Alfalfa, 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:

From 25 May to 22 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

deviations did not impact the validity of the study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in refrigerator in the original container
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: a primary stock solution was prepared for test item by dissolving 0.0312 g of extract in 3.9 mL of the 6-L solution of oxygen-rich water (that contained the mineral media solution). The extract was dissolved after an approximate 30-second sonication.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: nominal test concentration of 5 mg/L
- Final preparation of a solid: not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge was obtained from the Wareham Wastewater Treatment Facility, Wareham, Massachusetts, which treats primarily domestic sewage. Approximately 16 L of activated sludge was collected on 23 May 2018
- Preparation of inoculum for exposure: activated sludge was passed through a 2-mm stainless steel sieve and then centrifuged at 1000 rpm for 10 minutes. The supernatant (or secondary effluent) was kept and the solids further processed and stored. The resultant secondary effluent was filtered through coarse filter paper. Following filtration, the first 200 mL of secondary effluent was discarded. The next 500 mL of secondary effluent was retained and aerated in an environmental chamber set to maintain a temperature of 22 ± 2°C. The inoculum was used two days after preparation.

Duration of test (contact time):

28 d

Initial conc.:

5 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

A total of 72 biological oxygen demand (BOD) bottles were prepared for the blank control, inoculum control, reference substance, and test substance: eighteen bottles per treatment, two each for day 0 and three per sampling interval (i.e., days 7, 14, 21, and 28). The filled BOD bottles were maintained in a dark environmental chamber set to maintain a temperature of 22 ± 2°C.

Reference substance:

benzoic acid, sodium salt

Test performance:

The oxygen depletion of blank controls was <0.3 mg O2/L, thus meeting the requirement of =1.5 mg O2/L after 28 days. Biodegradation in the sodium benzoate test solutions was 77.6% of theoretical by day 7, thus meeting the validity criterion of reaching >=60% biodegradation within 28 days. Biodegradation in the sodium benzoate test solutions was 92.0% on day 28, which indicated that the inoculum contained a viable microbial population and that the test procedures used during the study were adequate.

Key result

Parameter:

% degradation (O2 consumption)

Value:

64.5

Sampling time:

28 d

Details on results:

The temperature of the environmental chamber ranged from 20 to 21°C throughout the study. Mean dissolved oxygen concentrations in the Alfalfa, ext. test solutions ranged from 8.8 mg O2/L on day 0 to 6.3 mg O2/L on day 28.
Mean dissolved oxygen concentrations in the blank control solutions generally remained stable and were 8.8 mg O2/L on day 0 and day 28. Mean dissolved oxygen concentrations in the inoculum control solutions generally remained stable and were 8.9 mg O2/L on day 0 and 8.8 mg O2/L on day 28. The BOD values of Alfalfa, ext. were 0.373, 0.433, 0.453, and 0.487 after 7, 14, 21, and 28 days, respectively.
Biodegradation of 64.5% was observed for Alfalfa, ext. on test day 28. Since Alfalfa is made of multiple components, according to the guideline, a 10- or 14-day window requirement “should not be used to interpret the results of the test” due to the possibility of sequential biodegradation instead of simultaneous biodegradation often seen in test substances that are mixtures.

Key result

Parameter:

ThOD

Value:

0.755 mg O2/g test mat.

Results with reference substance:

Mean dissolved oxygen concentrations for the sodium benzoate solutions decreased from 8.8 mg O2/L on day 0 to 1.0 mg O2/L on day 28.

See attached additional information on results.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In the Closed Bottle Test conducted according to the OECD test guideline 301D and to GLP, the ready biodegradability potential of Alfalfa, ext. was determined at 64.5% on Day 28, therefore, Alfalfa, ext. can be classified as readily biodegradable.

Executive summary:

The ready biodegradability potential of Alfalfa, ext. was determined in Closed Bottle Test conducted according to the OECD test guideline 301D and to GLP.

A total of 72 biological oxygen demand (BOD) bottles were prepared for the blank control, inoculum control, reference substance, and test substance: eighteen bottles per treatment, two each for day 0 and three per sampling interval (i.e., days 7, 14, 21, and 28). The filled BOD bottles were maintained in a dark environmental chamber set to maintain a temperature of 22 ± 2°C.

Mean dissolved oxygen concentrations in the Alfalfa, ext. test solutions ranged from 8.8 mg O₂/L on day 0 to 6.3 mg O₂/L on day 28. The mean dissolved oxygen concentrations for the sodium benzoate solutions decreased from 8.8 mg O₂/L on day 0 to 1.0 mg O₂/L on day 28. Mean dissolved oxygen concentrations in the inoculum control solutions remained generally stable and were 8.9 and 8.8 mg O₂/L on day 0 and day 28, respectively.

Biodegradation of 64.5% of theoretical was observed for Alfalfa, ext. by day 28.

Biodegradation in the sodium benzoate solutions was 77.6% of theoretical by day 7, thus meeting the validity criterion of reaching 60% or greater biodegradation within 28 days. Biodegradation in the sodium benzoate solutions was 92.0% on day 28, which indicated that the inoculum contained a viable microbial population and that the test procedures used during the study were adequate.

Alfalfa, ext. can be classified as readily biodegradable under the conditions of this test since it achieved >=60% biodegradation within 28 days.

Description of key information

Biodegradation: 64.5% at 28 days

Additional information

The ready biodegradability potential of Alfalfa, ext. was determined in Closed Bottle Test conducted according to the OECD test guideline 301D and to GLP.

A total of 72 biological oxygen demand (BOD) bottles were prepared for the blank control, inoculum control, reference substance, and test substance: eighteen bottles per treatment, two each for day 0 and three per sampling interval (i.e., days 7, 14, 21, and 28). The filled BOD bottles were maintained in a dark environmental chamber set to maintain a temperature of 22 ± 2°C.

Mean dissolved oxygen concentrations in the Alfalfa, ext. test solutions ranged from 8.8 mg O₂/L on day 0 to 6.3 mg O₂/L on day 28. The mean dissolved oxygen concentrations for the sodium benzoate solutions (reference substance) decreased from 8.8 mg O₂/L on day 0 to 1.0 mg O₂/L on day 28. Mean dissolved oxygen concentrations in the inoculum control solutions remained generally stable and were 8.9 and 8.8 mg O₂/L on day 0 and day 28, respectively.

Biodegradation of 64.5% was observed for Alfalfa, ext. by day 28.

Biodegradation in the sodium benzoate solutions was 77.6% by day 7, thus meeting the validity criterion of reaching 60% or greater biodegradation within 28 days. Biodegradation in the sodium benzoate solutions was 92.0% on day 28, which indicated that the inoculum contained a viable microbial population and that the test procedures used during the study were adequate.

Alfalfa, ext. can be classified as readily biodegradable under the conditions of this test since it achieved >=60% biodegradation within 28 days.