Tris(2-ethylhexyl) phosphate

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:

weight of evidence

Study period:

26 October 2016 to 23 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. Suitable measures were taken in study conduct to account for radiolabelled test item. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

Fresh samples of activated sludge are withdrawn on October 25th, 2016 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater.
The sludge was washed in mineral medium and centrifuged (at 1100 • g for 10 minutes). The supernatant was discarded. The concentrated sludge was suspended in mineral medium to a concentration of 2.4 g suspended solids/L. The sludge was aerated with CO2-free air over night to purge the system of carbon dioxide. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. The concentration was found to be 2.7 g suspended solids/L. The concentration used in the test was 10 mg dry mass/litre (30 mg dry mass/3 L).

Duration of test (contact time):

56 d

Initial conc.:

1 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Remarks:

14C-IC evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
For the preparation of the stock solutions for mineral medium the following reagents (analytical grade) were used:
(a)
KH2PO4. 8.50 g/L
K2HPO4. 21.75 g/L
Na2HPO4. 26.64 g/L
NH4Cl. 0.50 g/L
(b)
CaCl2 x 2 H2O. 36.40 g/L
(c)
MgSO4 x 7 H2O. 22.50 g/L
(d)
FeCl3 x 6 H2O 0.25 g/L.
The mineral medium applied in the test contained 10 mL/L and 1 mL/L of the mineral stock solution a and b–d, respectively.
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: The vessels were incubated at 22 °C (± 2 °C)
- pH: Before test start the pH value of the mineral medium was adjusted to the allowed range of 7.4 ± 0.2 using H2SO4 (50 g/L).
- Aeration of dilution water: Yes (during study)
- Suspended solids concentration: The concentration was found to be 2.7 g suspended solids/L. The concentration used in the test was 10 mg dry mass/litre (30 mg dry mass/3 L).
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5L flasks
- Number of culture flasks/concentration: 2 vessels containing test item and inoculum
- Method used to create aerobic conditions: CO2-free air
- Method used to create anaerobic conditions: Not applicable
- Test performed in closed vessels due to significant volatility of test substance: Not applicable
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Two absorption bottles, each containing 200 mL of 0.05 M sodium hydroxide solution were connected in series to each 5-litre flask.
- Other: The test item was 14C-Tris(2-ethylhexyl) phosphate. The concentration in the test vessels with test item (test suspension and abiotic control) was 1 μg test item ≡ 0.152 mCi ≡ 5.624 MBq per litre mineral test medium. A stock solution with a suitable concentration (223.7 MBq/10 mL) is prepared in acetonitrile.

SAMPLING
- Sampling frequency: Days 0, 2, 5, 7, 9, 14, 21, 28, 35, 42, 49, 56 and 57
- Sampling method: On the days of CO2 measurement, the sodium hydroxide absorbers were disconnected and aliquots of the solutions were measured regarding the total radioactivity (test assays and abiotic control) and TIC concentration (inoculum blank and procedural control), respectively. New absorbers (each containing 200 mL fresh 0.05 M sodium hydroxide) were connected to the test vessel. On the 56th day 1 mL of concentrated hydrochloric acid was added to each test vessel. Subsequently, they were aerated overnight to drive off the carbon dioxide present in the test suspensions. On day 57 the last analysis of evolved carbon dioxide was done. The samples at day 56 were taken before the concentrated hydrochloric acid was added.
- Sterility check if applicable: No
- Sample storage before analysis: Not applicable, measurements made on day of sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 vessels containing only inoculum
- Abiotic sterile control: 1 vessel containing test item and a sterilising agent
- Toxicity control: 1 vessel containing reference item and test item
- Other:

STATISTICAL METHODS:
The percentage transformation (Tt) and degradation (Dt) of test and reference item, respectively, at each time a sample was taken is calculated separately for each flask containing test or reference item using values of LSC or IC, respectively.
Tt =[(Ct –Cb)/TI]x100
Tt: Transformation (%) at time t;
Ct: Test item (mg) based on total radioactivity in the hydroxide absorbers of the
treatments at time t;
Cb: Test item (mg) based on total radioactivity in the hydroxide absorbers of the blanks
at time t;
TI: Test item content of the test suspension (mg).
Dt = [(Ct –Cb) / ThCO2] x 100
Dt: Degradation (%) at time t;
Ct: Inorganic carbon (mg) in the hydroxide absorbers of the treatments at time t; Cb: Inorganic carbon (mg) in the hydroxide absorbers of the blanks at time t; DOC: ThCO2 of the test suspension (mg).

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not applicable

Key result

Parameter:

% degradation (radiochem. meas.)

Value:

0.76

St. dev.:

0.05

Sampling time:

28 d

Key result

Parameter:

% degradation (radiochem. meas.)

Value:

1.22

St. dev.:

0.02

Sampling time:

56 d

Details on results:

The mineralization of 14C-Tris(2-ethylhexyl) phosphate (more precisely: [hexyl-1-14C]tris(2-ethylhexyl) phosphate) based on evolved 14C-labelled CO2 in the static test was found to be 1.2 % after 56 days on the basis of the nominal initial total radioactivity, considering the amount of 14C-IC measured after acid dissolving on day 56. Consequently, no mineralization within a 10-day-window could be calculated.

With 0.1 % no significant abiotic mineralization of 14C-Tris(2-ethylhexyl) phosphate was noticeable after 56 days of incubation.

Since only three carbon elements in the center of the molecule ([hexyl-1-14C]tris(2-ethylhexyl) phosphate) out of a total number of 24 carbon elements are 14C-labelled, it has to be stated that a possible primary degradation with a mineralization of up to 87.5 % of the lateral carbon elements could not be detected in the test design applied. This test design shows that there is a maximum of 1 % of total mineralization of 14C-Tris(2-ethylhexyl) phosphate.

Results with reference substance:

The reference item sodium benzoate was degraded to 99 % within the first 14 days. Total degradation after 56 days was found to be 103 %.

Table 1. Total radioactivity [mg 14C-IC evolved per flask] of the sodium hydroxide traps at each sampling date. Single values of the replicates; Test suspension and abiotic control: nominal 16872 kBq (= 3 μg)/3L; * acid dissolving.

Day	Test Flask 1	Test Flask 2	Abiotic Control Flask 1
2	12.95	11.48	1.27
5	19.98	19.49	2.18
7	12.96	11.94	1.23
9	10.22	9.18	1.04
14	24.75	21.57	0.97
21	28.89	25.70	1.97
28	23.11	21.42	1.05
35	18.04	17.27	0.78
42	16.66	23.16	0.93
49	15.87	27.54	0.68
56	13.76	17.45	0.63
57 *	3.79	3.91	0.20

Table 2. Cumulative inorganic carbon content [mg 14C-IC evolved per flask] of the sodium hydroxide traps at each sampling date. Single values of the replicates; Test suspension and abiotic ontrol: nominal 3 μg/3L; * acid dissolving.

Day	Test Flask 1	Test Flask 2	Abiotic Control Flask 1
2	12.95	11.48	1.27
5	32.93	30.97	3.45
7	45.89	42.91	4.49
9	56.11	52.09	5.53
14	80.86	73.66	6.50
21	109.75	99.36	8.47
28	132.86	120.78	9.52
35	150.90	138.05	10.30
42	167.56	161.21	11.23
49	183.43	188.75	11.91
56	197.19	206.20	12.54
57 *	200.98	210.11	12.74

Table 3. Transformation of the test item [%] on the basis of total radioactivity in the sodium hydroxide traps during the 56 days of incubation. Single values of the replicates; * acid dissolving.

Day	Test Flask 1	Test Flask 2	Abiotic Control Flask 1
2	0.08	0.07	0.01
5	0.20	0.18	0.02
7	0.27	0.25	0.03
9	0.33	0.31	0.04
14	0.48	0.44	0.05
21	0.65	0.59	0.06
28	0.79	0.72	0.06
35	0.89	0.82	0.07
42	0.99	0.96	0.07
49	1.09	1.12	0.07
56	1.17	1.22	0.07
57 *	1.19	1.25	0.08

Table 4. Inorganic carbon content [mg IC evolved per flask] of the sodium hydroxide traps at each sampling date. Single values of the replicates. Procedural control: nominal 60 mg/3L; * acid dissolving.

Day	Inoculum Blank Flask 1	Inoculum Blank Flask 2	Procedural Control (sodium benzoate) Flask 1
2	1.79	1.77	31.43
5	1.67	1.68	20.48
7	0.97	0.96	6.73
9	0.57	0.68	3.16
14	1.07	1.27	3.58
21	1.29	1.61	2.86
28	1.22	1.63	2.27
35	1.05	1.63	1.64
42	1.05	1.84	1.46
49	1.15	1.90	1.31
56	0.97	1.70	1.48
57 *	0.73	1.07	1.08

Table 5. Cumulative inorganic carbon content (mg IC evolved per flask) of the sodium hydroxide traps at each sampling date corrected for inoculum blank. Single values of the replicates. Procedural control: nominal 60 mg/3L; * acid dissolving.

Day	Inoculum Blank Flask 1	Inoculum Blank Flask 2	Procedural Control (sodium benzoate) Flask 1
2	1.79	1.77	31.43
5	3.46	3.45	51.91
7	4.43	4.41	58.64
9	5.00	5.09	61.80
14	6.06	6.36	65.38
21	7.36	7.97	68.24
28	8.58	9.60	70.51
35	9.63	11.24	72.16
42	10.68	13.07	73.61
49	11.83	14.97	74.93
56	12.80	16.67	76.41
57 *	13.53	17.74	77.49

Table 6. Degradation (%) on the basis of total radioactivity in the sodium hydroxide traps during the 56 days of incubation. Procedural Control. Single values of the replicates; * acid dissolving.

Day	Procedural Control (sodium benzoate) Flask 1
2	49.42
5	80.75
7	90.37
9	94.59
14	98.61
21	100.96
28	102.37
35	102.87
42	102.90
49	102.55
56	102.79
57 *	103.00

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The mineralization of [hexyl-1-14C]-Tris(2-ethylhexyl) phosphate based on evolved 14C-labelled CO2 in the static test was found to be 0.76 % after 28 days and 1.2 % after 56 days on the basis of the nominal initial total radioactivity. Since only three carbon elements in the center of the molecule out of a total number of 24 carbon elements are 14C-labelled, it has to be stated that a possible primary degradation with a mineralization of up to 87.5 % of the lateral carbon elements could not be detected in the test design applied. This test design shows that there is a maximum of 1 % of total mineralization of 4C-Tris(2-ethylhexyl) phosphate.

Executive summary:

The mineralization of [hexyl-1-14C]tris(2-ethylhexyl)phosphate based on evolved radiolabeled CO2 was investigated over a 56-day period in a CO2 Evolution Test according to EC method C.4-C (440/2008/EEC) and OECD guideline 301 B. The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater.

The test solutions were aerated by the passage of CO2 free air at a controlled rate in closed flasks at 22 °C for 56 days. The rate of degradation was monitored by measuring the 14C- carbon dioxide produced from degradation of the 14C-labelled test item. To monitor the validity of the test conditions, also carbon dioxide release of inoculum blank assays and a procedural control containing unlabelled sodium benzoate were applied. For the test item, the amount of 14C-carbon dioxide produced from the test substance was expressed as a percentage of the 14C-atoms present in the test item at test start. For the procedural control, the amount of carbon dioxide produced from the reference substance sodium benzoate (corrected for that derived from the blank inoculum) is expressed as a percentage of the ThCO2.

The mineralization of 14C-Tris(2-ethylhexyl) phosphate in the static test - based on the radiolabeled carbon elements in the molecule - was found to be 0.76 % after 28 days and 1.2 % after 56 days. Consequently, mineralization within the 10-day-window could not be calculated.

With 0.1 % after 56 days, no significant abiotic mineralization occurred.

Since only three carbon elements in the center of the molecule out of a total number of 24 carbon elements are labeled, it has to be stated that a possible primary degradation with a mineralization of up to 87.5 % of the lateral carbon elements could not be detected in the test design applied. This test design shows that there is a maximum of 1.2 % of total mineralization of 14C-Tris(2-ethylhexyl) phosphate.

The degradation of the reference substance sodium benzoate had reached 98.6 % within the first 14 days. The difference of extremes of replicate values of the removal of the test item at the end of the test is 0.1 %.Therefore, the test can be considered as valid.

No inhibitory effects of the test item were observed (more than 25 % degradation occurred within 14 days) in the toxicity control.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was well reported and did not appear to deviate from standard guidelines. Validity criteria were all met as per OECD 301 D test guidelines. However the method of analysis (i.e. oxygen meter or winkler titration) was not documented within the study report.

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, adapted

Details on inoculum:

secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)

Duration of test (contact time):

40 d

Initial conc.:

2.6 other: mL/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

(ThOD)

Details on study design:

Test item "ready biodegradability" was tested under aerobic conditions at 22 ± 2 °C in the dark over 40 days. The biodegradation was determined by analysing the reduction of dissolved oxygen. To each flask 0.6 mg of test item was added to ca. 300 mL of mineral medium, inoculum was then added to the flask and the volume was made up to 1.16 L. The test item concentration was 0.5 mg/L (0.6 mg/ 1.16 L). The flasks were closed with glass stoppers and free from air bubbles. A reference item (sodium benzoate) was also used to ensure test validity and that the test system was fully operational. The reference item was treated in a similar manner, the initial concentration was 2.9 mg/L (3.4 mg/1.16 L). For the test there were 28 test flask suspensions, 14 reference control (sodium benzoate) flasks, 14 inoculum blank flasks (medium and inoculum only) and 14 toxicity control flasks (containing test item, reference compound and inoculum).

Test performance:

Throughout the test pH remained between 6.8-7.2 for all test conditions. All validity criteria were met and were:

The oxygen depletion in the blank control did not exceed 1.5 mg dissolved oxygen/L after 28 days (table 1).

At the end of the test, at the plateau, or the end of the 10-d window, biodegradation in parallels with test item did not differ by more than 20 percentage points (table 6).

The reference compound reached the level of ≥ 60 percent for ready biodegradability within 14 days (table 7).

The toxicity control exhibited degradation rates > 25 % within 14 days.

Key result

Parameter:

% degradation (O2 consumption)

Value:

11

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

9

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

35 d

Details on results:

Tris(2-ethylhexyl) phosphate showed: 11 % degradation after 7 days, 14 % degradation after 14 days, 9 % degradation after 21 days, 4 % degradation after 28 days, 1 % degradation after 35 days and 3 % degradation after 40 days (not shown above).

Results with reference substance:

The reference compound sodium benzoate showed 73 % degradation after 14 days.

Tables 1: BOD values (mg O₂/L) of blank inoculum after n days

 

Flask	0 days	Flask	7 days	Flask	14 days	Flask	21 days	Flask	28 days	Flask	35 days	Flask	40 days
1	9.21	3	9.13	5	8.49	7	7.95	9	7.80	11	7.55	13	7.65
2	9.19	4	9.07	6	8.28	8	8.04	10	7.67	12	7.76	14	7.63
mean	9.20		9.10		8.39		8.00		7.74		7.66		7.64

 

Tables 2: BOD values (mg O₂/L) of reference compound after n days

 

Flask	0 days	Flask	7 days	Flask	14 days	Flask	21 days	Flask	28 days	Flask	35 days	Flask	40 days
15	9.19	17	5.02	19	5.03	21	4.12	23	3.86	25	3.77	27	3.71
16	9.18	18	5.12	20	4.66	22	4.16	24	4.07	26	3.34	28	3.67
mean	9.19		5.07		4.85		4.14		3.97		3.56		3.69

 

Tables 3: BOD values (mg O₂/L) of test item after n days

 

Flask	0 days	Flask	7 days	Flask	14 days	Flask	21 days	Flask	28 days	Flask	35 days	Flask	40 days
29	9.10	33	8.98	37	7.95	41	7.63	45	7.82	49	7.68	53	8.34
30	9.17	34	8.90	38	8.14	42	7.99	46	7.63	50	7.67	54	7.54
31	9.20	35	8.97	39	8.32	43	7.81	47	7.63	51	8.10	55	7.57
32	9.19	36	8.87	40	8.27	44	8.16	48	7.70	52	7.63	56	7.57
mean	9.17		8.93		8.17		7.90		7.70		7.77		7.76

 

Tables 4: BOD values (mg O₂/L) of toxicity after n days

 

Flask	0 days	Flask	7 days	Flask	14 days	Flask	21 days	Flask	28 days	Flask	35 days	Flask	40 days
57	9.11	59	5.00	61	4.57	63	4.39	65	3.86	67	3.89	69	3.80
58	9.13	60	4.91	62	4.70	64	4.49	66	3.96	68	3.94	70	3.63
mean	9.12		4.96		4.64		4.41		3.91		3.92		3.72

 

Tables 5: Degradation of the test item

 

Theoretical oxygen demand 2.651 mg O₂/mg

 

Test item	7	14	21	28	35	40
No.1:(mt0 - mtx) - (mb0 - mbx)	0.02	0.34	0.27	0.00	0.00	0.00
No.2:(mt0 - mtx) - (mb0 - mbx)	0.17	0.22	0.00	0.08	0.00	0.07
No.3:(mt0 - mtx) - (mb0 - mbx)	0.13	0.07	0.19	0.11	0.00	0.07
No.4:(mt0 - mtx) - (mb0 - mbx)	0.22	0.11	0.00	0.03	0.02	0.00
D1:% degradation	2	26	20	0	0	0
D2:% degradation	13	17	0	6	0	5
D3:% degradation	10	5	14	8	0	5
D4:% degradation	17	8	0	2	2	0
% degradation mean	11	14	9	4	1	3

 

Comments: Abbreviations according to test guideline.

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Tris(2-ethylhexyl) phosphate showed 4 -14 % degradation after 7-40 days. Tris(2-ethylhexyl) phosphate is considered not to be "Not Readily Biodegradable".

Executive summary:

The test item Tris(2-ethylhexyl) phosphate in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions for 40 days. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of theoretical oxygen demand (ThOD). Tris(2-ethylhexyl) phosphate showed 4 -14 % degradation after 7-40 days. All validity criteria were met.

The reference compound sodium benzoate showed 73 % degradation after 14 days.

Tris(2 -ethylhexyl) phosphate is considered to be "Not Readily Biodegradable".

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was well conducted and reported. Study was done to GLP standards using OECD 301 B test guidelines with acceptable deviation due to the low solubility of the test substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The inorganic carbon concentration of the test substance in the mineral medium was not properly quantified, because the mixture was not completely soluble, therefore the item of validity of test about this parameter was not verified.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Domestic sewage was collected at a sewage launching point, at Pircacicaba River. Samples were centrifuged (2000 rpm for 20 mins) and solid residue was resuspended in mineral medium. The number of microorganims were counted and after a 48 hour incubation period (36 °C) colony number was counted. There were 5.5 x 10^6 cfu/mL.

Duration of test (contact time):

28 d

Initial conc.:

15.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Biodegradation was tested over a 28 day period in a dark room at 21.4-23.6 °C. Three litres of mineral mediaum was added to each flask along with 15 mL of inoculum. Before addition of the reference substance or test substance the mixture was aerated with CO2 free air overnight. There after test substance or reference item was applied to the test system corresponding to 10 and 20 mg C/L, respectively. The test flasks were continually stirred for the duration of the test. Each test container was attached to 4 barium hydroxide traps with 100 mL barium hydroxide (0.0125M) solution to absorb CO2 released during the metabolism from the substances. There were 4 treatments tested in duplicate, as follows:

a) Blank: nourishing solution plus inoculum;

b) Reference substance: nourishing solution plus sodium benzoate solution, corresponding to the concentration of 20 mg of carbon pe
r liter and inoculum;

c) Test substance: nourishing solution plus the test substance at the concentration of 10 mg of carbon per litre and inoculum;

d) Inhibition control: nourishing solution plus sodium benzoate at the concentration of 20 mg of carbon per litre, the test substance at the concentration of 10 mg of carbon per litre and inoculum.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

9.6

Sampling time:

28 d

Details on results:

At the end of the 28 day screening the test substance degraded by 9.6 %. During the dyas prior the test substance slowly degraded with time apart from a plateau seen between days 5-14 (Table 1)

Results with reference substance:

Reference substance degraded by 78.0 and 77.4 % in each one of the 2 replicates.

Table 1 Degradation of varying test treatments over time

Day	Reference Substance		Test substance		Inhibition control		Abiotic control
	R1	R2	R1	R2	R1	R2	R1	R2
1	10.4	10.1	2.4	2.8	7.6	7.3	1.7	1.7
3	34.6	34.3	3.9	4.1	23.8	23.5	3.2	4.1
5	56.2	55.8	4.2	4.4	38.0	37.9	3.7	5.1
7	64.1	63.9	4.2	4.4	43.5	43.3	4.1	5.8
10	71.4	71.0	4.2	4.4	48.3	48.0	4.1	5.8
14	74.6	74.4	4.2	4.4	52.5	52.1	4.1	5.8
17	75.5	75.1	5.0	5.2	53.2	52.7	6.2	8.3
21	76.2	75.7	6.4	6.5	53.8	53.4	8.4	10.2
24	77.0	76.4	7.9	8.1	54.6	54.1	10.9	12.7
28	78.0	77.4	9.4	9.7	55.1	54.7	13.1	15.3

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Tris(2-ethylhexyl)phosphate showed 9.6 % of total biodegradation, under the test conditions.

Executive summary:

The assay was conducted to determine the ready biodegradation of Tris(2-ethylhexyl)phosphate using OECD 301B test guidelines. The test was incubated for 28 days with controlled temperature between 21.4 to 23.6 °C. Five treatments with two replicates were used: a. blank: basal culture medium; b. reference substance (sodium benzoate, as a biodegradable standard); c. test substance; d. inhibition control: sodium benzoate plus test substance ; e. abiotic control: test substance plus 3 mL/L of 5 % HgCl2. Each test flask was filled with 3 liters of nutritive solution inoculated with the microorganisms where appropriate.

The CO2 evolved from the respective treatments was captured using barium hydroxide solution (Ba(OH)2) and quantified through HCl titration.

The test was considered valid.

Tris(2-ethylhexyl)phosphate biodegradation was 9.6 %, after 28 days of incubation.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although not conducted in accordance with GLP, this published value is from a reliable peer reviewed source & contains supporting information on methodology.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

Principles of method if other than guideline:

OECD 301C, OECD 302C

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Duration of test (contact time):

>= 14 - <= 28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter:

other: BOD

Value:

0

Sampling time:

28 d

Details on results:

The degree of biodegradation by BOD is reported to be 0%.

The degree of biodegradation by BOD is reported to be 0%.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The degree of biodegradation by BOD is reported to be 0%.

Executive summary:

The biodegradation of the substance was investigated in a modified MITI test corresponding to OECD 301C or OECD 302C. The degree of biodegradation by BOD is reported to be 0%.

The substance can not be classified as readily biodegradable.

Description of key information

% Biodegradation = 0.76 % (28 d). 1.2 % (56 d); OECD 301B; Simon (2017)


% Biodegradation = 4-14 % (28 d); OECD 301D; Schroder (2014)

% Biodegradation = 9.6 % (28 d); OECD 301B; Bidinotto (2013)

% Biodegradation = 0 % (28 d); OECD 301C; Anon. (1992)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A weight-of-evidence is presented for biodegradation in water: screening tests, consisting of 4 ready biodegradability studies.  In a modified OECD 301B study (Simon, 2017) conducted with a radiolabelled test item, the biodegradability of the test item was assessed over a 56 day period.  The radiolabelled test item ([hexyl-1 -14C]tris(2 -ethylhexyl)phosphate) consisted of three 14C atoms located at the alpha position relative to the phosphate group on each alkyl chain.  This study was commissioned to investigate whether the low water solubility (0.14 μg/L) was the rate limiting factor in the other existing ready biodegradability studies (Schroder, 2014; Bidinotto, 2013; Anon, 1992), limiting the bioavailability of the test item to the organisms when the standard addition rate of 10-20 mg C/L is used.  In this study use of radiolabelled material permitted the use of a much lower addition rate (1 μg/L test item) without compromising detection of evolved carbon dioxide.

The test medium was inoculated with microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater. The test solutions were aerated by the passage of CO2 free air at a controlled rate in closed flasks at 22 °C for 56 days. The rate of degradation was monitored by measuring the 14C- carbon dioxide produced from degradation of the 14C-labelled test item. To monitor the validity of the test conditions, also carbon dioxide release of inoculum blank assays and a procedural control containing unlabelled sodium benzoate were applied. For the test item, the amount of 14C-carbon dioxide produced from the test substance was expressed as a percentage of the 14C-atoms present in the test item at test start. For the procedural control, the amount of carbon dioxide produced from the reference substance sodium benzoate (corrected for that derived from the blank inoculum) is expressed as a percentage of the ThCO2.

The mineralization of 14C-Tris(2-ethylhexyl) phosphate in the static test - based on the radiolabelled carbon elements in the molecule - was found to be 0.76 % after 28 days and 1.2 % after 56 days. Consequently, mineralization within the 10-day-window could not be calculated. Since only three carbon atoms in the centre of the molecule out of a total number of 24 carbon elements are labeled, it has to be stated that a possible primary degradation with a mineralization of up to 87.5 % of the lateral carbon elements could not be detected in the test design applied. This test design shows that there is a maximum of 1.2 % of total mineralization of 14C-Tris(2-ethylhexyl) phosphate.

The maximum biodegradation observed for the substance in ready biodegradability screening tests with standard addition rates was 14 % (Schroder, 2014).

Based on the available studies for this endpoint, we can conclude that the substance is not considered readily biodegradable under standard OECD 301 test conditions.  Additionally, we can conclude that up to 1 % of the carbon atoms located at the alpha position relative to the phosphate group on each alkyl chain will mineralise when the substance is added to an OECD 301B test design at 1 μg/L (7 times the water solubility, although the solubility in mineral media spiked with inoculum is expected to be lower).  The available data cannot rule out primary biodegradation and mineralisation of the alkyl chains beyond the alpha carbon when added to OECD 301B test system using test item addition rates closer to the solubility limit, nor can it rule out adsorption of the test item to glassware or organic particulates in the test system, which may also affect performance of this test.

The conclusion drawn is that the substance does not meet the criteria for ready biodegradability under the strict conditions of OECD 301 screening methodologies.

[Type of water: freshwater]