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Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted to recognised OECD guideline and GLP. However, significant challenges were observed on study as a result of the test substance physico-chemical properties.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
yes
Remarks:
: minor deviations to standard protocol to account for volatile substance
Principles of method if other than guideline:
An initial preliminary study for 7 days, using a setup similar to that planned for the main phase, found that the initial applied radioactivity had declined to 75.6% between Days 0 and 7 (total recoveries). A large portion of the radiolabelled material was found in the trapping solution and was determined by HPLC to be present mainly as parent compound. The Study Director attributed the loss of radioactivity to the volatility of the test material (both into the traps and generally). The unaccounted for loss of radiolabelled material, was deemed to be lost at sampling when the lids of the vessels were removed. No degradation was observed up to Day 7. The radioactive recovery was below the level deemed acceptable for a full study under the OECD 309 guidelines.

For the main study, a setup designed to minimise losses from volatilsation was used. Vessels were slighlty smaller than recomended in the guideline (250 ml) and had less headspace to reduce volatilisation losses. It was concluded that the reduced headspace would not adversely affect the aeration of the test vessels. A diagram of the study apparatus is attached below [Diagram of test system.pdf]
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
See 'Test material information' for full details.
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on source and properties of surface water:
- Details on collection: The river Great Ouse in Huntingdon, UK, (OS survey location 59° 19' - 0°10). Taken from a depth of 127 cm. Water source not known to be contaminated and has been fully characterised.
- Storage conditions: Water collected and transported in containers, and upon arrival at the test facility was passed through course filter paper.
- Storage length: Used within 1 day of collection
- Temperature (°C) at time of collection: 13.1°C
- pH at time of collection: 7.84
- Oxygen saturation (%): 68
- Hardness (CaCO3): 348 mg/L
- Dissolved organic carbon: 6.4 mg/L
- Biomass (CFU/g):
Day 0; 5.8 x 10^3, Day 60; 3.85 x 10^3 and 5.50 x 10^3 for aerobic bacteria in the untreated controls.
Day 60; 2.15 x 10^6 and 1.91 x 10^6 aerobic bacteria in the solvent treated controls.
- Water filtered: yes (using course filter paper)
- Type and size of filter used, if any: GF/A
Details on source and properties of sediment:
Pelagic test with no sediment required
Details on inoculum:
Pelagic test using natural surface waters.
Duration of test (contact time):
>= 0 - <= 60 d
Initial conc.:
20 µg/L
Based on:
test mat.
Initial conc.:
100 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 100 mL of filtered surface was added into 250 mL glass dreschel vessels. Portions of the required volumes of test stock solutions dissolved in ethanol were dispensed into individual flasks. These concentrated stock solutions, were diluted as appopriate to prepare treatment solutions. Aliquots (100 µL) were applied to samples of surface water. Determinations of radioactivity and t-Butyl acetate added to each sample were conducted by radioassay (LSC). An application rate of 21 µg/L and 105 µg/L was achieved.

- Composition of medium: Natural surface water (see 'details on source and properties of surface water' in IUCLID for further information).
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): Test material was dissolved in ethanol (concentrated stock solutions of 0.02 and 0.1 mg/mL)
- Test temperature: 12 ± 2°C (minor deviations from this range occured for short periods). The highest temperature observed was 14.3°C which was outside the range for a maximum of 5 minutes. This deviation was considered not to have affected the validity or integrity of the study.
- pH: The pH values ranged from 7.22 to 8.45 over the course of the study.
- pH adjusted: no
- CEC (meq/100 g): N/A
- Aeration of dilution water: Not aerated directly (sealed vessels). Aeration was maintained by continuous stirring and vessels were mainatined in an aerobic atmosphere as a result of air in the headspace. Oxygen measurements taken during the study confirmed a range of 82 to 98% air saturation was maintained. Oxygen measurements were not taken in error from days 5 to 33 of study. However, it was considered that the surface water remained aerobic during the incubation phase as the oxygen concentrations on Day 40 onwards provided acceptable levels.
- Suspended solids concentration: < 10 mg/L
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: None

TEST SYSTEM
- Culturing apparatus: N/A
- Number of culture flasks/concentration: See table 1 in 'Any other information on materials and methods incl. tables'.
- Method used to create aerobic conditions: See above 'aeration of dilution water'.
- Measuring equipment: Various depending on endpoint
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Ethyl digol

SAMPLING
- Sampling frequency: For both concentrations, duplicate replicates in surface water were taken for analysis immediately. After application, and after 5, 12, 19, 26, 33, 40, 47, 54 and 60 days of incubation, triplicate samples in surface water were analysed.
- Sampling method: Samples were taken for chromatographic and direct 14CO2 determination.
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A - analysed immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Included, blank flasks containing no test substance.
- Solvent blank: Included, blank flasks containing no test substance and solvent.
- Abiotic sterile control: Included and sterilized by autoclaving prior to addition of test substance.
- Toxicity control: Not required

STATISTICAL METHODS:
The decline of t-Butyl acetate in surface water were modelled using the Single First Order (SFO) kinetic model. The software ModelMaker (version 4.0, ModelKinetix, Oxford, UK) was used, employing default Marquardt optimisation and Runge-Kutta integration settings. An assessment of goodness-of-fit was made by visual inspection and by Chi-squared statistical analysis. Both replicate values were used in the kinetic modelling where appropriate.

SFO kinetics are described by the equation:
M = M0 x E^-KT

Where M is the amount of test substance at time t, M0 is the amount of test substance at zero-time and k is the rate constant. DT50 and and DT90 values are:

DT50 = ln2/k
DT90 = ln10/k

Statistical analyses was conducted on the results of surface water (including all data but not radio-activity losses through volatilisation to ethyl digol traps), surface water (including all data and radio-activity losses through volatilisation to ethyl digol traps), and surface water (including all data and radio-activity losses through volatilisation to ethyl digol traps, excluding anomalous data points (Day 47 single rep and Day 54 at 20 µg/L and Day 40 single rep and Day 54 at 100 µg/L)). The results from the latter assessment (all data included, removing anomalous data) are considered the most relevant, and as such results from the other analyses are not included in IUCLID.
Reference substance:
other: 14C-benzoic acid
Test performance:
Validity of the test:

Reference substance:
The total recoveries of radioactivity in the surface water treated at 9 µg/L of [14C]-benzoic acid were 90.5 – 100.7% applied radioactivity after 40 days. Direct volatile radioactivity, all associated with 14CO2, accounted for 77 to 93% applied radioactivity after 40 days. Therefore, the test was shown to be valid as the reference substance degraded within the expected time interval.

Mass balance
Samples for direct 14CO2 determination: The total recoveries of radioactivity (mass balance: ie the sum of radioactivity in the water layer and volatile radioactivity) from samples in the main experiment were 80.0 – 108.3% applied radioactivity. Except for the 20 µg/L at day 33 and 100 µg/L at day 12 where single replicates had anomalous low recoveries (70.7% AR and 64.1% AR respectively). In addition, for the 100 µg/L concentration at day 40 the sample was assumed not to have been treated.

The specific test vessels set up for the determination of mass balance at the end of test (Day 60) provided mass balance ranges of 91.5 - 106.7% (ranges at 20 and 100 µg/L) applied radioactivity indicating test validity.

The OECD 309 guideline states that 'the total recovery (mass balance) at the end of the experiment should be between 90-110% for radiolabelled substances. However, the indicated ranges should only be interpreted as targets and should not be used as criteria for acceptance of the test'.

Due to the physicochemical properties of the substance (high volatility) the achieved mass balance based on recoveries of 80-108.3% is considered acceptable based on the complexities of testing with this substance. The actual ranges from the mass balance vessels analysed at the end of study were also within the detailed guideline ranges.

Compartment:
natural water: freshwater
% Recovery:
99.7
St. dev.:
5.4
Remarks on result:
other:
Remarks:
Mean of mass balance vessel recoveries (Fm) at both test concentrations
Compartment:
natural water: freshwater
Half-life:
> 138 - < 252 d
Type:
(pseudo-)first order (= DT50)
Temp.:
12 °C
Remarks on result:
other: DT50
Compartment:
natural water: freshwater
Half-life:
> 459 - < 836 d
Type:
other: DT90
Temp.:
12 °C
Remarks on result:
other: DT90
Mineralization rate (in CO2):
3 other: max 2-3% mineralisation (as CO2) observed
Transformation products:
yes
No.:
#1
Reference substance:
Composition 1
No.:
#2
Reference substance:
Composition 1
Details on transformation products:
Two potential degradants were noted during the experimental phase (one present at greater than 10% after 60 days, and the other at ca. 6% applied radioactivity was seen only in one replicate on Day 26 and was not further investigated). Although considerable effort was expended to attempt identification, the identity of the degradant present at greater than 10% could not be confirmed.

Based on the known chemistry of the test substance and considering potential abiotic and biotic degradation products, attempts were made to identify the unknown by HPLC co-chromatography and GC-MS. However, by using co-chromatography with known reference standards, the similar substances t-butyl alcohol, acetic acid, t-butyl hydroperoxide, α-hydroxyisobutyric acid, 2-methyl propane-1,2 diol and 4,4-dimethyl-2-pentanone were ruled out as possible candidates for the unknown peak. Due to the volatile nature of the test substance, it was not possible to isolate sufficient material for GC-MS analysis.

Although beyond the remit of the study to investigate, it was considered possible that the unknown peak may have been the result of an analytical artefact. See attachment 'Summary document - Final' in 'attached background material' for more information.
Evaporation of parent compound:
yes
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes (except for minor deviations in temperature and oxygen measurements as per 'details on study design' section.
- Anomalies or problems encountered (if yes): Test was highly complex and issues were observed as a result of the high volatility of the test substance.

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: Maximum AR for the unknown (at 20 µg/L) was 62.2% after 47 days. (Most readings for unknown were between 3.9 and 32.3 %; one reading on Day 54 reached 89.3% AR but this was considered an anomalous result). Maximum AR for the unknown (at 100 µg/L) was 47.7% after 40 days. (Most readings for unknown were between 9.9 and 26.7%; readings on Day 54 reached 98.4-102.2% AR but were considered anomalous results).


MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: Only observed at ca. 6% AR in one replicate on Day 26 and was not further investigated.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: Total recoveries of radioactivity (mass balances) for samples treated at 20 µg/L and 100 µg/L were generally between 80.0 – 108.3% applied radioactivity, with minor anomalous values on a small number of days observed.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: max 2-3 % mineralisation (as CO2) observed

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Volatile radioactivity associated with t-Butyl acetate was recovered in the ethyl digol trap, at a maximum of 40.7% and 29.1% applied radioactivity within 60 days at 20 and 100 µg/L respectively.

STERILE TREATMENTS:
- Transformation of the parent compound: None observed (only 0.6 to 1 % mineralisation (as CO2) observed).
- Formation of transformation products: None observed
- Volatilization: Yes; volatile radioactivity associated with tert-butyl acetate, was recovered in the ethyl digol trap at a maximum of 39.3% applied radioactivity after 60 days.

RESULTS OF SUPPLEMENTARY EXPERIMENT: Two preliminary experiments were conducted. The first involved treatment at 20 µg/L of the test substance. This experiment was conducted with sealed vessels which provided a 7 day recovery value of 75.7% AR. Volatile radioactivity in the ethyl digol traps was shown to be tert-butyl acetate and no degradation was observed. The second experiment was conducted with sealed vessels that were purged with air through a series of flow-through traps before water sampling and the recovery at 2 days was improved on that obatined in experiment 1 (91.9% AR). Based on the preliminary experiments, the main experiment was conducted at 20 and 100 µg/L (nominal) using the setup determined to be most suitable from experiment 2.
Results with reference substance:
The total recoveries of radioactivity in the surface water treated at 9 µg/L of reference substance, [14C]-benzoic acid, were 90.5 – 100.7% applied radioactivity after 40 days. Direct volatile radioactivity, all associated with 14CO2, accounted for 77 to 93% applied radioactivity after 40 days. Therefore, the test was shown to be valid as the reference substance degraded within the expected time interval. The solvent used was determined not to have affected the test system.

See attachment 'Results Tables' in 'attached background material' for more information.

Validity criteria fulfilled:
yes
Conclusions:
The test substance t-Butyl acetate was shown not to significantly mineralise in the surface water, with calculated half-life (DT50) values of 138 days (at 20 µg/L) and 252 days (at 100 µg/L).

Two potential degradates were noted during the experimental phase (one present at greater than 10% after 60 days and the other at ca. 6% AR was seen only in one replicate on Day 26 and was not further investigated). Although considerable effort was expended to attempt identification, the identity of the degradant present at greater than 10% could not be confirmed.

Based on the known chemistry of the test substance and considering potential abiotic and biotic degradation products, attempts were made to identify the unknown by HPLC co-chromatography and GC-MS. However, by using co-chromatography with known reference standards, other similar substances were ruled out as possible candidates for the unknown peak.

Due to the volatile nature of the test substance, it was not possible to isolate sufficient material for GC-MS analysis. It was considered possible that the unknown peak may have been the result of an analytical artefact.

The experimental conduct of this study was challenging mainly due to the high volatility of the test substance. Although the Henry’s Law constant (83.2 Pa.m3/mol) was within the parameters considered acceptable for slightly volatile substances in the OECD 309 guideline (<100 Pa.m3/mol), under experimental conditions it was considered that the test substance was too volatile for this guideline test. Despite the nature of the test substance, the test design achieved acceptable mass balances and considerable efforts were undertaken to identify the possible unknown degradates. Therefore, it was considered that the study results and methodology met the requirements of the relevant study guideline.

Under the specific conditions of this test, mineralisation from surface (pelagic) water is not expected to be a significant method for removal of t-butyl acetate from the environment.

In isolation t-Butyl acetate could be concluded to be persistent in surface waters under the conditions of this test, however this ignores its main route of removal from fresh waters which will be from volatilisation to the atmospheric compartment and subsequent rapid photodegradation (estimated half-life for atmospheric degradation is 19.7 days).
Executive summary:

The test substance t-Butyl acetate was shown not to significantly mineralise in the surface water, with calculated half-life (DT50) values of 138 days (at 20 µg/L) and 252 days (at 100 µg/L).

Under the specific conditions of this test, mineralisation from surface (pelagic) water is not expected to be a significant method for removal of t-butyl acetate from the environment.

In isolation t-Butyl acetate could be concluded to be persistent in surface waters under the conditions of this test, however this ignores its main route of removal from fresh waters which will be from volatilisation to the atmospheric compartment and subsequent rapid photodegradation (estimated half-life for atmospheric degradation is 19.7 days).

Description of key information

OECD 309 pelagic study conducted as a result of ECHA dossier evaluation.

Key value for chemical safety assessment

Half-life in water:
138 d
at the temperature of:
12 °C

Additional information

The experimental conduct of this study was challenging mainly due to the high volatility of the test substance. Although the Henry’s Law constant (83.2 Pa.m3/mol) was within the parameters considered acceptable for slightly volatile substances in the OECD 309 guideline (<100 Pa.m3/mol), under experimental conditions it was considered that the test substance was too volatile for this guideline test. Despite the nature of the test substance, the test design achieved acceptable mass balances and considerable efforts were undertaken to identify potential degradants. Therefore, it was considered that the study results and the methodology met the requirements of the relevant study guideline.

Under the specific conditions of this test, mineralisation from surface (pelagic) water is not expected to be a significant method for removal of t-Butyl acetate from the environment.

In isolation t-Butyl acetate could be concluded to be persistent in surface waters under the conditions of this test, however this ignores its main route of removal from fresh waters which will be from volatilisation to the atmospheric compartment and subsequent rapid photodegradation (estimated half-life for atmospheric degradation is 19.7 days).

The test substance t-Butyl acetate was shown not to significantly mineralise in surface water in a pelagic water simulation test, with calculated half-life (DT50) values of 138 days (at 20 µg/L) and 252 days (at 100 µg/L).