Constructing Slow-Release Fungicide Formulations Based on

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Agricultural and Environmental Chemistry

CONSTRUCTING SLOW-RELEASE FUNGICIDE FORMULATIONS BASED ON POLY-3-HYDROXYBUTYRATE AND NATURAL MATERIALS AS A DEGRADABLE MATRIX Tatiana Grigorievna Volova, Svetlana Prudnikova, Anatoly Nikolayevich Boyandin, Natalia ZHILA, Evgeniy Kiselev, Anna Shumilova, Sergey Baranovsky, Aleksey Demidenko, Ekaterina Shishatskaya, and Sabu Thomas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01634 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 28, 2019

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Journal of Agricultural and Food Chemistry

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CONSTRUCTING SLOW-RELEASE FUNGICIDE FORMULATIONS BASED ON POLY-

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3-HYDROXYBUTYRATE AND NATURAL MATERIALS AS A DEGRADABLE MATRIX

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Tatiana Volova1,2, Svetlana Prudnikova1, Anatoly Boyandin1,2, Natalia Zhila1,2,*, Evgeniy Kiselev1,2, Anna Shumilova1, Sergey Baranovsky1, Aleksey Demidenko1,2, Ekaterina Shishatskaaya1,2, Sabu Thomas1,3

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1Siberian

Federal University, 79 Svobodnyi Av., Krasnoyarsk 660041, Russia of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, 50/50 Akademgorodok, Krasnoyarsk 660036, Russia 3International and Interuniversity Centre for Nano Science and Nano technology, Mahatma Gandhi University, Kottayam, Kerala, India 2Institute

*Corresponding author. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Slow-release fungicide formulations (azoxystrobin, epoxiconazole, and tebuconazole)

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shaped as pellets and granules in a matrix of biodegradable poly(3-hydroxybutyrate) and natural

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fillers (clay, wood flour, and peat) were constructed. IR spectroscopy showed no formation of

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chemical bonds between components in the experimental formulations. The formulations of

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pesticides had antifungal activity against Fusarium verticillioides in vitro. A study of

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biodegradation of the experimental fungicide formulations in the soil showed that the degradation

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process was mainly influenced by the type of the formulation without significant influence of the

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type of the filler. More active destruction of the granules led to a more rapid accumulation of

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fungicides in the soil. The content of fungicides present in the soil due to degradation of the

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formulations and fungicide release was determined by their solubility. Thus, all formulations are

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able to function in the soil for a long time, ensuring gradual and sustained delivery of fungicides.

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KEYWORDS: poly(3-hydroxybutyrate), fungicides, slow-release formulations, antifungal activity, degradation, fungicide release

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Introduction

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Modern efficient agriculture is impossible without fungicides. A most promising way to

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enhance agricultural production and improve its quality is to protect crops against diseases and

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pests. Fungicides hold the third place in sales volume and usage because phytopathogenic fungi are

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responsible for about 10% of the crop loss. Such important sources of plant protein as maize, wheat,

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and rice, whose yields constitute over 55% of the total yield of cereal crops, are particularly

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susceptible to the adverse effects of pathogens. Although chemicals and technological measures are

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widely used to protect plants, global losses caused by pests constitute between 35% and 48% of the

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potential crop yield. About one third of these losses are caused by diseases of plants. Numerous

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harmful fungi, bacteria, and viruses impair the quality of agricultural products. Fusarium infection

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is among the most common diseases of cereal crops, which is caused by fungi of the genus

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Fusarium. These fungi damage wheat, rye, barley, grasses, and many other crops. Fusarium fungi

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are producers of very potent mycotoxins such as deoxynivalenol (vomitoxin), zearalenone, and T-2

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mycotoxin.1 Fusarium root rot may affect the ears and grain of cereal crops, contaminating the grain

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with mycotoxins and making it unsuitable and even unsafe food for humans and animals.

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Application of fungicides decreases the incidence of fusarium infection and reduces the levels of

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mycotoxins in commercial grain,2 but yield losses caused by fusarium infection may still reach

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between 5 and 30%. New fungicide formulations need to be developed to increase the efficacy of

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fungicides and minimize their harmful effects on the environment.

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The newest trend in research is development of new-generation slow-release pesticides,

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including fungicides, embedded in degradable matrices. The use of such formulations will decrease

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the amounts of pesticides applied to soil, ensure slow and targeted release of the active ingredients,

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and reduce the risk of uncontrolled distribution of the pesticides in the biosphere. The main

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condition for constructing such formulations is the availability of appropriate materials with the

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following properties: degradability and compatibility with global biosphere cycles, safety for living

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organisms and their nonliving environment, long-term presence in the natural environment and


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controlled degradation followed by formation of non-toxic products, chemical compatibility with

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pesticides, and processability by available methods. Various natural and synthetic materials have

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been investigated as candidates for constructing pesticide carriers: pure polymers and polymers

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blended with low-molecular-weight components including different natural materials such as clays

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and clay-like silicates,3-7 cellulose derivatives,8,9 lignin,10,11 chitosan,12 etc. Research aimed at

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designing slow-release fungicide formulations was started quite recently. Materials tested as carriers

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for the tebuconazole fungicide included poly(methyl methacrylate),13 ethyl cellulose,9 silica,6

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chitosan,14 and cellulose/silica nanocomposites.15 Tang et al. (2019) produced tebuconazole

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porphyrinic metal-organic frameworks (MOFs) with pectin and chitosan.16 Azoxystrobin and

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difenoconazole were encapsulated in microspheres of poly(butylene succinate) (PBS) and

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poly(lactic acid) (PLA).17 Yao et al. (2018) reported the use of polylactide as a matrix for loading

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azoxystrobin.18 Campos et al. (2015) prepared and tested nanocapsules of polycaprolactone and

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glyceryl tripalmitate as carrier systems for tebuconazole and carbendazim.19

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Polyhydroxyalkanoates (PHAs) – degradable microbial polymers – hold promise as

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fungicide carriers. The properties of PHAs have made them potential material of choice in various

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applications such as medicine, pharmacology, electronics, municipal engineering, and agriculture.

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However, research on embedding agrochemicals in PHA matrices is still in its infancy.

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Homopolymer poly(3-hydroxybutyrate) [P(3HB)] – the most commonly occurring PHA – was first

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used as a matrix for embedding the tebuconazole fungicide in a study by Volova et al.20 The authors

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of another study prepared and investigated tebuconazole/poly(3-hydroxybutyrate) formulations

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shaped as films, microparticles, and pellets, showing their effectiveness against Fusarium.21,22

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These formulations were successfully tested in experiments with laboratory wheat stands infected

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by Fusarium.23

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Formulations for protecting crops should be not only effective against plant pathogens and

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safe for the environment but also cost-effective, as the amounts of pesticides produced and used in

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agriculture are enormous. Moreover, increasing importance is attached to industrial ecology and


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green chemistry – tools for creating new ecofriendly materials including those manufactured from

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renewable sources.24

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There are few published data on production of PHA blends with natural fillers. Several

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studies described results of filling polyhydroxyalkanoates with clay and its derivatives, plant fibers,

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lignin and holocellulose from a lignocellulosic biowaste, and wood chips and powder.25-28 Those

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blends were mainly considered as material for making furniture and as construction material.

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Therefore, the purpose of this study was to construct and investigate slow-release fungicide

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formulations, with the active ingredient embedded in the degradable matrix of P(3HB) blended with

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readily available natural materials (peat, clay, wood flour).

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Materials and Methods

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Materials

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Samples of P(3HB) polymer were synthesized by using the Cupriavidus eutrophus B10646

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strain and proprietary technology.29 Polymer was extracted from cells with chloroform, and the

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extracts were precipitated using hexane. The extracted polymers were re-dissolved and precipitated

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again 3-4 times to prepare homogeneous specimens.

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Three natural materials were used as fillers: peat, wood flour and clay. High-moor peat

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“Agrobalt-N”, state registration 0428-06-209-139-0-0-01, was produced by LLC “Akademiya

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tsvetovodstva”, Russia. Wood flour was produced by grinding wood of birch (Betula pendula Roth)

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using an MD 250-85 woodworking machine (“StankoPremyer” Russia). Then it was dried at 60°C

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for 120 h until it reached constant weight, and 0.5 mm mesh was used to separate the particle size

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fraction. Clay was taken from the mine “Kuznetsovskoye”, Krasnoyarsk Krai, Russia. It had the

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following composition: loss on ignition – 6.92 %; SiО2 – 60.1 %; Al2O3 – 19.17 %; Fe2O3 – 6.72 %;

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CaO – 2.02 %; MgO –2.12 %; SO3 – 0.65 %; Na2O – 0.88 %; K2O – 1.45 %.

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Three systemic pre-emergence fungicides for soil application were tested (Xi’an TaiCheng Chem Co., Ltd, China) (Table 1).


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Preparation of Blends of P(3HB) and Natural Materials and Fabrication of Pellets and Granules

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The polymer and fillers were pulverized by impact and shearing action in ultra-centrifugal

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mill ZM 200 (Retsch, Germany). To achieve high fineness of polymer grinding, the material and the

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mill housing with the grinding tools were preliminarily cooled at -80 °C for about 30 min in an

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Innova U101 freezer (NEW BRUNSWICK SCIENTIFIC, U.S.). Grinding was performed using a

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sieve with 2-mm holes at a rotor speed of 18000 rpm. The fractional composition of the powders

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was determined using vibratory sieve shaker AS 200 control (Retsch, Germany). Polymer + filler +

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fungicide (50:30:20 wt.%) powders were mixed in planetary mixer SpeedMixer DAC 250 SP

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(Hauschild Eng., Germany), for 1 min at 1000 rpm. The resulting homogenous mixtures were used

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to prepare pellets and granules. Pellets were prepared by cold pressing at a pressure of 36 bar using

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automatic press “Usilennyi Superpress” (Byelorussia). Granules were prepared by mixing polymer

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paste wetted with ethanol in screw granulator Fimar (Italy).

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SEM Microscopy

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The surface microstructure of pellets and granules was analyzed before and after incubation

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in soil using scanning electron microscopy (a TM-3000 Hitachi microscope with the QUANTAX

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70 program, Japan). Prior to microscopy, the samples were sputter coated with platinum (at 10 mA,

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for 40 s) using an Emitech K575XD Turbo Sputter Coater (Quorum Technologies Limited, U.K.).

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FTIR Spectroscopy

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The free fungicides and fungicide formulations, which differed in the composition of the

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composite matrix, were analyzed using FTIR spectroscopy. IR spectra were taken in the 400-4000

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cm-1 range using a “NICOLET 6700” FT-IR spectrometer (Thermo Scientific, U.S.) and a Smart

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Orbit accessory, by the attenuated total reflection (ATR) technique. The absolute value of the

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maximum permissible error was ±0.01 cm-1.

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Differential Scanning Calorimetry (DSC)


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Thermal analysis of the starting materials and experimental formulations was performed

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using a DSC-1 differential scanning calorimeter (METTLER TOLEDO, Switzerland). Powdered

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samples (4.0±0.2 mg each) were placed into the aluminum crucible and compressed prior to

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measurement. The specimens were heated at a rate of 5 °C/min to 200 °C, then cooled to -20 °C,

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held for 4 minutes and re-heated to 320 °C. Glass transition temperature (Tg), crystallization

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temperature (Tc), melting point (Tmelt), and thermal degradation temperature (Tdegr) were determined

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from peaks in thermograms using the “StarE” software.

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X-ray Diffraction

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X-Ray structure analysis and determination of crystallinity of starting materials and

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experimental formulations were performed employing a D8 ADVANCE X-Ray powder

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diffractometer equipped with a VANTEC fast linear detector, using CuKa radiation (Bruker, AXS,

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Germany). The degree of crystallinity was calculated as a ratio of the total area of crystalline peaks

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to the total area of the radiograph (the crystalline + amorphous components). Measurement

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accuracy: point measurement accuracy ± 0.4 pulses per second (PPS), with the lowest intensity 1.5

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PPS and the highest intensity 32 PPS.

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Degradation of Fungicide Formulations in Soil

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Biodegradation of the experimental fungicide formulations was studied in laboratory, using

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agro-transformed soil collected at field laboratory “Minderlinskoye” of the Krasnoyarsk State

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Agrarian University (56°N, 92°E). Soil was placed into plastic containers (100 g soil in each).

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Fungicide formulations were placed into fine-meshed gauze bags and incubated in soil at a

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temperature of 25 °C and constant moisture content of 50%. The experiment lasted 83 days. During

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the experiment, granules and pellets were collected from soil to measure their mass loss. In the first

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two weeks, no mass loss was recorded, and, thus, the first point corresponded to three weeks, when

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the mass loss was 5-15%. Seven weeks was approximately the midpoint of the experiment. The last

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point – 83 d – was the end of the experiment.


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Three samples of each type were collected periodically to investigate biodegradation of the

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polymeric matrix and fungicide release. Mass loss was measured to estimate degradation. Three

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specimens of each type were periodically taken out of the soil, rinsed with distilled water, dried to a

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constant weight, and weighed on the analytical balance of accuracy class 4 (Metler, Toledo). The

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mass (X) was determined as follows:

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Y1 - Y2 Х% = ______________ .100, Y1 where Y1 and Y2 denote the average mass of the samples (mg) before and after testing, respectively.

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Degradation of poly(3-hydroxybutyrate) granules and pellets was studied in the same soil

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and under the same conditions as degradation of the experimental fungicide formulations. During

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the experiment, which lasted 126-168 days (i.e. until P(3HB) granules and pellets had been

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completely degraded), the granules and pellets were periodically collected from the soil, dried until

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a constant weight had been reached, and weighed. The mass loss was calculated using the formula

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given above.

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A Microbiological Study

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Microbiological analysis was conducted by conventional methods, based on cultural and

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morphological properties of microorganisms and using standard biochemical tests mentioned in

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identification keys.31-33 The abundance of ammonifying copiotrophic bacteria (CFU/g soil) was

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determined on nutrient agar (HiMedia, India); the abundance of mineral nitrogen assimilating

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prototrophs was determined on starch and ammonia agar (SAA); nitrogen-fixing bacteria were

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counted on Ashby’s medium and oligotrophs on soil extract agar (SA).34 Mineralization coefficient

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was determined as a ratio between microorganisms assimilating mineral nitrogen and ammonifying

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bacteria. Oligotrophy coefficient was determined as a ratio of oligotrophic to ammonifying bacteria.

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Concentrations of microorganisms (CFU/g soil) were calculated for the control soil and the soil

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layer on the surface of the specimens. All platings were performed in triplicate from soil suspension

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to 107 dilutions. The plates were incubated for 3-7 days at 30 °C. The number of microorganisms ACS Paragon Plus Environment

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was determined taking into account the dilutions. Primary P(3HB) degraders were isolated using the

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clear-zone technique,35 by plating the samples on mineral agar that contained polymer powder as a

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sole carbon source. Dominant microorganisms and principal P(3HB) degraders were identified

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using time-of-flight MALDI mass spectrometry, with a MALDI-TOF mass spectrometer (Bruker,

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Germany).

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Antifungal activity analysis

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Fungal strain Fusarium verticillioides was extracted from the field soil and grown on malt

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extract agar (MEA, Sigma-Aldrich, U.S.) in Petri dishes at a temperature of 25 °C for 5-7 days.

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Then, 5-mm diameter slabs of agar were aseptically drilled from the culture regions with actively

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growing colonies. A slab with the fungal culture and one of the fungicide formulations were placed

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at opposite sides of the Petri dish containing sterile MEA. The dishes were incubated in the

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temperature-controlled cabinet at 25 °C for 7 days. Then, we analyzed results of colony growth by

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measuring the radius of the fungal mycelium and determining the degree of fungus growth

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inhibition relative to the control (the free active ingredient of the fungicide). As a negative control,

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we measured the radius of the fungal mycelium grown under the same conditions in the dish

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without the fungicide. All procedures were done in triplicate.

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Fungicide Analysis

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For fungicide concentration analysis in soil, 50 ml of chloroform was added to 10 g of dry

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soil and left for 24 h. Then, the soil was filtered and rinsed with chloroform; the operation was

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repeated 3 times. The solvent was removed in the rotary vacuum evaporator. To determine residual

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fungicides in the specimens, the granules and pellets collected from soil at different time point of

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the experiment were dissolved in 2 ml of chloroform. After the complete dissolution of the polymer,

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the solution was passed through a 0.45 μm PTFE filter (Agilent). Fungicide release from the matrix

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to soil (RA) was calculated using the formula RA = 100% - (Cres/Cin × 100%), where Сin is the

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initial fungicide content in the formulation (mg) and Cres is residual fungicide in the formulation

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(mg). Fungicide concentrations were analyzed using high-performance liquid chromatography, with


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an Agilent 1200 Infinity chromatographic system equipped with a gradient pump, autosampler, a

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column thermostat, and a diode matrix detector. An Eclipse XDB-C18 column was used.

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Chromatography was performed in the gradient of water acidified with 0.1% acetic acid and

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acetonitrile. Chromatography lasted 12 min. The solvents were delivered as follows: for 5 min after

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sample injection – acetonitrile gradient from 45% to 53% (from 55% to 47% water); between 5 min

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and 8 min – from 53% to 60% acetonitrile; after 8 min – 60% acetonitrile. Detection was done at

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the wavelengths located at the absorption maxima of the fungicides: at 220 nm for epoxiconazole

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and tebuconazole and at 230 nm for azoxystrobin. The reference values were recorded at a

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wavelength of 360 nm. The range of the concentrations detected was between 1 µg/ml and 500

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µg/ml or higher.

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Statistical Analysis

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Statistical analysis of the results was performed by conventional methods, using the standard

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software package of Microsoft Excel. Arithmetic means and standard deviations were found. The

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statistical significance of results was determined using Student’s t-test (significance level: P ≤ 0.05).

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Statistical analysis of surface properties of the samples was performed by using embedded methods

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of the DSA-4 software.

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Results and Discussion

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Characterization of P(3HB) and Starting Natural Materials

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Materials used as a composite matrix for embedding fungicides differed in some of their

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properties (Table 2). In contrast to the high-crystallinity P(3HB), whose Cx was 75%, all fillers had

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substantially lower degrees of crystallinity (from 9 to 53%). P(3HB) is a thermoplastic material,

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with a considerable difference between melting point (176 °C) and thermal decomposition

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temperature (287 °C). None of the fillers was a thermoplastic material. Their temperatures with the

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exception of clay for the onset of thermal decomposition were lower, and thermograms did not

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show any peaks corresponding to melting and crystallization. Concerning clay, even we detected a


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peak at 200 °C the degradation of clay minerals starts at much higher temperatures (above 400

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°C).36 The peak registered should be associated with clay dehydration.

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Characterization of Slow-Release Fungicide Formulations

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P(3HB) and filler powders were used to prepare fungicide formulations shaped as pellets

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(diameter 6 mm and height 3 mm) and granules (diameter 2 mm and length 2-4 mm) (Fig. 1).

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SEM images of formulations show differences in their surface microstructure, which are

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mainly associated with the type of the filler. The P(3HB)/wood flour specimens had solid surfaces

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with few voids for all fungicide formulations. The surface of the P(3HB)/peat specimens was more

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porous, with uneven topography. The surface of the P(3HB)/clay specimens had the loosest

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structure, with numerous pores and voids (Fig. 2). The structure of the matrix exerted some effect

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on the mixing and distribution of fungicides in it. P(3HB)/clay specimens contained clumps of

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powder and crystals of the three fungicides, but in the P(3HB)/wood flour and P(3HB)/peat

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specimens, fine particles of fungicides were uniformly distributed in the matrix.

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To determine how the loading of the fungicides into the matrix affected them, we

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investigated the physicochemical properties of the free fungicides and the experimental

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formulations (Table 3).

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The fungicides used in this study differed structurally and, thus, had dissimilar

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physicochemical properties (Mw and Tmelt). Loading of fungicides into the composite matrix resulted

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in a decrease in the degree of crystallinity and enthalpy of melting of the polymer (176 °C).

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Moreover, the presence of the pesticide in the specimens resulted in an earlier onset of thermal

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decomposition (Tables 2 and 3). All thermograms show two melting peaks corresponding to the

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pesticide and polymer. In a previous study20, with tebuconazole loaded into a matrix of pure

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P(3HB), the degree of crystallinity did not change, in contrast to the present study, but changes in

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the melting point were comparable (Table 3).

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To determine the type of interactions between the composite polymer matrix and fungicides

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loaded into it, we used IR spectroscopy, which showed that no chemical bonds formed between


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these components, i.e. their interactions were of physical nature. The absence of chemical

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interaction between P(3HB) and tebuconazole was also shown in the previous paper.20 Figure 3

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shows IR spectra of epoxiconazole loaded into degradable matrices with different compositions.

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The IR spectrum contains absorption bands characteristic of the fungicide and materials of

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the matrix. The spectra show distinct stretching vibrations of the carbonyl groups C=O of the

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polymer (1700-1760 cm-1) and superposition of vibrations of the epoxiconazole C=O groups as well

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as asymmetric stretching of СН3- and СН2-groups (2994, 2974, 2936 cm-1). The spectra of wood

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flour and peat show stretching and skeletal vibrations of the benzene ring (1422 cm-1, 1504 cm-1,

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1593 cm-1); in this region, the spectrum is superposition of vibrations of the epoxiconazole benzene

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rings. The presence of the characteristic band of the –NH–NH2 group (ν 1581 cm–1) for

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epoxiconazole indicates that no oxidation occurs during fungicide loading.

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Antifungal Activity of Slow-Release Fungicide Formulations

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Antifungal activity of the matrix-loaded fungicides and the free fungicides is shown in

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Figure 4. The in vitro study showed that all formulations had a significant inhibitory effect on F.

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verticillioides: the size of the fungus colonies was smaller in the presence of the fungicides than in

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the control group.

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Photographs in Figure 4 show F. verticillioides colonies on the nutrient medium in the

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presence of matrix-loaded fungicides and in the control group. The average radius of the fungus

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colonies in the Petri dishes with the experimental azoxystrobin formulations was comparable to the

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radius of the colonies in the dishes with pure fungicide (positive control) (1.3-1.7 cm) while in the

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dishes without any fungicide (negative control), the size of the fungus colonies was much larger

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(3.5-3.8 cm). Thus, the experimental formulations were as effective as the free fungicides. Similar

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results were obtained for the tebuconazole and epoxiconazole formulations (Fig. 4). Formulations

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with different fillers produced similar fungicidal effects. No significant differences in the inhibitory

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effects on the growth of fungal colonies were found between the pellets and granules. Similar

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results were obtained in a previous study.20


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Degradation of Slow-Release Fungicide Formulations in Soil

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Biodegradation of the experimental fungicide formulations was studied in laboratory soil

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microecosystems. The soil was chernozem with heavy loam particle-size composition, density of

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between 0.80 and 1.24 g/cm3, pH 7.3, with high contents of ammonium nitrogen (35 mg/kg) and

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nitrate nitrogen (9.2 mg/kg). The soil contained 280 mg/kg of phosphorus and 250 mg/kg of

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potassium. The total count of organotrophic bacteria in soil samples was about 1 million CFUg-1 of

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air-dry soil. The mineralization coefficient reached 4.72 and the oligotrophy coefficient was 6.76.

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The eco-trophic groups were dominated by oligotrophs: (76.712.4)106 CFUg-1. The abundances

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of prototrophs, copiotrophs, and nitrogen fixers were one order of magnitude lower: (5.31.8)106,

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(1.10.9)106, and (2.01.3)106 CFUg-1, respectively. The abundance of microscopic fungi was

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three orders of magnitude lower: (37.39.8)103 CFUg-1. The high abundance of oligotrophic

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microorganisms and the high oligotrophy and mineralization coefficients were indicative of soil

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maturity and low contents of organic nitrogen. The high abundance of nitrogen-fixing

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microorganisms suggested the lack of readily available nitrogen forms in the soil.

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Analysis of proportions of the cultured microorganisms in soil samples showed that 73.2%

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colonies on MPA were formed by Gram-positive bacteria. They were dominated by actinobacteria

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(38.6%, including 18.3% Streptomyces), which represented the autochthonic microflora of mature

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soils (Fig. 5). The second most abundant bacteria were spore-forming rods of the genera Bacillus

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(24.8%) and Paenibacillus (9.8%). Gram-negative bacteria were mainly represented by

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Pseudomonas (9.4%). Microscopic fungi were dominated by Penicillium species, which constituted

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about 70% of all isolates (Fig. 5). Fusarium species made up 8.4%, and other microscopic fungi

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were not abundant. The total percentage of potentially phytopathogenic microscopic fungi

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(Fusarium, Alternaria, Pythium, Verticillium) was rather high, reaching 15.3%, and this may pose a

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threat to plants grown without crop rotation and result in accumulation of phytopathogenic

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microscopic fungi in soil.


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The experimental fungicide formulations were incubated in soil for 83 days. The

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formulations contained 20% active ingredient, 30% filler, and 50% P(3HB). As the matrix was

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degraded, the surface of the specimens became more uneven and loose, and the number of

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defects and voids increased significantly, especially in the tebuconazole and azoxystrobin loaded

348

specimens whose matrix contained peat and clay (Fig. 6).

349

Degradation behavior of the experimental formulations and the release of fungicides to soil

350

were studied by determining the mass of the specimens during incubation of formulations in soil,

351

taking into account residual concentrations of polymer and fungicides. These processes are certainly

352

determined by various factors. The main factor is the activity of soil microflora, which makes the

353

major contribution to P(3HB) biodegradation; other factors include the composition of the matrix

354

and the structure and solubility of fungicides. The mass loss of the specimens incubated in soil

355

primarily occurred due to degradation of P(3HB), and other factors were fungicide dissolution and

356

leaching. The three types of degradable matrices (polymer/filler) loaded with three fungicides were

357

degraded slowly and at similar rates; their residual masses at the end of the experiment were 65-

358

80% of their starting masses (Fig. 7). The degradation rate of the granules was somewhat higher

359

than that of the pellets, and, at the end of the experiment, their residual mass was 55-70% of their

360

starting mass (or between 27.5 mg and 35 mg), depending on the filler type. The residual mass of

361

the pellets was about 65-80% (or between 32.5 mg and 40 mg) (Fig. 7).

362

The type of the filler did not produce any major effect on degradation of the specimens,

363

although specimens containing clay were degraded at a somewhat faster rate. Therefore, the

364

residual mass of clay-containing specimens was the lowest – 55% of their starting mass.

365

During the incubation of the experimental granules and pellets in soil, only the polymer was

366

degraded but the fillers (peat, clay, and wood flour) were not. Even if the formulations were

367

degraded completely, the fillers would stay in the soil. Therefore, a study was performed to

368

investigate degradation of poly(3-hydroxybutyrate) pellets and granules in soil (Fig. 8). The

369

granules were fully degraded in 126 days while degradation of pellets took longer – about 160 days.


14

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370

PHA biodegradation behavior is determined by various factors such as PHA chemical

371

composition, shape and technique of fabrication of polymer products, and the type of the incubation

372

medium, including climate and weather, soil structure and composition, and species composition of

373

the microbiota.37,38 Our previous studies showed that the rate of mass decrease of

374

P(3HB)/tebuconazole formulations in soil was determined by the geometry of the formulation

375

(pellets, granules, films) and fungicide loading.20,21 In the soil that had different composition and

376

microbial community, pellets of pure P(3HB) loaded with tebuconazole were 40% degraded in 35

377

days.20 That was comparable to the results obtained in the present study. Moreover, the addition of

378

the filler (wood flour, PCL, PEG) changed the degradation behavior of experimental metribuzin

379

formulations. The P(3HB)/PEG/metribuzin formulations were degraded faster than the

380

P(3HB)/wood flour/metribuzin specimens.39 The literature data on using PHAs to construct slow-

381

release fungicide formulations are scant. Savenkova et al., 2002 described experiments with film

382

systems made from poly-3-hydroxybutyrate and loaded with fungicides Sumilex and Ronilan.40 The

383

systems were incubated in soil and were nearly completely degraded in 4 weeks. The abundance of

384

pathogenic fungi in the soil was considerably reduced.

385 386

All experimental formulations were capable of functioning in soil longer than the experimental period of 83 d, enabling gradual and slow delivery of fungicides to plants.

387

Degradation behavior of the polymer is largely determined by the microbiological activity of

388

the soil in which it is incubated. Soil microbial communities are the main component in

389

detoxification and transformation of pesticides and self-purification of the environment. Pesticides

390

applied to soil interact with microorganisms, and there is a dose-effect relationship: the higher the

391

concentration of the pesticide applied to the soil, the more damaging it is to the microflora.41

392

Researchers noted a decrease in soil enzymatic activity, microbial respiration rate, biomass, and

393

diversity of bacterial communities after the application of fungicides such as iprodione,

394

carbendazim, spiroxamine, tebuconazole, triadimenol, etc.42-44 Fungicides with long half-lives such

395

as propiconazole are particularly toxic. When such fungicides accumulate in soil, after numerous


15


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396

applications, they may remain toxic to soil microflora for 40 days.45 Other data, however, suggest

397

that fungicides stimulate the development of certain groups of microorganisms such as

398

Proteobacteria.44 Application of organic amendments (compost, activated sludge, humic

399

preparation) to soil alleviated the toxic effect of pesticides on biota.46,47 However, there are very

400

few literature data on the effects of slow-release fungicide formulations on soil microflora.

401

Immobilization of picloram or diuron on silica gel resulted in a reduction in the toxicity of the

402

pesticides compared to the pesticides applied using conventional methods.48,49 Our previous studies

403

showed that matrix-loaded tebuconazole did not decrease the total counts of soil bacteria and

404

stimulated the development of certain groups of microorganisms such as bacteria capable of

405

degrading complex organic compounds – Pseudomonas, Stenotrophomonas, Variovorax and

406

Streptomyces.20

407

Soil application of the pesticides loaded into the degradable matrix containing poly-3-

408

hydroxybutyrate was, on the one hand, a source of supplementary substrate to microorganisms. On

409

the other hand, the fungicide released as the polymer matrix was degraded could exert adverse

410

effects on the microflora. In most soil samples, the abundance of copiotrophic bacteria increased

411

while the abundance of oligotrophs decreased by a factor of 2.5-10, resulting in a decrease in the

412

mineralization and oligotrophy coefficients by one or two orders of magnitude compared to the

413

original soil. These changes correspond to successions of the functional groups of microorganisms

414

when substrate is delivered to the soil. After application of the experimental fungicide formulations,

415

the composition of soil microflora changed as follows: the dominance of spore-forming bacteria,

416

including Bacillus, increased to 30%, and Streptomyces and Pseudomonas increased to 25.6% and

417

12.3%, respectively. Among mycelial fungi, species of the genus Penicillium constituted 88.2% on

418

average of the total abundance of microscopic fungi. Representatives of Pseudomonas, Bacillus,

419

Streptomyces, and Pseudarthrobacter were isolated as primary bacterial P(3HB) degraders and

420

Talaromyces and Penicillium – as primary fungal P(3HB) degraders. The increase in the abundance


16

Page 17 of 40


421

of these microorganisms could be their favorable response to soil application of P(3HB) as a

422

supplementary substrate.

423 424

Dynamics of Fungicide Release from Formulations and Dynamics of Fungicide Contents in Soil

425

Fungicide release from the polymer matrix was determined by measuring fungicide contents

426

in the formulations before and after incubation of the specimens in soil (Fig. 9). The active

427

ingredients were released rather uniformly, especially from the pellets. By Day 20, a significantly

428

higher amount of tebuconazole had been released from the granules than from the pellets.

429

Fungicide concentrations in the experimental formulations with different fillers and

430

fungicide concentrations in the soil were measured during incubation of the specimens in soil (Fig.

431

10). Fungicide release kinetics was influenced by various factors such as kinetics of degradation of

432

formulations in soil, shape of the specimens, composition of the matrix, solubility of the fungicides,

433

and stability of the fungicides in soil. The fungicides used in this study were poorly soluble

434

compounds, but their solubility varied in a wide range: 36 mg/L for tebuconazole, 6.7 mg/L for

435

azoxystrobin, and 6.6 mg/L for epoxiconazole.

436

The fungicides accumulation in the soil released from the granules was somewhat higher

437

than from pellets, especially in the early phase of incubation. The highest fungicide concentrations

438

in the soil with the experimental granules were measured by the third week of the experiment. By

439

contrast, the highest concentrations of the fungicides released from the experimental pellets were

440

observed later, between week five and week seven of the experiment.

441

The dynamics of accumulation of the three relatively poorly soluble fungicides were similar.

442

Measurements of their residual concentrations in the specimens suggested that the fungicides were

443

gradually released from both granules and pellets. After the fungicide concentrations in soil reached

444

their maxima, no dramatic changes in this parameter occurred until the end of experiment (83 days).

445

The highest concentration of tebuconazole, whose solubility was higher than the solubility of

446

azoxystrobin and epoxiconazole, varied between 20 and 26 µg/g soil, depending on the filler


17


Page 18 of 40

447

material (that is 20-26% of the fungicide content in the formulations before soil incubation) (Fig.

448

10) and the maximal concentrations of azoxystrobin and epoxiconazole in soil were 15 and 11 µg/g

449

soil, respectively (Fig. 10). These concentrations are corresponded to the recommended fungicide

450

rates. The differences in their concentrations in soil were most likely caused by their dissimilar

451

stabilities.

452

The fungicide concentrations in soil were measured at definite time points of the

453

experiment. However, soil is a complex and multifactor system, in which various biogenic and

454

abiogenic processes influence concentrations of the chemicals applied to the soil. The chemicals are

455

transformed by soil microflora in processes of co-metabolism and enzymatic oxidation.

456

Furthermore, they are dissolved and degraded due to natural abiogenic processes occurring in the

457

soil. It does not seem possible, however, to take into account all these processes and estimate their

458

contribution to fungicide concentrations in soil.

459

Rather few studies have reported the use of PHAs as matrix for pesticide loading and

460

pesticide release kinetics so far. Suave et al. (2010) showed that the rate of release of the malathion

461

pesticide from the P(3HB)/PCL microspheres was determined by the ratio of these components in

462

the blend.50 Another PHA type, namely P(3HB/3HV), was used in the studies by Lobo et al.

463

(2011)51 and Grillo et al. (2011).52 Ametryn encapsulated in P(3HB) and P(3HB/3HV)

464

microparticles was released at a slower rate than the free herbicide.52 Similar results were reported

465

in a study by Lobo et al. (2011) for the atrazine pesticide loaded into the polymer matrix.51 Our

466

previous research showed that encapsulation of tebuconazole into the P(3HB) matrix resulted in

467

slower release of the fungicide to water and soil, and the release rate was determined by the shape

468

of the formulation and the loading level.20,21 The types of the matrix and the filler have a very

469

strong effect on pesticide release rate. Asrar et al. (2004) found that addition of plasticizers and

470

PVA to the main substance, poly(methyl methacrylate) (PMMA) and poly(styrene-co-maleic

471

anhydride), resulted in a faster rate of tebuconazole release from microparticles to water.

472

Tebuconazole release from microspheres containing 0-30% PVA reached 25-80% of the loaded


18

Page 19 of 40


473

amount in 47-48 d.13 Tebuconazole release from ethyl cellulose microcapsules to water was slower:

474

40% pesticide in 50 d.9 Qian et al. (2013) described a study in which T50 of tebuconazole release

475

from porous hollow silica nanospheres to water varied between 3 and 18 d, depending on water

476

temperature and pH and size of the nanospheres.6 The effect of the size of polylactide microspheres

477

on azoxystrobin release kinetics was shown in a study by Yao et al. (2018).18

478

Thus, slow-release formulations of fungicides embedded in a matrix of P(3HB) and a filler

479

(clay, wood flour, and peat) were constructed. Fourier transform infrared spectroscopy showed that

480

the P3HB/filler/fungicide system was a mechanical mixture of the components. All fungicide

481

formulations had a pronounced inhibitory effect in vitro against the fungus Fusarium verticillioides.

482

Biodegradation behavior of the experimental fungicide formulations in the soil was mainly

483

determined by the shape of the specimens (granules or pellets) without significant influence of the

484

filler type. The content of fungicides present in the soil due to degradation of the formulations and

485

fungicide release was primarily determined by their solubility. The use of P(3HB) and fillers as a

486

matrix for embedding fungicides made it possible to develop slow-release formulations of

487

fungicides, which can function in soil for a long time, enabling gradual and slow delivery of

488

fungicides.

489

This study was financially supported by Project “Agropreparations of the new generation: a

490

strategy of construction and realization” (Agreement No 074-02-2018-328) in accordance with

491

Resolution No 220 of the Government of the Russian Federation of April 9, 2010, “On measures

492

designed to attract leading scientists to the Russian institutions of higher learning”.

493 494 495 496 497 498


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499

References

500

1. Binder, E.M.; Tan, L.M.; Chin, L.M.; Handl, J.; Richard, J. Worldwide occurrence of

501

mycotoxins in commodities, feeds and feed ingredients. Anim. Feed Sci. Technol. 2007, 137, 265-

502

282. 2 Schmale, D.G., Bergstrom, G.C. Fusarium head blight in wheat. The Plant Health

503 504

Instructor, 2003.

505

3. Undabeytia, T.; Recio, E.; Maqueda, C.; Morillo, E.; Gomez-Pantoja, E.; Sanchez-

506

Verdejo, T.; Reduced metribuzin pollution with phosphatidylcholine–clay formulations. Pest

507

Manag. Sci. 2011, 67, 271–278.

508

4. Hussein, M..; Rahman, N.S.S.A.; Sarijo, S.H.; Zainal, Z. Synthesis of a monophasic

509

nanohybrid for a controlled release formulation of two active agents simultaneously. Appl. Clay Sci.

510

2012, 58, 60–66.

511

5.

Ghazali,

S.A.I.S.M.;

Hussein,

M.Z.;

Siti

Halimah

Sarijo,

S.H.

3,4-

512

Dichlorophenoxyacetate interleaved into anionic clay for controlled release formulation of a new

513

environmentally friendly agrochemical. Nano. Res. Lett. 2013, 8, 362-369.

514

6. Qian, K.; Shi, T.; He, S.; Luo, L.; Liu, X.; Cao, Y. Release kinetics of tebuconazole from

515

porous hollow silica nanospheres prepared by miniemulsion method. Micropor. Mesopor. Mater.

516

2013, 169, 1-6.

517

7. Chevillard, A.; Angellier-Coussy, H.; Guillard, V.; Bertrand, C.; Gontard, N.; Gastaldi,

518

E. Biodegradable herbicide delivery systems with slow diffusion in soil and UV protection

519

properties. Pest Manag. Sci. 2014, 70, 1697-1705.

520

8. Sopeña, F.; Cabrera, A.; Maqueda, C.; Morillo, E. Ethylcellulose formulations for

521

controlled release of the herbicide alachlor in a sandy soil. J. Agric. Food Chem. 2007, 55, 8200–

522

8205.


20

Page 21 of 40


523

9. Yang, D.; Wang, N.; Yan, X.; Shi, J.; Zhang, M.; Wang, Z.; Yun, H. Microencapsulation

524

of seed-coating tebuconazle and its effects on physiology and biochemistry of maize seedlings.

525

Coll. Surf B: Biointer. 2014, 114, 241–246.

526

10. Fernández-Pérez, M.; Villafranca-Sánchez, M.; Flores-Céspedes, F.; Pérez-García, S.;

527

Daza-Fernández, I. Prevention of chloridazon and metribuzin pollution using lignin-based

528

formulations. Environ. Pollut. 2010, 158, 412-1419.

529

11. Fernández-Pérez, M.; Villafranca-Sánchez, M.; Flores-Céspedes, F.; Daza-Fernández, I.

530

Ethylcellulose and lignin as bearer polymers in controlled release formulations of chloridazon.

531

Carbohydr. Polym. 2011, 83, 1672–1679.

532

12. Grillo, R.; Clemente, Z.; de Oliveira, J.L.; Campos, E.V.R.; Chalupe, V.C.; Jonsson,

533

C.M.; de Lima, R.; Sanches, G.; Nishisaka, C.S.; Rosa, A.H.; Oehlke, K.; Greiner, R.; Fraceto, L.F.

534

Chitosan nanoparticles loaded the herbicide paraquat: The influence of the aquatic humic

535

substances on the colloidal stability and toxicity. J. Hazard. Mater. 2015, 286, 562–572.

536

13. Asrar, J.; Ding, Y.; La Monica, R.E.; Ness, L.C. Controlled release of tebuconazole from

537

a polymer matrix microparticle: release kinetics and length of efficacy. J. Agric. Food Chem. 2004,

538

52, 4814-4820.

539

14. Ding, X.; Richter, D.L.; Matuana, L.M.; Heiden, P.A. Efficient one-pot synthesis and

540

loading of self-assembled amphiphilic chitosan nanoparticles for low-leaching wood preservation.

541

Carbohydr. Polym. 2011, 86, 58-64.

542 543

15. Mattos, B.D.; Magalhães, W.L. Biogenic nanosilica blended by nanofibrillated cellulose as support for slow-release of tebuconazole. J. Nanopart. Res. 2016, 18, 274.

544

16. Tang, J.; Ding, G.; Niu, J.; Zhang, W.; Tang, G.; Liang, Y.; Fan, C.; Dong, H.; Yang, J.;

545

Li, J.; Cao, Y. Preparation and characterization of tebuconazole metal-organic framework based

546

microcapsules with dual-microbicidal activity. Chem. Eng. J. 2019, 359, 225–232.


21


Page 22 of 40

547

17. Wang, Y.; Li, C.; Wang, Y.; Zhang, Y.; Li, X. Compound pesticide controlled release

548

system based on the mixture of poly (butylene succinate) and PLA. J. Microencapsul. 2018, 35,

549

494-503.

550

18. Yao, J.; Cui, B.; Zhao, X.; Zhi, H.; Zeng, Z.; Wang, Y.; Sun, C.; Liu, G.; Gao, J.; Cui,

551

H. Antagonistic effect of azoxystrobin poly (lactic acid) microspheres with controllable particle size

552

on Colletotrichum higginsianum Sacc. Nanomaterials (Basel) 2018, 8, E857.

553

19. Campos, E.V.R.; de Oliveira, J.L.; da Silva, C.M.G.; Pascoli, M.; Pasquoto, T.; Lima,

554

R.; Abhilash, P.C., Fraceto, L.F. Polymeric and solid lipid nanoparticles for sustained release of

555

carbendazim and tebuconazole in agricultural applications. Sci. Rep. 2015, 5, 13809.

556

20. Volova, T.G.; Zhila, N.O.; Vinogradova, O.N.; Shumilova, A.A.; Prudnikova, S.V.;

557

Shishatskaya, E.I. Characterization of biodegradable poly-3-hydroxybutyrate films and pellets

558

loaded with the fungicide tebuconazole. Environ. Sci. Pollut. Res. 2016, 23, 5243-5254.

559

21. Volova, T.G.; Prudnikova, S.V.; Zhila. N.O.; Vinogradova, O.N.; Shumilova, A.A.;

560

Nikolaeva, E.D. Kiselev, E.G.; Shishatskaya, E.I. Efficacy of tebuconazole embedded in

561

biodegradable poly-3-hydroxybutyrate to inhibit the development of Fusarium moniliforme in soil

562

microecosystems. Pest Manag. Sci. 2017, 73, 925-935.

563 564

22. Shershneva, A.M.; Murueva, A.V.; Zhila, N.O.; Volova T.G Antifungal activity of P3HB microparticles containing tebuconazole. J. Environ Sci Health, Part B. 2019, 54, 196-204.

565

23. Volova, T.G.; Prudnikova, S.V.; Zhila, N.O. Fungicidal activity of slow-release

566

P(3HB)/TEB formulations in wheat plant communities infected by Fusarium moniliforme. Environ.

567

Sci. Pollut. Res. 2018, 25, 552-561.

568

24. Qaiss, A.; Bouhf, R.; Essabir, H. Characterization and use of coir, almond, apricot,

569

argan, shell, and wood as reinforcement in the polymeric matrix in order to valorize these products.

570

In Agricultural biomass based potential materials; Hakeem, K.R, Jawaid, M., Alothman, O.Y.,

571

Eds.; Springer, Cham, 2015, 305-339.

572

25. Torres-Giner, S.; Montanes, N.; Boronat, T.; Quiles-Carrillo, L.; Balart, R. Melt grafting


22

Page 23 of 40


573

of sepiolite nanoclay onto poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by reactive extrusion

574

with multi-functional epoxy-based styrene-acrylic oligomer. Eur. Polym. J. 2016, 84, 693–707.

575

26. Gunning, M.A.; Geever, L.M.; Killion, J.A.; Lyons, J.G.; Higginbotha, C.L. Mechanical

576

and biodegradation performance of short natural fibre polyhydroxybutyrate composites. Polym.

577

Test. 2013, 32, 1603–1611.

578

27. Angelini, S.; Cerruti, P.; Immirzi, B.; Scarinzi, G.; Malinconico, M. Acid-insoluble

579

lignin and holocellulose from a lignocellulosic biowaste: Bio-fillers in poly(3-hydroxybutyrate).

580

Eur. Polym. J. 2016, 76, 63–76.

581

28. Iewkittayakorn, J.; Khunthongkaew, P.; Chotigeat, W.; Sudesh, K. Effect of microwave

582

pretreatment on the properties of particleboard made from para rubber wood sawdust with the

583

addition of polyhydroxyalkanoates. Sains Malaysiana. 2017, 46, 1361–1367.

584

29. Volova, T.; Kiselev, E.; Vinogradova, O.; Nikolaeva, E.; Chistyakov, A.; Sukovatiy, A.;

585

Shishatskaya, E. A glucose-utilizing strain, Cupriavidus euthrophus B-10646: growth kinetics,

586

characterization and synthesis of multicomponent PHAs. PLoS One 2014, 9, 1–15.

587

30. Handbook of pesticides (toxicological-hygienic characterization) (Spravochnik po

588

pestitsidam (toksikologo-gigienicheskaya kharakteristika). Issue 1. Edited by RAMS Full Member

589

V.N. Rakitsky. Moscow. Agrorus Publishers. 2011 (in Russian).

590

31. Proteobacteria: Alpha and beta subclasses. In The Prokaryotes, Dworkin, M., Falkow,

591

S., Rosenberg, E., Schleifer, K.H., Stackebrandt, E., Eds.; Springer-Verlag: New York, 2006; Vol.

592

5, 910 p.

593

32. The Proteobacteria. In Bergey’s Manual of Systematic Bacteriology, Garrity, G.M.;

594

Brenner, D.J.; Krieg, N.R.; Staley, J.T., Eds.; Springer Science & Business Media, 2006; Vol. 2,

595

2791 p.

596

33. The Firmicutes. In Bergey’s Manual of Systematic Bacteriology, Vos, P., Garrity, G.,

597

Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H., Whitman, W., Eds.; Springer

598

Science & Business Media, 2009; Vol. 3, 1422 p.


23


Page 24 of 40

599

34. Netrusov, A.I.; Egorov, M.A.; Zakharchuk, L.M.; Kolotilova, N.N. Workshop on

600

microbiology: A textbook for students of higher educational institutions. Academy Publ.: Moscow,

601

2005 (in Russian).

602

35. Mergaert, J.; Webb, A.; Anderson, C.; Wouters, A.; Swings, J. Microbial degradation of

603

poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in soils. Appl.

604

Environ. Microbiol. 1993, 59, 3233-3238. 36. Guggenheim, S.; Groos, A.F.K. Baseline studies of the clay minerals society source

605 606

clays: thermal analysis. Clays Clay Miner 2001. 49, 433-443. 37. Emadian, S.M.; Onay, T.T.; Demirel, B. Biodegradation of bioplastics in natural

607 608

environments. Waste Manag. 2017, 59, 526-536.

609

38. Volova, T.G.; Prudnikova, S.V.; Vinogradova, O. N.; Syrvacheva, D.A.; Shishatskaya,

610

E.I. Microbial degradation of polyhydroxyalkanoates with different chemical compositions and

611

their biodegradability. Microbial Ecol. 2017, 73, 353-367.

612

39. Boyandin, A.N.; Zhila, N.O.; Kiselev, E.; Volova, T.G. Constructing slow-release

613

formulations of metribuzin based on degradable poly(3-hydroxybutyrate). J. Agricult. Food Chem.

614

2016, 64, 5625-5632. 40. Savenkova, L.; Gercberga, Z.; Muter, O.; Nikolaeva, V.; Dzene, A.; Tupureina, V. PHB-

615 616

based films as matrices for pesticides. Process Biochem. 2002, 37, 719-722. 41. Wu, X.; Cheng, L.; Cao, Z.; Yu, Y. Accumulation of chlorothalonil successively applied

617 618

to soil and its effect on microbial activity in soil. Ecotoxicol. Environmen. Saf. 2012, 81, 65-69.

619

42. Baćmaga, M.; Wyszkowska, J.; Kucharski, J. The effect of the Falcon 460 EC fungicide

620

on soil microbial communities, enzyme activities and plant growth. Ecotoxicology 2016, 25, 1575-

621

1587.

622 623

43. Wang, C.; Wang, F.; Zhang, Q.; Liang, W. Individual and combined effects of tebuconazole and carbendazim on soil microbial activity. Europ. J. Soil Biol. 2016, 72, 6-13.


24

Page 25 of 40


624

44. Zhang, M.; Wang, W.; Zhang, Y.; Teng, Y.; Xu, Z. Effects of fungicide iprodione and

625

nitrification inhibitor 3, 4-dimethylpyrazole phosphate on soil enzyme and bacterial properties. Sci.

626

Total Environ. 2017, 599, 254-263.

627

45. Fernández-Calviño, D.; Rousk, J.; Bååth, E.; Bollmann, U.E.; Bester, K.; Brandt, K.K.

628

Ecotoxicological assessment of propiconazole using soil bacterial and fungal growth assays. Appl.

629

Soil Ecol. 2017, 115, 27-30.

630

46. Pose-Juan, E.; Sánchez-Martín, M.J.; Herrero-Hernández, E.; Rodríguez-Cruz, M.S.

631

Application of mesotrione at different doses in an amended soil: dissipation and effect on the soil

632

microbial biomass and activity. Sci. Total Environ. 2015, 536, 31-38.

633

47. Bezuglova, O.S.; Gorovtsov, A.V.; Polienko, E.A.; Zinchenko, V.E.; Grinko, A.V.;

634

Lykhman, V.A., Demidov, A. Effect of humic preparation on winter wheat productivity and

635

rhizosphere microbial community under herbicide-induced stress. J. Soils Sediments 2019, 19,

636

2665-2675.

637 638 639 640

48. Prado, A.G.; Airoldi, C. A toxicity decrease on soil microbiota by applying the pesticide picloram anchored onto silica gel. Green Chem. 2002, 4, 288-291. 49. Prado, A.G.; Airoldi, C. The toxic effect on soil microbial activity caused by the free or immobilized pesticide diuron. Thermochim. Acta 2002, 394, 155-162.

641

50. Suave J.; Dall′Agnol, E.; Pezzin, A.; Meier, M.; Silva, D. Biodegradable microspheres of

642

poly(3-hydroxybutyrate)/poly(ε-caprolactone) loaded with malathion pesticide: preparation,

643

characterization, and in vitro controlled release testing. J. Appl. Polym. Sci. 2010, 117, 3419–3427.

644

51. Lobo, F.; Aguirre, C.; Silva, M.; Grillo, R.; de Melo, N.; Oliveira, L.; Morais, L.;

645

Campos, V.; Rosa, A.; Fraceto, L. Poly(hydroxybutyrate-co-hydroxyvalerate) microspheres loaded

646

with atrazine herbicide: screening of conditions or preparation, physico-chemical characterization,

647

and in vitro release studies. Polym. Bull. 2011, 67, 479–495.


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52. Grillo, R.; Pereira, A.; de Melo, N.; Porto, R.; Feitosa, L.; Tonello, P.; Filho, N.; Rosa,

649

A.; Lima, R.; Fraceto, L. Controlled release system or ametryn using polymer microspheres:

650

preparation, characterization and release kinetics in water. J. Hazard. Mater. 2011, 186, 1645–1651.

651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673


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674


Table 1 Characterization of the fungicides studied Fungicide Tebuconazole (RS)-1p-(4chlorophenyl)4,4-dimethyl-3(1H-1,2,4-triazol1ylmethyl)pentan3-yl Epoxiconazole (2RS,3SR)-1-(3(2-chlorophenyl)2,3-epoxy-2-(4fluorophenyl)prop yl)-1H-1,2,4triazole) Azoxystrobin (Methyl (2E)-2(2-{[6-(2cyanophenoxy)py rimidin-4yl]oxy}phenyl)-3methoxyacrylate

Structural formula

Use Effectively protects cereal crops from causal agents of root rot and rust

Toxic effects in vivo30 LD50*- oral dose for rats 3.9-5 g/kg ADI*-0.03 mg/kg of human bodyweight

Effectively protects cereal crops from causal agents of powdery mildew, all spot diseases, and rust

LD50*- for rats above 5 g/kg ADI*-0.004 mg/kg of human bodyweight

Effectively protects cereal crops, grape vines, and vegetables (tomatoes, cucumbers, onions, and potatoes) from causal agents of diseases resistant to triazoles and metalaxyl

LD50*- for rats above 5 g/kg ADI*-0.03 mg/kg of human bodyweight

675 676

LD50 – Median lethal dose

677

ADI - Acceptable daily intake

678 679 680 681 682 683 684


27


685

Page 28 of 40

Table 2 Physicochemical properties of starting materials and blends

686

Sample

Mw

Đ

Cx,%

Tmelt., °C

Tc, °С

687

Starting materials: 75 176 108 clay 53 peat 9 wood flour 26 (*) – the value should be associated with clay dehydration

688

(**) - onset of Tdegr.

P(3HB)

590

5.8

Tdegr, °C

287 200* 130** 220**

Melting enthalpy, (J/g)

89.3

689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 ACS Paragon Plus Environment

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708

Table 3 Physicochemical properties of the free fungicides and experimental formulations

709

Molecular weight, Cx,% Da Free fungicides: Tebuconazole 307.8 89 Epoxiconazole 329.8 88 Azoxystrobin 403.4 90 Slow-release fungicide formulations: Tdegr, °C (onset Composition Cx,% Tmelt, °C of sample decomposition) P(3HB)/wood flour/tebuconazole 57 104.7;166.7 259.6 P(3HB)/clay/tebuconazole 70 104.7;159.5 265.8 P(3HB)/peat/tebuconazole 59 104.9;162.9 266.9 P(3HB)/wood flour/epoxiconazole 60 135.84 165.1 250.9 P(3HB)/clay/epoxiconazole 70 136.0; 167.5 263.4 P(3HB)/peat/epoxiconazole 65 135.7; 165.1 272.9 P(3HB)/wood flour/azoxystrobin 54 118.7; 162.01 252.8 P(3HB)/clay/azoxystrobin 67 118.3; 168.6 274.5 P(3HB)/peat/azoxystrobin 56 118.2; 168.1 258.1 P(3HB)/tebuconazole* 80 164 283 *Data from Volova et al. (2016)20 Fungicide

Tmelt, °C 104.0 136.2 118.5 Melting enthalpy, ∆H, kJ/g 8.1; 29.2 27.8; 27.2 15.9; 20.3 14.81; 37.13 22.53; 28.57 27.16; 21.24 23.04; 19.85 20.59; 26.01 14.09; 37.9 70.1

710 711 712 713 714 715 716 717 718 719 720


29


721 722 723

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Figure 1 Photographs of slow-release formulations of fungicides embedded in degradable matrices of different compositions.

724 725


30

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726 727

Figure 2 SEM images of slow-release formulations of fungicides embedded in matrices of

728

degradable P(3HB) and natural materials: 1- epoxiconazole, 2 – tebuconazole, 3 – azoxystrobin;

729

bar = 200 µm. Arrows point to the filler.

730 731 732 733 734 735 736 737


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738 739 740

Figure 3 IR spectra of the experimental formulations of epoxiconazole loaded into the degradable matrix of P(3HB) and natural materials.

741 742 743 744 745 746 747 748 749 750


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751 752

Figure 4 Sensitivity of Fusarium verticillioides to the fungicides in different forms: K- –

753

negative control (without fungicide), K+ – positive control (active ingredient), 1 – pellets and

754

granules of P(3HB)/peat/fungicide, 2 – pellets and granules of P(3HB)/clay/fungicide, 3 – pellets

755

and granules of P(3HB)/wood flour/fungicide.



Page 34 of 40

756 757 758

Figure 5 Proportions of the dominant cultured microorganisms in soil samples

759 760 761 762 763 764 765 766 767


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768

769 770 771

Figure 6 Photographs of the slow-release fungicide formulations (granules and pellets) incubated in the soil for 83 days. 1 – epoxiconazole, 2 – tebuconazole, 3 – azoxystrobin.

772 773 774



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775 776

Figure 7 Mass loss kinetics of slow-release formulations of fungicides loaded into

777

degradable matrices (polymer/filler) with different compositions, shaped as pellets and granules,

778

during incubation in field soil

779 780 781 782 783 784 785 786 787 788 ACS Paragon Plus Environment

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789 790 791 792 793 794 795 796 797 798

Fig. 8 Degradation dynamics of poly(3-hydroxybutyrate) granules and pellets in soil

799


37


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800 801

Fig. 9 Fungicide release from the polymer/filler matrix in soil (% of the initial content).

802

I – tebuconazole, II – epoxiconazole, III - azoxystrobin

803 804 805 806


38

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807 808 809

Figure 10 Concentrations of fungicides released from the polymer/filler matrix in soil (µg/g soil). I – tebuconazole, II – epoxiconazole, III - azoxystrobin

810 811 812 813 814 815


39


816

Page 40 of 40

TOC Graphic

817


40

Constructing Slow-Release Fungicide Formulations Based on

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