Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

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:

08.02. – 10.02. 2016

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))

Deviations:

yes

Remarks:

no influence to the study results (see Any other information...)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

The stock solution was prepared by dissolution of 5.0 g of the test substance in 1000 mL deionized water on the day of the testing.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SYSTEM
- Laboratory culture: The 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 INOCULUM
The activated sludge was collected two days before the day of testing. After the sample collection the sludge was washed with potable water for 0.5 hour and subsequently decanted for 0.5 hour. This procedure was repeated three times in total.
Further the sludge was modified by addition of 50 mL of cultivation medium per 2 L of diluted sludge suspension at permanent aeration till the day of test.
The dry weight was determined from 10 mL of sludge suspension after 0.5 hour sedimentation.
Before the test the sludge was suspended in water up to concentration about 4 000 mg of sludge dry weight per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.

JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM
The activated sludge used in the test is in conformity with the recommendations of the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

approx. 7 – 8

Nominal and measured concentrations:

10, 100 and three replicates of 1000 mg·L-1 (Z1 – Z5 preliminary test)
The low level of inhibition of oxygen consumption by the test substance in the preliminary test demonstrated that a definitive test is unnecessary.
10, 100 and 1000 mg·L-1 (R1 – R3 reference substance)
Further two blank controls, the three abiotic controls with the highest concentration of the test substance and two beakers with specific nitrification inhibitor to determine the nitrification potential of sludge were included.

Details on test conditions:

TEST SYSTEM
- Test vessel: oxygen vessels (BOD bottles) with volume of about 280 mL for measurement of oxygen concentration
- Type (delete if not applicable): closed
- Aeration: by filtered pressured air
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge dose on concentration of 3000 mg.L-1 : 261 mL.L-1

WATER PARAMETERS
- deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: the pH adjustment to 6.0 was carried out.
- Photoperiod: daily lighting

TEST PROCEDURE
The defined dose of the test or reference substance, 100 mL of deionized water, 16 mL of the synthetic sewage feed and 250 mL of the prepared the sludge suspension were introduced into each test container. The volume was replenished up to 500 mL with deionized water.
The control samples were prepared only from the same volume of nutrient medium and sludge and replenished up to 500 mL with water.
The check of abiotic decomposition was performed at the highest concentration of the test substance and nutrient medium without sludge and with addition of 1 mL of mercury chloride solution.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The decline of oxygen concentration (respiration rate of activated sludge) was measured and recorded in 30-seconds intervals for 10 – 12 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The inhibition of oxygen consumption by the test substance in the preliminary test was lower than 50 % and demonstrated that a definitive test is unnecessary.
Range finding study
- Test concentrations: 10, 100 and 1000 mg.L-1

Results with positive control:
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 11.7 mg·L-1 (95% confidence interval: 11.2 – 12.2 mg·L-1)

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 11.7 mg·L-1 (95% confidence interval: 11.2 – 12.2 mg·L-1)

There was a deviation from the study plan. During the measuring of oxygen consumption of the test substance (Z3) the temperature was out of tolerable range (20 ± 2 °C) in the time 0.5 and 1.0 minute. The value of oxygen consumption measured in this time was not used for the evaluation in this container (Z3), so the deviation has not an influence to the study results.

Validity criteria fulfilled:

yes

Conclusions:

The highest inhibition the respiration rate of the activated sludge caused by the test substance Reactive Blue 234 was 5.8 % (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
The abiotic decomposition was not detected even at the highest concentration of the test substance.
The nitrifying activity of the activated sludge was detected in the preliminary test.

Executive summary:

The influence of the test substance, Reactive Blue 234, on the respiration rate 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. 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) were selected.

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

The nitrifying activity of the activated sludge was detected in the preliminary test.

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 substance Reactive Blue 234 was 5.8 % (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

Description of key information

The highest inhibition the respiration rate of the activated sludge caused by the test substance Reactive Blue 234 was 5.8 % 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^-1

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L