This has implications for the development of multi—purpose cropping systems of hemp.While low quality fibre can be extracted from plants gro̶wn primarily for their seeds or chemicals derived from inflorescences,the extraction of high-quality textile fibres requires harvesting when no seeds or inflorescences are present yet.Fibres from socalled multi—purpose hemp grown for seed and fibres will thus always contain a mixture of short and long fibres.Plants of increasing height and weight in Fig.6 could represent the same plant at different moments in time,but they can also be considered different plants in a crop at the same time,because with respect to primary and secondary fibre development,individual plant size is the key factor,not the moment a particular height or weight is reached,or the phenological stage.The stem part that is valuable for textiles,the stem part between the dotted lines in Fig.6,is located higher up along the stem with increasing plant size.It is supported by a stem base of increasing length and weight,which is unfit for yarn production due to the presence of secondary fibres.In short plants a share of the primary fibres,though fit for yarn spinning,is lost in the stubble,which in practice is 15–20 cm.Once the secondary fibre front surpasses the stubble height part of the primary fibres are contaminated by secondary fibres.The lower in the plant or the heavier the part of the stem above,the thicker this unwanted fibre layer is likely to be.For reasons of simplification,the inflorescences of the plants in Fig.6 have equal lengths for all plants,it could however be expected that this part becomes gradually longer with increasing plant size.However,vertical grow system no data were recorded.
Mediavilla et al. showed in a dioecious variety that the accelerated development of secondary fibres around flowering first takes place in female plants and later in male plants.This makes sense,when the increasing weight is considered the cause of secondary fibre formation.Male plants on average are longer,but the female inflorescences,where the seeds are gradually filled,are likely to have a higher weight.Bócsa and Karus also stated that female plants have a relatively higher secondary fibre content than male plants.The proposed conceptual model also implies that homogeneity with respect to plant size is important when growing hemp for textile uses.In stands with plants of different heights the valuable ‘middle section’ is at variable height,which would cause difficulties in harvesting and processing.The tops of the plants are the comparable parts,not the bottom parts.According to both Fig.4 and 5,secondary fibres above stubble height in Futura 75 can be avoided when the crop is cut when plants are around 1.3–1.4 m and not yet flowering.It is likely,however,that this height is different for varieties,as secondary fibre development is reported to be different between varieties.Reported differences,however,could partially be due to size differences as well.For this reason true varietal differences and differences caused by size differences should be disentangled in future experiments.A non–flowering crop with a height of 1.3–1.4 m can be grown in about two months when hemp is sown at a normal sowing date in April under Dutch growing conditions.However,a subsequent second fibre hemp crop,to compensate for the relatively low stem dry matter yield of the short crop,will be a challenge.For a first sown crop,varieties can be chosen based on excellent fibre qualities,for a possible second crop the variety should be late flowering and it should also have an inherently low secondary fibre content.Further the soil has to be prepared into a homogeneous seedbed again and in dry years irrigation might be necessary to achieve even emergence.Due to the drier summers and shorter days a second crop seems less realistic in Italy than in The Netherlands.However,even when technically possible,it should be economically sound as well,which is doubtful as yet.An advantage of a short crop is that the stems could be processed on existing flax processing lines as the limited market for high‒quality hemp yarns as yet does not justify the development of specialised hemp scutching and hackling lines.Such systems are dimensioned for flax ribbons with a length usually between 80 and 120 cm,which is about the length of the useful middle section of the stems in our experiment.Earlier attempts in Italy to produce smaller hemp plants by stopping their growth with glyphosate at the desired plant height of 1.2 m failed due to the environmentally unfriendly production methodology,low yields,low quality,and high costs.Westerhuis et al.,however,showed that normal amounts of scutched long fibres can be extracted from smaller plants.However,these fibres were not hackled.It is likely that the early harvested short crop that we need to avoid secondary fibre formation has relatively fine primary fibres as well.
Fibres have been reported to be finer with decreasing plant size or conditions that in general cause smaller plants,e.g.,earlier harvest or increasing sowing density.Also with respect to the unwanted lignification of the fibres an early harvest before flowering seems best.For future research on this topic it has to be considered that between individual plants of the same height or weight relatively large differences exist in the height of the secondary fibre front.This was not only caused by the fact we measured once every 10 cm stem length; in the field experiment where three cuts were made in every internode a comparable spread was observed.Differences between plants and,e.g.,wind would co-determine the actual forces along the stem and thus the mechano perception inducing fibre formation.Further,we should keep in mind that countering the forces the plant is subjected to,means countering the fresh weight,not the dry weight we measured hence plant fresh weight should be measured as well.To study the effect of weight on secondary fibre formation in more detail,plant weight could be increased artificially.Cannabis sativa L. has been used for more than 6000 years as a source of food, fiber, oil and medicine, as well as for recreational or religious purposes . Cannabis has a very complex chemical composition, with around 540 reported specialized metabolites, such as cannabinoids, terpenoids, flavonoids and alkaloids. The most active of these are by far the cannabinoids, a class represented by more than 100 known terpenophenolic compounds that accumulate mainly in the resin secreted from the trichomes of female plants . Based on its use, cannabis can be technically divided into two distinct groups: marijuana and hemp. Traditionally, marijuana is almost exclusively grown in greenhouses or other controlled environment facilities, being primarily bred for its main psychoactive cannabinoid, Δ9 -tetrahydrocannabinol . On the other hand, hemp serves more as an agricultural commodity, being valued for its fibers and seeds and, more recently, for its nonintoxicating medicinal compounds, notably cannabidiol . From a legal point of view, in most European and North American countries, cannabis is classified as hemp if the crop contains less than 0.2−0.3% THC . Due to their low THC content, hemp and CBD products have recently gained an increased popularity, as their attributed medical benefits are achieved without the “high” effects of marijuana . Consequently, a wide panel of products are nowadays marketed as “full-spectrum” formulas, dietary supplements or CBD-enriched products . The presence of pesticides in hemp crops is a very challenging issue nowadays, not only for cultivators, but also for regulators, consumers or public health researchers .
Many studies have revealed there is no clear relationship between pesticide use and augmentation of cannabis yields, being repeatedly claimed that their role in weed and pest control might be actually unnecessary . Beside their concealed use during cannabis growth, other reasons for pesticide contamination might be related to the spray drift from adjacent crops or assimilation from the contaminated soil; numerous pesticides are environmentally mobile, being carried away by groundwater and rain, whilst cannabis is recognized as a robust and fast growing plant able to absorb pollutants with a great efficiency . Thence, pesticide contamination of cannabis materials has been repeatedly brought into attention . A survey of 389 cannabis products from the state of Oregon found 24 residual pesticides, with piperonyl butoxide as the most commonly retrieved contaminant. Furthermore, 12 pesticides were found in up to 50 % of the cannabis samples collected from central Californian dispensaries . Out of 26 investigated cannabis samples, 84.6 % were confirmed to be positive for pesticides from various classes, such as insecticides, miticides, fungicides or growth regulators . In addition, a multi-screening study testing for 71 residual pesticides in various cannabis products from Italy revealed that amitraz, chlorpyrifos and trifluralin were above the acceptable residual limits set at 0.010 mg/kg . Nevertheless, occupational or non-occupational exposure to residual pesticides has become an important issue due to potential adverse health effects. For example, organophosphate pesticides, mobile grow systems such as chlorpyrifos, malathion, parathion methyl, ethoprophos or coumaphos, have been found to be highly toxic, inducing neurobehavioral and cognitive disorders, teratogenicity, immunotoxicity or endocrinal and metabolic disturbances . Daminozide and paclobutrazol are two plant growth regulators that have been banned in the USA and numerous European countries, due to their carcinogenetic properties; however, both have been found as contaminants in cannabis products . In this light, more and more regulatory agencies are addressing the residual pesticides issue in cannabis products. For example, in the USA, pesticide regulations are specific to each state, mostly due to the fact that cannabis cultivation is still not yet federally legal.
Among the states, California has the most severe requirements, monitoring 66 pesticides, followed by Oregon with 59 pesticides. In comparison to these, Canada has set wider and stricter controls of pesticides in cannabis products, demanding testing for 96 pesticides, with limits of quantification usually lower than those set in the USA . From a processing point of view, cannabis derived extracts are frequently obtained using CO2-supercritical fluid extraction followed by winterization , decarboxylation , distillation and, optionally, a chromatographic-based method for the isolation of specific cannabinoid compounds . For instance, CBD purification is mostly achieved by recrystallization, conventional chromatography with solid stationary phase or liquid-liquid chromatography. LLC, better known as centrifugal partition chromatography and counter current chromatography , is a preparative separation technique where the two phases of a biphasic solvent system are used as the mobile and stationary phase. One of the two phases is kept stationary inside the column with the help of a centrifugal field, while the other one is pumped through the column . In this sense, LLC enables high column loading, while expensive solid stationary phases and time intensive column packing procedures are not required. In addition, the almost limitless variety of possible biphasic solvent systems and easy preparation of tailor-made solvent systems make LLC a very flexible, scalable, as well as selective separation technique . Taking advantage of these benefits, Hazekamp et al. showed that CBD can be separated by CPC using n-hexane/acetone/acetonitrile 5/2/3 as a biphasic solvent system, with the acetonitrile-rich phase as the stationary phase . To this end, a considerable attention has been paid either on the large-scale purification of CBD by LLC or the ultra-trace detection of residual pesticides in cannabis products, but not to the removal of the contaminating pesticides. Therefore, the aim of this study was to establish a systematic computer-aided approach for the selection of biphasic solvent systems for the simultaneous purification of CBD from hemp extracts and removal of specific-contaminating pesticides. In this sense, a predictive thermodynamic model was used for solvent system screening, guiding the user toward the design of tailor-made solvent systems for the isolation of pesticide-free CBD from a particular hemp extract using LLC. Two experimental protocols were followed in the shake-flask experiments, depending on the purpose. For the initial screening of the partition coefficient of CBD in different solvent systems, 5 mg of hemp extract were added to upper and lower phases of preequilibrated solvent systems and placed into a 20 mL vial. Then, each phase was analyzed by HPLC-DAD . Once the three solvent systems were selected, the six pesticide mixes and CBD standards were combined together and a stock solution, in which CBD and each pesticide had a concentration of 50 ppm, was obtained by a proper dilution in acetonitrile. 20 μL of the stock solution were added to 20 mL of each of the three selected solvent systems, affording a nominal concentration of 50 ppb of each solute. Afterwards, 1 mL of upper phase and 1 mL of lower phase were taken, evaporated to dryness, re-dissolved in 1 mL methanol and analyzed by LC–MS/MS to determine the concentration of each analyte in the phases.