Category Archives: Agriculture

Microbial metabolism is a crucial process for the transformation of PPCP/EDCs in soils

While some compounds are readily degradable, their continual input causes these compounds to behave like pseudo-persistent pollutants . Direct, acute effects on wildlife are rare due to the low environmental concentrations typical of PPCP/EDCs. However, bio-accumulation of specific compounds in organisms creates the potential for toxic effects in susceptible populations. An example is the drastic decline of the South Asian vulture population, which suffered a species-specific toxicity to diclofenac in scavenged cattle carcasses . A wider concern is sub-acute toxicological effects . For instance, some fragrance compounds in personal products, such as polycyclic musks, as well as some cardiac pharmaceuticals, such as verapamil, have been shown to inhibit multi-drug transporters in cell membranes of aquatic organisms . These transporters are an integral part of an organism’s defense to xenobiotic compounds and their inhibition increases sensitivity to other compounds, like genotoxins . Selective serotonin reuptake inhibitors are a class of antidepressant pharmaceuticals that act by enhancing serotonin signaling in the brain by reducing reuptake of released serotonin. Low levels of SSRIs have been shown to initiate spawning in bi-valves and increase the aggression of subordinate lobsters, which may have subtle effects on ecological communities . Many PPCPs have non-specific toxicity mechanisms that require higher concentrations for acute effects , but EDCs act on specific cellular receptors of the endocrine system, and therefore even at extremely low levels can potentially cause toxicities by disrupting normal endocrine signaling . These compounds have varied modes of action, acting as agonists or antagonists for estrogen, androgen, or other receptors. For instance, bisphenol A and nonylphenol have agonistic effects on the estrogen receptor at cellular concentrations of 22.8 and 2.2 µg/L, respectively, and antagonistic effects on the androgen receptor at 137.0 and 550.9 µg/L, respectively ,vertical growing racks which are levels relevant to concentrations in treated wastewater and relevant to blood serum and urine concentrations in humans .

Exposure to bisphenol A, nonylphenol, and 17β-estradiol have all been shown to increase vitellogenin levels in fish and impact other endpoints like smolt development and survival . The endocrine activity of these and other PPCP/EDCs has contributed to detectable estrogenic and androgenic activity in WWTP effluent , which can cause increased vitellogenin levels and feminization in male fish exposed to effluent . Some of these effects have been observed in the environment , showing that current environmental levels of PPCP/EDCs are high enough to cause adverse effects in wildlife populations. Due to the nature of their environmental input, PPCP/EDCs usually exist as a complex mixture in environmental matrices. There is some evidence that these mixtures act additively, and perhaps synergistically, to elicit biological effects even at low levels . For example, the individual toxicities of the analgesics diclofenac, ibuprofen, naproxen, and acetylsalicylic acid were measured as 68 – 166 mg/L for Daphnia and 72 – 626 mg/L for algae, levels which are considerably higher than the typical ng/L environmental concentrations. However, when the 4 compounds were in a mixture, their toxicities were additive and the EC50 was reached at lower individual concentrations . While these concentrations were still in the low mg/L range, these results have implications for the potential toxicity of environmental matrices that may be contaminated by a multitude of individual PPCP/EDCs . There are also potential human health effects from PPCP/EDC exposures. While present at low levels, PPCP/EDCs are routinely detected in food, packaging, and other materials and the consumption of contaminated agricultural crops would contribute to the total exposure. Exposure to PPCP/EDCs may be detrimental to susceptible age and population groups . The potential impact of EDCs on developing organisms is especially concerning. As an example, cytokine secretion is an important process in human placenta tissue and disruption can lead to pregnancy loss. Nonylphenol was found to affect cytokine secretion in human placenta at cellular concentrations of 0.022 – 220 ng/L . Nonylphenol has been measured in human blood of non-occupational workers at 15.17 µg/L and in human urine at 1.57 µg/L , suggesting that humans are currently exposed to nonylphenol through environmental and other sources at levels sufficiently high to elicit this toxicity.

An emerging concern is the potential health effects of transformation products from the degradation of PPCP/EDCs in WWTP and environmental matrices . For many PPCP/EDCs, their complete fate in the environment and the types of transformation products formed are unknown. Of the limited information, some products of carbamazepine transformation in soil have been identified, which are known to have higher biological activity than the parent compound , a situation that was also known for nonylphenol ethoxylates . The effect on human health from unidentified transformation products with generally unknown behavior and toxicity requires further research. When treated wastewater, bio-solids, or manure is applied to soil, PPCP/EDCs may transfer into the soil compartment . Irrigation with treated wastewater may cause accumulation of PPCP/EDCs to higher levels in soil than in the irrigation water . An example is the 2.34 – 132 and 2.74 – 12.6 fold increase of mass in soils of the stimulant caffeine and the epileptic drug carbamazepine, respectively, as compared to the treated wastewater that was used for irrigation, suggesting accumulation from previous irrigation . bio-solids are applied to land less frequently than irrigation water, due to limitations on nutrient loading and run-off , which allows more time for PPCP/EDC degradation in between input events. Therefore, bio-solids applications typically result in lower levels in soil than in the amendment material .The potential of a compound in soil to be taken up by plants or transport off-site is largely governed by its partitioning between the soil matrix and soil-water. Weak sorption to soil implies enhanced mobility and availability, as in the rapid translocation of the antibiotic sulfachloropyridazine after land application, likely due to its low partitioning coefficient with soil . Adsorption of chemicals to soil is generally related to Kow . For example, in a leaching experiment, the antibiotic olaquindox was mostly recovered in the leachate while the more hydrophobic antibiotic tylosin was retained in the soil column . However, estimating partitioning coefficients from Kow may work well only for neutral PPCP/EDCs,vertical farming in shipping containers where hydrophobic partitioning is the dominant process. With ionizable PPCP/EDCs and in clayey soils, many other factors are likely to be important, including processes such as hydrogen bonding, surface complexation, and cation exchange . In addition, the partitioning behavior of ionizable PPCP/EDCs is highly susceptible to soil pH, as changes in pH may alter the ionic fraction. For instance, acidic chemicals have reduced affinity for clay minerals orsoil organic matter at pH levels above their pKa, resulting in increased availability and mobility .

Partitioning between soil and soil-water is best represented with Kd, which is specific to a compound and soil system and usually determined experimentally . Values can vary widely among soils and among compounds. For example, carbamazepine, diclofenac, and ofloxacin had log Kd values of 1.56, 2.21, and 3.55, respectively, in the same high organic content soil, but in a low organic content soil had values of -0.31, – 0.35, and 3.08, respectively . This specificity hinders the comparison of partitioning behavior between different compounds and different soils across studies. The calculation of a Koc value, by dividing a Kd by the organic fraction in the soil to produce an organic carbon content normalized distribution coefficient, has been used to address this limitation, although Koc values are available only for a limited number of PPCP/EDCs . Table 1.3 lists log Koc values for selected PPCP/EDCs. The persistence of the bio-available fraction of PPCP/EDCs in soil also affects their potential to be taken up by plants. This fraction is difficult to measure, so it is often approximated by the fraction that can be extracted using laboratory protocols . The time required for half of the extractable compound to dissipate is usually described with a half-life or 50% dissipation time , calculated by fitting the percent of a compound that is extractable at several time points to a regression curve or a first-order decay model . Soil half-lives for PPCP/EDCs can vary widely, ranging from hours, in the case of ibuprofen, to years, in the case of fluoxetine, depending on the compound and environmental conditions . One soil dissipation process for PPCP/EDCs involves sorption to the soil matrix and conversion to bound residue that is not recovered by solvent extraction procedures. Formation of bound residues is generally considered a decontamination pathway because the bound fraction is often unavailable for microbial metabolism or plant uptake . This has been shown to reduce or remove the toxicity of pesticides , but similar information is not available for PPCP/EDCs. The formation of bound residue involves several abiotic processes between a compound and the soil matrix, including hydrophobic partitioning, covalent bonding, ligand exchange, migration to micro-sites, and ionic bonding . The relative prevalence of these mechanisms is influenced by the characteristics of the compound and matrix, as well as the duration of compound exposure and concentration . In some cases it has been shown that a small portion of bound residue became available after a change in soil management or by mobilization by microbial metabolism or plant growth, but this may amount to only a few percent of the total residue . Because of the difficulty in assessing the bound fraction of a compound, many studies investigating this process use radio-labeled compounds, but this technique can be costly and is not available to all researchers. Another option is the use of a series of extractions employing progressively harsher solvents, though this approach makes it difficult to relate the various extracted fractions to bio-availability . The potential for PPCP/EDCs to form bound residues has been examined in a few studies. Fent et al. determined that about 80% of 14C-bisphenol A was quickly bound in 4 soils after a 3 d incubation, and the bound fraction persisted throughout a total of 120 d of incubation. Bound residues accounted for 44 – 78% of 14C-diclofenac after 40 d of incubation in a clayey silty soil and a silty sandy soil . Higher soil organic carbon content can enhance the formation of bound residues, which has been shown for diclofenac and carbamazepine . Overall, formation of bound residues is likely an important pathway to decrease the bio-availability of PPCP/EDCs in soil, although more experimental evidence is needed to validate the extent of this process for other PPCP/EDCs.In addition to abiotic processes, there is evidence that microbial activity is important in the formation of bound residues. Nowak et al. showed that 4.5% of ibuprofen was incorporated into fatty acids and amino acids of the soil biomass at 30 d, which decreased to 1.4% by 90 d. This decrease was attributed to population turnover, resulting in the incorporation of non-living fatty acids and amino acids into the soil matrix. Concurrently, at 30 d, 9.4% of ibuprofen was bound to the soil and at 90 d the bound residue fraction increased to 27.9%. Aerobic bio-degradation has been identified as the main route of transformation in soil for veterinary pharmaceuticals . Bacteria can directly use some PPCP/EDCs as growth substrate and can transform others through cometabolism . During cometabolism, the amount of soil organic matter may affect transformation rates since it acts as a substrate for overall microbial activity . Oxygen state affects the rate of microbial transformation . Under aerobic conditions, estrone had a half-life of 0.6 d in soil previously exposed to WWTP effluent, but under anaerobic conditions half-life increased to 6.3 d in the same soil . For triclosan, the effect of oxygen state was even more dramatic; its half-life increased from 5.9 d to 28.8 d. However, the degradation of 17β- estradiol was actually faster in anaerobic soils , showing compound specificity in microbial transformations. Other factors that affect the soil microbial community may also affect transformation rates of xenobiotics, including moisture content, temperature, amendment, and sterilization. For example, transformation of 14C-naproxen was inhibited in soils at cooler temperatures as compared to warmer temperatures . Degradation of naproxen was also reduced in air-dry soils as compared to soils at 15% or 30% water content . Prior exposure to a compound may also potentiate the transformation of a compound by selective enhancement of certain microorganisms .

This concentration was likely orders of magnitude higher than the environmentally relevant levels

The rhizosphere is usually considered an important player in the overall metabolism of xenobiotics by whole plants, as root exudates generally enhance the richness of microbial communities in the root zone, leading to a greater microbial abundance and accelerated microbial degradation.Although the rhizosphere in a hydroponic system may differ greatly from that in soil in terms of microbial community abundance, it was likely that some of the transformations of the target CECs or their TPs occurred in the solution due to rhizosphere-mediated microbial degradation.This could result in the occurrence of methylated or demethylated metabolites in the hydroponic solution and their subsequent uptake into the plant. In addition, previous studies also showed that some xenobiotics may be excreted from plant roots into their bathing solution. Analysis for the target CECs in the nutrient solution in this study, however, generally showed an absence of the corresponding methylation or demethylation products in the nutrient solution, except for acetaminophen and M-acetaminophen . The calculated compliance constants of the methylated CECs are summarized in Table 1, along with the calculated R-CH3 relaxed force constants. A stronger chemical bond is harder to break as it requires more energy, while it is easier to form as more energy may be released. The computation results of the relaxed force constants showed that the chemical bond strength between the methyl group and the major molecular fragment in the methylated CECs followed a general order of methylparaben < diazepam < naproxen < M-acetaminophen. Therefore,vertical garden indoor demethylation may be expected to occur more readily for methylparaben, but more slowly for M-acetaminophen.

Conversely, methylation of DM-methylparaben may be expected to be the hardest, while it is relatively easy for acetaminophen. The trends observed for the four pairs of CECs in A. thaliana cells generally followed the prediction from the bond strengths. For example, the demethylation of methylparaben in A. thaliana cells was the most extensive among the test compounds, followed by diazepam. In contrast, demethylation of M-acetaminophen or naproxen was negligible under the same conditions. Methylation from acetaminophen to M-acetaminophen was found to proceed more readily than the conversion from DMnaproxen to naproxen, while methylation of DM-diazepam was not observed. Due to the limited number of compounds considered in this study, a quantitative correlation between the calculated bond strength and transformation rates was not carried out. However, future studies may consider ascertaining such a relationship, with information from more compounds, in order to better understand the impacts of molecular structures on bio-transformation in plants. The demethylation and methylation processes involve distinct subfamilies of CYP450s, esterases and methyltransferases, which may depend on plant species specific enzyme activities, as well as the chemical structure of xenobiotics. The generally good agreement between the experimental results and bond strength-based predictions in this study suggests that evaluation of chemical characteristics such as the bond strength of R-CH3 may be used to identify CECs with a high tendency for specific transformation reactions. Given the large number of CECs, such a first-cut screening approach may be invaluable for developing a priority list of CECs that may undergo such conversions. The usefulness of such predictions may be further improved by considering more compounds and different plant species, and by developing and refining quantitative structural-activity relationships.To ensure confident identification and quantitative measurement of CECs and their TPs, an artificially high concentration was used in the growth media for A. thaliana and wheat seedlings.In addition, hydroponic cultivation was a simplified system, and the absence of soil should impart significant influences on the adsorption and hence the availability of CECs for plant uptake. Microorganisms in rhizosphere soil under field conditions likely play a great role in facilitating transformations of CECs, and therefore, the interconversion of CECs and their TPs in the soil-plant continuum may exhibit patterns different from observations from this study.

Nevertheless, results from the controlled experiments in this study clearly showed that plants can mediate transformations of CECs such as methylation and demethylation. In some cases, demethylated products were found at relatively high levels under experimental conditions. Given that a large fraction of TPs was likely non-extractable or conjugated, the actual occurrence of such transformations in plants may be much more pronounced than that detected in this study. Conjugated metabolites may become deconjugated upon ingestion, for example, by enzymes in the gastrointestinal tract, releasing bio-active molecules.The methylated or demethylated TPs likely retain or have even increased biological activity. For example, DM-diazepam , although a demethylated TP of diazepam, is itself a drug for treating anxiety. The addition or loss of a methyl group alters the physicochemical properties of a compound, leading to different environmental behaviors such as bio-accumulation, metabolism, and toxicity. For example, diclofenac methyl ether showed greater acute toxicity to aquatic invertebrates than diclofenac.Bisphenol A mono- and di-methyl ether also displayed greater developmental toxicity to zebrafish embryos than bisphenol A.Therefore, when considering the whole life cycle of CECs, e.g., along the entire human-wastewater-soil-plant-human continuum, such circular interconversions may effectively prolong the persistence of CECs and contribute to enhanced human and ecotoxicological risks, underscoring an urgent need to consider such interconversions for more comprehensive risk assessment. For the four pairs of CECs considered in this study, demethylation appeared to proceed more readily than methylation, and there were also differences among different compounds. A preliminary analysis showed a dependence of the methylation or demethylation rate on the bond strength of R-CH3 of the compounds. As CYP450s, esterases and methyltransferases are involved in the metabolism of many xenobiotics, CECs with similar functional groups like -OH, -OCH3, -NH-, and -NCH3- may also undergo the methylation and demethylation cycle. With more experimental observations, it is feasible to predict the likelihood of such transformations using basic chemical structures and molecular descriptors.

This is particularly valuable given that CECs and their TPs are numerous in numbers and identifying compounds or structural features conducive to interconversions constitutes an important first step to better understand the significance of this phenomenon for the overall environmental fate and risks of CECs.The occurrence of numerous contaminants of emerging concern in the effluent from wastewater treatment plants and impacted aquatic environments has been extensively reported.However, most research has focused on the parent form of CECs while generally neglecting their transformation products that are often in co-existence. Many CECs contain reactive functional groups, such as hydroxyl, carboxyl and amide groups, making them susceptible to various biotic and abiotic transformation reactions.Simple transformations, such as methylation and demethylation, have been observed in various environmental matrices for many CECs.For example, previous studies showed the presence of methylated TPs of triclosan and bisphenol A in wastewater effluents and receiving streams.The methyl ethers of tetrabromobisphenol A were formed in aquatic environments in the presence of background methyl iodide.Methylation of acetaminophen was observed in soil.On the other hand, demethylation is a major metabolism pathway for CECs in organisms. For example, after oral administration in humans, naproxen and diazepam are demethylated to 6-O-desmethyl naproxen and nordiazepam , respectively.Despite the fact that TPs seem to occur readily and co-exist with their parent forms in the environment,vertical garden indoor system the ecotoxicological consequences of such transformations have not been adequately considered. Transformations such as the addition or loss of a methyl group can significantly change a compound’s physicochemical properties, such as Kow that is known to influence its fate and bioaccumulation.Methylated products of diclofenac, BPA, and triclosan all displayed enhanced toxicity or bioaccumulation potential in aquatic organisms. In this study, we comparatively explored the behaviors of four typical CECs, i.e., acetaminophen, diazepam, methylparaben, and naproxen, and their methylated or demethylated TPs in Daphnia magna, by considering their bio-accumulation, acute toxicity, and interconversions. Quantitative structure-activity relationship models were further developed and used to describe the experimental results. The study findings highlight the importance of simple transformation reactions such as methylation and demethylation in understanding the overall ecological risks posed by CECs in aquatic environments. To further understand the effect of methylation and demethylation on the acute toxicity to D. magna, bio-accumulation of the CECs and their methylated or demethylated counterparts was measured in adult organisms. The concentrations of target compounds remained relatively constant in the aqueous media during the 24 h uptake phase, with RSDs ranging from 2.8% to 18.4% . Therefore, the mean measured concentrations of target compounds in the water phase were used as to fit Equations and to derive BCF values. The bio-accumulation kinetics of target compounds are shown in Figure 2. The concentrations of CECs and their methylated or demethylated TPs generally showed an increasing trend at the beginning of the uptake phase and reached an apparent equilibrium in 24 h.

Upon transferring the exposed D. magna to clean AFW to initiate the depuration phase, the concentration of test compounds gradually declined over time. With the exception of diazepam, methylated derivatives consistently showed much higher concentrations in D. magna than their demethylated counterparts. For example, after 2 h of exposure, the concentrations of acetaminophen and M-acetaminophen in D. magna were found at 308.7 ± 42.6 ng g-1 and 8730.7 ± 2900.9 ng g-1 , respectively, a 28-fold difference . This was consistent with the fact that methylated acetaminophen has a higher log Dlipw than acetaminophen . In addition, at pH 8.5, acetaminophen was expected to be partially ionized in the aqueous media, while M-acetaminophen should be completely in its neutral state . Methylparaben also displayed a much higher accumulation than DM-methylparaben in D. magna at the end of the uptake phase . The 3-fold change also coincided with the difference in log Dlipw between DM-methylparaben and methylparaben . The level of DM-naproxen in D. magna was below LOD, and therefore its bioaccumulation may be deemed negligible . In contrast, significant accumulation of naproxen in D. magna was observed, again suggesting a pronounced effect by hydrophobicity induced by methylation. It is also likely that DM-naproxen was rapidly metabolized due to the presence of an exposed hydroxyl group . The presence of the hydroxyl group in DM-naproxen may facilitate its conjugation with an amino acid or glucose in D. magna, contributing to its rapid metabolism and reduced bio-accumulation. Unlike the other three pairs, there was no significance in the bio-accumulation between DMdiazepam and diazepam in D. magna , with 6792.5 ± 1215.8 ng g-1 and 7599.7 ± 1470.3 ng g-1 detected in D. magna after 24 h, respectively. This may be attributed to the fact that methylation or demethylation does not result in a great change in their physicochemical properties and that both compounds have similar log Kow or log Dlipw values . The derived kinetic parameters of target compounds are given in Table S3. In general, the methylated derivative in each pair had a larger ku than the corresponding demethylated counterpart. The dynamic BCF values, calculated as the ratio of ku and kd, showed a strong correlation with the BCF values derived from the steady state , suggesting enhanced bioaccumulation for most methylated CECs. For example, the dynamic BCF of M-acetaminophen was 10.0 ± 0.0 in D. magna, which was significantly higher than the dynamic BCF of acetaminophen . For DMdiazepam and diazepam, however, the BCF values in D. magna were not significantly different from each other, which again coincided with their generally similar physicochemical properties. For aquatic organisms, increased bioaccumulation of contaminants is often attributed to a compound’s hydrophobicity, as bio-accumulation is driven by lipids in an organism and is positively related to hydrophobicity or log Kow for neutral compounds. Increased bioaccumulation after methylation was previously observed for diclofenac in aquatic invertebrates. Bioaccumulation of methylated diclofenac was found to be 25-110-fold that of diclofenac in H. azteca and G. pulex. In this study, methylation generally increased log Kow of CECs, and further log Dow and log Dlipw, although the relative increases are specific to the individual compounds. The generally enhanced bioaccumulation in D. magna was also in agreement with the effect of methylation on CEC bio-accumulation in plants.Methylation of CECs could occur in natural water bodies due to the presence of methyl iodide,during wastewater treatment, and during biological transformations in soil, plants ,and earthworms.Therefore, methylated derivatives of CECs may be prevalent in the environment and should be considered in a holistic risk assessment because of their different behaviors and biological activities, such as increased bio-accumulation potentials.

Two single benzenering metabolites of TBBPA were identified in pumpkin plants and rice cell cultures

Previous expression kinetics of CMG2-Fc in N. benthamiana suggested that protein accumulated in leaf increased from day 1 to day 6, supporting the first explanation. It is likely that kifunensine will remain stable even longer if longer incubation period is desired to maximize protein yield. Kifunensine is known to inhibit enzymatic activity of class I α-mannosidases, and thus should stop mannose trimming in the first place to yield single Man9 N-glycan structures. However, we observed multiple oligomannose-type N-glycans with mannose residues ranging from 3 to 9, although the most abundant structure was Man. This observation is consistent with cell culture kifunensine studies, where multiple oligomannose-type N-glycans were detected under kifunensine treatment. This could potentially due to the difference in inhibition efficacy of kifunensine towards class I α-mannosidases isoforms, which results in an incomplete inhibition of mannose trimming from Man9 structure. Also taking enzyme kinetics into consideration, depending on the ER concentrations of Man9 glycoprotein substrate, class I α-mannosidases, and kifunensine, enzymatic mannose trimming from Man9 could take place even if the amount of active ER α-mannosidase I is low. Although mannose trimming was not completely inhibited at Man9 structure, this method still showed the ability to significantly modify glycosylation using a simple bio-processing approach. It is likely that Man9 abundance can be further increased if treated with higher concentration of kifunensine, but it is not necessary if the goal is to eliminate the production of plant-specific complex N-glycans. Although in this case, CMG2-Fc can be purified easily from whole-leaf extracts through a one-step purification with Protein A chromatography, in many cases,vertical rack multiple steps of chromatography are required to purify a target protein from a large pool of host native proteins when a highly selective affinity tag is not present.

This could result in low protein yield and difficulties to achieve high purity, which is typically required for therapeutic recombinant protein products. Targeting proteins to the apoplast allows the collection of target protein in AWF, which contains much lower levels of plant native proteins than whole-leaf extract since only secreted proteins are collected, thus lowers the downstream process complexity. In this case, CMG2-Fc purity and concentration increased by 3.9-folds and 4.4-folds, respectively, when collected in AWF versus in whole-leaf extract. A similar trend was observed in kifunensine-treated samples, which confirms that kifunensine does not affect protein secretion, allowing secretion of CMG2-Fc with oligomannose-type glycoforms. The increase in purity and concentration was consistent with a previous study on harvesting a target protein from plant AWF. Hence, AWF collection is a feasible method for recombinant protein harvesting, which avoids contamination with intracellular host cell proteins, and is particularly valuable when target protein is hard to purify. Together with kifunensine treatment, apoplast-targeted recombinant protein without any plant-specific glycoforms can be transiently produced in N. benthamiana, and likely in other plants as well. Products can be collected at high concentration and purity from AWF, containing predominantly oligomannose-type N-glycans. Further studies should focus on determining how long the inhibition effect of kifunensine lasts after the one-time vacuum infiltration by monitoring the protein glycoform profile at multiple time points after vacuum infiltration, and the threshold concentration of kifunensine that results in a complete N-glycan shift from plant complex-type to oligomannose-type for other glycoproteins, particularly those with more N-linked glycosylation sites. In addition, the protein expression kinetics should be compared between kifunensine-treated and untreated groups to maximize target protein yield. Depending on the desired glycoform, this method can also be applied to other N-glycan processing inhibitors such as castanospermine, deoxynojirimycin, and swainsonine. Contaminants of emerging concern are chemicals and other substances with no regulatory standards but have been recently detected in the environment and have the potential to cause adverse effects at environmentally relevant concentrations.

CECs consist of many different types of chemicals based on their purposes of use, including flame retardants, pharmaceuticals and personal care products , endocrine- disrupting chemicals , nanomaterials, among others. Flame retardants such as polybrominated diphenyl ethers and tetrabromobisphenol A are added to manufactured materials to prevent or slow the development of ignition. Prescribed pharmaceuticals like amoxicillin and overthe-counter drugs like acetaminophen are widely used by individuals for personal health. PPCPs also contain many types of preservatives and anti-bacterial substances, like triclosan. Antibiotics and veterinary medicines are widely applied to improve the production of livestock. For a long time, these substances were unknown, unidentified, unexpected, or unsuspected pollutants due to limitations in analytical methodologies. It was also challenging to assess the impact of CECs on human health and the environment due to the lack of data or risk assessment tools. After emission from varied sources, including household sewers and industrial effluents, CECs are carried in contaminated wastewater to wastewater treatment plants . The removal efficiency of CECs during treatments depends on the design and performance of individual WWTPs, as well as the physicochemical properties of CECs.Many studies have shown that numerous CECs are present at trace levels in the treated effluent in the ng L -1 to µg L -1 range around the world, including Spain,Germany, the United States, China, and South Africa. The concentrations of CECs are generally higher in bio-solids because of the higher organic matter content, and are in the μg kg-1 to mg kg-1 range.For example, triclosan and triclocarban were detected at 2715 and 1265 μg kg-1 respectively, in bio-solids, in a study conducted in the U.S. The use of TWW and bio-solids in agriculture, and/or their direct discharge into the environment, can introduce CECs to agricultural ecosystems and surface aquatic ecosystems, posing potential risks to ecosystems and human health.Many CECs contain active functional groups such as hydroxyl, carboxyl, and amide groups in their chemical structures, and are susceptible to many biotic and abiotic transformations in the environment and organisms.

Transformation products of CECs can be directly introduced into WWTPs in municipal wastewater, leachates, and surface runoff. For instance, pharmaceuticals can be metabolized in the human body after consumption and are excreted in large portions as metabolites, particularly conjugates.TPs can also be formed during the treatment processes in WWTPs via microbial transformations, photochemical transformations and oxidation and halogenation by disinfection processes.These processes may also transform some TPs back to the parent CECs, such as hydrolysis/deconjugation of the conjugates of estrogens, leading to the “negative removal” for certain CECs in WWTPs.Transformations of CECs may also take place in agroecosystems and aquatic environments after TWW and bio-solids are discharged or applied. Soils,plants,terrestrial organisms,algae,aquatic organisms and photochemical degradation have all been reported to mediate CEC transformations. In some cases, TPs may pose higher ecological risks than their parent compounds, as they may have a greater bio-accumulation potential, increased toxicity to organisms, or longer persistence in the environment.Assuming the majority of irrigated TWW and applied bio-solids are received by soil, roots would serve as the major pathway for CEC uptake into plants.Mechanistic understanding of CEC uptake remains rather limited. Based on the current knowledge, root uptake of CECs occurs primarily through passive diffusion, although an energy dependent active process mediated by transporters is likely for certain hormone-like compounds such as naproxen, clofibric acid, hydrocinnamic acid and perfluoroalkyl acids.Translocation of CECs from roots to above-ground tissues, such as stems, leaves and fruits,vertical farming hydroponic has also been observed by previous studies, with concentrations of CECs generally being more substantial in roots.Both biotic and abiotic factors have been shown to affect the uptake, bio-accumulation and translocation of CECs by plants. These factors include plant physiology, soil pore water chemistry, the physicochemical properties of CECs, and the experimental conditions.Plant physiology plays an important role in plant uptake of CECs.Plants exposed to stressors such as drought, salinity and high temperature can respond with various adaptive mechanisms such as heightened antioxidant defense, hormone regulation, and metabolic modifications.The water and nutrient uptake and photosynthetic efficiency can decrease significantly in plants grown under stressed conditions.Therefore, it may be assumed that non-stressed plants have greater potential for CEC uptake and accumulation. Other than plant physiology, plant species within the same genus, even varieties of the same plant species, have shown different patterns of CEC uptake. For example, different carrot genotypes displayed distinct uptake patterns for metformin, ciprofloxacin and narasin.

Based on the current knowledge, the ability of crop plants to uptake and accumulate CECs in the edible tissues decreases in the following order: leafy vegetables > root vegetables > cereals and fodder crops > fruit vegetables.The physicochemical properties of CECs, such as hydrophobicity and speciation, can strongly affect their uptake and translocation in plants.Many CECs in TWW and bio-solids are polar compounds with low volatility and contain ionizable functional groups, like hydroxyl, carboxyl and amide groups.Only the dissolved CEC fraction in soil pore water would be considered available for root uptake.For neutral CECs, root uptake usually involves two pathways: 1) equilibrium between the aqueous phase in plant roots and the peripheral solution such as soil pore water; and 2) chemical sorption by the lipophilic root solids.Ionized CECs, on the other hand, may undergo disassociation in soil pore water depending on the solution pH.The electrical attraction or repulsion to the negatively charged root surface, along with the ion trap effects, which occur when CECs are neutral in the apoplast but ionized inside the cell , can greatly influence their uptake and translocation in plants.A linear relationship has been often observed between the hydrophobicity, e.g., log Kow, and the bioaccumulation of neutral CECs in plants.However, using log Kow to estimate the bio-accumulation of ionizable CECs is not accurate, partly because lipid bilayers can more easily accommodate charged organic species than n-octanol.Different experimental settings, such as hydroponic cultivation, greenhouse soil cultivation and field experiments, have also exhibited great influence on the uptake and accumulation of CECs in plants. Hydroponic experiments provide simplified conditions,while greenhouse soil cultivation and field experiments have more environmental relevance. The uptake of CECs by plants is usually evaluated by bio-concentration factor , which is calculated as the ratio of the concentration of CECs in plant tissues to that in soil pore water, or the growth media for hydroponic experiments. BCF values of CECs in roots can be high up to 840 L kg-1 in hydroponic settings, while the values obtained from soil experiments may be much smaller,suggesting the availability of CECs for plants decreased greatly in soil pore water during to phase partitioning. CECs with active functional groups, such as carboxyl, hydroxyl, and amide groups, are susceptible to metabolism in plants via various enzymatic activities after being taken up. This metabolic process is similar to the hepatic detoxification system and is known as the “green liver”.Three metabolic phases are usually involved in the metabolism of xenobiotics in plants: Phase I metabolism is an activation process that includes hydroxylation, dealkylation, oxidation and reduction, that are catalyzed by cytochrome P450s, esterase, peroxidase, or other enzymes to enhance reactivity and polarity of xenobiotics; Phase II metabolism is predominantly conjugation with polar bio-molecules, such as amino acids, sugars and glutathione, to further increase the hydrophilicity and mobility of xenobiotics; Phase III metabolism refers to the sequestration of conjugated metabolites in plant cells, including the storage in vacuoles and the incorporation into cell walls.There have been only a small number of studies focusing on the metabolism of CECs in plants. Plant cell systems, such as A. thaliana cell culture,carrot cell culture,rice cell cultures and horseradish hairy root cell culture,have been used as a simple and fast approach for characterizing metabolites of various CECs. Whole plants, either hydroponically cultivated or grown in soil, have also been used to understand plant metabolism of CECs. For example, phase I metabolites of carbamazepine, 10,11-epoxide-carbamazepine and 10,11-dihyro-10,11-dihydroxycarbamazepine, were observed in the leaves and fruits of tomato and cucumber, leaves and roots of sweet potato and carrot, and leaves of Typha spp.Diclofenac was found to be hydroxylated to 4’-OHdiclofeanc in barley,horseradish root cell culture and bulrush.Phase I metabolism was also reported for epimers of tetracycline in pinto bean leaves.Phase II metabolism has been found to occur extensively for some CECs in plants. For example, conjugation with amino acids was reported for naproxen,ibuprofen,diclofenac,and gemfibrozil in A. thaliana cells and whole plants.

The effect of bounce back appears to diminish with bidirectional QTL extension

Different execution strategies also make it difficult to compare validation results between papers. Additionally, there remain some problems with the RWR method, particularly its reliance on the known gene distribution, which is unlikely to reflect the true distribution of genes associated with the trait. It is also apparent that even with the bidirectional extension of QTL there remains a tendency to over represent the centers of chromosomes. Given the central importance of gene distribution to RWR, addressing this particular failing will produce a method that is much more effective at identifying QTL of interest, and will therefore improve the rate at which breeding and fine mapping can be accomplished. The probability associated with the selection of a particular marker, P, as the origin for a particular QTL is calculated by assessing the number of locations in the genome on which the QTL can be placed. P is determined by the number of markers that can be used as the origin O of the QTL of length L, a number which excludes all markers that would result in the QTL extending beyond the end of the chromosome. This origin-based model of QTL mapping is an approximation for the true process of QTL placement, wherein QTL are roughly centered on a marker and are terminated at a marker on either end which represent the 95% confidence intervals for that QTL. In the case where the original QTL were not terminated at markers, it is preferable to use a model in which the QTL is centered on the chosen marker. Models requiring that the QTL must both start and end on a marker are not feasible, vertical farming aeroponics because the distance between markers is not uniform; with this constraint, a QTL of a given length might have only one possible genomic location. Markers are used in a direction-independent manner to avoid under representation of the ends of the chromosomes.

Under the null hypothesis, the probability of using any marker is {0,1,2}/M, which can take on any value between 0 and 1, inclusive. Here, the numerator depends on whether the marker can serve as O with unidirectional QTL extension, bidirectional QTL extension, or with neither; M refers to the total number of markers from which the QTL can extend to the left plus the total number of markers from which the QTL can extend to the right . Although the denominator could be adjusted to take into account the fact that a single QTL can only be mapped to one chromosome, this is unnecessary, because a QTL is not equally likely to be mapped to every chromosome. To understand why, consider the most obvious strategy to account for differences in chromosome length and the number of usable markers: a weighting scheme. We would divide the number of usable markers on each chromosome by the total number of usable markers in the genome . If this weighting is then used to adjust the probability P of using any marker on that chromosome, which is already proportional to 1/MC, the chromosome-specific marker counts cancel out and leave only the whole-genome marker count. To best represent the topology of the genome, the SPQV simulates genetic loci by selecting genes at random from the whole genome gene distribution to represent the genetic basis of the trait of interest. The use of the whole genome gene distribution as a source accounts for the topology of the genome, including the decrease of gene density at the centromere and telomeres . This strategy assumes that the true distribution of genes associated with a particular trait is approximately the same as the distribution of genes on a whole, rather than the assumption used in the simplest instance of RWR: that the distribution of known, previously associated genes reflects the true distribution of genes associated with that trait. We argue that this novel assumption is more likely to be accurate because trait-related genes can easily be discovered in a spatially biased manner : tandem arrays promote clustered discovery, some transposons involved in transposon-mediated mutagenesis preferentially target certain sequences , and genes in regions close to the centromere tend to be difficult to identify through methods that rely on recombination .

Additionally, the genome is interconnected; many traits rely on the interaction between multiple, seemingly disparate biological processes. Use of a random distribution for simulating genes with the SPQV is also possible, but fails to capture the genomic topography. Because genes within functional groups are not randomly arranged, duplication events and gene clusters in the original set of known genes are taken into account by considering genes without a marker between them as one genetic unit. The SPQV values are clearly most similar to those produced by the RWR experiment that was closest to biological reality: marker-only QTL origins, bidirectional mapping, and no bounce back . This makes sense, as the SPQV method is designed as a smoothed version of an experiment with these characteristics. Restriction of QTL origin to the markers that were used in mapping leads to an increase in EGN for RWR . This effect occurs for all QTL lengths. It is likely that the increase of identified genes in the context of restricted QTL placement is attributable to the physical structure of chromosomes: the markers selected for QTL mapping have a similar distribution to the genome wide distribution of genes , and are therefore relatively sparse in gene-poor regions such as the centromere. Similarly, the use of bounce back leads to an increase in RWR identified genes for all lengths of QTL, though this increase is particularly noticeable for some of the larger QTL . It is likely that the relatively large number of genes situated close to the ends of chromosomes is the main contributor to the impact of bounce back on identified gene number. It is possible that this reduction is due to a smoothing of the distribution, as the occurrence of bounce back is effectively split in half over the two separate tails of the chromosome. The presence of long and short arms on chromosomes, and the corresponding lopsidedness of the gene distribution, might also contribute to this phenomenon .

The use of bidirectional mapping appears to result in fewer genes identified by RWR, though this effect is relatively minor when compared to the effects of origin restriction and bounce back. In spite of the prominence of dark colors on the left side of the heat map, the confidence intervals identified for small and medium QTL by the SPQV and by RWR are fairly similar regardless of RWR method . The CIs in this range were consistently far below 1 regardless of method. In all, the SPQV 95% confidence limit for small QTL tends to be slightly smaller than the one produced by the RWR method that takes the same biological realities into account . However, this makes little difference in practice, because they are both less than 1: because observed gene counts are integers, EGNs from either method will be rounded up . In other words, if an SPQV confidence limit is defined as 1.2, the QTL of interest must have an observed gene content of 2 or more genes to be considered significant during general use. For larger QTL, SPQV values tend to outsize those produced by RWR. These large QTL approach the size of a full chromosome, and can indeed be larger than several chromosomes within the S. italica genome. It is the authors’ opinion that this is not an overestimation for the true distribution of genes associated with the trait of interest, as the true distribution likely has more than the known number of genes. Because of this, significance is unlikely for very large QTL,vertical indoor hydroponic system except for in the case of a true distribution of genes that is extremely uneven at the chromosomal level. A reduction in tiller number is a classical domestication trait in maize . Modern maize lines have been bred to grow as single stalked plants to facilitate high-density planting, while the maize progenitor, teosinte, is highly tillered. The genetic network associated with tiller suppression is controlled by the teosinte branched1 gene that also controls several other aspects of maize morphology , including inflorescence and floral architecture. Several mapping populations made from crosses between the W22 maize inbred line and teosinte were recently described and used to map several domestication traits . As expected, several domestication traits associate tightly with tb1 pathway. Here, we use the QTL reported by Chen et al. 2019 to illustrate the utility of the SPQV. Only the QTL with the same effect direction in both maize/teosinte mapping populations were assessed. Seven genes closely associated with the tb1 pathway in maize were located in the Zm-W22 NRGene 2.0 assembly and analyzed using SPQV. Notably, these genes were selected based on their strong, known associations with the branching pathway in maize. Since only high-confidence genes can be used accurately with our method, if any gene were not truly associated with the trait of interest, its presence will render the SPQV more stringent than necessary.

The QTL identified for the traits BARE , EB , GLUM , KRN , STAM and TILN have previously been connected with the tb1 pathway in maize. These QTL were therefore assessed in relation to the seven genes in Table 1. The markers found in the W22 x TIL01 RIL sub-population were used to determine the base pairs associated with the CIs of these QTL. Where the end points of the QTL did not have an exact match to a marker, the next closest marker was used so as to mimic ‘extension’ style mapping. The results of this analysis are reported in Table 2-2. Four of the assayed QTL identified a gene from the tb1 pathway, corresponding to four out of six of the represented traits. If the various adjustments described in this paper are applied to the RWR-based assessment of QTL mapping experiments, a more apt confidence limit for the expected number of genes will be identified. These adjustments do not account, however, for all of the issues associated with the application of RWR to this particular variety of question. RWR not only continues to rely on the distribution of known genes, but also results in gene-count distributions that nearly always fail to meet the requirement for smoothness . These distributions, in other words, have a tendency to change abruptly, and are frequently binary in the case of small and very large QTL. The ‘unsmoothness’ of any given distribution will be exaggerated by small QTL size and short lists of known genes; a known gene list with fewer than one gene per chromosome, for example, would produce a binary distribution even for large QTL. Additionally, RWR continues to exhibit a reduced likelihood of the QTL falling in the regions [1,1+L] and [C–L, C] even with the adjustment for bidirectional QTL extension. Finally, a practical weakness of the application of considered RWR is that this procedure requires a great deal of thought, effort, and expertise, and there are many points in the procedure at which simple errors can produce dramatic changes in the confidence limits that are ultimately produced. In light of the flaws of naive RWR, and the complexity of making the suggested adjustments, we recommend using the SPQV to assess the quality of QTL mapping experiments. The function provided, SPQValidate, requires only a few lists of data; the function itself accomplishes the analytic work that might be a stumbling block in RWR. Many of the other problems inherent to RWR are overcome by the SPQV’s probabilistic nature. This tool is potentially overly conservative, however, in the case of short QTL. It is extremely unlikely that the SPQV will produce a value of 0 for the confidence limit, as any locus is likely to be within range of at least one marker for even the shortest identified QTL. Because of this, the minimum confidence limit is, in practice, 1, which might be misleading for small QTL. Additionally, the total number of genes in a QTL is not necessarily an authoritative measure of a QTL’s validity; one can imagine that a QTL located on a single gene of high impact might be considered non-significant if the SPQV is the only method of validation used.

Original leaves were designated as leaves present on the seedling at the beginning of the experiment

High concentrations of Na+ in the cytoplasm disrupt the ionic balance and the uptake of essential mineral nutrients, such as K+, which in turn causes adverse effects on many metabolic pathways. To cope with salt stress, plants have evolved various tolerance mechanisms including two transport processes at the single cell level. Either exporting Na+ out of the cell, or compartmentalizing excessive Na+ into the vacuole. These two transport mechanisms act in a coordinated manner to maintain a low Na+ concentration in the cytoplasm. However, it remains unknown if they are regulated by the same or different signaling pathways. The SOS pathway is generally viewed as a signaling mechanism for the activation of the Na+ efflux through SOS1, a NHX-type Na+/H+ exchanger in the plasma membrane. The loss of function of SOS genes thus results in hypersensitivity to NaCl, coupled with the Na+ over-accumulation in the cytoplasm. On the other hand, some Na+/H+ exchangers are localized in the tonoplast and may be involved in transporting Na+ from the cytoplasm to the vacuole. However, the exact role of different NHX isoforms responsible for salt tolerance remains unclear. Interestingly, the two distinct but inter-connected salt transport processes appear to be both regulated by calcium signaling, in which calcineurin B-like proteins are thought to be the primary calcium sensors during salt stress adaptation. Among them, CBL4 and CBL10 display distinct tissue expression patterns and subcellular localizations. The spatial specificity of these two calcium sensors may contribute to their functional diversification in salt stress adaptation. In order to understand how they work synergistically in the regulation of salt tolerance,plastic pots 30 liters we genetically analyzed the salt-sensitive phenotype of the cbl4 cbl10 double mutant in comparison with the single mutants.

The cbl4 cbl10 double mutant was dramatically more sensitive to salt as compared to the cbl10 and cbl4 single mutants, suggesting that CBL4 and CBL10 either functionally overlap or each directs an independent salt-tolerance pathway. If the two CBLs are functionally overlapping, they should regulate the same transport processes and then the double mutant should not only show more severe phenotype but also show more severe deviation in the Na+ and K+ contents as compared to the wild-type plants. However, that was not the case: cbl4 and cbl10 displayed generally opposite Na+ and K+ profiles. Although the cbl4 cbl10 double mutant plants showed Na+ over-accumulation compared to the wild type, but significantly lower Na+ content than the cbl4 single mutant . This suggests that CBL10 should not be involved in the CBL4-regulated Na+ extrusion process , although these two calcium sensors interact with a common downstream kinase CIPK24 . Instead, CBL10 should regulate a distinct Na+-transport process in response to high salt, probably the Na+ sequestration into the vacuole, as suggested by its tonoplast localization and the lower Na+ content in the cbl10 mutants. This is consistent with the general theme that the Na+ efflux or Na+ sequestration into the vacuole both contribute to salt tolerance and disrupting either may result in elevation of the Na level in the cytoplasm and thus leading to salt sensitivity. Certainly disrupting both transport processes would lead to more severe salt sensitivity, which match the more sensitive phenotype of cbl4 cbl10. Previous studies suggested that CIPK24 serves as the common downstream target of CBL4 and CBL10 by forming CBL4-CIPK24 or CBL10-CIPK24 complex at the plasma or vacuolar membrane separately. Although our findings in this study supported this hypothesis, they also suggested that other CIPKs, in addition to CIPK24, should be also involved in the CBL10-mediated pathway based on the genetic evidence that double mutants of cbl4 cbl10 and cipk24 cbl10 displayed a significant enhancement in Na+ sensitivity as compared to cipk24 .

Indeed, screened by the yeast two-hybrid assay, we found that CBL10 did interact with other CIPKs in addition to CIPK24 . Various combinations of CBL10 with different CIPKs may target different target proteins and exhibit diverse functions. To examine whether SOS1 is a downstream component of CBL10 in the pathway, we also compared the salt sensitivity between sos1 cbl10 and sos1. In our test conditions, the salt sensitivity of cbl4 cbl10 and sos1 cbl10 was comparable to sos1 , suggesting that SOS1 may serve as aconverging point for the two CBL pathways. However, the double mutants cbl4 cbl10 and sos1 cbl10 accumulated much lower Na+ content than the single mutants of cbl4 and sos1, respectively, under salt conditions , which implies that CBL10 and SOS1 functions in two different transport processes in regulating Na+ homeostasis. For instance, in the sos single mutants in which the Na+ efflux is blocked, the CBL10 pathway functions to transport Na+ into the vacuole leading to the over-accumulation of Na+ in plant tissues. When the vacuole sequestration is defective in the cbl10-associated double mutants, the Na+ uptake is inhibited as a feedback of lacking storage space, leading to less accumulation and thus lower Na+ content in these double mutants as compared to the sos single mutants . Despite overall lower Na+ content in plant tissues, the double mutants showed similar salt sensitivity as sos1 because the majority of Na+ in these double mutants is in the cytoplasm effectively causing toxicity. Our results thus provide an example where a two-tier evaluation system must be implemented for dissecting salt tolerance mechanism in plants: First by whole-plant phenotyping and further by the analysis of Na+/K+ homeostasis . Concerning the target transporters for CBL10, all evidence so far supports the hypothesis that the CBL10-CIPK pathway may regulate Na-transporters in the tonoplast. Sequestration of Na+ into the vacuole is presumably fulfilled by an array of Na + transporters that include the vacuole-localized NHX-type Na+ /H+ transporters.

However, recent genetic evidence indicates that vacuole-localized antiporters NHX1-4 have Na+-transport activities but may not contribute much to the vacuolar Na+ compartmentation, because the quadruple knockout mutant nhx1/2/3/4 is not more sensitive to NaCl than the wild type. Furthermore, vacuoles isolated from the quadruple mutant still retain the Na+ uptake that is independent to the pH gradient, implicating the presence of NHX-independent Na+ transporters in Arabidopsis vacuoles. We speculate that some of these unknown transporters may serve as CBL10-CIPK targets. On the other hand, endosomal compartments emerge as critical players that may be directly involved in controlling Na+ homeostasis. A possible but yet to be proved model is that the Na+ sequestration into the plant vacuole may actually be achieved, at least in part,round plastic pots through endosomal Na+ scavenging processes and subsequent fusion to the vacuole. NHX5 and NHX6 are localized to endosomal compartments and associated with protein trafficking from the Golgi/Trans-Golgi Network to vacuoles. Supporting this hypothesis is the finding that disruption of two endosomal NHXs in the nhx5 nhx6 double mutant showed increased sensitivity to salinity . Considering the fact that a proportion of the CBL10 protein was also localized to the dynamic endosomal compartments, NHX5/6 could also act as the candidate targets of the CBL10-CIPK complexes. In a recent work, translocon of the outer membrane of the chloroplasts 34 was identified as a novel interaction partner protein of CBL10 at the outer membrane of chloroplasts, clearly indicating that CBL10 can relay Ca2+ signals in more diverse ways than currently known.Identification of target transporter directly regulated by the CBL10-CIPK module is an important and challenging task for future research, which would also unravel the pathway through which Na+ is deposited into the plant vacuole. Treated wastewater, commonly called reclaimed or recycled water, is a valuable water source in arid and semi-arid areas where fresh water sources are becoming increasingly scarce due to urbanization and climate change . Reclaimed water may have many beneficial applications, including agriculture irrigation and landscape irrigation. In the state of California, these irrigation uses account for 37% and 18%, respectively, of the 650,000 acre-feet per year of water reuse . State policy calls to increase the use of reclaimed water to more than 2.5 million acre-feet per year by 2030 . Accompanying increased reuse, the presence and environmental risks of unregulated organic contaminants in reclaimed water are drawing attention . Pharmaceutical and personal care products and endocrine disrupting compounds are typically anthropogenic chemicals with known biological effects that may interfere with normal metabolism and behaviors of organisms .

Many PPCP/EDCs are routinely found in reclaimed water , as well as in surface water impacted by wastewater treatment plant effluent and in groundwater . When reclaimed water is used for irrigation, the associated PPCP/ EDCs may interact with the soil matrix and may contaminate groundwater and food crops . Accumulation of PPCP/EDCs into food crops that are consumed fresh, such as many leafy vegetables, is relevant due to the likelihood of unintentional human exposure. If research demonstrates that accumulation of PPCP/EDCs by crops is unlikely to result in human health risks, this will provide scientific basis to promote use of reclaimed water, as well as enhance positive public perception of water reuse. Many factors influence the uptake of organic compounds into plants, such as by affecting diffusion through cell membranes. Briggs et al. suggested that chemical hydrophobicity is an important factor affecting uptake by diffusion and that chemicals with a log Kow of 1 – 3.5 have the greatest plant uptake potential because lipid and aqueous solubility are balanced . In addition to hydrophobicity, molecular ionization has also been shown to influence plant accumulation, such as of herbicides . Charged molecules may have a reduced potential for plant uptake, since ionization may reduce their ability to permeate cell membranes . However, the role of ionization is poorly understood and exceptions have been noted . To date only a handful of studies have considered plant uptake of PPCP/EDCs . While these studies have clearly shown the ability for plants to take up PPCP/EDCs, the state of knowledge is limited to a few compounds or plant types. Due to the analytical challenges of detecting chemicals at trace levels in plant matrices, most studies also relied on the use of artificially high concentrations, with a few exceptions . In this study, we comparatively determined the accumulation of four commonly occurring PPCP/EDCs, i.e., bisphenol A , diclofenac , naproxen , or nonylphenol , at relevant environmental levels into two leafy vegetables, lettuce and collards, and examined the composition and distribution of accumulated residues. These compounds have been frequently detected in reclaimed water and surface water , and have different ionization states at neutral pH. To achieve realistically low concentrations while affording quantitative measurement, 14C-labeled compounds were used. Results were used to infer effects of plant type and compound characteristics on plant accumulation and estimate probable human intakes. Following 21 d of hydroponic cultivation, plants were sacrificed for analysis of 14C accumulation and distribution. Each whole plant was rinsed with DI water, and then separated into roots, stems, new leaves, and original leaves.Individual plant samples were placed in pre-weighed metal screen pouches, weighed to determine wet weight, and dried at 50 °C for 60 h. After drying, each plant sample was weighed to measure the dry weight, and then chopped and mixed in a stainless steel coffee grinder. The grinder was rinsed between samples with DI water and methanol to prevent cross contamination. Multiple 150 mg sub-samples of each plant sample were analyzed until standard deviation of the sub-samples was below 20%, due to notable variation in plant tissue activity. Sub-samples were combusted on an OX-500 Biological Oxidizer at 900 °C for 4 min, and the evolved 14CO2 was trapped in 15 mL of Harvey Carbon-14 cocktail . The 14C was measured on a Beckman LS 5000TD Liquid Scintillation Counter . Recovery was 91-96% for spiked standards,which was used to correct for the actual activity. The activity and weight of the sub-samples were used to determine the total radioactivity accumulated in different tissues of each plant. Analysis of 14C by combustion provided information on total residue in plant tissues. To better understand the nature of the residue, plant samples were solvent extracted using a method modified from Wu et al. . The fractions of 14C in solvent-extractable and nonextractable forms were separately determined.

Cuttings were placed in pots with approximately 400 cm3 of potting soil in a greenhouse

One and two weeks after inoculation, the roots were carefully removed from the Magenta jars, were rinsed, and were prepared either for GUS-staining or for viewing under a Zeiss Axiophot fluorescent microscope. Nodulation in potting soil. Stem cuttings of the transgenic alfalfa plants were made as described above and allowed to root.One week before inoculation, nitrogen nutrition was withdrawn from the plants, but other macronutrients were supplied. The potting soil was leached with large quantities of tap water four and one days before inoculation. Rm1021 cells were grown in RDM medium , containing 100 mg of streptomycin per liter to an OD600 of 0.11 or 0.13, depending on the experiment. Rhizobia were pelleted in a clinical centrifuge and were suspended in sterile milli-Q water to an OD600 of 0.1 . Rm1021 suspension was placed on the surface of the potting soil of each plant. The plants were grown for 21 dpi. Stems were cut off at the crown, and the potting soil was gently removed from the nodulated roots in standing tap water. The nodules were separated from the roots, were divided into pink and senescent types, and were counted. The external morphology of the nodules was also examined. Nodulation in Turface rooting medium. Rooted cuttings were placed in pots with approximately 400 cm3 of inert Turface rooting medium and were allowed to grow in the presence of a complete, dilute nutrient solution. One day before inoculation, nitrogen was withdrawn from the plants. Rm1021, grown to early stationary phase,barley fodder system was prepared as described above. Rm1021 suspension was inoculated onto each plant. The plants grew for 34 more days, and then, the stems were cut off at the crown. The Turface was removed from the nodulated roots in standing tap water. The external nodule morphology was examined. Nodulation under hydroponic conditions. Stem cuttings were allowed to root as described above.

Fluorescent light grates were covered with aluminum foil, and individual square openings of the grate, five to six squares apart, were cut out for placement of plants. The rooting medium was gently removed from the roots of the cuttings by placing them in standing tap water. The crown of each rooted cutting was wrapped in cotton and firmly wedged into an opening of a fluorescent light grate. Rooted cuttings were spaced evenly, with 30 cuttings per grate. Each grate was placed on top of a tank containing 30 liters of complete 1 /4-strength Hoagland’s medium. Tanks were continuously aerated with aquarium pumps. Six independent vector control, 12 independent LEC1AS, and 12 independent LEC2AS plant lines were used in each of six hydroponic tanks. The entire assembly of 30 plants could be removed and replaced relatively undisturbed from the medium. The complete nutrient solution was replaced with 30 liters of ¼-strength Hoagland’s medium lacking nitrogen. Five days after medium replacement, a suspension of Rm1021, prepared as described above, was uniformly mixed into the medium, and the roots were returned to the solution. Rm1021 inocula from mid-lag, early-exponential, late-exponential, early-stationary, or stationary phase were used. The liquid level of the hydroponic tanks was maintained by adding deionized water. The plants grew for an additional 28 to 37 dpi. Stems of nodulated plants were cut at the crown, were dried under vacuum at 45°C for 2 days, and were weighed. The nodulated roots were pooled for each plant type and were stored at –20°C; the nodules were later separated from the roots. Nodules and roots were dried under vacuum for 2 days at 45°C and were weighed. Some nodulated roots were left intact, were allowed to dry at room temperature under ambient conditions for 14 days, and then were weighed. Uninoculated plants were removed from the hydroponic conditions, were dried in the greenhouse for 1 week, and then, the roots and vegetative tissues were separated at the crowns and weighed. With the growing population and limited freshwater resources, there is increased interest in water conservation practices like using recycled wastewater and hydroponic agriculture. The presence of pathogens in the associated environmental compartments exposes a large fraction of the general populace to infection risks. Therefore, a need of the hour is ensuring that our infrastructure meets the safety requirements designed to protect human health.

Proper disposal and treatment of wastes generated at hospitals, industries and residences help meet this goal by reducing the pathogen loads in the environment. However, complete elimination of pathogens is not an option. Therefore, a framework to quantify the threat to human health is desired. The popularly adopted framework is called Quantitative Microbial Risk Assessment or QMRA. With the growing population and limited freshwater resources, there is increased interest in water conservation practices like using recycled wastewater and hydroponic agriculture. The presence of pathogens in the associated environmental compartments exposes a significant fraction of the general populace to infection risks. Therefore, a need of the hour is ensuring that our infrastructure meets the safety requirements designed to protect human health. Proper disposal and treatment of wastes generated at hospitals, industries, and residences help achieve this goal by reducing the pathogen loads in the environment. However, complete elimination of pathogens is not an option. Therefore, a framework to quantify the threat to human health is desired. The popularly adopted framework is called Quantitative Microbial Risk Assessment or QMRA. The main tenets of QMRA are as follows: 1) hazard identification; 2) exposure assessment; 3) dose-response modeling; 4) risk characterization, and 5) risk management. Hazard identification constitutes deciding on the system of interest and listing out the pathogens present/expected in that system. After identifying the hazard, the interaction of people with the system are modeled to quantify exposure to the pathogen. Suppose the system of interest is a particular lake used for recreation, and the hazard identified is E. coli. Exposure assessment would entail enumerating the E. coli finally ingested by the person . These processes have a lot of associated variability and uncertainty. Therefore, quantities are stratified by groups or represented by distributions rather than point estimates. Estimating the risk while accounting for these variabilities and uncertainties is done by Monte Carlo sampling. Dose-response models relate the number of the pathogen to the probability of a person falling ill . They are constructed with data from clinical trials in which a predetermined dose of pathogens is administered to a cohort of subjects and the number falling ill counted. The latter is then divided by the total number of subjects to reflect the probability of a single person falling ill.

This process is repeated for different pathogen doses to generate data for the models. While these clinical trials may use animals, datasets generated from human trials are preferred since they better reflect the human situation. Popular DRMs are the exponential and beta-Poisson models. DRMs for different pathogens may share the same functional form but differ in the numerical values of model parameters as a consequence of the biological differences between the pathogens. Risk characterization involves calculating the risk posed by the hazard by integrating the output of the exposure assessment with the DRM of choice for that pathogen. One then compares these estimates with guidelines established by the U.S. Environmental Protection Agency or the World Health Organization . Based on these comparisons, risk management measures can be investigated in an iterative process by computing the risk posed by the intervention measures. Understanding the risk posed by ARB has been stymied by the absence of DRMs parameterized for ARB. This difficulty arises from the clinical trials used to parameterize current DRMs, which were performed using antibiotic sensitive bacteria . While we have invitro kinetic information relating ARB to ASB, the biophysical/kinetic interpretation of the parameters of the popular exponential and beta-Poisson DRMs is not straightforward. Moreover,hydroponic barley fodder system the dose-response outcome is potentially complicated by the other processes at play, such as horizontal gene transfer and the differential influence of antibiotics on ASB and ARB death rates. The resulting illness may or may not respond to antibiotic treatment if the ARB sub-population persists. These challenges require a mathematical framework capable of handling the underlying processes, which can then be used to perform risk assessments of ARB and determine the best course of action. A point of longstanding debate in QMRA, and broadly the topic of disease progression, is the hypothesis of independent action. It proposes that pathogens act independently of one another, and each has a probability p of initiating infection. The alternative hypothesis is one of cooperation where infection is expected when more than one organism survives to overwhelm the host’s defenses collectively. DRMs assuming independent action have wider acceptance than DRMs which assume cooperativity. However, DRMs with cooperativity consider the cumulative effects of bacteria but not the potential synergistic interactions between bacterial cells or quorum sensing. I believe that incorporating cell-cell interaction in dose-response is an essential step to developing a better understanding of the development of disease and its treatment.Risk estimates for lettuce grown in the hydroponic tank or soil are presented in Fig. 2.4. Across these systems, the FP model predicted the highest risk while the 1F1 model predicted the lowest risk.

For a given risk model, higher risk was predicted in the hydroponic system than in the soil. This is a consequence of the very low detachment rates in soil compared to the attachment rates. Comparison of results from Sc1 and Sc2 of soil grown lettuce indicated lower risks and disease burdens under Sc1 . Comparing with the safety guidelines, the lowest risk predicted in the hydroponic system is higher than the U.S. EPA defined acceptable annual drinking water risk of 10−4 for each risk model. The annual burdens are also above the 10−6 benchmark recommended by the WHO. In the case of soil grown lettuce, neither Sc1 nor Sc2 met the U.S. EPA safety benchmark. Two risk models predicted borderline disease burden according to the WHO benchmark, for soil grown lettuce in Sc1, but under Sc2 the risk still did not meet the safety guideline. I found that neither increasing holding time of the lettuce to two days after harvesting nor using bigger tanks significantly altered the predicted risk . In comparison, the risk estimates of are higher than range of soil grown lettuce outcomes presented here for 2 of 3 models. The SCSA sensitivity indices are presented in Fig. 2.5. For hydroponically grown lettuce, the top 3 factors influencing daily risk are amount of lettuce consumed, time since last irrigation and the term involving consumption and ρshoot. Also, the risk estimates are robust to the fitted parameters despite low identifiability of some model parameters . For soil grown lettuce, kp appears to be the major influential parameter, followed by the input viral concentration in irrigation water and the lettuce harvest time. Scorr is near zero, suggesting lesser influence of correlation in the input parameters. To predict viral transport in plant tissue, it is necessary to couple mathematical assumptions with an understanding of the underlying biogeochemical processes governing virus removal, plant growth, growth conditions and virus-plant interactions. For example, although a simple transport model without AD could predict the viral load in the lettuce at harvest, it failed to capture the initial curvature in the viral load in the growth medium . An alternative to the AD hypothesis that could capture this curvature is the existence of two populations of viruses as used in, one decaying slower than the other. However, I did not adopt this approach as the double exponential model is not time invariant. This means that the time taken to decay from a concentration C1 to C2 is not unique and depends on the history of the events that occurred . Other viral models, such as the ones used in faced the same issues. Incorporating AD made the model time invariant and always provided the same time for decay between two given concentrations. This model fitting experience showcases how mathematics can guide the understanding of biological mechanisms. The hypothesis of two different NoV populations is less plausible than that of viral attachment and detachment to the hydroponic tank. While it appears that incorporating the AD mechanism does not significantly improve the accuracy of viral load predictions in the lettuce shoot at harvest, this is a consequence of force fitting the model to data under the given conditions.

Potential agricultural losses are exacerbated by a history of pesticide resistance development

Many antibiotics and other common contaminants of emerging concern can be excreted by both humans and animals with little change in their chemical structure . Not surprisingly, pharmaceuticals have been appearing in wastewater, surface waters, and in some cases tap water, over the past few years . Standard wastewater treatment facilities are not equipped to completely remove pharmaceuticals , resulting in these compounds being found in effluent. In addition, even higher concentrations of many pharmaceuticals are released during heavy storms in the untreated wastewater overflow, which then directly contaminate the environment . These pharmaceuticals have been found at biologically active concentrations in surface waters around the world . There is also an increasing effort to use reclaimed wastewater in drought-affected areas, such as Southern California . In agriculture/livestock operations, pharmaceuticals are found in manure that is used as fertilizer, effectively compounding the pharmaceutical concentrations . Current research shows these chemicals tend to be both pseudopersistent in soil and detrimental to soil and rhizosphere microbes . Our recent studies of the effects of pharmaceuticals on aquatic insects show that, at concentrations found in reclaimed water, these CECs can alter development of the mosquito Culex quinquefasciatus, its susceptibility to a common larvicide, and its larval microbial communities . Watts et al. showed 17α- ethinylestradiol, a common birth control agent, and Bisphenol-A, a common plasticizer, can cause deformities in the midge Chironomus riparius. However, because larval forms of aquatic insects develop directly in the contaminated water, their constant exposure is likely greater than most terrestrial insects. Interestingly, many CECs, which were not designed specifically to impact microbes, have been shown to affect microbial communities. For example, caffeine, a common mental stimulant, can alter biofilm respiration, and diphenhydramine, an antihistamine,hydroponic dutch buckets has been shown to modify the microbial community of lake biofilms . Due to such unexpected effects, accurately predicting the consequences of specific CECs, even in model insects, is not yet possible.

This problem is exacerbated by a lack of information regarding effects of pharmaceuticals and other CECs on the microbial communities of any terrestrial insects. Arthropods, such as insects and crustaceans, rely on hormones to grow, develop, mate, and produce pigmentation . However, many pharmaceuticals, especially mammalian sexhormones, are structurally similar to chemicals that these organisms rely on for growth and development. These pharmaceuticals then bind to receptors and either over express or suppress their counterparts’ natural function. This has been seen in birds, reptiles, and arthropods where endocrine disruption occurs, primary and secondary sexual characteristics are modified, and courtship behaviors are changed . Although most arthropod hormones do not closely match those of mammals, their molting hormone is very similar in structure to the mammalian female sex hormone 17β-estradiol. In crustaceans, mammalian hormones have been known to cause both increased molting events and inhibition of chitobiase, the enzyme responsible for digestion of the cuticle during insect molting . In insects, 17α-ethinylestradiol, a common synthetic birth control hormone, has been shown to alter molting and lead to deformities of C. riparius . In addition to these effects, pharmaceuticals have been shown to have delayed cross-generational effects . The cabbage looper is a well-studied polyphagous insect native to North America and is found throughout much of the world . T. ni are yellow-green to green in color and can complete their life cycle in as little as 21 d depending on temperature . This species is a pest on many agricultural crops including crucifers and a variety of other vegetables in both field and greenhouse settings .Currently, there is little to no information regarding pharmaceutical effects at the concentrations found in reclaimed water on the growth or microbial community composition of any terrestrial herbivore. Many herbivores can be exposed to these contaminants after the CECs enter surface waters, soil, and plants from wastewater reuse and unintended discharge. To investigate the function of the gut microbes in insects, several studies have used antibiotics applied at high doses . There is also no information regarding effects of CECs when translocated through plants to terrestrial insects.

To test the hypothesis that common pharmaceuticals affect mortality, development, and microbial communities of T. ni, we conducted a series of bio-assays in artificial diet and on a key host plant utilizing surface water concentrations of common important pharmaceuticals. We used a culture-independent approach by performing a 16S rRNA gene survey on both diet and whole-body insects. Any effects would have potentially important implications from agricultural perspectives. Also, as there is currently no information on effects of CECs on terrestrial insects acquired through a plant matrix, our findings would have possible interest for integrated pest management research.In our study, CECs at concentrations found in reclaimed wastewater were shown to increase mortality of T. ni, especially on artificial diets contaminated with antibiotics, hormones, and a mixture of the chemicals. The mortality effect was also evident when T. ni were reared on plants grown in antibiotic-containing hydroponic growth media. Because plants grown in the hydroponic system contained quantifiable levels of ciprofloxacin in the leaf tissue , and the antibiotic treatments significantly changed the microbial community of the insect , we think this is possibly a cause of the mortality but we cannot exclude direct effects of the CECs on the insects or indirect effects through the plants. Ciprofloxacin is a quinolone topoisomerase IV and DNA gyrase inhibitor that acts by stabilizing the DNA-topoisomerase IV and DNA-girase so that it is no longer reversible . This blocks DNA replication and eventually causes cell death of bacteria. However, unlike bacteria, when higher-level organisms evolved, the A and B subunits of the topoisomerases fused, creating homodimers that cannot be targets of ciprofloxacin , and thus damage to the ribosomes of insects is not a possible mechanism of toxicity. Interestingly, we did not see the increased time to adulthood in T. ni reared on plants compared with those reared on contaminated artificial diet. We postulate the discrepancy is possibly due to a number of factors such as dilution of CECs, as they were acquired from the water by the plants or there was bio-degradation of the chemicals occurring in the plant or by photodegradation. However, recent studies have shown pharmaceutical concentrations in surface waters, which appear to remain constant over the course of several years . More studies would be needed to determine how CECs at concentrations found in reclaimed water for agriculture would interact with current IPM strategies , and how soil matrices would affect the chemical acquisition and translocation by plants. Many insects rely on microbial communities and endosymbionts to grow and develop; however, it has been shown that Lepidoptera species do not have a vertically transmitted microbial community .

In addition, because the effects of microbial communities on T. ni survival and development have not been documented, we present these data only to show that microbial communities change when exposed to CECs, and not as a proven factor influencing survival. We found significant shifts in the microbial community in the various life stages examined within the control treatments notably from third instar to subsequent life stages. A similar result has been reported for mosquitoes and other insects . However, there is one family, Lactobacillaceae, which appears in all treatments and life stages in high proportions, except for adults. They are fairly common in insects and can be responsible for at least 70% of the bacterial community . Lactobacillaceae is responsible for ∼42% of the bacteria in all life stages, followed by Pseudomonadaceae, Alcaligenaceae, and Enterobacteriaceae. Lactobacillaceae have been shown to act as beneficial bacteria in Drosophila ; however, its function in T. ni is still unknown. Alcaligenaceae has been shown to be present in other moths ,bato bucket but Lepidopterans are not thought to have a functional microbiome . There are clear patterns regarding the changes in microbial community proportionality according to the heat map . In controls, third-instar microbial communities are relatively evenly spaced by family. The microbial community becomes predominately Lactobacillaceae for sixth instars and pupae. Once the insects reach the adult stage, their most predominant family is Pseudomonadaceae. This pattern holds in the acetaminophenand caffeine treatment groups as well. Interestingly, the other treatment groups do not share this pattern. For antibiotic- and hormone-treated T. ni, Lactobacillaceae is the predominant microbial family in the immature stages, but at the adult stage microbial community reverts to predominantly Pseudomonadaceae. We suspect that this is because, once the larvae undergo metamorphosis and shed their gut contents in preparation for pupation, they are no longer exposed to the pressures exerted by the CECs on the microbial community. Fig. 3 provides a visual indication of the changes in the bacterial communities over time. The increase in β diversity after eclosion could be due to the larvae no longer being exposed to CECs or diet-borne bacteria after being moved to sterile containers. Also, when bacteria are lost as larvae digest their gut contents during pupation, the microbial β diversity could change. Interestingly, the hormone-treated T. ni follow a similar pattern to those exposed to antibiotics, but their ellipses are always much smaller, suggesting the entire insect population is showing a uniform response within their microbial communities. However, in the mixture-treated insects, larvae displayed a greater average diversity in their microbial community structure than either pupae or adults. This finding has not been shown in any single category of treatment, and we suspect the microbes exposed to mixtures could be experiencing potential interactive effects among chemicals . Such interactions should be the focus of future studies along with investigations of plant rhizosphere bacteria, particularly since we found a difference in the Bradyrhizobiaceae family for all treatments. These results show that a terrestrial insect pest of commercial crops can be affected by CECs found in reclaimed wastewater for agricultural use. Our results suggest that CECs found in wastewater can impact T. ni growth and development, survivorship, and alter their microbial communities. Because T. ni is a common agricultural pest found around the world, feeds on a wide variety of plants, and has a history of developing pesticide resistance, its ability to deal with toxins is likely higher than many other insects. In addition, the responses we observed to CECs could have interesting implications for IPM practices on plants such as lowering the amount of pesticides needed or increasing susceptibility to insect pathogens, as has been shown in mosquitoes . These potential effects may be understated because some insects cannot detect the presence of the pharmaceuticals . However, we do not recommend purposefully exposing crops to CECs specifically for the control of insects because our study documented that these pharmaceuticals are translocated into crops and we do not yet know their possible effects on humans if consumed . We specifically want to note that ingestion of these compounds through uptake and translocation by a plant is not the only way T. ni or any other insect would be exposed to these compounds. Overhead sprinkler irrigation could cause contact absorption by the plants or insects, and simply drinking water on leaves at contaminated sites could expose insects to higher concentrations than were found in plant tissues. In fact, the ciprofloxacin concentration used was less than one-third of the highest rate . We urge caution in extrapolating to plants growing in soil, because variation in soil type and potential soil bacterial degradation could affect persistence [although soil bacteria are often negatively impacted by CECs ]. However, CEC exposures are considered pseudopersistent because they are reapplied with each irrigation. Thus, the effects reported here are likely to be conservative. Additional studies with other insects, particularly those with other feeding strategies, will be necessary before any patterns can be discerned.The growth of the human population places an ever-increasing demand on freshwater resources and food supply. The nexus of water and food is now well recognized. One promising strategy to sustain food production in the face of competing water demands is to increase the reuse of treated human wastewater. Municipal wastewater reuse for food production has been successfully adopted in some regions of the world. For example, Israel uses ~84% treated wastewater in agriculture production .

Each offset credit is equal to one metric ton of carbon dioxide equivalent

Offset programs make cap and trade programs “more attractive and palatable” to covered entities, as offset programs provide more flexibility to determine the lowest-cost method of reducing greenhouse gas emissions.As long as an offset project will be cheaper than internally reducing emissions, capped entities will likely seek out credit for emission reductions through offset programs. Offset programs are beneficial for governments implementing cap and trade programs because it shows that they are trying to work with industry to find lower cost means of reducing emissions to the mandated level. Additionally, now that offset programs are widely implemented, it would likely be more difficult to gain support for a cap and trade program that did not include offset programs. Offset programs are also beneficial for sectors that are target hosts for offset projects because offset projects are a source of improvements and an income opportunity for the host.Typically, the project host receives financial incentives or some sort of technology, facilities, or practice upgrade that they could not afford or would not have undertaken otherwise. Thus, the benefits of offset programs not only affect the capped entities, but also sectors that are otherwise unaffected by the cap and trade program.In the United States, the U.S. Environmental Protection Agency estimates that agriculture accounts for 8% of the country’s greenhouse gas emissions.The EPA estimates that half of these agricultural emissions come from management practices of agricultural soils, including fertilizer application, irrigation, and tillage methods,hydroponic nft and that livestock manure management accounts for 15% of the agricultural emissions.The livestock digestion process accounts for about one third of the agricultural emissions and the remainder comes from smaller sources, such as rice cultivation and burning crop residues.

These estimates do not include carbon dioxide emissions from on-farm energy use.CARB’s Scoping Plan estimates that the agricultural sector contributes to about 6% of the total greenhouse gas emissions in California.CARB also includes estimates of emissions based on the end use rather than the actual source of emissions.When calculated in this manner, 9% of California’s greenhouse gas emissions can be attributed to agriculture and food processing industries.In general, the agricultural sector provides at least two strong avenues for reducing greenhouse gases through offset programs: decreasing emissions from raising livestock and sequestering carbon in agricultural soil.California has already incorporated an offset program that takes advantage of the opportunity to decrease livestock emissions by installing biogas control systems , which capture and destroy methane, on dairies and swine farms.An offset program that takes advantage of the second opportunity to decrease carbon concentrations in the agricultural sector by sequestering carbon in agricultural soil has been used in conjunction with other cap and trade programs and may provide an opportunity for an expansion of California’s offset programs in the future.Many livestock operations manage livestock waste by using anaerobic liquid-based systems in lagoons, ponds, tanks, or pits.61 Manure that is stored in this fashion emits methane,a powerful greenhouse gas that is estimated to have a radiative forcing power, or global warming potential, twenty-five times that of carbon dioxide.Manure management accounts for 15% of the agricultural greenhouse gas emissions in the United States, and CARB’s most recent estimates indicate that manure management accounts for 1% of California’s total greenhouse gas emissions.Even though manure management is not the largest source of agricultural emissions, California’s cap and trade program includes the Livestock Projects Compliance Offset Protocol , an offset program that issues offset credits in exchange for installing biogas control systems , a type of manure digester, on dairies and swine farms.BCSs capture methane from the livestock operation’s manure storage facility before it is released into the atmosphere.The Livestock Protocol permits the captured methane to be destroyed on-site, transferred offsite, or used to power vehicles, but “the ultimate fate of the methane must be destruction.”

The dairies and swine farms may then sell the offset credits that they produce through this offset program on AB 32’s carbon market.The Livestock Protocol is considered a standards-based offset protocol, as it “creates additionality thresholds for particular categories of projects instead of determining additionality individually for each project.”CARB’s standards-based approach for its current offset protocols came under attack in Citizens Climate Lobby et al. v. California Air Resources Board.70 Citizens Climate Lobby argued that CARB’s standards based approach results in non-additionality by issuing offset credits for greenhouse gas reducing projects that would have been completed anyway, which impermissibly enlarges the scope of AB 32, and that CARB should have adopted a project-byproject approach instead in order to perfectly determine whether each offset project is indeed additional to business-as-usual.In addition to determining that using a standards-based approach for offset protocols was within CARB’s authority, the court in Citizens Climate Lobby determined “that the Livestock Protocol is reasonably necessary to effectuate the purpose of [AB 32] and [CARB] was neither arbitrary nor capricious in its promulgation.”The court made this determination by reviewing evidence presented by CARB that less than 1% of dairies and swine farms in the United States install BCSs to dispose of manure, installing BCSs is not a standard or common practice, and that the cost of installing a BCS is the primary barrier to installation.Because farms were not installing BCSs despite other favorable conditions for installation, the court stated that it is not arbitrary for CARB to use installation as the standard to determine additionality.Thus, CARB maintains a standards-based approach, rather than a project-by-project approach, for its Livestock Protocol. The court reached a similar decision regarding CARB’s three other offset protocols.Agricultural soil carbon sequestration offset programs function like other offset programs, but the projects can include switching to conservation practices including no till, conservation tillage, planting cover crops, utilizing high-diversity crop rotation, and other agricultural practice changes in order to increase the amount of carbon sequestered in the agricultural soil and reduce the amount of emissions from farm machinery.

All of these practice changes increase carbon sequestration by differing amounts. The U.S. Environmental Protection Agency estimated that conservation tillage can sequester between .6 and 1.1 metric tons of carbon dioxide per acre per year.The U.S. Department of Agriculture estimated that planting cover crops and improving crop rotations and fallowing practices can sequester between .2 and .4 metric tons of carbon dioxide per acre per year.78 One assessment of the effects of conservation practices on cropland in the Missouri River Basin estimated that the studied area sequesters 9.9 million tons of carbon dioxide per year.Estimates of the global potential of soil sequestration vary greatly, but one estimate says .9 petagrams of carbon per year may be sequestered globally, which is enough to offset one-fourth to one-third of the annual global increase in carbon dioxide concentrations.Soil’s sequestration properties occur naturally when organic compounds produced by plants cycle through plants, animals, and microorganisms to create soil organic matter,hydroponic channel which is where carbon is stored in the soil.Carbon is released from the soil into the atmosphere when it is disturbed due to changes in water, air, and temperature conditions.Thus, reducing tillage increases the carbon sequestered in the soil, and the level of sequestration depends on many variables including soil type, weather, precipitation, temperature, and other factors. Aside from decreasing atmospheric carbon levels, the practices that encourage carbon sequestration boast local benefits such as reducing soil erosion and nutrient depletion while increasing water retention rates.A USDA project that ran from 2003-2006 assessed the effects of cropland conservation practices, including tillage management along with a host of other conservation practices that also sequester carbon, in the Missouri River Basin.84 The assessment determined that conservation practices decreased loads delivered from cropland to rivers and streams by 76% for sediment, 54% for nitrogen, and 60% for phosphorous.85 These dramatic reductions cannot all be attributed to changes in tillage management or other carbon sequestering practices, as those were only some of the measures among many diverse strategies for decreasing sediment and nutrient loss from agricultural soil. Additionally, the assessment noted that carbon that is sequestered in agricultural soil “improves the soil’s ability to function with respect to nutrient cycling, improves water holding capacity, and reduces erodibility through enhanced soil aggregate stability.”So, in addition to sequestering carbon, the conservation practices that are typically implemented in agricultural soil carbon sequestration offset projects provide many important benefits incidental to sequestering carbon.The EU ETS, the Kyoto Protocol, and RGGI, some of the most major carbon markets in the world, currently do not recognize offset credits that are generated from soil carbon sequestration projects.This is most likely “because soil carbon is viewed as difficult to measure, verify, and track.”88 However, some smaller markets recognize this opportunity for carbon sequestration and income for farmers, so some offset programs that generate credits for agricultural soil carbon sequestration are already in existence. In 2010, the World Bank implemented the Kenya Agricultural Carbon Project, which encourages “covering crops, crop rotation, compost management, and agro-forestry.”This method of farming generates credits that are sold to the World Bank’s BioCarbon Fund.Additionally, in 2012, the World Bank created a new farming methodology, approved by the Verified Carbon Standard,that encourages less plowing.Before the Chicago Climate Exchange’s closure in 2010, it verified and traded soil carbon offset credits generated by farmers in the United States using no-till practices.

The Oklahoma Carbon Program currently operates a voluntary program that verifies and issues credits for farmers who use conservation tillage.Canada’s guidance for its future offset programs indicates that it would include an agricultural soil carbon sequestration offset program to address the intensity of tillage operations, adopting crop rotations, and increasing cover crops.If the American Clean Energy and Security Act of 2009, better known as the Waxman-Markey bill, had been approved by both houses of the United States Congress, it is likely that agricultural soil carbon sequestration offset programs would have played a role through that legislation’s proposed nationwide cap and trade program.The agricultural industry was concerned that other industries’ products used by agriculture—fertilizer, diesel, electricity, etc.—would increase in price if the proposed legislation passed, in turn affecting the agricultural sector’s expenses.The National Corn Growers Association devised nine principles relating to cap and trade that had to be met before it would support any climate legislation bill.The first principle is that “[t]he agricultural sector must not be subject to an emissions cap.”The second principle asks cap and trade to “fully recognize the wide range of carbon mitigation or sequestration benefits that agriculture can provide.”The fourth principle expects the USDA to create the regulations and oversee an agricultural offset program.The fifth principle provides that “[t]he use of domestic offsets” is not “artificially limited.”This principle is directly at odds with current caps on offset credits that can be used to meet compliance obligations as in RGGI and AB 32’s cap and trade program. Additionally, the Illinois and Iowa Corn Growers Associations owned Novecta, a group that was working on standardizing a program to reward farmers for no-till practices.Agricultural soil carbon sequestration offset programs were also proposed and discussed in relation to the Lieberman-Warner bill, the cap and trade climate change bill that was introduced in the 110th Congress, just before the Waxman-Markey bill was introduced in the 111th Congress.Considering this significant support for agriculture offset programs, it is likely that an agricultural soil carbon sequestration offset program could have been implemented on a nationwide scale if Waxman-Markey Bill had passed. Overall, agricultural soil carbon sequestration offset programs prove to be attractive because the farmers implementing the change are paid to change their practices.This can be a welcome source of income, especially at a time when farmers, especially small-scale and those most affected by droughts and the changing climate, are having a difficult time maintaining productivity and income.Considering all the factors discussed above, it seems that a future natural step may be to adopt an agricultural soil carbon sequestration program to link to AB 32’s cap and trade program. The possibility has already been recognized in a bill that was proposed to the California State Assembly. The proposed bill stated that new offset programs will be needed in order to supply the highest number of useable offset credits allowed under AB 32.An early version of the proposed bill listed possible offset programs, including offset programs that maintain agricultural productivity while emitting less greenhouse gases—the idea behind soil carbon sequestration offset programs.

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.

Farmers can respond by shifting their production into less labor-intensive crops

However, domestic food demands continue to increase and diversify, creating important employment opportunities in the off-farm AFS. These changes mean that both traditional and new digital technologies can be leveraged to induce a productive exit out of agriculture in Sub-Saharan Africa while maintaining a competitive agricultural workforce on and off the farm in the chains elsewhere. Three key policy implications emerge. First, productivity-enhancing investment in agriculture must accelerate in the lower-income countries and proceed at least in tandem with the movement of workers off the farm elsewhere. Populations will continue to grow despite slowing birthrates, and food production will have to expand to keep pace. The movement of workers off the farm to meet the demand for other goods requires producing more food with fewer workers, once underemployed labor has been activated. Historically in today’s high-income countries, agricultural extension and public investments in infrastructure, from irrigation to information, marketing institutions, and roads, played a critical supporting role in facilitating the labor exit out of agriculture. They enabled the remaining farmers to earn a living commensurate with non-farm sectors, as competition for workers with the non-farm sectors and downstream food processors intensified. This agenda holds as much today as then. In Sub-Saharan Africa, the agricultural share of public spending continues to be well below that in East Asia . Myriad input, factor, and output market constraints hold agricultural labor productivity back, and integrated solutions that simultaneously overcome a number of these constraints are needed. Inclusive value chain development ,dutch bucket hydroponic which links farmers with buyers in contracting arrangements, offering knowledge, access to credit and inputs , and higher prices in exchange for a consistent volume of high-quality products , provides a market-based solution to do so, though smallholders’ lack of legal protections can be an obstacle .

Given the challenge to develop self-enforcing incentive compliant contracts, iVCD typically does not work well for raising staple crop productivity. Yet, in low income countries, this is where the need and scope for raising labor productivity and poverty reduction is highest. For raising labor productivity in staple crops, more and better public investment in public goods is needed . Second, the scope for iVCD to raise smallholder incomes and benefit the poor and women is greater for non-staples. iVCD also creates jobs off the farm, in the chains and beyond . Success factors of iVCD include careful diagnosis of the competitiveness and sustainability of the product value chain chosen, starting small, involving financial institutions, monitoring producer-buyer relationships, and sustaining capacity building. This is in addition to creating an economic environment that is conducive to investment generally. Developing systems to monitor and enforce food quality standards in the AFS is equally critical. There is clearly a role for agricultural ministries, as well as for the private sector, to ensure that the development and use of labor-saving technologies keeps pace with the movement of workers off-farm. Many questions remain, however, especially on the best entry points for support: through farmer organizations/cooperatives, large anchor firms and/or SMEs, or externally initiated stakeholder platforms. More experiments are needed. In the meantime, appropriate measures will be needed to help SMEs in the transformative food chains see through the decline in liquidity caused by COVID-19 and avoid undue concentration of activity in the long run. Labor-market regulations and other social protections can also be useful in protecting vulnerable populations from exploitation as they transition into non-farm work . Third, investment in people is critical to raise agricultural labor productivity and to make sure that those leaving can access the new jobs in the AFS, as well as other non-farm sectors, and meet the rising economic aspirations of rural youth.

Continued investment in quality rural education, which continues to largely underperform in developing countries, is needed . Increasing educational attainment in rural areas facilitates technology adoption, as well as occupational mobility, and reduces income inequality. This is also important for young women facing social norms that make it difficult to escape from traditional gender roles. Nontraditional skill-building programs and effective agricultural extension systems will be equally needed to build up human capital in regions where traditional education has proven ineffective. The extension system is particularly weak in Sub-Saharan Africa and has been largely neglected for the past couple of decades by governments and donors alike. The 2010s have witnessed a surge in studies on social network or farmer-to-farmer technology extension, which proves more promising especially in combination with public extension than traditional public-sector extension approaches. But several issues remain such as the choice and compensation of appropriate lead farmers . Policy implications are different, but just as immediate, in high income countries. Rich-country farmers will be required to produce more and higher-quality fresh and processed foods for a growing, and increasingly affluent, domestic and global population, and they will be required to do so under increasingly stringent environmental and animal welfare standards. However, they will have to do this with fewer workers. The transition of domestic workers out of farm work largely has run its course in rich countries. The option of importing foreign workers is gradually closing, due to a declining farm labor supply in farm labor exporting countries and a less supportive political environment for immigration, particularly of low-skilled workers, in high-income countries. Three key policy implications emerge for high-income countries in this era of growing farm labor scarcity: First, farmers in high income countries will increasingly need to look beyond immigration policy as an answer to farm labor scarcity —especially in the medium and long run.

Guest worker programs can expand as a short-run response to farm labor scarcity. However, as the structural transformation progresses in farm labor-exporting countries and political resistance to importing low-skilled farm workers intensifies, the immigration solution to the farm labor problem becomes less of an option. This does not mean that immigration will not continue to play a central role in farm labor markets throughout the developed world for some time. But farmers will need to take steps to retain an aging, mostly immigrant, workforce while pursuing available options to contract new workers from abroad. International farm labor migration could continue to be a much-needed channel for sharing prosperity across nations and reducing poverty in the world’s poorest countries. For this, however, a counternarrative needs to take hold rapidly. If not, its days may be numbered prematurely, especially now that the COVID-19 pandemic so clearly exposed the agri-food sector’s dependence on immigrant labor and the logistical challenges this may entail, eroding support for reliance on immigrant agricultural labor even further. Second, increasingly sophisticated technological change is going to be a fundamental feature of the food supply chain,dutch buckets system from farming to food processing. Productivity-enhancing investments likely will include the use of highly-advanced robotic systems that will dramatically reduce the need for workers . Scouring the landscape in today’s high-income countries, we find automation success stories like the ones described earlier in this paper, as well as major challenges. There is a danger that automation will not happen quickly enough to enable farmers to maintain their competitiveness in a high wage, labor-scarce, world.However, more affluent consumers will demand fresh, locally-grown fruits and vegetables, as well as specific qualities like organics, environmentally friendly production practices, fair trade, and possibly better labor practices, all of which tend to increase labor demands compared to field crops where automation is more advanced. Prices of these fresh fruits and vegetables will rise, causing farmers to think twice about abandoning production as wages rise while intensifying pressure on public and private researchers and policy makers to accelerate the development of labor-saving technologies and deploy the necessary digital infrastructure to run it, including in remote rural areas.

Policymakers will need to keep an eye out for undue concentration of power in the supply of these new technologies and devise adequate policies to ensure competition . Third, a technologically advanced AFS requires a technology-savvy workforce, with more engineers and people capable of working with increasingly complex technologies. As agricultural and food processing technologies become more IT intensive, so do human capital demands all along the AFS. To some extent, developments in IT can help respond to human capital shortages; viz. bar codes in supermarkets and hamburger buttons at fast-food restaurants. Nevertheless, the numbers of workers with little education who pick themselves a living wage will diminish. As new technologies become available for relatively easy-to mechanize crops and routine tasks, the farm workforce will move out of those crops and tasks into ones that have not yet been mechanized and are non-routine . A major policy challenge is to prepare the future farm workforce for technological change while also ensuring that employment opportunities expand as new technologies release workers from crop production. There is no magic bullet to guarantee that automation, human capital formation, and new job creation move apace. It is undeniable that the future holds far-reaching changes in mechanization and automation in developing and developed countries alike. Without it, agriculture and the AFS generally will not be able to keep up with rising food demands and a declining farm labor supply. Inevitably, many farms and farm workers will have difficulty adjusting. Some farms and farmers, particularly larger, wealthier and better educated ones, are in a far better position to experiment with and adopt new labor-saving technologies, including advanced robotics. And some farmers and farm workers, particularly older ones, will have a difficult time shifting to new commodities and tasks; the more technology-savvy farm workforce of the future is likely to be younger and better educated than current workers. Decoupling social insurance from employment, as proposed in Packard et al. , could be a worthwhile social insurance model to mitigate adverse consequences of this transition and avoid the introduction of ineffective agricultural and food policies. The need for greater food system resilience, highlighted by the COVID-19 experience, would also be better served by food trade diversification instead of a reversal to protectionism and food self-sufficiency. Yet, without successful social insurance schemes to help mitigate the adjustment costs and rapid ramp up in agricultural education and extension, the ongoing evolution in the agricultural labor force is bound to raise inequality as well as anti-trade sentiment, including in agri-food. Agricultural production has grown to meet the demands of an increasingly large and wealthy human population. The development of high-yield crop varieties combined with the widespread use of irrigation, synthetic fertilizers, pesticides, and land use changes that marked the “Green Revolution” have enabled an enormous increase in crop production per area . As a result of these technologies, cereal production has doubled . This increased production is credited with reducing poverty and improving nutrition intake for millions of people worldwide . However, this increase in production also has costs. There are concerns that the loss of natural enemies and biodiversity caused by the increased size and connectivity of agricultural land, the trend toward monocultures, and the conversion of natural habitat—termed “landscape simplification”—makes farms more susceptible to pest outbreaks . With increased risk of pest outbreaks comes enhanced pesticide use. Although other aspects of intensive farming also have negative externalities, such as synthetic fertilizers and eutrophication, pesticides have received some of the most widespread scrutiny and their reduction has become a priority for policy makers, as evidenced by integrated pest management . The emphasis on pesticide use stems from serious human health concerns related to pesticide exposure in farm workers , pesticide residues in food and water sources , and bio-accumulation of pesticides in higher trophic levels . Despite popular ecological thinking that the connection between landscape simplification and pesticide use is clear, both theoretical and empirical studies have found ambiguous results. Agroecological theory holds that landscapes composed of a high proportion of cropland are more susceptible to pest outbreaks because of their habitat homogeneity and reduced natural enemy populations. Therefore, more simplified landscapes would experience more pest problems and consequently use more pesticides. Conversely, economic theory of pesticide use suggests that the application of pesticides by a neighboring farm may have positive externalities for surrounding farms as a result of pesticide drift or pest suppression .

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