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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Remarks:
test item and reference compound concentrations were based on nominal concentrations
Vehicle:
no
Details on test solutions:
I. Pre-treatment of test item without ATU
- Method of application: direct weighing
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water
- Stirring period of test item before start of incubation time: 17 hours
- pH was measured and ranged between pH 6.6 - 6.7
- pH was adjusted to pH 7.3 - 7.4 with NaOH

II. Pre-treatment of reference compound without ATU
- Method of application: stock solution (500 mg/L prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluted to 0.5 litre with deionised water)
- pH was adjusted to pH 7-8 with HCl

III. Pre-treatment of test item with ATU
- Method of application: direct weighing
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water
- Stirring period of the test item before start of incubation time: 17 hours
- pH was measured and ranged between pH 6.5 - 6.6; pH was adjusted to pH 7.2 -7.3 with NaOH

- 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
- The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C.
- Control vessels (inoculated sample without test item) were prepared the same way.

- For the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period.

- To determine the heterotrophic oxidation four additional controls and two replicates with the test item concentration 100 mg/L, all containing 1.25
mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L, were prepared.

- Vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no
activated sludge.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test organism
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Date of collection: 2017-07-10
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of suspension before application: 7.6
- Synthetic sewage feed: a synthetic waste water feed was prepared
- pH of the synthetic sewage feed: 7.5 ± 0.5
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
19.5 - 20.4 °C
pH:
8.1 - 8.3
Physico-chemical oxygen consumption control: 7.0
Dissolved oxygen:
n.a.
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
- Test item concentration (nominal): 100 mg/L
- reference substance concentrations 3,5-Dichlorophenol (nominal): 2.5, 5, 10, 20 and 40 mg/L
Details on test conditions:
Exposure conditions
- Test item concentration/s: 100 mg/L, 3 replicates; 100 mg/L with ATU, 2 replicates
- Control: 6 replicates, 4 replicates with ATU
- Test item concentration in physico-chemical oxygen consumption control: 100 mg/L
- Test vessels: 300 mL glass Erlenmeyer flasks
- Method of application: direct weighing
- Test concentration of the activated sludge: 1200 mg/L suspended solids
- Test temperature: 20 ± 2°C
- Stirring period of the test item before start of incubation time: 17 hours
- Incubation time: 3 hours with permanent aeration

- Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (Purity: 99.9%; Acros Organics)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Solvent Violet 59 showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit concentration of 100 mg/L active ingredient.
As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50.
The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).
Results with reference substance (positive control):
In the present test the EC50 of the reference compound 3,5-Dichlorophenol was 11.74 mg/L

The oxygen consumption in the presence of N-allylthiourea was determined in four controls without test item and in two replicates of the test item concentration 100 mg/L. As no inhibition was observed for the total oxygen consumption at 100 mg/L no differences between the heterotrophic and the nitrification oxygen uptake rates have to be calculated.

Validity criteria fulfilled:
yes
Remarks:
oxygen uptake rate >=20 mg oxygen per gram of activated sludge in an hour; coefficient of variation of oxygen uptake in the control replicates <= 30 % at the end of the test; EC50 of reference compound between 2–25 mg/L for total respiration
Conclusions:
Solvent Violet 59 showed 0.0 % respiration inhibition of activated sludge at the highest test concentration of 100 mg/L after 3 hours. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.
Executive summary:

According to OECD Guideline 209, which is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008), the activated sludge was exposed to a limit concentration of Solvent Violet 59 of 100 mg/L. After 3 hours of exposure the substance showed 0.0 % respiration inhibition of activated sludge. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed. To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed aerated Erlenmeyer flasks. The oxygen content was measured with an oxygen meter (redox electrode). Six controls without Macrolex Rot B were included in the test design, three at the start and the others at the end of the test series. The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated. The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration. The EC50 of the reference compound 3,5-Dichlorophenol was 11.74 mg/L.

Description of key information

Solvent Violet 59 showed 0.0 % respiration inhibition of activated sludge at the highest test concentration of 100 mg/L after 3 hours. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Should read: EC50 > 100 mg/L, NOEC >= 100 mg/L