Hexyl hexanoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2018 to 14 Jun 2018

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)

Version / remarks:

17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number 202462
Expiry date: 19 November 2019
Clear colourless liquid with a purity of 99.29%

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

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. 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 3 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 1 mL per litre of mineral medium, leading to a final concentration SS of 3 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

29 mg/L

Based on:

test mat.

Initial conc.:

80 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Concentration and Preparation of Test Solutions:
Hexyl Caproate was a clear colourless liquid with a purity of 99.29%. The test item was tested in duplicate at a target concentration of 29 mg/L, corresponding to a ThOD of 80 mg O2/L. The ThOD was based on the molecular formula (2.72 mg O2/mg).

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.

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 3 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 1 mL per litre of mineral medium, leading to a final concentration SS of 3 mg/L.

Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.

Testing Strategy and Experimental Design:
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 mineral components:

A) 8.50 g KH2PO4
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.

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.

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 0-3-7-11-14-18-21-25-28. 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.

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.

INTERPRETATION
Acceptability of the Test:

1. The reference item was biodegraded by at least 60% (actual value: 88%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (5% on day 18 and 1% 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: 13 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 (7.7).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

ANALYSIS
ThOD, expressed as mg O2 required for full biodegradation of one mg of test item, was calculated from the elemental composition of Hexyl Caproate. For test item CcHhXxNnNanaOoPpSs, with Xx being all halogens and assuming full nitrification, ThOD was calculated using the ThOD equation (see "Any other information on materials and methods incl. tables"):

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 = O2 uptake 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 O2⁄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.

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.

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

Reference substance:

other: Octan-1-ol

Test performance:

In the toxicity control, more than 25% biodegradation occurred within 14 days (51%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item 1-octanol, which showed a normal biodegradation curve

Key result

Parameter:

% degradation (CO2 evolution)

Value:

63

Sampling time:

28 d

Details on results:

Theoretical Oxygen Demand:
The ThOD of Hexyl Caproate was calculated to be 2.72 mg O2/mg.
The ThOD of 1-octanol was calculated to be 2.95 mg O2/mg.

Biodegradation:
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. All data are presented in Appendix 3. 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 Hexyl Caproate in bottles A and B. Figure 1 shows the curves for biodegradation of the two bottles with Hexyl Caproate, the procedure control and the toxicity control.

The relative biodegradation values calculated from the measurements performed during the test period revealed 63% and 64% biodegradation of Hexyl Caproate, for A and B, respectively (based on ThOD). However, biodegradation of Hexyl Caproate of at least 60% was not reached within a 10-day window. Thus, the criterion for ready biodegradability was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (51%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item 1-octanol, which showed a normal biodegradation curve.

Monitoring of Temperature and pH:
The temperature recorded in a vessel with water in the same incubator varied between 22 and
24°C. The pH values of the different test media are presented in Table 2.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

In conclusion, Hexyl Caproate was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed. Since the results of the present test indicate that the pass level criterion was fulfilled but the 10-day window criterion was not fulfilled, the results can be used to support inherent biodegradability.

Executive summary:

The objective of the study was to evaluate the test item Hexyl Caproate 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.

Hexyl Caproate was a clear colourless liquid with a purity of 99.29%. The Theoretical Oxygen Demand (ThOD) of Hexyl Caproate was determined to be 2.72 mg O2/mg. The test item was tested in duplicate at a target concentration of 29 mg/L, corresponding to a ThOD of 80 mg O2/L. The ThOD was based on the molecular formula.

The study consisted of six bottles:

2 inoculum blanks (no test item),

2 test bottles (Hexyl Caproate),

1 procedure control (1-octanol) and

1 toxicity control (Hexyl Caproate plus 1-octanol).

In order to transfer Hexyl Caproate to the test vessels, the required amounts were pipetteddirectly 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 63% and 64% biodegradation of Hexyl Caproate, for A and B, respectively (based on ThOD). However, biodegradation of Hexyl Caproate of at least 60% was not reached within a 10-day window. Thus, the criterion for ready biodegradability was not met.

In the toxicity control, Hexyl Caproate was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, Hexyl Caproate was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed. Since the results of the present test indicate that the pass level criterion was fulfilled but the 10-day window criterion was not fulfilled, the results can be used to support inherent biodegradability.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater