Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-27 to 2016-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.24 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Source and lot/batch No.of test material: Source – Blychem Ltd; Lot No. – 12BLY066-Re

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98%
- Specific activity: 0.81 MBq/mg
- Locations of the label: within six carbon benzene ring
- Expiration date of radiochemical substance:2016-08-27

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -20 deg C
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Miscible with and stable in selected solvent
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Non-reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Re-purified by HPLC to achieve radiochemical purity of 98%

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Used in normal physical form (liquid)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

Sample 1 Swiss Lake:
- Details on collection - Location: Swiss Lake, Chatsworth, Derbyshire, England
Sampling depth: 10-15 cm
Contamination history: Lake fed by surface water run-off mainly originating from moorland and woodland. No pesticide use
in area over last 5 years
- Storage conditions: circa +4 deg C, in the dark
- Storage length: 7 days
- Temperature (°C) at time of collection: 11.3 deg C
- pH at time of collection: 6.75
- Electrical conductivity: 105 uS/cm
- Redox potential (mv) initial/final: 250.5 at start of acclimatisation; 264.1 at end of test
- Oxygen concentration: 83%
- Hardness (mg/L): 30
- Dissolved organic carbon (mg/L): 5.1
- Biomass (as % TOC): - 3% at start of system acclimatisation
- Water filtered: Yes
- Type and size of filter used: 212 um sieve

Sample 2 – Calwich Abbey Lake:
- Details on collection - Location: Calwich Abbey Lake, Calwich, Staffordshire
Sampling depth: 40-50cm
Contamination history: Perennial lake fed by a stream from a weir on river Dove. Fringed by woodland. Ley grassland sloping
away from the lake to the south. No pesticide use in area over last 5 years.
- Storage conditions: circa +4 deg C, in the dark
- Storage length: 7 days
- Temperature (°C) at time of collection: 12.7 deg C
- pH at time of collection: 8.03
- Electrical conductivity:508 uS/cm
- Redox potential (mv) initial/final: - 201.9 at start of acclimatisation; 188.6 at end of test
- Oxygen concentration: 75-76%
- Hardness (mg/L): 230
- Dissolved organic carbon (mg/L): 4.6
- Biomass (as % TOC): - 1% at start of system acclimatisation
- Water filtered: Yes
- Type and size of filter used: 212 um sieve

Details on source and properties of sediment:

Sample 1 - Swiss Lake:
- Details on collection - Location: Swiss Lake, Chatsworth, Derbyshire, England
Sampling depth: 10-15 cm (water); top 5 cm (sediment)
Contamination history: Lake fed by surface water run-off mainly originating from moorland and woodland. No pesticide use
in area over last 5 years
- Storage conditions: circa +4 deg C, in the dark
- Storage length: 7 days
- Textural classification (i.e. %sand/silt/clay): 93.8% sand; 3.7% silt; 2.5% clay
- pH: - 6.6
- Organic carbon (%): 0.5%
- Redox potential (mv) initial/final: - -255.3 at start of acclimatisation; -382.3 at end of test
- CEC (meq/100 g): - 3.3
- Biomass: 3.0% (as % of total organic carbon)
- Sediment samples sieved: Yes - 2 mm sieve

Sample 2 - Calwich Abbey Lake:
- Details on collection - Location: Calwich Abbey Lake, Calwich, Staffordshire
Sampling depth: 40-50cm (water); top 5 cm (sediment)
Contamination history: Perennial lake fed by a stream from a weir on river Dove. Fringed by woodland. Ley grassland sloping
away from the lake to the south. No pesticide use in area over last 5 years.
- Storage conditions: circa +4 deg C, in the dark
- Storage length: 7 days
- Textural classification (i.e. %sand/silt/clay): 36.5% sand; 55.8% silt; 7.7% clay
- pH: - 7.0
- Organic carbon (%): 7.3%
- Redox potential (mv) initial/final: - -263.5 at start of acclimatisation; -407.1 at end of test
- CEC (meq/100 g): - 27.8
- Biomass: 1.0% (as % of total organic carbon)
- Sediment samples sieved: Yes - 2 mm sieve

Duration of test (contact time):

100 d

Initial conc.:

0.3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 0.12 mL (radiolabelled TOTM in acetonitrile) equating to 44.28 µg (35.86 KBq) TOTM
- Test temperature: 12 +/- 2 deg C
- pH: 7 - 8
- pH adjusted: No
- CEC (meq/100 g): 3.3 / 27.8
- Continuous darkness: Yes
- Any indication of the test material adsorbing to the walls of the test apparatus: Vessel rinse indicated ca. 13% TAR on vessels walls on dosing the test system. Rapid desorption occurred as the test system equilibrated and ca. 1% TAR remained adsorbed on vessel walls for the majority of the study (Days 7 to 100)

TEST SYSTEM
- Culturing apparatus: Bespoke glass test vessel
- Number of culture flasks/concentration: 14 for each sediment type
- Method used to create aerobic conditions: Pre-moistened air passed over surface of water
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Series of trapping vessels containing solutions of ethylene glycol, sulphuric acid (0.05 M) and sodium hydroxide (2 M) per replicate.

SAMPLING
- Sampling frequency: Days 0, 1, 7, 14, 28, 59 and 100 after application of test substance
- Sampling method: Destructive

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, duplicate
- Abiotic sterile control: No
- Toxicity control: No

STATISTICAL METHODS: The kinetic modelling software KinGUII (version 2.1) was used alongside the guidance of Boesten et al (Guidance Document on Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on Pesticides in EU Registration, Sanco/10058/2005, version 2.0, June 2006) to estimate the kinetics of the parent compound in the water, sediment and total water-sediment system. For metabolites the formation and degradation was estimated in the total system only. The KinGUII optimizer used was IRLS (Iteratively Reweighted Least Squares) and the optimization method was LM (Levenberg-Marquardt).
Goodness of fit was assessed visually and statistically, a chi2 value of < 15 % generally being considered to be a good statistical fit.
A t-test probability value of < 0.05 was sufficient to indicate that the slope of the determined regression line was significantly different from zero

Compartment:

natural water / sediment: freshwater

% Recovery:

90.66

Remarks on result:

other:

Remarks:

Swiss Lake

Compartment:

natural water / sediment: freshwater

% Recovery:

94.34

Remarks on result:

other:

Remarks:

Calwich Abbey Lake

Key result

% Degr.:

17.99

Parameter:

CO2 evolution

Sampling time:

100 d

Remarks on result:

other:

Remarks:

Swiss Lake

Key result

% Degr.:

16.45

Parameter:

CO2 evolution

Sampling time:

100 d

Remarks on result:

other:

Remarks:

Calwich Abbey Lake

Key result

Compartment:

natural water: freshwater

DT50:

0.04 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Swiss Lake

Key result

Compartment:

natural water: freshwater

DT50:

0.07 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Calwich Abbey lake

Key result

Compartment:

natural sediment: freshwater

DT50:

39.31 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Swiss Lake

Key result

Compartment:

natural sediment: freshwater

DT50:

53.82 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Calwich Abbey Lake

Key result

Compartment:

natural water / sediment: freshwater

DT50:

15.62 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Swiss lake

Key result

Compartment:

natural water / sediment: freshwater

DT50:

25.23 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: Calwich Abbey Lake

Mineralization rate (in CO2):

0.166 w-1

Other kinetic parameters:

pseudo-first order rate constant

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Formation and decline of each transformation product during test: One metabolite was formed in both water-sediment systems over the course of the 100 days of the study at levels greater than 10 % of the applied radioactivity. This metabolite was identified in both systems as the di-esters of the parent substance, a tri-ester. Minor metabolites were identified as the mono-esters and trimellitic acid together with one other, unidentified, minor metabolite. There was evidence that trimellitc acid was further degraded and for mineralisation to occur, significant levels of radio-labelled carbon dioxide being measured.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: Sampling on Days 59 and 100 from Swiss Lake and Day 100 from Calwich Abbey revealed a mass balance < 90 % TAR. A large amount of radioactivity was detected in the NaOH traps at these time-points. It is believed that the low mass balance may be due to leakage from these traps.

RESULTS
SWISS LAKE
MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
The di-ester metabolites detected in water phase at >10% TAR – Day 1 1.42%; Day 7 5.93%; Day 14 10.66%; Day 28 6.81%; Day 59 5.12% and Day 100 2.31%. A second, unidentified metabolite detected in the water phase at >5% TAR – Day 7 2.04%; Day 14 5.26%; Day 28 6.27%; Day 59 1.74% and Day 100 0.70%.
In the sediment phase, the di-ester metabolites detected >10% TAR – Day 1 0.96%; Day 7 13.01%; Day 14 17.49%; Day 28 37.37%; Day 59 19.90% and Day 100 20.57%. A second, unidentified metabolite detected in the sediment phase at <5% TAR – Day 59 3.38% and Day 100 3.64%.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
The mono-ester metabolites detected in water phase from Day 7 - Day 7 0.13% TAR; Day 14 0.92% TAR; Day 28 1.99% TAR. Trimellitic acid detected at 0.11% TAR on Day 28.
In the sediment phase, trimellitic acid was detected at 2.64% Tar on Day 59 and 2.77% TAR on Day 100.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
In addition to the unknown major metabolite, above, % TAR of other unknown metabolites ranged from 0.05% to 2.88% in the water phase and 0.84% to 3.47% in the sediment phase.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 46.67% TAR
- % of applied amount at end of study period: 61.16% TAR

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.04% TAR
- % of applied amount at end of study period: 5.32% TAR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: For Swiss Lake, total of 17.99% TAR over the course of the study.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: <0.3% TAR

CALWICH ABBEY
MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
The di-ester metabolites detected in water phase at >10% TAR – Day 1 2.15%; Day 7 8.92%; Day 14 7.70%; Day 28 4.41%; Day 59 2.54% and Day 100 4.60%. A second, unidentified metabolite detected in the water phase at >5% TAR – Day 14 0.67%; Day 28 4.94%; Day 59 6.77% and Day 100 5.19%.
In the sediment phase, the di-ester metabolites detected >10% TAR – Day 1 1.84%; Day 7 14.66%; Day 14 25.10%; Day 28 33.76%; Day 59 27.53% and Day 100 18.80%. A second, unidentified metabolite detected in the sediment phase at >5% TAR – Day 59 0.69% and Day 100 0.56%.


MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
The mono-ester metabolites detected in water phase from Day 14 - Day 14 0.91% TAR; Day 28 0.43% TAR. Trimellitic acid detected from Day 14 – Day 14 0.16% TAR; Day 28 1.65% TAR; Day 59 1.15% TAR and Day 100 1.54% TAR.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
In addition to the unknown major metabolite, above, % TAR of other unknown metabolites ranged from 0.06% to 2.81% in the water phase and from 0.12% to 0.38% in the sediment phase.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 46.97% TAR
- % of applied amount at end of study period: 43.65% TAR

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.22% TAR
- % of applied amount at end of study period: 6.77% TAR

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: A total of 16.54% TAR over the course of the incubation period

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: <1.00% TAR

Validity criteria fulfilled:

yes

Conclusions:

The rate and route of degradation of the registered substance under aerobic conditions was investigated in two aquatic sediment systems and the disappearance time 50 % (DT50) of the substance in the two systems The rate and route of degradation of the registered substance under aerobic conditions was investigated in two aquatic sediment systems, in the absence of light, at 12±2°C.

Degradation and dissipation of the substance was demonstrated to be bi-phasic within the water-sediment systems. Dissipation was driven by multiple processes - partition into the water and the sediment phase, initial degradation to di-esters of the parent compound followed by further degradation and formation of carbon dioxide. The registered substance does not fulfil the persistence criterion according to REACH, Annex XIII for fresh water sediment (degradation half-life for fresh water sediment > 120 days) and would not therefore require classification as persistent (P) or very persistent (vP).

Executive summary:

The rate and route of degradation of the registered substance under aerobic conditions was investigated in two aquatic sediment systems, in the absence of light, at 12±2°C. Methods used were in accordance with OECD Guidelines for the Testing of Chemicals. Test No 308: Aerobic and anaerobic transformation in aquatic sediment systems OECD (2002) and C.24 (Commission Regulation (EC) No. 440/2008).

 

Water sediment from Swiss Lake and Calwich Abbey Lake was treated with the radio-labelled substance at a single concentration in the water phase. At intervals, samples were taken and analysed. This data was then used to determine the disappearance time 50 % (DT50) of the substance in the two systems.

 

One metabolite was formed in both water-sediment systems over the course of the 100 days of the study at levels greater than 10% of the applied radioactivity. This metabolite was identified in both systems as the di-esters of the parent substance, a tri-ester. Minor metabolites were identified as the mono-esters and trimellitic acid together with one other, unidentified, minor metabolite. There was evidence that mineralisation occurs, significant levels of radio-labelled carbon dioxide being measured.

 

The DT50 of the parent substance from the two systems was <1 day from the water phase, 39-54 days from the sediment phase and 16-25 days from the total system.

 

Degradation and dissipation of the substance has been demonstrated to be bi-phasic within a water sediment system. Dissipation is driven by multiple processes, partition into the water and the sediment phase, initial degradation to di-esters of the parent compound followed by further degradation and formation of carbon dioxide. The registered substance does not fulfil the persistence criterion according to REACH, Annex XIII for fresh water sediment (degradation half-life for fresh water sediment > 120 days) and would not therefore require classification as persistent (P) or very persistent (vP).

Description of key information

DT 50 in water and sediment

Key value for chemical safety assessment

Half-life in freshwater:

0.07 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

53.82 d

at the temperature of:

12 °C

Additional information

In accordance with REACH Regulation 1907/2006, Annex IX, Column 2, 9.2.1.3, simulation tests in water and sediments should be considered unless the substance is readily biodegradable. While the substance cannot be regarded as readily biodegradable there is evidence that ultimate degradation can occur. A value for the half-life of the substance in sediment has been calculated using a Fugacity model according to Mackay, Level III using EPIWIN (v.4.10). Data are available on long-term toxicity to sediment dwelling organisms indicating toxicity not to be a concern and further testing beyond the water screening tests is not considered appropriate.