Octahydro-2H-1-benzopyran-2-one

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:

31 May 2016 - 30 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Version / remarks:

1994

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

a) ammonium chloride was omitted from the medium to prevent O consumption due to nitrification (omission does not result in N limitation as shown by the biodeg of the ref. compound), b) river water instead of effluent/extract/mixture was used as inoculum

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate issued on October 6, 2014

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Remarks:

relatively low number of microorganisms

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
River water was sampled from the Rhine near Heveadorp, The Netherlands (26-05-2016). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992).
River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

- Preparation of inoculum for exposure:
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: direct addition of nutrients to the river water used (see field 'details on inoculum')
- Test temperature: 22-24°C
- pH: 8.0 at test start
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: low (not further specified)
- Continuous darkness: yes
- Other: Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top and the content was mixed vigorously. Subsequently 0.2 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Subsequently the bottles were filled with nutrient medium with inoculum and closed.
Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

TEST SYSTEM
- Culturing apparatus: not further specified; 0.3 L BOD bottles with glass stoppers
- Number of culture flasks/concentration: 10 bottles (only river water), 10 bottles (river water and blank silica gel), 10 bottles (river water and silica gel with test substance), 6 bottles (river water and sodium acetate)
- Measuring equipment:
* The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW TrioXmatic EO 200) and meter (WTW OXI 530).
* The pH was measured using a Eutech Cyberscan pH11 pH meter.
* The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels due to significant volatility of test substance: yes
- Other: All bottles were completely filled without air bubbles.

SAMPLING
- Sampling frequency: days 0, 7, 14, 21 and 28
- Sampling method: The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at specified sampling days.
- Sterility check if applicable: no
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: river water; river water with blank silica gel
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:

Reference substance:

acetic acid, sodium salt

Remarks:

anhydrous, purity > 99.0%; concentration in test: 6.7 mg/L

Key result

Parameter:

% degradation (O2 consumption)

Value:

71

Sampling time:

28 d

Remarks on result:

other: Over 60% biodegradation was achieved in a period of approximately 7 days immediately following the attainment of 10% biodegradation at day 15

Remarks:

the substance therefore fulfilled the time window criterion for ready biodegradable substances under guideline OECD 301

Details on results:

Theoretical oxygen demand (ThOD):
The calculated theoretical oxygen demand (ThOD) of bicyclononalactone (multiconstituent) is 2.4 mg/mg.
The ThOD of sodium acetate is 0.8 mg/mg.

Toxicity:
Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (see Table I attached in the field 'Attached background material').
Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions:
The pH of the media was 8.0 at the start of the test. The pH of the media at day 28 was 8.0 (test and controls).
Temperatures were within the prescribed temperature range of 22 to 24°C.

Results with reference substance:

89% biodegradation at day 14

Oxygen consumption and percentages biodegradation are presented in Table II in the field 'Attached background material'.

A graphical representation of the results is provided in the field 'Illustration (picture/graph)'.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 1.1 mg/L at day 28 (Table I).

Furthermore, the differences of the replicate values at day 28 were less than 20%.

Biodegradation of the reference compound, sodium acetate, at day 14 was 89% (Table II and Figure).

Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The result tables are included below.

Table IDissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Ocs	Ot	Oc	Oa
0	8.7	8.7	8.7	8.7
	8.7	8.7	8.7	8.7
Mean (M)	8.7	8.7	8.7	8.7
7	7.9	8.0	8.1	3.9
	8.0	8.0	8.1	3.7
Mean (M)	8.0	8.0	8.1	3.8
14	7.9	7.5	7.9	3.1
	7.9	7.6	7.9	3.0
Mean (M)	7.9	7.6	7.9	3.1
21	7.5	4.7	7.6
	7.6	4.9	7.7
Mean (M)	7.6	4.8	7.7
28	7.4	3.9	7.5
	7.5	4.2	7.6
Mean (M)	7.5	4.1	7.6

O_cs        River water with nutrients and silica gel.

O_t         River water with nutrients, test material (2.0 mg/L) and silica gel.

O_c         River water with nutrients.

O_a          River water with nutrients and sodium acetate (6.7 mg/L).

Table IIOxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.0	4.3	0	80
14	0.3	4.8	6	89
21	2.8		58
28	3.4		71

Validity criteria fulfilled:

yes

Remarks:

see field 'Any other information on results'

Interpretation of results:

readily biodegradable

Conclusions:

The substance showed 71% biodegradation after 28 days in river water, meeting the criteria for the 10-day time window in a Closed Bottle Test, following the procedures of the OECD TG 301 D.

Executive summary:

The ready biodegradability of the substance was investigated in a study conducted in river water following the procedures of OECD TG 301 D (Closed Bottle Test) and GLP. The concentration tested was 2.0 mg/L substance in river water. After 28 days, the substance was biodegraded for 71% and the 10 -day window was met. Therefore the substance is considered readily biodegradable.

Description of key information

In the key study: The ready biodegradability of the substance was investigated in a study conducted in river water following the procedures of OECD TG 301 D (Closed Bottle Test) and GLP. The concentration tested was 2.0 mg/L substance in river water. After 28 days, the substance was biodegraded for 71% and the 10 -day window was met. Therefore the substance is considered readily biodegradable.

Other available information: In another study at 10 mg/l absence of biodegradation was observed possibly due to toxicity (OECD TG 301B). A third study was not considered because sludge adaptation was used.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable