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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-02 to 2013-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Deviations did not impact the scientific validity and integrity of the study.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Test material was described with the generic name "Cyprinal", chemical name "alpha-methylcinnamaldehyde" and described with the generic CAS 101-39-3/EC 202-938-8. As manufactured by Innospec, the product met the criteria of > 80% trans-isomer, supporting that the test material was the isomer better described as CAS 15174-47-7.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Innospec Limited (Cheshire, UK); Batch No. A101112A
- Expiration date of the lot/batch: 2012-11-12
- Purity test date: 2010-11-16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C ± 3°C and N2 atmosphere
- Stability under test conditions: Stable under normal conditions of storage and use, hazardous polymerisation will not occur

FORM AS APPLIED IN THE TEST (if different from that of starting material): Yellow Clear Liquid

OTHER SPECIFICS:

Purity: >99%
Solubility in water: 490 mg/L
Density: 1.036 - 1.040 at 20°C
Volatility: 3.33 Pa at 25°C
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: 25-400 mg/L
- Sample storage conditions before analysis: Storage at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:

The tested concentration range was 25-400 mg/L. Because total liquid volume in the vessels was 500 mL and the density of the test material was 1.036 - 1.040 g/cm^3 following direct addition of the test material was performed:

Concentration (mg/L) Volume Test Material (µL)

400 193.2
200 96.6
100 48.4
50 24.1
25 12.1

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge from a municipal Sewage treatment plant "AZV Staufener Bucht“
- Pretreatment: The activated sludge was lept aerated and fed with synthetic sewage (50 mL per Litre and day) until use. Dry solids of the activated sludge were determined a 4.3 g/L by weight measurements before and after 3 hour drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant, and re-suspending the sludge in aerated tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.
- Initial biomass concentration: 1.5 g/L dry solids
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
21- 22°C
pH:
7.3-7.4
Nominal and measured concentrations:
400, 200, 100, 50 and 25 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1000 mL
- Aeration: Yes, all vessels were stirred and aerated throughout the contact time with room air at a rate of 0.5-1 litre per minute using a membrane pump
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.5 g/L

TEST MEDIUM
Synthetic Sewage Feed:

Bacto-Peptone 16g
Meat Extract 11g
Urea 3g
NaCl 0.7g
CaCl2*2H2O 0.4g
MgSO4*7H2O 0.2g
K2HPO4 2.8g

dissolved in demineralized water and made up to 1 litre.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration Rate

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: To find a concentration range a limit test with 1000 mg/L Cyprinal was first prepared. Here the inhibition was about 90%. Therefore, a full test with 5 concentrations bewteen 1000 and 62.5 mg/L was prepared and a second full test with 5 concentrations between 100 and 6.25 mg/L was prepared, but the results showed high negative inhibitions (until 53%) and a relative low start value of oxygen at the beginning of the measurement. Also, a high variation of the inhibition of results could be observed. Therefore the aeration of the test was optimized and the test repeated.

With a third test and a concentration range of 5 concentrations between 25 and 400 mg/L the concentration range was met and the results conclusive.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
ca. 366 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
76.53 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The EC50 (3h) of the test material was 366.18 mg/L.

Blanks: The co-efficient of variation of oxygen uptake rate in blank control replicates was below 30% (12%) at the end of the test (calculation of the co-efficient: standard deviation of the blanks divided by the arithmetic average of the blanks). The blank controls oxygen uptake rate was not less than 20 mg oyygen per one gram of activated sludge in an hour (22.6-33.1 mg / (l*h)).


Results with reference substance (positive control):
The EC50 (3h) of 3,5-Dichlorophenol was 13.79 mg/L and within the Guideline range of 2-25 mg/L.

Criteria Met for the Validity of the Study:

1) The mean blank controls oxygen uptake rate was more than 20 mg oxygen per one gram of activated sludge in an hour.

2) the co-efficient of variation of oxygen uptake rate in blank control replicates was less than 30% at the end of the definitive test.

3) The EC50 of the reference material was within the acceptable range of 2 -25 mg/L in both tests.

The study is valid.

Table 1. Respiration Rate of Activated Sludge after application of Cyprinal

Vessel

O2Concentration in mg/L

Consumption Rate (mg / (l*h))

Inhibition (%)

First Measurement

Last Measurement

Blank 1

6.86

5.67

28.6

 

Blank 2

5.69

4.47

29.3

 

Blank 3

6.72

5.41

31.4

 

Blank 4

6.90

5.96

22.6

 

Blank 5

6.94

5.60

32.2

 

Blank 6

6.81

5.43

33.1

 

Test 400 mg/L 1

7.35

6.70

15.6

47

Test 400 mg/L 2

8.18

7.77

9.8

67

Test 400 mg/L 3

8.01

7.47

13.0

56

Test 200 mg/L 1

6.48

5.36

26.9

9

Test 200 mg/L 2

7.20

6.30

21.6

27

Test 200 mg/L 3

7.35

6.44

21.8

26

Test 100 mg/L 1

7.82

6.91

21.8

26

Test 100 mg/L 2

5.99

2.80

76.6

-159*

Test 100 mg/L 3

6.88

5.98

21.6

27

Test 50 mg/L 1

6.73

5.38

32.4

-10

Test 50 mg/L 2

6.61

5.24

32.9

-11

Test 50 mg/L 3

6.77

5.59

28.3

4

Test 25 mg/L 1

6.53

5.12

33.8

-15

Test 25 mg/L 2

6.69

5.10

38.2

-29

Test 25 mg/L 3

6.28

4.77

36.2

-23

 

Table 2. Respiration Rate of Activated Sludge after application of 3,5-Dichlorophenol

Vessel

O2Concentration in mg/L

Consumption Rate (mg / (l*h))

Inhibition (%)

First Measurement

Last Measurement

Blank 1

6.86

5.67

28.6

 

Blank 2

5.69

4.47

29.3

 

Blank 3

6.72

5.41

31.4

 

Blank 4

6.90

5.96

22.6

 

Blank 5

6.94

5.60

32.2

 

Blank 6

6.81

5.43

33.1

 

3,5-D 1 mg/L

5.00

2.91

50.2

-70

3,5-D 10 mg/L

7.93

7.21

17.3

41

3,5-D 30 mg/L

8.12

7.77

8.4

72

 

Table 3. ECx (3h) Analysis of Cyprinal

Parameter

EC10

EC20

EC50

Value (mg/L)

76.53

130.98

366.18

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (3h) of Cyprinal was determined to be 366.18 mg/l.
Executive summary:

In a key Guideline (OECD 209) Activated Sludge Respiration Inhibition Test, the effects of the test material (Cyprinal; CAS# 101-39-3) on micro-organisms of activated sludge was evaluated by measuring the respiration rate at different concentrations (0, 25, 50, 100, 200, and 400 mg/L) of the test material.

 

The respiration rate corresponding to the oxygen consumption of aerobic activated sludge after a contact time of 3 hours was determined and the inhibitory effect of the test material was expressed as a percentage of the mean respiration rate of six controls. 3,5-dicholorphenol was used as the positive control in this study.

 

The calculated EC50 (3h) of the test material was 366.18 mg/L while the calculated EC50 of 3,5-Dichlorophenol was 13.79 mg/L.

Description of key information

In a key Guideline (OECD 209) Activated Sludge Respiration Inhibition Test (Hydrotox GmbH, 2013a), the effects of the test material (Cyprinal; CAS# 101-39-3) on micro-organisms of activated sludge was evaluated by measuring the respiration rate at different concentrations (0, 25, 50, 100, 200, and 400 mg/L) of the test material.

 

The respiration rate corresponding to the oxygen consumption of aerobic activated sludge after a contact time of 3 hours was determined and the inhibitory effect of the test material was expressed as a percentage of the mean respiration rate of six controls. 3,5-dicholorphenol was used as the positive control in this study.

 

The calculated EC50 (3h) of the test material was 366.18 mg/L.

Key value for chemical safety assessment

EC50 or LC50 for microorganisms:
366.18 mg/L
EC10, LC10 or NOEC for microorganisms:
76.53 mg/L

Additional information

In a reliable study performed to OECD 209, the EC50 (3h) of Cyprinal was 366.18 mg/L.