2,5-di-tert-butylhydroquinone

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:

03/08/2020-20/11/2020

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item : YAPOX®2245 (2,5-di-tertiary butyl hydroquinone)

Chemical name (CAS) : 2,5-di-tertiary butyl hydroquinone

CAS No. : 88-58-4

Mfg date : June-2020

Batch manufactured by : Plot No. 2514/2515, Phasse IV, G.I.D.C.
Vapi – 396195, Gujarat, India

Batch supplied by : Yasho Industries Limited, Office No.101/102, Peninsula Heights, C. D. Barfiwala Marg,
Juhu Lane, Andheri (W), Mumbai – 400058

Best before use : MAY 2022

Purity : 99.22%

Batch No. : 20015011220

Physical appearance : White crystalline powder

Storage conditions : Ambient (+15C to +25C)
Note:
a) The test item was received at the test facility on 08 July 2020.
b) Test item code by test facility: Y002-03

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on source and properties of surface water:

7.5 Test System and its Justification
Test system consisted of treated surface water in multiple glass vessels (conical flasks) of 250 mL capacity closed with glass stoppers. The test vessels were sterilized by autoclaving at 121ºC for 20 minutes before use to avoid microbial contamination. All test vessels were incubated in dark.
The test system was chosen to simulate degradation of the test item in surface water as per guideline requirement.
7.6 Test System Identification
Each test system was uniquely identified by study number, treatment, test item, tentative scheduled sampling time and other appropriate study identifiers.
7.7 Collection of Surface Water
The surface water was collected in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection. Furthermore, the depth of sampling and the appearance of the water sample (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions.
The test water was collected by the test facility on 15 September 2020.
7.8 Transportation of Surface Water
The water was transported to the test facility within 3 hours from collection. The water samples were not frozen.
7.9 Storage of Surface Water
The test water was stored about 4 to 6°C with aeration before use for a period not more than 4 weeks.
7.10 Preparation of Surface Water
The water sample was stored at about 4 to 6C with aeration until use. Prior to use, the coarse particles were removed by filtration through a 100 m mesh sieve.

Duration of test (contact time):

168 h

Initial conc.:

10 µg/L

Based on:

test mat.

Initial conc.:

100 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

a) Test concentration – 0.01 and 0.1 g/mL
A 20 mL aliquot of 1.0146 g/mL of YAPOX 2245 treatment solution was transferred into a 2000-mL volumetric flask, diluted to the mark using test water. The solution was mixed thoroughly to get a solution of nominal concentration of 0.01 g/mL.
A 20 mL aliquot of 10.416 g/mL of YAPOX 2245 treatment solution was transferred into a 2000-mL volumetric flask, diluted to the mark using test water. The solution was mixed thoroughly to get a solution of nominal concentration of 0.1 g/mL.
b) Test concentration – 1 g/mL
A 20 mL aliquot of 109.0425 g/mL of YAPOX 2245 treatment solution was transferred into a 2000-mL volumetric flask and diluted to the mark using test water. The solutions were mixed thoroughly to get nominal concentration of 1.0 g/mL.
7.16 Preparation of Test Solutions for Incubation
A 100 mL aliquot of test water and sterile water treated with test item at 10 µg/L (0.01 µg/mL) concentration was transferred into 20 test vessels of 250 mL capacity.
A 100 mL aliquot test water and sterile water treated with test item at 100 µg/L (0.1 µg/mL) concentration was transferred into 20 test vessels of 250 mL capacity.
A 100 mL aliquot test water and sterile water treated with test item at 1000 µg/L
(1 µg/mL) concentration was transferred into 10 test vessels of 250 mL capacity.
7.17 Test Temperature
The test systems were incubated in the dark at 20  2C.
7.18 Agitation
The test vessels were placed on an incubator shaker at 20C to maintain a homogeneous suspension of particles and microorganisms. Agitation was also helpful to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained.
7.19 Test Duration
The treated surface water was incubated for a period of about 7 days.
7.20 Measurement of pH and Oxygen Concentration
pH and oxygen concentration in the test system treated with YAPOX 2245 was measured at each sampling interval.
7.21 Sampling
Duplicate test vessels from each test concentration was collected for analysis at zero-time (immediately after treatment), 2, 4, 6, 12, 18, 24, 48, 72, 96 and 168 hr during the incubation period. Shaking was continued at 20 ± 2C in dark for using in other sampling intervals.

Reference substance:

aniline

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

ca. 1

St. dev.:

0

Parameter:

test mat. analysis

Sampling time:

168 h

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

ca. 2.9

St. dev.:

0

Parameter:

test mat. analysis

Sampling time:

168 h

Key result

Compartment:

natural water

DT50:

14.8 h

St. dev.:

0

Type:

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

Temp.:

20 °C

Key result

Compartment:

natural water

DT50:

13.7 h

St. dev.:

0

Type:

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

Temp.:

20 °C

Other kinetic parameters:

first order rate constant

Transformation products:

no

Details on results:

8.1 Properties of Test System
8.1.1 Properties of Test Water
The date of sample collection, depth of collection, appearance (e.g., colour and turbidity), temperature, oxygen concentration and pH were recorded. The location map of the collection site is presented in Figure 6. The test water sample was sent to Eurofins Analytical Services India Pvt. Ltd., # 540/1, Doddanekundi Industrial Area 2, Hoodi, Whitefield, Bangalore – 560048, Karnataka, India for analysis of total organic carbon (TOC), dissolved organic carbon (DOC), Nitrate (NO3-), Nitrite (NO2-), Ntot, Ptot, orthophosphate (PO43-), ammonia (NH4+) and BOD.
A summary of characterization of test water is presented in Table 1.
8.1.2 Experimental Design
Details of the experimental design are provided in Table 2.
8.1.3 Test Item Solubility
With a targeted concentration of 0.1 g/mL with 1% methanol as co-solvent, the initial concentration of the test solution prepared in Milli-Q water was 0.0956 g/mL, and the concentration was found to be 0.0920 g/mL when the test solution was centrifuged.
Considering the solubility of YAPOX 2245 as 0.0956 g/mL, which was sufficient to prepare treatment solutions for test concentrations of 10 g/L (0.01 g/mL) and 100 g/L (0.1 g/mL).
8.2 Determination of YAPOX 2245 in Test water Samples
Analysis of 0 hr samples at 10 g/L (0.01 g/mL) and 100 g/L (0.1 g/mL) test concentrations demonstrated quantitative recovery of YAPOX 2245.
Following application of YAPOX 2245 to test water at 10 g/L, the average amount of YAPOX 2245 present was 84.5% and 2.9% at 0 hr and 168 hr, respectively.
Following application of YAPOX 2245 to test water at 100 g/L, the average amount of YAPOX 2245 present was 86.7% and 1.0% at 0 hr and 168 hr, respectively.
Following application of YAPOX 2245 to sterile test water at 100 g/L, the average amount of YAPOX 2245 present was 87.5% and 1.1% at 0 hr and 168 hr, respectively.
No metabolites were observed at this test concentration during the incubation period.
Based on the above results, YAPOX 2245 degrade rapidly in natural water and no metabolite was found in both unsterile and sterile test water samples treated at 1000 µg/L.
8.3 Validity of the Test
The percent recovery of aniline reference standard was 0.0% on day 14, indicated its degradation in the test water within the expected time interval of two weeks. Therefore, the validity of the test is acceptable. Results are presented in Table 9.
8.4 Identification of Metabolites
All test water samples were initially analyzed by reverse phase HPLC with on-line radiochemical detector. During HPLC analysis, the test item in test water were assigned by comparing the retention time of that of authentic standard (Figure 11). No metabolite was found the samples analysed up to 7 days by HPLC. An effort was made to identify YAPOX 2245 and metabolites (if any) in all samples till day 7 by LC-MS.
Representative HPLC chromatograms along with reference standard are presented in Figure 10 through Figure 17.
8.5 Kinetic Analysis of Data
The YAPOX 2245 data generated from 0, 2, 4, 6, 12, 18, 24, 48, 72, 96 and 168 hr after test item application to test water was used for degradation kinetics by CAKE version 3.3 (Release) software.
The details of optimized parameters, Chi square and r2 values from each model are included in Appendix 4. The fit model and its calculated DT50 and DT90 values were shown in Table 12.
The calculated DT50 (hours) and DT90 (hours) for each concentration are summarized in the table below:
Concentration DT50 (hours) DT90 (hours) Rate constant (d-1)
10 µg/L 14.8 49 0.04699 ± 0.005629
100 µg/L 13.7 45.7 0.05043 ± 0.002245
100 µg/L (sterile) 22.1 73.4 0.09105 ± 0.006926
Plots of the observed and fitted data and parameter estimates from the best-fit model for test water treated with YAPOX 2245 are presented in Appendix 4
8.6 Degradation Pathway
YAPOX 2245 degrade rapidly in natural water during the study and no metabolites were found the samples. Hence, no degradation pathway was proposed for the test item YAPOX 2245.
8.7 Method Validation
The chromatography method was validated with respect to below mentioned parameters.
8.7.1 Specificity
Due to the selective nature of the detection of this method (two individual ion transitions monitored), interference peaks were not observed at the retention time of the analyte.
8.7.2 Linearity
Linearity solutions prepared in diluent used for quantitative and confirmatory analysis of YAPOX 2245 ranged from 0.0010-0.200 µg/mL in concentration. Typical LC-MS/MS chromatogram for standard analyzed during method validation is presented in Figure1.
8.7.3 Accuracy (Recovery) and Precision (Repeatability)
During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was <20% at each fortification level. Recovery data from these samples demonstrated that YAPOX 2245 was stable during analysis. The recoveries of all the samples analysed were in the range of 70-110% with %RSD ≤ 20%. Results are presented in Table 5.

Results with reference substance:

0%

Validity criteria:

percent recovery of aniline reference standard

Observed value:

0%

Validity criteria fulfilled:

yes

Conclusions:

The biodegradation of YAPOX 2245 was studied at two concentrations (10 g/L and 100 g/L) in aerobic natural water using YAPOX 2245 in dark at 20  2C and the observations were quantified in the form of kinetic rate expressions. The degradation was also studied at 1000 g/L for identification of metabolites. This simulation test was laboratory shake flask batch test to determine rates of aerobic biodegradation of YAPOX 2245 in samples of natural surface water.
Test systems consisted of treated surface water in sterilized conical flasks closed with glass stoppers. The surface water was treated at 10 µg/L (0.01 µg/mL) and
100 µg/L (0.1 µg/mL). Additionally, the sterile surface water was treated at
100 µg/L (0.1 µg/mL). The treated water was housed in an incubator shaker at
20 ± 2C in dark. Aerobic condition was maintained in the system by continuous shaking.
Duplicate test vessels from each test concentration were removed at each sampling occasion and analyzed at zero-time (immediately following test item application) and at 2, 4, 6, 12, 18, 24, 48, 72, 96 and 168 hr. At each sampling occasion, duplicate aliquots from each test concentration were subjected to LC-MS/MS analysis.
Analysis of 0 hr samples at 10 g/L (0.01 g/mL) and 100 g/L (0.1 g/mL) test concentrations demonstrated quantitative recovery of YAPOX 2245.
Following application of YAPOX 2245 to test water at 10 g/L, the average amount of YAPOX 2245 present was 84.5% and 2.9% at 0 hr and 168 hr, respectively.
Following application of YAPOX 2245 to test water at 100 g/L, the average amount of YAPOX 2245 present was 86.7% and 1.0% at 0 hr and 168 hr, respectively.
Following application of YAPOX 2245 to sterile test water at 100 g/L, the average amount of YAPOX 2245 present was 87.5% and 1.1% at 0 hr and 168 hr, respectively.
Based on the above results, YAPOX 2245 degrade rapidly in natural water and no metabolite was found in both the non-sterile and sterile test water samples treated at 1000 µg/L.
The DT50 (hours) and DT90 (hours) values calculated for YAPOX 2245 are summarized below:
Concentration DT50 (hours) DT90 (hours) Rate constant (d-1)
10 µg/L 14.8 49 0.04699 ± 0.005629
100 µg/L 13.7 45.7 0.05043 ± 0.002245
100 µg/L (sterile) 22.1 73.4 0.09105 ± 0.006926

Executive summary:

The aerobic mineralization in water of YAPOX 2245 was studied at two concentrations (low concentration of0.01mg/mL and high concentration of
0.1mg/mL)in natural water using YAPOX 2245 (2.5-di-tertiry butyl hydroquinone) in dark at 20±2°C under aerobic conditions and the observations were quantified in the form of kinetic rate expressions. Additionally, the degradation was also studied at 1mg/mLin natural water using in dark at 20±2°C under aerobic conditions for identification of metabolites. This simulation test was laboratory shake flask batch test to determine rates of aerobic biodegradation of YAPOX 2245 in samples of natural surface water. The characteristics of water used in this study was as follows:

Parameter	Results
pH	7.31
Temperature (°C)	23.4
Depth of sampling (feet)	1 – 2
Appearance (colour / turbidity)	Clear with no turbidity
Oxygen concentration (mg/L)	7.9
Total organic carbon (TOC), mg/L	3.0
Dissolved organic carbon (DOC), mg/L	3.5
Nitrate (NO3^-), mg/L	<1
Nitrite (NO2^-), mg/L	0.2
Ntot (mg/L)	<0.5
Ptot (mg/L)	<0.1
Orthophosphates (PO4^3-), mg/L	<0.1
Ammonia (NH4^+), mg/L	<0.3 BOD, mg/L <2 The study was conductedusing procedures outlined in OECD Guideline 309 (13^thApril 2004). Test systems consisted of treated surface water in sterilized conical flasks closed with glass stoppers. The surface water was treatedat 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL). Additionally, the sterile surface water was also treated at 100 µg/L (0.1 µg/mL) concentration. The treated water was housed in an incubator shaker at 20 ± 2°C in dark.Aerobic condition was maintained in the system by continuous shaking. Duplicate test vessels from each test concentrationwere removed at each sampling occasion and analyzed at zero-time (immediately following test item application) and at2, 4, 6, 12, 18, 24, 48, 72, 96 and 168 hr.At each sampling occasion, duplicate aliquots from each test concentration were subjected to analysis by a validated LC-MS/MS method.     Analytical method based on LC-MS/MS was validated for the determination of test itemYAPOX 2245 in surface water sampleswith respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The surface water samples were analyzed for the residues of test item by liquid chromatography with negative-ion electrospray ionization (ESI) tandem mass spectrometry using the transitionm/z221.1 -> 205.2 for primary quantification and the transitionm/z221.2 -> 149.1 for qualitative confirmation. The recoveries of fortified samples at 0.001 µg/mL (LOQ) and 0.01 µg/mL (10 LOQ) support the satisfactory performance of this method. The below table summarized the average recovery results from the fortified water samples. Statistics N YAPOX 2245 Surface water fortified at 0.001µg/mL(LOQ) level (m/z221.1 -> 205.2) (%) Mean 5 93.8 (±) SD 5.9 (%) RSD 6.3 Surface water fortified at 0.01µg/mL(10 LOQ) level (m/z221.1 -> 205.2) (%) Mean 5 100.1 (±) SD 0.8 (%) RSD 0.8   Analysis of the 0 hour samples at 10mg/L and 100mg/Ltest concentrations demonstrated quantitative recovery ofYAPOX 2245.

Following application of YAPOX 2245 to test water at 10mg/L, the average amount of YAPOX 2245 present was 84.5% and 2.9% at 0 hr and 168 hr, respectively.

Following application of YAPOX 2245 to test water at 100mg/L, the averge amount of YAPOX 2245 present was 86.7% and 1.0% at 0 hr and 168 hr, respectively.

Following application of YAPOX 2245 to sterile test water at 100mg/L, the average amount of YAPOX 2245 present was 87.5% and 1.1% at 0 hr and 168 hr, respectively.

For identify the possible metabolite during study period the test item YAPOX 2245 was treated at 1000mg/L to test water and samples were analysed at pre-determined sampling intervals. There were no transformation products/ metabolites found.

Based on the above results,YAPOX 2245degrade rapidly in natural water and no transformation product was found.

The DT₅₀(hours) and DT₉₀(hours) values calculated for YAPOX 2245 are summarized below:

Concentration	DT50(hours)	DT90(hours)	Rate constant (d^-1)
10 µg/L	14.8	49	0.04699 ± 0.005629
100 µg/L	13.7	45.7	0.05043 ± 0.002245
100 µg/L (sterile)	22.1	73.4	0.09105 ± 0.006926

 

Key value for chemical safety assessment

Half-life in freshwater:

14.8 h

at the temperature of:

20 °C