Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jan 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
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): MTDID 7831
- Physical state: Waxy solid
- Analytical purity: 93.96 w%
- Storage condition of test material: At room temperature in the dark

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: Because of low solubility, test substance was added to reverse osmosis-purified water at 2.5X final loading rate, with 25.5 - 27 hours' stirring in closed, dark brown 1-L test bottles. For the test, synthetic sewage feed (16 mL) and prepared, activated sludge (250 mL) were added to bring the final volume to 500 mL and the loading rate to 1000 mg/L. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

-Control: blanks, 3,5-Dichlorophenol as reference substance

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Sludge was collected from municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, the Netherlands.The sludge was coarsely sieved, washed and diluted with ISO medium. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The sludge was collected one day before use. Before use the pH was checked (measured value: 7.9).

ISO medium (in reverse osmosis [RO]-purified tap water)
211.5 mg/L CaCl2∙2(H2O)
88.7 mg/L MgSO4∙7(H2O)
46.7 mg/L NaHCO3
4.2 mg/L KCl

ISO medium was used only for sludge preparation, not during the test.

- Method of cultivation: Because the sludge was used one day after collection, 50 mL of synthetic sewage feed* was added to each litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use (*synthetic sewage feed: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCI, 0.4 g CaCI2·2(H2O), 0.2 g MgSO4·7(H2O), 2.8 g K2HPO4 dissolved in 1 L RO water and filtered, final pH 7.5±0.5).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
combined limit/rangefinding test

Test conditions

Hardness:
Not reported
Test temperature:
18.8 - 21.7
pH:
7.6 - 8.4
Dissolved oxygen:
Continuous aeration throughout exposure period. Range of measured dissolved oxygen [DO] values at start of observation period were 7.1 - 8.7 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations 10 mg/L, 100 mg/L or 1000 mg/L during exposure period. No analytical monitoring.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass bottles/vessels.
- Type (delete if not applicable): Open during exposure. Closed during observation.
- Material, size, headspace, fill volume: 200 mL test solution, 16 mL synthetic sewage, and 250 mL sludge suspension plus sufficient RO water to bring total volume to 500 mL.
- Aeration: Continuous aeration with pipette, and stirring.
- Nitrification assessed: yes, 11.6 mg/L N-allylthiourea used as nitrification inhibitor.
- No. of vessels per concentration (replicates): three for highest test substance loading rate, one each for test substance with nitrification inhibitor, lower (rangefinding) test substance concentrations, and for reference substance 3,5-dichlorophenol.
- No. of vessels per control (replicates): Six blanks, one nitrification blank, one abiotic blank.
- Biomass loading rate: 1.5 g/L suspended solids in the final test mixture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges.
- Intervals of water quality measurement: Not specified. Water quality data not provided.

OTHER TEST CONDITIONS
- Adjustment of pH: none.
- Light intensity: not specified.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Dissolved oxygen concentration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10.
- Justification for using less concentrations than requested by guideline: Combined limit and rangefinding test of poorly soluble substance.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: no statistically significant inhibition, α=0.05
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance was not completely soluble in test medium at the loading rates initially prepared.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes (see Table 1)
- Relevant effect levels: EC50 7.6 mg/L
Reported statistics and error estimates:
Normality of data: Chi-square test for normality: Chi-square value 1.6226. Pass at α = 0.01, 4 degrees of freedom (df = 4).
Equality of variance (control v. 1000 mg/L): F-Test for Equality of Two Variances: F-value 4.3056. Fail to reject equality of variance at α = 0.01, df = 2, 5.
Statistically significant inhibition of respiration (control v. 1000 mg/L): t-Statistic 1.5119. Fail to reject equal inhibition at α = 0.05, df = 7. Test had a minimum statistically detectable inhibition of 14.9%.

Any other information on results incl. tables

Table 1, Study results

Sample

Conc. or loading rate

(mg/L)

Oxygen conc (mg O2/L)

Respiration rate

% Inhibition relative to control mean

start

end

mg O2

L∙h

mg O2

g∙h

Blank controls

C1

0

7.4

0.3

41

27

C2

0

8.3

1.0

41

27

C3

0

8.7

0.3

45

30

C4

0

8.1

1.8

38

25

C5

0

7.9

0.0

45

30

C6

0

7.5

0.6

42

28

Mean

42

28

SD

3

2

CV (%)

6

6

ATU¹ control

0

7.1

3.3

24

16

43

Reference sub.²

ref 1

5.0

7.1

3.0

26

17

38

ref 2

12

7.4

5.1

15

10

64

ref 3

30

8.3

7.4

7

5

83

Test sub.

test sub. 1

10

8.4

0.0

48

32

-14

test sub. 2

100

7.9

-0.2

46

31

-10

test sub. 3a

1000

7.9

-0.1

43

29

-2

test sub. 3b

1000

7.8

0.8

39

26

7

test sub. 3c

1000

8.3

3.0

32

21

24

test sub. 3 (mean)

38

25

10

Test sub., abiotic

1000

8.1

8.3

1

1

98

Test sub. w/ ATU

1000

8.1

2.7

31

21

-29 ³

1. ATU, nitrification inhibitor N-allylthiourea, 11.6 mg/L final concentration

2. Reference substance, 3,5-dichlorophenol

3, Percent inhibition calculated relative to ATU control

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control respiration rate ≥ 20 mg/(g*h), variability < 30% (20 mg/(g*h), variability 6%). Reference substance 3-h EC50 within 2-25 mg/L (7.6 mg/L).
Conclusions:
C4 acrylate is not inhibitory to activated sludge respiration (3-h EC50 >1000 mg/L)
Executive summary:

The toxicity of C4 acrylate to activated sludge organisms was assessed in an assay performed according to OECD TG209. The test solution was made by stirring C4 acrylate 25.5 - 27 hours in a closed vessel at a loading rate of 2500 mg/L, with subsequent dilution to 1000 mg/L for the test. Undissolved material presumably remained in the test bottles. The exposure period was for three hours with continuous aeration. No statistically significant inhibition was observed upon exposure to C4 acrylate at the maximum loading rate (α = 0.05, df = 7). The 3-hour EC50 to sludge is therefore >1000 mg/L.

The study was conducted in accord with GLP criteria and satisfies the guideline requirements for test method OECD209, activated sludge respiration inhibition test. It is classified as reliable and is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.  

Results Synopsis  

Test Type: Static                          

EC50:  >1000 mg/L                       95% C.I.:  not applicable

NOEC:  1000 mg/L                        Probit Slope:  not applicable

Endpoint(s) Effected:  Respiration inhibition