(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

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:

2010-04-28 to 2010-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

Adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Aerobic activated sludge from a domestic wastewater treatment plant

- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated. This suspension was used the same day for the experiment.

Duration of test (contact time):

28 d

Initial conc.:

102.3 mg/L

Based on:

test mat.

Initial conc.:

101.8 mg/L

Based on:

ThOD

Remarks:

NH4

Initial conc.:

123.3 mg/L

Based on:

ThOD

Remarks:

NO3

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Reconstituted Test Water
- Test temperature: 22°C ± 1°C
- pH: 7.1 (test item flask, measured at the start of the test), the test item flasks and the abiotic control were adjusted to pH 7.3 with NaOH solution. 7.4 (control, procedure control), measured at the start of the test) 7.2 to 7.6 (measured at the end of the test)
- pH adjusted: yes
- Aeration of dilution water: no
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD Sensor-System
- Test performed in closed vessels: The test flasks were closed gas-tight by a measuring head
- Test performed in open system: no

SAMPLING
- Sampling frequency: every day of the exposure period

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

106

Sampling time:

28 d

Remarks on result:

other: Based on ThODNH4

Key result

Parameter:

% degradation (O2 consumption)

Value:

87

Sampling time:

28 d

Remarks on result:

other: Based on the consideration of Nitrification (ThODNO3) in the evaluation of the results.

Results with reference substance:

The reference item was sufficiently degraded to 79% after 14 days and to 80% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Validity Criteria

Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O₂/L and thus not greater than 60 mg O₂/L within 28 days as required by the test guideline.

pH-Value: The pH-value of the test item flasks at the end of the test was 7.2 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (> 60%) within 14 days as required by the test guideline.

The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.

Test Item: The difference of duplicate values for the degradation of the test item at the end of the 10-day window and at the end of the test was less than 20%. The difference of duplicate values at days 14 and 28 was 5.0% and 1.0%. The validity criterion was fulfilled

Toxicity Control: If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on ThOD_NH4) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 91% at day 14; the test item was not inhibitory.

 

Biodegradation of Test Item

Nitrogen is part of the molecular structure of the test item; therefore nitrification (ThOD_NO3) was considered for the evaluation of the results. The mean biodegradation percentage based on ThOD_NO3 at the end of the 10-day window was 75%; the criterion of the 10 day window was passed. The mean biodegradation percentage at the end of the 28-day exposure period was 87% (ThOD_NO3).

If no nitrification is considered, the mean biodegradation was 106% (ThOD_NH4) after 28 days of incubation. The criterion of the 10 day window was fulfilled on day 14, when the degradation was 91%.

The nitrate-N formation rate after 28 days of incubation in the controls was 1.294 mg/L (mean). The nitrate-N formation rate in the test item treated vessels was 5.478 mg/L (mean). Therefore, a nitrification occurred. In the case of a nitrification, or transformation of ammonium to nitrate the degradation of the test item was 86.8% after 28 days.

 

Biodegradation of Reference Item

The reference item sodium benzoate was sufficiently degraded to 79% after 14 days and to 80% after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Biodegradation in the Toxicity Control: In the toxicity control containing both, the test item and the reference item sodium benzoate, 91% (ThOD_NH4) biodegradation was noted within 14 days and 97% (ThOD_NH4) biodegradation after 28 days of incubation (84% and 90% based on ThOD_NO3). According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25% within 14 days.

 

Oxygen Demand in Abiotic Control

The oxygen demand in the abiotic control was 0 mg/L during the test duration.

 

Table 1: Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

 

Time Days	Flask no.
	1	2	3	4	5	6	7
1	5	5	0	0	55	0	65
2	10	10	5	5	80	0	120
3	10	10	5	5	95	0	145
4	20	15	5	5	110	0	170
5	25	30	10	5	115	0	200
6	45	55	10	5	125	0	210
7	70	70	10	10	135	0	230
8	75	80	10	10	135	0	235
9	85	90	10	10	140	0	245
10	90	95	15	10	145	0	245
11	95	100	15	15	145	0	250
12	95	100	15	15	145	0	250
13	100	105	15	15	150	0	255
14	105	110	15	15	150	0	260
15	110	110	20	15	155	0	260
16	110	110	20	20	155	0	265
17	110	115	20	20	155	0	265
18	115	115	20	20	155	0	270
19	115	120	20	20	155	0	270
20	115	120	25	20	160	0	270
21	120	120	25	20	160	0	270
22	120	120	25	20	160	0	275
23	125	120	25	20	160	0	275
24	125	120	25	20	160	0	275
25	125	120	25	20	160	0	280
26	130	125	25	20	160	0	280
27	130	125	25	20	160	0	280
28	130	130	25	20	160	0	285

 

Flasks 1 and 2: Test item

Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control);

Flask 6: abiotic control

Flask 7: toxicity control

 

Table 2: Percentage Biodegradation of Test Item, of Reference Item and of the Toxicity Control based on ThOD_NH4

 

Time (Days)	Percentage Biodegradation
	Test item^1		Reference time^2	Toxicity Control^1,2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	5	5	32	24
2	5	5	44	43
3	5	5	53	52
4	15	10	61	61
5	17	22	63	71
6	37	47	69	77
7	59	59	73	81
8	64	69	73	83
9	73	79	76	87
10	76	81	77	86
11	78	84	76	87
12	78	84	76	87
13	83	89	79	89
14	88	93	79	91
15	91	91	80	90
16	88	89	79	91
17	88	93	79	91
18	93	93	79	92
19	93	98	79	92
20	91	96	80	92
21	96	96	80	92
22	96	96	80	93
23	100	96	80	93
24	100	96	80	93
25	100	96	80	95
26	105	101	80	95
27	105	101	80	95
28	105	106	80	97

 

¹ ThOD_NH4 of Test item: 0.996 mg O₂/mg test item

² ThOD_NH4 of Reference Item: 1.666 mg O₂/mg reference item

 

Table 3: Percentage Biodegradation of Test Item and of the Toxicity Control based on ThOD_NO3 and of Reference Item based on ThOD_NH4

 

Time (Days)	Percentage Biodegradation
	Test item^1		Reference time^2	Toxicity Control^1,2
	Flask 1 [%]	Flask 2 [%]	Flask 5 [%]	Flask 7 [%]
1	4	4	32	22
2	4	4	44	39
3	4	4	53	48
4	12	8	61	57
5	14	18	63	66
6	30	39	69	71
7	49	49	73	75
8	53	57	73	77
9	61	65	76	81
10	63	67	77	80
11	65	69	76	81
12	65	69	76	81
13	69	73	79	82
14	73	77	79	84
15	75	75	80	83
16	73	73	79	84
17	73	77	79	84
18	77	77	79	86
19	77	81	79	86
20	75	79	80	85
21	79	79	80	85
22	79	79	80	87
23	83	79	80	87
24	83	79	80	87
25	83	79	80	88
26	87	83	80	88
27	87	80	88
28	87	87	80	90

 

¹ ThOD_NO3 of Test item: 1.206 mg O₂/mg test item

² ThOD_NH4 of Reference Item: 1.666 mg O₂/mg reference item

 

Table 4: Correction of Percentage Biodegradation of the Test Item based on ThOD underconsideration of nitrification on day 28

 

	Nitrite-N				Nitrate-N				Total^3	Meas.^4	Corr.^5	Day 28
	Day 0	Day 28	∆ Nitrate-N^1	O2 Equi.^2	Day 0	Day 28	∆ Nitrate-N^1	O2 Equi.^2	O2 Equi.	ThODNH4	ThODNH4	Degradation^6
	mg NO2-N/L			mg	mg NO3-N/L			mg	mg	mg	mg	%
Test Item Rate 1	0.004	0.004	0.000	0.000	0.109	5.549	5.440	4.623	4.623	26.230	21.607	86.8
Test Item Rate 2	n.d.	0.004	0.000	0.000	n.d.	5.625	5.516	4.708	4.708	26.230	21.522	86.8
Control (mean)	0.004^+	0.004	0.000	---	0.109^+	1.403	1.294	---	---	---	---	---
Test Item (mean)	0.004^+	0.004	0.000	0.000	0.109^+	5.587	5.478	4.666	4.666	26.230	21.565	86.8

 

--- = Not applicable; Meas: Measured; Corr: Corrected; n.d.: not determined; ⁺: determined in one replicate

1: Δ _{nitrite-N /} Δ _nitrate-N: Difference of Nitrite-N / Nitrate-N between day 28 and day 0

2: Δ _{nitrite-N /} Δ _nitrate-N multiplied by empirically determined conversion factor (NH₄-N to NO₂-N: 3.43; NH₄-N to NO₃-N: 4.57), corrected by test volume (0.244 L) and concentration of control

3: Sum of nitrite-NO₂-equivalent and nitrate-NO₂-equivalent

4: BOD value test item day 28 [mg/L] - BOD value control day 28 [mg/L] multiplied with test vessel volume [0.244L]

5: Corrected ThOD_NH4 = ThOD_NH4 - O₂-Equivalent

6: Degradation = Corrected ThOD_NH4 / Calculated ThOD_NH4 * 100

+: Limits of quantification: nitrite: 0.004 mg nitrite-N/L; nitrate: 0.109 mg nitrate-N/L

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the test conditions according to OECD 301 F, the test item is considered to be readily biodegradable based on ThODNH4 and ThODNO3.

Executive summary:

The ready Biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 102.3 mg/L in duplicate, corresponding to an oxygen demand of about 101.8 mg/L (ThOD_NH4) and 123.3 mg/L (ThOD_NO3) while the reference item was tested at a concentration of 102.9 mg/L corresponding to an oxygen demand of about 171.4 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. Since nitrogen is part of the molecular structure of the test item, the evaluation of biodegradation is based on ThOD_NH4 and ThOD_NO3. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 79 % after 14 days and to 80 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 91 % (ThOD_NH4) biodegradation was noted within 14 days and 97 % (ThOD_NH4) biodegradation after 28 days of incubation (84 % and 90 % based on ThOD_NO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25% within 14 days. The mean biodegradation of 10% or more of the test item was reached at day 4 (ThOD_NH4, ThOD_NO3). At the end of the 10-day window at day 14, the mean degradation of the test item was 91 % (ThOD_NH4) and 75 % (ThOD_NO3) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 106 % (ThOD_NH4) and 87 % (ThOD_NO3). Therefore, the test item is considered to be readily biodegradable based on ThOD_NH4 and ThOD_NO3.

Description of key information

Under the test conditions according to OECD 301 F, the test item is considered to be readily biodegradable based on ThOD_NH4 and ThOD_NO3 (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready Biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and Regulation (EC) No. 440/2008 Method C.4-D under GLP was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test item was tested at a concentration of 102.3 mg/L in duplicate, corresponding to an oxygen demand of about 101.8 mg/L (ThOD_NH4) and 123.3 mg/L (ThOD_NO3) while the reference item was tested at a concentration of 102.9 mg/L corresponding to an oxygen demand of about 171.4 mg/L (ThOD_NH4). The oxygen concentration was measured using a BSB/BOD-Sensor-System and the test flasks were closed gas-tight by a measuring head. Since nitrogen is part of the molecular structure of the test item, the evaluation of biodegradation is based on ThOD_NH4 and ThOD_NO3. The validity criterion was fulfilled. The reference item sodium benzoate was sufficiently degraded to 79 % after 14 days and to 80 % after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 91 % (ThOD_NH4) biodegradation was noted within 14 days and 97 % (ThOD_NH4) biodegradation after 28 days of incubation (84 % and 90 % based on ThOD_NO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was > 25% within 14 days. The mean biodegradation of 10% or more of the test item was reached at day 4 (ThOD_NH4, ThOD_NO3). At the end of the 10-day window at day 14, the mean degradation of the test item was 91 % (ThOD_NH4) and 75 % (ThOD_NO3) and therefore the 10 day window criterion was passed. The mean biodegradation at test end after 28 days was 106 % (ThOD_NH4) and 87 % (ThOD_NO3). Therefore, the test item is considered to be readily biodegradable based on ThOD_NH4 and ThOD_NO3.