test material has been prepared. Therefore, 1.022 g of the test material have been dissolved in 100 mL of deionized water in a 100 mL volumetric flask, Class A using agitation, so we have a stock solution of 10220 mg/L. Because the test material contains in average 97.87% of the active ingredient, the stock solution contains 1000 mg/L of active ingredient. The different solutions used in the trial have been prepared from this stock solution of 1000 mg/L.
- Actual concentration of the stock solution: 1001 mg/L of active ingredient.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Source: domestic waste water treatment plant in Manresa (Spain)
- Preparation of inoculum for exposure: After knowning the concentration of dried activated sludges (7.2 g/l) from a domestic waste water treatment plant, a suspenstion is prepared by adding water until the concentration os suspended matter is 4g/l. Then, 200 ml of this suspended sluge solution, is added to the vessels with the rest of components up to a final volume of 500 mL. Therefore, the final concentration of activated sludge (supended solids) is 1.6 g/L

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20±2 °C
ca. 7.0-7.5
Dissolved oxygen:
After the exposure period of 3hr. each sample has been measured with an oxigen electrode cell for a period of 10 minutes.
Nominal and measured concentrations:
Concentration of the test are: 0.1 mg/l; 0.32mg/l; 1 mg/l; 3.2 mg/l; 10 mg/l; 32 mg/l; 100 mg/l and two controls.

Details on test conditions:
- Test vessel: beaker, 500 mL
- Aeration: continuous aeration, approximate flux 1 L/min
- No. of vessels per concentration (replicates): 1
- Sludge concentration (weight of dry solids per volume): The dry weight of the activated sludge from the urban treatment plant is calculated (7.2 g/L) and is suspended in water to reach a concentration of 4 g/L of suspended matter, so that by adding 200 mL of these suspended sludge the final concentration of activated sludge is 1.6 g/L.

- Source/preparation of dilution water: declorated water
- Nutrients: synthethic residual water (as described in OECD 209), composed of 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4 diluted to 1000 mL with deionized water.

- Adjustment of pH: no
- Temperature: 20+-2ºC
- Details on termination of incubation: The desired concentration of test material and 16 mL of synthetic residual water are added to each vessel. The incubation period begins when 200 mL of activated sludge is added to each vessel at 15 minute interval to each of the test vessels. All vessels are aerated for a period of 3 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Oxygen measurements (mg/L) are collected during the trial ata 1-minute interval and breath rate (mg/L.h) obtained for each of the concentrations and controls.
Measurement apparatus: Oximeter

Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
481 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
Concentration of dissolved oxygen during the inhibition respiratory test where analyzed at 1 minute intervals.
Calculation of oxygen uptake rates are measured for each concentration and control.
Calculation of percentage of inhibition respiration rate is also reported.
Results with reference substance (positive control):
Respiration rate of the test control are: 26.82 mgO2/L.h and 26.70 mgO2/L.h. The difference between both values is 0% which is inside the acceptation criteria (15%).
EC50 (3h) of 3,5 Dichlorophenol is 12.1 mg/L, which is between the acceptation range of the OECD 209 guide (5mg/L - 30mg/L).

Table 1: pH values at the begining of the study:



Table 2: Oxygen measurements (mg/L) during the respiration inhibition test.

Conc (mg/L)mg O2/L in the defined time interval (minutes)




Tabla 3: Respiration rates (mg·L-1h-1) for each concentration and control

Respiration rate (mgL-1h-1)26.8228.9226.1622.9817.288.126.70



Table 4: % of respiration inhibition

 conc (mg/L) % respiration inhibition (3h)
 control 0
 10 0
 32 2.2
 100 14.1
 320 35.4
 1000 69.7
Validity criteria fulfilled:
The concentration of substance that produces an inhibition of the breathing rate in activated sludge (EC50 3h) is 481 mg/L
Executive summary:

The study of bacterial inhibition in activated sludge has been carried out for the substance. For the determination of bacterial inhibition the OECD guide 209 has been followed (1984) together with the Royal Decree 363/1995 (BOE No. 133 of June 1995).
This method is used to evaluate the effect that the substance under test has on microorganisms, measuring the rate of respiration under certain conditions and in presence of different concentrations of the substance.
Activated sludge from an urban treatment plant has been subjected to the substance at test concentrations: 10, 32, 100, 320 and 1000 mg/L of active ingredient. The total duration of the trial was 3 hours.
The result obtained in this test indicates that the substance presents a value of EC50 (3h) equal to 481 mg/L (act. ingr.)

Description of key information

One study conducted according to OECD 209 is available to determine the inhibition growth of micro-organisms of the substance. The study gives a value of EC50 (3h) of 481 mg/L, obtained by calculations based on experimental results.

Key value for chemical safety assessment

EC50 for microorganisms:
481 mg/L

Additional information