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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-08-2019 to 25-09-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: August 2017 ; signature: September 2017
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, 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.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.4 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L.
- 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.:
2 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 litre of deionized water; 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Solubilising agent (type and concentration if used): None.
- Test temperature: Temperatures were within the prescribed temperature range of 22 to 24°C (Actual: 22.5 to 22.9°C).
- pH: The pH of the media (activated sludge) was 7.2 at the start of the test. The pH of the medium at day 28 was 7.2 (controls and control with silica gel) and 7.1 (test)
- pH adjusted: No.
- Aeration of dilution water: Not reported
- Suspended solids concentration: 2 mg/L dry weight
- 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: 2 bottles containing only inoculum per time point (10 bottles total) ; 2 bottles containing only inoculum and silica gel per time point (10 bottles total) ; 2 bottles containing test item, silica gel with inoculum per time point (10 bottles total) ; 2 bottles containing test item and reference item and inoculum per time point (up to 10 bottles total)
- Measuring equipment: See details on analytical methods. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The pH was measured using a 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.

SAMPLING
- Sampling frequency: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
- 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.
- Abiotic sterile control: No.
- Toxicity control: Yes (where applicable)
- Other: Positive reference control (Sodium Acetate)
Reference substance:
acetic acid, sodium salt
Remarks:
6.7 mg/L
Test performance:
1. The endogenous respiration was 0.65 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 on day 28.
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and test item vessels)
4. Sodium Acetate attained 85% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.
5. Oxygen concentrations were > 0.5 mg/L in all bottles during the test period
6. 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 at day 7 was not detected.
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
28 d
Details on results:
- The test item was biodegraded by 63% at day 28 in the Closed Bottle test
- Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation.
- The test item did not exhibit a percentage biodegradation exceeding the pass level of 60% within 10-day window. However, the test item is a multi-constituent substance composed of - two structurally similar constituents and therefore the 10-day window is not applied (UN GHS 4th edition, 2011 and further revisions and OECD, 2006). Further technical details based on growth-linked processes and biodegradation kinetics (lag period, growth rate and yield) within ready biodegradability tests, along with test item limited water solubility and anticipated slow test item desorption and dissolution rates which make the time-window concept inapplicable, are provided in the full study report with citations.
Results with reference substance:
Sodium Acetate attained 85% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Table 1. Oxygen consumption (mg/L) and the percentages biodegradation of the test item (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

 

Test substance

Acetate

Test item

Acetate

0

0.00

0.00

0

0

7

0.40

4.10

7

78

14

1.55

4.45

27

85

21

2.35

 

41

 

28

3.65

 

63

 

- The test item was biodegraded by 63% at day 28 in the Closed Bottle test

- Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation.

- The test item did not exhibit a percentage biodegradation exceeding the pass level of 60% within 10-day window. However, the test item is a multi-constituent substance composed of - two structurally similar constituents and therefore the 10-day window is not applied (UN GHS 4th edition, 2011 and further revisions and OECD, 2006). Further technical details based on growth-linked processes and biodegradation kinetics (lag period, growth rate and yield) within ready biodegradability tests, along with test item limited water solubility and anticipated slow test item desorption and dissolution rates which make the time-window concept inapplicable, are provided in the full study report with citations.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The mean biodegradation in duplicate was 63 % at day 28.
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 activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage in sealed BOD vessels and in the dark at 22°C ± 1°C for 28 days. The inoculum concentration was 2 mg/L DW in the BOD bottles. 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. Control solutions with inoculum, inoculum and silica gel, the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.65 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item on day 28. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and test item vessels). Sodium Acetate attained 85% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. Oxygen concentrations were > 0.5 mg/L in all bottles during the test period. The mean biodegradation for the test item at 28 days was 63%. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because the test item is a substance consisting of two isomers and biodegradation kinetics are considered to be affected by limited bioavailability (OECD, 2006). Under the conditions of the study, test item is considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
not specified
GLP compliance:
no
Remarks:
The study was conducted prior to 01 June 2008.
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): A mixed population of activated sewage sludge micro-organisms (Test System) was obtained from the secondary treatment stage of the sewage treatment plant at Villette (Geneva, Switzerland)), which treats predominantly domestic sewage.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was filtered through a coarse filter paper. The filtrate was centrifuged/washed twice with deionised water, resuspended at pH 6.5 and then oxygenated (stream of pure oxygen) for 6 h to reduce the carbon load (organic and inorganic). The final inoculum concentration in test bottles was 20 mg/L dry weight activated sludge solids.
- Concentration of sludge: The sludge was diluted in the test bottles to 20 mg DW/L.
Duration of test (contact time):
28 d
Initial conc.:
25 mg/L
Based on:
test mat.
Remarks:
18.75 mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 10 mL of Solution A [KH2PO4: 8.50 g/L; K2HPO4: 21.75g/L; Na2HPO4,2H2O: 33.40 g/L; NH4Cl: 0.50 g/L]; 1 mL of Solution B [CaCl2,2H2O: 27.50 g/L]; 1 mL of Solution C [MgSO4,7H2O: 22.50 g/L]; 1 mL of Solution D [FeCl3,6H2O: 0.25 g/L] and purified water diluted to 1 L.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 24 ±2 °C
- pH: Starting pH 7.4
- pH adjusted: N
- Aeration of dilution water: Not reported
- Suspended solids concentration: 20 mg/L dry weight
- Continuous darkness: No. The test was conducted in diffuse light.

TEST SYSTEM
- Culturing apparatus: 5500 mL glass flasks with continuous stirring (fill volume ca. 2000 mL)
- Number of culture flasks/concentration: In duplicate (test item); In duplicate (Inoculum blank); duplicate flasks (reference item) ; N/A (Abiotic Sterile Control and toxicity control)
- Method used to create aerobic conditions: Gas tight syringe/teflon septum.
- Measuring equipment: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (10 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered on a 0.2 m filter prior to analysis. Analysis conducted in triplicate.

SAMPLING
- Sampling frequency: Every three or four days to day 28.
- Sampling method: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (10 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered on a 0.2 m filter prior to analysis. Analysis conducted in triplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: No.
- Other: Positive reference control (Methyl dihydrojasmonate).
Reference substance:
other: methyl dihydrojasmonate
Remarks:
25.2 mg/L or 17.37 mg C/L
Parameter:
% degradation (inorg. C analysis)
Value:
82.19
Sampling time:
28 d
Details on results:
The 10-day window criteria may have been met, but due to TOC analyser failure a reading was not taken and therefore was considered not met. Although the 10-d window need not apply.

Applicant assessment indicates: The test item was biodegraded by 82.19% at day 28 in the CO2 evolution test. The multi-constituent test substance is therefore classified as readily biodegradable. The 10-day window that can be applied to Ready Biodegradability Tests, where 60% (O2 consumption or CO2 evolution) or 70% (dissolved carbon generation) biodegradation must be achieved following the attainment of 10 % biodegradation, is not applied to multi-constituent substances consisting of structurally similar constituents (OECD, 2006).
References:
1. OECD (2006), Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation
2. GHS revision 4 (2011), Globally harmonized system of classification and labelling of chemicals (GHS). Chapter 4.1. Hazardous to the Aquatic Environment. United Nations, 2011
Results with reference substance:
The reference item attained 65% degradation at 5 days and 79.9% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item mean biodegradation in duplicate was 82.19 % at day 28. 10-d window does not apply to multi-constituent substances consisting of structurally similar constituents (OECD, 2006).
Executive summary:

The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline. The test item, at a concentration of 25 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 24°C ± 2°C for 28 days. The sludge was diluted in the test bottles to 20 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 and 28. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (10 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered on a 0.2 m filter prior to analysis. Analysis conducted in triplicate. The reference item attained 65% degradation after 5 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 82.19%. It was considered the 10-day window was not met due to technical issue with the TOC analyser. However, the 10-d window does not apply to multi-constituent substances consisting of structurally similar constituents (OECD, 2006). Under the conditions of the study, test item is considered as readily biodegradable.

Description of key information

Weight of evidence : Readily Biodegradable, 2019

1. Biodegradation: readily biodegradable, mean biodegradation 63% (28-days, 14-day window not met). 14-day window not applicable to multi-constituent substances consisting of structurally similar constituents (OECD, 2006) and/or when biodegradation kinetics are affected by limited bioavailability; OECD TG 301D, 2019

2. Biodegradation: readily biodegradable, mean biodegradation 82.19% (28-days, 10-day window not met). 10-day window not applicable to multi-constituent substances consisting of structurally similar constituents (OECD, 2006); eq. or similar to OECD TG 310, 2003

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key Study : OECD TG 301D, 2019 : 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 activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage in sealed BOD vessels and in the dark at 22°C ± 1°C for 28 days. The inoculum concentration was 2 mg/L DW in the BOD bottles. 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. Control solutions with inoculum, inoculum and silica gel, the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.65 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item on day 28. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.1 to 7.2 for controls and test item vessels). Sodium Acetate attained 85% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. Oxygen concentrations were > 0.5 mg/L in all bottles during the test period. The mean biodegradation for the test item at 28 days was 63%. Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. The time window criterion should however not be applied because the test item is a substance consisting of two isomers and biodegradation kinetics are considered to be affected by limited bioavailability (OECD, 2006). Under the conditions of the study, test item is considered to be readily biodegradable.

Key Study : eq. or similar to OECD TG 310, 2003 : The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline. The test item, at a concentration of 25 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 24°C ± 2°C for 28 days. The sludge was diluted in the test bottles to 20 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 and 28. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (10 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered on a 0.2 m filter prior to analysis. Analysis conducted in triplicate. The reference item attained 65% degradation after 5 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 82.19%. It was considered the 10-day window was not met due to technical issue with the TOC analyser. However, the 10-d window does not apply to multi-constituent substances consisting of structurally similar constituents (OECD, 2006). Under the conditions of the study, test item is considered as readily biodegradable.