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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15.08.-17.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
published in O.J. L 54/1 (2016)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
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 (with pH adjustment) and three replicates of 1000 mg/L (without pH adjustment) of the test substance were tested in the preliminary test.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
TEST SYSTEM
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 TEST SUBSTANCE
The 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 next day. The next day the sludge was washed with potable water and decanted according to the procedures described for the first day. This procedure was repeated three times in total again. The suspended solids concentration was determined from 10 mL of sludge suspension after the last 30-minutes sedimentation. The sludge was fed with the 100 mL synthetic sewage feed per 2 L of diluted sludge suspension at permanent aeration till day of the test. Before the test the sludge was suspended in water up to concentration about 3000 mg of suspended solids per litre. The pH adjustment to 6.1 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 obtained is in conformity with the recommendations of the test guidelines.

JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM
The activated sludge obtained is in conformity with the recommendations of the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
6.7 - 7.9 without pH adjustment
7 - 8 with pH adjustment
Nominal and measured concentrations:
Nominal concentration: 10, 100 and 1000 mg/L
Details on test conditions:
SOLUTIONS AND CHEMICALS
The stock solution of synthetic sewage feed:
Peptone: 16 g
meat extract: 11 g
urea: 3 g
NaCl: 0.7 g
CaCl2·2H2O: 0.4 g
MgSO4·7H2O: 0.2 g
K2HPO4: 2.8 g
deionized water up to volume of 1000 mL
The 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
H2SO4 0.5 mol/L
NaOH 1.0 mol/L

Sterilizing solution
Mercury 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 nitrification
N-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.

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)

PREPARATION 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 (with pH adjustment) and three replicates of 1000 mg/L (without pH adjustment) 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: 3 hours
Lighting: daily light
Stirring and aeration: with filtered pressured air
The pH value of reaction mixtures during the preliminary test: approx. 7 – 8

TEST PROCEDURE
Experimental design
The 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. Because the solutions of the test substance with the concentration 1000 mg/L had pH value 6.7, the pH of these solutions were adjusted (to pH value 7.0). Since neutralisation may change the chemical properties of the test substance, were included three test vessels with a concentration of 1000 mg/L at the end of the test, which were tested without pH adjustment.
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 mixtures
Test/reference substance
100 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 control
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 control
The 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 sludge
The 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 was performed in the test mixtures on the concentration 1000 mg/L in the preliminary test.

Measurement
The pH was determined in the reaction mixtures at the beginning of the test.
The 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 11 minutes. The pH was determined in the remaining part of the reaction mixture.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (CAS: 591-35-5)
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:
see Table 1
Results with reference substance (positive control):
EC50 = 6.7 mg/L (3,5-dichlorophenol)

Table 1: Evaluation of inhibition of respiration rates

Container number

Label

Concentration

mg/L

Total inhibition
(%)

1

K1

0

-

2

Z1

10

1.4

3

Z2

100

4.3

4

Z3

1000

16.9

5

Z4

1000

20.5

6

Z5

1000

22.2

7

R1

5

39.0

8

R2

10

66.6

9

R3

20

84.2

10

Zab1

1000

-

11

Zab2

1000

-

12

Zab3

1000

-

13

KN1

0

-

14

KN2

0

-

15

K2

0

-

16

Z6

1000

20.9

17

Z7

1000

23.4

18

Z8

1000

19.2

K1, K2 – control experiment

Z1 – Z5 – test substance with pH adjustment

Z6 – Z8 – test substance without pH adjustment

R1 – R3 – reference substance

Zab1 – Zab3 – test substance – abiotic decomposition

KN1 – KN2 – nitrification potential of sludge

Validity criteria fulfilled:
yes
Conclusions:
The highest inhibition the respiration rate of the activated sludge caused by the test substance, Acid Brown 235, was 22.2 % (with the pH adjustment) and 23.4 % (without pH adjustment) in the preliminary test. These low levels 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 nitrification respiration rate 25.9 % 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 highest inhibitory effect of the test substance is 22.2 % (with the pH adjustment) and 23.4 % (without pH adjustment). This low level of inhibition demonstrated that further experiments are not needed.
Executive summary:

The influence of the test substance, Acid Brown 235, on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The test was performed according to 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. Because the solutions of the test substance with the concentration 1000 mg/L had pH value 6.7, the pH of these solutions were adjusted (to pH value 7.0). Since neutralisation may change the chemical properties of the test substance, three more test vessels were included with a concentration of 1000 mg/L at the end of the test, which were tested without pH adjustment.

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 25.9 % 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 highest inhibitory effect of the test substance was 22.2 % (with the pH adjustment) and 23.4 % (without pH adjustment). These low levels of inhibition demonstrated that further experiments are not needed.

The pH value during the preliminary test was in the range of 6.7 to 7.9.

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 Acid Brown 235 was 22.2 % (with the pH adjustment) and 23.4 % (without pH adjustment)in the preliminary test. These low levels 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

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information