A handful of previous studies have suggested that subjective experiences for different drugs may share a common etiology

Tashkin et al measured FEV1 serially in 255 habitual male and female smokers of marijuana and/or tobacco and nonsmokers of either substance on up to seven occasions at intervals of $ 1 year over a total period of up to 8 years. Random effects models were used in men and women separately to estimate mean rates of decline in FEV1 and to compare these rates between smokers of marijuana or tobacco alone, smokers of both substances, and nonsmokers of either substance. Smoking marijuana was not found to be associated with greater declines in FEV1 than nonsmoking nor was a relationship seen between the daily number of marijuana joints smoked and rate of decline in FEV1. In contrast, smoking tobacco had a significant effect on FEV1 decline in men in a dose-responsive manner. Findings are illustrated in Figure 3. Hancox et al performed serial lung function measurements in 779 members of a birth cohort in Dunedin, New Zealand, at 18, 21, 26, and 32 years of age. They assessed changes in FEV1, FVC, and FEV1/FVC associated with tobacco and marijuana smoking using regression analyses and estimates of both joint-years and pack-years as predictors, adjusting for sex and height at 32 years of age, changes in height between 15 and 32 years of age, and concomitant tobacco or marijuana use. The results are shown in Table 3. Among marijuana smokers, FVC increased significantly over time, whereasFEV1 showed a non significant increase and FEV1/FVC exhibited a non significant decline. In contrast, among tobacco smokers, FEV1 and FEV1/FVC both showed significant declines, whereas FVC showed no change.The smoke contents of marijuana cigarettes contain the same procarcinogenic components, including the very potent human carcinogen, benzpyrene, found in tobacco smoke.Bronchial biopsies obtained from heavy, habitual marijuana smokers have shown the same widespread histopathologic epithelial abnormalities noted in tobacco smokers, including squamous metaplasia and cellular disorganization,that are widely thought to be precancerous.

Immunohistologic examination of bronchial biopsies from marijuana smokers has shown higher levels of the protein products of oncogenes, including Ki67 and EGFR,whereas laryngeal cancer specimens from marijuana smokers have been shown to exhibit increased expression of oncoproteins compared both with tobacco smokers and nonsmokers.A few older epidemiologic studies from North Africa have shown a positive association with lung cancer; however,greenhouse rolling racks the common practice of admixing marijuana and tobacco within the same cigarette in these societies precludes disentangling the effects of marijuana from those of tobacco. A population-based cohort study of men 18 to 20 years of age conscripted into the Swedish military in 1969 to 1970 tracked these men until 2009 for incident lung cancer cases using linked nationwide medical registries.Cox regression was used to assess the relationship between lifetime use of marijuana self-reported at the time of conscription and risk of subsequent lung cancer diagnosis over the ensuring 40 years, adjusting for tobacco use also up to only 18 to 20 years of age.The hazard ratio for lung cancer was significantly positive in relation to lifetime marijuana use of > 50 times. However, because neither marijuana nor tobacco use was determined after the baseline assessment, the authors were unable to adjust for true lifetime use of tobacco, a crucially important residual confounder.Several investigators have demonstrated a tumor suppressive effect of THC and other cannabinoids on a variety of malignancies, including lung, in both cell culture systems and animal models, as previously reviewed by Bifulco et al and Velasco et al.These findings appear to reflect antiproliferative, proapoptotic, and antiangiogenic properties of THC that might counteract the tumor-initiating or tumorpromoting effects of the carcinogens contained with the smoke of marijuana. A large cohort study of health plan participants in Northern California failed to show an increased risk of tobacco-related cancers in association with self-reported marijuana use.A pooled analysis of six well-designed case-control studies of the association between habitual marijuana smoking and lung cancer that totaled 2,159 cases and 2,985 control subjects did not find any increased risk of lung cancer in association with marijuana use.Only one of the six studies included in the pooled analysis showed a significantly positive association between lung cancer and marijuana use, mainly in the heaviest marijuana use tertile, but the latter tertile included only four matched control subjects, making the estimates of risk imprecise.

Marijuana smoking might predispose to lower respiratory tract infection in at least three ways. First, the destruction of ciliated epithelium in the large central airways and the associated hyperplasia of mucus-secreting surface epithelial cells demonstrated in bronchial biopsies of habitual marijuana smokers may lead to increased production of mucus in the face of a diminished capacity to cleanse the lung of the excess mucus because of ciliary loss, thereby providing a substrate for potentially pathogenic microbial organisms colonizing the lower respiratory tract.Second, the immunosuppressive effect of THC leading to impairment of the bactericidal and fungicidal activity of AMs,further compromises the lung’s defense against microbial infection. Finally, marijuana has been shown to be frequently contaminated with Aspergillus fumigatus and potentially pathogenic gram-negative bacteria.Therefore, introduction of these microorganisms into the lung in the face of marijuana-related impairment in the lung’s host defense provides another possible mechanism for increasing the risk of pneumonia. The possible association of marijuana with an increased risk of pneumonia is supported by older case reports of Aspergillus pneumonia in smokers of marijuana immunocompromised by AIDS,chronic granulomatous disease,bone marrow transplantation,renal transplantation, or lung cancer treated with chemotherapy.In addition, a cluster of five patients with cavitary TB who used a marijuana water pipe was reported in Australia in 2003, followed by another report from Australia 10 years later of three additional cases of open cavitary TB in marijuana bong users.In these cases, it is not clear whether the spread of TB infection was because of close contact with patients with cavitary TB who might have shared a water pipe to smoke marijuana and/or to marijuana-related impairment in the lung’s defense against infection. Although a few older epidemiologic studies have suggested that marijuana use might be a significant independent risk factor for opportunistic infection in individuals who are HIV positive,an early analysis of data from the Multi-center AIDS Cohort Study failed to find evidence that marijuana was a risk factor for progression of individuals who are HIV positive to full-blown AIDS.Further preliminary analysis of the possible association of marijuana use with pneumonia risk using the Multi-center AIDS Cohort Study data set updated to 2013 has not shown a significant association of marijuana use with increased risk of either community-acquired or opportunistic pneumonia in either the HIV-negative or HIV-positive members of the cohort, after adjustment for tobacco, age, and among the individuals who are HIV positive, cluster of differentiation 4 cell counts and viral load.

A long-standing observation in clinical and epidemiological research into substance use has been that users of one drug typically do not limit their use to a single substance. For example, alcohol and tobacco are commonly used by the same person and often in the same setting, as are tobacco and marijuana. Though the synergistic effects of these particular drugs have been suggested as a potential explanation , another interesting possibility is that individuals have an underlying liability to drug use within and across different pharmacological classes. Support for this notion has been shown for both licit and illicit drugs in a variety of populations and drug use phenotypes. As poly-substance use is associated with problematic use and reduced treatment efficacy , identifying informative precursors to the onset of abuse and dependence remains a priority. Among the variety of factors that have been examined as an early indicator of later, more problematic use patterns, how someone experiences a drug , is one of the most interesting. Subjective experiences are thought to reflect individual differences in the pharmacological effects of a drug. Factor analytic studies of these experiences frequently yield two main factors: pleasant or positive and unpleasant or negative. Positive subjective experiences often include euphoria, relaxation, and feeling less inhibition. Negative subjective experiences include nausea,vertical grow difficulty inhaling, dizziness and sadness. Though weakly correlated , users of a drug sometimes report both positive and negative experiences. Alcohol, tobacco, and marijuana are the most commonly used licit and illicit drugs. Studies examining the subjective experiences to these drugs have generally found that how a person responds to a particular drug is predictive of more problematic use of the same drug. For example, dependent cigarette users more frequently endorse positive experiences than regular smokers and moderate-to-heavy drinkers report experiencing greater stimulant-like effects to alcohol than lighter drinkers. A similar relationship has been demonstrated for marijuana use. Although results are mixed, negative experiences to tobacco and marijuana have also been positively associated with problematic use. For alcohol consumption, low levels of response, primarily measured using negative subjective effects, have been associated with an increased risk of an alcohol use disorder as lower thresholds to the sedative effects of alcohol protected against developing abuse later in life.In particular, subjective experiences to a variety of drugs are correlated and can predict levels of involvement for substances in other pharmacological classes. This observation has been shown for pleasurable experiences of alcohol and tobacco where both drugs were predictive of current alcohol use in a college aged sample. Further, marijuana use has been shown to increase a sense of “liking” among non-smokers whereas alcohol has no effect on the subjective experiences of cigarettes. Lastly, greater rates of alcohol dependence and illicit drug use have been observed among high marijuana users as defined by greater rates of sensitivity to positive and negative subjective experiences. Though additional studies are needed, these cross-drug results indicate that how a person responds to a drug is predictive of how they will respond to other drugs. In this report we detail findings from a study of subjective experiences to alcohol, tobacco, and marijuana in a sample of young adults participating in the Colorado Center for Antisocial Drug Dependence. Subjective experiences were collected from both clinical and community participants using a questionnaire developed by Lyons et al.. Our analyses were designed to address three questions. First, how do positive and negative subjective experiences across alcohol, tobacco, and marijuana compare? Second, to what extent do subjective experiences to alcohol,tobacco, and marijuana overlap? Lastly, to what degree do subjective experiences to one drug associate with more problematic use behaviors for a different drug?Participants were drawn from the Colorado Center on Antisocial Drug Dependence [CADD] and consisted of 3853 participants between the ages of 11 and 30 years old and included both community and clinical participants. Our community-based sample was drawn from those participating in the Colorado Twin Registry , Colorado Adoption Project , with clinical controls drawn from the Colorado Adolescent Substance Abuse Family Study. Our clinical sample was drawn from adolescents in treatment for substance abuse and delinquency as a part of the ASA study. Additional clinical participants were drawn from an adjudicated sample from the Denver metropolitan area. Siblings of the clinical subjects were also included. All participants in the current study met one or more of the following criteria: they had consumed at least six drinks in their lifetime, had used tobacco daily for at least one month, or had used marijuana six or more time in their lifetime.Patterns of alcohol, tobacco and marijuana use, abuse and dependence symptomatology were collected using the Composite International Diagnostic Interview-Substance Abuse Module. Abuse and dependence status as defined by the Diagnostic and Statistical Manual of Mental Disorders was determined using scoring algorithms based on whole life substance related problems. Retrospective subjective experiences were collected using a 23- item questionnaire developed by Lyons et al.. The original Lyons questionnaire was comprised of 23 items. As discussed in Zeiger et al. , due to the CADD interview length the original Lyons questionnaire was shortened after wave 1; a factor analysis was conducted on the Lyons questionnaire and 10 items with lower or mixed loadings were dropped. Subsequently, most subjects received the shortened 13-item questionnaire, thus these analyses were conducted on the 13-item response set from all subjects.

Functional annotation clustering in DAVID was used to minimize redundancy in the GO terms

As an extension of our previous work, the objective of the present study is to employ a toxicogenomics approach to compare and contrast the molecular pathways that are perturbed by MSC and TSC. However, subtle differences in gene expression provide insight into mechanisms underlying the observed differences in toxicities.A reference design with arrays as blocks of size 2 was used to analyze the median signal intensities of the two-color micro-array data. Five biological replicates per condition were used for each of the eight conditions, for a total of 80 micro-arrays. Six MSC and four TSC “outlier” micro-arrays were removed based on quality control checks , leaving a minimum of 3 replicates per group. The background signal intensity for each array was estimated using the 1533xSLv1 negative controls present on each array. All pre-processing of the data was conducted using R. The data were normalized using the LOWESS normalization method in the R library “MAANOVA”. Differential expression between the control and exposed samples for each of the three dose levels at each of the two time points was tested using the MAANOVA library. The ANOVA model was fitted to include the main effects of dose and time, with a dose by time interaction term and the array as a blocking variable. The Fs statistic , a shrinkage estimator, was used for the gene-specific variance components, and the associated p-values for all the statistical tests were estimated using the permutation method. These p-values were then adjusted for multiple comparisons using the false discovery rate approach. The least squares mean , a function of the model parameters, was used to estimate the fold change for each pairwise comparison of the six pairwise comparisons of interest among the eight treatment-by-time groups. The micro-array data for this experiment has been submitted to the Gene Expression Omnibus repository and can be accessed under record number GSE44603. Visualization and analysis of significantly changing genes was performed using Gene Spring GX 7.3. Important pathways containing significantly expressed genes were identified using Ingenuity Pathway Analysis. 

Genes were assigned to functional categories using gene ontology in the Database for Annotation, Visualization and Integrated Discovery. Analyses were performed on genes that were identified as statistically significant by one-way ANOVA using four models: Hill, Power, Linear and 2◦ Polynomial. Models that described the data with the least complexity were selected. A nested chi-square test,vertical rack with cutoff of 0.05, was first used to select among the linear and 2◦ polynomial model, followed by comparison of Akaike information criterion , which measured the relative goodness of fit of a statistical model, between nested models and the power model. The model with the lowest AIC was selected as the best fit. A maximum of 250 iterations and a confidence level of 0.95 were selected. For functional classifications and analyses, the resulting BMD datasets were mapped to KEGG pathways with promiscuous probes removed. BMDs that exceeded the highest exposure dose were removed from the analysis.Three RT-PCR pathway specific arrays were used to validate the expression of specific micro-array genes. Eight nanograms of total RNA, from the same samples that were used for the micro-array study, were reverse transcribed to cDNA using an RT2 First Strand Kit. cDNA was mixed with the RT2 qPCR Master Mixes and aliquoted into 96-well plates containing primers for 84 pathway specific genes. Expression levels were evaluated using a CFX96 real-time Detection System. Relative gene expression was normalized to the Gapdh housekeeping gene, which remained unaffected under experimental conditions. Fold changes and statistical significance were calculated using the REST method for statistical significance.For the micro-array study, FE1 cells were exposed to 2.5, 5 and10 g/ml of MSC and 25, 50 and 90 g/ml of TSC. Exposed samples were compared to their matched controls, and genes were considered significantly differentially expressed if they had a fold change ≥2 with an FDR-adjusted p-value ≤0.05. A total of 1020 unique probe identifiers were significantly differentially expressed following exposure to MSC, and of these, 979 were deemed “present”. 

Following exposure to TSC, 557 probes were significantly differentially expressed and 527 were deemed “present”. Of these, 356 were common to both MSC and TSC exposures. The number of significantly up- and down-regulated genes at each time point and concentration is shown in Table 1. Overall, there was an increase in the number of differentially expressed genes with increasing concentration of condensate, and there were more genes changing after the four hour recovery. At the highest concentration for both time points, cells exposed to MSC had a greater number of changing genes as compared to cells exposed to TSC. Gene expression was most altered for cells exposed to the highest concentration of MSC at the 6 + 4 h time point. Whether separated by dose or considered all together , Venn diagrams show considerable overlap in the genes that are significantly expressed at each time point following MSC or TSC exposure. Hierarchal clustering using all genes that were statistically significant revealed that the controls and the marijuana high concentration clustered independently from the rest of the samples. The remaining samples clustered first by concentration ,then by condensate type , with the last branching resulting from time. When cells exposed to TSC and MSC were analyzed separately, samples clustered first by concentration and then by time point, suggesting that concentration has the largest overall effect on gene expression. For MSC, the high concentration samples were on the first main branch, followed by control, low and medium concentrations. The results indicate that the expression profiles of the high concentration MSC exposed cells are quite distinct. For TSC, the controls branched separately from all the treatment groups. The top 10 genes with the largest overall fold changes are listed in Table 2. All of the top 10 genes were significantly up-regulated with the exception of low density lipoprotein receptor , which was down-regulated in MSC exposed cells. Of the top 10 changing genes, five genes were common to both MSC and TSC. The GO terms associated with these commongenes included multicellular organismal development, vasculogenesis, regulation of transcription, and regulation of inflammatory response. Ingenuity Pathway Analysis was used to define the pathways that were significantly altered following exposure to MSC or TSC. Fig. 3 shows the overlap in all the significant pathways between the two condensate types.

The top five most significantly altered pathways for cells treated with MSC or TSC are listed in Table 3. NRF2-Mediated Oxidative Stress Response was the most significant pathway for cells exposed to TSC at all concentrations and time points, with the exception of lowest concentration at time 6 + 4 h where LXR/RXR Activation was the most significant. For cells exposed to MSC, the most significantly altered pathways were Biosynthesis of Steroids, as well as NRF2-Mediated Oxidative Stress Response, Aminoacyl-tRNA Biosynthesis and HMGB1 Signaling. Some ofthe top five pathways were common to both the MSC and TSC including those related to oxidative stress and xenobiotic metabolism. However, inflammation pathways were more predominant for the MSC, whereas cell cycling and cancer signaling pathways were more predominant for the TSC. To further elucidate differences between the two smoke condensates, the genes that were uniquely expressed following TSC exposure or uniquely expressed following MSC exposure at the highest concentrations for the two separate time points were compared in IPA. The findings confirm the importance of inflammation and steroid biosynthesis pathways in MSC exposed cells and highlight the significance of apoptotic pathways particularly at the 6 h time point. For cells exposed to TSC,Mphase cell cycle pathways appear to be of particular importance. Gene Ontology in the Database for Visualization, Annotation and Integrated Discovery was used to apply functional annotation to all the significantly differentially expressed genes for each condensate. The full results are shown in Supplementary Tables 1 and 2. For cells exposed to MSC, significant perturbations were associated with steroid/cholesterol/lipid biosynthesis, NOD like receptor signaling , tRNA aminoacylation, transcription regulation, unfolded protein response and DNA binding. Like MSC, cells exposed to TSC had significant perturbations in transcription regulation, unfolded protein response and DNA binding. In addition, perturbations in cell cycle, p53 signaling, oxidative stress, and cancer signaling were also noted in TSC exposed cells. Fig. 5 shows the overlap of all the significantly affected ontologies between the two condensate types.This analysis revealed 19 clusters with enrichment scores greater than 2 for MSC and 19 clusters for TSC. The top clusters for MSC relevant to toxicological processes include lipid/steroid biosynthesis , RNA processing ,indoor hydroponic system cellular response to unfolded protein , tRNA aminoacylation , and positive regulation of transcription. 

The top clusters for TSC relevant to toxicological processes include cellular response to unfolded protein , cell cycle , positive regulation of transcription , response to steroid hormone stimulus , and positive/negative regulation of apoptosis and cell death. To investigate early versus downstream effects, functional annotation was applied to significantly differentially expressed genes at the two separate time points. The results are shown in Supplementary Tables 5–8. For cells exposed to MSC at the 6 h time point, the analyses revealed 79 significant terms including those related to transcription activity, DNA binding, and steroid/cholesterol biosynthesis. Four KEGG pathways and 1 Biocarta pathway were also deemed significant at this time point. At the 6 + 4 h time point, 76 significant terms were identified. These terms included unfolded protein response, and tRNA aminoacylation, as well as steroid/cholesterol biosynthesis which was found at the 6 h time point. Three KEGG pathways were significant at this time point including Steroid Biosynthesis, Terpenoid Backbone Biosynthesis, and Aminoacyl-tRNA Biosynthesis. Analyses of cells exposed to TSC at the 6 hr time point revealed 67 significant terms including those associated with oxidative stress, cell death, protein unfolding, transcription regulation, DNA binding and cell cycle. In addition, 2 KEGG pathways were significant. At the 6 + 4 h time point, 32 GO terms were identified as significant with oxidative stress being the only relevant toxicological endpoint. In addition, only one KEGG pathway was significant. Overall for MSC, the DAVID analyses confirmed many of the significant pathways identified by IPA including steroid biosynthesis, tRNA aminoacylation, inflammation and apoptosis. In addition, the analyses highlighted transcription regulation, DNA binding and unfolded protein response as also significant. For TSC, the DAVID analyses confirmed the significance of IPA pathways related to oxidative stress and cell cycle. As with the MSC, the DAVID analyses also further highlighted the importance of transcription regulation, DNA binding and unfolded protein response, as well as cell death. Transcription regulation and DNA binding were significant terms common to both MSC and TSC at the 6 h time point, whereas no common terms existed for the two condensates at the 6 + 4 h time point.In our previous genotoxicity study we showed that MSC and TSC were both cytotoxic and genotoxic. However, quantitatively, MSC was more cytotoxic and mutagenic than TSC, and TSC appeared to induce chromosomal damage in a concentration-dependent manner whereas MSC did not. Our earlier chemical analyses of MSC and TSC noted that aside from the nicotine in tobacco and the cannabinoids in marijuana, the two smoke condensates contained mixtures of chemicals that were qualitatively similar though quantitatively different. The similarities in the chemical profiles and some of the toxicity findings suggested that the two smoke condensates might elicit somewhat comparable gene expression profiles. Hierarchal clustering of all the MSC and TSC exposed samples in the present study supported this notion and samples clustered first by concentration as opposed to smoke type. In addition, analysis of the top ten greatest gene expression changes relative to control revealed that half of the genes were common to both marijuana and tobacco. A number of previous studies have examined gene expression changes in pulmonary cells following exposure to tobacco smoke. Generally, these studies have shown thattobacco smoke stimulates xenobiotic metabolism, and that metabolized smoke constituents contribute to DNA damage. Following early insult, DNA damage leads to disruptions in the cell cycle such as arrest at the G2 checkpoint to allow time for response. Cellular response can include DNA repair, mutation induction through faulty repair or lack of repair, and programmed cell death of heavily damaged cells.

Hemp oil contains high contents of unsaturated fatty acids

They promote extraction by breaking down the cell walls of the structures containing the oils and hence can also lead to the extraction of essential nutraceutical and antioxidant compounds with lesser degenerative effects on the cells.Ultrasound assisted extraction of hemp seed oils was performed by Lin and his group as well as and Esmaeilzadeh Kenari and associates on hemp seeds and increased extraction yields were reported as compared to traditional extraction methods.A slightly modified ultrasound extraction method was also proposed in which solvent extraction using a Soxhlet apparatus was supplemented using ultrasound applied using an ultrasound probe.This coupled effect of conventional Soxhlet extraction along with ultrasound extraction led to an increased oil yield.Rezvankhah and his team studied the effects of Microwave assisted extraction on hemp seed oils and reported that the method was more time efficient as compared to traditional Soxhlet extraction.The oils produced were also of higher quality.Similarly,it was also reported that Microwave assisted extraction of oils from pongamia pinnata seeds was quicker and had no adverse effect on the quality of oils as compared to other conventional methods.Previously,hemp did not receive significant attention because of legal issues concerned with hemp cultivation.According to the 2018 reports by Health Canada,77,800 acres of industrial hemp were planted in Canada out of which 33,000 acres were planted in Alberta,27,100 acres in Saskatchewan,and 11,500 acres in Manitoba.It is estimated that roughly 90% of the total seed for hemp cultivation is produced in Canada which contains mostly Canadian developed varieties.Thus,it is evident that with increasing awareness there is a significant increase in hemp cultivation as well as the number of hemp processing plants leading to more research focusing on hemp seeds,grow rack especially oils extracted from hemp hearts.

Although there are previous research reports concerning the extraction of hemp oils,the literature is scanty and the scientific community lacks a thorough comparison between the effect of different extraction techniques on the quality of oils produced from hemp seeds.Previous research has mostly focused on the extraction and analysis of hemp seed oil using a single technique or a comparison between two techniques.However,a comprehensive comparative analysis of different extraction techniques for hemp heart oils operating at different temperature ranges have not been performed previously.Thus,the major objectives of the study were to Compare the extraction efficiencies of three different extraction techniques-microwave,ultrasound and cold pressing for the extraction of hemp seed oils by varying particle size and extraction conditions to obtain maximum yield.Analyze the physicochemical properties,fatty acid composition,free radical scavenging activity,and inherent chemical composition of the extracted oils to determine the quality and,understand the nature of the oils in order to suggest probable areas of application.These results indicated that the ultrasound is more effective in the first 30 min and with an increase in time,a decreasing trend in the yield was noticed.This may be attributed to the fact that due to increased time periods; ultrasound waves can disrupt more hemp heart cell walls leading to increased penetration of the solvent inside the cells resulting in more extraction.Again,the slight decrease in the extraction yield can be hypothesized to have been caused by prolonged exposure time as a result of which ultrasound oxidation of the edible oils take place either by thermal degradation or sonolysis,which can be attributed to the phenomenon of “cavitation”.Cavitations are micro-mechanical shocks affecting the structural and functional components of the cells and such an increased phenomenon in the cells lead to lipid oxidation and deterioration.This phenomenon may have resulted in a slight decrease in the oil yield upon increasing the ultrasound time.Further,an increase in the rate of cavitation leads to excessive breakdown of the cell membranes and cell walls,thus exposing the hydrophobic fatty acid tail of the phospholipids.

This might cause binding of the oil to the phospholipid and lower its extractability by the solvent.Therefore,excessive ultrasound can have a delirious effect on extraction.Again,the ultrasound time was optimized at 30 min and 60 min respectively to understand the effect of UAE power on the yield.Ultrasound powers of 20 W,130 W,and 200 W were used for UAE time of 60 min which is represented in Figure S3.On the other hand,130 W and 200 W were used for 30 min.The yields obtained by varying the ultrasound power at 60 min was not found to be significantly different.With an increase in power,the yield gradually increased and the maximum yields of 46.8 ± 3.25% and 44.9 ± 3.11% were produced at 200 W for 30 min and 60 min respectively.It can be hypothesized that when the ultrasound power is low,the energy produced by the ultrasound probe is not enough to cause the propagation of ultrasound pressure waves to cause cavitation.The results obtained are on par with previous research led by Lin et al.,who also reported that the maximum extraction yield is achieved for 200 W.The slight decrease in the yield% for 130 W and 60 min run time may be attributed to sonolysis and the cavitation phenomena.The maximum extraction yield was obtained for ultrasound power of 200 W and 30 min which was considered for further experiments.Means in the same column followed by different superscripts are significantly different.the yields except for the minimum and maximum values obtained.A comparative analysis of the extraction yields is presented in Figure S6.Results reported that there existed a significant difference between the yield obtained by CP and MW,but not between CP and UAE or UAE and MW.MW extraction produced the maximum oil yield of 54 ± 3.8% followed by 47 ± 1.97% for UAE,and 41 ± 2.82% for CP.This can be attributed to microwaves causing increased rupture of the cell walls resulting in lipolysis and proteolysis of the cell wall materials,and thus enhancing the penetration of the solvent inside the cells to extract the oil.Moreover,a temperature of 70 °C and MW exposure time of 15 min led to a movement in the media due to a reduction in solvent viscosity which enhanced the extraction yield.The mechanical effect of ultrasound promotes the release of soluble compounds from the plant material disrupting the cell walls,enhancing mass transfer,and facilitating the solvent to access the cellular contents.On the other hand,microwaves lead to an increased solvent penetration into the matrix due to the movement of dissolved ions and as a result,causes increased extraction of intracellular materials.It can further be hypothesized that ultrasound waves may have caused the lipoprotein cell membrane to break and retain some of the oils,thus reducing its extractability which led to a lower yield as compared to MW extraction.Cold pressing is a mechanical extraction technique and hence the yield is fairly less compared to the other two techniques.Overall,it can be concluded that the yield obtained by each extraction technique was more compared to the yields produced by hemp cultivars in Turkey and Pakistan.

The presence of antioxidants like tocopherols in higher concentrations in CP oils can be a reason for a higher induction time since we know that these antioxidants act as free radical scavengers and singlet oxygen quenchers.It is also known that factors like the processing and storage treatment as well as the fatty acid composition of the oil play a key role in determining the oxidative stability.Since cold pressing took place at room temperature ,it was highly likely that the oxidation reaction did not initiate during its storage leading to a higher induction time during the analysis.Electro paramagnetic resonance spectroscopy has found a wide variety of applications in detecting free radicals owing to its high sensitivity to the unpaired electrons.The close interaction of DPPH with the free radical scavengers present in the hemp seed oils presented a reduced signal intensity relative to the DPPH stock solution EPR spectrum.DPPH scavenging activity of the hemp seed oils is reported in Table 4; the addition of 0.2 g of hemp seed oils to 1 ml of DPPH stock solution quenched more than 93% of free radicals that are present in DPPH for all the oil samples in the first two hours.The declining trend in the spectral intensity for the oil sample is observed from Fig.5 and relative to the DPPH spectra.Among the three extraction techniques,UAE and MW oils have lower antioxidant activity relative to CP oil.During the extraction,cold-pressed seeds and oil were not exposed to the higher temperature like MW and UAE techniques.Due to the high-temperature processing,thermally sensitive antioxidant compounds were destroyed in MW and UAE hemp seed oils.On the other hand,CP hemp seed oil was extracted at room temperature,thereby,greenhouse grow tables the antioxidant activity of the CP is higher than MW and UAE.Further,the antioxidant activity of the oils was evaluated at 20 h,therefore the DPPH stock solution allows the dissolution of both DPPH and lipid-associated antioxidants that are present in the hemp seed oils.Upon analyzing the spectra at 20 h,enhanced scavenging activity was observed by the antioxidant compounds present in the hemp seed oils.After 20 h,1 – 3% increment in the antioxidant activity was noticed relative to at 2 h,i.e.,within 20 h of reaction with DPPH,hemp seed oils quenched or neutralized the synthetic-free radicals present in the DPPH,primary and secondary oxidation products from the hemp seed oils.As discussed in the previous section,GC–MS analysis of the hemp seed oils revealed the presence of -tocopherols,sitosterols,stigmasterol that may be hypothesized to be primarily responsible for the antioxidant properties shown by the oils.The free radical scavenging activity of the hemp seed oils showed that they are capable of quenching the reactive oxygen species and their derivatives in short time owing to the fast reactivity of the antioxidant compounds in hemp seed oils.Therefore,it was anticipated that the presence of antioxidants in the hemp seed oils,could be a value-added feedstock for the production of pharmaceuticals and nutraceuticals as reported by Polovka in his research.

Hemp has a long cultivating history all over the world,with the earliest report in Chinese ancient literatures dating back to 4000 BC.Traditionally,hemp has been widely used in textile,building materials,and paper,as well as herb medicines.In general,there are two types of hemp plants: the fifi ber type and the cannabinoids type.The fiber type hemp plants are tall and are harvested for fibers and seeds.However,they are very low in cannabinoids contents.On the other hand,the cannabinoids type hemp plant is shorter in heights and is rich in cannabinoids contents.Depending on the level of tetrahydrocannabinol in the plant,the hemp can be further classified into industrial hemp and marijuana.In recent years,the bioactive compounds,including oils,protein,cannabinoids and terpenes,etc.,in hemp plants have drawn increasing interests in both scientific research and product development.As the results,the cultivation area and production of hemp have been increasing.In 2019,the bearing acreage of hemp in the US,China,Europe and Canada were 465 787,164 819,138 863,and 92 504 acres,respectively.The global hemp industry is projected to reach $10.6 billion by 2025.Hemp seed protein contains the full essential amino acids profile required by humans,and the hemp protein isolate shows higher nutritional values than the soy protein isolates.Particularly,the contents of omega-6 to omega-3 fatty acids in hemp oil have a ratio of 3:1,which is recommended for healthy human diet.Cannabinoids such as cannabidiol ,THC,cannabigerol and cannabinol ,etc.,are gaining increasing interests in biomedical and pharmaceutical applications.Among them,CBD is the most popular topic in scientific research and hemp industry,which shows functionality in facilitating pain relief,reducing anxiety/distress,and promoting relaxation without any psycho-activities.CBD has already been applied in cosmetics industry and food products.In addition,terpenes,another group of bio-active compounds that define the characteristic aroma of the plant,are also drawing more attention together with CBD.Hemp terpenes show excellent anti-inflammatory,anti-cancer values and antioxidant activities,etc.,and have been proven to have an ‘entourage effect’ with CBD,improving CBD functionality.With these excellent pharmaceutical and nutritional values,hemp CBD and terpenes are expected to be used as future functional food ingredients,catching the wind of booming functional foods market.Currently,applications of CBD in foods are limited by several important factors: 1) Global legal regulations are still strict,which in general,prohibit the addition of CBD in any foods,beverages or supplements.However,these ever-changing regulations are opaque and varies from country to country; 2) The consumer base of hemp CBD is relatively weak.The word ‘CBD’ is unfamiliar to food consumers,and for most cultures in the world,habitual thinking associates the word ‘hemp cannabinoids’ to ‘drug’,insinuating that it is illegal and bad for human health.This further illustrates the need for the functionality and health benefits of CBD to be studied and verified; 3) Effective incorporation of the hemp CBD in conventional food products has not been extensively studied and reported,which are essential for the development of potential products; 4) Hemp plants have high yield and high moisture contents at harvest,which increases vulnerability to quality deterioration and microbial spoilage.Conventional natural drying process has low efficiency and causes product loss.

The grafting with BTCA was carried out in the laboratory using a previously published procedure

The variations in the metabolic composition of the aqueous extracts of leaves and stems were investigated by performing an OPLS-DA.A clear separation between the two classes of samples along the first two components was obtained and the analysis of the Splot allowed for the detection of the most significant x-variables and,thus,the metabolites contributing to such clustering.Specifically,the buckets having values of VIPpredictive >1 and values of p1 >|0.5| were analyzed more deeply.The spectral regions containing signals related to glucose,fructose,and galactose moieties contributed significantly to the clustering of the stems.Also,the buckets containing the signals assigned to the organic acids were characterized by significant values of VIP and p1.An analogous trend was observed for the buckets related to the signals assigned to γ-aminobutyric acid at 1.89–1.93 ppm and 3.01–3.05 ppm,and glutamine at 2.13–2.17 ppm.Interestingly,the clustering of the leaves was affected by some regions of the spectra containing very broad signals at 0.88,1.29,2.23,and 5.31 ppm ,presumably attributable to the lipids of the cell membranes and/or walls.The comparison of the 1D 1 H NOESY spectra of the leaves collected from control plants ,vigorous plants ,weak plants indicated that such broad signals were predominant in vigorous plants.Such broad signals were not observed in the aqueous extracts of stems.Also,the buckets containing the signals of ethanol at 1.17–1.25 ppm contributed relevantly to the grouping of the leaves towards the stems.These data could be explained by the fact that the leaves and,particularly the leaves sampled from vigorous green plants,have active photosynthetic processes and intact cell structures,grow racks system whereas stems of weakest plants begin to enrich with lignin and have less active photosynthesis.

The spectral regions containing these broad signals correlated positively with the calculated amounts of residual copper in the leaves.Under physiological conditions,Cu is found in two common forms,Cu preferentially binding sulfur-containing compounds having a thiol or a thioether group,and Cu ,that coordinates mainly with oxygen or imidazole nitrogen groups.The main functions of Cu are the transport of electrons in mitochondria and chloroplasts,the control of the cellular redox state ,and the remodeling of the cell wall,which is one of the major Cuaccumulation sites in hyper accumulating plants.Moreover,Cu leaf content is inversely associated with the intensity of the buckets relating to glucose and fructose and ethanol.In weakest plants,sucrose,the main soluble component of the phloem sap and translatable product of photosynthesis might be hydrolyzed into the two constituting monomers and fermented by yeast and/or bacteria producing ethanol.In a second task,the effect of the treatment with spirulina during the cultivation of hemp towards the remediation of contaminated soil was investigated.Thus,a deeper look was taken at the higher components of the OPLS model described in paragraph 3.1.Indeed,a good clustering of samples according to the treatment was visible in the t[2] vs t[5] scores plot.The second component and the fifth one explained together 11.75% of the x-variance predictive of the y-variance ,suggesting that the treatment with spirulina affected slightly the metabolic composition of the plant.Such evidence is relevant to make possible a re-utilization of the hemp,which does not change its metabolome during the phytoremediation stage.Specifically,the samples collected from plants grown in contaminated soil and the samples obtained from plants treated with a higher concentration of spirulina tended to cluster at values of t[2] >0.Conversely,the samples derived from plants treated with a lower concentration of the blue-green alga and the control ones were distributed preferentially at values of t[2] <0.A further piece of evidence was that samples C and the samples CS1 tended to separate into two clear groups along t[5].Analysis of the loading plot suggested that the uptake of all the metals,except for lead,contributed relevantly to the clustering of the plants treated with a higher dose of spirulina.Also,the average plant biomass was generally higher in contaminated plants compared to the uncontaminated ones.In addition,the plants that grew in contaminated soil and were irrigated with a higher dose of spirulina presented a higher value of biomass,confirming the activity of these cyanobacteria to promote plant growth.

The average plant contents of Ni ,Zn ,and Cd were greater in contaminated plants grown in soils added with spirulina than those grown without it.To get deeper insights into the effects deriving from the dose of spirulina,OPLS was applied to CS1 and CS0.5 samples.The observations,namely CS1 and CS0.5 ,separated along the second predictive component P2,explaining about 9.5% of the predictive x-variance.The distribution was strongly affected by the part of the plant the samples derived from,leaves or stems,with a noticeable clustering along the first predictive component P1.The corresponding loading plot showed important dose-dependent variations.Indeed,nickel,zinc,copper,and chromium were more abundant in samples treated with a higher dose of spirulina.Generally,as the concentration of spirulina increased,the residual metal content increased both in the leaves and in the stems ,whereas the Cr content increased in leaves and decreased in stems.While no dose-effect was observed for Cd,noticeable variations were exerted by increased amounts of spirulina on the quantities of Pb incorporated into the plant.Specifically,the average content of Pb in the plant was higher in CS0.5 than in CS1,suggesting that a higher amount of the blue-green alga contained in the soil might compete with hemp to sequester this metal.A piece of further evidence was that the average soil residual of all heavy metals resulted higher when it was irrigated with a higher concentration of spirulina ,suggesting that the cyanobacteria stationing in the soil may exert a strong action of metal chelation,avoiding leaching of them during the watering stage and acting as a reservoir for hemp to absorb the chelated metals.Such results find fundamentals in the reported ability of Arthrospira platensis to adsorb and accumulate all the six metals here tested through various mechanisms.Very small effects were observed on the metabolic profile when samples CS1 were compared with CS0.5.Interestingly,according to the loading plot ,the buckets at 2.66 and 2.74 ppm,containing the signals assigned to citric acid,contributed importantly to the observed grouping between the samples treated with the two different doses of spirulina.Specifically,the signals of citric acid were more intense in samples treated with a higher dose of spirulina.Considering that citric acid is one of the main chelating agents in plants,this evidence may support the hypothesis that,at a higher concentration,the cyanobacteria can chelate the heavy metals sequestering them into the soil.

As a result,less quantity of heavy metal is available for the plant to uptake,and,thus,more chelating agents inside the plant are in the free form not coordinating any metal.Industrial hemp is an annual crop grown for its seed and fibers.The three main global producers of hemp are China,Europe,and North America.Hemp is found in countless daily products: foods ,beverages ,cosmetics,and personal care products ,textiles ,papers and cardboard ,jewelry and fashion,leisure products ,animal feed,animal litter,mulching and horticulture,detergents,bio-fuel,building materials,insulation,composites ,etc.Hemp is also the object of numerous fundamental studies for innovative potential applications in bio-pesticides,biotechnology,pharmacy and medicine,paints,energy production,and wastewater treatment.Hemp shives,as valuable co-products produced during the process of extracting fiber from hemp straw,are constituted from the xylem tissue of the stem.Long considered as a by-product of the industry,used for plant mulch or animal bedding ,hemp shives are now used for house insulation,energy,and fuel production.There is also an interest in using these co-products as adsorbent materials to complex environmental pollutants since this resource is abundant,cheap,and easily burned.The last one is interesting in the context of recovery of adsorbed metals and their revalorization for example.Moreover,these materials have a highly reactive lignocellulosic macromolecular structure capable of interacting with other substances.Finally,the industry also needs to find new outlets because the volume of shives produced annually is constantly increasing.Recently,hemp fibers have been studied as adsorbent for metal removal and for the preparation of activated carbons for similar applications.However,work on hemp in shives/hurds form for environmental applications is rare.Furthermore,the identification of interactions involved in liquid-solid adsorption phenomena is often carried out using empirical mathematical models that allow the modeling of experimental results and the drawing of conclusions from the assumptions of these models.Another approach is to couple different microscopic and spectroscopic techniques to obtain information on these mechanisms.This study is part of the European research project FINEAU which brings together academics and industrialists focused on the potential use of hemp shives to recover metals from industrial effluents aiming their valorization.Two types of materials were targeted in this study by comparing shives washed with water with those chemically modified using polycarboxylic agent ,and with shives washed with water and activated with sodium carbonate.

The last one was the most efficient biosorbent of copper in our previous study among investigated raw shives and those treated with H2O,H3PO4,or KOH.The chemical treatment with Na2CO3 was carried out on a laboratory scale by copying the process already used by one of the industrialists of the project on other cellulosic materials.The objective of these two modifications is to purify and increase the capacity of raw shives towards metals,rolling flood tables while presenting high selectivity and performances independently of the pH and ionic strength representing two important issues for wastewater from the surface treatment industry.Recently,Mongioví et al reported that in the context of copper recovery by SHI-C and SHI-BTCA samples,these two materials had similar high adsorption capacities and fast adsorption kinetics,while their adsorption behaviors were very different.For example,the pH of the solution after copper adsorption onto the SHI-BTCA sample increased significantly,while that of the SHI-C sample varied much less,suggesting different interactions between these shives samples and the adsorbed copper.In addition,the amounts of sodium released into the solutions after an exchange with metal ions increased strongly in the case of the SHI-BTCA sample due to the presence of COONa groups in this material,also confirming different adsorption mechanisms.This study has two objectives: the first is to characterize the surface state of the materials before and after copper adsorption and the second is to assess the interactions between the materials and the metal ions involved in the adsorption process using microscopic and spectroscopic tools,namely computed nano-tomography ,energy-disperse X-ray spectroscopy,X-ray photoelectron spectroscopy ,Fourier-transform infrared spectroscopy,Raman spectroscopy,and X-ray absorption near-edge structure spectroscopy.Three different treatments were applied to SHI-R samples.A water washing ,an activation using sodium carbonate ,and a grafting reaction using 1,2,3,4-butanetetracarboxylic acid were carried out to study their role in the performance of samples which were further used as adsorbents for copper presents in aqueous solution.To prepare SHI-W,SHI-R samples were simply washed with water for 2 days at room temperature.After that,they were filtered and dried in an oven at 80◦C until a constant mass was obtained.

The SHI-C shives were prepared by treating SHI-W samples in 1 M Na2CO3 for 4 h at 40◦C.Thereafter,the samples were washed extensively with water until a neutral pH was obtained and dried in an oven at 80◦C until a constant mass was obtained.To obtain SHI-BTCA,SHI-W samples were pre-treated with 1 M NaOH for 3 days at ambient temperature under mechanical stirring.The shives suspension was then filtered on a filtering funnel and thoroughly washed with distilled water until the water was colorless.After drying at 60◦C in a ventilated oven for 15 h,the samples were immersed in an aqueous solution containing 100 g/L of BTCA and 30 g/L of NaPO2H2 for 15 h at ambient temperature under mechanical stirring.After draining,shives were spread on the bottom of a glass crystallizer put in a ventilated oven at 160◦C for 90 min.They were suspended in distilled water overnight under stirring,filtered,thoroughly washed with distilled water,and dried in a ventilated oven at 60◦C for 15 h.The shives were finally activated by converting the acidic form of carboxylic groups into their basic carboxylate form by immersing materials in a NaHCO3 solution under mechanical stirring for 24 h.SHI-BTCA samples were filtered,washed with distilled water until neutral pH was reached,and dried in a ventilated oven at 60◦C for 15 h.The interactions between copper and hemp-based samples were assessed by using different microscopic and spectroscopic techniques such as computed nano-tomography ,energy-disperse X-ray spectroscopy,Fourier-transform infrared spectroscopy,Raman spectroscopy,X-ray photoelectron spectroscopy ,and X-ray absorption near-edge structure spectroscopy.Nano-CT images given in Fig.3a show the typical cross-section of raw hemp shives.Namely,different tissues such as the woody part constituting the main part of the shives and some remaining pith on the right side can be observed.The woody part is made of different cell types: i) isolated or grouped in groups of two or three vessels which have a quite thin cell wall and a diameter of approximately 50-150 µm; ii) the fibers with a thick cell wall,an irregular polygonal cross-section and a diameter of only a few µm and iii) the rays which are oriented in the radial direction and are generally composed of one cell in width.

The TBA value of all treatments increased gradually up to the end of the storage time

A similar experiment was performed with THC,this time only evaluating the FBBB stability at refrigerated temperatures.The characteristic red color indicative of the presence of THC did form; however,like the hemp,the color was duller than when performing the usual procedure.Finally,FBBB was preloaded onto PSPME substrates and left in refrigerated temperatures for a week and were then used to evaluate 1000 ppm THC and 1000 ppm CBD solutions.Red and orange colors did form,respectively,but they were duller like all the other chromophores formed using FBBB as a preloaded salt.There is a growing interest in natural bioactive compounds both by producers and consumers in the food and pharmaceutical industries.Particularly,consumers are looking for foods without artificial and harmful preservatives that can promote their health.Essential oils as natural food preservatives are,generally,extracted from medicinal plants and herbs for their noticeable biological activities.EOs are complex combination of secondary metabolites,including terpenoid hydrocarbons,phenol derivatives and oxygenated terpenoids ; however,due to having volatile components,their usage is limited; the volatile compounds can be degraded easily by adverse external conditions such as light,oxygen,temperature,pressure,and pH.In addition,controlled release of EOs is needed when used for specific purposes like food additives.In recent years,the isolation and utilization of EOs from agroindustrial wastes and by-products have gained much research interest.Industrial hemp’s inflorescences are usually discharged during the conventional hemp processing,resulting in an underused biomass for future uses.They are a rich source of EOs and contain mainly monoterpenes and sesquiterpene hydrocarbons like -caryophyllene,α-pinene,myrcene and α-humulene,which exhibit important biological activities.Valorization of hemp by-products is a matter of interest for producers,allowing them to increase the market value of hemp cultivation.Some studies have recently been carried out on the functional and pharmacological applications/properties of industrial hemp essential oil due to their antioxidant,trimming trays for weed antimicrobial,and anti-inflammatory activities.

There is much interest in developing biodegradable nanoparticles as an effective delivery system for delivering lipophilic food bioactives.Proteins are attractive options for designing polymeric NPs as suitable wall materials thanks to their amphiphilic nature compatible with many active substances,as well as excellent functional properties.Whey protein,as a typical cheese processing by-product,is commonly used to foods due to its great nutritional value and techno-functional properties such as gelling,foaming and emulsifying.Protein fibrillation is used nowadays for fabrication of protein fibrils having novel functionalities and improved structure stability.Heating the protein above 80 ◦C for 5–24 h at low ionic strength and acidic conditions can result in production of WPI fibrils ,which are more suitable as delivery carriers comparing to native proteins,due to having multiple functional groups,and thus,promoting different interactions with numerous and drugs and nutrients.Protein complexation has recently been proposed as a promising and efficient way for improving the bio-availability,chemical stability and dispersion of bio-active compounds in an aqueous environment.Mung bean protein is a significant plant protein that demonstrates high potential as sustainable protein source for its availability,nutritional value,hypoallergenic,and desirable foaming,emulsifying,gelling,and film-forming capabilities.The rainbow trout as an extremely perishable food belongs to the Salmonidae family.Recently,the demand for rainbow trout has increased remarkably,and this could be due to its desirable characteristics.Due to the demand for fresh refrigerated fish with extended shelf life,notable study has been directed toward prolonging the shelf life of this fish.Although,there are scarce studies on the characterization and fabrication of IHEO encapsulated in complexation of WP nanofibrils and MBP NPs.The present study is to manufacture and characterize WPNFMBP NPs as a novel wall material for loading IHEO.Furthermore,application of IHEO-reinforced WPNF-MBP NPs as a potential natural additive for increasing the shelf-life of Rainbow trout fillets during refrigerated storage is going to be evaluated.The chemical changes of Rainbow trout fillets were monitored through the measurement of TVB-N,TBA and PV values of the fish oil during storage.Hydroperoxides are the main products of lipid oxidation; thus,measurement of peroxide values is helpful for indicating oxidative rancidity.As depicted in Fig.3a,PV in the control samples increases from 1.33 to 8 meq O2/kg at day 10 and declines after this day,maybe due to the reaction of hydroperoxide with protein as well as the collapse of primary oxidation products into secondary oxidation products.PV level tends to increase toward the end of the storage period.

A similar pattern of hydroperoxide content has also been reported in Rainbow trout fillets during storage.The samples coated with WPNF-MBP and IHEO-reinforced WPNF-MBP NPs showed significantly lower PV levels than the control during the storage period.The literature considers a PV value of 20 meq O2/kg oil is as the maximum limit for fish.Our results indicated that the IHEO-reinforced WPNF-MBP NPs coating is significantly reduced the amount of primary lipid oxidation in fish during storage; this is in agreement with the results of other similar investigations.TBA index is a common indicator for evaluating the lipid oxidation level by measuring such oxidation products as aldehydes like malondialdehyde.The recommended perceivable level of TBA in food as objectionable odour is about 1–2 mg MDA/kg ; however,Raeisi et al.proposed the maximal acceptable level 5 mg MDA eq/kg in Rainbow trout with no negative effects on its safety and quality.As indicated in Fig.3b,the TBA level remained below the maximal acceptable level during the 14-day storage.However,in the uncoated samples,the TBA value was much higher than in the coated samples ; however,the initial value of TBA was in the range of 0.09–0.13 mg MDA/kg,consistent with reports of other researchers for fresh Rainbow trout.The slightly lower oxidation rate in the IHEO-reinforced WPNFMBP NPs coatings can be because of the oxygen barrier and antioxidant activity features of IHEO and WPNF.The antioxidant mechanism of IHEO can be due to its polyphenols,which show scavenging activity against free radicals through providing hydrogen atoms to free radicals,preventing radical chain initiation,and thus,preventing the formation of metal catalyzed free radicals.On the other hand,the WP coatings provided a great protection against oxidation.IHEO addition improved the WPNF-MBP NPs coatings’ antioxidant properties.WPs exhibit antioxidant activity through different ways: 1) formation of a coating,which is a good barrier for O2 permeability coated samples during storage,2) enjoying a free radical scavenging capacity by some amino acids and metal chelation by bovine serum albumin and lactoferrin,3) having sulfhydryl groups partially responsible for their antioxidant properties,and 4) containing β-lactoglobulins and α-lactalbumin with good antioxidant activity for having amino acid residues.Furthermore,particle size reduction of IHEO and WPNF-MBP coatings after nanoencapsulation can increase these ingredients’ specific surface; thereby achieving an efficient amount of free radical absorption would be achieved.

TVB-N is produced from degradation of proteins and non-protein nitrogenous compounds,mainly as a result of microbial and enzymatic activities,as an indicator of meat and fish spoilage.The TVB-N level in the control samples was initially 6.68 mg N/100 g,and there was no significant difference among the different samples at the first day of storage.By increasing the bacterial counts,the TVB-N value increased gradually in all groups; however,in the samples coated with WPNF-MBP and IHEO-reinforced WPNF-MBP NPs,the TVBN value was significantly lower than in the controls.Considering the maximal acceptable level of 25 mg N/100 g in fish flesh ,the TVB-N limit was achieved at day 8 of storage in the controls,while in the WPNF-MBP coated samples,this limit was reached by the 10th days,and in the IHEO-reinforced WPNF-MBP NPs coated samples,it was lower than this limit during the whole period of storage.At the last day of storage,the TVB-N value for the IHEO-reinforced WPNF-MBP NPs coated samples was 12.1 and 9 mg N/100 g lower than the control and WPNF-MBP coated samples,respectively.We previously mentioned that TVB-N is produced mainly because of the bacterial degradation of the nitrogenous compounds of proteins and non-protein products,its low value in the coated samples can be due to the microbial inhibitory effects of treatments that decrease the formation of TVB-N.Similarly,Shokri et al.found that coating of Rainbow trout fillets with chitosan-Ferulago angulate EO nano-emulsion retarded the increasing rate of TVB-N index during storage at 4 ◦C.Ozogul et al.found that nano-emulsions based on plant EOs significantly inhibited the TVBN formation in Rainbow trout fillets during ice storage.To the best of our knowledge,there are no studies on the effect of IHEO-reinforced WPNFMBP NPs coating on the formation of TVB-N in Rainbow trout fillets; however,trimming tray with screen the lower TVB-N in the samples coated with IHEO-reinforced WPNF-MBP NPs in the present work could result from the antibacterial efficiency of WPNF-MBP coating facilitated by nano-encapsulation of IHEO.Fig.4 illustrates the overall acceptable scores of the control and coated Rainbow trout fillets with WPNF-MBP and IHEO-reinforced WPNF-MBP NPs.In the present study,appearance,color,odor,and texture were taken into consideration in the overall acceptance scoring.The overall acceptance scores were in the range of 1–5.High preference levels represent high element scores.All samples exhibited high sensory and quality scores at the first day of analysis.An overall acceptance below 3 of fish is considered to be unacceptable for human consumption.The overall acceptance scores of the coated and control fish samples showed a decreasing trend up to the end of storage time.The samples coated with IHEO-reinforced WPNF-MBP NPs exhibited a higher score comparing to the other samples during the refrigerated storage.The control samples’ sensory properties were ‘unacceptable’ by the 10th day.Also,at the day 12,the samples coated with WPNF-MBP did not achieve acceptable scores due to their unpleasant appearance and sticky surface; however,the incorporation of IHEO reduced these defects.Hence,the samples coated with IHEO-reinforced WPNF-MBP NPs had better sensory scores than the others for control the lipid oxidation and bacterial population.

These results are consistent with the findings of O˘ guzhan Yıldız and Yangılar and Farsanipour et al.for Rainbow trout WP-based coated samples incorporated with EOs stored at refrigerator condition.Industrial hemp,Cannabis sativa L.is a multifaceted crop that is increasingly being considered by farmers looking to diversify and reduce their ecological footprint.Industrial hemp is grown for both fiber and food,and is also used in cosmetics and for medicinal and nutraceutical/ pharmaceutical purposes.A major factor contributing to the sustainability of hemp is that most parts of the plant can be used,and innovation is contributing to additional uses of hemp for biofuels,high-performance textiles and natural insecticides.Hemp has long been used as a food source and in traditional medicine ,and this has stimulated research into its use as a functional food.In addition to its healthy oil and protein profiles,several bioactive peptides have been found in hemp seed,as well as polyphenols and other effective antioxidants.Hemp seed should be relatively easy to get to food markets as seed processing can use existing production facilities with some adjustments.Hemp seed is actually an achene or nut,with a hard outer shell ,a papery testa,and an inner seed.Industrial hemp is classified as Cannabis plants with low levels of Δ9- tetrahydrocannabinol ,depending on the country and state.It was recently estimated that the global production of hemp will double in the four years from 2016 to 2020,and some states in Australia have already more than doubled their production.This resurgence of interest in hemp has led to an increasing number of states in the USA growing low THC hemp,and as of 2017 all states in Australia have legalized cultivation of industrial hemp for food and fiber applications.In comparison to other crops hemp has been described as sustainable because it is relatively resistant to biotic and abiotic stresses thereby reducing agronomic inputs.In temperate Australia industrial hemp is grown as a summer crop and requires supplemental irrigation.Hemp has a relatively high water requirement compared to dryland cereals,although much less than cotton.A major advantage of hemp from an agronomic point of view is the rapid growth of seedlings such that it effectively outcompetes weeds.This and the fact that it has relatively few pests and diseases makes hemp better suited for organic production than many other row crops.In Australia,some pests and diseases have been observed,but in most cases there has been no significant impact on productivity.Industrial hemp is suited to a wide variety of soil types as long as they are fertile with good drainage and water holding capacity.When C.sativa is grown for fiber,tall varieties are used because of their increased biomass.Shorter varieties are being bred with higher harvest index that are specifically targeted to food markets.Characterization of FINOLA seed showed that it contains approximately 30% oil and 25% protein.Analysis of lipids of FINOLA seed revealed that over 80% are polyunsaturated including two essential fatty acids,linoleic acid and α-linolenic acid.Hemp seed contains a ratio of omega-6 to omega-3 in a desirable range between 2:1 and 3:1.Current western diets are generally deficient in omega-3 fatty acids,with a high ratio of omega-6:omega-3,due to the increased intake of vegetable oils including sunflower and corn oils,which have ratios of >50:1 omega-6:omega-3.

It confirms that there was indeed a direct effect of hemp fabric on the Izod impact energy of the composites

Considering the average lengths before and after extrusion-injection molding , a reduction of about 2 times in the case of HF, 2.5 times for HF and 4 times for HF was observed. It follows that the reduction of fiber length was greater for longer initial fibers, similar to the results on simple PP/HF composites. Moreover, their length in the final composites was greater than for shorter initial fibers, which is consistent with previous results. For a deeper evaluation of fiber length reduction in PPM/HF composites after extrusion and injection molding, the length histograms of the initial fibers, collected after the mechanical treatment and before the incorporation in composites are given in Fig. 5. The representative images of the initial hemp fibers, HF, HF and HF are also shown in this figure. A thorough analysis of the initial fiber length shows that about 60% of HF are under 1mm in length, compared to 90% after processing, and only 27% in the case of HF and 4.5% in the case of HF, compared to 72% and 47% after processing. It follows that the proportion of “fines” is drastically increased in the case of longer fibers but it is maintained lower than 50% for HF. A side-by-side analysis of optical microscope images of HF before and after incorporation in composites highlights the intense defibrillation of the fibers. This is due to the concerted action of temperature and shear forces during processing in the double-screw extruder and injection molding machine. Average diameters of 91 ± 64, 99 ± 48 and 119 ± 69 m were obtained for the initial fibers, HF, HF and HF, after mechanical treatment. The lower dm value for HF and HF was caused by the more intensive mechanical treatment provided by the automatic cutting. The dm of HF decreased to 17.1 ± 11.1, 18.8 ± 9.8 and 23.8 ± 12.4 m after incorporation in PPM 30HF, PPM 30HF and PPM 30HF composites. Therefore, a rough calculation of the aspect ratio of the fibers,grow tent kit the length to diameter ratio, shows an increase from 12, 26 and 35 for mechanically treated HF,and to 33, 39 and 43 for HF in the composites containing these fibers.

Although both the length and the diameter of HF diminished after the melt processing of composites, the defibrillation seems more intense, which leads to an increase in the aspect ratio of the fibers. The analysis of fiber size after processing will be further used to understand the thermo-mechanical behavior of these composites.Natural fibers used as reinforcement of polymer matrices have been intensively investigated in recent decades and considered for diversified applications, from automotive, civil construction, sports, furniture, and packaging industries as well as in ballistic armors. The interest in the use of natural fibers ranges from their availability and renewability to intrinsic features, such as low density, biodegradability, cost-effectiveness, and low processing energy. In addition, the current need for replacing synthetic materials due to problems related to sustainability and non-renewable sources of energy led to a surging number of works to optimize their composite properties. Among the well-known natural fibers, the hemp fiber has historically been used in several applications such as textiles, manufacture of papers, and even in the pharmaceutical industry. Archaeologically, hemp is the oldest discovered natural fiber. The Columbia History of the World stated that the most antique relics of human industry are bits of hemp fabric discovered in tombs dating back to approximately 8000 BC. Nowadays, the plant Cannabis sativa, from which the industrial hemp fibers are extracted, is again widely grown in China, Europe, and Central Asia, after years of restriction in some countries. In fact, the C. sativa species produces less than 0.2 wt% of tetrahydrocannabinol , the, which is too low for use as a recreational drug, as in the case of marijuana, a popular denomination of Cannabis indica. The C. sativa fibers, responsible for keeping the trees upright, have its structural properties studied for reinforcement composites due to their remarkable strength and stiffness. Some historical applications corroborate its potential, such as when Henry Ford tried to adopt hemp-polymers composites in the automotive industry in 1941. Another example was the constructive details of the window frames and floor coverings, made of hemp fiber reinforced polymer composites used in facilities during the 2008 Beijing Olympics. Indeed, due to a relatively larger amount of cellulose and hemicellulose and lower microfibrillar angle,which are related to mechanical properties, the hemp fiber displays a remarkable tensile strength , elastic modulus and total strain. As such, after sisal, hemp is the world’s most applied natural fiber as reinforcement in composites.

However, a recognized disadvantage is the aforementioned variability in their properties, which is inherent to natural fibers in general and requires that each lot of fiber, obtained from a given supplier, to be preliminarily tested. This procedure will be carried out in the present work regarding basic mechanical and thermal properties. According to Shahzad, the main hemp fiber reinforced composite matrices are polypropylene and unsaturated polyester due to easy processing and cost. On the other hand, with comparable properties and cost of UP, epoxy is another thermoset polymer that has not been often used as a composite matrix for hemp fibers. As shown in our literature survey in Fig. 1, there is an emerging tendency to study epoxy composites reinforced with hemp fiber. In this decade an approximately exponential rise is occurring in the related number of articles. According to Fig. 1, today seven new publications already have a DOI number. In particular, flexural strength and modulus of 30 vol% of hemp fiber epoxy composites were found to surpass the corresponding flexural strength and modulus of polyester composites with an equal amount of same hemp fibers. In spite of the raising interest, Fig. 1, for epoxy composites reinforced with hemp fiber, no work has so far specifically investigated epoxy composites with hemp fabric. A surging area, where natural fiber/fabric reinforced polymer composites are attracting increasing attention, is that of personal ballistic protection. In addition to the numerous works cited in the aforementioned review papers, several recent publications reported on the ballistic protection provided by NFCs. As part of multilayered armor system , these NFCs display ballistic performance superior to commonly applied Kevlar and Dyneema laminates. Although much weaker than synthetic aramid and ultra-high molecular weight polyethylene, natural fibers possess the same capacity of absorbing the ballistic energy by capturing the fragments after the bullet impact against the MAS front ceramic. In view of these disclosed NFCs superior ballistic performance combined with lower density, cost effectiveness and sustainability, one might expect that hemp fabric reinforced epoxy composite could be used for ballistic protection. In a pioneer works, Wambua et al. investigated the response of 46 vol% of hemp fabric reinforced PP composite to ballistic impact by 1.1 g fragment simulating projectile . Their main result revealed an absorbed kinetic energy of 36 J associated with a limit ballistic impact velocity of 260 m/s.

Therefore, the primary objective of the present work was to investigate for the first time the ballistic performance of epoxy composites reinforced with up to 30 vol% of hemp fabric against 0.22 ammunition. As a preliminary investigation, the mechanical and thermal properties of the aforementioned composites are also investigated to characterize the specific reinforcement effect of our Brazilian supplied hemp fabric. The possible variability of results was statistically evaluated using the analysis of variance and the Tukey test.Table 1 shows the results for all conditions: composites reinforced with 10, 20, and 30 vol% of hemp fabric and neat epoxy, which was used as a control group. For better visualization, the average values were plotted in Fig. 4. All tested specimens were completely fractured, validating the results obtained as required by the standard. The graph in Fig. 4 presents a continuous increase in the impact energy along with the fabric volume fraction and a lower standard deviation for the plain epoxy resin compared to the composites due to the non-uniform proprieties of natural fiber. This behavior was observed in previous works for different composites and is expected as the amount of reinforcement is related to the increase of energy necessary to break the samples. Comparatively, the 30 vol% hemp fabric-epoxy composite presented only about 36% lower absorbed energy than the 30 vol% curaua-polyester composite, which, in another work, was presented as an acceptable ballistic performance. Fig. 5 shows the broken specimens after the Izod impact test. By visual analysis, the fracture surface of specimens with 0 and 10 vol%, and , respectively, reveal a smoother surface related to a brittle fracture tendency. On the other hand, the fracture surface of the samples reinforced with 20 and 30 vol% hemp fabric,indoor grow tent and gets more irregular as the volume fraction of reinforcement increases, which could indicate a brittleeductile transition. From the results, in Table 1 and Fig. 4, Table 2 presents the ANOVA analysis for the impact energy absorbed. The equality hypothesis with a confidence level of 95% was rejected, as F was higher than the Fc.The Tukey test honest significant difference for the absorbed impact of the plain epoxy result and the composite values above this result present a significant difference. Hence, the results in Table 3 show that the impact strength of the 30 vol% hemp fabric composites is the best amid the tested composites. It is important to note that the values are always greater than the HDS calculated, which suggests that the incorporation of hemp fabric provides an effective reinforcement to the epoxy resins for all volume fractions.

This points toward what was already presented in early works, in which the reinforcement contributed to a greater rupture surface area by interrupting or deviating the crack’s propagation.Table 4 presents the average results for the tensile strength of the hemp fabric-reinforced composites. From these values and the strain of the samples, it was also possible to calculate the elastic modulus. These results are plotted in Fig. 6 and , which correspond to tensile strength and elastic modulus, respectively, for better visualization. Table 4 also shows literature values for the tensile strength of the same neat epoxy resin. The results in Table 4 and Fig. 6 display relatively poor tensile properties for the 10 and 20 vol% hemp fabric reinforced composites comparatively with the neat epoxy resin. It suggests that these two fractions of fabric do not act as reinforcement when tensile loads are applied to the material. Consequently, the epoxy matrix bears most of the load applied during the test. As such, the hemp fabric incorporation has a negative effect on the composites and acts more as flaws in the material’s structure. However, the composites with 30 vol% hemp fabric improved considerably the tensile strength compared to the neat epoxy resin, which means that this amount of hemp fabric acts as an effective reinforcement for the material due to the mechanisms of restrict rupture of the fibers in the fabric that impart the tensile strength. This good performance, compared to other composites reinforced with hemp fibers, is an important parameter for the ballistic performance of the composites, as the tensile strength is associated with the penetration resistance of the target material. Also, it is possible to observe a considerable improvement of the elastic modulus for the composites reinforced with 30 vol% hemp fabric, which could be related to the higher stiffness of the hemp fiber. Although, the 10 and 20 vol% hemp fabric show lower values with a slight decrease attributed to a nonuniformity of the reinforcement’s properties. To better understand the behavior of the analyzed composites under tensile stress, Fig. 7 presents SEM images of the broken specimens, in which it can be noted different fracture mechanisms. The “river marks” present on the composites reinforced with 10 and 20 vol% hemp fabric, shown in Fig. 7 and , reveal low effectiveness of reinforcement of these composites due to limited fabric content and causes a brittle fracture for the material as the epoxy matrix bears most of the load.

Absolute and normalized organ weights were determined on selected tissues

Sixty male rats weighing 224-286 g and 60 female rats weighing 170-218 g were distributed to treatment groups stratified by body weight among the dose and control groups . For both the 14-day and the 90-day studies, body weights were recorded twice during the acclimation period and weekly for the duration of the study. Feed intake was determined at the same day body weights were determined. Filtered potable water and feed were provided ad libitum. Feed and water were assayed for detrimental substances and none were found at levels that would alter study results. In the 90-day study, sentinel rats were kept in the animal rooms. Serology done on samples collected at the end of the study from the sentinel rats were negative for Rat Parvovirus, Toolan’s Virus , Kilham Rat Virus, Rat Minute Virus, Parvovirus NS-1, Rat Coronavirus, Rat Theilovirus, and Pneumocystis carinii. The animals in the 14-day and the 90-day study were observed daily for clinical evidence of ill health and given physical exams weekly corresponding to body weight determinations. The physical exam included observing for changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity . The exam also included changes in gait, posture, and response to handling, as well as the presence of clonic or tonic movements, stereotypies , or bizarre behavior . All abnormal observations were recorded. Rats in the 90-day study received a Functional Observation Battery in an open field for excitability, autonomic function, gait and sensorimotor coordination , reactivity and sensitivity and other abnormal clinical signs including, but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration and general appearance. Additionally, during week 12 rats in the 90-day study underwent a Motor Activity Assessment using a Photobeam Activity System [San Diego Instruments, Inc ] following recommended procedures. Investigators doing the physical examinations, Functional Observation Battery, and Motor Activity Assessment were blind to the treatments the rats were receiving. The clinical chemistry parameters for the 14-day and 90-studies are given in Table 5.

For the 14-day study, blood,vertical grow after overnight fasting, was collected before necropsy from the inferior vena cava while the rats were anesthetized with isoflurane. For the 90-day study, blood was collected from all groups for hematology and clinical chemistry on study day 94 for males and study day 95 for females in Groups 1 to 4 and on study day 124 for Groups 5 to 8 . Blood samples for hematology and clinical chemistry were collected by sublingual bleeding after the rats were anesthetized with isoflurane. Approximately 500 μL of blood was collected for hematologic parameters in a pre-calibrated tube containing Potassium EDTA4 anticoagulant and 1000 μL of whole blood was collected in tubes for clinical chemistry parameters . Whole blood samples were kept cold until examined in the laboratory using standard hematology methods. For clinical chemistry, blood was allowed to coagulate, and the samples were centrifuged in a refrigerated centrifuge. The serum supernatant was harvested and placed in cryotubes, and frozen and stored at -80 ◦C until thawed and assayed. Hematology parameters were determined on an ADVIA 120 Hematology System and clinical chemistry parameters were determined on a COBAS C311 autoanalyzer . Blood samples used to determine the prothrombin time and activated partial thromboplastin time were collected immediately before terminal sacrifice by venipuncture of the inferior vena cava during anesthesia with isoflurane. Approximately 1.8 mL of blood was collected in a pre-calibrated tube containing anticoagulant . These samples were centrifuged in a refrigerated centrifuge and the plasma was transferred to labeled tubes. Plasma samples were frozen and stored in a -80 ◦C freezer until thawed and analyzed on a Sysmex CA620 . The day before collection of samples for the clinical chemistry evaluations, the animals were placed in metabolism cages. Food was withheld for at least 15 hours prior to blood collection, and voided urine was collected from each animal. Urine samples were refrigerated until analyzed . Urine volume was measured, the appearance was recorded, chemical parameters were measured by Multistix® 10 SG Reagent Strips and urine sediment was evaluated by light microscopy. A full necropsy was done on each study animal including animals removed from the studies. Included in the necropsy were examination of the external body surface, body orifices, and the thoracic, abdominal and cranial cavities inclusive of contents. All surviving animals were weighed, anesthetized with isoflurane and exsanguinated from the abdominal aorta. All gross lesions were recorded.The eyes, epididymides, optic nerve and testes were fixed in modified Davidson’s fixative and then stored in ethanol.

All other tissues were fixed in 10% neutral buffered formalin. Specified tissues were embedded in wax, thin sections cut and stained with hematoxylin and eosin, and examined by light microscopy for histopathology . For the 14-day study, liver and adrenal glands from all treatment and control animals, and the kidneys from Groups 1 and 4 were examined by histopathology. For the 90-day study, tissues from all animals removed from the study, tissues from Groups 1 and 4 and the livers from Groups 2 and 3 and groups 5 to 8 were examined for histopathologic changes by light microscopy . All gross lesions observed were described, the tissues taken and examined by histopathology. All pathology procedures were under the supervision of aveterinary pathologist5 . Mean and standard deviations were calculated for all quantitative data. For all in-life endpoints that were identified as multiple measurements of continuous data over time , treatment and control groups were compared using a two-way analysis of variance , testing the effects of both time and treatment, with methods accounting for repeated measures in one independent variable. Significant interactions observed between treatment and time, as well as main effects, were further analyzed by a post hoc multiple comparisons test; e.g. Dunnett’s test of the individual treated groups to control. When warranted by sufficient group sizes, all endpoints with single measurements of continuous data within groups were evaluated for homogeneity of variances and normality. Where homogeneous variances and normal distribution was observed, treated and control groups were compared using a one-way ANOVA. When one-way ANOVA was significant, a comparison of the treated groups to control was performed with a multiple comparisons test, e.g., Dunnett’s test . Where variance was considered significantly different, groups were compared using a nonparametric method, e.g., Kruskal-Wallis non-parametric analysis of variance. When non-parametric analysis of variance was significant, a comparison of treated groups to control was performed, e.g., Dunn’s test. Significance was a probability value of p < 0.05. For hematology and clinical chemistry, the data from male and female rats were analyzed separately. Means and standard deviations were calculated for all quantitative clinical pathology parameters using Pristima® version 7 . These data were analyzed in a sequential manner. First, Bartlett’s test for homogeneity and Shapiro-Wilk test for normality was done.

If the Bartlett’s test for homogeneity and Shapiro-Wilk test for normality were not significant, a one-way analysis of variance followed with Dunnett’s test was performed. If the Bartlett’s test for homogeneity and Shapiro-Wilk test for normality were significant then data transformations to achieve normality and variance homogeneity were done. The order of transformations attempted was log, square root, and rankorder. If the log and square root transformations fail, the rank-order was used. When an individual observation was recorded as being less than a certain value, e.g., below the lower limit of quantitation, calculations were performed on one-half of the recorded value. For example, if bilirubin was reported as <0.1 or ≤0.1, then 0.05 was used for all calculations performed with that bilirubin data. When an individual observation was recorded as being greater than a certain value, e.g., above the upper limit of quantitation, then a greater value was used in place of the recorded value. For example, if specific gravity was reported as >1.100 or ≥1.100, then 1.100 was used for all calculation performed using that specific gravity value. For all statistical testing, significance was a probability value of p < 0.05. The mutagenicity potential of the test article as well as undiluted extracts were evaluated in the Bacterial Reverse Mutation Assay in accordance with FDA GLP and US FDA Redbook 2000 and ICH guidelines. Four strains of Salmonella typhimurium and one strain of Escherichia coli were used. The studies were conducted in the presence and absence of a metabolic activation system from male Sprague-Dawley rats which had been induced with phenobarbital and benzoflavone . The overlay agar and minimal glucose agar plates were purchased . The fresh bacterial suspension cultures in the nutrient broth were prepared so that they were in the late exponential phase of growth when used. The test article in olive oil was formulated as a solution in dimethyl sulfoxide to provide the required dose levels of up to 76,335 μg/plate to account for the 6.55% of active ingredient . For the undiluted extract prepared by isopropanol or supercritical CO2 extraction, indoor growers the extract was formulated as a solution in DMSO to provide the required dose levels up to 5000 μg/plate. Positive controls were used, both in the presence and absence of a metabolic activation system. The positive control substances included were sodium azide, ICR 191, daunomycin and methyl methanesulfonate for S. typhimurium strains TA100 and TA1535, TA1537, TA98 and E. coli WP2 uvrA, respectively in the absence of metabolic activation and 2-aminoanthracene for all strains in the presence of metabolic activation. The initial test for all test articles utilized the plate incorporation method in which the following materials were mixed and poured onto the minimal agar plate; 100 μL of the prepared test substance solutions/negative control/positive control substance, 500 μL of S9 mix or substation buffer, 100 μL bacterial suspension or 2000 μL overlay agar.

The plates were then incubated at 37 ◦C until the growth was adequate for enumeration. A confirmatory test for all test articles was conducted utilizing the pre-incubation method. The test or control substances, bacterial suspensions and the S9 mix or substitution buffer were incubated under agitation for approximately 30 minutes at 37 ◦C prior to mixing with the overlay agar and pouring onto the minimal agar plates and proceeding as for the initial test. The strains used and dose levels were the same as that in the initial test for all test articles. The plates for both tests were prepared in triplicate for each experimental point. For the undiluted isopropanol extract, the final doses utilized for both the initial and confirmatory tests were 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 μg/plate. For the undiluted supercritical CO2 extract, the final doses utilized were 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 μg/plate for the initial test and 0.5, 2.5 and 25 μg/plate for the confirmatory test. Due to toxicity noted for strains TA100 and TA1537 with the supercritical CO2 extract, a supplemental test was conducted to ensure five concentrations could be assessed without toxicity. Both the plate incorporation and pre-incubation methods were used as previously described at final doses of 0.5, 2.5 and 25 μg/plate. Following incubation, the number of colonies per plate was counted manually and/or with the aid of a plate counter. The mean and standard deviation were calculated for each set of triplicate plates. The test was considered valid if the control plates had normal background lawn; the mean revertant colony counts for each strain treated with vehicle was close to or within the expected laboratory historical control range or published values; and the positive controls should produce substantial increases in revertant colony numbers with the appropriate bacterial strain. The plates were also evaluated for cytotoxicity which is indicated by the partial or complete absence of a background lawn on non-revertant bacteria or a substantial dose-related reduction in revertant bacteria. Recently, there has been an increasing interest regarding the health benefits of CBD and other phytocannabinoids and with this increased interest, more research is also being conducted to assess the safety of these compounds for human consumption. The current studies were performed to better understand the toxicological profile of a CBD rich proprietary hemp extract and to assess the results in tandem with information currently available regarding the toxicity and safety of CBD. Marx et al. reports on a battery of GLP compliant toxicological studies which were conducted on a supercritical CO2 extract of the aerial parts of the C. sativa plant.

The values of initial adsorption rate and desorption constant varied in function of the copper concentration

Values of ki and correlation coefficients are listed in Table 3,from which it can be seen that the kinetics of copper adsorption on the SHI-W and SHI-C samples followed this model with R2 values higher than 0.95,indicating that intraparticle diffusion was involved in the adsorption process.For these samples,it seems that physisorption played the main role in the adsorption mechanism.However,for the SHI-BTCA,the R2 values did not support the fact that the metal-adsorption data closely follow this model,suggesting that,in this case,the diffusion mechanism is not the main interaction,and the process was mainly controlled by chemisorption.Nevertheless,for the three selected materials and the entire concentration range studied ,it was noted that the adsorption process tends to be followed by two linear regions with non-zero intercepts: the initial curved portion of the plots indicated a boundary layer effect while the second linear portion is due to intraparticle diffusion.Indeed,the data did not pass through the origin,indicating that intraparticle diffusion was not the only rate-limiting mechanism and that some other interactions also played an important role.The calculated ki values for each initial concentration indicated that,when the metal concentration was augmented,the rate constant increased for all the tested samples.The values of C also increased with copper concentration.However,the analysis of the data indicated that deviations from Weber and Morris model mainly occurred at high initial metal concentrations.Similar interpretations on this were reported before.The Elovich model,inspired by Zeldowitsch’s model and developed for gaseous systems,helps to predict the mass and surface diffusion,activation and deactivation energy of a given system,pot drying determining the nature of adsorption on the heterogeneous surface of the adsorbent,whether chemisorption or not.

This model is expressed by Eq.where a and b are the initial rate and desorption constant,respectively,during any experiment.Fig.6 shows that,for the three selected samples,the metal adsorption also fitted the Elovich equation,suggesting a chemisorption mechanism such as surface complexation formation and that shives surfaces are energetically heterogeneous.Table 3 lists the kinetic constants.In particular,when the concentration increased,the b constant decreased for both SHI-C and SHI-BTCA samples,suggesting a decrease in the availability of adsorption sites for copper adsorption.The analysis of the data clearly indicated that the Elovich model gave the best fit for the experimental data obtained for the SHI-BTCA sample,confirming the involvement of the carboxylate groups in the adsorption mechanism.Nevertheless,the data were also well simulated by the Elovich model for SHI-W and SHI-C samples,indicating the mechanism was complicated with the presence of both chemisorption and physisorption.By applying the Lagergren,Ho and McKay,and Elovich models,it is assumed that the overall adsorption process rate is governed by the rate of binding,whereas when applying the Weber and Morris model,the rate of mass transport is expected to govern the overall process rate.The Boyd model is also an adsorption diffusion model used to predict the mechanistic steps involved in an adsorption process,i.e.,whether the rate of removal of the metal takes place via film diffusion or particle diffusion mechanism.If the plot is a straight line passing through the origin,then adsorption is governed by a particle diffusion mechanism,otherwise governed by film diffusion.An analysis of the literature shows that,in numerous works,film diffusion is the limiting step during the initial stages of the adsorption process followed by intraparticle diffusion when pollutant species reach the material surface.However,it is difficult to estimate appropriate values of Bt for the entire time scale,so the data should be carefully interpreted.The good correlation obtained between the experimental data and predicted curves proved the validity of the Boydmodel only for the SHI-W sample,with R2 values higher than 0.95.For the two other samples,the Boyd model did not adequately fit the data.In addition,as shown in Fig.7,the plots for the three materials did not pass through the origin,revealing that the film diffusion controls the process for the copper adsorption onto three samples.And the properties of AC are also affected by the type of activator, the ratio of added activator, activation temperature, activation time and so on. In consideration of activation difficulty, supply stability and the price of raw materials, AC can be prepared by a wide range of different raw materials.

The use of agriculture byproducts or lignocellulosic materials, such as rice husks and waste newspapers to produce AC has been widely studied. Kazemipour et al., respectively used some agricultural products such as almond, hazelnut, walnut, and apricot to prepare AC Hemp, which can grow quickly in various climates and has various applications is an good choice owing to low cost, economic, and environmental considerations. Hemp, an annual herbaceous plant, as a result of hemp fiber processing technology is rapidly expanding, is widely cultivated in China . However, compared to hemp seed can be refined into biological diesel and hemp bast can be applied in the clothing, respectively. The hemp stems are almost useless and often burned through post-harvest burning of cultivation fields, which not only wastes the resources, but also polluts the environment. Hemp stems are very rich in cellulose and lignin, and have natural nano pore structure. Accordingly, the utilization of hemp stems to produce AC reduce the cost, enhance economic efficiency and contribute to comprehensive utilization of agricultural wastes. There are two processes for the preparation of activated carbons, the so-called physical and chemical activation. The results show that chemical activation has the advantage of high effective and well-controlle porosity. In spite of all kinds of activating agents are used, KOH which can promote AC to produce more pore structure is one of the most widely applied. Rosas et al. adopted phosphoric acid activation to product AC from hemp. A.H. Basta et al. used 2-steps KOH activation to prepare AC from rice straw. However, straw-based material such as hemp stems often have high ash content compared with other agricultural wastes, which are not benefit for the preparation of super AC. As a consequence, preparation and characterization of high performance AC from straw material was studied by few researchers. Not to mention the preparation mechanisms of straws materials-derived AC were investigated. Only Ru Yang et al. investigated characterization of AC derived from biomass source hemp stem via N2, CO2 and H2 adsorption.

Anthropogenic activities, especially those of the industrial and mining sectors, have left considerable areas of heavy metal – contaminated areas worldwide. Due to their nonbiodegradable and persistent characteristics, HMs cause serious soil and water contamination and severe health hazards for living beings upon exposure. In Europe, potentially polluting activities have taken place at an estimated 2.8 million sites, and only 24% of the sites have been inventoried. Currently, only 28% of all registered sites have been investigated, which is a powerful precondition for deciding whether remediation is needed. The term soil remediation refers to actions that are undertaken to limit the extent of soil contamination near hazardous waste sites to prevent exposure to harmful chemicals to people and other life forms. Currently, there are many soil remediation techniques where the most appropriate method depends on the soil characteristics, contamination type, treatment depth and costs involved. As reported by Dhaliwal et al., among the different technologies that are used to ameliorate contaminated soils, phytoremediation is the cheapest and fastest technique to decontaminate soil with HMs. Moreover, phytoremediation has been shown to be a cost-effective and eco-friendly technology compared to physicochemical soil reclamation methods. Metal hyperaccumulator plants can accumulate large amounts of concentrated HMs in their biomass and remain healthy, which makes them suitable for the phytoremediation of HMs in contaminated soil. Among the promising species that have been used for remediating HM-contaminated soils, the use of industrial hemp has shown promising phytoremediation potential in terms of morphophysiological and metal accumulation responses, remediation capacity, trace element phytoextraction and the phytoremediation capability of wild weed. In addition, industrial hemp grows in different climates and produces high biomass yields, and its roots grow deep into the soil, which allows the plant to penetrate deep into the soil and increases the efficiency of removing widespread contamination. Linger et al. found that industrial hemp accumulates HMs in all of its plant parts and that the highest contaminant concentrations accumulate in the leaves. This finding emphasizes the importance of plant management, treatment and disposal of contaminated biomass to avoid secondary pollution, since the methods that are used to recover HMs from plant biomass and/or the safe disposal of harvested plants are still limited. The same study also suggested that in the future, more research must be conducted to provide technological improvements regarding the proper disposal of harvested contaminated biomass. Wu et al. indicate that industrial hemp has been extensively applied, with appropriate pretreatments and investigations, to fields including bioenergy, paper production and construction materials. According to Zhang et al., the postharvest management of plant biomass can be developed, which would include energy production, cannabis drying where the coupling of bioremediation techniques to bioenergy production yields far-reaching social and economic benefits.

Notwithstanding the fact that the phytoremediation of HMcontaminated soil is perceived as an eco-friendly technique, such phytoremediation activities might generate considerable environmental impacts, mainly due to the on-field activities, transportation and disposal or treatment of the biomass. To evaluate the environmental burdens of phytoremediation techniques, life cycle assessment represents a valuable methodology to analyze the in-depth impacts. Other studies have underlined the environmental implications of several bioremediation technologies. Witters et al.found that phytoremediation technologies do not have the energy-consuming and CO2- emitting drawbacks of conventional remediation activities that consist of soil excavation, chemical stabilization, incineration, vitrification and soil washing. Ali et al. also emphasize that conventional remediation technologies may lead to the alteration of soil properties and the disturbance of microflora. In another work, Vigil et al.argued that if the contaminated biomass was not valorized, then the sustainability of phytoremediation was questionable. An LCA methodology was also applied by Vocciante et al. to evaluate the environmental sustainability of phytoremediation technology. The results emphasized the importance of correctly managing the disposal of the contaminated biomass that was produced, where biomass incineration could be more onerous than direct land filling but would be viewed as a more sustainable choice if combined with energy production. Moreover, O’Connor et al. found that phytoremediation at a site was resilient when faced with moderate sea level rises and other hydroclimatic effects that were induced by climate change. Despite the aforementioned studies conducted on phytoremediation, there is a lack of knowledge on assessing the energy and environmental impacts of such remediation techniques. Although the technical issues and suitability of industrial hemp for phytoremediation have been deeply analyzed, less attention has been given to the energy and environmental aspects of the entire supply chain. Despite the growing interest in industrial hemp cultivation, which is also supported by national and European founding and regulations, this ’new crop’ suffers from current limitations, which start with the lack of knowledge of cultivation techniques, concerns by farmers about the legality of cultivation, and the scarcity of product processing plants, especially for the textile and fiber sectors, which limits its diffusion. Moreover, new studies are needed to overcome the current limitations related to the development of more sustainable industrial hemp supply chains. The aim of this study was to assess and evaluate the energy and environmental impacts of growing industrial hemp for the phytoremediation of HM-polluted soil. Additionally, the use of contaminated biomass as an energy resource in 4 different valorization scenarios was also analyzed. The three novel aspects of the performed analysis can be summarized as follows: i) to the authors’ knowledge, none of the previous LCA studies evaluated the energy and environmental impacts of industrial hemp at each phase of the cleanup process; ii) the energy and environmental benefits of phytoremediation were evaluated under 4 different industrial hemp biomass valorization scenarios, which included analyses of different products harvested and different subsystems, such as industrial hemp cultivation in HM-contaminated soils, with and without hemp seed processing, with and without anaerobic digestion of the contaminated products for electricity generation and biomass/digestate incineration in a biomass-fired power plant for electricity production; and iii) the work applied a broad approach in the analyses by including the entire supply chain of industrial hemp for phytoremediation in real sites that were contaminated by HMs.

Selection for genetically stable cultivars with larger seeds will be important for increasing hemp grain yields

The gel point was taken as the time when the sol did not show any movement on turning the container upside down.For analysis of the binder,the left-over sol contaminated with leached out hemp shiv extractives was aged in a container until the gel point was reached and the specimen was termed the “binding matrix”.A schematic illustration of silica glass has been presented in Fig.1.The EDX analysis shows the surface composition of the silica specimens.The percentage of carbon is significantly higher in the binding matrix than the silica glass.The presence of carbon in the silica glass is due to the alkyl groups providing functionalisation.The chemical composition of the silica glass and the binding matrix was determined by X-ray photoelectron spectroscopy.The atomic percentage of various elements present at the sample surface was determined by a low-resolution survey scan.The relative elemental composition for the specimens is listed in Table 2.The main elements detected for both the silica specimens were carbon,oxygen and silicon.The binding matrix showed higher content of carbon as seen in Fig.3.A high-resolution scan was performed on the C1s region for the silica glass and the binding matrix to determine the type of oxygen-carbon bonds present.The chemical bond analysis of carbon was performed by curve-fitting the C1s peak and deconvoluting it into four sub peaks corresponding to unoxidized carbon C1,and various oxidised carbons C2,C3 and C4.The binding energy,corresponding bond type and their relative percentage are listed in Table 3.The silica based binder shows additional oxidised carbon sub peaks,C3 and C4.The C1s high resolution spectra with the deconvoluted peaks for silica glass and binding matrix are represented in Fig.4.The C1 peak is related carbon-carbon or carbon-hydrogen bonds whereas C2,C3,and C4 peaks are associated with carbon-oxygen bonds.The identification of the extracted compounds was performed using GCMS.The polar components of the extractives were analysed for identification of the lipophilic extractives which are responsible for their tacky nature and would contribute to the adhesive properties of the binding matrix.

The yield of total extractives in hemp shiv was 6.23%.The hexane yield and methylene chloride yield in the total extract was 9.05% and 5.00% respectively.The chromatographs for hexane extract and methylene chloride extract are presented in Figs.6 and 7 respectively.All the compounds identified by GCMS are listed in Tables 4 and 5.The individual compounds were identified based on a comparison with GC retention times and mass spectra from the NIST library.Over twenty compounds were identified in the hexane extract and twelve compounds were identified in the methylene chloride extract.For the analysis of the GCMS data,cannabis square pot peaks lower than 30000 counts were rejected.From the chromatograms,it was determined that fatty acids esters,mainly lauric acid and phthalic acid,gave the highest peaks.The compression testing of the composite samples prepared with hemp shiv and binding matrix is imaged in Fig.8 and stress versus strain curves for the before and after immersion samples are presented in Fig.9.The moisture sensitivity of the composite was determined by comparing the mechanical properties of the hemp shiv composite before and after immersion in water for 24 h.Preparation of composite samples using hemp shiv and ethanol-water solution was unsuccessful as the hemp shiv particles were not able to bind.From Fig.9,the results from three test samples before immersion reveal that the composite reaches an average compressive stress of 0.48 ± 0.02 MPa at 30% strain.After the immersion test,a slight reduction in compressive stress by 15% was observed for the three samples and the average reading was 0.41 ± 0.01 MPa at 30% strain.It was noted that further compression led to densification of the sample.After compression,the sample showed some elastic behaviour as seen in Fig.8.In the present study,hemp shiv based composites have been manufactured by using silica sol as a binder.The binding matrix has been characterised and its morphology,chemical composition and physical properties have been studied in comparison with silica glass.

The binder is prepared by the hydrolysis and condensation of TEOS in water in the presence of ethanol as the mediator solvent.HDTMS is added for functionalisation thereby providing hydrophobic alkyl groups in the silica network.The formulation has been used earlier for treatment of hemp shiv particles for imparting hydrophobicity to the material.Here we report the binding properties of silica when mixed with hemp shiv.The silica sol interacts with hemp shiv leaching out extractives and waxes which leads to visual changes turning the silica matrix fromcolourless transparent to yellowish opaque.The silica is able to covalently bond to hemp shiv through the hydroxyl groups of cellulose.During the drying process,the gel starts condensing,releasing ethanol and water and develops a silica network.The extracts from the shiv that are entrapped in the silica network alter the characteristics of the silica.From the SEM analysis,it was seen that the silica morphology is modified.The structure of the new modified silica with incorporated extracts is less brittle when compared to the pure silica glass.The chemical composition of the silica specimens is mainly composed of carbon,oxygen and silicon.Chemical characterisation using EDX reveals that the modified silica has a higher carbon content than the pure silica.Detailed XPS analysis indicates that due to sol interaction with hemp shiv,the silica chemistry has been significantly altered.The surface carbon content of the binding matrix increased by 27%.On the other hand,the oxygen content decreased by 21%.This change in C/O ratio and increase in the surface carbon content can be attributed to the additional extracts that have been identified in the modified network of the binding matrix.The decrease in surface oxygen content can be related to the masking effect of the hemp shiv extracts reducing the detectability of the oxygen bonds in the silica network.The C1s high resolution XPS spectra reveal that the hemp shiv extracts have modified the silica network leading to the appearance of C3 and C4 peaks which are not present in the pure silica glass.Furthermore,the increase in the intensity of the C1 component for the binding matrix from 68% to 84% indicates the presence of CeC and CeH bonds from the incorporated extracts.To analyse the extracts that were leaching out from hemp shiv during the silica based treatment,the process was simplified by using a solution of ethanol and water for the extraction process.

Ethanol is able to dissolve waxes and isolate lipophilic extractives.These ethanol-soluble extractives were analysed using GCMS and it was found that the extract was mainly composed of lauric acid and phthalic acid with many other fatty acids.The majority of the compounds identified using GCMS belong to the group of lipophilic extractives which are hydrophobic in nature.This could possibly be one of the factors for the compatibility between the lipophilic extractives and the sol-gel chemistry due to their hydrophobic nature.The thermal decomposition patterns of the silica specimens were studied by TGA.The binding matrix had a higher weight loss below 100 °C and a greater endothermic peak that can be attributed to the presence of fatty acids in addition to the physically adsorbed water.The embedded extracts in the silica network changed the decomposition range of the organic fragments of the silane corresponding to the temperature range of 270–600 °C.Due to the higher percentage of the organic compounds in the binding matrix,the weight loss was greater and a peak shift was observed in the first derivative of the weight loss thermogram.The maximum decomposition rate in the DTG curve for silica glass was at 520 °C attributed to the loss of silanol groups.The modification of silica network with hemp shiv extracts lowered the thermal stability of the binding matrix.Composites were prepared using hemp shiv and silica sol and their mechanical performance was evaluated.The composites were light weight with a density of 175 kg/m3 and the compressive stress of 0.48 MPa attained at 30% strain is relatively good when compared to other hemp shiv based composites such as hemp-lime,hemp-starch and hemp-clay.Higher strains corresponded with higher compressive stresses leading to densification of the sample without reaching a failure point.This suggests that the interfacial adhesion between the shiv and binding matrix is good and the shear forces are low.After the immersion test,the decrease in mechanical strength can be related to the swelling of the shiv when placed in water for 24 h.Since the binder also provides hydrophobicity to the hemp shiv,the compressive stress versus strain characteristics are not compromised to a great extent.However,the swelling could be related to the slow penetration of water through micro-cracks on the coated surface or due to the presence of small uncovered pores within the hemp shiv.The binder can provide hydrophobicity to the hemp shiv but it cannot fully protect the hemp shiv against long-term water interaction.The slight decrease in compressive stress reached at 30% strain can be attributed to the weakening of the interfacial bonding between the hemp shiv and the binding matrix.However,composites produced using an ethanol-water mixture instead of silica sol was unsuccessful as the hemp shiv fell apart on demoulding.The ethanol is responsible for isolation of the extractives and waxes from hemp shiv but the extractives cannot bind hemp shiv on their own.

The extractives modify the silica chemistry and the binding matrix holds the hemp particles together resulting in the production of coherent composite blocks.When compared to conventional hemp-lime composites,it is evident that the production costs of the hemp-silica composites would be higher due to the hydrophobic treatment on hemp shiv.However,this cost could be off-set by savings elsewhere,both in production ingredients as well as an extension in service life,potentially reducing the whole life cost.Moreover,trim tray the commercial availability of sol-gel solution on an industrial scale would significantly lower the cost of this novel composite.The preparation of hemp-silica composite results in the reduction of 2 L of mixing water per 1 kg of hemp shiv when compared to a conventional hemp-lime composite.The thermal performance of the new composite is expected to be better due to their significantly lower density than hemp-lime.Overall early indications are that the global warming potential of this composite would be approximately 5% lower than that of a conventional composite.The life span is expected to increase by 50% due the improved resistance to water that is responsible for degradation of the composite.Hemp is a genetically diverse and variable crop that produces raw products in three distinct categories: seed/oil,fiber,and metabolites.Within each category,hemp can be improved by multiple avenues of research.We highlight key research areas which increase grower yield or product quality for processors.These topics are not exhaustive,but are intended to guide research to areas which are of the highest priority.Notably,due to the diverse nature of raw products produced from hemp,research targeting hemp yield traits will improve our understanding of basic plant biology.Seed and oil research will enrich our comprehension of grain yield and composition.Research into hemp fiber will enhance our knowledge of stem development and composition,genetic regulation of fiber traits,and biofuel production.Studies targeting metabolite yield will expand insights into both Cannabis-specific and shared plant chemistries,interaction with biotic stresses,and trichome development.Investigations into the plasticity of hemp’s sexual phenotype will contribute to identifying mechanisms underlying plant sex determination.Importantly,unlike previously domesticated crops,selection for increased hemp yield provides a unique opportunity to study plant domestication for grain,fiber,and chemistry traits.With hemp,unlike most other crops,these valuable characteristics can be studied within a single species for which they are essential to sustainable and profitable production.As a semi-domesticated crop,many traits for hemp seed and oil yield require improvement; these include seed size consistency and improved shattering resistance.Significant advances in hemp seed production occurred with the development of FIN-314 ,an auto flowering grain variety with a short stature,adaptation to high latitudes,and high yield,resulting in it presently being the most popular cultivar grown in Canada.However,seed size is highly variable among hemp cultivars and ‘Finola’ seeds are 50% the size of many commercial varieties.During domestication,hemp has retained little resistance to shattering.However,hemp field trials have revealed that significant grain is lost due to shattering prior to and during harvesting as a result of inconsistent inflorescence maturity ,particularly if collected outside of the optimal harvest time windows.To mitigate this problem,growers harvest seeds at 70% maturity ii.Hemp inflorescences are large multi-seeded heads in which each individual seed is partially surrounded by a bract,and an abscission zone connects the hull to the pedicle.Selection for a stronger-walled abscission zone or the prevention of bracts releasing seeds are possible physiological traits to target to reduce hemp seed loss due to shattering.Furthermore,immature seeds are similar in size,but weighed only half that of mature seeds due to incomplete embryo development.

The addition of heavy metals and Sb significantly affected the growth of all three hemp varieties

Compared with the control,Ni110 significantly reduced the hemp emergence rates for the three hemp cultivars,and Ni220 even completely inhibited germination.Unlike Ni stress,Pb1500 had no marked effects on the emergence rates for any of the hemp cultivars compared with the control,however,Pb3000 significantly increased the rates of Guangxibama and Yunma No.1.Both Sb50 and Sb100 significantly increased the germination rate of Guangxibama compared with the control,however,no marked differences between treatments for rates were found for the other two cultivars.Husain et al.also reported a reduction in seed germination of six hemp varieties sown in mine soils and most of the germinated plantlets died one to two weeks after their emergence.Heavy metal and Sb stresses significantly negatively affected the plant height,stem diameter,and the number of leaves of flax.For Cd,Ni,and Pb,most measurements of the plant height,stem diameter,and the number of leaves of the three flax cultivars were significantly reduced by both treatment levels,and the negative effects increased with increasing stress level.In general,compared with the control,Y2I329 was affected only slightly by the different levels of Cd because the three morphological indices of Y2I329 changed the least under Cd stress.Similarly,under the stress of Pb,Y2I329 still showed the best endurance.The plants under high Ni treatment died,and similar negative effects of Ni in flax were also reported by Amna and Syed.The effects of Sb on flax are complex and there is no uniform rule.Plant height was the least affected.The plant height,stem diameter,cannabis grow equipment and the number of leaves of Guangxibama and Yunma No.1 were significantly or slightly reduced by Cd stress compared with that of the control.Similar results were also observed by Luyckx et al..Treatment Cd40 increased the plant height of Zhongdama No.1 but Cd80 significantly reduced it; no significant difference was found for stem diameter and number of leaves.Treatment Ni220 completely inhibited the growth of the three hemp varieties; Ni110 significantly reduced the three morphological parameters for all varieties of hemp.

Pb addition at both levels significantly or slightly reduced plant height,stem diameter,and the number of leaves of all varieties.Sb did not significantly affect the plant height of Guangxibama and Yunma No.1 but significantly increased this characteristic for Zhongdama No.1 compared with that of the control; Sb had no marked effects on the stem diameter of hemp except for the significant reduce in Yunma No.1 by the Sb50.Treatment with Sb50 showed no significant effects on the number of leaves for Guangxibama and Zhongdama No.1 compared with the control but significantly reduced that for Yunma No.1; Sb100 did not significantly affect the number of leaves for Guangxibama and Yunma No.1 compared with Sb50,but notably reduced them compared with the control; Sb100 markedly increased the number of leaves compared with that for Sb50.Cd stress significantly reduced the fresh biomass of HP,but no significant reduction was found for Hongyou No.2 and GGS compared with the control.No significant difference in fresh biomass of the three kenaf varieties was found between Cd40 and Cd80.The addition of Ni and Pb notably or slightly reduced the fresh biomass of all three kenaf varieties compared with the control,and the reduction of biomass increased with increasing metal concentration.Similarly to its effects on flax,Sb50 significantly improved the biomass of all kenaf varieties compared with the control; Sb100 notably or slightly reduced the biomass compared with Sb50 but still increased the biomass compared with the control.Nickel was not lethal for kenaf varieties,however,it significantly inhibited plant growth.Lead was the second most toxic element,especially Pb3000,followed by Cd and lastly by Sb.Concrete is the single most widely used material in the world.Due to the worldwide substantial expansion of our built environment the demand for cement,as a par excellence material used for concrete production,will significantly increase in the near future.For cement production,huge exploitation of Earth’s minerals precedes.Its production is also an energy-intensive process,which contributes to high pollution,especially carbon dioxide emission.Since the increase in global carbon dioxide emissions is unacceptable,a search for other binder alternatives is necessary.

The solution for cement substitution or even its total replacement could be found in industrial by-product pozzolanic materials.Until now,the mostly used pozzolanic material for cement substitution is fly ash that comes from thermal power plants after coal combustion.When fly ashes are mixed with an alkali activator,they form alkali-activated material.This group of materials is comparable in physical and mechanical properties with cementitious materials,but has better durability and can come even at a cheaper price,leaving a much lower environmental footprint.However,these materials still do not have a broad usage in the building construction.The standardization of their solid based materials and activators should be firstly defined.Besides,there are also some disadvantages of the materials that should be solved before they find a broad usage on construction sites.AAMs are brittle or quasi-brittle materials and to move toward more ductile behaviour they need to be reinforced,similarly to cementitious materials.In the field of fibre reinforced materials,natural fibres have attracted a lot of attention in the last decade due to their low production cost and low consumption of energy during their production.Natural fibres are fully biodegradable,renewable resources and globally available.Their tensile strength and E-modulus are comparable to those of synthetic fibres.However,the mechanical properties of natural fibre reinforced composites are somewhat weaker compared to their counterpart reinforced with synthetics fibres.Natural fibres are generally nonhomogeneous,have high hydrophilicity and reduced long term durability in the matrix.One of the most distinctive parameters influencing the properties of the composite is the characteristic of the fibre/matrix interface itself.The quality of the bond is related to the fibres’ surface morphology and topography.To optimise the bond,so far in cementitious composites various chemical treatments on the surface of natural fibres have been applied.In terms of its simplicity and effectiveness,sodium hydroxide treatment could be a viable treatment also in AAMs.There are no results of the hemp fibres’ surface roughness changes and their influence on the pre-and post-peak of the flexural stress–strain curve,after the usage of the sodium hydroxide treatment.However,the enhancement of the fibres’ physical and mechanical properties when the fibres are treated with sodium hydroxide is confirmed.The researches on the alkali-treated fibres show that 5% sodium hydroxide solution treatment of hemp fibres resulted in the fibres’ density increase ,since the treatment cleans the impurities from the fibres’ surfaces.

The treatment also increased the tensile strength and Young’s modulus of the hemp fibres for ca.4% and 28% respectively.Mwaikambo and Ansell tested mechanical characteristics of the hemp fibres after treatment with 0.03%,0.08%,0.16%,0.24% and 0.32% of sodium hydroxide solution.They stated that there was an increasing trend in the tensile strength of the fibres after increasing the concentration of sodium hydroxide.The fibres’ highest tensile strength is reached after the usage of 0.24% of sodium hydroxide.The usage of the concentration of 0.16% of sodium hydroxide solution resulted in the fibres’ highest Young’s modulus.The highest concentration of sodium hydroxide provided the highest fibres’ strain at failure.Alawar et al.showed that data palm tree fibres treated with 1% of sodium hydroxide increased their tensile strength by 300% compared to non-treated fibres.Up to 11%,higher tensile strength of the kenaf fibres after the treatment with 6% sodium hydroxide is achieved in the research conducted by Edeerozey et al..After treating the jute fibres with 0.5% sodium hydroxide solution,the tensile strength of fibres increased by 82%.The sodium hydroxide treatment of natural fibres proved to be very efficient in enhancing fibres’ mechanical characteristics.However,there is no published work on the influence of the sodium hydroxide treated fibres on any group of alkali-activated mortars.According to the authors’ knowledge,there are only the works conducted by Amalia et al. and by Sean et al.where previously treated short natural fibres are used in alkali-activated matrices.In both works,natural fibres were used,but none of them used hemp fibres.Additionally,both studies focused on the alkali-activated pastes,having no aggregates in the matrix.Due to the pure mechanical characteristics and high cost of the pastes they should be upgraded to composites containing aggregate to be considered as building materials in civil engineering applications.Very importantly,the work conducted in this study is the first research dealing with the durability of the natural fibre reinforced alkaliactivated materials in terms of wet/dry cycles.Composites reinforced with natural fibres show comparable mechanical properties tested at an early age as their counterparts reinforced with synthetic fibres.However,after some months,natural fibres degrade in the alkaline environment and their long-term physical and mechanical properties are questioned.Therefore,it is extremely important to estimate the durability of the natural fibre reinforced high alkaline materials.Only with a proof of their durability,they can find a broader usage in the replacement of some traditional fibres and the usage where high energy absorption capacity,mobile grow system resistance to impact- and dynamic loading or prevention of cracking is required.The objective of this research was to study the physical and mechanical properties of hemp fibre reinforced alkali-activated materials before and after wet/dry cycles.The constant change of the relative humidity can seriously attack and destroy the fibre–matrix interface,which reduces the composite’s mechanical properties.With the wet/dry cycles we wanted to simulate a real weather change for the specimens that could be exposed to outdoor environmental conditions and to examine the composites’ properties change.Additionally,the influence of sodium hydroxide surface treatment of hemp fibres on the physical and mechanical properties of fly ash-based alkali-activated mortars was examined.Three different concentrations,i.e.,3%,6% and 9% of sodium hydroxide treatment were applied on fibres and the mortars’ porosity,water absorption,bulk density,compression- and flexural strength,as well as energy absorption capacity under flexure,prior and after the wet/dry cycles,were measured.Besides,the X-ray Powder Diffraction and Thermogravimetry-Differential thermal analysis were conducted on the mortars.

To estimate the fibres surface changes after the treatment,Scanning Electron Microscope and Atomic Force Microscope are used.In addition,the water absorption of the fibres was measured.For fibre reinforcement,primary bast hemp fibres were used Hemp fibres were selected since they are widely used as fibre reinforcement in cementitious materials and have a long cultivation tradition in Europe.Their properties are listed in Table 1.The fibres length and dosage were chosen based on the literature review,as optimal values for a compromise between mechanical properties in hardened state and fresh state properties such as work ability and fibres good dispersion within the matrix.To cut fibres to the length of 10 mm,a small bundle of fibres were taken,measured with a ruler and cut with scissors manually.Due to their hydrophilic nature,the fibres were added to the mixture in water-saturated dry surface condition.They were completely soaked in water for 24 h,taken out,softly squeezed by hand and their surface was wiped with a cloth.The water absorption was measured on 200 g of fibres.From a fibre bundle,fibres were separated manually,and their weight is measured firstly in a dry condition.Afterwards they were put in water and on defined time intervals their water absorption was measured.Even though it could be seen that the fibres’ highest water absorption happens within the first 20 min,they were left in water for 24 h in order to be fully sure that they absorbed maximum water they could.It is measured that the weight of the water-saturated non-treated fibres is 2.3 times the weight of the dry fibres.Due to the ease of application,effectiveness of the fibres’ structure and surface change,availability of the material for fibres treatment,the sodium hydroxide was used for fibres treatment.The sodium hydroxide was also used within the composites’ activator and it was not necessary to additionally provide a new material for fibres treatment.It is true that sodium hydroxide has a negative environmental footprint.However,there is a very minor amount of the sodium hydroxide that could be used for the fibre treatments.In this context,it should be noted that NaOH is the main activating agent for other types of alkali-activated materials bound in a hardened matrix,which has been documented in the publications or even applied in practice.Besides,natural fibres have enormous advantages over traditional fibres.Not only regarding the environmental footprint,but also that they are renewable materials.Therefore,even though they are not used as raw materials,their usage within the composites in the research is still justified.For fibre treatment three different concentrations of sodium hydroxide solution were used: i) 3%-; ii) 6%- and iii) 9% sodium hydroxide solution.To obtain the required sodium hydroxide concentrations,185.57 g,382.98 g and 593.41 g of sodium hydroxide were dissolved in 6000 g of distilled water respectively and left for 24 h to homogenize.Three plastic containers were half-filled with respective sodium hydroxide solutions.Afterwards,in each container 100 g of hemp fibre bundles were added,the containers were filled completely with sodium hydroxide solutions and tightly closed.The containers were placed in a drying oven at 80 ◦C temperature for one hour.