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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2018
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))
Version / remarks:
2010
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: Ecological Effects Test Guidelines of the United States Environmental Protection Agency (EPA 712-C-014): OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test"
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Balatonfüred, Hungary on 16 January 2018 (one day before the main test)
- Pretreatment: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (6.244 g wet weight), dried and the ratio of wet sludge to dry weight (0.5126 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (27 g dry weight that was equivalent to 329 g wet sludge) was suspended in isotonic saline solution (ad. 9 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis). (In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.) The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.41), additional pH adjustment of the inoculum was considered not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
7-8
Nominal and measured concentrations:
Nominal concentrations: 160, 64, 25.6, 10.24 and 4.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Material, fill volume: glass, 300 mL
- Aeration: yes, 0.5 L/min
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per reference control (replicates): 3
- No. of vessels per nitrification control (replicates): 3
- Sludge concentration: of 3 g/L (on dry weight basis)
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM PARAMETERS
- Preparation of synthetic sewage: Ratio of composition referring to 1000 mL:
Peptone 16 g
LAB-LEMCO Powder (BEEF extract) 11 g
Urea 3 g
NaCl 0.7 g
CaCl2 x 2H2O 0.4 g
MgSO4 x 7H2O 0.2 g
K2HPO4 2.8 g
Distilled water ad. 1000.0 mL
Analytical grade reagents were used in the study. The pH of the prepared synthetic sewage solution was in the range of 7.5 ± 0.5. The synthetic sewage was prepared one day before the treatment. The pH of the synthetic sewage was checked after preparation (pH=7.41) and additional pH adjustment was not performed. The prepared synthetic sewage was stored in the dark, in refrigerator at the temperature range of 3.2-5.3 °C.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED
after 3h: oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and the results were recorded for about 7-10 minutes. The measurement was carried out in completely filled Winkler bottles.
pH and the oxygen concentrations were determined at the start and at the end of the incubation period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
13.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CI: 10.8–17.0 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
48.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CI: 42.2–56.3 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption: no
- Blank controls oxygen uptake rate: 27.90 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 2.87 %.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50: 15.5 mg/L (within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary))
Reported statistics and error estimates:
The 3-hour EC50 values of the test and reference items (and the EC10 and EC80 values of test item) and their 95 %-confidence limits were calculated by appropriate statistical test (Probit analysis by IBM® SPSS® Statistics, Version 24 (2016) statistical software program). For the determination of the NOEC the calculated specific respiration rates were tested on significant differences to the control values by Dunnett’s Test (2-sided, α = 0.05) by IBM® SPSS® Statistics, Version 24 (2016) statistical software program. Statistical analysis of the homogeneity of replicate results was tested by adequate test statistics.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 48.6 mg/L (95% confidence limits: 42.2–56.3 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 10.24 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions according to OECD 209. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 4.1, 10.24, 25.6, 64 and 160 mg/L. Defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test was performed without pH adjustment. All validity criteria of the study were met. The inhibition of the oxygen consumption rates showed clear dose-related tendency in the whole examined concentration range 4.1-160 mg/L. At the lowest examined concentration of 4.1 mg/L inhibition was not noticed, at 10.24 mg/L 4.0 % inhibition, at 25.6 mg/L 29.5 %, at 64 mg/L 63.2 % inhibitions were calculated. At the highest examined concentration of 160 mg/L 86.2 % inhibition was obtained. Based on the available data the 3-hour EC10, EC50 and EC80 values and their 95% confidence limits were calculated by Probit analysis using IBM® SPSS® Statistics software. The values are the following: EC10: 13.9 mg/L (10.8–17.0), EC50: 48.6 mg/L (42.2–56.3), EC80: 110.6 mg/L (92.3–138.6). The specific respiration rates were compared with the blank control values using Dunnett t-test (α=0.05). Consequently the NOEC can be statistically and biologically determined as 10.24 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 48.6 mg/L (95% confidence limits: 42.2–56.3 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 10.24 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
48.6 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions according to OECD 209. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the nominal concentrations of 4.1, 10.24, 25.6, 64 and 160 mg/L. Defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test was performed without pH adjustment. All validity criteria of the study were met. The inhibition of the oxygen consumption rates showed clear dose-related tendency in the whole examined concentration range 4.1-160 mg/L. At the lowest examined concentration of 4.1 mg/L inhibition was not noticed, at 10.24 mg/L 4.0 % inhibition, at 25.6 mg/L 29.5 %, at 64 mg/L 63.2 % inhibitions were calculated. At the highest examined concentration of 160 mg/L 86.2 % inhibition was obtained. Based on the available data the 3-hour EC10, EC50 and EC80 values and their 95% confidence limits were calculated by Probit analysis using IBM® SPSS® Statistics software. The values are the following: EC10: 13.9 mg/L (10.8–17.0), EC50: 48.6 mg/L (42.2–56.3), EC80: 110.6 mg/L (92.3–138.6). The specific respiration rates were compared with the blank control values using Dunnett t-test (α=0.05). Consequently the NOEC can be statistically and biologically determined as 10.24 mg/L.

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