4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2020 – Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD (2002). Guidelines for the Testing of Chemicals. TG 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems. OECD, Paris.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

[14C]Test Substance
Test substance name: [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline
Scymaris reference number: 1108TS002
Molecular weight: 407.44 g/mol
Batch/Lot number: 11468YXC011-4
Supplier: Selcia
Radiochemical Purity: 99.0%
Specific activity: 5297 Bq/µg
Physical state and appearance: White solid
Storage conditions: Freezer

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural sediment

Details on source and properties of sediment:

The two aquatic sediments chosen differed in carbon content and texture. One sediment (collected from Tarnock (Somerset, UK)) had a high organic carbon (HOC) content of 4.8% and a fine texture (clay and silt fractions > 50%). The second sediment (collected from Ashprington (Devon, UK)) had a low organic carbon (LOC) content of 1.57% and a coarse texture (clay and silt fractions < 50%).
The high organic carbon sediment and associated water was collected on the 14 October 2020 by a third party, Ecospan Environmental Ltd, Plymouth, UK (a non-GLP supplier). Using a pole grab sampler, the sediment was collected from the 10 cm surface layer before being passed through a 2 mm sieve into plastic containers. Overlying water was added to the containers to ensure the sediment remained water-logged.
The low organic carbon sediment and associated water was collected by Scymaris Ltd. on the 15 October 2020. Using a hand shovel, the sediment was collected from the 10 cm surface layer into plastic containers. Overlying water was added to the containers to ensure the sediment remained water-logged. At the laboratory, the sediment was passed through a 2 mm sieve.
The temperature, pH and oxygen concentration were measured in the overlying water at the time of collection, along with the depth of the sediment layer.
Particle size distribution (% sand, silt and clay) of the sediments were determined by Smithers, Harrogate, UK as a separate GLP study.

Duration of test (contact time):

101 d

Initial conc.:

0.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Test system
The test vessels were cylindrical glass bottles connected to a series of traps, labelled with the appropriate study number and test vessel content information. The glass vessels used for each sediment were of different dimensions, 1 L glass bottles with ground glass necks were used for high organic carbon sediment and 250 mL glass bottles with ground glass necks were used for low organic carbon sediment. This provided the correct depth of sediment while ensuring at least 50 g dry weight of the sediment in each vessel. Humidified ambient air was drawn through each test vessel using negative pressure provided by a vacuum system. Aeration of the test vessel was provided just below the water surface. Gases exiting the test system passed through two types of sorbent material in the form of ORBO tubes (ORBO-32 followed by ORBO 91) to trap volatiles produced, followed by duplicate tubes of 2 M sodium hydroxide (NaOH) solution to capture evolved 14CO2. During the acclimation period the ORBO tubes were not present and the traps were empty. These were added to the test rig on Day 1.
The test design comprised of the following test vessels:
Transformation vessels: Duplicate vessels of each sediment type treated with 0.1 mg/L [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and sacrificed at each timepoint of the 6 pre-determined timepoints, with duplicate spare vessels to provide an additional point of analysis, if required.
Transformation vessels: Duplicate vessels of each sediment type treated with 1 mg/L [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline to aid identification of any transformation products, if required.
Characterisation vessels (redox, microbial biomass, organic carbon): Four blank control vessels (two of each sediment type) with a two-neck adapter fitted. One of the two necks had an adapter with a cone screw thread, to hold a redox probe. The second neck had the air inlet and outlet, with aeration supplied beneath the water surface. Each redox probe was positioned to record the redox potential of the water and the sediment layer. Selected single vessels from these control vessels were used for the determination of microbial biomass and organic carbon on Day 101 for measurement of microbial biomass.
Characterisation vessels (dissolved oxygen, pH): Two blank control vessels (one of each sediment type) were used to monitor dissolved oxygen (DO) and pH (in water and sediment) throughout the study.
Characterisation vessels (microbial biomass, organic carbon): Two blank control vessels (one of each sediment type) were prepared for measurement of microbial biomass and total organic carbon content of sediments and waters on Day 0.
Characterisation vessels (microbial biomass in presence of test substance): Two test vessels (one of each sediment type) were prepared and acclimated, they were dosed on Day 0 with non-radiolabelled 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and sacrificed for measurement of sediment microbial biomass on Day 101.
Characterisation vessels (microbial biomass in presence of carrier solvent): Two test vessels (one of each sediment type) were prepared and acclimated, they were dosed on Day 0 with the same volume of solvent used to dose the exposure vessels and sacrificed on Day 101 for measurement of microbial biomass.

The 1.0 mg/L transformation vessels and spare 0.1 mg/L vessels were not analysed during this study, and therefore are not included in the results section of the report.

Acclimation period
All test and control vessels were acclimated prior to the addition of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline for an 18 day period at the test temperature of 12 ± 2°C. Redox and DO of the water and pH in both sediment and water were measured 3 times weekly throughout this period.

Stock solution preparation
A 0.54 mL aliquot containing 8.03 MBq of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline from a bulk stock in acetonitrile was made up to 25 mL volume with acetonitrile, to give a final concentration of 60.7 mg [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline/L (Stock H1).
7.01 mg of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was weighed into a vial and approximately 7 mL acetonitrile was added to give a final concentration of 1 g 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline/L (Stock C1). This stock was used as a HPLC retention marker.
0.00301 g of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was weighed into a 50 mL glass volumetric and made up to volume with acetonitrile, to give a final concentration of 60 mg 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline/L (Stock C2).
A 0.107 mL aliquot containing 1.59 MBq of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline from a bulk stock in acetonitrile and 2.70 mg of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were made up to 5 mL volume with acetonitrile, to give a final concentration of 600 mg 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline/L (Stock H2).

Test substance application
1.25 mL or 0.3 mL (HOC or LOC respectively) of Stock H1 or Stock H2 were applied to the water phase of the transformation test vessels, in a single aliquot, to give a test concentration of either 0.1 mg/L [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline or 1.0 mg/L [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline. Characterisation vessels for microbial biomass in the presence of test substance or solvent were dosed with an equivalent volume of either Stock C2 or acetonitrile. The amount of solvent added to the test vessel did not exceed 1% v/v.
The actual concentration of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the dosing solution, at the time of application to the test vessels, was measured by LSC of at least triplicate aliquots and calculated using the specific activity. The radiochemical purity of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was determined prior to dosing the study in Stock H1 by high performance liquid chromatography with ultraviolet and radiochemical detection (HPLC-RAD/UV).
The test solution was applied to the test vessels just above the overlying water surface using a pipette of an appropriate volume. The vessel was gently swirled to mix into the aqueous phase with minimum disturbance of the sediment. Vessels being sacrificed on Day 0 were dosed last to minimise time between dosing and separating the water and sediment.

Incubation of test vessels
All test vessels were incubated in darkness at 12  2C. The temperature in the room was monitored continuously throughout the acclimation and exposure phase using a digital max-min thermometer, and was recorded on working days. Incubation was for a maximum of 101 days after dosing of the test substance.

Sampling intervals
Duplicate transformation vessels treated with 0.1 mg/L [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were sacrificed on sampling Days 0, 7, 14, 28, 70 and 101.

Measurement of key parameters
Key parameters were measured in blank test vessels. One vessel per sediment type of the four prepared for parameter measurement was opened during the test to measure pH (in water and sediment) and DO (in water only), thrice weekly though the acclimation period and on sampling occasions during the exposure period. The other two vessels had redox probes in-situ for the duration of the test to measure redox of the water and sediment at the same sampling frequency. The total organic carbon content of the water and sediment phases was measured on Day 0 and test end in blank control vessels.

Measurement of sediment microbial activity
Sediment microbial activity was measured as microbial biomass. This was measured on blank sediment at the start and end of the exposure phase. The microbial activity of sediment treated with non-radiolabelled 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and in the solvent control vessels was measured at the end of the exposure phase to assess the effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and dosing solvent on the microbiota.
The sediment microbial biomass was determined using the chloroform fumigation-extraction method.

SAMPLING PROCEDURE
Overlying water
The overlying water was removed (by pipette) with minimal disturbance to the sediment layer into a high-density polyethylene (HDPE) bottle containing 70 mL acetonitrile (HOC) or 20 mL acetonitrile (LOC). The bottle was reweighed and triplicate 5 mL aliquots were taken for LSC analysis.
If the overlying water contained ≥ 5% of the applied radioactivity, further analysis was carried out on this phase to determine whether transformation products were present.
On Day 0 only, each overlying water sample was extracted by LLE using DCM, by the following procedure:
• 50 mL of the overlying water was transferred to a separating funnel, and extracted in three 10 mL portions, shaking each time for 2 minutes and then transferring the lower layer into a pre-weighed vial. The vial was reweighed, and triplicate aliquots taken for LSC. The resulting DCM extract was then evaporated to dryness under nitrogen at ~30°C and reconstituted in 2 mL of acetonitrile/water (1:1 v/v). Triplicate aliquots of the concentrate were taken for LSC.
On Day 7 the HO waters were extracted by LLE using DCM, by the following procedure:
• 100 mL of the overlying water was transferred to a separating funnel, and extracted in three 20 mL portions, shaking each time for 2 minutes and then transferring the lower layer into a glass container.
• The DCM layer from Vessel 6 was collected in a volumetric flask and made up to 100 mL with DCM. Triplicate aliquots were taken for LSC.
• The DCM layer from Vessel 5 was collected in a glass vial, and reweighed. Due to an emulsion forming, the extract was centrifuged, and the bottom layer pipetted out into a pre-weighed vial. The centrifuge tube was rinsed with DCM into the same vial. A further two 20 mL DCM extractions were carried out on the post-extracted waters, collected into a pre-weighed vial, and triplicate aliquots taken for LSC.
Both DCM extracts from Day 7 were then transferred to round bottomed flasks and evaporated to dryness by rotary evaporation at 30°C. They were then reconstituted in 2 mL of acetonitrile/water (1:1 v/v) and triplicate aliquots were taken for LSC. A further 2 mL rinse was carried out on the flasks due to low recoveries and triplicate aliquots taken for LSC.
Day 14 and Day 28 HO waters were directly analysed by HPLC via fraction collection into well plates and scintillation counting by TopCount. This was also the procedure for Vessel 18, 22 and 24 (on Days 70 and 100).

Sediment extraction
On all sampling occasions the sediment was extracted using acetonitrile. After removal of the overlying water, the test vessel was rinsed with acetonitrile, and the sediment and acetonitrile slurry transferred to a HDPE bottle. The bottle was mechanically rolled for approximately 30 minutes, centrifuged, and the supernatant decanted into a pre-weighed glass bottle. This was reweighed and weighed aliquots were taken for LSC.
Room temperature acetonitrile extractions were carried out sequentially until either > 90% of applied radioactivity (AR) was recovered or <5% AR was extracted.
If there was still > 10% AR not recovered, the sediment was then extracted with acetonitrile under high temperature and pressure using an accelerated solvent extractor (Dionex ASE200) until > 90% AR recovered or < 5% AR was extracted.
The ASE extractions were carried out as follows.
The post extracted sediment bottles were weighed. For LOC sediment, a portion of this sediment was packed into a 33 mL ASE cell (total weight recorded). For HOC sediment, approximately 25 g w/w of sediment was transferred to a pot and mixed with approximately 10 g diatomaceous earth. These were homogenised by hand and a 33 mL ASE cell was packed with the mixture (total weight recorded).
The samples were then extracted with acetonitrile using the Dionex ASE200 (conditions below) into a pre-weighed 60 mL vial. The vial was reweighed, and triplicate 1 mL aliquots were weighed for LSC.
Heat: 6 min
Flush: 50% volume
Purge: 30 sec
Cycles: 2
Pressure: 1500 psi
Temp: 125°C
HOC sediment was mixed with diatomaceous earth prior to extraction to reduce the pressure, due to the fine particle size. The stability of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline under the ASE200 conditions was checked by HPLC-RAD/UV in method development to ensure no degradation occurred.
Room temperature extractions and ASE extractions were combined separately, concentrated by rotary evaporation and HPLC-RAD/UV was performed if ≥ 5% of the applied radioactivity was measured in this phase to determine whether transformation products were present at a proportion of ≥ 5% of the applied radioactivity.

Glassware Extract
From Day 7 onwards the test vessels each had 100 mL of acetonitrile added to extract any glassware residue, due to low mass balances. The HOC bottles were mechanically rolled for approximately 30 minutes, and the LOC bottles mechanically shaken for approximately 30 mins (due to the shape of the test vessel). The solvent was then decanted into pre-weighed vials, reweighed, and triplicate aliquots taken for LSC. Day 7 and Day 14 LOC vessels had a further glassware rinse carried out as the mass balance was still low – the entire vessel was filled with acetonitrile, left for approximately 1 hour. The extract was then decanted into a pre-weighed bottle which was then re-weighed and triplicate weighed aliquots were taken for LSC.
HPLC-UV/RAD was performed on the glassware extracts if ≥ 4% of the applied radioactivity was measured in this phase. 50 mL of the extract was concentrated by rotary evaporation in a round bottomed flask, and the concentrate transferred into a pre-weighed vial. The flask was rinsed with 2 mL acetonitrile which was added to the concentrate, the vial reweighed, and triplicate aliquots taken for LSC prior to HPLC analysis.

Quantification of bound radioactivity
The bound radioactivity was quantified through combustion of dried homogenised sediment residue aliquots in triplicate, using a Packard 307 sample oxidiser followed by LSC. Recovery checks of a known amount of radioactivity were run on the oxidiser prior to samples each day, and samples were only combusted if recovered radioactivity was between 95 and 105%. Data was not corrected for recovery.

Quantification of volatile radioactivity
The 14CO2 traps were sampled by decanting the contents of each trap into a pre-weighed HDPE bottle, reweighing and triplicate aliquots taken for LSC.
At each sampling occasion, the ORBOTM tubes of the sacrificed transformation vessels were removed. Volatile organic radioactivity trapped in the ORBOTM tubes was extracted with 10 mL of methanol and analysed by LSC.

Mass balance
A mass balance was obtained by the summation of radioactivity measured in the overlying water, sediment extracts, glassware extracts, volatile traps and bound (non-extractable) radioactivity.

Liquid scintillation counting
Radioactivity in the prepared sample/scintillant mixtures was quantified using a Tri-Carb liquid scintillation counter (PerkinElmer) with automatic quench correction using an external standard. The analyser has the optimal channel settings and quench correction curves for the relevant isotope/scintillant combinations.
Samples were counted for 10 min or to a 2 sigma counting error of 0.5% (whichever was reached soonest). Disintegration rates per second (Bq) of appropriate blank sample vials were subtracted from each sample measurement to give a net Bq value for each sample. A radioactivity measurement of less than twice the mean background count was considered below the limit of accurate quantification.

HPLC-RAD/UV Analysis
Radioactivity in samples analysed by HPLC was quantified by either on-line radiodetection using a Beta-Ram 3.0 with peak evaluation using Laura v 6.1.1.20 software, or by fraction collection with TopCount and subsequent evaluation in Laura v 6.1.1.20.
The HPLC conditions used were:
HPLC system: Agilent 1100/1200
Column: Gemini-NX C18 150 x 4.6 mm, 5 µm
Eluent flow rate: 1.0 mL/min (if required)
Mobile phase A: LC-MS Water & 0.1% phosphoric acid
Mobile phase B: LC-MS Acetonitrile & 0.1% phosphoric acid
Column Temperature: 30°C
UV wavelength 290 nm


Regions of background and regions of interest (ROI) were manually added to chromatograms. Percentage of sample radioactivity per ROI was calculated. The proportions of sample radioactivity in each ROI was converted to % of applied radioactivity.
Day 101 V21 HOC sediment extracts were analysed in duplicate by radio-HPLC to show the repeatability of the analytical method.
Column recovery checks were carried out for the HPLC method used on both HOC and LOC sediment extracts. V22 and V23 samples were injected onto the HPLC column twice and the eluent was collected pre and post column. Triplicate aliquots were taken for LSC in each case and the recovery percentage was calculated.

Identification of transformation products
The confirmation/identification of transformation product(s) detected was attempted by the analysis of Day 101 Vessel 24 concentrated sediment extracts by suitable LC-MS/MS techniques. Identification of transformation products was attempted when observed:
• At greater than 10% applied radioactivity at any single sampling interval
• At greater than 5% applied radioactivity at any two consecutive sampling intervals
• At greater than 5% applied radioactivity and steadily rising at the end of the study.
The selected sample was analysed by LC-RAD-MS/MS, including accurate mass analysis to attempt to identify the chromatographically separated radiolabelled components. Structural information on the observed radiochemical components in the samples was based on molecular ion and product ion information.
Using a Dionex U3000 liquid chromatography system and Thermo Scientific LTQ Orbitrap mass spectrometer along with a Raytest Mirastar radio detector, the LC-MS conditions used for the qualitative analysis of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline and any [14C]metabolites.
Online LC-MS radioactivity data in chromatogram format, with associated mass spectrometry data, was captured directly by Xcalibur v 2.0.7 software. Any structural elucidation work required was carried out using Compound Discoverer v 3.1.
Limit of detection and limit of quantification
Values of limit of detection (LOD) and limit of quantification (LOQ) for both LSC and HPLC-RAD were calculated for samples generated at Day 14 (as Vessel 10 had the smallest %AR equivalent to a peak in the HPLC-RAD chromatogram); this was considered representative of all study sample data.
Calculations
Analytical results were calculated using Microsoft Excel. The values presented in the tables and appendices of the report were rounded. Manual calculations using the rounded values may produce slightly different results.
For each sample by LSC, the mean Bq/mL or Bq/g was calculated from the measured radioactivity in each aliquot. The sample mean was multiplied by the aliquot size (mL or g) to determine the total radioactivity in each sample. The total radioactivity was divided by the amount of radioactivity applied to determine the % AR in each sample. The total radioactivity in the water layers, sediment extracts, sediment solids, plus the cumulative amounts in the traps were used to determine the total radioactivity recovered from each test chamber. The total radioactivity recovered was divided by the applied radioactivity to determine the mass balance for each test chamber.
The total amount of radioactivity in the sediment layers was calculated as the sum of the total radioactivity in the room temperature sediment extracts, ASE sediment extracts, and the sediment solids after extraction.
For each sample analysed by HPLC-RAD/UV, the percentage of radioactivity in each ROI was multiplied by the % AR in each sample to determine the relative distribution of radioactivity within each sample.
Kinetics calculation
The behaviour of [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the total system, and separately in the overlying water and sediment layers, was evaluated to determine the dissipation/transformation rate. The DT50 and DT90 (i.e. time taken for 50 or 90% of parent [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline to disappear by dissipation and transformation processes) in overlying water, sediment extracts, and whole test system was calculated using the %AR attributable to parent [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline. The methods to determine transformation rate followed FOCUS recommendations. CAKE v 3.4 was used to fit the experimental data to different kinetic models – simple first order (SFO), double first-order in parallel (DFOP), hockey stick (HS) and first-order multiple component (FOMC). The r2 and χ2 values from each model was compared and the graphs visually assessed to determine the best fit. Acceptable values are typically r2 > 0.7 and χ2 < 15%. The best fit model was used to derive the DT50 and DT90 values.

Partition coefficient (kd)
If possible, the partitioning coefficient (Kd) value was calculated using the following equation:
K_d= (concentration equivalents in the sediment (µg/kg))/(concentration equivalents in the overlying water (µg/L))

Assuming the water has a density of 1.0 kg/L, then the Kd value is dimensionless.

Test performance:

See "details on results".

Key result

Compartment:

sediment

DT50:

40.6 d

Type:

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

Temp.:

12.2 °C

Remarks on result:

other: LOC sediment

Key result

Compartment:

sediment

DT50:

37.5 d

Type:

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

Temp.:

12.2 °C

Remarks on result:

other: HOC sediment

Transformation products:

yes

No.:

#1

No.:

#2

Details on transformation products:

LC-MS/MS data and transformation product discussion for Day 101 Vessel 21 overlying water is detailed below. In summary, two transformation products were observed and structures based on LC-MS/MS were proposed with molecular weights of 434.24 and 808.48 g/mol.

Evaporation of parent compound:

no

Remarks:

The mean % AR captured in the volatile traps was <0.1% and 0.1% for the HOC and LOC vessels, respectively, on Day 7. For the remainder of the study the mean % AR remained below 1% for both HOC and LOC vessels.

Volatile metabolites:

no

Remarks:

The mean % AR captured in the volatile traps was <0.1% and 0.1% for the HOC and LOC vessels, respectively, on Day 7. For the remainder of the study the mean % AR remained below 1% for both HOC and LOC vessels.

Residues:

yes

Remarks:

The amount of bound or non-extractable residues (NER) increased over time in both HOC and LOC vessels. See below, "details on results"

Details on results:

Chemical and physical analysis of sediment and water
During the test, pH values of the sediment phase measured in the HOC vessels were between 7.26 and 8.13 and between 6.72 and 8.57 in the LOC vessels. The pH values of the overlying water were between 7.35 and 9.19 in the HOC vessels and the LOC vessels between 6.32 and 9.13. Neither phase showed a trend of increasing or decreasing pH during the test.
The DO readings in the overlying waters were greater than or equal to 7.07 mg/L and 7.27 mg/L over the test period in the HOC or LOC vessels, respectively.
The redox values measured in the HOC vessels were in the range of 449.7 to 622.0 mV (Eh) in the overlying water and -201.2 to -47.0 mV (Eh) in the sediment.
The redox values measured in the LOC vessels were in the range of 443.2 to 585.6 mV (Eh) in the overlying water and -291.4 to -246.4 mV (Eh) in the sediment.
TOC in the sediment of HOC vessels was 4.8 mg/kg post handling, 4.6 mg/kg on Day 0 and 5.2 mg/kg on Day 101. TOC in the sediment of LOC vessels was 1.6 mg/kg post handling, 1.1 mg/kg on Day 0, and 1.4 mg/kg on Day 101.

Sediment microbial activity
Microbial biomass measured in the sieved HOC sediment at the start of acclimation was 178.21 mg C/kg (dry weight). In the designated blank characterisation vessels, biomass was 109.79 and 389.28 mg C/kg (dry weight) on Day 0 and Day 101, respectively. On Day 101, biomass in the characterisation vessels dosed with 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and acetonitrile were 286.75 and 349.75 mg C/kg (dry weight), respectively.
Biomass in the sieved LOC sediment at the start of acclimation was 145.77 mg C/kg (dry weight); and 53.58 and 95.97 mg C/kg (dry weight) in the designated blank characterisation vessels on Day 0 and Day 101, respectively. On Day 101, biomass in the characterisation vessels dosed with 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline and acetonitrile were 49.71 and 103.62 mg C/kg (dry weight), respectively.

Temperature monitoring
The room temperature, monitored with a min/max thermometer, remained within 12 ± 2°C throughout the test.

Analysis of dosing solutions
The total amount of radioactivity applied to each vessel was calculated from the mean of the six replicates (triplicate pre and post dosing), and then adjusted to the volumes dosed to the test vessels (1.25 mL to the HOC vessels and 0.30 mL to the LOC vessels).
The total amount of radioactivity dosed to the transformation high and low organic carbon vessels was 411558 Bq and 98774 Bq respectively. This gave a concentration of 0.10 mg/L in both water phases. These values were used for the calculating the mass balance.
The total amount of radioactivity dosed to the high concentration transformation high and low organic carbon vessels was 408100 Bq and 97944 Bq respectively. This gave a concentration of 1.0 mg/L in both the high and low organic carbon vessels.
HPLC-RAD/UV on Stock H1 showed 98.8% AR was present as [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the stock solution.

Overlying water
The overlying water contained a mean of 93.2% of applied radioactivity (AR) on Day 0 in the HOC vessels, and 95.4% AR in the LOC vessels. Radioactivity in the HOC overlying water was low at all other sampling occasions and by Day 101 had decreased to a mean of 5.4% AR. Overlying water in the LOC vessels decreased to < 5% AR from Day 7, with the exception of Day 101 which had a mean of 8.0% AR.
On Day 0, HPLC-RAD/UV analysis of the overlying water samples with > 5% AR showed a mean of 89.9% AR was attributed to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in HOC vessels and 92.2% AR in LOC vessels. By Day 101, there was no [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the HOC overlying water analysed, and only 0.5% in LOC overlying water analysed. On Day 101, Vessel 24 was the only LOC overlying water after Day 0 that had > 5% AR, and only 0.46% AR was attributed to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline.
Two transformation products were measured in the overlying waters that were > 10% AR. One transformation product appeared in both HOC and LOC waters, at a relative retention time (RRT) of 0.96-0.98 minutes, increased throughout the test from not appearing in HOC and 0.3% AR in Vessel 3 (LOC) on Day 0 to 3.7% AR (HOC) and 10.0% AR (LOC) in individual vessels on Day 101. Another transformation product appeared sporadically in both overlying waters throughout the test (RRT 1.43-1.46 min). LC-MS/MS analysis confirmed that both transformation products were different from parent 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline.

Sediment extraction
Total extractable radioactivity from the HOC sediment stayed constant from Day 7 to Day 101, with a mean of 0.3% on Day 0 and recoveries ranging from 69.8% to 74.0% between Days 7 and 101.
Total extractable radioactivity from the LOC sediment also stayed constant from Day 7 to Day 101, with a mean of 3.2% on Day 0 and recoveries ranging from 74.2% to 72.7% between Days 7 and 101.
HPLC-RAD/UV analysis of the ambient temperature sediment extraction samples was carried out on all samples with >5% AR, which included all sampling points with the exception of Day 0. Day 7 HOC sediment extracts had a mean of 63.6% AR attributed to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, which had all disappeared by Day 101. Day 7 LOC sediment extracts had a mean of 52.7% attributed to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, which had decreased to 12.8% by Day 101.
Two transformation products, the same as were observed in the overlying waters were measured in both HOC and LOC ambient sediment extracts at > 5% AR. One, at a RRT of 0.96-0.98, first appeared on Day 7 at a mean of 0.87% AR in HOC and 15.7% AR in LOC. This had increased by Day 101 to 60% and 49.9% AR in the HOC and LOC sediment extracts respectively. The second transformation product at a RRT of 1.43-1.46 was present on Day 7 in HOC sediment extracts at a mean of 0.82% AR and 1.04% AR in the LOC sediment extracts and had increased to 8.3% and 4.7% AR by Day 101 in the HOC and LOC sediment extracts respectively.
Sediment extractions carried out on ASE200 for HOC vessels contributed to 3.3% AR on Day 7, increasing to 7.3% by Day 14. This decreased again by Day 101, with a mean of 1.6% AR. ASE200 sediment extractions carried out on LOC vessels ranged from 0.6% and 1.83% AR between Day 7 and Day 101.
HPLC-RAD/UV analysis of the ASE extractions was carried out on all samples with >3% AR. Day 7, Day 14, and Day 28 ASE extracts had a mean of 3.15% AR, 7.08% AR, and 6.76% AR attributed to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline respectively. There were no transformation products in the ASE extracts that were > 5% AR.

Glassware Extract
The amount of radioactivity in the glassware extract was < 5% AR on all occasions. Day 14 HOC glassware extracts however had a mean of 4.6% AR and were analysed by HPLC-RAD/UV. A mean of 4.35% AR was attributable to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline.

Bound radioactivity
The amount of bound or non-extractable residues (NER) increased over time in both HOC and LOC vessels. On Day 0 the mean AR in the sediment was 4.0% AR and 0.88% AR in the HOC and LOC vessels, respectively. This increased to a mean of 8.8% AR by Day 101 in HOC vessels and 7.6% AR in LOC vessels.

Volatile radioactivity
The mean % AR captured in the volatile traps was <0.1% and 0.1% for the HOC and LOC vessels, respectively, on Day 7. For the remainder of the study the mean % AR remained below 1% for both HOC and LOC vessels.
The mean % AR in the high and low organic carbon Orbo™ tubes remained below 0.1% on all sampling occasions.

HPLC repeatability and column recovery
Duplicate injections of Day 101 V21 HOC sediment extracts showed repeatability of the method, with the integrated areas of interest within 10% of each other. The HPLC column recoveries were 103.9% and 105.6% AR for the Day 101 HOC and LOC sediment extracts respectively, showing that all radioactivity injected was measured by the appropriate detector for both matrices.

Mass balance
A mean mass balance of 90-110% was achieved for the HOC vessels on all sampling occasions except Day 70 when the mean %AR was 87. The LOC vessel mass balances ranged from 77% AR to 99% AR. The low mass balance values are thought to be inefficient combustion of the sediment, as the LOC contains a greater amount of sand.

Identification of transformation products
In summary, two transformation products were observed and structures based on LC-MS/MS were proposed with molecular weights of 434.24 and 808.48 g/mol.
Identification of transformation products
The limit of detection (LOD) and limit of quantification (LOQ) were deemed to be equivalent. The LOQ was below 1% of applied radioactivity for both LSC and HPLC-RAD.
An example LOQ for LSC analysis was calculated for each sample matrix using samples from Day 14 and are displayed in the table below. As a Bq value is not determined for the blank vial, the CPM value is converted to Bq using the lowest efficiency (therefore the worst-case scenario) for that set of samples.
For the HPLC-RAD analyses, the smallest applied radioactivity equivalent peak quantified was for HOC Day 14 glassware rinse (Vessel 10), which was equivalent to 0.09% AR.

Kinetics
Since the ASE extractions in both test systems contained less than or equal to 8.6% AR, the bound residues were considered to be irreversibly bound and subsequently not bioavailable. Irreversible binding was considered a path of disappearance of parent [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline from the water-sediment systems. However, DT50 and DT90 values are degradation kinetics, therefore the amounts in the total system were the sum of the amounts in the water layers plus sediment extracts and any radioactivity remaining in the sediment to represent a worst-case scenario.
The HOC overlying water data were fit to SFO, FOMC, DFOP, and HS; all models provided similar fit, therefore the simplest model (SFO) was chosen. The HOC sediment data were fit to SFO, FOMC, DFOP and HS, the only fit with χ2 <15% was HS therefore this fit was chosen. The HOC total system data were fit to SFO, FOMC, DFOP and HS; HS provided the best χ2 and r2 values therefore the this model was chosen.
The LOC overlying water data were fit to SFO, DFOP, FOMC and HS; the SFO model was chosen because this was the best fit (no χ2 value could be determined due to only having 2 data points). The LOC sediment data were fit to SFO, DFOP, FOMC and HS; all models provided similar fits but no models had χ2 <15% or r2 >0.7, therefore SFO was chosen as the best fit with caution. The LOC total system data were fit to SFO, DFOP, FOMC and HS; all models provided a similar fit, therefore the simplest (SFO) was chosen, although χ2 >15% (this was the lowest χ2 value of all fits).
Partition coefficient (kd)
The partitioning coefficient (Kd) value could not be calculated due to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline degradation over time.

Experimental design

Vessel number	Organic carbon content	Vessel type^1	Test substance^2 or solvent^3	Sampling day	Parameter measured
					Sediment	Water	Gas traps
1 / 2	High	Trans	0.1 mg [^14C]TS/L	0	TR, P, TP	TR, P, TP	-
3 / 4	Low	Trans	0.1 mg [^14C]TS/L	0	TR, P, TP	TR, P, TP	-
5 / 6	High	Trans	0.1 mg [^14C]TS/L	7	TR, P, TP	TR, P, TP	VOC, CO2
7 / 8	Low	Trans	0.1 mg [^14C]TS/L	7	TR, P, TP	TR, P, TP	VOC, CO2
9 / 10	High	Trans	0.1 mg [^14C]TS/L	14	TR, P, TP	TR, P, TP	VOC, CO2
11 / 12	Low	Trans	0.1 mg [^14C]TS/L	14	TR, P, TP	TR, P, TP	VOC, CO2
13 / 14	High	Trans	0.1 mg [^14C]TS/L	28	TR, P, TP	TR, P, TP	VOC, CO2
15 / 16	Low	Trans	0.1 mg [^14C]TS/L	28	TR, P, TP	TR, P, TP	VOC, CO2
17 / 18	High	Trans	0.1 mg [^14C]TS/L	70	TR, P, TP	TR, P, TP	VOC, CO2
19 / 20	Low	Trans	0.1 mg [^14C]TS/L	70	TR, P, TP	TR, P, TP	VOC, CO2
21 / 22	High	Trans	0.1 mg [^14C]TS/L	100 ± 2	TR, P, TP	TR, P, TP	VOC, CO2
23 / 24	Low	Trans	0.1 mg [^14C]TS/L	100 ± 2	TR, P, TP	TR, P, TP	VOC, CO2
25 / 26	High	Trans	0.1 mg [^14C]TS/L	Spare	TR, P, TP	TR, P, TP	VOC, CO2
27 / 28	Low	Trans	0.1 mg [^14C]TS/L	Spare	TR, P, TP	TR, P, TP	VOC, CO2
29 / 30	High	Trans	1 mg [^14C]TS/L	If required	TR, P, TP	TR, P, TP	VOC, CO2
31 / 32	Low	Trans	1 mg [^14C]TS/L	If required	TR, P, TP	TR, P, TP	VOC, CO2
33	High	Charac	1 mg TS/L	100 ± 2	BM	-	-
34	Low	Charac	1 mg TS/L	100 ± 2	BM	-	-
35	High	Charac	≤ 1% acetonitrile	100 ± 2	BM	-	-
36	Low	Charac	≤ 1% acetonitrile	100 ± 2	BM	-	-
37^5 / 38^5	High	Charac	-	100 ± 2	Redox, BM, TOC	Redox, TOC	-
39^5 / 40^5	Low	Charac	-	100 ± 2	Redox, BM, TOC	Redox, TOC	-
41^4	High	Charac	-	100 ± 2	pH	pH, DO	-
42^4	Low	Charac	-	100 ± 2	pH	pH, DO	-
43^6	High	Charac	-	0 ± 2	BM, TOC	TOC	-
44^6	Low	Charac	-	0 ± 2	BM, TOC	TOC	-

Abbreviations: TS: test substance 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline; TR: total radioactivity; P: Parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline; TP: [¹⁴C]transformation products; VOC: evolved [¹⁴C]volatile organic compounds; CO2 evolved [¹⁴C]carbon dioxide; DO: dissolved oxygen; BM: biomass; TOC: total organic carbon

1 Vessels types are transformation (trans) or characterization (charac)

2 Concentration of test substance in overlying water layer as [14C]4-(1-methyl-1-phenylethyl)-N-(1-methyl-1-phenylethyl)phenyl]aniline or non-radiolabelled 4-(1-methyl-1-phenylethyl)-N-(1-methyl-1-phenylethyl)phenyl]aniline

3 Concentration of organic carrier solvent in overlying water will be ≤ 1% of total volume

4 Characterisation vessels used for pH and DO measurements during the acclimation period and during the test

5 Characterisation vessels used for redox potential measurements during the acclimation period and during the test, and for biomass and TOC determination at the end of the test

6 These blank control vessels were used for biomass and TOC determination at the start of the test

 

Analysis of overlying water

High Organic Carbon Overlying water MUD/2020/W Tarnock	Matrix parameters	Day of collection	Post handling^a	Acclimation		End of test
				Start	End^b
	pH	7.58	7.41	8.88	7.39	8.68
	DO (mg/L)	1.33	3.55	7.33	8.22	8.25
	Redox (Eh) (mV)	-	-	465.6	579.1	592.5
	Organic carbon (mg/L)	-	24.404^c	-	16.1	11.592
	Temperature (°C)	11.7	-	-	-	-
Low Organic Carbon overlying water MUD/2020/09/W Ashprington	Matrix parameters	Day of collection	Post handling^a	Acclimation		End of test
				Start	end^b
	pH	8.21	8.21	8.35	6.73	8.58
	DO (mg/L)	10.55	7.54	7.79	8.05	8.74
	Redox (Eh) (mV)	-	-	506.9	505.2	504.1
	Organic carbon (mg/L)	-	2.002^c	-	<3.0^d	5.178
	Temperature (°C)	9.6	-	-	-	-

^a After sieving of sediment

^b End of acclimation phase was also Day 0 of dosed phase

^c Organic carbon not measured on start date of acclimation in error

^d Value below limit of detection 3.0 mg/L

 

Analysis of sediment

High Organic Carbon Sediment MUD/2020-10/S Tarnock	Matrix parameter		Post handling^a	Acclimation		End of test
				Start	End^b
	pH		7.10	7.26	7.51	7.96
	Redox (Eh) (mV)		-	-53.2	-174.7	-161.5
	Organic carbon (% dry weight)		4.8^c	-	4.6	5.2
	Moisture content (% wet)		74.5	-	-	-
	Biomass (mg C/kg dry weight sed)	Control	178.21	-	109.79	389.28
		Dosed	-	-	-	286.75
		Solvent control	-	-	-	349.75
	% distribution of particle size (USDA particle size distribution)^d		Sand		12
			Silt		41
			Clay		47
Low Organic Carbon Sediment MUD/2020/09/S Ashprington	Study phase		Post handling^a	Acclimation		End of test
				Start	End^b
	pH		7.76	8.02	6.96	8.51
	Redox (Eh) (mV)		-	-250.5	-286.5	-265.8
	Organic carbon (% dry weight)		1.5^c	-	1.1	1.4
	Moisture content (% wet)		28.0	-	-	-
	Biomass (mg C/kg dry weight sed)	Control	145.77	-	53.58	95.97
		Dosed	-	-	-	59.71
		Solvent control	-	-	-	103.62
	% distribution of particle size *USDA particle size distribution)^d		Sand		87
			Silt		5
			Clay		8

^a After sieving of sediment

^b End of acclimation phase was also Day 0 of dosed phase

^c Organic carbon not measured on start date of acclimation in error

^d Analysis by Smithers ERS Ltd, 108 Woodfield Drive, Harrogate, North Yorkshire, HG1 4LS, UK

 

Mass balance (recovered radioactivity shown as percentage of applied)

Day	Sediment	Overlying water (%)	Total extracted from sediment (%)	Sediment residue (%)	Mineralisation (%)	Volatiles (%)	Glassware extract (%)	Total (%)	Mean (%)
0	HOC	92	0.3	5	-	-	-	97	97
		95	0.2	3	-	-	-	98
	LOC	94	4.1	1	-	-	-	99	99
		97	2.3	1	-	-	-	100
7	HOC	9	76	3	<0.1	<0.1	2.2	90	93
		31	63	1	<LOQ	<LOQ	0.3	96
	LOC	3	76	2	0.1	0.1	<0.1	81	79
		3	72	2	0.1	0.1	<0.1	78
14	HOC	19	68	1	<0.1	<0.1	4.7	93	93
		9	79	1	<0.1	<0.1	4.5	93
	LOC	4	71	7	0.6	0.6	<0.1	83	83
		4	71	8	0.5	0.5	<0.1	82
28	HOC	15	65	9	0.3	0.3	1.2	90	92
		17	71	5	0.1	0.1	0.9	93
	LOC	4	79	3	0.3	0.3	0.5	87	86
		4	75	6	0.6	0.6	0.2	85
70	HOC	4	71	10	0.4	0.4	0.5	87	87
		6	71	9	0.3	0.3	0.8	87
	LOC	3	67	8	0.9	0.9	0.4	79	77
		4	64	6	0.7	0.7	0.2	75
101	HOC	5	76	7	<0.1	<0.1	1.4	89	90
		6	72	10	0.2	0.2	1.6	91
	LOC	2	75	7	0.3	0.3	0.2	84	89
		14	71	8	0.5	0.5	<LOQ	93

<LOQ: below limit of quantification (less than twice background)

 

Mass balance (expressed in dosing concentration of 0.10 mg/kg equivalents)

Day	Sediment	Overlying water (mk/kg eq.)	Total extracted from sediment (mg/kg eq.)	Sediment residue (mg/kg eq.)	Mineralisation (mg/kg eq.)	Volatiles (mg/kg eq.)	Glassware extract (mg/kg eq.)	Total (mg/kg eq.)	Mean (mg/kg eq.)
0	HOC	0.092	0.0003	0.005	-	-	-	0.097	0.097
		0.095	0.0002	0.003	-	-	-	0.098
	LOC	0.094	0.0041	0.001	-	-	-	0.099	0.099
		0.097	0.0023	0.001	-	-	-	0.100
7	HOC	0.009	0.076	0.003	<0.0001	<0.0001	0.0022	0.09	0.093
		0.031	0.063	0.001	<LOQ	<LOQ	0.0003	0.096
	LOC	0.003	0.076	0.002	0.0001	0.0001	<0.0001	0.081	0.079
		0.003	0.072	0.002	0.0001	0.0001	<0.0001	0.079
14	HOC	0.019	0.068	0.001	<0.0001	<0.0001	0.0047	0.093	0.093
		0.009	0.079	0.001	<0.0001	<0.0001	0.0045	0.093
	LOC	0.004	0.071	0.007	0.0006	0.0006	<0.0001	0.083	0.086
		0.004	0.071	0.008	0.0005	0.0005	<0.0001	0.082
28	HOC	0.015	0.065	0.009	0.0003	0.0003	0.0012	0.090	0.092
		0.017	0.071	0.005	0.0001	0.0001	0.0009	0.093
	LOC	0.004	0.079	0.003	0.0003	0.0003	0.0005	0.087	0.086
		0.004	0.075	0.006	0.0006	0.0006	0.0002	0.085
70	HOC	0.004	0.071	0.01	0.0004	0.0004	0.0005	0.087	0.087
		0.006	0.071	0.009	0.0003	0.0003	0.0008	0.087
	LOC	0.003	0.067	0.008	0.0009	0.0009	0.0004	0.079	0.077
		0.004	0.064	0.006	0.0007	0.0007	0.0002	0.075
101	HOC	0.005	0.076	0.007	<0.0001	0.0014	0.089	0.090	90
		0.006	0.072	0.010	0.0002	0.0002	0.0016	0.091
	LOC	0.002	0.075	0.007	0.0003	0.0003	0.0002	0.084	0.089
		0.014	0.071	0.008	0.0005	0.0005	<LOQ	0.093

<LOQ: below limit of quantification (less than twice background)

 

HPLC profiling of overlying water expressed in % applied radioactivity (AR)

Sample Day	Sediment	Vessel	Relative Retention Time (RRT)
			0.18	0.86-0.88	0.96-0.98	1.0	1.06-1.08	1.15	1.39-1.42	1.43-1.46	1.48-1.51
0	HO	1		1.49		88.50	0.84			0.88
		2		2.54		91.37				0.73
	LO	3	0.40	1.24	0.29	90.36	0.57			0.53	0.43
		4		1.89		94.01					1.02
7	HO	5				8.73	0.36
		6		0.55	0.38	30.31
	LO^a	7^a
		8^a
14	HO	9			0.80	12.66			0.39	5.07	0.44
		10				3.01				4.68	0.88
	LO^a	11^a
		12^a
28	HO	13				7.44	0.95			6.20
		14				1.90		3.43		10.30	0.94
	LO^a	15^a
		16^a
70	HO	17
		18			5.56
	LO^a	19^a
		20^a
101	HO	21^a
		22			3.70					2.34
	LO	23^a
		24			10.04	0.45	1.85			1.55

^a No quantification was performed on these samples as < 5% of the applied was present in the water

^b RRT of 1.0 is parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

 

Sediment Extractions (% applied radioactivity)

Day	Vessel No	Sediment	Extract 1	Extract 2	Extract 3	Extract 4	Extract 5	Extract 6	Extract 7
			% Applied Radioactivity
0	1	HOC	0.3	-	-	-	-	-	-
	2		0.2	-	-	-	-	-	-
	3	LOC	4.1	-	-	-	-	-	-
	4		2.3	-	-	-	-	-	-
7	5	HOC	4.5	15.9	22.0	16.2	9.8	4.4	3.4
	6		2.5	22.2	12.4	10.0	9.0	4.2	3.1
	7	LOC	59.3	13.2	2.9	0.6	-	-	-
	8		55.6	13.2	3.0	0.6	-	-	-
14	9	HOC	2.5	36.4	8.5	7.3	4.9	8.2	0.3
	10		1.5	44.5	10.6	10.0	6.4	5.8	0.3
	11	LOC	54.9	12.1	2.8	0.9	-	-	-
	12		53.1	13.1	2.9	1.6	-	-	-
28	13	HOC	3.7	34.7	9.2	9.0	4.9	3.6	-
	14		2.8	42.5	9.2	8.2	4.8	3.8	-
	15	LOC	61.0	13.3	2.9	1.3	-	-	-
	16		55.3	14.2	3.0	2.3	-	-	-
70	17	HOC	3.3	32.4	7.5	15.4	7.8	2.7	2.2
	18		2.9	28.1	7.1	18.3	9.8	3.5	1.4
	19	LOC	51.7	12.0	2.6	0.8	-	-	-
	20		49.2	11.4	2.6	0.3	-	-	-
101	21	HOC	2.4	18.0	10.5	23.3	14.0	5.9	1.6
	22		2.5	16.9	10.8	21.3	13.7	5.7	1.6
	23	LOC	58.2	12.4	3.4	0.7	-	-	-
	24		53.5	13.47	3.4	0.5	-	-	-

All extracts were in acetonitrile

Extract 4 for Day 7-Day 101 LOC sediment were carried out on Dionex ASE200. Extract 6 for Day 14 and 28 HOC sediment was carried out on Dionex ASE200. Extract 7 was carried out on ASE200 on all occasions.

 

HPLC profiling of sediment extracts (ambient) expressed in % applied radioactivity (AR)

Sample Day	Sediment	Vessel	Relative Retention Time (RRT)
			0.43-0.44	0.86-0.88	0.96-0.98	1.0	1.06-1.08	1.22	1.39-1.42	1.43-1.46	1.48-1.51
0	HO	1^a
		2^a
	LO	3^a
		4^a
7	HO	5		0.69	0.84	69.23	0.40			1.04	0.55
		6		0.80	0.91	57.99				0.60
	LO	7	0.90		6.68	63.17	1.85			0.90	1.86
		8	1.15		24.74	42.23	1.31	0.53		1.18	0.66
14	HO	9			0.88	57.71				0.98
		10			2.58	68.69				1.69
	LO^a	11			12.71	57.05
		12			7.83	59.24				2.05
28	HO	13			1.99	42.71	1.41			12.67	2.70
		14			0.93	56.54				8.27	1.69
	LO^a	15			9.58	62.58				2.74	2.28
		16			5.38	67.09
70	HO	17			54.84	3.09				8.22	2.89
		18			55.49	0.95	1.95		2.26	9.17
	LO^a	19			52.76		1.46		2.54	9.53
		20			46.56	9.15	1.08			6.46
101	HO	21			63.96				3.61	6.47
		22			56.05				4.76	10.02
	LO	23			42.64	23.86	2.09			4.05	1.33
		24			57.17	1.82	3.74		2.22	5.37

^a No HPLC-RAD/UV analysis  was performed on these samples as < 5% of the applied radioactivity was present in the sediment extract

^b RRT of 1.0 is parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

 

HPLC profiling of sediment extracts (ASE) expressed in % applied radioactivity (AR)

Sample Day	Sediment	Vessel	Relative Retention Time (RRT)
			0.96-0.98	1.0^c	1.06-1.08	1.43-1.46	1.48-1.51
0	HO	1^a
		2^a
	LO	3^a
		4^a
7	HO	5	0.12	3.27
		6	0.11	3.03
	LO	7
		8
14	HO	9	0.12	8.41
		10	0.31	5.75
	LO	11
		12
28	HO	13	0.62	2.57		0.26	0.12
		14	0.37	3.34	0.14
	LO	15
		16
70	HO	17
		18
	LO	19
		20
101	HO	21
		22
	LO	23
		24

^a No extracts carried out on the Dionex ASE200

^b No HPLC-RAD/UV analysis was performed on these samples as < 3% of the applied radioactivity was present in the sediment extract

^c RRT of 1.0 is parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

 

HPLC profiling of glassware extracts expressed in % applied radioactivity (AR)

Sample Day	Sediment	Vessel	Relative Retention Time (RRT)
			0.96-0.98	1.0^c	1.43-1.46
0	HO	1^a
		2^a
	LO	3^a
		4^a
7	HO	5^b
		6^b
	LO	7^b
		8^b
14	HO	9	0.13	4.48	0.10
		10	0.18	4.21	0.09
	LO	11^b
		12^b
28	HO	13^b
		14^b
	LO	15^b
		16^b
70	HO	17^b
		18^b
	LO	19^b
		20^b
101	HO	21^b
		22^b
	LO	23^b
		24^b

^a No glassware extracts carried out

^b No HPLC-RAD/UV analysis was performed on these samples as < 3% of the applied radioactivity was present in the sediment extract

^c RRT of 1.0 is parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

 

HPLC profiling of total system expressed in % applied radioactivity (AR)

Sample Day	Sediment	Vessel	Relative Retention Time (RRT)
			0.18	0.43-0.44	0.86-0.88	0.96-0.98	1.0^a	1.06-1.08	1.15	1.22	1.39-1.42	1.43-1.46	1.48-1.51
0	HO	1			1.49		88.50	0.84				0.88
		2			2.54		91.37					0.73
	LO	3	0.40		1.24	0.29	90.36	0.57				0.53	0.43
		4			1.89		94.01						1.02
7	HO	5			0.69	0.96	81.24	0.76				1.04	0.55
		6			1.35	1.40	91.34					0.60
	LO	7		0.90		6.68	63.17	1.85				0.90	1.86
		8		1.15		24.74	42.23	1.31		0.53		1.18	0.66
14	HO	9				1.93	83.27				0.69	6.15	0.44
		10				3.07	81.66					6.46	0.88
	LO	11				12.71	57.05
		12				7.83	59.24					2.05
28	HO	13				2.62	52.72	2.36				19.13	2.82
		14				1.75	65.90	0.31	3.43			18.57	2.62
	LO	15				9.58	62.58					2.74	2.28
		16				5.38	67.09
70	HO	17				54.84	3.09					8.22	2.89
		18				61.05	0.95	1.95			2.26	9.17
	LO^a	19				52.76		1.46			2.54	9.53
		20				46.56	9.15	1.08				6.46
101	HO	21				63.96					3.61	6.47
		22				59.75					4.76	12.36
	LO	23				42.64	23.86	2.09				4.05	1.33
		24				67.21	2.29	5.60			2.22	6.92

^a RRT of 1.0 is parent [¹⁴C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Conclusions:

[14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline dissipated over time from the water phase to the sediment phase in both sediment types, with overlying water DT50 ¬values of 3.35 and 13.2 days in the HOC and LOC test systems respectively. [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline degraded more rapidly in the HOC sediment than the LOC sediment, with total system, reflected in the total system DT50 values of 37.5 and 40.6 days respectively.
Almost no mineralisation occurred, with <1% measured in both the HOC and LOC systems on Day 101.
8.8% and 7.6% of applied radioactivity remained in the sediment on Day 101 as bound radioactivity in the HOC and LOC test systems respectively after all extractions had been carried out. ASE extractions removed less than 10% of applied radioactivity. Therefore, these residues are not considered to be bioavailable and binding to sediments was considered a pathway for disappearance. As the degradation half life is < 120 days, the substance is not considered to be "P" or "vP" in accordance with the REACH Annex XIII.

Executive summary:

STUDY SUMMARY
Materials & Methods
Test Substance	Name:		[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline
	Lot Number:		11468YXC011-4
	Specific activity:		5297 Bq/µg
	Radiochemical purity:		99.0%
Test Systems			Tarnock		Ashprington
	Sediment	Sand/Silt/Clay (USDA) (%):	12/41/47		87/5/8
		Organic Carbon (%):	4.8		1.57
		Moisture Content (%):	74.5		28.0
		pH:	7.10		7.76
		Microbial Biomass (µg/g):	109.79		53.58
	Water	pH:	7.39		6.73
		Dissolved Oxygen (mg/L):	1.33		10.55
		Total Organic Carbon (mg/L):	24.404		2.002
Incubation Conditions	Ratio:		3.5:1 water:sediment
	Duration:		100 ± 2 days
	Dates of Incubation:		19 November 2020 to 1 March 2021
	Temperature/Light:		12 ± 2 °C in dark
	Concentration:		0.1 mg [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline /L
	Replication:		2 vessels per sampling interval
	Aeration:		Flow-through air
Analysis	Sampling Intervals:		0, 7, 14, 28, 70, 101 days (water + sediment)
	Processing:		Decant overlying water
			Extract sediments sequentially with acetonitrile at room temperature until <5% extracted, followed by accelerated solvent extraction using acetonitrile
	Methods:		Total radioactivity in water, sediment extracts, combusted post-extraction sediments, and volatile solutions by LSC
			Radioactivity profile of water and sediment extracts by HPLC-RAD/UV
Controls	Solvent Control		Microbial biomass in presence of solvent only
Results
Distribution of Radioactivity				Tarnock (HOC)		Ashprington (LOC)
				% Applied Radioactivity
Total Test System
Mean Mass Balance				92		86
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 0				90		92
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 101				<LOQ		13
%^14CO2 (cumulative) at Day 101				0.1		0.4
% Volatile organic compounds (cumulative) at Day 101				<0.1		<0.1
Total transformation products in total test system at Day 101				75		66
Major transformation products observed (>10% AR)				2		1
Water Layer
% Radioactive residues in water layer at Day 101				18		31
% [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in water layer at Day 101				<LOQ		0.46
Sediment Layer
% Extractable radioactive residues from sediment at Day 101				74		73
% [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC/RAD in sediment extracts at Day 101				9		20
% Non-extractable radioactive residues from sediment on Day 101				9		8
Kinetic Analysis				Days at 12°C
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in water layer (SFO)				3.35		13.2
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in sediment extracts (HS, SFO)				43.2		49.7
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in total test system (HS, SFO)				37.5		40.6
DT50 = half-life using simple first-order kinetics (SFO) or hockey stick (HS)

 

[14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline dissipated over time from the water phase to the sediment phase in both sediment types, with overlying water DT50 ­values of 3.35 and 13.2 days in the HOC and LOC test systems respectively.  [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline degraded more rapidly in the HOC sediment than the LOC sediment, with total system, reflected in the total system DT50 values of 37.5 and 40.6 days respectively.

Almost no mineralisation occurred, with <1% measured in both the HOC and LOC systems on Day 101.

8.8% and 7.6% of applied radioactivity remained in the sediment on Day 101 as bound radioactivity in the HOC and LOC test systems respectively after all extractions had been carried out.  ASE extractions removed less than 10% of applied radioactivity.  Therefore, these residues are not considered to be bioavailable and binding to sediments was considered a pathway for disappearance.

Description of key information

[14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline dissipated over time from the water phase to the sediment phase in both sediment types, with overlying water DT50 ­values of 3.35 and 13.2 days in the HOC and LOC test systems respectively.  [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline degraded more rapidly in the HOC sediment than the LOC sediment, with total system, reflected in the total system DT50 values of 37.5 and 40.6 days respectively.

Almost no mineralisation occurred, with <1% measured in both the HOC and LOC systems on Day 101.  As the degradation half life is < 120 days, the substance is not considered to be "P" or "vP" in accordance with the REACH Annex XIII.

Key value for chemical safety assessment

Half-life in freshwater sediment:

37.5 d

at the temperature of:

12.2 °C

Additional information

STUDY SUMMARY
Materials & Methods
Test Substance	Name:		[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline
	Lot Number:		11468YXC011-4
	Specific activity:		5297 Bq/µg
	Radiochemical purity:		99.0%
Test Systems			Tarnock		Ashprington
	Sediment	Sand/Silt/Clay (USDA) (%):	12/41/47		87/5/8
		Organic Carbon (%):	4.8		1.57
		Moisture Content (%):	74.5		28.0
		pH:	7.10		7.76
		Microbial Biomass (µg/g):	109.79		53.58
	Water	pH:	7.39		6.73
		Dissolved Oxygen (mg/L):	1.33		10.55
		Total Organic Carbon (mg/L):	24.404		2.002
Incubation Conditions	Ratio:		3.5:1 water:sediment
	Duration:		100 ± 2 days
	Dates of Incubation:		19 November 2020 to 1 March 2021
	Temperature/Light:		12 ± 2 °C in dark
	Concentration:		0.1 mg [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline /L
	Replication:		2 vessels per sampling interval
	Aeration:		Flow-through air
Analysis	Sampling Intervals:		0, 7, 14, 28, 70, 101 days (water + sediment)
	Processing:		Decant overlying water
			Extract sediments sequentially with acetonitrile at room temperature until <5% extracted, followed by accelerated solvent extraction using acetonitrile
	Methods:		Total radioactivity in water, sediment extracts, combusted post-extraction sediments, and volatile solutions by LSC
			Radioactivity profile of water and sediment extracts by HPLC-RAD/UV
Controls	Solvent Control		Microbial biomass in presence of solvent only
Results
Distribution of Radioactivity				Tarnock (HOC)		Ashprington (LOC)
				% Applied Radioactivity
Total Test System
Mean Mass Balance				92		86
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 0				90		92
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 101				<LOQ		13
%^14CO2 (cumulative) at Day 101				0.1		0.4
% Volatile organic compounds (cumulative) at Day 101				<0.1		<0.1
Total transformation products in total test system at Day 101				75		66
Major transformation products observed (>10% AR)				2		1
Water Layer
% Radioactive residues in water layer at Day 101				18		31
% [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in water layer at Day 101				<LOQ		0.46
Sediment Layer
% Extractable radioactive residues from sediment at Day 101				74		73
% [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC/RAD in sediment extracts at Day 101				9		20
% Non-extractable radioactive residues from sediment on Day 101				9		8
Kinetic Analysis				Days at 12°C
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in water layer (SFO)				3.35		13.2
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in sediment extracts (HS, SFO)				43.2		49.7
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in total test system (HS, SFO)				37.5		40.6
DT50 = half-life using simple first-order kinetics (SFO) or hockey stick (HS)

 

[14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline dissipated over time from the water phase to the sediment phase in both sediment types, with overlying water DT50 ­values of 3.35 and 13.2 days in the HOC and LOC test systems respectively.  [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline degraded more rapidly in the HOC sediment than the LOC sediment, with total system, reflected in the total system DT50 values of 37.5 and 40.6 days respectively.

Almost no mineralisation occurred, with <1% measured in both the HOC and LOC systems on Day 101.

8.8% and 7.6% of applied radioactivity remained in the sediment on Day 101 as bound radioactivity in the HOC and LOC test systems respectively after all extractions had been carried out.  ASE extractions removed less than 10% of applied radioactivity.  Therefore, these residues are not considered to be bioavailable and binding to sediments was considered a pathway for disappearance.  As the degradation half life is < 120 days, the substance is not considered to be "P" or "vP" in accordance with the REACH Annex XIII.