ABA plays a vital role in signaling water stressed conditions from the roots to the shoots to initiate stomatal closure resulting in water-saving, anti-transpirational activity . The defense response is also partially mediated by ABA, with ABA playing both negative and positive roles. For example, exogenous application of ABA on Arabidopsis plants increased its susceptibility to fungus, Fusarium oxysporum , by suppressing the transcription of defense genes . In contrast, ABA plays a more positive role in pre-invasive defense response against pathoPgens by initiating the closure of stomata, therefore increasing penetration resistance . Several studies have considered ABA to gain a mechanistic understanding of how environmental stressors effect the physiological response to a plant , and has also been a target of manipulation from a bioengineering standpoint to increase a plant’s tolerance to environmental stressors such as drought or disease .Jasmonic acids are multifunctional signaling compounds involved in senescence, tendril coiling, and reproductive processes such as flowering and fruiting . JA levels differ amongst plant tissues and stage of development, with highest levels being found in flowers and reproductive tissues . JA is involved in response to environmental stressors such as salinity, drought, and low temperature ,drainage collection pot as well as plant response to pathogens by inducing genes that enhance production of compounds used in plant defense . In response to salinity stress, an increase in endogenous JA levels in the roots reportedly alleviated the deleterious effects of salinity stress .

This observation was in agreement with Yoon et al.who observed that exogenous application of a jasmonic acid metabolite decreased the adverse effects of salinity stress in soybean seedlings. Since several studies have linked exposure of CECs to phytotoxicity and oxidative stress , it may be hypothesized that phytohormone homeostasis is also influenced by CECs to mediate a plant’s stress response to such xenobiotics. Carter et al. observed a significant decrease in leaf ABA concentrations in response to verapamil and carbamazepine exposure in soil . IAA concentrations significantly increased in response to verapamil treatments, however, a hormesis effect was found in the carbamazepine treatments, where IAA concentrations were elevated from the controls at treatment concentrations from 0 to 2 mg kg-1 , followed by a rapid decrease at carbamazepine concentrations of 4-10 mg kg-1 . This work was one of the first to link uptake of CECs such as pharmaceuticals to effects on plant hormone homeostasis, which at high concentrations, resulted in reductions in biomass . Several studies have effectively utilized a phytohormone profile to explore a plant’s response to environmental stressors, such as salinity and drought. Since the phytohormones discussed above have active roles in multiple areas of the physiological response, it is believed that their response to chronic exposure of a mixture of CECs would help elucidate the stress response to CECs and mechanistically explain the visual phytotoxicity that is measured in endpoints such as root length, germination, and biomass. Therefore, recognizing how low-dose, repeated exposure to CEC mixtures would add greatly to our understanding of how beneficial reuses of treated wastewater, bio-solids, and animal wastes may affect crop productivity and food security.In nature, crops are often simultaneously exposed to diverse biotic and abiotic stresses.

Controlled laboratory and greenhouse experiments focusing on only single stresses are often not reflective of true environmental conditions due to combinational stresses that occur in the field . Extreme temperatures affect several biochemical processes and the stability of proteins, RNAs, membranes, and cytoskeletal structures . Heat stress disrupts cellular homeostasis, respiration, and photosynthesis leading to decreased plant growth and productivity . However, plants have developed sophisticated mechanisms that allow plant cells to sense changes in temperature and activate defense mechanisms to protect against damage imposed by heat stress, known as the heat stress response . Phytohormones, including ABA, JA, and IAA, have been reported to play pivotal roles in plant response to heat stress . For example, exogenous application of ABA increased thermotolerance of Agrostis stoloniferalikely by controlling water movement via stomatal closure and inducing the expression of protective compounds and proteins to heat stress. Dobrá et al. observed a significant increase in ABA leaf content for 30 min exposure to heat stress after a 30 min delay, followed by a significant decrease from the initial levels after being under heat stress for over 1 h. A decrease in leaf ABA content increases the transpiration rate and consequently decreases leaf temperature to alleviate heat stress. However, water deficits can occur upon prolonged enhanced transpiration rates, therefore forcing stomatal closure as to not impose water-stress to the plant . JA plays a positive regulatory role in basal thermotolerance and has been shown to improve heat tolerance in wild-type Arabidopsis thaliana . With a changing climate and projected increases in water scarcity, it is expected that irrigation with TWW will be adapted to meet the needs of agriculture. The effect of trace contaminants present in TWW can have negative or positive impacts on a plant’s physiological response to other natural environmental stressors, such as heat. Extreme high temperatures are one of the most frequent abiotic stresses experienced by plants .

With a rate of warming of 0.05 °C per decade , the frequency and magnitude of temperature change will be a future environmental stressor that can affect crop productivity. Battisti and Naylor predicted that every 1 °C increase in seasonal temperature would cause 2.5-16% loss in direct crop yields. Therefore, heat was chosen as the combinational stressor in this study because arid and semi-arid environments that are more prone to use TWW for irrigation are the same areas that typically experience extreme temperatures and excessive heat.A total of 10 CECs were selected based on their detection frequency and concentrations in treated wastewater. These CECs included 4 antibiotics, 3 analgesic antiinflammatory drugs, 1 anti-epileptic, 1 beta-blocker, and 1 antimicrobial. Standards of naproxen, diclofenac, atenolol, trimethoprim, and tetracycline were purchased from Sigma Aldrich . Sulfamethoxazole was obtained from MP Biomedicals . Ibuprofen and triclosan were purchased from Alfa Aesar . Azithromycin was purchased from Tokyo Chemical Industry . Isotope labeled standards azithromycin-d5, diclofenac-d4,10 liter pot and trimethoprim-d9 were purchased from Toronto Research Chemicals . Triclosan-d3, atenolol-d7, sulfamethoxazole-d4, and carbamazepine-d10 were purchased from CDN Isotopes . Hormone standards indole-3-acetic acid and jasmonic acid were purchased from Santa Cruz Biotechnologies and abscisic acid was purchased from Chem-Impex International . Abscisic acid-d6 was purchased from Toronto Research Chemicals and indole-3-acetic acid-d7 was purchased from Cambridge Isotope Laboratories. Stock solutions were prepared in 25% DMSO or HPLC grade methanol and stored at -20 °C before use. HPLC grade acetonitrile and methanol were used for extraction. Ultrapure water was obtained from a Milli-Q system . Solvents for UPLC analysis including methanol and formic acid were Ultima grade .After 30-d cultivation, cucumber plant tissues were extracted and analyzed for various hormones. Plant tissues were immediately frozen using liquid nitrogen upon sampling and then ground to a fine powder using a mortar and pestle. The samples were extracted with 2 mL of 80% methanol with 0.1% formic acid containing 50 ng of labeled standards as recovery surrogates. The extract was transferred to 2 mL microcentrifuge tubes and kept at -20 °C for 12 h, after which the sample extract was vortexed, and centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was filtered through a 0.22 µm polytetrafluoroethylene membrane , transferred to a clean 2 mL microcentrifuge tube and evaporated to near dryness on ice under nitrogen. The samples were resuspended in 200 µL of 50% methanol and centrifuged at 10,000 g for 3 min. Supernatants were transferred to 300 µL inserts housed in 2 mL glass vials and stored at – 20 °C until analysis.Freeze dried plant tissue samples were extracted and analyzed following a previously published method . Briefly, approximately 0.2 g of plant tissue was added to 50-mL polypropylene centrifuge tubes and spiked with deuterated standards of the CECs as recovery surrogates. Methyl tert-butyl ether  was added to each sample, vortexed for 30 s, and placed in an ultrasonic water bath for 20 min. The samples were centrifuged at 3000 rpm for 20 minutes and the supernatant decanted into a 40 mL glass vial.

Samples were re-extracted with methanol following the above procedure and their resulting supernatants combined. The extracts were evaporated under a steady flow of nitrogen until near dryness, resuspended in 1 mL of methanol, and diluted with DI water to a volume of 20 mL. Clean-up was performed by loading the sample on 150-mg Oasis© HLB cartridges pre-conditioned with 7 mL methanol and 14 mL DI water. After loading, the cartridge was dried on a vacuum manifold and eluted with 20 mL methanol under gravity. Samples were filtered through 0.22 µm polytetrafluoroethylene filters into 2 mL glass vials and stored at -20 °C until instrumental analysis. Freshly prepared nutrient solution containing CECs at different levels were extracted to determine the initial concentrations of CECs in the medium. Additional spiked nutrient solutions were exposed to the plant growth conditions for 3 d without plants and others with plants to determine the dissipation of CECs between nutrient solution renewal. The nutrient solution volume was recorded and loaded on to pre-conditioned 150 mg Oasis HLB cartridges and prepared as described above. Samples were stored in 2 mL glass vials at -20 °C until instrumental analysis. Hormone and CECs in sample extracts were quantified on a Waters ACQUITY ultra-performance liquid chromatography combined with a Waters Micromass Triple Quadrupole mass spectrometer equipped with electrospray ionization interface Separation was achieved on an ACQUITY UPLC BEH C18 column at 40 °C using a 5 µL injection volume. Hormones and CECs analyzed in the ESI+ mode were separated using mobile phase A . Separation of CECs in the ESI- mode was carried out using mobile phase A and mobile phase B . For hormone separation, the following mobile phase gradient was used with respect to mobile phase A: 0-0.5 min, 95%; 0.5-1 min, 60%; 1-2 min, 10%; 2-4 min, 95%; 4-5 min, 95%. For CECs, the following mobile phase program was used with respect to mobile phase A: 0-0.5 min, 95%, 0.5-3 min, 10%; 3-6.5 min, 95%; 6.5-7 min, 95%. The flow rate was 0.3 mL min-1 . The TQD parameters were as follows: source temperature, 120 °C; desolvation temperature, 350 °C; capillary voltage, 3.0 kV; cone voltage, 20 V; desolvation gas flow, 600 L hr-1 ; cone gas flow, 50 L hr-1 . Quantitative analysis was performed in the multiple reaction monitoring mode . Details on monitored ions and their respective collision energies can be found in Table 3. All data were processed using MassLynx 4.1 software . All plant and hydroponic treatments were set-up in triplicate. Controls, solvent controls, and negative controls were included with each sample extraction. A methanol water blank was run between treatments during analysis on the UPLC-MS/MS to assess and eliminate contamination. Labeled surrogate standards were used in all sample extraction and analysis to estimate analyte recovery and account for matrix effects during instrumental analysis. Surrogate recoveries can be found in Table 4. Calibration curves were used for quantification with r2 values of at least 0.98 for all analytes. Limits of detection and limits of quantitation can be found in Table 5 and were defined as the concentration at which a signal to noise ratio of 3 and 10 was achieved, respectively. Statistical analysis of data including means, standard deviations, t-tests, and ANOVA with post hoc Scheffe’s test were performed using SPSS Statistics . Data are represented as mean ± standard deviation.To assess the effect on germination, lettuce seeds were exposed to the mixture of ten CECs at concentrations 1X of that in treated wastewater up to 20X. DMSO was used as a carrier solvent to solubilize the CECs. To ensure that the observed effects were attributed to the presence of CECs, two types of controls DMSO were included. No significant differences between controls and solvent controls were observed. Germination, root length, and biomass were used as biological endpoints for the germination bio-assay. No significant differences were observed in germination rate across all treatments.