Collectively, our findings provide genetic and physiological evidence that AVP1 is a new component required for plant growth under high external Mg2+ concentrations and functions in regulating Mg2+ tolerance via PPi hydrolysis. For on-plate growth assays, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water for three times, and sown on Murashige and Skoog medium containing 2% sucrose and solidified with 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically at 22 ◦C in growth chamber with a 14 h light/10 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agarose-solidified media containing various ions as indicated in the figure legends and were grown under 14 h light/10 h dark photoperiod. For phenotypic assay in the hydroponics, 10-day-old seedlings geminated on MS plate were transferred to 1/6 strength MS solution and were grown under the 14 h light/10 h dark condition in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with 15 mM MgCl2. Increasing populations and shifting precipitation patterns put pressure on freshwater and food systems and incentivize governments and industries to exploit historically underutilized resources, such as recycled water and bio-solids. Use of recycled water and bio-solids in agricultural systems, however, comes with the potential risks of environmental and food contamination by trace organic contaminants including, contaminants of emerging concern . These compounds pose potential threats to humans and environmental health because they are designed to be biologically active at low concentrations and are considered “pseudo-persistent” due to their continuous release into the environment. Using 14C tracing, mass spectrometry, stable-isotope labeling,planting blueberries in pots and enzyme extractions to assess the fate, metabolism, and biological effects of four environmentally prevalent CECs in terrestrial organisms in hydroponic and artificial soil cultivations.

These organisms included a model terrestrial invertebrate , a model plant and two crop plants . Compounds were selected based on environmental prevalence and test organisms were selected due to their use in the literature, commercial availability, and global range. Sulfamethoxazole was metabolized in A. thaliana cell cultures, and E. Fetida forming phase I and II metabolites. Diazepam was also metabolized in A. thaliana cell cultures and radish and cucumber seedlings, forming the phase I metabolites nordiazepam, temazepam and oxazepam, with the longevity corresponding to that of human metabolism. The major metabolites of naproxen and methyl paraben, Odesmethylnaproxen and p-hydroxybenzoic acid, respectively, were detected in treatment soils containing E. fetida and N4-acetylsulfamethoxazole was detected in E. fetida tissues, indicating CEC metabolism and excretion. Exposure to CECs resulted in changes to enzymes associated with detoxification and oxidative stress in crop plants and E. fetida. Our research indicates that terrestrial organisms can absorb and transform CECs and that CECs can change biochemistry of the exposed organisms. Accordingly, it is crucial to consider CEC fate, transformation and effects on non-target organisms of CECs when assessing risk in the agro-environment. Climate change and human population growth threaten sustainable development by placing stress on limited resources, including freshwater and agricultural productivity. As a result, the use of historically underutilized resources, such as treated wastewater and bio-solids , is now being harnessed to combat freshwater scarcity, improve soil fertility, and increase crop growth. Currently, ~54% of the bio-solids produced in the United States are land applied, predominantly in agriculture, landscaping, and forestry . Only ~8% of TWW in the United States is currently reclaimed and implemented for direct potable reuse or indirect potable reuse . The use of these underutilized resources is likely to increase in the future as governments and industries are also promoting their use to help communities adapt to a shifting climate, rising population, and increasing urbanization . Population growth and increasing urbanization are giving rise to megacities around the globe. In 2016 there were an estimated 31 megacities and the number is projected to reach 41 by 2030 . Such megacities face substantial challenges when it comes to waste disposal and water management, making the recycling of bio-solids and TWW essential . The need for TWW and bio-solid recycling is also heightened in arid and semi-arid areas where these resources are increasingly being used to improve agricultural output and conserve limited potable water resources .

The recycling of TWW and bio-solids could be environmentally and economically beneficial, but significant concerns remain about their use in terrestrial environments . TWW and bio-solids have been shown to contain “contaminants of emerging concern” . CECs are defined by the EPA as “chemicals and other substances that have no regulatory standard, have been recently discovered in natural water and may potentially cause deleterious effects in aquatic life at environmentally relevant concentration”. CECs include various compound classes such as antibiotics, non-steroidal anti-inflammatory drugs, preservatives, and flame-retardants. The concentrations of various CECs generally range from ng L-1 to low µg L-1 in TWW and µg kg-1 to mg kg-1 in bio-solids . Literature reportedly documents the fate and toxicity of many CECs in various organisms in aquatic systems and raises concerns about the unintended consequences of the widespread use and release of CECs into the aquatic environment . For example, endocrine disruption and changes in male to female sex ratios have been reported in several fish species .Data on environmental fate and toxicity of CECs in terrestrial environments are relatively scarce but crucial for understanding the risks to terrestrial organisms after TWW or bio-solids application.There is increasing information on the effects of CECs in terrestrial plants, invertebrates, and vertebrates . For example, studies have shown that pharmaceuticals can induce oxidative stress in plants , exhibit phytotoxicity in crops and increase the time to adulthood in agricultural pests .Several studies report the uptake of emerging contaminants from hydroponic solutions, spiked soils and soil irrigated with TWW or amended with bio-solids or animal manure . However, these studies often report conflicting or contradictory results concerning the rate of uptake and the extent of translocation. Hydroponic studies often exhibit higher rates of uptake than those observed in spiked or amended soils studies. For example, in a study conducted by Boonsaner and Hawker the maximum concentration of antibiotics in plant tissues was reached within 2 d in spiked water but took 6-8 d when plants were grown in contaminated soils.In hydroponic systems, plant uptake and translocation are largely driven by the contaminants water solubility, log Kowand/or the pH of the hydroponic solution and the potential for ionization, log Dow, . Whereas in soils, soil-specific processes such as soil-pore water partitioning, and transformations in soil, also contribute to contaminant uptake and accumulation in plants. Thus, the uptake rate and translocation of a contaminant in plants can vary widely depending upon soil and environmental conditions.

For example, a soil with higher organic matter content can limit plant uptake of organic contaminants, due to stronger contaminant adsorption than a soil with lower organic matter content . Also, shifts in soil pH can result in ionization of ionizable organic contaminants,blackberries in containers affecting the rate of plant uptake . Antibiotics constitute one of the most extensively used pharmaceuticals classes for both human and livestock and as such are nearly ubiquitously detected in wastewater effluent, bio-solids and livestock manure . Relatively more studies have been reported on terrestrial plant uptake and translocation of antibiotics than other pharmaceuticals in the agro-environment, including studies conducted in hydroponic growth solutions, greenhouses, and under field conditions .In hydroponic growth solution, the antibiotic sulfamethoxazole was taken up in the roots and translocated to leaves of four vegetable plants, including lettuce , spinach , cucumber , and pepper plants , with the concentration report to be significantly greater in the roots . In a 55 day hydroponic study, three antibiotics, i.e., tetracycline, cephalexin, and sulfamethoxazole, were found to be taken up and translocated into edible tissues of pakchoi , with concentrations ranging from 6.9 – 11.8, 26.4 – 48.1, and 18.1 – 35.3 µg kg-1 for tetracycline, cephalexin and sulfamethoxazole, respectively . Several studies have also explored plant uptake of antibiotics from spiked soils . For example, Boxall et al., exposed carrot and lettuce plants to soils spiked with 1 mg kg-1 of 7 antibiotics, i.e., sulfadiazine, trimethoprim, tylosin, amoxicillin, enrofloxacin, florfenicol, and oxytetracycline. After 103 d and 152 d cultivation, antibiotics were quantified in both crops. However, the concentrations varied considerably among different antibiotics and between plant species. For example, amoxicillin was detected at < 1 µg kg-1 in lettuce tissues but was 24 µg kg-1 in carrot tissues . Three sulfonamides, i.e., sulfadiazine, sulfamethazine, and sulfamethoxazole, were also reported to be taken up by pakchoi cultivated in spiked-soils, with sulfamethoxazole having the highest concentration among the three antibiotics throughout the 49 d cultivation . To better predict environmentally relevant risks from antibiotic uptake to human consumption, several studies have been carried out on crops grown in soils irrigated with spiked TWW and/or amended with livestock manure . These studies showed that food crops were capable of taking up and accumulating antibiotics from wastewater and/or manure-amended soils; however, the levels were often very low. For example, chlortetracycline was taken up by corn , green onion , and cabbage that were grown in soils amended with antibiotic spiked manure . However, the concentrations were low . Sulfamethoxazole and lincomycin were found to accumulate in lettuce tissues at concentrations up to 125 µg kg-1 and 822 µg kg-1 , respectively, after irrigation with antibiotic-spiked synthetic wastewater at 1 mg L-1 , .

Similarly, in field studies, crops irrigated with TWW were found to take up antibiotics, including but not limited to, roxithromycin, clindamycin, ciprofloxacin, sulfamerazine, and sulfamethoxazole . However, in nearly every case the concentration of antibiotics in plant tissues was negligibly low. Nonsteroidal anti-inflammatories are the most commonly consumed class of pharmaceuticals in the world . As such they are ubiquitously found in TWW, bio-solids, and surfaces water . They have been reported to accumulate in soils that receive TWW or bio-solids . Several studies have explored the potential for uptake and translocation of NSAIDs in plants, including in hydroponic systems, amended soils, and field studies . NSAIDs have a wide range of physicochemical range properties and, as such, have displayed vastly different uptake and translocation rates . For example, in a hydroponic study the NSAID diclofenac was observed to accumulate only in the roots of four vegetables while relatively high levels of acetaminophen were detected in the leaves . Similarly, a study exploring plant uptake of 14C labeled naproxen and diclofenac from hydroponic solutions showed that two vegetables, i.e., lettuce and collard greens , were capable of accumulating both compounds, and both plants accumulated significantly more diclofenac than naproxen . Radish and ryegrass were shown to absorb and accumulate diclofenac from soils spiked with the chemical at an initial concentration of 1 mg kg-1 . However, after 40 d cultivation, the concentration of diclofenac in the plants was very low < 1 µg kg-1 . Greenhouse studies using soils amended with bio-solids and field studies using TWW irrigation considered the uptake of NSAIDs under environmentally relevant conditions. For example, Cortés et al. conducted a greenhouse study in which soybeans and wheat were cultivated in bio-solids-amended soils for 60 and 110 d. However, none of the four NSAIDs was detected in the plant shoots. On the other hand, in a long-term field study , diclofenac was found relatively high levels in the fruits of tomato plants after prolonged irrigation with TWW, as compared to sulfamethoxazole and trimethoprim . Further, in another field study, naproxen was detected in the edible tissues of various vegetables irrigated with TWW or TWW fortified with the chemical at 250 ng L-1 and grown until maturity . Several NSAIDs have also been considered in the investigation of potential metabolism of pharmaceuticals in plant cell cultures and whole plants . The metabolism of diclofenac was investigated in four different plant systems, including a horseradish hairy root culture , barley , Arabidopsis thaliana cell culture, and Arabidopsis thaliana whole plants . However, the formation of diclofenac metabolites differed significantly by plant systems. For instance, while phase I hydroxylation was observed in all the systems, the horseradish hairy root cultures and barley formed a glucopyranoside as the major Phase II metabolite .