2H-Pyran-2-one, tetrahydro-5-pentyl-

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:

From 27 March 2019 to 24 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 301F and EU Method C.4-D with GLP certificate. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/08/2018; Date of issue: 19/11/2018

Specific details on test material used for the study:

- Storage conditions: Approximately 4°C, in the dark
- Water solubility: 1.95 g/L at 25°C (iSafeRat® v1.7)
- Vapour pressure: 0.853 Pa at 25°C (iSafeRat® v1.7)
- Log Kow: 2.39 (Phytosafe 2006, Shake flask method EEC A8)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 25 March 2019 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at approximately 20°C prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

MINERAL MEDIUM:
The mineral medium used in this study was that recommended in the OECD Guidelines. The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

TEST ITEM PREPARATION:
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to the pH values being measured using a Hach HQ40d Flexi handheld meter and the addition of inoculum (5 mL). The volume was adjusted to 500 mL with mineral medium to give the test concentration of 100 mg/L. The inoculum control vessels were prepared by inoculating mineral medium (495 mL) with inoculum (5 mL). The pH values were measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum. A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.

REFERENCE ITEM PREPARATION:
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving a nominal amount of the reference item (500 mg) directly in mineral medium (500 mL) with the aid of ultrasonication for approximately 20 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (350 mL) prior to measurement of the pH value and then addition of inoculum (5 mL) and adjusting to a final volume of 500 mL with mineral medium, to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity. The pH of the reference item stock solution was 7.4. The pH value was measured using a Hach HQ40d Flexi handheld meter.

TOXICITY CONTROL:
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effects of the test item on the sewage treatment micro-organisms used in the test. A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution and measurement of the pH value using a Hach HQ40d Flexi handheld meter. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

PREPARATION OF TEST SYSTEM:
The following test preparations were prepared and inoculated in 500 mL amber glass bottles:
a) Three replicate bottles containing inoculated mineral medium alone to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control.
c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, aniline, at a concentration of 100 mg/L to act as toxicity control vessels.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0, the test and reference items were added to the mineral medium and the pH of all vessels was measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum and adjusting to the final volume of 500 mL with mineral medium.
In order to confirm that the aniline stock solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was also sampled for Total Organic Carbon (TOC) analysis.
All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at temperatures of between 20 and 21 °C.
On Day 28, two inoculum controls, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

TEMPERATURE MEASUREMENTS:
The temperature of the water bath was recorded daily.

PH MEASUREMENTS:
In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.

Reference substance:

aniline

Preliminary study:

Information provided by the Sponsor indicated that the water solubility of the test item was 1.95 g/L at 25 ºC (iSafeRat ® v1.7). Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) it was concluded that the best testable dispersions were found to be obtained when using the high shear mixing method and solvent with filter paper and high shear mixing method of preparation. As there was no difference between the test item dispersions using these methods of preparation, it was considered appropriate to just use high shear mixing to disperse the test item into the test system.

Test performance:

The mean BOD of the inoculated mineral medium (control) was 22.56 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 were 7.4 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test and at the end of the 10-Day window was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The toxicity control attained 70% biodegradation after 14 days and 74% biodegradation after 28 days, thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

74

Sampling time:

28 d

Remarks on result:

other: satisfied the 10-Day window validation criterion

Details on results:

The test item attained 74% biodegradation after 28 days, calculated from the oxygen consumption values, and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained with 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
See biodegradation curve in "Illustration"

Results with reference substance:

Aniline (procedure control) attained 72% biodegradation after 14 days in a 10-Day Window and 76% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
TOC of the diluted aniline stock solution confirmed that it had been prepared correctly.

Table 5.2.1/1: Biological Oxygen Demand (BOD) values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test item		Toxicity Control
	R1	R2		R1	R2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28	0.00 0.50 1.34 2.74 3.92 4.96 5.74 6.66 7.16 8.28 9.08 9.82 11.00 11.70 12.50 13.28 13.96 14.78 15.50 16.36 17.46 18.40 19.00 20.12 20.70 21.62 22.58 23.24 24.00	0.00 0.54 1.20 2.16 3.16 3.96 4.70 5.50 6.04 7.28 8.16 8.96 9.96 10.66 11.32 12.04 12.66 13.50 13.92 14.70 15.62 16.42 16.74 17.74 18.24 18.96 19.86 20.40 21.12	0.00 0.46 1.66 9.38 67.64 138.86 192.54 210.20 217.04 221.62 224.42 226.86 229.24 231.70 234.74 237.08 239.12 241.36 243.02 245.24 247.32 249.24 250.56 252.32 253.48 254.86 256.56 257.74 258.72	0.00 1.46 2.58 3.50 4.54 5.34 6.24 7.88 11.70 23.28 53.40 86.26 109.74 126.36 140.20 152.94 165.36 177.88 188.38 195.50 199.80 202.96 205.26 207.70 209.58 211.46 213.66 215.46 216.70	0.00 1.66 2.42 3.74 4.62 5.62 6.66 7.78 10.24 19.12 39.82 71.98 95.30 111.38 124.16 134.36 143.10 151.36 158.10 165.06 172.10 178.42 183.72 188.92 192.84 196.04 199.26 201.80 203.80	0.00 1.80 2.96 17.90 49.94 117.36 180.88 215.04 261.64 315.84 361.94 386.60 395.72 401.34 407.18 411.38 414.16 416.50 418.08 420.20 423.08 426.54 428.08 430.24 432.36 434.62 436.74 438.58 440.20

Table 5.2.1/2: Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test item			Toxicity Control
		R1	R2	Mean
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28	0 0 0 2 21 43 61 66 68 69 70 70 71 71 72 73 73 74 74 74 75 75 75 76 76 76 76 76 76	0 0 1 0 0 0 0 1 2 6 18 30 39 45 51 55 60 64 68 71 72 73 74 74 75 75 76 76 76	0 0 0 1 0 0 1 1 1 4 12 25 33 39 44 48 51 54 56 59 61 63 65 67 68 69 70 71 71	0 0 1 1 0 0 1 1 2 5 15 28 36 42 48 52 56 59 62 65 67 68 70 71 72 72 73 74 74	0 0 0 3 8 20 31 37 45 55 63 67 68 69 70 71 71 71 72 72 72 73 73 73 73 74 74 74 74

Table 5.2.1/3: pH values of the test preparations on day 0 and 28

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1 Inoculum Control R2 Procedure Control Test item R1 Test item R2 Toxicity Control	7.4 7.4 7.4 7.4 7.4 7.4	7.6 7.6 8.1 7.4 7.4 7.2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 74% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110.

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 20 and 21 ºC for 28 days.

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

The test item attained 74% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 76% biodegradation was attained.

Description of key information

OECD Guideline 301F, GLP, Key study, validity 1:

74% biodegradation after 28 days, within the 10-day window.

The substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

To assess the ready biodegradability of the registered substance, one experimental study is available.

This study, from Covance, 2019, assessed as the key study (klimisch 1), was performed on the registered substance according to OECD Guideline 301F and EU Method C.4-D with GLP compliance. The substance, at a concentration of 100 mg/L, was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 20 and 21°C for 28 days. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes. The test substance attained 74% biodegradation after 28 days and satisfied the 10 -day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.