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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to the OECD Testing Guideline and GLP compliant . All validity criteria are fulfilled. (and no deviaton)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 10707
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum:The source of test organisms was secondary effluent freshly obtained from a municipal sewage treatment plant:'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: Secondary effluent was filtered through a coarse filter paper, the first 200 ml was discarded. The filtrate was kept aerated until inoculation.
- Inoculation: 4 ml filtrate of secondary effluent per litre of final volume.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST SOLUTIONS:
- Test concentrations and preparation of test solutions:
1,6-divinylfluorohexane was a clear colourless liquid with a purity of >99%. Nominal concentrations of 2 and 5 mg/l were tested. No correction was made for the purity/composition of the test substance. Preparation of the test media started with a stock solution of 10 mg/l by adding 29.6 mg test substance to 3000 ml of mineral medium. Thorough mixing was used to accelerate dissolution of the test substance in mineral medium. Preparation was as much as possible performed under low light conditions.

- Reference substance concentrations and preparation of test solutions:
For the preparation of the test media a stock solution of 2 g/l was prepared in mineral medium. Exact volumes of the stock solution corresponding to the final sodium acetate concentration (2 mg/l) were added to the test medium of the procedure control and the toxicity control.

TEST CONDITIONS:
- test duration: 28 days for the inoculum blank and test suspension, 14 days for the procedure and toxicity control
- Mineral medium: As prescribed in the OECD guideline 301D. The concentration of dissolved oxygen was measured for control purposes. The mineral medium was left at test temperature to obtain a saturated solution at the start of the test.
- Additional substrate: No
- Test temperature: 22 +/- 2°C
- pH: The pH values at the start of the test were 7.4 or 7.5
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes, The test bottles were protected from light.
- Other:

TEST SYSTEM:
- Culturing apparatus: 250-300 ml BOD bottles with glass stoppers.
- Number of culture flasks: 42 flasks set up as following:
- 10 Inoculum blank (only inoculum, no test substance);
- 6 Procedure control (sodium acetate at 2 mg/l and inoculum);
- 10 Test substance (1,6-divinylfluorohexane at 2 mg/l and inoculum);
- 10 Test substance (1,6-divinylfluorohexane at 5 mg/l and inoculum);
- 6 Toxicity control (sodium acetate at 2 mg/l, 1,6-divinylfluorohexane at 2 mg/l and inoculum).
Individual BOD bottles were prepared for each measuring point, except that the bottles measured at the start were also measured on day 7.
Parallel groups were prepared to allow duplicate measurements of oxygen consumption at the test intervals.

- Measuring equipment:Oxygen meter
- Test performed in closed vessels due to significant volatility of test substance: yes


SAMPLING AND MEASUREMENTS:
- Determination of oxygen concentration:
Frequency: In duplicate; immediately at the start of the experiment (day 0), and at day 7, 14, 21 and 28.
Measurement method: WTW oxygen meter supplied with a WTW CellOx 325 oxygen electrode, electrolyte type ELY/G.
Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,USA)
- Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.
- pH : At the start of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing only inoculum (no test substance)
- Abiotic sterile control: No
- Toxicity control: Containing test substance at the lowest concentration, reference substance and inoculum.

Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Remarks on result:
other: test substance 2 mg/L
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Remarks on result:
other: test substance 5 mg/L
Details on results:
The relative biodegradation values calculated from the O2 measurements performed during the test period of 28 days revealed no significant biodegradation of 1,6-divinylfluorohexane at both concentrations.
In the toxicity control more than 25% biodegradation occurred within 14 days (46%, based on ThOD). Thus, the toxicity control showed that 1,6-divinylfluorohexane did not inhibit microbial activity.


Results with reference substance:
The reference substance was degraded by at least 60% (104%) within 14 days.

Theoretical Oxygen Demand:

The ThOD of 1,6-divinylfluorohexane was calculated to be 0.77 mg O2 per mg.

The ThOD of sodium acetate (reference substance) was calculated to be 0.78 mg O2/mg.

The pH values at the start of the test were 7.4 or 7.5.

The temperature recorded in a vessel with water in the same room varied between 21.8 and 22.5°C.

Table 1: Biodegradation percentages at different points in time

Test medium

Concentration (mg/l)

% Biodegradation after x days1)

 

 

0

7

14

21

28

Procedure control A2)

Procedure control B2)

Procedure control mean A and B

Difference between duplicate values

2

0

0

0

0

85

87

86

2

109

98 104

11

 

 

Test substance low A3)

Test substance low B3)

Test substance low mean A and B

Difference between duplicate values

2

0

0

0

0

-2

0

-1

2

-4

-1 -3

3

-2

-1

-2

1

5

9

7

4

Test substance high A3)

Test substance high B3)

Test substance high mean A and B

Difference between duplicate values

5

0

0

0

0

2

2

2

0

2

5

4

3

6

8

7

2

-4

5

1

9

Toxicity control A

Toxicity control B

Toxicity control mean A and B

Difference between duplicate values

2/24)

0

0

0

0

33

36

35

3

45

47 46

2

 

 

1): Calculations were performed with th e mean oxygen concentrations of t=0 and the oxygen depletion as given in

Table2 . Except for the percentages biodegradation, all calculations were performed without rounding off.

2): ThOD positive control sodium acetate: 0.78 mg O2/mg

3): ThOD test substance: 0.77 mg O2/mg

4): Toxicity control contains sodium acetate and test substance.

Table 2: pH at the start and oxygen concentration during the test

Series

Content

pH at t=0

Flask No.

Oxygen concentration (mg/L) afterx days

 

 

 

 

0

7

14

21

28

Inoculum blank

 

Mineral medium, inoculum

 

7.5

A

B mean

 

8.64 8.64

8.64

7.38 7.54

7.46

 

8.87

8.68

8.78

8.22

8.15

8.19

8.40

7.94

8.17

Procedure control

 

Mineral medium, inoculumsodium acetate (2 mg/l)

 

7.5

A

B

mean

 

8.70 8.68

8.69

 

6.18 6.16

 

7.12

7.30

 

 

Test

substance

low

 

Mineral medium, inoculum1,6-divinylfluorohexane (2 mg/l)

 

7.4

 

A

B

mean

 

8.68 8.69

8.69

7.54 7.50

 

8.88

8.83

8.26

8.24

8.14

8.07

Test

substance

high

 

Mineral medium, inoculum1,6-divinylfluorohexane (5 mg/l)

 

7.4

A

B

mean

 

8.79 8.72

8.76

7.51

7.50

8.81

8.71

8.08

7.98

8.43

8.10

Toxicity

Toxicity control

Mineral medium, inoculums

1,6-divinylfluorohexane

(2 mg/l)

sodium acetate (2 mg/l)

7.4

A

B mean

 

8.75 8.70

8.73

6.51 6.42

7.46

7.41

 

 

Table 3: Mean values of oxygen depletion at different points in time

Test medium

Concentration

(mg/l)

Oxygen depletion (mg BOD/L) after x days1)

 

 

7

14

21

28

Procedure control A

Procedure control B

2

2

1.33

1.35

1.71

1.53

 

 

Test substance low A

Test substance low B

2

2

-0.04

0.00

-0.06

-0.01

-0.03

-0.01

0.07

0.14

Test substance high A

Test substance high B

5

5

0.06

0.07

0.08

0.18

0.22

0.32

-0.15

0.18

Toxicity control A

Toxicity control B

 

1.04

1.13

1.40

1.45

 

 

1): Corrected for oxygen depletion in the blank control (mean value).

Validity criteria fulfilled:
yes
Remarks:
according to the OECD TG 301D
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
1,6-divinylfluorohexane was not readily biodegradable under the conditions of the closed bottle test.
Executive summary:

The evaluation of the ready biodegradability of the 1,6 -Divinylperfluorohexane was investigated in a 28-days closed bottle test according to the OECD guideline No. 301 D, 1992 and the EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test).

The Theoretical oxygen demand (ThOD) of 1,6-divinylfluorohexane was calculated to be 0.77 mg O2/mg.

The test was perfomed using the following test preparations:

- Two inoculum blank (only inoculum, no test substance);

- Two procedure control (sodium acetate at 2 mg/l and inoculum);

- Two test substance (1,6-divinylfluorohexane at 2 mg/l and inoculum);

- Two test substance (1,6-divinylfluorohexane at 5 mg/l and inoculum);

- Two toxicity control (sodium acetate at 2 mg/l, 1,6-divinylfluorohexane at 2 mg/l and inoculum).

The nominal concentrations of 2 and 5 mg/l were tested.

Measurements of the oxygen concentrations were performed at the start of the experiment (day 0) and at day 7, 14, 21 and 28 and allowed to the determination of the Biochemical Oxygen Demand (BOD) values.

The relative biodegradation values (BOD/ThOD) calculated from the O2 measurements performed during the test period of 28 days revealed no significant biodegradation of 1,6-divinylfluorohexane at both concentrations.

The toxicity control showed that 1,6-divinylfluorohexane did not inhibit microbial activity.

The study was considered to be valid since all validity criteria were fulfilled.

In conclusion, 1,6-divinylfluorohexane is designated as not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 2015 to December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, Degradation and Accumulation (the Second Edition), No. 301F "Manometric Respirometry Test". (2013.9)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Appearance: Clear colourless liquid
- Test item storage: At room temperature protected from light
- Purity/Composition correction factor: No correction factor required
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Inoculum was activated sludge derived from a conventional treatment plant for domestic waste in Shenyang.
- Preparation of inoculum for exposure: A fresh sample of activated sludge was collected from the aeration tank of Shenyang Northem Sewage Treatment Plant one day before the test item exposure was started.
- Concentration of sludge: less than 30 mg/L (as suspended solids (SS))
Duration of test (contact time):
28 d
Initial conc.:
98.2 mg/L
Based on:
ThOD
Initial conc.:
127.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
MINERAL MEDIUM
- Stock solution A: 8.5 g/L KH2PO4, 21.75 g/L K2HPO4, 33.4 g/L Na2HPO4.2H2O, 0.5 g/L NH4Cl
- Stock solution B: 36.4 g/L CaCl2. H2O
- Stock solution C: 22.5 g/L MgSO4.7H2O
- Stock solution D: 0.25 g/L FeCl3.6H2O
- Mineral medium: 10 mL solution A + 1 mL solution B + 1 mL solution C + 1 mL solution D + ultrapure water up to 1L (pH adjusted to 7.4+/- 0.2 with a diluted hydrochloric acid solution)


TEST CONDITIONS
- Preparation of lnoculated Mineral Medium:
6.00 ml of the prepared activated sludge was added to flask and made up to volume to give a suspended solid concentration of 30 mg/L. And then inoculated minerai medium was transferred into each sample flask respectively .

- Preparation Test Item / Reference Item / Toxicity control test vessel:
The appropriate amount of test item an reference item were added directly into sample flask.

TEST SYSTEM
- Inoculum blank: 2 replicates (mineral medium + inoculum)
- Test substance: 2 replicates (mineral medium + test substance (96.4 and 99.9 mg/L ThOD + inoculum)
- Procedure control (reference): 1 replicates (mineral medium + 94.6 mg/L ThOD reference substance + inoculum)
- Toxicity control: 2 replicates (mineral medium + 95.4 mg/L ThOD test substance + 95.5 mg/L ThOD reference substance + inoculum)


SAMPLING AND MEASUREMENT:
- The BOD values in all test vessels were recorded every 12 hours for the 28 day period by the automated respirometer.
- Incubation temperatures (min to max values) were measured and recorded every working day.


Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d

Test Substance:

The average biodegradation percentage in the test substance bottles containing the test substance was 0% on Day 14, 0% on Day 28.

Toxicity Control:

The average biodegradation percentage in the toxicity control bottles containing the test substance was 42.5% on Day 14. Therefore, the test item was not inhibitory to the activity of the microbial inoculum based on the test criteria (≥ 25% ThOD).

validity criteria:

1) The oxygen uptake of the inoculum blank was 24.95 mg O2/L in 28 days, which did not exceed 60 mg O2/L.

2) The pH values were in the range of 7.29-7.57.

3) The difference of extremes of replicate values of the removal of the test item at the end of the test was 0%, less than 20%.

4) The percentage degradation of the reference item reached 61.8% by day 4.5, and was 85.5% by Day 14 which exceeded the required pass level (> 60% within 14 days)

5) In Toxicity Control (TC) test mixture, containing both test item and reference item, 42.5% ThOD degradation within 14 days. (> 25% ThOD) ,  it  was  concluded  that  the  test  item  is  not inhibitory  to sludge micro-organisms

Thus, the test is considered valid.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No biologically-relevant degradation of the test item was observed during the 28-day test period. Therefore, the test item should be considered not readily biodegradable under the conditions of the test
Executive summary:

The ultimate aerobic biodegradability of the test item was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301F. No biologically-relevant degradation of the test item was observed during the 28-day test period. Therefore, the test item should be considered not readily biodegradable under the test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
not specified
Inoculum or test system:
other: Secondary effluent from a domestic waste water treatment plant
Details on inoculum:
Inoculum was collected on measurement start date, February 10, 2000, by Mitsubishi Chemical Safety Institute of Science.
Duration of test (contact time):
28 d
Initial conc.:
5.97 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
other: BOC degradability
Details on study design:
TEST CONDITIONS
- Composition of medium:Dimethyl sulfoxide (DMSO)
- Additional substrate: none
- Test temperature: 20+/- 1oC
- pH:6.9
- pH adjusted: o
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: Incubator bottle
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: closed system
- Measuring equipment: DOC testor, pH meter, GC
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system:No
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance:
aniline
Remarks:
2.00 mg/L
Test performance:
Degradabilities of aniline based on BOD on 14th day were 62% (average 62%) respectively and acitivity of the inoculum was not changed.
The decrease of dissolved oxygen concentration in the blank system was less than 1.5 mgO2/L (0.2 mgO2/L).
In addition to that, during BOD measurement period,
Parameter:
other: based on BOD
Value:
0
Sampling time:
7 d
Parameter:
other: Based on BOD
Value:
0
Sampling time:
14 d
Parameter:
other: based on BOD
Value:
0
Sampling time:
21 d
Key result
Parameter:
other: based on BOD
Value:
>= 0 - <= 2
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
0
Sampling time:
28 d
Details on results:
Degradation based on BOD was calculated according to the following equation;
Biodegradation (%) = BOD/ThOD x 100
BOD: Biological oxygen demand for test substance or aniline (mgO2/mg)

BOD was calculated according to the following equation;
BOD (mgO2/mg) = {(DO0 - DOx ) - (DOb0a - DObxa)}/C
C: Concentration of test sample or aniline in the incubation flask (mg/L)
DO0; Average dissolved oxygen concentration on 0 day (mgO2/L)
DOx; Dissolved oxygen concentration on x day (mgO2/L)
DOb0a; Average dissolved oxygen concentration in the blank system on 0 day (mgO2/L)
DObxa: Average dissolved oxygen concentration in the blank system on x day (mgO2/L)

Calculation of theoretical oxygen demand
Aniline ; 2.41 mgO2/mg based on chemical equilibrium; C6H7N + 7O2 -> 6CO2 + 2H2O + NH3
Test substance : 1.04 mgO2/mg base on chemical equilibrium: C10H6F12 + 23/2O2 -> 10CO2 + 3H2O + 12F

Biodegradability based on residual test substance
BIodegradability (%) = (1-C28/C)x100
C: Initial concentration of test substance 8mg/L)
C28: Concentration of test substance on 29 day in the test system (mg/L)
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Test substance was considered not readily biodegarable and no structure change was occured, for average biodegradability based on BOD was 1% and average biodegaradability based on residual test material was 0% on 28 day.
Executive summary:

Test substance was considered not readily biodegarable and no structure change was occured, for average biodegradability based on BOD was 1% and average biodegaradability based on residual test material was 0% on 28 day.

Description of key information

The ready biodegradability of the test item was investigated in a GLP-compliant studies performed in accordance with etiher OECD Guideline No. 301D, or 301F without deviations. All the studies are consistent showing that no significant biodegradtion occurred under the test conditions.

1,6 -divinylfluorohexane is not readily biodegradable.

All the available studies have comparable reliability, the more recent study (SYRICI 2015) is selected as key.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

In both studies of M.J.E Desmares-Koopmans (2014) and unko Koyasu (2000) , the ultimate aerobic biodegradability of the test item 1,6 -Divinylperfluorohexane was investigated in a 28-days closed bottle test according to the OECD guideline No. 301 D (Closed Bottle Test).

In both studies, no significant biodegradation of 1,6-divinylfluorohexane was observed.

In another test ( SYRICI 2015), the ultimate aerobic biodegradability of the test item was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301F . No relevant degradation of the test item was observed during the 28-day test period.

In conclusion, 1,6 -divinylfluorohexane is considered not readily biodegradable.

The more recent study (SYRICI 2015) is selected as key.