Degradation of the anilide fungicide inpyrfluxam in illuminated water–sediment systems

Abstract

The degradation behavior of inpyrfluxam (1) was investigated in aerobic aquatic water–sediment systems exposed to continuous artificial sunlight (λ>290 nm). Under irradiation in the presence of sediment, 1 preferentially underwent oxidation at the 1′- and 3′-positions of the indane ring, followed by cleavage of the amide linkage with the half-life of 16–18 days, and finally mineralized to carbon dioxide or exhibited extremely strong adsorption to sediment. Especially, as compared to systems kept in darkness, carboxylation at the 1′-position of the indane ring was remarkably accelerated in illuminated water–sediment systems and the aqueous photodegradation study due to the presence of photosynthetic microorganisms in the sediment soil. No significant degradation products were observed in the water–sediment in darkness and in the sterilized water–sediment under irradiation throughout the study. The fate of 1 and its degradation products in illuminated water–sediment systems was considered to better reflect realistic conditions, as it accounts for various effects attributed to sunlight, such as the presence of photosynthetic microorganisms.

Introduction

Pesticides have been used to improve crop productivity and quality, as well as to prevent disease. Pesticides applied to agricultural fields exhibit various degradation or dissipation behaviors depending on distinctive factors such as crop metabolism, soil microorganisms activity, and sunlight exposure.^1–3) Particularly, in aquatic systems, factors such as hydrolysis, direct photolysis, and indirect photoreaction under light irradiation, with the latter involving light absorption by photosensitizers generated from dissolved substances in water, contribute to the degradation of pesticides.^4–6) In order to clarify the fundamental behavior of pesticides in aqueous environments, simulation studies of their degradative processes, such as hydrolysis and aqueous photolysis, are mandatory for the registration of pesticides in accordance with Organization for Economic Co-operation and Development (OECD) Guidelines 111 and 316, respectively.^7,8) Furthermore, OECD Guideline 308 describes a test simulating field conditions, i.e., water–sediment systems under dark conditions,⁹⁾ assuming scenarios such as pesticide application to rice fields and its drift or run-off into rivers and lakes. These studies are crucial to understand the behavior of pesticides in the natural environment. However, even when these studies are performed individually, fully elucidating the degradation or dissipation behavior remains challenging due to the complexity arising from multiple factors in the agricultural fields.^10–13) For example, in an aqueous photolysis study of the fungicide mandestrobin, unique compounds such as rearranged and cyclized products were rapidly produced with photoreaction.¹⁴⁾ In contrast, in the irradiated water–sediment systems, the total amount of photoproducts were decreased by the effect of light shielding, and therefore, no specific cyclized photoproducts were observed.¹⁵⁾ Similarly, the herbicide flumioxazin produced a significant amount of a unique four-membered ring compound in the aqueous photolysis study, however, it was rapidly decomposed in the illuminated water–sediment systems.¹⁰⁾ To clarify the photodegradation behavior of pesticides in surface water in the presence of sediment, which is not specified in the guidelines, would be extremely useful for conducting precise risk assessment of aquatic organisms under conditions better reflecting the natural environment.

Inpyrfluxam (INDIFLIN, 3-Difluoromethyl-N-[(R)-2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl]-1-methylpyrazole-4-carboxamide), 1, is an anilide fungicide developed by Sumitomo Chemical Co., Ltd. (Fig. 1). Moreover, 1 is resistant to hydrolysis in aqueous solutions with pH 4–9, and undergoes almost no degradation when exposed to light in a buffer solution. However, it exhibits gradual degradation under light irradiation in natural water.^16,17) Additionally, 1 absorbs light and undergoes oxidation mainly at the 3′-position of the indane ring as well as a cleavage of the amide bond. The presence of nitrate ions, which function as photosensitizers, promotes the degradation of 1 in a concentration-dependent manner as reported previously.¹⁸⁾ Furthermore, in soil metabolism and water–sediment studies conducted under dark conditions, the formation of carboxylic acid derivatives has been mainly observed due to oxidation at the 1′-position of the indane ring.¹⁶⁾ However, the behavior of 1 under light irradiation in the presence of sediment remains unknown.

Fig. 1. Proposed photolysis pathways of 1 under the illuminated condition in the water–sediment system. ¹⁴C labeled positions of 1: *, [PY-¹⁴C]-1; #, [PH-¹⁴C]-1.

This research aims to elucidate the photodegradation pathway of 1 and the behavior of its photodegradation products in agricultural fields. This is achieved by using xenon (Xe) lamp irradiation as simulated sunlight in a system containing natural water and sediment. In addition, to confirm the contribution of microorganisms present in the bottom sediment and surface water to the degradation of 1, a similar test was conducted under sterilized conditions. These original systems provide intrinsic data that are considered to better reflect realistic surface water environments compared to regulatory studies.

Materials and methods

1. Chemicals

Two radiolabels of 1, ¹⁴C-labeled at the 4-position of the pyrazole ring and uniformly at the phenyl ring, abbreviated as [PY-¹⁴C]-1 and [PH-¹⁴C]-1 (Fig. 1), respectively, were synthesized by Pharmaron UK Limited (Cardiff, United Kingdom). The specific radioactivities of [PY-¹⁴C]-1 and [PH-¹⁴C]-1 were 6.33 and 13.5 MBq/mg, respectively, and their radiochemical purities were determined to exceed 99% just before the study. Non-radiolabeled 1 and its related five reference standards 2–6 were used for the structural identification. Among them, 1, 3, and 5 were prepared in our laboratory,¹⁸⁾ and 4 and 6 were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and AURUM Pharmatech (Howell, New Jersey, USA), respectively. The reference standard of (1RS,3RS;1RS,3SR)-2,3-dihydro-1,3-dimethyl-4-{[1-methyl-3-(difluoromethyl)-1H-pyrazole-4-ylcarbonyl]amino}-1H-indene-1-carboxylic acid, 2, a metabolite produced in water–sediment systems, was prepared as follows. The mixture of [PY-¹⁴C]-1 (2.74 µg) and non-radiolabelled 1 (274 µg) was applied to the overlying water (depth of 6 cm) in the presence of sediment collected from Sharkey (50 g, Mississippi, USA) into the cylindrical glass vessel (4.4 cm i.d.). The vessel was continuously irradiated using an Atlas Suntest XLS+ 2-kW Xe arc lamp (Toyo Seiki, Tokyo, Japan) through the Pyrex inner filter to remove ultraviolet (UV) light at wavelengths below 290 nm. The light intensity with its irradiance of 40.6 W/m² at 300–400 nm was almost constant throughout the study. After 28 days of irradiation, the aqueous layer was acidified with hydrochloric acid (1 N HCl) and was extracted two times with 80 mL of ethyl acetate. The combined organic phase was evaporated to dryness and purified by high performance liquid chromatography (HPLC, LC-20A series, Shimadzu, Kyoto, Japan) to obtain the fraction containing compound 2. Compound 2 had an asymmetric carbon at the 1′-position and was divided into two peaks in HPLC analysis. The higher polar component was designated as 2 (A), and the lower polar component as 2 (B). The chemical structure of 2 was confirmed by liquid chromatography-mass spectroscopy (LC-MS, Q Exactive HF spectrometer, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and ¹H-nuclear magnetic resonance (NMR, Ascend 400, Bruker, Kanagawa, Japan) analyses, and the chemical purity was determined to be >97%.

Compound 2 (A): MS m/z 362 (M–H)⁻; 1H-NMR (400 MHz, in deuterated chloroform [CDCl₃]) δ 8.06 (s, 1H, 5-position of the pyrazole ring), 7.19–7.31 (m, 3H, 5′–7′-positions of the indane ring), 6.86 (t, 1H, J=54.2 Hz, –CHF₂), 3.96 (s, 3H, –NCH₃), 3.37–3.42 (m, 1H, –CHCH₃), 2.55 (dd, 1H, J=2.6, 13.4 Hz, –CH₂–), 2.24 (dd, 1H, J=8.6, 13.4 Hz, –CH₂–), 1.59 (s, 3H, –CCH₃), 1.25 (d, 3H, J=7.0 Hz, –CHCH₃).

Compound 2 (B): MS m/z 362 (M−H)⁻; 1H-NMR (400 MHz, in CDCl₃) δ 8.04 (s, 1H, 5-position of the pyrazole ring), 7.15–7.28 (m, 3H, 5′–7′-positions of the indane ring), 6.86 (t, 1H, J=54.3 Hz, –CHF₂), 3.94 (s, 3H, –NCH₃), 3.49–3.54 (m, 1H, –CHCH₃), 3.03 (dd, 1H, J=8.5, 13.4 Hz, –CH₂–), 1.67 (dd, 1H, J=5.0, 13.4 Hz, –CH₂–), 1.64 (s, 3H, –CCH₃), 1.28 (d, 3H, J=7.0 Hz, –CHCH₃).

Phosphate buffer solution (100 mM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was diluted two-fold with pure water supplied from a Direct-Q 3UV water purification system (>18 MΩ cm, Merck Japan, Tokyo, Japan) to prepare a 50 mM, pH 7 buffer solution. The solvents, CDCl₃ with 0.03% tetramethylsilane for NMR, was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All the other chemicals and solvents were of analytical grade.

2. Spectroscopy and chromatography

A HPLC system equipped with a 150 mm×6.0 mm i.d., 5 µm, SUMIPAX ODS A-212 column (Sumika Chemical Analysis Service, Osaka, Japan) was operated at a flow rate of 1 mL/min using the following gradient program for analysis of the degradation profile: solvent A, 0.05% formic acid; solvent B, acetonitrile; 0–5 min; 5% of B and 5–50 min; with a linear gradient of 5–100% of B. The retention times of reference standards were 39.6 min (1), 29.2 and 29.9 min (2 as isomer), 36.6 min (3), 17.3 min (4) and 12.5 min (5). The radioactivity of column effluents was monitored with a Radiomatic 150TR detector equipped with a 500 µL liquid cell using Ultima-Flo AP liquid scintillation cocktail (Revvity, Kanagawa, Japan). Each ¹⁴C peak was identified by HPLC co-chromatography by comparing its retention time with those of the corresponding non-radiolabeled reference compounds detected at 254 nm with an SPD-20A UV detector (Shimadzu, Kyoto, Japan). To further confirm the chemical identity of each degradation product detected by HPLC, two-dimensional thin-layer chromatography (2D-TLC) was conducted using silica gel 60F₂₅₄ thin-layer plates (200×200 mm, 0.25-mm layer thickness; Merck Japan, Tokyo, Japan) with the solvent systems of A (chloroform/methanol; 9 : 1, v/v) and B (toluene/ethyl acetate/acetic acid; 5 : 7 : 1, v/v/v). The R_f values of reference standards were 0.75 (1), 0.23 and 0.33 (2), 0.55 (3), 0.14 (4) and 0.25 (5) for solvent system A and 0.52 (1), 0.37 and 0.40 (2), 0.41 (3), 0.45 (4) and 0.21 (5) for solvent system B. Radioactive regions on TLC plates were exposed to BAS III_S imaging plates (Fuji Photo Film, Tokyo, Japan) and signal intensities were quantitated by a Typhoon 9200 Variable Mode Imager (GE Healthcare Japan, Tokyo, Japan). Non-radiolabeled references co-developed on TLC were visualized using UV light.

LC-MS in negative ion mode was performed with an electrospray ionization (ESI) interface, which was connected to a SHIMADZU HPLC system (Shimadzu, Kyoto, Japan). Samples were eluted using the same gradient system as used in the HPLC method for degradation profile analysis, except with the flow rate of 0.2 mL/min.

An ion chromatography system ICS-5000 (Thermo Fisher Scientific) was employed to determine the concentration of inorganic substances in the water samples before and after the irradiation. The operating parameters and equipment were as follows: a DIONEX Ion Pak AS23 (250 mm×4 mm i.d., Thermo Fisher Scientific) was used as analytical column with a DIONEX Ion Pak AG23 (50 mm×4 mm i.d., Thermo Fisher Scientific) as guard column; a solution containing 4.5 mmol/L Na₂CO₃ and 0.8 mmol/L NaHCO₃ was used as eluent at a flow rate of 1 mL/min; chemical suppression; sample loop volume of 10 µL. The typical retention times of fluoride, chloride, nitrite, nitrate, and sulfate ions were 4.3 min, 7.1 min, 9.0 min, 12.8 min, and 20.2 min, respectively.

3. Radioassay

The radioactivity in aliquots of photolysis test solutions, trapping media, and rinsates of glass surfaces was individually determined in duplicate by mixing each aliquot with 10 mL of Emulsifier Scintillator Plus (Revvity, Kanagawa, Japan) and then analyzed by liquid scintillation counting (LSC) for 5 min in a Tri-Carb 5110 TR liquid scintillation spectrometer (Revvity, Kanagawa, Japan). The amounts of ¹⁴C collected in 0.5 M NaOH traps were determined as ¹⁴CO₂ by adding 1.0 M BaCl₂, resulting in quantitative precipitation as Ba¹⁴CO₃. The background level of all samples was 0.5 Bq.

4. Test system

Bottom sediment and overlying water were collected from Sharkey (Mississippi, USA) in August 2021 and stored in a refrigerator at 4°C until use. The textural class of the used sediment was clay with organic carbon 3.1%, organic matter 5.3% and pH 6.7 in soil/water=1/1 (v/v) ratio. The test apparatus was prepared in accordance with the previous method.¹⁵⁾ A 50-g sediment sample was placed into a two-necked cylindrical glass vessel (4.4 cm i.d.) to a depth of 2 cm, and then the overlying water was added to each vessel to a depth of 6 cm. The applied solutions were prepared in acetonitrile with [PY-¹⁴C]-1 and [PH-¹⁴C]-1 at concentrations of approximately 346 kBq/mL and 740 kBq/mL, respectively. For each label, a test system for the light irradiation and the dark control was prepared in singlicate. After a pre-incubation period of a month for water–sediment samples, 50 µL of the above ¹⁴C-1 solutions, was applied to the water surface in each vessel at a rate of 2.74 µg/vessel using a microsyringe, which is equivalent to the field application rate of 300 g a.i./ha, assuming its uniform distribution in the water layer, to a depth of 100 cm. The glass vessel containing each water–sediment system was immersed in a water bath maintained at 20±2°C and swirled on an orbital shaker (MK-200D, Yamato Scientific, Tokyo, Japan) at 80 rpm to moderately mix the water phase without disturbance of the sediment. Humidified air was successively passed over the water surface to ethylene glycol and alkaline traps in sequence for collection of organic volatiles and carbon dioxide, respectively, in the same manner as previously reported.¹⁵⁾ Each vessel was continuously irradiated using an Atlas Suntest XLS+ 2-kW Xe arc lamp (Toyo Seiki, Tokyo, Japan) through the Pyrex inner filter to remove UV light at wavelengths below 290 nm. The light intensity was almost constant throughout the study with its irradiance at 300–400 nm measured as 30.0, 72.5, and 54.6 W/m² for the [PY-¹⁴C]-1 and [PH-¹⁴C]-1 labeled non-sterilized study and the [PY-¹⁴C]-1 labeled non-sterilized study, respectively. Since the light intensity of the Xe lamp was different in each study, it was converted to the typical natural sunlight intensity according to Ministry of Agriculture, Forestry and Fisheries of Japan (J-MAFF) (Tokyo, 35°N, spring)¹⁹⁾ and OECD test guidelines (EU/US, 30–50°N, summer).²⁰⁾ The same test system, established under non-sterilized and dark conditions, was maintained at 20±2°C in a SR-30VE2 incubator (Nagano Science, Osaka, Japan). As the sterilization test, the soil thin layer and surface water were sterilized at 120°C for 20 min once a day for three times. The overlying water and associated sediment were separately analyzed at 0, 7, 14, 21, and 28 days after application (sterilized study was analyzed at 0, 14, and 28 days after application). At each sampling, the pH, oxidation-reduction potential (ORP) values of the water and sediment layers and the dissolved oxygen content (DO) in the water layer were measured using a D-210PD meter (Horiba, Kyoto, Japan).

The overlying water was collected by decantation, directly radioassayed by LSC, and then extracted twice with ethyl acetate. A portion of the combined extracts (ethyl acetate layers) was concentrated in vacuo for HPLC analysis. The sediment was transferred to a centrifugal bottle and extracted by shaking with 100 mL acetone/water/concentrated HCl (60/40/1, v/v/v) in a mechanical shaker for 10 min, centrifuging the bottle for 10 min at 8,000 rpm, and collecting the supernatant by decantation. The extraction was repeated twice in the same manner. All the collected supernatants were put into a single flask. Then, an aliquot was evaporated to remove acetone (from approximately 200 mL to 80 mL) and extracted twice with 50 mL ethyl acetate. A portion of the combined ethyl acetate layers was radioassayed by LSC, then concentrated in vacuo for HPLC analysis after filtration and dehydration with magnesium sulfate. The aqueous layers obtained by extracting surface water and soil with ethyl acetate were also subjected to LSC and HPLC analyses. The chemical identity of each radiolabeled compound was confirmed by comparing its HPLC retention time and TLC R_f value with those of the non-radiolabeled reference standards. The unextractable sediment residue (bound residue ¹⁴C) was air-dried and combusted for radioassay. The selected unextractable residue was further extracted under harsh conditions including: (1) 100 mL of acetonitrile/1 M ammonium solution (4 : 1, v/v), (2) 100 mL of acetone/water/concentrated HCl (60/40/1, v/v/v) using the Soxhlet method at 70–80°C for 15 cycles. After the above basic and acid extraction, (3) the 28-day soil samples of [PH-¹⁴C] label were subjected to the following silylation method to release the target chemicals from sediment by breaking hydrogen bond interactions between ¹⁴C-labeled compounds and the sediment constituents.²¹⁾ A 5.0-g sample of dried matrix material was taken and mixed with 50 mL of chloroform, 6.0 g of NaOH, and 20 mL of trimethylchlorosilane. The mixture was then stirred at room temperature under nitrogen during the reaction. After 3 hr of stirring, an additional 6.0 g of NaOH and 20 mL trimethylchlorosilane were added for one-day reaction. Finally, the chloroform layer was removed by decantation from the reaction mixture and the soil residue was extracted twice with 40 mL of acetone. These chloroform and acetone extracts were combined and centrifuged, and the radioactivities therein were measured by LSC.

Degradation curves of 1 were fitted to a single first-order (SFO) model according to Forum for the coordination of pesticide fate models and their use (FOCUS) recommendations²²⁾ and half-life values were estimated by using Computer Assisted Kinetic Evaluation (CAKE v. 3.1, Tessella, Oxford, United Kingdom) software.

Results

1. Ion chromatography analysis

The concentrations of inorganic ions in the test solutions before and after irradiation were measured by ion chromatography. Prior to the experiment, the concentrations of fluoride, chloride, nitrate, and sulfate ions were observed to be 0.18, 4.55, 0.65, and 6.74 mg/L, respectively. Nitrite ions, which could serve as photosensitizers,²³⁾ were not detected throughout the study. After irradiation, the concentrations of fluoride (0.23 mg/L), chloride (3.53 mg/L), and sulfate (1.26 mg/mL) ions showed almost no change, and nitrate ion completely disappeared.

2. Illuminated water–sediment study of 1

2.1. Conditions of the water–sediment system

The measured pH, ORP, and DO values in the overlying water and sediment throughout the studies are listed in Tables 1 and 2. The DO value for each label did not significantly change during the study period. A gradual increase of pH value was observed with irradiation under non-sterilized conditions from pH 8.49–8.56 to 9.48–9.65 throughout the study. The ORP values were approximately 150–200 mV in the water phase and below 0 mV in the sediment.

Table 1. Distribution of 1 in an illuminated water–sediment system.

	% of the applied 14C
	[PY-14C]					[PH-14C]
Days	0	7	14	21	28	0	7	14	21	28
Volatile 14C	na	0.2	0.4	0.2	0.5	na	1.6	2.2	5.2	2.8
14CO2	na	0.1	0.4	0.2	0.5	na	1.5	2.2	5.2	2.7
Others	na	<0.1	<0.1	<0.1	<0.1	na	<0.1	<0.1	<0.1	<0.1
Water 14C	97.2	54.2	47.3	47.1	53.8	95.4	46.7	33.9	21.0	23.1
Ethyl acetate layer	97.0	53.5	43.8	45.4	52.0	95.4	43.6	28.5	16.9	18.8
1	96.4	38.6	20.0	9.5	nd	94.8	30.5	10.0	8.5	4.8
2	nd	8.1	10.5	24.1	40.2	nd	8.5	14.9	6.9	13.2
3	nd	0.9	2.4	1.0	0.8	nd	2.2	1.6	1.4	0.9
4	nd	2.8	10.8	9.4	11.0	—	—	—	—	—
5	nd	nd	nd	nd	nd	—	—	—	—	—
6	—	—	—	—	—	nd	nd	nd	nd	nd
Others	0.6	3.2	nd	1.5	nd	0.6	2.4	1.9	nd	nd
Aqueous layer	0.1	0.7	3.5	1.7	1.8	0.1	3.1	5.4	4.1	4.3
Sediment 14C	5.6	46.7	52.2	50.7	45.4	5.2	51.3	59.4	67.9	68.0
Ethyl acetate layer	4.4	41.1	43.7	41.9	36.5	4.9	43.9	37.5	37.7	35.5
1	4.4	39.6	40.9	33.1	21.8	4.9	41.5	33.2	33.8	31.3
2	nd	1.5	2.8	6.1	11.2	nd	1.4	2.7	1.6	2.7
3	nd	nd	nd	1.2	nd	nd	0.9	1.6	1.6	1.5
4	nd	nd	nd	0.7	1.6	—	—	—	—	—
5	nd	nd	nd	nd	nd	—	—	—	—	—
6	—	—	—	—	—	nd	nd	nd	nd	nd
Others	nd	nd	nd	0.8	1.9	nd	nd	nd	0.6	nd
Aqueous layer	nd	0.6	1.0	1.1	0.8	nd	1.2	1.7	2.4	1.9
Bound residue 14C	1.1	4.9	7.4	7.7	8.2	0.3	6.3	16.4	24.4	26.6
Acetonitrile layer (1)	na	na	na	na	na	na	na	3.7	3.5	3.9
Total 14C	102.8	101.1	99.9	98.1	99.7	100.6	99.6	95.4	94.1	93.9
Total 1	100.9	78.2	60.9	42.6	21.8	99.7	72.1	47.0	45.8	39.9
Aqueous ORP (mV)	200	150	110	175	174	206	174	158	158	184
pH	8.56	9.22	9.98	9.70	9.48	8.49	9.63	9.93	10.02	9.65
DO (ppm)	8.45	10.24	12.94	10.19	10.27	7.34	11.8	7.94	11.44	7.76
Sediment ORP (mV)	−127	−213	−148	−133	−140	−142	−141	−127	−159	−96
pH	7.17	7.33	7.16	7.34	7.20	6.96	7.06	7.20	7.18	7.32

[PY-¹⁴C]-1, ¹⁴C-labeled at the 4-position of the pyrazole ring; [PH-¹⁴C]-1, ¹⁴C-labeled uniformly at the phenyl ring; DO, dissolved oxygen content; ORP, oxidation-reduction potential; na, not analyzed; nd, not detected; —, not detected due to labeled position. Maximum unknown: Consist of multiple components, none of which exceed 1.9% AR.

Table 2. Distribution of 1 under the dark condition and sterilized illuminated condition in water–sediment systems.

	% of the applied 14C [PY-14C]
	Dark condition					Illuminated and sterilized
Days	0	7	14	21	28	0	14	28
Volatile 14C	na	nd	<0.1	<0.1	<0.1	na	0.6	0.4
14CO2	na	nd	nd	nd	nd	na	0.2	0.3
Others	na	<0.1	<0.1	<0.1	<0.1	na	0.4	0.1
Water 14C	96.2	48.2	40.1	25.3	23.7	92.3	29.6	24.4
Ethyl acetate layer	96.1	48.2	40.1	25.3	23.7	92.1	29.0	23.6
1	95.2	48.2	40.1	25.3	23.7	91.4	29.0	23.6
2	nd	nd	nd	nd	nd	nd	nd	nd
3	nd	nd	nd	nd	nd	nd	nd	nd
4	nd	nd	nd	nd	nd	nd	nd	nd
5	nd	nd	nd	nd	nd	nd	nd	nd
6	—	—	—	—	—	—	—	—
Others	0.9	nd	nd	nd	0.1	0.7	nd	nd
Aqueous layer	0.1	<0.1	<0.1	<0.1	<0.1	0.3	0.6	0.8
Sediment 14C	2.5	48.7	62.0	75.8	73.3	10.9	68.9	74.2
Ethyl acetate layer	2.3	44.1	56.3	65.1	63.6	10.4	64.3	67.1
1	2.3	44.1	56.3	65.1	63.6	10.4	64.3	66.6
2	nd	nd	nd	nd	nd	nd	nd	nd
3	nd	nd	nd	nd	nd	nd	nd	nd
4	nd	nd	nd	nd	nd	nd	nd	nd
5	nd	nd	nd	nd	nd	nd	nd	nd
6	—	—	—	—	—	—	—	—
Others	nd	nd	nd	nd	nd	nd	nd	0.5
Aqueous layer	0.1	0.2	0.2	<0.1	0.1	0.1	0.3	0.3
Bound residue 14C	0.2	4.4	5.6	<0.1	1.9	0.4	4.3	6.8
Acetonitrile layer (1)	na	na	na	10.7	7.8	na	na	na
Total 14C	98.8	96.9	102.1	101.1	97.0	103.2	99.2	98.9
Total 1	97.5	92.4	96.4	101.1	94.9	101.8	93.3	90.1
Aqueous ORP (mV)	131	135	142	202	214	148	160	172
pH	8.44	8.41	8.38	7.85	8.31	8.38	8.16	8.06
DO (ppm)	8.42	6.22	7.74	7.25	7.03	6.85	8.32	6.60
Sediment ORP (mV)	−138	−209	−147	−98	−116	−94	−83	−125
pH	7.17	7.44	7.24	7.23	7.19	6.97	6.92	7.06

[PY-¹⁴C]-1, ¹⁴C-labeled at the 4-position of the pyrazole ring; DO, dissolved oxygen content; ORP, oxidation-reduction potential; na, not analyzed; nd, not detected; —, not detected due to labeled position. Maximum unknown: Consist of multiple components, none of which exceed 0.9% AR.

2.2. Distribution of the radioactivity

The ¹⁴C distributions within the test system individually treated with [PY-¹⁴C]-1 (sterilized and non-sterilized systems) or [PH-¹⁴C]-1 (non-sterilized) under irradiated conditions, and [PY-¹⁴C]-1 (non-sterilized) under dark conditions are summarized in Tables 1 and 2. Good ¹⁴C recovery in the range of 93.9–103.2% of the applied radioactivity (AR) was obtained throughout the study. The ¹⁴C adsorbed onto the inner vessel wall was <1.0% AR, therefore, the adsorption of 1 and its degradation products to the apparatus was considered negligible. Under irradiation in the non-sterilized system, most of 1 degraded or completely disappeared from the aqueous layer, while it gradually and steadily degraded to 21.8–39.9% AR in the sediment by the end of the study (28 days). The half-lives of 1 were estimated to be 4.5–5.7 days in the aqueous layer and 16–18 days in the total test system where the latter is equivalent to 55–156 days (Tokyo, 35°N, spring)¹⁹⁾ and 19–53 days (EU/US, 30–50°N, summer)²⁰⁾ under natural sunlight (Table 3). The maximum amounts of volatile and adsorbed ¹⁴C in the sediment were 0.5–5.2% and 8.2–26.6% AR, respectively. In contrast, under darkness and sterilized irradiation, more than 90% of the unchanged 1 remained in total system with no degradation products exceeding 0.9% AR. In addition, the maximum amounts of volatile and adsorbed ¹⁴C were 0.6% and less than 6.8% AR, respectively. Therefore, 1 was assumed to be comparatively stable in darkness and sterilized irradiation throughout the study (Fig. 2).

Table 3. Half-lives of 1 under the illuminated condition and dark condition in water–sediment systems.

Test condition		Xe lamp (day)	J-MAFF19) (day)	OECD20) (day)
[PY-14C] light	Aqueous	5.7	—	—
	Total	16	55	19
[PH-14C] light	Aqueous	4.5	—	—
	Total	18	156	53
[PY-14C] Sterilized light	Aqueous	11	—	—
	Total	156	>1000	339
[PY-14C] Dark	Aqueous	11	—	—
	Total	>1000	—	—

Xe, xenon; [PY-¹⁴C]-1, ¹⁴C-labeled at the 4-position of the pyrazole ring; [PH-¹⁴C]-1, ¹⁴C-labeled uniformly at the phenyl ring; J-MAFF, Ministry of Agriculture, Forestry and Fisheries of Japan. OECD, Organization for Economic Co-operation and Development.

Fig. 2. Total recovery rate of 1 in the irradiated system (solid line, ●: [PY-¹⁴C] and ■: [PH-¹⁴C]), the dark control system (dashed line, ▲: [PY-¹⁴C]) and the sterilized irradiated system (◆: [PY-¹⁴C]).

2.3. Identification and quantification of the degradation products

The chemical structures of all major degradation products were completely confirmed by HPLC and TLC co-chromatography with the corresponding reference standards. In the non-sterilized system, the profile of degradation products under irradiation is summarized in Table 1. Two main photoproducts, 2 and 4, were observed and reached maximum levels of 51.4% and 12.6% AR, respectively, in the whole system for both [PY-¹⁴C]-1 and [PH-¹⁴C]-1 labeled studies. A minor product, 3, was observed (maximum amount of 3.2% AR) to be present in the aqueous photolysis study, however, 5 was not detected in the illuminated water–sediment systems. The expected product, 6, i.e., the structural counterpart of 4, was not detected throughout the study. There were numerous unidentified peaks which did not co-elute with any of the reference substances, but none of them amounted to >1.9% AR. In the harsh extraction with acetonitrile and ammonia solution, a maximum ¹⁴C of 10.7% AR was detected, which consisted solely of 1. The combustion analysis showed that the amount of bound residue ¹⁴C in the sediment after acid extraction at room temperature (Table 1) was maximumly 26.6% AR at the end of the study for the [PH-¹⁴C]-1 label. Successive exhaustive harsh extraction using the same acid solvent at high temperature released only a trace amount of ¹⁴C from the sediment (5.2% AR) which primary consisted of 1.9% of 1 and 3.0% of highly polar components. This indicated no significant products were present in bound residue. In addition, bound residue ¹⁴C was characterized using a different method, i.e., silylation, and as a result, only an insignificant amount of the radioactivity was detected in the extracted solution (<2.3% AR).

The degradation behavior under darkness and sterilized irradiation is summarized in Table 2. Interestingly, no degradation products were observed in water layer and sediment soil throughout the study. The maximum amount of volatile and bound residue was remarkably low as 0.6% and 6.8%, respectively.

Discussion

The half-life of 1 in the total illuminated water–sediment system (55–156 days, Tokyo, 35°N, spring) was remarkably shorter than that of the system in darkness (> 1000 days), indicating that the light irradiation significantly contributed to the degradation of 1 in the water–sediment system (Fig. 3 and Table 3). In this system, 1 primarily underwent oxidation at the 1′- and 3′-positions of the indane ring to form 2 and 3, respectively. In addition, cleavage of the amide bond proceeded to form 4 which has been reported to decompose into carbon dioxide under light exposure in an aqueous environment through photoreaction.^18,24) Finally, these degradation products are likely to undergo mineralization or strongly bind to the soil (Fig. 1). The structural counterpart of 4, referred to as 6, which is expected to be generated via amide bond cleavage, was not detected throughout the study. Aniline derivatives similar to compound 6, such as dicloran, are known to photodegrade very rapidly, with an approximate half-life of <1 day.^25,26) This degradation occurs through the ring cleavage of the 1,4-benzoquinone form produced by the oxidation of amino moiety.²⁷⁾ The volatile generated from the PH label (5.2% AR) was clearly higher than that from the PY label (0.5% AR), supporting the conclusion that 6 rapidly decomposed and mineralized after the amide bond cleavage. No specific major degradation products, other than those identified in registration guideline studies (water, soil, or photodegradation studies),¹⁶⁾ were detected.

Fig. 3. Degradation behavior of [PY-¹⁴C]-1 in the irradiated system (solid line, ●: water phase and ■: sediment) and the dark control system (dashed line, ▲: water phase and ◆: sediment).

Incidentally, in the absence of sediment, 1 degrades very slowly even under light irradiation (223–549 days, Tokyo, 35°N, spring).¹⁷⁾ Moreover, in the presence of sediment, it is hardly metabolized under illumination in the sterilized water–sediment system (>1000 days). This implies that, in addition to light irradiation as an abiotic degradation factor, microbial metabolism in the sediment also contributed to the biotic degradation of 1. The remarkable major degradation product, the carboxyl derivative 2, increased up to a maximum of 51.4% AR (PY-labeled 28 days, illuminated non-sterilized conditions, Table 1), which was also detected at 26.2% AR in the regulatory soil metabolism study.²⁸⁾ However, 2 was not detected in the aqueous photodegradation or nitrate ion water photolysis studies.¹⁸⁾ The above data may indicate that the presence of algae and macrophytes can accelerate the degradation of pesticides under light irradiation, serving as one of the degradation sources, compared to clear buffer solutions through indirect photolysis. In fact, in natural environments, the relative abundance of cyanobacteria was observed in surface water in response to increased sunlight exposure,²⁹⁾ which contributed to the degradation of isopyrazam, possessing an N-methyl difluoropyrazole ring similar to 1.³⁰⁾ Carboxylation of the side chain at the indane ring also occurred in the illuminated water–sediment study of isopyrazam, which was not observed in the direct photoreaction.²⁸⁾ These observations indicated that the increase in photosynthetic microorganisms and/or heterotrophic organisms in the sediment soil promoted the degradation of 1, resulting in the significant production of compound 2 under light irradiation as a biotic reaction. In this study, an increase in pH and DO was observed under the light exposed condition (Table 1, pH: 8.56 to 9.70, DO: 8.45 to 12.94 in [PY] labeled study), suggesting that CO₂-fueled photosynthesis by the related bacteria occurred under light irradiation.^31,32) The absence of a pH increase in the dark control or sterilized conditions (Table 2) further supported the contribution of photosynthetic microorganisms in the transformation of 1 to 2. Although 2 was maximally increased up to 51.4% AR in the system, its soil adsorption coefficient (K_OC=11–44) is significantly lower than that of 1 (K_OC=500–913), implying that the portion generated in the sediment may dissolve into the surface water. Since 2 retains a structure similar to that of the parent compound, it may undergo direct photoreaction, resulting in ester cleavage and mineralization in the surface water. Furthermore, 2 is known to gradually degrade in soil under aerobic metabolic conditions,²⁸⁾ suggesting that it would also be steadily degraded by microorganisms. Overall, it is scientifically plausible to conclude that the degradation products of 2 will not persist in the environment.

In general, the photodegradation of pesticides can be slower in natural water due to the light shielding effect caused by the presence of dissolved substances,¹⁵⁾ meanwhile, the nitrate ions are known to contribute to the indirect photodegradation of 1 in a concentration-dependent manner under light exposure.¹⁸⁾ In this study, 0.65 mg/L of nitrate ion, acting as a photosensitizer, was detected by ion chromatography analysis in the surface water, suggesting that dissolved inorganic ions in the sediment also promoted the photodegradation of 1 via indirect reactions besides direct ones. In the aqueous photodegradation study, hydroxylation at the 3′-position of the indane ring to produce minor degradation product 3,¹⁸⁾ amide bond cleavage, and eventual mineralization were observed. Similar reactions proceeded with analogous compounds such as isopyrazam and bistrifluron as the direct photoreaction.^24,33)

It has been confirmed that the major product 2 and the minor component 3, generated in this study, exhibit significantly lower toxicity to aquatic organisms compared to 1.²⁸⁾ The acute toxicity value of 1 to rainbow trout, i.e., the most sensitive species, is LC₅₀=0.031 mg/L, and those of 2 and 3 are LC₅₀>50 and 6.2 mg/L, respectively.²⁴⁾ These results suggest that, even if 2 and 3 are produced as photoproducts in surface waters, their toxic effects on aquatic organisms are likely to be negligible. Therefore, there is no concern regarding the formation of more toxic compounds from 1 in aquatic environments under sunlight.

Although photodegradation progresses in irradiated water–sediment systems, a significant increase in bound residue (up to 26.6% AR) was observed in the sediment. Similarly, in the water–sediment systems of mandestrobin and esfenvalerate, the bound residue of ¹⁴C also increased over time under light exposure, while no significant amount was observed in the dark condition. This implies that the radical cleavage photodegradation products of 1 adsorb to the sediment soil.^12,15) In this study, only trace amounts of ¹⁴C were extracted from the bound residue even when using harsh extraction and silylation methods. These observations indicate that the bound residue of ¹⁴C forms strong bonds with the sediment soil, suggesting that the risk of its release into the environment is extremely low.³⁴⁾

In conclusion, we elucidated the photodegradation behavior of 1 in the original irradiated water–sediment system. The main degradation pathways of 1 were considered to be oxidation at the 1′-position of the indane ring by photosynthetic microorganisms and/or heterotrophic organisms, and cleavage of the amide bond and oxidation at the 3′-position of the indane ring by the direct photoreaction. The observed major degradation products were consistent with those identified in existing environmental studies, and no specific degradation products were generated in the irradiated water–sediment system. Furthermore, the toxicity of the major degradation products to fish was significantly lower compared to that of 1 and they were considered to dissipate and mineralize under continuous light exposure. Compound 1 and its degradation products can be adsorbed to the sediment, forming a very strong bonds with the soil, which makes their release back into the environment unlikely. These results from the irradiated water–sediment system study are considered to more closely reflect real agricultural field conditions compared to results from defined studies (e.g., aqueous photodegradation or soil metabolism studies), demonstrating the behavior of compound 1 in aquatic environments and indicating that its residual risk is low.