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Uptake of Pharmaceuticals Influences Plant Development and Affects Nutrient and Hormone Homeostases Laura J. Carter,† Mike Williams,*,† Christine Böttcher,‡ and Rai S. Kookana† †

CSIRO Land and Water, Waite Campus, Adelaide, South Australia, 5064 CSIRO Agriculture, Waite Campus, Adelaide, South Australia, 5064

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S Supporting Information *

ABSTRACT: The detection of a range of active pharmaceutical ingredients (APIs) in the soil environment has led to a number of publications demonstrating uptake by crops, however very few studies have explored the potential for impacts on plant development as a result of API uptake. This study investigated the effect of carbamazepine and verapamil (0.005−10 mg/kg) on a range of plant responses in zucchini (Cucurbita pepo). Uptake increased in a dose-dependent manner, with maximum leaf concentrations of 821.9 and 2.2 mg/kg for carbamazepine and verapamil, respectively. Increased carbamazepine uptake by zucchini resulted in a decrease in above (100 mg/kg with very few studies demonstrating effects at lower concentrations in the soil environment.21 However, more recently, it has been demonstrated that other classes of APIs including nonsteroidal anti-inflammatory drugs (NSAIDs) and the antidepressant venlafaxine can affect plant growth as well as a number of biological end points such as reduction in the mitochondrial activity.25,26 While studies have observed a toxic effect due to API exposure, very few have offered an explanation for why this was seen and link this to the presence of the API in the soil-plant system. For example, it is not yet clear whether the negative effects on plant biomass result from the direct damage of the plant by antibiotic Received: July 16, 2015 Revised: September 22, 2015 Accepted: September 29, 2015

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DOI: 10.1021/acs.est.5b03468 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

described.41 Jasmonic acid (JA)-D5, JA-isoleucine (JA-Ile)-D2, isopentenyladenine (iP)-D6 and trans-zeatin (t-Z)-D5 were obtained from OlChemIm Ltd. (Olomouc, Czech Republic). Abscisic acid (ABA)-D6 and its glucose ester (ABA-GE-D5) were purchased from the NRC Canada (Ottawa, Ontario). Soil was obtained from the Cooke Plains agricultural region in South Australia (pH 7.8, EC 0.03 dS/m, OC 0.46%, CEC 3.9 cmol (+)/kg, 0.002% moisture, clay 4.8%, 16.5% silt, and 78.7% sand). The soil was not cropped and had not previously received biosolids or wastewater applications. Prior to use in experiments the soil was air-dried and then sieved to 2 mm to ensure homogeneity. Zucchini seeds (Cucurbita pepo, Midnight F1 container garden variety) were obtained from Mr Fothergills (Sydney, Australia). Dose Response Experiment. For each API treatment, plastic pots containing 1500 ± 15 g of soil were prepared in replicates of four. A 10 g portion of the soil was removed from each pot and placed in a culture tube. This was spiked with 75− 1500 μL of stock solution (10 mg/mL in methanol) for the 0.5− 10 mg/kg treatments and 150 μL of stock solution (0.05 mg/mL in methanol) for the 0.005 mg/kg treatment. In addition to an unspiked negative control, soil was also spiked with the maximum solvent volume used for the solvent control. The methanol was evaporated under a stream of nitrogen until the soil was dry, after which the soil was replaced in the respective pots to create nominal concentrations of 0.005, 0.5, 1, 2, 4, 8, and 10 mg/kg. The pots were lidded and then placed in an endoverend shaker for 3 h to thoroughly homogenize the spiked soil. The moisture content was adjusted to 60% of the maximum water holding capacity (MWHC) by addition of ultrapure water (18.2 MΩcm) and pots were left to equilibrate for 48 h. Before seeds were sown, 2 ± 0.2 g of soil, fresh weight (FW) was removed from each pot (from three random positions) to confirm nominal start concentrations and equal distribution of the API (Table SI 2, Supporting Information). Three seeds were then sown per pot which was thinned down to one seedling after germination in excess of 80% in all treatments was reached. Pots were incubated under controlled conditions (65% relative humidity, 12 h light (23 °C)/ 12 h dark (15 °C)), arranged in a completely randomized design (Microsoft Excel) and rerandomized on a weekly basis. Moisture content adjustments were made on a daily basis to ensure the MWHC remained at 60% until harvest (4 weeks). To ensure the plants received an adequate amount of nutrients, Ruakura nutrient solution was applied to the soil twice a week (5 mL per 250 g soil) instead of water for the first 3 weeks followed by one application in the final week (nutrient regime and preparation previously outlined in Carter et al.8) At harvest loose soil was removed from around the roots to allow for the intact removal of the zucchini plant. Each plant was then thoroughly rinsed in ultrapure water to remove any soil residues, patted dry with paper towel, weighed and divided up into above and below ground biomass, and these were reweighed individually. The leaf material was detached from the above ground biomass and reweighed separately. After the seedling leaves were discarded the remaining leaf material was combined and cut into smaller pieces. For chlorophyll analysis, 1 ± 0.1 g of leaf material (FW), from each treatment (n = 4) was placed in a 2 mL Eppendorf vial. To determine API residues, 1 ± 0.1 g of zucchini leaf (FW) from each replicate was placed in a glass culture tube to which 0.1 μg of deuterated internal standard (1 μg/mL in methanol) was added to each sample to account for recoveries and matrix interference. Any excess leaf material was

APIs or whether antimicrobial action on soil microorganisms is responsible for the damage by affecting the plant-microorganism symbiosis.27,28 Studies are yet to comprehensively explore the potential for impacts on plant homeostases, such as changes in cellular metabolism and signaling, as a result of API uptake. A number of small signaling molecules collectively known as plant hormones are crucial for the integration of cellular signals and environmental cues, such as abiotic and biotic stressors.29 It is crucial to understand the effects of API uptake on the finely balanced regulation of plant hormone production and metabolism, because changes in concentration of these signaling molecules can alter the physiological response of a plant, for example by triggering the closure of stomata30 or decreasing cell division in the shoot and root meristems.31 Such hormonetriggered changes may be the foundation of the more long-term visual phytotoxic responses, for example, reduced root length. Previous research has demonstrated that bioactive xenobiotics affect key plant biological parameters including carbon balance, hormone balance and antioxidant defense in addition to reducing plant growth32,33 and thus there is the potential that bioactive chemicals such as APIs may also elicit similar responses. Therefore, this study was designed to investigate potential effects on plant hormone concentrations, chlorophyll pigments, and nutritional status of the plant after uptake of the antiepileptic, carbamazepine and the antihypertensive, verapamil from a spiked soil. These measurements were in addition to the archetypical end point of biomass, which is more commonly measured in API phytotoxicity studies. It was hypothesized that effects on the homeostasis of plant hormones may also be initiated at much lower concentrations than observed for biomass effects. The APIs were chosen specifically due to their physiological mode of action, namely their regulation of calcium and sodium ion flow in humans (Table SI 1). As studies have previously demonstrated verapamil can regulate ion flux across root membranes there is the potential that uptake of these APIs may influence ion transfer across cell membranes in the zucchini, and thus alter the nutritional composition of the leaf.34,35 Considering their detections in biosolid samples from WWTPs at 1200 and 551 μg/kg respectively, there is the potential for both carbamazepine and verapamil to enter the soil environment.1,36 While carbamazepine uptake has been documented in various plant species,8,37−39 verapamil uptake has not been extensively characterized; this research will therefore reveal new insights into the uptake capacity of this particular API. Exposure at a range of soil concentrations was used to determine where effects may be seen, and included a representative soil concentration of “environmentally realistic” API soil contamination (0.005 mg/ kg).40 In addition, this study reveals how API uptake is influenced by an increasing concentration in the exposure medium.

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MATERIALS AND METHODS Analytical grade carbamazepine (≥98% purity) and verapamil hydrochloride (≥99% purity) were obtained from Sigma-Aldrich (Sydney, Australia). Deuterated isotopes (carbamazepine-D10 (99.4% purity) and verapamil-D7 (99.5% purity) were purchased from TLC Pharmachem (Vaughan, Canada) for use as internal standards in the API analyses. Certified reference materials for nutrient analysis were purchased from the U.S. Department of Commerce, National Institute of Standards and Technology, Gaithersburgh, MD (apple leaves (1515) and spinach leaves (1570a)). Indole-3-acetic acid (IAA)-D5 was purchased from Cambridge Isotope Laboratories (Andover, MA) and IAAaspartic acid (IAA-Asp)-D5 was synthesized as previously B


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Shapiro-Wilk and Levene-Mediane test were used to test for normality and equal variance, respectively. A one-way analysis of variance (ANOVA) (significance level 0.05) was employed to assess differences in values for plant biomass, plant hormone concentrations and leaf nutrient concentrations between treatments (all on a dry weight (DW) basis). Where necessary, a Holm-Sidak pairwise comparison was used to establish which treatments were significantly different from each other. If normality failed ANOVA was performed using a Kruskal−Wallis analysis on ranks and additional multiple comparisons were carried out using Student−Newman−Keuls method or Dunn’s method where appropriate. In addition, a two-way ANOVA (significance level 0.05) was used to explore the differences in photosynthetic pigments among treatments keeping study type (control or treatment) and pigment type as the independent variables.

then placed in extra storage vessels which would be subsequently used for nutrient and hormone analysis. In addition, soil was sampled from each pot to confirm API residues remaining at the end of the experiment (2 ± 0.2 g of soil (FW)) and 0.1 μg of deuterated internal standard was added to each sample. All samples were then freeze-dried, and dry weights of the plant material subsequently calculated, then stored at −20 °C until extraction apart from the chlorophyll samples which were stored at −80 °C until required. Pharmaceutical Analysis. Extraction from the Pore Water, Soil, and Plant. Soil pore water was extracted from the soil immediately after harvest following methods outlined previously.8 Pharmaceutical compounds were extracted from soil and plant material using validated methods chosen for their high percentage recoveries (Table SI 3, Supporting Information). Extraction procedures were similar to those outlined in Carter et al.,8 and are provided in Supporting Information for reference. Extracts were analyzed for API residues by LC-MS/ MS using a ThermoFinnigan TSQ Quantum Discovery Max (Thermo Electron Corporation). Specific details of the LC-MS/ MS analytical method for the detection of APIs in soil, plant and pore water matrices, including limits of detection (LOD) and quantitation (LOQ) are provided in Supporting Information (SI Table 3 and 4). Determination of Chlorophyll a, Chlorophyll b, and Carotenoid Pigments. Zucchini leaf tissue was extracted using 80% cold acetone, to determine the absorbance measurements at a range of wavelengths, which enabled chlorophyll a and b concentrations to be calculated using equations previously described.42 A detailed description of the method is provided in the Supporting Information Plant Hormone Analysis. Freeze-dried leaf material from each replicate was extracted to determine levels of the auxin IAA and the auxin conjugate IAA-Asp (20 ± 0.5 mg), cytokinins t-Z and iP (25 ± 0.5 mg), JA and its bioactive conjugate JA-lle (20 ± 0.5 mg), and ABA (20 ± 0.5 mg) together with its glucose ester using methods previously described elsewhere.41,43−45 Plant Nutrient Analysis. To determine nutrient levels, freeze-dried leaf material from each replicate (0.2 ± 0.02 g) was digested in 7 mL of concentrated nitric acid and 3 mL of hydrogen peroxide. The samples were heated in a microwave system, following a temperature profile similar to EPA method 3052.46 A range of elements (major cations, trace elements and nonmetallic elements) were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES; Thermo iCAP 6000) or inductively coupled plasma-mass spectroscopy (ICPMS; Agilent 7700). Each batch of digests contained a number of certified reference materials (CRM) to check for recoveries and several duplicates were run to account for consistency in the results. Blanks were also included to track any potential contamination of samples. Supporting Information contains detailed information on plant nutrient analysis including ICP methods and CRM recovery (Table SI 6, 7). Statistical Analysis and Laboratory QA/QC Protocols. Each treatment consisted of four replicates to allow for the mean and standard error to be calculated. Data quality objectives for analysis were assured by use of blanks (unspiked negative controls and solvent controls were also included in the study design), use of an internal standard (or CRM for nutrient analysis), analysis of duplicates, determination of spike recovery, analysis of a matrix standard and monitoring of response factors, for example, retention time. Statistical analysis of the data was performed using SigmaPlot (v. 12.5). Prior to all analyses, a

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RESULTS After 2 weeks, an average of 80% germination had been achieved across all treatments except for one replicate in the 0.005 mg/kg verapamil treatment. This replicate was therefore not included in the rest of the analysis. As all other treatments germinated successfully this was most likely due to an experimental artifact and not an effect of the API. API Residue in Soil, Plant, and Pore Water. Confirmation of the starting residue concentrations can be found in Supporting Information (Table SI 2). The pore water concentrations of carbamazepine and verapamil, measured at the end of the study, were found to increase with increasing soil concentration (Figure 1). Average soil water partition coefficient (Kd) calculated using soil and pore water concentrations across all treatments were 1.5 ± 0.09 and 1426.3 ± 553.0 L/kg for carbamazepine and verapamil, respectively (Table SI 8). The relatively low sorption coefficients in comparison to those previously reported in literature8,40,47 are likely to be due to the lower organic carbon and clay content of the soil used in this study. Carbamazepine and verapamil were taken up by zucchini in a dose-dependent manner, at all exposure concentrations including the environmentally realistic 0.005 mg/kg. A correlation plot between pore water and leaf concentration, suggests that the increased bioavailability of the APIs in the pore water is largely responsible for this increased uptake by the zucchini (Figure SI 1). For carbamazepine, leaf concentrations increased from 0.2 ± 0.02 mg/kg to 821.9 ± 120.2 mg/kg (DW) in the highest treatment (10 mg/kg) (Figure 1). Even though verapamil uptake increased with increasing exposure medium concentration, verapamil accumulated in zucchini leaf to a lesser extent than carbamazepine (1 mg/kg treatments in comparison to the control (p < 0.05) (Figure 2). No effects on plant biomass were observed in the verapamil treatments (Figure 2). C


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Figure 1. Carbamazepine (a,b) and verapamil (c,d) residues in soil (open bar), pore water (black dot) and zucchini leaf (dry weight) (striped bar) at the end of the experiment. Values provided are the average of four replicates ± SE.

Figure 2. Zucchini biomass results (dry weight) for total plant material harvested as well as, above ground, roots and leaf material separately for carbamazepine (a) and verapamil (b). Results presented are the average of four replicates ± SE with statistically significant (p < 0.05) treatments in comparison to the control denoted by a letter.

Plant Nutrient Status and Chlorophyll Pigments. In all verapamil treatments, concentrations of chlorophyll a, b and carotenoid pigments remained relatively constant, independent of verapamil concentrations. In comparison, a 50% decrease in the photosynthetic pigments was observed for the 8−10 mg/kg carbamazepine treatments, although this was only statistically significant for chlorophyll a (p < 0.05) (Figure 3). A decrease in chlorophylls such as this is indicative of a reduced photosynthetic ability of the plant. The higher carbamazepine treatments (>8 mg/kg) resulted in a statistically significant (p < 0.05) increase of the essential

macronutrients, potassium and phosphorus, in the leaf material in comparison to the control (Figure 4). For the 10 mg/kg treatment specifically, there was a 48% increase in the macronutrient composition of the plant (Figure 4). A similar trend was also observed for the secondary essential macronutrient magnesium, as well as the essential micronutrients silicon and zinc, albeit to a lesser extent. At the lowest treatment (0.005 mg/kg), significantly (p < 0.05) increased concentrations of magnesium, phosphorus and manganese were also noted in comparison to the controls (Figure 4, Figure SI 3). The presence of APIs in the plant or soil therefore appears to be influencing the D


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Figure 3. Average (n = 4, ± SE) concentration of photosynthetic pigments measured in the plant material (fresh weight) including chlorophyll a, b and carotenoids for carbamazepine (a) and verapamil (b). Treatments denoted by a letter are significantly different to the control, treatments with a different letter are significantly different to each other and treatments with the same letter are not significantly different (p < 0.05).

Figure 4. Results from nutrient analysis of zucchini leaf (dry weight) after exposure to carbamazepine (CBZ) and verapamil (VRP). Average (n = 4) concentrations (±SE) provided for essential macronutrients ((a) phosphorus and (b) potassium) and secondary macronutrients ((c) sodium, (d) calcium, (e) sulfur, (f) magnesium). Treatments denoted by a letter are significantly different to the control, treatments with a different letter are significantly different to each other and treatments with the same letter are not significantly different (p < 0.05).

essential macro and micronutrient composition of zucchini leaves, although this only occurred in the carbamazepine treatments (Figure 4, Figure SI 3). Plant Hormones. Carbamazepine and verapamil uptake significantly altered the concentrations of auxins (IAA and IAAAsp), cytokinins (iP and t-Z), jasmonates (JA and JA-lle), ABA and ABA-GE in zucchini leaf. Exposure at 0.005 mg/kg resulted in a significant decrease in ABA concentrations in response to both API treatments, however a similar decrease in the glucose ester of ABA, ABA-GE was only significantly different after verapamil exposure (p < 0.05) (Figure 5). For jasmonates, JA and JA-lle decreased in comparison to the controls after exposure to carbamazepine and verapamil at 0.005 mg/kg and 0.5 mg/kg respectively (p < 0.05). The concentration of the most common, naturally occurring plant hormone of the auxin class, IAA, was

increased significantly in response to all verapamil treatments while the concentrations of its conjugate, IAA-Asp, remained unchanged. For IAA, a similar response was observed in leaves from the carbamazepine treatment, however at 4−10 mg/kg the IAA concentration decreased rapidly (Figure 5). This decrease may be explained, at least in part, by the increase (p < 0.05) in IAA-Asp which suggests a change in IAA metabolism. The concentrations of iP, a member of the cytokinin family, were in excess of the controls for low concentrations of the carbamazepine treatment and for the entire concentration range of verapamil treatments. For the most part, concentrations of plant hormones changed in comparison to the controls for both API exposures with the exception of t-Z. E


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Figure 5. Average (n = 4, ± SE) concentrations of plant hormones in zucchini leaf (dry weight) ((a) abscisic acid: ABA and ABA-GE, (b) auxins: IAA and IAA-Asp, (c) cytokinins: t-Z and iP, (d) jasmonates: JA and JA-lle) after exposure to carbamazepine and verapamil, with treatments denoted by a letter are significantly different to the control, treatments with a different letter are significantly different to each other and treatments with the same letter are not significantly different (p < 0.05). Results for carbamazepine are on the left and verapamil on the right.

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DISCUSSION

realistic exposure, carbamazepine concentrations of approximately 400 ng/g in shoots have been published after application of spiked biosolids and wastewater, which were comparable to leaf concentrations obtained in the present study at the “environmentally realistic” exposure (0.005 mg/kg).7,9 Although

Concentration-Dependent Uptake. The increased uptake of both carbamazepine and verapamil with increasing concentration in exposure medium suggests the potential for greater API uptake by zucchini at higher soil concentrations. In terms of F


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feedback loops between the production of plant hormones and the pathways initiated by the external cues.58 A large and complex intracellular signaling cascade begins, including the generation of secondary signaling molecules, such as calcium or ABA, which ultimately leads to alterations in expression of defense-related genes and activation of defense responses.57 In this study, changes in plant hormone concentrations were observed in comparison to the controls after exposure to the APIs (Figure 5). As plant hormones play integral roles in plant growth processes as well as in biotic and abiotic stress responses, alterations in their concentrations may result in significant effects on plant development. Specifically, auxin (IAA) and cytokinin (iP) production increased in response to carbamazepine and verapamil exposure from 0.005 mg/kg (Figure 5). Both of these plant hormones are responsible for numerous processes including proliferation of undifferentiated cells in the shoot and root apical meristems as well as cell differentiation and organ out growth in the peripheral zone.31 Production of excess iP and IAA, may therefore be responsible for shoot and root growth stimulation, perhaps as a compensatory mechanism for effects induced by API uptake, and might explain why no biomass effects ensued at low exposure concentrations. In line with this hypothesis, at >4 mg/kg where deleterious effects on plant growth were noted in the carbamazepine treatment, IAA and iP concentration decreased sharply. As cytokinins delay leaf senescence and promote staygreen phenotypes enabling plants maintain their leaves for a longer period of time59 a reduction in iP such as observed in this study corresponds with the observed reduction in chlorophyll pigments in the leaf material at the higher treatment concentrations (Figure 3). The reduction in chlorophylls observed in this study is also analogous to previously published research which found that atorvastatin, gemfibrozil, tamoxifen, and sildenafil caused a reduction in chlorophylls and carotenoids in lettuce.60 Jasmonates and ABA are also known as important signaling molecules regulating plant defense responses.56,57 The decrease of JA and ABA concentrations in both carbamazepine and verapamil exposures at lower concentrations, suggests that the plant defense capabilities in response to a stressor may be impacted (Figure 5). An effect such as this has wider implications for plant disease survival, because if the plant was to encounter additional stress factors, such as pathogens or wounding, it may not be capable of translating the stress signal into the required stress response. Visual inspection of the leaves from the high carbamazepine treatments suggested the plants were suffering deviation from the critical nutrient concentration (Figure SI 2, Supporting Information).61 As this was primarily observed in mature leaves, typically this would be associated with a deficiency in plant mobile nutrients. However, the results from the nutrient analysis revealed that rather than being deficient, the plants exposed to the higher treatments had significantly increased mobile nutrient concentrations in their leaves (potassium and phosphorus, p < 0.05) (Figure 4). Therefore, consistent with the mechanism of action of carbamazepine and verapamil in humans, respectively sodium and calcium ion flow regulation, may be responsible for controlling ion flux across membranes in the zucchini. Previous research has shown that verapamil can bind to zucchini membrane fractions62 and, in a separate study, inhibit calcium flux at very high concentrations (500 or 1000 μM) as well as stimulate calcium influx at lower concentrations (90% ionised at soil pH 7.8) and lipophilic nature (log Dow, of 2.7 at pH 7.8).8,48 The low verapamil plant uptake is therefore analogous to other ionizable APIs with similar lipophilic characteristics such as diphenhydramine, propranolol, triclosan, and diclofenac.7,8,49 Plant Biomass. A clear reduction in above and below ground biomass was observed at higher carbamazepine concentrations. The greatest reduction in plant biomass was observed in the below ground plant material (8 mg/kg, p < 0.05), this was considerably less than previously reported effect-concentrations (EC10, root length) which were in excess of 300 mg/kg for a range of antibiotics.21 While deleterious effects on plant growth were observed in the carbamazepine treatments, no such effects were noted in the verapamil exposures. This demonstrates the importance of API physicochemical properties in influencing plant uptake and toxicity. Differences in the level of toxic effect between APIs belonging to distinct therapeutic classes have been noted previously due to the lipophilic characteristics influencing uptake behavior; for example diclofenac and ibuprofen were more toxic to horseradish (Armoratia rusticana) and flax (Linum usitatissimum) than acetaminophen.51 Plants can convert carbamazepine to its active metabolite, 10,11-epoxide-carbamazepine. Approximately 40% of the molar fraction of carbamazepine was found in the form of the epoxy metabolite in a previous study.9 It is possible that in our study the active epoxy carbamazepine may have been present in the zucchini leaf at concentrations similar to that of carbamazepine. While verapamil is also metabolized, this results in inactivation of the parent compound in humans.52 This may therefore present less of a risk to the plant in comparison to epoxy carbamazepine. Additional studies are required to better understand the contribution of metabolites toward plant stress. Relationship to Pore Water Concentrations. A majority of phytotoxic effects resulting from API uptake have been previously observed in hydroponic experiments, using a wide range of concentrations (10,000 μg/L).22,24 Hydroponic exposures are of10 orders of magnitude higher than environmentally relevant concentrations. In the current study, pore water concentrations reached a maximum of 3994.4 ± 102 μg/L and 11.8 ± 1.7 μg/L for carbamazepine and verapamil, respectively, and therefore were lower than typical concentrations used in hydroponic studies but higher than characteristically observed in WWTP effluents.3 Effects on Nutrient and Hormone Homeostasis in Zucchini Plants. Evaluation of biomass is a common measurement of stress response as a reduction in plant growth typically represents the overall sum of responses within the entire plant. Prior to observing whole plant level effects, stress during plant development can also lead to physiological, biochemical and molecular changes within the plant.53−57 To date, with respect to API induced toxicity, there is very little work investigating the effects in plants from mechanistic perspective. When a plant is under biotic or abiotic stress this induces a complex network of G


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taken up from environmentally relevant soil exposures (0.005 mg/kg), resulted in a deviation from the normal concentrations of plant hormones and nutrients. In the case of ABA and jasmonates, a suppressed immune response may make the plant more susceptible to environmental stressors. As this study only evaluated the impact of two APIs, future research is required to investigate how a plant responds to supplementary stresses after exposure to a wider range of APIs and their metabolites to allow for more comprehensive generalizations to be made.

regulating ion influx across membranes in the zucchini leaf, this may explain the relatively high micronutrient concentrations in comparison to the controls (boron, p < 0.05) (Figure SI 3). Although the main target of carbamazepine in humans is voltagegated sodium channels it has been previously demonstrated that carbamazepine can also interact with voltage-gated calcium and potassium channels in animal cells.63 Therefore, carbamazepine uptake in plants may result in regulation of ion flux across various transport channels, and not exclusively those for sodium transport. If carbamazepine can potentiate channels causing ion influx across membranes, this may explain the increase in nutrient levels in the leaves for potassium, phosphorus and magnesium in particular (Figure 4). In order to confirm this, additional studies are required to explore the mode of action of both APIs in plant cells, using in vitro assays to demonstrate direct effects on ion transporters by these compounds. In addition to the observed increase in the nutritional composition of leaves exposed to higher carbamazepine concentrations, a similar increase was also measured at the lower end of the exposure scale for a number of nutrients, most notably for phosphorus and magnesium (p < 0.05) (Figure 4). This may be an adaptive response following a moderate stress, such as API uptake, and thus form part of a weak biphasic dose response. The antibiotic flumequine has been previously observed to induce hormesis in plants as low concentrations (50 μg/L) of this API led to increased growth of the aquatic weed Lythrum salicaria L. but higher concentrations reduced biomass.64 However, in the context of this study, the results do not support the concept of hormesis as opposite effects were not observed at high and low exposure concentrations; for example an increased nutrient composition at the lower exposure concentrations did not result in a significantly reduced nutrient composition at higher exposure concentrations (Figure 4). Additional studies are required to investigate this initial disruption of homeostasis caused by the API at ecologically relevant concentrations. If the presence of the APIs in the plant are disturbing ion transport, whether this is by directly by binding to the transport channels, indirectly by influencing plant hormones and thus secondary signaling molecule production (e.g., calcium) or by other mechanisms this may induce a change in cell membrane potentials.65,66 Membrane potentials ultimately enable sugars to be transported from source organs (e.g., mature leaves) to sink organs (e.g., young leaves and fruit)67 and disturbing this could explain why the plants treated with carbamazepine had reduced biomass and eventually went necrotic at the highest concentrations, displaying symptoms akin to sugar deficiency (Figure SI 2). Additional studies are required to explore this further and evaluate potential differences in sugar concentrations of the source and sink organs in API exposed plants to validate impacts on sugar transport. While published findings so far suggest the accumulation of pharmaceuticals in edible plants presents little risk to human health6,8 our study indicates that API uptake can result in a risk to plant health by interfering with cell signaling and plant defense pathways. When harvested, the plants were displaying necrotic symptoms after exposure to carbamazepine at concentrations >4 mg/kg which manifested in a reduction of plant biomass (Figure SI 2, Supporting Information). This is some of the first work to link the uptake of APIs to effects on plant hormone concentrations and the nutritional status of the plant which ultimately, at higher concentrations, results in effects on plant biomass. Even low concentrations of the APIs in the plant matrix,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03468. Properties of the APIs, confirmation of starting carbamazepine and verapamil residues, pharmaceutical extraction and analysis methods, and detailed methods of chlorophyll and nutrient analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 8 83038515; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank CSIRO for the funding support. Thanks are also due Sheridan Martin for her technical support and Jason Kirby for his assistance with the nutrient analysis. We also thank the anonymous reviewers for their comments and suggestions. Any use of trade, firm, or product names in this paper does not imply endorsement by the authors.

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REFERENCES

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