Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Jul - 02 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

EC Number:

817-761-7

Cas Number:

765293-81-0

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Test material form:

other: waxy paste

Details on test material:

Appearance: White to pale yellow waxy paste
Test item storage: At room temperature
Stable under storage conditions until: 30 May 2019 (retest date)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Source
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 3 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 8 mg/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

19 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

16 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test Concentration and Preparation of Test Solutions
Plandool-H was a white to pale yellow waxy paste (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. A sample of the test item was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma, Steinheim, Germany) as total carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as inorganic carbon (IC) standard. The Total Organic Carbon (TOC) content of the test item was determined to be 85.37%. The test item was tested in duplicate at a target concentration of 19 mg/L, corresponding to 16 mg TOC/L. No correction was made for the purity/composition of the test item.
On the day of testing weighed amounts of the test item were added to the 2 litre test bottles containing medium with microbial organisms and mineral components (test item bottle A: 38.3 mg; test item bottle B: 37.7 mg and toxicity control bottle: 38.4 mg). To this end, small watch glasses were used to transfer the weighed amounts of test item to the respective test bottles. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.

Testing Strategy and Experimental Design
Test Procedure and Conditions
Test duration: 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the procedure and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre brown coloured glass bottles
Milli- RO water: Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
Stock solutions of mineral components:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl, dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2.
B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
Illumination: The test media were excluded from light.
Preparation of Bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles).
Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

29 d

Remarks on result:

other: 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 HCl.

Any other information on results incl. tables

Acceptability of the Test

1. The reference item was biodegraded by at least 60% (actual result: 74%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20 (actual result:≤1%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (56.0 mg CO2 per 2 litres of medium, corresponding to 28.0 mg CO2/L).

4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

RESULTS

Theoretical CO2 Production

The ThCO2 of Plandool-H was calculated to be 3.13 mg CO2/mg.

The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation

CO2 Production in the Blank

Day	HCl (0.05 N) titrated (mL) for untreated 0.0125 M Ba(OH)2 solution	HCl (0.05 N) titrated (mL) for Blank (mean)	Produced CO2(mL HCl)	Produced CO2(mg)	Cumulative CO2(mg)
2	51.47	49.23	2.25	2.5	2.5
5	51.37	47.04	4.33	4.8	7.2
8	51.28	46.19	5.09	5.6	12.8
12	51.42	44.96	6.46	7.1	19.9
15	51.61	46.10	5.51	6.1	26.0
19	51.41	45.97	5.44	6.0	32.0
23	51.36	46.09	5.27	5.8	37.8
29 ^1)	51.25	43.03	8.23	9.0	46.8
29 ^1)	51.17	46.66	4.51	5.0	51.8
29 ^1)	51.79	47.97	3.82	4.2	56.0

¹⁾: 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 HCl.

CO2 Production and Percentage Biodegradation of the Reference Item

Day	HCl (0.05 N) titrated (mL) for untreated 0.0125 M Ba(OH)2solution	HCl (0.05 N) titrated (mL) for Procedure control	Produced CO2(mL HCl)	Produced CO2(mg)	Cumulative CO2(mg)	Biodegradation^1)Blank (%)
2	49.23	37.40	11.83	13.0	13.0	15
5	47.04	28.52	18.52	20.4	33.4	39
8	46.19	33.30	12.89	14.2	47.6	55
12	44.96	36.35	8.61	9.5	57.0	66
15^2)	46.10	40.26	5.84	6.4	63.5	74

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 85.8 mg CO₂/2L.

²⁾: CO2 measured on day 15 is actually part of CO2 production of day 14, since microbial activity was ended on day 14 by addition of HCl.

CO2 Production and Percentage Biodegradation of the Test Item (Bottle A)

Day	HCl (0.05 N) titrated (mL) for Blank (mean)	HCl (0.05 N) titrated (mL) for Bottle A	Produced CO2(mL HCl)	Produced CO2(mg)	Cumulative CO2(mg)	Biodegradation^1)(%)
2	49.23	49.26	0.00	0.0	0.0	0
5	47.04	47.28	0.00	0.0	0.0	0
8	46.19	46.59	0.00	0.0	0.0	0
12	44.96	45.80	0.00	0.0	0.0	0
15	46.10	46.62	0.00	0.0	0.0	0
19	45.97	45.61	0.36	0.4	0.4	0
23	46.09	45.57	0.52	0.6	1.0	1
29^2)	43.03	43.82	0.00	0.0	1.0	1
29^2)	46.66	46.73	0.00	0.0	1.0	1
29^2)	47.97	49.29	0.00	0.0	1.0	1

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 119.9 mg CO₂/2L.

²⁾: CO₂measured on day 29 is actually part of CO₂production of day 28, since microbial activity was ended on day 28 by addition of HCl.

CO2 Production and Percentage Biodegradation of the Test Item (Bottle B)

Day	HCl (0.05 N) titrated (mL) for Blank (mean)	HCl (0.05 N) titrated (mL) for Bottle B	Produced CO2(mL HCl)	Produced CO2(mg)	Cumulative CO2(mg)	Biodegradation^1)(%)
2	49.23	48.92	0.31	0.3	0.3	0
5	47.04	47.60	0.00	0.0	0.3	0
8	46.19	46.70	0.00	0.0	0.3	0
12	44.96	45.78	0.00	0.0	0.3	0
15	46.10	46.82	0.00	0.0	0.3	0
19	45.97	45.47	0.50	0.6	0.9	1
23	46.09	45.39	0.70	0.8	1.7	1
29^2)	43.03	43.26	0.00	0.0	1.7	1
29^2)	46.66	47.22	0.00	0.0	1.7	1
29^2)	47.97	48.96	0.00	0.0	1.7	1

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test item: 118.0 mg CO₂/2L.

²⁾: CO₂measured on day 29 is actually part of CO₂production of day 28, since microbial activity was ended on day 28 by addition of HCl.

CO2 Production and Percentage Biodegradation of the Toxicity Control

Day	HCl (0.05 N) titrated (mL) for Blank (mean)	HCl (0.05 N) titrated (mL) for Toxicity control	Produced CO2(mL HCl)	Produced CO2(mg)	Cumulative CO2(mg)	Biodegradation^1)(%)
2	49.23	40.79	8.44	9.3	9.3	5
5	47.04	26.97	20.07	22.1	31.4	15
8	46.19	36.31	9.88	10.9	42.2	21
12	44.96	39.14	5.82	6.4	48.6	24
15^2)	46.10	39.61	6.49	7.1	55.8	27

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test item and reference item: 206.0 mg CO₂/2L (ThCO₂test item: 120.2 mg CO₂/2L + ThCO₂sodium acetate: 85.8 mg CO₂/2L).

²⁾: CO₂measured on day 15 is actually part of CO₂production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Comparison of Biodegradation of the Test Item in Bottles A and B

Day	Biodegradation (%): Bottle A	Biodegradation (%): Bottle B	Biodegradation (%):Mean A and B	Biodegradation (%): ΔA-B^1)
2	0	0	0	0
5	0	0	0	0
8	0	0	0	0
12	0	0	0	0
15	0	0	0	0
19	0	1	1	1
23	1	1	1	0
29^2)	1	1	1	0
29^2)	1	1	1	0
29^2)	1	1	1	0

¹⁾: Absolute difference in biodegradation between bottles A and B

²⁾: Biodegradation is ended on day 28 by addition of HCl. Therefore, differences observed on day 29 are actually differences of day 28.

Note: Negative produced CO₂-values are expressed as 0.00 mL HCl.

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of Plandool-H (1% in both test vessels).

In the toxicity control, more than 25% biodegradation occurred within 14 days (27%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

Monitoring of Temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C. The pH values of the different test media are presented in the following table.

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.7→7.6^1	-	7.6
Blank control (B)	7.7^2	-	7.6
Procedure control	7.6	7.8	-
Plandool-H (A)	7.6	-	7.6
Plandool-H (B)	7.6	-	7.6
Toxicity control	7.6	7.8	-

¹: Adjusted using 1 M HCl (Merck, Darmstadt, Germany)

²: Value was recorded as 7.7 but not adjusted

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The substance is not readily biodegradable according to OECD criteria