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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 25th, 2015 - February 25th, 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: SE7B
Lot No. of test item: 0000140602 (information from the sponsor)
Purity: 98% (information from the sponsor)
Test concentration: 1000 mg/l
Sample preparation: The test item was added directly into the test vessels.
Test system: Activated sludge, municipal wastewater treatment plant Staufener Bucht, 1.5 g dry solids per litre
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
500 mg of the test item were added directly into the test vessels to obtain final concentration of 1000 mg/L.
Test organisms (species):
activated sludge
Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Staufener Bucht was used as test system. The treatment plant has a capacitiy of 140 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was February 23rd, 2015. The activated sludge was kept aerated and fed with synthetic sewage (50 ml per litre and day) until use. Dry solids of the activated sludge was determined as 6.5 g/l by weight measurements before and after 3 h 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20.7 - 22.1°C
pH:
7.0 - 7.3
Nominal and measured concentrations:
1000 mg/L
Details on test conditions:
The different vessels must be started separately with an interval of exactly 15 min in order to be able to measure the respiration rates one by one while making sure that the aeration time is 3 h in every vessel. At time "0", 16 ml synthetic sewage and the test item or reference compound was added to the test vessel and made up to 250 ml with tap water. After a measurement and adjustment of pH 250 ml of activated sludge (3 g/l dry solids) were added thereby obtaining a total volume of 500 ml with 1.5 g/l dry solids. For the controls the same procedure was made as in the test vessels just without test or reference substance. 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. Before starting the test the aeration rate was measured in all test vessels.
After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.
This determination was repeated for each vessel at 15-min intervals in such a way, that the contact time in each vessel was 3 h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorphenol
Key result
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
Details on results:

Test item
At a concentration of 1000 mg/L there was no inhibition of SE7B on the respiration rate of the inoculum. On the contrary, there was a negative inhibition of -8 to -15% in the test vessels (1000 mg/L) which is caused by a slight promotion of the respiration.


Blanks
The respiration rate of the six blanks were within 24.0 – 35.3 mg/(l*h) and differed less than 30% from each other.
The temperature range was between 20.7°C and 22.1°C, the pH in the vessels were between 7.0 and 7.3 in all vessels.
Results with reference substance (positive control):
Reference item
The EC50 of 3,5-dichlorphenol was calculated as 8.58 mg/l (ToxRat software by probit analysis using linear max. likelihood regression) and was therefore within the acceptable range of 2 to 25 mg/l.
Reported statistics and error estimates:
Criteria met for the validity of the study
> The respiration rates of the six blanks were less than 30% different from each other.
> The blank controls oxygen uptake rate was higher than 20 mg oxygen per one gram of activated sludge per hour.
> The EC50 of the reference item was within the acceptable range of 2 to 25 mg/l.

 Oxygen concentration in the test- and reference vessels and in the blanks

Vessel

 Concentration

Ineterval of measurement [sek]

Measurement after 180 Min

O2-concentration [mg/l] at time [sec]

1

Blank 1

30

6.25

5.99

5.72

5.46

5.22

4.99

2

Blank 2

30

5.92

5.65

5.42

5.24

5.08

4.90

3

Blank 3

30

6.26

6.02

5.81

5.62

5.43

5.26

4

test item 1 (1000 mg/l)

30

4.85

4.50

4.21

3.94

3.70

3.48

5

test item 2 (1000 mg/l)

30

4.25

3.90

3.59

3.30

3.03

2.79

6

test item 3 (1000 mg/l)

30

4.28

3.98

3.70

3.40

3.12

2.86

7

3,5-D 1 mg/l

30

6.44

6.25

6.06

5.88

5.71

5.52

8

3,5-D 10 mg/l

30

7.00

6.83

6.72

6.59

6.46

6.34

9

3,5-D 30 mg/l

30

7.77

7.65

7.56

7.48

7.41

7.35

10

Blank 4

30

6.00

5.65

5.33

5.04

4.78

4.53

11

Blank 5

30

6.70

6.52

6.34

6.17

5.78

5.26

12

Blank 6

30

6.51

6.15

5.83

5.54

5.30

5.06

 

 

Respiration rate of activated sludge after application of SE7B

Vessel

O2 concentration in [mg/l]

Consuption rate [mg/(l*h)]

Inhibition [%]

First measurement

Last measurement

Blank 1

6.25

4.99

30.2

 

Blank 2

5.92

4.90

24.5

 

Blank 3

6.26

5.26

24.0

 

Blank 4

6.00

4.53

35.3

 

Blank 5

6.70

5.26

34.6

 

Blank 6

6.51

5.06

34.8

 

test item 1 (1000 mg/l)

4.85

3.48

32.9

-8

test item 2 (1000 mg/l)

4.25

2.79

35.0

-15

test item 3 (1000 mg/l)

4.28

2.86

34.1

-12

coefficient of variation of blanks:

0.16


 

 

Respiration rate of activated sludge after application of 3,5-dichlorphenol

Vessel

O2 concentration in [mg/l]

Consuption rate [mg/(l*h)]

Inhibition [%]

First measurement

Last measurement

Blank 1

6.25

4.99

30.2

 

Blank 2

5092.00

4.90

24.5

 

Blank 3

6.26

5.26

24.0

 

Blank 4

6.00

4.53

35.3

 

Blank 5

6.70

5.26

34.6

 

Blank 6

6.51

5.06

34.8

 

3,5-D 1 mg/l

6.44

5.52

22.1

28

3,5-D 10 mg/l

7.00

6.34

15.8

48

3,5-D 30 mg/l

7.77

7.35

10.1

67

 

Validity criteria fulfilled:
yes
Conclusions:
At a concentration of 1000 mg/L there was no inhibition of SE7B on the respiration rate of the inoculum. On the contrary, there was a negative inhibition of -8 to -15% in the test vessels (1000 mg/L) which is caused by a slight promotion of the respiration.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-Across Justification is attached below
Reason / purpose:
read-across source
Key result
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
Conclusions:
Read-Across is claimed between BT4 (target) and SE7B (Source), according to the justification attached to the target record, and based on structural and physical/chemical similarities.

Analogue diesters (SE7B, SE6B) and BT4 contain the same functional groups, i.e the ester group adjacent to the ethylhexane side chain, and the ester group at the opposite end of the molecules. The carbon range in the main backbone of the molecules is all the same (C18) though the acetate moiety is attached at slightly different positions (C12 for BT4, C9/10 for the analogue diesters). The analogue diesters have the additional alkane chain attached to the acetate cap. The alkane chains themselves are not typically considered to be functional groups, per se, as they are relatively inactive biologically. Thus the parent molecules BT4 and the analogue diesters are similar enough to allow for read across in that there are no differences with respect to functional groups, and their only real difference is number of, and length of, saturated hydrocarbon chains.

The Water Solubility of SE7B was measured as 0.778 mg/L @ 30 °C, whereas BT4 is found to be not stable in water but forms micelle structures resulting in suspension.

Hydrolysis of BT4 would yield acetic acid plus 12-hydroxystearic acid (C18), versus either lauric acid (C12) or the coconut oil fatty acid mixture (C8-18) for SE6B and SE7B, respectively.
These substances are not expected to persist in the environment, consistent with the hazard assessment presented in the OECD SIDS (2009) for the category “Aliphatic Acids Category” where aliphatic fatty acids with a carbon chain length in the range of C8 – C22 were judged to be readily biodegradable.

In an OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)), at a concentration of 1000 mg/L there was no inhibition of SE7B on the respiration rate of the inoculum. On the contrary, there was a negative inhibition of -8 to -15% in the test vessels (1000 mg/L) which is caused by a slight promotion of the respiration.

Considering the structural similarity and low soubility of BT4, this result is also considered relevant for the read-across target BT4.

Description of key information

Key value for chemical safety assessment

Additional information

Read-Across is claimed between BT4 (target) and SE7B (Source), according to the justification attached to the target record, and based on structural and physical/chemical similarities.

Analogue diesters (SE7B, SE6B) and BT4 contain the same functional groups, i.e the ester group adjacent to the ethylhexane side chain, and the ester group at the opposite end of the molecules. The carbon range in the main backbone of the molecules is all the same (C18) though the acetate moiety is attached at slightly different positions (C12 for BT4, C9/10 for the analogue diesters). The analogue diesters have the additional alkane chain attached to the acetate cap. The alkane chains themselves are not typically considered to be functional groups, per se, as they are relatively inactive biologically. Thus the parent molecules BT4 and the analogue diesters are similar enough to allow for read across in that there are no differences with respect to functional groups, and their only real difference is number of, and length of, saturated hydrocarbon chains.

The Water Solubility of SE7B was measured as 0.778 mg/L @ 30 °C, whereas BT4 is found to be not stable in water but forms micelle structures resulting in suspension.

Hydrolysis of BT4 would yield acetic acid plus 12-hydroxystearic acid (C18), versus either lauric acid (C12) or the coconut oil fatty acid mixture (C8-18) for SE6B and SE7B, respectively.

These substances are not expected to persist in the environment, consistent with the hazard assessment presented in the OECD SIDS (2009) for the category “Aliphatic Acids Category” where aliphatic fatty acids with a carbon chain length in the range of C8 – C22 were judged to be readily biodegradable.

In an OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)), at a concentration of 1000 mg/L there was no inhibition of SE7B on the respiration rate of the inoculum. On the contrary, there was a negative inhibition of -8 to -15% in the test vessels (1000 mg/L) which is caused by a slight promotion of the respiration.

Considering the structural similarity and low soubility of BT4, this result is also considered relevant for the read-across target BT4.