Phosphoric acid, mono- and di-C16-18-alkyl esters

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This experimental study is a preliminary test performed to establish the feasibility of the full and definitive test according the TG OECD 308.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The study is a preliminary test performed to establish the feasibility of the full and definitive test.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

Phosphoric acid octadecyl ester, [14C] dose solution (93.2%)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

- Details on collection : for more details please refer to the aquatic sediment sampling information (Table 1 and 2) which encompass the values measurements made at the time of collection.
Location : Staffordshire for both batches
Sampling depth: over sample area (cm) 25-35 cm (H161117D : Water batch) ; 20-30 cm (H280618C : Water batch)
Contamination history : None for 5 years for both batches numbers ( H161117D : Water batch, H280618C : Water batch).
Procedure : The ‘Calwich Abbey Lake’ aquatic sediments were provided by LRA Labsoil, Lockington, Derby, UK. The sediment was passed through a 2 mm sieve and the water was passed through a 0.2 mm sieve. Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 4 Degree. Characterization of the sediment and water was carried out, not to GLP, by CEM Analytical Services Ltd, Wokingham, UK or NRM Ltd, Bracknell, UK. Sampling details are shown in Table 1 and characterization data are shown in Table 2.
- Storage conditions: Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 4°C.
- Storage length: not reported
- Temperature (°C) at time of collection: Temperature (°C) just below surface = 7,2°C (H161117A : Sediment; H161117D : Water); Temperature (°C) just below surface = 22,9 °C (H280618A : Sediment; H280618C : Water).
- pH at time of collection: pH = 7.62 for batch H161117D (Water); pH = 8.28 H280618C (Water)
- Electrical conductivity: not reported
- Redox potential (mv) initial/final: not reported
- Oxygen concentration (mg/l) initial/final: Just below water surface : 78.4 % for H161117D water batch. 153% for H280618C water batch. 5 cm above sediment : 75.2 % for H161117D water batch. 152.4% for H280618C water batch.
Not reported for fnal concentrations.
- Hardness (CaCO3): Hardness as CaCO3 (mg/L) 226 - 275 (fro more details, see table 2).
- Dissolved organic carbon (%): 4.55 % (H161117A-a) - 2.7 % (H280618A-b)
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): not reported
- Water filtered: yes.
- Type and size of filter used, if any: the water was passed through a 0.2 mm sieve.

Details on source and properties of sediment:

- Details on collection : for more details please refer to the aquatic sediment sampling information (Table 1 and 2) which encompass the values measurements made at the time of collection.
Location : Staffordshire for both batches
Sampling depth: Depth of sediment layer (cm) = 100 cm ; Depth of sampled layer (cm) = 5 cm for both sediment batches (H161117A, H280618A).
Contamination history : None for 5 years for both sediment batches.
Procedure : The ‘Calwich Abbey Lake’ aquatic sediments were provided by LRA Labsoil, Lockington, Derby, UK. The sediment was passed through a 2 mm sieve and the water was passed through a 0.2 mm sieve. Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 4 degree. Characterization of the sediment and water was carried out, not to GLP, by CEM Analytical Services Ltd, Wokingham, UK or NRM Ltd, Bracknell, UK. Sampling details are shown in Table 1 and characterization data are shown in Table 2.
- Storage conditions: Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 4°C.
- Storage length: not reported
- Textural classification (i.e. %sand/silt/clay): H161117A batch : Silt loam/Silty clay loam. H280618A : Silt loam.
- Temperature (°C) at time of collection: Temperature (°C) just below surface = 7,2°C (H161117A : Sediment; H161117D : Water); Temperature (°C) just below surface = 22,9 °C (H280618A : Sediment; H280618C : Water).
- pH at time of collection: H161117A batch : 7.2, H280618A batch : 6.1
- Organic carbon (%): H161117A batch = 4.55 %, H280618A batch = 2,7 %
- Redox potential (mv) initial/final: not reported
- CEC (meq/100 g): H161117A batch = 15.7 , H280618A batch = 14,2
- Bulk density (g/cm³): not reported.
- Biomass (e.g. in mg microbial C/100 mg, CFU or other):
- Sediment samples sieved: yes. The sediment was passed through a 2 mm sieve.

Details on inoculum:

Not Applicable

Duration of test (contact time):

14 d

Initial conc.:

20.3 µg/L

Based on:

act. ingr.

Initial conc.:

20.9 µg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: Portions of Calwich Abbey Lake sediment (108.2 g, dry weight equivalent) were added to one-litre glass vessels.
- Composition of medium: Portions of Calwich Abbey Lake water (565.3 g) were added to the vessels to achieve the volume ratio of approximately 1 : 3, where the sediment layer was 3 cm deep.
- Additional substrate: none
- Solubilising agent (type and concentration if used): Treatment solutions of phosphoric acid octadecyl ester, [14C] were prepared in acetonitrile : water (1 : 1, v/v) at concentrations of 32.7 and 34.3 µg/mL. ither 0.51 or 0.46 mL was applied to the test vessels. This achieved application rates of 20.35 or 20.91 µg/L in water based on the total volume of water in the vessel including water contained within the sediment layer.
- Test temperature: Samples were incubated under aerobic conditions at 12 ± 2°C in the dark.
- pH: not reported
- pH adjusted: no
- CEC (meq/100 g): 15.7 meq/100 g (H161117A), 14.2 meq/100 g (H280618A) (initial values)
- Aeration of dilution water: The end of the glass stem bringing the air flow into each test vessel was just below the surface of the water. Air was drawn through each system at a flow rate of approximately 50 mL/minute.
- Suspended solids concentration: 2.0 mg/L (Batch H161117D), 19 mg/L (Batch H280618C)
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: /
- Other:

TEST SYSTEM
- Culturing apparatus: flow-through systems
- Number of culture flasks/concentration: six vessels/treatment
- Method used to create aerobic conditions: Test vessel containing the aquatic sediment test system, aerated through a dip tube immersed just below the surface of the water. The end of the glass stem bringing the air flow into each test vessel was just below the surface of the water. Air was drawn through each system at a flow rate of approximately 50 mL/minute. Samples were incubated under aerobic conditions at 12 ± 2¿C in the dark.
- Method used to create anaerobic conditions: not reported
- Method used to control oxygen conditions: not reported
- Measuring equipment: HPLC (see more details in analytical method part)

- Test performed in closed vessels due to significant volatility of test substance: yes.
(i) Humidifying vessel (with sintered stem for uniform gas dispersion) containing water to humidify the air-flow.
(ii) Test vessel containing the aquatic sediment test system, aerated through a dip tube immersed just below the surface of the water.

- Test performed in open system: no.
- Details of trap for CO2 and volatile organics if used:
(iii)Vessel containing ethyl digol to trap volatile organic compounds (Trap 1);
(iv) Vessel containing 1 M aqueous potassium hydroxide solution with phenolphthalein indicator to trap 14CO2 (Trap 2);
(v) Vessel 3 containing 1 M aqueous potassium hydroxide solution with phenolphthalein indicator as a backup 14CO2 trap (Trap 3);
- Other:
(vi) A non-return valve to prevent accidental backflow through the test apparatus.

SAMPLING
- Sampling frequency: Initially single samples of aquatic sediment were taken for analysis immediately after application, and after 7 and 14 days of incubation. Trapping media were taken for analysis with the associated samples at sampling. Additional samples were then set up for sampling at 4 hours, 1 day and 2 days following the start of incubation.
- Sampling method used per analysis type:
Water sampling analysis:
The water was decanted into a bottle and acetonitrile (100 mL) was added to the same bottle. The weight of the water was recorded and duplicate weighed aliquots (1.0 mL) taken for radioassay.
Water samples for Day 7 and Day 14 were concentrated by centrifugal evaporation. Acetonitrile (3 mL) was added to the concentrated sample and sonicated for 5 minutes. Duplicate aliquots (100 µL) were taken for radioassay. The recovery of radioactivity through the concentration procedure was low (62.4 - 67.3%), however these samples were analysed by HPLC.
Sediment sampling analysis:
The sediment was extracted with acetonitrile (200 mL) by sonicating for 15 minutes then shaking for 15 minutes. The sediment and solvent were separated by centrifugation (3000 rpm for 15 minutes). The extraction was repeated using acetonitrile : water, 1 : 1, v/v, (200 mL). Each extract was weighed and duplicate weighed aliquots (1.0 mL) of each extract solution were taken for radioassay. The sediment debris remaining after extraction was air-dried, weighed, milled and duplicate weighed aliquots (approximately 0.3 g) were taken for combustion and radioassay.
Air sampling analysis:
The volumes of trapping solutions were measured and duplicate aliquots (1.0 mL) taken for radioassay.

Sodium carbonate was added (1 mL trap solution : 0.1 g sodium carbonate) to pools of the potassium hydroxide trapping solutions from the 7 and 14 day samples. Saturated barium chloride solution was then added (1.5 mL barium chloride : 1 mL trap solution) and the resulting precipitate separated from the mixture by centrifugation. Duplicate aliquots of the supernatant solution were taken for radioassay. The trapped radioactivity was shown to be associated with 14CO2 by precipitation of the insoluble barium 14C-carbonate.

- Sterility check if applicable: not precised
- Sample storage before analysis: samples of aquatic sediment were taken for analysis immediately after application.
- Other:

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: none
- Abiotic sterile control: none
- Toxicity control: none
- Other:

STATISTICAL METHODS: none

Reference substance:

not specified

Remarks:

There were no references available to aid identification or establish co-chromatographic correspondence with the authentic non-radiolabelled phosphoric acid octadecyl ester, which is a UVCB substance.

Test performance:

Although the purity was < 95% this was considered sufficient to obtain the objectives of this feasibility study. It was possible to follow the dissipation of parent from the water however very little radioactivity was extracted from the sediment. Therefore the relatively low radiochemical purity did not impact the overall outcome of the study.

Compartment:

natural water: freshwater

% Recovery:

10.9

Compartment:

sediment

% Recovery:

35.7

Remarks on result:

other: Total in sediment (extracted 3.1% + non extracted 32.6%)

Compartment:

other: Trapped (KOH)

% Recovery:

40.8

Compartment:

entire system

% Recovery:

87.4

Remarks on result:

other: Total recovery

Remarks:

Sum of water + total sediment + volatilised

Key result

% Degr.:

10.9

Parameter:

radiochem. meas.

Remarks:

degradation product (polar)

Sampling time:

14 d

Key result

% Degr.:

< 0

Parameter:

radiochem. meas.

Remarks:

Parent constitaunt (Phosphoric acid octadecyl ester)

Sampling time:

14 d

Remarks on result:

not determinable because of methodological limitations

Remarks:

Not apparent or below the limit of detection (LOD < 0.006% AR)

Key result

Compartment:

natural water: freshwater

DT50:

0.93 d

Type:

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

Temp.:

12 °C

Key result

Compartment:

natural sediment: freshwater

Type:

not specified

Temp.:

12 °C

Remarks on result:

not determinable because of methodological limitations

Remarks:

Based on the results of the feasibility trial, the following challenges were identified that preclude the conduct of a full study according to OECD Guideline 308, “Aerobic and Anaerobic Transformation in Aquatic Sediment Systems”: • The radiolabelled test item cannot be extracted from sediment and DT50 values therefore cannot be determined for sediment fraction or total system; • Samples cannot be concentrated due to the production of volatile degradates; • Degradates cannot be identified due to their low molecular weights and low concentrations in samples.

Key result

Compartment:

entire system

Type:

not specified

Temp.:

12 °C

Remarks on result:

not determinable because of methodological limitations

Remarks:

Based on the results of the feasibility trial, the following challenges were identified that preclude the conduct of a full study according to OECD Guideline 308, “Aerobic and Anaerobic Transformation in Aquatic Sediment Systems”: • The radiolabelled test item cannot be extracted from sediment and DT50 values therefore cannot be determined for sediment fraction or total system; • Samples cannot be concentrated due to the production of volatile degradates; • Degradates cannot be identified due to their low molecular weights and low concentrations in samples.

Transformation products:

not specified

Remarks:

Phosphoric acid octadecyl ester was shown to degrade to unidentified polar material (up to 40.5% AR) and one low level unidentified degradate (= 4.7% AR).

Details on transformation products:

- Formation and decline of each transformation product during test: Some phosphoric acid octadecyl ester and its degradates were incorporated into bound residues in sediment and mineralised to carbon dioxide.

In the overlying water of Calwich Abbey Lake sediment system, phosphoric acid octadecyl ester declined from 82.2% AR at zero-time to 19.8% AR after 2 days and was not detected after 7 days (Table 4). The unidentified polar material increased to a maximum of 40.5% AR after 2 days and declined to 10.9% AR after 14 days. One low level unidentified degradate (= 4.7% AR) was detected in water samples. In the water samples, the dissipation rate of phosphoric acid octadecyl ester, DT50, was estimated by 1st order kinetics and was 0.9 days (Table 5). The decline of radiolabelled test item (DT50) in the sediment or total system cannot be described because the residues could not be extracted from the sediment.

- Pathways for transformation: none
- Other: The polar component(s) would be difficult to identify due to the low molecular weight and complexity. The samples also were unstable upon concentration. Further investigation was concluded to be not technically feasible.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Remarks:

The test vessels were incorporated into air flow lines with traps to collect volatile radioactivity and incubated in the dark at 12ºC. Volatile radioactivity, all associated with 14CO2, accounted for 40.8% AR after 14 days.

Residues:

yes

Remarks:

Non-extractable radioactivity increase to a maximum of 37.9% AR over 14 days. The decline of radiolabelled test item (DT50) in the sediment or total system cannot be described because the residues could not be extracted from the sediment.

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Not reported
- Anomalies or problems encountered (if yes): /

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: In sediment, the maximum extractable radioactivity accounted for 3.5% AR. Non-extractable radioactivity in the sediment (bound residues) increased to a maximum of 37.9% AR after 7 days. The total radioactivity in sediment was maximum at day 7 with 41,4% AR observed.

In the overlying water of Calwich Abbey Lake sediment system, phosphoric acid octadecyl ester declined from 82.2% AR at zero-time to 19.8% AR after 2 days and was not detected after 7 days. The unidentified polar material in water layer increased to a maximum of 40.5% AR after 2 days and declined to 10.9% AR after 14 days.

- Range of maximum concentrations in % of the applied amount at end of study period: In the sediment, the total radioactivity were 35.7% AR after 14 days (3,1 % AR total extracted sediment and 32,6% AR Non Extractable sediment).

In the water layer, the unidentified polar material declined at the end of the test to 10,9% (after 14 days). In the water samples, the dissipation rate of phosphoric acid octadecyl ester, DT50, was estimated by 1st order kinetics and was 0.9 days. The polar component(s) would be difficult to identify due to the low molecular weight and complexity. The samples also were unstable upon concentration.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: One low level unidentified degradate (= 4.7% AR) was detected in water samples. The maximum level of this unidentified degradate is 4,7 % AR at day 2 (Results from additional vessels set up for earlier sampling intervals).
- Range of maximum concentrations in % of the applied amount at end of study period: At days 7 and 14, the unidentified degradate were not detected in the water layer of the system because were found below the limit of detection (LOD, <0.006% AR).

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: The parent substance, phosphoric acid octadecyl ester, was shown to degrade to unidentified polar material (up to 40.5% AR) and one low level unidentified degradate (= 4.7% AR).

EXTRACTABLE RESIDUES
- % of applied amount at day 0: the extractable radioactivity accounted for 0,6 % AR at the start of the test (t=0) in the sediment.
- % of applied amount at end of study period: the extractable radioactivity accounted for 3.5% AR at day 7 and decreased at 3,1 % AR at day 14 (end of the test) in the sediment samples, therefore these extracts were not analysed.

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: The non-extractable residues at the start of the test were mesured at 13,6 % AR in the sediment.
- % of applied amount at end of study period: The non-extractable residues at the end of the test were mesured at 32,6 % AR in the sediment.
Non-extractable radioactivity in the sediment (bound residues) increased to a maximum of 37.9% AR after 7 days.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Volatile radioactivity, all associated with 14CO2, accounted for 40.8% AR after 14 days.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Not detected

STERILE TREATMENTS (if used) : none
- Transformation of the parent compound:/
- Formation of transformation products: /
- Formation of extractable and non-extractable residues: /
- Volatilization: /

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any): none

Results with reference substance:

none

The mean total recoveries of radioactivity (‘mass balances’, i.e. the sum of radioactivity in the water layer, extractable and non-extractable sediment radioactivity and volatile radioactivity) were in the range 86.2 to 106.1% applied radioactivity (Table 3). The radioactivity from the water layer declined from 91.9% applied radioactivity (AR) at zero-time to 10.9% AR after 14 days. In the sediment, the radioactivity increased to 35.7% AR after 14 days. The extractable radioactivity accounted for 0.6 – 3.5% AR for all samples, therefore these extracts were not analysed. Non-extractable radioactivity in the sediment (bound residues) increased to a maximum of 37.9% AR after 7 days. Volatile radioactivity, all associated with 14CO2, accounted for 40.8% AR after 14 days. In the overlying water of Calwich Abbey Lake sediment system, phosphoric acid octadecyl ester declined from 82.2% AR at zero-time to 19.8% AR after 2 days and was not detected after 7 days (Table 4). The unidentified polar material increased to a maximum of 40.5% AR after 2 days and declined to 10.9% AR after 14 days. One low level unidentified degradate (= 4.7% AR) was detected in water samples.

In the water samples, the dissipation rate of phosphoric acid octadecyl ester, DT50, was estimated by 1st order kinetics and was 0.9 days (Table 5). The decline of radiolabelled test item (DT50) in the sediment or total system cannot be described because the residues could not be extracted from the sediment.

Aquatic sediment	Compartment	DT50 (days)	DT90 (days)
Calwich Abbey Lake	Water	0.93	3.09
	Sediment	-	-
	Total System	-	-

For more details on the tabulated results, please see the attached background documents.

Validity criteria fulfilled:

no

Remarks:

Quality criteria : Recoveries should range from 90% to 110% for labelled chemicals. The total recovery of radioactivity was in a range of 86.2 – 106.1% applied radioactivity (AR).

Conclusions:

Phosphoric acid octadecyl ester dissipated rapidly from the water of aquatic sediment with a DT50 value of 0.9 days. The decline of radiolabelled test item (DT50) in the sediment or total system cannot be described because the test item could not be extracted from the sediment.

Phosphoric acid octadecyl ester was shown to degrade to unidentified polar material (up to 40.5% AR) and one low level unidentified degradate (= 4.7% AR). Some phosphoric acid octadecyl ester and its degradates were incorporated into bound residues and mineralised to carbon dioxide.

The results of this feasibility study demonstrate that conducting a full OECD Guideline 308 study would not generate any further information and it is unlikely that a full study would meet the objectives of the OECD Guideline 308 in aerobic or anaerobic aquatic sediment systems.

Executive summary:

This non GLP study was designed to provide information on the feasibility of conducting a transformation study in aerobic aquatic sediments systems (OECD Guideline 308). Aerobic aquatic sediment, collected from Calwich Abbey Lake and described as a neutral silt loam with a high organic carbon content, was treated with phosphoric acid octadecyl ester, [14C] at a nominal concentration of 22 µg/L based on the total volume of water in the test vessel including that present in the sediment layer. The test vessels were incorporated into air flow lines with traps to collect volatile radioactivity and incubated in the dark at 12ºC. Individual samples of treated aquatic sediment were taken for analysis at zero-time (0 hours), after 4 hours, and at 1, 2, 7 and 14 days.

Total recovery of radioactivity was in a range of 86.2 – 106.1% applied radioactivity (AR). The parent substance, phosphoric acid octadecyl ester, dissipated rapidly from the water of the aquatic sediment system with a DT50 value of 0.9 days (1st order kinetics). The volatile radioactivity increased to 40.8% AR and non-extractable radioactivity increase to a maximum of 37.9% AR over 14 days.

The parent substance, phosphoric acid octadecyl ester, was shown to degrade to unidentified polar material (up to 40.5% AR) and one low level unidentified degradate (= 4.7% AR). Some phosphoric acid octadecyl ester and its degradates were incorporated into bound residues and mineralised to carbon dioxide.

Based on the results of the feasibility trial, the following challenges were identified that preclude the conduct of a full study according to OECD Guideline 308, “Aerobic and Anaerobic Transformation in Aquatic Sediment Systems”:

• The radiolabelled test item cannot be extracted from sediment and DT50 values therefore cannot be determined for sediment fraction or total system;

• Samples cannot be concentrated due to the production of volatile degradates;

• Degradates cannot be identified due to their low molecular weights and low concentrations in samples.

Although an OECD Guideline 308 study would demonstrate that radioactivity detected in bound residues can be mineralised to CO2 in two aerobic sediment systems, the results of this feasibility study demonstrate that conducting a full study would not generate any further information and it is unlikely that a full study would meet the objectives of the OECD Guideline 308 in aerobic or anaerobic aquatic sediment systems.

Reference 2

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Data waiving:

study technically not feasible

Justification for data waiving:

the study does not need to be conducted because the substance is highly insoluble in water

Transformation products:

not specified

Description of key information

Phosphoric acid octadecyl ester dissipated rapidly from the water of aquatic sediment with a DT50 value of 0.9 days. The decline of radiolabelled test item (DT50) in the sediment or total system cannot be described because the test item could not be extracted from the sediment.

Phosphoric acid octadecyl ester was shown to degrade to unidentified polar material (up to 40.5% AR) and one low level unidentified degradate (= 4.7% AR). Some phosphoric acid octadecyl ester and its degradates were incorporated into bound residues and mineralised to carbon dioxide.

The results of this feasibility study demonstrate that conducting a full OECD Guideline 308 study would not generate any further information and it is unlikely that a full study would meet the objectives of the OECD Guideline 308 in aerobic or anaerobic aquatic sediment systems.

Key value for chemical safety assessment

Half-life in freshwater:

0.93 d

at the temperature of:

12 °C

Additional information

Based on information provided in the dossier the registered substance (CAS 39471-52-8) appears to have low solubility in water (< 0,162 mg/L according TG OECD Guideline 105, and adsorption potential (Log Koc = 4.35 according a WoE, QSAR based upon the main constituents, KOCWIN v2). Therefore, it is relevant to waive the simulation testing on ultimate degradation in surface water according TG OECD 309.

According the Decision TPE-D-2114349030-64-01/F and physico-chemical properties, it is considered relevant to investigate the aerobic and anaerobic transformation of the registered substance in sediment at 12°C and its degradation products.