Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 December 2009 to 1 September 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:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (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): "3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE"
- Molecular formula (if other than submission substance):C5H10O3
- Lot No.: CH 138067/001.
- CAS No.: 185146-18-2.
- Appearance: uncolored and high viscous.
- Purity test date:
~ 82 % Dimethylpropanale
~ 10 % Hydroxymethanesulfonic acid, Na-salt (CAS No: 870-72-4)
~ 6 % water *)
~ 2 % Potassiumformiate (CAS No: 590-29-4).
- Solubility: In water: Soluble
- pH: 7.25.
- Conditions of storage: Room temperature.
- Stability at conditions of storage: Min. 1year.
- Expiry date: 30 November 2010.

*) The results presented in the report refer to the dry weight of the test substance not including the 6 % water content.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
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 on the day before the test. The container for transportation was only maximally 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (less than 1 hour after sample collection), the supernatantover the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthetic sewage concentrate were added per litre each day. On the day of the test the sludge was allowed to sediment and the supernatant was decanted.
- Initial biomass concentration: On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L
with tap water. 200 mL of this adjusted inoculum are added per test vessel, resulting in 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

Test conditions

Test temperature:
19°C
pH:
The pH of the incubation mixtures after the 3 hour incubation was between 8.12 and 8.27, the pH of the negative control samples was 8.26 and 8.31. The test substance did not influence the pH.The pH of the standardised activated sludge was 7.76.
Dissolved oxygen:
The oxygen concentration was determined with a DO-meter "OXI Level 2" and an oxygen electrode (Wissenschaftlich-technische Werkstätten G.m.b.H., Dr. rer.nat. K. Slevogt, D-82362 Weilheim i. OB, Trifthofstr. 57a). The readout was set to 100 % in water-saturated air.
Nominal and measured concentrations:
final conentrations*: 7.7 mg/l, 19.3 mg/l, 48.2 mg/l, 120.5 mg/l and 301.2 mg/l
*) The amount of test substance stated refers to the dry weight ("3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE"contains 6 % water)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers with a nominal volume of 1000 mL
- Type (delete if not applicable): open during 3h incubation.
- Material, size, headspace, fill volume: All incubation mixtures were prepared by adding 200 mL of microbial inoculum and 200 mL
of synthetic sewage solution to 100 mL of tap water and/or the appropriate amount of the test or control substance in glass beakers with a nominal volume of 1000 mL.
- Aeration: oil free compressed air, flow rate 0.7 L/min
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

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

OTHER TEST CONDITIONS
- Adjustment of pH: None.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (nominal concentrations: 5.0, 12.1 and 29.8 mg/L)

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The actual values of the EC20, EC50 and EC80 for 3,5-dichlorophenol were:
EC20 = 8.1 mg/L EC50 = 17.5 mg/L EC80 = 37.8 mg/L
- Results with reference substance valid? yes

Any other information on results incl. tables

Respiration rate and inhibition of samples


sample

final
concentr. (mg/L)*)

linear range (minutes)


mg O2/ L
at begin


mg O2/ L
at end


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

percent
respiration
inhibition

neg. control 1

0.0

7.40

5.48

2.15

27.00

0.11

test substance 1

7.7

7.36

6.23

3.06

25.84

4.40

test substance 2

19.3

7.84

5.89

2.24

27.93

-3.34

test substance 3

48.2

7.28

5.87

2.59

27.03

-0.01

test substance 4

120.5

7.52

6.08

2.66

27.29

-0.95

test substance 5

301.2

7.28

5.21

1.54

30.25

-11.90

neg. control 2

0.0

7.76

6.18

2.68

27.06

-0.11

DCP 1

5.0

8.12

5.79

2.08

27.41

-1.42

DCP 2

12.1

7.04

7.00

5.11

16.11

40.41

DCP 3

29.8

8.12

7.86

6.70

8.57

68.29

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
EC10: greater than 300 mg/L
EC20: greater than 300 mg/L
EC50: greater than 300 mg/L
EC80: greater than 300 mg/L
Executive summary:

The study was performed to estimate possible effects of "3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE" 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 (7.7, 19.3, 48.2, 120.5 and 301.2 mg/L) were

tested versus two negative controls (tap water).

As positive control substance 3,5-dichlorophenol was used and tested in three

concentrations (5.0, 12.1 and 29.8 mg/L).

The microbial inoculum was a preparation of activated sludge collected on the day before the

test. 50 mL of synthetic sewage concentrate were added per litre and the sludge was kept

areated. 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 at 19 °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 respiration rates using the

mean value of the negative controls as 100 %.

EC10: greater than 300 mg/L

EC20: greater than 300 mg/L

EC50: greater than 300 mg/L

EC80: greater than 300 mg/L