2-benzyl-4-methyl-4a,5,6,7,8,8a-hexahydro-4H-benzo[d][1,3]dioxine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Test dates: May. 02 to Jul. 12, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on: 11 January 2011; Signed on: 11 March 2011

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge: derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage
- Storage conditions: 20 ± 1°C.
- Storage length: 5 days
- Preparation of inoculum for exposure: The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 5 days at the test temperature in the mineral medium used for testing.

Duration of test (contact time):

28 d

Initial conc.:

2.5 mg/L

Based on:

ThOD

Remarks:

6.6 mg

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS

- Composition of medium:

Mineral stock solutions were prepared as follows:
Stock solution A:
Monopotassium dihydrogen orthophosphate, KH2PO4: 8.50 g/L
Dipotassium hydrogen orthophosphate, K2HPO4: 21.75 g/L
Disodium hydrogen orthophosphate, dihydrate, Na2HPO4 2H2O: 33.40 g/L
Anunonium chloride, NH4Cl: 0.50 g/L
Stock solution B:
Calcium chloride anhydrous, CaCl2: 27.50 g/L
Or Calcium chloride dihydrate, CaCl2 2H2O: 36.40 g/L
Stock solution C:
Magnesium sulphate heptahydrate, MgSO4 7H2O: 22.50 g/L
Stock solution D:
Iron (III) chloride hexahydrate, FeCl3 6H2O: 2.5 g/L
The mineral medium was prepared using 1 mL/L of each the stock solutions A, B, C and D. The mineral medium was strongly aerated for at least 20 minutes, and then allowed to stand for approximately 20 h at the test temperature before use.

- Test temperature: 20 ± 1 °C
- Aeration of mineral medium: at least 20 minutes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 250 mL BOD bottles
- Number of culture flasks/concentration: 2
- Measuring equipment: electrode


SAMPLING
- Sampling frequency: every 3-4 day throughout the 28-day incubation period.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 300 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
- Toxicity control: 300 mL of the mineral medium mineral medium were added with 6 mL of the test item aqueous stock solution, 100 µL of the Aniline stock solution and 0.6 mL of pre-conditioned inoculum.
- Reference group: A stock solution was prepared using 0.1219 g Aniline for 20 mL of water. 300 mL of mineral medium were added with 100 µL of the stock solution and 0.6 mL of pre-conditioned inoculum.

Reference substance:

aniline

Remarks:

Sigma Reference A9880 (> 99.0%), batch No.034K0126.

Test performance:

The test was terminated after 28 days of incubation.

The test was considered as valid:
- Mean oxygen uptake in the blank vessels < 1.5 mg/L on day 28,
- The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time,
- Differences of extremes of replicate values of the removal of the test item was less than 20 % of mean value at the end of test,
- Percentage biodegradation of the reference item (aniline) had reached the pass-level by day 14,
- In the toxic control series more than 25 % biodegradation had occurred on day 14.

Key result

Parameter:

% degradation (O2 consumption)

Value:

17.15

Sampling time:

28 d

Details on results:

The test was performed using a 2.43 mg/L test solution. In the test item treatment, the oxygen up-take was similar to that of the controls for the entire test period. At the end of test, analytical check of the test item in the test solutions and in the toxic control proved that the initial value still remained. Test item was thus considered as not easily biodegradable.

Results with reference substance:

Aniline: The beginning of the 10-day window was observed between day 6 and day 9 of the test penod The pass-level was reached on day 9: mean value for biodegradation was 72.7 %. The biodegradatlon stabilized at approximately 80% afterwards and the measurements were not continued after day 23.

Active Ingredient Content

The measured initial concentrations represented 96 - 107 % the nominal value (2.43 mg/L). The treatment application was considered as valid.

At the end of the 28-day test period, the measured concentrations still represented more than 90 % of the nominal value in the test item solutions and in the toxic control.

Table 5.2.1.1: Mean Measured concentrations of test item throughout the test period

Day		Test item (mg/L)	% of nominal value
Day 0	Test group	2.50	102.7
Day 28	Test group	2.54	104.4
	Toxic control	2.34	96.1

 

Oxygen Uptake and Biodegradation

The initial value for dissolved oxygen concentration was 8.37 mg/L of the control, 8.80 mg/L for the test item treatment, and 8.53 mg/L for the reference item treatment. The initial value was thus taken as mean from the three measured values: 8.57 mg/L.

The consumption of oxygen in the controls was less than 1.5 mg/L over the 28-day test period, as required.

For the test item treatment, the measured concentrations for dissolved oxygen were approximately the same as for the controls up to day 28 of the test period.

For the reference item treatment and for the toxic control, the difference with the controls was more than 1 mg/L up from day 9 of the test period.

 

Table 5.2.1.2: Calculated mean biodegradation for test item based on oxygen uptake, %

Day	Test item	Toxic control	Anilline (reference item)
0	0	0	0
3	7.02	4.14	1.43
6	10.42	5.98	1.74
9	11.65	34.49	72.65
12	11.27	42.06	72.65
16	14.12	43.55	76.43
19	12.66	41.88	84.21
23	15.12	48.13	82.27
28	17.13	45.31	-

 

Test item: The calculated values for biodegradation were less than 20% for the entire test penod Toxic control: Based on total ThOD, the biodegradation was more than 25 % up from day 9.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

For the test item the pass-level (60 %) was not reached within the 28-day observation period. Thus, test item should be regarded as not readily biodegradable according to this test.

Executive summary:

The biodegradation of the test substance was assessed according to the Closed-bottle test method.

The solution of the test substance (2.43 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely filled, closed bottles in the dark at constant temperature (20 +/- 1 °C).

Degradation was followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the theorical oxygen demand (ThOD).

Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge the potential for primary degradation over the test period.

The calculated values for biodegradation of the test substance were less than 20% for the entire test period (mean of 17.13%). Analytical measurement at the end of the test verified that the initial concentration of parent still remained. Therefore, the test substance was considered as not readily biodegradable (removal within 28 days < 20%).

 

Thus, test item should be regarded as not readily biodegradable according to this test.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 November to 28 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according 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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 12 - 13 November 2019 / signed on 09 december 2019

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 103.0 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.0 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992). The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar.
Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.9 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 9.105 CFU/L.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.41 mg Na2HPO4.2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2 2H2O, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification that is not related to the biodegradation of the test substance.
Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of 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 cap and the content was mixed vigorously until there were no more lumps in the silica gel, indicating homogeneous distribution of the test substance. Subsequently, 0.20 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. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel (0.2 g silica gel / bottle), 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease at day 42 and 60 using the bottles of day 28 and a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).
Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.8%; Batch n° BCCD4856

Preliminary study:

A non-GLP screening test was performed prior the GLP final test in order to determine the most suitable inoculum for the degradation of the test substance, in the Closed Bottle test.
Substance is classified not ready biodegradable based on the 21% biodegradation achieved at day 56 in the OECD 301D test using activated sludge as inoculum and 21% biodegradation achieved at day 56 with river water.
For the final GLP test it is recommended to use activated sludge as inoculum.

Parameter:

% degradation (O2 consumption)

Value:

27

Sampling time:

60 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

23

Sampling time:

28 d

Details on results:

Theoretical oxygen demand (ThOD)
The molecular formula of the known isomer constituents of the test substance was used as representative for all organics present in the test substance (assuming 100% purity of test substance). The theoretical oxygen demand (ThOD) of test substance calculated from this molecular formula and used to calculate the biodegradation percentages is 2.67 g oxygen/g test substance. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

Toxicity

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test is optional in the OECD guideline and was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition can be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance (higher oxygen concentration in bottles with test substance compared to the concentration in the control bottles). Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions

At the start of the test the pH of the media in the duplicate control, reference substance, control with silica gel, and test substance bottles was 7.0. The pH of the medium in the duplicate reference bottles measured at day 14 was 6.9. The pH of the medium in the duplicate bottles at day 60 was 7.1, 7.1 and 7.0 for the control, the control with silica gel and the test substance bottles, respectively. The temperature ranged from 22.4 to 22.9 °C which is within the prescribed temperature range of 20 to 24°C.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 0.80 mg/L at day 28 . Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 79%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.


Biodegradability

TEst item was biodegraded by 23% at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 27% at day 60. Biodegradation percentages below 20% suggest that there is only minor partial degradation of the test substance achieved in the (prolonged) Closed Bottle test. Test item is therefore regarded not biodegradable in the OECD 301D ready biodegradation screening test. The lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Test item is classified as not biodegradable in ready biodegradation screening test based on <20% biodegradation achieved in the OECD 301D test at day 60
The test item is considered as not readily biodegradable but showed primary inherent biodegradation with degradation rate of 27% in the OECD 301D test after 60 days.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 79%

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)	Oxygen concentration (mg/L)
	Mc	Ma	Mcs	Mt
0	9.00	9.00	9.00	9.00
	9.00	9.00	9.00	9.00
Mean (M)	9.00	9.00	9.00	9.00
7	8.6	4.7	8.3	8.5
	8.5	4.6	8.4	8.5
Mean (M)	8.55	4.65	8.35	8.50
14	8.3	4.3	8.2	7.4
	8.4	4.1	8.3	7.4
Mean (M)	8.35	4.20	8.25	7.40
21	8.4		8.2	6.8
	8.4		8.2	6.9
Mean (M)	8.4		8.2	6.85
28	8.2		8.0	6.7
	8.2		8.0	6.8
Mean (M)	8.2		8.00	6.75
42	8.1		7.9	6.4
	8.0		7.9	6.6
mean	8.05		7.90	6.50
60	8.0		7.8	6.5
	7.9		7.9	6.3
Mean	7.95		7.85	6.40

 

Mc = Mineral nutrient solution with only inoculum.

Mcs = Mineral nutrient solution with inoculum, and silica gel 

Mt = Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance) and silica gel

Ma = Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

 

 

Table 5.2.1/2: Oxygen 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	0.00	0.00	0	0
7	-0.15	3.90	-3	75
14	0.85	4.15	16	79
21	1.35		25
28	1.25		23
42	1.40		26
60	1.45		27

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was biodegraded by 23% at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 27% at day 60. Biodegradation percentages below 60% suggest partial degradation of the test substance. The lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
The test item is considered as not readily biodegradable but showed primary inherent
biodegradation with degradation rate of 27% in the OECD 301D test after 60 days.

Executive summary:

To assess the biotic degradation of the test item a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.
The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles, and incubated in the dark at 22.4 to 22.9 °C for 60 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance did cause a slight inhibition of the endogenous respiration of the inoculum at day 7 of the test and therefore may have hampered the biodegradation at the start of the test. The test item was biodegraded by 23% at day 28 in the Closed Bottle test. The test substance should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 27% at day 60. Biodegradation percentages below 60% suggest partial degradation of the test substance. The lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
The test item is considered as not readily biodegradable but showed primary inherent biodegradation with degradation rate of 27% in the OECD 301D test after 60 days. The test is valid as shown by an endogenous respiration of 0.80 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 79% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

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

23% biodegradation after 28 days and 27% biodegradation after 60 days, with activated sludge

Not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

To assess the biodegradation potential of the registered substance, two experimental studies are available. 

The most recent study (Nouryon, 2022), assessed as the key study, was performed on the registered substance to assess the ready biodegradability of the test item in an aerobic aqueous medium, according to OECD Guideline 301D, performed under GLP (with statement included in the report). The silica gel with test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles, and incubated in the dark at 22.4 to 22.9 °C for 60 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test item did cause a slight inhibition of the endogenous respiration of the inoculum at day 7 of the test and therefore may have hampered the biodegradation at the start of the test. The test item was biodegraded by 23% at day 28 in the Closed Bottle In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance was biodegraded by 27% at day 60. Biodegradation
percentages below 60% suggest partial degradation of the test substance. The lack of complete biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. The test item is considered as not readily biodegradable but showed primary inherent biodegradation with degradation rate of 27% in the OECD 301D test after 60 days. The test substance is classified as not biodegradable in ready biodegradation screening test based on <20% biodegradation achieved in the OECD 301D test at day 60. The test is valid.

The second study (Phytosafe, 2012) was also performed on the registered substance according to the closed-bottle test method. The solution of the test substance (2.43 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely filled, closed bottles in the dark at constant temperature (20 +/- 1 °C). Degradation was followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the theorical oxygen demand (ThOD).

Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge the potential for primary degradation over the test period. The calculated values for biodegradation of the test substance were less than 20% for the entire test period (mean of 17.13%). Analytical measurement at the end of the test verified that the initial concentration of parent still remained. Therefore, the test substance was considered as not readily biodegradable (removal within 28 days < 20%).

Thus, test item should be regarded as not readily biodegradable according to these test.