An additional experiment was carried out in an airtight container without aeration

Briefly, 400 mg sub-samples of the dried, ground plant matter were freeze-dried for 12 h, weighed, and extracted in polypropylene tubes by sequential sonication and centrifugation with 20 mL methyl tert-butyl ether and then again with 20 mL acetonitrile. The combined extracts were evaporated under nitrogen to less than 1 mL, and mixed with 5 mL methanol and 20 mL water. A 6 mL aliquot of the extract was taken for analysis by LSC to determine the fraction of activity as extractable residue. Selected 150 mg sub-samples of the solventextracted plant matter were combusted on the Biological Oxidizer as described above to determine the fraction of 14C present as non-extractable residue. When nutrient solution and jars were exchanged, the volume of remaining nutrient solution in each jar was gravimetrically determined. A 9 mL aliquot of the solution was mixed with 13 mL Ultima Gold scintillation cocktail and the 14C was quantified by LSC. Water loss from evaporation during each 3 d period was found to be negligible in the no-plant control containers. It is likely that microbial activity in the nutrient solution may have resulted in transformation of the spiked 14C-compounds and that plants may have accumulated both parent PPCP/EDCs and transformation products. To discern the contribution of transformation products to plant accumulation, the used nutrient solution from day 21 was preserved with 2 g sodium azide and 100 mg ascorbic acid, extracted, and fractionated using high performance liquid chromatography . Solutions from 14C-BPA, DCL, or NPX treatments were first filtered through a What man #4 filter paper and then passed through a HLB solid phase extraction cartridge . Before use, the cartridges were sequentially conditioned with 5 mL each of MTBE, methanol , and water. The filtered solution was drawn through the conditioned HLB cartridges under vacuum and followed by 50 mL deionized water. A sub-sample of the filtrate that passed through the cartridge was collected for analysis by LSC to quantify 14C that was not retained by the cartridge.

The cartridges were dried with nitrogen gas,hydroponic bucket and then sequentially eluted with 5 mL of MeOH:MTBE and 5 mL MeOH. The collected eluent was dried under nitrogen to 100 μL. The concentrated eluent was transferred to an HPLC vial equipped with a 250 μL insert. The condensing vial was rinsed with 130 μL of methanol, and the rinsate and 20 μL of non-labeled parent standard were added to the HPLC vial. Preliminary experiments showed that the recovery of this extraction procedure from the initial solution to HPLC analysis was 81.5 ± 7.1% for BPA, 85.8 ± 2.5% for DCL, and 74.0 ± 1.9% for NPX. Nutrient solutions from the 14C-NP treatment were extracted by a simple liquid-liquid extraction method. Each nutrient solution sample was shaken with 50 mL hexane for 30 min, and then the upper layer of the sample was transferred to a centrifuge tube and centrifuged at 3500 rpm for 30 min to reduce emulsification. The hexane phase was transferred to a 15 mL glass tube, concentrated under nitrogen to 300 μL, and transferred to an HPLC vial. The condensing vial was rinsed with 180 μL of methanol, and the rinsate and 20 μL of non-labeled NP standard were added to the HPLC vial. The recovery of this extraction method from the initial solution to HPLC analysis for NP was determined to be 66.8 ± 12.0%. Young plants of lettuce and collards were grown for 21 d in nutrient solution containing one of the four 14C-labeled PPCP/EDCs. No significant differences in plant mass were observed between treatments at the end of the experiment. During the experiment, three plants died . Figure 2 shows the mean mass balance for the systems at the end of the experiment, depicting the fractions of the spiked 14C present in plant tissues, in the used nutrient solution, and as unaccounted activity. The unaccounted activity reflected the 14C that was not found in the nutrient solution at the time of solution renewal or in the plant tissues after harvest and may include losses via unidentified processes, such as volatilization, microbial mineralization in the nutrient solution , or stomatal release. Activity in each fraction varied across compounds and to a lesser degree across plant species, suggesting specificity to uptake. Figure 3 shows the cumulative 14C dissipation from the nutrient solution as calculated from the difference in activity in the solution at the beginning and end of each 3 d interval of solution renewal, representing 14C loss from plant uptake and other processes. Dissipation followed the decreasing order of BPA > NP > DCL > NPX for all treatments and occurred at a similar rate throughout the 21 d cultivation. The presence of plants significantly increased the dissipation of PPCP/EDCs from the nutrient solution, except for NP.

For example, the initial concentration of 14C-DCL in the nutrient solution was 105.3 ± 0.3 dpm/ mL, but it decreased to only 32.8 ± 1.9 dpm/mL after 3 d in the presence of lettuce, while 91.2 ± 3.2 dpm/mL remained in the no-plant control. Lettuce and collards treatments had different levels of chemical dissipation in the nutrient solution. For example, the overall dissipation of BPA in the lettuce treatment was 69.1 ± 8.7%, as compared to 88.4 ± 5.3% in the collards treatment . Different compounds also dissipated at different rates. For instance, in the presence of collards, the cumulative loss was 88.4 ± 5% for BPA, 55.6 ± 11.8% for DCL, and 45.5 ± 4.3% for NPX.The dissipation of NP in the solutions with plants was found to be similar to that in the noplant control, especially for the lettuce treatment . The loss of NP from the noplant control was likely associated with volatilization, as continuous aeration was used to maintain the oxygen level in the nutrient solution throughout the experiment. The Henry’s Law constant for NP is 1.09 ×10−4 atm m3 mol−1 , suggesting a tendency for volatilization. The loss of NP in the solution was found to be insignificant, as all of the spiked 14C was found in the solution , and a solvent rinse of the system showed little sorption of 14C-NP on the container wall . Doucette et al. found that in a hydroponic set up, about 13% of the spiked NP was lost to volatilization in the absence of plants. The increased volatilization losses in the current study were likely due to specific aeration and temperature conditions used. Despite volatilization losses, significant amounts of 14C were detected in plant tissues, suggesting that both collards and lettuce accumulated NP . Noureddin et al. studied the uptake of 5 mg/L BPA from hydroponic solution by water convolvulus and found that approximately 75% of the spiked BPA was removed after 3 d. This removal was comparable to that observed for BPA with lettuce in this study, but was smaller than that with collards . Calderón-Preciado et al. evaluated hydroponic uptake of triclosan, hydrocinnamic acid, tonalide, ibuprofen, naproxen, and clofibric acid by lettuce and spath and showed that the removal of NPX from solution was about 70% for lettuce and 10% for spath after 3 d. In comparison, Matamoros et al. observed less than 10% removal of NPX after 3 d of hydroponic growth with wetland plants , while 46% removal of NPX was measured in the collards treatment in the present study.

Matamoros et al. also showed that DCL did not dissipate appreciably in treatments with wetland plants, which was in contrast to the high removal of DCL by leafy vegetables observed in this study . It is likely that the smaller plant mass and the use of non-aerated nutrient solution in the earlier study contributed to the limited plant uptake. The range of variation suggests that plant species,stackable planters along with other factors such as plant mass and environmental conditions, affect the actual accumulation of PPCP/ EDCs into plant tissues. Plant tissues were collected after 21 d of cultivation, rinsed with deionized water, and separated into roots, stems, new leaves, and original leaves for analysis of both extractable and non-extractable 14C. Table 1 shows concentrations of 14C in plant tissues, expressed as parent-equivalents. In agreement with the dissipation trends in solution, plant accumulation followed the decreasing order of BPA > NP > DCL > NPX. Concentrations based on dry plant mass ranged from 0.22 ± 0.03 to 12.12 ± 1.91 ng/g in leaves and stems. Statistical analysis showed that the accumulation in leaves and stems was not significantly different between lettuce and collards, or among the different compounds. In contrast, roots accumulated significantly more 14C than all the other plant tissues, with concentrations that ranged from 71.08 ± 12.12 to 926.89 ±212.89 ng/g. Accumulation of 14C in plant tissues exhibited several apparent trends. In whole collards plants, significantly greater accumulation was found for the neutral compounds BPA and NP than the anionic compounds DCL and NPX , suggesting that the charge state of PPCP/EDCs may greatly influence plant uptake . Similar effects have been frequently observed for anionic herbicides, and are attributed to exclusion of negatively charged molecules by cell membranes . Between lettuce and collards, lettuce significantly accumulated less PPCP/EDC when all test compounds were pooled , although the interaction effect for individual compounds was not significant . Accumulation of BPA or NP in plant roots was significantly higher for collards than lettuce , while portion of DCL accumulated into lettuce and collards roots was not significantly different. Analysis of tissue extracted with solvent showed that essentially all of the 14C was non-extractable; only the root samples from NP-collards treatment contained a detectable fraction of 14C in extracts .

Combustion of extracted plant tissues confirmed that almost all 14C remained as non-extractable residue, one possible endpoint for xenobiotics taken up by plants . Only a few studies have examined the plant uptake of some of the same PPCP/EDCs considered in this study. Wu et al. grew iceberg lettuce and spinach for 21 d in hydroponic solution initially spiked with a suite of 19 PPCPs, including DCL and NPX, each at 500 ng/L and found no detectable residues of DCL or NPX, except for NPX in spinach at 0.04 ng/g. Calderón-Preciado et al. analyzed apple tree leaves and alfalfa from fields irrigated with water containing BPA, DCL, and NPX. DCL was detected at 0.354 ng/g in apple leaves and 0.198 ng/g in alfalfa; NPX was detected at 0.043 ng/g and 0.04 ng/g, respectively. The low concentrations found in these studies generally agree with the findings of this study, but there is some variation in the tendency for specific compounds to accumulate. This variation may be partly attributed to the different analytical approaches. In other studies, uptake of PPCP/EDCs by plants was evaluated using non-labeled compounds, and accumulation was measured by targeted chromatographic analysis for the extractable parent compound. The use of 14C-labeled compounds in the current study should have provided “worst-case” estimates of human exposure, as the concentrations included non-extractable residue and likely also included transformation products. Transformation products may be an important component of potential risk since the metabolites of some PPCP/EDCs have higher biological activity than their parents and studies have shown that a large portion of PPCP/EDCs that are taken up by plants may be transformed in vivo . A translocation factor , which was the total 14C in stems, new leaves, and original leaves divided by the 14C in roots, was calculated . The derived TFs were consistently very small, demonstrating the poor translocation of these PPCP/EDCs from roots to upper tissues after uptake. The TF values followed the decreasing order of NPX > DCL > NP > BPA, the opposite observed for plant accumulation. Lettuce displayed lower TFs than collards for the same PPCP/EDCs. For example, the mean TF for BPA was only 0.010 ± 0.003 for lettuce, but was 0.051 ± 0.008 for collards. The much greater accumulation of PPCP/EDCs in roots, as compared to leaves, has been observed in previous studies. For instance, Herklotz et al. found that the levels in leaves were 0.00952 – 0.00503 of those in roots for cabbage grown in nutrient solution spiked with carbamazepine, salbutamol, sulfamethoxazole, and trimethoprim. Doucette et al. reported that the accumulation of NP in leaves was 0.0233 – 0.0167 of that in the roots of crested wheatgrass grown in solution.