Fatty acids, C18-unsatd., phosphates

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:

26th June 2017-17th September

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:

In addition, the following ISO standard was followed: 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", (1995).

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: 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.
- Pretreatment and inoculum concentration: The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Duration of test (contact time):

28 d

Initial conc.:

21 mg/L

Based on:

TOC

Remarks:

The test item was tested in duplicate at a target concentration of 21 mg/L, corresponding to 12 mg TOC/L.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- Solubilising agent (type and concentration if used): No
- Test temperature: 22.0 – 23.1 °C
- pH: 7-6-7.8. At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
- Suspended solids concentration: The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge.
- Other: During the test period, the test media were aerated and stirred continuously.

TEST SYSTEM
-Test duration: 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the positive 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 sparged 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.

PROCEDURE AND SAMPLING
-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 (1% of final volume) 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)
Positive 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.
-Experimental CO2 production. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
-Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).
-Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.

Reference substance:

acetic acid, sodium salt

Remarks:

A solution of CH3COONa was prepared by dissolving 799.77mg in Milli-RO & making this up to a vol. of 200mL. Vols. of 20mL from it were added to 2l of the test med.of the + control bottle and the toxicity control bottle, final conc.of 40mg CH3COONa per l.

Test performance:

The ThCO2 of the test item was calculated to be 2.09 mg CO2/mg. The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg. Please see section "Any other information on results incl. tables" for details.

Parameter:

% degradation (CO2 evolution)

Value:

3

St. dev.:

1

Sampling time:

4 d

Parameter:

% degradation (CO2 evolution)

Value:

7

St. dev.:

1

Sampling time:

6 d

Parameter:

% degradation (CO2 evolution)

Value:

10

St. dev.:

4

Sampling time:

8 d

Parameter:

% degradation (CO2 evolution)

Value:

18

St. dev.:

5

Sampling time:

11 d

Parameter:

% degradation (CO2 evolution)

Value:

24

St. dev.:

2

Sampling time:

15 d

Parameter:

% degradation (CO2 evolution)

Value:

28

St. dev.:

0

Sampling time:

18 d

Parameter:

% degradation (CO2 evolution)

Value:

31

St. dev.:

2

Sampling time:

22 d

Parameter:

% degradation (CO2 evolution)

Value:

32

St. dev.:

2

Sampling time:

25 d

Parameter:

% degradation (CO2 evolution)

Value:

33

St. dev.:

0

Sampling time:

29 d

Remarks on result:

other:

Remarks:

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

Details on results:

All data are presented in tables found in section "Any other information on results incl. tables". The results of CO2 production and biodegradation in blank bottles, background bottles and each test bottle are listed in Table 2 to 8. Biodegradation of the test item (bottle A&B), positive control (sodium acetate) and toxicity control are presented in figure 1.

Results with reference substance:

1 The positive control item was biodegraded by at least 60% (85%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 5%).
3.The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (50.6 mg CO2 per 2 litres of medium, corresponding to 25.3 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.

Table 2. HCl Titrated in Duplicate Blank Bottles

Day	HCl (0.05 N) titrated (mL)
	Blank A	Blank B	Mean Value
1	47.44	47.9	47.67
4	46.05	46.86	46.46
6	47.05	47.64	47.35
8	46.81	47.26	47.04
11	47.27	47.39	47.33
15	46.65	46.3	46.48
18	46.22	47.21	46.72
22	46.3	46.6	46.45
25	45.66	45.52	45.59
29^1)	44.4	45.21	44.81
29^1)	46.44	46.93	46.69
29^1)	48.21	47.98	48.1

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.

Table 3. HCl Titrated in Ba(OH)₂Solution (Background Bottles)

Day	HCl (0.05 N) titrated (mL)
	Bottle A	Bottle B	Mean value
1	50.18	49.89	50.04
4	50.33	50.53	50.43
6	50.51	50.92	50.72
8	51.11	51.22	51.17
11	51.36	51.32	51.34
15	50.81	50.31	50.56
18	50.76	50.85	50.81
22	50.61	51.59	51.1
25	50.66	49.6	50.13
29	50.41	50.63	50.52
29	49.79	50.13	49.96
29	49.92	49.85	49.89

Table 4. CO2 Production in the Blank

Day	HCl (0.05 N) titrated (mL)		Produced	Produced CO2	Cumulative CO2
			CO2	(mg)	(mg)
	Ba(OH)2^1)	Blank (mean)	(mL HCl)
1	50.04	47.67	2.36	2.6	2.6
4	50.43	46.46	3.98	4.4	7
6	50.72	47.35	3.37	3.7	10.7
8	51.17	47.04	4.13	4.5	15.2
11	51.34	47.33	4.01	4.4	19.6
15	50.56	46.48	4.09	4.5	24.1
18	50.81	46.72	4.09	4.5	28.6
22	51.1	46.45	4.65	5.1	33.7
25	50.13	45.59	4.54	5	38.7
29^2)	50.52	44.81	5.72	6.3	45
29^2)	49.96	46.69	3.28	3.6	48.6
29^2)	49.89	48.1	1.79	2	50.6

1): "Strength" of untreated 0.0125 M Ba(OH)2 solution

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 HCl.

Table 5. CO2 Production and Percentage Biodegradation of the Positive Control Item

Day	HCl (0.05 N) titrated (mL)		Produced	Produced CO2	Cumulative CO2	Biodegradation^1)
			CO2	(mg)	(mg)	(%)
	Blank	Positive	(mL HCl)
	(mean)	control
1	47.67	48.11	0	0	0	0
4	46.46	26.7	19.76	21.7	21.7	25
6	47.35	32.93	14.42	15.9	37.6	44
8	47.04	37.32	9.72	10.7	48.3	56
11	47.33	37.57	9.76	10.7	59	69
15^2)	46.48	33.98	12.5	13.7	72.8	85

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 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.

Table 6. CO2 Production and Percentage Biodegradation of the Test Item (Bottle A)

Day	HCl (0.05 N) titrated (mL)		Produced	Produced	Cumulative	Biodegradation^1)
			CO2	CO2	CO2	(%)
	Blank	Bottle A	(mL HCl)	(mg)	(mg)
	(mean)
1	47.67	48.41	0	0	0	0
4	46.46	44.6	1.86	2	2	2
6	47.35	44.72	2.63	2.9	4.9	6
8	47.04	44.88	2.15	2.4	7.3	8
11	47.33	41.85	5.48	6	13.3	15
15	46.48	39.85	6.62	7.3	20.6	23
18	46.72	42.98	3.74	4.1	24.7	28
22	46.45	44.62	1.83	2	26.7	30
25	45.59	44.83	0.76	0.8	27.6	31
29^2)	44.81	43.26	1.55	1.7	29.3	33
29^2)	46.69	46.18	0.51	0.6	29.8	34
29^2)	48.1	48.06	0.03	0	29.9	34

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 87.8 mg CO2/2L.

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 HCl.

Table 7. CO2 Production and Percentage Biodegradation of the Test Item (Bottle B)

Day	HCl (0.05 N) titrated (mL)		Produced	Produced	Cumulative	Biodegradation^1)
			CO2	CO2	CO2	(%)
	Blank	Bottle B	(mL HCl)	(mg)	(mg)
	(mean)
1	47.67	47.85	0	0	0	0
4	46.46	44.32	2.14	2.3	2.3	3
6	47.35	44.01	3.34	3.7	6	7
8	47.04	42.75	4.29	4.7	10.7	12
11	47.33	40.75	6.58	7.2	18	20
15	46.48	42.58	3.9	4.3	22.3	25
18	46.72	44.41	2.31	2.5	24.8	28
22	46.45	43.7	2.75	3	27.8	32
25	45.59	44.84	0.75	0.8	28.6	33
29^2)	44.81	44.5	0.31	0.3	29	33
29^2)	46.69	46.91	0	0	29	33
29^2)	48.1	48.3	0	0	29	33

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test item: 87.8 mg CO2/2L.

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 HCl.

Table 8. CO2 Production and Percentage Biodegradation of the Toxicity Control

Day	HCl (0.05 N) titrated (mL)		Produced	Produced CO2	Cumulative CO2	Biodegradation^1)
			CO2	(mg)	(mg)	(%)
	Blank	Toxicity	(mL HCl)
	(mean)	control
1	47.67	48.37	0	0	0	0
4	46.46	25.53	20.93	23	23	13
6	47.35	30.97	16.38	18	41	23
8	47.04	31.97	15.07	16.6	57.6	33
11	47.33	33.67	13.66	15	72.6	42
15^2)	46.48	26.38	20.1	22.1	94.7	54

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test item and positive control: 174.7 mg CO2/2L (ThCO2 test item: 89.1 mg CO2/2L + ThCO2 sodium acetate: 85.6 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.

Table 9. Comparison of Biodegradation of the Test Item in Bottles A and B.

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A and B	∆ A-B^1)
1	0	0	0	0
4	2	3	3	1
6	6	7	7	1
8	8	12	10	4
11	15	20	18	5
15	23	25	24	2
18	28	28	28	0
22	30	32	31	2
25	31	33	32	2
29^2)	33	33	33	0
29^2)	34	33	34	1
29^2)	34	33	34	1

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.

Validity criteria fulfilled:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Interpretation of results:

not readily biodegradable

Conclusions:

The test fulfills all validity criteria mentioned in OECD 301B guideline and it was conducted in compliance with GLP. After 28 days biodegrability of the substance was 34%. Based on the results it was concluded that the test substance is not readily biodegradable according to OECD 301B.

Executive summary:

Fatty acids, C18-unsatd., phosphates was a yellow liquid UVCB of unknown purity, and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test item was determined to be 57.01%. Based on the TOC content the ThCO₂of the test item was calculated to be 2.09 mg CO₂/mg. The test item was tested in duplicate at a target concentration of 21 mg/L, corresponding to 12 mg TOC/L.

The study consisted of six bottles:

·        2 inoculum blanks (no test item),

·        2 test bottles (Fatty acids, C18-unsatd., phosphates),

·        1 positive control (sodium acetate) and

·        1 toxicity control (Fatty acids, C18-unsatd., phosphates plus sodium acetate).

Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days for the inoculum blank and test item (last CO₂measurement on day 29) and 14 days for the positive and toxicity control (last CO₂measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 34% and 33% biodegradation of Fatty acids, C18-unsatd., phosphates (based on ThCO₂), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, the test item was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion,the test item was designated as not readily biodegradable.

Description of key information

The biodegradation study was conducted for the test item according to OECD 301 B "Ready Biodegradability: CO2 Evolution Test". The test fulfills all validity criteria mentioned in OECD 301 B guideline and it was conducted in compliance with GLP. The test determined 34% degradation after 28 days. Therefore the test item can be concluded as “not readily biodegradable”.

The key value for CSA was selected based on the result from the OECD 301B, the substance is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

EPISuite, Model BIOWIN v4.10 estimated aerobic and anaerobic biodegradability of organic chemicals using 7 different models. Two of these are the original Biodegradation Probability Program (BPP™). The seventh and newest model estimated anaerobic biodegradation potential. It was concluded as not biodegradable.