Dioctyl maleate, branched

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:

14 September 2017 to 20 October 2017

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:

OECD Guideline 301B, Ready Biodegradability
(Adopted 1981, Revised 1992)

GLP compliance:

yes

Specific details on test material used for the study:

Bernel Ester DCM
CAS Number 85566-63-8
Retest July 2018

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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 6.2 g/L.
The activated sludge used in this study was not deliberately acclimatised or adapted to Isotridecyl 3,5,5-trimethylhexanoate before exposure under test conditions.

Duration of test (contact time):

ca. 29 d

Initial conc.:

ca. 15 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

reparation of Test Vessels:
The study consisted of four treatment groups as described in the following table:

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 withRO water.
(c) magnesium sulphate heptahydrate (22.50 g, Sigma-Aldrich), dissolved in andmade up to 1 L with RO water.
(d) ferric chloride hexahydrate (0.25 g, Sigma-Aldrich) and concentrated hydrochloricacid (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

Test material was accurately weighed 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. During the course of the study, the temperature decreased to 17.6°C. Thereafter temperature settled back to target range. This did not appear to affect the study integrity.

- 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 7.54 to pH 7.66 on Day 0 and pH 7.44 to pH 7.61 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.

- Calculations:
Theoretical CO2 Yields
The theoretical yield of carbon dioxide (TCO2 in mg) from cultures containing the test and/or reference substances was calculated as shown below.

TCO2 = D(abs) x Pc x 3.667 where:

D(abs) = the absolute dose i.e. the amount (mg) of test or reference substance added to the culture
Pc = the percentage carbon content of the test or reference substance 3.667 = the weight (mg) of CO2 produced from 1 mg of carbon.

The theoretical CO2 yield for the quantity of Isotridecyl 3,5,5-trimethylhexanoate applied to the test vessels and toxicity control vessel was 82.5 mg CO2. Theoretical yields for the quantities of applied sodium benzoate in the toxicity control vessel and reference vessels were also 82.5 mg CO2. The combined theoretical yield from the toxicity control vessel was 165 mg CO2. The purpose of this control was not to assess the extent of degradation of the entire mixture, but instead to assess the impact of the presence of the test substance on the degradation of the reference material. The theoretical yield in this case was, therefore, limited to the 82.5 mg CO2 expected from the sodium benzoate alone and the actual yield corrected for that from the test substance.

Measured CO2 Yields as a Percentage of Theoretical

Biodegradation (Dt) of the reference substance and of Isotridecyl 3,5,5trimethylhexanoate expressed in terms of percentage theoretical CO2 yield was calculated by applying the formula:

Dt = (cumulative mf CO2 produced at time (t))/82.5 x 100

The cumulative CO2 values for the test substance, reference substance and toxicity control were corrected for the mean CO2 generated by the blank controls, to determine the percent biodegradation.

- Validity Criteria:
The following criteria were required for the study to be considered valid:
• the IC content of the mineral medium was less than 5% of the total carbon content at the beginning of the test
• degradation of the reference material reached 60% by Day 14
• duplicate percentage biodegradation values for vessels containing the test substance differ from one another by less than 20%
• total CO2 production from each blank control vessel did not exceed 70 mg/L

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The inorganic carbon content of the test medium was determined to be 1.93 mg carbon/L, corresponding to 4.29%.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

47

Sampling time:

29 d

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 in Figure 1. The mean percentage biodegradation had exceeded 60% by Day 7 (66%), 85% by the end of the incubation phase on Day 28 and 86% by the end of the study on Day 29. The validity criterion of 60% biodegradation at 14 days was therefore met.

- Toxicity Control:
Assessment of biodegradation in the toxicity control was confined to the sodium benzoate fraction. The rate of biodegradation of the reference substance in the presence of Isotridecyl 3,5,5-trimethylhexanoate (63% at Day 7, 75% at the end of the incubation phase on Day 28 and 76% by the end of the test on Day 29) was higher than that of the reference substance alone. This suggests that Isotridecyl 3,5,5trimethylhexanoate had no inhibitory effect on the sludge microorganisms under the test, the test was considered to be valid.

Day	% Biodegradation
	Reference			Test Substance			Toxicity Control *
	5	6	Mean	7	8	Mean	9
0	0	0	0	0	0	0	0
1	13	13	13	0	1	0	12
4	48	46	47	4	7	6	50
6	65	63	64	14	22	18	82
8	75	71	73	24	39	31	106
11	79	77	78	38	60	49	129
13	79	79	79	49	70	60	138
15	82	80	81	58	76	67	141
18	84	80	82	64	79	71	144
20	87	82	85	66	81	74	146
22	90	82	86	67	81	74	147
25	92	83	88	68	82	75	150
*	Calculated on Reference substance only
Note: Data not fully QC checked so please treat with caution

 

 

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 the test material.
Mean carbon dioxide evolution from the test material was >70% of the theoretical carbon dioxide yield throughout the test. The level of biodegradation did not meet the requirements for ready biodegradability and test material cannot, therefore, be classified as readily biodegradable.

Executive summary:

The ready biodegradability of the test material 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,, was added to the test system as a direct addition. 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.

The mean total CO₂production in the blank control vessels was 43 mg/L at the end of the test, satisfying the validity criterion of < 70 mg/L.

The test material showed < 60% biodegradation during the incubation. As a result, it cannot be considered readily biodegradable.

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.

Description of key information

Study conducted to recognised testing guidelines and with GLP certification.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater