Tetradecafluorohexane

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

Minor: Concentration of suspended solids below the range stated in the Study Plan. Evaluation: High biodegradation percentages were observed in the procedure control toxicity control, and both test item vessels. No impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetradecafluorohexane

EC Number:

206-585-0

EC Name:

Tetradecafluorohexane

Cas Number:

355-42-0

Molecular formula:

C6F14

IUPAC Name:

tetradecafluorohexane

Test material form:

liquid

Specific details on test material used for the study:

Identification: Tetradecafluorohexane
Appearance: Clear colourless liquid
Test item storage: At room temperature protected from light
Stable under storage conditions until: 21 February 2020 (expiry date)
Additional information
Test Facility test item number: 209384/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or
trade name:
1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane
CAS number: 355-42-0
Molecular formula: C6F14
Molecular weight: 338
Highly reactive to water: Not indicated
Volatile: Yes, vapour pressure: 309 hPa at 20 °C
Specific gravity / density: 1.669 g/cm3 (25°C)
Solubility in water: Not soluble
Stability in water: Stable

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, adapted

Details on inoculum:

Source The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
After treatment, the concentration of suspended solids (SS) was determined to be 1.4 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 2 mL per litre of mineral medium, leading to a final concentration SS of 2.8 mg/L.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial conc.:

ca. 333 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

3. INTRODUCTION
The objective of the study was to evaluate the test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.
The design of this study was based on the following study guideline:
• OECD Guideline 301F, Section 3. Degradation and Accumulation. "Ready Biodegradability: Manometric Respirometry Test", (adopted July 17, 1992).
The Study Director signed the study plan on 02 May 2018. The experimental start date was 25 Jun 2018, and the experimental completion date was 06 Sep 2018. The Study Plan, Last Amended Study Plan, and deviations are presented in Appendix 2.
3.1. Definitions
Biochemical oxygen demand (BOD) is the amount of oxygen consumed by microorganisms when metabolizing a test item; also expressed as mg oxygen uptake per mg of test item.
Readily biodegradable are those test items giving a result of at least 60% biodegradation within 28 days. This pass level must be reached within the 10 days immediately following the attainment of 10% biodegradation (10-day window).
Suspended solids (SS) are the solids removed from activated sludge by filtration and dried to a constant mass, generally expressed in grams per litre.
Theoretical oxygen demand (ThOD) is the total amount of oxygen required to oxidize a chemical completely; it is calculated from the molecular formula and is also expressed as mg oxygen required per mg test compound.
4.1.2. Reference Item
4.1.2.1. Reference Item Information
Identification number: RS186
Container: F1
Identification: Sodium acetate
CAS Number: 127-09-3
Molecular formula: CH3COONa
Molecular weight: 82.03
Appearance: White powder (determined at Charles River Den Bosch)
Batch: AM1176568
Purity: 99.7%
Storage conditions: At room temperature Stable under storage conditions until: 31 May 2019
Supplier: Merck KGaA, Darmstadt, Germany
Article number: 1.06268
Certified: Yes
ThOD: 0.78 mg O2/mg
4.1.2.2. Reference Item Concentration and Preparation of Test Solutions
For the preparation of the test media a stock solution of 74 g/L sodium acetate was prepared in Milli-RO water, by adding a weighed amount of 738.8 mg and making this up to a volume of 10 mL. Aliquots of 500 µL and 340 µL of this stock solution were added to the test medium of the procedure control (final volume 360 mL) and toxicity control (final volume 244 mL), respectively. The resulting concentration of sodium acetate corresponded to a ThOD of 80 mg O2/L.
4.2. Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility and is presented in Appendix 3.
4.3. Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.
4.4. Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

4.5. Test Concentration and Preparation of Test Solutions
Tetradecafluorohexane was a clear colourless liquid with a purity of 99.25%. The test item was tested in duplicate at a target concentration of 333 mg/L, corresponding to a ThOD of 80 mg O2/L. The ThOD was based on the molecular formula.
Volumes of test item, reference item, and mineral medium added to the test vessels can be found in Table 1. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms. Test duration was 28 days.
Any residual volumes were discarded.

1): Density: 1.669 g/mL
4.6. Test System
Source The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Treatment The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment, the concentration of suspended solids (SS) was determined to be 1.4 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 2 mL per litre of mineral medium, leading to a final concentration SS of 2.8 mg/L. See also deviation in Appendix 2.
Reason for selection The test has been accepted internationally for determining
the 'ready' biodegradability of test items under aerobic conditions.
4.7. Testing Strategy and Experimental Design
4.7.1. Test Procedure and Conditions
Test duration 28 days. During the test period, the test media were aerated and stirred continuously.
Test vessels 500 mL brown colored glass bottles. The end volume of medium was 360 mL or 244 mL, in order to obtain the required headspace-to-volume ratio.
Milli-RO water Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Stock solutions of A) 8.50 g KH2PO4 mineral components 21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
Mineral medium 1 litre mineral medium contains: 10 mL of solution (A),
1 mL of solutions (B) to (D) and Milli-RO water.
CO2 absorber Sodium hydroxide pellets (Merck, Darmstadt, Germany).
Illumination The test media were excluded from light.
4.7.2. Preparation of Bottles
Inoculation At the start of the test (day 0) mineral medium was inoculated with activated sludge.
Type and number of bottles Test suspension: containing test item and inoculum
(2 bottles).
Inoculum blank: containing only inoculum (2 bottles) Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing inoculum, and test item and reference item in amounts contributing equally to the total ThOD (1 bottle).
Preparation At the start of the test (day 0), test and/or reference item were added to the respective bottles. The volumes of suspensions were made up to 360 mL or 244 mL using inoculated mineral medium.
Rubber gaskets containing two pellets of sodium hydroxide were inserted into each bottle. Subsequently, each bottle was sealed by screwing an automated respirometer head on top.
4.7.3. Determination of Biodegradation
Experimental CO2 production The CO2 produced in each test bottle reacted with the CO2
absorbent in the rubber gaskets. As gaseous O2 was converted into gaseous CO2 that was absorbed, the gas pressure in the test system slowly decreased. This decrease in air pressure was measured by the respirometer heads and automatically converted into oxygen consumption
(mg O2/L).
Measurements Measurements were recorded on day 0-4-7-11-14-18-21-25-
28. After recording the oxygen consumption on day 28, the pH was determined in all test vessels.
Theoretical Oxygen Demand The ThOD was calculated from the molecular formula.

4.8. Measurements and Recordings
pH At the start of the test (day 0) and on the last day (day 28), after the last measurement.
Respirometer Lovibond BD600-GLP manometric respirometry system, equipped with an inductive stirring system.
Temperature of medium Continuously in a vessel with Milli-RO water in the same
climate controlled incubator.
5. INTERPRETATION
5.1. Acceptability of the Test
1. The reference item was biodegraded by at least 60% (actual value: 81%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (0% at the end of the test).
3. The total oxygen uptake in the blank at the end of the test did not exceed 30 mg O2/L (average actual value: 8 mg O2 per litre).
4. The pH value in the test item bottles at the end of the test was within the range 6-8.5 (7.4). Since all criteria for acceptability of the test were met, this study was considered to be valid.

6. ANALYSIS
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
ThOD, expressed as mg O2 required for full biodegradation of one mg of test item, was calculated from the elemental composition of Tetradecafluorohexane. For test item CcHhXxNnNanaOoPpSs, with Xx being all halogens and assuming full nitrification, ThOD was calculated as follows:
ThOD = ((16(2c + 1⁄2 (ℎ − x) + 5⁄2 n + 3s + 5⁄2 p + 1⁄2 na − o)/Molecular weight) mg O2⁄mg test item
The first step in calculating the amount of O2 consumed was to correct for background O2 consumption. Thus, the amount of O2 consumed for biodegradation of the test item (BOD) was determined using the following equation:
BOD = O2uptake by test item (mg O2/L) − O2 uptake by blank (mg O2/L)
Relative biodegradation values were then calculated using the ThOD of the added amount of test item, expressed as mg O2/L, as follows:

% degradation = (BOD (mg O2⁄L)/ThOD (mg O ⁄L)) × 100
The relative biodegradation values were plotted versus time together with the relative biodegradation in the procedure control and the toxicity control. If not clear from the experimental data the number of days was calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10-day window), then the test item is designated as readily biodegradable.
Toxicity control: if less than 25% biodegradation (based on combined ThOD of the test item and reference item) occurred within 14 days, the test item was assumed to be inhibitory.
7. COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.
Text Table 1
Critical Computerized Systems

System name Version No. Description of Data Collected and/or Analyzed
REES Centron SQL 2.0 Temperature, relative humidity and/or
atmospheric pressure monitoring

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

Test performance:

5.1. Acceptability of the Test
1. The reference item was biodegraded by at least 60% (actual value: 81%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (0% at the end of the test).
3. The total oxygen uptake in the blank at the end of the test did not exceed 30 mg O2/L (average actual value: 8 mg O2 per litre).
4. The pH value in the test item bottles at the end of the test was within the range 6-8.5 (7.4). Since all criteria for acceptability of the test were met, this study was considered to be valid.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

Sampling time:

14 d

Details on results:

9.1. Theoretical Oxygen Demand
The ThOD of Tetradecafluorohexane was calculated to be 0.24 mg O2/mg. The ThOD of sodium acetate was calculated to be 0.78 mg O2/mg.
9.2. Biodegradation
All data are presented in Appendix 1. The results of O2 consumption and biodegradation in blank bottles, procedure and toxicity control, and each test bottle are listed in Table 3 to 7. Table 8 contains the comparison of biodegradation of Tetradecafluorohexane in bottles A and B.
Figure 1 shows the curves for biodegradation of the two bottles with Tetradecafluorohexane, and results up to Day 14 for the procedure control and the toxicity control.
The relative biodegradation values calculated from the measurements performed during the test period revealed no biodegradation of Tetradecafluorohexane, based on ThOD (0% in both test vessels).
In the toxicity control, more than 25% biodegradation occurred within 14 days (40%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity. After Day 14 there was a decline in the measurements for the toxicity control. However, since this effect was only observed after the 14-day period used to assess the presence of inhibitory effects and a similar effect was not observed in any of the other vessels within this study, it can be concluded that this did not observe the outcome of the study or the assessment of the toxicity control.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

BOD5 / COD

Key result

Parameter:

ThOD

Value:

ca. 0 other: mgO2/L

Results with reference substance:

1. The reference item was biodegraded by at least 60% (actual value: 81%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (0% at the end of the test).
3. The total oxygen uptake in the blank at the end of the test did not exceed 30 mg O2/L (average actual value: 8 mg O2 per litre).
4. The pH value in the test item bottles at the end of the test was within the range 6-8.5 (7.4). Since all criteria for acceptability of the test were met, this study was considered to be valid.

Any other information on results incl. tables

Table 2

pH Values of Different Test Media

Test medium:	On day 0:	On day 28:
Blank control (A)	7.6	7.4
Blank control (B)	7.6	7.5
Procedure control	7.6	8.1
Tetradecafluorohexane (A)	7.6	7.4
Tetradecafluorohexane (B)	7.6	7.4
Toxicity control	7.6	8.2

Table 3

Mean Oxygen Consumption in the Blanks

Day	Oxygen consumption (mg O2/L)
	Blank A	Blank B	Mean Value
0	1	1	1
4	01)	01)	0
7	0	1	1
11	2	4	3
14	2	5	4
18	3	7	5
21	3	9	6
25	4	10	7
28	4	11	8

1): Value was below the selected measurement range of the equipment, and is therefore represented as 0.

 

Table 4

O2Consumption and Percentage Biodegradation in the Procedure Control

Day	Oxygen consumption (mg O2/L)		BOD (mg O2/L)	Biodegradation (%)	1)
	Blank (mean)	Procedurecontrol
0	1	02)	0	0
4	0	51	51	64
7	1	60	60	74
11	3	66	63	79
14	4	68	65	81
18	5	70	65	81
21	6	71	65	81
25	7	72	65	81
28	8	73	66	82

1): Calculated as the ratio between BOD and ThOD of reference item added: 80.0 mg O2/L.

2): Value was below the selected measurement range of the equipment, and is therefore represented as 0.

 

Table 5

O2Consumption and Percentage Biodegradation of the Test Item (Bottle A)

Day	Oxygen consumption (mg O2/L)		BOD (mg O2/L)	Biodegradation (%)	1)
	Blank(mean)	Bottle A
0	1	02)	0	0
4	0	02)	0	0
7	1	02)	0	0
11	3	2	0	0
14	4	3	0	0
18	5	4	0	0
21	6	6	0	0
25	7	7	0	0
28	8	7	0	0

1): Calculated as the ratio between BOD and ThOD of test item added: 80.0 mg O2/L.

2): Value was below the selected measurement range of the equipment, and is therefore represented as

Table 6

O2Consumption and Percentage Biodegradation of the Test Item (Bottle B)

Day	Oxygen consumption (mg O2/L)		BOD (mg O2/L)	Biodegradation (%)	1)
	Blank(mean)	Bottle B
0	1	02)	0	0
4	0	02)	0	0
7	1	02)	0	0
11	3	02)	0	0
14	4	02)	0	0
18	5	1	0	0
21	6	2	0	0
25	7	3	0	0
28	8	4	0	0

1): Calculated as the ratio between BOD and ThOD of test item added: 80.0 mg O2/L.

2): Value was below the selected measurement range of the equipment, and is therefore represented as 0.

 

Table 7

O2Consumption and Percentage Biodegradation in the Toxicity Control

Day	Oxygen consumption (mg O2/L)		BOD (mg O2/L)	Biodegradation (%)	1)
	Blank(mean)	Toxicitycontrol
0	1	9	8	5
4	0	51	51	32
7	1	62	62	38
11	3	67	64	40
14	4	68	65	40
18	5	49	44	27
21	6	51	45	28
25	7	50	43	27
28	8	51	44	27

1): Calculated as the ratio between combined BOD/ThOD of test item and reference item:

160.2 mg O2/L (ThOD test item: 79.9 mg O2/L + ThOD reference item: 80.3 mg O2/L)

 

Table 8

Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B	1)
0	0	0	0	0
4	0	0	0	0
7	0	0	0	0
11	0	0	0	0
14	0	0	0	0
18	0	0	0	0
21	0	0	0	0
25	0	0	0	0
28	0	0	0	0

1): Absolute difference in biodegradation between bottles A and B

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Tetradecafluorohexane was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed.

Executive summary:

The objective of the study was to evaluate the test item Tetradecafluorohexane for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Manometric Respirometry Test.

The study procedures described in this report were in compliance with the OECD guideline No. 301 F, 1992.

Tetradecafluorohexane was a clear colourless liquid with a purity of 99.25%. The TheoreticalOxygen Demand (ThOD) of Tetradecafluorohexane was determined to be 0.24 mg O2/mg.

The test item was tested in duplicate at a target concentration of 333 mg/L, corresponding to aThOD of 80 mg O2/L. The ThOD was based on the molecular formula.

The study consisted of six bottles:

·       2 inoculum blanks (no testitem),

·       2 test bottles(Tetradecafluorohexane),

·       1 procedure control (sodium acetate)and

·       1 toxicity control (Tetradecafluorohexane plus sodiumacetate).

In order to transfer Tetradecafluorohexane to the test vessels, the required amounts were pipetted directly into the test bottles containing medium with microbial organisms and mineral components. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days.

The relative biodegradation values calculated from the measurements performed during the test period revealed no biodegradation of Tetradecafluorohexane, based on ThOD (0% in both test vessels).

In the toxicity control, Tetradecafluorohexane was found not to inhibit microbial activity. After Day 14 there was a decline in the measurements for the toxicity control. However, since this effect was only observed after the 14-day period used to assess the presence of inhibitory effects and a similar effect was not observed in any of the other vessels within this study, it can be concluded that this did not observe the outcome of the study or the assessment of the toxicity control.

Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, Tetradecafluorohexane was designated as not readily biodegradable.