Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jul 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP, volatile test substance in open test system

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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 16437
- Physical state: Clear colorless liquid
- Analytical purity: 98.5 area %
- Storage condition of test material: At room temperature in the dark under nitrogen
- Other: Reactive to moisture and oxygen

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: The test substance is highly volatile (vapor pressure elsewhere in this study). Because of low solubility, test substance was pipetted below the surface of reverse osmosis-purified water at 2.5X final loading rate. Subsequently, synthetic sewage feed (16 mL) and prepared, activated sludge (250 mL) and additional RO-purified water 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, nitrification control, abiotic control, 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 (to 1 mm), 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: 7.4).

ISO medium (in reverse osmosis [RO]-purified tap water)
211.5 mg/L CaCl2∙2(H2O)
88.8 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

Test temperature:
15.9 - 19.5
pH:
7.3 - 8.2
Dissolved oxygen:
Continuous aeration throughout exposure period. Range of measured dissolved oxygen [DO] values at start of observation period were 7.26 - 9.61 mg/L
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):
blank controls, six
nitrification inhibitor controls (only substance added), two
abiotic control, one
highest concentration test substance (1000 mg/L), three
highest concentation test substance plus nitrification inhibitor, three
other test substance concentrations, one each
reference substance concentrations, one each

- 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).
- 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 concentrationsopen allclose all
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
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:
- 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. Also, the test substance is volatile (vapor pressure elsewhere in this dossier)
Results with reference substance (positive control):
- Results with reference substance valid?: Yes (see Table 1)
- Relevant effect levels: EC50 4.57 mg/L
Reported statistics and error estimates:
Normality of data: Shapiro-Wilks test for normality: 9 residues. W = 0.931. Pass at α = 0.05.
Homogeneity of variance (control and 1000 mg/L): Levene's Test on variance homogeniety: df = 8, F-value 0.029. p(F) = 0.869. Pass at α = 0.05.
Statistically significant inhibition of respiration (control v. 1000 mg/L): Student's t-test with homogeneous variance: df = 7, t-statistic 0.08. p(t) - 0.531. Pass at α = 0.05

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

7.50

1.56

44

29

C2

0

7.33

1.97

38

25

C3

0

8.70

3.03

41

27

C4

0

7.94

3.84

32

21

C5

0

7.73

2.33

40

27

C6

0

8.78

5.28

29

19

Mean

 

 

 

37

25

 

SD

 

 

 

6

4

 

CV (%)

 

 

 

15

15

 

ATU¹ controls

 

 

 

 

 

 

CN1

0

7.34

4.96

21

14

44

CN2

0

8.31

5.78

18

12

52

Mean

 

 

 

20

13

 

 

 

 

 

 

 

 

Reference sub.²

ref 1

2.0

8.65

5.46

24

16

36

ref 2

5.0

8.08

5.57

20

13

46

ref 3

12

8.75

7.50

10

7

73

Test sub.

T1

10

7.26

1.83

43

29

-15

T2

100

7.26

1.88

39

26

-4

T3a

1000

7.81

1.47

44

29

-18

T3b

1000

7.92

2.49

37

25

1

T3c

1000

7.79

3.56

32

21

14

T3 (mean)

 

 

 

38

25

-1

 

 

 

 

 

 

 

Test sub. w/ ATU

TN1

1000

9.61

7.42

19

13

TN2

1000

8.39

5.47

21

14

-8³

TN3

1000

9.05

6.24

21

14

-8³

TN (mean)

 

 

 

20

14

-4³

 

 

 

 

 

 

 

Test sub., abiotic

1000

8.75

8.77

0

0

100

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% (25 mg/(g*h), variability 15%). Reference substance 3-h EC50 within 2-25 mg/L (4.57 mg/L).
Conclusions:
PPVE is not inhibitory to activated sludge respiration (3-h EC50 >1000 mg/L)
Executive summary:

The toxicity of PPVE to activated sludge organisms was assessed in an assay performed according to OECD TG209. The test solution was made by adding the volatile test substance under the surface of the test solution. The exposure period was for three hours with continuous aeration and stirring. No statistically significant inhibition was observed upon exposure to PPVE 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. However, the test substance is highly volatile and was tested in an open, aerated system. It is classified as reliable with restriction, 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