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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 2017
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: OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
For correct administration, ~3 g test item was ground with a pestle and mortar (as fine as possible). Thereafter, defined amounts of the ground test item (1 x 3; 1 x 30; 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each test flask. The subsequent calculations refer to the initial weighed nominal concentration.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Balatonfüred, Hungary, 07 August 2017
- Method of cultivation: The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 48 hours (2 days) and fed daily with 50 mL synthetic sewage/L activated sludge.
- Preparation of inoculum for exposure: 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 (6.613 g wet weight), dried and the ratio of wet sludge to dry weight (0.5668 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (60 g dry weight that was equivalent to 700 g wet sludge) was suspended in isotonic saline solution (ad. 20 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
pH:
7.55 - 8.30
Dissolved oxygen:
7.14 - 8.15
Nominal and measured concentrations:
Nominal concentration: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass bottles
- Material, fill volume: glass, 300 mL
- Aeration: yes, with compressed air (0.5 L/minute)
- No. of vessels per concentration (replicates):5
- No. of vessels per control (replicates): 8
- No. of vessels per nitrification control (replicates): 3
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Ultrapure water
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED:
after exactly 3 hours incubation time: measurement of respiration rate for 5 - 10 min (O2 electrode)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 3, 30, 300 mg
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 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: 33.69 mg O2 / g activated sludge (based on dry weight) in an hour with a coefficient of variation of 2.87 %.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 13.87 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 EC50 value of the reference item was calculated using Probit analysis. The analysis was done using the IBM® SPSS® Statistics, Version 24 (2016) statistical software program. The specific respiration rates of the highest test item concentration level were compared to the blank control values by 2 Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics, Version 24 (2016) software
Validity criteria fulfilled:
yes
Conclusions:
In the study according to OECD 209 with áctivated sludge the test item hab no influence on the specific respiration rates up to 1000 mg/L. The EC50 (3h) > 1000 mg/L, NOEC (3h) = 1000 mg/L
Executive summary:

The purpose of the 3-hour test according to OECD 209 was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. 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. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. 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 were investigated. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 32.65 mg O2/gh). No inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Description of key information

In the study according to OECD 209 with the test item no influence on the specific respiration rates up to 1000 mg/L were shown. The EC50 (3h) > 1000 mg/L, NOEC (3h) = 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The purpose of the 3-hour test according to OECD 209 was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. 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. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. 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 were investigated. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 32.65 mg O2/gh). No inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.