Author: ecogrow

Evaluation of both upregulated and downregulated genes among the experimental groups were in the direction of inflammation resolution. For example, in the Fat-1 genotype the observed differences in the downregulated genes were associated with cytokine production in macrophages, regulation of tight junctions and ECM disassembly, suggesting a move towards resolution of inflammation in tissue . Cytokine production and tight junctions contribute significantly to the maintenance of epithelial cell integrity and mucosal immunity of the lung in response to environmental insults as well as pathogens . Similarly, changes in ECM are important for recruitment of immune cells into and within the lung . Altogether, changes in these processes promoted by elevated levels of ω-3 fatty acids as in Fat-1 transgenic mice might affect how the lung responds to DE exposure. Particularly with TPPU, we identified down regulation of genes involved in IL-33 mediated signaling, as well as IL-5, IL-13, and IL-6 secretion. Overall, these data identified differences in immune clearance between the two genotypes and TPPU appeared to contribute to T-cell differentiation and regulation of PMN activation in part by downregulating the IL-33-mediated signaling pathway. IL-33 has previously been reported as an important regulator of Th- 2 immune response in allergic inflammation . The studies described herein do have numerous limitations. As with all transgenic animal models, the use of the Fat-1 mice to increase total body tissue levels of ω-3 PUFA and achieve an ideal ∼1:1 ratio of ω-6:ω-3 PUFA does not fully recapitulate the human condition. For example, differences in PUFA intakes are seen not only between individuals,nft hydroponic but temporal fluctuations in diet tendencies for each individual also undoubtedly alter daily to monthly PUFA levels, and the impacts of this will vary across different tissues based on PUFA uptake kinetics .

As PUFA substrate utilized during inflammatory events likely comes from both tissue sources as well as circulating blood and associated inflammatory cell infiltration, the impacts of recent dietary intake versus long-term dietary patterns on PUFA substrate availability are difficult to ascertain. Also, there are numerous recognized health benefits of diets high in ω-3 PUFA, due at least partly to their role in the endogenous production of SPM that regulate inflammation resolution and repair activities . SPM are produced temporally during an inflammatory response; their levels increase within hours to days following an inflammatory insult, and their production is critical to inflammation catabasis, including promoting neutrophil clearance, reducing inflammatory cytokine production, activating M2-like pro-resolution macrophages, promoting regulatory T cell recruitment, and activating tissue repair . Deficiencies in SPM generation pathways have been identified in asthma, and SPM are decreased in lavage, sputum, and/or exhaled breath condensates of COPD and asthma patients compared to individuals without lung disease . Therefore, an in-depth analysis of both ω -3 and ω -6 PUFA derived SPMs is necessary, and lipid metabolomics analyses of our data are currently underway in our laboratory. Another limitation is that we have used a model of organic dust exposure that utilizes a sterile-filtered aqueous extract of environmental dusts that is intranasally instilled to mice in a saline solution. This model will not fully recapitulate the inhalantinjury that is experienced by an individual working in a swine confinement facility, as it does not consider certain components, including live pathogens or gaseous components that have recognized respiratory impacts in these workers . It has been reported that airway inflammation in swine confinement workers differ from naïve subjects even after repetitive exposures, one displaying neutrophilic airway inflammation and the other neutrophilic and eosinophilic inflammation, respectively .

We have characterized a number of components that are likely to be involved in neutrophilic inflammation. Proteases are one of the components of agricultural dust that has been shown to be in part responsible for this type of inflammation . Our results are consistent with neutrophilic inflammation observed in people. In addition, since we delivered agricultural dust extract under light isoflurane anesthesia, the previously reported sex-related differences of isoflurane might confound some of the sex-related effects we observed in our study; however, given all the mice in each group underwent this light anesthesia the significant differences between the groups would still stand. Compelling data indicate the importance of having a balanced ω-6:ω-3 PUFA ratio for optimal health , yet the typical Western diet has a ratio of ∼10–20:1 . This imbalance is considered a driving or compounding factor in chronic inflammatory diseases, with many pro-inflammatory lipids formed from the ω-6 PUFA arachidonic acid, such as leukotrienes, thromboxanes and prostaglandins . It was recently reported that Veterans with COPD and an occupational history that include agricultural work had an ω-6:ω-3 PUFA ratio of ∼50:1 , underscoring the potential vulnerability of this population. Supplementation with ω-3 PUFA has demonstrated health benefits in clinical studies of cystic fibrosis, COPD, and other lung diseases . Thus, increasing ω-3 FA intake, possibly in combination with a reduced intake of ω-6 PUFA, could be an accessible and effective means of preventing and ameliorating airway disease in patients with inflammatory lung diseases such as those caused by agricultural dust exposures. Taken together, our investigations utilizing the Fat-1 mouse model in conjunction with sEH inhibition highlight the potentials of targeting repair/ resolution pathways therapeutically to promote lung protection from environmental dust exposures, and also highlight differences based on sex in the protectiveness offered by these interventions.

In the case of agriculture workers who are chronically inhaling inflammatory dusts, these outcomes hold promise for improving lung health outcomes via both limiting lung inflammation but also promoting repair following injury in this vulnerable population.The current projection for the world population to reach 9 billion by 2050 strikes an alarm when considering global food security. This drives a need, even greater than already present, to ensure sustainable and resilient agricultural systems with maximal crop production. For decades, countless studies have exemplified the benefits of using plant growth promoting rhizobacteria as soil inocula to improve agronomic productivity . Various strains of PGPR are able to fix nitrogen, solubilize phosphorus, sequester iron, suppress stress ethylene production by roots, produce plant growth hormones, antibiotics and anti-fungal compounds, and to enhance competitive exclusion of plant pathogens. The first recorded commercialization of PGPR was the use of rhizobia in 1895 . Since then, over a dozen different genera have been introduced to soils or seeds and many are sold commercially . In fact, Popular Science named Bio-Soil™, a cocktail of over 300 species of PGPR developed at Michigan State University, as one of the top 10 solutions for the future of farming . PGPR can be categorized based on their beneficial traits. For example; a nitrogen fixer would be termed a biofertilizer,nft system an organism emitting plant growth hormones is a phytostimulator, and ones secreting antibiotics and antifungal compounds are bio-control agents. While PGPR offer a multitude of benefits for healthy plant growth and development, this body of work focuses on mineral phosphate solubilizing bacteria and phytostimulators. Root colonization by phytostimulators has been shown to enhance root development, resulting in greater total root surface area and enhanced nutrient and water absorption. In turn, phytostimulators play the greatest role in increasing crop yields in stressed agriculture, as plant-hormone interference can induce plant systemic tolerance to drought, flooding, salinity, and heavy metals . Phosphorus is a macronutrient essential for plant growth and development and current agricultural practices have become reliant on the application of P fertilizers, especially phosphate rock. Consequently, current reservoirs of phosphate rock are expected to become depleted in 50– 100 years . Inorganic P accounts for approximately 35– 70% of total soil P . Plants are able to uptake Pi in its soluble forms . In soils with neutral to slightly acidic pH’s an abundant amount of Pi is available in these soluble compounds, but unfortunately this can result in the loss of P from plant root zones via leaching. Also, Pi forms highly insoluble mineral complexes with Ca in alkaline soils and with Al and Fe in strongly acidic soils, such that it has maximum availability to plants only at near neutral pH . These issues present a challenge as approximately 75% of P added to soil via fertilizers becomes non-plant available Rodr g e and raga, 1999. To release mineral-bound Pi some bacteria have MPS capabilities, typically achieved by the release of organic anions and correspondingly free protons . This lowers the pH of the soil solution and can release P from Ca, Al, and Fe complexes Rodr g e and raga, 1999). Hence, MPS bacteria can function as biological fertilizers, releasing mineral-bound Pi currently present in soils and reducing the need for additional P fertilization. Plant growth promoting microorganisms can affect root development by generating growth hormones or depleting hormones that would otherwise cause stunted root development.

Ethylene, a gaseous phytohormone, is critical for many plant developmental stages. While an initial peak in ethylene concentration in plant tissues is essential for root and stem growth and flower and fruit development, its continued accumulation triggers a cascade of responses including inhibition of root elongation, induction of hypertrophies, acceleration of aging, promotion of senescence and abscission, and inhibition of auxin transport . Ethylene can also inhibit stimulation of cell proliferation and elongation by repressing auxin response factor synthesis . The ethylene-mediated stress response can be activated by many environmental factors such as heavy metal contamination, high salinity, flooding, drought, and phytopathogens. The pathway to ethylene biosynthesis in plants involves the conversion of methionine into S-adenosyl-L-methionine by an enzyme SAM synthetase followed by the hydrolysis of SAM to form 1-aminocyclopropane-1-carboxylic acid by ACC synthase. In the next step, plant produced ACC oxidases bind to ACC with high affinity, with Km values range from about 50– 120 µM, and catalyze the conversion of ACC to ethylene, carbon dioxide and hydrogen cyanide . Conversely, if ACC is exuded from root tissues, root colonizing bacteria can uptake the ACC and use it as a nitrogen source for growth. Microorganisms with ACC deaminase cleave the propane ring of ACC to produce ammonia and α-ketobutyrate, both of which are then metabolized by bacteria . However, ACC deaminase enzymes have low binding affinities for ACC, with Km values ranging from 1.5– 17.5 mM . Thus, it has been suggested that ACC deaminase must be available at 100– 1000 fold greater amounts than ACC oxidase to reduce the accumulation of deleterious ethylene levels in plant tissues . The genetic regulation of the ACC deaminase structural gene, acdS, has been well described for Pseudomonas putida UW4, detailing an intricate regulation promoted by an ACC-bound complex and inhibited by a protein transcribed upstream of acdS, termed the ACC deaminase regulatory protein, AcdR. The intergenic space between these genes has been identified as the P. putida UW4 promoter region for AcdS, where binding of both regulatory complexes occurs . In particular, bacterial strains that generate an auxin compound, indole-3-acetic acid , stimulate total root length, root hair formation, and root branching when colonizing plant rhizospheres . When applied as a seed coat, Enterobacter cloacae UW5 promoted increased root branching and total root length in both mung bean and canola in growth pouch assays . This activity was explicitly linked to IAA production by UW5 . The indole-3-pyruvate pathway is considered to be the major pathway for IAA synthesis in plants and is also utilized by many bacteria . The genetic regulation of a key enzyme, indole-3-pyruvate decarboxylase , involved in this pathway has been well described. Strain UW5 has been shown to have a pathway to IAA production in which the genetic regulation of ipdC is induced by tryptophan . The promoter region of the IpdC gene has a tyrosine transcriptional repressor recognition box . The transcription of ipdC is promoted by the binding of the TyrR box by tryptophan, phenylalanine, and tyrosine, all of which are plant root exudates . The development of commercial bio-fertilizers is difficult as a result of poor or inconsistent survival rates of soil inocula, making quality assessment and verification of any given product very difficult . For example, Acea et al.effectively demonstrated that declines in population densities of soil inocula, such as Pseudomonas, Rhizobium, and Bacillus spps. corresponded to increases in populations of bacterial predators and bacterial competitors . Additionally, direct incorporation of liquid inoculum into soils is complicated by bacterial adhesion to soil particles, which greatly reduces their vertical transport and the ability to colonize roots located in the subsurface soil profile .

Photodynamic therapy has emerged as an efficacious adjuvant treatment modality for several types of cancer.In PDT, light is used to locally excite a photosensitizer to generate reactive oxygen species. The resulting oxidative stress disrupts organelle functions, promotes cell apoptosis, and damages the tumor vasculature that supply oxygen and nutrients required for the tumor to survive.While a few PDT therapies have received FDA approval , efficient delivery of the PS to the target site remains challenging. Tumor accumulation of the PS is generally poor due to the physicochemical properties of the PS.Therefore, large doses are administered to compensate for the poor drug accumulation at the target site. This is particularly unfavorable because most PS suffer from slow in vivo clearance, which increases toxicity. For example, as skin is highly vascularized and easily exposed to light, the long circulation time of PS promotes skin phototoxicity. As a result, patients are required to limit their exposure to the sun several weeks post-treatment. Therefore, there is a critical need to develop delivery systems with enhanced clearance that promote the accumulation of the PS in the tumor site. To this end, I turned toward the development of plant virus-based nanoparticles for the delivery of PS. VNPs have been developed as carriers for the delivery of contrast agents, chemotherapeutics, protein therapies, epitopes, agro-pesticides, as well as PS . Plant VNPs have several attributes that are favorable for nanomedicine delivery and in particular PS delivery. Bio-manufacturing is well established and the biologic platform offers well-defined,hydroponic channel monodisperse structures that can be tailored with molecular precision.

Plant VNPs are non-infectious toward mammals, and most importantly the proteinaceous nanoparticles are cleared rapidly from circulation and from tissue,thus making this a particularly attractive platform for PS delivery. Plant VNPs as well as bacteriophage-derived nanoparticles have been developed for PS delivery;in most instances PS agents are covalently coupled to viral carriers. However, covalent binding of the PS to nanoparticles may impair their photoactivity due to quenching and reduced molecular freedom, and in turn limit their intracellular activity. Therefore, non-covalent drug delivery may be advantageous to enhance and control steady release of the PS within the tumor environment. This strategy relies on hydrophobic-hydrophilic and electron charge interactions between the PS and its carrier. In this work, I utilized two high aspect ratio, soft matter tubular nanostructures for PS delivery, namely tobacco mosaic virus and tobacco mild green mosaic virus . TMV and TMGMV were selected as carrier platforms based on their well-established surface chemistry and elongated shape. Elongated nanoparticles have enhanced blood margination, transport across tissue membrane, cell adherence, and macrophage avoidance, promoting their accumulation in the tumor tissue.TMV and TMGMV self assemble helically around a single-stranded RNA genome to form a 300 x 18 nm rod with a 4 nm wide hollow interior channel . As described in chapter II, both particles are made of 2,130 identical copies of coat protein units; TMV and TMGMV share 86% sequence homology.Of particular interest, the interior channels of TMV and TMGMV are covered with solvent exposed glutamic acids that are readily available for electrostatic loading of positively charged guest molecules .While TMV has been extensively studied for clinical applications, including the delivery of PS,this is the first study investigating TMGMV for medical applications. To probe drug loading and release, I studied the monocationic, dicationic, tricationic and tetracationic version of a zinc porphyrin photosensitizer. Lastly, we selected one formulation and developed a cancer cell targeting strategy to further enhance treatment efficacy.The TMV results can be attributed to the combined effect of electrostatic and hydrophobic/hydrophilic interactions; the greater the positive charge the better stabilization inside the TMV interior channel.

In addition, the increased hydrophobic nature of the monocationic and dicationic Zn-Por formulations in combination with their electrostatic properties led to the formation of more aggregates compared to their tricationic and tetracationic counterparts, thereby reducing the loading efficiency. Several factors may explain the differential loading results between TMGMV and TMV. In chapter II, I have previously compared the amino acids sequences of TMV and TMGMV and analyzed their distribution of charged residues on both the inner and outer surfaces of the virus.While it has been shown that only two glutamic acid residues are chemically available on TMV , our analysis revealed that in addition to the Glu 95 and Glu 106 in the interior channel of TMGMV, Glu 145 and aspartic acid 66 were also exposed on the outer surface and could be available for electrostatic charge interactions. The difference in the amino acid sequences of TMV and TMGMV could also play a role in the difference in loading by changing the charge and hydrophobicity surrounding the glutamic acid residues. Furthermore, the virus coat proteins are not rigid structures, and therefore small molecules could diffuse in between coat proteins.Based on the above studies, I prepared drug-loaded VNPs using the 2000:1 Zn-Por:VNP ratio. I studied whether changing the pH of the 10 mM KP buffer solution would influence the loading efficiency of Zn-Por into TMV and TMGMV . At pH 3, VNPs aggregated and disassembled, which led to lower yields. The corresponding loading efficiency was low due to the protonation of carboxylate groups, resulting in weak electrostatic interaction. At pH 5, the reaction yields and loading efficiency were improved compared to pH 3, and reached their maximum at pH 7.8. Increasing the pH to 10 did not increase the loading yield, but rather just slightly decreased loading efficiency and reaction yields. While ~60−75% of starting materials were recovered at pH 7.8, the yield dropped to ~40% at pH 10.

Based on the findings of the pH studies, I conducted the remaining experiments at pH 7.8 due to the relatively high loading efficiency and recovery observed at this pH. Next, I analyzed the drug release profile of each Zn-Por:VNP formulation . 1 mg of particles was resuspended in 300 μL PBS and loaded in triplicate in 10,000 MW cutoff Slide-A-Lyzer MINI dialysis units for 72 hrs. To mimic physiological conditions, samples were dialyzed against 3 L of PBS adjusted to pH 7.4 as well as pH 5 at 37°C. At time t = 0, 1, 3, 6, 18, 24, 48, and 72 h, 10 μL was extracted from each dialysis unit and the remaining Zn-Por entrapment was measured by UV/Visible spectroscopy. The half-life t1/2, defined as the time required for 50% of the drug to be released from the VNPs, decreases as the electropositivity of Zn-Por increases. At pH 7.4, TMV: Zn-Por4+ and TMGMV: Zn-Por4+ formulations had the lowest t1/2 . In contrast, only 20% and 25% of ZnPor1+ was released from TMV and TMGMV respectively within 72 hrs. The release profiles of Zn-Por3+ were similar to that of Zn-Por 4+, while the release rates of Zn-Por2+ were in between those of Zn-Por4+/3+ and Zn-Por1+. While the t1/2 values of each Zn-Por:VNP formulation were slightly lower at pH 5, the trend remained the same. These results indicate that the dominant force of interaction between TMV/TMGMV and Zn-Por is not electrostatic,hydroponic dutch buckets but rather hydrophobic/hydrophilic interactions. Since Zn-Por becomes more hydrophobic as its electropositivity is reduced, its ability to solubilize in PBS surrounding the VNP is impaired, thereby decreasing the rate of drug release. I also tested stability of the Zn-Por:VNP formulations under storage conditions , and observed a slow and constant release of Zn-Por from TMV and TMGMV over a period of 6 weeks Less than 45% of Zn-Por was released from the other formulation within 6 weeks. To evaluate in vitro efficacy of Zn-Por:VNP formulations, I first compared TMV and TMGMV’s uptake by B16F10 melanoma cells. Melanoma was chosen as a model because PDT has shown promise in melanoma.While most melanomas are removed by surgery supplemented with adjuvant chemotherapy and/or immunotherapy, some melanomas remain unresponsive to these therapies. A growing body of data indicates that PDT could be applied as an adjuvant therapy for those melanomas not responsive to traditional therapies.For cell uptake studies, TMV and TMGMV were conjugated with the fluorophore Cyanine 5 using solvent exposed tyrosine side chains click chemistry,followed by the purification of the reaction mixture as previously described. The covalent attachment of Cy5 was confirmed by UV-vis and denaturing SDSNuPAGE gel electrophoresis . We have previously demonstrated that a minimum conjugation of Cy5 to ∼8% of TMV coat proteins is sufficient to yield maximum fluorescence intensity.237 TMV and TMGMV particles displayed ~160 and ~490 dyes respectively. The higher dye conjugation efficiency in TMGMV could be due to differences in the chemical micro-environment and greater surface exposure of the tyrosine side chain.

The corresponding average distances between fluorophores are equal to 2.7 nm and 1.6 nm for TMV and TMGMV respectively, which are large enough to prevent quenching due to energy transfer between dye molecules and trapping by dimers.Therefore these Cy5-TMV and Cy5-TMGMV constructs are suitable for imaging experiments. To assess VNP–cell interactions, B16F10 melanoma cells were incubated with 100,000 VNPs per cell at 37 °C and 5% CO2 for 1 h and 8 h in Dulbecco’s modified Eagle’s media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were washed thoroughly with FACS buffer 0.5 M EDTA, 1% FBS and 2.5% 1 M HEPES pH 7.0 in DPBS and fixed with 2% paraformaldehyde. Cells were then analyzed using a BD Accuri C6 Plus flow cytometer and 1 x 104 events were recorded. Data were analyzed using FlowJo v8.6.3 software. After 1 h of incubation, 85% and 100% of TMV and TMGMV were taken up by B16F10 cells, respectively . This is reflected by an increase in mean fluorescence intensity compared to cells only . The slightly higher uptake of TMGMV may be attributed to greater particle instability during viral production and purification, which causes some of the particles to be broken218; a shorter TMGMV rod would have a faster rate of cell penetration. Nonetheless, the cellular uptake of TMV and TMGMV reached 93% and 100%, respectively, after 8 h of incubation. This time point was selected to allow VNPs to traffic through the cells before proceeding with the photodynamic treatment. I evaluated efficacy of the drug delivery approach against B16F10 cells using previously established white light therapy.The following samples were tested: Drug-free VNPs, free Zn-Por, and Zn-Por-loaded VNPs, and a dark control for each sample was included. Cells were incubated with 0.001, 0.01, 0.1, 1, 5, and 10 μM of Zn-Por, Zn-Por:VNP, or controls for 8 h at 37 °C and 5% CO2. Cells were washed to remove any Zn-Por that was not endocytosed and samples were illuminated under white light for 30 min . Control samples were kept in the dark at 37 °C and 5% CO2. In all experiments, neither dark controls nor any of the VNP only controls showed significant cell toxicity. Free Zn-Por1+ was 1.8 to 2.8-fold more effective compared its TMV/TMGMV formulation; this reduced efficacy was even more dramatic for the Zn-Por3+ loaded particles which showed a 30-50 fold decrease in efficacy. The decreased drug activity of VNPs loaded with Zn-Por vs. free Zn-Por is expected. The reactive oxygen species produced by PS drugs have a very short half-life and act locally from their generation site. Therefore, the subcellular localization of the PS greatly influences its phototoxicity. Like most nanoparticles, TMV and TMGMV are internalized by endocytosis and follow the endosomal-lysosomal pathway. Previous data suggest the phototoxicity of PS localized in lysosomes is significantly reduced compared to PS localized in other organelles, in particular in mitochondria.On the other hand, hydrophobic PS with cationic charges such as free Zn-Por is likely to localize in mitochondria.Nonetheless, TMV and TMGMV are here used to improve the bio-availability and tumor accumulation of Zn-Por while reducing non-specific tissue toxicity. TMV and TMGMV can be further chemically or genetically modified to display moieties such as cancer cell targeting ligands, cell penetrating ligands, and chemotherapeutics for combined therapy, which would further improve the treatment efficacy. As a proof of concept, we set out to develop a targeted Zn-Por delivery system. We chose Zn-Por3+ and TMV, in particular we used the well-established and characterized Lys-added mutant denoted as TMVlys. While TMGMV showed greater toxicity than TMGMV, the genetic engineering of TMGMVlys mutant has yet to be established in the future. TMVlys offers amine functional groups for bio-conjugation: targeting ligands synthesized with a terminal Cys side chain can be conjugated using heterobifunctional NHS-maleimide linkers. Here we chose the F3 peptide as the ligand.

They demonstrated that abamectin encapsulated in RCNMV had increased stability and superior mobility in soil compared to free abamectin, which resulted in enhanced bio-availability and treatment efficacy in tomato seedlings. As expected, no viral infection in the tomato seedlings was observed, as tomato is not a RCNMV host species. In the present work, I propose the use of tobacco mild green mosaic virus , also known as the U2 strain of tobacco mosaic virus , as a carrier to deliver nematicides. TMGMV self-assembles into a 300 by 18 nm rod-shaped virus with a 4 nm wide hollow channel. The high aspect ratio soft-matter nanotube may provide a promising alternative over the spherical platform technologies, i.e. most synthetic nanoparticles as well as RCNMV. The nanomedicine field has demonstrated that carrier shape impacts the in vivo fates with elongated materials conferring advantages with enhanced margination, diffusion, and penetration through tissue.Whether high aspect ratio materials perform better in soil remains to be seen. Another advantage of the TMGMV platform is the high surface area , 3.9 times larger than RCNMV , which may allow for higher payload delivery. TMGMV is already EPA-approved: Solvinix, a formulation of TMGMV mass-produced by BioProdex, is commercially available as an herbicide in the state of Florida for the treatment of the invasive weed tropical soda apple.TMGMV is not transmitted by insects, pollen, or other vectors; it is not seed borne and cannot self-disseminate. While TMGMV is capable of infecting solanaceous plants , TMGMV is unable to penetrate and infect healthy plants in the absence of a lesion wound.

Furthermore, Solvinix was tested on 435 plants representing 311 species,blueberry box among which only 8% of plants were killed.It is therefore safe to conclude that TMGMV can be applied in the field with little to no risk to the environment or the crop itself. Although there are a few species of plants to which TMGMV is lethal,it is important to remember that the remaining 3000 species infected by parasitic nematodes are not susceptible to TMGMV. While its structure is known to atomic resolution ,the chemistry of TMGMV has not yet been established. Making use of the structural information and the well established chemistries for tobacco mosaic virus , I developed bio-conjugation techniques and non-covalent drug loading strategies for TMGMV. As a proof of concept, I used the anthelmintic drug Crystal Violet in our studies.The supernatant was subsequently analyzed by UV/visible spectroscopy .TMGMV is the U2 strain of tobacco mosaic virus ; the latter has been extensively studied in plant pathology and structural biology since the 1900s and more recently in nanomedicine, biotechnology and energy research.173 Therefore, the surface chemistry of TMV is well understood. Here I set out to establish the chemistry of TMGMV. The amino acid sequences of the coat proteins of TMV and TMGMV present 72% homology; also the structural overlay of a single CP of TMV and TMGMV reveals a high degree of structural similarity, only 14% of the amino acids do not overlap in the crystal structures . This is also reflected when comparing the assembled nucleoprotein complexes of TMV and TMGMV . Just like TMV, TMGMV forms a cylindrical structure measuring 300 by 18 nm with a 4 nm-wide hollow interior channel. The TMGMV particles consist of 2130 identical copies of CP units arranged helically around a single-stranded RNA genome .

Analysis of the structure reveals the amino acid profile on the exterior and interior surface: because LYS, CYS, TYR, ASP, and GLU are often targeted for bio-conjugation or electrostatic drug loading, I analyzed the TMGMV structure for presence of these residues. While solvent-exposed LYS and CYS side chains were not identified in TMGMV , several TYR, ASP and GLU residues were found to be solvent-exposed on the exterior/interior TMGMV surfaces. Structural data indicate TYR2 to be exposed on the exterior surface – this is different from the structure of TMV, for which both TYR2 and to a greater extent TYR139 are solvent-exposed on the exterior surface . The TYR2 side chain of TMGMV could provide a potential target for bio-conjugation, e.g. the introduction of a fluorescent label for imaging and tracking studies as described below. Further, I identified ASP66 and GLU95, 106, and 145 to be solvent exposed, with GLU145 and ASP66 located on the exterior surface and GLU95 and GLU106 on the interior surface . This is similar to the structure of TMV, for which GLU145, ASP64 and ASP66 are solvent-exposed on the exterior surface while GLU97 and GLU106 are solvent-exposed on the interior surface . However, it should be noted that previous research identified GLU97 and GLU106 to be the only carboxylates in TMV that are reactive toward carboxylate-specific chemistries; GLU145 and ASP64 and 66 were not found to be reactive.The presence of GLU/ASP residues in TMGMV would allow for functionalization through bio-conjugate chemistry or electrostatic loading of positively charged guest molecules, as we previously described in the case of TMV.Lastly, I analyzed the surface charge of TMGMV and determined that the inner and outer surfaces carry a net negative coulombic charge with the interior being more electronegative than the exterior . Together these data indicate solvent-exposed TYR side chains on the exterior surface of TMGMV and addressable carboxylates – possibly on the exterior and interior surfaces. With the structural information in hand, I set out to develop TMGMV as a carrier for nematicide delivery.

Specifically, I chose to work with crystal violet as a proof-of-concept, because this therapeutic compound is fluorescent and thus streamlines the analysis. The positively charged CV was loaded into TMGMV making use of electrostatic interactions and concepts that were previously developed to load positively charged platinum drug candidates and porphyrin derivatives into TMVThe reaction mix of 6000:1 CV:TMGMV resulted in the highest loading efficiency while still yielding dispersed TMGMV particles: 68% of the CPs were modified with a CV molecule. Assuming a full length TMGMV particle , each TMGMV would carry ~ 1500 drug molecules. This formulation was subsequently used for all following studies. When compared with TMV-drug formulations, the TMGMV formulation yielded comparable results: I previously reported the loading of 2,000 phenanthriplatin per TMV206 and 900 copies of a porphyrin derivative ZnPr per TMV.235 In those cases, the loading procedure was similar, in which a positively-charged guest molecule was loaded via electrostatic interaction with TMV’s interior carboxylates. As in the case of TMV, the interior channel of TMGMV is lined with a dense layer of carboxylates – this, in combination with the more electronegative interior surface,blueberry package may suggest that drug loading occurs on the inside channel. However further studies would be needed to rule out drug association with the exterior surface in both the cases of TMV and TMGMV. To compare the drug loading efficiency of the rod-shaped TMGMV system to the icosahedral RCNMV-based nematicide carrier, the number of drug molecules was normalized to the molecular weight of the nanocarrier yielding ~3.6 × 10-5 CV per dalton of TMGMV protein, while only ~1.8 × 10-5 abamectin molecules were loaded per dalton of RCNMVprotein. In other words, when normalized per molecular weight, twice as much drug molecule can be loaded per TMGMV than compared to RCNMV. The structural integrity of non-modified TMGMV and CVTMGMV were assessed by size exclusion chromatography and transmission electron microscopy . SEC measurements revealed no significant difference comparing native TMGMV and CVTMGMV; both particles showed the same elution profile . Further, TEM imaging of TMGMV and CVTMGMV revealed rod-shaped samples with no apparent differences when comparing TMGMV and CVTMGMV ; TEM imaging indicates that the average length of TMGMV and CVTMGMV is comparable measuring 146 ± 97 nm and 136 ± 76 nm, respectively .It is possible that this is an artifact from the TEM grid preparation, i.e. the particles may break during the drying process. However, it is important to note that there are no apparent differences comparing the TMGMV and CVTMGMV, indicating that the nucleoprotein complex withstands the loading and purification process. To gain insights into whether CV- loading into TMGMV is indeed via electrostatic interactions with GLU and/or ASP residues, chemically modified TMGMV in which the carboxylates were neutralized was prepared. To do so, EDC coupling was used to introduce alkyne ligands at the carboxylates, for subsequent addition of biotin labels using Cu-catalyzed alkyneazide cycloaddition . The protocols are detailed in the methods and were adapted from previous methods established for bioconjugation to TMV.Biotinylation was confirmed by western blot , yet quantitative data could not be obtained. To quantify the degree of labeling, the fluorescent Cy5 dye was conjugated to GLU/ASPTMGMV, yielding ~275 dyes per full length TMGMV, or about 13% of CPs were modified with Cy5 . Biotinylated and alkyne-labeled TMGMV were then used in CV-loading experiments, and I observed a 40% decrease in CV loading when using alkyne-labeled TMGMV compared to unmodified GLU/ASPTMGMV . Severe aggregation was observed when biotinylated TMGMV was used in CV loading experiments .

This phenomenon may be explained as follows: if the chemistry of TMGMV and TMV is matched, then biotins will be displayed along the interior channel, preventing the positively charged guest molecules to be loaded and protected inside the TMGMV channel – instead CV may cross link the particles through interactions with the while less negative, also negatively-charged exterior surface. Because the data indicate that CV-loading is mediated through the solvent-exposed GLU/ASP acids, and in light of the TMGMV structure and its similarities to the known biochemistry to TMV,I therefore expect that interior loading of CV is achieved by this method. Next, I evaluated the release profile of CV from the TMGMV nanocarrier. The release rate of CV from CVTMGMV is expected to be proportional to the pH of the bathing conditions as well as temperature. Based on thermodynamics, the rate of diffusion should increase with temperature. Furthermore, as pH decreases, a larger number of carboxylate groups become protonated and carry a net neutral charge that can no longer interact with the positively charge CV and consequently, free CV should diffuse away from TMGMV. Therefore the release rate of CV should be higher at lower pH and higher temperatures. To test this experimentally, 1 mg of a 1 mg.mL-1 solution of CVTMGMV was prepared as described above and dialyzed against various buffers for 72 h . I tested the release profile at room temperature and 4°C to evaluate two extreme upper soil thermal conditions.Sodium acetate and PBS buffer solutions were chosen to mimic the acidic and neutral soil environments respectively. Diffusion of CV from CVTMGMV was also evaluated in KP buffer, which was used during loading and storing conditions of the sample. Free CV, in a concentration matched to the concentration and number loaded into TMGMV, was also dialyzed in KP buffer at 4°C as a positive control. As expected, increased release rates of CV from CVTMGMV were observed at low pH and high temperature . Approximately half of the free CV was dialyzed within 1.6 h and complete release was observed in less than 18 hrs, while delayed release profiles were observed for the CVTMGMV nanoparticle formulations. For CVTMGMV, 50% of CV was released only after 5 h in acidic conditions at room temperature , with complete release achieved after about 24 hrs. These conditions most realistically mimic the soil environment. In stark contrast, release in storing conditions was significantly slower, with 50% of CV released within 13 hrs, and complete release was not observed within 72 h. This is promising for application of these nanoparticles, however it would be advised to prepare fresh formulations before application in the field. We have previously reported similar results with the release of the cancer drug phenanthriplatin from TMV;half of the encapsulated chemotherapeutic was released after 1 h at pH 5 and 24 h at pH 7.4. On the other hand, encapsulated porphyrin derivatives loaded in TMV were found to be stably encapsulated for at least one month when stored at 4°C and pH 7.I hypothesize that the increase in stability of the porphyrin drug was due to its higher electropositivity: the compound used carries 3 positive charges. In contrast, phenanthriplatin and CV carry 2 and 1 positive charges, respectively. Compared to the previously reported RCNMV carrier, the release rate of CV from TMGMV is slightly faster than that of abamectin from RCNMV in acidic soil conditions. 50% of abamectin was released within 8 and 7 h at pH 5.2 and 7.4 respectively .

When immobilization processes are considered retention is increased to an even greater extent and peak in-stream concentrations range from approximately 8.1×10−3 to 1.8×10−5 oocysts/mL at 100 m and 700 m, respectively. Thus, oocyst in-stream concentrations quickly decreased when immobilization processes were considered, which can then remain immobilized within the sediments and remobilize at a later time .In scenario 1, 99.7% of the oocyst inputs already passed 100m at 1 month with only 0.1% of the inputs inactivated, however when immobilization processes were considered only 33.8% were observed in the mobile phase prior to the stop of the input . Although in-stream concentrations decrease relatively quickly , pathogen accumulation within immobile zones results in high counts within these areas for long periods of time . After relatively quick immobilization, Cryptosporidium is slowly released back to the water column, where at 100m the Cryptosporidium immobilized decreases from 2.2×109 to 1.4×109 oocysts, 66.2% to 43.0% of the inputs, from 1 to 6 months . These results demonstrate that Cryptosporidium persists for months at 100m and may rework its way downstream, but will remain within the first 700m for years after the input under base flow conditions .When considering a pathogen with such a low infectious dose,grow bag gardening the retention within streambeds can dramatically increase the time during which thresholds for safe consumption are exceeded. These results demonstrate that both hyporheic exchange and immobilization processes within sediments and other slower moving areas increase pathogen persistence in streams.

Cryptosporidium oocysts are readily immobilized within the first 100 m and stay for days to years within transient storage areas. In fact, at 100 m Cryptosporidium in-stream levels do not reduce to below 10−5 oocysts/mL until 280 days after the start of a 1-month input. At 700 m from the simulated input of Cryptosporidium, in-stream concentrations only slightly exceeded 10−5 oocysts/mL for a short period of time, but the majority were immobilized in transient storage areas within the 700 m reach downstream of the source. Te numbers of viable oocysts remaining in the 100m reach downstream of the input decreased over time, while the numbers of viable oocysts from 100–300m increased and then decreased from 0 to 24 months and the reaches further downstream showed a consistent increase in the number of viable oocysts. The constant reworking of sediment beds has previously been observed, and is a combination of flushing, trapping, and accumulation of fne sediment and microbes that occurs both at base flow and during high flow events. The model scenario was for 1 month and only one stream inlet, but the landscape is continuously adding Cryptosporidium to the stream, which can result in even more accumulated pathogens within the stream. As non-point sources are distributed among the watershed, there will be constant and sporadic inputs simultaneously, but the majority of the input will be deposited within the first 100 m, at least initially before remobilization processes move the pathogenic microorganisms further downstream. Cryptosporidiosis remains an important waterborne disease in both the United States and Europe, posing significant public health and economic problems. Likely the high risk is due to a combination of a low consistent input of Cryptosporidium, long retention times, and low inactivation rates that can result in long-term accumulation within streams.

Accumulated pathogens can subsequently resuspend slowly back into the water column during base flow or as a pulse in response to a storm event. Previous cryptosporidiosis outbreaks have occurred following a high flow event, where microbes are rapidly resuspended due to scour of streambed sediments. The extent of microbial resuspension is dependent on the frequency of high flow events, microbial colonization of benthic bioflms, macrophytes, and sediments, and potential for erosion or remobilization from these reservoirs. Implications of pathogen persistence within stream storage areas, and in particular sediments, includes increased risk especially when climate change is expected to increase the duration of drought periods and lead to intensified rain events. This model framework can be used to help predict response to storm events as pathogens remobilized during high flow conditions are directly related to the in-stream source of pathogens accumulated during base flow. Furthermore, this model framework can incorporate new information to improve predictions of specific Cryptosporidium species. The use of molecular diagnostic tools has significantly improved our understanding of cryptosporidiosis epidemiology and now it is known that human cryptosporidiosis is predominantly caused by C. hominis and C. parvum. It has also recently been discovered that Cryptosporidium can multiply without host cell encapsulation , which potentially poses an even greater environmental risk. Overall net inactivation was observed in our study case, but specific sub groups and growth can be incorporated into the model framework as needed for specific cases or applied to other pathogenic bacteria of interest.

This could be especially useful if supporting lab-scale experiments have been conducted to parameterize the retention within the sediments, previously shown to control the late-time tailing of observed in-stream breakthrough curves. Here we presented how the mobile-immobile model framework is a first step for accurately characterizing pathogen transport and retention in stream. A mobile-immobile framework can accurately characterize pathogen dynamics in streams and incorporate key processes that lead to long residence times. The fate of the pathogens after entering a stream and the long-term retention has been underestimated if hyporheic exchange and immobilization processes within sediments and other storage areas is not considered. The multi-scale model enables information on pathogen retention in column experiments to be related to net downstream transport at the stream-reach scale. Therefore, while it is not possible to conduct field scale tracer experiments with pathogens, lab scale studies, such as column experiments can be used to parameterize reach-scale estimates,plastic grow bag as demonstrated within this modeling study. The combination of stream reach-scale analysis and multi-scale modeling improves assessment of Cryptosporidium transport and retention in streams to predict downstream exposure to human communities, wildlife, and livestock. The mobile-immobile model framework can be modified to any system of interest to estimate base flow pathogen accumulation and therefore help predict the potential loads of resuspended pathogens from the streambed to the water column in response to a storm event.Bio-solids are the nutrient-rich byproduct of wastewater treatment operations and large quantities are generated. For example, approximately 750,000 dry tons is produced annually in California and 54% of these bio-solids are applied on agricultural lands, 16% are composted and the remaining 30% goes to landfills . Concerns about potential health and environmental effects of land application of bio-solids include possible off-site transport of pathogens, heavy metals, and trace organic constituents such as TCS . A less explored set of potential impacts is how TCS and other bio-solid-borne contaminants affect ecosystem processes and associated soil microbial communities. Potential impacts on soil microorganisms are important to assess since these organisms mediate much of the nitrogen, carbon and phosphorous dynamics in soil, biodegrade contaminants, create soil structure, decompose organic compounds, and play a major role in soil organic matter formation . We hypothesized that bio-solids containing TCS would have detrimental effects on soil microbial communities by decreasing biomass and altering community composition in agricultural soil. Our objectives were to evaluate the effects of increasing amounts of TCS on soil microbial community composition in the presence and absence of bio-solids. We used phospholipid fatty acid analysis to characterize the response of microbial communities; the method provides information about microbial community composition, biomass, and diversity . Experiments in which TCS was added to soil without bio-solids allowed the relative effects of bio-solid and TCS addition on microbial community composition and function to be compared and also provided a “secondary control” because TCS-free municipal bio-solids are essentially unavailable in the United States .

Triclosan was purchased from Fluka . Yolo silt loam was collected from the Student Experimental Farm at the University of California, Davis at a depth of 0 to 15 cm. The soil was passed through a 2 mm sieve and stored at 4 °C until use. Bio-solids originated from a municipal wastewater treatment plant in Southern California that employed a conventional activated sludge treatment system followed by aerobic sludge digestion. Bio-solids from this system were selected for study because they had the lowest concentration of TCS among those collected from 10 different wastewater treatment plants in California . The soil and bio-solid physiochemical properties are reported in Table 1 and were determined using standard techniques . The soils were moistened to 40% water-holding capacity, which is equivalent to 18% water content in our experiments, and pre-incubated for 7 days at 25°C to allow time for normal microbial activity to recover to a constant level after disturbance. The pre-incubated 50 g of soil was weighed into 200 ml glass bottles to make three replicates per treatment. For the bio-solid amended soil sample, 20 mg/g of bio-solid was added. Each treatment sample was then spiked with TCS to achieve final concentrations of 10 or 50 mg/kg using TCS stock solutions prepared in acetone, as recommended by Waller and KooKana . This spiking level was chosen as a conservative upper bound on anticipated soil concentrations in the field. The lower spiking level is below the mean concentration observed in US bio-solids and the higher level is below the 95th percentile for US bio-solids ; adding bio-solids to soils at typical application rates would produce soil concentrations ~50–200 times lower. Control samples were also prepared with acetone only. After that, the solvent was allowed to evaporate inside the fume hood before the samples were thoroughly mixed. The microcosms were incubated in the dark at 25°C for 0, 7 and 30 days. Every week, each vial was opened to help keep conditions aerobic and the water content of each set of samples was measured and water was added as needed to maintain target moisture levels. At each sampling time, the remaining TCS was measured by drying 3–5 g samples at 70°C for 24 hours and homogenizing with a mortar and pestle. Replicate 1 g sub-samples of each dried sample were placed in centrifuge tubes, spiked with deuterated trichlorocarban in methanol, air dried under a fume hood to remove the methanol, and then mixed well. Extraction was performed by adding 15 mL of 1:1 acetone and methanol to the centrifuge tube. Samples were extracted on a shaker table for 24 hours at 295 rpm and 55 °C and then centrifuged for 30 min at 4,100 g. The supernatant was diluted as needed to ensure that the concentration remained within the linear portion of the calibration curve. The extracts were analyzed for TCS using LC-MS/MS. Additional details regarding the extraction and analysis procedures can be found in Ogunyoku & Young . As expected, the bio-solids contained far larger amounts of nitrogen and carbon than the Yolo soil . Even though the bio-solids constituted less than 2% of the amended soil, they contributed nearly 50% of the total nitrogen and 40% of the total carbon in the amended soil system. The bio-solids contained an abundance of nutrients accumulated as by-products of sewage treatment in forms likely to be more labile than equivalent nutrients present in the soil. As will be discussed further, the greater availability of C and N in the SB than soil treatments had a strong influence on some of the results, especially at the early time points. In the following section, therefore, it is useful to remember that all SB treatments contain more available C and N than all soil treatments. The initial concentration of TCS in unspiked SB samples was very low , fell below the quantitation limit for TCS after 7 days, and was not detectable after 30 days of incubation. Significant TCS bio-degradation was observed in spiked soil and SB samples during incubation and the data were well described using a first order model as indicated by linear plots of ln against time . Degradation trends were consistent at the two spiking levels for each sample type but bio-solid addition significantly reduced degradation rates at both spiking levels compared with un-amended samples. The percentage of TCS removed was approximately two times greater in soil than in SB samples. Approximately 80% of the TCS was removed over 30 days in soil treated with either 10 mg/kg or 50 mg/kg of TCS, but no more than 30% was transformed in the corresponding SB microcosms.

By using cohorts defined by narrow age groups, I am able to analyze how wages of such overall similar workers change as their cohort faces a higher rural out-migration shock. My investigation is challenged by the fact that information on the rural/urban status of a migrant’s locality of origin is not available in most population censuses and in the Brazilian censuses in particular. While the administrative region of a migrant’s origin may be known, these regions are often too large to meaningfully use data on population size or density to construct a proxy for the rural/urban status of the locality. In this paper I use the size of a cohort relative to a baseline year to measure rural net out-migration rates. More specifically a cohort’s rural out-migration in 1991 is measured as the change in the size of the cohort between 1980 and 1991. This net-migration method has been shown to perform well in the Mexico-US immigration by Martinez and Woodruff and Hanson and McInstosh. My empirical strategy is implemented using population census data from the Brazilian census of 1980, 1991 and 2000. The basic identification strategy is based on the fact that cohorts of workers have differential rural net out-migration rates and that these differences vary over time. I also address two key concerns about the potential sources of bias in the estimates. First, rural out-migration and wages may be simultaneously determined by, for instance,cut flower bucket pull factors such as urban employment growth or wages. Following the literature I apply an instrumental variables strategy using past rural out-migration rates as an instrument for current out-migration rates.

The results show that the instrument is strongly related to rural out-migration rates and reinforce the OLS estimates. Second, I address the issue of selectivity bias which arises because the measured wages in rural areas are obtained from selected workers who decided not to migrate. This is because, in the presence of self-selection into migration, rural out-migration may both reduce overall rural labor supply and affect the composition of rural labor supply. I show that the OLS and IV results are robust to controlling for key worker characteristics that may affect wages. Although addressing the issue only partially, this approach provides evidence that the OLS and IV results are robust to controlling for key characteristics that determine the composition of rural labor. My results show that a 10% increase in rural out-migration rate in a cohort raises wages in that cohort by 1 to 5%. This result suggests that rural out-migration flows in Brazil between 1991 and 2000 have increased wages by 3 to 6.5%. Moreover the impact on wages differs substantially by cohort. I find that rural out-migration between 1991 and 2000 led to a 4.5 to 5.6% increase in wages for the older cohorts of workers and between 7 to 9% for the younger cohorts. The remainder of this chapter is organized as follows: Section 2.2 provides an overview of the theoretical literature and presents the empirical framework. Section 2.3 describes the data, the basic results and some robustness checks. Section 2.4 discusses the issue of selection bias and changes in the composition of rural labor. Section 2.5 concludes.In this section I present the implications of existing models that could explain the effect of rural emigration on rural wages.

As discussed by Mishra, partial equilibrium models with closed economies suggest that, under certain hypotheses on the production function and uniform inputs, rural out-migration will raise real wages in rural areas leading to a welfare gain for workers, a welfare loss for owners of capital and an aggregate welfare loss. Models such as those in Findlay and Davis and Einstein extend the Ricardian model of international trade to allow for migration. In these models, under free trade, rural out-migration from will increase the real wage in rural areas in terms of the importable goods and remain unchanged in terms of the exportable good, leading to an increase of the real wage. However in the presence of a non-traded labor-intensive good, the real wages in rural areas could decrease with the degree of rural out-migration as shown in Quibria and Rivera-Batiz. This arises if migrants possess a large fraction of the capital or when there are increasing returns to the production of the non-traded good. Beyond the labor supply push, rural out-migration may also affect rural wages by increasing the rate of capital inflow. Remittances may increase consumption and investment of households at origin thus potentially raising local demand for goods and services and pushing prices and wages upward. In this paper I examine empirically how rural out-migration measured by changes in cohort size residing in a rural area affects the wage of rural workers in the same cohort. In light of the discussion above such analysis will measure, in general, the combined effect of changes in labor supply, capital inflow, and local demands for goods and services following a higher out-migration shock.

An important question is whether one can disentangle these effects or provide evidence on the main channels. The empirical model developed below presents a framework to estimate the effect of rural out-migration on rural wages. Due to the absence of data on rural capital stock, I assume that the stock of capital is fixed. As such, the results presented here provide long-run estimates of the effect of rural out-migration on rural wages in a general equilibrium setting. Where cohortsize is the size of cohort c living in rural areas of state s at time t. The variable shockemigrationcst measures the number of individuals which belong to cohort c of state s that have migrated out of rural areas as a share of the size of that cohort. The first issue is related to data limitations. While census data often includes information related to an individual’s migration status and administrative region of origin, they do not generally record whether the migrant was originally from a rural or urban area. Moreover, the administrative regions of origin are often too large to, meaningfully,flower display buckets use information on population size or density to construct measures of the rural/urban status of the locality of origin. The second issue is related to the identification of the effect of rural out-migration on rural wages. With information on individual’s migration status and rural/urban status of the locality of origin, one could measure the out-migration shock as the size of the rural locality residing elsewhere. However this will raise the concern that higher rural out-migration in a state could increase rural in-migration of individuals from other areas . Nonetheless while the approach in this paper deals with such concern, it doesn’t allow to separately estimate the effect of rural out-migration of native population and rural in-migration. The last issue is related to the definition of a rural area and whether this definition changes over time. In Brazil, the status of a locality as an urban or rural area is defined by municipal law and doesn’t adhere to any specific thresholds based on population size or density for instance. Moreover, the rural/urban status of localities did not change between the 1980 and 2000 censuses limiting concerns that rural/urban classification may be endogenous to living standards or wages. Lastly, the official rural/urban classification captures the differences in occupational structure that characterizes these two areas. In our sample over 80% of individuals residing in areas classified as rural are engaged in agricultural activities against 5% in localities classified as urban. Figure 2.1 shows smoothed densities of the measure of rural net out-migration in 1991 and 2000. Except for three states all states experienced net rural out-migration during this period. Our results are not affected by excluding the three states with positive net rural in-migration. Figures 2.2 to 2.8 show the distribution of out-migration by cohort. Both the theoretical and the empirical literature suggest that migration decisions are endogenous to wage differential between source and destination regions. As a consequence falling rural wages and job prospects in urban areas may lead to differential propensity to migrate across cohorts. To control for this reverse causality I instrument the emigration shock by lagged emigration shock .

This instrument is motivated by evidence on the importance of networks and social in migration decision . Since networks are usually based on blood relationship and friendship, our instrument which varies by cohort and state should be able to pick-up a large fraction of the movements in rural out-migration within the same cohort-by-state group. The data from my analysis are 5% random samples from the 1980, 1991 and 2000 Public Use Microdata Samples of the decennial census of Brazil. Unfortunately the 1980 census doesn’t contain reliable data on wages. However the 1980 census is used to construct the emigration shocks in 1991 and 2000. Individuals are divided in 7 cohorts using their age in 1991. The cohorts are individuals aged 20-24, 25-29, 30-34, 35-39, 40-44, 45-49 and 50-54 in 1991. The sample is restricted to individuals aged 20 to 54 in 1991 who earn between no less than 200 and no more 50,000 Reais per month in real terms which constitutes about 86% of the original sample. Table 2.1 reports basic summary statistics for the measure of rural net emigration shock and the logarithm of real monthly earnings. The degree of rural out-migration is considerable. In 1991, the rural emigrants shock was about 48% of the rural population. By 2000 this number increased to over 95%. In other words for every 100 individuals still living in rural areas in 2000, over 95 individuals had left rural areas since 1980. The emigrant shock is strikingly different across cohorts with the largest shocks appearing for the younger cohorts of individuals. Moreover these shocks are large compared to international immigration figures. By way of comparison Mishra finds that, in the case of MexicoUS migration, the largest immigration shock across skill groups is a little above 50% of the Mexican labor force. The real wages of individuals in the sample increased slightly between 1991 and 2000 despite several periods of hyperinflation. The increase in real wages range between 1.6 to 3.4 percentage points across cohorts with the largest increase occurring for the younger cohorts . As discussed by Mishra , in the presence of self selection of individuals into migration, the parameter estimated in equations and do not provide un-biased estimates of an exogenous change in rural labor supply. This is because, in the presence of self-selection into migration, rural out-migration affects rural labor markets in two distinct ways: a reduction in overall rural labor supply, and a change in the composition of rural labor supply. The instrumental variable strategy used in the analysis described above provides exogenous changes in rural labor supply but may not provide exogenous changes in the composition of rural labor supply. With pooled cross-sections I cannot model or control for individual’s self selection into migration. I provide some evidence that the results are robust to controlling for some of the observable characteristics of changes in workforce composition. I estimate again controlling for the share of a cohort that is male, with some primary education and that have completed primary education. The results from this estimation are reported in Table 2.4. Comparison the results in Table 4 to the coefficient estimates in columns to of Table 2 provides some indication on the robustness the previous results are to changes to workforce composition. I find that the OLS estimates are not affected by the educational attainment and gender composition of the population that remains in rural areas. However the IV estimates are lower by an order of 2 to 3 points of standard errors. This result is consistent with models of positive selection into migration, where out-migrants have a higher educational attainment and tend to be males. The results show that a 10% increase in out-migration increases rural wages by 1.4 to 2.1%. This suggests that after controlling for changes in workforce composition, the wage effect of rural out-migration flows between 1991 and 2000 drops from 6.5% to about 3%. This points to the fact that changes in workforce compositions resulting from higher rural out-migration could explain up to half of the effect of rural out-migration on rural wages.

A farm is placed in one of three tiers based on its risk to water quality: tier 1 being the lowest risk and tier 3 being the highest. Just as monitoring requirements vary, so too does the usefulness of the data collected. The variance in monitoring information value depends on four factors: where, when, and what parameters are collected, as well as what test organisms are used to assess water and sediment toxicity. There are also noteworthy differences in the usefulness of data depending on whether data is collected from surface water or groundwater. While this chapter largely focuses on surface water, the challenges of groundwater monitoring and assessment will also be addressed. The location of the monitoring station is an important element in identifying what farm is polluting and to what extent. For example, upstream edge-of-field monitoring on a tributary offers more precise and detailed data on the pollutants discharging from surrounding farm; whereas data from a monitoring station located downstream along the main stem of a river are a composite of all upstream sources, making the task of teasing out probable non-point sources of pollution near-impossible The timing of data collection is also important because water quality can change during and after storm or irrigation events, or during or after fertilizer or pesticide applications, increasing pollutant concentrations. If monitoring data were taken before applying a pesticide, for example,cut flower transport bucket that information might not accurately portray the pollutants present in the waterways for the next several months. The issue of timing is even more problematic when testing groundwater; research has shown that it can often take decades for leached pollutants, particularly, nitrates, to show up in groundwater .

Thirdly, the parameters limit the extent of knowledge about the health of a given waterway. For example, collecting basic parameters, such as dissolved oxygen, turbidity, temperature, nutrients, metals, and pH, provides a good baseline by which to assess water health, but other important parameters, such as the amount of a specific prevalent pesticide in the water column, might be overlooked, and could be the key to more accurate problem identification. Finally, certain test organisms are more appropriate for certain pollutants. For example, Ceriodaphnia dubiais an appropriate test organism for testing water toxicity, while Hyalella aztecais more suited for assessing sediment toxicity. These two are commonly used in regulatory monitoring programs, however, other new classes of pesticides in the offing will require new test organisms, ones that are not part of the current cadre of test organisms used for regulatory monitoring in the Central Coast region and California in general. For example, a more suitable test organism to use for burgeoning classes of pesticides, such as neonicotinoids, but one that was not incorporated into monitoring programs until 2015, is Chironomous dilutus. Tier 1 and 2 water quality monitoring is conducted in the main stem of a tributary and reported as an aggregate twice a year. The usefulness of these data pales in comparison with that of individual discharge data taken at the edge-of-field from tier 3 farms. One of the biggest concerns among growers during the drafting and implementation of the 2012 Ag Waiver was public disclosure. Farmers soon to be classified in tier 3 were concerned about reporting individual discharge water quality data due to matters of privacy and value of information, but also due to fear of being regulated as point source dischargers.

Three factors that weigh heavily on tier criteria are a farm’s size, risk to nitrate pollution, and risk to water column toxicity. Mounting scientific evidence of nitrate groundwater contamination as well as pressure from environmentalists and environmental justice groups elevated the nitrate issue to the top of the agenda during the 2012 Ag Waiver negotiation process. Additionally, a series of several scientific studies from the Granite Canyon Laboratory pointed to two particular types of organophosphate pesticides—diazinon and chlorpyrifos—as the culprits of water toxicity in the region. While pesticides did not receive specific attention in the first Agricultural Waiver, these two pesticides found their place at the top of the agenda during the 2012 regulatory process over a long list of other potentially harmful pesticides used in the region. Agricultural use of chlorpyrifos and diazinon has decreased dramatically in California’s Central Coast region over the past decade .However, while chlorpyrifos use in the region has plummeted, statewide use has stayed relatively consistent over the past decade, with minor fluctuations. What causal factors drove the regional chlorpyrifos and diazinon decline, and how much of the decrease can be attributed to the 2012 Agricultural Waiver? What conditions made the chlorpyrifos decline possible in the Central Coast region, but not in other regions or California as a whole? What societal, environmental and regulatory implications have resulted from farmers’ decisions to stop using both chemicals? The following section explores these questions by assessing data collected from interviews, survey responses, a thorough review of policy related documents, water quality information, organic production data and pesticide use records in three different California regions. Survey responses were collected from a subset of questions in a 2015 study on growers’ opinions of water quality management practices and policies in the Central Coast Region .

The set of survey questions relevant to this study asked growers if and how their use of chlorpyrifos and diazinon had changed since the Agricultural Waiver was implemented. Sixty-five growers responded to this optional part of the survey. Forty-seven of the 65 reported a change in chlorpyrifos and diazinon practices; their responses are reported below. Results from the survey and other data suggest that a grower’s decision to apply the two pesticides or not depends on several factors, including specific regulatory requirements embedded in the 2012 Ag Waiver as well as regulations generally, concerns over workers safety, harm to the environment, the cost of pesticides and their application, the availability of alternatives to manage pests, as well as the extent of pest damage and the value of the crop; each of these will be discussed in more detail. Another motivation for the decline in diazinon and/or chlorpyrifos use was that growers simply did not like using the two pesticides knowing they caused harm to their workers and the environment. It appears that growers were aware of the growing scientific evidence documenting the impacts of chlorpyrifos and diazinon on the environment, ranging from water and air quality to small invertebrates to human health. Of the survey responses, 5 stated they stopped using the pesticides because of worker safety, while 10 cited environmental factors as their motivation. Selected quotes from survey responses are reported below.Several pesticides are popping up as the next big threat to water quality . Malathion is the third most commonly used OP insectidide next to diazinon and chlorpyrifos with similar chemical characterists, yet interestingly,procona flower transport containers was not targeted in the 2012 Ag Waiver. Imidicaloprid is in the neonicatinoid class, and studies have linked the pesticide to bee colony collapse disorder . And third, pyrethroid chemicals, such as bifthenthin and lambda-cyhlalothrin, are being linked to sediment toxicity issues in the region. While less toxic than OP pesticides, neonicotinoids and pyrethroids have longer halflives. As chlorpyrifos and diazinon have steadily decreased, many of these new pesticides, which are differently or equally as harmful as OPs, have increased. To address if growers have switched to other chemicals to replace diazinon and chlorpyrifos in the Central Coast region, growers’ use of possible substitutes were assessed. Two datasets were reviewed: the University of California Integrated Pest Management reports, CDPR pesticide use data, as well as scientific literature. In Monterey County , chlorpyrifos use on broccoli declined by 86% from 2000 to 2013, yet the total pesticide use on broccoli only declined by 47% over those years. Were growers substituting chlorpyrifos for another pesticide to control cabbage maggots? UC IPM reports show that one of the only viable alternatives for use on a commonly targeted pest is diazinon.

From pesticide use records, it is apparent that broccoli growers are not readily switching to diazinon, since it comes with the same baggage of regulatory and environmental problems as its counterpart. In response to the growing demand for an alternative pest management strategy to control cabbage maggot, a new study by Joseph and Zarate in the Journal of Crop Protection explored at least eleven other insecticides with similar or superior efficacy to chlorpyrifos on cabbage maggots; of these five of these are pyrethrins plus pyrethroids, and one is a neonicotinoid. To identify if growers were switching over to any of these five promising, but potentially environmentally-harmful, new pesticides, CDPR use data on broccoli was assessed for each chemical. Results from this analysis showed that while these alternative pesticide numbers are still relatively small, growers might be increasingly turning to them in the future, especially if cabbage maggot pest problems escalate and the value of broccoli continues to rise. Diazinon’s demise has been even starker in lettuce than chlorpyrifos on broccoli in the Central Coast, as well as in the two other regions assessed in this study and the state as a whole. From 2000 to 2013, diazinon use on lettuce in Monterey County dropped by 99% compared to a 26% drop in the total pesticide use on lettuce, with diazinon removed. However, data from UC IPM reports and CDPR pesticide use data, as well as scientific literature do not suggest a widespread switch to other pesticides, rather organic practices might be the larger force. Diazinon use on lettuce crop pests is more diversified, making any switch from diazinon to another chemical more dispersed. With broccoli, because chlorpyrifos use is limited to controlling cabbage maggots, a switch to another chemical was more easily identifiable. Diazinon use on lettuce, on the other hand, has historically been used to control at least six different pests , each opening up a pandora’s box of alternative chemicals. CDPR pesticide data on diazinon’s use on Monterey County lettuce shows no dramatic chemical-for-chemical switch. For example, as diazinon dramatically falls, no single pesticide rises up to take its place. Because diazinon’s use on lettuce was so varied, it is logical that several different chemicals might be used in its stead to fit one or more specific needs pest needs.The lack of readily available pesticide alternatives for use on broccoli’s cabbage maggot, or the scarce use of them thus far, alludes to growers simply using fewer chemicals to grow broccoli, and perhaps switching to organic farming practices. The same appears to be true for lettuce growers, although the data are more limited. The option of switching to organic production with higher profit margins and the consumer demand for less chemical use offer appealing motives for many growers to curtail their diazinon and chlopryifos use, in addition to other pesticides. The number of farms, value and acreage of organic production has blossomed over the past decade in the Central Coast. The top three agricultural producing counties in the region have steadily increased the amount of land in organic production every year. In San Luis Obispo County, the conversion to organic was even more staggering: In 2005, 4,493 acres were dedicated to organic production and by 2014 50,636 acres were grown organically—an eleven-fold increase. In Santa Cruz County, 2,700 acres were under organically production in 2005, and by 2014 4,058 acres were grown organically. In Monterey County, the organic production nearly tripled, from 16,410 acres in 2005 to 46,570 acres in 2014. More specifically, in Monterey County organic production of broccoli doubled over the past decade and a half: 1,430 acres of broccoli were grown organically in 2000, increasing to 2,862 acres in 2015 . Organic’s proportion of the total broccoli grown in Monterey County also grew: in 2000, organic production accounted for 2.3 % , and by 2015, roughly 4.5% of all broccoli production was devoted to organic. Although organic still does not account for a significant portion of total Monterey County broccoli production and cannot explain the chlorpyrifos decline alone, corroborated with growers’ survey responses , it is safe to conclude that a transition to organic has played a role in the declining use of the two pesticides. While long-term longitudinal data was not available for organic head and leaf lettuce production in Monterey County to assess whether or not organic production played a role in diazinon’s decline, a related crop, spring mix, was available.

The 2010 Draft Waiver proposed that all farms should be required to implement a 50-100-foot buffer; by November of that year the mandate was reduced to only Tier 3 farms and the buffer width was reduced to 30 feet, and by the final 2012 Waiver the buffer requirement was left largely to the discretion of the agricultural operator, stating that either a buffer or a proposed alternative must be implemented to protect adjacent polluted waterbodies. With the E. coli event still fresh on the public’s minds, water quality temporarily faded from the regulatory spotlight. But not for long: the 2004 Ag Waiver was due to expire in July 2009, forcing the Regional Board staff to launch a new stakeholder process for the updated Ag Waiver. Unfortunately, the proposed public input process was deemed “not transparent or open to the public” by a California Farm Bureau representative, and did not keep pace with the 2009 deadline. The Waiver was extended for another year. In addition to the pending deadline, mounting scientific evidence of water pollution sources and mobilization of several interest groups pushed agricultural water pollution back on the agenda. Water quality data collected over the preceding five years from the 2004 Ag Waiver Cooperative Monitoring Program clearly showed discharges from agricultural lands were a cause of pesticide toxicity as well as a contributing source of nitrate and sediment impairments in the region . Due to growing concerns about one contaminant in particular, nitrate, a 2008 Senate Bill was passed,25 liter pot plastic requiring the State Water Resource Control Board to prepare a report addressing nitrate groundwater contamination.

The Center for Watershed Sciences at the University of California, Davis conducted the report, and one of the watersheds they chose to study was in the Central Coast region. Additionally, the 2010 State Water Resource Control Board Report found that the Central Coast Region had the highest percentage of toxic water sites statewide. Furthermore, several scientific reports found that pesticide use in the Central Coast was contributing to water column and sediment toxicity , as well as cause human health problems, such as developmental delays in infants and children . The Regional Board staff had the scientific evidence and momentum it needed to develop an ambitious 2010 Draft Waiver. Among the many sweeping reforms, the 2010 Draft Waiver required all dischargers to conduct individual surface water discharge monitoring, required Farm Plans to be accompanied by monitoring and site evaluation results, prohibited the use of excess fertilizer, required a comprehensive list of pesticides to be regulated, and required all farms to implement vegetative buffers. Members of the agricultural community voiced their concerns with the Draft in Regional Water Board meetings, through comment letters, on the web, and in newspapers. In a December 2009 meeting, several agricultural representatives reiterated their frustrations about the public input process, their worries regarding the mounting costs, and their opinions that the existing 2004 Ag Waiver was working well and did not need to be amended. Environmentalists, on the other hand, believed the proposed Order should be adopted without further delay. At a standstill, the Board re-issued the existing Conditional Waiver four more times: November 2010, March 2011, July 2011, and August 2011. Environmental groups, with agendas ranging from environmental justice to marine ecosystem protections to urban stormwater programs, were highly disappointed that the 2010 Draft Waiver was not adopted.

The environmental community was strongly represented by the Santa Barbara Channel keeper, The Otter Project, and Monterey Bay Keeper, providing extensive comments at Regional Board meetings up until the adoption of the 2012 Agricultural Order. In 2012, published results from the State commissioned nitrate contamination study, although controversial among the agricultural community, found that cropland was the primary source of human-generated nitrate contamination in the Tulare Lake Basin and the Salinas Valley , and that 254,000 people in the area are at risk for nitrate contamination in their drinking water. Because nitrate-contaminated drinking water is a well-known human health effects, including “blue baby syndrome” , the results of this study became a rallying-cry for the Department of Health to encourage a more stringent Agricultural Waiver. The California Department of Health shed light on nitrate groundwater contamination, echoing concerns reported from the UC Davis report. The United Farm Workers and a coalition of groups rallied behind environmental justice concerns, representing the voice of people most affected by nitrate contaminated drinking water. At a Central Coast Board meeting in February of 2012, Marcela Morales of the Central Coast Alliance United for a Sustainable Economy explained that contaminated water is disproportionately impacting low-income populations and people of color. She strongly urged the Board to take action and not delay the updated Waiver, claiming that communities affected by drinking water contamination are in urgent need of basic protection to ensure clean drinking water. Another impetus arose from water quality regulators in urban areas. Municipalities, facing ever-stringent regulations, began to question the fairness of waiving the agricultural water quality requirements .

City managers voiced their concern about pollutants from agricultural areas being deposited into receiving waterbodies within city boundaries, which cities are required to clean up through stormwater National Pollutant Discharge Elimination System permits. As the City Manager of Monterey, for example, suggested that agricultural industries and municipalities should be held to the same standard . On the other side, Farm Bureaus, individual growers and the Growers and Shippers Association represented the agricultural interests. California’s $43.5 billion agriculture industry comprised of 81,500 farms spread over 25.4 million acres is one of the largest and most influential interest groups in the state . Historically, the California Farm Bureau has had success at regional and national lobbying efforts. Between the two Agricultural Waivers , there were grumblings within the agricultural community that the Regional Board was not involving the growers in the deliberation process as much as during the 2004 Ag Waiver negotiations. As one farm stakeholder explained, growers felt they were not involved when figuring out solutions to water quality improvements, rather “[the Regional Board] set the rules without much input and expected growers to comply.” As a lettuce grower in the Salinas Valley stated, “the Regional Board didn’t take into account stakeholder opinion…The elephant in the room…[was] that there was no collaboration between the grower community and the regional water board staff… Discussions about the [Agricultural Waiver] and how to implement it should have been happening during the past four years, but it did not” . Several board meetings leading up to the March vote were packed with testimonies from agricultural interests assembling to delay the vote and water quality interest groups, encouraging the Board to pass a more stringent updated Agricultural Waiver. Steve Shimek , 25 litre plant pot spearheading the environmental interests, described the dualistic nature of the unfolding politics: “on one side are community activists seeking tougher pollution limits and public access to water quality data. On the other side are too many farmers trying to avoid cleaning up the waste from their operations.” At the March 15, 2012 Board meeting, the three-year long debate culminated in the passage of an updated Agricultural Waiver. But the process was not over. As mentioned earlier, five groups requested a deferral on several provisions of the 2012 Ag Waiver. In September of 2013 the State Board adopted the existing Ag Waiver, which made some modifications to the 2012 version passed by the Regional Board. A few months later, environmentalists filed a lawsuit in Sacramento Superior Court challenging the modified 2012 Ag Waiver as being too weak. The modified waiver and lawsuit will be discussed in more detail in the next section. Overall, the policy process leading up to the 2012 Ag Waiver was fraught with tension between a variety of stakeholders, including agriculture, cities, environmentalists, scientists and environmental justice groups. Consequently, the Waiver that ultimately passed was more robust than its 2004 predecessor, but weaker than ambitious draft orders that came to the fore during negotiations . The next part of this chapter will analyze the effectiveness of the resultant provisions embedded in both Ag Waivers.Public policy literature presents several means to assess the efficacy of a policy. The criteria chosen for policy analysis is important, as it could influence the direction of the policy as well as future budget allocations.

Cass Sunstein , former Administrator of White House Office of Information and Regulatory Affairs for the Obama administration, asserts that determining the success or failure of a regulation depends on its goals and scope. Dowd and his colleagues echo this claim in their paper on agricultural non-point source pollution policy in the Central Coast, stressing that the success of the Agricultural Waiver largely depends on the evaluative criteria used. Six parameters were carefully selected to measure the effectiveness of the 2004 and 2012 Ag Waivers: 1) complying with mandates set in the Agricultural Waiver, 2) evaluating quantifiable water quality improvements, 3) evaluating the requirements themselves, 4) assessing the significance of monitoring data, 5) comparing costs to growers vs. broader societal and environmental benefits, and 6) evaluating the equity of compliance across growers, including the distributive consequences.A logical place to begin evaluating the success of the 2004 and 2012 Agricultural Waiver is by measuring the degree to which growers met the compliance requirements. Based on the high level of enrollment in the 2004 Agricultural Waiver the 2004 Waiver has been labeled a success by simple participation among growers. The number that completed The 2012 Ag Order boasts roughly the same enrollment numbers: 1,796 operations managing 94% of farm acreage in the region. Evaluating compliance based on specific 2012 requirements, however, is more variable. As Table 3-1 indicates, there is a high compliance rate for simply enrolling in the program, but slightly less so in regards to more complex requirements. For example, close to a quarter of all farms have not reported groundwater monitoring at the individual level for both domestic drinking water and agricultural wells. On the other hand, every farm that is required to report total nitrogen applied to their farm has done so. The Clean Water Act Section 303 list1 of Impaired Waterbodies for the Central Coast Region can be an indication, albeit a limited one, of how water quality has changed over time. Two relevant listing cycles, 2006 and 2010, indicate a dramatic increase in the number of polluted waterways in the Central Coast. Over these four years, Regional Board staff added 515 listings of impaired waterbodies, totaling 707 in the 2010 listing cycle . While these numbers are striking, trends using these data should be made with caution for at least two reasons: 1) the number of waterbodies assessed for the 303 list varies from year-to-year and 2) there may be a latency period between when a waterbody was surveyed and when it is listed. The most commonly cited monitoring databases used to assess water quality in the region also indicate degrading water quality. Reports from these two agencies suggest that many of the same waterbodies, especially in the two areas responsible for most water pollution, are more polluted than they were a decade ago . While some waters have improved—47 waterbodies were de-listed as impaired in 2010—the vast majority have not. The lower Salinas watershed and the lower Santa Maria area are responsible for most of the region’s polluted waters; these areas are also the leading agricultural producers in the Central Coast . The 303 list, CMP, CCAMP, CWC, and scientific studies from the UC Davis Marine Pollution Studies Laboratory at Granite Canyon, identify a number of water quality concerns, in particular, dissolved oxygen, elevated pH, elevated nitrate and ammonia, water and sediment toxicity, and habitat disturbances. Monitoring patterns show that these pollution parameters are variable throughout the region, and that particular watersheds are hotspots for certain pollutants. When listed together, these parameters are responsible for impairments to the beneficial uses of drinking water, recreation, aquatic life, and agricultural uses. Of these concerns, nitrate contamination is the most serious and widespread problem in the region. Regional water quality reflects a larger state and national trend of degrading and variable water conditions. California Water Boards’ Annual Performance Report found half of all surveyed streams in the state to be degraded or very degraded, as measured by the health of aquatic organism communities that live in the state’s streams.

Citing passages where one of the characters compares planting and harvesting to death and rebirth, he concludes that “Norris’s utterly idealized account of the production of wheat as the emergence of a spiritual body out of a natural one can coexist peacefully with an utterly materialist account of the growing wheat as a mechanical force” . Thus the more important binary in the novel for Michaels is not between nature and machine but between the ideal and the material. Conlogue helpfully cuts through the binaries that accompany the idea of the pastoral, whether they are conceived of as country and city, nature and machine, or ideal and material. He focuses instead on labor and management, arguing that the novel is part of a minor tradition of “American georgic” concerned with historical shifts in agricultural practice. In support of this he advances a more robust definition of industrialization as a confluence of technologies: not only machines, the focus of so much criticism on The Octopus, but also management technologies such as record-keeping, mapping, and other quantifications together make up what he calls “the new agriculture.” In redirecting the discussion to industrialization as a whole, Conlogue emphasizes that what Norris is describing is itself an emergent phenomenon. Many critics treat the mechanical and managerial technologies depicted in the novel as typical of the time period— indeed taking them as the hallmarks of this era—reserving their detailed analysis for how Norris, as a naturalist writer, makes sense of them.

Conlogue, on the other hand,30 plant pot gives significant attention to the history of farming practices to argue that these technologies of industrial agriculture were largely unknown to the American public at the time. Thus the book should be read as playing a role in reporting, popularizing, and celebrating them. The novel is not a reflection of historical changes in agricultural labor practices, but can be seen as itself a part of that history. It promotes new forms of labor organization by, among other things, celebrating new farm equipment in ecstatic sexual terms. In this way, The Octopus advocates for the future development of both the China trade and agricultural industrialization, suggesting that neither can exist without the other. Norris pays special attention to the record-keeping and communications technologies as new forms of writing necessary for the new agriculture, and we can add these to the several modes of representing the land that saw Presley and the narrator experiment with. We have already quoted some of the description of the Los Muertos ranch office whose, “appearance and furnishings were not in the least suggestive of a farm” but rather of the headquarters of an international farm . In this book about the mechanization of agriculture, moreover, the first machine we encounter on the ranch is the “typewriting machine,” and the most significant object, again, is the stock-ticker, which makes the farmers feel themselves part of a global whole as they watch updates being printed before their eyes . Writing as record-keeping does not passively reflect an external reality but by identifying imbalances and inefficiencies will spur on new enterprises. Between the scales of the typewriter to keep accounts and the stock-ticker to know the world, the text features a map as a medium-scale technology of representing the farm as a whole. Because the ranch is a business enterprise, every feature of the landscape needs to be measured and recorded, accessible at any time.

But the reader, like the farmer, can also refer to this information at any time: at Norris’s request, the first printed edition contained a “map of the locality” in the “frontmatter,” and this map has generally been read as part of the overall reality effect of the book.And just as the printed novel incorporates the map, so this textual description of a map is curiously suggestive of narrative: “A great map of Los Muertos with every watercourse, depression, and elevation, together with indications of the varying depths of the clays and loams in the soil, accurately plotted, hung against the wall between the windows” . Here, plotting is the process of spatially representing the exact conditions of the soil so that it can be best utilized. But surprisingly, every description of a map in the novel contains this same phrase; later, in the local railroad office we see “a vast map of the railroad holdings… the alternate sections belonging to the Corporation accurately plotted,” and in the San Francisco office of Lyman Derrick, again on the wall “the different railways of the State were accurately plotted in various colours, blue, green, yellow” . Here we can note that the word “plot” condenses three central elements of The Octopus: the graded plots of land under dispute, the conspiracy plot to gain their ownership, and the narrative plot which decides the outcome. A final form of writing that The Octopus highlights as playing a decisive role in the development of the West is advertising, and this is in fact the most central to the plot. Conlogue has shown that The Octopus reproduces almost verbatim many sentences from the actual pamphlets that the Southern Pacific Railroad circulated to encourage ranchers to begin leasing the land. He interprets these circulars within a contrast between the “paper value” of abstract legal content, on the side of the railroad, and the “work values” of those actually improving the land, on the side of the ranchers.

I would argue, however, that such a contrast does not hold in the novel, as the ranchers both approach farming as an industry like any other and also make their case in terms of this “paper value” of the written text. Instead, we should see the competing interpretations of the advertisements as different ways of valuing the land before and after the close of the frontier. The exaggerated and even confusing syntax in which these “circulars” are introduced in the novel foreshadows their grand role at the center of the legal case: “Long before this the railroad had thrown open these lands, and, by means of circulars, distributed broadcast throughout the State,grow raspberries in a pot had expressly invited settlement thereon” . Like the newspaper and the stock ticker, this early broadcasting medium disseminates business information throughout the country. The term circular links writing to the theme of circulation epitomized by the railroad itself, and to empire circling back to China where it began, by way of America. The legal question of ownership, and so the outcome of the plot, hangs on the interpretation of these texts. It is the newspaperman Genslinger—his Wild West name juxtaposed to his profession as a writer—who first suggests that the railroad will sell at a higher price than the ranchers anticipated. To this, the rancher Annixter responds that their writing is their bond: “Haven’t we got their terms printed in black and white in their circulars? There’s their pledge” . But a closer reading of the black text on the white sheet reveals an ambiguity: “‘When you come to read that carefully,’ hazarded old Broderson, ‘it—it’s not so very reassuring. ‘Most is for sale at two-fifty an acre,’ it says. That don’t mean ‘all,’ that only means some’” . Here the entire Mussell Slough incident, indeed the entire plot of the novel and the future of California wheat farming, comes down to textual interpretation. The importance of reading for this narrative arc is brought home when the ranchers learn the outcome of their court case, the authoritative interpretation of the circular’s text. Tellingly, this comes at the conclusion of Cedarquist’s speech, when Magnus in the midst of imagining his son in Hong Kong, having marched with the course of empire full circle back to its origin in China. At this point he overhears a stranger reading aloud the afternoon newspaper: “It was in the course of this reading that Magnus caught the sound of his name,” . Called by name by the text, he listens on to the full reading of the verdict: the League’s plot has failed to secure the plots of land, and his ultimate fate will be to lose the ranch. By quoting these circulars verbatim, then, Norris enfolds authentic material from the historical incident into the text of the novel, to tell the true story of the West. Furthermore, by announcing the verdict at the moment that Magnus is listening to Cedarquist’s speech, Norris suggests the link between the land dispute and the China market, which is the close of the frontier.

As we saw above, in “The Frontier Gone at Last” Norris dated the decisive end of the frontier to the arrival of U.S. marines in China in 1900, the year before The Octopus was published. This was the moment America finally reached and bordered on another civilization to the west, with no wilderness separating them. The railroad’s case in the novel is not the victory of paper values over work values, but rather the historical transformation of the land from an open space of prospecting into a closed loop of industrial production. Indeed, Magnus and the others are already operating their farms as businesses, and Norris portrays this economic contradiction as moral hypocrisy, which weakens Presley’s identification with them. In understanding The Octopus’s representation of the new agriculture, then, Conlogue corrects earlier scholars’ focus on the machines themselves and redirects attention to changes in the organization of agricultural labor. Namely, he points to the technical management of large scale wage labor. Still we can connect this insight to larger economic changes by employing the more precise vocabulary of Marxist analysis, as well as examining the role of racialization in U.S. labor history. Palumbo-Liu, for example, has argued that Americans have long understood their own modernization through reference to Asia. In the case of The Octopus, Lye has given perhaps the fullest account of the importance of racialization for the novel, in which all of the cooks working on the ranch appear to be Chinese. These men are represented as docile workers, feminized by their labor in food production. Chinese prepare the food for both the owners of the ranch, these minor capitalists, and also for the workers planting in the fields. Their presence working at the Derricks’ ranch underscores the modernity of industrial production, its post frontier character. While Norris is notorious as one of the most unrelentingly racist “major” writers in the history of American literature, Lye’s work illuminates the importance of food in anti-Chinese racism of the time and how Norris engages with it. In particular she points to the American Federation of Labor pamphlet Meat vs. Rice, Anglo-Saxon Manhood Against Asiatic Coolieism, authored chiefly by Samuel Gompers, which explicitly links Chinese labor to modernization and industrialization. It does so, moreover, by asserting a biological connection between body and food. The original Exclusion Act, issued in 1882, prohibited immigration for ten years, and was renewed in 1892 for another ten. During this period anti-immigrant writers presented the “Asiatic” and the “Anglo-Saxon” as separate biological types. In fact one of the words that Turner himself used when speaking of the frontier was the Anglo-Saxon “organism.” Turner’s form of white supremacy was slightly different, however, as he used this term metaphorically, with Anglo-Saxon cultural spirit moving through history as an organism. The anti-immigrant writers, on the other hand, use organism in a direct physical sense: the Anglo-Saxon and the Chinese are separate biological entities in Darwinist competition for resources. The root of the problem was that Chinese supposedly ate less food, which indicated a superior, more efficient body. This is not yet a quantitative discourse of calories, but the qualitative differences among foods are seen to give different levels of sustenance.It is not just that there is no question of labor solidarity, but this lack of solidarity is blamed on the Chinese themselves. The rice standard is a kind of bare human subsistence, on which “the beef-and-bread man” cannot survive, an instance of what Eric Hayot has identified as a tradition of Western views of the Chinese as the limit case of humanity.

Cotton species was modeled as a fixed effect, since there are only two possible categories – not enough to meaningfully estimate a random effects distribution. We also included 15 real-valued fixed effect predictor variables that indicate the number of fields, out of the 8 surrounding fields, planted with each of the 15 crops we analyzed. The goal was to control for effects of the surrounding landscape, and thereby avoid spurious correlations between rotational history and yield. Our Bayesian modeling approach required the specification of priors for all parameters whose posteriors were estimated using MCMC. Non-informative priors were used for all fixed effects. The random effects for both field and year were assumed to follow a normal distribution with mean 0 and variance hyperparameters estimated from the data. Since the support of variance parameters is constrained to positive realnumbers, non-informative inverse gamma distributions with shape and scale parameters set to 0.001 were used as the prior for the variance parameter of the top-level stochastic node, and as the priors for the variance hyperparameters of the field and year random effects distributions. Model 2. To help us understand whether any effects of the crop grown in the field the previous year on cotton yield could be due to effects on L. hesperus, we fit the same model as Model 1, but with average June L. hesperus abundance as the response variable. Model 3. Next, to formally assess whether there was an association between the effects of crop rotation on yield and the effects of crop rotation on L. hesperus abundance, we performed a linear regression of the estimated effects on yield against the estimated effects on L. hesperus abundance .

Noninformative N priors were used for the mean and intercept,container raspberries and a noninformative inverse gamma distribution with shape and scale parameters set to 0.001 was used as the prior for the variance. Model 4. A great deal of experimental evidence has demonstrated that crop rotation leads to increased yield compared to successive plantings of a single crop; therefore, we explored whether or not a yield loss was incurred by cotton crops grown in fields where cotton was grown in previous years. For the 782 fields that had complete crop rotational records for the previous 4 years, we calculated the number of consecutive cotton plantings in the 4 years preceding the focal cotton crop. We then fit a model, with yield as the response variable, using the number of consecutive prior cotton plantings as a predictor. Field, year, and cotton type were included as they were in Models 1 and 2. Since the number of prior consecutive cotton plantings could be correlated with the number of cotton fields in the surrounding landscape during the focal year, we avoided a possible spurious correlation between consecutive cotton plantings and yield by also including a fixed effect for the number of cotton fields in the 8 fields adjacent to the focal field. We chose not to explore rotational histories of specific crops for longer than one previous year, since the number of possible rotational histories becomes very large and the number of records for each possible history becomes too small to allow for robust statistical analysis. Model 5. To see if the number of consecutive years of cotton cultivation preceding the focal year was associated with June L. hesperus densities, we fit the same model as Model 4, but with June L. hesperus as the response variable.

Capitalizing on a large existing set of crop records from commercial cotton fields in California, we employed an ecoinformatics approach to explore the effects of crop rotational histories on cotton yield. Our hierarchical Bayesian analyses revealed evidence that several crops, when grown in the same field the year before the focal cotton planting, were associated with either decreased or increased cotton yield , and either increased or decreased early season densities of the pest L. hesperus . Furthermore, crops associated with decreased yield were generally also associated with increased L. hesperus densities, while those associated with increased yield were also associated with decreased L. hesperus densities . These results suggest a possible mechanism for the observed yield effects of these rotational histories. Since L. hesperus preferentially attacks certain crops, a field cultivated with a crop that is heavily attacked by L. hesperus may, if L. hesperus disperse from the focal field, increase the abundance of L. hesperus in nearby fields. These populations may subsequently attack the crop planted in the focal field the following year, explaining the increase in early-season L. hesperus densities that we detected following certain crops. In turn, these increased L. hesperus populations may exert strong herbivorous pressure on focal cotton crops, possibly explaining the corresponding decrease in yield. We believe that the effect of rotational history on early-season L. hesperus likely operates at a landscape scale that is larger than the within-field scale. If cotton was grown in a field the previous year, then farmers in the San Joaquin Valley are required to maintain a 90-day plant-free period prior to 10 March of the following year. This prevents L. hesperus, which overwinter as adults on live host plants, from overwintering in a focal field where cotton was grown the year before.

If a crop other than cotton was grown the previous year, then it could be possible for L. hesperus to overwinter in the focal field on residual plant or weed populations; however,since fields are completely plowed prior to planting cotton in the spring, L. hesperus adults would still need to temporarily leave the focal field. Therefore, we believe that the preferred host crops for L. hesperus increase L. hesperus populations at a landscape scale. Then, when cotton, another target of L. hesperus, is planted in the same field the following year, the cotton field is attacked by this regional population. If regional populations are already large due to lingering effects from crops grown the previous year, L. hesperus populations may move into cotton early in the growing season; this could be particularly damaging given research suggesting that cotton yield is particularly sensitive to L. hesperus densities early in the growing season. Using our data, we were not able to determine at exactly what scale the effects of rotation on L. hesperus likely operate. We do not believe a within-field scale is plausible, but determining a more precise spatial scale for these effects could be an interesting topic for future research. Our findings match expectations of crop yield effects based on previous research on L. hesperus host crop preferences, lending support to our hypothesis that yield effects of crop rotational histories are, at least partially, mediated by effects on L. hesperus. Alfalfa and sugarbeets, both crops for which we found negative effects on yield and positive effects on L. hesperus when grown in a field the previous growing season, are all considered preferred hosts for L. hesperus , and have been shown to also increase L. hesperus populations in nearby cotton fields during an individual growing season. Presumably,draining pots this effect is due to these crops supporting large L. hesperus populations. Large L. hesperus populations are known to build up in alfalfa, and their dispersal following alfalfa harvesting can threaten nearby cotton crops. L. hesperus is also known to emigrate to nearby cotton fields when safflower begins to dry in mid-summer. While the potential for nearby alfalfa and safflower fields to increase L. hesperus populations in cotton fields in a given year has been recognized, our results are the first indication that these landscape effects may extend temporally, affecting L. hesperus populations, and yield, in the next growing season. Tomatoes, associated with increased yield and decreased pest abundance in our data, have likewise been shown to decrease L. hesperus abundances in nearby cotton fields within a given year. While previous experimental work has examined the effects of crop rotations on cotton yield, our work expands on these studies in several ways. First, we explore a much wider diversity of possible crop rotational histories, providing quantitative estimates of the cotton yield effects of cultivating 14 different crops the previous year. Second, since we analyze records from commercial cotton fields, our data have the potential to capture yield effects that could only be detected at this realistic spatial scale. Third, since we have collected data on pest abundances, not only yield, we have also been able to use our data to generate and build evidence for a hypothesized mechanistic explanation of the yield effects we identify. We also found that farmers incurred a decline in cotton yield of about 2.4% for every additional year cotton was grown consecutively in a field preceding the focal season . This is consistent with previous research suggesting that continuous cultivation of cotton in the same location can reduce yield compared to interspersing cotton with other crops.

We also found some evidence that the number of years cotton was grown consecutively in a field was associated with higher June L. hesperus densities: the posterior probability of there being a positive association was about 95%. Identifying the actual mechanism underlying this yield effect is beyond the scope of this study, but would be an interesting avenue for future research. It is possible that the yield decline is not caused by changes in L. hesperus densities, and instead results from the buildup of soil pathogens, especially in light of previous research showing that continuous cotton cultivation increases the densities of fungal pathogens in the soil When interpreting our results, it is important to remain cognizant of the challenges of drawing causal inferences from observational data. The key assumption required to make causal inferences from regression coefficients is that all variables that affect both the treatment assignment and the response variable are included in the model; this ensures that the probability of receiving each treatment becomes, conditional on the predictor variables included in the model, conditionally independent of the response variable. In experimental studies, the treatment assignment is typically controlled by the experimenter, so one can be confident that the only difference between treatment and control groups is in fact the treatment. However, in observational studies, it is impossible to prove definitively that there was no other factor that affected both the treatment assignment and the response variable . As such, we want to be very clear that our hypothesis that the effects of rotation on yield are mediated by effects on L. hesperus densities is exactly that – a hypothesis. While our data do support a negative association between effects on L. hesperus and effects on yield, we cannot prove with observational data that the varying effects on yield are caused by the varying effects on L. hesperus. This could be a fruitful topic for future experimental work. Although causality is impossible to prove using observational data, ecoinformatics paves the way for implementing data-driven agricultural strategies and allows us to mine large datasets to explore important questions that are difficult to address experimentally. While by no means a replacement for experimentation, ecoinformatics can be a cost-effective and realistic complementary approach. In particular, our result identifying the effects of crop rotation on L. hesperus density would have been extremely difficult to reach experimentally. Since L. hesperus readily disperse across spatial scales of more than 1000 meters, an experimental study would have required massive plots comparable to the size of commercial fields in order to adequately capture their spatial dynamics. Growers with knowledge of the crop rotations associated with depressed cotton yield could make more informed decisions, selecting the sequence of crop cultivations that lead to maximized yield. When feasible, cotton plantings could be avoided following crops that decrease cotton yield, and instead limited to fields where crops that increase cotton yield were previously planted. In some cases, market conditions may lead a grower to plant cotton following a yield-depressing crop, even given the knowledge of likely yield loss. In those situations, our results may still be helpful, as an early warning sign of a potential pest problem in a particular field could allow the grower and PCA to focus pest detection efforts on that field and provide time to eliminate the problem before severe yield loss was incurred.

Although there are extensive resources in the state, most urban population reside in the water-scare coastal and southern region and most agricultural activities are in semi-arid lands. To accommodate the growth in population, California and the federal government, built a complex and expansive network of dams, aqueducts, and pumping facilities to harness California’s water supplies and deliver them to its cities and agricultural areas . Today, California’s water resources support over 38.3 million people , a $2.2 trillion economy , and the largest agricultural sector in the country . California’s rivers, streams, lakes, and estuaries are also home to a vast array of aquatic species and habitats, and support substantial aquatic recreation. The state’s water system has a total storage capacity of about 43 million acre-feet and includes hundreds of miles of aqueducts to deliver supplies to places of need and hundreds of thousands of wells to tap the state’s vast groundwater resources . The system is comprised of federal, state and local projects and it’s operated by federal, state, regional, and local organizations as shown in Figure 2-1. The Central Valley Project was authorized in 1935 by the federal government to increase the Central Valley’s resilience to drought and protect it from flooding. Shasta Dam was the first dam to be built as part of the CVP and was initiated in 1938. In 1979, the last dam, New Melones, was completed. The CVP system includes 18 other dams and reservoirs, 11 power plants, and 500 miles of conveyance and related facilities .

The CVP has facilities on the Trinity, Sacramento, American, Stanislaus, and San Joaquin Rivers,blueberry plant size and it serves over 250 long-term water contractors in the Central Valley, Santa Clara Valley, and the San Francisco Bay Area . The total annual contract exceeds 9 MAF . Historically, 90% of CVP deliveries serve agricultural users. In 2000, the CVP and other smaller federal projects delivered about 7.5 MAF to users. About 35% went to the Sacramento River region, 31% went to the Tulare Lake region, and 24% went to the San Joaquin region. Smaller shares went to the North Coast, San Francisco and Central Coast regions . Agricultural users served by the CVP will likely experience additional price increases . CVP contractors are currently behind on repaying the project costs. Under the original contracts, which were negotiated and signed in the late 1940s, the project was to be paid off 50 years after its construction . By 2002, however, irrigators had repaid only 11 percent of the project cost . Based on an analysis of 120 CVP irrigation contracts and a review of full cost rates, which include cost of service and interest on unpaid capital costs since1982 , water contractors will need to pay on average an additional 196 percent to be brought up to full cost rates. Combining the estimated price increases for CVP contractors with rising cost of service rates for the remainder of agricultural water users, Gleick et al 2005 projected that overall agricultural water price will increase by 68 percent statewide between 2000 and 2030. The State Water Project was the first stage of an ambitious strategy outlined in 1957 State Water Plan to improve the reliability and capacity of water delivery throughout California. The SWP captures large amounts of water behind 28 different dams in the Western Sierra Nevada. The Oroville Dam, the largest in the system with a capacity of 3.5 MAF, began construction in 1961 and was completed in 1967.

The dams control the flow of water through the Sacramento River system, in order to maximize the amount of fresh water that can be pumped out of the Bay-Delta into the California Aqueduct. The California Aqueduct then transports the supply south through the San Joaquin Valley to Southern California and the Central Coast. The transport of water is facilitated by 26 pumping and generating plants and about 660 miles of aqueducts. The last major component of the system – the Coastal Branch, which delivers supply to Santa Barbara and San Luis Obispo counties, was completed in 1997. Prior to the commencement of construction of the SWP, contracts were signed between the DWR, the managers of SWP, and urban and agricultural water districts. Since the signing of the contracts in the 1960s, the capabilities of the system have not fully developed, and the SWP regularly does not meet all of its obligations. In 1998, existing long-term SWP water supply contracts totaled about 4.1 MAF , and these contracts are scheduled to increase to about 4.2 MAF by 2020 . In the year 2000 , however, the SWP delivered only 2.9 MAF of Table A water . DWR’s State of Water Project Delivery Reliability Report confirms that without additional facilities, the SWP will consistently be unable to meet its obligations to Table-A contractors. The Department of Water Resources administers long-term water supply contracts to 29 local water agencies for water service from the State Water Project. These water supply contracts are central to the SWP construction and operation. In return for State financing, construction, operation, and maintenance of Project facilities, the agencies contractually agree to repay all associated SWP capital and operating costs. To provide a convenient reference, SWP Analysis Office has prepared consolidated contracts for several contracting agencies.

These contracts contain the amendments integrated into the language of the original contract. Listed below, under the names of the contracting agencies, are the consolidated contracts and original contracts. DWR plans to add more consolidated long-term water supply contracts as they are completed. The 29 State Water Project contractors are shown in Table 2-1.The Bay-Delta ecosystem is a major hub of the state’s water re-distribution system. In order for the large freshwater of the Sacramento River and its tributaries to be made available to users in the southern half of the state, they must flow from north through the Sacramento-San Joaquin Delta and then be pumped out of the Delta in South into the aqueducts of the State Water Project. An extensive system of levees has also developed over the years to protect agricultural and urban land holdings within the delta from water intrusion and flooding. Together, the pumping of freshwater from the south to the delta and the artificial support of the Delta’s numerous islands has dramatically altered the natural hydrology and ecosystem function of the Bay-Delta system. In response to dramatic declines in Delta ecosystem quality during the 1987-1992 drought, a Federal and State partnership was established in 1994. The purpose of the multibillion dollar restoration and management effort, now managed by the California Bay-Delta Authority is to restore ecosystems within the Delta, improve the quality and reliability of water supplies from the Delta, and stabilize the Delta’s levee systems . The challenge of this mandate is immense, particularly when considered along with potential climate change . The incongruent nature of the program’s objectives has arguably hampered its effectiveness to date,plant raspberry in container yet the effort continues and will remain a significant consideration in future California water management and planning. Prior to extensive human development, the San Francisco Bay-Delta was largely marsh, river channels, and islands, bounded in the west by the Golden Gate Strait and Pacific Ocean and in the East by the confluence of the Sacramento and San Joaquin Rivers which drain the Sierra Nevada Mountains to the Pacific Ocean. The Bay-Delta in its natural state was an enormous estuary and supported extensive habitat for fish, birds, and other terrestrial animals. Water flowing through the Delta is the main source of supply for two major California water delivery projects, the SWP and the Federal CVP. From these projects, a majority of Californian relies on water flowing through the Delta for all or part of their drinking water. In addition, approximately one third of the state’s cropland uses water flowing through the Delta . Figure 2-2 shows the Bay Delta water distribution during a typical hydrological year. The Colorado River supplies Southern California with more than 4 MAF a year of water via the Colorado River Aqueduct and the Coachella and All American Canals.

The Colorado River Compact, signed six states bordering the Colorado River in 1922, established California’s base water entitlement to be 4.4 MAF a year. In recent years, however, California has relied upon the unused allocation of upstream states, importing more than 0.8 MAF a year of additional supply some years . Due to growing water use by other states, California was forced to reach an agreement to gradually eliminate its use of surplus water. The Colorado River Quantification Settlement Agreement resolves much of the uncertainty over Colorado River allocations, but an on-going drought in the Colorado River basin still threatens future Colorado River water availability to California. The iconic Colorado River supplies water to millions of people in fast-growing cities in Colorado River’s watershed, such as Las Vegas, Mexicali, Phoenix, and St. George, Utah. Tens of millions of people outside the watershed, from Denver to Albuquerque and from Salt Lake City to Los Angeles, San Diego and Tijuana, also receive water exported from the basin to meet at least some of their residential and commercial water needs. More than half of the people receiving water from the basin live in Southern California . Figure 2-3 shows historical water supply and usage for the Colorado River Basin from 1914 to 2007. Local cities in California have also taken initiative to develop water supplies. The cities of Los Angeles, San Francisco, and several in the East Bay region have all financed and constructed infrastructure to capture, store, and transport water from sources far away from the municipalities. Specifically, the Los Angeles Aqueduct transports water over 200 miles from the Owens Valley to the Los Angeles area; the O’Shaughnessy Dam captures and stores water in the Hetch Hetchy Valley for delivery to San Francisco and surrounding cities; and the PardeeReservoir and Mokelumne Aqueducts supply the East Bay Municipal Water District service area with supplies from the western slopes of the Sierra Nevada . Groundwater is a major source of water for California’s agricultural industry and municipalities. During an average year a third of the state’s water supply comes from groundwater. Some regions are entirely dependent on groundwater, and 40-50% of Californians use some amount of groundwater . Much of the state’s groundwater resources have been developed locally by individual landowners or municipalities. Such decentralized management has led to unsustainable groundwater use in California. Estimates by DWR in 1980 suggest that use of groundwater exceeds recharge by between 1 and 2 MAF per year . Such overuse has led and will continue to lead to many serious problems including land subsidence, sea water intrusion, and degradation of groundwater quality. Groundwater is currently managed through local water agencies, local groundwater management ordinances, and court adjudication. Importantly, state and regional planning agencies have little influence or control over the management of groundwater, making it difficult to implement integrated surface and groundwater management plans. The total groundwater storage in California is estimated to be about 1.3 billion acre-feet and about 140 MAF of precipitation percolates into the state’s aquifer annually . These estimates however, do not characterize the potential water supply for the region – many other factors limit the development potential of an aquifer . Most of the state’s groundwater is located in the aquifers beneath the Central Valley, although Southern California also has considerable amount of groundwater. Groundwater is a major contributor to the state’s water supply and even more so in dry years. As shown in Figure 2-4, groundwater supplies on average 30 percent of California’s overall demand and up to 40 percent in dry years . In some areas where surface water supplies are not accessible or economically feasible, groundwater provides 100 percent of a community’s public water . During years where surface water deliveries are not available, groundwater may also provide up to100 percent of irrigation water for certain areas. About 43 percent of Californians obtain at least some of their drinking water from groundwater sources. Local municipalities and regional water agencies are increasingly turning to alternative sources of water supply. Treated urban wastewater is becoming an important source of water for agriculture, industry, landscaping, and some non-potable uses in commercial and institutional buildings. In many regions it is discharged into rivers and streams and thus used by downstream users. In some regions it is also blended with conventional sources and is injected or allowed to percolate into groundwater basins.