Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-treatment of test item without ATU:
Direct weighings were prepared to give the test item concentrations (100 mg/L active ingredient equalling to 115.9 mg/L test item). The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 6.2 – 6.3. The pH was adjusted to pH 7.2 – 7.4 with NaOH.
- Pre-treatment of test item with ATU:
Direct weighings were prepared to give the test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 6.2 – 6.3. The pH was adjusted to pH 7.3 – 7.4 with NaOH.
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

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Method of cultivation: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Preparation of inoculum for exposure: 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.
- Initial biomass concentration: 1200 mg/L suspended solids
- Date of collection : 2017-10-09

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

Test without allyl thiourea (ATU): 19.4 - 20.2 °C
Test with allyl thiourea (ATU): 19.5 - 20.4 °C

pH:

Test without allyl thiourea (ATU): 7.4 - 8.4
Test with allyl thiourea (ATU): 8.2

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Typ: closed
- Aeration: 3 hours with permanent aeration
- No. of vessels per concentration (replicates): 3 replicates (without ATU, 2 replicates (with ATU)
- No. of vessels per control (replicates): 6 replicates (without ATU), 4 replicates (with ATU)
- Sludge concentration (weight of dry solids per volume): 1200 mg/L suspended solids
- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
-Control vessels (inoculated sample without test item) were prepared
- Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge.
- 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.
- The exposure medium with the reference substance was prepared to achieve the test concentrations.
Pre-treatment of reference compound without ATU: a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 - 8 with HCl.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption, temperature and ph were measured after an aeration time of 3 hours

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol , concentration (2.5, 5, 10, 20, 40 mg/L)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

- Bayscript Gelbkomponente showed 2.0 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L active ingredient.
- Bayscript Gelbkomponente showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L active ingredient.
- 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.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50: 13.281mg/L
:

As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L active ingredient 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).

Validity criteria fulfilled:

yes

Remarks:

(oxygen uptake rate blank controls differ not less than 20 mg oxygen -coefficient of variation of oxygen uptake in control not more than 30 % at end of test - EC50 reference compound in the range of 2–25 mg/L total respiration)

Conclusions:

After an incubation period of 3 hours Bayscript Gelbkomponente showed an EC50 > 100 mg/L and a NOEC >= 100 mg/L active ingredient. Bayscript Gelbkomponente showed 2.0 % respiration inhibition of activated sludge at the limit test item concentration of 100 mg/L active ingredient.

Executive summary:

To asses the effect of Bayscript Gelbkomponente on microorganisms a study was performed in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02). The activated sludge was exposed to Bayscript Gelbkomponente at a limit test item concentration of 100 mg/L active ingredient equalling to 115.9 mg/L test item (purity 86.3 %). The respiration rate of each mixture was determined after aeration periods of 3 hours.

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. 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. Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge. 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.

Oxygen consumption, temperature and pH were measured and recorded after an aeration time of 3 hours.

After an incubation period of 3 hours Bayscript Gelbkomponente showed an EC50 > 100 mg/L and a NOEC >= 100 mg/L active ingredient. Bayscript Gelbkomponente showed 2.0 % respiration inhibition of activated sludge at the limit test item concentration of 100 mg/L active ingredient. Bayscript Gelbkomponente showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L active ingredient.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

After an incubation period of 3 hours Bayscript Gelbkomponente showed an EC50 > 100 mg/L and a NOEC >= 100 mg/L active ingredient.

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