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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:

01-02-2012 to 01-06-2012

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: non-GLP guideline study which is well documented. All relevant validity criteria were met. The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Enhanced screening tests were conducted utilising: the prolonged Closed Bottle tests inoculated with (i) acclimatized microorganisms from semi-continuous activated sludge (SCAS) ; (ii) acclimated river water exposed to the test item at a low concentration and/or compared with test results from those inoculated with (iii) unacclimated sludge and/or (iv) unacclimated river water

Deviations:

no

GLP compliance:

no

Remarks:

non-GLP guideline study which is well documented. All relevant validity criteria were met. The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: ambient temperature in the dark
- Other: colourless liquid

Oxygen conditions:

aerobic

Inoculum or test system:

other: acclimated sludge and/or acclimated river water (refer to 'details on inoculum' for further details)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Enhanced screening tests were conducted utilising: the prolonged Closed Bottle tests inoculated with (i) acclimatized microorganisms from semi-continuous activated sludge (SCAS) ; (ii) acclimated river water exposed to the test item at a low concentration and/or compared with test results from those inoculated with (iii) Unacclimated sludge and/or (iv) unacclimated river water
The source of inoculum, respectively was as follows:
(i) Acclimatized microorganisms from semi-continuous activated sludge (SCAS): Semi-Activated Sludge (SCAS) tests were performed according to Test Guidelines (OECD, 1981). Secondary activated sludge and primary settled sewage were obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater.
(ii) Acclimated river water: River water was sampled from the Rhine near Heveadorp, The Netherlands and used for semi-continuous exposure. One third of the river water was replaced weekly by freshly collected river water.
(iii) Unacclimated sludge: Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. 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. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992 : Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotoxicol. Environmen. Saf. 24, 319-327).
(iv) Unacclimated river water: River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days before use to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions:
(i) Acclimatized microorganisms from semi-continuous activated sludge (SCAS): See ‘preparation of inoculum for exposure’. Adapted sludge from the SCAS unit was used upon sampling.
(ii) Acclimated river water: Used upon sampling, with one third of the river water was replaced weekly by freshly collected river water.
(iii) Unacclimated sludge: Aerated for 7 days before use
(iv) Unacclimated river water: Aerated for 7 days before use
- Storage length: up to 7 days
- Preparation of inoculum for exposure:
(i) Acclimatized microorganisms from semi-continuous activated sludge (SCAS): The primary settled sewage was collected weekly and stored at -20°C until required. 150 mL of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit. The tests were performed in 150 mL SCAS units. At the start the SCAS test, a unit was filled with 150 mL of activated sludge and the aeration was started. After 23 hours the aeration was stopped, and the sludge was allowed to settle for 45 minutes. After settling 100 mL of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml of a test item stock suspension were added to the respective test units. Stock suspensions of test item samples (1.0 g/L) and Tween 80 (1.0 g/L) in water was prepared by ultrasonic dispersion at 200 W for 5 minutes using a Vibra-cell. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test. Adapted sludge from the SCAS unit was used upon sampling.
(ii) Acclimated river water: Semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water. The test item(s) (liquid) were directly added with the help of silica gel. The concentration of the test item spiked to the river water was be 100 μg/L. One third of the river water was replaced weekly by freshly collected river water. The flasks were agitated and incubated at a temperature of 20±1°C.
(iii) Unacclimated sludge: 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. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
(iv) Unacclimated river water: The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating.
The closed bottle tests were conducted according OECD TG 301D, nutrients were added to either a liter of deionized water or river water: 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 omitted from the medium to prevent nitrification.
- Pretreatment: N/A
- Concentration of sludge: N/A
- Initial cell/biomass concentration: N/A
- Water filtered: no. Deionized water was used for preparing test medium. River water particles were removed by sedimentation. Then spiked with nutrients.
- Type and size of filter used, if any: N/A

Duration of test (contact time):

>= 28 - <= 112 d

Initial conc.:

2 mg/L

Based on:

test mat.

Remarks:

(i) Acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Initial conc.:

100 µg/L

Based on:

test mat.

Remarks:

(ii) Acclimated river water

Initial conc.:

2 mg/L

Based on:

test mat.

Remarks:

(iii) Unacclimated sludge

Initial conc.:

2 mg/L

Based on:

test mat.

Remarks:

(iv) Unacclimated river water

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The closed bottle tests were conducted according OECD TG 301D, nutrients were added to either a liter of deionized water or river water: 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 omitted from the medium to prevent nitrification.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used):N/A
- Test temperature: for (i), (ii) and (iii) and (iv) 22 to 24°C
- pH: For (i) SCAS unit effluent 6.0 to 7.5 and for (ii) 7.9 to 8.3 within 56 days for (iii) and (iv) not reported
- pH adjusted: no
- CEC (meq/100 g): not measured
- Aeration of dilution water: 7 days before use
- Suspended solids concentration: See “Preparation of inoculum for exposure”, as applicable.
- Continuous darkness: yes
- Other: Ammonium chloride was not added to the media to prevent nitrification. Administration of test item for (i) and (ii) was accomplished by dosing 0.04 g of the test item on 10 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 item. The serum flask was closed with a screw top with aluminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with the respective test item were added to the test bottles. The resulting concentration of test item in the bottles was 2.0 mg/L. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. Administration of the test item for (iii) and (iv) was accomplished by dosing 0.03 g of test item on 20 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 with aluminium foil and the content was mixed vigorously. Subsequently 0.4 g of silica gel dosed with test item was added to the test bottles. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. For (i), (ii), (iii) and (iv): use was made of 3 bottles containing only inoculum, and 3 bottles containing inoculum and test item administered using silica gel. 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 closed and incubated in the dark at 23±1°C in the dark.

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: Use was made of 3 bottles containing only inoculum, 3 bottles containing inoculum, test item and silica gel
- Method used to create aerobic conditions: 7 days of aeration before use
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a Eutech 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: No.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: 0, 7, 14, 21, and 28 plus day 42, 56, 84 and 112 days.
- Sampling method: Triplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28 plus day 42, 56, 84 and 112. 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).
- Sterility check if applicable: N/A
- Sample storage before analysis: ambient temperature, in the dark
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: inoculum only
- Abiotic sterile control: N/A
- Toxicity control: No.
- Other: N/A

STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test item, and reference item (where applicable) were calculated from their molecular formulae and molecular weights as follows.

ThODNH3(mgO2 /mg)= 16(2C+12 (H-Cl-3N)+3S+2(0.5*P)+(0.5*Na-O))/ MW

Oxygen consumptionn (mg/L) by test item = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles with and without silica gel n-days after the start of the test.

Mt or a is the mean oxygen concentration in the bottles containing the test item (t) or the reference compound (a), n-days after the start of the test.
The BOD mg/mg of the test item and reference item (if applicable) was calculated by dividing the oxygen consumption by the concentration of the test item and reference item in the closed bottle, respectively.

Calculation of the biodegradation percentages:
The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD).

Parameter:

% degradation (O2 consumption)

Remarks:

(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Value:

1

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Value:

4

Sampling time:

42 d

Parameter:

% degradation (O2 consumption)

Remarks:

(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Value:

13

Sampling time:

56 d

Parameter:

% degradation (O2 consumption)

Remarks:

(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Value:

42

Sampling time:

84 d

Parameter:

% degradation (O2 consumption)

Remarks:

(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)

Value:

44

Sampling time:

112 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(ii) acclimated river water

Value:

48

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(ii) acclimated river water

Value:

61

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(ii) acclimated river water

Value:

67

Sampling time:

42 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iii) unacclimated sludge

Value:

0

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iii) unacclimated sludge

Value:

1

Sampling time:

56 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iii) unacclimated sludge

Value:

45

Sampling time:

112 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iv) unacclimated river water

Value:

0

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iv) unacclimated river water

Value:

20

Sampling time:

56 d

Parameter:

% degradation (O2 consumption)

Remarks:

(iv) unacclimated river water

Value:

59

Sampling time:

112 d

Table 1.0 : Details on results

Test item concentration	Inoculum	Biodegradation at day
		7	14	21	28	42	56	84	112
2 mg/L	(i) acclimatized microorganisms from semi-continuous activated sludge (SCAS)	0	1	1	1	4	13	42	44
100 µg/L	(ii) acclimated river water: subjected to semi-continuous preexposure	3	22	48	61	67	-	-	-
2 mg/L	(iii) unacclimated sludge	-1	-2	-2	-1	-2	1	21	45
2 mg/L	(iv) unacclimated river water	-3	-3	-2	0	6	20	55	59
2 mg/L	GLP OECD TG 301D study (2012) (v) unacclimated river water	-2	-2	0	3	8	24 #1	64 #2

#1: Information from GLP study for comparative purposes

#2: measured at 60 days

#3: measured at 100 days

The study was to determine if potential persistence is not the result of toxic effects of the test item and/or of prescribed low inoculation size of the test. To this end, prolonged Closed Bottle tests inoculated with acclimatized microorganisms from semi-continuous activated sludge (SCAS) were performed. Tests inoculated with river water exposed to the test item at a low concentration were also carried out. Specifically, semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water (Rhine). The test items (liquid) were directly added with the help of silica gel. The concentration of the test item spiked to the river water was 100 μg/L. One third of the river water was replaced weekly by freshly collected river water.

A limited variation of the biodegradation percentages at the end of the tests was observed demonstrating that acclimatization did not result in enrichment of additional microorganisms, which might be able to degrade the substance completely. Shortening of the lag period does show increase of the number of competent microorganisms already present in the unacclimated inocula. With the test item: biodegradation percentages in excess of 60% were reached with both acclimated and unacclimated inocula. As a result of the preexposure short (no) lag periods were observed.

Acclimatization of river water compared to activated sludge does seem to give better results. The test item do inhibit the endogenous respiration. Toxic effects of the test item may also cause a delay of the onset of the biodegradation. This inhibition was not detected with acclimated microorganisms. In conclusion, lag periods were not found with preexposed microorganisms (acclimatisation) especially with river water. Exposure at low concentrations of river water is therefore a method to show non persistence of test item.

Validity criteria fulfilled:

yes

Interpretation of results:

other: See "conclusions". The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Conclusions:

The study was to determine if potential persistence is not the result of toxic effects of the test item and/or of prescribed low inoculation size of the test. To this end, prolonged Closed Bottle tests inoculated with acclimatized microorganisms from semi-continuous activated sludge (SCAS) were performed. Tests inoculated with river water exposed to the test item at a low concentration were also carried out. Specifically, semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water (Rhine). The test item (liquid) were directly added with the help of silica gel. The concentration of the test item spiked to the river water was 100 μg/L. One third of the river water was replaced weekly by freshly collected river water. With the test item: biodegradation percentages in excess of 60% were reached with both acclimated and unacclimated inocula. As a result of the preexposure short (no) lag periods were observed. With acclimated river water and at 100 μg/L test item concentration, the % degradation was 61% at 28 days and 67% at 42 days, respectively. The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D guideline. Enhanced screening tests were conducted utilising: the prolonged Closed Bottle tests inoculated with (i) acclimatized microorganisms from semi-continuous activated sludge (SCAS) ; (ii) acclimated river water exposed to the test item at a low concentration and/or compared with test results from those inoculated with (iii) Unacclimated sludge and/or (iv) unacclimated river water. The test systems were prepared respectively, as follows: (i) The primary settled sewage was collected weekly and stored at -20°C until required. 150 mL of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit. The tests were performed in 150 mL SCAS units. At the start the SCAS test, a unit was filled with 150 mL of activated sludge and the aeration was started. After 23 hours the aeration was stopped, and the sludge was allowed to settle for 45 minutes. After settling 100 mL of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml of a test item stock suspension were added to the respective test units. Stock suspensions of test item samples (1.0 g/L) and Tween 80 (1.0 g/L) in water was prepared by ultrasonic dispersion at 200 W for 5 minutes using a Vibra-cell. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test. Adapted sludge from the SCAS unit was used upon sampling. (ii) Semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water. The test item(s) (liquid) were directly added with the help of silica gel. The concentration of the test item spiked to the river water was 100 μg/L. One third of the river water was replaced weekly by freshly collected river water. The flasks were agitated and incubated at a temperature of 20±1°C. (iii) 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. The sludge was diluted in the BOD bottles. (iv) The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating. Within (i) through (iv), the closed bottle tests were conducted according to OECD TG 301D, nutrients were added to either a liter of deionized water or river water: 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 omitted from the medium to prevent nitrification. Administration of test item for (i) and (ii) was accomplished by dosing 0.04 g of the test item on 10 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 item. The serum flask was closed with a screw top with aluminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with the respective test item were added to the test bottles. The resulting concentration of test item in the bottles was 2.0 mg/L. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. Administration of the test item for (iii) and (iv) was accomplished by dosing 0.03 g of test item on 20 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 with aluminium foil and the content was mixed vigorously. Subsequently 0.4 g of silica gel dosed with test item was added to the test bottles. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. For (i), (ii), (iii) and (iv): use was made of 3 bottles containing only inoculum, and 3 bottles containing inoculum and test item administered using silica gel. 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 closed and incubated in the dark at 23±1°C in the dark. As a result of the preexposure short (no) lag periods were observed. With acclimatized microorganisms from semi-continuous activated sludge (SCAS) and at 2 mg/L test item concentration, the % degradation was 1% at 28 days, 4% at 42 days, 13% at 56 days, 42% at 84 days and/or 44% at 112 days. With acclimated river water and at 100 μg/L test item concentration, the % degradation was 61% at 28 days and 67% at 42 days, respectively.

The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-10-2011 to 29-01-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2010 ; signature: March 2010

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: ambient temperature in the dark
- Other: colourless liquid

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Rhine near Heavedrop, The Netherlands (14-10-2011). The nearest sewage treatment plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Not reported, assumed ambient
- Storage length: 8 days
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating. river water used in the Closed Bottle test was spiked per liter of water with 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.
- Pretreatment: N/A
- Concentration of sludge: N/A
- Initial cell/biomass concentration: N/A
- Water filtered: no
- Type and size of filter used, if any: N/A

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: River water spiked with 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 per litre.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used):N/A
- Test temperature: 22 - 24 °C
- pH: 8.2 (starting pH) and 8.2 on day 28 in controls
- pH adjusted: no
- CEC (meq/100 g): not measured
- Aeration of dilution water: 7 days before use
- Suspended solids concentration: N/A
- Continuous darkness: yes
- Other: Ammonium chloride was not added to the river water to prevent nitrification.

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and test substance (2.0 mg/L), and 6 bottles containing river water and sodium acetate (6.7 mg/L).
- Method used to create aerobic conditions: 7 days of aeration before use
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a Eutech 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: No.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: 0, 7, 14, 21, and 28 plus day 42, 60 and 100 days.
- Sampling method: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28 plus day 42, 60 and 100.
- Sterility check if applicable: N/A
- Sample storage before analysis: ambient temperature, in the dark
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: river water only and river water and silica gel
- Abiotic sterile control: N/A
- Toxicity control: Sodium acetate
- Other: N/A

STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights as follows.

ThODNH3(mgO2 /mg)= 16(2C+12 (H-Cl-3N)+3S+2(0.5*P)+(0.5*Na-O))/ MW

Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles with and without silica gel n-days after the start of the test.

Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.
The BOD mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages:
The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD).

Reference substance:

acetic acid, sodium salt

Remarks:

6.7 mg/L

Test performance:

1. The endogenous respiration was 1.5 mg/L at day 28 (i.e. < 30 mgO2/L after 28 days)
2. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled.
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels)
4. Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.
5. Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test item in the Closed Bottle test was not determined because possible toxicity of the test item to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test item could not be excluded.

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

24

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Value:

64

Sampling time:

100 d

Results with reference substance:

Sodium Acetate attained 83% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mean biodegradation was 3% at day 28, 24% at day 60 and 64 % at day 100 (within the enhanced biodegradation screening test).

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The test item at a concentration of 2 mg/L was exposed to river water which was spiked with nutrients, in sealed BOD vessels and in the dark at 23°C ± 1°C for 28 days and then extension of the exposure for up to 100 days in an enhancement to the test method. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28, plus 42, 60 and 100. Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 1.5 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels). Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 3%. Under the conditions of the study, test item is not considered to be readily biodegradable.In the prolonged phase of the Closed Bottle test, the test item was biodegraded by 24% at day 60 and 64% at day 100. The biodegradation found demonstrates that this test item should be classified as inherently biodegradable. The biodegradation percentage of 64% reached at day 100 offers evidence that the test item is ultimately (completely) biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 3% (28 -days), 24% (60 -days) and 64% (100 -days), OECD TG 301D, enhanced closed bottle test, 2012

Supporting study: mean biodegradation (n=3) was 61% (28 -days) and 67% (28 -days) using acclimated river water at 100 μg/L test item concentration. The acclimated river water was prepared by semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water (Rhine). The test items (liquid) were directly added with the help of silica gel. One third of the river water was replaced weekly by freshly collected river water. OECD TG 301D - enhanced biodegradation screening test, non-GLP, 2012

The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.

Key value for chemical safety assessment

Type of water:

freshwater

Additional information

Key study: OECD TG 301D, 2012 : The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The test item at a concentration of 2 mg/L was exposed to river water which was spiked with nutrients, in sealed BOD vessels and in the dark at 23°C ± 1°C for 28 days and then extension of the exposure for up to 100 days in an enhancement to the test method. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28, plus 42, 60 and 100. Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 1.5 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels). Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 3%. Under the conditions of the study, test item is not considered to be readily biodegradable. In the prolonged phase of the Closed Bottle test, the test item was biodegraded by 24% at day 60 and 64% at day 100. The biodegradation found demonstrates that this test item should be classified as inherently biodegradable. The biodegradation percentage of 64% reached at day 100 offers evidence that the test item is ultimately (completely) biodegradable.

 

Supporting Study: OECD TG 301D, 2012: The ready biodegradability test was carried out according to OECD TG 301D guideline. Enhanced screening tests were conducted utilising: the prolonged Closed Bottle tests inoculated with (i) acclimatized microorganisms from semi-continuous activated sludge (SCAS) ; (ii) acclimated river water exposed to the test item at a low concentration and/or compared with test results from those inoculated with (iii) Unacclimated sludge and/or (iv) unacclimated river water. The test systems were prepared respectively, as follows: (i) The primary settled sewage was collected weekly and stored at -20°C until required. 150 mL of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit. The tests were performed in 150 mL SCAS units. At the start the SCAS test, a unit was filled with 150 mL of activated sludge and the aeration was started. After 23 hours the aeration was stopped, and the sludge was allowed to settle for 45 minutes. After settling 100 mL of the supernatant liquor was withdrawn from the tap. Subsequently, 95 mL of primary settled sewage and 5 ml of a test item stock suspension were added to the respective test units. Stock suspensions of test item samples (1.0 g/L) and Tween 80 (1.0 g/L) in water was prepared by ultrasonic dispersion at 200 W for 5 minutes using a Vibra-cell. Aeration was started again and continued for 23 hours. The above fill and draw procedure was repeated 6 times per week throughout the test. Adapted sludge from the SCAS unit was used upon sampling. (ii) Semi-continuous preexposure was carried out in closed 5 L serum flasks filled with 5 L of river water. The test item(s) (liquid) were directly added with the help of silica gel. The concentration of the test item spiked to the river water was 100 μg/L. One third of the river water was replaced weekly by freshly collected river water. The flasks were agitated and incubated at a temperature of 20±1°C. (iii) 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. The sludge was diluted in the BOD bottles. (iv) The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating. Within (i) through (iv), the closed bottle tests were conducted according to OECD TG 301D, nutrients were added to either a liter of deionized water or river water: 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 omitted from the medium to prevent nitrification. Administration of test item for (i) and (ii) was accomplished by dosing 0.04 g of the test item on 10 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 item. The serum flask was closed with a screw top with aluminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with the respective test item were added to the test bottles. The resulting concentration of test item in the bottles was 2.0 mg/L. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. Administration of the test item for (iii) and (iv) was accomplished by dosing 0.03 g of test item on 20 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 with aluminium foil and the content was mixed vigorously. Subsequently 0.4 g of silica gel dosed with test item was added to the test bottles. The tests were performed in 0.3 L BOD (biological oxygen demand) bottles with glass stoppers. For (i), (ii), (iii) and (iv): use was made of 3 bottles containing only inoculum, and 3 bottles containing inoculum and test item administered using silica gel. 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 closed and incubated in the dark at 23±1°C in the dark. As a result of the preexposure short (no) lag periods were observed. With acclimatized microorganisms from semi-continuous activated sludge (SCAS) and at 2 mg/L test item concentration, the % degradation was 1% at 28 days, 4% at 42 days, 13% at 56 days, 42% at 84 days and/or 44% at 112 days. With acclimated river water and at 100 μg/L test item concentration, the % degradation was 61% at 28 days and 67% at 42 days, respectively.

The information is added as supporting evidence of potential for ultimate (complete) biodegradation in the environment.