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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 25, 2012 - November 26, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge as collected but coarse particles were removed by settling for a short period and decanting the upper layer of finer solids. Afterwards, the sludge was decanted for a period (15 minutes). The supernatant was discarded and the sludge was resuspended in chlorine-free tap water, with shaking and aeration. The washing and decantation process was repeated four times. The dry mass of a known volume (100 mL) of the re-suspended sludge was determined and the sludge concentrated by removing liquor or diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/L. The activated sludge was continuously aerated (3 L/minute) at the test temperature, and was used on the next day. The sludge was fed daily with the synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) for two additional days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
7.26-7.99
Nominal and measured concentrations:
0 (control) 1, 7, 49, 343 and 2401 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel: 1000 mL beaker- Fill volume: 500 mL- Aeration: Forced aeration (0.5 to 1 L/min)- No. of vessels per concentration (replicates): 3 (test item), 1 (reference substance)- No. of vessels per control (replicates): 6 (test item), 2 (reference substance)TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Synthetic wastewater (filled up to a final volume of 1 liter with deionized water): 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.OTHER TEST CONDITIONS- Adjustment of pH: Synthetic sewage feed pH was adjusted from 7.15 to 7.52.Test substance stock solution pH was adjusted from 4.75 to 7.53.Reference substance stock solution pH was adjusted from 4.75 to 7.53.Activated sludge stock suspension 3g/L: pH = 7.75.EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At the end of 3 hour incubations, samples were withdrawn to measure the rate of decrease of the concentration of dissolved oxygen in a completely filled BOD bottle. The concentration of dissolved oxygen was continuously measured and recorded for a 10 minute period, until the oxygen concentration falls below 2 mg/L. From the data collected, the specific respiration rates of the control and test mixtures were calculated; the percentage inhibition was then calculated. With the use of the specific nitrification inhibitor (N-allylthiourea, ATU), the heterotrophic respiration was measured, and the differences between these and the corresponding total respiration rates represent nitrification.TEST CONCENTRATIONS- Range finding study: A preliminary test was performed to estimate to determine the main test concentrations based on the inhibition of oxygen consumption (3 hour exposure, 10 g/L test item or reference substance, 3 g/L of activated sludge).- Test concentrations: 4.62, 46.2 and 462 mg/L (3 replicates of the highest dose), blank control (without test item, 2 replicates), abiotic control (1000 mg/L test item in test medium without activated sludge). The same procedure was performed with the reference substance 3,5-dichlorophenol at 0.30, 3.0, 30.0 mg/L concentrations to check the sensitivity of the sludge.- Results used to determine the conditions for the definitive study:Test substance: The total respiration inhibition was determined to be about 800 mg/L, the heterotrophic respiration inhibition was about 1000 mg/L and the nitrification inhibition was about 500 mg/L. These results were in accordance with the validity criteria.Reference substance: The total respiration inhibition was determined to be about 15 mg/L, the heterotrophic respiration inhibition was about 50 mg/L and the nitrification inhibition was about 1.8 mg/L. These results were in accordance with the validity criteria.Abiotic control: No significant oxygen uptake occurred and the total respiration rate due to abiotic processes was assumed to be 0. Therefore, the abiotic control was not included in the main test.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol at 0, 0.05, 0.5, 5.0, 50 mg/L)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
530 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Total respiration inhibition
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Heterotrophic respiration inhibition
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
236 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Nitrification inhibition
Details on results:
The estimated end points EC50 for the test substance were found to be:1. total respiration inhibition: 530 mg/L 2. heterotrophic respiration inhibition: 1240 mg/L 3. nitrification inhibition: about 236 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? Yes.The EC50 of the reference inhibitory substance 3,5-dichlorophenol were found to lie in the ranges allowed by the validity criteria.The estimated end points EC50 for the reference substance were found to be:1. total respiration inhibition: 14.5 mg/L which lies in the range 2 mg/L to 25 mg/L according to validity criteria2. heterotrophic respiration inhibition: 31.0 mg/L which lies in the range 5 mg/L to 40 mg/L of validity criteria3. nitrification inhibition: about 1.25 mg/L which is in the range 0.1 mg/L to 10 mg/L according to validity criteria.

Values of the end points ECx (mg/L test item) for total, heterotrophic and nitrification inhibition:

             ECx, mg/L

Inhibition, %

EC10

EC20

EC50

EC80

EC90

Total

20.9

91

530

1760

>2000

Heterotrophic

146

377

1240

2300

>2000

Nitrification

< 1

15

236

1230

2060

The NOEC value is expected to lie below 1 mg/L.

Validity criteria fulfilled:
yes
Remarks:
(Blank controls O2 uptake rate was about 20 mg O2/g of activated sludge in 1 hour; in control replicates the coefficient of variation of O2 uptake rate are <30% at the end of definitive test)
Conclusions:
The 3h-EC50 values for total, heterotrophic and nitrification inhibition were determined to be 530, 1240, 236 mg/L respectively in activated sludge.
Executive summary:

Activated sludge respiration inhibition test was carried out according to OECD 209 Guideline. The inhibition of three different oxygen uptakes was determined, total, heterotrophic only and due to nitrification. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode. 3 g/L SS of activated sludge were exposed to 0 (control) 1, 7, 49, 343 and 2401 mg/L test item for 3 hours incubation period. The chemical 3,5-dichlorophenol was used as the reference inhibitory substance. The chemical N-methylaniline was used as a specific reference inhibitor of nitrification. All the validity criteria were fulfilled.

The following end points EC50 were determined for total, heterotrophic and nitrification inhibition for test substance:

EC50(total) = 530 mg/L

EC50 (heterotrophic) = 1240 mg/L

EC50 (nitrification) = 236 mg/L

The estimated NOEC values are expected to be below 1 mg/L.

Description of key information

Key study: Test method OECD 209. GLP study. The 3h-EC50 in activated sludge were determined to be 530 mg/L for total respiration inhibition, 1240  mg/l for heterotrophic respiration inhibition and 236 for nitrification inhibition.

Key value for chemical safety assessment

EC50 for microorganisms:
530 mg/L

Additional information

Key study: Activated sludge respiration inhibition test was carried out according to OECD 209 Guideline. 3 g/L SS activated sludge were exposed up to 2401 mg/L under static conditions for 3 hours incubation period and the total, heterotrophic only and due to nitrification oxygen uptakes were determined. The EC50 were determined to be as follows:

EC50 (total) = 530 mg/L

EC50 (heterotrophic) = 1240 mg/L

EC50 (nitrification) = 236 mg/L