At the end of the 7 d incubation, root growth of lettuce seedlings was found to increase with increasing rates of the CEC mixture with significant differences observed at the 2X, 10X, and 20X concentration . Compared to the control, root length was found to increase by 16 ± 3, 24 ± 6, and 32 ± 8 % at the 2X, 10X, and 20X CEC concentration levels, respectively. This was in contrast with studies that showed negative root length effects when plants were exposed to other CECs such as tetracyclines and sulfonamides . However, in those studies, high concentrations of a single CEC were generally considered. For example, Liu et al. observed inhibition of root growth in oats, rice, and cucumbers when the seedlings were exposed to oxytetracycline concentrations at 5-10 mg L-1 . Chemical mixtures may involve more complex interactions, where various chemicals may have different but related targets that can have an additive or nonadditive effect . This may be the reason for the observed stimulatory effect by the CEC mixtures at environmentally relevant concentrations in this study. It could also be the result of a biphasic response where a favorable biological response at low dose and inhibition at high dose is observed, a phenomenon known as hormesis . Primary root length after 7 d germination is indicative of the plant’s ability to establish itself and obtain nutrients during this critical period of development. The stimulatory effect on root length observed in response to the low-dose exposure of a mixture of CECs in this study suggested that a low-dose mixture may help the plant establish itself better and increase its ability to obtain water and nutrients during the beginning stages of growth. However,10 liter drainage collection pot it must be noted that only a small set of CECs were considered in this study, and a similar response may not necessarily occur for other CECs or for these CECs with a different species.

Roots, stems, and leaves each maintains their own dynamic balance in biomass that is indicative of the relative above-ground resources and below-ground resources . The root to shoot biomass ratio provides insight into the overall health of the plant. A lower root to shoot ratio suggests greater investment in above-ground tissues possibly due to interference with photosynthetic mechanisms or interference in root functioning, resulting in reduced nutrient uptake and therefore growth . A greater root to shoot ratio is typically influenced by below ground conditions, suggesting reduced water and nutrient availability. Although no significant differences in root to shoot ratio with respect to biomass during the 7 d study, a positive correlation between CEC treatment levels and root to shoot ratio was observed , suggesting a possible interference with nutrient or water uptake by the roots. This was in agreement with Carter et al. , who found that carbamazepine and verapamil exposure caused changes in sodium and calcium ion flow regulation in zucchini plants, demonstrating the influence of CECs on nutrient transport. Since toxicity can only be elicited when a chemical has reached its target site, we monitored bio-accumulation of the target CECs into various cucumber tissues. The starting concentrations and their dissipation in the nutrient solution after 3 d with and without plants are found in Table 6. The 20X CEC treatment was used because the higher concentrations facilitated qualitative evaluation of CEC bio-accumulation and translocation. Among the various cucumber tissues, only one flower sample per treatment was collected due to the limited growth duration and plant tissue. Samples of flowers had to be pooled from replicates for each treatment, and therefore some standard deviations could not be calculated for the CEC concentrations in flower samples. Concentrations of CECs in plant tissues increased with increasing concentrations in the hydroponic solution . All CECs except triclosan were detected in the roots .

The absence of triclosan in the root samples could be due to its relatively high quantification limit of triclosan , active metabolism , or suppressed uptake of triclosan in the presence of other CECs. Above-ground and below-ground biomass were measured for cucumber plants at the end of a longer-term exposure to the same CEC mixture in hydroponic solution at incremental levels. Biomass measurements are useful in measuring stress response, as deviations in growth from the control are indicative of the overall sum of response of the plant . Although there were no significant differences in the biomass among the different CEC levels, there appears to be a dose dependent response when change in biomass, expressed as the percentage difference relative to the control, was considered . At the 20X treatment, the relative percentage differences in the average below ground, above ground, and total biomass from the control were -51.2 ± 20.9, -26.3 ± 34.1, and -33.2 ± 41.7%, respectively . The greatest reduction in plant biomass occurred in the roots, and this finding was similar to Carter et al. who also observed a ~30% reduction in the below ground plant tissues of zucchini from the control when the plant was exposed to 10 mg kg-1 carbamazepine in soil. The observed reduction in above-ground biomass and total biomass along the dose-response curve suggested that there was not simply further investment in photosynthetic or aerial tissues due to interferences in photosynthetic mechanisms, but rather that multiple aspects of the plant were affected without ways to mitigate the stress . It was also possible that the roots could not support an increase in aerial tissues, the common stress mitigation mechanism, because the roots were also under stress and were unable to take up the necessary nutrients to promote growth.A hormone profile was analyzed to further understand the dose-response effect of chronic exposure to a mixture of CECs on cucumber plants . In this study, we focused on three phytohormones; auxin , jasmonic acid , and abscisic acid because of their critical roles in regulation of a plant’s development and stress-response. The auxin profile was characterized by a hormesis effect along the dose-response curve when the leaves and stems were considered . The solvent control did appear to have some stimulatory effect on auxin concentrations in the stems , but the change was not statistically significant . A 6-fold increase was observed in the stem auxin concentrations at the 1X CEC treatment level as compared to the control .

The trend, however, was followed by a gradual decrease to 2-fold the control at the 10X CEC treatment rate . The leaf auxin concentrations significantly increased at the 1X and 10X CEC treatment rates to 16 and 11-fold, respectively,10 liter drainage pot which was followed by a decrease at the 20X CEC treatment rate. A similar pattern in leaf auxin content was also observed by Carter et al. along a dose-response treatment of carbamazepine for zucchinis grown in soil. A similar pattern, however, was not visible for the auxin content in the roots or fruits at the end of 30 d cultivation in this study. Auxin is known to be involved in cell elongation and division of meristematic tissues. The observed increase of auxin inthe stems and leaves at CEC levels as low as the 1X treatment rate suggested that the stems and leaves were being signaled to grow in order to gain increased light exposure because of interferences with photosynthetic mechanisms, and/or decrease heat stress by allowing for more air flow. Jasmonates are phytohormones that are involved in flower development, fruiting, reproduction, and plant defense. No clear trends or significant differences were observed in JA levels in any of the plant tissues along the dose-response curve . This could be due to the time of sampling, as the plant was still in an early stage of development , when flowering and fruiting was not the primary focus of the plant. Instead, at this point in development, increasing photosynthetic tissues was likely of the upmost importance. ABA is a signaling hormone that communicates water stress to the plant. ABA levels were significantly elevated in the leaves with exposure to increasing levels of CECs and significantly decreased in the roots at environmentally relevant concentrations of the CEC mixture . ABA in the roots dropped from 56.5 ± 17.3 ng g-1 in the controls to 8.23= ± 9.5, 5.6 ± 2.2, and 11.8 ± 13.7 ng g-1 at the 1X, 10X, and 20X CEC treatment rates. Low ABA levels in the roots could indicate over-saturation by water at the root tips. In this study, we observed an approximate 20% decrease in root ABA levels when the plant was exposed to the CEC mixture at the 1X level as compared to the control, demonstrating that even exposure to CECs at low levels could significantly affect the homeostasis of this hormone. The decrease in the ABA levels coincided with visual symptoms of the roots, where the roots appeared to be over-saturated and less rigid structurally, which also resulted in a ‘shedding’ of some small roots into the hydroponic medium. In the leaves, ABA was found to increase significantly at the 1X, 10X, and 20X CEC treatment levels from the control 42.6 ± 12.8 ng g-1 . Elevated ABA levels can cause stomatal closure, thereby reducing transpiration in a plant’s efforts to conserve water . The increase of ABA in the leaves and its resulting effect on anti-transpiration and therefore decreased pulling force of nutrients to aerial tissues could be the reason for the reduction in above-ground biomass observed in this study. Antitranspiration activity may also impose an impediment on plant growth by limiting gas exchange and impairing the plant’s ability to adapt to additional stressors such as extreme temperatures . With stomatal closure, the plant’s ability to mitigate heat stress by transpiring is also impacted, threatening its survival. 2.3.5. Phytohormone response to multiple stressorsFollowing cultivation in CEC-containing nutrient solution, a subset of cucumber plants was exposed for 4 d to heat stress at temperatures up to 41 °C in a greenhouse, and the plants were then sampled for hormone analysis.

JA content was not significantly altered with the additional heat stress in the roots and stems . Although, a significant effect on the JA content in the leaves was not observed following any of the lower level CEC treatments, JA content in the 20X treatment was significantly increased from JA content in plants exposed to heat without CEC exposure . A consistent trend across CEC treatments was a decrease in the JA content in the leaves in response to heat stress.Jasmonates are important signaling molecules in plant defense, and therefore a decrease in JA content in leaves in response to heat stress across all CEC treatments has implications for plant survival when exposed to disease, wounding, or pathogens . Although heat-stress exposed plants did not have statistically significant changes in JA content, this decreased trend in the JA content in leaves exposed to excessive heat conflicts with a study where heat shock was found to result in an upregulation of JA pathway genes and its consequentially enhanced production in agarwood cells . This could be due to the type of heat exposure , the range of temperature exposure, or the difference at the cellular level . Auxin concentrations in the roots of the control treatments with heat stress were significantly elevated as compared to the 1X and 20X CEC treatments . This finding showed how different types of stress may have opposing effects on plant hormone levels. In the stems, heat stress resulted in a significant decrease in auxin concentrations in plants in the 1X CEC treatment as compared to the 1X CEC treatment without heat stress , however the 1X CEC treatment with the added heat stress was not statistically different from the control or the 20X CEC treatment. The role of auxins in cell division and elongation has recently been associated with being an adaptive growth response to high temperature tolerance as seedlings elongate to elevate photosynthetic and meristematic tissues away from the heat-absorbing soil, thereby allowing increased air circulation and cooling effects . Leaf auxin concentrations were not affected by heat stress . Between the control and 20X CEC treatments, heat stressed plants displayed only slightly elevated auxin contents from their respective CEC treatments without the additional heat exposure. ABA is integral to how plants mitigate heat stress.