Certain plants have been identified with the capability to take up metallic and metal oxide nanoparticles (ENPs), thus suggesting their potential role in phytoremediation. The reported study evaluated the response of two aquatic plants, sedge (Carex rostrata) and cattail (Typha latifolia), on their exposure to Ag, ZnO, TiO2, BiVO4/Pd, and Cu2O/Pd nanoparticles over 15 weeks. Plant physiological responses (chlorophyll content, carbonic anhydrase (CA) activity, leaf area, production of new shoots, and root length) varied according to the plant species and ENP type. By week 15, sedge treated with BiVO4/Pd ENP had a high chlorophyll content and increased CA activity and leaf area compared to the control. In contrast, cattail had reduced chlorophyll levels and number of new shoots when exposed to exogenously applied BiVO4/Pd. Highest sedge chlorophyll content at week 15 was measured in the mixed-ENPs, Cu2O, and Ag (53.2, 35.8, and 32.7%, respectively, greater than the control). The ZnO ENPs were beneficial for sedge chlorophyll content, cattail shoot production and root length. The mixed-ENPs treatment imparted positive effects to several sedge properties (CA and new shoots) and cattail (chlorophyll, leaf area, and root length). Additional research is needed to assess the capabilities of different aquatic plant species to tolerate metal-based ENPs for remediation purposes.

  • Sedge and cattail plants were exposed to five nanoparticle types over 15 weeks.

  • Plant physiological response varied with plant species and ENP type.

  • Cu2O nanoparticles resulted in the reduction of several physiologic variables.

  • ZnO ENPs were beneficial for shoot production and root growth in cattail.

  • BiVO4 ENPs were often beneficial to sedge but detrimental to cattail.

Engineered nanoparticles (ENPs) are defined as synthetic entities measuring between 1 and 100 nm. By virtue of unique characteristics including magnetism, catalytic capacity, and optoelectronic properties, ENPs are incorporated into myriad industrial, commercial, and domestic products. The manufacture and usage of ENPs are expected to continue to increase at an exponential rate.

As a consequence of their extensive usage, surface waterways receive ENPs via a release in wastewater, from accidental spills during manufacture and transport, through direct weathering of nano-functionalized products, aerial deposition, and other routes (Yu et al. 2013; Cornelis et al. 2014). ENPs are also lost by washout from landfills and via runoff from agricultural lands that have received biosolids (Limbach et al. 2008; Gottschalk et al. 2013). Using computer models, Gottschalk et al. (2013) estimated potential environmental concentrations of nano-sized silver (Ag), titanium dioxide (TiO2), zinc oxide (ZnO), and others. Mode values for Ag ENPs in surface water ranged from 0.116 ng·L−1 in the US to 0.764 ng·L−1 in Europe; values for TiO2 ENPs in water ranged from 0.002 (US) to 0.015 μg·L−1 (Europe); and ZnO in water from 0.001 (US) to 0.010 μg·L−1 (Europe).

The release of nanoparticles into the biosphere has raised concern regarding potential phytotoxic effects. Accumulation of soluble metals by plants is documented to lead to cellular toxicity and physiological changes resulting in altered morphology, decreased growth, and sometimes plant death (Gill 2014; Riyazuddin et al. 2022). The effects of various metal and metal oxide ENPs (herein collectively termed ‘metal-based ENPs’) on aquatic plants, however, are not entirely clear. Several papers have demonstrated adverse impacts of metal-based ENPs on plant physiology and growth (Badawy et al. 2021; Yusefi-Tanha et al. 2022); other researchers, however, have reported beneficial effects of certain ENPs on specific plant properties (Jasim et al. 2017).

Many aquatic plants possess the capability to take up metals from contaminated water and have thus been employed for phytoremediation (Olkhovych et al. 2016). Phytoremediation uses plants to remove and/or sequester hazardous materials from water and soil. There is growing evidence that aquatic plants can accumulate substantial quantities of certain nanoparticles (Ansari et al. 2020; Ali et al. 2021; Ebrahimbabaie et al. 2023). Positive experience and technical methods are documented for phytoremediation of natural surface waters using aquatic macrophytes (Rahman & Hasegawa 2011; Sasmaz & Obek 2012).

Bottle sedge (Carex rostrata) is a perennial grass-like plant that grows up to 50 cm in height and has a fibrous root system. Sedge grows well in a wide range of soil types but tends to be found in wetlands such as marshes and riparian zones (Chadde 2011). Cattail (Typha latifolia), also known as bulrush, is an obligate wetland species and is generally found in flooded locations where it grows rapidly. Its wide leaves can grow to almost 2 m in height. Roots (rhizomes) are thick with small fibrous roots (Chadde 2011). These two species were chosen for study as they are widespread in aquatic ecosystems, exhibit robust growth, are highly competitive, and readily adapt to diverse environments.

In order to optimize the phytoremediation of aquatic systems, detailed investigation is necessary to understand the physiological response of target plants when exposed to metal-based nanoparticles. Research to date has focused on plant response to commonly utilized ENPs, such as Ag, TiO2, and ZnO. Little information is available, however, on environmental effects, including impacts on plants, of many innovative ENPs. For example, both BiVO4 and Cu2O ENPs have gained popularity in commercial usage (Zahran et al. 2014), but their behavior in the biosphere is essentially unknown.

Sedge (C. rostrata) and cattail (T. latifolia) were recently evaluated for uptake of Ag, TiO2, ZnO, Pd/BiVO4, and Pd/Cu2O nanoparticles (Ebrahimbabaie et al. 2023). The purpose of the current study was to evaluate plant physiologic variables (e.g., chlorophyll content, enzyme activity, leaf area, shoot and root growth) of both species in the presence of these five ENP types. Such data are essential for the identification of resilient plant species for the design of effective water remediation operations.

Plant collection and experimental setup

Sedge (C. rostrata) plants were purchased from a commercial supplier, and cattail (T. latifolia) plants were collected from a wetland in central Indiana. Plants were prepared as described in Ebrahimbabaie et al. (2023). Plants as individual bundles were placed into polyethylene containers with 500 mL of water from a local reservoir. 50-mL nutrient solution (General hydroponics FloraGro™, containing nitrogen, phosphate, potash, and magnesium) was added once every 2 weeks, and insecticide (Sevin™) was sprayed as needed. Plants were allowed to acclimate to the new conditions for 2 weeks. All plants were incubated in the growth chamber at 22 ± 2 °C with illumination of about 465 cd (16 h light/8 h dark photoperiod) and 80 ± 5% relative humidity for 15 weeks. Fresh reservoir water was added to the containers on a weekly basis to account for evaporative losses.

Plants were exposed to metal-based ENPs or the corresponding metals in ionic form. Nanoparticles used in this study were Ag, TiO2, ZnO, BiVO4/Pd, and Cu2O/Pd. The Ag, TiO2 and ZnO nanoparticles were obtained from US Research Nanomaterials (Houston, TX). The BiVO4/Pd and Cu2O/Pd ENPs were synthesized as described below.

Synthesis of nanoparticles

BiVO4/BiOBr was synthesized by a modified surfactant-assisted aqueous method according to Palmai et al. (2017). Briefly, 117 mg of NH4VO3 was dissolved in 10 mL of DI H2O at 90 °C, stirred vigorously for 10 min, and then cooled to room temperature. The NH4VO3 solution was added dropwise into a two-neck flask containing 20 mL of 0.05 M CTAB (cetyl trimethyl ammonium bromide) solution under vigorous stirring (500 rpm) at 60 °C. Next, 970 mg Bi(NO3)3·xH2O was dissolved in 10 mL of DI H2O and stirred for 10 min at room temperature. The newly-formed suspension was added dropwise into the two-neck flask, the temperature was set to 80 °C, and the mixture was stirred for 16 h. The m-BiVO4/BiOBr yellow product was filtered and washed with DI water and ethanol several times, and air-dried at 60 °C. To deposit palladium (Pd) nanoparticles on the surface of the material, 219.3 mg of m-BiVO4/BiOBr composite was dispersed in 300 mL of ethanol. Then, 21.9 mg of Pd(CH3COO)2 was added and stirred overnight in the dark. The product was filtered, washed with ethanol several times, and dried at 60 °C (Palmai et al. 2017).

In order to prepare Cu2O nanostructures, a total of 85 mL of DI water, 5 mL of CuSO4 (0.68 M), and 1.5 g of polyvinyl pyrrolidone (PVP) were added to a 250-mL round-bottom flask. The mixture was stirred for 20 min, and then a mixture of 5 mL of 0.74 M sodium citrate and 5 mL of 1.2 M sodium carbonate was added dropwise. The color of the solution changed to a clear deep blue. After 15 min of stirring, 10 mL of 1.4 M glucose solution was added dropwise. The round-bottom flask was placed in an oil bath at 80 °C for 2 h and the solution was filtered and dried overnight at 60 °C (Zahran et al. 2014). To prepare the Pd-decorated nanomaterial, 100 mg of Cu2O composite was dispersed in 300 mL of 200-proof ethanol; then, 8 mg of Pd(CH3COO)2 was added and mechanically stirred overnight in the dark. The material was collected via vacuum filtration and dried at 60 °C.

The two synthesized multicomponent nanomaterials will be denoted as ‘BiVO4’ and ‘Cu2O’ throughout this paper for Pd/BiVO4/BiOBr and Pd/Cu2O, respectively.

The size and morphology of all ENPs were determined via transmission electron microscopy using a JEOL model JEM-1400 transmission electron microscope operating at 120 kV. The commercially purchased ENPs (Ag, TiO2, ZnO) ranged in size from 100, 30, and 60 nm, respectively. The synthesized Cu2O and BiVO4 ENPs measured approximately 700 and 1,000 nm, respectively (Ebrahimbabaie et al. 2023).

For the metal treatments, soluble salts were used, i.e., silver (Ag) as AgNO3, bismuth (Bi) as Bi2(SO4)3, vanadium (V) as VOSO4·xH2O, zinc (Zn) as ZnSO4·7H2O, copper (Cu) as Cu(NO3)2·3H2O, and titanium (Ti) as TiO2. In the case of Ti, bulk TiO2 was dissolved in DI water and then sonicated.

Each week, ENPs or metals were added to containers to increase total concentration by an additional 1.5 mg·L−1. Plants were grown in the contaminated media for a total of 15 weeks; the final concentration was therefore 22.5 mg·L−1. The mixed-ENPs treatment was prepared by combining all five ENP types, resulting in a total of 22.5 mg·L−1 of each. In the growth chamber, treatments were set up in triplicate (i.e., three independent replicates) in a randomized complete block design.

Chlorophyll production

Each week, the chlorophyll content of three leaves of cattail and sedge (mature leaves, entire length) was measured using a hand-held SPAD 502 chlorophyll meter.

Leaf carbonic anhydrase activity

At week 15, the carbonic anhydrase (CA) activity of fresh leaf samples was measured by the method of Dwivedi & Randhawa (1974). Leaf samples were cut into small pieces with a stainless-steel blade in a cold mortar and pestle (<25 °C). Leaf pieces were weighed (100 mg) and transferred to a Petri dish. The leaf pieces were further cut into fine pieces in 5 mL of 0.2 M cysteine hydrochloride and left at 4 °C for 20 min. Samples were transferred to test tubes containing 2 mL of phosphate buffer (pH 6.8). To this, 2 mL of 0.2 M sodium bicarbonate solution and 0.1 mL of 0.002% bromothymol blue were added. Test tubes were shaken gently and left at 4 °C for 20 min. Carbon dioxide liberated by the catalytic action of CA on NaHCO3 was estimated by titrating the reaction mixture against 0.05 N HCl using methyl red as an indicator. The volume of HCl used to develop a light purple color was recorded. A blank consisting of the above reaction components (except the leaf sample) was run simultaneously with each set of samples. Enzyme activity was calculated as follows (Dwivedi & Randhawa 1974):
formula
where V is the difference in volume (mL of HCl used in control versus test sample during titration), N is the normality of HCl, W is the fresh mass of tissue, and 22 is the equivalent weight of CO2.

Shoot and root growth

Numbers of new sedge and cattail shoots in all treatments were observed and counted weekly. The root length of both species was measured weekly using standard measuring tape. Every 5 weeks, three of the longest leaves in each container were excised and their area was measured manually using standard grid paper (Jadon et al. 2016).

Statistical analysis

Statistical analysis of experimental data was carried out using one-way and two-way Analysis of Variance (ANOVA) on a Windows-based PC. The homogeneity of variance assumption was checked using Levene's test. As the sample size (n) for the analysis was equal, the ANOVA is robust to normality issues in regards to Type I error and is conditionally robust to variance assumption violations. As needed, post hoc Duncan's Multiple Range and Least Significant Difference tests were conducted on means found to be significantly different at p < 0.05. SPSS® ver. 23 was employed (SPSS Inc., Chicago, IL) for statistical analysis.

Chlorophyll content

Chlorophyll content is considered among the most sensitive parameters of toxicity to plants. Earlier studies have reported that exogenously added ENPs alter the chlorophyll content of plants and the efficiency of photosynthesis in both positive and negative directions.

In ENP-treated sedge, chlorophyll content decreased during weeks 4–6 in most treatments but subsequently recovered (Figure 1(a)). From weeks 12–15, chlorophyll content was consistently highest in the BiVO4 treatment (one-way ANOVA p > 0.05). At week 15, the lowest chlorophyll values were in the mixed-ENPs treatment (17.1 SPAD units), which was 14.6% below the control. Olkhovych et al. (2016) found that a solution of mixed metal nanoparticles (Ag, Ag2O, Zn, Cu, and Mn) negatively affected the content of pigments (chlorophylls and carotenoids) in seven species of wetland plants. It must be noted that the plants in their study were submerged hydrophytes and free-floating plants, whose physiology may differ from sedge, which grows in saturated soil; and cattail, which is an emergent hydrophyte.
Figure 1

Chlorophyll content in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

Figure 1

Chlorophyll content in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

Close modal

Published data on the effects of novel ENPs such as BiVO4 on plant growth are not available. BiVO4 ENPs are insoluble at neutral pH and somewhat soluble at acid pH. Water in the mesocosms had a pH of 7.6 (Ebrahimbabaie et al. 2023). Plant roots, however, exude a suite of organic acids and BiVO4 solubility may have increased when in proximity to roots. Solubilized Bi3+, VO3 and/or VO43− may have therefore supported chlorophyll production.

When sedge was treated with metals in ionic form, chlorophyll content gradually decreased over 15 weeks (Figure 1(b)). Chlorophyll content in all ionic metal treatments was below the control from week 8 and beyond (one-way ANOVA p < 0.05 for weeks 10, 13, 14, and 15). As a consequence of repeated weekly metal additions, a limit of tolerance may have been exceeded in sedge for one or more soluble metals, following which plants experienced toxic effects. Beyond week 10, chlorophyll content in sedge plants treated with metals in ionic form was consistently below that in plants treated with ENPs (Figure 1(a) and 1(b)). This effect may be due, in part, to the presence of excess soluble metals in the former; in contrast, nanoparticles, being of relatively lower solubility, released only limited soluble metals in microcosms.

ENP treatments to cattail had significant effects on chlorophyll concentration over the study period (two-way ANOVA, F(66,48) = 1.60; p = 0.044). By week 11 and beyond, all cattail ENP treatments had greater chlorophyll levels compared to control (Figure 1(c)). These data imply a beneficial effect of ENPs for the synthesis of photosynthetic organelles in cattail, at least at the application rate chosen for this study. The highest chlorophyll content at week 15 was measured in the mixed-ENPs, Cu2O, and Ag (30.0, 25.0, and 24.2 SPAD units, respectively). These values are 53.2, 35.8, and 32.7%, respectively, greater than the control value (17.4 SPAD units). The lowest chlorophyll readings were in the BiVO4, ZnO and control (18.7, 18.4, and 17.4 SPAD units, respectively).

Trends of chlorophyll content in the BiVO4 and ZnO treatments were opposite those for sedge, where values were above control (Figure 1(a)).

Previous work on the interaction of CuO ENPs with plants has shown species-dependent results. Adverse effects of CuO ENPs on photosynthetic machinery and photosynthesis and shoot and root production have been reported. In the current study, decreased chlorophyll levels in sedge treated with ionic Cu support such findings. It is suggested that Cu+ ions released directly from Cu2O ENPs (and Cu2+ ions from subsequent oxidation) impede certain plant physiologic processes. Copper ions can cause protein dysfunction, tissue necrosis and stunting. The decline in chlorophyll content by Cu2O ENP-exposed sedge may be linked with inhibition of enzymes involved in chlorophyll biosynthesis. Limited studies have, however, noted the beneficial effects of low concentrations of exogenous CuO ENPs on morphological and physiological aspects of plants with no indication of toxicity or repression of growth.

Chlorophyll content in cattail during 15 weeks of chronic dosing of ionic metals fluctuated somewhat but overall was relatively consistent for most treatments (Figure 1(d)). These data are in contrast to that for sedges treated with ionic metals (Figure 1(b)), where values were initially stable and then declined after week 8. These results suggest that cattail is more tolerant to several ionic metals than sedge. In the latter case, a threshold of tolerance to one or more metals was reached, following which plant physiological processes were adversely affected. Many studies have documented the resilience of cattails in the presence of heavy metals.

Plant response to exogenously added nanoparticles is documented to vary as a function of plant factors (e.g., terrestrial versus aquatic); monocotyledonous versus dicotyledonous; species; growth stage; nanoparticle factors (chemical composition; ENP size; ENP shape; presence of coatings); and environmental factors (pH; ionic strength of medium; influence of organic matter and minerals).

Carbonic anhydrase

The CA enzyme is abundant in the chloroplast, where it converts CO2 to HCO3 during initial photosynthetic reactions. This enzyme is involved in other biological functions including stomatal closure, ion exchange, respiration, and acid–base buffering. CA activity can be assayed to gauge plant health. Limited research has involved the study of the effects of exogenously applied ENPs or metals for effects on CA activity.

At 15 weeks, the highest sedge CA activity occurred in the BiVO4, mixed-ENPs and control (0.57 mol CO2·g FM/s), and values were significantly lowest (one-way ANOVA p < 0.05) in the TiO2 and ZnO treatments (0.01 and 0.07 mol CO2·g FM/s, respectively) (Table 1). These data correspond with those for chlorophyll, i.e., highest chlorophyll content in sedge was determined in the BiVO4 treatment during weeks 12–15 (Figure 1(a)).

Table 1

CA activity at week 15 as a function of plant species and ENP treatment

Plant speciesNanoparticleMol CO2/g1 (FM)/s
Sedge Ag 0.41b ± 0.03 
TiO2 0.01a ± 0.02 
ZnO 0.07a ± 0.09 
BiVO4 0.57b ± 0.23 
Cu20.49b ± 0.05 
Mixed-NPs 0.57b ± 0.15 
Control 0.57b ± 0.08 
Cattail Ag 0.26a ± 0.09 
TiO2 0.30a ± 0.01 
ZnO 0.23a ± 0.01 
BiVO4 0.30a ± 0.11 
Cu20.22a ± 0.02 
Mixed-NPs 0.24a ± 0.09 
Control 0.33a ± 0.18 
Plant speciesNanoparticleMol CO2/g1 (FM)/s
Sedge Ag 0.41b ± 0.03 
TiO2 0.01a ± 0.02 
ZnO 0.07a ± 0.09 
BiVO4 0.57b ± 0.23 
Cu20.49b ± 0.05 
Mixed-NPs 0.57b ± 0.15 
Control 0.57b ± 0.08 
Cattail Ag 0.26a ± 0.09 
TiO2 0.30a ± 0.01 
ZnO 0.23a ± 0.01 
BiVO4 0.30a ± 0.11 
Cu20.22a ± 0.02 
Mixed-NPs 0.24a ± 0.09 
Control 0.33a ± 0.18 

n = 3. Data are shown as mean ± standard deviation.

Mean values followed by the same letter are not significantly different (p < 0.05).

Chlorophyll content in ZnO-ENP-treated sedge was also among the lowest in the ENP treatments (Figure 1(a)). The mechanisms responsible for the toxicity of ZnO ENPs to different plants likely differ. One study suggests that the Zn2+ released from ZnO ENPs altered photosynthetic pigment concentrations and caused oxidative stress (García-Gómez et al. 2017). Zhang et al. (2015) showed that the phytotoxicity of ZnO ENPs in maize was primarily caused by the ENPs, whereas for cucumber, it was mainly caused by dissolved Zn2+.

When sedge was treated with ionic metals, the greatest CA activity was in the Bi treatment (0.94 mol CO2/g FM/s,) followed by mixed metals and the control (0.81 and 0.79 mol CO2/g FM/s, respectively) (Table 1). These trends follow those for sedge treated with ENPs, i.e., the highest CA occurred in the BiVO4, mixed-ENPs and control. In addition, the highest chlorophyll content was determined in the BiVO4 treatment during weeks 12–15 (Figure 1(a)).

The lowest CA values were observed in the Cu and Zn treatments (0.398 mol CO2/g FM/s). Accumulation of CuO NPs results in a disturbed ultrastructure in leaves, especially in the photosynthetic apparatus, decreased numbers of thylakoids per granum and plastoglobules, as well as reduced stomatal aperture.

In cattail, highest CA activity after 15 weeks was in the control (0.33 mol CO2·g FM/s) followed by BiVO4 and TiO2 (each 0.30 mol CO2·g FM/s) (one-way ANOVA p > 0.05) (Table 1).

When cattail was treated with ionic forms of metals, the highest CA activity was measured in the Cu (0.83 mol CO2), and lowest in the mixed metal treatment (0.47 mol CO2) (p > 0.05).

Production of new shoots

Key processes that determine the quality and quantity of plant growth include cell division, enlargement and differentiation, which are manifest in shoot and root growth, fruit production, and other products.

Exogenous application of ENPs results in inconsistent effects on plant growth. Several ENPs are known to enhance shoot and root formation. In the case of other ENPs and plants, however, shoot and root growth is adversely affected to varying degrees.

When sedge was treated with ENPs, the number of new shoots increased consistently over 15 weeks for all treatments (Figure 2(a)). By week 15, the greatest number was in the mixed-ENPs (8 new shoots/week) and the Ag ENP treatment (6.3 new shoots/week) (p < 0.05). All treatments except Cu2O experienced higher shoot numbers compared to the control. In the mixed-ENPs treatment, soluble nutrient ions (e.g., Zn and Cu) plus other, possibly beneficial ions (e.g., Ag+, Bi3+, VO3 and/or VO43−) may have been released at rates suitable for vigorous shoot production.
Figure 2

Number of new shoots in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; and (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

Figure 2

Number of new shoots in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; and (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

Close modal

The lowest number of new shoots was in the Cu2O treatment (4 shoots/.week), which was similar to the control (4.2 shoots/.week). Many researchers have reported adverse effects of CuO ENPs on plant morphology, shoot and root production, and quality of produce.

Ionic metal treatments had significant effects on new shoots in sedge over the study period (two-way ANOVA F(91,70) = 1.48; p = 0.045) (Figure 2(b)). The highest number of new sedge shoots by week 15 occurred in the Ag and Zn treatments (9.1 new shoots/.week for each). The lowest values of new shoots were noted in the Cu (5.1 new shoots/.week) and control (4.4). It is suggested that Cu+/Cu2+ ions released from Cu2O ENPs impede the production of new shoots.

In plants, Cu ions can cause enzyme inactivation as a result of interaction with sulfhydryl groups of proteins, with consequent protein dysfunction, tissue necrosis, stunting, and root growth inhibition. Oxidative stress induced by CuO ENPs or their released Cu ions is also commonly recognized as a key mechanism for CuO ENP toxicity. Copper ions can elevate the generation of ROS (reactive oxygen species) through the Fenton () or the Haber–Weiss () reactions (Letelier et al. 2010). Oxidative stress via the generation of ROS damages lipids, carbohydrates, proteins and DNA.

It must be emphasized that nearly all published literature on the effects of copper oxide nanoparticles on plants has addressed the cupric oxide (CuO) form, while the current study is investigating the effects of cuprous oxide (Cu2O) ENPs. Data on the effects of cuprous oxide ENPs on plant growth are very limited.

When cattail was grown with ENPs, both ZnO- and Cu2O-treated plants experienced rapid growth of new shoots after week 6 and remained highest by week 15 (Figure 2(c)). By week 15, the mean number of new shoots in the ZnO and Cu2O treatments (3.0 and 2.0, respectively) were significantly (one-way ANOVA p < 0.05) greater than that in the control (1.4/.week).

The behavior and effects of ZnO ENPs in plants are not completely clear. In many reported cases, the presence of ZnO ENPs up to a certain concentration has the capability to enhance growth, where they provide Zn2+ as a micronutrient. ZnO ENPs have imparted beneficial effects to shoot growth of terrestrial plants; this may be due to the regulation of gene expression levels of related hormones (gibberellic acid and indole-3-acetic acid) and transcriptional proteins (zinc transporter proteins) (El-Badri et al. 2021). Benefits may also accrue from the modulation of the content of relevant enzymes and carbohydrates in planta (Liu et al. 2022). ZnO ENPs also have an important role in reducing oxidative stress caused by ROS.

New cattail shoots in the TiO2 and ionic Zn treatments were found to differ by week (two-way ANOVA p = 0.004 and p = 0.014, respectively) (Figure 2(d)) – highest values by week 15 were noted in TiO2- and Zn-treated plants (4.0 and 3.7 shoots per week, respectively), compared with 2.4 per week for the control. In several treatments, shoot production stalled and leaf necrosis was noted.

When cattail was treated with ionic forms of metals, the Zn-treated plants had among the highest values for new shoots by week 15 (3.7 shoots per week), compared with 2.4 per week for the control (Figure 2(d)).

The high number of new cattail shoots in the Cu2O ENP-treated cattail (Figure 2(c)) was opposite to that found in sedge treated with Cu2O (Figure 2(a)). A preponderance of published literature indicates the detrimental effects of CuO ENPs on plant growth; few have reported beneficial effects of CuO ENPs. These results suggest that further investigation of the effect of CuO, Cu2O, and ZnO ENPs on plants at both the sub-cellular level and at field-scale is needed.

Leaf area

When sedge was treated with ENPs, the greatest leaf area was measured at week 15 in the Cu2O treatment (23.6 cm2) followed by BiVO4 (20.2 cm2) (one-way ANOVA p > 0.05) (Table 2).

Table 2

Leaf area at week 15 as a function of plant species and ENP treatment

Plant speciesENPArea (cm2)
Sedge Ag 8.97 ± 5.95 
TiO2 18.02 ± 6.72 
ZnO 18.30 ± 11.28 
BiVO4 20.20 ± 11.20 
Cu223.63 ± 11.39 
Mixed-NPs 16.86 ± 1.52 
Control 13.53 ± 7.74 
Cattail Ag 12.73 ± 6.45 
TiO2 12.26 ± 10.02 
ZnO 9.47 ± 3.17 
BiVO4 13.35 ± 2.30 
Cu26.06 ± 4.46 
Mixed-NPs 18.00 ± 2.36 
Control 14.12 ± 3.03 
Plant speciesENPArea (cm2)
Sedge Ag 8.97 ± 5.95 
TiO2 18.02 ± 6.72 
ZnO 18.30 ± 11.28 
BiVO4 20.20 ± 11.20 
Cu223.63 ± 11.39 
Mixed-NPs 16.86 ± 1.52 
Control 13.53 ± 7.74 
Cattail Ag 12.73 ± 6.45 
TiO2 12.26 ± 10.02 
ZnO 9.47 ± 3.17 
BiVO4 13.35 ± 2.30 
Cu26.06 ± 4.46 
Mixed-NPs 18.00 ± 2.36 
Control 14.12 ± 3.03 

n = 3. Data are shown as mean ± standard deviation.

No mean values were significantly different (p > 0.05).

Copper combines with specific proteins and enzymes that have essential functions in plant growth. Copper is involved in mitochondrial respiration, photosynthetic electron transport, oxidative stress response, and hormone signaling. Various studies have noted the beneficial effects of low concentrations of exogenous CuO ENPs on the morphological and physiological aspects of plants with no indications of toxicity or repression of growth.

The lowest leaf area was in the Ag ENP treatment (8.9 cm2), which was below that for the control (13.5 cm2). The leaf area of plants treated with soluble Ag+ did not significantly differ from that of control plants. This trend has been corroborated by other studies. Various authors suggest that growth inhibition and cell damage from Ag ENPs are directly attributed to the nanoparticles themselves or to dissolved Ag+ affecting critical biotic receptors. Ag ENPs can affect the fluidity and permeability of the cell membrane and, consequently, influence water and nutrient uptake.

In cattail grown with ENPs, all leaf area values were below control (14.1 cm2) except for the mixed-ENPs treatment (18.0 cm2) (p > 0.05). The lowest leaf area was in the Cu2O treatment (6.1 cm2) (p < 0.05), which is in contrast to the data for sedge, where the beneficial effects of Cu2O ENP treatment were demonstrated. Previous work on the interaction of CuO ENPs with plants has shown species-dependent results.

It is suggested that trends for leaf area (and possibly other variables) were not significant due to the high variation among replicates.

Root growth

In ENP-treated sedge, the mean root length by week 15 was greatest in the TiO2 treatment (15.0 cm) (Figure 3(a)). All other values for the root length were below control (11.2 cm) (one-way ANOVA p > 0.05). The reported effects of TiO2 ENPs on plant growth are contradictory. Some studies have noted inhibitory effects, while others have revealed beneficial impacts of TiO2 ENPs to plants which may directly or indirectly enhance growth. TiO2 ENPs have been determined to increase nitrogen metabolism and photosynthesis, which ultimately enhances plant growth.
Figure 3

Mean root length in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; and (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

Figure 3

Mean root length in (a) sedge treated with ENPs; (b) sedge treated with ionic metals; (c) cattail treated with ENPs; and (d) cattail treated with ionic metals. n = 3. * indicates the extreme value; o indicates outlier value.

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By weeks 10 and 15, sedge root length in all ionic metal treatments was below control (p < 0.05) (Figure 3(b)), which suggests that roots experienced toxicity when in contact with high concentrations of soluble metals.

When cattail was grown with ENPs, average root length by week 15 was greatest in the ZnO and mixed ENP treatments (10.7 and 10.3 cm, respectively) (p > 0.05) (Figure 3(c)). These values are 41.8 and 38.2%, respectively, greater than the control (7.0 cm). ZnO ENPs have been proven beneficial to the growth and development of roots of a number of terrestrial plants. The mean cattail root length by week 15 was lowest in the Ag ENP treatment, which was below control (p > 0.05).

The impact of Ag ENPs is both species- and concentration-dependent. Ag ENPs are documented to inhibit photosystem II quantum yield. Other studies have determined that Ag ENPs inhibit plant growth by impairing various stages of cell division and collapsing root cortical cells, epidermis, and root caps. However, it remains difficult to fully understand the mechanism of Ag ENP toxicity in aquatic plants due to the limited number of studies.

When grown with ionic metals, the greatest mean cattail root length in weeks 5, 10, and 15 was in the Zn-treated plants (Figure 3(d)). These data, plus data for the uptake of Zn in cattail tissue (Ebrahimbabaie et al. 2023), suggest the importance of the dissolved Zn2+ ion from dissolution of ZnO nanoparticles.

No overall difference in root length was determined for interaction for ENPs and sampling date for sedge or cattail (two-way ANOVA p = 0.24 and 0.68, respectively).

Given the extensive usage of ENPs combined with their losses to the environment, it is probable that biota is exposed to a suite of ENPs, rather than just one. The mixed-ENPs treatment imparted positive effects to sedge CA and new shoots, and cattail chlorophyll, leaf area, and root length. In the mixed-ENPs treatment, soluble nutrient ions (e.g., Zn, Cu) plus other, possibly beneficial ions (e.g., Ag+, Bi3+, VO3− and/or VO43−) may have been released at rates favorable for vigorous production of shoots and other positive results. Results were not consistent, however: the mixed-ENPs resulted in low sedge chlorophyll content. One study (Olkhovych et al. 2016) found that mixed metal nanoparticles (Ag, Ag2O, Zn, Cu, and Mn) imparted variable effects on the content of chlorophylls and carotenoids in wetland plants; the response was a function of species.

When mixed metals in ionic form were applied weekly, negative impacts to sedge and cattail root length, and new shoots in cattail were noted. In those cases, a threshold limit may have been exceeded for one or more soluble metals. In contrast, nanoparticles, being of relatively lower solubility, released only limited soluble metals in the microcosms. This paper is one of very few that has investigated the impacts of multiple ENPs on plant response.

The current study demonstrates that the response of aquatic plants exposed to metallic ENPs must be considered species-specific. Additionally, toxicity mechanisms between ionic and ENP forms may differ in plants. Sedge and cattail tolerated a range of concentrations of metal-based ENPs, including light-activated particles (i.e., BiVO4 and Cu2O), with modest adverse effect, suggesting potential for phytoextraction (Ebrahimbabaie et al. 2023). Virtually all research on plant responses to ENPs involves the application of a single nanoparticle to pristine water. In countless locations worldwide, however, natural waters are contaminated by multiple chemicals. This study reveals that treatment with mixed-ENPs was often not detrimental to the test plants, and was sometimes even beneficial. The effect is suggested to be due to the gradual release, via particle dissolution, of nutrient metal ions.

The reported study is among the few to apply ENPs over a prolonged period (15 weeks) rather than as a single dose. Long-term application more accurately reflects real-world conditions (i.e., gradual inputs of a pollutant to an ecosystem). It is possible that such prolonged application may allow plants to acclimate to continued exposure to ENPs. The mechanisms involved in nanoparticle interaction with plants are still unknown and require further study. Many gaps persist in our understanding of these processes, and more collaborative research is necessary including expertise from fields such as plant physiology, geochemistry, biochemistry, agricultural engineering, and genetic engineering.

Funding from the Sigma Xi Scientific Society, Indiana Academy of Science and the Ball State University Aspire program is gratefully acknowledged.

P.E. contibuted to conceptualization; writing original draft; preparation of figures and tables; editing of manuscript; A.S. performed data collection; J.P. was involved in conceptualization; writing the original draft; preparation of figures and tables; editing of the manuscript; J.J. did statistical analysis and interpretation of data; E.Z. was involved in directing the synthesis of nanoparticles and editing of manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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