[No public or meaningful name is available]

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:

22 January 2018 to 06 December 2018

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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

-Purity: >95% (based on 1H and 13C NMR)
-Description: Amber Liquid
- Storage: room temperature, in the dark
- Carbon content: 73.3% (determined by lab; used for dosing)
75.2% (determined by Sponsor/Supplier)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste, supplemented with a filtered soil/sediment suspension prepare from fresh soil (Station Street), Taunton River sediment, and Weweantic River sediment (Wareham, MA)
- Method of cultivation: The sludge was passed through a 2-mm sieve, centrifuged at 1000 rpm for 10 minutes. The supernatant discarded and the sludge was washed with mineral medium and centrifuged, and the supernatant was discarded. The moisture content of the activated sludge was determined to be 96.00% and the percent solids was determined to be 4.00%. The soil and sediments were suspended in Weweantic River water by hand shaking and the suspension was filtered through glass wool to remove all solids.
- Preparation of inoculum for exposure: An inoculum solution with 15 mg suspended solids/mL was prepared, stirred with a Teflon magnetic stir bar at 22 ± 2 °C, and aerated until used. The sludge was used within 24 hours of collection.

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg C/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The aqueous mineral salts medium used for testing was prepared according to OECD Guideline 301B.
- Test temperature: 20.0 to 21.1 °C
- pH: The pH of the medium used for test vessels and toxicity control upon preparation was 7.43 and required no adjustment. The pH of the pH check vessel on day 0 was measured to be 7.22. The pH values of the study vessels ranged from 7.30 to 7.44 at the end of the study on Day 28.
- DOC content of the reagent grade water prior to use: 0.58 ppm (Shimadzu TOC-L/ASI-L/SSM analyzer)
- Aeration of dilution water: purged with CO2-free air
- Suspended solids concentration: 30 mg solids/L (inoculum)
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 4-L glass bottle, each containing 3 L, fitted with a rubber stopper into which one stainless steel needle with a Luer-Lok connection and two pieces of glass tubing were inserted. A stainless-steel needle was extended through the stopper into the test solution serving as a sampling port for solution samples. The outlet port of each system was connected to two CO2 effluent gas traps, the first containing 200 mL of 0.2 N potassium hydroxide (KOH) and the second trap containing 100 mL of 0.2 N KOH.
- Number of culture flasks/concentration: Six; test substance (2); inoculum blanks (2), sodium benzoate procedural control (1), and toxicity control (1). An additional vessel was established for pH measurements and sampling for TIC and total carbon (TC) on Day 0; this vessel was not attached to KOH traps following pH, TC, and TIC determination.
- Method used to create aerobic conditions: continuous aeration; CO2 free air was pumped under positive pressure through a hydration flask before entering the test system.

SAMPLING
- Sampling frequency: Days 1, 2, 5, 7, 9, 12, 14, 19, 23, and 26
- Sampling method: 7-mL sample was removed from the first KOH carbon dioxide trap on each test system and analyzed for CO2 evolution.
- Terminal sampling: On Day 28, 1 mL of concentrated hydrochloric acid was added to each test vessel following terminal pH measurements. After overnight aeration to drive any residual inorganic carbon from the test vessels, 7-mL samples for analysis of CO2 evolution were removed from the first and second traps.
- Sterility check: none
- Sample storage before analysis: none

CONTROL AND BLANK SYSTEM
- Inoculum blank: two flasks containing mineral medium and inoculum
- Procedure control: one flask containing mineral medium, inoculum, reference substance (10 mg C/L)
- Toxicity control: one flask containing mineral medium, inoculum, test and reference substance (10 mg C/L of each)
- Abiotic sterile control: none

Reference substance:

benzoic acid, sodium salt

Remarks:

10 mg C/L

Test performance:

- The inorganic carbon content of the test material suspension in mineral medium was less than 5% of the total organic carbon, meeting the validity criterion of the test.
- The toxicity control produced a cumulative net percent CO2 evolved of 34.98% by Day 14, demonstrating that the test material is not toxic to the inoculum as defined by OECD Guideline 301B (i.e., <25% on Day 14 is considered toxic).
- The cumulative net percent CO2 evolved from the procedural control was 64.42% of theoretical by Day 14. This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and test system integrity.
- The difference between replicates at the plateau and at the end of the test did not exceed 20%.
- The control blanks did not exceed 40 mg evolved CO2 per liter.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

4.59

Sampling time:

28 d

Details on results:

The mean cumulative CO2 production in the test material, toxicity control, and blank control test vessels on Day 28 were 24.16, 51.11 and 22.47 mg/L, respectively. The cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, for the test material and toxicity control were calculated to be 4.59 and 39.05%, respectively.

Results with reference substance:

The mean cumulative CO2 production in the procedural control vessels on Day 28 were 46.75 mg/L. The cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, for the procedural control was 66.19%.

Cumulative net percent CO2 evolved (% ultimate biodegradation) 

Vessel	Replicate	Biodegradation (% CO2 Evolved) by Study Day
		1	2	5	7	9	12	14	19	23	26	28
1	Test Suspension A	4.76	6.63	7.82	6.10	7.26	6.81	5.12	9.04	8.21	10.71	6.87
2	Test Suspension B	5.23	2.55	2.54	-0.81	0.89	2.50	2.98	3.25	3.65	4.50	2.30
	Mean	4.99	4.59	5.18	2.65	4.07	4.65	4.05	6.14	5.93	7.60	4.59
	Std. Dev.	0.33	2.89	3.74	4.89	4.50	3.05	1.52	4.09	3.22	4.39	3.23
5	Procedural Control	5.40	21.49	47.34	57.72	64.11	65.47	64.42	66.47	66.67	65.01	66.19
6	Toxicity Control	2.82	13.46	32.57	31.39	35.26	36.91	34.98	31.60	39.05	39.79	39.05

 * The rounded values presented in this table were calculated based on unrounded experimental results.

 All values were corrected for mean inoculum blank CO2 production (vessels 3 and 4).

Biodegradation is calculated as (mg CO2 produced / mg TOC added x 3.667) x 100; where 3.667 is the molecular weight conversion factor for carbon to carbon dioxide (CO2).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Based on the extent of CO2 evolution during this study (4.59% on Day 28), the test material cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since 60% CO2 evolution was not achieved within a 10-day window of reaching 10% biodegradation.

Executive summary:

This study was performed to determine the potential biodegradation of the test material in an aerobic aqueous medium by the carbon dioxide evolution method following OECD Guideline 301B. The amount of carbon dioxide (CO2) released upon biodegradation of the test substance and a reference substance, sodium benzoate, was measured to assess the potential for ultimate biodegradation. Two blank controls (containing inoculum), a procedural control (containing inoculum and reference substance), and a toxicity control (containing inoculum, reference substance, and the test material) were established to account for background CO₂production, viability of the inoculum, and the toxicity of the test material, respectively. An additional vessel was established without traps for pH measurements and sampling for TIC and total carbon (TC) on Day 0. Test flasks were incubated at 22 +/- 2˚C with continuous aeration, in the dark for a period of 28 days. 

The total inorganic carbon measured in the KOH traps was used to calculate the cumulative CO2 evolved from the test vessels. The mean cumulative CO2 values from the test material, procedural control, toxicity control, and blank control test vessels at Day 28 were 24.16, 46.75, 51.11, and 22.47 mg/L, respectively. The cumulative net percent CO2 production (blank control values subtracted), or percent ultimate biodegradation, values for the test material, procedural control, and toxicity control were calculated to be 4.59, 66.19, and 39.05%, respectively. The cumulative net percent CO2 evolved from the procedural control was 64.42% of theoretical by Day 14, meeting the acceptance criteria of the test (reaching 60% or greater CO2 evolution within 14 days). This rapid biodegradation of sodium benzoate confirmed the presence of an active microbial population and test system integrity. The toxicity control produced a cumulative net CO2 percentage of 34.98% by Day 14, demonstrating that the test substance was not toxic to the inoculum as defined by OECD Guideline 301B.

Based on the extent of CO2 evolution during this study, the test material cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since it did not achieve 60% CO2 evolution within a 10-day window of reaching 10% biodegradation. 

Description of key information

Based on the extent of CO2 evolution during this study (4.59% on Day 28), the test substance cannot be classified as readily biodegradable by the criteria set forth in OECD Guideline 301B since 60% CO2 evolution was not achieved within a 10-day window of reaching 10% biodegradation. 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater