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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.09.2018 - 19.12.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Due to the tendency of the test substance to be lost by evaporation in typically used flow-through test systems, the guideline had to be adapted and a closed system has been used.
Qualifier:
according to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Principles of method if other than guideline:
Due to the tendency of the test substance to be lost by evaporation in typically used flow-through test systems, the guideline had to be adapted and a closed system has been used.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical Name: 2,6-di-tert-butyl-4-methyl[U-14C]phenol
Specific activity: 2.22 GBq/mmol corresponding to 9.9865 MBq/mg
Molecular weight: 222.3 g/mol (at this specific activity)
Radiochemical purity: 98.5 % (CoA)
Label position: benzene ring
Impurities: not determined
Radiolabelling:
yes
Remarks:
14C-labelling in the phenyl ring
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Remarks:
surface water was sampled from Biggesee (Bigge reservoir), Germany (51°4’42” N, 7°50’3” E)
Details on source and properties of surface water:
The study was conducted using lake water sampled by the testing facility. The surface water was sampled from Biggesee (Bigge reservoir), Germany (51°4’42” N, 7°50’3” E). After receipt, the test water was filtered through a 100 µm mesh filter and filled into the test vessels.

Details on sampling and storage:

Name Biggesee lake water
Sampling location Biggesee lake, 57462 Olpe-Sondern, North-Rhine-Westphalia, Germany
Site description A freshwater lake fed by a stream from a weir on the river. Wood- and grassland around the lake.
Geographical region/ global co-ordinates Central Europe / 7°50’ East and 51°4’ Nord
Date of collection 06 May 2019, 9:30 to 10:30 a.m.
Sampling depth 15 – 20 cm
Collection procedure Immersion of container
Duration of transportation (to test facility) 06 May 2019, 10:30 to 11:30 a.m.
Duration of storage (prior to use) No storage

Physico-chemical properties of test surface water

Temperature at collection (oC) 6.3 °C
pH at collection 7.46
Oxygen concentration at collection (mg/L) 12.21
Optical appearance at collection No turbidity, colourless
Redox potential after filtration (mV) 203.8
Oxygen concentration after filtration (mg/L) 9.39 (17.6 °C)
pH after filtration 7.49
TOC (mg/L) 2.12
DOC (mg/L) 2.13
Nitrogen (total, mg/L) 8.3
Nitrate (mg/L) 8.0
Nitrite (mg/L) 0.020
Ammonium-N (mg/L) < 0.01

Dissolved Orthophosphate (total, mg/L) < 0.10
Details on source and properties of sediment:
Sediment was taken from the same sampling site.
Details on inoculum:
The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to biological activity control samples (reference controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were incubated in a flow-through test system or in a closed test system and at 12°C or 20 °C. The formation of 14CO2 was followed until 28 days (flow-through system) or 62 days (closed system).


The biological activity of filtered lake water was characterised by the addition of the easily degradable compound 14C-sodium benzoate to biological activity control samples (reference controls). Concentration of 14C-sodium benzoate was 10 µg/L. Control samples were incubated in a flow-through test system or in a closed test system and at 12°C or 20 °C. The formation of 14CO2 was followed until 28 days (flow-through system) or 62 days (closed system).
Duration of test (contact time):
62 d
Initial conc.:
10 µg/L
Based on:
test mat.
Remarks:
corresponding to 15.0 kBq per 150 mL surface water
Initial conc.:
90 µg/L
Based on:
test mat.
Remarks:
corresponding to 134.8 KBq per 150 mL surface water
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Due to volatilastion the test was performed in a closed system. The results of the reference control samples treated with 14C-sodium benzoate indicate that the mineralisation rate determined in a closed system is generally slower than in a flow-through system even when the oxygen concentrations in the test vessels were sufficiently high.

- Sterile control samples
For the preparation of the sterile (abiotic) surface water samples, the test water, test vessels, and all required equipment was autoclaved at 121 °C for 20 minutes and the preparation of the samples was performed under a laminar flow workbench under sterile conditions. The sample weight of the sterilized water samples was adjusted after sterilization by means of sterilized water under a clean bench using sterilized equipment.

- Incubation conditions
The incubation of the surface water samples treated with 14C-labelled 2,6-di-tert-butyl-p-cresol was carried out on orbital shakers in a temperature controlled room at a test temperature of 12 ± 2 °C. Incubation was performed in 0.5 L Erlenmeyer flasks which were closed gas tight. Trapping of the exhaust gases was carried out by NaOH absorption traps integrated in the flasks and by Tenax tubes. Oxygen sensors were placed within the gas phase of the Erlenmeyer flask of two representative samples per concentration level to monitor the oxygen concentration. No areation of the surface water samples was performed.
The reference control samples were incubated at 12 ± 2°C or at 20 ± 2°C in the dark in gas tight glass vessels (closed test system) and in flow-through test systems, respectively.

The incubation of surface water samples applied with the test or reference item at 12 ± 2 °C was carried out from May 07, 2019 to July 08, 2019. However, the cooling of the 12 °C incubation room was not working from May 24, 2019 to May 27, 2019 (day 17 to day 20 of incubation) so that the temperature range of 12 ± 2 °C was exceeded. Maximum temperature during this time period was 17 °C (for 4 hours), minimum temperature 11.5 °C (for 3 hours). Temperature was monitored throughout the study and also during this period of time .

- Absorption traps
Surface water samples treated with 2,6-di-tert-butyl-p-cresol:
Trapping of the exhaust gases was carried out by solutions of sodium hydroxide and Tenax tubes.
The sodium hydroxide absorption solution consisted of 5 mL 2 M NaOH with 5 drops of phenolphthalein indicator solution in glass vessels which were located inside the test vessels. The phenolphthalein did not indicate a decrease of the pH-value of the absorption solution throughout the incubation period of 62 days. The absorption solutions were sampled at each sampling time. The total radioactivity in each solution was determined by LSC.
Tenax tubes were attached to the test vessels to trap organic volatile compounds. Tenax tubes were sampled at the respective sampling time and were extracted using in total 2 mL acetonitrile with 1% formic acid. The radioactivity in the acetonitrile with 1% formic acid eluate was determined by LSC.

Surface water samples treated with reference item sodium benzoate:
Trapping of 14CO2 in reference control samples was carried out by solutions of sodium hydroxide. Tenax tubes were not used.
When using the closed test system, the sodium hydroxide absorption solution consisted of 5 mL 2 M NaOH with 5 drops of phenolphthalein indicator solution. The phenolphthalein did not indicate a decrease of the pH-value of the absorption solution throughout the incubation period of 62 days in case of those samples which were sampled only after the end of the incubation period. The absorption solutions were sampled at each sampling time. The sodium hydroxide traps were removed from the Erlenmeyer flask and replaced by fresh sodium hydroxide solution. The total radioactivity in each solution was determined by LSC.
When using the flow-through system, the sodium hydroxide absorption solution consisted of two traps of 100 mL 1 M NaOH in series per sample. The absorption solutions were sampled at each sampling time. The traps were removed from the flow-through system and were replaced by new traps. The volume of the traps were determined and the total radioactivity in each solution was determined by LSC.

- Sampling
Sampling was performed after the following incubation times: 0 d (immediately after application), 3 d, 7 d, 14 d, 21 d, 28 d, 42 d and 62 d after application. Sterilised surface water samples were taken after 62 d. The surface water was partintioned with ethyl acetate before and after acidification. Extracts were analysed for the test substance and possible degradation products by HPLC and TLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity except for 5 replicates of the concentration level 10 µg/L and for 3 replicates of the higher concentration level of 90 µg/L. These replicates were all in the range of 83.0% and 89.4%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during incubation and/or sample preparation. The amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system (Tenax® traps and rinse of inner test vessel surfaces) were always < 5 % AR.
The results show that mineralisation of the test item occurred during the aerobic incubation of 2,6-di-tert-butyl-p-cresol demonstrating complete degradation of the test item. Maximum amounts of 23.7 % AR (concentration level of 10 µg/L) and 36.3 % (concentration level of 90 µg/L) were detected in the sodium hydroxide traps. However, also under abiotic conditions 14CO2 formation was detected indicating that also abiotic transformation led to a complete destruction of the parent molecule.
The amount of radioactivity extracted from the surface water by ethyl acetate decreased continuously in concentration levels from 94.2 % – 98.6 % AR at the beginning of incubation to 45.9 % – 46.9 % AR until the end of incubation. The amount of radioactivity remaining in the water phase after the partitioning with ethyl acetate increased to maximum values of 9.0 % (concentration level of 90 µg/L) – 21.7 % AR (concentration level of 10 µg/L) until the end of incubation. In sterile samples after 62 days of incubation, radioactive amounts in the organic extract and aqueous phase were found in ranges which are comparable to the respective microbial active samples. By acidification of the aqueous phase to a pH-value of ≤ 2, further amounts of radioactivity couldan be extracted additionally byusing ethyl acetate afterwards.

The amount of parent compound in the organic extracts decreased continuously from maximum levels between 94.2 % - 98.6 % AR immediately after application to amounts in the to amounts between 8.1 % AR (concentration level of 90 µg/L) to non-detectable levels (concentration level of 10 µg/L) after 62 days of incubation. In extracts of sterilised samples, 2,6-di-tert-butyl-p-cresol could not be found at the end of the incubation period of 62 days.

Degradation of 2,6-di-tert-butyl-p-cresol in surface water was observed by the formation of metabolites. During 62 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the organic extracts as well as an unknown transformation product exceeding 10 % AR.

The obtained data sets were analysed using the program CAKE version 1.4. The kinetic models considered for the analysis of 2,6-di-ter-butyl-p-cresol were SFO (Single First Order), DFOP (Double First Order in Parallel), HS (Hockey Stick), and FOMC (First Order Multi Compartment). According to the results the best fitting results were obtained when considering SFO kinetics.


2,6-di-tert-butyl-p-cresol (BHT) was incubated in test water using the test concentrations 10 µg/L and 90 µg/L under dark, aerobic conditions. Sterile samples were additionally prepared to enable differentiation between biotic and abiotic degradation.
The mineralisation of 2,6-di-tert-butyl-p-cresol (BHT) was studied over an incubation time of 62 days at 12°C ± 2°C.
Reference substance:
benzoic acid, sodium salt
Remarks:
14C-labelled
Test performance:
The total radioactivity in the absorption trap present during partitioning was determined by LSC. The top part of the test vessel was rinsed with 20 mL acetonitrile with 1% formic acid. The volume of the rinsing solution was determined and aliquots were analysed by LSC.
The surface water was partitioned with ethyl acetate before and after acidification. Extracts were analysed for the test substance and possible degradation products by HPLC and TLC. By acidification of the aqueous phase to a pH-value of ≤ 2, further amounts of radioactivity could be extractedusing ethyl acetate.

Compartment:
natural water: freshwater
% Recovery:
91.4
St. dev.:
3.9
Remarks on result:
other: 10 µg/L
Remarks:
low dose
Compartment:
natural water: freshwater
% Recovery:
95
St. dev.:
4.2
Remarks on result:
other: 90 µg/L
Remarks:
high dose
% Degr.:
94.2
Parameter:
test mat. analysis
Sampling time:
0 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
54
Parameter:
test mat. analysis
Sampling time:
3 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
25.4
Parameter:
test mat. analysis
Sampling time:
7 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
6.4
Parameter:
test mat. analysis
Sampling time:
14 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
2.4
Parameter:
test mat. analysis
Sampling time:
21 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
2
Parameter:
test mat. analysis
Sampling time:
28 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
1.9
Parameter:
test mat. analysis
Sampling time:
62 d
Remarks on result:
other: 10 µg/L concentration
% Degr.:
98.6
Parameter:
test mat. analysis
Sampling time:
0 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
69.2
Parameter:
test mat. analysis
Sampling time:
3 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
56.7
Parameter:
test mat. analysis
Sampling time:
7 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
34.2
Parameter:
test mat. analysis
Sampling time:
14 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
14.5
Parameter:
test mat. analysis
Sampling time:
21 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
8
Parameter:
test mat. analysis
Sampling time:
28 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
7.3
Parameter:
test mat. analysis
Sampling time:
42 d
Remarks on result:
other: 90 µg/L concentration
% Degr.:
8.1
Parameter:
test mat. analysis
Sampling time:
62 d
Remarks on result:
other: 90 µg/L concentration
Key result
Compartment:
natural water: freshwater
DT50:
8.616 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: concentration level of 90 µg/L
Compartment:
natural water: freshwater
DT50:
3.706 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: concentration level of 10 µg/L
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
No.:
#5
No.:
#6
Details on transformation products:
During 62 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the organic extracts as well as an unknown transformation product exceeding 10 % AR.
Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in surface water.
Evaporation of parent compound:
no
Remarks:
Results of pre-tests in flow-through system revealed a high portion of radioactivity lost by evaporation; Therefore the main tes was performed by incubation in closed gas tight glass vessels
Volatile metabolites:
yes
Remarks:
Thus, the test was performed in a closed system.
Residues:
no
Remarks:
Surface water samples were filtered through 100 µm mesh prior to testing. No solid residues reported after testing.
Details on results:
Test water properties
Throughout the incubation time pH-value and oxygen concentration of the test water was measured regularly in untreated surface water. PH value and the oxygen concentration in the surface water samples remained stable over the incubation time. Details are listed in the overall remarks.

Mass balance
For each sample a mass balance was performed by summing the radioactivity detected in the sodium hydroxide trap, in the Tenax trap, in the organic soil extracts plus the radioactivity detected in the remaining water phase. In addition, this sum was compared with the total radioactivity which had initially been applied to the samples determined by means of application controls. According to this, the surface water samples of the test item concentration of 10 µg/L were applied with 13.9 kBq/sample of [14C]-labelled 2,6-di-tert-butyl-p-cresol and surface water samples of the concentration of 90 µg/L were applied with 127.8 kBq/sample. The applied radioactivity corresponds to 92.5 % and 94.8 % of the target application rate, respectively.

Distribution of radioactivity
The amounts of radioactivity (radiolabelled test item and transformation products) and its distribution in organic and aqueous phase and volatile substances were calculated as % of initially applied radioactivity (AR).

Distribution of radioactivity in non-volatile extractables and organic volatiles to parent and metabolites
The amount of test item and metabolites at each sampling time was calculated from determined radioactivity in the extract (LSC) in combination with the relative distribution of parent compound and metabolites in the extract analysed by means of HPLC. The sum of each individual (parent of metabolite) gives the total amount of test item and metabolite at the respective sampling date as % of the initially applied radioactivity (AR) in each compartment.

Volatiles:
In the Tenax® traps of the samples, the radioactivity was always ≤ 3.2 % AR. In addition, radioactivity in amounts of ≤ 2.3 % AR were found in the rinsing solutions of the inner surfaces of the test vessels. Thus, only small amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system (< 5 % AR).
In the sodium hydroxide traps, radioactivity increased rapidly during the first days up to values of 21.1 % AR (concentration level of 10 µg/L) or 13.4 % AR (concentration level of 90 µg/L at day 3. Afterwards, the radioactivity in the sodium hydroxide traps remained relative stable (concentration level of 10 µg/L) or increased to values of 30.3 % AR – 36.3 % AR (concentration level of 90 µg/L) until the end of incubation. In the sodium hydroxide traps of the sterile samples radioactivity in similar amounts of 25.0 % AR (concentration level of 10 µg/L) and 32.8 % AR (concentration level of 90 µg/L) were detected. The results show that mineralisation of the test item occurred during the aerobic incubation of 2,6-di-tert-butyl-p-cresol demonstrating complete degradation of the test item. However, also under abiotic conditions 14CO2 formation was detected indicating that also abiotic transformation led to a complete destruction of the parent molecule.

Extractable radioactivity:
The amount of radioactivity extracted from the surface water by ethyl acetate decreased continuously in concentration levels from 94.2 % – 98.6 % AR at the beginning of incubation to 45.9 % – 46.9 % AR until the end of incubation. In sterilised surface water samples the extractable radioactivity was always in the range of 39.9 % - 42.3 % AR for both concentration levels at the end of incubation period.

Aqueous phase after first extraction step:
Generally, the amount of radioactivity remaining in the water phase after the partitioning with ethyl acetate increased from 0.3 % - 0.6 % AR at day 0 to maximum values of 9.0 % (concentration level of 90 µg/L) – 21.7 % AR (concentration level of 10 µg/L) until the end of incubation. In sterile surface water radioactive amounts of radioactivity in the range of 12.8 % – 16.0 % AR were found in the aqueous phase which are comparable to the microbial active samples.

Additional extraction of the aqueous phase after acidification:
During the aerobic incubation of 2,6-di-tert-butyl-p-cresol the amounts of radioactivity remaining in the aqueous phase after the first partitioning with ethyl acetate increased during the incubation to values > 5 % AR (concentration level of 90 µg/L) or > 10 % AR (concentration level of 10 µg/L). Therefore, samples taken at incubation day 7 and thereafter (including sterile samples) were submitted to an additional extraction step with ethyl acetate after acidification of the existing aqueous phase. By acidification of the aqueous phase to a pH-value of ≤ 2, further amounts of radioactivity can be extracted additionally usingby ethyl acetate afterwards.
The radioactivity found in the additional ethyl acetate extracts (organic extract 2) was always in the range of 4.4 % - 11.0 % AR for the concentration level of 10 µg/L and between 1.8 % AR and 8.4 % AR for the concentration level of 90 µg/L. The radioactivity remaining in the aqueous phase also after this second extraction step after acidification was 2.3 % AR – 9.5 % AR (concentration level of 10 µg/L) and 0.9 % AR – 3.7 % AR (concentration level of 90 µg/L).
The radioactivity found in the ethyl acetate extracts 2 of the sterile samples was in a comparable range of 11.5 % AR (concentration level of 10 µg/L) and 9.1 % AR (concentration level of 90 µg/L). In the resulting aqueous phases radioactivity amounted to 4.3 % AR (concentration level of 10 µg/L) and 3.5 % AR (concentration level of 90 µg/L).

Identification of extractable radioactivity
Determinations are based on radio-HPLC analysis of the extracts. The values are expressed in percent of the total initially applied radioactivity (AR). Radio-HPLC was chosen as primary (quantitative) analytical method due to the sensitivity of the parent compound towards oxygen. HPLC ensured chromatographic conditions avoiding oxidation or transformation of the test item due to the low contact with atmospheric oxygen compared to TLC. The amount of parent compound in the organic extracts decreased continuously from maximum levels between 94.2 % - 98.6 % AR immediately after application to amounts in the to amounts between 8.1 % AR (concentration level of 90 µg/L) to non-detectable levels (concentration level of 10 µg/L) after 62 days of incubation.
In extracts of sterilised samples 2,6-di-tert-butyl-p-cresol could not be found at the end of the incubation period of 62 days.
Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in surface water. During 62 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or, if no reference compounds were available - by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the organic extracts as well as one unknown transformation product exceeding 10 % AR which was specified as unassigned metabolite with the retention times of 33.7 min.
After 3 days of incubation the known metabolites BHT-OH and BHT-CH2OH were detected in the organic extracts. During the further incubation BHT-OH was detected in increasing amounts up to the end of incubation. Maximum levels were 27.1 % AR at 42 days of incubation (concentration level of 10 µg/L) or 21.3 % AR at 62 days of incubation (concentration level of 90 µg/L). Maximum levels of BHT-CH2OH were found after 28 days of incubation for the concentration level of 10 µg/L (10.8 % AR) or at 14 days of incubation for the concentration level of 90 µg/L (8.2 % AR). BHT-CH2OH decreased thereafter until the end of incubation to amounts in the range of 4.4 % AR (concentration level of 90 µg/L) and 7.9 % AR (concentration level of 10 µg/L).
The known metabolite BHT-quinone was detected after 7 – 14 days of incubation and reached maximum amounts during the incubation after 21 -28 days of 14.0 % AR (concentration level of 10 µg/L) or 6.7 % AR (concentration level of 90 µg/L). BHT-CHO was detected firstly after 14 – 21 days of incubation in both test concentration levels. A maximum amount of 7.4 % AR was reached after 28 days in samples of the concentration level 10 µg/L. However, in samples of the concentration level 90 µg/L amounts never exceeding 2.6 % AR were found for BHT-CHO during the incubation. The known metabolite BHT-COOH was detected only sporadically in minor amounts ≤ 3.1 % AR during the incubation of the surface water.
In extracts of sterilised samples BHT-OH, BHT-quinone and BHT-CHO were detected at the end of the incubation period of 62 days. The known metabolite BHT-COOH was not detected and BHT-CH2OH was detected only in one sterilised replicate in minor amounts.
An unknown transformation product exceeding 10 % AR in both concentration levels was detected in the organic extracts and was specified as unassigned metabolite with the retention time of 33.7 min. The retention time of this metabolite indicate a more non-polar character of the transformation product compared to the parent compound. From radio-HPLC and HPLC-UV analyses and comparison to reference standard it could be concluded, that the metabolite is different from 2-BHT. Studies regarding the elucidation of this unknown metabolite are ongoing. In addition, unknown transformation product(s) with a retention time of 3.6 min never exceeding 5 % AR was found indicating the further degradation to polar metabolites.
Results with reference substance:
Reference control samples treated with the easily degradable compound 14C-sodium benzoate were incubated under the same conditions as the samples (closed test system at 12 ± 2 °C) to verify the biological activity of the test water. In addition, further test sets were incubated to compare the different test setups and incubation conditions and to monitor their effect on mineralization and degradation rate of the reference compound. For this reason, further reference control samples of the same surface water were incubated contemporaneously at 20 ± 2 °C in a closed system as well as at 12 ± 2 °C and 20 ± 2 °C in a flow-through system. The formation of 14CO2 was followed until 28 days (flow-through system) or 62 days (closed system).
The time course of mineralization can only be shown for replicates where sampling during the incubation occurred i.e. replicates of the flow-through test sets as well as for replicates 1-4 (12 °C) and 1-2 (20°C) of the closed system test sets, respectively.

At the end of the incubation period more than 70% of the applied radioactivity was mineralised in all reference control samples. Therefore, the test water can be classified as sufficiently biologically active in the different test setups.

Mineralization of the control samples

Reference control samples treated with the easily degradable compound 14C-sodium benzoate were incubated under the same conditions as the samples (closed test system at 12 ± 2 °C) to verify the biological activity of the test water. In addition, further test sets were incubated to compare the different test setups and incubation conditions and to monitor their effect on mineralization and degradation rate of the reference compound. For this reason, further reference control samples of the same surface water were incubated contemporaneously at 20 ± 2 °C in a closed system as well as at 12 ± 2 °C and 20 ± 2 °C in a flow-through system. The formation of 14CO2 was followed until 28 days (flow-through system) or 62 days (closed system).

The mineralisation results are summarized in the following table (Mineralisation is given in % of applied radioactivity, %AR):

 

  Mineralisation in the reference control samples treated with 14C-sodium benzoate and incubated in a closed test system after 62 days of incubation

 Sample     Replicate     Mineralisation* [%AR]
  Incubation at 12 +- 2 °C  Incubation at 20 +- 2 °C
    

Reference without solvent (sampling during incubation)

 1  77.0  79.4
 2  77.7  80.2

 Reference with solvent addition (sampling during incubation)

 3

 76.7

n.m. (not measured)

 4

 76.7

 n.m.

Reference without solvent (sampling at test end only)

 5

 71.7

 75.0

 6

 73.9

 71.0

*Mineralisation as sum of radioactivity found in the absorption traps including acidification of the the surface water at the end of the incubation period.

 

 Mineralisation in the reference control samples treated with 14C-sodium benzoate and incubated in a flow-through test system after 28 days of incubation.

 Sample     Replicate     Mineralisation* [%AR]
  Incubation at 12 +- 2 °C  Incubation at 20 +- 2 °C
    

Reference without solvent

 1 82.9  75.5
 2 71.7  74.0

*Mineralisation as sum of radioactivity found in the absorption traps including acidification of the the surface water at the end of the incubation period.

 

The time course of mineralization can only be shown for replicates where sampling during the incubation occurred i.e. replicates of the flow-through test sets as well as for replicates 1-4 (12 °C) and 1-2 (20°C) of the closed system test sets, respectively.

At the end of the incubation period more than 70% of the applied radioactivity was mineralised in all reference control samples. Therefore, the test water can be classified as sufficiently biologically active in the different test setups.

 

The distribution of the applied radioactivity (expressed as % AR) during the aerobic mineralisation of 2,6-di-tert-butyl-p-cresol in surface is presented in the following tables. The tables show the distribution of the test item (as mean value of two replicates) found in organic extracts, in aqueous solutions after partitioning, in the sodium hydroxide traps, Tenax® tubes and rinsing solutions.

 

  Distribution of radioactivity in surface water treated with 2,6-di-tert-butyl-p-cresol at a concentration of 10 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, the aqueous phases, traps, rinsing solutions and the overall recovery.

Mean values of two replicates.

 

 Sampling time  Organic extract  Aqueous Phase  Mineralisation (NaOH trap)  Volatile Compounds (Tenax tube)  Volatile Compounds (rinse of inner surfaces)  Recovery
                   [% AR]
 0d  94.2  0.6  -  -  -  94.9
 3d  65.1  3.4 21.1 0.9  0.8  91.3
 7d  57.2  7.5       

 20.3 

 1.0

 1.7

 87.6

 14d

51.0 

 14.8

 23.7

 1.4

 2.1

 93.0

 21d

 46.4

 22.8

 22.9

 1.7

 2.3

 96.1

 28d

 46.3

 19.2

 22.5

 1.9

 1.9

 91.6

 42d

 43.8

 19.5

 21.0

 1.4

 2.1

 87.7

 62d

 46.9

 21.7

 19.8

 1.7

 2.5

 92.7

 62d sterile  42.3  16.0  25.0  2.8  1.5  87.6

 

 

 

Distribution of radioactivity in surface water treated with 2,6-di-tert-butyl-p-cresol at a concentration of 90 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, the aqueous phases, traps, rinsing solutions and the overall recovery.

Mean values of two replicates.

 

 Sampling time  Organic extract  Aqueous Phase  Mineralisation (NaOH trap)  Volatile Compounds (Tenax tube)  Volatile Compounds (rinse of inner surfaces)  Recovery
                   [% AR]
 0d 98.6  0.3  -  -  -  98.9
 3d  82.6  2.5 13.4 0.7  0.3  99.3
 7d  71.2  2.9       

 22.1 

 1.0

 0.3

 97.6

 14d

56.6

 7.8

 28.7

 1.2

1.2

 95.5

 21d

 53.1

 8.3

 31.3

 1.9

 2.3

 97.0

 28d

 50.6

 8.3

 32.5

 2.2

 1.8

 95.3

 42d

 42.8

 8.6

 36.3

 2.0

 1.6

 91.4

 62d

 45.9

 9.0

 30.3

 2.4

 2.2

 89.8

 62d sterile  39.9  12.8  32.8  3.2  1.0  89.7

 

The overall recoveries ranged between 90 and 110 % of initially applied radioactivity for all samples except for 5 replicates of the concentration level 10 µg/L and for 3 replicates of the higher concentration level of 90 µg/L. These replicates were all in the range of 83.0% and 89.4%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during incubation and/or sample preparation and that this is the reason for the reduced recoveries.

 

Detailed results of the analytical measurements

Detailed distribution of radioactivity in surface water treated with 2,6-di-tert-butyl-p-cresol at a concentration of 10 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, aqueous phases after partitioning, traps, rinsing solutions and the overall recovery.

Sampling time

Replicate

Organic extract

Aqueous phase

Mineralisation

(NaOH trap)

Volatile compounds

(Tenax tube)

Volatile compounds

(rinse of inner surfaces)

Recovery

   

[% AR]

0d

1

90.8

0.5

-

-

-

91.4

2

97.7

0.7

-

-

-

98.4

3d

1

60.2

3.4

27.7

1.3

0.7

93.2

2

70.1

3.5

14.5

0.5

0.8

89.4

7d

1

55.8

6.8

19.6

1.0

1.2

84.3

2

58.5

8.2

20.9

1.0

2.2

90.9

14d

1

50.2

14.7

24.0

1.3

2.0

92.2

2

51.8

14.9

23.4

1.5

2.3

93.8

21d

1

46.3

23.7

22.0

1.7

2.0

95.7

2

46.6

21.8

23.8

1.7

2.6

96.5

28d

1

46.9

20.2

22.3

2.0

1.9

93.3

2

45.7

18.3

22.8

1.8

1.9

90.5

42d

1

40.5

18.5

20.6

1.3

2.2

83.0

2

47.1

20.4

21.4

1.5

2.0

92.4

62d

1

48.2

24.5

18.3

1.1

3.0

95.1

2

45.6

18.9

21.4

2.4

2.1

90.4

62d sterile

1

41.4

15.1

27.3

2.6

1.7

88.1

2

43.1

17.0

22.8

3.0

1.3

87.2

Detailed distribution of radioactivity in surface water treated with 2,6-di-tert-butyl-p-cresol at a concentration of 90 µg/L and incubated at 12°C in % of applied radioactivity (% AR) in the extracts, aqueous phases after partitioning, traps, rinsing solutions and the overall recovery.

Sampling time

Replicate

Organic extract

Aqueous phase

Mineralisation

(NaOH trap)

Volatile compounds

(Tenax tube)

Volatile compounds

(rinse of inner surfaces)

Recovery

   

[% AR]

0d

1

95.8

0.3

-

-

-

96.2

2

101.4

0.4

-

-

-

101.7

3d

1

82.4

3.6

12.4

0.7

0.3

99.4

2

82.8

1.3

14.3

0.6

0.2

99.3

7d

1

75.4

2.8

19.3

0.9

0.3

98.7

2

67.1

3.0

24.9

1.2

0.3

96.5

14d

1

59.4

10.3

22.5

0.9

1.3

94.4

2

53.7

5.3

34.8

1.6

1.1

96.7

21d

1

50.2

6.6

34.7

2.1

2.8

96.4

2

56.1

10.0

27.9

1.7

1.8

97.6

28d

1

54.3

9.4

27.0

2.1

1.7

94.5

2

46.8

7.2

37.9

2.4

1.9

96.2

42d

1

46.5

9.3

36.5

2.1

1.4

95.9

2

39.1

7.9

36.1

1.9

1.9

86.9

62d

1

48.8

10.0

25.1

1.8

2.1

87.8

2

42.9

8.0

35.5

2.9

2.3

91.7

62d sterile

1

39.9

12.2

30.5

3.1

0.9

86.6

2

39.9

13.4

35.1

3.3

1.1

92.8

Detailed results of additional partitioning with ethyl acetate after acidification of remaining aqueous phase after the first extraction step of surface water samples treated with 2,6-di-tert-butyl-p-cresol at a concentration of 10 µg/L.

Values in % of applied radioactivity (AR).

Sampling time

Replicate

Aqueous phase

Additional extraction after acidification

(before acidification)

Organic extract 2
ethyl acetate

Aqueous phase
(after acidification and second partitioning)

7d

1

6.8

4.1

2.1

2

8.2

4.7

2.5

14d

1

14.7

9.3

7.0

2

14.9

9.2

6.4

21d

1

23.7

11.3

12.4

2

21.8

10.4

11.1

28d

1

20.2

10.5

8.3

2

18.3

10.7

7.8

42d

1

18.5

8.7

8.3

2

20.4

9.5

9.0

62d

1

24.5

11.3

12.2

2

18.9

10.7

6.9

62d sterile

1

15.1

10.9

4.1

2

17.0

12.1

4.4

Detailed results of additional partitioning with ethyl acetate after acidification of remaining aqueous phase after the first extraction step of surface water samples treated with 2,6-di-tert-butyl-p-cresol at a concentration of 90 µg/L.

Values in % of applied radioactivity (AR).

Sampling time

Replicate

Aqueous phase

Additional extraction after acidification

(before acidification)

Organic extract 2
ethyl acetate

Aqueous phase
(after acidification and second partitioning)

7d

1

2.8

1.8

0.9

2

3.0

1.9

0.9

14d

1

10.3

9.9

4.6

2

5.3

3.7

1.7

21d

1

6.6

4.2

2.3

2

10.0

5.6

4.2

28d

1

9.4

6.2

3.1

2

7.2

5.0

2.3

42d

1

9.3

5.0

4.2

2

7.9

4.4

3.3

62d

1

10.0

11.2

3.2

2

8.0

5.5

2.6

62d sterile

1

12.2

8.7

3.3

2

13.4

9.5

3.6

 

Detailed results on concentrations for 2,6 -di-tert-butyl-p-cresol (BHT) used for CAKE calculations

Sampling time

Replicate

Test concentration
10 µg/L

Test concentration
90 µg/L

   

[% AR]

0d

1

90.8

95.8

2

97.7

101.4

3d

1

50.1

62.7

2

57.8

75.6

7d

1

23.4

58.5

2

27.3

54.9

14d

1

6.6

41.9

2

6.2

26.5

21d

11

2.3

11.6

2

2.5

17.3

28d

1

2.5

3.2

2

1.5

12.9

42d

1

n.d.

9.7

2

n.d.

4.8

62d

1

3.8

15.1

2

n.d.

1.1

n.d. not detected

 

 

 

Results of the measurments regarding the degradation products

 

The following tables contain detailed information on the degradation products measured in the samples in the organic extracts.

The degradation product BHT-COOH was too low in concentration. No calculation of half-lives could be performed.

The amounts of relevant metabolites in the organic extracts of each replicate are compiled in the following tables.

The results of the individual replicates are the basis of CAKE calculations of the metabolites.

Metabolite BHT-CHO in surface water treated with 2,6-di-tert-butyl-p-cresol at concentrations of 10 µg/L and 90 µg/L determined in the organic extracts. Values are given in percent of the applied radioactivity (% AR).

Sampling time

Replicate

Test concentration
10 µg/L

Test concentration
90 µg/L

   

[% AR]

0d

1

n.d.

n.d.

2

n.d.

n.d.

3d

1

n.d.

n.d.

2

n.d.

n.d.

7d

1

n.d.

n.d.

2

n.d.

n.d.

14d

1

6.4

n.d.

2

6.5

n.d.

21d

1

7.4

1.4

2

6.6

n.d.

28d

1

7.2

2.1

2

7.5

n.d.

42d

1

5.8

3.0

2

5.8

2.2

62d

1

4.2

2.4

2

6.4

2.3

n.d. = not detected.

 

Metabolite BHT-OH in surface water treated with 2,6-di-tert-butyl-p-cresol at concentrations of 10 µg/L and 90 µg/L determined in the organic extracts. Values are given in percent of the applied radioactivity (% AR).

Sampling time

Replicate

Test concentration
10 µg/L

Test concentration
90 µg/L

   

[% AR]

0d

1

n.d.

n.d.

2

n.d.

n.d.

3d

1

7.5

4.1

2

3.8

3.9

7d

1

6.3

4.7

2

8.1

3.4

14d

1

11.3

11.0

2

11.0

7.4

21d

1

17.1

10.9

2

18.1

11.5

28d

1

18.5

9.8

2

17.7

11.3

42d

1

22.7

18.0

2

31.6

14.2

62d

1

23.7

20.7

2

26.3

22.0

n.d. = not detected.

Metabolite BHT-CH2OH in surface water treated with 2,6-di-tert-butyl-p-cresol at concentrations of 10 µg/L and 90 µg/L determined in the organic extracts. Values are given in percent of the applied radioactivity (% AR).

Sampling time

Replicate

Test concentration
10 µg/L

Test concentration
90 µg/L

   

[% AR]

0d

1

n.d.

n.d.

2

n.d.

n.d.

3d

1

n.d.

7.9

2

4.5

3.2

7d

1

7.7

5.1

2

10.0

3.8

14d

1

7.9

9.5

2

11.3

6.8

21d

1

10.5

6.3

2

10.0

7.9

28d

1

10.5

8.1

2

11.1

5.8

42d

1

6.9

5.8

2

6.2

3.5

62d

1

4.7

4.4

2

11.1

4.4

n.d. = not detected

Metabolite BHT-quinone in surface water treated with 2,6-di-tert-butyl-p-cresol at concentrations of 10 µg/L and 90 µg/L determined in the organic extracts. Values are given in percent of the applied radioactivity (% AR).

Sampling time

Replicate

Test concentration
10 µg/L

Test concentration
90 µg/L

   

[% AR]

0d

1

n.d.

n.d.

2

n.d.

n.d.

3d

1

n.d.

n.d.

2

n.d.

n.d.

7d

1

5.8

n.d.

2

3.7

n.d.

14d

1

10.0

3.8

2

8.0

4.4

21d

1

15.6

6.7

2

12.5

6.1

28d

1

11.6

7.7

2

11.4

5.7

42d

1

8.3

6.4

2

13.1

5.1

62d

1

5.6

5.7

2

12.5

3.8

n.d. = not detected.

 

Detailed results on the determination of the DT50 values

Five transformation products were calculated which reached 10% AR or two times 5% AR. Due to the complexity of the metabolism scheme, the optimisation tool is not able to handle all five transformation products in parallel. Therefore, two separate optimisation were performed to cover all substances.

Model 1 considers BHT-CH2OH (=A1) , BHT-CHO (=B1).

Model 2 considers BHT-OH (=A1), BHT-quinone (=A2) and an unknown metabolite with a retention time of 33.7 min (= B1).

The analyses for the metabolites were based on the best fit kinetics for the parent compound. For the metabolites always SFO (single first order) was considered. The results of the optimisations are presented in the following tables:

Calculated DT50 and DT90 for BHT-CH2OH (= Model 1, A1) based on SFO kinetics.

 

chi2

f

DT50

DT90

 

(%)

P → A1

(d)

(d)

Concentration level of
90 µg/L

22.66

0.146

22.66

28.29

Concentration level of
10 µg/L*

13.28

0.1192

91.87

305.2

Geomean DT50 (d)

   

45.63 (n=2)

 

* Final step failed, results from next to last step considered

Calculated DT50 and DT90 for BHT-CHO (= Model 1, B1) based on SFO kinetics.

 

chi2

f

DT50

DT90

 

(%)

A1 → B1

(d)

(d)

Concentration level of
90 µg/L

29.41

0.2397

-**

-**

Concentration level of
10 µg/L*

69.68

1

-**

-**

Geomean DT50 (d)

       

* Final step failed, results from next to last step considered

** no degradation found in optimisation

Calculated DT50 and DT90 for BHT-OH (= Model 2, A1) based on SFO kinetics.
 

chi2

f

DT50

DT90

 

(%)

P → A1

(d)

(d)

Concentration level of
90 µg/L

29.58

0.208

80.87

268.6

Concentration level of
10 µg/L*

41.61

0.2673

52.63

174.8

Geomean DT50 (d)

 

 

65.24 (n=3)

 

** no degradation found in optimisation

Calculated DT50 and DT90 for BHT-quinone (= Model 2, A2) based on SFO kinetics.
 

chi2

f

DT50

DT90

 

(%)

A1 → A2

(d)

(d)

Concentration level of
90 µg/L

54.94

1

-**

-**

Concentration level of
10 µg/L*

46.76

1

-**

-**

Geomean DT50 (d)

       

** no degradation found in optimisation

Calculated DT50 and DT90 for the unknown metabolite with Ret. 33.7 min (= Model 2, B1) based on SFO kinetics.

 

chi2

f

DT50

DT90

 

(%)

P → B1

(d)

(d)

Concentration level of
90 µg/L

30.94

0.2171

49.36

164

Concentration level of
10 µg/L*

25.74

0.1704

11.04

36.66

Geomean DT50 (d)

   

23.34 (n = 2)

 

** no degradation found in optimisation

The chi2 values are relatively high as can be seen in the tables with values in the range of 13.3 % to 69.7 % indicating a relative high uncertainty of the calculations. Ideally, the error value at which the chi2-test is passed for the metabolite should be below 15 %, like for parent substance, and the fit must be visually acceptable.

However, this value should only be considered as guidance and not absolute cut-off criterion. There will be cases where the error value to pass the chi2-test for a metabolite is higher, but the fit still represents a reasonable description of its formation and degradation behaviour. This is especially the case when metabolite residues are low.

According to the results of the fitting following geometric mean DT50 values were found:

BHT-CH2OH 45.63 d (n=2)BHT-CHO no degradation

BHT-OH 65.24 d (n=2)

BHT-quinone no degradation

Unkonwn metabolite with Ret. 33.7 min: 23.34 (n=2).

Validity criteria fulfilled:
yes
Remarks:
The degradation rates of the test item might be a worst-case result since a close test setup had to be used due to the given test item properties. It is the responsibility of regulation to consider those results in an appropriate way for risk assessment.
Conclusions:
1. The parent compound 2,6-di-tert-butyl-p-cresol degrades rapidly with half lifes of 8.6 days and 3.7 days at 12°C for an initial concentration level of 90 µg/L and 10 µg/L, respectively.
2. Mechanism of degradation follows two pathways:: a) Oxidation of the p-methyl group to BHT-CH2OH, BHT-CHO and BHT-COOH, and b) oxidative cleavage of the p-methyl group forming BHT-OH amd BHT-quinone. Half-lives of metabolites are reported but have to be used with care due to high uncertainties (chi2 >15% in most cases)
3. Mineralisation is 25 to 33% at the end of the study indicating further cleavage of the aromatic ring.
4. Comparison of results with a sterile control indicates that the degradation is mainly abiotic (oxidative). This behavior reflects the intended property of the substance as an oxidation inhibitor. The observed fate is based on the property of the substance and does not harm the validity of the test.
Executive summary:

In the present study, the aerobic mineralisation of 2,6-di-tert-butyl-p-cresol (BHT) according to the OECD-Guideline 309 " Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test" in surface water was studied. The incubation was performed using 14C-labelled test item in test water using the test concentrations 10 µg/L and 90 µg/L under dark, aerobic conditions. Sterile samples were additionally prepared to enable differentiation between biotic and abiotic degradation. The mineralisation of the test item was studied over an incubation time of 62 days at 12°C ± 2°C.

The properties of the test water used were suitable for studying aerobic mineralisation in water judged by the extensive mineralisation (> 70% AR) of a control reference substance, 14C-sodium benzoate within the incubation time of 62 days. In addition, further test sets were incubated to compare the different test setups and incubation conditions and to monitor their effect on mineralisation and degradation rate of the reference compound.

Replicate samples were taken for analyses at 0, 3, 7, 14, 21, 28, 42 and 62 days after application. The surface water was partintioned with ethyl acetate before and after acidification. Extracts were analysed for the test substance and possible degradation products by HPLC and TLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity except for 5 replicates of the concentration level 10 µg/L and for 3 replicates of the higher concentration level of 90 µg/L. These replicates were all in the range of 83.0% and 89.4%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during incubation and/or sample preparation .

The amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system (Tenax® traps and rinse of inner test vessel surfaces) were always < 5 % AR.

The results show that mineralisation of the test item occurred during the aerobic incubation of 2,6-di-tert-butyl-p-cresol demonstrating complete degradation of the test item. Maximum amounts of 23.7 % AR (concentration level of 10 µg/L) and 36.3 % (concentration level of 90 µg/L) were detected in the sodium hydroxide traps. However, also under abiotic conditions 14CO2 formation was detected indicating that also abiotic transformation led to a complete destruction of the parent molecule.

The amount of radioactivity extracted from the surface water by ethyl acetate decreased continuously in concentration levels from 94.2 % – 98.6 % AR at the beginning of incubation to 45.9 % – 46.9 % AR until the end of incubation. The amount of radioactivity remaining in the water phase after the partitioning with ethyl acetate increased to maximum values of 9.0 % (concentration level of 90 µg/L) – 21.7 % AR (concentration level of 10 µg/L) until the end of incubation. In sterile samples after 62 days of incubation, radioactive amounts in the organic extract and aqueous phase were found in ranges which are comparable to the respective microbial active samples. By acidification of the aqueous phase to a pH-value of ≤ 2, further amounts of radioactivity could be extracted using ethyl acetate.

The amount of parent compound in the organic extracts decreased continuously from maximum levels between 94.2 % - 98.6 % AR immediately after application to amounts in the to amounts between 8.1 % AR (concentration level of 90 µg/L) to non-detectable levels (concentration level of 10 µg/L) after 62 days of incubation. In extracts of sterilised samples, 2,6-di-tert-butylp-p-cresol could not be found at the end of the incubation period of 62 days.

During 62 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the organic extracts as well as an unknown transformation product exceeding 10 % AR. The detailed pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in surface water is summarized in the following tables:

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in surface water treated with 2,6-di-tert-butyl-p-cresol a concentration of 10 µg/L determined in the organic extracts.

      Mean values of two replicates; values are given in percent of the applied radioactivity (% AR)

   Incubation time [d]                     
 Radioactive fraction  0  3  7  14  21  28  42  62
BHT   94.2  54.0  25.4  6.4  2.4  2.0  *  1.9
 BHT-CHO  *  *  *  6.5  7.0  7.4  5.8  5.3
 BHT-OH  *  5.7  7.2 11.1 17.6   18.1  27.1  25.0
 BHT-COOH  *  *  *  *  *  *  *  3.1
 BHT-CH2OH  *  2.2  8.9  9.6  10.3  10.8  6.6  7.9
 BHT-quinone  *  *  4.7  9.0  14.0  11.5  10.7  9.1
 Unassigned Ret. 33.7 min  *  3.3  11.0  8.4  6.0  4.8  *  *
 Unassigned Ret. 3.6 min  *  *  *  *  *  2.2  2.8  *

* = not detected

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in surface water treated with 2,6-di-tert-butyl-p-cresol a concentration of 90 µg/L determined in the organic extracts.

      Mean values of two replicates; values are given in percent of the applied radioactivity (% AR).

   Incubation time [d]                     
 Radioactive fraction  0  3  7  14  21  28  42  62
 BHT  98.6  69.2  56.7  34.2  14.5  8.0  7.3  8.1
 BHT-CHO  *  *  *  *  0.7  1.1  2.6  2.3
 BHT-OH  *  4.0  4.1  9.2  11.2  10.6  16.1  21.3
 BHT-COOH  *  *  *  *  *  *  1.0  *
 BHT-CH2OH  *  5.6  4.5  8.2  7.1  6.9  4.7  4.4
 BHT-quinone  *  *  *  4.1  6.4  6.7  5.8  4.8
 Unassigned Ret. 33.7 min  *  3.8  6.0  6.9  16.7  21.0  8.0  10.6
 Unassigned Ret. 3.5 min  *  *  *  0.8  1.5  1.9  2.0  2.7

* = not detected

The obtained data sets were analysed using the program CAKE version 1.4. The kinetic models considered for the analysis of 2,6-di-ter-butyl-p-cresol were SFO (Single First Order), DFOP (Double First Order in Parallel), HS (Hockey Stick), and FOMC (First Order Multi Compartment). According to the results the best fitting results were obtained when considering SFO kinetics.

The results are 8.6 days and 3.7 days for an initial concentration level of 90 µg/L and 10 µg/L, respectively. The geometric mean DT50 of the recommended optimisation was found to be 5.65 days for the parent compound.

The mechanism of degradation follows two pathways: a) Oxidation of the p-methyl group to BHT-CH2OH,

BHT-CHO and BHT-COOH, and b) oxidative cleavage of the p-methyl group forming BHT-OH and BHT-quinone. Half-lives of metabolites are reported but have to be used with care due to high uncertainties (chi2 >15% in most cases). Further degradation results in cleavage of the phenyl ring and formation of CO2. A sterile control sample run in parallel yielded similar results compared to the active surface water tests indicating that the degradation is mainly abiotically, i.e. oxidative. This behavior can be explained by the technical function of the substance which is intended to act as an oxidation inhibitor.

The validity criterion of reference substance degradation within 2 weeks was met with the test water used at standard conditions defined by the guideline(flow-through system at 20 °C) and also at 12 °C, which is beyond guideline requirements. The results from the flow-through system clearly demonstrate that the biological activity of the surface water used was in line with guideline requirements.

Under the modified test conditions required due to the test substance properties, the mineralisation of the reference substance was much slower. However, since the validity criteria are stated for standard flow-through conditions, their applicability in the closed setup is unclear.

The determined degradation rates of the test item 2,6-di-ter-butyl-p-cresol might be a worst-case result since a close test setup had to be used due to the given test item properties. It is the responsibility of regulation to consider those results in an appropriate way for risk assessment.

Endpoint:
biodegradation in water: sediment simulation testing
Data waiving:
other justification
Justification for data waiving:
other:
Transformation products:
not measured

Description of key information

A simulation study in surface water according to OECD 309 (Derz 2019) yielded the following key results:

1. The parent compound 2,6-di-tert-butyl-p-cresol degrades rapidly with half lifes of 8.6 days and 3.7 days at 12°C for an initial concentration level of 90 µg/L and 10 µg/L, respectively.

2. Mechanism of degradation follows two pathways:: a) Oxidation of the p-methyl group to BHT-CH2OH, BHT-CHO and BHT-COOH,  and b) oxidative cleavage of the p-methyl group forming BHT-OH amd BHT-quinone. Half-lives of metabolites are reported but have to be used with care due to high uncertainties (chi2 >15% in most cases)

3. Minearalisation is 25 to 33% at the end of the study indicating further cleavage of the aromatic ring.

4. Comparison of results with a sterile control indicates that the degradation is mainly abiotic (oxidative). This behavior reflects the intended property of the substance as an oxidation inhibitor.

Key value for chemical safety assessment

Half-life in freshwater:
8.6 d
at the temperature of:
12 °C

Additional information

In the present study (Derz 2019), the aerobic mineralisation of 2,6-di-tert-butyl-p-cresol (BHT) according to the OECD-Guideline 309 " Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test" in surface water was studied. The incubation was performed using 14C-labelled test item in test water using the test concentrations 10 µg/L and 90 µg/L under dark, aerobic conditions. Sterile samples were additionally prepared to enable differentiation between biotic and abiotic degradation. In a pre-test, relevant losses of th test substance under flow-through conditions were found. Therefore the test had to be performed under closed conditions.

Replicate samples were taken for analyses at 0, 3, 7, 14, 21, 28, 42 and 62 days after application. The surface water was partintioned with ethyl acetate before and after acidification. Extracts were analysed for the test substance and possible degradation products by HPLC and TLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity except for 5 replicates of the concentration level 10 µg/L and for 3 replicates of the higher concentration level of 90 µg/L. These replicates were all in the range of 83.0% and 89.4%. It is assumed that the formed volatiles, e.g. 14CO2 could not be trapped completely during incubation and/or sample preparation .

The amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system (Tenax® traps and rinse of inner test vessel surfaces) were always < 5 % AR.

The results show that mineralisation of the test item occurred during the aerobic incubation of 2,6-di-tert-butyl-p-cresol demonstrating complete degradation of the test item. Maximum amounts of 23.7 % AR (concentration level of 10 µg/L) and 36.3 % (concentration level of 90 µg/L) were detected in the sodium hydroxide traps. However, also under abiotic conditions 14CO2 formation was detected indicating that also abiotic transformation led to a complete destruction of the parent molecule.

The amount of radioactivity extracted from the surface water by ethyl acetate decreased continuously in concentration levels from 94.2 % – 98.6 % AR at the beginning of incubation to 45.9 % – 46.9 % AR until the end of incubation. The amount of radioactivity remaining in the water phase after the partitioning with ethyl acetate increased to maximum values of 9.0 % (concentration level of 90 µg/L) – 21.7 % AR (concentration level of 10 µg/L) until the end of incubation. In sterile samples after 62 days of incubation, radioactive amounts in the organic extract and aqueous phase were found in ranges which are comparable to the respective microbial active samples. By acidification of the aqueous phase to a pH-value of ≤ 2, further amounts of radioactivity could be extracted using ethyl acetate.

The amount of parent compound in the organic extracts decreased continuously from maximum levels between 94.2 % - 98.6 % AR immediately after application to amounts in the to amounts between 8.1 % AR (concentration level of 90 µg/L) to non-detectable levels (concentration level of 10 µg/L) after 62 days of incubation. In extracts of sterilised samples, 2,6-di-tert-butylp-p-cresol could not be found at the end of the incubation period of 62 days.

During 62 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the organic extracts as well as an unknown transformation product exceeding 10 % AR.

The obtained data sets were analysed using the program CAKE version 1.4. The kinetic models considered for the analysis of 2,6-di-ter-butyl-p-cresol were SFO (Single First Order), DFOP (Double First Order in Parallel), HS (Hockey Stick), and FOMC (First Order Multi Compartment). According to the results the best fitting results were obtained when considering SFO kinetics.

The results are 8.6 days and 3.7 days for an initial concentration level of 90 µg/L and 10 µg/L, respectively. The geometric mean DT50 of the recommended optimisation was found to be 5.65 days for the parent compound. The higher value was chosen as a key value as worst case.

The mechanism of degradation follows two pathways: a) Oxidation of the p-methyl group to BHT-CH2OH, BHT-CHO and BHT-COOH, and b) oxidative cleavage of the p-methyl group forming BHT-OH and BHT-quinone. Half-lives of metabolites are reported but have to be used with care due to high uncertainties (chi2 >15% in most cases). Further degradation results in cleavage of the phenyl ring and formation of CO2. A sterile control sample run in parallel yielded similar results compared to the active surface water tests indicating that the degradation is mainly abiotically, i.e. oxidative. This behavior can be explained by the technical function of the substance which is intended to act as an oxidation inhibitor.

The properties of the test water used were suitable for studying aerobic mineralisation in water judged by the extensive mineralisation (> 70% AR) of a control reference substance, 14C-sodium benzoate within the incubation time of 62 days. In addition, further test sets were incubated to compare the different test setups and incubation conditions and to monitor their effect on mineralisation and degradation rate of the reference compound. For this reason, further reference control samples of the same surface water were incubated contemporaneously at 20 ± 2 °C in a closed system as well as at 12 ± 2 °C and 20 ± 2 °C in a flow-through system. The results of the reference control samples treated with 14C-sodium benzoate indicate that the mineralisation rate determined in a closed system is generally slower than in a flow-through system even when the oxygen concentrations in the test vessels were sufficiently high.

The validity criterion of reference substance degradation within 2 weeks was met with the test water used at standard conditions defined by the guideline(flow-through system at 20 °C) and also at 12 °C, which is beyond guideline requirements. The results from the flow-through system clearly demonstrate that the biological activity of the surface water used was in line with guideline requirements.

Under the modified test conditions required due to the test substance properties, the mineralisation of the reference substance was much slower. However, since the validity criteria are stated for standard flow-through conditions, their applicability in the closed setup is unclear.

The determined degradation rates of the test item 2,6-di-ter-butyl-p-cresol might be a worst-case result since a close test setup had to be used due to the given test item properties. It is the responsibility of regulation to consider those results in an appropriate way for risk assessment.