Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-04-27 to 2010-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP experiment conducted under OECD guideline.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Mexoryl SAB
- Structural formula attached as image file (if other than submission substance): no data
- Substance type: white solid powder
- Physical state: solid
- Analytical purity: see confidential details
- Impurities (identity and concentrations): none
- Composition of test material, percentage of components: none
- Isomers composition: none
- Purity test date: 2010-02-25
- Lot/batch No.: see confidential details
- Expiration date of the lot/batch: 2013-02-28
- Stability under test conditions: no data
- Storage condition of test material: at room temperature at about 20°C, away from direct sunlight.
- Other: none
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not applicable

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The study was performed with aerobic activated sludge from a wastewater treatment plant treating predominantly domestic wastewater. The sludge was washed twice with tapwater by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of 3 days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 7.3
- Eluate: tap water
- Differential loading: Test item amounts of 5.02, 16.05, 50.02, 160.93, and 500.50 mg were weighed and transferred to the designated test vessels with 284 mL tap water.
- Controls: 2 controls containing only tap water, synthetic sewage feed and inoculum.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: The study was performed with aerobic activated sludge from wastewater treatment plant (ARA Ergolz II, Füllinsdorf/Switzerland).
- Method of cultivation: The sludge was washed twice with tapwater by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter.
- Preparation of inoculum for exposure: During the holding period of 3 days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test.
- Pretreatment: The test item was mixed into tap water by ultrasonic treatment for 15 min and intense stirring for 24 h at room temperature in the dark to dissolve a maxium amount of the test item and.or disperse it as homogeneously as possible. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.
- Initial biomass concentration: no data

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data

Test conditions

Hardness:
no data
Test temperature:
20°C
pH:
6.9-8.5
Dissolved oxygen:
7.6-8.5 mgO2/L
Salinity:
not applicable
Nominal and measured concentrations:
- final nominal conc.: 10, 32, 100, 320, and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 2000 mL glass beakers.
- Aeration: yes by intense stirring
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 16 mL of synthetic sewage feed and 200 mL of activated sludge
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 16 mL of synthetic sewage feed and 200 mL of activated sludge

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: in the dark
- Light intensity: not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: not applicable
- Test concentrations: 10, 32, 100, 320 and 1000 mg/L
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.:
238 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 221-254 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
413 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 394-434 mg/L
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
716 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 663-785 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
179 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 161-195 mg/L
Details on results:
No data
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50(3h) = 13 mg/L. The EC50(3h) is whitin the guideline recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.
- Other: None.
Reported statistics and error estimates:
No data

Any other information on results incl. tables

Table 1: Influence of Mexoryl SAB and 3,5-dichlorophenol on the oxygen consumption of activated sludge

Vessel no.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate (mg O2/L/min)

Inhibition (%)

pH values

Oxygen concentration (mg O2/L)

 

 

 

 

Start

End

Start

end

                    Control

A

0

1.378

 

7.8

8.5

8.3

8.2

B

0

1.437

 

7.5

8.1

8.2

7.8

Mean

Deviation (%)

1.407

4.3

 

                    Test item

4

10

1.413

-0.4

7.5

8.1

8.3

8.5

5

32

1.528

-8.6

7.5

7.9

8.2

8.1

6

100

1.460

-3.8

7.5

8.1

8.1

7.6

7

320

0.913

35.1

7.5

7.9

8.0

7.9

8

1000

0.129

90.8

6.9

7.7

8.2

8.3

                    Reference item

1

5

1.307

7.1

7.5

8.2

8.2

8.5

2

16

0.527

62.5

7.4

8.2

8.2

8.4

3

50

0.135

90.4

7.5

8.2

8.3

8.5

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3 -hour respiration inhibition test according to the EU commission directive 88/302/EEC, Part C.11, commission regulation (EC) No. 440/2008, Part C.11 and the OECD guideline for testing of chemicals, No. 209. The calculated 3 -hour EC values were determined as follows:
NOEC: 179 mg/L 95% confidence limits: 161 -195 mg/L
EC20: 238 mg/L 95% confidence limits: 221 -254 mg/L
EC50: 413 mg/L 95% confidence limits: 394 -434 mg/L
EC80: 716 mg/L 95% confidence limits: 663 -785 mg/L
Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3 -hour respiration inhibition test according to the EU commission directive 88/302/EEC, Part C.11, commission regulation (EC) No. 440/2008, Part C.11 and the OECD guideline for testing of chemicals, No. 209.

The following nominal concentrations of test item were tested: 10, 32, 100, 320, and 1000 mg/L.

In addition, 2 controls and 3 different concentrations of the reference item 3,5 -dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the test item concentration 100 mg/L, the test item had no significant inhibitory effect (=< 10%) on the respiration rate of activated sludge after the incubation period of 3 hours. At the higher test item concentrations of 320 and 1000 mg/L, the inhibitory effects were 35 and 91%, respectively.

The calculated 3 -hour EC values were determined as follows:

NOEC: 179 mg/L 95% confidence limits: 161 -195 mg/L

EC20: 238 mg/L 95% confidence limits: 221 -254 mg/L

EC50: 413 mg/L 95% confidence limits: 394 -434 mg/L

EC80: 716 mg/L 95% confidence limits: 663 -785 mg/L