Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

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:

25 Jan 2022 - 24 Mar 2022

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

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634

Version / remarks:

"Water Quality - Preparation and treatment of poorly
water-soluble organic compounds for the subsequent evaluation of their biodegradability
in an aqueous medium", (2018)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Laboratory culture: no
- Storage conditions: continuous aeration
- Preparation of inoculum for exposure: Before use, sludge was coarsely sieved (1 mm2 mesh) and homogenized using a blender on medium speed for approximately 2 minutes.
After treatment, concentration of suspended solids (SS) was determined to be 4.47 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum
at an amount of 2.2 mL per liter of mineral medium, leading to a SS concentration of 10 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

12 mg/L

Based on:

TOC

Initial conc.:

16 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to OECD Guideline 301B
- Test temperature: 22 - 23°C
- pH: 7.4 - 7.8
- pH adjusted: no
- Suspended solids concentration: 10 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2-liter amber glass bottles
- Number of culture flasks: Test suspension 2 vessels
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20 %) and nitrogen (ca. 80 %) was passed through a vessel at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min)
- Measuring equipment: CO2 produced in each test vessel reacted with barium hydroxide in the gas scrubbing vessel and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
- Details of trap for CO2: Three CO2-absorbers (vessels filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit aeration line of each test vessel.


SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until Day 29, for inoculum blank and test material. Titrations for procedural and toxicity control were made over a period over a period of at least 14 days.
- Sampling method: Each time the CO2-absorber nearest to the test vessel was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test vessel. A new CO2-absorber was placed at the far end of the series. On the penultimate day, pH of respective test suspensions was measured and 1 mL of concentrated HCl (37 %, Merck) was added to the inoculum blank and test suspension. Vessels were aerated overnight to drive off CO2. Final titration was made on Day 15 (procedural and toxicity control) and on Day 29 (remaining vessels).

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 vessels
- Abiotic sterile control: no
- Toxicity control: yes, 1 vessel
- Procedural control: ontaining procedural control material and inoculum, 1 vessel

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

74 - 78

Sampling time:

28 d

Details on results:

The test substance was degraded between 74% and 78% (2 replicates) after 28 d. Since the test material consists of a mixture of structurally similar molecules, the 10-day window was not applicable. Therefore, the test substance is readily biodegradable.

Results with reference substance:

78% within 14 d, 60% pass level was reached.

Table 1
CO2 Production and Biodegradation of Test Material (Vessel A)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation1) (%)
	Blank (mean)	Vessel A
2 5 8 12 15 19 23 292) 292) 292)	45.50 40.62 42.69 42.98 43.75 43.22 43.91 42.47 45.12 47.94	44.55 31.76 29.50 30.00 35.73 34.45 38.52 37.53 44.11 47.36	0.95 8.86 13.19 12.98 8.02 8.77 5.39 4.94 1.01 0.58	1.0 9.7 14.5 14.3 8.8 9.6 5.9 5.4 1.1 0.6	1.0 10.8 25.3 39.6 48.4 58.0 64.0 69.4 70.5 71.1	1 12 28 44 53 64 70 76 78 78

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test material: 90.9 mg CO2/2 L.

2): CO2 measured on Day 29 is actually part of CO2 production of Day 28, since microbial activity was ended on Day 28 by addition of concentrated HCl

Table 2
CO2Production and Biodegradation of Test Material (Vessel B)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation1) (%)
	Blank (mean)	Vessel B
2 5 8 12 15 19 23 292) 292) 292)	45.50 40.62 42.69 42.98 43.75 43.22 43.91 42.47 45.12 47.94	44.70 29.50 29.76 32.26 36.51 37.41 36.97 38.21 43.89 47.48	0.80 11.12 12.93 10.72 7.24 5.81 6.94 4.26 1.23 0.46	0.9 12.2 14.2 11.8 8.0 6.4 7.6 4.7 1.4 0.5	0.9 13.1 27.3 39.1 47.1 53.5 61.1 65.8 67.1 67.6	1 14 30 43 51 58 67 72 73 74

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test material: 91.5 mg CO2/2 L.

2): CO2 measured on Day 29 is actually part of CO2 production of Day 28, since microbial activity was ended on Day 28 by addition of concentrated HCl

Table 3
CO2 Production and Biodegradation in Toxicity Control

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation1) (%)
	Blank (mean)	Toxicity control
2 5 8 12 152)	45.50 40.62 42.69 42.98 43.75	29.93 18.46 21.53 25.44 23.13	15.57 22.16 21.16 17.54 20.62	17.1 24.4 23.3 19.3 22.7	17.1 41.5 64.8 84.1 106.7	10 24 37 48 60

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test material: 91.5 mg CO2/2 L.

2): CO2 measured on Day 29 is actually part of CO2 production of Day 28, since microbial activity was ended on Day 28 by addition of concentrated HCl

Table 4: Validity criteria for OECD 301B

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	4%	yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	78%	yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	60% degradation based on ThCO2	yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.	Test medium was prepared in tap-water purified by reverse osmosis, therefore IC was less than 5% of TC.	yes
The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	25.4 mg CO2/L	yes

Validity criteria fulfilled:

yes

Remarks:

Please refer to Table 4 at "Any other information on results incl. tables"

Interpretation of results:

readily biodegradable

Description of key information

Readily biodegradable: 74 - 78% after 28 d (CO2 evolution, OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

One experimental study is available investigating the ready biodegradability of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol. The study was conducted according to OECD 301B (GLP) using non-adapted activated sludge as inoculum. 16 mg of the test substance were used and incubated for 28 d. The CO2 produced was absorbed in Ba(OH)2 solution and analysed. On day 28 concentrated hydrochloric acid was added to each vessel in order to drive off any inorganic carbon formed. The final measurement was done on day 29 but refers to the degradation of the substance after 28 d. After 28 d the test substance was degraded to 74 - 78% (two replicates). Thus, the substance is concluded to be readily biodegradable according to the OECD criteria.
The toxicity control attained 60% biodegradation after 14 d confirming that the test substance is not inhibitory to STP microorganisms.