tris(2-octyldodecyl) citrate

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:

15 August 2017 to 14 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)

Version / remarks:

Adopted 1981, Revised 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-tris(2-octyldodecyl) 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

603-121-2

Cas Number:

126121-35-5

Molecular formula:

C66H128O7

IUPAC Name:

1,2,3-tris(2-octyldodecyl) 2-hydroxypropane-1,2,3-tricarboxylate

Test material form:

liquid

Details on test material:

Tris(octyldodecyl) citrate 99.87 %
Bis(octyldodecyl) citrate 0.13 %

Specific details on test material used for the study:

Test substance name: Citmol 320
Synonym: 2,3-Propanetricarboxylic acid, 2-hydroxy-, 1,2,3-tris(2-octyldodecyl) ester, trioctyldodecyl citrate
CAS Number: 1265121-35-5
Molecular formula: C66H128O7
Molecular weight: 1033.71 g/mol
Batch number: P7329
Purity: 99.39%
Date received: 1 March 2017
Retest date: 31 August 2018
Storage conditions: Room temperature (15-30°C) protected from light

All test substance details were supplied by the Sponsor. An analysis report is presented in the attachments.
The carbon content, 77%, was calculated from the molecular weight provided by the Sponsor. This carbon content was used in setting the test concentration of Citmol 320. No allowance was made for purity.
A solubility trial was performed by weighing a sub-sample (294 mg calculated quantity to ensure 225 mg carbon) of Citmol 320 into a suitable flask and making up to 100 mL with reverse osmosis (RO) water. The test substance did not appear to be in solution and was sonicated for 15 minutes and stirred for 15 minutes. As the test substance was not in solution, it was deemed insufficiently soluble to be added as an aqueous stock solution. Citmol 320 was therefore added directly to the test vessels.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic waste-water catchment. The sample was transported in a closed container, but with an adequate headspace, to prevent the sample becoming anaerobic. On arrival, the sample was aerated by means of a compressed air supply.
The suspended solids concentration of the activated sludge was determined by filtering a subsample (25 mL) through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C). The filter and retained solids were then dried in an oven (nominally 105°C) and re-weighed. The weight of the sludge solids was determined from the difference in the weights before and after drying. The concentration of suspended solids was calculated to be 3.96 g/L.
The activated sludge used in this study was not deliberately acclimatised or adapted to Citmol 320 before exposure under test conditions.

Details on study design:

Preparation of Test Vessels
The study consisted of four treatment groups as follows:
Treatment Group Vessel Contents
Blank control inoculated mineral salts medium
Test substance inoculated mineral salts medium and test substance
Reference substance inoculated mineral salts medium and sodium benzoate
Toxicity control inoculated mineral salts medium, test substance and sodium benzoate

The purpose of the toxicity control was to assess the biodegradation of the reference substance in the presence of the test substance.
Duplicate vessels were prepared for the test substance, reference substance and blank control groups. A single vessel was prepared for the toxicity control.

Preparation of Test Medium
The test was conducted in an aqueous, synthetic, mineral salts medium. A test medium concentrate was prepared in RO water containing 30 mL/L solution (a) and
3 mL/L of each of solutions (b), (c) and (d). Solutions (a) to (d) were prepared as follows:

(a) potassium dihydrogen phosphate (8.50 g, VWR); dipotassium hydrogen phosphate (21.75 g, VWR); disodium hydrogen phosphate dihydrate (33.40 g, Fisher); ammonium chloride (0.50 g, Fisher), all dissolved in and made up to 1 L with RO water.
(b) calcium chloride dihydrate (36.40 g, VWR), dissolved in and made up to 1 L with RO water.
(c) magnesium sulphate heptahydrate (22.50 g, Sigma-Aldrich), dissolved in and made up to 1 L with RO water.
(d) ferric chloride hexahydrate (0.25 g, Sigma-Aldrich) and concentrated hydrochloric acid (1 drop, VWR), dissolved in and made up to 1 L with RO water.

On the basis of the suspended solids determined to be 6.2 g/L, the medium was inoculated with activated sludge (116 mL in a total volume of 8 L) to give a suspended solids concentration of 90 mg/L. This provided a nominal final solids concentration of 30 mg/L in each test vessel (500 mL added to a total volume of 1.5 L).
The inorganic carbon (IC) concentration of the inoculated mineral salts medium was determined using an InnovOx carbon analyser. In this analysis, IC in the samples was released as CO2 by acidification with hydrochloric acid. The CO2 was then passed to a non-dispersive infra red (NDIR) detector. The concentration of carbon dioxide was determined in the NDIR detector, by measuring the amount of infra-red energy absorbed by the sample. A calibration check was performed on each occasion by injecting a series of sodium hydrogen carbonate standards. The existing calibration curve was used to quantify the IC present in the samples. Each sample was analysed in triplicate/quadriplicate.

Treatment of Test Vessels
Citmol 320 was accurately weighed (29.40 to 29.45 mg) for direct addition to test substance and toxicity control vessels, to give a nominal test substance concentration corresponding to 15 mg carbon/L.
A reference substance stock solution (2.25 g carbon/L) was prepared by dissolving sodium benzoate (1.93 g) in RO water (500 mL). Reference and toxicity control vessels were treated with the stock solution (10 mL), to give a nominal sodium benzoate concentration corresponding to 15 mg carbon/L.

Test Initiation
Following all test and reference substance treatments and addition of the inoculated medium concentrate, the volume in each vessel (including the blank control vessels) was made up to 1.5 L by addition of RO water. Each vessel was sealed, connected to a series of three traps containing aqueous barium hydroxide (nominally 0.0125 M), and the carbon dioxide-free air supply initiated.

Incubation Conditions
The test vessels were incubated in the dark under the conditions below.

Temperature
The incubation and test measurements were conducted at a target temperature range of 22 ± 2°C.

Measurement of pH
Measurements of pH were made in the blank control and reference substance vessels at the start of incubation and in all vessels at the end of the test prior to the addition of the hydrochloric acid. Measured pH values ranged from pH 7.42 to pH 7.49 on Day 0 and pH 7.43 to pH 7.58 on Day 28 (Table 1).

Air Flow
The air used in this study was delivered from a cylinder of CO2-free air (Air Products) and was regulated in two stages. Initial control was provided by a gas regulator and the air flow to each vessel controlled by individual needle valves. Measurements of the flow rate exiting each test vessel were made at intervals not exceeding seven days, with a bubble flow meter and stopwatch. Adjustments were made as necessary to maintain a flow rate of ca 50 mL per minute.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (CO2 evolution)

Value:

<= 0

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable based on experimental result

% Degradation 2

Parameter:

other: Activated sludge inhibition

Value:

<= 0

Sampling time:

28 d

Remarks on result:

other: No inhibitory effect observed

Details on results:

The inorganic carbon content of the test medium was determined to be 1.68 mg carbon/L, corresponding to 3.73%.

Carbon dioxide evolution and percentage biodegradation data are presented in Table 2, Table 3 and Figure 1 (attached). The mean total carbon dioxide production in the blank control vessels was 47.7 mg/L at the end of the test which was above the 40 mg/L recommended in the guidance document, but lower than the acceptable maximum limit of 70 mg/L. The data and the study are therefore considered valid.

BOD5 / COD results

Results with reference substance:

Sodium Benzoate
Rapid carbon dioxide generation commenced immediately and declined to a more gradual rate over the period of the incubation as shown inFigure 1. The mean percentage biodegradation had exceeded 60% by Day 7 (67%), 86% by the end of the incubation phase on Day 28 and 87% by the end of the study on Day 29. The validity criterion of 60% biodegradation at 14 days was therefore met.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

All validity criteria were satisfied (inorganic carbon content, degradation of reference material, CO2 production from blank controls) and the results of this study are therefore considered to be valid.
No inhibitory effect was observed on the biodegradation of the reference substance in the presence of Citmol 320.
Mean carbon dioxide evolution from Citmol 320 was < 60% of the theoretical carbon dioxide yield throughout the test. The level of biodegradation did not meet the requirements for ready biodegradability and Citmol 320 cannot, therefore, be classified as readily biodegradable.

Executive summary:

The ready biodegradability of Citmol 320 was assessed by measurement of carbon dioxide (CO₂) evolution under standard conditions. The procedure followed was that of OECD Guideline 301B, Ready Biodegradability (Adopted 1981, Revised 1992).

The test substance, Citmol 320, was added to the test system asa solid[BS1] . Buffered mineral salts medium was added to give a test substance concentration equivalent to 15 mg organic carbon/L. The medium was inoculated with microorganisms derived from a sample of activated sludge not previously intentionally exposed to the test substance. Test vessels were incubated in darkness at 22 ± 2°C for 28 days and their contents continuously sparged with a supply of CO₂‑free air. The exhaust air from each vessel was passed through a series of traps containing a barium hydroxide solution to trap evolved CO₂.

At regular intervals during the incubation, traps were detached and their contents titrated with hydrochloric acid to determine the quantity of CO₂evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO₂that may have remained in solution. Titration of the traps was performed following overnight aeration.

The procedure and the activity of the inoculum were checked by measuring the CO₂evolved from vessels containing a reference substance, sodium benzoate. An additional vessel containing a combination of the test and reference substances served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration. Two blank control vessels were also prepared containing inoculated medium only. The results of these vessels were used to check the validity of the test and to correct the evolved CO₂values.

Citmol 320 showed 0% biodegradation during the incubation. As a result, Citmol 320 cannot be considered readily biodegradable.

The mean total CO₂production in the blank control vessels was 47.7 mg/L at the end of the test which was above the 40 mg/L recommended in the guidance document, but lower than the acceptable maximum limit of 70 mg/L. The data and the study are therefore considered valid.

Mean biodegradation of the reference substance had exceeded 60% by Day 7 and had reached a maximum mean of 86 % by the end of the incubation phase on Day 28 and87% at the end of the test on Day 29. The rate of biodegradation of the reference substance in the presence of Citmol 320 (57 % at Day 7, 78% by the end of the incubation phase on Day 28 and 82 % by the end of the test on Day 29) was similar to that of the reference substance alone, suggesting that Citmol 320 did not have an inhibitory effect on the sludge microorganisms under the test conditions and the test was considered to be valid.

The inorganic carbon content of the test medium was determined to be
1.68 mg carbon/L, corresponding to 3.73% satisfying the validity criterion of < 5%.

 

All validity criteria were satisfied, the blank controls and reference vessels performed as expected, and the results of this study are therefore considered to be valid.

 [BS1]Citmol 320 is a liquid not solid. Please amend