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The primary objective of this study is to examine the relationship between early biomass smoke exposure and atopy among a cohort of children enrolled in a HAP-reducing chimney stove intervention trial among a population living in the western highlands of Guatemala. In the study community, women often carry their youngest child on their back during cooking, until the child is approximately 18 months old, exposing the newborn children to high levels of HAP. We hypothesize that the availability of a vented chimney stove would reduce the children’s HAP exposure compared to those who use open fires for cooking and would be associated with risks of allergic sensitization.Participating households and children were recruited from the Randomized Exposure Study of Pollution Indoors and Respiratory Effects cohort and its follow-up study, the Chronic Respiratory Effects of Early Childhood Exposure to Respirable PM study. Details of the RESPIRE and CRECER cohorts have been published elsewhere . Briefly, 518 rural Guatemalan women with newborn children who cooked exclusively over an open fire were recruited for the RESPIRE study between October 2002 and December 2004. Households were randomized to either receive a chimney stove , which improves combustion and uses a chimney to vent emissions outdoors, or to continue to cook with their typical open fires until the end of the trial, when they also received the intervention plancha stove. CRECER, the follow up study, took place from 2006 to 2009 and revisited RESPIRE households and recruited 169 new households that were from the same geographical region, hydroponic rack system exclusively used open fires, had one child in the same age range as the RESPIRE study children and one infant less than 6 months old .

For equity purposes, these new households received a chimney stove at the end of the CRECER study when all exposure and outcome information had already been collected. All households in the study thus received a chimney stove at different time periods: RESPIRE intervention households received the stove when the index children were less than 6 months old; RESPIRE control households received the stove when the index children were approximately 18 months old; new CRECER households received the stove when the index children were approximately 5 years old, and their proxy infant siblings were 18–24 months old. The grouping and study timeline are illustrated in Figure 1.The plancha stoves provided in this study reduced the children’s biomass smoke exposure by improving combustion and venting cooking smoke outdoors. Although this may result in an increase in outdoor exposure, we would expect participants in households with plancha stoves to have lower overall biomass smoke exposure, because the total amount of smoke produced would not increase, and outdoor biomass smoke would affect children for shorter duration and lower intensity. We also hypothesized that group 1 index study children would have the lowest cumulative biomass smoke exposure because they were provided the plancha stoves earliest, followed by groups 2 and 3, respectively. To test these hypotheses, we measured carbon monoxide exposure for the study children. Personal CO exposure was used as a proxy for personal biomass smoke exposure: CO has been shown to correlate well with fine particulate matter exposure in this population, in homes using open fires or chimney stoves. Study participants wore small, passive CO diffusion tubes for 48 h every 3 months during RESPIRE and every 6 months during CRECER. Since group 3 index study children did not have CO measurements obtained when they were <18 months of age, the personal CO exposures of their younger infant siblings were used as a proxy for their early life exposures. Details on exposure assessment methodology, validation, and quality control and assurance have been extensively described elsewhere.

We combined data from different measurements, including the aforementioned 48-h samples, to estimate cumulative CO exposure. We used RESPIRE CO tube measurements to estimate cumulative exposure during the first 600 days of life for groups 1 and 2 and used CRECER infant sibling CO tube measurements to estimate cumulative exposure during the first 600 days of life for group 3. Cumulative CO exposure from 600 days old to first allergy questionnaire was estimated based on CRECER CO tube measurements for the study children. We conducted a sensitivity analysis using three alternative calculations for cumulative CO exposure, two of which did not use the 600 days cut point. Details of these calculations can be found in the Table A1.Five rounds of skin prick tests were performed on 539 participants to determine allergic sensitization to six common indoor and outdoor aeroallergens . During each round of SPT, a positive control and a negative control were also performed on each child. SPT results were considered invalid if the histamine control was negative or the saline control was positive. Results from children who reported taking antihistamine or cold medications prior to testing were also excluded.Atopic symptoms were assessed via quarterly respiratory questionnaires completed by the study children’s mothers. The QRQs were conducted three times during CRECER to ascertain the occurrence of symptoms associated with asthma, allergic rhinitis, and eczema. The questions were developed based on the International Study of Asthma and Allergies in Childhood questionnaire. All QRQs were conducted by field workers fluent in the participating mothers’ primary language . Details of the questionnaire development and translation processes have been published elsewhere. All questions were close ended and began with “in the last three months”. A child’s final allergic outcome was recorded as positive if the mother reported him/her having positive symptoms in any of the three QRQ rounds.Logistic regression models were used to analyze the relationship between biomass smoke exposure and the risks of developing allergic outcomes.

Primary statistical analysis used study group as a categorical exposure variable based on the length of having a chimney stove in the household. Odds ratios and 95% confidence intervals were reported. In this analysis, group 1 is the baseline level, groups 2 and 3 represent intermediate and highest levels of biomass smoke exposure, respectively. Secondary statistical analysis used the estimated cumulative CO exposure as a linear continuous exposure variable. Age, sex, second-hand smoke exposure, the number of children in the family, kitchen structure , child’s average weekly temazcal use in minutes, number and species of pets and farm animals at home, maternal history of allergic outcomes, parental education, and socioeconomic status were collected from the CRECER baseline questionnaire, and were adjusted for in the logistic regression models. We did not adjust for race because the study population was homogenous, self-identifying as Mam indigenous.Among the 557 households participating in CRECER, 20 lacking valid SPT or QRQ results were excluded. For the two households with twins that both participated in the study, only the first child recorded was kept in the analysis to ensure independence among observations. Respiratory outcomes were available for 537 children, 188 from group 1, 192 from group 2 and 157 from group 3. Valid SPT results were available for 526 children, 184 from group 1, 187 from group 2 and 155 from group 3 . The quality and precision of the five rounds of SPTs during CRECER improved with additional training and more experience of the staff, indicated by the significantly lower number of invalid tests in the later rounds. As such, rolling tables grow the results of the last valid round of SPT for each child was recorded as the child’s final allergic sensitization outcome. Among the 526 children with valid SPT results, results for 496 participants were taken from the 5th round of SPT tests. The household-level and child-level demographic characteristics were similar among the three study groups , especially between groups 1 and 2 that were randomized in the RESPIRE study. Study children were on average 3.6 years old at the first QRQ.This prospective cohort study followed more than 500 children in rural Guatemala over 7 years and examined associations between cooking-related biomass smoke exposure and childhood atopy outcomes. Children from households that received chimney stoves when the children were approximately 5 years old had higher risks of maternal-reported allergic asthma and rhinitis symptoms compared to children from households that received a chimney stove intervention within the first 6 months of life. A 1 ppm-year higher cumulative CO exposure and its related cumulative biomass smoke exposure was associated with 6–12% higher odds of maternal reported allergic rhinitis and conjunctivitis symptoms. No significant association was found between biomass smoke exposure and eczema or skin prick test outcomes. Notably, the overall prevalence of sensitization to cockroach in this population of rural Guatemalan children was high , which is similar to that reported for inner city children in the U.S. A summary of main results from studies that looked at household biomass smoke exposure and allergic or respiratory outcomes is presented in Table A2.

Our finding that higher biomass smoke exposure was associated with maternal-reported respiratory symptoms such as wheezing, sneezing, nasal congestion and rhinorrhea in their children is consistent with other studies that looked at exposure to biomass stove use and self reported or clinically diagnosed respiratory diseases among children. Prior studies that looked at exposure to cooking and heating-related HAP and atopy outcomes in children residing in high-income countries did not find significant associations after adjusting for lifestyle and socioeconomic factors. In this study, we did not find a significant association between biomass smoke exposure and maternal-reported eczema symptoms. Additionally, there was no significant association between biomass smoke exposure and allergic sensitization as measured by SPT. Part of the reason might be that important windows for atopic sensitization such as prenatal exposures were not captured in the study. The number of cases of allergic sensitization to dogs, cats, and ragweed were small among the study children , resulting in compromised statistical power. The low number of dog and cat allergies might have been due to the high dog ownership and medium cat ownership in the study population : living in proximity to animals is associated with lower sensitization to allergens among children. No significant differences in allergic sensitization or symptoms were found between children in group 1 and group 2, among whom chimney stoves were installed around birth and around 18 months old, respectively. This might have been due to the gradual deterioration of chimney stoves during the 2-year gap between the RESPIRE and CRECER studies, during which group 1 might have been exposed to higher HAP than group 2 because of the older stoves. Another reason might be insufficient exposure reduction, which was also found in a previous analysis of the RESPIRE study: a larger reduction in mean CO exposure was associated with reduction in pneumonia risks, but the moderate difference in group mean CO levels between groups 1 and 2 was not enough to yield a statistically significant difference in pneumonia risk between the groups. The high percentages of reported allergic symptoms and high prevalence of cockroach sensitization among the children in this study is contrary to the “hygiene hypothesis” or “microbial deprivation hypothesis” that early life exposure to microorganisms shapes the Th1 , Th2, and regulatory T cell responses and alters immune response patterns. For instance, children exposed to enteric pathogens have higher resistance to allergic sensitization compared to those living in pathogen-free environments. While the study population was exposed to abundant microorganisms, it is possible that exposure to HAP prenatally, in early life, or even in reduced amounts after the stove upgrade intervention, could promote a shift toward Th2 responses and thus increase risk for atopy. Previous studies have demonstrated that exposure to PM2.5 may increase the risk of asthma via airway inflammation, increase in oxidative stress, changes in immune signaling, and subsequent disruptions of airway epithelial cells and mucosal barrier function. Studies on rhesus monkeys have also found that co-exposure to a pollutant that causes oxidative stress, ozone, and allergen altered airway structural development and increased risk of an asthma-like phenotype. Another consideration is that the increased wheezing and rhinitis symptoms reported by their mothers among children exposed to higher HAP could also be due to the direct irritating effects of biomass smoke to the upper and lower airway epithelium rather than an underlying allergic mechanism. During study design, we hypothesized that group 3 index study children would have the highest cumulative biomass smoke exposure because they were provided the upgraded chimney stoves the latest, thus were exposed to higher levels of biomass smoke for the longest period of time.

While this phenotype does not exclude more nuanced forms of social anxiety, it does support the idea that the modulation of stress reactivity and social behavior can be dissociable. In line with this notion, CB1 over expression in the medial prefrontal cortex alters social interactions without overtly changing the anxiety-related phenotype. In contrast to socially impaired mice, parallel experiments in normal mice indicated that increasing anandamide does not alter social approach. One explanation could be technical – namely, that the social approach test has a ceiling effect or is unable to capture more subtle qualities of social interaction. Indeed, the task is typically used as a screening tool rather than a continuous-scale measure of sociability. Another explanation could be biological – that signaling systems in a healthy brain are able to compensate for endocannabinoid enhancement in a way that socially impaired brains cannot. These explanations are in line with previous reports of different social situations in which faah-/- mice demonstrated increased direct reciprocal interactions, as well as URB597-treated juvenile rats engaging in more social play. Therefore, expanded investigation is warranted into how anandamide contributes to different social contexts and the qualities of social interactions. Nevertheless, our set of results suggests that the prosocial action of FAAH blockade is selective for social impairment in certain contexts, which may be therapeutically advantageous for the spectral nature of ASD. Based on our results and the available literature, plant grow trays we can reasonably speculate on two possible scenarios improved by anandamide signaling that may underlie social impairment in BTBR and fmr1-/- mice.

First, oxytocin-driven anandamide activity in the nucleus accumbens, which we previously demonstrated to be important for social reward, may be impaired in these mice. Consistent with this idea, BTBR mice were found to have abnormal oxytocin expression in the hypothalamus45. BTBR mice were also reported to be deficient in conditioned place preference to social interactions. However, because social conditioned place preference is a relatively new construct, and the learning impairments in these mice make interpretation problematic, further support from the literature is lacking. A second possible scenario is that anandamide might correct an imbalance of excitatory and inhibitory neurotransmission in the cortex, which has been postulated to underlie ASD. Enhancing GABAergic activity in BTBR mice ameliorates their social impairment, and negative allosteric GABA modulation in C57Bl6J mice recapitulates social impairment. This suggests that a loss of balance between inhibitory and excitatory activity might contribute to social impairment. A simplified view of this result orients us to interpret our findings as indicating that anandamide could modulate such balance. This view is consistent with the presence of CB1 receptors on presynaptic terminals of both glutamatergic projection neurons and GABAergic interneurons.In conclusion, the present study provides new insights into the role of endocannabinoid signaling in social behavior and validates FAAH as a novel therapeutic target for the social impairment of ASD.The use of marijuana, the most widely consumed illicit drug in the world, and synthetic cannabinoid drugs typically starts in adolescence. Its usage pattern is striking. According to the 2014 NIDA survey, Monitoring the Future, past-month usage of marijuana was at 6.5% among 8th graders, 16.6% among 10th graders, and 21.2% among 12th graders . The percentage of high school seniors who think marijuana is harmful has declined from 52.4% five years ago to only 36.4% today, indicating a possible prelude to increasing usage in the coming years .

In addition to recreational use, the accelerating prevalence of cannabinoids as a medicine, e.g. in child epilepsy and autism, is exposing growing numbers of young people to the drug. These usage trends are especially concerning within a rapidly changing social sphere, consisting of technological advancements in marijuana cultivation and cannabinoid synthesis, widespread cultural acceptance, as well as novel politics and economies. Long-term effects are likely as a consequence of the roles of the endocannabinoid system in the developing brain. This signaling system has three main components: two lipid-derived local messengers – 2-arachidonoyl-sn-glycerol and anandamide , enzymes and transporters that mediate their formation and elimination, and cannabinoid receptors that are activated by endocannabinoids and regulate neuronal activity . Endocannabinoid activity regulates processes such as neuronal genesis, migration, and differentiation, as well as in synaptic pruning and glial cell formation . With regard to the adolescent period, developmental fluctuations have been reported on the expression levels of CB1 cannabinoid receptors in corticostriatal areas and, to a lesser extent, levels of endocannabinoids. For example,levels of CB1 receptors increased in the cortex and striatum during this period , while 2-AG is reduced in these regions and anandamide continuously increases in the prefrontal cortex . These lines of evidence each emphasize the vulnerability of the developing brain to consequences of early cannabinoid exposure. However, they also highlight the knowledge gap regarding the molecular mechanisms responsible for these consequences, especially with regard to brain reward systems . In fact, it remains unknown whether disrupted endocannabinoid signaling is responsible for impairment. We hypothesized that non-physiological activation of cannabinoid receptors during adolescence persistently impairs the endocannabinoid regulation of social reward in early adulthood.We first administered a synthetic cannabinoid receptor agonist, WIN55212-2 for 2 weeks to adolescent mice at postnatal day. We chose the WIN compound at this dose because it models the effects of marijuana on cannabinoid receptors over other bio-active constituents. After a two-week washout period, at PND58, we found that whereas vehicle-treated mice developed a social place preference, WIN treated mice developed no such preference . This result suggests that improper cannabinoid receptor activation during adolescence impairs the later expression of social reward in adulthood.It is reasonable to expect that socially stimulated anandamide mobilization, which we previously found to be important in social reward , would also be disrupted by nonphysiological activation of cannabinoid receptors in early life. Indeed, we found that after a twoweek washout, at PND58, mice previously treated with WIN had increased levels of anandamide in the nucleus accumbens , medial prefrontal cortex , and ventral hippocampus . Thus, socially stimulated anandamide signaling in these regions may no longer occur properly following WIN treatment.These preliminary results require additional confirmation. Nevertheless, the results are a proof in principle that early, custom grow rooms non-physiological activation of cannabinoid receptors can persistently impair social reward, and that this impairment is due to an underlying disruption in anandamide signaling.

Future investigation should be aimed at: the critical window that leads to persistent impairment; other persistent molecular changes, such as 2-AG levels and CB1 surface expression; and whether oxytocin-stimulated anandamide signaling is also impaired. These investigations would provide a more complete picture of the molecular changes responsible for persistent impairment. They would be relevant to the long-term effects of early cannabinoid exposure on mental illness and addiction.Social interactions and social support protect against physical pain. Such protection exerts powerful effects in adaptive as well as pathological contexts, with profound consequences for pain and pain-related health outcomes . Basic neural mechanisms linking a social signal to an analgesic one, however, are poorly defined. We recently identified a mechanism through which social stimulation mobilizes the endocannabinoid neurotransmitter anandamide, which in turn enhances the saliency of the social interaction . As endocannabinoid signaling is known for its analgesic effects independently of social factors, we therefore tested the idea that social stimulation might also recruit the endocannabinoid system as a mediator of analgesia. We targeted a key area for descending pain modulation, the periaqueductal grey .We used juvenile rats in order to analyze subdivisions of the periaqueductal grey and because of previous investigation of opioids in separation distress . These models, however, have not been extensively replicated. In preliminary experiments, we isolated juvenile rats for 24 h, then re-socialized half while keeping the other half isolated for 3 additional hours. This social stimulation protocol is consistent with that which elicits an increase in anandamide in the mouse nucleus accumbens, ventral hippocampus, and medial prefrontal cortex . We found that acute social stimulation in this manner increases levels of anandamide in the ventral periaqueductal grey , but not the dorsal PAG . In a survey of other regions, we found that the cingulate and insular cortices trended toward an increase, but these results were not significant, and the nucleusaccumbens did not show a change in anandamide . Paralleling this phenomenon, we used the hot-plate test to assess for acute pain response. Animals were placed inside a large beaker which had been heated to 55 degrees on a hot plate, and their latency to withdrawal or lick their hindpaw was measured. We found that social stimulation increased the threshold for paw withdrawal . These results suggest that social stimulation induces anandamide-mediated endocannabinoid signaling in order to exert social protection against pain.These preliminary experiments illustrate the concept that socially mobilized endocannabinoid signaling is recruited to buffer against physical pain. Further investigation should be directed toward: establishing the stimulation protocol, as the profile of endocannabinoid changes between mice and rats are clearly different – it is possible that rats may be more sensitive to the rewarding effects of play and thus require a shorter stimulation; this would be needed to understand whether endocannabinoids modulate an affective component of pain through reward signaling, or actually modulates descending pain at the level of the periaqueductal grey; whether isolation and social stimulation are qualitatively different or simply opposite effects; the confounding effects of stress on pain; and whether endocannabinoid signaling elicited here is also oxytocin-stimulated and, if so, the responsible circuitry.According to the United Nations, the population of the world is expected to grow in the next century, which in turn encourages the development of innovative techniques to ensure agricultural sustainability. Agriculture on productive land is threatened not only by high levels of urbanization, uneven water distribution, and inclement weather, but also is threats to biodiversity that have unfavorable environmental impacts. Due to the anticipated drastic population growth and constraints on resources in the upcoming decades, only 10% of the demand for food is estimated to be met by expansion of productive lands, with the remainder relying on new techniques that can achieve higher yields. Therefore, developing novel methods to augment the ratio of crop production over used land is a vital issue. In recent years, the indoor vertical farming systems with artificial light are found to be a viable solution to resolve the in-creasing demands of future agricultural products. The IVFS are promising alternatives to open field or greenhouse agriculture because they have precisely monitoring environmental parameters and are insensitive to outdoor climates, which can boost annual sales volume per unit area up to 100 times compared to that of open lands. Furthermore, employment of light emitting diodes as light sources can initiate and sustain photosynthesis reactions and the optical wavelength, light intensity, and radiation intervals can further enhance growth quality . Recently, many studies have been carried out to investigate how environmental parameters, such as closed-loop control, ultrasound, and electro-degradation, affect hydroponic cultivation of leafy vegetables in these systems. One of the most influential factors affecting growth in IVFS is to maintain a uniform air flow at an optimal air current speed over plants canopy surfaces. Poor flow uniformity or variation in air velocity over culture beds destabilizes crop production rates. It has been found that inducing a horizontal air speed of 0.3–0.5 m s−1 boosts photosynthesis through more efficiently exchanging species between the stomatal cavities in plants and the flow of air. Lee et al. studied the effects of air temperature and flow rate on the occurrence of lettuce leaf tip burn in a closed plant factory system. Furthermore, it was observed that the relative humidity of the air flow can significantly influence calcium transportation in lisianthus cultivars. According to Vanhassel et al., higher levels of relative humidity can significantly decrease the occurrence of tip burn. Therefore, it is vital to maintain relative humidity in the desired range to ensure even distribution of calcium in lettuce leaves. Over the past few years, researchers have been trying to develop techniques for improving uniformity over cultivation zones. Regardless of the recent progress, the control and automation systems of IVFS bring additional costs, which makes systematic experimental investigation and optimization a challenge. Computational fluid dynamics has been utilized as a reliable tool to numerically simulate complex physical phenomena. Markatos et al. developed a CFD procedure to study velocity and temperature distribution in enclosures using buoyancy-induced physics.

In humans, marijuana can either facilitate or impair social interactions and social saliency, possibly depending on dose and context . Analogously, in animal models, cannabinoid receptor activation with direct-acting agonist drugs disrupts social interactions, whereas FAAH inhibition enhances them, which is suggestive of a role for anandamide in socialization . While important, these data leave unanswered two key questions. The first is whether the anandamide, whose functions in the modulation of stress-coping responses are well recognized , might influence social behavior by modulating stress reactivity . We addressed this question with two complementary sets of experiments. In one, we used a model of socially conditioned place preference that focuses specifically on the acquisition of incentive salience . Mice were conditioned to social contact with familiar cage-mates for 24 h, an intervention that does not cause stress. When this conditioning procedure was paired with FAAH inhibition, or faah-/- mice were used instead of wild-type mice, the animals displayed a markedly increased preference for the social context. In separate studies, we evaluated the impact of genetic FAAH deletion or pharmacological FAAH blockade in the social approach task, in which mice are given the option of interacting with a novel conspecific or a novel object for a relatively short period of time . Socially normal mice spend more time with their conspecific than with the object. We found that neither pharmacological nor genetic FAAH blockade had any effect in this model.

Collectively, hemp drying racks our findings support the conclusion that anandamide signaling at CB1 receptors specifically regulates the incentive salience of social interactions, and that this effect is independent of anandamide’s ability to modulate anxiety. A second question addressed by the present work pertains to the neural circuits responsible for recruiting endocannabinoid neurotransmission during socialization. We used convergent experimental approaches to show that OTR activation by endogenously released oxytocin triggers anandamide mobilization in the NAc. This result is consistent with evidence indicating that oxytocin acts as a social reinforcement signal within this limbic region, where it elicits a presynaptic form of long-term depression in medium spiny neurons . Thus, a plausible interpretation of our findings is that oxytocin triggers an anandamide-mediated paracrine signal in the NAc, which influences synaptic plasticity through activation of local CB1receptors. Activation of these receptors is known to induce presynaptic LTD at corticostriatal synapses . While providing an economical explanation for our results, the hypothesis formulated above also raises several new questions. Particularly important among them are questions about the roles that other modulatory neurotransmitters may play in regulating the interaction between oxytocin and anandamide. Previous studies point toward serotonin, which is needed for the expression of oxytocin-dependent plasticity in the NAc , and dopamine, which has been implicated in striatal anandamide signaling . Defining such roles will require, however, further investigation. In conclusion, our results illuminate a novel mechanism underlying the prosocial actions of oxytocin, and provide unexpected insights on possible neural substrates involved in the social facilitation caused by marijuana.

Pharmacological modulation of oxytocin-driven anandamide signaling – by utilizing, for example, FAAH inhibitors – might open new avenues to treat social impairment in autism spectrum disorders.We dissolved URB597 and AM251 in a vehicle of saline/propylene glycol/Tween-80 . L-368,899 was dissolved in saline for intraperitoneal injections or in DMSO for intracerebroventricular injections. WAY-267464 was dissolved in DMSO. Clozapine-N-oxide and cocaine were dissolved in saline. For lipid analyses, L-368,899 was administered 0.5 h before the start of socialization , WAY-267464 was administered by i.c.v. injection to 24-h isolated animals 0.5 h prior to sacrifice, and CNO to 24-h isolated animals 1 h before sacrifice. For the socially conditioned place preference test, animals were habituated to injections for 3 days leading up to the experiment. URB597, L-368,899 and AM251 were administered twice a day during social conditioning . Balancing vehicle treatments were given during isolation conditioning . For the social approach task, URB597 was administered i.p. 3 h before starting the test.We anesthetized mice with ketamine-xylazine and stereotaxically implanted a 22-gauge guide cannula positioned 1 mm above the right lateral ventricle at coordinates from bregma: AP -0.2, ML -1.0 and DV -1.3 mm . Animals were allowed to recover 10 days after surgery, during which they were maintained in social housing . Infusions were made in wake animals through a 33-gauge infusion cannula that extended 1 mm beyond the end of the guide cannula. The injector was connected to a 10- µL Hamilton syringe by PE-20 polyethylene tubing. The syringe was driven by an automated pump at a rate of 0.66 µL/min to provide a total infusion volume of 0.5 µL. Cannula placements were verified histologically.We used a serotype 2 AAV vector that was previously constructed and validated to express a modified muscarinic receptor and the fluorescent protein mCherry .

The hM3Dq receptor is exclusively activated by the otherwise inert compound clozapine-N-oxide. Expression of the virus is restricted by the oxytocin promoter, which directs oxytocin cell-specific expression of hM3Dq receptors and mCherry. mCherry expression in the paraventricular nucleus was verified using a Leica 6000B epifluorescence microscope.We bilaterally injected the AAV2-oxt-hM3Dq-mCherry construct into the PVN using the following coordinates: AP -0.70, ML ±0.30 and DV – 5.20 mm. We used an adaptor with 33-G needle connected via polyethylene tubing to a 10-µL Hamilton syringe driven by an automated pump . We waited for 5 min prior to infusion in order for tissue to seal around the needle, infused a total volume of 0.5 µL over 5 min and waited 10 min after infusion before removing the needle. Experiments were conducted 3 weeks after viral injections to allow for recovery and adequate expression. During this period, animals were maintained in social housing .Whole brains were collected and flash-frozen in isopentane at -50 to – 60 °C. Frozen brains were maintained in liquid nitrogen on the day of sacrifice until they were transferred to storage at -80 °C. To take micropunches of brain tissue, we first transferred frozen brains to -20°C in a cryostat and waited 1 h for brains to attain local temperature. We then cut to the desired coronal depth and collected micropunches from bilateral regions of interest using a 1×1.5-mm puncher . The micropunches weighed approximately 1.75 mg. A reference micropunch was taken to normalize each punch to the brain’s weight. Bilateral punches were combined for lipid analyses.Procedures were described previously . Briefly, tissue samples were homogenized in methanol containing internal standards for H2 -anandamide , H2 -oleoylethanolamide and 2 H8-2-arachidonoyl-sn-glycerol . Lipids were separated by a modified Folch-Pi method using chloroform/methanol/water and open-bed silica column chromatography. For LC/MS analyses, we used an 1100 liquid chromatography system coupled to a 1946D-mass spectrometer detector equipped with an electrospray ionization interface . The column was a ZORBAX Eclipse XDB-C18 . We used a gradient elution method as follows: solvent A consisted of water with 0.1% formic acid, and Solvent B consisted of acetonitrile with 0.1%formic acid. The separation method used a flow rate of 0.3 mL/min. The gradient was 65% B for 15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. Under these conditions, Na+ adducts of anandamide/H2 -anandamide had retention times of 6.9/6.8 min and m/z of 348/352, OEA/H2 -OEA had Rt 12.7/12.6 min and m/z 326/330, and 2-AG/2 H8-2-AG had Rt 12.4/12.0 min and m/z 401/409. An isotopedilution method was used for quantification .Ketamine-xylazine anesthetized mice were perfused through the heart left ventricle, industrial rolling racks first with ice-cold saline solution and then with a fixation solution containing 4% paraformaldehyde in 0.1 M phosphate buffered saline . Brains were collected, post-fixed for 1.5 h and cryoprotected using 30% sucrose in PBS. Coronal sections were cut using a microtome and mounted on Superfrost plus slides . For cFos immunostaining, sections were washed in 0.1 M glycine solution to quench excess PFA. Sections were incubated for 1 h in blocking solution . Washed sections were incubated for 48 h at 4°C with anti-cFos antibody .

After washing with 0.1M PBS to remove unbound primary antibody, sections were then incubated for 1 h at room temperature with donkey anti-rabbit IgG conjugated to Alexa Fluor 594. Slides were cover-slipped with Vectashield plus DAPI . Images were captured using a 10x objective on a Leica 6000B epifluorescence microscope with PCO Scientific CMOS camera and Metamorph acquisition software.cFos quantification. Image montages were stitched together using FIJI . Variability in background fluorescence was standardized by subtracting a gaussian-blurred image of each image from itself. Objects of cellular size and shape were then detected using Python 2.7 and FIJI. Brain regions were traced by hand in FIJI using an atlas reference , and resulting coordinates were used to restrict cell counts. Because immunostaining varies across animals and experiments, values were normalized as a ratio to the dorsal striatum of the same animal. The dStr was selected as an internal control because it did not vary across compared groups . Socially conditioned place preference . Following previously described procedures , mice were placed in an opaque acrylic box , divided into two chambers by a clear acrylic wall with a small opening. In the box, a 30-min pre-conditioning test was used to establish baseline non-preference to two types of autoclaved, novel bedding , which differed in texture and shade . Individual mice with strong preference for either type of bedding were excluded – typically, those that spent more than 1.5x time on one bedding over the other. The next day, animals were assigned to a social cage with cage-mates to be conditioned to one type of novel bedding for 24 h, then moved to an isolated cage with the other type of bedding for 24 h. Bedding assignments were counterbalanced for an unbiased design. Animals were then tested alone for 30 min in the two-chambered box to determine post conditioning preference for either type of bedding. Fresh bedding was used at each step and chambers were thoroughly cleaned between trials with SCOE 10X odor eliminator to avoid olfactory confounders. Volumes of bedding were measured to beconsistent – 300 mL in each side of the two-chambered box and 550 mL in the home-cage. Animals from the same cage were run concurrently in four adjacent, opaque CPP boxes. Scoring of chamber time and locomotion were automated using a validated image analysis script in ImageJ – the static background image was subtracted, moving objects of mouse shape and size were thresholded out and frames were counted in a position restricted manner.Three-chambered social approach task. Test mice were habituated to an empty three-chambered acrylic box , as previously described . Habituation consisted of a 10-min trial in the center chamber with doors closed, and then a 10-min trial in all chambers with doors open. Then, during the 10-min testing phase, subjects were offered a choice between a novel object and a novel mouse in opposing side-chambers. The novel object was an empty inverted pencil cup and the novel social stimulus mouse was a sex, age and weight matched 129/SvImJ mouse. These mice were used because they are relatively inert, and they were trained to prevent aggressive or abnormal behaviors. Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting was used – all chambers were measured to be 5 lux. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator between trials to preclude olfactory confounders. Object/mouse side placement was counterbalanced between trials. Chamber time scoring was automated as in social CPP. Subjects with outlying inactivity or side preference were excluded.Cocaine and high-fat diet CPP. These paradigms were largely similar to social CPP, including unbiased and counterbalanced design, cleaning and habituation, exclusion criteria, and scoring, except for the following key differences which followed established methods . Mice were conditioned and tested in a two-chambered opaque acrylic box with a small opening. Pre- and post-conditioning tests allowed free access to both chambers and each had durations of 15 min and 20 min . For conditioning, animals underwent 30-min sessions alternating each day between saline/cocaine or standard chow pellet/high-fat pellet . The two chambers offered conditioning environments that differed in floor texture and wall pattern – sparse metal bars on the floor and solid black walls vs. dense-wire-mesh floors and striped walls.

Complete centroids were generated for 42 monitored fishers, 12 fishers from the northwestern California population and 30 from the southern Sierra Nevada population . Of these fishers, 3-month MCP centroids were generated for 39 fishers, and 6-month centroids for 27 . Spatial analysis for 6-month centroids from the KRFP could not be conducted because all fishers in the data set were AR exposed. Sixteen fishers were excluded from the analysis due to lack of monitoring data. No spatial clustering of AR exposure was detected for any of the temporal periods, specific AR compounds, generation class of AR, or distribution of numbers of ARs per fisher in any of the study areas .Cause-specific mortality factors for all 58 fishers sampled ranged widely and included predation, infectious and non-infectious disease processes and vehicular strikes . The cause of death for four of these fishers was attributed to lethal toxicosis, indicated by AR exposure with simultaneous coagulopathy and bleeding into tissues or cavities and ruling out any concurrent processes that might cause hemorrhaging. Two of the four fishers killed by ARs were from the southern Sierra Nevada population, and two were from northern California and the case details are described below.An adult male fisher was recovered on 15 April 2009, grow racks with lights in the southern Sierra Nevada at the SNAMP project area. The fisher showed no signs of predation or scavenging . Gross necropsy determined that the fisher was in good nutritional and fair postmortem condition.

Frank blood was observed in both the thoracic and abdominal cavities , and in the pericardial sac . The stomach and lower gastrointestinal tract contained some blood but no prey or formed feces, and no mucosal changes were noted. There were no other findings on gross examination. Histopathologically, no significant changes were observed in any tissues. Brodifacoum and BRM were detected and quantified in the liver sample at 0.38 ppm and 0.11 ppm, respectively, and CHL at trace levels . The second fisher mortality was a lactating adult female recovered on 2 May 2010 in the center of a paved rural highway in the SNAMP project area approximately 3.7 km from Yosemite National Park. Vehicular strike was initially suspected as the cause of mortality due to the location of the carcass but lacerations, abrasions and visual evidence of trauma were not seen on gross examination of the intact carcass. The post-mortem state of the carcass was good and the nutritional state was poor . Shallow subcutaneous hemorrhage was noted over the hindquarters and spinal column with no associated fractures, punctures or abrasions. There was approximately 20 ml of frank blood within the thoracic cavity. There was no evidence of pneumothorax, vessel ruptures, or visceral tearing. No blood or visceral damage was seen in the abdominal cavity. Stomach contents contained various rodent parts with formed feces in the descending colon. Histopathologically, no significant changes were observed in any tissues. Brodifacoum and BRM were detected and quantified at 0.60 ppm and 0.17 ppm, while one first generation AR, DIP was detected at a trace level within the liver tissue .

No evidence was present to suggest that this fisher died due to vehicular trauma, despite its location on the highway.A sub-adult male fisher was recovered on 4 May 2010 at the base of several riparian shrubs near a watercourse in northwestern California at the HVRFP. Severe ectoparasitism on the carcass was noted in the field with ticks in both replete and non-replete stages. Predation was not suspected due to absence of external wounds. The gross necropsy determined that this fisher was in poor nutritional condition with no subcutaneous or visceral fat. Frank blood was present in the right external ear canal, nasal and oral cavities, within the lumen of the trachea and within the periorbital tissue with no associated skull fractures or punctures.The stomach was devoid of prey. The colon only contained semiformed feces. Ectoparisitism was severe with approximately 48 female and 10 male American dog ticks and 8 female and 2 male western black-legged ticks removed from various regions of the fisher. The liver sample from this fisher had quantifiable levels of BRD at 0.04 ppm as well as a trace level of CHL . The second northern California fisher AR death, was an adult male recovered on 26 May 2010 at the HVRFP. Field observations included no evidence of predation or scavenging. The nutritional state as well as the postmortem condition were poor. Gross necropsy determined that the fisher had no body fat present in any of the tissues. Frank blood was present in both thoracic and abdominal cavities. The stomach contained red and black fluid but no prey. Ectoparasitism was severe with 204 female and 27 male adult American dog ticks in both replete and non-replete stages on areas of the muzzle, chest, tops of fore-and hind-limbs as well as inguinal sections. Severe nematodiasis was seen in skeletal muscle throughout the body . Pulmonary nematodiasis was also noted in the marginal portions of the lungs. Histopathologically, no notable disease processes were seen but severe parasitism was noted. The liver sample for this fisher had quantifiable levels of BRD at 0.61 ppm and trace levels of BRM .Necropsies and AR testing was performed on four kits who were all still dependent on mother’s milk when they died following maternal abandonment from their mothers death. One kit, a female fisher from KRFP tested positive for AR exposure.

This kit was approximately six weeks of age and was recovered within a monitored maternal den tree shortly after maternal abandonment. Cause of death was determined to be acute starvation and dehydration. The liver tissue contained trace level of BRD but there was no associated hemorrhaging in any tissues, body cavities or lumina, suggesting that this finding was not clinically significant.Our findings demonstrate that anticoagulant rodenticides, which were not previously investigated in fishers or other remote forest carnivores, are a cause of mortality and may represent a conservation threat to these isolated California populations. This isthe first documentation of exposure to ARs and of direct mortality from ARs in fishers anywhere in their geographic range. Earlier studies suggest ARs posed little or no additive mortality effects on non-target populations. The shortfall of many of these studies was the utilization of common cosmopolitan species so they did not take in consideration that AR mortality may be additive in otherwise compromised populations. The spatially ubiquitous exposure observed within all post-weaning age classes and across the project areas in their contemporary range in California is of significant concern especially considering the recent work of Spencer et al. , who demonstrated that even a small increase in human-caused mortality of 10–20% in the isolated Southern Sierra Nevada fisher population would be enough to prevent population expansion if other restrictive habitat elements were removed. The high rate of exposure to second generation AR compounds in these populations is surprising and cause for concern. This generation of ARs are not only more acutely toxic, but have long retention through biphasic elimination in mammal tissues. Second-generation ARs are more toxic because death can occur from a single primary ingestion by a rodent. However, rolling benches for growing rodents can receive a lethal dose of second-generation ARs in one feeding bout and it can take up to 7 days before clinical signs manifest. Therefore, prey that have consumed a ‘‘super-lethal’’ dose of AR can pose a substantial risk to predators for several days prior to death. In one study, a group of Norway rats was given a choice between BRD bait and untreated food and another group had access only to the BRD bait. Both groups consumed 10 and 20 median lethal doses on the first day and 40 to 80 LD50 doses by day 6.5, respectively. If sources for these toxicants are maintained for even short periods, exposed rodents, the main prey source for fishers in these populations can pose significant threats to their predators. Many manufactures use ‘‘flavorizers’’ since the AR compound may be bitter and unpalatable to rodent pests. Emulsions used to increase palatability include sucrose, bacon, cheese, peanut butter, and apple flavors , and thus could be palatable to generalist carnivores like fishers. Although we did not visually detect AR bait in the stomach or GI tracts of any fishers that died, primary poisoning cannot be completely ruled out.In addition to the risk from lethal toxicosis, sub-lethal AR exposure may compromise fishers through a reduction in the function of normal clotting. The occurrence of AR -exposed wildlife dying from minor wounds that otherwise might have easily resolved themselves if ARs were not present suggests contributory lethal effects. Several cases describe raptors receiving minor defensive lacerations or trauma from prey that lead to the raptor’s death by exsanguination or hemorrhaging.

Fishers actively pursue a wide array of terrestrial and arboreal prey. Hence, it is conceivable that a fisher could receive similar wounds or trauma from prey, or during the pursuit of prey. Consequently, if clotting mechanisms were compromised due to ARs, benign injuries could lead to serious complications. The leading causes of mortality within the USFWS DPS is intraguild predation . It is possible that some of these cases, AR exposure could have compromised clotting mechanisms at the predation attempt and this deserves further study. High levels of tick infestations were noted in two of the AR mortalities when compared to other sympatric species within the same project area. In addition, locations of of these replete ticks were in infrequent regions in other captures, most likely due to a lack of regular grooming. Whether ARs played a role by allowing more ticks to obtain a blood meal due to immobilization due to compromised clotting factors is unknown. Furthermore, sublethal AR exposure may decrease an animal’s resilience to environmental stressors. In a study on rabbits and rats subjected to stressors such as severe decreases in ambient temperature , approximately 10% of test animals died; however when animals were exposed to low non-lethal doses of anticoagulants and subjected to the same stressors, mortality rates increased to 40–70%. It is unknown if stressors or injuries from environmental, physiological or even pathogenic factors could predispose fishers to elevated mortality rates when coupled with AR exposure.The documentation of neonatal or lactational transfer of AR to a dependent fisher kit was unexpected, and the effects of AR exposure to a kit during fetal development or shortly after birth are unstudied. AR exposure in pregnant or whelping domestic canids varied, causing no clinical signs in some cases but death due to coagulopathy immediately after delivery in other cases. The female fisher who gave birth to this kit did not exhibit clinical signs at pre- or postpartum captures and monitoring of her maternal den site verified that one kit survived from that litter . Nevertheless, clinical signs including hemorrhaging, inappetence and lethargy have been seen in domestic canid puppies of AR-exposed mothers. Mild to severe manifestations such as low birth weight, stillbirth or eventually neonatal death has been documented in several cases. In one human study where pregnant women received low doses of warfarin due to severe risk of thromboembolic events, 33% of them had stillbirths, 28% had abortions, and 11% of the neonates died shortly after birth. The range for congenital anomalies and miscarriages in pregnant females for prescribed doses of warfarin varied from 15 to 56% and long-term neurological symptoms have been reported in children that were exposed in-utero. In addition, because fishers exhibit delayed implantation of the blastocyst, whether ARs may cause pregnant females to abort or reabsorb the fetus merits further research. The transfer of first generation ARs from mother to offspring in milk is not well-understood and there are no data on lactational transfer of second-generation ARs.The quantity of AR we observed in fisher liver tissues varied and overlapped extensively in both sublethal and lethal cases with no clear indication of a numeric threshold that might indicate an amount leading to morbidity or mortality. This lack of predictive ability has been shown in numerous wildlife cases. For example, Brodifacoum, the most prominent AR compound detected in fishers in this study ranged considerably in lethal cases among individual mustelid species, with 0.32– 1.72 ppm in stoats, 0.7 ppm in least weasels, 1.47–1.97 in ferrets and 9.2 ppm in American mink.

Our verification efforts, confined to 47 delivery businesses serving 4 diverse block groups, found that only half actually delivered to designated locations. Cao et al. show value in combining listings from multiple crowd sourced platforms plus official license listings to increase sensitivity and specificity, but the address search function appears unique to Weedmaps: official license listings and other websites do not indicate the region to which each retailer delivers. Duplicative listings were also more common for delivery retailers than for brick-and-mortar outlets. Listings of delivery services may therefore require special de-duplication procedures. Retailers reporting to deliver statewide—and those operating exclusively through websites, apps, or text messaging—may require additional verification. Further, missingness must be assessed: although Weedmaps’ name recognition means that businesses are strongly incentivized to list themselves, some delivery businesses may not register on Weedmaps, and reported availability of home delivery should be compared to that from individual companies offering home delivery . The challenges for measuring cannabis delivery are also conceptual. Standard methods assume that availability is fixed in space and time, drain trays for plants based only on physical proximity between residential addresses and brick-and-mortar outlets. Yet we found that home delivery varies dynamically as a function of staffing levels, order sizes, time of day, competition, and demand.

Researchers will therefore need to adopt standard definitions sensitive to this variation in availability, along with differences in price between the two sources. In the absence of fixed geographic delivery zones, researchers must think carefully about how to operationalize cannabis availability in light of home delivery by revising existing conceptual models . Different conceptualizations of cannabis availability for brick-and-mortar versus delivery may also have differing implications for cannabis use and related harms. Whether such differences exist and why should be evaluated. In future research, consumer surveys of cannabis purchasing patterns should be used to quantify variation in home delivery utilization and to validate Weedmaps data. Such surveys, although costly, should also examine the implications of home delivery for enforcement of public health regulations. As a proprietary business, Weedmaps could change its data on delivery services at any time. Researchers should therefore encourage state regulatory agencies to monitor the geographic footprint of cannabis delivery businesses by incorporating defined delivery zones as a component of licensing. Other states permitting home delivery do so only with restrictions such as these. Monitoring the scale of the cannabis home delivery is warranted because more states are legalizing cannabis. Growing availability of cannabis through home delivery could translate into increases in cannabis-related benefits and harms. Methodological work to measure home delivery also has implications for other commercial substances, including alcohol and tobacco, for which home delivery is also increasingly common, understudied, and challenged by measurement.

Generalizabilty of this study may be limited because the scale of home delivery in California is unique. Thus, varying contexts must also be evaluated, as the accuracy of our method may vary by social context and cannabis market characteristics.Humboldt County has been an epicenter of cannabis cultivation for decades, and an element of social division has characterized the region: the “back-to-the-landers” versus the born-and-raised locals, the “hippies” versus the “rednecks,” and the pot growers versus the loggers and ranchers . However, as cannabis cultivation has been decriminalized in California, the social dynamics around cannabis have become more complex. Over the last 20 years, new growers from different parts of California, the United States and even outside the United States have moved to Humboldt County and surrounding areas to grow cannabis — a so-called green rush of growers hoping to strike it rich . Growers have come from a host of countries beyond the United States, including Bulgaria, Russia, Mexico and nations in Southeast Asia . Some work independently while others work together in operations that may qualify as more organized. For many Humboldt County residents — “mom-and-pop” cannabis growers and more traditional agricultural producers alike — the near-exponential growth of the industry has been a shock, and it has unleashed numerous social, economic and environmental concerns.This situation is not unique to Humboldt — cannabis cultivation has increased rapidly throughout rural California . California voters, when they legalized cannabis for adult recreational use in 2018, created conditions for competition among agricultural interests and changes in rural social dynamics.

Indeed, because new cannabis farming is often conducted near traditional ranching and timber-producing lands , the potential for conflict — or collaboration — between traditional land uses and cannabis production has grown. But little research documents the effects of cannabis production on traditional agricultural producers, and therefore we know little about such producers’ adaptation to change. Understanding this dynamic is important for local governments as they develop land use policies to govern when, where and how much cannabis production is permissible . Cannabis production’s effects on neighbors is an important point for local government officials to consider as they develop and adopt new policies to encourage the transition of black-market cannabis operations into compliant operations. The effects of cannabis production on neighbors is also important to consider while formulating policies to mitigate unintended consequences — such as unwanted odors and nighttime lights — which can exacerbate land use and social conflicts. For example, should cannabis be allowed on lands zoned for timber production or prime agriculture? Should cannabis production be allowed in cities and in unincorporated towns? What areas are compatible or incompatible with cannabis? Increased cannabis production can directly or indirectly affect traditional agriculture and timber producers. Over the last decade, cannabis cultivation has expanded rapidly in rural communities, with many cannabis farmers having moved only recently to the areas where they grow . These new arrivals are sometimes described as green rush growers. Conflicts can arise if new growers, who are often unaware of community norms, don’t manage workers appropriately, control dogs, close gates, help maintain shared roads — or if, in other ways, they complicate operations for traditional agricultural producers. Likewise, even cannabis producers who have been in business for many years — including some whose families have grown cannabis for two generations — may hold different views of rural life than do traditional agriculture and timber producers . In addition, while cannabis is now legal in California, many cannabis farmers still grow outside the regulated system, and some traditional agricultural producers may retain the sense that illegal activity is negatively affecting their community. In recent years, the environmental impacts of cannabis cultivation have been a matter of increasing focus in California, and traditional agricultural producers and other community members have voiced concerns about water diversions , pollution from chemical fertilizers , the impacts of pesticides on wildlife , light pollution and forest fragmentation . Concerns have also arisen regarding negative impacts on local livestock producers and challenges for public land managers attempting to control trespass growing operations . At the same time, 4 x 8 grow tray cannabis cultivation can contribute to community well-being in a variety of ways. It can bring economic gains to rural areas where the timber, livestock and fisheries industries have experienced declines. For example, cannabis cultivation can provide new business opportunities to traditional agricultural producers in the form of heavy equipment work, firewood sales, trucking, forest management or construction services. In addition, cannabis production may help buffer population declines such as those experienced in many of California’s rural areas over the last 20 years; in particular, rural schools may benefit from the enrollment of cannabis growers’ children. More broadly, cannabis farmers can bring new energy to rural communities through engagement at schools, volunteer fire departments and other points of gathering. Traditional growers’ perceptions of cannabis farmers can vary based on several factors, including the scale at which cannabis farmers operate. Scales of operation have expanded greatly over the last 20 years. Some cannabis farmers produce a few plants for personal use, others augment their incomes by growing moderate amounts of cannabis and still others grow on an industrial scale, with multiple operations on numerous parcels. One might expect traditional agricultural producers to regard these different varieties of cannabis growers differently. But large landowners are themselves not homogenous — for example, some are absentees.

In this research we hypothesized that absentee landowners would have different experiences and perceptions of the cannabis industry than do traditional producers who live on their land. Humboldt County and many communities around California are currently setting ordinances to manage legal cannabis production. But as they do so, little is known about the potential interaction of cannabis with traditional agriculture and timber producers and whether these industries are compatible. Information about the effects of cannabis production on traditional agricultural producers may be helpful to policy makers because traditional producers are often important contributors to rural economies and stewards of public trust resources such as wildlife and clean water. We conducted this research with the goal of determining how larger landowners — who, in Humboldt County, are generally timber or beef producers — experience and perceive cannabis production. We surveyed by mail all landowners in Humboldt County who own at least 500 acres . We asked a series of questions about landowner experiences with the cannabis industry and how the industry directly affected landowners’ economic well-being, community, property and personal safety. We also asked how, in their view, the cannabis industry influences the community and the environment. We asked landowners to provide their views on grower demographics and on changes in their communities over time. In addition, we compared the experiences and perceptions of absentee and non-absentee landowners.Humboldt County has long been among the leading cannabis-producing regions in the United States . Located on the North Coast of California, Humboldt County is characterized by steep terrain and a Mediterranean climate; a climatic gradient runs from the cooler and wetter coastline to the drier and warmer in lands . Humboldt County’s agricultural and timber industries are significant in scale, with agricultural production amounting to $326 million in 2016 and timber production amounting to $70 million in the same year — although the timber numbers are down from a decade ago. These agricultural production numbers do not include cannabis production revenues, but recent estimates put cannabis production in the larger Humboldt, Trinity and Mendocino region, known as the “Emerald Triangle,” at $5 billion annually . Humboldt County is home to numerous species of concern — including threatened and endangered salmonids, spotted owls, marbled murrelets, fishers and so on — that are protected under the U.S. Endangered Species Act . Cannabis cultivation occurs within these species’ habitat areas, including in locations near and adjacent to old-growth redwood and Douglas fir forests.The intent of the survey was to understand how cannabis production in Humboldt County was affecting traditional agricultural producers, and therefore we focused only on landowners with enough property to derive a large percentage of their income from agriculture and timber activities. We identified landowners with at least 500 acres by combining land use and tax roll data. In total, 211 landowners fit this description. Landowners were mailed a paper survey, along with a stamped, pre-addressed envelope in which to return it, in January 2018. After 3 weeks, follow-up postcards were sent to landowners who had not returned their surveys. In total, 71 landowners responded to the survey . Of these, two landowners reported owning less than 500 acres and one landowner did not confirm meeting this minimum standard; we did not include these three surveys in our analysis. All survey responses were anonymous.Surveys were organized into three sections. One portion of the survey asked landowners about their direct experiences with the cannabis industry, asking them to agree or disagree with 22 statements that corresponded to four themes: how the cannabis industry has affected the economics of their operations ; how cannabis has impacted their local community ; how cannabis has affected their properties and how cannabis has affected their safety . The surveys asked landowners to respond to each statement using a five-point Likert scale, with responses ranging from strong disagreement to strong agreement . Respondents could also respond “NA” to statements that did not apply to them. Additionally, respondents were given space at the end of each subsection to provide comments or examples. In another section of the survey, we tested respondents’ perceptions of cannabis by asking them how they felt about certain cannabis-related issues and whether cannabis cultivation has had positive or negative impacts on their communities, specifying that their responses should not necessarily be based on their personal experiences.

The study’s strength is that it provides a novel and timely snapshot of practices that may influence cannabis consumption in a rapidly evolving context. Surveillance of cannabis websites can complement and help to explain findings of the behavioral studies reporting increased cannabis use. This aspect of cannabis marketing may be more agile and difficult to capture with traditional marketing surveillance; the COVID-19 announcements appeared soon after lockdowns. By October 2021, all of these announcements had disappeared from all but one of the dispensary websites . This study captured a unique transient marketing activity, suggesting that during future societal events or stressors that might impact cannabis consumption, rapid surveillance of cannabis dispensary websites is warranted. The study has several limitations: it has a small sample size, this cross-sectional analysis of dispensary websites does not represent other types of cannabis websites or places without legal cannabis, and it addresses only dispensary COVID-19 announcements, not consumer behavior. This study adds perspectives on new COVID-19 related content to prior analyses of cannabis advertising, which also found retailers positioning themselves as healthcare providers. While we did not find explicit claims that cannabis could treat COVID-19, some announcements suggested ways to use cannabis to avoid infection, 4×4 grow tray or suggested cannabis products to use if experiencing anxiety; these implicit connections between cannabis and health are consistent with prior studies finding cannabis dispensary health benefit claims. 

This analysis was limited only to the COVID-19 announcements; a subsequent analysis of the full website content found that anxiety was the most common mental health claim, present on 80% of websites in this sample. This limited analysis of the COVID-19 announcements from a single time point early in the pandemic likely underestimates the potential influence dispensary marketing could have on cannabis consumption. While COVID-19 announcements had largely disappeared from dispensary websites by October 2021, an informal review of the dispensary websites included in this analysis in October 2021 found that one featured a blog post on the topic, “Can marijuana cure coronavirus?” and several websites featured blog posts about using cannabis for anxiety, a topic indirectly relevant to the stress of the ongoing pandemic. While this study does not address consumer behavior, another study found in states where cannabis is legally sold, people with mood or anxiety disorders were more likely to use cannabis to self medicate. The marketing tactics we observed to maintain cannabis availability and align with health authorities might contribute to increased cannabis use, particularly since other studies show stress and decreased access to medical services19 were associated with the pandemic.This study demonstrates that cannabis dispensary websites are a timely source of data on industry responses to rapidly changing events and public health policies that may impact cannabis consumption.

The study identified two tactics in the COVID-19 announcements preserved which might contribute to increased cannabis consumption: preserving ready access to cannabis as an essential service, and reinforcing perceptions of cannabis as a medicine and dispensaries as health services. While use of online ordering and delivery services has been documented in prior analyses of cannabis websites15 we found these services became nearly universal after stay-at-home orders. The increase in home delivery of cannabis and other intoxicants that accompanied the COVID-19 pandemic may persist, posing new challenges for surveillance research. Future studies should address the impact of cannabis marketing and messaging on consumer cannabis consumption and health-care seeking behavior during stressful events.The ancient hexactinellid sponges are associated with the Ediacaran Period, and they are one of the basal metazoans . Despite great interest in the group, the natural history of the Hexactinellida is poorly known, and the life history is largely derived from inferences. The Antarctic hexactinellid fauna is well described; however, they are generally found at depths greater than 30 m, making it very difficult to study them in situ. Thus, life history patterns of recruitment, growth and reproduction are poorly understood for most hexactinellid sponges, although they have been thought to be very slow in comparison to the more common demosponges. Much of this characterization of low recruitment and growth rates of Antarctic sponges is based on early research at McMurdo Sound. Three species of hexactinellid sponges comprise the bulk of the benthic biomass: the relatively small Rossella antarctica is very common, reproduces by budding and exhibited growth during the study, while the massive, volcano-shaped hexactinellids, Anoxycalyx joubini and Rossella nuda/racovitzae, had no recruitment or growth.

Anoxycalyx joubini is the largest and most conspicuous sponge in the Antarctic and although it has been observed as much as 2 m in height , it has never been observed to settle or grow which has led to estimates of extreme longevity. Here we present observations of remarkable episodic recruitment and growth and apparently high mortality of the hexactinellid, A. joubini, in McMurdo Sound, Antarctica. These observations call into question the validity of previously held generalizations, at least for this conspicuous species.The observations of recruitment and growth occurred on artificial, experimental structures located on each side of McMurdo Sound . The area around McMurdo Station is normally influenced by southerly currents bringing phytoplankton from a region north of Ross Island, which is often dominated by a large and productive polyna. Slow northerly currents sourced beneath the Barrier Ice bathe the Explorers Cove region of New Harbor. The currents at Explorers Cove advect very different and minimal plankton because the water mass has circulated under the Ross Ice Shelf. With the exception of a wooden gangplank dropped from a ship in 1960, all structures were purposefully placed in the 1960s and early 1970s. The gangplank is at a depth of 25–30 m located just north of Hut Point, McMurdo Station . During the 1970s, the gangplank and the surrounding area were subject to massive settlement of the demosponge, Homaxinella balfourensis; however, essentially all of these sponges were removed by anchor ice in the mid-1980s. Various cages in the vicinity of McMurdo Station were placed in 1967. Settling surfaces either supported off the substratum by iron and wood posts or suspended beneath floats as much as 30 m off the bottom were established along the Hut Point Peninsula from Cape Armitage to Cape Evans in 1974. Various types of settling substrates composed of PVC plates and pipes were suspended in the water column by floats at Explorers Cove in 1974–75. These floaters, or settling structures suspended by floats up to 20 m above the floor were at bottom depths of 24– 43 m . In addition, greenhouse racking platforms placed in the 1970s for experiments also served as habitat for recruitment and growth of sponges. This paper is based on regular observations from 1967 at Ross Island sites and from 1974 at Explorers Cove through the end of 1989. The Ross Island sites were re-visited in 1998, and all sites were re-visited in 2004. In 2004, photographs were taken of A. joubini that had settled on the gangplank and on Explorers Cove structures. Detailed sponge photographs were obtained in 2010 and one site was revisited and photographed in 2012. While the specific sites discussed in this paper were not visited between 1989 and 2004, no recruitment of the very conspicuous A. joubini was observed in the late 1990s on Ross Island. Therefore it is reasonable to assume that during the extensive diving by Kathy Conlan and others in that period, there would have been observations of some new sponges, had there been a strong recruitment event much prior to 1998.There was no sponge colonization on the gangplank in the 1960s, but in 1974–1978 there was virtually no anchor ice formation, facilitating heavy recruitment and settlement of the demosponge, H. balfourensis over the entire area.

Anchorice returned in the 1980s removing H. balfourensis, and by 1989 the gangplank was clean of sponges . From 1967 through 1989, there were no hexactinellid sponges on the gangplank, but sometime between 1989 and 2004, A. joubini settled and grew there. In 2010, 19 A. joubini were photographed on the gangplank, which together had an estimated mean biomass of 30 kg, with the largest sponge weighing over 76 kg . While several new A. joubini were observed on the bottom in the vicinity of McMurdo Station, only one other A. joubini had settled on an old predator exclusion cage at Cape Armitage . This sponge was observed to have grown almost 30% when the site was revisited in 2012, only two years later, demonstrating the potential of fast growth rates relative to the 1967 through 1989 period when no growth was observed. In Explorers Cove, no sponges were observed on settling surfaces from 1974 until 1989 when a few H. balfourensis were found on some floating surfaces and racks. In addition, one large floater that had been suspended about 15 m above the bottom in 1975 was observed to have two small hexactinellids, possibly A. joubini in 1989. In 2004 A. joubini were found on nearly all artificial substrates and by 2010, the racks and floats at Explorers Cove included individual sponges over 40 kg . It is important to note that the Explorers Cove data are underestimates of total sponges that have recruited to the floater and rack structures, because very large sponges observed in 2004 had fallen off their substrata by 2010. Massive sponges unbalanced and tipped one floater, dumping the sponges sometime before 2010 and several other floaters had simply sunk from the weight of the sponges. In all cases, piles of A. joubini spicules were found on the bottom where the sponges had landed. In addition, some of the very large sponges observed on racks had become large enough to push other sponges off the structure, again reducing the total estimate of recruited individuals and biomass to the structures over this time period . We do not know how many of these sponges either fell off their structures or sank their floater, nor do we know how they died; however, in a few cases sponges apparently were dislodged recently and appeared still alive but were infested with the amphipod, Seba antarctica and being consumed by Acodontaster conspicuous. While the exact cause of death is uncertain, it is clear that essentially all of them die after landing on the bottom. Even with many of the large sponges lost before we could measure them, the estimated mean sponge weight of A. joubini found on the floaters was over 13 kg and nearly 18 kg on the rack . We observed a high mortality of A. joubini. Nearly every sponge that fell from artificial substrates to the bottom after 2004 was deadn 2010– the exceptions were 2–3 sponges that were mostly dead and two sponges estimated to be 8 kg and 17 kg that had recently sunk a float. Moreover, all seven A. joubini observed in the 60 m basin area at Cape Armitage during 1967–68 were dead by 1977. We marked 35 large A. joubini at Cape Armitage, Hut Point and Explorers Cove in 1974 and 6 of these were dead by 1977 and none were alive in 2010. In addition, between 1974 and 1975 approximately 30 transect lines were laid at depths ranging from 20 to 60 m at Cape Armitage, Hut Point and Explorers Cove to follow long-term changes. When possible, transects were started near the large, conspicuous A. joubini to facilitate relocation. In total, transects included 15 large A. joubini at Cape Armitage and 10 at Explorers Cove, and none of these sponges were found alive in 2010. We do not know the ages of any of these sponges nor do we know precisely when they died, but all 67 of them died over 43 years. Finally, photographs taken in 2012 of the big sponges on the gangplank revealed that several appear to be dying, possibly from infestations of the amphipod S. antarctica, suggesting a much more rapid turnover of A. joubini than previously assumed. We have observed platelet ice formation on A. joubini at Cape Armitage and Hut Point in waters less then 33 m and anchor ice has been observed to kill sponge tissue of H. balfourensis, so it might also kill patches of A. joubini tissue.Anoxycalyx joubini is one of the dominant, structure-forming species in the McMurdo Sound region of Antarctica. Here we report a highly episodic, massive and Sound-wide recruitment event and subsequent growth spurt, which occurred mainly on artificial structures.

The trend held for all of the major components of the particle phase as well, including PG, VG, and nicotine. Clearly, PG is easier to aerosolize than VG. This is due to the differences in chemical structure, and correspondingly, viscosity, vapor pressure, and boiling point. VG has one more OH group than PG, which results in stronger hydrogen bond intermolecular forces in the e-liquidsolution. The order of magnitude higher viscosity of VG at room temperature, requires more energy for vaporizing the solution. Moreover, the coil temperatures in Table 3.2 already surpass the boiling point of PG but are below the boiling point of VG . This is consistent with the high aerosol production when pure PG was used, and the high PG fraction in the gas phase The difference in total aerosol mass when vaping pure PG versus pure VG e-liquids at 375 °F suggests that PG was lost from the e-liquid at 8 times the rate of VG. This observation was corroborated in the mixed e-liquid using the gaseous CIMS and particle filter GC-MS data for PG and VG. The combined analytical uncertainties from CIMS and CG-MS are larger than for the pure gravimetric analysis, but also suggested a significant acceleration of PG loss compared to VG by a factor of ~9. Figure 3.7 shows representative carbonyl compounds and nicotine emitted from vaping e-liquid with different PG:VG ratios. Generally, grow trays carbonyl and nicotine concentrations decrease as the VG percentage increases in the mixture; although the hydroxycarbonyls do not decrease as dramatically as the simple carbonyls. This is likely due to the lower total aerosol and particle mass production overall as VG content increases in the e-liquid .

A notable exception is acrolein , which is the only compound whose formation increased with increasing VG, even as total aerosol mass decreased. Thus, for the 100% VG e-liquid, acrolein was one of the most concentrated carbonyls inhaled, and its relative production exceeded that of formaldehyde. The enhancement of acrolein between 30:70 and 0:100Mechanistic differences were more apparent when carbonyl formation was normalized by the total aerosol mass . The normalized trends reverse the absolute trends for some compounds, such as hydroxyacetone . Although hydroxyacetone can be generated from both PG and VG , the increase in relative aerosol fraction of hydroxyacetone with higher VG percentage in the e-liquid suggests that it is more efficiently formed from VG through the dehydration mechanism. This is consistent with the previous discussion that the heat-induced pathway is much more favorable for VG at the vaping temperatures we tested, and supports the exponential temperature dependence of hydroxyacetone . Likewise, formaldehyde emissions were inversely proportional to VG content , but increased slightly when normalized by aerosol mass . Formaldehyde can originate from both PG and VG and from either thermal or radical pathways. The data suggest that it is formed at similar efficiencies from both precursors, perhaps slightly favoring VG, which is consistent with more pathways available from VG . The relative production trends clearly showed that PG decomposition was responsible for all of the propionaldehyde and most of the acetaldehyde , while VG decomposition was responsible for all of the dihydroxyacetone and nearly all of the acrolein .

The VG source of acrolein is well-studied, and is leveraged in the conversionof biomass to fuels. Although acrolein can be formed by PG, it is a secondary product of a minor compound that is formed by the primary alkyl radical intermediate instead of secondary , which limits the importance of the PG source. The isomeric lactaldehyde and 1- hydroxypropanal likely had opposite trends that overlapped since they are solely formed by PG and VG, respectively . Given the higher formation of the sum of lactaldehyde and 1-hydroxypropanal with increased VG percentage , it appears that the formation of 1-hydroxypropanal from VG dominates over lactaldehyde formation from PG. These data support the exponential temperature trends of the lactaldehyde/1-hydroxypropanal pair, given that 1-hydroxypropanal is formed via heat-induced dehydration from VG. So far, most of the data are consistent with PG/VG mechanisms from the literature as shown in Scheme 3.1. However, notable deviations may exist for acetaldehyde and acetone. Acetaldehyde is thought to be a coproduct of formaldehyde in the VG dehydration,80 which would elevate it to be a major VG product, yet it appeared to be formed almost exclusively from PG . From PG, there was a suggested acetaldehyde source via radical reaction, instead of heat-induced dehydration. Thus, there was no reason to expect such a large abundance, or an exponential temperature curve . These observations, together with the fact that propionaldehyde . Acetone is known to be formed by PG; however, the data suggest that it can be formed by both PG and VG at roughly equal efficiencies. The temperature results also suggest a radical mechanism is dominant for acetone formation. Combined with the relative production trends, it would suggest that a radical formation mechanism from VG is missing from Scheme 3.1.

We are not aware of any proposed mechanism in the literature stemming from VG, especially one that is radical-initiated. The nicotine percentage in the particle phase at the same vaping temperature fluctuated with different PG:VG ratios . The nicotine concentration range observed in the particle phase is comparable to that in the original e-liquid . These results are consistent with those of Baassrir et al. and the trials organized by the Cooperation Centre for Scientific Research Relative to Tobacco .A 3-mg/mL concentration of nicotine in the e-liquid translated to 1.2 – 3.4 mg/mL nicotine in the particle phase, with the lowest nicotine percentage for the 50:50 mixture and increasing in both directions . More research is needed to understand the robustness of, and underlying reasons for, this trend and whether it is conserved with different nicotine content in the e-liquid. Approximately 0.3 mg/mL nicotine was observed in the total aerosol compared to 3 mg/mL used in eliquid.Figure 3.9 shows that the mass of the particles and representative carbonyl compounds generally increased with puff duration, as expected. Given the simultaneous increase in both particle mass and carbonyl mass with puff duration at the same flow rate, which would increase the puff volume, the carbonyl mass yield as normalized by aerosol mass would more or less be invariable. Both linear and non-linear fits would have yielded acceptable correlation coefficients within the studied range of only three data points. As puff durations in realistic use cases are unlikely to exceed this range, we did not test further. The relative increase between carbonyl compounds were roughly the same, within uncertainty. These results agree with Son et al., who found that increases in puff duration will increase the formation of carbonyl compounds, OH radicals, and nicotine.Most of the aerosol mass ended up in the gas phase , i.e., not captured on the hydrophilic PTFE filter, regardless of the temperature or PG:VG ratio tested . It is challenging to understand how the carbon mass from the e-liquid loss was distributed in the gas phase because there is noconventional analytical technique to quantify PG and VG in the gas phase due to the semivolatile nature of these compounds. The results from CIMS , pruning cannabis demonstrated that the majority of the gas phase was PG and VG instead of unknown compounds that are not well-measured by targeted techniques. This is consistent with findings that CO and CO2 are not abundant e-cigarette emissions. The CIMS spectra also showed that PG and VG were orders of magnitude larger in concentration than hydroxycarbonyls, a result that is consistent with the carbonyl-DNPH analysis. For the 30:70 PG:VG condition at 375 °F coil temperature, the sum of PG and VG obtained by CIMS in the gas phase accounted for the missing mass that was not captured by the particle filter within uncertainty . As discussed in Section 3.2.4, CIMS may overestimate semivolatile distribution in the gas phase due to evaporation during the sample dilution . However, it is clear from the CIMS spectra that the gas phase was dominated by mainly PG . GC-MS analysis of filters also showed that PG and VG were dominant components in the particle phase, with nicotine making up much less than 1%. In the 30:70 sample , approximately three-fourths of the particulate fraction was VG and three-fourths of the gaseous fraction was PG. The particle-phase composition roughly mirrored the e-liquid composition . The particle phase content of nicotine at 2.2 mg/mL was also similar to the eliquid composition. Our particle-phase results are consistent with other accounts that PG, VG, water and nicotine are the main components of e-cigarette droplets, and that nicotine is a small fraction of the total aerosol and only found in the particle phase . Thermal degradation products of nicotine have been reported in other works, but were not found in the present study even though the GC-MS method we used can detect nicotine products.It can be assumed that regular e-cigarette users intake a median of 200 puffs per day. This translates to an exposure dose of approximately 4.5 g PG/day and 0.8 g VG/day through inhalation of e-cigarette aerosols produced from vaping 30:70 PG:VG e-liquid at a coil temperature of 375 °F for a duration of 3 seconds. Although the PG exposure is fairly high compared to other aerosol components, animal and human studies demonstrate that PG has low toxicity even at relatively high doses. Mild sensory and respiratory irritation effects may result at concentrations of > 871 mg/m3 for particle plus gas phase PG, which translates to ~ 17.5 g/day exposure assuming 20 m3 air intake per day for a 70 kg adult. 

VG has similarly weak irritation effects, which is supported by the German occupational exposure limit of 200 mg glycerin/m3 to protect against sensory irritation effects in the workplace. In contrast, the thermal degradation products, such as carbonyls, are a concern for potential risk of acute and chronic adverse human health effects despite their low absolute concentration in our study . Carbonyls may be further enhanced in flavored e-liquid, and may approach or exceed unhealthy doses for toxicological exposure with or without additional flavors. Formaldehyde, acetaldehyde, and acrolein are classified as known or probable human carcinogens. The more abundant hydroxycarbonyls in e-cigarette aerosols, such as hydroxyacetone, do not have available toxicology data. Carbonyls are also found in combustible cigarettes, so it is informative to discuss carbonyl exposure risk compared to combustible cigarettes and normalized to nicotine, as e-cigarette users have been reported to self-titrate for nicotine intake. At a VG content of 100% in the e-liquid, exposure to VG products such as hydroxyacetone, 1-hydroxypropanal, and acrolein become increasing important. At 100% VG, the acrolein/nicotine ratio range increased by a factor of 20 compared to the 30:70 e-liquid at the same coil temperature range of 315 – 510 °F , which exceeds the acrolein/nicotine ratio in combustible cigarettes under some temperature conditions. A Chronic Reference Exposure Levels value of 0.35 μg/m3 was set by the California Office of Health Hazard Assessment for acrolein. If this is multiplied by 20 m3 inhaled volume of per day for a 70 kg adult, then a threshold of 7 μg/day may be considered safe for chronic exposure. However, at 100% VG, the acrolein e-cigarette exposure that is equivalent to replacing only 1 cigarette/day exceeds chREL threshold at all tested temperatures. Given the lower aerosol and nicotine production at high VG ratios in the e-liquid , users may increase temperatures, puff duration, or puff frequencies to achieve higher aerosolization rates, which will significantly increase carbonyl exposure.Although e-liquids with 100% VG can be readily found commercially, they also may be formed during the dynamic vaping process. Our data suggest, because the total e-liquid mass loss from PG was 8 times that of VG , the e-liquid will be more enriched in VG as vaping continues. This will shift the e-cigarette aerosol composition toward VG and its degradation products, particularly acrolein, as VG enrichment occurs. Likewise, total aerosol mass and total nicotine will decrease during the lifespan of the e-liquid. We can build a simple model to predict the e-liquid mass remaining when 100% enrichment occurs. The model assumes that, as the PG and VG ratio changes during vaping, the total amount of e-liquid lost also changes in accordance with the total aerosol data =16.92e. Thus, for an 8:1 aerosolization ratio for PG:VG, and for a 30:70 ratio PG:VG mixture, it can be estimated that approximately 30-40% of e-liquid mass will be consumed by the time the e-liquid reaches 100% VG .

Gas chromatography and liquid chromatography coupled with different detectors including mass spectrometry, UV-Vis, and others are usually applied for the characterization of components in e-cigarette aerosol. Besides chromatography, nuclear magnetic resonance has also been applied to the characterization of e-cigarette thermal degradation products. Jensen et al.74 have published a library of 1H NMR spectra for many thermal degradation products in ecigarette aerosol that are derived from PG and VG. These technologies are also applied for thecharacterization of including volatile organic compounds , carbonyls , nicotine and tobacco specific nitrosamines , polycyclic aromatic hydrocarbons , heavy metals , and flavoring compounds. VOCs are normally detected by GC-FID, GC-MS and related instruments. Lee et al.99 detected VOCs including ethanol, acetonitrile, isopropyl alcohol, benzene and toluene using the GC method from National Institute for Occupational Safety and Health that employ evacuated canisters lined with fused silica for sample collection. Carbonyl compounds are usually derivatized by 2,4 – dinitrophenylhydrazine in solution or on silica gel cartridges impregnated by 2,4 – DNPH, followed by analysis on HPLC-UV or HPLC-MS. Nicotine, TSNAs, and PAHs are usually captured on filter pads and analyzed by GC-MS. Trace metals are usually captured on quartz filters and analyzed by ICP-MS. The e-cigarette aerosol includes a liquid-like particle phase and a gas phase. Most e-cigarette emissions are semivolatile, pots for cannabis plants which can partition to both the gas and particle phases depending on different environmental conditions and chemical properties .

Pankow et al. predicted that the phase distribution of various components in ecigarette aerosol are related to the mass concentration of particles , the composition of particles, the vapor pressure of the chemical, and the ambient temperature. For example, formaldehyde can be found mainly in the gas phase even at the highest level of total particular level of e-cigarette aerosol, while formaldehyde hemiacetals partition into the particle phase, even at the lowest total particulate matter levels.Although simple thermal degradation products such as formaldehyde, acetaldehyde, and acrolein are commonly quantified in the literature, the reported levels of specific components significantly vary in different publications. This might due to the diversity and complexity of ecigarette products, as devices from different brands and design generations vary in power settings, coil type and puff topography . Another potential reason is that there is no standard sampling and analytical methods for the target analytes in e-cigarettes aerosols. For example, Eddingsaas et al. found that the use of limited collection methods will not identify all aerosol components. This suggests a more comprehensive approach, using a combination of techniques, is needed for the collection of e-cigarette aerosols. Moreover, thousands of e-liquid formulations with different PG:VG ratios and different flavoring compounds introduce further challenges to a unified understanding of the e-cigarette chemistry in the literature. This suggests a need to isolate key e-liquid ingredients to study their fundamental chemistry, instead of sampling from commercial e-liquid blends that are proprietary in composition, in order to have a more predictive understanding of thermal degradation in e-cigarette vessels.

Since its introduction to the United States in 2007, the electronic cigarette market has expanded significantly. The prevalence of e-cigarette use was 3.2% for adults and 7.6% for young adults in 2018. The prevalence of e-cigarette use among high school students increased from 1.5% in 2011 to 27.5% in 2019, eclipsing conventional cigarettes among youth. With the growing population of e-cigarette users, the evidence that e-cigarette use is related to higher frequency of cigarette smoking, and the lack of historical governmental regulation, there is a significant need to fill existing data gaps on chemistry, toxicology, and clinical/behavioral patterns to inform on e-cigarette consumer safety and risk. E-cigarettes have been suggested as a reduced harm alternative to traditional tobacco-based products due to the reduced presence of well-studied toxicants formed during tobacco combustion. However, the use of e-cigarette may have its own risk, such as electronic cigarette or vaping-associated lung injury , respiratory function impairment, inhalation of carcinogenic carbonyls, and changes in gene expression. Furthermore, as e-cigarette emissions are not completely inhaled, there is potential for bystander or secondary exposure to non-users from the exhaled aerosol to the environment. Recent works have provided insights into how e-cigarette components and emissions affect indoor air quality and exposure pathways. Yet to date, there remain majorgaps in our knowledge of a complete chemical profile generated from the vaping process, as well as detailed mechanisms producing those chemicals. Moreover, the astonishing variety of ecigarette products and innumerable flavors available on the market, combined with the fast pace of product alterations due to the steady increase in e-cigarette popularity, present significant challenges in e-cigarette research and the estimation of user risk. The thermal degradation of propylene glycol and vegetable glycerin , the primary components of e-liquid, can generate complex chemical products through a series of reactions. Laino et al. showed that the thermal degradation of VG can form formaldehyde, acetaldehyde, acrolein by dehydration via the formation of glycidol, while PG can generate propionaldehyde and acetone via the intermediate formation of propylene oxide.Diaz et al. suggested PG could also participate in a heat-induced radical-mediated degradation pathway, initiated by O2 insertion to C-H bonds to generate the OH radical that further propagate the radical chain, forming at least five degradation products.

The radical-mediated pathway of VG has also been proposed by other researchers, and at least seven thermal degradation products have been observed in the process. Some degradation products can react further to form simple carbonyls, and accretion reactions between carbon-centered radicals or stable products can further complicate the chemistry of e-cigarette aerosols.The fragmentation of aliphatic alcohols tend to produce compounds that have a carbonyl moeity; However, since PG and VG are polyols, their degradation will also result in carbonyls functionalized with hydroxyl groups in addition to the simple types. Organic acid formation may also occur to an extent, possibly as a carbonyl oxidation process. Some thermal degradation products have well-documented toxicity to humans , while others have suspected toxicity . In addition to thermal degradation products, hundreds of flavoring ingredients may be added to e-liquids and vaporized in e-cigarette aerosol, which can potentially lead to adverse health impacts. Jensen et al. identified the largest variety of thermal degradation products to date from aerosolized e-liquid using Nuclear Magnetic Resonance ; however, the data are not quantitative in that work. Since most compounds in e-cigarettes have a carbonyl moiety, quantification is conventionally done by derivatizing with 2,4-dinitrophenylhydrazine to produce hydrazone adducts , followed by analysis with liquid-chromatography or gas chromatography using authentic carbonyl-DNPH standards for calibration of chromatographic peak areas. Even so, authentic carbonyl-DNPH standards are not available for many complex products. Synthesis of carbonyl-DNPH standards may be done; however, the process to synthesize, purify, indoor cannabis grow system and purity-check is laborious, requires specialty equipment, and requires reasonable syntheticchemistry skills. Synthesis of DNPH hydrazones of multi-carbonyls require additional purification steps to isolate the mono- and multi-hydrazones. In addition, some carbonyls are not commercially available as starting material, requiring their own separate synthesis. Thus, an approach to quantify without chemical standards is an attractive alternative. Furthermore, spectroscopic chromatography methods that rely on retention time and UV-visible absorbance spectra may be limited by co-elution or indistinctive spectra, even when utilizing authentic chemical standards. The coupling between chromatography and high-resolution mass spectrometry is a powerful tool for chemical identification, as it removes the co-elution limitation by enabling molecular formula assignments from exact mass. The goals of this work are twofold: use high mass resolving power coupled to chromatography to better identify DNPH hydrazones of functionalized and simple carbonyls and acids, and develop a method to quantify e-cigarette chemical products for which analytical standard are unavailable.First-generation disposable e-cigarettes from blu® , a popular e-cigarette brand,144 with “Classic Tobacco” e-liquid cartridges were used for this study. The blu® e-cigarettes are comprised of a rechargeable battery with a capacity of 140 mAh, an atomizer with coil resistance of 3.5 ohm, and a disposable, non-refillable e-liquid cartridge with proprietary ingredients. Batteries were charged after every 20 min of usage and the e-liquid cartridge was replaced after 400 puffs. A TE-2B smoking machine was used to generate e-cigarette aerosol for the analysis. The apparatus puffed two e-cigarettes, in alternating turns, at a frequency of 8 puffs/min for a 2 second puff duration. The average flow rate was 2.3 L/min and the puff volume was 77 mL, quantified by a primary flow calibrator . Ecigarette aerosol samples were collected through 2,4-dinitrophenylhydrazine cartridges for carbonyls/acids and 47 mm Polytetrafluoroethylene filters for nicotine. Atotal of 200 puffs were collected for each analysis, which is within the linear dynamic range of the analysis . The emission profile was stable, within the uncertainty of the analysis, for the first and second 200-puff collection of each cartridge. After collection, DNPH cartridges were extracted with 2 mL acetonitrile into 1.5 mL auto-sampler vials . Consecutive extractions of DNPH cartridges for 40-, 80-, and 200-puff samples confirmed that >97% of both DNPH and its hydrazones were extracted after the first 2 mL volume.

The samples were diluted using LC-MS acetonitrile to the desired concentrations for directinfusion HRMS and MSn analyses . Extracts were used for HPLC-HRMS analyses without dilution. All samples were promptly analyzed after preparation; sample collection and analyses were performed in triplicate.Diluted carbonyl-DNPH extracts were analyzed for molecular composition using a linear-trapquadrupole Orbitrap mass spectrometer at a mass resolving power of ~ 60,000 m/Δm at m/z 400. The extracts were directly infused into a capillary nano-electrospray ion source and the spectra taken in the negative ion mode. An external mass calibration was performed using the ESI-L tuning mix immediately prior to the MS analysis, such that the mass accuracy was adjusted to be approximately 1 ppm for standard compounds. Insights into molecular structure were obtained using collision induced dissociation multistage tandem mass spectrometry in the LTQ-Orbitrap. CID energy was tuned for each mass, such that the precursor ion has 10 – 20% normalized abundance. Thermo Xcalibur software was used for data processing.DNPH hydrazones were quantified by HPLC coupled to the same LTQ-Orbitrap in 2.2 with an electrospray ionization source, operating in the negative ion mode at a mass range of m/z 150 – 500 to cover the mass range of carbonyl-DNPH and dicarbonyl-2 adducts observed in this work. Separation by HPLC was performed using a C18 column end-capped with dimethyln-octadecyl silane and a mobile phase of LC-MS grade water with 0.1% formic acid and acetonitrile . The analytes were eluted over the course of 37 minutes at 0.27 mL/min with the following gradient program: 40% B , 50% B , 60% B , 100% B , 40% B . After separation by chromatography, single ion chromatography for the accurate m/z of DNPH adducts that were identified by the methods in 2.2.2 was used for quantification. A carbonyl-DNPH standard solution , comprised of 13 carbonyl-DNPH analytes was used to obtain the concentration standard curves for calculating the concentration of formaldehyde, acetaldehyde, acetone, acrolein and propionaldehyde in e-cigarette aerosol . From application of the standard curves and propagating the remaining errors of the analysis , the ±1σ uncertainty for calibrated compounds is 10%-20%. The concentration of the remaining carbonyls and organicacids are calculated by their SIC peak areas and the calculated sensitivities to the ESI negative ion mode in 2.2.4. The concentration of nicotine was also measured by the same method using the positive ion mode.The chemical structures of the DNPH hydrazones affect their deprotonation efficiency in the ESI negative ion mode, and thus, their calibration sensitivity in HPLC-HRMS. The Gibbs free energy change of the deprotonation reaction of carbonyl-DNPH compounds that occurs in the ESI negative ion mode was calculated by Gaussian 09 in both the gas phase and solution phase. The structural geometry optimization and frequency calculation was performed by density functional theory using the M06-2X functional and 6-31g+ basisset, which has been recommended for the study of main-group thermochemistry in recent years. 145-147 First, the ΔGd values for the 13 carbonyl-DNPH compounds in the analytical standard mixture were calculated to obtain a relationship to their measured ESI sensitivities. The relationship between the ΔGd and ESI sensitivities was then extended to calculate the relative theoretical ESI sensitivities for the DNPH hydrazones for which commercial standards are not available. Calculated sensitivities were then used to estimate the concentrations of carbonyl-DNPH hydrazones in e-cigarette aerosol extractions with the method described in 2.2.3.

The relationships between racial composition and garden proximity remain relatively unchanged with interaction terms added to the Philadelphia model: an increase in the share of Black or Hispanic residents predicts that the nearest garden would be closer, while the share of Asian and Pacific Islander residents is not significantly associated with garden proximity. The interaction between percent Black and year also has a significant and positive coefficient, suggesting that like housing costs, the relationship is gradually disappearing over time. The model estimates that in 1980, a 1% increase in the share of Black residents would be associated with a decrease of about 19 meters to the nearest garden, all other factors being equal. However, every additional year would see the predicted distance to the nearest garden increase by about 0.3 meters for every 1% increase in the share of Black residents. Taken together, these coefficients suggest that the greater proximity of gardens to neighborhoods with more Black residents will disappear after about 65 years, or around 2044. The model cannot determine whether this change in the relationship results from more garden attrition in Black neighborhoods than other neighborhoods, or from the proportion of Black residents decreasing in neighborhoods in which gardens have more durability , cannabis equipment but either scenario seems plausible given the shift in organizational priorities at the Pennsylvania Horticultural Society.

With little grant funding to help maintain existing gardens, many of the projects that were developed through Philadelphia Green did decline over time. At the same time, the Neighborhood Gardens Trust has begun to target its garden preservation efforts at rapidly gentrifying neighborhoods where gardens appear to be the most threatened; however, preserving the gardens does not prevent the demographic change and possible displacement that are associated with gentrification. Results for Seattle provide a contrasting example of what can happen when garden preservation becomes the rule rather than the exception, and when organizational priorities shift over time toward developing gardens closer to people who ostensibly need them more. As in Philadelphia, with interaction terms added to the Seattle model, a higher poverty rate is associated with the nearest P-Patch being further away . However, unlike Philadelphia, the interaction term is also significant, and it moves in the opposite direction. That is, for every passing year, a 1% increase in the poverty rate is associated with a decrease of about 0.7 meters to the nearest P-Patch, with all other factors being equal. Taken together, these coefficients suggest that Seattle’s gardens were originally distributed further away from high-poverty neighborhoods and closer to low-poverty ones, but the relationship reversed after about 22 years, and from 2002 onwards gardens are increasingly likely to be found closer to high-poverty neighborhoods than low-poverty ones. As explained in chapter 3, the leaders of the P-Patch program in the 1990s were responsive to the public concern that the gardens were a private use of public space and to city officials’ appreciation for evidence showing how the gardens benefitted low-income and other marginalized residents. The program leaders undertook a concerted effort to expand the program in neighborhoods with greater socioeconomic need, an effort which gained traction especially after the 2000 Pro-Parks Levy infused the program with $2 million.

This effort included working with the Seattle Housing Authority to build gardens in low-income housing developments specifically for use by their residents. The changing organizational priorities in the 1990s and influx of resources in 2000 would logically explain why the interaction model shows gardens’ proximity to poor neighborhoods equalizing around 2002 and growing gradually closer since then. The interaction model’s results suggest that P-Patch gardens have become more accessible to poor neighborhoods over time, but also that they have become less accessible to immigrants. Similar to the pattern observed with percent Black residents in Philadelphia, the coefficient for percent foreign born in Seattle is negative and significant while the coefficient for interaction between year and percent foreign born is positive and significant—in fact, it is the largest coefficient of any interaction term across the three models, suggesting a relatively fast pace of change. The model estimates that in 1980, the nearest garden would be about 60 meters closer for every 1% increase in the immigrant population, with all other factors held constant. With every additional year, a 1% increase in the immigrant population relative to otherwise identical tracts would predict 1.4 meters further to the nearest garden, suggesting that after about 42 years the percent foreign born in a tract will have no impact on garden proximity, and ultimately after 2022 gardens will be further away from communities with higher shares of immigrants. As in Philadelphia, the gradual attenuation of garden accessibility for immigrants in Seattle may be linked to gentrification, as higher housing costs push vulnerable groups further from the more desirable areas, but this explanation cannot be verified from the model alone. What the model can tell us is the relationship between housing costs and garden locations in Seattle, as well as how this relationship changed over time. The coefficient for housing costs is significant and positive, suggesting that gardens were originally built further from high-demand real estate. With the coefficient for interaction between year and housing costs being negative, this relationship appears to be gradually weakening over time. Chapter 3 describes the widespread garden preservation that P-Patch advocates accomplished with the passage of Initiative 42, which offers a plausible explanation for why this pattern would be seen in Seattle: gardens were initially built where more land was available, and most of them have not been removed as property values in the surrounding neighborhoods have increased.

Spatial analysis indicates that the citywide programs in Milwaukee, Philadelphia, and Seattle have generally developed gardens closer to marginalized communities than to more privileged ones. That said, significant historical trends and a few deviations from the overall pattern are important to note, especially given their apparent relation to organizational decisions and political-economic factors described in previous chapters. First, while the models suggest that community gardens in all three cities have generally been closer to neighborhoods with more Black and Hispanic residents, their accessibility for Asian and Pacific Islander residents and for immigrants is not as consistent. On the one hand, vertical grow shelf studies of urban food access indicate that Black and Hispanic communities are the ones most impacted by lack of healthy, affordable food options , so if organizations are prioritizing the food-security benefits of urban agriculture, then building access for Black and Hispanic residents more than for Asian and Pacific Islander populations may genuinely reflect understandings of local need and equitable use of the organizations’ resources. On the other hand, food insecurity is just as acute in some Asian American and immigrant communities, and there is a chance these communities are being overlooked. Furthermore, food access isn’t the only benefit that community gardens bring; the organizations in this study have also emphasized social, cultural, and economic benefits of urban agriculture. Advocates for Seattle’s P-Patch program were the most explicit in touting the ability of gardens to build community among diverse people and to provide cultural continuity for immigrants from agrarian backgrounds. Perhaps because of this recognition, Seattle’s gardens have been the most accessible to immigrants according to the spatial error models. However, the P-Patches’ proximity to immigrants is eroding over time. In Milwaukee, gardens appear to be further away from communities with higher foreign-born populations, and in Philadelphia the relationship does not register as significant in the spatial error models. According to my interviews and review of organizational documents, in all three cities, immigrants—and in particular Southeast Asian immigrants—have been heavily involved in building gardens and organizing the labor required to keep them going. Yet it appears that immigrant gardeners may have to travel further than others to reach their sites, and they may lose access altogether if they or their garden is displaced when a neighborhood gentrifies. Qualitative researchers have drawn attention to some ways that immigrant gardeners may be undervalued by urban agriculture organizations and media accounts . My research suggests that this oversight may influence organizational priorities in development and preservation efforts, extending inequity to the physical siting of gardens. When community garden organizations do identify the benefits they want their spaces to provide and identify neighborhoods to prioritize in receiving those benefits, they can achieve desired outcomes over time. One example is the expansion of Seattle’s P-Patch network through the 1990s and 2000s, which was undertaken with conscious attention to increasing garden access for the city’s low-income residents. The spatial error model with interaction terms shows that initially, P-Patches were less accessible for communities with higher poverty rates, but this relationship flipped over time such that communities with higher poverty rates are now likely to be closer to the nearest garden than otherwise similar communities with lower poverty rates. Philadelphia Green provides another example of how programs can achieve clear outcomes by prioritizing a certain benefit that they want urban agriculture to provide in their city. In this case, the benefit has been economic.

As explained in Chapter 2, Pennsylvania Horticultural Society secured grant funding for its Philadelphia Green program to undertake concentrated neighborhood greening initiatives in the 1980s and 1990s; these initiatives included tracking how the greening affected the target neighborhoods, which helped the Society to make a broader case for public investment in their greening services. Scholarship based on the greening initiatives and organizational publications from the time highlight how the program’s community gardens and greening intervention improved neighborhood attractiveness and increased local property values. One organizational brochure includes a map showing, for different neighborhoods, the percentage of vacant lots involved in the program which had subsequently been sold and developed. Building and preserving green space for the benefit of disadvantaged neighborhoods was not the goal, and it has not been the primary outcome. Compared to Milwaukee and Seattle, Philadelphia has seen the highest rate of garden attrition, so a longitudinal analysis based on the distribution of existing gardens at given points in time misses some of the story. Still, even by examining the spatial error models and maps of garden locations over time, we can see distributional outcomes that are likely related to Philadelphia Green’s prioritization of economic benefits and limited efforts toward long-term garden preservation. The program’s gardens tended to be developed in neighborhoods with higher housing costs and lower poverty rates, but this relationship with housing costs has gradually diminished over time. Maps of garden locations in successive decades show that gardens have disappeared in neighborhoods where housing costs have increased and poverty rates have decreased. This pattern reflects the program’s overall weak commitment to maintaining gardens for the long term, allowing market forces to displace gardens from more desirable areas. Meanwhile, garden proximity to neighborhoods with a higher share of Black residents has decreased over time, which suggests that either gardens are disappearing at higher rates in neighborhoods with more Black residents, or that as neighborhoods themselves are changing through gentrification, those that keep their gardens are nevertheless seeing decreases in the proportion of Black residents. The pattern of garden distributions over time in Milwaukee demonstrates an outcome likely to result from program management without the resources or a clear strategy to direct garden development toward specific communities. The model with interaction terms did not yield any significant interactions with year, suggesting that garden distributions over time have not moved toward or away from communities with any of the characteristics analyzed . Instead, the static model shows that the nearest garden is likely to be closer to neighborhoods with higher poverty rates, higher percentages of Black, Hispanic, and/or Asian and Pacific Islander residents, and lower percentages of immigrants. Given what we know about the potential benefits of urban gardens and the communities most in need of those benefits, the distribution of Milwaukee’s gardens seems to produce equitable outcomes other than the lower proximity to neighborhoods with more immigrants. However, the historical analysis in preceding chapters demonstrates the ongoing vulnerability of most gardens in Milwaukee to potential removal in the face of development pressure. In other words, the gardens are close to the populations where they are needed because that is where land is available and development pressure is low; the process of displacement and disappointment that unfolded in Philadelphia is likely to be repeated in Milwaukee if and when market conditions change.

While the LBA was constituted of entities more closely associated with the typical centers of power in growth coalitions, the breadth of constituencies sharing a common goal in this effort highlights how widely shared urban growth goals tend to be, despite the uneven share in returns on growth . The LBA and the CTBVL worked toward the same goal of establishing a land bank that would consolidate the city’s vacant property holdings and streamline its disposition process. According to development professionals interviewed who were active in each of the groups, the LBA emphasized insider strategies to advocate for the land bank in private meetings with elected officials, while the CTBVL used an outsider strategy by mobilizing large numbers of people to pressure key decision makers—in this case the members of city council who would put forth and vote on the Land Bank bill. Characteristic of their respective organizational orientations, PHS participated in the LBA while Soil Generation participated in the CTBVL. The Public Interest Law Center’s Garden Justice Legal Initiative was a member of both coalitions. While the interests of their member organizations were slightly different, both coalitions framed the need for a land bank in essentially the same way, amplifying a narrative that PHS had been constructing for decades. As PHS contracted to green vacant lots throughout the city in the 1990s, they developed and helped disseminate arguments for the city to invest more in urban greening. They collaborated with local researchers and the Pew Charitable Trusts to produce Urban Vacant Land: Issues and Recommendations, 4×8 grow table with wheels a 1995 report that highlighted their successes in greening vacant lots and also stressed the need for city agencies to “simplify and depoliticize the acquisition process by establishing public policy that supports the transfer of city-owned vacant land into community or private ownership” .

Reiterating these findings, PHS’s 2000 Managing Vacant Land report advocated for the creation of an “Office of Vacant Land Management” within the Redevelopment Authority , and the 2002 Reclaiming Vacant Lots report was published as a technical manual for anyone looking to repurpose vacant lots that highlighted the work that PHS had already accomplished in collaboration with community groups across the city . Through these reports and other communications at the time, PHS framed the city’s vacant lots as public problems that could become assets if community groups and developers faced fewer barriers to access and ownership. Similarly, in 2010, one of PHS’s collaborators in the Land Bank Alliance, the Philadelphia Association of Community Development Corporations, commissioned a report that found the city was spending $20 million a year to maintain vacant lots, while losing $2 million annually in uncollected property taxes as the blighted lots dragged down overall property values by an estimated $3.6 billion. Prominent voices from both the housing and greening constituencies highlighted the same issues, framing vacant lots as an economic drag that could be lifted through government reorganization. Both of the land bank coalitions argued that with 40,000 vacant lots around the city, some could be preserved as gardens and open spaces while others could be developed into housing at various price points. The abundance of vacant property made possible a shared vision among groups who might otherwise have been competitors, but who instead were all in agreement that the city’s process for land disposition was too slow and uncoordinated. The details of who would acquire what land did not need to be worked out until the Land Bank bill was passed.

With the widely representative coalitions calling for change and a relatively large constituency mobilized, the Land Bank bill made it into the public discourse and onto the legislative agenda. Newspaper coverage was largely supportive, although skeptical of a provision in the bill that would codify the tradition of council manic prerogative . Articles and editorials frequently cited the $20 million annual maintenance figure, which worked to underscore the economic inefficiency of letting so much land go unused and the liability this land had become for the city government. In October 2013, when the Land Bank bill got its hearing in the Committee on Public Property and Public Works, attendance overflowed. Compared to Milwaukee and Philadelphia, Seattle has had both political and economic conditions more favorable to community garden development and preservation. The economy and the revenue-generating tools available in Seattle created opportunities for the city to fund desired public investments, including gardens. With the city’s tech sector thriving, Seattle has been a “winner” in the global competition for urban growth for the last 30 years. In this time, public investments in community gardens, greenspace and other neighborhood amenities have redoubled Seattle’s appeal to the “creative class” . The favorable political economy in recent decades has helped solidify the status of community gardens as a legitimized, permanent feature of the urban landscape. That said, the popularity and security of Seattle’s gardens do not ensure that they are providing the potential benefits most needed by the city’s marginalized residents. If the city were facing the kinds of budget crises that Milwaukee and Philadelphia currently confront, open space improvements might not win approval from voters or City Council when tax revenue was direly needed for basic services such as police and schools. Seattle’s city budget contracted in 2000 with the bursting of the dot-com bubble, and again in 2008-2010 during the Great Recession. Otherwise, since the early 1990s, the city budget has increased fairly steadily.

The growing technology sector has served as a stronger economic base than more traditional industrial manufacturing during this period, in which outsourcing has led to significant economic impacts in cities like Milwaukee and Philadelphia as described above. Seattle faced population loss between 1960 and 1980, including a steep economic downturn during the “Boeing Bust” when the city’s major manufacturer shed thousands of jobs. However, Seattle began to grow again as the information technology sector expanded, with major companies like Microsoft and Amazon headquartered in the area. The city’s population grew 4.5% from 1980 to 1990, then 9% from 1990 to 2000 , 8% from 2000 to 2010, and a whopping 21% between 2010 and 2020. Economic conditions in Seattle differ significantly from the other case-cities: the poverty rate is 11% , and the median household income of $92,263 is greater than that of Milwaukee and Philadelphia combined. A stronger economy and reasonably comfortable city budget have made allocating public resources to community gardens easier in Seattle than in Milwaukee or Philadelphia. The P-Patch Program is administered by the City, as explained in chapter 2, grow tray stand and public resources have undergirded its entire existence. Seattle has supported gardens as part of its budget since 1973, at first agreeing to pay $950 to cover the property taxes of Rainie Picardo so that his land could continue serving neighbors as a community gardening space. City Council then expanded the program to 10 other sites around the city and took over administration . For the P-Patch program’s first two decades, the city budget allocated roughly $15,000-50,000 to the program for 1-2 staff positions, plowing costs, and money for tools and materials. In 1983—in part due to contracting federal support for local governments that affected all of the cities in this study—a municipal budget crunch forced cuts in the P-Patch program that led to the first notable site vacancies in the program’s ten-year history. With two part-time staff working far more than the hours they were paid for, and significant volunteer contributions to make up the difference, the program survived and continued to add new sites through the late 1980s. When the city was facing budget cutbacks again in 1992, gardeners organized a letter-writing campaign and visited council members to advocate for fully funding the program. Successful in this effort, they received a $50,000 budget increase for 1993. For the next 14 years, as Seattle’s economy and city budget saw gradual but nearly uninterrupted growth, the P-Patch program garnered increases in staff and funding that enabled them to administer more and more sites. During this period, the program more than doubled in size—from 30 gardens and 2 staff positions in 1993, to almost 70 gardens and 7 staff in 2007. Although the city froze the program staff size during the Great Recession, funding from open space tax levies continued to facilitate expansion in the number of gardens. As of 2021, there are nearly 90 P-Patches reaching across every neighborhood in Seattle. The program is well known and popular, in part because of its expanse and its stable administrative capacity; these features result from the substantial public resources that the City of Seattle has been able to dedicate to the program over the last 40 years. In addition to the annual budget allocation that supports P-Patch administration, the garden program has been able to expand because of funding from tax levies. Seattle and King County give citizens official decision-making powers in regard to certain tax policies. Washington state allows cities and counties to raise revenue through taxes of different types; many such tax increases require voter approval with turnout requirements and at least 60% support at the ballot. Seattle voters typically see at least one tax levy question on their ballots every year, either for the City of Seattle or for King County. Not all of these measures receive the necessary 60% support, but since 2000 voters have approved several tax levies related to parks and open space improvements at both the city and county levels. These measures have raised hundreds of millions of dollars for parks and open space, including at least $4 million specifically for the acquisition and improvement of P-Patches. Such an infusion of cash into citywide community gardening efforts has only been possible because a) the P-Patch program is a public entity; b) county and city governments in Washington state have the ability to raise revenue with tax levies; and c) the citizens of Seattle and King County are willing to pay higher taxes in order to improve and secure open spaces. The levy funds have been used for the City to acquire land for P-Patches in high-demand parts of the city and, importantly, levy funds have also been used to enhance existing P-Patches with features such as picnic tables, gazebos, or benches designed to make the sites more inviting for the general public. As discussed in chapter 3, the P-Patch gardeners and program administrators undertook a concerted effort to design community gardens so that they are accessible, usable and therefore valued by the general public. This effort ramped up in 1998, shortly before the first of the munificent open space bonds was approved in 2000, putting P-Patch advocates in a perfect position to apply the flush funding in a way that would yield visible returns for the public at-large. Seeing the benefits of improved P-Patch gardens likely made voters more amenable to approving the next open space tax levy that came before them—a positive feedback loop made possible by the particular political-economic conditions in Seattle.The City of Seattle was willing to dedicate resources to the P-Patch community gardens in part because of the stable city budget and revenue from tax levies, and in part because of how local garden advocates have framed the value of urban agriculture. In addition to legitimizing urban agriculture as a community-building tool and source of food for those in need, leaders of the P-Patch nonprofit built a narrative around the value of community gardens as an amenity that would keep Seattle neighborhoods green and livable as the city took on more residents. Building off of existing ideas about what made Seattle special, such as its environmental amenities and pleasant neighborhoods, the P-Patch advocates constructed an effective framing for the value of community gardens in contributing to Seattle’s place-legacy . As the city grew and neighborhoods densified, community garden advocates argued that the P-Patch program should also grow as a way to maintain residents’ quality of life . Essentially, garden advocates used a framing that would appeal to the growth coalition: exchange value could continue to increase along with concession of a relatively small amount of the city’s land preserved for use value. Seattle’s garden advocates had constructed this sophisticated narrative by the mid- 1990s, and in the early 2000s Richard Florida outlined a theory of “creative cities” that essentially describes the alignment of certain kinds of use value with exchange value.