Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Evaluation of thermo-chemical conversion temperatures of cannabinoid acids in hemp (Cannabis sativa L.) biomass by pressurized liquid extraction.

BACKGROUND: Cannabinoids are increasingly becoming compounds of medical interest. However, cannabis plants only produce carboxylated cannabinoids. In order to access the purported medical benefits of these compounds, the carboxylic acid moiety must be removed. This process is typically performed by heating the plant material or extract; however, cannabinoids being thermolabile can readily degrade, evaporate, or convert to undesired metabolites. Pressurized liquid extraction (PLE) operates using a pseudo-closed system under pressure and temperature. While pressure is maintained at 11 MPa, temperature can be varied from ambient to 200 °C. METHODS: Temperatures were evaluated (80 to 160 °C) using PLE for the thermo-chemical conversion of cannabinoid acids utilizing water as the solvent in the first step of extraction with subsequent extraction with ethanol. Optimum temperatures were established for the conversion of 6 cannabinoid acids to their neutral cannabinoid forms. Cannabinoid acid conversion was monitored by HPLC. RESULTS: The use of PLE for thermo-chemical decarboxylation has resulted in a rapid decarboxylation process taking merely 6 min. The temperatures established here demonstrate statistically significant maxima and minima of cannabinoids and their parent cannabinoid acids. One-way ANOVA analysis shows where individual cannabinoids are statistically different, but the combination of the maxima and minima provides temperatures for optimum thermo-chemical conversion. CBC, CBD, CBDV, and CBG have an optimum temperature of conversion of 140 °C, while THC was 120 °C for 6 min. DISCUSSION: Decarboxylation of cannabinoid acids is necessary for conversion to the bioactive neutral form. The pseudo-closed chamber of the PLE makes this an ideal system to rapidly decarboxylate the cannabinoid acids due to pressure and temperature, while minimizing loss typically associated with conventional thermal-decarboxylation. This study established the optimum temperatures for thermo-chemical conversion of the cannabinoid acids in water and provides the groundwork for further development of the technology for industrial scale application.

Sourcing Cannabis sativa L. by thermogravimetric analysis.

Marijuana, dried and ground Cannabis, is the most consumed illicit drug in the world. Many undesirable and risky effects to human health are caused by its use. The medicinal use or legal recreational use of Cannabis has also been rising in many countries. These facts make traceability methodologies increasingly important whether for forensic use, such as drug trafficking eradication, or for quality control purposes of legal medicinal Cannabis. Consequently, the objective of this study was to analyze Cannabis by means of thermogravimetric analysis (TGA) in order to assess the capability of this technique to trace the geographical origin of Cannabis cultivated in Colorado, United States of America. TGA appears to be sensitive enough to detect the degradation/decarboxylation of cannabinoids and terpenes, at least to some extent; also, the degradation of cellulose, hemicellulose and lignin was indicated. Overall, the temperature ranges we analyzed using linear discriminant analysis showed high accuracies, with the 200 to 300 °C and 600 to 700 °C ranges achieving 100% accuracy.

Occurrence, temporal variation, and estrogenic burden of five parabens in sewage sludge collected across the United States.

Five parabens used as preservatives in pharmaceuticals and personal care products (PPCPs) were measured in sewage sludges collected at 14 U.S. wastewater treatment plants (WWTPs) located in nine states. Detected concentration ranges (ng/g, dry weight) and frequencies were as follows: methyl paraben (15.9 to 203.0; 100%), propyl paraben (0.5 to 7.7; 100%), ethyl paraben (<0.6 to 2.6; 63%), butyl paraben (<0.4 to 4.3; 42%) and benzyl paraben (<0.4 to 3.3; 26%). The estrogenicity inherent to the sum of parabens detected in sewage sludge (ranging from 10.1 to 500.1pg/kg 17ß-estradiol equivalents) was insignificant when compared to the 106-times higher value calculated for natural estrogens reported in the literature to occur in sewage sludge. Temporal monitoring at one WWTP provided insights into temporal and seasonal variations in paraben concentrations. This is the first report on the occurrence of five parabens in sewage sludges from across the U.S., and internationally, the first on temporal variations of paraben levels in sewage sludge. Study results will help to inform the risk assessment of sewage sludge destined for land application (biosolids).

Occurrence of Triclocarban and Triclosan in an Agro-ecosystem Following Application of Biosolids.

Triclocarban (TCC) and triclosan (TCS), two of the most commonly used antimicrobial compounds, can be introduced into ecosystems by applying wastewater treatment plant biosolids to agricultural fields. Concentrations of TCC and TCS were measured in different trophic levels within a terrestrial food web encompassing land-applied biosolids, soil, earthworms (Lumbricus), deer mice (Peromyscus maniculatus), and eggs of European starlings (Sturnus vulgaris) and American kestrels (Falco sparverius) at an experimental site amended with biosolids for the previous 7 years. The samples from this site were compared to the same types of samples from a reference (biosolids-free) agricultural site. Inter-site comparisons showed that concentrations of both antimicrobials were higher on the experimental site in the soil, earthworms, mice (livers), and European starling eggs, but not American kestrel eggs, compared to the control site. Inter-species comparisons on the experimental site indicated significantly higher TCC concentrations in mice (TCC: 12.6-33.3 ng/g) and in starling eggs (TCC: 15.4-31.4 ng/g) than in kestrel eggs (TCC: 3.6 ng/g). Nesting success of kestrels only was significantly lower on the experimental site compared to the reference site due to nest abandonment. This study demonstrates that biosolids-derived TCC and TCS are present throughout the terrestrial food web, including secondary (e.g., starlings) and tertiary (i.e., kestrels) consumers, after repeated, long-term biosolids application.

Transformation products and human metabolites of triclocarban and triclosan in sewage sludge across the United States.

Removal of triclocarban (TCC) and triclosan (TCS) from wastewater is a function of adsorption, abiotic degradation, and microbial mineralization or transformation, reactions that are not currently controlled or optimized in the pollution control infrastructure of standard wastewater treatment. Here, we report on the levels of eight transformation products, human metabolites, and manufacturing byproducts of TCC and TCS in raw and treated sewage sludge. Two sample sets were studied: samples collected once from 14 wastewater treatment plants (WWTPs) representing nine states, and multiple samples collected from one WWTP monitored for 12 months. Time-course analysis of significant mass fluxes (α=0.01) indicate that transformation of TCC (dechlorination) and TCS (methylation) occurred during sewage conveyance and treatment. Strong linear correlations were found between TCC and the human metabolite 2'-hydroxy-TCC (r=0.84), and between the TCC-dechlorination products dichlorocarbanilide (DCC) and monochlorocarbanilide (r=0.99). Mass ratios of DCC-to-TCC and of methyl-triclosan (MeTCS)-to-TCS, serving as indicators of transformation activity, revealed that transformation was widespread under different treatment regimes across the WWTPs sampled, though the degree of transformation varied significantly among study sites (α=0.01). The analysis of sludge sampled before and after different unit operation steps (i.e., anaerobic digestion, sludge heat treatment, and sludge drying) yielded insights into the extent and location of TCC and TCS transformation. Results showed anaerobic digestion to be important for MeTCS transformation (37-74%), whereas its contribution to partial TCC dechlorination was limited (0.4-2.1%). This longitudinal and nationwide survey is the first to report the occurrence of transformation products, human metabolites, and manufacturing byproducts of TCC and TCS in sewage sludge.

Presence and transport of the antimicrobials triclocarban and triclosan in a wastewater-dominated stream and freshwater environment.

The presence of the antimicrobials triclocarban (TCC) and triclosan (TCS)in Fountain Creek, a wastewater-dominated stream, and the Arkansas River, Colorado, USA was measured in the surface water, suspended sediments, and bed sediments during spring runoff (high flow) and summer base flow (low flow) conditions. Fountain Creak is a tributary of the Arkansas River. Passive polar organic chemical integrative samplers (POCIS) were used along with active sampling (water grab samples) to measure and TCS concentrations in these surface waters. The concentration of TCC and TCS, based on POCIS measurements, ranged from 4.5 to 47.3 ng/L and 3.9 to 28.3 ng/L, respectively, at the five sample sites monitored in this study under both flow conditions. The range of concentrations of TCC and TCS in suspended sediments was 0.7-57.3 ng/g and 0.7-13.3 ng/g, respectively, and was closely tied to the quantity of organic carbon in the suspended sediment, which ranged from 1.6 to 14.5%. The quantity of organic carbon in suspended sediment during the summer base flow was influenced by runoff from the burn area of a large forest fire that occurred between the two sampling periods. The primary transport mechanism of TCC and TCS in these surface waters was in the dissolved phase, with 64-99% of TCC and 68-99% of TCS transported in the dissolved phase. The total amount of TCS and TCC in bed-sediments was relatively low, with the maximum amount at any one site being 0.38 ± 0.15 ng/g TCS and 4.09 ± 5.26 ng/g TCC. Fountain Creek contributed up to 76% and 69% of the TCC and TCS, respectively, that is transported directly below its confluence with Arkansas River. Fountain Creek drained approximately 3.0 g/day TCS (in spring), 2.9 g/day TCS (in summer) and 1.9 g/day TCC (in spring), 2.0 g/day TCC (in-summer) into the Arkansas River, which suggests consistent input of TCC and TCS into Fountain Creek, such as in discharge of treated wastewater that is independent of changing creek flow conditions.

Uptake of human pharmaceuticals and personal care products by cabbage (Brassica campestris) from fortified and biosolids-amended soils.

Human pharmaceuticals and personal care products (PPCPs) are routinely found in biosolids from wastewater treatment plants (WWTPs). Once land applied, the PPCPs in biosolids are potentially available for plant uptake and bioaccumulation. This study used a greenhouse model to investigate uptake of PPCPs commonly detected in biosolids by the agricultural plant Chinese cabbage (Brassica campestris). Two series of greenhouse experiments were conducted as part of this project. In the first set of experiments, four pharmaceuticals were added to an organic matter-rich soil in environmentally relevant concentrations based on typical biosolids application rates, resulting in final soil concentrations of 2.6 ng g(-1) carbamazepine, 3.1 ng g(-1) sulfamethoxazole, 5.4 ng g(-1) salbutamol, and 0.5 ng g(-1) trimethoprim. In the second set of experiments, the cabbage was grown in soil amended with an agronomic rate of biosolids from a local WWTP. The ambient concentration of PPCPs in the biosolids resulted in final soil concentrations of 93.1 ng g(-1) carbamazepine, 67.4 ng g(-1) sulfamethoxazole, 30.3 ng g(-1) salbutamol, 433.7 ng g(-1) triclosan, and 24.7 ng g(-1) trimethoprim. After growing to maturity, the aerials of the plants were separated from roots and the two tissue types were analyzed separately. All four human pharmaceuticals were detected in both tissues in the cabbage grown in the soil fortified with the four pharmaceuticals with median concentrations of 255.4 ng g(-1) aerials and 272.9 ng g(-1) roots carbamazepine; 222.8 ng g(-1) aerials and 260.3 ng g(-1) roots sulfamethoxazole; 108.3 ng g(-1) aerials and 140.6 ng g(-1) roots salbutamol; and 20.6 ng g(-1) aerials and 53.7 ng g(-1) roots trimethoprim. Although all study compounds were present in the biosolids-amended planting soil, only carbamazepine (317.6 ng g(-1) aerials and 416.2 ng g(-1) roots), salbutamol (21.2 ng g(-1) aerials and 187.6 ng g(-1) roots), and triclosan (22.9 ng g(-1) aerials and 1220.1 ng g(-1) roots) were detected in the aerials of the cabbage. In addition to the study compounds detected in the aerials, sulfamethoxazole was detected in the roots of one of the plants in the biosolid-amended soil. In comparison to many previous studies that have utilized PPCP concentration that exceed environmentally relevant concentrations, plants in this study were exposed to environmentally relevant concentrations of the PPCPs, yet resulted in uptake concentrations similar to or greater than those reported in comparable studies. We suggest that rhizosphere conditions, particularly the presence of dissolved organic matter in the planting matrix, might be one of the critical factors determining mobilization and bioavailability of xenobiotic compounds such as PPCPs.

Earthworm bioassays and seedling emergence for monitoring toxicity, aging and bioaccumulation of anthropogenic waste indicator compounds in biosolids-amended soil.

Land application of biosolids (treated sewage sludge) can be an important route for introducing xenobiotic compounds into terrestrial environments. There is a paucity of available information on the effects of biosolids amendment on terrestrial organisms. In this study, the influence of biosolids and biosolids aging on earthworm (Eisenia fetida) reproduction and survival and lettuce (Lactuca sativa) seedling emergence was investigated. Earthworms were exposed to soils amended with varying quantities of biosolids (0, 1, 2, 3, or 4% dry mass). To investigate the influence of biosolids aging, the biosolids used in the study were aged for differing lengths of time (2 or 8 weeks) prior to exposure. All of the adult earthworms survived in the biosolids-amended soils at all concentrations that were aged for 2 weeks; however, only 20% of the adults survived in the soil amended with the highest concentration of biosolids and aged for 8 weeks. Reproduction as measured by mean number of juveniles and unhatched cocoons produced per treatment correlated inversely with biosolids concentration, although the effects were generally more pronounced in the 8-week aged biosolids-soil samples. Latent seedling emergence and reduced seedling fitness correlated inversely with biosolids concentration, but these effects were tempered in the 8-week aged versus the 2-week aged soil-biosolids mixtures. Anthropogenic waste indicator compounds (AWIs) were measured in the biosolids, biosolids-soil mixtures, and earthworm samples. Where possible, bioaccumulation factors (BAFs) were calculated or estimated. A wide variety of AWIs were detected in the biosolids (51 AWIs) and earthworm samples (≤19 AWI). The earthworms exposed to the 8-week aged biosolids-soil mixtures tended to accumulate greater quantities of AWIs compared to the 2-week aged mixture, suggesting that the bioavailability of some AWIs was enhanced with aging. The BAFs for a given AWI varied with treatment. Notably large BAFs were determined for some AWIs. For example, the maximum BAF determined for para-cresol, methyl salicylate, bisphenol-A, and cholesterol was greater than 100 in some treatments.

Uptake of human pharmaceuticals by plants grown under hydroponic conditions.

Cabbage (Brassica rapa var. pekinensis) and Wisconsin Fast Plants (Brassica rapa) were chosen for a proof of concept study to determine the potential uptake and accumulation of human pharmaceuticals by plants. These plants were grown hydroponically under high-pressure sodium lamps in one of two groups including a control and test group exposed to pharmaceuticals. The control plants were irrigated with a recirculating Hoagland's nutrient solution while the test plants were irrigated with a Hoagland's nutrient solution fortified with the pharmaceuticals carbamazepine, salbutamol, sulfamethoxazole, and trimethoprim at 232.5 microg L(-1). When plants reached maturity, nine entire plants of each species were separated into components such as roots, leaves, stems, and seedpods where applicable. An analytical method for quantifying pharmaceuticals and personal care products was developed using pressurized liquid extraction and liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) in positive and negative ion modes using single ion monitoring. The method detection limits ranged from 3.13 ng g(-1) to 29.78 ng g(-1) with recoveries ranging from 66.83% to 113.62% from plant matrices. All four of the pharmaceuticals were detected in the roots and leaves of the cabbage. The maximum wet weight concentrations of the pharmaceuticals were detected in the root structure of the cabbage plants at 98.87 ng g(-1) carbamazepine, 114.72 ng g(-1) salbutamol, 138.26 ng g(-1) sulfamethoxazole, and 91.33 ng g(-1) trimethoprim. Carbamazepine and salbutamol were detected in the seedpods of the Wisconsin Fast Plants while all four of the pharmaceuticals were detected in the leaf/stem/root of the Wisconsin Fast Plants. Phloroglucinol staining of root cross-sections was used to verify the existence of an intact endodermis, suggesting that pharmaceuticals found in the leaf and seedpods of the plants were transported symplastically.

Bioaccumulation of pharmaceuticals and other anthropogenic waste indicators in earthworms from agricultural soil amended with biosolid or swine manure.

Analysis of earthworms offers potential for assessing the transfer of organic anthropogenic waste indicators (AWIs) derived from land-applied biosolid or manure to biota. Earthworms and soil samples were collected from three Midwest agricultural fields to measure the presence and potential for transfer of 77 AWIs from land-applied biosolids and livestock manure to earthworms. The sites consisted of a soybean field with no amendments of human or livestock waste (Site 1), a soybean field amended with biosolids from a municipal wastewater treatment plant (Site 2), and a cornfield amended with swine manure (Site 3). The biosolid applied to Site 2 contained a diverse composition of 28 AWls, reflecting the presence of human-use compounds. The swine manure contained 12 AWls, and was dominated by biogenic sterols. Soil and earthworm samples were collected in the spring (about30 days after soil amendment) and fall (140-155 days after soil amendment) at all field sites. Soils from Site 1 contained 21 AWIs and soil from Sites 2 and 3 contained 19 AWls. The AWI profiles at Sites 2 and 3 generally reflected the relative composition of AWls present in waste material applied. There were 20 AWls detected in earthworms from Site 1 (three compounds exceeding concentrations of 1000 microg/kg), 25 AWls in earthworms from Site 2 (seven compounds exceeding concentrations of 1000 microg/ kg), and 21 AWls in earthworms from Site 3 (five compounds exceeding concentrations of 1000 microg/kg). A number of compounds thatwere present in the earthworm tissue were at concentrations less than reporting levels in the corresponding soil samples. The AWIs detected in earthworm tissue from the three field sites included pharmaceuticals, synthetic fragrances, detergent metabolites, polycyclic aromatic hydrocarbons (PAHs), biogenic sterols, disinfectants, and pesticides, reflecting a wide range of physicochemical properties. For those contaminants detected in earthworm tissue and soil, bioaccumulation factors (BAF) ranged from 0.05 (galaxolide) to 27 (triclosan). This study documents that when AWls are present in source materials that are land applied, such as biosolids and swine manure, AWls can be transferred to earthworms.

Application of ultrasonic assisted extraction of chemically diverse organic compounds from soils and sediments.

The objective of this research was the development, optimization, and demonstration of an ultrasonic assisted extraction (UAE) based method for organic anthropogenic waste indicators (AWIs) with a range of physicochemical properties from soil and sediment samples. The optimized method was designed to be cost effective compared to existing extraction methods, which may require large quantities of consumables, produce substantial volumes of organic waste, or require costly instrumentation or equipment. Reagent grade sand, soil collected from the native grassland in proximity to Eastern Washington University (EWU), and sediment samples collected from the Spokane river were used as sample matrices during method development. These matrices were fortified with eight AWIs of varying chemical properties that are representative of a variety of household, industrial, and agricultural sources. The recoveries of the AWIs spiked onto these matrices were determined in the extracts using gas chromatography/mass spectrometry (GC/MS). These values reflect the efficiency of the method for extraction of these analytes from representative environmental matrices. Recoveries ranged from 46.1% to 110% in the fortified soil and from 49.2% to 118.6% in the fortified sediment samples, which is comparable with existing methods for the study analytes. The optimized method was then used to quantify AWIs in a biosolid-amended soil. Indole and p-cresol were detected in the biosolid-amended soil.

Survey of organic wastewater contaminants in biosolids destined for land application.

In this study, the presence, composition, and concentrations of organic wastewater contaminants (OWCs) were determined in solid materials produced during wastewater treatment. This study was undertaken to evaluate the potential of these solids, collectively referred to as biosolids, as a source of OWCs to soil and water in contact with soil. Nine different biosolid products, produced by municipal wastewater treatment plants in seven different states, were analyzed for 87 different OWCs. Fifty-five of the OWCs were detected in at least one biosolid product. The 87 different OWCs represent a diverse cross section of emerging organic contaminants that enter wastewater treatment plants and may be discharged without being completely metabolized or degraded. A minimum of 30 and a maximum of 45 OWCs were detected in any one biosolid. The biosolids used in this study are produced by several production methods, and the plants they originate from have differing population demographics, yet the percent composition of total OWC content, and of the most common OWCs, typically did not vary greatly between the biosolids tested. The summed OWC content ranged from 64 to 1811 mg/kg dry weight. Six biosolids were collected twice, 3-18 months apart, and the total OWC content of each biosolid varied by less than a factor of 2. These results indicate that the biosolids investigated in this study have OWC compositions and concentrations that are more similar than different and that biosolids are highly enriched in OWCs (as mass-normalized concentrations) when compared to effluents or effluent-impacted water. These results demonstrate the need to better describe the composition and fate of OWCs in biosolids since about 50% of biosolids are land applied and thus become a potentially ubiquitous nonpoint source of OWCs into the environment.

Presence and distribution of wastewater-derived pharmaceuticals in soil irrigated with reclaimed water.

Three sites in the Front Range of Colorado, USA, were monitored from May through September 2003 to assess the presence and distribution of pharmaceuticals in soil irrigated with reclaimed water derived from urban wastewater. Soil cores were collected monthly, and 19 pharmaceuticals, all of which were detected during the present study, were measured in 5-cm increments of the 30-cm cores. Samples of reclaimed water were analyzed three times during the study to assess the input of pharmaceuticals. Samples collected before the onset of irrigation in 2003 contained numerous pharmaceuticals, likely resulting from the previous year's irrigation. Several of the selected pharmaceuticals increased in total soil concentration at one or more of the sites. The four most commonly detected pharmaceuticals were erythromycin, carbamazepine, fluoxetine, and diphenhydramine. Typical concentrations of the individual pharmaceuticals observed were low (0.02-15 microg/kg dry soil). The existence of subsurface maximum concentrations and detectable concentrations at the lowest sampled soil depth might indicate interactions of soil components with pharmaceuticals during leaching through the vadose zone. Nevertheless, the present study demonstrates that reclaimed-water irrigation results in soil pharmaceutical concentrations that vary through the irrigation season and that some compounds persist for months after irrigation.