Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

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:

other: simulation study with the relevant constituent of the registered UVCB substance

Adequacy of study:

key study

Study period:

2021-03-30 - 2023-01-19

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:

The test substance is a component of the registered UVCB substance:
Name of test substance: 14C-Bis(4-tert-butylphenyl)amine
Test substance No.: 18/0435-1
Label name: phenyl-U-C14
Batch No: 1254-1101
Chemical purity: 97.3 %
Radiochemical Purity: 98.7 %
Identity: confirmed
Specific activity: 72.8 MBq/g
Specific activity of AI: 7.8 MBq/mg
Concentration of AI:
AI=active ingredient (test item)
9.21 mg/g
Homogeneity: Homogenous
Solvent: Acetonitrile
IUPAC name: Bis(4-t-butylphenylamin)
Reg. No.: 6151103

Name of unlabeled test
substance: Bis(4-tert-butylphenyl)amine
Chemical name Bis(4-tert-butylphenyl)amine
Synonym Bis(4-tert-butylphenyl)amine
Test substance No: 21/0138-1
Batch No: HDMPF
CAS No: 4627-22-9
Purity: 93.9 % (Certificate of Analysis) or 95.1 corr. Area-%
Water solubility: 4 µg/L at temperature =20 °C ±0.5 °C
Appearance
- physical state: Solid/
- color: white to yellow
Storage conditions Ambient (room temperature)
Storage stability: 08 April 2023

Radiolabelling:

yes

Remarks:

14C- labelling

Inoculum or test system:

effluent/surface water mixture, untreated

Details on source and properties of surface water:

Surface water from river arm “Ranschgraben’’ west of Schifferstadt was collected on 13th April 2021 for this study. After reaching the laboratory, the coarse particles were removed from the water with a nylon sieve (100 µm) prior to use in the test. Subsamples from the collected water were taken for determination of physico-chemical and biological parameters. Afterwards, fresh surface water sample was stored under dark conditions for one day at a temperature of ~ 5.8 °C – 6.9 °C before preparation of test assays.

Duration of test (contact time):

91 d

Initial conc.:

40 µg/L

Based on:

test mat.

Initial conc.:

4 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

test mat. analysis

Details on study design:

3.6. EXPERIMENTAL PROCEDURE
3.6.1. SETTING UP OF TEST ASSAYS
The test was set up as a batch-test “pelagic test” in multiple test assays. Mass of
samples corresponding to the volume in water were determined during setting up of test assays as well as during sampling procedure unless otherwise indicated.
Preparation of test vessels The test was performed in 1 L GL45 cylindrical spinner flasks with 2 angled sidearms (dimension (H x B ): 23 cm x 10 cm) as show below). In total, 82 test vessels were set up for this study (see table 3a and 3b for the preparation scheme of test assays).
To each of these 1 L test vessels, about 500 mL surface water was added and also a Teflon coated magnet bar. Separate closed tests vessels were set up for planned sampling point.

Stripping vessel for trapping the dissolved CO2 (schematic diagram)
Through the sampling port inlet, air was supplied after acidification process to remove the dissolved CO2.
Test assays with test substance: Two different concentrations of test substance differed by a factor of 10 were used. Low concentration of test substance designated by acronym ‘L’ is intended for measuring the degradation kinetics and high concentration designated by ‘H’ for identification and quantification of major transformation products.

Low concentration: 4 μg test substance per liter
High concentration. 40 μg test substance per liter

Test assays with test substance for biodegradation kinetics and metabolite
identification (FT): These test assays were set up in duplicates at two different
test concentrations, namely low concentration FT-L assays and high concentration FT-H assays.
Test assays with test substance for mass balance calculation (FM): Additionally, test flasks with test substance were set up for mass balance calculation at the end of exposure in duplicate flasks for each test concentration (low concentration FM-L, high concentration FM-H)).
The test substance is added from the stock solution prepared as described in section 3.4., into the test substance assays (FT and FM). The organic solvent will not exceed 1% by volume in the water phase.
Low concentration flasks (FT-L & FM-L): 45.8 μL from corresponding test substance stock solution per 500 mL test water
High concentration flasks (FT-H & FM-H): 45.8 μL from corresponding test substance stock solution per 500 mL test water
Sterile control assay (FS) with test substance for abiotic transformation: Duplicate test vessels containing sterile test system and high-test concentration test substance (40 μg/L) were set up as sterile controls. For this, surface water was autoclaved at 121°C for 20 minutes prior to the addition of test substance. These test assays were sampled only at the end of exposure to examine the possible abiotic degradation or other nonbiological removal of the test substance. No water exchange was conducted on day 59 as these are sterile samples.
Sterile control flasks (FS): 45.8 μL from corresponding test substance stock solution per 500 mL test water.
Reference control assay (FC): 14C reference substance was added from the stock
solution (see section 3.4) to the test system to confirm a minimum of microbial activity.

This assay was set up in duplicates for each sampling day.
Reference assay flasks (FC): 550 μL of the reference substance stock solution
Solvent control assay (FOC): Duplicate flasks containing test system containing reference substance at a inal nominal concentration of 10 μg/L treated with the approximate same amount of solvent (50 μL acetonitrile) that was present in the highest test substance concentration used in this test (40 μg/L test substance). This was to examine possible adverse effects of the solvent by determining the degradation of the reference substance.
Solvent control (FSC) : Test flasks containing test system and used solvent but without test substance. The highest solvent volume used for adding the test substance to the test assays (50 μL) was tested in these assays.
Blank control assay (FB): Test flask with only test system was set up and was used only for the physico-chemical analysis on the samplings days as well as for the microbiological analysis at the end of exposure.
Parameter control assay with test substance (FP): These test assays were dosed with high concentration test substance (40 μg/L) and test system.
High-12C (FTMET-H): These additional test assays were prepared with 49.8 μL of non labelled test substance at high concentration of 40 μg/L and test system.
Solvent control (FSC-MET): Additional set of solvent control test flasks containing test system and solvent (50 μL) but without test substance for 12C experiment. The highest solvent volume used for adding the test substance to the test assays was dosed to these assays.
After setting up the test assays mentioned above, about 5 mL of 2 M NaOH solution was placed in the internal container as the absorbing solution in each test flask. The flasks were closed with polypropylene matching screw cap with pouring ring impermeable to air and CO2 and were placed on magnetic stirrers and gently and continuously stirred while still maintaining a homogeneous suspension (approx. 100 rpm agitation) until the end of exposure. No contact between test solution and lid of the test flask is made during the preparation of test assays and also during incubation. The test vessels were connected
with two CO2 traps filled with 2 M NaOH of 100 mL and a trap filled with ethylene glycol of 50 mL to capture organic volatiles.

3.6.2. Test temperature, duration and agitation
Test vessels were filled with 500 mL surface water on 14th April 2021 and incubated 12 ± 2°C in the dark for 5 days and was aerated by a gentle orbital shaking of the test vessel on magnetic stirrer under constant air flow. This equilibration procedure was done due to very low temperature in the test system (about 7.2 °C) at the time of collection. All these test flasks were incubated at 12 ±1 °C from day 0 of exposure in a thermostat enabled water bath under dark conditions for 91 days. The test flasks were placed on magnetic stirrers and gently and continuously stirred while still maintaining a homogeneous suspension (approx. 100 rpm agitation) until the end of exposure. Temperature was checked and documented from the water bath on sampling days.

3.6.3. Sampling from the test assays
Samplings were conducted from the corresponding test assays by harvesting the whole flasks at the start of exposure (Day 0, after complete homogenization), Day 1, Day 7, Day 14, Day 21, Day 28 and on Day 49. Because of very low 14CO2 evolution detected within the first 49 days, it was decided to extend the batch test from 60 to 91 days as a semicontinuous procedure. Reserve bottles (FTLA and FTHA, FML and FMH, FMET-H and FSC-MET) were used for sampling in the remaining 31 days. On day 59 before surface water removal and water replenishment, direct LSC, evolved 14CO2 from NaOH traps and organic
volatiles measurements were conducted from the reserve flasks. After these measurements, water (1/3, about 165 mL, see table 7) was removed from the
test flasks and the residual test substance (theoretic) concentration lost in this 1/3rd water will be replenished for the initial amounts of test substance by adding adequate aliquots (approx.15.3 μL of 14C labelled test substance stock solution and approx. 16.6 μL unlabeled test substance stock solution) from corresponding stock solution with freshly collected water. Every two weeks. Three additional samplings after Day 59 were conducted such as on Day 71, Day 80 and on Day 91. Since the test substance is degrading slower without significant CO2 production, long interval between sampling days is chosen after water change. No water change was needed after day 59 as the metabolite formation is detected (preliminary info from NOACK institute) and the pH and oxygen
parameters were stable. Analytical samples from these additional sampling points were also sent to NOACK for substance specific analytics.
A general scheme of sampling is provided in table 3a and 3b. Sampling was conducted as described in the following steps after harvesting the whole flasks at each sampling time from the corresponding test assays and the samples are represented in mass instead of volume unless otherwise stated.

Direct LSC measurements in the test substance assays (FTL, FTLA, FTH, FTHA, FS, FSA, FML, FMH) and reference assays (FC, FCA, FOC) For direct LSC measurements, duplicate samples (about 5 mL) were taken (except on
day 7 in FCA where only 2.73 g was taken) through the inlet and after combining with 15 mL of Ultima Gold scintillation cocktail, these samples were assayed by LSC.

Sampling of 14CO2 for quantification of mineralization in the test substance
assays (FTL, FTLA, FTH, FTHA, FS, FSA, FML, FMH) and reference assays (FC, FCA, FOC)
The mineralization of the test substance was determined by “direct 14CO2 measurement” from duplicate test assays on each sampling day. The 14CO2 generated from mineralization of test substance during experimental exposure is trapped into the absorption liquid (2 M NaOH solution) placed in the internal container attached in the test vessel.
a. Dissolved 14CO2
In addition to this, there can be CO2 dissolved in the test solution. In order to remove this dissolved CO2 from the surface water, about 50 mL of the water phase in the test flasks was removed through the sampling port constructed of Teflon tubing that extended to the bottom of all test vessels connected to a stopcock. Prior to the removal of samples, the stopcock was opened, and the test mixture was repeatedly pulled up and pushed back into the flask using a syringe to clear the line of the test flask. The samples were added to another flask (see figure in section 3.6.1) containing a compartment filled with 5 mL of 2
M NaOH and were acidified by adding about 1.5 mL of 6N HCl to lower the pH of the samples to ~ 1 without opening the vessels to the atmosphere. After acidification, the test vessel was connected to the air supply and is lightly aerated for 24 hours to allow CO2 to diffuse from solution into the headspace. The dissolved CO2 was captured in the absorption liquid (2 M NaOH solution) placed in the internal container in the test vessel.
After 24 hrs., a suitable aliquot from this NaOH solution (~ 1 mL) was mixed with 15 mL LSC scintillation cocktail and was measured by LSC as double determination for radioactivity. Remaining radioactivity in the acidified sample was measured in duplicates without any further treatment.

b. Evolved 14CO2 from NaOH traps
After closing the valve in the gas transferring tubes, two NaOH traps with 100 mL of 2 M solution were removed and quickly capped. Duplicate samples from the sodium hydroxide traps were transferred to scintillation vials and combined with a suitable scintillation cocktail and analyzed by LSC.
Confirmation of the analyte were originally planned to be performed in representative samples through precipitation with barium chloride if [14C]-CO2 occurred in amounts higher than 5% of applied radioactivity. For this, NaOH sample (40 mL) on each day of sampling were retained in freezer. These NaOH samples were however, not sent for substance specific analytics since the activity was very low.

Organic volatiles in the test substance assays (FTL, FTLA, FTH, FTHA, FS, FSA, FML,FMH) and reference assays (FC, FCA, FOC)
Organic volatiles that might have been formed are absorbed into the 100 mL ethylene glycol absorption flask connected to the test flasks. The aeration was stopped, and a suitable volume from this was taken after opening the valve. The samples were mixed with LSC scintillation cocktail and was measured by LSC as double determination for radioactivity. Retain sample (40 mL) on each day of sampling were stored in the freezer. These samples were discarded without further analysis, since the activity was very low.

Chemical analyses for the measurement of parent and degradation products (test
substance assays only ((FTL, FTLA, FTH, FTHA, FS, FSA)
After sampling for direct LSC measurement and dissolved 14CO2 sample from each of the test substance assays FTL, FTH, FS, the rest test water was transferred to 500 mL polypropylene flasks. After keeping in the freezer (about -18°C) until shipment, the processed samples were sent in dry ice or under cooling conditions to the test site Noack Laboratorien GmbH Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany for substance specific analysis .

Chemical analyses for the measurement of parent and degradation products (test
substance assays on Day 91, FM (FML & FMH):
After direct LSC analysis, following additional extraction steps were performed mass balance test assays on Day 91, FM (FML & FMH). Due to the affinity of the test item to the test vessel, on day 60 about 8 g of C18 adsorbent powder (Agilent C18 End capped)) was added as adsorber to the water phase and shaken vigorously for five minutes. The mixture of water, sediment and C18 powder were separated from the water phase by filtration through a funnel attached with fluted filter to separate the C18 material from the water phase. The funnel attached with fluted filter containing the C18 adsorbent was placed on a centrifuge vessel. The test vessel was first flushed with 100 mL of dichloromethane (DCM) and later this liquid was transferred to the centrifuge vessel through the filter containing the C18 adsorbent.
The filter containing the C18 powder was extracted additionally by adding it to the
centrifuge vessel and by shaking the mixture for five minutes. This vessel was centrifuged for 4 minutes at 4000 rpm to separate the extract from filter and C18 adsorbent. The above extraction step was repeated two more times (3 extraction steps in total) with 100 mL dichloromethane in each step. An aliquot of each extraction step was analyzed by LSC and after pooling all the three extracts, the pooled extract was measured by LSC.
An aliquot was withdrawn from the water phase after filtration and analyzed by LSC. Both the combined dichloromethane extracts and water phase after filtration were sent at room temperature to the test site Noack Laboratorien GmbH Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany for substance specific analysis as precautionary samples, if the analytics of the FTH do not work out due to technical issues. The extraction phases were not analyzed due to the poor mass balance recovery after this extraction procedure which is comparable to the recovery derived from direct analysis of water phase.

Residual activity in the test flasks on Day 91, FM (FML & FMH after dichloromethane
extraction
Since the mass balance recovery after summing up the contents in the test solution with the contents in the absorption bottles does not reach the required areas of activity (90%- 100%), it was assumed that the test substance and possibly formed metabolites adhered to the walls of the test vessels. Therefore, these additional following extractions were conducted intending to close the mass balance. The test vessels of the test assays FML1, FML2, FMH1 and FMH2 were extracted with various solvents to remove the attached test substance and possible metabolites from the walls of the test vessels. For this purpose, 50 mL of the corresponding solvent was heated to about 50 °C and given to the appropriate test vessel. Then the test vessels were shaken for about 30 minutes at 125 rpm on a horizontal lab shaker. This procedure was done 3 times per solvent. The individual extracts were measured in the LSC. After sampling for LSC measurement, the extracts for each solvent were combined separately. The combined extracts were also measured in the LSC. These extracts were stored at <-18°C. The weights of the amounts of solvent added and those of the extracts were documented.The extractions are carried out with the following solvents in the order indicated. 1.Acetone, 2.Acetonitrile 3.Ethanol 4.Chloroform 5.Toluene 6.Heptane. Since the recovery was poor (1%) after each solvent step except with acetone where up to 5%TAR was recovered in both concentration groups, no additional analyses were conducted on these extracts.

Chemical analyses for the measurement of Parent and degradation products (FMETH and FSC-MET)
These samples are from additional test assays with unlabeled test material (FMET-H) and its control (FSC-MET). On each sampling day (including day 0), about 5 mL sample from the duplicate test vessels of FMET-H and FSC-MET were taken and were transferred to a polypropylene vial. After keeping in the freezer (about -18°C) until shipment, the processed samples were sent in dry ice or under cooling.

Reference substance:

benzoic acid, sodium salt

Remarks:

Name of reference substance: Benzoic acid, sodium salt, [ring-14C(U)]- Reference substance No.: 19/0395-3 Batch No: 393-095-0644-A-20160108-SBA Radiochemical Purity: 100% Concentration of AI: 0.1 mCi/ml; 226.96 µg/ml Specific activity :64.4 mCi/mmol

Test performance:

low concentration 4µg/l:
Radioactivity remaining in the test solution after the addition of test substance on the start day of exposure was determined by direct LSC measurement of test solution (without centrifugation). In the FTL assays, about half of the applied radioactivity was remained in the water phase on each sampling days until the end of exposure. Average radioactivity detected in the water phase (direct LSC) of FTL assays was in the range of 44%-94% during the 91 days of incubation. The maximum TAR value 94% was found on day 0 directly after application of test substance and thereafter, the recovery from water phase was declined by adsorption to the test flasks. Similarly, in the low concentration mass balance test substance assay FML, about 62% TAR was measured in the water phase (Table 25).

Compartment:

entire system

% CO2:

0.2

% Other volatiles:

0

% Recovery:

80.4

Remarks on result:

other: FS (=Sterile control assay with test substance in high concentration) at day 59

Compartment:

entire system

% CO2:

0.2

% Other volatiles:

0

% Recovery:

55

Remarks on result:

other: FS (=Sterile control assay with test substance in high concentration) at day 91

Compartment:

entire system

% CO2:

0

% Other volatiles:

0

% Recovery:

91.2

Remarks on result:

other: FTH (=Test substance assay in high concentration for regular sampling) for day 0

Compartment:

entire system

% CO2:

0.7

% Other volatiles:

0

% Recovery:

80.2

Remarks on result:

other: FTH (=Test substance assay in high concentration for regular sampling) for day 59

Compartment:

entire system

% CO2:

1.1

% Other volatiles:

0

% Recovery:

80.4

Remarks on result:

other: FTH (=Test substance assay in high concentration for regular sampling) for day 91

Compartment:

entire system

% CO2:

0.8

% Other volatiles:

0

% Recovery:

29.5

Remarks on result:

other: FMH (=Test substance assay in high concentration for mass balance) at day 91

Compartment:

entire system

% CO2:

0

% Other volatiles:

0

% Recovery:

96.6

Remarks on result:

other: for FTL (=Test substance assay in low concentration for regular sampling ) at day 0

Compartment:

entire system

% CO2:

3

% Other volatiles:

0

% Recovery:

82.8

Remarks on result:

other: for FTL (=Test substance assay in low concentration for regular sampling ) at day 59

Compartment:

entire system

% CO2:

5.1

% Other volatiles:

0.2

% Recovery:

65.2

Remarks on result:

other: for FTL (=Test substance assay in low concentration for regular sampling ) at day 91

Compartment:

entire system

% CO2:

3

% Other volatiles:

0

% Recovery:

65.2

Remarks on result:

other: for FML (=Test substance assay in low concentration for mass balance ) at day 91

Key result

Remarks on result:

not determinable because of methodological limitations

Transformation products:

yes

No.:

#3

No.:

#2

No.:

#1

Details on transformation products:

Metabolite Identification and Transformation Pathway
Metabolites could be detected in the samples of the test group FT H and FS. A literature review indicated that diphenylamine can be degraded by icroorganisms under formation of Aniline and Catechol (1,2-Dihydroxybenzene) [Shin and Spain 2009, Papadopoulou et al. 2018]. This information was adapted to this test item and 4-tert-Butylaniline (CAS-No. 769-92-6) and 4-tertButylcatechol (CAS-No. 98-29-3) were hypothesized as possible metabolites according to Figure 5. These metabolites were also postulated by the software CATALOGIC 301C (v.11.15, LMC Oasis). Samples from the extracts of the storage vessels for the water phase of the groups FTH and FS were selected for the metabolite analysis.

4-tert-Butylaniline could be detected as metabolite in the storage vessel extract of the water samples from day 59. This is in accordance with the radiometric analysis which showed an increase in % of M2 between day 49 and day 59.

Metabolites:
3 metabolites have been detected:
Metabolite 1: 4-tert-Butylaniline (CAS-No. 769-92-6); confirmed by analysis
Metabolite 2: 4-tertButylcatechol (CAS-No. 98-29-3) ; suggested metabolite which was finally not confirmed by analysis
Metabolite 3: metabolite, which could not be characterized

Details on results:

Maximum mineralization (14CO2) found in the low concentration test assay was 5% TAR whereas in high concentration was only 1% TAR.

No organic volatiles were detected in high concentration test assays but up to 0.5% organic volatiles were observed in low concentration test assays.

Kinetic evaluation of the biodegradation test results was not performed in this study due to the poor mass balance recovery achieved in the substance specific analytics and also because of the insufficient data for metabolites and test substance in each sampling points due to the loss of activity through adsorption and possibly other abiotic transformation.

Conclusions:

The objective of this study is to determine the time course of biodegradation of the test item with two different concentrations in natural aerobic surface water and to evaluate the major pathway of its degradation. Mean recoveries of total applied radioactivity (TAR) in the water phase of the tests with the low (4.0 μg/L) and high (40 μg/L) test concentrations were within the range of 44% - 94% TAR and 40 - 90% TAR in LSC measurements, respectively. 14CO2 was found at a maximum of 5% TAR in the low concentration test assay whereas in high concentration, the maximum measured 14CO2 was only 1% TAR. A maximum of 0.5% organic volatiles was found in FTL but no organic volatiles were detected in high test concentration group. On Day 0 and on day 28, a mass balance recovery in the range of 90 - 100% TAR was achieved in low test concentrations and in high concentration, on day 0, day 28 as well as on day 71. Loss of radioactivity by adsorption to the test vessels in the range of 3 - 26% in FTL and 1 - 29% in FTH test assays were observed during the exposure period. In the sterile control FS, 36% activity was found on test flask surface and 45% in water and 0.2% 14CO2 and thus an 80% total recovery was measured on day 59. About 55% TAR in total from test water and 14CO2 were measured in FS on day 91. Since no test vessel extraction was taken place for the FS test assays on day 91, no exact mass balance can be given. In mass balance test assays with low, FML and high concentration, FMH respectively, an overall recovery of 93% and 94% TAR was achieved on day 91 after rigorous extraction procedure using different solvents.
The test substance14C-Bis(4-tert-butylphenyl)amine was removed from the surfacewater after application by degradation and abiotic elimination processes such as adsorption to the test vessel surface. During storage a significant amount of the test substance was absorbed to the storage vessel. The analysis of FTH and FS indicated the formation of metabolites and in total, three metabolites (M1, M2 and M3) were detected. The test item concentration declined from 58% to 1% and the concentration of M1 and M2 increased during the exposure. A maximum of 13% metabolite M1, 36% metabolite M2 and 12% metabolite M3 was observed (single values) during the exposure. In the low concentration test assay, metabolite determination was analytically not possible.
Substance specific analysis of the sterile control FS test assays indicated that about 66% of the total applied radioactivity was retrieved on day 59. Test substance of about 34% as well as about 3% M1, 27% M2 and 4% M3 were determined from this assay.
Radiolabeled benzoic acid, sodium salt was used as the reference substance in
biological control assay for determining the presence of biological activity in the test water system used for this study. Mineralization of about 69% after 14 days in the reference substance assays indicated that the surface water test system was biologically active and therefore, the test is valid. Reference assay with solvent indicated a cumulative total mineralization of 67% of the total applied reference item after 14 days in presence of acetonitrile as solvent. This result indicated that the solvent did not pose a toxicity to the microorganisms present in surface water test system and the test system was biologically active during exposure. Kinetic analysis was not performed in this study due to the poor mass recovery after substance specific analytics and insufficient data for metabolites and test substance in each sampling points due to the loss of activity through adsorption and possibly other abiotic transformation.

Executive summary:

The objective of this study is to determine the time course of biodegradation of the test item with two different concentrations in natural aerobic surface water and to evaluate the major pathway of its degradation. Mean recoveries of total applied radioactivity (TAR) in the water phase of the tests with the low (4.0 μg/L) and high (40 μg/L) test concentrations were within the range of 44% - 94% TAR and 40 - 90% TAR in LSC measurements, respectively. 14CO2 was found at a maximum of 5% TAR in the low concentration test assay whereas in high concentration, the maximum measured 14CO2 was only 1% TAR. A maximum of 0.5% organic volatiles was found in FTL but no organic volatiles were detected in high test concentration group. On Day 0 and on day 28, a mass balance recovery in the range of 90 - 100% TAR was achieved in low test concentrations and in high concentration, on day 0, day 28 as well as on day 71. Loss of radioactivity by adsorption to the test vessels in the range of 3 - 26% in FTL and 1 - 29% in FTH test assays were observed during the exposure period. In the sterile control FS, 36% activity was found on test flask surface and 45% in water and 0.2% 14CO2 and thus an 80% total recovery was measured on day 59. About 55% TAR in total from test water and 14CO2 were measured in FS on day 91. Since no test vessel extraction was taken place for the FS test assays on day 91, no exact mass balance can be given. In mass balance test assays with low, FML and high concentration, FMH respectively, an overall recovery of 93% and 94% TAR was achieved on day 91 after rigorous extraction procedure using different solvents.
The test substance14C-Bis(4-tert-butylphenyl)amine was removed from the surfacewater after application by degradation and abiotic elimination processes such as adsorption to the test vessel surface. During storage a significant amount of the test substance was absorbed to the storage vessel. The analysis of FTH and FS indicated the formation of metabolites and in total, three metabolites (M1, M2 and M3) were detected. The test item concentration declined from 58% to 1% and the concentration of M1 and M2 increased during the exposure. A maximum of 13% metabolite M1, 36% metabolite M2 and 12% metabolite M3 was observed (single values) during the exposure. In the low concentration test assay, metabolite determination was analytically not possible.
Substance specific analysis of the sterile control FS test assays indicated that about 66% of the total applied radioactivity was retrieved on day 59. Test substance of about 34% as well as about 3% M1, 27% M2 and 4% M3 were determined from this assay.

Description of key information

An OECD 309 on the relev constituent of the registered UVCB substance is avaialble. Half lifes of the parent could not be determined due to an insufficient measurement of the mass balance.

Metabolite Identification and Transformation Pathway
Metabolites could be detected in the samples of the test group FT H and FS. A literature review indicated that diphenylamine can be degraded by microorganisms under formation of Aniline and Catechol (1,2-Dihydroxybenzene) [Shin and Spain 2009, Papadopoulou et al. 2018]. This information was adapted to this test item and 4-tert-Butylaniline (CAS-No. 769-92-6) and 4-tertButylcatechol (CAS-No. 98-29-3) were hypothesized as possible metabolites according to Figure 5. These metabolites were also postulated by the software CATALOGIC 301C (v.11.15, LMC Oasis). Samples from the extracts of the storage vessels for the water phase of the groups FTH and FS were selected for the metabolite analysis.

4-tert-Butylaniline could be detected as metabolite in the storage vessel extract of the water samples from day 59. This is in accordance with the radiometric analysis which showed an increase in % of M2 between day 49 and day 59.

Metabolites:
3 metabolites have been detected:
Metabolite 1: 4-tert-Butylaniline (CAS-No. 769-92-6); confirmed by analysis
Metabolite 2: 4-tertButylcatechol (CAS-No. 98-29-3) ; suggested metabolite which was finally not confirmed by analysis
Metabolite 3: metabolite, which could not be characterized