Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

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 October 11, 2017 to November 10, 2017

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 17, 1992

Deviations:

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.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

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 used immediately. The concentration of suspended solids was determined to be 3.5 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (43 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test substance under aerobic conditions.

Duration of test (contact time):

28 d

Initial conc.:

98 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test concentration and preparation of test solutions:
Test substance was a white turbid liquid (UVCB). The carbon content was based on TOC-analysis of an aqueous stock solution. A weighed amount of 499.38 mg of the test substance was dissolved in Milli- RO water and made up to 500 mL. Magnetic stirring (60 minutes) was used to accelerate dissolution and to ensure homogeneity. The TOC concentration of the slightly glassy and foaming solution was determined to be 122.4 mg/L. The test substance was tested in duplicate at a target concentration of 98 mg/L, corresponding to 12 mg TOC/L.

Analyses of the solution were performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V autosampler (Shimadzu, Kyoto, Japan).

Calibration solutions:
TC standard: Potassium hydrogen phtalate solution (p.a., C8H5KO4, Merck, Darmstadt, Germany).
IC standard: Sodium hydrogen carbonate (p.a., NaHCO3, Merck, Darmstadt, Germany) / sodium carbonate solution (p.a., Na2CO3, Merck, Darmstadt, Germany).
The test substance was added directly to the test bottles. Weighed amounts of test substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 196.03 mg; test substance bottle B: 195.82 mg and toxicity control bottle: 195.72 mg). To this end, 10 mL of Milli- RO water was added to each weighing bottle containing the test substance. The glassy and slightly foaming solution was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Testing strategy and experimental design

Test procedure and conditions:
- Test duration: 28 days for the inoculum blank and test substance (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 L.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 L.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 L.

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).

iIllumination: 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 (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 substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: At the start of the test (day 0), test and reference substance 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.

Determination of CO2
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 substance. 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.


Measurements and recordings
pH: 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 substance), before addition of concentrated HCl.
Temperature of medium: Continuously in a vessel with Milli- RO water in the same room.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottle A

Value:

31

Sampling time:

28 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Bottel B

Value:

33

Sampling time:

28 d

Results with reference substance:

Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

Theortical CO2 production:

The TOC concentration of the 1 g/L stock solution was determined to be 122.4 mg/L (= 12.24%).The ThCO₂of the test substance was calculated to be 0.45 mg CO₂/mg. The ThCO₂of sodium acetate was calculated to be 1.07 mg CO₂/mg.

Toxicity control:

In the toxicity control, more than 25% biodegradation occurred within 14 days (53%, based on ThCO₂). Therefore, the test substance was assumed not to inhibit microbial activity.

HCl Titrated in Duplicate Blank Bottles

Day	HCl (0.05 N) titrated (mL)
	Blank A	Blank B	Mean Value
1	48.99	49.68	49.34
4	47.08	47.8	47.44
6	46.85	47.89	47.37
8	47.38	47.97	47.68
11	46.15	47.29	46.72
15	44.64	46.79	45.72
18	43.93	46.55	45.24
22	43.84	45.62	44.73
25	40.87	42.66	41.77
29^1)	41.73	44.36	43.05
29^1)	46.1	46.41	46.26
29^1)	48.95	48.15	48.55

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

HCl Titrated in Ba(OH)₂Solution (Background Bottles)

Day	HCl (0.05 N) titrated (mL)
	Bottle A	Bottle B	Mean value
1	50.86	50.77	50.82
4	50.98	51.12	51.05
6	50.99	50.94	50.97
8	51.31	51.5	51.41
11	50.66	50.5	50.58
15	49.66	49.7	49.68
18	50.28	50	50.14
22	50	49.8	49.9
25	47.06	46.93	47
29	48.87	48.66	48.77
29	49.45	49.39	49.42
29	49.73	49.43	49.58

CO₂Production 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	50.82	49.34	1.48	1.6	1.6
4	51.05	47.44	3.61	4	5.6
6	50.97	47.37	3.6	4	9.6
8	51.41	47.68	3.73	4.1	13.7
11	50.58	46.72	3.86	4.2	17.9
15	49.68	45.72	3.97	4.4	22.3
18	50.14	45.24	4.9	5.4	27.7
22	49.9	44.73	5.17	5.7	33.3
25	47	41.77	5.23	5.8	39.1
29^2)	48.77	43.05	5.72	6.3	45.4
29^2)	49.42	46.26	3.17	3.5	48.9
29^2)	49.58	48.55	1.03	1.1	50

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

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.

CO₂Production and Percentage Biodegradation of the Positive Control Substance

Day	HCl (0.05 N) titrated (mL)		Produced	Produced CO2	Cumulative CO2	Biodegradation^1)
			CO2	(mg)	(mg)	(%)
	Blank	Positive	(mL HCl)
	(mean)	control
1	49.34	49.49	0	0	0	0
4	47.44	31.41	16.03	17.6	17.6	21
6	47.37	32.08	15.29	16.8	34.5	40
8	47.68	32.78	14.9	16.4	50.8	60
11	46.72	35.51	11.21	12.3	63.2	74
15^2)	45.72	37.57	8.15	9	72.1	84

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 85.4 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.

CO₂Production and Percentage Biodegradation of the Test Substance (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	49.34	50	0	0	0	0
4	47.44	48.13	0	0	0	0
6	47.37	46.86	0.51	0.6	0.6	1
8	47.68	45.28	2.4	2.6	3.2	4
11	46.72	44.1	2.62	2.9	6.1	7
15	45.72	42.9	2.82	3.1	9.2	10
18	45.24	39.11	6.13	6.7	15.9	18
22	44.73	39.86	4.87	5.4	21.3	24
25	41.77	40.2	1.57	1.7	23	26
29^2)	43.05	41.13	1.92	2.1	25.1	28
29^2)	46.26	45.49	0.76	0.8	25.9	29
29^2)	48.55	47.51	1.04	1.1	27.1	31

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 88.2 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.

CO₂Production and Percentage Biodegradation of the Test Substance (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	49.34	49.89	0	0	0	0
4	47.44	46.97	0.47	0.5	0.5	1
6	47.37	45.74	1.63	1.8	2.3	3
8	47.68	45.84	1.83	2	4.3	5
11	46.72	43.54	3.18	3.5	7.8	9
15	45.72	43.06	2.66	2.9	10.7	12
18	45.24	41.74	3.5	3.8	14.6	17
22	44.73	37.7	7.03	7.7	22.3	25
25	41.77	39.51	2.26	2.5	24.8	28
29^2)	43.05	41.2	1.85	2	26.8	30
29^2)	46.26	45.39	0.86	1	27.8	32
29^2)	48.55	47.6	0.95	1	28.8	33

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of the test substance: 88.1 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.

CO₂Production 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	49.34	49.8	0	0	0	0
4	47.44	32	15.44	17	17	10
6	47.37	31.42	15.95	17.5	34.5	20
8	47.68	33.99	13.69	15.1	49.6	29
11	46.72	29.86	16.86	18.5	68.1	39
15^2)	45.72	23.37	22.35	24.6	92.7	53

¹⁾: Calculated as the ratio between CO₂produced (cumulative) and the sum of the ThCO₂of the test item and positive control: 173.5 mg CO₂/2L (ThCO₂test item: 88.1 mg CO₂/2L + ThCO₂sodium acetate: 85.4 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	Bottle B	Mean A and B	∆ A-B^1)
1	0	0	0	0
4	0	1	1	1
6	1	3	2	2
8	4	5	5	1
11	7	9	8	2
15	10	12	11	2
18	18	17	18	1
22	24	25	25	1
25	26	28	27	2
29^2)	28	30	29	2
29^2)	29	32	31	3
29^2)	31	33	32	2

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

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the substance was not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to the OECD Guideline 301 B, in compliance with GLP. Activated sludge was exposed to 98 mg/L test substance in duplicates for 28 d. Test and reference substances (positive control: sodium acetate and toxicity control; test substance plus sodium acetate) were added to the bottles containing the microbial organisms (domestic sludge) and mineral components followed by sampling three times a week during the first 10 days and thereafter twice a week until Day 28, to measure the CO2 evolution. The positive control achieved 60% biodegradation within 10 d. The toxicity control, achieved 25% biodegradation occurred within 14 d (53%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. The relative biodegradation values calculated from the measurements of bottle A and B performed during the test period revealed 31% and 33% biodegradation of the test item (based on ThCO2). Under the study conditions, the substance was not readily biodegradable (Timmer, 2018).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], according to the OECD Guideline 301 B, in compliance with GLP. Activated sludge was exposed to 98 mg/L test substance in duplicates for 28 d. Test and reference substances (positive control: sodium acetate and toxicity control; test substance plus sodium acetate) were added to the bottles containing the microbial organisms (domestic sludge) and mineral components followed by sampling three times a week during the first 10 days and thereafter twice a week until Day 28, to measure the CO2 evolution. The positive control achieved 60% biodegradation within 10 d. The toxicity control, achieved 25% biodegradation occurred within 14 d (53%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. The relative biodegradation values calculated from the measurements of bottle A and B performed during the test period revealed 31% and 33% biodegradation of the test item (based on ThCO2). Under the study conditions, the substance was not readily biodegradable (Timmer, 2018).