Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

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:

07.03.-09.03.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance is soluble in deionized water. The stock solution was prepared by dissolution of 5.0 g of the test substance in 2000 mL deionized water on the day of the testing. The concentrations 10, 100 and three replicates of 1000 mg.L-1 of the test substance were tested in the preliminary test.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SYSTEMThe activated biological sludge containing mixed culture of microorganisms obtained from the second step of sewage treatment plant of Pardubice was used for the testing. The wastewater processed by the sewage treatment plant is predominantly municipal.PREPARATION OF THE APPLICATION OF TEST SUBSTANCEThe activated sludge was collected two days before the day of testing.After the sample collection the sludge was washed with potable water for 30 minutes and subsequently decanted for 30 minutes. This procedure was repeated three times in total. Further the sludge was fed with the 100 mL synthetic sewage feed per 2 L of diluted sludge suspension at permanent aeration till the day of the test. The suspended solids concentration was determined from 10 mL of sludge suspension after 30-minutes sedimentation.Before the test the sludge was suspended in water up to concentration about 3000 mg of suspended solids per litre. The pH adjustment to 6.3 was carried out. In that way modified sludge suspension was aerated until the use.JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEMThe activated sludge obtained is in conformity with the recommendations of the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

180 min

Test temperature:

20± 2 °C

pH:

7-8

Nominal and measured concentrations:

10, 100, three replicates of 1000 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel: aerated apparaturs with glass ends- Type (delete if not applicable): closed- Aeration: with filtered pressured air, airflow rate 0.5 L.min-1- control experiment 1, (K1)- 3 concentrations of the test substance: 10, 100 mg.L-1and 1000 mg.L-1, (three replicates of highestconcentration), (Z1 – Z5)- 3 concentrations of the reference substance 5, 10 and 20 mg.L-1, (R1 – R3)- abiotic decomposition check at test substance highest concentration 1000 mg.L-1, (three replicates ),(Zab1 – Zab3)- nitrification potential of sludge, (KN1-KN2)- control experiment 2, (K2)WATER PARAMETERS- deionized waterOTHER TEST CONDITIONS- Adjustment of pH: was not carried out- Photoperiod: daily lighting- Stirring and aeration: with filtered pressured air, airflow rate 0.5 L.min-1.- The pH value of reaction mixtures during the preliminary test: approx. 7 – 8TEST PROCEDURETest/reference substance100 mL of deionized water, the defined dose of the test or reference substance, 16 mL of the synthetic sewage feed and 250 mL of the prepared the sludge suspension were introduced into each test conta iner. The volume was replenished up to 500 mL with deionized water.Blank controlThe blank controls were prepared at the beginning and the end of the exposure period. The blank controls contained only 16 mL of synthetic sewage feed and 250 ml of the sludge suspension and replenished up to 500 mL with deionized water.Abiotic controlThe check of abiotic decomposition was prepared at the highest concentration of the test substance, 16 mL synthetic sewage feed without the sludge suspension and with addition of 1 mL of sterilizing solution (mercury chloride solution). The volume was replenished up to 500 mL with deionized water.Nitrification potential of sludgeThe mixtures to determine the nitrification potential of sludge included 16 mL of the synthetic sewage feed, 250 mL the sludge suspension and 2.32 mL of specific inhibitor of nitrification (N-allylthiourea solution). The volume was replenished up to 500 mL with deionized water.The activated sludge concentration in all test, reference and blank (but not abiotic control) mixtures is nominally 1.5 g.L-1 of suspended solid.The test mixtures were prepared in turns in 15 minutes time intervals and aerated/incubated at 20 °C ± 2 °C for 3 hours. The aeration was carried out by means of glass pipettes.EFFECT PARAMETERS MEASURED: respiration rate of activated sludgeTEST CONCENTRATIONS- Test concentrations: 10, 100, 1000 mg.L-1

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? yes - Relevant effect levels: EC50 = 9.9 mg.L-1 (95% confidence interval: 7.0 – 13.9 mg.L-1)

Validity criteria fulfilled:

yes

Conclusions:

The highest inhibition the respiration rate of the activated sludge caused by the test substance Direct Blue 78 was 16.9 % (see table 4) in the preliminary test. This low level of inhibition of oxygen consumption by the test substance in the preliminary test demonstrated that a definitive test is unnecessary. The result could be used as final one, because preliminary test design includes three replicates required by guidelines. Exact value of EC50 could not be calculated, the value of EC50 is given in the form of a range:EC50 > 1 000 mg.L-1The abiotic decomposition was not detected even at the highest concentration of the test substance.The nitrification respiration rate 18.5 % of the total respiration rate in the blank controls was detected in the preliminary test. In the case of observing the inhibitory effect of the test substance on the respiration of activated sludge it would be necessary to take into account the activity of nitrifying.

Executive summary:

The influence of the test substance, Direct Blue 78, on the respirationrate of activated sludge was investigated after a contact time of 3 hours.

Test performance

Test was performed according to:

OECD Test Guideline No. 209 – Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation). Adopted July 22, 2010

Method C.11 – Activated Sludge Respiration Inhibition Test, Council Regulation (EC)
No. 440/2008, published in O.J.L 142, 2008.

The preliminary test was performed using 3 concentrations 10, 100 and 1000 mg·L^-1. Further two blank controls, the three abiotic controls with the highest concentration of the test substance was included and two beakers with specific nitrification inhibitor to determine the nitrification potential of sludge.

 

3,5-dichlorophenol was used as reference substance. Three concentrations in geometric progression with a factor of 2.0 (5, 10 and 20 mg·L^-1) were selected.

 

The abiotic decomposition was not detected even at the highest concentration of the test substance.

The nitrification respiration rate 18.5 % of the total respiration rate in the blank controls was detected in the preliminary test.In the case of observing the inhibitory effect of the test substance on the respiration of activated sludge it would be necessary to take into account the activity of nitrifying.

The pH value during the preliminary test was in the range of 7.0 to 8.0.

 

Validity of the test

The prescribed validity criteria in the test were fulfilled.

Since all criteria of acceptability were met, this study is considered to be valid.

 

Results of the respiration inhibition test

 

The highest inhibition the respiration rate of the activated sludge caused by the test substanceDirect Blue 78was 16.9 % (see table 4) in the preliminary test. This low level of inhibition of oxygen consumption by the test substance in the preliminary test demonstrated that a definitive test is unnecessary.

The result could be used as final one, because preliminary test design includes three replicates required by guidelines.Exact value of EC₅₀could not be calculated, the value of EC₅₀ is given in the form of a range:

 

EC₅₀> 1 000 mg·L^-1

Description of key information

Test was performed according to: OECD Test Guideline No. 209 – Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation). Adopted July 22, 2010 Method C.11 – Activated Sludge Respiration Inhibition Test, Council Regulation (EC)
No. 440/2008, published in O.J.L 142, 2008.

Only one study is available.

GLP study.

Klimish score 1.

EC₅₀> 1 000 mg·L^-1

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L