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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:

03-10-2019 to 03-02-2020

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

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: December 2019

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): River water sampled from Rhine near Heavedrop, The Netherlands (26-09-2019). 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: 7 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 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification.
- 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 from the Rhine spiked with 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51mg 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 (Actual: 22.4 to 22.9°C).
- pH: 7.8 (starting pH) and 8.0 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 silica gel and test item (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 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.
- 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 0.75 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 for the flasks containing test item at end of 14-day window and on day 28.
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.0 to 8.1 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. 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:

83

Sampling time:

28 d

Remarks on result:

other: 14-day window met

Details on results:

- The test item was biodegraded by 83% at day 28 in the Closed Bottle test
- Over 60% biodegradation was achieved within a period of 9 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation

Results with reference substance:

Sodium Acetate attained 83% 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 item	Acetate	Test item	Acetate
0	0.00	0.00	0	0
7	0.45	3.65	8	70
14	2.85	4.35	50	83
21	4.60		81
28	4.75		83

- The test item was biodegraded by 83% at day 28 in the Closed Bottle test

- Over 60% biodegradation was achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. Actual start day 7 = 10% degradation and end day 16 = 60% degradation)

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean biodegradation in duplicate was 83 % at day 28 the 10-day window (14 days for the Closed Bottle test) was met.

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. 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 and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.75 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10- or 14-day window and on day 28. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.0 to 8.1 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 83% and the 10-day window (14-days for the Closed Bottle test) was met. Under the conditions of the study, test item is considered to be readily biodegradable.

Description of key information

Biodegradation: readily biodegradable, mean biodegradation 83 % (28 -days; 14-day window met), OECD TG 301D, 2020

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Key study: OECD TG 301D, 2020 : 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. 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 and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 0.75 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10- or 14-day window and on day 28. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.0 to 8.1 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 83% and the 10-day window (14-days for the Closed Bottle test) was met. Under the conditions of the study, test item is considered to be readily biodegradable.