Willow, Salix alba, ext.

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:

2019-03-26 to 2019-04-25 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Stability: Stable at room temperature
- Carbon content: 39.3% (according to information from the sponsor)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal wastewater treatment plant AZV Breisgauer Bucht. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600,000 inhabitant equivalents.
- Sampling date: 25 March 2019
- Dry solid content of activated sludge: 4.8 g/L; determined by weight measurements after drying at 105°C for 3.5 hours (mean of triplicate measurements).
- Preparation of inoculum for exposure: the activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and resuspending the sludge.
- Concentration of inoculum: 30 mg dry solids per litre

Duration of test (contact time):

28 d

Initial conc.:

76.2 - 76.5 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of mineral medium: Stock solutions (A) - (D) were prepared according to OECD Guideline 301 B. For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.
- Composition of CO2-absorption medium: 64.03 g NaOH was dissolved in 8000 mL deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined (IC = 2.738 mg/L).
- Addition of test item: the test item was added directly into the test vessels
- Test temperature: 20.8 – 22.7°C throughout the whole study
- Concentration of inoculum: 30 mg dry solids per litre
- Continuous darkness: no; diffuse light

TEST SYSTEM
- Culturing apparatus:
Gas wash bottles (2000 ml volume) with lateral connecting pieces for butyl rubber septa were used as reactors.
- Number of culture flasks/concentration: 3 reactors containing the test item; concentration of organic carbon: 20 mg/L
- Method used to create aerobic conditions: aerated by the passage of carbon dioxide-free air; the aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second); incubation under aerobic conditions for 28 days.
- Measuring equipment: IC-measurement with Total carbon analyzers TOC-L and TOC-5050A (with autosamplers)
- Test performed in closed vessels: yes, the system was sealed
- Details of trap for CO2: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH.

SAMPLING
- Sampling frequency: on the 3rd, 7th, 10th, 14th, 21st and 28th day
- Sampling method:
+ For each sampling 4 mL were taken from the first of two CO2-absorber flasks connected in line and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air.
+ On the 28th day, sampling of each reactor was performed to measure the CO2 dissolved in water as inorganic carbon (IC), through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringe.
+ The IC in the second CO2-absorber flasks, used as protective flasks, was determined on day 30.

CONTROL AND BLANK SYSTEM
- 3 reactors containing only inoculum (blank)
- 1 reactor containing test and reference item (toxicity control)
- 3 reactors containing the reference compound

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

89.4

Sampling time:

28 d

Remarks on result:

other: mean of 3 replicates

Parameter:

% degradation (CO2 evolution)

Value:

75.2

Sampling time:

7 d

Remarks on result:

other: mean of 3 replicates

Details on results:

Validity criteria:
+ The IC content in the test vessel before introducing the test item was less than 5% of the TOC introduced with the test item.
+ The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.
+ The difference of extremes of replicate values of the test item at the end of the test was less than 20%.
+ The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 7.
+ The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2.

The test is valid according to OECD Test Guideline 301 B (July 1992).

Results with reference substance:

See IUCLID section "Any other information on results incl. tables"

Table 1: Ultimate biodegradation after x days [% of ThCO2]
reactor	day	0	3	7	10	14	21	28	28 with IC reactor
15	Test flasks	0	54.2	75.8	80.8	87.5	89.1	94.1	91.7
16		0	54.5	76.8	80.8	85.5	86.1	91.2	88.2
17		0	49.1	73.1	78.5	83.3	85.0	91.2	88.2
mean		0	52.6	75.2	80.0	85.4	86.8	92.2	89.4
4	reference flasks	0	60.2	81.4	85.3	90.3	90.0	94.6	95.0
5		0	61.6	82.6	87.5	93.1	94.1	100.9	101.5
6		0	57.0	78.6	82.9	88.6	89.2	94.1	93.5
mean		0	59.6	80.9	85.2	90.7	91.1	96.5	96.7
	Toxicity control
18	Test item	0	51.5	70.4	74.9	80.8	81.3	84.1	82.9
	Reference item

Reference item: The reference compound sodium benzoate reached the pass level for ready biodegradability within seven days.

Toxicity control: The degradation extent in the toxicity control was 80.8% within 14 days.

Table 2: Mean CO2-evolution of blank flasks after x days
Day		0	3	7	10	14	21	28	28 with IC reactor
CO2-evolution [mg/l]		0	8.4	14.8	17.2	21.0	23.8	24.1	27.3

Inoculum blanks: The mean CO2-evolution of the blank flasks was 27.3 mg/L on day 28 with considering the IC in the liquid phase.

Test parameters

+ Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.192 mg/L) were found.

+ The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 15 ppm and was not considered in the data processing, because CO2 absorption from room air was its source

Validity criteria fulfilled:

yes

Remarks:

The test is valid according to OECD Test Guideline 301 B (July 1992). For details see IUCLID section "Details on results"

Interpretation of results:

readily biodegradable

Conclusions:

Based on a reliable CO2 Evolution Test according to OECD Guideline 301 B (adopted July 1992) the test item "White Willow Bark Extract" is readily biodegradable, fulfilling the 10-day window requirement.

Executive summary:

The test item "White Willow Bark Extract" was tested for ready biodegradability in a reliable CO2 Evolution Test according to OECD Guideline 301 B (adopted July 1992). All relevant data are given in the study report. Biodegradation extent (ThCO2) reached 89.4% (mean value of 3 replicates) within 28 days.

The 10 day-window requirement was met; the biodegradation was 75.2% on day 7.

All validity criteria according to OECD Guideline 301 B (adopted July 1992) are fulfilled.

Accordingly, the study is considered valid and ready biodegradability can reliably be concluded for the test item "White Willow Bark Extract".

Description of key information

Based on a reliable CO2 Evolution Test according to OECD Guideline 301 B (adopted July 1992) the test item "White Willow Bark Extract" is readily biodegradable, fulfilling the 10-day window requirement.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The test item"White Willow Bark Extract" was tested for ready biodegradability in a reliable CO2 Evolution Test according to OECD Guideline 301 B (adopted July 1992). All relevant data are given in the study report. Biodegradation extent (ThCO2) reached 89.4% (mean value of 3 replicates) within 28 days.

The 10 day-window requirement was met; the biodegradation was 75.2% on day 7.

All validity criteria according to OECD Guideline 301 B (adopted July 1992) are fulfilled.

Accordingly, the study is considered valid and ready biodegradability can reliably be concluded for the test item "White Willow Bark Extract".