Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

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:

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))

Version / remarks:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

SOURCEThe 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 INOCULUMThe 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 mgof suspended solids per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.JUSTFICATION FOR THE SELECTIONThe 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:

3 h

Test temperature:

20 ± 2°C

pH:

7 - 8

Nominal and measured concentrations:

10, 100 and 1000 mg/L (nominal concentrations)

Details on test conditions:

INSTRUMENTS AND EQUIPMENT - oxygen vessels (BOD bottles) with volume of about 280 mL for measurement of oxygen concentration - graduated cylinders of volume 1000 mL - analytical balance AG135 (Mettler Toledo) - analytical balance Kern 870-15 - oximeter WTW Inolab OXI 730 with membrane probe CellOx 325 and with printer - stirring adapter for electrode - pH meter WTW- pH 539 with pH-electrode WTW SenTix 81 with temperature probe - magnetic stirrers - aerated apparatus with glass ends (pressured air is passed through filter for odour and oil removal) - air-conditioned room, free of dust, with automatic temperature recording - common laboratory equipment (pipettes, beakers, volumetric cylinders, volumetric flasks and further ones) SOLUTIONS AND CHEMICALSThe deionized water was used for the preparation of solutions. Analytical grade reagents were used.The stock solution of synthetic sewage feed:- peptone: 16 g- meat extract: 11 g- urea: 3 g- sodium chloride (NaCl): 0.7 g- calcium chloride dehydrate (CaCl2·2H2O): 0.4 g- magnesium sulphate heptahydrate (MgSO4·7H2O): 0.2 g- anhydrous dipotassium hydrogen phosphate (K2HPO4): 2.8 gdeionized waterup to volume of 1000 mLThe pH value of this solution is: 7.0 - 8.0.The synthetic sewage feed for sludge adaptation was always prepared fresh on the day of the use.The solutions for dissolution and pH adjustment- sulphuric acid (H2SO4), of concentration 0.5 mol/L- sodium hydroxide (NaOH), of concentration 1.0 mol/LSterilizing solutionMercury chloride (HgCl2), of concentration of 5 g/L; 1 mL of this solution per 500 mL of reaction mixture was used for elimination of microbial activity.Specific inhibitor of nitrificationN-allylthiourea (ATU), of concentration of 2.5 g/L; 2.32 mL of this solution per 500 mL of reaction mixture was used for inhibition of nitrification in a activated sludge.Reference substanceName: 3,5-dichlorophenol (CAS No.: 591-35-5)Manufacturer: Sigma-Aldrich Chemie GmbH, Riedstr. 2, D-89555 Steinheim, GermanyPurity: 99.7 %Stability/Expiration: 07/2017PREPARATION OF SOLUTIONS FOR THE TEST Test substance The test substance is soluble in deionized water in concentrations needed for the test. The stock solution was prepared by dissolution of 5.0 g of the test substance in 1000 mL deionized water on the day of testing. The concentrations 10, 100 and three replicates of 1000 mg/L of the test substance were tested in the preliminary test. Reference substance The stock solution of the reference substance was prepared by dissolution of 0.1 g of 3,5-dichlorophenol in 200 mL deionized water. The ultrasound for 60 minutes to accelerate the dissolution was used. The resulting solution has a concentration of 500 mg/L. The stock solution was prepared on the day of testing. The concentration range used was 5 - 20 mg/L. It was calculated by geometric progression with factor of 2.0.TEST CONDITIONS - Temperature: 20 ± 2°C - Exposition time: 180 minutes - Lighting: daily - Stirring and aeration: with filtered pressured air, airflow rate 0.5 L/min.The pH value of reaction mixtures during the preliminary test: approx. 7 – 8 TEST PROCEDUREExperimental designThe respiration rates of the activated sludge in standard nutrient medium were measured in the presence of three concentrations of the test substance after contact time of 3 hours. 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 and two beakers with specific nitrification inhibitor to determine the nitrification potential of sludge were included. 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 inhibition effects of the test and reference substance at each concentration were expressed as the percentage of respiration rate from the average of control respiration rates determined at the beginning and the end of the test. The low level of inhibition of oxygen consumption by the test substance in the preliminary test demonstrated that a definitive test is unnecessary. Preparing of the test mixturesTest/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 container. 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 the 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 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. The pH adjustment of the test mixtures in the preliminary test was carried out in abiotic controls. MeasurementThe measurement of the oxygen content was performed in aliquots of the reaction mixtures after 3-hour exposure period. Bottles with narrow neck and volume of about 280 mL were filled with the sample and positioned on a magnetic stirrer. Then, the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottleneck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured and recorded in 30-seconds intervals for 10 minutes. The pH was determined in the remaining part of the reaction mixture.

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

Details on results:

The nitrification respiration rate 18.7 % 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. In this case, the inhibitory effect of the test substance is only 8 %. This low level of inhibition demonstrated that further experiments are not needed.

Results with reference substance (positive control):

see Table 1

Table 1: Evaluation of total inhibition of respiration rates in the preliminary test

Container number	Label	Concentration (mg/L)	Total inhibition (%)
1	K1	0	-
2	Z1	10	2.7
3	Z2	100	1.4
4	Z3	1000	3.4
5	Z4	1000	8.0
6	Z5	1000	4.1
7	R1	5	23.4
8	R2	10	57.3
9	R3	20	82.8
10	Zab1	1000	-
11	Zab2	1000	-
12	Zab3	1000	-
13	KN1	0	-
14	KN2	0	-
15	K2	0	-

K1, K2: control experiments

Z1 - Z6: test substance

R1 - R3: reference substance

Zab1 - Zab3: abiotic decomposition with the test substance

KN1, KN2: nitrification potential of the sludge

Validity criteria fulfilled:

yes

Conclusions:

The highest inhibition the respiration rate of the activated sludge caused by the test substance Direct Black 112 was 8.0 % 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/LThe abiotic decomposition was not detected even at the highest concentration of the test substance.

Executive summary:

The influence of the test substance, Direct Black 112, on the respiration rate of activated sludge was investigated after a contact time of 3 hours. 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 (Carbon and Ammonium Oxidation), Council Regulation (EU) No. 2016/266, published in O.J. L 54/1, 2016.

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 nitrification respiration rate 18.7 % 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. In this case, the inhibitory effect of the test substance is only 8 %. This low level of inhibition demonstrated that further experiments are not needed.

The pH value during the preliminary test should be in the range of 7.0 to 8.0. The pH values of abiotic controls was higher than this prescribed range, therefore the pH values were adjusted.

The prescribed validity criteria in the test were fulfilled. Since all criteria of acceptability were met, this study is considered to be valid.

The highest inhibition the respiration rate of the activated sludge caused by the test substance Direct Black 112 was 8.0 % 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

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L