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Environmental fate & pathways

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:
2/8/2017 to 12/10/2017
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:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
Adopted 1981, Revised 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test substance name: Bernel Ester TCC
CAS Number: 16502-99-1
Molecular formula: C30H56O7
Molecular weight: 528.771 g/mol
Batch number: P7803
Purity: 94%
Date received: 12 June 2017
Retest date: 23 January 2019
Storage conditions: Room temperature (15-30°C)

A solubility trial was performed by weighing a sub-sample (330 mg calculated quantity to ensure 225 mg carbon) of Bernel Ester TCC and made up to 100 mL with reverse osmosis (RO) water. The test substance did not appear to be in solution and was sonicated for 5 minutes and stirred for 5 minutes. As the test substance was not in solution, it was deemed insufficiently soluble to be added as an aqueous stock solution. Bernel Ester TCC 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:
Activated Sludge 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 2.76 g/L. The activated sludge used in this study was not deliberately acclimatised or adapted to Bernel Ester TCC before exposure under test conditions.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
ca. 15 mg/L
Based on:
other: Carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation 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 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 2.76 g/L, the medium was inoculated with activated sludge (261 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:
Bernel Ester TCC was accurately weighed (33.07 to 33.18 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 incubationa and in all vessels at the end of the test prior to the addition of the hydrochloric acid. Measured pH values ranged from pH 7.45 to pH 7.51 on Day 0 and pH 7.49 to pH 7.64 on Day 28.

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
Key result
Parameter:
% degradation (CO2 evolution)
Value:
<= 7
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Details on results:
Inorganic Carbon (IC) Content of the Test Medium
The inorganic carbon content of the test medium was determined to be 1.62 mg carbon/L, corresponding to 3.6%.

Measured CO2 Yields as a Percentage of Theoretical
Carbon dioxide evolution and percentage biodegradation data are presented in Table 2, Table 3 and Figure 1. The mean total carbon dioxide production in the blank control vessels was 43.3 mg (Test end), satisfying the validity criterion of less than 120 mg/L.

BERNEL ESTER TCC
To be considered readily biodegradable, a test substance must achieve 60% biodegradation by the end of the test. Additionally, the test substance must biodegrade by at least 60% within 10 days after 10% biodegradation has been achieved.
Bernel Ester TCC showed limited biodegradation during the study with a maximum mean biodegradation of 7%. Bernel Ester TCC cannot, therefore, be considered readily biodegradable.
Percent biodegradation values at each sampling interval, for the two replicates containing Bernel Ester TCC did not vary by more than 2%, therefore satisfying the validity criterion of less than 20% difference.

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 Bernel Ester TCC did exceed the 25% recommended by the guidance document on day 2 (32%) and reached a maximum of 37%, therefore the test was considered to be 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 in Figure 1. The mean percentage biodegradation had reached 60% by Day 7, 78% at the end of the incubation phase on Day 28 and by the end of the study on Day 29, the mean biodegradation had reached 79%. 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:
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 testb
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 120 mg/L
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 Bernel Ester TCC.
Mean carbon dioxide evolution from Bernel Ester TCC was ≤7% of the theoretical carbon dioxide yield throughout the test. The level of biodegradation did not meet the requirements for ready biodegradability and Bernel Ester TCC cannot, therefore, be classified as readily biodegradable.
Executive summary:

The ready biodegradability of Bernel Ester TCC 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, Bernel Ester TCC, was added to the test system directly. 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 CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that 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 CO2 evolved 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 CO2 values.

Bernel Ester TCC showed < 10% biodegradation during the incubation. As a result, Bernel Ester TCC cannot be considered readily biodegradable.

The mean total CO2 production in the blank control vessels was 43.3 mg/L at the end of the test, satisfying the validity criterion of < 120 mg/L.

Mean biodegradation of the reference substance had reached 60% by Day 7 and had reached a mean of 78 % by the end of the incubation phase on Day 28 and 79% at the end of the test. The rate of biodegradation of the reference substance in the presence of Bernel Ester TCC did exceed the 25% recommended by the guidance document on day 2 (32%) and reached a maximum of 37%, therefore the test was considered to be valid. The observed values are noted to be low, however this seems likely to be indicative of the variability of the biological nature of the test system.

The inorganic carbon content of the test medium was determined to be 1.62 mg carbon/L, corresponding to 3.6 % 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.