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CB has been deemed persistent in the environment but with a low potential for bioaccumulation and toxicity

Root exudation may also be altered after nanomaterial exposure.In addition, adsorption of nanomaterials to bacterial cell surfaces has been reported to disperse nanomaterial agglomerates.Such processes and other soil characteristics could cause temporal variations in CNM behavior within the natural soil environment, including differentially over the course of plant growth. The results of the CNM concentration-dependent agglomeration in aqueous soil extracts qualitatively explained the observed inverse dose–response trends, which deviate from typical sigmoidal dose–response relationships reported for toxicants that dissolve in soil water , but quantitative tests are not possible because of the complex soil characteristics and dynamic processes described above. In this study, with nondissolving but agglomerating CNMs, small amounts of CNMs in moist soil did not agglomerate but rather remained suspended in soil water where they were more bio-available and impactful to soil microbes and plant roots. With larger amounts of CNMs in moist soil, large agglomerates formed, which led to a sharp decrease in their bio-availability and observed impacts . Although the inverse dose–response patterns were mostly shared across CNMs, the relationships were linear for CB and fit a power function for MWCNTs . Differences in agglomeration and possibly differing toxicity mechanisms could explain the differing model fits. Our results demonstrate that not only the mass concentration and primary particle size but also the level of agglomeration may play critical roles in determining CNM effects on plants and their root symbioses in soils. In prior microbial toxicity and hydroponic phytotoxicity studies, it was recognized that nanomaterial effects would increase as nanomaterial size decreases but would decrease as nanomaterials agglomerate. For instance, antimicrobial activity was found to be higher for smaller versus larger graphene oxide sheets,while debundled, short,nft system and dispersed MWCNTs were demonstrated to have relatively higher bacterial cytotoxicity due to enhanced MWCNT–cell contact.Depicted as “nano darts”, individually dispersed single-walled carbon nanotubes were reported to induce more bacterial death than SWCNT aggregates, as dispersed SWCNTs directly damaged bacterial cell membranes.

In hydroponic studies, dispersed MWCNTs were found to have stronger effects on tomato plants than MWCNT agglomerates.Even when comparing among agglomerates, small MWCNT agglomerates exerted stronger impacts to Arabidopsis T87 cells than large agglomerates.Still, the dose–response relationship for unstudied low concentrations, which are, across the herein unstudied range of 0 to 0.1 mg kg−1, is uncertain. It is possible that the whole-plant N2 fixation potential decreased continuously with CB concentration until 0.1 mg kg−1 . Alternatively, there could be a threshold concentration somewhere between 0 and 0.1 mg kg−1, possibly close to the lowest studied dose , above which the inhibition of the whole-plant N2 fixation potential occurred but below which it did not . There is uncertainty in such untested low concentration regimes. Such uncertainty reinforces the challenges in extrapolating toxicological results from studies using only high nanomaterial concentrations to low concentration exposure scenarios, owing to influential effects of nanomaterial physicochemical structuring.We chose multi-walled carbon nanotubes and graphene nanoplatelets as two representative engineered CNMs, with industrial carbon black for comparison. CB has been commercialized for decades in the rubber and pigment manufacturing industries,with annual production of over 10 million metric tons.However, there is evidence that CB may have similar or higher toxic effects on soil bacterial communities and amphipods compared with other CNMs.Therefore, assessing whether CB affects soybean and N2 fixing symbioses and comparing how the effects differ from those of MWCNTs and GNPs are important from an environmental regulatory standpoint. MWCNTs and GNPs were purchased from Cheap Tubes Inc. ; carbon black was purchased from Dorsett & Jackson Inc. . Besides the manufacturer reported properties , CNMs were characterized by transmission electron microscopy , thermogravimetric analysis , and inductively coupled plasma optical emission spectroscopy for material morphology, thermal stability, overall purity, and metal composition, following previously reported methods.The CNMs were used as received without further purification.

Three concentrations of MWCNTs, GNPs, and CB were evaluated in this study. A sequential 10-fold dilution method accompanied by mechanical mixing was used to prepare homogenized soil and CNM mixtures as reported previously.The mixing was performed using a hand-held kitchen mixer, from the low to the high CNM concentration treatments, with the mixer cleaned between different CNMs to avoid contamination. The cleaning procedure followed guidelines recommended by the National Institute for Occupational Safety and Health for cleaning surfaces contaminated with carbon nanotubes.CNM dry powder was weighed and amended directly into soil in concentrations of 0.01, 10, and 100 g kg−1 . Each mixture was blended thoroughly using the mixer for at least 10 min. These CNM–soil stocks were then diluted ten times by the addition of unamended soil and mixing by the mixer similarly as above, resulting in concentrations of 0.001, 1, and 10 g kg−1. The dilution and mixing were repeated again to achieve the final CNM working concentrations of 0.1, 100, and 1000 mg kg−1. The CNM–soil mixtures were stored prior to planting.Bradyrhizobium japonicum USDA 110 was initially streaked from frozen stock glycerol onto solid modified arabinose gluconate medium24 with 1.8% agar in a Petri dish, then cultivated in the dark. Following incubation, several discrete colonies were dispersed into 4 mL of liquid MAG medium. An aliquot was inoculated into a 500 mL glass flask containing 100 mL of liquid MAG medium and incubated in the dark for 5 d until stationary growth phase. Aliquots of the culture were dispensed into centrifuge tubes and centrifuged , and the supernatant was discarded. Cell pellets were resuspended in a 1 M MgSO4 solution to an optical density at 600 nm of 1.0 to serve as the inoculum during seed planting. Soybean seeds were purchased from Park Seed Co. . Seeds were inoculated with B. japonicum following the method of Priester et al.Specifically, seeds were soaked in the B. japonicum inoculum for 10 min and deposited into rehydrated peat-filled seed starter pellets at 1/4-in. depth using forceps. An aliquot of the B. japonicum inoculum was dispensed into the pellet holes over the planted seed; the seed plus additional inoculum were then covered with a thin layer of the peat pellet substrate. The pellets were watered daily and incubated on a heating mat . Each planting pot was comprised of a 3 qt high density polyethylene container with bottom perforations, which was lined with polyethylene WeedBlock fabric at the bottom, and overlain by 400 g of washed gravel to allow water drainage.

A polyethylene bag punched with 40 evenly spaced 5 mm holes was placed over the gravel, and 2.3 kg of soil was weighed into each bag. Perforation of the bags allowed for water drainage, thereby preventing root rot within the soil-filled bags. Overall, there were 10 treatments, including three concentrations for each of CB, MWCNTs, and GNPs, plus a control soil without nanomaterial amendment. There were eight replicate pots per treatment. Ten days after seed sowing, 80 VC stage 59 seedlings were transplanted into potted soils. Prior to transplanting, the outside mesh of the starter pellets was removed carefully to minimally disturb the seedling roots. A central planting hole was formed in the soil, into which B. japonicum inoculum was dispensed. One seedling was inserted into the hole, and another aliquot of B. japonicum inoculum was dispensed onto the surface. Both inoculation steps were deemed necessary for adequate contact between B. japonicum and the soybean roots and thus effective inoculation. The filled transplanting hole was covered by a thin layer of soil, and the potted soil surface covered by a layer of WeedBlock fabric to minimize soil surface crusting and weed growth. A wooden support stake was inserted against the inside wall of each pot for later plant support by tying, as needed. After transplanting, the plants were grown for another 39 d to the R6 stage in the Schuyler Greenhouse at the University of California at Santa Barbara. The greenhouse climate was controlled using VersiSTEP automation under full sunlight. The indoor air temperature ranged from 15 to 34 °C,hydroponic gutter and the indoor photosynthetically active radiation fluctuated between 21 and 930 μmol m−2 s −1 from nighttime to daytime. Soil moisture sensors were inserted to a depth of 13 cm into the soil of seven pots to monitor soil volumetric water content, electrical conductivity, and temperature. Data were recorded at least twice daily using a ProCheck data display . Pots were watered to retain an average soil volumetric water content of 0.25 m3 m−3 .Midori Giant is a determinate soybean variety, which stops vegetative growth soon after flowering initiates.Also, N2 fixation will accelerate when plants initiate pod development. Therefore, plants were harvested at each of two stages: intermediate or final , aimed at capturing CNM effects on plant vegetative growth with early nodule formation, and then reproductive development with highest N2 fixation potential. Three replicate plants from each of the ten treatments were sacrificed at the intermediate harvest , and five replicates were sacrificed at the final harvest , when plants reached stage R6 .At harvest, plants were separated, above ground from below ground, by cutting the stem at the soil surface using a single edge razor blade. The above ground part was further divided into stem, leaves, and pods . Leaves and pods were counted and arranged according to their sizes, then photographed. Total leaf area and pod size were further quantified by analyzing the images using Adobe Photoshop software.Sub-samples of fresh leaves and pods were weighed and then stored for future analyses.

The remaining tissues were transferred to separate paper bags, then weighed before and after drying to determine wet and dry biomass plus gravimetric moisture content. The below ground plant parts were removed from the pot within the polyethylene bag surround. The soil in the bag was gently loosened from around the roots and nodules using a metal Scoopula , while minimizing root system disturbance. The relatively intact below ground parts, including roots and nodules, were rinsed in deionized water thoroughly to remove remaining attached soil, then air-dried. The nodules were carefully excised from the roots using a single edge razor blade and forceps as reported previously.Nodules were counted; sub-samples were weighed and refrigerated for later TEM analysis. The remaining nodules were weighed and then analyzed immediately for N2 fixation potential. Roots were dried and massed as above, to determine gravimetric moisture content and dry biomass. After N2 fixation potential measurements, nodules were also similarly dried and massed. After acquiring dry masses, all dried plant parts were archived for future analyses. Sub-samples of soil from each pot were collected and stored for future analyses. The N2 fixation potentials of root nodules were measured as nitrogenase activity by the acetylene reduction assay, according to standard methods with some modifications.Pure acetylene gas was generated by the reaction of calcium carbide and deionized water in a 1 L Erlenmeyer flask, with C2H2 collected into a 1 L Tedlar bag . Intact nodules that were freshly excised from cleaned plant roots were placed into a 60 mL syringe with a LuerLok Tip and incubated with 10% C2H2 . At 0, 15, 30, 45, and 60 min, 10 mL of the gas sample in the syringe was injected into an SRI 8610C gas chromatograph with a sample loop to measure the C2H2 reduction to ethylene over time. The GC was equipped with a flame ionization detector and a 3 ft × 1/8 in. silica gel packed column. Helium was used as the carrier gas at a pressure of 15 psi . Hydrogen gas and air were supplied for FID combustion at 25 and 250 mL min−1, respectively. The oven temperature was held constant . The C2H4 peak area and retention time were recorded using PeakSimple Chromatography Software . Chemically pure C2H4 gas was diluted by air and measured to establish a C2H4 standard curve . The C2H4 peak area values were converted to C2H4 concentrations against the standard curve and further to moles of C2H4 using the ideal gas law assuming ambient temperature and pressure. For each analysis, the moles of C2H4 produced were plotted over time, and the relationship was evaluated for linearity, then fitted by a linear regression model to calculate the C2H4 production rate. The N2 fixation potential was calculated as the C2H4 production rate normalized to the assayed dry nodule biomass.

Pests and diseases are another uncertainty for which little published literature exists

Of the five initial variables, the fraction of land in cropland, the soil Storie index, and the land fraction converted to urban had high positive loading values on PC1. The close relationship between these variables is consistent with other studies that show high rates of urbanization on some of the highest quality cropland in the state . Soil salinity and the fraction of land in the 100‐yr floodplain had high positive loadings on PC2. Figure 2.4 shows the spatial distribution of land use vulnerability throughout California as measured by the sub‐ index. While relatively high land use vulnerability occurs throughout the Central Valley, areas of particular concern are the Sacramento‐San Joaquin Delta, and the corridor between the Sacramento and Fresno. In these areas of rapid change from agricultural to urban land uses, sub‐index values were frequently > 2.5 standard deviations above the mean. In the Delta region, the high vulnerability was largely due to the risks posed by both urbanization and flooding on highly productive agricultural soils. In contrast, a combination of increasing urbanization and high soil salinity were the important drivers of vulnerability further south in the San Joaquin Valley. Conversion of prime farmland to urban uses is essentially a permanent loss of agricultural potential, with many consequences for agricultural livelihoods and society at large. When urban development fragments agricultural land, farmers often lose the benefits associated with being part of an integrated farming economy; for example, sources for inputs, information sources, and processing facilities . Farming activities occurring along the urban edge can raise concerns about noise, odor, dust, and spray drift among new suburban residents, while vandalism of farm fields can cause problems for farmers . Regional and local strategies to preserve farmland and manage urban growth include strengthening agricultural zoning policies,hydroponic gutter acquisition of conservation easements on farmland, establishment of urban growth boundaries, and prioritizing infill development .

Given that greenhouse gas emissions from urban land can be more than 70 times greater per unit area than cropland , policies that preserve agricultural land will also help achieve the mitigation targets set by California’s recent suite of climate policies, namely AB 322 and SB 375.3 While the risks of flooding and soil salinization are not new to California farmers, they are likely to be exacerbated by climate change. Declining snow water storage in the Sierra Nevada is expected to increase the frequency and severity of flooding in the Central Valley . As such, efforts to help regional and district water resource managers develop accurate flood forecasts and flexible reservoir operations will further improve adaptive capacity .More than 3 million acres of irrigated farmland in California have soils with an electrical conductivity above 4 dS m‐1, a standard threshold for the occurrence of agricultural impacts . Of the acreage affected, more than two‐thirds is located in the San Joaquin Valley. In these areas, various irrigation methods can be used to leach salts out of the crop’s rooting zone . But since salts can still accumulate along the margins of the wetted area, growers must often apply water in excess of crop needs to ensure that salts are sufficiently leached . The installation of systems to drain, reuse, and dispose of saline effluent are also options, though high costs and a lack of suitable disposal sites remain important barriers .Results of the PCA for the socioeconomic vulnerability sub‐index indicate that 70.3 percent of the cumulative variance among grid cells is accounted for by retaining three principal components . Seasonal and migrant farm workers, loss of farms, and farm disaster payments all had high positive loadings on PC1, while loss of farm jobs and the social vulnerability index loaded highly on PC2. The commodity concentration was largely independent of these other factors, as indicated by its high positive loading on PC3. Three counties along California’s Central Coast all had socioeconomic sub‐index values greater than 1.5 standard deviations above the mean . The high vulnerability of these counties was due to two main factors: the high rate of disaster payments per unit of cropland; and the large number of seasonal and migrant farm workers per unit of cropland. A closer look at the agriculture in these counties reveals that while each have only a small amount of cropland, the mild coastal climate allows them to devote a large fraction to vegetable and berry crops. Since these tend to be high‐value crops that require more labor, it follows that disaster payments and the number farm workers per unit of cropland area are also higher.

Larger counties such as Monterey, San Joaquin, Imperial, and San Bernardino had moderately high socioeconomic vulnerability due to some of the same factors. In Yuba, Sutter, and Madera counties vulnerability was driven by a combination of high disaster payments and a loss of farm jobs. The main factor influencing the high vulnerability in Mendocino County and the moderately high vulnerability in Napa and Sonoma counties was their high Herfindahl index values, which captured the heavy concentration of wine grape production in this region. While disaster payments are used here as an indicator of vulnerability, the federal programs that provide these payments are generally seen as a way to help farmers cope with risk and strengthen their adaptive capacity. Since many fruit and vegetable crops receive no federal subsidies, disaster payments and crop insurance are among the few remaining options for specialty crop producers . However, as agricultural support programs receive greater scrutiny under tightening state and federal budgets, studies that examine the impact of potential reforms and their effects on vulnerability are needed. In contrast to government programs, the advantage of diversification to new crops, products, markets, or income sources is that farmers have more control over the outcome. But while diversification can help spread risk and facilitate a shift toward new crops should the need arise, concerted efforts to improve knowledge‐sharing among stakeholders will be needed to overcome the risks and trade offs associated with unfamiliar cropping systems and market opportunities .Figure 2.6 provides an illustration of total agricultural vulnerability statewide by integrating the four sub‐indices into one total AVI index. Based on this analysis, moderate vulnerability exists in most of California’s agricultural lands, which suggests that there is a need for all agricultural communities to begin to develop adaptation plans that address the potential impact of changing climate, land use and economic factors. Many local and regional governments are now developing climate action plans that accompany updates to their general plans . To date, these climate action plans have mostly focused on greenhouse gas mitigation, but the results presented here suggest that adaptation should hold an equally important place in local planning activities. The total AVI also suggests that there are several regions of concern that merit careful consideration.

These include the Sacramento‐San Joaquin Delta, the Salinas Valley, the corridor between Merced and Fresno, and the Imperial Valley, which all had a mix of high and very vulnerability. While the sub‐indices discussed above help to highlight the location‐specific factors contributing to these regions’ overall vulnerability, the indexing method used in this study is inherently coarse. Given this limitation, future studies that follow a “place‐based” approach will be needed in order to understand the unique local characteristics, both biophysical and socioeconomic, that may contribute to improved resilience within agricultural communities. The recently completed case study of agricultural adaptation to climate change in Yolo County, summarized in Section 3 below, is an early example of how to integrate these elements .While the AVI presented above represents an early a proof of concept, significant gaps remain in the set of potential variables that could be included in the index. In particular,u planting gutter future iterations of the AVI will need to consider additional variables that more fully assess the vulnerabilities to California’s water resources and livestock systems in a spatially explicit manner. For livestock, studies that evaluate statewide spatial variation in the season length of adequate forage and its links with winter precipitation may be a useful addition . These are but a few of the many types of spatial datasets that might be integrated in to the California AVI. In its current form, the AVI is designed to assess “present” agricultural vulnerability. However, going forward there is potential to modify the AVI so that it can accommodate future projections of climate, land use, and socioeconomic variables. For example, integrating down scaled climate projections into the climate vulnerability sub‐index, or integrating statewide UPlan runs into the land use vulnerability sub‐index, are very feasible next steps . Yet, since many of the biophysical and socioeconomic factors included in the sub‐indices can vary unpredictably over time, and in some cases have not been accurately modeled into future, use of the AVI to examine future scenarios may have inherent limitations. To overcome the potential limits, contributions of expertise and data from a broad range of stakeholders, government agencies, and academic disciplines will no doubt be required.Preservation of agricultural land is a priority in Yolo County, and planning is focused on regional land use guidelines that maintain land in agricultural production and concentrate new development into urban areas. Regions within Yolo County are distinguished by their land forms , proximity to the Sacramento River and Delta , water availability , and the influence of small towns and cities . There is greater prevalence of wine grapes along the river, processing tomatoes in the alluvial plains, and organic fruits and vegetables in an isolated, narrow valley to the north. Flooding along the Sacramento River poses the most significant regional hazard from climate change; water flows will increase by at least 25 percent by 2050 due to a decrease in snow pack in the Sierra Nevada . As for most of California during the past few decades, there has been a trajectory toward less crop diversity, larger farm sizes, but fairly stable markets for commodities . Most commodities are managed with high intensification of agricultural inputs . The number of organic farms, however, is growing. A recent survey showed that many riparian corridors have low scores for soil quality and riparian health , and there is concern about transport of pesticides to the San Francisco Bay delta .

Environmental quality is now receiving more attention with active grower participation in programs from several agencies.Phase I of this case study examined possible effects of increased temperature and decreased precipitation on Yolo County crops . The horticultural “warm‐season” crops in the county will experience more stress than field crops, due to greater environmental sensitivity of their reproductive biology, water content, visual appearance, and flavor quality. New horticultural crops may include “hot‐season’ crops in summer, and “cool‐ season” crops that prefer warmer winters. Expansion of citrus and of heat and drought‐tolerant trees are likely partly because fewer winter chill hours will be difficult for some stone fruits and nuts . Forage production for livestock in upland grasslands may increase with warmer temperatures during the winter rainy season, but field experiments with elevated carbon dioxide do not corroborate this expectation . More nitrogen limitation will likely occur under eCO2 , unless N‐fixing legumes become more abundant. During the past 25 years, crop diversity has decreased across Yolo County , but resilience to extreme events, such as heat waves, may be enhanced in the future by a more diverse crop mix that varies in stress tolerance. Water supply has been considered the most uncertain aspect of climate change for farmers in Yolo County, who rely on groundwater for about 30 to 40 percent of their supply in a normal water year .Discussions with the Yolo County UC Cooperative Extension farm advisors indicate special concern for stripe rust on wheat , insectpests on nuts, medfly, corn earworm on tomato, tomato spotted wilt virus, stem nematode on alfalfa, and earlier activity of perennial weeds such as bindweed . Crop management is subject to change to improve production and environmental quality. Phase I evaluated a set of practices and found that most practices either benefitted GHG mitigation or benefitted adaptation to a changing climate. More comprehensive analysis of these complex relationships is needed.

Increased reliance on groundwater resources makes evaluation of new potential sources a priority

While this choice can be painted as paying the class in question not to work and thus may face push back from the public or even within government, it avoids the myriad complications that stem from subsidizing production, including surplus crises, fights over price adjustments, and conflicts with international trade agreements. In short, leaving production to the most efficient, while supporting the less competitive via income payments allows policymakers to open up more market share for the most efficient while protecting the uncompetitive from impoverishment. A second critical juncture concerns rules structuring benefits, specifically the imposition of benefit limits. A decision to forego benefit limits is arguably more inclusive as it does not discriminate based on some measure of success or size of operation but is also more expensive. Alternatively, the decision to restrict benefits can be seen as discriminatory, splintering the target group into the privileged and unprivileged while saving costs in the long run. This decision has important consequences for the scope and type of future reform. Despite repeated attempts, a reform that the CAP has never been able to impose due to ardent opposition from a few member states is a benefit limit. As a result, some farmers receive hundreds of thousands of Euros in CAP income payments every year. Policymakers wishing to minimize the costs of support to extent possible will want to impose benefit limits, whether they be yearly or lifetime, at the time the policy is created. Benefit levels can always be extended and expanded, but it is much harder to retrench these policies. By imposing limits early, policymakers can better contain overall costs. In addition, these choices position politicians to make popular reforms rather than unpopular,nft hydroponic and likely unsuccessful reforms . However, it may be quite difficult to get the target’s representative groups and unions to agree to a policy that essentially imposes a two-tier system.

The third critical decision concerns qualification and/or behavior standards and requirements. On the one hand, avoiding or limiting standards and rules increases the odds of compliance and support from the target population. On the other hand, imposing standards and rules early controls costs and makes future reform easier. The CAP has struggled to evolve over time, largely failing to impose even the most basic of standards on payment recipients. Policymakers who decide to manage class decline through a CAP-style income support system must think carefully about what types of standards and requirements make sense and impose them early. For example, two reforms CAP officials have struggled to impose are environmental standards and eligibility rules . Neither of these ideas, that farmers should have to meet basic environmental standards or that one’s primary profession must be agricultural in order to receive aid for those employed in agriculture, is particularly radical, but since no real rules or standards for good environmental practices or eligibility existed when the CAP was created, it has been nearly impossible to impose them in a meaningful way in subsequent reforms. Taken together, these three junctures identify moments when policymakers must think carefully about their decisions for how to manage a declining class because these decisions are sticky, proving hard to reverse, and carry important long-term implications. My dissertation and its core findings speak to a broader question of what happens to declining social classes. By investigating the current status of farmer power, I cast light on how and when the political power of a social class is affected by a decline in numbers. Farmers are not the only class to have experienced a dramatic reduction in its share of the population; the blue-collar industrial working class has shrunk dramatically with the shift to a service sector oriented economy. European governments have buffered workers against the effects of deindustrialization with generous disability and early retirement benefits. In addition, in many countries, unemployment programs for workers were structured to ensure that benefits would not run out. As this brief example illustrates, the framework I have used to examine the decline of Europe’s farmers can also be deployed to examine and explain the decline of other social classes. For both blue-collar workers and farmers, policy took the same path. It started with an effort to preserve the class by subsidizing employment and ultimately ended up with a policy that paid individuals not to work.

The path to this final policy outcome in both cases was long and expensive. The lesson, then, for reformers is move to an income support policy as quickly as possible. While these policies are expensive and often unpopular, costs can be contained and crises avoided if these types of programs are adopted first, and reasonable benefit restrictions are imposed early. In sum, my project is not only about the intricacies of CAP reform, but also about the conditions that permit or forestall EU and welfare state policy reform, the techniques for overcoming resistance to policy change, and ultimately the politics of and strategies for managing social class decline. This body of research beginning with my study of farmers and expanding to other threatened social classes will further clarify the important puzzle of why some groups are able, against all odds, to exercise strength without numbers.The rising prevalence of drought conditions in California and elsewhere has dramatically increased demands on groundwater for irrigation and human consumption.Organic and inorganic contaminants in the water supply are prevalent in many human impacted sites such as agricultural, industrial, and municipal. However, simply measuring known sources of contamination has the potential to miss the complex effects of microbial communities in the soil and groundwater. Diverse microbial communities in subsurface environments including groundwater systems exhibit extraordinary phylogenetic diversity and metabolic complexity that has only recently become apparent using culture-independent sequencing-based analytics. The impact of changes in water chemistry on these aquifer microbial communities, and ultimately on groundwater quality, is unknown. Nitrogen as ammonia and nitrate are among the most ubiquitous groundwater contaminants due to widespread use in agriculture as fertilizers, as unintentional discharge in septage and effluent. While crops absorb much of the applied fertilizers, significant amounts leach to groundwater. In certain regions of California’s Central Valley, over 40% of drinking water aquifers have elevated levels of nitrates. The impact of these nitrogen compounds on environmental and groundwater microbial communities is not well understood, including the secondary effects on human, livestock, and wildlife health, and the potential for naturally occurring microbial populations to mineralize ammonia and nitrate to non-toxic forms. There thus exists an urgent need to understand these processes and how they may interact with remediation strategies to protect the quality of groundwater supplies.

To explore these important issues, we sampled groundwater from three adjacent wells completed at different depths that are part of a long term study on agricultural groundwater. The wells are affected to different degrees by manure, a common source of aqueous agricultural contamination. We subjected these samples to chemical analytics as well as next-generation sequencing, assembly,nft system and genomic analysis. Our genomic analysis revealed a highly diverse microbial community dominated by many new lineages of the Candidate Phyla Radiation and the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota,Nanohaloarchaea superphyla and new lineages of the Planctomycete phylum with metabolic potential for both bio-remediation of the contamination as well as production of potentially hazardous secondary metabolites.We collected individual samples from each of four sites , the contamination source water as well as three wells. The domestic well water sample was clear and colorless in appearance with no odor. This water is pumped from ~100 m depth and used for human and cow consumption. Cow waste is pumped into the effluent lagoon , which was cloudy and brown in appearance with an apparent odor of ammonia and feces. After settlement of particulates, the lagoon water is used as a fertilizer source for the surrounding corn fields. Monitoring well 5 and monitoring well 6 are located immediately down gradient and upgradient respectively from a corn field receiving lagoon water . These wells are screened from 3 m to 10 m below ground surface . Monitoring well samples were clear and yellowish-green in appearance with a slight organic odor. Depth to the water table for the monitoring wells was 3.4 m bgs, and the wells were sampled at a depth of 4.3 m bgs. A previous hydrologic analysis indicated that MW5 is primarily recharged from the manured corn field, MW6 receives partial recharge from the manured corn field and partial recharge from an adjacent unmanured orchard, and DOM is primarily recharged from the adjacent orchard with slight impact from the manured field.Each water sample was tested for the presence of USDA pathogenic bacteria by inoculating liquid enrichment media and plating on selective nutrient media. The specific pathogens tested for were Salmonella, Enterococcus, Escherichia coli, and E. coli O157. Of these, Enterococcus, Escherichia coli, and E. coli O157 were detected in the LAG sample, but no pathogens were detected in any of the groundwater samples. Previous samplings from these and other similar monitoring wells on dairies did reveal the presence of USDA pathogens. However, it is not known how long these pathogens remain viable in the groundwater, and lagoon water had not recently been applied to the field where the monitoring wells are located. Our failure to detect these pathogens in groundwater suggests that they have a limited residence time.We asked whether the microbial composition of the water samples matched the chemical and culture-based observations. We analyzed the water microbial communities for DOM, LAG, MW5, and MW6 by constructing a whole metagenome library for each water sample and shotgun sequencing to a depth of ~50 million paired end 101 bp reads. We analyzed taxonomic makeup of the samples both by 16S rRNA gene profiling and whole metagenome assembly. First, we used EMIRGE to do reference-guided assembly of 16S ribosomal subunit genes and abundance estimation for each of our shotgun sequencing libraries.

We then assigned taxonomy to the 16S assemblies using the RDP web interface. Second, we assembled all of our reads and binned genomes from the assembled contigs and then assigned taxonomy to the genomic bins using RAPSEARCH to the UniProt UniRef100 database. The two metagenomic approaches we took are in good agreement with each other and with the water chemistry, however, we did not detect any of the cultured pathogens from the surface water by sequencing, suggesting they are rare. The shallow groundwater communities of MW5 and MW6 have similar species composition and are similar to the activated sludge bioreactor communities recently reported by Speth et al , a community sampled from the nitrogen removal stage of sewage wastewater treatment. However, in addition to observing 10 of the 12 phylogenetic groups reported by Speth, we additionally see 13 more in the groundwater. Specifically enriched are prokaryotes from the recently described nano-bacterial Parcubacteria and Microgenomates , nano-archaeal DPANN and ThaumarchaeotaAigarchaeota-Crenarchaeota-Korarchaeota superphyla as well as two distinct clades of Planctomycetes: the OM190 group, and the anammox Brocadiaceae group. Examining the EMIRGE data at abundances over 5%, the Archaea dominate, with the Crenarchaeote, Thermocladium , Woesearchaeota , and Methanomassiliicoccus . The Bacteria include anammox Planctomycetes from the Brocadia group , Acanthopleuribacter , Microgenomates genera , Dehalogenimonas , Parcubacteria genera , and Opitutus . The other major lineages in these samples include many known as nitrifiers, denitrifiers and methylotrophs as well as the heterotrophic eukaryote, Chlorella , at 7.4% and 5.4% respectively. In contrast to the similarities seen between the two shallow groundwater samples, the DOM and LAG samples each have their own distinct communities. The DOM sample is dominated by Domibacillusfollowed by Sphingomonasand Nitrospira . Anammox genomes are more rare in the deep groundwater, matching the trend seen in nitrate concentrations. The surface water is dominated by Rikenella ,which is known from animal feces. Several other likely animal-associated genera are abundant, including Anaerorhabdusand Acholeplasma , as well as a photosynthetic bacterium, Halochromatium . Overall, the taxonomic representation in the water samples matches well with expectations based on the chemical data. We note that several taxa appear unexpectedly in both the DOM and LAG samples, and we suspect these are contaminants in DOM from airborne dust. Specifically, Rikenella, the most dominant member of LAG, is present at 2.9% abundance in DOM. Likewise Anaerorhabdus, Coprobacillus, Halochromatium, and Acholeplasma are abundant at >5% in LAG and ~1% in DOM.