Triethyl phosphonoacetate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-07-18 to 2000-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

2000-04-26

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed poulation of activated sewage sludge micro-organism was obtained on 17 july 2000 from the aeration stage of the severn Trent Water Plc sewage treatment plant at Belper Derbyshire, UK, which treats predominantly domectic sewage.

- Storage conditions:The sample of activated sewage slugde was maintained on continious aeration upon receipt
- Storage length: No data
- Preparation of inoculum for exposure: The activated sewage sludge was washed 3 times by settlement and resuspension in culture medium to remove excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.
- Pretreatment: No
- Concentration of sludge: 28.1 ml in 3 liters corresponding to 30mg/L suspended solid
- Initial cell/biomass concentration: No data
- Water filtered: No data
- Type and size of filter used, if any: No data

Duration of test (contact time):

29 d

Initial conc.:

23.3 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
10 mL of the stock solution: KH2PO4 (8.50 g/L) + K2HPO4 (21.75 g/L) + Na2HPO4, 2H2O (33.40 g/L) + NH4Cl (0.50 g/L) -and 1ml of each solution MgSO4, 7H20 (22.50 g/L) - CaCl2, (27.50 g/L) - FeCl3, 6H2O (0.25 g/L), were combined and made up to 1000 mL with purified water (Reverse osmosis purified and deionised water (Elga Optima 15+)).
- Solubilising agent (type and concentration if used): No
- Test temperature: 21°C
- pH: No data
- pH adjusted: No data)
- CEC (meq/100 g): No data
- Aeration of dilution water: CO2-free air through the solution at a rate of approximately 40 ml/min. The CO2-free air is product by passing compressed-air through a glass column containing self-indicating soda lime (carbosorb®) granules.
- Suspended solids concentration: No data
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter glass culture vessels containing 3 liters of solution
- Number of culture flasks/concentration: 2 for control and test item and 1 for reference item
- Method used to create aerobic conditions: Sealed vessels with bubbled through the solution
- Method used to create anaerobic conditions: NA
- Measuring equipment: For CO2, Tekmar-Dohrmann Apollo 9000 TOC analyser and an ionic 1555B TOC analyser. For the DOC, a shimadzu TOC-5050A TOC analyser
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: The CO2 product by degradation was collected in two 5OO ml Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solution were prepared using purified de-gassed water.

SAMPLING
- Sampling frequency: on day 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28, and 29 in the first CO2 absorber and D0 and 29 in the second absorber.
- Sampling method: 2 mL every day. on Day 28, 1ml of concentrated hydrochloric acid was added to each vessels to drive off any inorganics carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on day 29.
- Sample storage before analysis: Analyse provided immediately except on day 12 and 18 sored deep frozen at -20°C

CONTROL AND BLANK SYSTEM
- Toxicity control: TEPA 23.3 mg/L equivalent to 10 mg C/L and Sodium benzoate 17.1 mg/L equivalent to 10 mg C/L. Final concentration of 20 mg C/L

Reference substance:

benzoic acid, sodium salt

Remarks:

sigma lot N° 77H05005

Parameter:

% degradation (CO2 evolution)

Value:

27

Sampling time:

28 d

Details on results:

The result of the inorganic carbon analysis of sample of the first absorber vessels on D29 showed an increase in all replicates vessels with the exception of control replicate 2, standard material replicate 2 and test material replicate 2. these increases were considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on day 28 and resulted in an increase in the percentage degradation value from 27% on day 28 to 36% on day 29.

The toxicity control attained 67% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment-micro-organism used in the study. The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value from 67% on day 28 to 73% on day 29.

Inorganic carbon analysis of the sample from the second absorber vessels on day 29 confirmed that no significant carry-over of CO2 into the second vessels occurred.

Analysis of the test media from the test material culture vessels on day 0 and 28 for DOC (see table 4), gave percentage degradation values of 32% and 21% respectively for replicate R1 and R2 and 63% for the toxicity control. Sodium benzoate attained 98% and 98% respectively for the replicates R1 and R2, calculated from the DOC analysis.

The degradation rate calculated from the results of the DOC analysis, were higher than those calculated from the inorganic carbon analysis in all replicate vessels, with the exception of test material R2 and the toxicity control. This was considered to be due to incorporation of test material/sodium benzoate into the microbial biomass prior the degradation, and hence CO2 evolution occurring. The slightly lower degradation rate calculated from the result of DOC analysis for the test material replicate R2 and the toxicity control were considered to be due to sampling and/or analytical variation.

Results with reference substance:

Sodium benzoate attained 90% of degradation after 28 days thereby confirming the suitability of the inoculum test conditions. The increase in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value from 90% on day 28 to 92% on day 29.

Table 1: Inorganic carbon value on each analysis occasion

DAY	Control (mg IC)				Sodium benzoate (mg IC)				Test material (mg IC)				Test material + sodium benzoate toxicity control (mg IC)
	R1		R2		R1		R2		R1		R2
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.57	2.80	2.45	2.45	2.80	2.45	2.45	2.45	2.45	2.45	2.45	2.80	2.45	2.45
1	4.29	-	6.15	-	9.63	-	10.09	-	4.76	-	4.41	-	9.86	-
2	8.07	-	9.57	-	19.95	-	24.45	-	9.46	-	8.76	-	23.64	-
3	10.43	-	11.93	-	23.74	-	29.58	-	10.21	-	11.24	-	28.21	-
6	13.45	-	15.96	-	28.27	-	35.91	-	15.73	-	15.84	-	37.39	-
8	14.51	-	16.32	-	31.28	-	38.53	-	15.98	-	15.41	-	38.76	-
10	16.11	-	16.22	-	37.63	-	40.67	-	17.80	-	18.36	-	41.24	-
14	19.15	-	19.04	-	41.19	-	43.53	-	21.93	-	25.49	-	44.20	-
16	18.15	-	19.59	-	41.50	-	43.38	-	22.58	-	25.23	-	49.58	-
20	21.43	-	20.56	-	43.73	-	45.92	-	25.48	-	27.55	-	53.46	-
22	17.82	-	20.10	-	42.71	-	44.12	-	23.80	-	28.69	-	51.83	-
24	22.25	-	22.25	-	48.71	-	48.49	-	27.00	-	31.65	-	54.86	-
27	22.47	-	23.61	-	50.66	-	51.95	-	31.34	-	33.06	-	60.64	-
28	2475	-	25.07	-	51.63	-	52.05	-	32.21	-	33.71	-	64.85	-
29	25.34	3.71	24.27	3.37	54.59	3.59	49.93	3.94	35.62	5.33	32.12	5.33	66.57	5.80

R1-R2 = Replicate 1 and 2

Abs = CO₂absorber vessels

“-“ = Value not determined

 

Table 2: Percentage biodegradation value

 

Days	% degradation Sodium Benzoate	% degradation test material	% degradation Test material + Sodium Benzoate Toxicity control
0	0	0	0
1	15	0	8
2	45	1	25
3	52	0	28
6	58	4	38
8	65	1	39
10	77	6	42
14	78	15	42
16	79	17	51
20	79	18	54
22	82	24	55
24	88	24	54
27	91	27	61
28	90	27	67
29*	92	36	73

 

* Day 29 value corrected to include any carry-over of CO₂detected in Absorber 2

 

 

 

 

Table 3: Percentage biodegradation value

 

Test vessels	Total Carobn* (mg/L)	Inorganic Carbon* (mg/L)	IC/TC Ratio (%)
Sodium benzoate 10 mg C/L R1	8.95	-0.16	0
Sodium benzoate 10 mg C/L R2	9.72	-0.24	0
Sodium benzoate 10 mg C/L R1	10.21	-0.02	0
Sodium benzoate 10 mg C/L R2	9.55	-0.25	0
Test material + sodium benzoate toxicity Control 20 mg C/L	19.68	-0.03	0

R1 – R2 = Replicate 1 and 2

Corrected for control values. Negative values are due to measured concentration value being less than control values

 

 

 

Table 4: Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

 

 

DOC* Concentration
Test vessels	Day 0		Day 28
	Mg C/L	% nominal Carbon Content	Mg C/L	% of Initial Carbon Concentration	% degradation
Sodium benzoate 10 mg C/L R1	9.11	91	0.19	2	98
Sodium benzoate 10 mg C/L R2	9.96	100	0.25	2	98
Sodium benzoate 10 mg C/L R1	10.23	102	7.07	69	31
Sodium benzoate 10 mg C/L R2	9.81	98	7.76	79	21
Test material + sodium benzoate toxicity Control 20 mg C/L	19.71	99	7.33	37	63

 

 

R1 – R2 = Replicate 1 and 2

* Corrected for control values

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 27% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of the OECD guideline 301B.

Executive summary:

In this study, during 28 days, the biodegradation of TEPA at an initial concentration of 10 mg carbon/L (23.3 mg 4 -TBC/L) was studied according to OECD guideline 301B and according to GLP.

The inoculum used in the test was an activated sludge at a concentration of 30 mg/L suspended solid taken in the aeration basin of a sewage treatment plant of domestic effluents. The percentage of biodegradation was estimated by the following parameter CO₂ evolution. The percentage of biodegradation obtained after 28 days was 27%.

Under the test conditions, TEPA is therefore considered as not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-17 to 2012-05-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-08-31

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- A mixed population of activated sewage sludge micro-organism was obtained on 3 January 2012 from the aeration stage of the Seven Trent Water Plc sewage treatment plant at Loughborough, Leceistershire, UK which treats predominantly domestic sewage
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed twice by settlement and resuspension in mineral medium to remove any excessive amount of dissolved organic carbon (DOC) that may have been present. A sub-sample of washed sewage sludge was then removed and the suspended solids concentration determined.

- Concentration of sludge: 300 mg dry material per liter

Duration of test (contact time):

28 d

Initial conc.:

233.3 mg/L

Based on:

test mat.

Initial conc.:

100 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium:
Solution a°): KH2PO4 (8.50 g/L) + K2HPO4 (21.75 g/L) + Na2HPO4, 2H2O (33.40 g/L) + NH4Cl (0.50 g/L) à pH 7.4
Solution b°) : CaCl2, (27.5 g/L)
Solution c°): MgSO4, 7H20 (22.50 g/L)
Solution d°): FeCl3, 6H2O (0.25 g/L)

Preparation of mineral medium for washing the activated sewage sludge:
To 10 liters (final volume) of purified water was added as following:
100 ml of solution a°)
10 ml of each solution b°), c°) and d°)

Preparation of mineral medium for biodegradation test:
To approximately 500ml of purified water* was added as following:
20 ml of solution a°)
2 ml of each solution b°), c°) and d°)

Activated sludge to give 300 mg suspended per liter final volume.
Sufficient test item to give an initial DOC of 100mg C/L
The volume was then adjusted to 2 liters using purified water
*: ion exchange and reverse osmosis treated tap water (Elga Optima 15+ or Elga Purelab Option – R15 BP)
- Additional substrate:

- Test temperature: 19°C to 22°C
- pH:7 to 8
- pH adjusted: yes
- CEC (meq/100 g):
- Aeration of dilution water: Yes
- Suspended solids concentration: 300 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: No data
- Number of culture flasks/concentration:
- Method used to create aerobic conditions: Culture vessels were aerated with compressed air via narrow bore glass tube
- Measuring equipment: HI 93510 digital thermometer, WTH pH 340I pH and dissolved meter, shimadzu TOC-L CSH , shimadzu TOC-V CPH
- Test performed in closed vessels due to significant volatility of test substance: Yes

SAMPLING
- Sampling frequency: 0h and 3h, Days, 1, 7, 14, 21, 27 and 28
- Sampling method: No data
- Sample storage before analysis: No, analysis performed immediately after sampling

CONTROL AND BLANK SYSTEM
- Toxicity control: test item (100mg Carbon/L) and reference item (100mg Carbon/L)

Reference substance:

diethylene glycol

Remarks:

220 mg/L corresponding to 100 mg Carbon/L

Parameter:

% degradation (DOC removal)

Value:

30

Sampling time:

28 d

Details on results:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not absorb to filter matrices or to activated sewage sludge.
The toxicity control attained 64% degradation after 14 days and 62% after 28 days, therefore confirming that the test item was no toxic to the sewage treatment micro-organism used in the study. the slight decrease between day 14 and day 28 was considered to be due to sampling/analytical variation.

Results with reference substance:

Diethylene glycol attained 100% degradation after 14 days and 28 days, thereby confirming the suitability of the inoculum and culture conditions

Table 1: Dissolved Organic Carbon (DOC) Values on each sampling occasion

 

Vessels		DOC (mg C/L)
		Day
		O hours	3 hours	1	7	14	21	27	28
Inoculum Control	R1	3.87	4.41	2.33	3.77	4.03	4.07	4.04	4.47
	R2	3.41	4.02	2.30	3.82	3.83	3.67	3.93	3.47
	Mean	3.64	4.22	2.32	3.80	3.93	3.87	3.99	3.97
Diethylene glycol (≡100 mg C/L)	R1	96.60	93.01	102.10	4.08	4.21	4.36	4.03	3.75
	R2	89.99	100.10	103.90	4.07	4.48	4.51	4.41	3.93
	Mean	93.30	96.56	103.00	4.08	4.35	4.44	4.22	3.84
Test item (≡100 mg C/L)	R1	87.19	109.90	107.40	77.98	75.66	79.64	77.31	77.45
	R2	100.40	108.00	101.80	76.18	73.24	77.89	76.56	76.92
	Mean	93.80	108.95	104.60	77.08	74.45	78.77	76.94	77.19
Toxicity control (≡100 mg C/L)		199.50	196.70	207.80	76.19	73.29	78.90	76.06	76.34

R1-R2 = replicate 1 and 2

 

Table 2: Percentage degradation value

 

Days	% degradation *
	Diethylene glycol	Test item	Toxicity control
1	0	2	0
7	100	30	62
14	100	33	64
21	99	28	61
27	100	30	63
28	100	30	62

*: Corrected for control DOC values

Validity criteria fulfilled:

yes

Interpretation of results:

other: evidence for inherent, primary biodegradability

Conclusions:

From DOC analysis, the test item attained 30% degradation after 28 days. OECD guideline 302B does not give any definitive pass levels for test items. However the "summary of consideration in the report from AECD expert Group on Degradation and Accumulation" suggest that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability.

The test item can therefore be considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Executive summary:

The inherent biodegradability of triethyl phosphono acetate (TEPA) (initial concentration: 233.3 mg/L), was tested in a Zahn-Wellens test during 28 days, according to OECD guideline for Testing of Chemicals 302B (1992).

The inoculum used in the study was 300 mg/L of activated sewage sludge from a wastewater treatment plant.

The percentage of biodegradation was estimated by following the DOC removal.

-TEPA did not adsorb on activated sludge.

-TEPA attained 30% degradation after 28 days

Under the test conditions, TEPA is considered to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in the study.

This study is classified as acceptable and satisfies the guideline requirements for inherent biodegradability study (OECD 302B).

Description of key information

Ready biodegradation: 27% in 28 days; Not readily biodegradable
Inherent Biodegradation (Zahn Wellens test): Not inherent biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Ready biodegradation

The biodegradation of TEPA at an initial concentration of 10 mg carbon/L (23.3 mg 4 -TBC/L) was studied according to OECD guideline 301B.

The inoculum used in the test was an activated sludge at a concentration of 30 mg/L suspended solid taking in an aeration basin of a sewage treatment plant of domestic effluents. The percentage of biodegradation was estimated by the following parameter: CO2evolution. The percentage of biodegradation obtained after 28 days was 27%.

Under the test conditions, TEPA is therefore considered as not readily biodegradable.

Inherent biodegradation

The inherent biodegradability of triethyl phosphono acetate (TEPA) (initial concentration: 233.3 mg/L) was tested in a Zahn-Wellens test during 28 days, according to OECD guideline 302B (1992).

The inoculum used in the study was 300 mg/L of activated sewage sludge from a wastewater treatment plant.

The percentage of biodegradation was estimated by following the DOC removal.

-TEPA did not adsorb on activated sludge.

-TEPA attained 30% degradation after 28 days

Under the test conditions,TEPA is therefore considered as not inherently biodegradable