2,5-di-tert-pentylhydroquinone

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jun 2019 to 28 Jan 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Radiolabelled Test Item
Identification: [phenyl-U-14C]LOWINOX AH25
Physical Description: Pale Yellow Solid
Batch (Lot) Number: 10532JLM009-8
Radiochemical Purity: 97.8 area%
Chemical Purity: 95.6 area%
Specific Activity: 34.1 mCi/mmol (1262 MBq/mmol)
Conditions: In freezer (≤-15°C)
Expiry Date: Not indicated
Supplier: Selcia Limited, Fyfield Business and Research park, Fyfield Road, Ongar, Essex, CM5 0GS, UK
Radiochemical purity was determined by the supplier (see Appendix 6) and was verified by Charles River Den Bosch at the start of the study.

Additional information
Test Facility test item number: 210342/A (file 206774)
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular formula: C16H26O2
Molecular weight: 251.47 g/mol

Justification for Position and Type of Labelling
Carbon-14 is the isotope of choice in environmental fate studies. Test item is labelled in the most stable part of the molecule (i.e. in the ring).

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

This surface water originated as a result of a dike (levee) failure in the 16th century. The force of the water flowing through the dike created a low area behind the dike. After closure of the dike, a small freshwater pond remained. "Schoonrewoerdse Wiel" (SW) is located in the province Zuid-Holland, in Leerdam, the Netherlands (N51.9168, E005.1331).
Water was sampled on 24 June 2019 from the upper layer (up to 1 meter depth) at the bank of the pond. Oxygen content, pH and water temperature were measured at sampling. The water was transported to the laboratory at ambient temperature. Upon arrival in the laboratory, the water was sieved through a 150 μm sieve. The sieved water was stored refrigerated under aerobic conditions (open lid) in the dark until use.
A sample was shipped to CEMAS, UK, for characterization of the test water.

Details on source and properties of sediment:

Not required

Details on inoculum:

Not required

Duration of test (contact time):

60 d

Initial conc.:

10 µg/L

Based on:

test mat.

Initial conc.:

98 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Preparation of Flasks
On 26 Jun 2019, approximately 300 mL SW water was weighed into 1 L amber-colored conical flasks. A total of 16 flasks was prepared. Two flasks were filled with sterile SW water. Sterilization was performed by autoclaving for 3 hours at 160°C (empty flasks) and 20 minutes at 121°C (water).
The flasks were placed on stirring devices in a climate room at 12°C ± 2°C. The flasks were ventilated gently with humidified air. The air was passed through the flasks just above the water surface. The water was kept in continuously agitation by a stirring bar and a magnetic stirrer.

Test Item Concentration
The target concentrations in the water phase were 10 μg/L (low concentration) and 100 μg/L (high concentration). The target concentration for the reference control was 50 μg/L.

Preparation of Spike Solutions
A spike solution was prepared by dissolving radiolabeled Lowinox ® AH25 in acetonitrile.
Based on LSC of three 10 μL aliquots, the spike solution contained 1.31 MBq/mL (RSD 1.3%), which is equivalent to 260 mg/L. This spike solution was used to treat the water phases of the high test concentration flasks.
A second spike solution was prepared by diluting the first spike solution approximately 10 times with acetonitrile. Based on LSC of three 10 μL aliquots, the spike solution contained 126 kBq/mL (RSD 0.5%), which is equivalent to 25.1 mg/L. This spike solution was used to treat the water phases of the low test concentration flasks.
The reference spike solution was prepared by diluting 14C-labeled benzoic acid in acetonitrile.
Based on LSC of three 10 μL aliquots, the reference spike solution contained 1.34 MBq/mL (RSD 0.4%), which is equivalent to 69.9 mg Benzoic acid/L.
The spike solutions were prepared on the day of spiking. The radiochemical purity of the spike solutions was determined by LC on the same day.

Spike Procedure
A volume of 120 μL of the test item spike solution, was added to ten flasks (low test concentration). To another ten flasks, 115 μL test item spike solution was added (high concentration). To two flasks, 215 μL reference spike solution was added. The spike volume was chosen based on a water layer volume of 300 mL. Two flasks were not treated and were used to monitor oxygen content and pH.
In the beginning, halfway through and at the end of the spiking procedure, the same volume of spike solution was analyzed on LSC (by pipetting the spike solution directly into scintillation vials for the low test concentration and by pipetting the spike solution into separate 5 mL volumetric flasks and made up to volume using acetonitrile for the high test concentration). The results showed that 14.7 kBq (n=3, RSD 0.3%) had been added to the low test concentration flasks (equivalent to 9.8 μg/L), 147 kBq (n=3, RSD 0.5%) had been added to the high test concentration flasks (equivalent to 98 μg/L) and 280 kBq had been added to the reference control flasks (equivalent to 49 μg/L).

Incubation
Immediately after spiking, the metabolism flasks were placed in a climate-controlled room (12 ± 2 °C) in the dark and connected to a series of traps; a polyurethane foam (PUF) plug inserted in the neck of the metabolism flask, a liquid trap containing ethylene glycol methyl ether (EGME) and two liquid traps containing 2 N NaOH. During incubation, aeration took place continuously. The ingoing air was allowed to flow gently above the surface water before leaving the flasks.
The incubation lasted 60 days. The temperature in the climate room was continuously monitored. Dissolved oxygen and pH were every two weeks in the water of an untreated flask (SW 1).

Reference substance:

other: benzoic acid

Remarks:

14C radiolabelled.

Key result

Compartment:

natural water: freshwater

DT50:

0.05 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Low Test Concentration (10 μg/L)

Key result

Compartment:

natural water: freshwater

DT50:

4 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: High Test Concentration (100 μg/L)

Mineralization rate (in CO2):

0 w-1

Transformation products:

yes

No.:

#1

Details on transformation products:

One major transformation product (TP-1) was detected. TP-1 was tentatively identified by LC-MS/MS. In the water layer, TP-1 increased to 59% (low concentration) and 20% (high concentration) within 2 hours after treatment. On Day 11, less than 10% of applied radioactivity was recovered as TP-1 in the water layers of both test concentrations. In the PUF, TP-1 increased to 39% (low concentration) and 16% (high concentration) on Day 4, further increased to 77% (Day 28, low concentration) and 73% (Day 18, high concentration) and then decreased to 38-42% at the end of the incubation period (Day 60).

Evaporation of parent compound:

not specified

Volatile metabolites:

not specified

Residues:

not specified

Details on results:

Radiochemical Purity of Test Item in the Stock Solution
The radiochemical purity of the spike solution was 96.9 % on the day of spiking as determined by LC.

Temperature during Equilibration/Incubation
During the total equilibration and incubation period, the temperature was within the range 12.1 – 12.7 °C.

pH and Oxygen Concentration Measurements during Incubation
During incubation, the pH ranged between 8.1 and 8.4 (mean 8.2) indicating stable, slightly alkaline conditions. The dissolved oxygen concentrations in the water layer fluctuated between 8.3 and 9.1 mg/L (mean 8.9 mg/L). The measurements indicate aerobic conditions in the water layer throughout the incubation period.

Radioactivity in the Surface Water
The amount of radioactivity in the surface water quickly decreased in the test systems: to 12% (low concentration) and 15 % (high concentration) of applied radioactivity on Day 11 and then remained stable in the range 5- 18% up to the end of the incubation period (Day 60). In the sterile controls, 1 % of applied radioactivity was recovered in the surface water on Day 60.

Volatilized Radioactivity
In the PUF extracts of the test systems treated at the low concentration up to 91% of applied radioactivity was recovered on Day 28 and then decreased to 65% of applied radioactivity on Day 60.
In the PUF extracts of the test systems treated at the high concentration up to 84% of applied radioactivity was recovered on Day 18 and then decreased to 65% of applied radioactivity on Day 60 (Day 28 result was considered an outlier).
No significant amounts of radioactivity (< 1% of applied radioactivity) were detected in the ethylene glycol monoethyl ether traps and in the NaOH traps, indicating that no other organic volatiles were formed than trapped in the PUF and that mineralization did not occur.
In the PUF extracts of the sterile controls, 21% (low concentration) and 58% (high concentrations) of applied radioactivity was recovered.

Overall Mass Balance
The total amount of radioactivity recovered in the test systems was between 71% and 102% of applied radioactivity, with the exception of the metabolism flask treated at the high concentration and sampled on Day 28. There was no trend of decreasing mass balances observed.
The mass balance in the sterile controls was much lower. This is likely due to losses of test item due to volatilization. It is not unlikely test item was better retained in the biotic test vessels due to the presence of microbial biomass.

Identification/Characterization of Radioactivity
Lowinox ® AH25 quickly degraded in the surface water. On Day 7, no parent could be detected in the water layer of the low test concentration and less than 10% of applied radioactivity was recovered on Day 11 in the high test concentration. From Day 18 to Day 60, up to 29% parent was recovered in the PUF.
One major transformation product (TP-1) was detected.
In the water layer, TP-1 increased to 59% (low concentration) and 20% (high concentration) within 2 hours after treatment. On Day 11, less than 10% of applied radioactivity was recovered as TP-1 in the water layers of both test concentrations.
In the PUF, TP-1 increased to 39% (low concentration) and 16% (high concentration) on Day 4, further increased to 77% (Day 28, low concentration) and 73% (Day 18, high concentration) and then decreased to 38-42% at the end of the incubation period (Day 60).
Transformation product TP-1 was identified by LC-MS/MS,

DT50 and DT90 Calculations Lowinox ® AH25
The DT50 and DT90 calculations for the decrease of Lowinox ® AH25 in surface water are based on the individual LC results as shown in Table 6 and Table 7. The SFO and FOMC model were fitted to the data. Additionally, HS and DFOP models were fitted to the data for the low concentration.
No good fits were obtained by the SFO and FOMC models for Lowinox ® AH25 low concentration, based on visual and statistical (χ2) assessment. Therefore, biphasic models were tested. The fits obtained by the DFOP and HS models resulted in a better fit.
Considering that the p-values for estimated DFOP model parameters are statistically not significant or higher compared to HS model, the results from the HS fits are reported.
The fits obtained by the SFO and FOMC models for Lowinox ® AH25 high concentration were equally good based on visual fit and residuals. Therefore, further modelling beyond the FOMC model was not performed. Considering that the p-values for estimated FOMC model parameters are statistically not significant or higher compared to SFO model, the results from the SFO fits are reported.

Results with reference substance:

In the NaOH traps connected to the test systems spiked with the reference control significant amounts of radioactivity were detected, increasing to 38 and 80 % of initially applied radioactivity after 60 days of incubation. These results indicate sufficiently viable conditions for the test system (SW water).

Distribution of Radioactivity in Test System at Low Test Concentration (% of Applied Radioactivity; Mean Values)

Time (days)	PUF	EGME	CO2	Water layer	Flush container	Total
0.083 0.25 1 4 7 11 18 28 41 60	0.3 4.1 5.5 39.4 47.2 57.2 70.0 91.1 73.8 64.6	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4	0.0 0.0 0.0 0.1 0.0 0.2 0.2 0.1 0.0 0.1	95.9 84.7 89.2 56.0 43.1 11.7 9.5 9.7 17.9 16.2	1.9 0.4 1.6 2.0 2.3 2.7 1.7 0.7 1.1 0.6	98 89 96 98 93 72 81 102 93 82
60 (sterile)	20.5	0.0	0.0	1.0	0.4	22^1)

na: not applicable; PUF: polyurethane foam; EGME: ethylene glycol monoethyl ether

¹⁾: low value likely caused by losses from the vessel due to volatilisation

 

Distribution of Radioactivity in Test System at High Test Concentration (% of Applied Radioactivity; Mean Values)

Time (days)	PUF	EGME	CO2	Water layer	Flush container	Total
0.083 0.25 1 4 7 11 18 28 41 60	0.3 2.1 3.8 15.7 50.5 63.7 84.0 55.6^1) 78.2 64.6	0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.1 0.1 0.7	95.8 95.5 93.9 81.8 31.3 15.0 7.3 7.9 17.7^2) 4.9	1.6 0.7 2.7 1.6 2.3 1.6 1.5 0.5 1.1 0.4	98 98 100 99 84 80 93 64 97 71
60 (sterile)	57.8	0.0	0.0	1.2	1.0	60^1)

na: not applicable; PUF: polyurethane foam; EGME: ethylene glycol monoethyl ether

¹⁾: much lower recovery from PUF trap than other replicates; not clear what may have caused this

²⁾: deviating chromatogram with significant activity in peaks not defined previously

³⁾: low value likely caused by losses from the vessel due to volatilisation

 

Distribution of Radioactivity in Test System at Low Test Concentration (% of Applied Radioactivity; Mean Values)

Time (days)	Flasks	PUF	EGME	CO2	Water layer	Total
7 11 18 28 41 60	22 / 23 22 / 23 22 / 23 22 / 23 22 / 23 22 / 23	na na na na na na	na na na na na na	8.5 / 30.0 31.2 / 58.4 33.3 / 73.3 36.0 / 76.9 37.0 / 78.5 37.6 / 79.5	na na na na na 4.1 / 8.3	na na na na na 42 / 88

na: not applicable; PUF: polyurethane foam; EGME: ethylene glycol monoethyl ether

 

Parent and Transformation Products in Test System at Low Test Concentration (% of Applied Radioactivity)

Time (days)	Parent in water	Parent in PUF	TP-1 in water	TP-1 in PUF
0	100	0	0	0
0.083 0.25 1 4 7 11 18 28 41 60	38.7 32.3 34.1 18.4 0.0 0.0 0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0 0.0 0.0 5.4 9.2 28.0 26.3	59.0 52.8 56.7 39.3 45.4 2.7 1.7 0.0 17.9^1) 6.3^2)	0.0 4.1 3.8 39.4 45.7 57.2 63.2 76.5 41.7 38.3
60 (sterile)	0.0	0.0	0.0	20.5

na: not applicable

¹⁾: Peak in chromatogram is relatively small; this value should be considered as extrapolated

²⁾: Peak in chromatogram is relatively small; this value should be considered as extrapolated

 

Parent and Transformation Products in Test System at High Test Concentration (% of Applied Radioactivity)

Time (days)	Parent in water	Parent in PUF	TP-1 in water	TP-1 in PUF
0	100	0	0	0
0.083 0.25 1 4 7 11 18 28 41 60	76.0 76.6 72.2 63.0 16.9 6.0 0.1 3.2 2.4 4.9	0.0 0.0 0.0 0.0 0.2 0.2 9.9 0.7^1) 28.6 21.8	20.4 19.4 24.1 19.3 16.4 9.0 5.4 2.0 1.7 0.4	0.3 2.1 3.8 15.7 49.6 63.1 73.4 54.9^2) 48.4 42.2
60 (sterile)	0.0	7.4	1.0	49.9

na: not applicable

¹⁾: Peak in chromatogram is relatively small; this value should be considered as extrapolated

²⁾: Peak in chromatogram is relatively small; this value should be considered as extrapolated

 

Goodness of Fit Assessment (Parent)

Model	Visual fit	χ^2	p-value^1	Residuals
Low concentration
SFO	Moderate	53	<0.05 (k)	Poor
FOMC	Moderate	30.1	Not applicable	Poor
HS	Good	13.4	<0.05 (k1; k2)	Good
DFOP	Good	13.2	>0.1 (k1); <0.05 (k2)	Good
High concentration
SFO	Good	18.7	<0.05 (k)	Good
FOMC	Good	19.5	Not applicable	good

¹p<0.05: estimated parameter(s) statistically significant (t-test) at 0.05 level. p>0.01: estimated parameter(s) not statistically significant (t-test) at 0.1 level.

 

Parameter Estimates for Low Concentration (Parent)

Model	Parameter estimates	DT50(d)	DT90(d)
SFO	M0= 94.0 ± 14.3 k = 6.68 ± 2.65 d^-1(1.6e-2)	0.10	0.35
FOMC	M0= 99.7 ± 8.5 α = 0.39 ± 0.12 β = 0.012 ± 0.013	0.06	4.34
HS	M0= 100 ± 3.8 k1= 13.9 ± 1.0 d^-1(1.6e-6) k2= 0.260 ± 0.047 d^-1(4.3e-4) tb= 0.069 ± 0.008	0.05	5.24
DFOP	M0= 100 ± 3.8 k1= 51.6 ± 70.7 d^-1(2.4e-1) k2= 0.258 ± 0.049 d^-1(5.8e-4) g = 0.61 ± 0.04	0.03	5.25

 

Parameter Estimates for High Concentration (Parent)

Model	Parameter estimates	DT50(d)	DT90(d)
SFO	M0= 87.4 ± 5.4 k = 0.172 ± 0.035 d^-1(3.9e-4)	4.0	13.4
FOMC	M0= 89.9 ± 5.7 α = 141 ± 1990 β = 642 ± 2380	3.2	10.6

 

Validity criteria fulfilled:

yes

Conclusions:

Lowinox ® AH25 quickly degraded in the surface water to less than 10 % of applied radioactivity within 7 days of incubation in the test systems treated at a low test concentration (10 μg/L) and within 11 days of incubation in the test systems treated at a high test concentration (100 μg/L). From Day 18 to Day 60, up to 29% parent was recovered in the PUF.
One major transformation product (TP-1) was detected. TP-1 was tentatively identified by LC-MS/MS. In the water layer, TP-1 increased to 59% (low concentration) and 20% (high concentration) within 2 hours after treatment. On Day 11, less than 10% of applied radioactivity was recovered as TP-1 in the water layers of both test concentrations. In the PUF, TP-1 increased to 39% (low concentration) and 16% (high concentration) on Day 4, further increased to 77% (Day 28, low concentration) and 73% (Day 18, high concentration) and then decreased to 38-42% at the end of the incubation period (Day 60).
Mineralization to CO2 did not occur.
These values are based upon fitting HS kinetics to parent compound in surface water spiked at 10 μg/L (low concentration) and fitting SFO kinetics to parent compound in surface water spiked at 100 μg/L (high concentration).

Executive summary:

The objectives of the study were to determine the mineralisation and rate of degradation of LOWINOX ® AH25 in surface water under aerobic conditions at 12 ± 2 °C. An additional objective of this study was to identify the transformation product TP-1 detected during the aerobic transformation of the test item in surface water.

 

Study Set-Up

¹⁴C-labeled Lowinox ® AH25 was incubated under aerobic conditions in the laboratory in surface water (“pelagic test”) at 12 ± 2 °C in the dark for 60 days. The initial Lowinox ® AH25 concentrations in the test system were 10 μg/L and 98 μg/L. Per test concentration, ten flasks were prepared (including a sterile control). Additionally, two flasks were treated with benzoic acid as reference control. Two flasks were not treated and were used for monitoring temperature, pH and oxygen concentration.

 

Oxygen concentration measurements indicated aerobic conditions in the water throughout the test. The results of the reference control (benzoic acid) showed that the test system was sufficiently viable.

 

Dedicated flasks were sampled after 2, 6 hours, 1, 4, 7, 11, 18, 28, 41 and 60 days after spiking.

 

Volatiles were trapped by polyurethane foam, ethylene glycol monoethyl ether and NaOH traps. The surface water was analyzed by LSC and LC. A selection of samples was analysed by a second chromatographic method.

 

Fate of Lowinox ® AH25

Lowinox ® AH25 quickly degraded in the surface water to less than 10 % of applied radioactivity within 7 days of incubation in the test systems treated at a low test concentration (10 μg/L) and within 11 days of incubation in the test systems treated at a high test concentration (100 μg/L). From Day 18 to Day 60, up to 29% parent was recovered in the PUF.

 

One major transformation product (TP-1) was detected. TP-1 was tentatively identified by LC-MS/MS. In the water layer, TP-1 increased to 59% (low concentration) and 20% (high concentration) within 2 hours after treatment. On Day 11, less than 10% of applied radioactivity was recovered as TP-1 in the water layers of both test concentrations. In the PUF, TP-1 increased to 39% (low concentration) and 16% (high concentration) on Day 4, further increased to 77% (Day 28, low concentration) and 73% (Day 18, high concentration) and then decreased to 38-42% at the end of the incubation period (Day 60).

 

Mineralization to CO2 did not occur.

 

Kinetics

The DT50 and DT90 values of Lowinox ® AH25 in surface water are shown in the table below.

 

DT₅₀and DT₉₀of Lowinox® AH25 in Surface Water

Compartment	Initial test concentration (μg/L)	Kinetics	DT50 (days)	DT90 (days)
Surface water	10 100	HS SFO	0.05 4.0	5.24 13.4

 

Description of key information

DT₅₀and DT₉₀of Lowinox® AH25 in Surface Water

Compartment	Initial test concentration (μg/L)	Kinetics	DT50 (days)	DT90 (days)
Surface water	10 100	HS SFO	0.05 4.0	5.24 13.4

 

Key value for chemical safety assessment

Half-life in freshwater:

4 d

at the temperature of:

12 °C

Additional information

The objectives of the study were to determine the mineralisation and rate of degradation of LOWINOX ® AH25 in surface water under aerobic conditions at 12 ± 2 °C. An additional objective of this study was to identify the transformation product TP-1 detected during the aerobic transformation of the test item in surface water.

Fate of Lowinox ® AH25

Lowinox ® AH25 quickly degraded in the surface water to less than 10 % of applied radioactivity within 7 days of incubation in the test systems treated at a low test concentration (10 μg/L) and within 11 days of incubation in the test systems treated at a high test concentration (100 μg/L). From Day 18 to Day 60, up to 29% parent was recovered in the PUF.

 

One major transformation product (TP-1) was detected. TP-1 was tentatively identified by LC-MS/MS. In the water layer, TP-1 increased to 59% (low concentration) and 20% (high concentration) within 2 hours after treatment. On Day 11, less than 10% of applied radioactivity was recovered as TP-1 in the water layers of both test concentrations. In the PUF, TP-1 increased to 39% (low concentration) and 16% (high concentration) on Day 4, further increased to 77% (Day 28, low concentration) and 73% (Day 18, high concentration) and then decreased to 38-42% at the end of the incubation period (Day 60).

 

Mineralization to CO2 did not occur.

 

Kinetics

The DT50 and DT90 values of Lowinox ® AH25 in surface water are shown in the table below.

 

DT₅₀and DT₉₀of Lowinox® AH25 in Surface Water

Compartment	Initial test concentration (μg/L)	Kinetics	DT50 (days)	DT90 (days)
Surface water	10 100	HS SFO	0.05 4.0	5.24 13.4