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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.08 - 03.09.2009
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:
2008
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
The test substance is non-soluble in water in concentrations needed for the test. For this reason the direct dosing of test substance was used.
The substance was weighed into reaction container where 100 mL of water were introduced.
In the preliminary test the concentrations were in the range of 46 - 1000 mg.L-1 with a geometric progression factor of 2.2.
In the main test the concentrations were in the range of 100 - 1000 mg.L-1 with a geometric progression factor of 1.8.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
PREPARATION OF THE INOCULIM
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 4000 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2°C
pH:
The pH value of reaction mixtures during the main test: approx. 6 - 8
Nominal and measured concentrations:
Preliminary test: 46, 100, 220, 460 and 1000 mg.L-1
Main test: 100, 180, 320, 560 and 1000 mg.L-1
Details on test conditions:
TEST CONDITIONS
Temperature: 20 ± 2 °C
Exposition time: 180 mins
Lighting: daily
Stirring and aeration: with filtered pressured air, airflow rate 0.8 L/min.
The pH value of reaction mixtures during the main test: approx. 6 – 8.

TEST PROCEDURE
100 mL of deionized water, the defined dose of the test or reference substance, 16 mL of the synthetic nutrient medium and 200 mL of the prepared 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.
The test mixtures were prepared in turns in 15 minutes time intervals.
The pH adjustment of mixtures in the preliminary and in the main test was not carried out.
The aeration was carried out by means of glass pipettes.

MEASUREMENT
The measurement of oxygen content was performed in aliquots of the reaction mixtures after 3-hour incubation 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 at 10-minute intervals.
The pH was determined in the remaining part of the reaction mixture.
Reference substance (positive control):
yes
Remarks:
3,5-DICHLORPHENOL
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
Remarks:
respiration rate
Details on results:
See tables No. 3 and No. 6
Results with reference substance (positive control):
- Results with reference substance valid? yes
The EC50 value of the reference substance 3,5-dichlorophenol was: EC50 = 10.2 mg.L-1 (95 % confidence interval: 9.3 – 11.1 mg.L-1).

Table No. 3 - Performance of the main test

Container No.

Label

Test/Ref.

substance

 dose

/500 mL

Test/Ref.

substance

concentration

mg.L-1

Medium

dose

 

mL

Sludge dose

 

mL

Water

dose

 

mL

pH

 

 

start

pH

 

 

end

1

K1

0

0

16

200

up to 500 mL

7.07

7.72

2

R1

19 mL

19

16

200

up to 500 mL

7.09

7.74

3

R2

14 mL

14

16

200

up to 500 mL

7.09

7.75

4

R3

10 mL

10

16

200

up to 500 mL

7.08

7.78

5

R4

7.2 mL

7.2

16

200

up to 500 mL

7.07

7.84

6

R5

5.2 mL

5.2

16

200

up to 500 mL

7.06

7.75

7

Z1

50 mg

100

16

200

up to 500 mL

7.15

7.68

8

Z2

90 mg

180

16

200

up to 500 mL

7.17

7.67

9

Z3

160 mg

320

16

200

up to 500 mL

7.18

7.72

10

Z4

280 mg

560

16

200

up to 500 mL

7.25

7.75

11

Z5

500 mg

1000

16

200

up to 500 mL

7.40

7.78

12

K2

0

0

16

200

up to 500 mL

7.03

7.68

13

Zab

500 mg

1000

16

0

up to 500 mL

7.82

7.65

Table No. 6 - Evaluation of inhibition of respiration in the main test

Containernumber

Label

Conc.

mg.L-1

p1

mg.L-1

p2

mg.L-1

t1

min

t2

min

R

mg.L-1. h-1

Inhibition

%

1

K1

0

6.91

3.58

2.5

10

26.64

0

2

R1

19

8.42

7.34

0.5

10

6.82

74.3

3

R2

14

8.14

6.93

2.0

10

9.08

65.9

4

R3

10

8.01

6.26

2.0

10

13.13

50.6

5

R4

7.2

7.94

5.48

1.5

10

17.36

34.7

6

R5

5.2

7.28

3.95

0.5

10

21.03

20.9

7

Z1

100

6.81

3.04

1.5

10

26.61

-0.1

8

Z2

180

6.43

2.53

1.5

10

27.53

-3.5

9

Z3

320

6.46

2.80

2.0

10

27.45

-3.2

10

Z4

560

7.53

3.18

0.5

10

27.47

-3.3

11

Z5

1000

7.92

3.70

0.5

10

26.65

-0.2

12

K2

0

6.82

3.06

1.5

10

26.54

0

13

Zab

1000

8.76

8.66

0.5

10

0.63

-

Validity criteria fulfilled:
yes
Conclusions:
On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Semi Dry Absorption (SDA) Product, the EC50 was found to be:
EC50 > 1000 mg.L-1

Under the current experimental conditions, all tested concentrations in the main test provided no inhibition of respiration rate (see table 6). From available data it was not possible to calculate the EC50 value.
Executive summary:

The influence of the test substance, Semi Dry Absorption (SDA) Product, on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

 

Test performance

Test was performed according to:

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 5 concentrations in range 46 - 1000 mg.L-1with a geometric factor of 2.2 to determine the concentrations to be used in the main test.

In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1000 mg.L-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

3,5-dichlorophenol was used as reference substance. Five concentrations in geometric progression with a factor of 1.4 (5.2, 7.2, 10, 14 and 19 mg.L-1) were selected.

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

The pH value during the main test was in the range of 7.0 to 7.8.

 

Validity of test

The test met the following validity criteria:

-       the respiration rates of two control experiments should not differ from each other by more than 15 %

-       the EC50value of the reference substance should be in the range of 5 – 30 mg.L-1

The difference of two respiration rates of control experiments at the beginning and the end of the main test from each other was:

         (0.10 mg.L-1.h-1= 0.37 % (if the value of lower control 26.54 mg.L-1.h-1= 100 %)

The 3h-EC50value of reference substance, 3,5-dichlorophenol:

EC50= 10.2 mg.L-1

(95 % confidence interval: 9.3 – 11.1 mg.L-1)

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

 

Results of the respiration inhibition test

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Semi Dry Absorption (SDA) Product, the EC50was found to be:

EC50> 1000 mg.L-1

Under the current experimental conditions, all tested concentrations in the main test provided no inhibition of respiration rate (see table 6). Therefore it was not possible to calculate the EC50value.

Description of key information

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

GLP Study.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
83 mg/L

Additional information

Test results:

24–hour EC50 > 100 mg·L-1 (nominal concentration)

48–hour EC50 > 100 mg·L-1 (nominal concentration)

24–hour NOEC = 100 mg·L-1 (nominal concentration)

48–hour NOEC = 83 mg·L-1 (nominal concentration)

24–hour EC100 > 100 mg·L-1 (nominal concentration)

48–hour EC100 > 100 mg·L-1 (nominal concentration)

Classification of the test substance on the basis of hazard to aquatic environment was performed according to the Directive 93/21/EEC, Annex IV (article 5.2.1.).

According to the classification criteria given in quoted Directive the test substance, Semi Dry Absorption (SDA) Product, is not assigned to any toxicity categories.