Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2009 - 23 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Green liquor 1
- Chemical name: Sulphite liquors and Cooking liquors, green
- Molecular formula (if other than submission substance): UVCB
- Appearance: Yellowish liquid
- Solubility: In water: the substance is a water solution (82.3 % water)
- Impurities (identity and concentrations): UVCB
- pH: over 11
- Lot/batch No.: Not stated.
- Expiry date: Not available.
- Stability under test conditions: No data available.
- Storage condition of test material: Room temperature. Storage in the dark but may be used under light.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the test substance (nominally 8.45 g/L, substance wet weight) was prepared by dissolving 12 756 mg in 1 500 mL tap water. Appropriate amounts of this stock solution were added to the different samples at the beginning of the incubation .
- Controls: tap water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: A sample of activated sludge was collected three dys before the test. At arrival in the laboratory the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthethic sewage concentrate were added per litre per day. On the day of the test the sludge was allowed to sediment and the supernatant was decanted. The concentration of suspended solids was determined.
- Pretreatment:
- Initial biomass concentration: 4 g suspended solids per litre activated sludge, 200 mL sludge were added per test vessel resulting in a final concentration of 1.6 g dry weight per litre in the samples.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None.

Test conditions

Hardness:
Not determined.
Test temperature:
19 °C
pH:
8.5 to 9.4 in test vessels, 8.4 to 8.6 in control vessels
Dissolved oxygen:
Not determined.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 298, 595, 1208, 2415, 4830 mg/L wet weight, solvent water included (82.3%)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers with a nominal volume of 1000 mL (filled with 500 mL)
- Type: open
- Material, size, headspace, fill volume: glass beakers, filled with 500 mL (200 mL inoculum, 200 mL diluted sewage feed, 100 mL test solution)
- Aeration: aeration with a flow rate of 0.7 L/min using oil free compressed air
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.6 g suspendid solids/L in final test solutions (500 mL/vessel)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local tap water

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: A range finding study using 1 000, 10 000, and 100 000 mg/L was performed to reveal a concentration range in which an EC50 value can be found.
- Results used to determine the conditions for the definitive study: The results of this experiment indicated that the test substance itself consumes oxygen, therefore an additional experiment (not according to GLP) with blank solutions was performed. Two blanks containing 1 000 and 5 000 mg test substance per litre but no sludge were incubated for three hours and the oxygen consumption was measured. A clear indication of oxygen consumption was observed, therefore the second main experiment was performed with one blank solution for each test substance concentration.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
950 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight, solvent water included (82.3%)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 460 to 1400 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 700 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight, solvent water included (82.3%)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 980 to 2400 mg/L
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
3 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight, solvent water included (82.3%)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 1700 to 4200 mg/L
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
3,5-dichlorophenol: EC20: 7.3 mg/L, EC50: 16.1 mg/L, EC80: 35.5 mg/L

Any other information on results incl. tables

Respiration rate and inhibition of samples

sample

final
concentr. (mg/L wet wt.)

linear range (min)

mg O2/ L
at begin

mg O2/ L
at end

respiration rate
(mg O2.L-1.h-1)

Net respiration rate*)

(mg O2.L-1.h-1)

percent
respiration
inhibition

neg. control 1

0.0

7.60

5.06

1.20

30.47

-

1.40

test sub. 1

297.6

7.96

6.73

2.48

32.04

31.65

-2.40

test sub. 2

595.3

7.76

5.16

0.86

33.25

32.89

-6.41

test sub. 3

1207.6

8.08

7.04

4.27

20.57

19.70

36.26

test sub. 4

2415.1

7.64

7.94

6.42

11.94

10.07

67.42

test sub. 5

4830.2

7.64

6.94

4.37

20.18

13.25

57.13

neg. control 2

0.0

7.60

5.77

1.80

31.34

-

-1.40

DCP 1

5.0

7.88

6.29

2.37

29.85

-

3.43

DCP 2

12.2

7.24

7.47

5.39

17.24

-

44.23

DCP 3

30.0

7.84

8.53

7.37

8.88

-

71.28

blank 1

297.6

7.64

8.75

8.70

0.39

-

-

blank 2

595.3

8.24

9.18

9.13

0.36

-

-

blank 3

1207.6

8.24

9.26

9.14

0.87

-

-

blank 4

2415.1

7.72

9.20

8.96

1.87

-

-

blank 5

4830.2

7.88

8.60

7.69

6.93

-

-

Validity criteria

The respiration rates of the two negative control samples were within 15 % of each other, the actual individual values were 98.6 and 101.4 % of their calculated mean value.

  The EC50of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
EC50: 1700 mg/L wet weight, 300 mg/L dry weight,
Executive summary:

The study was performed to estimate possible effects of"GREEN LIQUOR 1"on aerobic microbial sewage treatment plants. The test was performed according to the OECD Guideline for Testing of Chemicals 209 "Activated Sludge, Respiration Inhibition Test" and the EU-COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Five concentrations of the test substance (298, 595, 1 208, 2 415 and 4 830 mg/L (wet weight)) were tested versus two negative controls (tap water).

As the test substance itself consumes oxygen, blank values of the test substance (containing tap water instead of microbial inoculum) were determined and subtracted from the results obtained with the test solutions.

As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.0, 12.2 and 30.0 mg/L).

The microbial inoculum was a preparation of activated sludge collected three days before the test. 50 mL of synthetic sewage concentrate were added per litre per day and the sludge was kept aerated. On the day of the experiment, the concentration of the microbial inoculum was adjusted to 4 g of dry weight per litre which results in a final concentration of 1.6 g dry weight per litre in the samples.

After mixing the inoculum with synthetic sewage solution and appropriate dilutions of control or test substance solutions the samples were aerated for a contact time of 3 hours at19 °C. Negative controls were run before the first and after the last test substance sample. After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the net respiration rates (respiration rates corrected for blank values) using the mean value of the negative controls as 100 %.

EC50: 1700 mg/L (wet weight), 300 mg/L (dry weight)