In contrast, Watsonville Slough had its highest SRP concentrations from fall through spring, with concentrations declining to an annual low point in mid summer . High SRP concentrations in the winter rainy season may be associated with increased surface runoff from agricultural fields located along the slough. Tile drains may also facilitate subsurface loses of phosphorus . In Carneros Creek, which is dry from approximately May until December each year, a third seasonal pattern emerged . SRP concentrations were moderately elevated at both upstream and downstream sites following the first winter rains, which suggests that soil phosphorus accumulates over the summer months and is flushed into the creek with the first rains. At Dunbarton Road where there is little cultivation upstream, sources may include natural decomposition in grasslands, cattle grazing, and rural residential land use; at downstream sites sources also include agricultural land use. At San Miguel Canyon Road, the downstream location, SRP concentrations increased again in the late winter and spring of 2002 and 2003, reaching very high levels that frequently exceeded 1 mg/L. Nutrient concentrations were highly erratic in 2002 and 2003, and subsequently declined in 2004, suggesting that nutrients originated from a point-source that has ceased to discharge. No seasonal concentration trends were observed in the upstream tributaries of the Pajaro River . At these locations SRP concentrations remained low throughout the year. We calculated the SRP load discharged by each tributary ,procona buckets and found loads varied seasonally corresponding with discharge . The SRP load was greatest at Chittenden during January and February, when discharge was also greatest. San Juan Creek was not sampled during this period, but likely accounts for a significant portion of the unaccounted load because it has elevated SRP concentrations and year round flow. In the Pajaro River, there is a strong seasonal trend in SRP concentrations .

Concentrations decline after the rainy season ends. Because SRP concentrations remain relatively high in the winter, rainfall probably transports SRP to surface waters. Furthermore, the loss of SRP from Santa Clara/San Benito Counties is highest during these rainfall periods . Because concentrations and export of SRP in the Pajaro River are rainfall dependent, it is difficult to determine long-term trends independent of recent rainfall patterns.Elevated phosphorus concentrations can cause excessive algal growth in waterways, and preventing excessive growth is the primary reason phosphorus concentrations are regulated. Algal biomass in the water column can be determined from the concentration of chlorophyll a, which indicates the degree of excessive algal growth. We monitored chlorophyll a concentrations at several sites in the Pajaro River watershed on a biweekly basis and compared these concentrations to phosphorus. The lack of a direct correlation between chlorophyll a and phosphorus levels indicates that P availability is only one of the factors controlling algal growth. Canopy cover and turbidity , algae-eating organisms , substrates that allow different types of algae to attach, and algae sources also play a role in algal growth and chlorophyll a concentrations. Furthermore, additions of nitrogen can stimulate algal growth in streams and rivers, which challenges the commonly held belief that phosphorus is the nutrient that controls the growth of algae in freshwater ecosystems. Our research group from the Center for Agroecology and Sustainable Food Systems has begun efforts to assess the growth patterns of algae in order to determine how elevated phosphorus and nitrogen levels influence these patterns. Under state legislation known as the Agricultural Discharge Waiver that took effect in January 2005, farmers are required to develop farm water quality plans to protect surface waters along the Central Coast. One goal of our research is to inform growers of current water quality conditions in waterways adjacent to their land so that they can take steps to reduce their impacts on waterways while continuing to farm profitably. Because phosphorus is transported to waterways in storm and irrigation runoff, reducing soil erosion and surface runoff is an important step in reducing phosphorus losses from the farm .

Subsurface flow is also an important mechanism of phosphorus losses from the farm . Growers can address these losses by matching P demand in plants with fertility management, keeping P concentrations in soils at agronomically responsive levels , and managing irrigation to minimize or eliminate runoff. It is important to note that many growers on the Central Coast and throughout the state have already initiated practices to reduce the loss of phosphorus from their farms. The University of California has several research projects in progress to document the impacts of changes in farm management, and a number of government agencies and NGOs are working with growers to improve water quality .Despite growers’ efforts, the target level of 0.12mg/L or lower for ortho-P set by the RWQCB may be difficult to attain in the lower Pajaro River and Elkhorn Slough watersheds, where natural sources of phosphorus may be high, and tile drainage systems facilitate losses of phosphorus to surface waters. Even with changes in agricultural practices, the ambient water quality improvements may not be detected for several years, which makes enforcing the water quality regulations difficult on the short-term. Thus, long-term monitoring programs are important to determine the success of changing management practices.Nitrate contamination of freshwater resources from agricultural regions is an environmental and human health concern worldwide . In agriculturally intensive regions, it is imperative to understand how management practices can enhance or mitigate the effect of nitrogen loading to freshwater systems. In California, managed aquifer recharge on agricultural lands is a proposed management strategy to counterbalance unsustainable groundwater pumping practices. Agricultural managed aquifer recharge is an approach in which legally and hydrologically available surface water flows are captured and used to intentionally flood croplands with the purpose of recharging underlying aquifers . AgMAR represents a shift away from the normal hydrologic regime wherein high efficiency irrigation application occurs mainly during the growing season. In contrast, AgMAR involves applying large amounts of water over a short period during the winter months. This change in winter application rates has the potential to affect the redox status of the unsaturated zone of agricultural regions with implications for nitrogen fate and transport to freshwater resources.

Most modeling studies targeting agricultural N contamination of groundwater are limited to the root zone; these studies assume that once NO3 – has leached below the root zone, it behaves as a conservative tracer until it reaches the underlying groundwater or, these studies employ first order decay coefficients to simplify N cycling reactions . However, recent laboratory and field-based investigations in agricultural systems with deep unsaturated zones have shown the potential for N cycling, in particular denitrification, well below the root zone . For example, Haijing et al. found denitrifying enzyme activity as deep as 12 metersin an agriculturally intensive region in China. Lind and Eiland reported N2O production in sediments taken from 20 meter deep cores. Other studies have reported the capability of deep vadose zone sediments to denitrify in anerobic incubations with or without the addition of organic carbon substrates . Moreover,procona florida container in agricultural settings, especially in alluvial basins such as in California with a history of agriculture, large amounts of legacy NO3 – has built up over years from fertilizer use inefficiencies and exists within the deep subsurface . It is not yet clear how this legacy nitrogen may respond to changing hydrologic regimes and variations in AgMAR practices, and more importantly, if flooding agricultural sites is enhancing nitrate transport to the groundwater or attenuating it by supporting in situ denitrification. Denitrification rates in the subsurface have been reported to vary as a function of carbon and oxygen concentrations, as well as other environmental factors . While total soil organic carbon typically declines with depth , dissolved organic carbon can be readily transported by water lost from the root zone to deeper layers and can therefore be available to act as an electron donor for denitrification . Oxygen concentration in the vadose zone is maintained by advective and diffusive transport, while oxygen consumption occurs via microbial metabolic demand and/or abiotic chemical reactions . The effects of drying and wetting cycles on oxygen concentrations in the deep subsurface are not well documented. However, in 1 meter column experiments, there is some evidence that O2 consumption proceeds rapidly as saturation increases and rebounds quickly during dry periods . These variations in oxygen concentration can influence N cycling and thus, transport to groundwater. Variability in nitrate concentration has also been linked to heterogeneous subsurface properties, rainfall events, seasonality of flow and other local geochemical conditions across a diversity of settings However, a gap currently exists in quantifying N attenuation and transport from agriculturally intensive regions with a “deep” vadose zone while accounting for the many competing N cycle reactions and transformations, as impacted by different hydrological regimes imposed under AgMAR.

The application of AgMAR itself can vary in terms of the hydraulic loading and rates used, as well as the duration between flood water applications. These can in turn affect water retention times, O2 availability, consumption of electron donors and consequently, denitrification rates . For example, denitrification rates were found to increase with increased hydraulic loading and with shorter times between flood applications within the vadose zone of a rapid infiltration basin system used for disposing of treated wastewater . In shallow, sandy soils, high flow rates – above an infiltration threshold – were negatively correlated with denitrification rates, suggesting that an optimum infiltration rate exists for a given sediment stratigraphy to maximize NO3 – reduction . Given the immense stratigraphic heterogeneity in alluvial basins, such as in California’s Central Valley, a range of optimum infiltration rates may exist with implications for managing AgMAR differently based on the geologic setting of the intended site. Therefore, the objectives of this study are to: a) understand the effects of varying stratigraphy and hydrologic regimes on denitrification rates, and b) identify AgMAR management scenarios that increase denitrification rates, such that the potential for N leaching to groundwater is decreased. Herein, we focus on an agricultural field site in Modesto, California located within the Central Valley of California, which is responsible for California’s $46 billion-dollar agricultural economy . The field site typifies the deep vadose zones prevalent in this region, which are characterized by heterogenous layered alluvial sediments intercalated with discontinuous buried clay and carbon rich paleosols . These discontinuous, layered features, especially the paleosols and areas of preferential flow, are typically associated with enhanced biogeochemical activity, higher carbon content and availability of metabolic substrates such as nitrogen . These regions respond to and change depending on environmental conditions such as water content and oxygen concentration in situ that are influenced by the hydrologic regime at the surface and may be important for NO3 – attenuation and reduction prior to reaching the water table. Therefore, this study considers varying hydrologic regimes and stratigraphic variations that are prevalent in the region. More specifically, at the Modesto field site , large amounts of legacy N already reside in the vadose zone, while N fertilizer application and irrigation occurs throughout the spring and summer months. AgMAR, if implemented, occurs during the winter months as water becomes available. Therefore, we focus here on quantifying the effects of AgMAR on N cycling in the deep vadose zone and implications for NO3 – contamination of groundwater at this characteristic agricultural field site. We also investigate the specific AgMAR application rates that would increase the effectiveness of in situ denitrification under different stratigraphic configurations through the development and testing of a reactive transport model. We believe such an analysis provides important insights for the successful application of AgMAR strategies aimed at improving groundwater storage in a changing climate. Reactive transport models can be beneficial tools to elucidating N fate and transport in deep vadose zone environments. Herein, we develop a comprehensive reaction network incorporating the major processes impacting N transport and attenuation, such as aqueous complexation, mineral precipitation and dissolution, and microbially mediated redox reactions. While using the same reaction network, we implement several numerical scenarios to quantify the range of denitrification rates possible under different AgMAR implementation strategies and stratigraphic configurations . For the latter, we used four different stratigraphic configurations with a low permeability layer on top including i) two homogeneous textural profiles, ii) a sand stratigraphy with a discontinuous silt band, iii) a silt stratigraphy with a discontinuous sand band, and iv) a complex stratigraphy more representative of the field conditions investigated by electrical resistance tomography .

Our environmental correlation results provide evidence for a habitat filtering process in which agricultural adapters are promoted by vineyard expansion, while co-occurrence of and negative interactions between agricultural adapters and oak woodland birds provide evidence for indirect detrimental effects of land conversion. These findings support a more pluralistic hypothesis that community structure is not influenced solely by habitat filtering or by species interactions, but rather is influenced by both mechanisms and interactions between them. It is not surprising that agricultural adapters and woodland dependent birds co-occur in oak woodlands where they can take advantage of woodland resources, such as food or nesting sites that may not be abundant in the vineyard matrix. Habitat filtering can act as a driver of change in communities ; considering that vineyards favor agricultural adapters , the relatively high abundance of these species within the agricultural matrix may increase the probability of percolation of these species into oak woodlands. Accounting for the influence of the vineyard matrix and other environmental site and landscape variables, our results reveal negative interactions between several agricultural adapters and a host of native woodland species – some of which are species of conservation concern. To the best of my knowledge this is the first report of these types of indirect interactions, in addition to predation and parasitism, related to birds near agroecosystems. Vineyards appear to influence bird communities at different scales, with increased vineyard influence in small fragments of oak woodland, which are more likely to be used by agricultural adapters and perhaps more vulnerable to indirect spillover effects that could devalue the remaining wild lands. The species identified by our study as agricultural adapters are among the most commonly recorded species in Californian vineyards suggesting that vineyards likely promote these species more broadly. My results suggest that vineyard expansion threatens bird diversity not only directly via habitat conversion, but also by promoting changes in community composition and species abundance that then have indirect, negative consequences for species in remaining natural habitat. Over time,plastic plant pot competitive interactions could change the community composition of oak woodlands, undermining their conservation value.

The combined impact of habitat conversion and reduced habitat quality is concerning for bird conservation. For instance, body condition and fitness are important for successful migration for Neotropical and altitudinal migratory species and are related to habitat quality. Abundance of some Neotropical migrant species is associated with large forest patches and negatively associated with agricultural/urban landscapes . Equally important are impacts to species with declining populations, in our context Spotted Towhee, Orange-crowned Warbler, and Bewick’s Wren or oak woodland specialist species as Nuttall’s Woodpeckers. The method I used does not explicitly model species unidirectional interactions, instead it directly estimates reciprocal interactions . I assume negative interactions to be negative effects of agricultural adapters on oak woodland birds. Several mechanisms documented in other studies may help interpret our results. Firstly, direct biotic interactions may be at play. For instance, starlings are extremely aggressive birds that displace other species from nest sites they want to use, which are mainly cavities , and are considered among the most spread invasive species worldwide that threaten species with extinction . Aggressive behavior by native birds towards European Starlings has also been observed during nesting . Similarly, nest usurpation and/or depredation by starlings has been recorded . Studies in fragmented forest reported a higher predation by birds or parasitism by Brown headed cowbirds on Neotropical migrants birds in small than in larger fragments . I suggest that European starlings can have similar effects on oak woodland birds, but with a different mechanism. There is at least three mechanism of species loss, high predation, low dispersal abilities, and detrimental conditions of the environment .Other evidence of these direct interactions include American crows, which aggressively chase away other birds and eating the eggs and nestlings of other birds , and Brown headed cowbirds, which are a brood parasite of several bird species . Secondly, indirect biotic interactions also be a role but are more difficult to observe and understand than direct interactions and could make community dynamics difficult to predict . In some cases, the mere presence of one bird can affect another’s feeding behavior or the inter specific interactions of the community .

This gap in behavioral knowledge for some of these species could be elucidated with future research. Thirdly, changes in bird community composition could affect the ecosystems functions and services through promote or attenuate complex interactions, that can cause cascade effects that change the ecosystem properties and affect biodiversity . Changes in composition of the landscape matrix, in our study vineyard extension, influence the bird community composition. Other studies previously documented the importance of landscape context and habitat quality for wildlife communities. Species benefitting from exotic plantations were promoted by mechanisms such as habitat filtering , temporal changes in the surrounding matrix causing shifts in species interactions , and new species introductions that change co-occurrence patterns in a community . Some of the relationships in our analysis appear to have a complex pattern of interactions with an unclear mechanism, for instance, the negative relationship of Orange-crowned Warbler with shrubs at the plot scale. Previous studies report varying bird-habitat relationships depending on the landscape context or interaction between species, such as in the case of a negative relationship between some Neotropical migrants and forest patch size . It is probable that for some species the proportion of oak woodland forest will be a better predictor than the proportion of vineyards, but these variables have a highly negative correlation at the landscape scale so are hard to address independently. Different landscape scales may more accurately predict the relationship between landscape variables and different species according to biological and ecological traits . Fragmentation and agricultural intensification have led to reductions in beta diversity and biological community homogenization in many temperate and tropical systems . These changes in native communities can have cascading effects on ecosystem function and resilience . Here, I show that vineyards can lead to regional biotic homogenization of neighboring natural areas through the expansion of the vineyard matrix, by increasing the abundance of birds adapted to vineyards, and by increasing competition between agricultural adapter and oak woodland birds. It is unlikely that all species will be equally affected , as habitat specialists are more likely to be threatened than generalists .

The underappreciated, indirect effects need to be considered as the agricultural footprint continues to expand across California’s coastal ranges and vineyards regions globally. Agriculture land conversion is recognized as one of the main threats to biodiversity due to habitat loss and fragmentation. In this study I found that the effects of agricultural land can spill over into natural areas. I see a clear relationship between the extent of agricultural land and bird species detection rates in adjacent natural areas. More surprisingly, I detected strong negative interactions through co-occurrence patterns between agriculturally adapted bird species and oak woodland associated birds. This research provides evidence that competition from species adapted to agricultural land use could be another driver of biotic homogenization in addition to habitat loss and fragmentation associated with habitat conversion. To the best of our knowledge, this is the first report of these types of indirect interactions beyond predation or parasitism between birds related to agroecosystems. Conserving biodiversity in agroecosystems is a global challenge . Agricultural impacts on biodiversity are widely recognized, and land use change and fragmentation are major drivers of species extinction and ecosystems degradation . Wine grapes are one of the most important crops in terms of land cover surface and economic relevance in Mediterranean type ecosystems worldwide,plastic planter pot which also overlapping with a designated biodiversity hotspot . Future projections for vineyards predict advancement to new areas, where climatic conditions will be favorable for wine grape production, in addition to demand from new markets, can increase vineyard expansion . Different strategies are proposed to increase biodiversity conservation in agricultural landscapes . Some studies indicate that biodiversity can best be conserved while increasing yield by dividing surface area into conservation zones and intensively managed agriculture . Others propose that increasing the quality of agroecosystem as habitat via agroecological management, enhances biodiversity. Still others propose an integration of both aforementioned strategies according the specific traits of the area of study/species . Finding new alternative approaches that enhance biodiversity within the agricultural matrix is part of the current policy efforts of “protect wildlife beyond the protected areas” of Chilean President M. Bachelet . The spatial configuration of the natural vegetation surrounding agriculture can significantly influence in the occurrence and abundance of bird species . It is recognized that increasing landscape complexity within agroecosystem can increase niche availability and thus host higher levels of biodiversity . An increasing number of studies indicate that remnant native vegetation can contribute to the persistence of birds. Birds can be favored by the presence of forest fragments , forest edges, and riparian vegetation in agroecosystems . Assessing the impacts of the native habitats on biodiversity in a dominant agroecosystem such as vineyards in central Chile is vital for conservation management within this biodiversity hotspot, and could provide management insight for other Mediterranean type ecosystems.

I used a natural experiment based on a gradient of landscape complexity within vineyards to investigate: whether bird trophic guilds change between continuous forests, fragments and vineyards, if changes in the presence and/or proportion of forest fragments influence birds communities, and if the proportion of continuous native vegetation influence birds communities. I hypothesized that vineyards promote some species, and that these functional guilds are different between vineyards and fragments within vineyards. My aim is to disentangle the changes in bird communities due to vineyards at different landscape scales. Conserving biodiversity in agricultural landscapes is an unresolved challenge, in particular in Mediterranean type ecosystems in where agricultural expansion and unique wildlife habitat overlap. I found that fragments of native vegetation within vineyards significantly retained bird communities in vineyard landscapes. Fragments significantly increased abundance and richness compared to the vineyards in which they are located, and also affected assemblages of endemics, insectivores, granivores and omnivores . An equally important finding is that the proportion of fragment native vegetation within a given landscape area affected similarly species than the mere presence of fragments. This highlights the important role of remnants of native vegetation within agroecosystem, particularly remnant size. Our evidence supports a land sharing approach, where more structurally diverse habitat within agroecosystems and habitat heterogeneity at landscape scale can enhance biodiversity in working landscapes . The results coincide with other studies finding that fragments, as well as hedge proportion and small woods within vineyards, support higher bird richness and abundance . In particular, the observed increase in insectivore birds due to large fragments is in agreement with other studies in vineyards . Insectivore species as Chilean swallow and House Wren, were more abundant in vineyards with fragments. Higher insectivore abundance could be associated to ecosystem services such as insect predation relevant to vineyard pest management . However, further research is required to better assess the impacts of biological control via bird predation of insects in Chilean agroecosystems. Fragments were particularly relevant for endemic species. Although fragments could be expected to favor mainly more mobile species such as Chilean Mockingbird and Dusky-tailed Canastero, this was observed also for the Dusky tapaculo, a rhinocryptid species which is less mobile . Other endemic rhinocryptids were excluded from the analysis due to their low rates of detectability within fragments and vineyards , indicating that these species are less likely to be found in anthropogenic environments. More focused research on these less mobile species would be needed to study their behavior. Forest patches were also relevant for Neotropical migratory species as White-crested Elaenia, showing that agroecosystems with large forest patches were indispensable for birds, and in particular for species that need connectivity at landscape scales . The results are consistent with existing literature demonstrating that vineyards support bird communities across the globe . Vineyards monocultures, without fragments of native vegetation favored a subset of species, all of them associated with open habitat and higher tolerance to anthropogenic environments.

We observed substantially higher odds of low neurobehavioral scores , among children living within 100m of a plantation compared to children living at >500m; the odds ratios were weaker among participants living within 101m to 500m of a plantation. Finally, we inspected GAM plots for the relationship between continuous distance and neurobehavior scores for the three subdomains with the largest effect sizes . These are qualitatively similar to the results of analyses of distance by category , exhibiting a decrease in Attention & Inhibitory Control and Language scores at close proximity but do not provide strong evidence of nonlinear effects. Only 12% of the study population lived within 100m of a plantation , which limited the power of our analyses of associations within very close distances to plantations. All results from linear regression analyses comparing growing area within 100m of participant residences to those living further than 100m from floricultural crops were null . In logistic regression analyses, children with the most growing area within 100m of their residence had higher odds of low scores in the Language domain , compared to children without any plantation land within 100m of their residence .We observed that close residential proximity to floricultural crops was associated with poorer neurobehavioral outcomes in the domains of Attention & Inhibitory Control, Language and Memory & Learning. Associations were strongest among children living within 50m of a flower crop and present to some extent among children living between 51 and 100m. These findings were partially corroborated by sensitivity analyses using areas of floricultural crops near homes as a related construct of pesticide drift from flower crops. Unlike short-lived biomarkers of exposure, proximity of a child’s home to agricultural crops may approximate the child’s ongoing and historical low dose exposure to pesticides through off-target drift or direct access to pesticide-treated areas. In the ESPINA study,black plastic nursery pots we previously described positive associations between AChE activity and the domains of Attention & Inhibitory Control, Memory & Learning, and borderline associations with the Language domain affecting boys more than girls .

Alterations in the same domains were observed in the present study, which is consistent with previous findings. Epidemiologic studies provide increasing evidence that pesticide exposure during key developmental periods may be a risk factor for a range of neurocognitive deficits later in life, including attention deficit and hyperactivity disorder, autism spectrum disorder, developmental delay, slowed reaction time, and slowed motor control, poor verbal comprehension People living closest to agricultural crops are at increased risk of pesticide exposure. In our analyses, children living within 100m of a flower crop, and especially within 50m, had lower neurobehavioral scores compared to children living farther than 500m. These findings suggest that the amount of pesticide drift from crops onto nearby homes can especially affect the neurobehavioral performance of children living within 100m. However, alterations in neurobehavioral performance may also be present at greater distances but the limited statistical power of our study to detect smaller differences precluded us from assessing this further. In previous analyses of the ESPINA study, we observed positive associations between residential distances to flower crops and AChE activity, with the lowest AChE levels observed among children living within 232m of a greenhouse floricultural crop . This supports the construct of residential distance to flower plantations as a pathway of exposure to pesticides Furthermore, we previously observed that children living closer to flower crops had higher systolic blood pressures, which indicates that additional physiologic processes may be affected among children living near pesticide spray sites . Multiple investigations have studied the association between proximity of homes to agricultural crops and pesticide exposure . While maximal exposure attributable to pesticide drift, among these studies, varied from 60 to 750 meters, this collection of studies rather consistently indicates that homes residing closer to pesticide treated fields tend to have higher pesticide levels and that children residing closer to pesticide treated fields tend to reflect higher pesticide exposure levels using bio-monitoring studies. In this study, exposure was modeled as distance to the nearest plantation in the primary analyses, based on the assumption that increased distance reflects lower exposures. An alternative measure, area of plantations within varying buffer areas, which is likely a better proxy for exposure to pesticide drift, was also explored.

As expected, results showed consistent associations between these two related but different constructs of pesticide exposure, which strengthens our findings. Several studies have utilized residential proximity to agriculture as a metric of exposure to pesticides when studying its associations with neurodevelopment . A number of these studies used data from California State’s Pesticide Use Reporting System, finding positive correlations between proximity of prenatal residence to areas of agricultural pesticide applications and neurodevelopmental outcomes in early childhood, specifically ASD . In our analyses the observed effect size in the logistics models were small, but the magnitude may have been attenuated by the non-linear dose-response relationship shown in Figure 3. Furthermore, the linear regression models indicated that a difference of 100m in residential proximity to floricultural crops is associated with a greater likelihood of the child scoring in the sub-clinical ranges for the Language and the Memory and Learning domains by 9% and 24% respectively. In the context of measurable outcomes, this is clinically significant in that identifying children with delayed development warrants early intervention by clinicians as well as educators. The expected distance of pesticide drift from flower crops to nearby homes was smaller in our study population compared to those of other studies likely because rose production is enclosed within greenhouses. Greenhouses in Pedro Moncayo County have air circulation vents or windows, which could allow the escape of fumigated pesticides during and after spraying. However, these analyses suggest that pesticide drift, even in this setting, could still be problematic in the context of pesticide exposure affecting the neurodevelopment of children living nearby. This body of evidence coupled with the growing number of studies describing neurobehavioral alterations associated with pesticide exposures suggests that extending buffer zones or protective areas that separate the industry from the neighboring communities, could be an effective way to protect developing children from the adverse effects of pesticide exposure. The present study was subject to several limitations. Though a crude exposure assessment, the use of this exposure metric is supported by the existing literature and validated within our study population . Prevailing winds were not accounted for in the present analyses. This provides potential for non-differential misclassification of the amount of pesticide drift from plantations to homes and may have biased our findings towards the null . Also, while the vast majority of the floricultural production in Pedro Moncayo County includes roses, which are grown inside greenhouses, a small amount of production of other flowers also occurs in nonenclosed fields typically located near the greenhouses. For this reason, it is plausible that some of the pesticide drift from crops, and hence the associations observed in this study, may be a result of both greenhouse and open field floricultural production. Nonetheless, residential proximity to crops is a useful construct of exposure as it is an indicator of chronic pesticide exposure, and provides practical information about the distances in which populations may have an increased risk of pesticide exposure and/or adverse health. While it does not allow us to determine which specific chemicals are influencing this association,greenhouse pot it indirectly accounts for a mixture of the various agrochemicals used in floriculture. The floricultural industry in Pedro Moncayo frequently uses various pesticides including insecticides , many classes of fungicides and to lesser extent, herbicides .

Many of the studies assessing neurodevelopment and pesticide exposure, including the ESPINA Study, are limited in that they study bio-monitoring of few pesticides, even though it is unusual for one pesticide to be used without multiple others. It is plausible that pesticides or other neurotoxicant agrochemical exposures explain the neurobehavioral deficits seen among participants living near the flower crops. Determining the quantity and types of agrochemicals used over time and by location would improve precision but would be very difficult to ascertain in this agricultural setting. Another limitation related to exposure assessment is that we were not able to account for all potential routes of exposure to pesticides, including dietary intake. We did not have information on time-activity patterns, which would have provided better insight into participants’ outdoor exposures . There is also some uncertainty associated with using home location only to estimate exposure to environmental pesticides. Some children in the cohort went to school during the day, while younger children attended daycare or stayed home with a relative. Modeling exposure experienced across all daily activities and locations is beyond the scope of this current study; we choose to focus on exposures at the children’s home locations. Another limitation of this study is that neurobehavioral outcomes were assessed at only one point in time, and thus we are unable to assess if the neurodevelopmental effects are permanent. Additionally, the NEPSY-2 is based on a US normative sample. Although this does not affect the internal validity of our findings, it is unclear how accurately the cut-off values for “low performance” apply to this study population. This study has several strengths and thus contributes to our understanding of the effects of pesticide use in floricultural communities. This study is unique in that there was a wide distribution of participants’ residential distance to crops, and we had a considerable number of children living in close proximity to flower crops, which allowed us to estimate effect sizes at short distances. Additionally, all participants were examined during a period of relatively homogeneous flower production and pesticide use. Children in this study were examined during a period of lower flower production and pesticide use compared to other times of the year. In theory, this would reduce the off-target pesticide drift potential from crops, with resulting lower exposures to children living nearby. Considering that pesticide spray seasons may also have short-term neurobehavioral alterations in children , it is plausible that these observed associations would be stronger during peak exposure periods. Lastly the existing studies have focused on residential distance to agricultural open fields. To our knowledge, the present study is the first to characterize the associations of neurobehavior in relation to greenhouse agricultural production, which is generally though to result in reduced pesticide drift from crops. Many types of crops involve the use of greenhouses such as flowers, tomatoes, cucumber, a variety of herbs, lettuce, bell peppers, and eggplants. The present study findings may be applicable to such agricultural production. High rates of ecosystem modification and subsequent consequences for biota, climate and geochemical natural cycles characterize the Anthropocene . To date, agriculture has modified nearly 40% of terrestrial ecosystems and drives biodiversity depletion via multiple direct and indirect effects associated with wildlife decline . These mechanisms include intensive use of agrochemicals, conversion of natural areas to simplified monocultures, fragmentation associated with land use change, soil erosion and sediment runoff, and greenhouse gas emissions, among others . These negative effects of agriculture consequently impact on species, habitat loss, and lack of connectivity, generating synergic detrimental effects on biodiversity, ecosystem functionality and provision of ecosystem services . Reconciling agricultural production and biodiversity conservation goals is a global priority in light of future scenarios pertaining to food consumption and land use change . However, agricultural management is not homogenous and may differentially influence wildlife. Agricultural intensification which entails a shift from diversified small farming practice to large monoculture production relies on the use of agrochemicals and has taken a toll on biodiversity . Adoption of agroecological principles and wildlife friendly farming can favor habitat suitability for wildlife and maintain sustainable crop yields . Strategies to balance agricultural food production and wildlife conservation include land sharing and land sparing strategies . Land sparing proponents have focused efforts on increasing food productivity per land area often requiring high levels of agrochemical input, and argue that with high production in target crop areas, natural areas can be spared from conversion to crop production . One impact that this approach does not address is the agrochemical spill over and its negative impacts on non-target wildlife species leading – another detrimental externality resulting from agrochemicals .

Like other emerging donors, Brazil follows the global standard of providing cooperation in the project format. As I will argue in Chapter 3, however, it does so through a mode of engagement that is more hands-off and based on demonstration, in contrast with traditional aid, based on more bureaucratized and large scale kinds of intervention. Here I will approach this question through an idiom closer to my relational analytics, that of robustness.At least since Ferguson’s insightful chapter on the politics of knowledge in World Bank reports in The Anti-Politics Machine, discourse lingered for a long while as a prevailing analytical angle in the anthropology of development , remaining important even after potent critiques during the late nineties that continue to resonate today . To refuse discursive determinism is not however to deny the importance of discourse, but to pay close, empirically grounded attention to its relations with history and practice. The first three chapters will begin by approaching South-South cooperation discourse in three domains: Chapter 1, South-South / North-South politics; Chapter 2, culture and history in Brazil-Africa relations; and Chapter 3, nature and agricultural development in the tropics. Each will seek to show how official discourse participated in context-making efforts, and then move on to look at its relations with front line practice. In this dissertation, however I will refer to discourse in two senses, which I try to differentiate. Most of the time, discourse will refer to a working tool consciously deployed by certain groups of actors in the field – most notably that of the diplomats, but also those in politics,raspberry container size academia and other intellectual circles. I tried to mark this specificity by qualifying it as official discourse rather than discourse in general. Official discourse in this sense is mostly concerned with a self-account of Brazilian cooperation. But one of my most forceful observations during fieldwork was how distant it could be from the practice of front liners.

The various chapters will suggest how, rather than describing the latter accurately or even shaping it directly, official discourse is more often than not disconnected from it: it follows a logic and productivity of its own that is largely circumscribed, by organizational and sociality lines, to diplomatic and more political and intellectual kinds of circles. Not that there are no relations between diplomats and front liners ; they not only exist as may play a significant part in cooperation activities. But as will be seen, they unfold in ways that do not follow a linear, coherent referential bridge between discourse and practice. The other way in which I talk about discourse here draws on the Saidian-Gramscian Foucauldian analytics found in much of the U.S. literature in the anthropology of development . In it, the Foucauldian view on knowledge production as part of the apparatus of power is refracted by Said’s postcolonial inflection and/or by Gramsci’s deeply historical approach to hegemony and special attention to political economy. Here, I will largely follow these refractions. Some of the discursive elements I will approach are long lasting and do seem to provide a common grammar that is shared by virtually everyone on the Brazilian side. I traced discourse in this sense to certain historical processes, in special those involved in shaping Brazil’s postcolonial condition. This discussion, which I have also started to entertain elsewhere , will be made explicit in Chapter 2. There I draw, besides on Said himself , on works on the question of post coloniality and modernity in Latin America in general, and Brazil in particular . In particular, some notions put forth by Portuguese sociologist Boaventura de Sousa Santos such as double colonialism, internal coloniality, and situated post colonalisms were highly productive for making sense of Brazil’s postcolonial condition as well as of its past and contemporary relations with Africa . Here, I have coalesced these and other insights into an attention to how coloniality11 operates in two, interrelated directions: both externally and internally to postcolonial nation-states.

What is framed as the postcolonial condition in general usually focuses on the international dimension. Some Latin American authors, on the other hand, have sought to specify its domestic dimension through the term internal colonialism . Few however have made a sustained, empirical and theoretical, investment in looking at the relations between these two . While this dissertation will focus on how this double directionality has played out on the Brazilian side, this perspective could also be useful for looking at equivalent processes on the African side.12 I will introduce it in Chapter 2, through a discussion about a kind of hegemonic discourse on Africa that I term Brazil’s nation-building Orientalism. But like coloniality itself, this double directionality can be found in dimensions beyond discourse, from political economy to culture, from agricultural development to geopolitics. Some of these will be brought in the other chapters, albeit not as explicitly as in Chapter 2. Finally, the postcolonial inflection will reappear in Chapters 4 and 5, which will provide an account of an ongoing technical cooperation project between Brazil and four countries in West Africa. In these final chapters, I will try to bring these insights to bear on questions raised by science and technology studies and vice-versa – not unlike those who have been working at the scholarly interface some have been calling postcolonial science and technology studies.13 This discussion will bring us back full circle to the question of North-South difference raised in the first chapter, but now hopefully enriched by the analytics of relationality deployed more broadly here.This Introduction has already drawn on various analytical idioms of relationality found in anthropology and science and technology studies : interfaces, emergence, scaling, assemblages, context-making, socio-technical networks, situatedness, or robustness. These and others evoke works from science studies, such as those by Marilyn Strathern , Bruno Latour and Donna Haraway . To these I add insights from works that tread the path opened up by these authors, but introduce important new twists such as De Laet and Mol , Hayden , Da Costa Marques , or the Deleuzian approach put forth by Jensen and Rödje . Less frequently, a similar perspective has been brought to bear on discussions on development, although rarely incorporating the techno-scientific dimension of projects . We do not need to delve too deep into micro-practice to realize the centrality of relations to the phenomenon approached in this dissertation: it is in the very hyphen in South-South. As Chapter 1 will suggest, the duplication of the term brought into relation, “South”, is meant to evoke horizontality: a leveling opposition to the asymmetry explicit in the North-South configuration. As the hyphen in African- or Native-American, however, the one in South-South denotes less hybridism than an interface – which, I have been arguing here, is characterized by being in emergence. The character of this relation is therefore largely underdetermined; it is a work in progress being actively, and in some cases reflexively, performed by those involved in practicing and thinking it . As was already indicated,raspberry plant container the ways in which this interface is being worked will be approached here most frequently through an analytics of context-making, scaling and domaining, after some of Strathern’s discussions on gender, kinship and audit cultures.

This emphasis on the production of context came out of the empirical observation that interactions between actors from both sides of the Southern Atlantic have unfolded through relational channels which are much less consolidated than the ones underlying relations between, say, Mali and France. Correspondingly, given the largely unprecedented character of these relations, much of my field interlocutors’ efforts have been directed towards making a context for them, in a more intensive, less bureaucratized, and reflexive way than its Northern counterparts. In Strathern’s prolific oeuvre, context-making has appeared alongside related operations such as analogy-making, scaling, and domaining, all of which were also salient in the discourses and practices observed during fieldwork. As Brazilians and Africans are brought together into South-South cooperation’s emerging interfaces, their relational effort proceeds largely through analogies based on their respective experiences. In this process, some contextual elements are differentially assigned to preexisting domains and scales; some are brought to the fore, while others are left to evanesce in the background or are altogether eclipsed. Although these operations strive to coherence, quite often they lead to contradiction and ambivalence, especially as they straddle different interfaces and the lag between official discourse and cooperation practice. Indeed, when there is an over investment in certain analogies at a discursive level – most notably, between Brazil’s and Africa’s peripheral conditions, cultural outlooks, natural environments, developmental paths –, they not always correspond to practical relations. But as I will argue in Chapter 2, this does not mean, as those who have remarked some of these mismatches before me suggested , that official discourse is false, deluding, or naïve. There is, rather, certain diffuse functionality to it, including as an effort to open up a path for turning – to use a classic organizing duality in anthropology –metaphor into metonym: that is, to incite the establishment of mutually transformative, exchange-intensive interactions between Brazilians and their African counterparts. However, those who come up with the most explicit discursive analogies are not necessarily the ones who will work the hardest in practice to entice and nourish metonymic relations. Chapters 3 and 4 will focus on the work of the latter – the cooperation front liners – as they strove to make a productive context for their relations with their African counterparts during capacity-building trainings and technology transfer efforts. In these activities, as Chapter 3 will suggest, demonstration has been the prevalent mode of engagement. Here, demonstration is evinced from a contrast with the notion of intervention, which denotes conventional views on the global North’s prevalent mode of engagement with Africa .They show how capacity-building has been performed less as the imposition of abstract, authoritative techno-scientific knowledge than as the demonstration of a particular kind of experience in agricultural development and research, making explicit its socio-technical entanglements and enticing the audience to participate in context-making. Demonstration is the basis of key modalities of TT in agriculture in Brazil, African countries, and elsewhere. Context describes the site to which technologies will be transferred, which, in common policy views, denotes an inert background for a bounded object. Chapter 4 will draw on part of the STS literature, especially Latour’s actor-network theory and works on technology transfer inspired by it , to recast the process of technology transfer as a co-production between contexts and technologies. Vital to this end will be to bring more emphatically to the fore the question of power, which is not readily evident in Latour himself . For this, I will recruit in Chapter 5 the notion of sociotechnical controls. “Socio-technical” draws on an epistemological-methodological assumption that has by now become part of STS’s commonsense: that there is nothing essential about nature or society, the task being to trace how this ontological boundary is empirically made by scientists and those with whom they interact. Controls, on the other hand, are part of an idiom that came to the fore during fieldwork, especially during my time in Mali. It was enticed by a perception that what Brazilian and African researchers and technicians were doing in their experimental activities was less about constructing scientific facts than about deploying, or trying to deploy, practical controls – experimental controls, most obviously, but also sociopolitical controls. In fact, I came to see one type of control as inextricably linked to the other, and both as linked to the question of power at large: it matters where and when techno-science is being carried out, after all. Latour’s Salk Institute is not the same as Mali’s Institut d’Economie Rurale and even Embrapa research units – and yet these are part of a common global techno-scientific assemblage, but unequally so. The last chapter will foreground this paradoxical aspect of the cotton project, intimately tied especially to Sub-Saharan Africa’s postcolonial predicament, by proposing to view techno-science as being about controlling the flow of vitalities in both nature and society, in a multi-scalar network ranging from subterranean mineral molecules to the global rules of the World Trade Organization.

A total of 294 participants were included in either the prenatal or postnatal analyses. Participants included in this analysis did not differ significantly from the original full cohort on most attributes, including maternal asthma, maternal education, marital status, poverty category, and child’s birth weight. However, mothers of children included in the present study were slightly older and more likely to be Latino than those from the initial cohort. Women were interviewed twice during pregnancy , following delivery, and when their children were 0.5, 1, 2, 3.5, 5, and 7 years old. Information from prenatal and delivery medical records was abstracted by a registered nurse. Home visits were conducted by trained personnel during pregnancy and when the children were 0.5, 1, 2, 3.5 and 5-years old. At the 7-year-old visit, mothers were interviewed about their children’s respiratory symptoms, using questions adapted from the International Study of Asthma and Allergies in Childhood questionnaire . Additionally, mothers were asked whether the child had been prescribed any medication for asthma or wheezing/whistling, or tightness in the chest. We defined respiratory symptoms as a binary outcome based on a positive response at the 7- year-old visit to any of the following during the previous 12 months: wheezing or whistling in the chest; wheezing, whistling, or shortness of breath so severe that the child could not finish saying a sentence; trouble going to sleep or being awakened from sleep because of wheezing, whistling, shortness of breath, or coughing when the child did not have a cold; or having to stop running or playing active games because of wheezing, whistling, shortness of breath, or coughing when the child did not have a cold. In addition, a child was included as having respiratory symptoms if the mother reported use of asthma medications,plastic gardening pots even in the absence of the above symptoms.Three identical EasyOne spirometers were used . Routine calibration was performed every morning and 92% of tests were conducted by the same technician.

The expiratory flow-volume curves were reviewed by two physicians experienced in pediatric spirometry, and only adequate quality data were included in the statistical analyses. Some participants with adequate quality data for FEV1 did not provide adequate quality data to calculate FVC or FEF25–75. Young children have difficulty sustaining forceful exhalation after a deep breath that is required to produce a plateau in airflow and calculate FVC and subsequently FEF25–75. Each child performed a maximum of eight expiratory maneuvers and up to three best acceptable tests were saved by the spirometric device software. Latitude and longitude coordinates of participants’ homes were collected during home visits during pregnancy and when the children were 0.5, 1, 2, 3.5 and 5 years old using a handheld Global Positioning System unit . At the 7-year visit, mothers were asked if the family had moved since the 5-year visit, and if so, the new address was recorded. We used Geographic Information System software to geocode the new addresses and obtain coordinates. Residential mobility was common in the study population. We estimated the use of agricultural fumigants near each child’s residence using a GIS based on the location of each child’s residence and the Pesticide Use Report data . Mandatory reporting of all agricultural pesticide applications is required in California, including the active ingredient, quantity applied, acres treated, crop treated, and date and location within 1-square-mile sections defined by the Public Land Survey System . Before analysis, the PUR data were edited to correct for likely outliers with unusually high application rates using previously described methods . We computed nearby fumigant use applied within each buffer distance for combinations of distance from the residence and time periods . The range of distances best captured the spatial scale that most strongly correlated with concentrations of methyl bromide and 1,3-DCP in air . We weighted fumigant use near homes based on the proportion of each square-mile PLSS that was within each buffer surrounding a residence.

To account for the potential downwind transport of fumigants from the application site, we obtained data on wind direction from the closest meteorological station . We calculated wind frequency using the proportion of time that the wind blew from each of eight directions during the week after the fumigant application to capture the peak time of fumigant emissions from treated fields . We determined the direction of each PLSS section centroid relative to residences and weighted fumigant use in a section according to the percentage of time that the wind blew from that direction for the week after application. We summed fumigant use over pregnancy , from birth to the 7-year visit and for the year prior to the 7-year visit yielding estimates of the wind-weighted amount of each fumigant applied within each buffer distance and time period around the corresponding residences for each child. We log10 transformed continuous fumigant use variables to reduce heteroscedasticity and the influence of outliers, and to improve the fit of the models. We used logistic regression models to estimate odds ratios of respiratory symptoms and/or asthma medication use with residential proximity to fumigant use. Our primary outcome was respiratory symptoms defined as positive if during the previous 12 months the mother reported for her child any respiratory symptoms or the use of asthma medications, even in the absence of such symptoms . We also examined asthma medication use alone. The continuous lung function measurements were approximately normally distributed, therefore we used linear regression models to estimate the associations with residential proximity to fumigant use. We estimated the associations between the highest spirometric measures for children who had one, two or three maneuvers. We fit separate regression models for each combination of outcome, fumigant, time period, and buffer distance. We selected covariates a priori based on our previous studies of respiratory symptoms and respiratory function in this cohort. For logistic regression models of respiratory symptoms and asthma medication use, we included maternal smoking during pregnancy and signs of moderate or extensive mold noted at either home visit . We also included season of birth to control for other potential exposures that might play a causal role in respiratory disease , pollen , dryness , and mold. We defined the seasons of birth as follows: pollen , dry , mold based on measured pollen and mold counts during the years the children were born . In addition, we controlled for allergy using a proxy variable: runny nose without a cold in the previous 12 months reported at age 7. Because allergy could be on the causal pathway, we also re-ran all models without adjusting for allergy. Results were similar and therefore we only present models controlling for allergy. Additionally, for spirometry analyses only, we adjusted for the technician performing the test, and child’s age, sex and height. We included household food insecurity score during the previous 12 months , breastfeeding duration , and whether furry pets were in the home at the 7 year visit to control for other factors related to lung function.

We also adjusted for mean daily particulate matter concentrations with aerodynamic diameter ≤ 2.5 µm during the first 3 months of life and whether the home was located ≤150m from a highway in first year of life determined using GIS,blueberry pot size to control for air pollution exposures related to lung function. We calculated average PM2.5 concentration in the first 3 months of life using data from the Monterey Unified Air Pollution Control District air monitoring station. In all lung function models of postnatal fumigant use, we included prenatal use of that fumigant as a confounder. To test for non-linearity, we used generalized additive models with three-degrees of-freedom cubic spline functions including all the covariates included in the final lung function models. None of the digression from linearity tests were significant ; therefore, we expressed fumigant use on the continuous log10 scale in multi-variable linear regression models. Regression coefficients represent the mean change in lung function for each 10-fold increase in wind-weighted fumigant use. We conducted sensitivity analyses to verify the robustness and consistency of our findings. We included other estimates of pesticide exposure in our models that have been related to respiratory symptoms or lung function in previous analyses of the CHAMACOS cohort. Specifically, we included child urinary concentrations of dialkylphosphate metabolites , a non-specific biomarker of organophosphate pesticide exposure using the area under the curve calculated from samples collected at 6-months, 1, 2, 3.5 and 5 years of age . We also included agricultural sulfur use within 1-km of residences during the year prior to lung function measurement . We used similar methods as described above for fumigants to calculate wind-weighted sulfur use, except with a 1-km buffer and the proportion of time that the wind blew from each of eight directions during the previous year. The inclusion of these two pesticide exposures reduced our study population with complete data for respiratory symptoms and lung function . Previous studies have observed an increased risk of respiratory symptoms and asthma with higher levels of p, p’– dichlorodiphenyltrichloroethylene or p, p’-dichlorodiphenyldichloro-ethylene measured in cord blood . As a sensitivity analysis, we included log10- transformed lipid-adjusted concentrations of DDT and DDE measured in prenatal maternal blood samples . We also used Poisson regression to calculate adjusted risk ratios for respiratory symptoms and asthma medication use for comparison with the ORs estimated using logistic regression because ORs can overestimate risk in cohort studies . In additional analyses of spirometry outcomes, we also excluded those children who reported using any prescribed medication for asthma, wheezing, or tightness in the chest during the last 12 months to investigate whether medication use may have altered spirometry results. We ran models including only those children with at least two acceptable reproducible maneuvers . We ran all models excluding outliers identified with studentized residuals greater than three. We assessed whether asthma medication or child allergies modified the relationship between lung function and fumigant use by creating interaction terms and running stratified models. To assess potential selection bias due to loss to follow-up, we ran regression models that included stabilized inverse probability weights . We determined the weights using multiple logistic regression with inclusion as the outcome and independent demographic variables as the predictors. Data were analyzed with Stata and R . We set statistical significance at p<0.05 for all analyses, but since we evaluated many combinations of outcomes, fumigants, distances and time periods we assessed adjustment for multiple comparisons using the Benjamini-Hochberg false discovery rate at p<0.05 . Most mothers were born in Mexico , below age 30 at time of delivery , and married or living as married at the time of study enrollment . Nearly all mothers did not smoke during pregnancy. When cohort participants were 6 and 12 months old, most households showed signs of moderate or extensive mold at either visit. At age 7, based on maternal report, the majority of families was living below the Federal Poverty Level, 15.7% of cohort children experienced a runny nose without a cold within the past year, 16.3% displayed asthma symptoms, and 6.1% were currently taking asthma medication. Table 2 shows the distributions of wind-weighted fumigant use within 8 km of CHAMACOS residences during the prenatal and postnatal exposure periods. Methyl bromide and chloropicrin were the most heavily used fumigants during the prenatal period, with mean ± SD wind-adjusted use of 13,380 ± 10,437 and 8,665 ± 6,816 kg, respectively. Reflecting declines in methyl bromide use, the use of chloropicrin was greater than the use of methyl bromide during the postnatal period, with median values of 127,977 and 109,616 kg during the 7 years, respectively. When we examined correlations within each fumigant, use within 3, 5, and 8 km from the home was highly correlated for each fumigant . Fumigant use during the prenatal and postnatal periods was also highly correlated for methyl bromide and chloropicrin, but was not correlated for metam sodium use and was inversely correlated for 1,3-DCP use . We also examined correlations among fumigants and observed high correlations between prenatal methyl bromide and chloropicrin use and between prenatal metam sodium and 1,3-DCP use . There were negative correlations between prenatal methyl bromide and chloropicrin use with prenatal metam sodium and 1,3-DCP use .

While only including tillage treatments with residue incorporation establishes systems with similar residue input levels, it arguably poorly reflects farmers’ predominant practices in mixed crop-livestock farming systems – especially in sub-Saharan Africa and South Asia – in which residues tend to be exported from fields for feed, fuel, housing materials or other purposes . As such, the applicability of meta-analytical results to smallholder farming conditions in either sub-Saharan Africa and South Asia may be questioned. Given the large variation in crop management practices that result from differences in the scale of farming operations, the nature of farm enterprises and cropping patterns in different farming systems, one may therefore ask: Does the presentation of average results from ‘global meta-analyses’ in agronomy make sense? Our case studies show the ways in which the practical value of meta-analyses to provide comprehensive evidence on topics of development relevance is undermined by the social construction of treatment categories that may be decoupled from the conditions faced by farmers themselves.Most meta-analyses reviewed in this study used primary data from small-plot agronomic trials. The problems associated with extrapolating results from small plot experiments to whole fields, cropping systems and farming systems have however been widely acknowledged . These problems also affect meta-analysis. Many manage multiple separate fields – each of which may be environmentally heterogeneous – across landscapes. Farmers may therefore not be able to rigorously and evenly implement recommended crop management practices across fields and farm units with the same precision as researchers managing small-plot trials. This therefore casts some doubt about the usefulness of data from small-plot trials. Kravchenko et al. ,for example,blackberries in containers demonstrated that yield results from small-plot OA experiments were not always consistent with field-scale measurements of the same treatments.

Caution is therefore needed when extrapolating results from small-plot research to the field, farming system, landscape and global levels. These problems are most apparent in the OA case study. Badgley et al. , for example, extrapolated OA yield responses from plot studies to the global agricultural system, concluding that OA could feed the world’s population with nitrogen requirements supplied in situ by legumes, without expanding the footprint of agriculture. Connor conversely pointed out that soil moisture deficits would likely constrain the productivity of legumes in arid environments. He also noted that rotations with legumes may also not be feasible where legumes are less profitable or important than other crops for income generation and food production. Assessing productivity on a yield per unit of time basis, rather than yield alone, may therefore be an appropriate alternative in such comparisons . Leifeld also referenced landscape-scale considerations when contesting data presented by Ponisio et al. . He contended that OA is unable to cope with high-fecundity and rapidly dispersing pests, which could result yield losses more severe than observed in isolated, small-plot experiments. Leifeld also evoked ‘Borlaug hypothesis’ arguments that low-yielding farming systems may require the conversion of natural ecosystems to meet expanding food demand, thereby negatively affecting biodiversity. Ponisio and Kremen countered with evidence of the positive effects of organic and ecologically managed farmland on pest suppression at the landscape scale. They also highlighted the study of Meyfroidt et al. , who showed that higher yields and profitability can also drive agricultural expansion and deforestation under conventional practices. Considering the complexity of these problems, Brandt et al.proposed that bias could be reduced and science quality increased if researchers using meta analysis make their research protocols and intended methods publically available, for example, through online posting or journal publication, prior to undertaking meta-analysis. ‘Pre-registration’ of planned studies may be a logical suggestion , though it implies serious changes in research practice and re-thinking of how journals accept papers and conduct peer-review. This proposition has therefore not yet been widely applied in agronomy or other disciplines.

While there is no easy answer to how to rectify this conundrum, our review presents and important step in challenging underlying assumptions that meta-analysis can provide definitive and unifying conclusions as proposed by Garg et al. , Borenstein et al. , Rosenthal and Schisterman and Fisher .Agricultural expansion is the main cause of tropical deforestation , highlighting the trade offs among ecosystem services such as food production, carbon storage, and biodiversity preservation inherent in land cover change . Expansion of intensive agricultural production in southern Amazonia, led by the development of specific crop varieties for tropical climates and international market demand , contributed one third of the growth in Brazil’s soybean output during 1996–2005 . The introduction of cropland agriculture in forested regions of Amazonia also changed the nature of deforestation activities; forest clearings for mechanized crop production are larger, on an average, than clearings for pasture, and the forest conversion process is often completed in o1 year . How this changing deforestation dynamic alters fire use and carbon emissions from deforestation in Amazonia is germane to studies of future land cover change , carbon accounting in tropical ecosystems , and efforts to reduce emissions from tropical deforestation . Fires for land clearing and management in Amazonia are a large anthropogenic source of carbon emissions to the atmosphere . Deforestation fires largely determine net carbon losses , because fuel loads for Amazon deforestation fires can exceed 200 Mg C ha
1 . Reductions in forest biomass from selective logging before deforestation are small, averaging o10 Mg C ha
1 . In contrast, typical grass biomass for Cerrado or pasture rarely exceeds 10 Mg C ha
1 and is rapidly recovered during the subsequent wet season . Yet, the fraction of all fire activity associated with deforestation and combustion completeness of the deforestation process remain poorly quantified . Satellite fire detections have provided a general indication of spatial and temporal variation in fire activity across Amazonia for several decades . However, specific information regarding fire type or fire size can be difficult to estimate directly from active fire detections because satellites capture a snapshot of fire energy rather than a time-integrated measure of fire activity .

Overlaying active fire detections on land cover maps provides a second approach to classify fire type. Evaluating fire detections over large regions of homogenous land cover can be instructive , but geolocation errors and spurious fire detections may complicate these comparisons, especially in regions of active land cover change and high fire activity such as Amazonia . Finally, postfire detection of burn-scarred vegetation is the most data-intensive method to quantify carbon emissions from fires. Two recent approaches to map burn scars with Moderate Resolution Imaging Spectroradiometer data show great promise for identifying large-scale fires , yet neither algorithm is capable of identifying multiple burning events in the same ground location typical of deforestation activity in Amazonia. Deriving patterns of fire type, duration and intensity of fire use, and combustion completeness directly from satellite fire detections provides an effi- cient alternative to more data and labor-intensive methods to estimate carbon emissions from land cover change. We assess the contribution of deforestation to fire activity in Amazonia based on the intensity of fire use during the forest conversion process,blackberry container measured as the local frequency of MODIS active fire detections. High confidence fire detections on 2 or more days in the same dry season are possible in areas of active deforestation, where trunks, branches, and other woody fuels can be piled and burned many times. Low-frequency fire detections are typical of fires in Cerrado woodland savannas and for agricultural maintenance, because grass and crop residues are fully consumed by a single fire. The frequency of fires at the same location, or fire persistence, has been used previously to assess Amazon forest fire severity , adjust burned area estimates in tropical forest ecosystems , and scale combustion completeness estimates in a coarse-resolution fire emission model . We build on these approaches to characterize fire activity at multiple scales. First, we compare the frequency of satellite fire detections over recently deforested areas with that over other land cover types. We then assess regional trends in the contribution of high frequency fires typical of deforestation activity to the total satellite-based fire detections for Amazonia during 2003–2007. Finally, we compare temporal patterns of fire usage among individual deforested areas with different post clearing land uses, based on a recent work to separate pasture and cropland following forest conversion in the Brazilian state of Mato Grosso with vegetation phenology data . The goals of this research are to test whether fire frequency distinguishes between deforestation fires and other fire types and characterize fire frequency as a function of post clearing land use to enable direct interpretation of MODIS active fire data for relevant information on carbon emissions.We analyzed active fire detections from the MODIS sensors aboard the Terra and Aqua satellite platforms to determine spatial and temporal patterns in satellite fire detections from deforestation in Amazonia during this period.

Combined, the MODIS sensors provide two daytime and two night-time observations of fire activity. Figure 1 shows the location of the study area and administrative boundaries of the nine countries that contain portions of the Amazon Basin. For data from 2002–2006, the date and center location of each MODIS active fire detection, satellite , time of overpass, 4 micron brightness temperature , and confidence score were extracted from the Collection 4 MODIS Thermal Anomalies/Fire 5-min swath product at 1-km spatial resolution . Beginning in 2007, MODIS products were transitioned to Collection 5 algorithms. Data for January 1–November 1, 2007 were provided by the Fire Information for Resource Management System at the University of Maryland, College Park based on the Collection 5 processing code. Seasonal differences in fire activity north and south of the equator related to precipitation were captured using different annual calculations. North of the equator, the fire year was July–June; south of the equator, the fire year was January–December. Our analysis considered a high-confidence subset of all MODIS fire detections to reduce the influence of false fire detections over small forest clearings in Amazonia . For daytime fires, only those 1-km fire pixels having 4330 K brightness temperature in the 4-mm channel were considered. This threshold is set based on a recent work to identify true and false MODIS fire detections with coincident high-resolution satellite imagery , comparisons with field data , and evidence of unrealistic MODIS fire detections over small historic forest clearings in Mato Grosso state with 420 days of fire detections per year in 3 or more consecutive years, none of which exceeded 330 K during the day. Daytime fire detections 4330 K correspond toa MOD14/MYD14 product confidence score of approximately 80/100. The subset of high-confidence fires includes all night-time fire detections, regardless of brightness temperature. Differential surface heating between forested and cleared areas during daylight hours that may contribute to false detections should dissipate by the 22:30 or 01:30 hours local time overpasses for Terra and Aqua, respectively. Subsequent references to MODIS fire detections refer only to the high-confidence subset of all 1-km fire pixels described earlier.The simple method we propose for separating deforestation and agricultural maintenance fires is based on evidence for repeated burning at the same ground locations. The spatial resolution of our analysis is de- fined by the orbital and sensor specifications of the MODIS sensors and the 1-km resolution bands used for fire detection. The geolocation of MODIS products is highly accurate, and surface location errors are generally o70 m . However, due to the orbital characteristics of the Terra and Aqua satellite platforms, the ground locations of each 1-km pixel are not fixed. We analyzed three static fire sources from gas , mining , and steel production in South America to identify the spatial envelope for MODIS active fire detections referencing the same ground location. Over 98% of the high-confidence 2004 MODIS active fire detections from Terra and Aqua for these static sources were within 1 km of the ground location of these facilities. Therefore, we used this empirically derived search radius to identify repeated burning of forest vegetation during the conversion process. High-frequency fire activity was defined as fire detections on two or more days within a 1-km radius during the same fire year.

In this study we have shown that feed backs are significant in both directions and have also shown that money and exchange rate shocks affect prices. Thus, any reduction in government expenditure in agriculture affects the path by which price shocks feedback on money and the exchange rate. From a policy perspective, this is very important, since it implies that any change in government support of the farm sector should be evaluated from an integrated market point of view. This more integrated or global perspective is needed because expenditures and budget deficits, monetary, exchange rate, and farm policies are significantly related and their interactions far too strong to be neglected. Triclosan is a non-agricultural pesticide widely used as an antibacterial agent in common medical, household and personal care products in the range of 0.1%–0.3% . The use of TCS has increased worldwide over the last 30 years . The broad household use of products containing TCS results in the discharge of TCS to municipal wastewater treatment plants, and it has been detected in effluents and sewage sludge in Europe and the United States .The mode of action of TCS on bacteria is through inhibition of fatty acid synthesis by targeting enzymes specific to bacteria . Since fatty acid biosynthesis is a fundamental process for cell growth and function; the ability to inhibit this makes TCS a particularly effective antimicrobial compound. 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,growing raspberries in containers 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 subsamples 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 . Recoveries of deuterated TCC ranged from 63–115% during extraction and analysis.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 ,large plastic pots for plants 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.

The reduced bio-degradation in the SB microcosms may have resulted from the ~40% higher carbon content in the SB microcosms, which would be expected to increase the soil-water distribution coefficient by a comparable amount. Reduced TCS concentration in soil pore water would be expected to slow bio-transformation, potentially in a nonlinear fashion. Another possible contributor to the slower degradation of TCS in SB is the greater availability of alternative, likely more easily degradable, carbon sources in SB than soil microcosms, reducing the use of TCS as a substrate. Selective bio-degradation of one carbon source, and inhibition of the degradation of other chemicals also present, has been observed for mixtures of chemicals in aquifers . To assess which of these mechanisms was controlling, measured Freundlich isotherm parameters for TCS adsorption on bio-solid amended Yolo soil were used to calculate equilibrium pore water concentrations in the soil and SB microcosms over the course of the experiment. Using estimated pore water concentrations of moistened soil and SB samples, instead of total soil concentrations to perform half-life calculations, resulted in modest increases in the rate constants and decreases in half-lives of soil samples and did not narrow the significant gap between half lives in soil and SB . This suggests that the primary reason for the slower degradation of TCS in bio-solid amended soils is the increase in more labile forms of carbon because organic material is highly porous and has a lower particle density. Previous research shows that TCS biodegrades within weeks to months in aerobic soils , although Chenxi et al., found no TCS degradation in bio-solids stored under aerobic or anaerobic conditions, Kinney et al., observed a 40% decrease in TCS concentrations over a 4-month period following an agricultural bio-solids application. Because the slopes of the lines in Fig. 1 are not significantly different as a function of spiking level , the slopes were averaged for each treatment type, yielding apparent first order rate constants of 0.093±4% d−1 for soil samples and 0.024±41% d−1 for SB samples where the percent error represents the relative percent difference between the 10 mg/kg and 50 mg/kg degradation curves. These apparent rate constants translate to half-life estimates of 7.5 d in soils and 29 d in bio-solid amended soil. The estimated half-life of TCS in soil is within the range of previously reported half-lives of from 2.5 to 58 d in soil . The half-life determined here in bio-solid amended soils is lower than the one available literature value of 107.4 d . The microbial biomass decreased in the TCS spiked samples after 7 or 30 days of incubation in comparison with the unspiked controls, for both soil and SB, and the decline was statistically significant at 50 mg/kg . Although exposure to TCS caused declines in biomass in both soil and SB microcosms, the total microbial biomass was two times higher in SB than soil probably due to the increased availability of nutrients and/or possibly due to addition of bio-solid associated microorganisms in the latter . The total number of PLFAs ranged from 42–47 in soil and 48–59 in SB . No significant change in numbers of PLFAs was evident with increasing dosage of TCS for any incubation time suggesting that TCS addition did not adversely affect microbial diversity. Microbes respond to various stresses by modifying cell membranes, for example by transforming the cis double bond of 16:1ω7c to cy17:0, which is more stable and not easily metabolized by the bacteria, reducing the impact of environmental stressors . Consequently, the ratio of cy17 to its precursor has been employed as an indicator of microbial stress that has been associated with slow growth of microorganisms . Increases in this stress biomarker were observed in both soil and SB samples as TCS concentrations increased , suggesting that TCS has a negative effect on the growth of soil microorganisms. The overall level of cy17 to its precursor is lower in SB than soil samples, suggesting that nutrients contributed by the bio-solids reduce stress on the microbial community. Our study agreed with a previous study showed that carbon added to soil led to a reduction in the cy17 fatty acid TCS additions, however, increased the stress marker compared with that detected in the corresponding samples with no added TCS. A broader implication of this result is that presence of bio-solids may mitigate the toxic effects of chemicals in soil, or chemicals added in combination with bio-solids, on soil microbial communities. Groupings of microbial communities, based on CCA analysis of their composition as estimated by PLFA, were distinguished primarily by whether they were in soil or SB treatments and secondarily by time since spiking .

The other aspect of private sector involvement is perhaps more mixed in its consequences, compared to individual farmers’ efforts. Indian agriculture has long been heavily influenced by powerful intermediaries, who may combine participation in credit and input, and even output and land markets, to earn economic rents associated with market power, in a phenomenon wellstudied as interlinkage . Market intermediaries and other private actors in the agricultural supply chain certainly provide essential products and services for the success of Punjab’s present agricultural system, but it is not clear that their incentives for enabling innovation are aligned with maximizing social welfare, just as, with imperfect competition, static resource allocation may not satisfy that optimality property. Given the foregoing discussion, as well as the issues highlighted in previous sections, it is reasonable to suggest that beneficial innovation in Punjab agriculture will not occur solely through the private sector. At an abstract level, the problems of asymmetric information, externalities, the public good nature of innovations and imperfect competition in various markets along the agricultural value chain all point towards some public sector involvement in facilitating greater innovation, especially that which incorporates crop diversification. It is arguably the case that the state government can make targeted interventions that provide effective nudges towards innovation, as well as adoption and diffusion of innovations, even in the face of the severe constraints imposed by the state’s own fiscal situation, and the conduct of national food procurement policy. Some of the barriers to innovation have to be overcome by relatively large financial investments in physical infrastructure, but the state government can catalyze the private sector to undertake these investments by improving the ease of doing business in the state. The public sector’s focus can and should be on improving the knowledge available to farmers, finding ways to overcome their switching costs, and providing them with better insurance as they move towards activities that involve greater risk and uncertainty.Myoblast, myocytes, and fibroblasts are cells of greatest interest for the field of cellular agriculture. For texture and taste, plastic pots for planting adipocytes may be used and grown either separately or co-cultured with muscle cells. The choice of animal will also have an effect on the final product and production process because cells from different animals will have different growth characteristics, morphology, and product qualities.

The majority of these cell lines are adherent, meaning they require a suitable substrate to grow. Ideally, cells may be grown in suspension culture , bringing cellular agriculture in line with typical pharmaceutical practice such as CHO cells. Micro-carriers may also be used to increase the surface area of the total surface. Proliferating many cells is not the only consideration in cellular agriculture. Stem cells differentiate into more complex tissue structures depending on time and environmental conditions, which is critical in forming final products that consumers are willing to purchase. For example, C2C12 immortalized murine skeletal muscle cells differentiate into myotubes at high density and when exposed to DMEM + 2% horse serum . However, because cell differentiation often precludes further proliferation, cells must be periodically pass aged to provide more physical space for growth. This is typically done by detaching the cells from the substrate using trypsin enzyme and physically placing the cells onto additional surface area. Fundamental techniques in cell culture can be found in and a general overview of mammalian cell culture for bio-production uses can be found in . Figure 1.1b shows a high level overview of the cellular agriculture process. Throughout this entire process, media is used to support cells by providing them with nutrients, signal molecules, and an environment for growth. We are focused on reducing the cost of the media while supporting cell proliferation. This is because the media has been identified as the largest contribution to cost . The main considerations for the design of cell culture media in cellular agriculture are the media must be inexpensive, it must be free of animal products, and it must support long-term proliferation of relevant cell lines and final differentiation into relevant products. The most basic part of a cell culture medium is the basal component, which supplies the amino acids, carbon sources, vitamins, salts, and other fundamental building blocks to cell growth. The optimal pH of cell culture media is around 7.2 – 7.4 which is achieved through buffering with the sodium bicarbonate – CO2 or organic buffers like HEPES. Temperature should be maintained at around 37◦C at high humidity to prevent evaporation of media. Osmolarity around 260 – 320 mOsm/kg is maintained by the concentration of inorganic ions salts such as NaCl as well as hormones and other buffers. Inorganic salts also supply potassium, sodium, and calcium to regulate cell membrane potential which is critical for nutrient transport and signalling.

Trace metals such as iron, zinc, copper, and selenium are also found in basal media for a variety of tasks like enzyme function. Vitamins, particularly B and C, are found in many basal formulations to increase cell growth because they cannot be made by the cells themselves. Nitrogen sources, such as essential and non-essential amino acids, are the building blocks of proteins so are critical to cell growth and survival. Glutamine in particular can be used to form other amino acids and is critical for cell growth. It is also unstable in water so is typically supplemented into media as L-alanyl-L-glutamine dipeptide . Carbon sources, primarily glucose and pyruvate, are essential as they are linked to metabolism through glycolysis and the pentose-phosphate pathway. Fatty acids like lipoic and linoleic acid act as energy storage, precursor molecules, and structural elements of membranes and are sometimes supplied through a basal medium like Ham’s F12. Having a sufficient concentration of all of these components is required for proliferating mammalian cells across multiple passages as per above. Having a robust basal media is a necessary but not sufficient condition for long-term cell proliferation and differentiation. Serum is a critical aspect of cell culture because it provides a mix of proteins, amino acids, vitamins, minerals, buffers and shear protectors . Serum stimulates proliferation and differentiation, transport, attachment to and spreading across substrates, and detoxification. Serum has large lot-to-lot variability, zoonotic viruses and contamination , as well as the ethical issues associated with collecting serum from animals. Therefore, while it often simplifies cell growth and differentiation, it is critical to remove serum as per point . Supplementation with growth factors like FGF2, TGFβ1, TNFα, IGF1, or HGF is a common way to induce growth of mammalian muscle cells without the use of serum. Transferrin, another protein found in serum, fulfills a transport role for iron into the cell membrane. PDGF and EGF are polypeptide growth factors that initiate cell proliferation. Such components enhance cell growth but are expensive and comprise the vast majority of the cost of theoretical cellular agriculture processes. Much work has been done on developing serum-free media. The E8 / B8 medium for human induced pluri potent stem cells is based on Dulbecco’s Modified Eagle Medium / F12 supplemented with insulin, transferrin, FGF2, TGFβ1, ascorbic acid, and sodium selenite. Beefy-9 by is similar to E8 but with additional albumin optimized for primary bovine satellite cells. The approach we will take in this dissertation is to use prior knowledge of biological processes to construct a list of potential media components, and use design-of-experiments methods to optimize component concentrations based on cell proliferation. This will be particularly useful for cellular agriculture because by developing and using these statistical tools, as we will see in the next section, DOEs will help develop media quickly and efficiently.

One of the most difficult aspects of this work is measuring the quality of media. Viable cells must be counted after a period of time over which the scientist believes the medium will have an effect, which changes depending on cell type, media components, cell density, ECM, pH, temperature, osmolarity, and reactor configuration. If cells grow by adhering to a substrate, then sub-culturing / passaging may play a role on the health of a cell population,drainage for plants in pots so discounting this effect may have deleterious effects on media design quality. Counting using traditional methods like a hemocytometer or more advanced automatic cell counters using trypan blue exclusion are labor-intensive and prone to error. Cell growth / viability assays are chemical indicators that correlate with viable cell number such as metabolism or DNA / nuclei count and can also be used to quantify the effect of media on cells. In chapter 5 we conducted many experiments with different assays and show the inter-assay correlations in Figure 1.3. Notice no assay is perfectly correlated with any other assay because they are collected with different methodologies and fundamentally measure different physical phenomena. For example, Alamar Blue measures the activity of the metabolism in the population of cells, so optimizing a media based on this metric might end up simply increasing the metabolic activity of the cells rather than their overall number. As some of these measurements can be destructive / toxic to the cells , continuous measurements to collect data on the change in growth can be tedious. Collecting high-quality growth curves over time may be accomplished using image segmentation and automatic counting techniques. Using fluorescent stained cells and images, segmentation can be done using algorithms like those discussed. Cells may even be classified based on their morphology dynamically if enough training data is collected to create a generalizable machine learning model.The primary means by which this dissertation will improve cell culture media is through the application of various experimental optimization methods, often called design-of-experiments . The purpose of DOEs are to determine the best set of conditions xto optimize some output yby sampling a process for sets of conditions in an optimal manner. If an experiment is time / resource inefficient, then optimizing the conditions of a system may prove tedious. For example, doing experiments at the lower and upper bounds of a 30-dimensional medium like DMEM requires 2 30 ≈ 109 experiments. This militates for methods that can optimize experimental conditions and explore the design space in as few experiments as possible. DOEs where samples are located throughout the design space to maximize their spread and diversity according to some distribution are called space-filling designs. The most popular method is the Latin hypercube , which are particularly useful for initializing training data for models and for sensitivity analysis. Maximin designs, where some minimum distance metric is maximized for a set of experiments, can also allow for diversity in samples, with the disadvantage being that in high dimensional systems the designs tend to be pushed to the upper and lower bounds. Thus, we may prefer a Latin hypercube design for culture media optimization because media design spaces may be >30 factors large. Uniform random samples, Sobol sequences, and maximum entropy filling designs, all with varying degrees of ease-of-implementation and space-filling properties, also may be used. It cannot be known a priori how many sampling points are needed to successfully model and optimize a design space because it is dependent on the number of components in the media system, degree of non-linearity, and amount of noise expected in the response. Because of these limitations, DOE methods that sequentially sample the design space have gained traction, which will be talked about in the next section. A more data-efficient DOE is to split up individual designs into sequences and use old experiments to inform the new experiments in a campaign. One sequential approach is to use derivative-free optimizers where only function evaluations y are used to sample new designs x. DFOs are popular because they are easy to implement and understand, as they do not require gradients. They are also useful for global optimization problems because they usually have mechanisms to explore the design space to avoid getting stuck in local optima. The genetic algorithm is a common DFO where a selection and mutation operator is used to find more fit combinations of genes . In Figure 1.7, notice the GA was able to locate the optimal region of both problems regardless of the degree of multi-modality.

Although we did not directly compare skin equivalents without adipose to AVHSEs here or directly compare culture time points, we have not observed any obvious changes in epidermal coverage compared to our previous work in vascularized human skin equivalents that do not contain a subcutaneous adipose compartment. While the model is customizable to study the effects of intrinsic and extrinsic aging factors, as a test case we have demonstrated suitability for studies in UVA photo aging due to the strong literature base of both in vitro and in vivo studies available for comparison. Finally, we demonstrated the accessibility of the model for both molecular and morphological studies . A key aspect of any HSE model is a differentiated and stratified epidermis. Here, N/TERT-1 keratinocytes were used to generate skin epidermis as previously completed. Importantly, N/TERTs are a suitable and robust substitute to primary keratinocytes which have disadvantages including limited supply, limited in vitro passage capabilities, and donor variability. HSEs generated with N/TERT keratinocytes demonstrate comparable tissue morphology, appropriate epidermal protein expression, and similar stratum corneum permeability when compared to HSEs generated with primary keratinocytes. Similar to prior models, we demonstrate AVHSEs appropriately model the skin epidermis with correct localization of involucrin , and cytokeratin , and nuclei localized in the lower stratified layers . Further, volumetric imaging and automated analysis allows for epidermal thickness to be robustly calculated. AVHSE present with median epidermal thicknesses within 90-100 µm, similar to values in both prior in vitro studies 100-200 µm and in vivo optical coherence tomography imaging of adult skin 59±6.4 to 77.5±10 µm 194. Consistent with prior in vitro and in vivo results showing UVA wavelengths predominantly impact dermal rather that epidermal layers, UVA photoaging resulted in no observable changes in epidermal thickness or expression of differentiation markers in AVHSE . In the dermis and hypodermis,square plastic plant pot skin is highly vascularized with cutaneous microcirculation playing important roles in thermal regulation and immune function.

Many prior HSE models have not included a vascular component4; however, there is increasing recognition of its importance. In the present work, we used collagen IV as a marker of the vascular basement membrane, enabling the automated segmentation and mapping of a vascular network within AVHSEs. The vascular VF of AVHSEs is lower than in vivo dermis , but prior work has shown this is tunable by using different cell seeding conditions . Optimizing the VF may be more involved in the AVHSE, since the ratio of adipose and vascular cells has been shown to be important in regulating tissue morphology. Thus, ratio of adipose and vascular cells would need to be optimized again for new cell and collagen densities. Adipose tissue is densely vascularized and the ability of adipocytes to generate lipid droplets and adipokines in the presence of endothelial cells is important to replicate the in vivo environment. Previous work has shown that in co-culture of endothelial cells and mature adipocytes can lead to dedifferentiation of mature adipocytes, but in homeostatic cultures ECs and adipocyte crosstalk is important. Through soluble factor release, ECs regulate lipolysis and lipogenesis and adipocytes regulate vasodilation and contraction. Secretion of adipokines by adipocytes aids vascular formation and adipose tissue stability. In prior work, Hammel & Bellas demonstrated that 1:1 is the optimal ratio for vessel network within 3D adipose, and we matched the 1:1 cell ratio in the present work. Quantification of vessel diameter in the Hammel & Bellas study shows that a 1:1 ratio of adipocytes to endothelial cells gives an average vessel diameter of ~10 µm235, our work supports this finding with a median inner vessel diameter of ~6 µm. Importantly, these data are within the range of human cutaneous microvascular of the papillary dermis . We did not observe morphological changes of VF and diameter within the vasculature due to photoaging. This is not entirely unexpected, as UVA exposure and its effects on vasculature are still poorly understood. While it is established that chronic UVA exposure can contribute to vascular breakdown, the duration of our studies may be too short to see this effect in diameter and VF . However, photoaging did induce an increase in diffusion length . Rk is a measure of the 90th percentile of distance from the vascular network and so a higher value corresponds to less coverage; values presented here match previous studies of vascularized collagen.

Rk of the vascular network for both control and photoaged samples was within the range of 51 – 128 µm which is importantly below the 200 µm diffusion limit. Upon photoaging, AVHSEs did demonstrate a significant increase in Rk compared to controls.In vascularized tissue, a high VF and low Rk is preferable and the Rk increase demonstrated indicates a loss in vascular coverage in photoaged AVHSEs. These findings conflict with studies of acute UV exposure in skin, which show stimulation of angiogenesis. It has been proposed that UV light exposure may improve psoriasis by normalizing disrupted capillary loops through upregulation of VEGF by keratinocytes. The AVHSE model could be used to more thoroughly test the effects of UV light and other molecular mechanisms it induces in future studies. The vascular networks extend from adipose to the epidermal-dermal junction , consistent with previous literature and to normal human skin histology/stereography. Further, we observed vasculature colocalized with the lipid droplet BODIPY staining , indicating recruitment of the vascular cells to the hypodermis. Importantly, the vascular networks in prior studies and the present AVHSE are self-assembled. While there are advantages to self-assembly, especially the simplicity of the method, it is important to note the limitations. Cutaneous microcirculation in vivo has a particular anatomical arrangement with two horizontal plexus planes, one deep into the tissue in the subcutaneous fat region and one just under the dermal-epidermal junction. Between these two planes are connecting vessels running along the apicobasal axis that both supply dermal tissues with nutrients and are an important part of thermoregulation. Although the AVHSEs presented here are fully vascularized up to the epidermal junction they do not recapitulate this organization. While not covered in this work, future studies could incorporate layers of patterned or semi-patterned vasculature to more closely match the dermal organization, depending on the needs of the researcher. In contrast to the epidermal and some vascular components, photoaging impacted the hypodermis. Volumetric imaging of BODIPY, which stains lipid droplets, was used to identify the adipose. While small reductions in the morphological parameters were observed, they were not significant, suggesting there was not large-scale necrosis or loss of fat mass. However, there was a significant decrease in the intensity of BODIPY staining, indicating decreased lipid levels. This is consistent with photoaging of excised human skin showing that UV exposure decreases lipid synthesis in subcutaneous fat tissue228. We further collected culture supernatant and tested for the presence of adiponectin, IL6, and MMP-1. The data collected through ELISA show that this AVHSE model secretes both adiponectin and IL6, which are also present innative skin and both considered important adipokines. Elevated serum adiponectin levels are linked to anti-inflammatory effects in humans and centenarians have elevated levels of adiponectin.Decreased adiponectin has previously been associated with photoaging in both excised human skin that was sun-exposed compared to protected skin and in protected skin that was exposed to acute UV irradiation. Conversely,25 liter square pot IL6 is a key factor in acute inflammation in skin, and has been shown to regulate subcutaneous fat function. In prior studies of photoaging, IL6 has demonstrated an increase after UVA irradiation in monolayer fibroblast cultures and excised human skin. IL-6 is released after UV irradiation and has been linked to decreased expression of adipokine receptors and mRNA associated with lipid synthesis, decreases in lipid droplet accumulation, and enhanced biosynthesis of MMP1. However, after one week of photoaging we did not observe an increase in IL-6 or MMP-1 via ELISA. The absence of changes in IL-6 and MMP-1 expression but decreases in lipid accumulation and adiponectin are not expected results but they could be due to methodology differences in UVA exposure. We determined our UVA dose and exposure based on literature values. The dose used here was 0.45 ± 0.15 mW/cm2with exposure for 2 hours daily for 7 d which roughly converts to 3.24 J/cm2 per day and a total of 22.68 J/cm2.

Many studies do not report exposure time and/or present ambiguous time points. This, compounded with the practice of using doses based on sample pigmentation threshold and broad definition of UVA wavelengths is likely contributing to the discrepancy in IL-6 and MMP-1 expressions. Previous work has shown that neutralizing anti-IL-6 antibody prevents UV induced decrease of important fat associated mRNA and that IL-6 secreted from keratinocytes and fibroblasts following UV irradiation inhibits lipid synthesis. From previous work, it is clear that IL-6 secretion is upregulated by UVA and presence contributes to negative adipose function but more investigation is necessary to understand what UVA doses and exposures induce IL-6 and further at what time points after photoaging are these expressions quantifiable. In this model, it is possible that there were increases in IL-6 that contributed to adiponectin decreases in photoaged samples, these trends may have been caught with different media collection time points. Alternatively, other analysis of inflammatory responses and adipokines may show generalized inflammatory responses identified in literature and further, changes in dose/exposure or continued photo aging may mimic the previously shown effects. There are notable limitations of the AVHSE model presented. Although we have presented a skin model that is closer to both anatomy and biology of human skin in comparison to past HSEs, we have not modeled skin fully through inclusion of other features of in vivo skin such as immune and nerve components. Including a functional immune system is important in understanding autoimmune diseases, cancer, wound healing, and decline of immune function in aged skin. Additionally, neuronal cell inclusion will allow for modeling of sensory processes necessary for grafting and modeling of skin disorders associated with nerve dysregulation. Further, while the cell lines used in this study were chosen for their low cost and accessibility, primary cells or populations differentiated from induced pluripotent stem cells would more closely match the physiology in vivo. While changing cell populations would likely require some adjustment to the culture system, we have previously demonstrated that cell types can be replaced with minimal changes. We model epidermis, dermis, and hypodermis here, but we do not model the depth that is present in thick skin tissue; to mimic thicker skin the model would need to be taller. As nutrient and waste diffusion in tissues is limited to ~200 µm, thick tissues will likely require perfusion to maintain throughout culture. Vasculature in thicker skin has higher diameters, especially in the lower dermis and hypodermis, these can be up to 50 µm. Finally, for ease of use, initial collagen density in the AVHSE model is 3 mg/mL, much lower than in vivo densities. Decline of collagen density is an important aspect of skin aging, correlating with skin elasticity and wound healing. Varying collagen density influences vascular self-assembly, but higher collagen densities are possible through a variety of techniques, including dense collagen extractions146, and compression of the collagen culture. By incorporating these tools, AVHSE could be modified to more closely represent the in vivo dermal matrix. Further, the AVHSE method was demonstrated with low serum requirements; but serum was used for initial growth and the cultures are maintained for weeks without serum. Serum replacements during the growth phase could potentially provide a chemically defined xeno-free culture condition in beginning culture stages for greater reproducibility and bio-compatibility. The presented AVHSE model provides unique capabilities compared to cell culture, ex vivo, and animal models. Excised human skin appropriately models penetration of dermatological products but there is limited supply and high donor variability; replacing excised human skin with animal models or commercially available skin equivalents is not the best course of action because of the differences such as varying penetration rates, lipid composition, lipid content, morphological appearance, healing rates, and costs; and limitations of customization. AVHSE can be cultured using routinely available cell populations, are cost effective, and are customizable for specific research questions. Further, the model is accessible for live imaging, volumetric imaging, and molecular studies, enabling a wide range of quantitative studies.

We report three analyses: trends in the time slice characterizing the baseline time period ; the calibration and validation of basin discharge by comparing post‐processed runoff and recharge measures to derive discharge, and comparing that value to streamgage measurements; and a comparison of the historical and future conditions for BCM variables—precipitation, potential evapotranspiration, runoff, recharge, and climatic water deficit. We present the map‐based assessments, using the difference in magnitude for each variable; the number of standard deviations by which projected future conditions will differ from the standard deviation of baseline conditions; and the geographic variations across California of both historical and future projections. Temperature values are available, but for brevity, and because temperature has previously been more widely reported, this paper focuses on hydrological components.The process used to estimate hydrologic impacts of climate change at fine scales involved down scaling climate data for model input.The BCM then generated outputs as a series of hydrologic and associated variables. This section discusses: precipitation, air temperature, PET, snow pack, runoff, recharge, and climatic water deficit.During the 30‐year baseline period of 1971–2000, precipitation generally increased, with the exception of the deserts and eastern Sierra Nevada . Largest percentage increases are in the Great Valley, Central Western California, and Sierra Nevada. Both minimum and maximum air temperatures increased for all ecoregions, ranging from 0.5°C to 1.6°C for minimum air temperature and much less of an increase for maximum air temperature . Potential evapotranspiration increased throughout the state by about 3 percent. Recharge decreased by up to 24 percent in southwestern California, and by 11 percent in northwestern California, while all other ecoregions increased in recharge. Recharge in the Mojave Desert increased by 51 percent ,pot with drainage holes and in the Modoc Plateau by 42 percent . The change in climate over the 30‐year period is exemplified by the changes in snow pack in California, which integrates effects of precipitation and air temperature on the dominant water resource in California for water supply.

The snow pack in this region is the warmest in the western United States and is the most sensitive to small changes in air temperature. This is illustrated by the change in April 1 snow pack , where snow pack has diminished the most in extent in the northern portions of the state; whereas, the highest elevation snow pack in the southern Sierra Nevada mountains and Mount Shasta have actually increased in some locations. However, the dominant loss of April 1 snow pack results in less runoff to extend surface water resources throughout the summer season. This situation has implications for recharge and climatic water deficit as well. Corresponding to increases in precipitation, runoff increased over the baseline period in most locations in the state, notably the northern Sierra Nevada Mountains and parts of the Trinity Mountains in the northwestern ecoregion . Some declines are noted in the northwest, where the smallest change in precipitation occurred. Decreases in recharge are notable in the northwest portions of the state, with moderate decreases in the Sierra Nevada foothills and southern California mountains . Generally locations with little to no recharge, such as areas with deep soils or arid climate, also had little to no change in recharge indicated. Detailed views of basins in the Russian River watershed and Santa Cruz mountains are shown in Flint and Flint , illustrating the dominance of runoff in the Russian River watershed, where water supply relies heavily on reservoirs, in contrast to the reliance on groundwater resources and recharge in the Santa Cruz mountains. Increases in runoff in snow‐dominated regions, due to warming air temperatures, diminishes recharge, which is more likely to occur during the slow snowmelt season. This is confirmed for the northwestern ecoregion, where the Trinity Alps decreased in snow pack, and shows small increases for the Sierra Nevada, in contrast to other regions .Figure 9a shows the average annual climatic water deficit for 1971–2000. There is high climatic water deficit in the southern Central Valley and Mojave and Sonoran Deserts, and low climatic water deficit in the north coast and Sierra Nevada. Climatic water deficit declined over the baseline period in the central and northwestern California ecoregions and the Great Valley, while in all other regions, despite the increases in precipitation, climatic water deficit increased . This variable integrates energy loading and moisture availability from precipitation with soil water holding capacity. The distribution of moisture conditions that generally define the amount of water in the soil that can be maintained for plant use throughout the growing season and summer dry season corresponds very well to the established distribution of vegetation types. However, in many locations, shallow soils limit the contribution of precipitation. The lowest climatic water deficits in California are in regions with snow pack that, as it melts in the springtime, provides a longer duration of available water, thus maintaining a lower annual climatic water deficit, even despite shallow soils.

Locations in the south with higher PET have higher climatic water deficits.Precipitation has increased in most locations, but has declined in the desert and eastern Sierra Nevada. Air temperature and PET have increased in all ecoregions . This translates into increases in climatic water deficit in nearly all locations, and particularly those dominated by snow pack, such as the Sierra Nevada ecoregion and Trinity Mountains in the northwestern California ecoregion. The recorded increases in air temperature, particularly minimum air temperature, result in earlier snowmelt and reduce the ability of the snow pack to sustain the water available throughout the summer season. The deserts all increased in deficit with declining precipitation and increasing air temperature. However there are some small areas in the Great Valley ecoregion that experienced small decreases in deficit because of the ability of the deep soils to store the additional precipitation rather than result in recharge or runoff. Some moderating effects of coastal climatic conditions are seen in small valleys along the coast with decreases in deficit.In the analysis of the impacts from historic to future climate on hydrology, we characterized the changes in precipitation, PET, runoff, recharge, and climatic water deficit from the BCM for watersheds and for ecoregions, and compared changes in variables from historical to baseline periods and from the baseline period to the end of the twenty‐first century. Three types of map analyses were applied to this comparison: assessment of the difference in magnitude for each variable; the number of standard deviations of baseline conditions by which historic and projected future conditions differ; and a geographic review of the variations in hydrologic conditions across California for both historical and future time periods. A summary of variables by modified Jepson ecoregion and for the HUC 12 watersheds averaged for the extent of California was calculated. Overall, mean precipitation increased by 80 millimeters between 1911–1940 and 1971–2000 . Under the PCM scenarios, precipitation continued to increase to 2070–2099 , but it decreased under the GFDL scenario . Potential evapotranspiration increased 10 mm from historic to baseline time frames,large pot with drainage and increased under all future time frames between 51 and 104 mm. Runoff increased historically 36 mm. It increased under future PCM projections by 51 to 77 mm, but decreased under GFDL projections by 38 to 42 mm.

Finally, climate water deficit decreased by 16 mm from historic to baseline time; however, it increased under all projections between 40 and 174 mm, indicating increases in PET and decreases in available soil moisture resulting in lower actual evapotranspiration.While most of northern California got wetter from the historic to baseline time, only the northeast, an eastern area representing the high Sierra Nevada and Inyo/White mountains, and a few scattered watersheds saw an increase that was even one‐half a standard deviation from the baseline SD for the 30‐year mean, a pattern that is mostly repeated when looking at the statistically significant trends . This suggests that the trend in increased moisture is well within the baseline variability of precipitation from year to year. The same is true for the southern half of the region, which mostly shows a drying trend. As expected, given the GCMs selected, the PCM future scenarios forecast increased precipitation, and GFDL forecasts a drier future . However, compared to baseline precipitation variability and statistically significant change, only the desert ecoregions receive more than 0.5 SD more precipitation under PCM, while under GFDL A2, the northern half of California loses precipitation mostly between 0.5 and 0.9 SD . The calculation of PET using the Priestley‐Taylor equation assumes that PET is a function of, and is non‐linearly related to, air temperature. The application of PET in the BCM assumes that plants are in equilibrium with their environment and will transpire at maximum rates until the soil reaches the wilting point. Potential evapotranspiration increased from historical to baseline time periods in most of California, with the exception of a few places in the Sierra Nevada, where it decreased between 0.5 and > 2 SD of baseline PET values, with similar patterns in the significance values . The extreme change in these locations is due to cooling air temperature, but because PET is already low in these locations, due to the non‐linear relation between PET and air temperature, the change is greater than if the PET were initially high. Potential evapotranspiration is projected to increase under all scenarios and for all ecoregions and shows one of the strongest spatial patterns of all the variables, with nearly the entire region increasing by at least 1 SD, and statistically significant under the PCM projections, and by > 2 SD under the GFDL projections .Annual runoff values increased slightly in California between 1911–1940 and 1971–2000 , a change driven by increases throughout the northwest ecoregion, and in the northern Sierra Nevada. Looking at this difference relative to the standard deviation during the baseline time period, none of the watersheds had runoff increase by more than one standard deviation, but a few in the desert ecoregions decreased by more than one. This is because the annual runoff in these watersheds was less than 3 mm in 1911–1940 and less than 1 mm in 1971–2000. Comparing the baseline conditions to future scenarios , the PCM model shows an increase in runoff for all ecoregions except the Modoc Plateau , and especially in the Sierra Nevada and the coast ranges, while the GFDL model shows an almost inverse pattern of drying . Because of the very low runoff values in the baseline time period, the incremental increases in the desert regions of the study show future runoff to be above 1 SD under the PCM model. For the GFDL model, parts of the Sierra Nevada and the northeast region of the state show decreases in runoff above 0.5 SD of baseline . Note that statistically significant change differs from the SD view under the future scenarios, particularly in the desert systems, where much of the change while high in terms of standard deviations is not significant at the 0.05 level .Annual recharge values increased throughout the mountains and coast of northern California between 1911–1940 and 1971–2000 , similarly to runoff in distribution, but at a lower magnitude. Declines in recharge in the southern parts of the state and the Central Valley are at a similar magnitude. The difference between the time periods relative to the standard deviation during the baseline time period indicated very small changes outside the normal variability. The differences between recharge and runoff are more pronounced in the changes between baseline and the future scenarios . This difference is exemplified by a very important characteristic that results from warming, regardless of the direction of change in precipitation in future projections, and that is the alteration of seasonality, with a shorter wet season and longer dry season.For the wet scenarios , there are slight increases in recharge in the Central Western and Great Valley ecoregions , and the Cascade and Sierra Nevada , but in contrast to runoff there are declines in recharge in the Sierra foothills and the northwestern part of the state. Because of the compression of the wet season with warming, , in addition to the earlier onset of springtime snowmelt, there is less time with conditions conducive to recharge.