Dialkyl C18 and C18-unsaturated phosphonates

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:

From 19 April 2017 to 25 september 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dialkyl C18 and C18-unsaturated phosphonates

EC Number:

701-298-1

Cas Number:

64051-29-2

Molecular formula:

Not applicable for a UVCB Substance

IUPAC Name:

Dialkyl C18 and C18-unsaturated phosphonates

Test material form:

liquid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 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.

- Preparation of inoculum for exposure: 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 test substance concentration

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

63

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

85% degradation within 14 days

Any other information on results incl. tables

Theoretical CO2 Production

The ThCO2 of Alkenyl phosphonate was calculated to be 2.33 mg CO2/mg.

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

 

Biodegradation of Alkenyl phosphonate and Sodium Acetate in the Modified Sturm Test

The relative biodegradation values calculated from the measurements performed during the test period revealed 63% and 60% biodegradation of Alkenyl phosphonate, for A and B, respectively (based on ThCO2).

Since the test item is a UVCB and sequential degradation of the components is to be expected, the 10-day window need not be applied. Therefore, the test item can be classified as readily biodegradable.

In the toxicity control, more than 25% biodegradation occurred within 14 days (59%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

 

pH Values of Different Test Media

Test medium:	At the start of the test:	On day 14:	On day 28:
Blank control (A)	7.7 → 7.6^1	-	7.8
Blank control (B)	7.7 → 7.6^1	-	7.8
Positive control	7.7 → 7.6^1	7.9	-
Alkenyl phosphonate (A)	7.7 → 7.6^1	-	7.6
Alkenyl phosphonate (B)	7.7 → 7.6^1	-	7.7
Toxicity control	7.7 → 7.6^1	7.8	-

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

CO₂Production in the Blank

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)
	Ba(OH)2^1)	Blank (mean)
1	50.04	47.67	2.36	2.6	2.6
4	50.43	46.46	3.98	4.4	7.0
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	51.10	46.45	4.65	5.1	33.7
25	50.13	45.59	4.54	5.0	38.7
29^2)	50.52	44.81	5.72	6.3	45.0
29^2)	49.96	46.69	3.28	3.6	48.6
29^2)	49.89	48.10	1.79	2.0	50.6
^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 Item

Day		HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
		Blank (mean)
1		47.67	48.11	0.00	0.0	0.0	0
4		46.46	26.70	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	59.0	69
15^2)		46.48	33.98	12.50	13.7	72.8	85
^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.6 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 Item (Bottle A)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle A
1	47.67	47.42	0.25	0.3	0.3	0
4	46.46	44.87	1.59	1.7	2.0	3
6	47.35	44.45	2.90	3.2	5.2	7
8	47.04	45.93	1.11	1.2	6.4	9
11	47.33	39.30	8.03	8.8	15.3	20
15	46.48	35.59	10.89	12.0	27.2	36
18	46.72	37.94	8.78	9.7	36.9	49
22	46.45	40.81	5.64	6.2	43.1	58
25	45.59	43.44	2.15	2.4	45.4	61
29^2)	44.81	43.60	1.21	1.3	46.8	63
29^2)	46.69	46.76	0.00	0.0	46.8	63
29^2)	48.10	48.00	0.09	0.1	46.9	63
^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 74.7 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 Item (Bottle B)

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Bottle B
1	47.67	49.15	0.00	0.0	0.0	0
4	46.46	47.76	0.00	0.0	0.0	0
6	47.35	46.03	1.32	1.4	1.4	2
8	47.04	45.85	1.19	1.3	2.7	4
11	47.33	41.17	6.16	6.8	9.5	13
15	46.48	36.25	10.23	11.2	20.8	28
18	46.72	39.38	7.34	8.1	28.8	39
22	46.45	40.30	6.15	6.8	35.6	48
25	45.59	41.73	3.86	4.2	39.9	54
29^2)	44.81	40.99	3.82	4.2	44.0	60
29^2)	46.69	46.86	0.00	0.0	44.0	60
29^2)	48.10	48.51	0.00	0.0	44.0	60
^1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 74.0 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.

CO₂Production and Percentage Biodegradation of the Toxicity Control

Day	HCl(0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Toxicity control
1	47.67	48.80	0.00	0.0	0.0	0
4	46.46	30.23	16.23	17.8	17.8	11
6	47.35	34.00	13.35	14.7	32.5	20
8	47.04	36.61	10.43	11.5	44.0	28
11	47.33	31.82	15.51	17.1	61.1	38
15^2)	46.48	15.83	30.65	33.7	94.8	59
^1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and positive control: 159.6 mg CO2/2L (ThCO2test item: 74.0 mg CO2/2L + ThCO2sodium acetate: 85.6 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	3	0	2	3
6	7	2	5	5
8	9	4	7	5
11	20	13	17	7
15	36	28	32	8
18	49	39	44	10
22	58	48	53	10
25	61	54	58	7
29^2)	63	60	62	3
29^2)	63	60	62	3
29^2)	63	60	62	3
^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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In conclusion, Alkenyl phosphonate was readily biodegradable under the conditions of the modified Sturm test

Executive summary:

The objective of the study was to evaluate the non-volatile test item Alkenyl phosphonate for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2) evolution test (modified Sturm test).

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995. Alkenyl phosphonate was a colourless liquid UVCB of high purity. The Total Organic Carbon (TOC) content of the test item was determined to be 63.52%. Based on the TOC content the ThCO2 of the test item was calculated to be 2.33 mg CO2/mg. The test item was

tested in duplicate at a target concentration of 16 mg/L, corresponding to 10 mg TOC/L.

The study consisted of six bottles:

 2 inoculum blanks (no test item),

 2 test bottles (Alkenyl phosphonate),

 1 positive control (sodium acetate) and

 1 toxicity control (Alkenyl phosphonate plus sodium acetate).

Since Alkenyl phosphonate 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 CO2 measurement on day 29) and 14 days for the positive and toxicity control (last CO2 measurement on day 15). The relative biodegradation values calculated from the measurements performed during the test period revealed 63% and 60% biodegradation of Alkenyl phosphonate, for A and B,

respectively (based on ThCO2). Since the test item is a UVCB and sequential degradation of the components is to be

expected, the 10-day window need not be applied. Therefore, the test item can be classified as

readily biodegradable. In the toxicity control, Alkenyl phosphonate 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, Alkenyl phosphonate was designated as readily biodegradable.