Chromium tris((2-ethylhexanoate)

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:

April 13, 2017 - July 13, 2017

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:

July 17, 1992; §14 corrected July 26, 1993

Deviations:

yes

Remarks:

The stirring of one measuring system (test item replicate) was not done correctly, data corresponding to this flask were not used. The data corresponding th the 2 other flasks were used. This deviaiton has no incidence on study results.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 14 January 2016

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Laboratory preparation:
A filtration through a fine sieve (about 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in a mineral medium until sludge was considered free from excess substrate or inhibitor.

Inoculum pre-conditioning:
Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST ITEM PREPARATION
According to the study monitor, the test item is poorly soluble in water; it was introduced directly in the test Flasks. The test item was tested at 100 mg/L in mineral medium.
The test medium will be the mineral medium, a weakly saline aqueous medium.

PROTOCOL
Principle:
A measured volume of inoculated medium containing a known concentration of the test item (100 mg/L) as the nominal sole source of Carbon, was stirred in a closed vessel at a constant temperature up to 28 days.
Microbial respiration consumes O2 and releases CO2 which was absorbed by soda lime pellets present in the headspace of flasks. This produces a decrease in pressure in the flask proportional to the amount of oxygen taken up by the microbial population during biodegradation, which was expressed as a percentage of ThOD (Theoretical Oxygen Demand).
Allowance was made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without the test item.
Furthermore, in order to check the procedure, a reference item control (sodium benzoate 60.2 mg/L + inoculum) and a toxicity control (sodium benzoate + inoculum + test item) were run in parallel.
The pH of the contents was measured at the start and the end of the experiment.

Test design:
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline.

Incubation:
The test was performed in darkness between 20 to 24°C (constant temperature ±1°C).

MATERIAL AND METHOD
Inoculum preparation:
Sample site = Water treatment plant 'STEP de Lille', 33290 Blanquefort (France) - Tank 2
Date of sampling = April 20, 2017
Inoculum physico-chemical characteristic at the beginning of the test:
Suspended solid = 3.11 g/L
pH = 6.9
Dissolved oxygen = 8.8 mg/L
Activated sludge preparation:
1. Filtration through a 1 mm sieve
2. 3 successive decantations into mineral medium
3. Activated sludge aeration during 5 days

Preparation of the test solutions:
The test item was weighed and introduced directly in each flasks to obtain a nominal concentration of 100 mg/L.
A solution of sodium benzoate was produced: weighing of 200.03 mg in 200 mL (final volume) mineral medium.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The theorical Oxygen Demand is 2.366 mg O2 per mg of test item.

Test performance:

All validity criteria were successful.

Key result

Parameter:

% degradation (O2 consumption)

Value:

19.65

St. dev.:

1.68

Sampling time:

28 d

Remarks on result:

other: See 'Remarks'

Remarks:

The test item contains no nitrogen according to the elementary analysis, the evaluation of biodegradation has to be based as ThOD(NH4).

Details on results:

The degradation for the 10-day window failed.
The degradation rate of the test item reached 18.47% to 20.84%, following the ThODNH4, after 28 days of incubation.
According to the OECD guideline 301F, the test item cannot be considered readily biodegradable.

Biodegradation of the reference item: “Sodium benzoate”:
The reference item “Sodium benzoate” was degraded up to 87.86% after 14 days.
Therefore, it confirms the suitability of the used aerobic sludge inoculum.

Biiodegradation of the Toxicity Control:
A biodegradation of 33.10% after 14 days of incubation was noted in the Toxicity control (containing both of the test item and the reference item).
At the end on the test, 28 days of incubation, the results were 34.82% and 40.47%.
Therefore, the test item was not inhibitory at the tested concentration on the aerobic activated sludge micro organism because degradation was >25% within 14 days (validity criterion).

Results with reference substance:

The reference item “Sodium benzoate” was degraded up to 87.86% after 14 days.
Therefore, it confirms the suitability of the used aerobic sludge inoculum.

Percentage (%) Biodegradation during 28 days:

Time (days)	Reference item - Sodium benzoate		Toxicity control		Test item
Measuring system	13	9	3	16	11	21	Average
0	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1	23.94	22.45	0.83	0.42	0.00	0.00	0.00
2	54.87	57.67	10.03	8.79	0.00	0.00	0.00
3	60.46	64.65	15.88	14.64	4.18	5.96	5.07
4	66.51	69.40	18.55	18.55	4.97	6.75	5.86
5	71.73	73.13	21.35	21.35	5.96	6.55	6.26
6	75.46	78.25	23.29	24.98	6.95	7.54	7.24
7	78.72	81.61	24.70	27.19	7.73	8.96	8.35
8	81.14	81.14	25.39	28.18	8.17	9.35	8.76
9	82.08	84.87	26.50	29.83	9.16	10.34	9.75
10	83.94	85.33	27.89	31.15	9.95	11.13	10.54
11	83.90	85.30	28.35	32.21	9.93	11.71	10.82
12	85.30	88.09	28.65	33.70	11.12	13.52	12.32
13	85.73	87.13	29.55	34.30	11.30	13.12	12.21
14	87.16	88.56	30.57	35.62	12.50	14.91	13.70
15	88.53	88.53	30.44	36.08	12.48	14.89	13.69
16	88.49	88.49	30.61	36.25	13.10	15.47	14.29
17	88.96	88.96	31.64	36.98	13.30	16.26	14.78
18	89.42	89.42	31.36	37.00	14.09	16.46	15.27
19	89.49	87.10	31.38	37.32	14.88	16.06	15.47
20	89.89	88.49	32.69	38.03	15.47	17.24	16.36
21	90.09	88.09	32.15	38.09	15.47	17.24	16.36
22	91.49	89.49	33.16	38.80	16.06	19.06	17.56
23	89.69	86.70	32.33	38.57	16.06	18.47	17.27
24	91.09	90.09	33.04	39.87	16.65	19.65	18.15
25	91.02	89.03	34.21	40.15	17.05	20.05	18.55
26	89.69	86.70	33.52	39.75	17.29	19.65	18.47
27	90.56	88.56	34.37	40.31	18.08	20.44	19.26
28	91.09	90.09	34.82	40.47	18.47	20.84	19.65

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under conditions of OECD guideline 301F, the test item cannot be considered readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 2017 - January 2018

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:

version 17 July 1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A secondary effluent source that treates primarily domestic sewage was used as the inoculum. To each liter of medium, 10.0 mL of effluent were added to achieve a fina lconcentration od approximately 23.4 mg/L of solids. All treatments except the abiotic sterile control were inoculated.

Duration of test (contact time):

28 d

Initial conc.:

15 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test substance:
Attempts were made to dissolve the test substance in deionized water to make a concentrated stock solution, and then at successively lower concentrations directly in mineral medium. Howver, the test substance would not solubilize in water at any measurable concentration. Subsequently, carrier solvents were used in attempts to solubilize the test substance in water. Acetone was moderately successful in dissolving the test substance, but addtion of water to the test substance-acetone solution immediately caused the test substance to precipitate back out, even at successessively lower concentrations (as low as around 50 µL/L). Hexane dissolved the test substance well, but the addition of water brought the same effect.
The test substance was ultimately tested using direct addtion to the experimental flasks, in which a known mass of the test substance was dissolved in a small volume of hexane. The aliquot of the test substance/hexane was then poured into the flasks and swirled to coat the bottom and lower walls with a thin film of test substance. The hexane was completely evaporated before test initiation.At the study conclusion, some of this film remained, indicating that the test substance did not completely dissolve in the mineral medium during the study.

Apparatus:
A gas cylinder containing 80% Nitrogen and 20% Oxygen was connected to each apparatus with flow-valves distributing CO2-free air through a sealed system at a fixed rate of around 50 mL/min to each treatment. Each apparatus (seven total) consisted of one five-liter flask containing three liters of treatùent solution connected in-line to three 250 mL gas-absorption flasks filled with 100 mL of 0.0125 M barium hydroxide. The five-liter flasks were continuously agitated using a magnetic stir bar and plate. A 50 mL glass burette was used for titrations.

Mineral medium and stock solutions:
Mineral medium was the growth medium and dilution water used for this study. IT was prepared from four stock solutions in accordance with OECD 301B using BMI deionized water and appropriate reagent grade compounds.
Blacksburg, Virginia municipal water was dechlorinated using activated carbon, deionized using mixed bed deionization tanks to a resistivity of > 1 megohm/cm, and polished with a Millipore Milli-Q UV Plus system with a 0.22 µ filter to a fina lresistivity of ≥ 18.1 megohm/cm. This is referred to as BMI deionized water.
Appropriate concentrations of minerals components (potassium and sodium phosphates plus ammonium chloride, calcium chloride, magnesium sulphate and iron (III) chloride) were added to BMI deionized water as specified in the OECD method.

Flask preparation:
1. Each of the six 5-liter treatement flasks contaitend 2 400 mL mineral medium. Inoculum was added to achieve a concentration of about 23.4 mg/L of suspended solids. Mineral medium was then added to make up three liters. A seventh treatement flask was filled with three liters of uninoculated mineral medium for the abiotic sterile control. All seven solutions were aerated with CO2 free air overnight to purge the flasks of carbon dioxide.
2- The refereence compound (sodium acetate) was added to one treatment as a positive control.
3- Possible inhibitory effects were evaluated by adding both the reference and test substance at concentrations equivalent to the other flasks.
4- An abiotic sterile control was prepared by addiding 300 mg cadmium chloride as a sterilizing agent to three liters of uninoculated mineral medium and test substance.
5- The pH was determined and no adjustments were necessary (pH = 7.25 - 7.47).
6- For CO2 traps, three 250 mL absorption flasks were filled with 100 mL of 0.0125 M barium hydroxide solution and joined each 5-liter flask in line. The solution was free of precipitated sulphate and carbonate, and the molar strength was evaluated.
7- Flasks 1&2 contained test substance and inoculum (test solution).
Flasks 3&4 contained only inoculum (inoculum blank).
Flask 5 contained reference compound and inoculum ( procedure control).
Flask 6 contained test substance and sterilizing agent (abiotic sterile control).
Flask 7 contained test substance, reference compound and inoculum (toxicity control).

Reference substance:

acetic acid, sodium salt

Preliminary study:

The ThCO2 is 2.19 mg CO2 evolved per mg of test item.

Test performance:

All validity criteria were successful.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

32.6

Sampling time:

28 d

Details on results:

The degradation for the 10-day window failed. The test substance 10-day window of degradation began just after day 6 (around 10.0% biodegradation) and ended just after day 16 (around 23.6% degradation).
The test substance reached 32.6% biodegradation by test termination.
According to the OECD guideline 301B, the test item cannot be considered readily biodegradable.

The controls for this study (inoculum blank, abiotic sterile control, toxicity control) behaved as expected. The inoculum blank did not exceed the method suggested maximum CO2 production of 40 mg/L. The abiotic sterile control yielded results similar to the inoculum blank as expected. The toxicity control indicated that the test substance was likely not inhibitory.

Results with reference substance:

The reference substance degradation reached the required 60% degradation just before Day 14.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under conditions of OECD guideline 301B, the test item cannot be considered readily biodegradable.

Description of key information

Biodegradation in water was tested in a Manometric Respirometry test according to OECD 301F test method. Under these test conditions, Chromium 2-ethylhexanoate is not readily biodegradable. This test result was confirmed by the result of a second test ran according to OECD 301B method (CO2 Evolution test).

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable