Juniper, Juniperus mexicana, ext., isomerized, acetylated

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:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline screening study; non-GLP. Minor deviations from guideline not considered to impact the overall reliability of the study. No positive or toxicity controls utilised.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301) were introduced: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation; the study was extended using established inherent biodegradation determination literature methodology. A second series of test was carried out with bottles (triplicate) which were sacrificed at day 28, 42, and 56 (standard procedure described in the OECD test guideline) plus 84 and 112 days.

Deviations:

yes

Remarks:

minor deviations not considered to have an impact on the reliability of the study.

Qualifier:

according to guideline

Guideline:

other: van Ginkel and Stroo (1992) Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotox Environ Saf 24 319-327.

Deviations:

not applicable

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301) were introduced: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation; the study was extended using established inherent biodegradation determination literature methodology. A second series of test was carried out with bottles (triplicate) which were sacrificed at day 28, 42, and 56 (standard procedure described in the OECD test guideline) plus 84 and 112 days.

GLP compliance:

no

Remarks:

non-GLP; well documented study report. Conclusion: ‘not biodegradable’. The GLP study to OECD TG 301D would only be required to conclude if the test item were to be concluded as ‘readily biodegradable’ or ‘inherently biodegradable’.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 1. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. 2. River water was sampled from the Rhine near Heveadorp, The Netherlands. This river water was aerated for 7 days before use and particles were removed by sedimentation. The river water spiked with mineral salts was used undiluted.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week.
- Concentration of sludge: The sludge was diluted in the BOD bottles to 2 mg DW/L

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4•2H2O, 22.5 mg MgSO4•7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3•6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Accurate administration of the water-insoluble test item was accomplished with a solid stock of 3.0 mg substance/g silica gel. The silica gel dosed with test substance was subsequently added to the respective test bottles. An aqueous stock emulsion of the test item with a concentration of 1.0 g/L were prepared with Tween 80 1.0 g/L. To this end, approximately 80 mL deionized water with the surfactant and test substance were ultrasonically dispersed at 400 W for a few minutes of using a Vibra-cell (Recognised supplier, The Netherlands). Volumes of 0.3 mL of this emulsion were added to bottles already filled with river water. Control bottles with Tween 80 or silica gel were included.
- Test temperature: Temperatures were within the prescribed temperature range of 22 to 24°C.
- pH: The pH of the media (activated sludge) was 7.3 and 8.0 (river water) at the start of the test. The pH of the medium at day 28 was 7.2 (activated sludge) and 7.9 (river water).
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 3 bottles containing only inoculum, 3 bottles containing test substance with inoculum
- Measuring equipment: See details on analytical methods
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: No.

SAMPLING
- Sampling frequency: Biodegradation was measured by following the course of the oxygen decrease in the bottles with 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.
- Sampling method: Analyses of the dissolved oxygen concentration
- Sterility check if applicable: No.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Toxicity control: No.

Reference substance:

not required

Key result

Parameter:

% degradation (O2 consumption)

Value:

41

Sampling time:

28 d

Remarks on result:

other: Sludge: Silica Method

Parameter:

% degradation (O2 consumption)

Value:

42

Sampling time:

84 d

Remarks on result:

other: Sludge; Silica Method

Key result

Parameter:

% degradation (O2 consumption)

Value:

38

Sampling time:

28 d

Remarks on result:

other: River water; Slica Method

Parameter:

% degradation (O2 consumption)

Value:

40

Sampling time:

84 d

Remarks on result:

other: River water; Silica Method

- Theoretical oxygen demand (ThOD): The calculated theoretical oxygen demand (ThOD) with the brutto formula of an identified constituent 3.0 g/g. Another identified constituent had a ThOD 3.3 g/g. The authors acknowledged that the ThOD 3.0 g/g is regarded as an estimate (references reported in the present study report).

- Toxicity: Inhibition was not detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance. The test substance is therefore considered as not toxic to microorganisms present in both inocula.

- Biodegradability: The test substance was degraded approximately 40% at day 28 in the prolonged Closed Bottle test with activated sludge and river water as inoculum (Table). The biodegradation did not increase after day 28. A ThOD of <2 g/g is required to obtain biodegradation percentages of >60. Such a low ThOD for the test item is considered unlikely. Additional screening tests were carried out to exclude that the incomplete biodegradation found was due to loss of the test item during opening and closure of the bottles necessary to determine the oxygen concentration. To this end the bottles were sacrificed. A comparable result was obtained (presented in the present study report). The test item is partially degraded in the prolonged Closed Bottle test. The test substance cannot be classified as (readily) biodegradable.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was biodegraded by ca. 40 % at day 28 in the Closed Bottle test with activated sludge and river water as inoculum

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D guideline, consistent with EU and ISO Test Guidelines but but without parallel positive or toxicity controls in a limited screening study using activated sludge and river water as inocula and minor modifications according to Van Ginkel and Stroo, and OECD (1992). The study was extended in accordance with established inherent biodegradation determination literature methodology. Accurate administration of the water-insoluble test item was accomplished with a solid stock of 3.0 mg test item/g silica gel. The silica gel dosed with test item was subsequently added to the respective test bottles. An aqueous stock emulsion of the test item with a concentration of 1.0 g/L were prepared with Tween 80 1.0 g/L. To this end, approximately 80 mL deionized water with the surfactant and test item were ultrasonically dispersed at 400 W for a few minutes of using a Vibra-cell. Volumes of 0.3 mL of this emulsion were added to bottles already filled with river water. Control bottles with Tween 80 or silica gel were included. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only inoculum, and 3 bottles containing test item and inoculum. The concentration of the test item in the bottles was 3.0 mg/L. 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 bottles were incubated in the dark at a temperature of 23˚C. The biodegradation was measured by following the course of the oxygen decrease in the bottles with 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). A second series of test was carried out with bottles (triplicate) which were sacrificed at day 28, 42, and 56 (standard procedure described in the OECD test guideline). Inhibition was not detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test item. The test item is therefore considered as not toxic to the microorganisms present in both inocula. The pH of the media was 7.3±0.1 (activated sludge) and 8.0±0.1 (river water) at the start of the test. The pH at day 28 was 7.2±0.2 (activated sludge) and 7.9±0.1 (river water). Temperatures ranged from 22 to 24°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The calculated theoretical oxygen demand (ThOD) with the brutto formula of an identified constituent 3.0 g/g. Another identified constituent had a ThOD 3.3 g/g. The authors acknowledged that the ThOD 3.0 g/g is regarded as an estimate (references reported in the present study report). The test item was degraded approximately 40% at day 28 in the prolonged Closed Bottle test with activated sludge and river water as inoculum. The biodegradation did not increase after day 28. A ThOD of <2 g/g is required to obtain biodegradation percentages of >60%. Such a low ThOD for the test item is considered unlikely. Additional screening tests were carried out to exclude that the incomplete biodegradation found was due to loss of the test item during opening and closure of the bottles necessary to determine the oxygen concentration. To this end the bottles were sacrificed. A comparable result was obtained The test item is partially degraded in the prolonged Closed Bottle test. The test item cannot be classified as (readily) biodegradable. Under the conditions of this study, the test item degraded approximately 40% at 28 days. The biodegradation did not increase after day 28. The test item can therefore not be classified as readily biodegradable.

Description of key information

Biodegradation: ca. 40% (28-days) ; not readily biodegradable, OECD TG 301D, 2015

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

Key study : OECD TG 301D, 2015 :  The ready biodegradability test was carried out according to OECD TG 301D guideline, consistent with EU and ISO Test Guidelines but but without parallel positive or toxicity controls in a limited screening study using activated sludge and river water as inocula and minor modifications according to Van Ginkel and Stroo, and OECD (1992). The study was extended in accordance with established inherent biodegradation determination literature methodology. Accurate administration of the water-insoluble test item was accomplished with a solid stock of 3.0 mg test item/g silica gel. The silica gel dosed with test item was subsequently added to the respective test bottles. An aqueous stock emulsion of the test item with a concentration of 1.0 g/L were prepared with Tween 80 1.0 g/L. To this end, approximately 80 mL deionized water with the surfactant and test item were ultrasonically dispersed at 400 W for a few minutes of using a Vibra-cell. Volumes of 0.3 mL of this emulsion were added to bottles already filled with river water. Control bottles with Tween 80 or silica gel were included. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only inoculum, and 3 bottles containing test item and inoculum. The concentration of the test item in the bottles was 3.0 mg/L. 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 bottles were incubated in the dark at a temperature of 23˚C. The biodegradation was measured by following the course of the oxygen decrease in the bottles with 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). A second series of test was carried out with bottles (triplicate) which were sacrificed at day 28, 42, and 56 (standard procedure described in the OECD test guideline). Inhibition was not detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test item. The test item is therefore considered as not toxic to the microorganisms present in both inocula. The pH of the media was 7.3±0.1 (activated sludge) and 8.0±0.1 (river water) at the start of the test. The pH at day 28 was 7.2±0.2 (activated sludge) and 7.9±0.1 (river water). Temperatures ranged from 22 to 24°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The calculated theoretical oxygen demand (ThOD) with the brutto formula of an identified constituent 3.0 g/g. Another identified constituent had a ThOD 3.3 g/g. The authors acknowledged that the ThOD 3.0 g/g is regarded as an estimate (references reported in the present study report). The test item was degraded approximately 40% at day 28 in the prolonged Closed Bottle test with activated sludge and river water as inoculum. The biodegradation did not increase after day 28. A ThOD of <2 g/g is required to obtain biodegradation percentages of >60%. Such a low ThOD for the test item is considered unlikely. Additional screening tests were carried out to exclude that the incomplete biodegradation found was due to loss of the test item during opening and closure of the bottles necessary to determine the oxygen concentration. To this end the bottles were sacrificed. A comparable result was obtained The test item is partially degraded in the prolonged Closed Bottle test. The test item cannot be classified as (readily) biodegradable. Under the conditions of this study, the test item degraded approximately 40% at 28 days. The biodegradation did not increase after day 28. The test item can therefore not be classified as readily biodegradable.