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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-14 to 2017-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
, signed 2016-05-31
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Samples from test start (exactly 20 mL; without inoculum) were filteres (0.2 µm, PES membrane, DIA Nielsen, Germany), transferred into disposable 25 mL scintillation vials (Sarstedt, Nuembrecht, Germany), and acidified by addition of 0.2 mL of concentrated nitric acid.
Samples from test end (with inoculum) were filtered twice with a 1.2 µm PES Membrane (VWR, Germany) followed by a 0.2 µm PES membrane (DIA Nielsen). Afterwards the filtrates were centifuged (9000 x g, 10 min, 20 °C). The supernatant was taken and acidified with 0.15 mL conc. nitric acid into 15 mL of sample
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The solid was added directly on a weight basis due to unkonw water solubility. The required mass to obtain accurate test concentrations was applied on pieces of Teflon, which were subsequently added to the dilution water. To achieve a maximum dilution of the test item, the assay was stirred for 20 ± 2 h.
- Controls: Reference item (3,5-Dichlorophenol), Inoculum blank (control), abiotic control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Fresh samples of activated sludge are withdrawn on 14-08-2017 (range finding test) and 11.09.2017 (main test) from sewage treatment plant "Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany"; which is mainly fed with municipal wastewater
- Preparation of inoculum for exposure: Supernatant was decanted after a settling phase. A stock solution was prepared with washed activated sludge in chlorine-free tap water with a mixed liquor suspended solids level of 3.8 g dry mass sludge/L (main test)
- Pretreatment: Sludge stock solution was fed with 50 mL/L synthetic sewage feed and incubated areated overnight at 20 ± 2°C in the dark.
- Initial biomass concentration: Mixed liquor suspended solids level in the test vessel was nominally 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20°C either at test start and end
pH:
pH at test start: 7.5 ± 0.5
pH after 3 h of incubation: 8.2 - 8.5
Nominal and measured concentrations:
Nominal test concentrations in the main test: 0 (control), 3.2, 8.0, 20, 50, 125 mg test item/L
The nominal loading was confirmed at test start with mean recovery rates of 110 -124 %. At test end (after incubation) the recovery was 57 - 70 %.
Details on test conditions:
TEST SYSTEM
- Test vessel: 1L Erlenmeyer flask
- Material, size, headspace, fill volume: 500 mL before addition of the inoculum
- Aeration: stirred and aerated (air flow 0.5 - 1 L/min) during incubation
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3.5 g/L
- Nutrients provided for bacteria: synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+)

EFFECT PARAMETERS MEASURED: respiration rate (total oxygen uptake including heterotrophic and nitrification uptake) after 3 h

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (control), 10, 100, 1000 mg test item/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
25.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: CL 95%: 4.3 - 40.9 mg/L / analytically verified concentrations
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
111.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: CL 95 %: 78.8 - >125.0 mg/L / analytically verified concentrations
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: analytically verified concentrations
Details on results:
The EC10, EC20 and EC50 were found to be 24.5 mg Zinc-5-nitroisophtalate per liter (CL 95: 4.3 – 40.9 mg/L), 41.2 mg Zinc-5-nitroisophtalate per liter (CL 95: 14.3 – 60.2 mg/L) and 111.4 mg Zinc-5-nitroisophtalate per liter (CL 95: 78.8 – > 125 mg/L), respectively, based on nominal concentrations. The EC80 was found to be higher than the highest concentration tested.
Results with reference substance (positive control):
The EC50 of the reference item was found to be 18 mg/L and therefore within the accepted range of 2-25 mg/L (OECD 209).
Validity criteria fulfilled:
yes
Conclusions:
The NOEC, EC10 and EC50 concentrations for the 3-h respiration inhibition of activated sludge by Zinc-5-nitroisophthalate were determined with 20, 24.5 and 111.4 mg/L (nominal, analytically verified), respectively in a GLP-conform test according to OECD TG 209.

Description of key information

Respiration inhibition of activated sludge was observed after 3 h incubation with zinc-5 -nitroisophthalate in a GLP-conform test according to OECD TG 209 [Simon, 2018] resulting in NOEC, EC10 and EC50 values of 20, 24.5 and 111.4 mg/L (nominal, analytically verified), respectively.

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
111.4 mg/L
EC10, LC10 or NOEC for microorganisms:
24.5 mg/L

Additional information