In this context, we recently studied a collection of environmental samples in Eastern France to determinate whether sawmills and market gardens , recognized as huge consumers of triazole molecules, were affected by the presence of azole-resistant A. fumigatus. The higher rate of resistant strains was found in market gardens, which mainly used difenoconazole as fungicide, available since 2008 in Europe . Thus, to reduce resistance, limiting the contact between A. fumigatus and triazole molecules clearly appears necessary, while maintaining their efficient action against phytopathogenic molds. Chemical treatments were recently proposed for the removal of pesticides present in wastewaters . To our knowledge, studies on their elimination in soils are however rare. An alternative would be to limit the difenoconazole diffusion in soils. For this, we studied the interaction of difenoconazole with a hemp-based material used as adsorbent. Hemp, an annual plant, is an interesting raw material due to its ease of production , low-cost and versatility. Hemp has numerous applications, e.g. textile and paper industries, building and insulation, cosmetics, food, and composites . However, applications in environmental chemistry are rare. Recently, hemp in fibre or felt forms has been proposed for metal removal from aqueous solutions . In this work, we propose for the first time the use of hemp to capture triazole fungicides before reaching soil. Studies concerning the effects of contact time, material dose, and difenoconazole concentration were evaluated using two analytical methods, i.e. batch method and percolation technique. The effect of organic content of soils was also investigated in order to confirm our previous hypothesis that more resistant strains in soils could be related with huge rate of organic matter . This could be explained by the stronger selection pressure exerted by fungicides more retained in soils containing high levels of organic matter .
The experiments were conducted using two adsorption-oriented methods: a batch method and a percolation technique. The first set of experiments was realized using a batch method detailed in previous works ,grow tent indoor for which kinetic and adsorption capacities of hemp were determined. For kinetic experiments, a hemp disk was added to 50 mL of the solution 12 in a tightly closed glass flask and stirred on a thermostatic mechanical shaker operating at 250 rpm for various times, ranging from 5 to 240 min. The experiments were conducted at 25.1C without changing the initial pH of the solution. For adsorption experiments, the effect of difenoconazole concentration and of disk mass was tested. Thus, 3 masses of hemp disk, i.e. 0.35 g , 0.70 g and 1.75 g , were added to 50 mL of solution S1 and solution S12 and stirred on a thermostatic mechanical shaker operating at 250 rpm during 15 min. Our objective was to demonstrate that the percentage of difenoconazole removal increased with the hemp dose, even for a low contact time . The removal of difenoconazole was expressed in percentage of abatement, representing the ratio between the amount of adsorbed difenoconazole and its initial amount. Experiments were performed in triplicate. The repeatability has been validated . For the percolation procedure, the system illustrated in Scheme 3 has been realized thanks to a funnel put on a flask to percolate solution 12 through the hemp. This technique is similar to an open column method. First, 50 mL of solution 12 was poured in the funnel onto 2 masses of hemp disk . The flow-through was collected after 1, 10 or 15 passes on the same hemp disk. Secondly, the same experiments were performed in presence of soils. 50 mL of solution 12 was then poured on the 3 types of soils with or without hemp disk.The removal of difenoconazole was also expressed in percentage of abatement/removal. To correct any adsorption of difenoconazole on container and cotton, control experiments were also carried out in the same conditions. Experiments were performed in triplicate. The pH of all solutions was measured before and after experiments.
It was noted during the experiments a slight pH variation did occur at the end of each experiment, i.e. an increase of between 0.2 and 0.3.The analytical methodology for the liquid-liquid extraction and quantification of difenoconazole is based on a method recently developed by our group . Ten milliliters of the solution after adsorption were added to 20 mL of acetonitrile and 3 g of NaCl and stirred by a magnetic plate for 10 min. The supernatant organic layers were recovered using a separating funnel and adjusted to 20 mL with acetonitrile. Then sample extracts were analyzed on a system composed of gas chromatography apparatus and a triple quadrupole spectrometer . The GC-MS/MS optimized parameters of the triazole studied are: precursor ion 265, product ion 202 and 139, collision energy 36 and 40 V, retention time 34.366 and 34.485 min, and limit of detection 0.28 μg/L. Soils of the percolation procedure have also been extracted for analysis. Ten grams of soil were added to 5 mL of water, 20 mL of acetonitrile and 3 g of NaCl, stirred by a vortex for 3 min. The supernatant organiclayers were recovered using centrifugation at 3500 x g for 5 min and adjusted to 20 mL with acetonitrile. Sample extracts were then analyzed as previously described . The portion of difenoconazole retained by hemp has been deducted by subtracting the concentration found in the soil from the concentration found in the flow through.In order to optimize the design of an adsorption system to remove fungicide from solutions, it is important to establish the most appropriate contact time used in batch experiments. Fig. 1 shows the amount of difenoconazole adsorbed by a hemp-based material versus the contact time for concentration of difenoconazole of 12 mg/L . The amount of fungicide adsorbed increased with contact time until reaching a constant value where no more fungicide was removed from the solution. These kinetic results indicated that adsorption process was uniform with time and can be considered very fast because of the largest amount of difenoconazole adsorbed to the material within the first 60 min. The process could be divided in three regimes: the removal is increased instantly at initial stages, from 5 to 60 min, e.g. after only 5 min, 33.3% of fungicide was removed; then the removal keeps increasing gradually from 60 to 120 min, until the equilibrium is reached and remains constant.
For a contact time of 240 min, the removal was 93.5%, indicating strong interactions between difenoconazole and binding sites present in the main fiber constituents, i. e cellulose, hemicellulose and lignin. The remaining concentration of difenoconazole become asymptotic to the time axis after 90 min of shaking and the amount of difenoconazole showed no significant difference when the contact times were longer than this. Similar results were obtained with a concentration of 1.2 mg/L.Industrial hemp is a multipurpose crop, whose fibre has a wide range of industrial applications . The stem of C. sativa is used to extract natural fibres. The stem contains two types of fibres, known as bast and hurd , which differ in their biological, chemical, and physical properties . Bast fibres are crystalline cellulosic fibre bundles located in the phloem at the periphery of the C. sativa stem . They consist of primary bast fibres, which are generated from the procambium, and secondary bast fibres, which are generated from the vascular cambium . The woody core, which contains xylem vessels, makes up the inner hurd fibres and is rich with lignin . During development, C. sativa stems exhibit basipetal gradient of lignification. Compared to the younger parts, the older parts of the stems show both the primary and secondary bast fibers, and the xylem are more developed . Retting is a process used to extract bast fibres in the phloem tissues through dissolving certain cells and constituents surrounding the fibre bundles, such as hemicellulose, lignin, and pectin . The main retting methods are chemical, mechanical, enzymatic, field retting , and microbial approaches, and they influence the quality and quantity of the fibres . Other factors that also reportedly contribute to fibre quality and quantity are morpho-anatomical traits and the cellular biochemical composition of C. sativa stems . Changes in the C. sativa stem from vegetative to flowering stage affect fibre quality and quantity owing to significant chemical and structural changes . However, other variables, such as genotype, environment, management, and their interaction, also affect raw C. sativa fibre quality and yield . Quantity and quality parameters of C. sativa have been extensively studied. For instance, fibre yield was known to be largely influenced by agronomic practices, environment, and genotype . Similarly, biochemical quality of fibre bundles, their lengths, and stem processability vary between C. sativa genotypes .
Physical properties and mechanical behavior of C. sativa fibres such as tensile strength , compression , elastic modulus , thermal properties , grow tent hydroponic moisture retention , colour , crystallinity , surface properties , and bundle architecture have been widely evaluated. However, there are limited studies that examine the effect of the genotype on physico-mechanical properties. Therefore, further research on this area will identify suitable genotypes that form different end products. The C. sativa genotype has been shown to be an important factor in determining several quantity and quality parameters of fibres . However, C. sativa germplasm is complex. Genetic reticulation from prolonged domestication and haphazard breeding have made it hard to identify genotypes . Generally, the genotypes are classified according to a wide range of attributes: i) population types–for instance, wild, landraces, and cultivars; ii) gender as they are dioicous or monoecious; iii) stem colours as yellow and green; iv) flowering time, which include early, intermediate, and late flowering genotypes; v) application–fibre, seed, dual , phytochemical , and ornamentals . Dioecious C. sativa genotypes have shown higher fibre yields than monoecious C. sativa . Mechanical properties and the morphology of the fibres of C. sativa are also reportedly affected by gender . Another study has shown that the C. sativa genotypes with yellow stems exhibit greater mechanical processability than green-stemmed genotypes and contain more bast fibre yield . Further, late-flowering genotypes have shown a high fibre yield compared with early flowering genotypes . While dual-purpose or multipurpose genotypes have been produced in hemp-breeding programs , there is no clear split between fibre, seed, and phytochemical type male plants , and their potential for fibre production has not been investigated. Therefore, more studies are needed to unveil the effect of genotypes on fibre production, linking stem anatomy and morphology with fibre properties. Moreover, the identification of C. sativa genotypes belonging to non-fibre categories, which potentially produce quality fibres, is important for the fibre industry. The objectives of the present study were to; 1) identify the morphoanatomical differences of the stems of C. sativa genotypes, 2) compare the physico-mechanical fibre properties of different C. sativa genotypes, 3) understand the effect of the stem colour of genotypes on biological and/or mechanical properties of hemp fibres, and 4) test the fibre quality of non-fibre type C. sativa genotypes. To achieve the objectives, we evaluated the physical properties of hurd and bast fibres from various C. sativa genotypes extracted using chemical, microbial, and enzymatic retting methods and tested the stem morpho-anatomical differences between the C. sativa genotypes.To score the morphological characters of stems, accessions were randomly chosen. A total of 10 fibre-relevant discrete and continuous characters were scored for each genotype. Morphological characters, character-state definitions, and methods of scoring are presented in Mendeley Data: Table S1. Heights of live plants were measured using a measuring tape and the number of grooves on the stem, branch number, and leaf number were counted after uprooting at the full flowering stage . Stem cross-sections were measured using a digital reticule of the Olympus DP73 stereo microscope at 63 on fresh stem sections. The stem curvature and inflorescence position of the herbarium specimens and colours of the freshly peeled stems were visually inspected. The internodal lengths of the digitised herbarium specimens were measured using ImageJ version 2 . Only continuous variables were used for statistical analyses.