Alumina nanoparticles enhance growth of Lemna minor

Aquatic Toxicology 105 (2011) 328–336

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Alumina nanoparticles enhance growth of Lemna minor Guillaume Juhel a,c,1 , Emeline Batisse a , Quitterie Hugues a , Deirdre Daly a , Frank N.A.M. van Pelt b,c , John O’Halloran a,c , Marcel A.K. Jansen a,c,∗ a b c

Department of Zoology, Ecology and Plant Sciences, School of Biological, Earth and Environmental Sciences, University College Cork, Distillery Field, Cork, Ireland Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland Environmental Research Institute, University College Cork, Cork, Ireland

a r t i c l e

i n f o

Article history: Received 18 April 2011 Received in revised form 10 June 2011 Accepted 21 June 2011 Keywords: Lemna minor Alumina Nanoparticle Toxicity Biomass

a b s t r a c t The industrial use of nanoparticles is rapidly increasing, and this has given rise to concerns about potential biological impacts of engineered particles released into the environment. So far, relatively little is known about uptake, accumulation and responses to engineered nanoparticles by plants. In this study, the effects of alumina nanoparticles on growth, morphology and photosynthesis of Lemna minor were quantified. It was found that alumina nanoparticles substantially increase biomass accumulation of L. minor. Such a stimulatory effect of alumina nanoparticles on growth has not been reported previously. Enhanced biomass accumulation was paralleled by morphological adjustments such as increased root length and number of fronds per colony, and by increased photosynthetic efficiency. Metal nanoparticles have previously been shown to enhance the energy transfer efficiency of isolated reaction centres; therefore it is proposed that the mechanism underlying the alumina mediated enhancement of biomass accumulation in L. minor is associated with increased efficiencies in the light reactions of photosynthesis. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The industrial use of nanoparticles is rapidly increasing, and this has given rise to concerns about the environmental fate and potential biological impacts of such engineered particles. Nanoparticles have distinctly different physico-chemical characteristics compared to both individual molecules and bulk materials with the same chemical composition (Handy et al., 2008). These distinct characteristics relate to their small size, and relatively large specific surface area, and surface energy. Consequently, engineered nanoparticles are characterised by, among others, chemical composition, primary particle size, particle size distribution, hydro-

Abbreviations: ROS, reactive oxygen species; SIMR, stress-induced morphogenic responses; PSII, photosystem II; Fo, minimum fluorescence in the dark adapted state; Fm, maximum fluorescence in the dark adapted state; Fv, variable fluorescence; Y(II), steady state quantum yield of photosystem II; Qp , coefficient of photochemical quenching; Qn , coefficient of non-photochemical quenching; Fm , maximum fluorescence under light adapted conditions; Fo , minimum fluorescence under light adapted conditions; TEM, Transmission Electron Microscopy; NTA, Nanoparticle Tracking Analysis; SA, surface area. ∗ Corresponding author at: School of Biological, Earth and Environmental Sciences, University College Cork, Distillery Field, Cork, Ireland. Tel.: +353 021 490 4553; fax: +353 021 490 4664. E-mail address: [email protected] (M.A.K. Jansen). 1 Current address: Tropical Marine Science Institute, National University of Singapore, Singapore. 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.06.019

dynamic diameter, and zeta potential (Handy et al., 2008). The special physico-chemical characteristics of engineered nanoparticles are exploited in a broad range of applications as diverse as cosmetics, pharmaceuticals, textiles, electronics, biosensors and catalysts (Handy et al., 2008). Due to the widespread use of engineered nanoparticles, it is expected that substantial amounts of these particles will ultimately end up in aquatic, terrestrial and atmospheric environments (Nowack and Bucheli, 2007). To understand the risks associated with such releases, there is a clear need for studies that investigate the biological impacts of engineered nanoparticles on living organisms. In parallel, further information is required about the fate of released engineered nanoparticles, including degradability and bioavailability in the environment. A broad range of engineered nanoparticles displays toxicity towards human tissues, fish and invertebrates (Handy et al., 2008). Acute toxicity data are available for some model species such as Daphnia magna, for which impacts of metal nanoparticles such as zinc oxide, titanium oxide and aluminium oxide on mobility and mortality have been reported (Zhu et al., 2009). Disruptive effects of metal nanomaterials on biochemical processes and induction of genotoxicity have been reported, among others, for fish cells (Vevers and Jha, 2008). Studies on mammals have especially focused on respiratory toxicity and have established links with the generation of Reactive Oxygen Species (ROS), and oxidative stress (Nel et al., 2006). Oxidative stress was also found to be a major component of titanium oxide nanoparticle induced toxicity

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Fig. 1. (A) representative image of alumina nanoparticles visualized using Transmission Electron Microscopy (TEM). Scale bar: 50 nm. (B) Histogram of alumina nanoparticle diameters determined by TEM. (C and D) Size distribution of alumina nanoparticles (10 mg/L and 1 g/L, respectively) analysed by Nanoparticle Tracking Analysis (NTA) in half-strength Hutner’s medium.

in carp (Cyprinus carpio) (Hao et al., 2009). Plants are also exposed to nanoparticles that can be present in the terrestrial, aquatic and atmospheric environments (Navarro et al., 2008). The specific exposure route will determine the uptake pathway into the plant. Foliar uptake of atmospheric metal nanoparticles has been demonstrated and is affected by leaf morphological parameters such as the structure of trichomes, and the presence of a hypodermis and stomata (Da Silva et al., 2006). Root hairs have been shown to play a role in uptake of particles from terrestrial and aquatic media, and this is thought to involve endocytosis (Oveˇcka et al., 2005). There are only limited data on the biological consequences of contact and/or uptake of engineered nanoparticles by plants and many questions concerning the fate and behaviour of nanoparticles in plants remain (Ma et al., 2010). Metal-based nanoparticles such as titanium oxide nanoparticles were found to be toxic to the green alga Desmodesmus sp., impeding growth (Hund-Rinke and Simon, 2006). Zinc and zinc-oxide nanoparticles impede germination of ryegrass (Lolium perenne) and corn (Zea mays) seeds (Lin and Xing, 2007). Zinc oxide nanoparticles are particularly phytotoxic, and exposure of radish

(Raphanus sativus), rape (Brassica napus), ryegrass (L. perenne), lettuce (Lactuca sativa) or cucumber (Cucumis sativus) seedlings to this nanomaterial resulted in the nearly complete obliteration of plant roots (Lin and Xing, 2007). Alumina (Al2 O3 ) nanoparticles were also found to impede root elongation in a variety of species, including corn, cucumber and cabbage (Brassica oleracea) (Yang and Watts, 2005; Lin and Xing, 2007). In contrast, aluminium (Al) nanoparticles enhanced root elongation growth of radish and rape (Lin and Xing, 2007). Growth stimulation was also observed in experiments with titanium oxide nanoparticles which were found to enhance biomass accumulation of spinach (Spinacia oleracea) (Gao et al., 2008). It has previously been shown that a broad range of low concentration chemical stressors can stimulate plant growth (Kovács et al., 2009), questioning whether some of the observed growth stimulating effects are nano-specific. The diversity of responses to nano-metals is not surprising. Different nanomaterials can, among others, have a different chemical composition, chemical structure, crystalline structure, particle size, and surface area. In addition, plant responses to stressors can vary dramatically depending on

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Table 1 Physico-chemical characterisation of alumina nanoparticles. Particle characterisation

Alumina nanoparticles

Nominal size (nm) Shape (TEM) Average size (nm) (TEM) Hydrodynamic diameter (nm) (NTA) 10 mg/L 1 g/L Particle number/mL (NTA) 10 mg/L 1 g/L Zeta potential in stock solution (mV) Zeta potential in half-strength Hutner medium (mV)

20.0 Spherical to ovoid 9.01 ± 3.2 165 ± 38 189 ± 23 (2.75 ± 1.4) × 107 (7.06 ± 2.54) × 107 −4.6 ± 1.5 5.3 ± 1.4

whether exposure is acute or chronic, concentrations used, specific plant species, as well as a range of other environmental parameters. Typically, stress exposure causes a complex mixture of damage and acclimation responses, the balance of which is dependent on the exposure conditions. Generic plant acclimation responses which may decrease damage (i.e. increase stress tolerance) include increased antioxidant defences and signalling (Foyer and Noctor, 2005), as well as stress-induced morphogenic responses (SIMR) (Potters et al., 2007). Lemnaceae or duckweeds are ideally suited to study the development of damage (toxicity), and/or stress acclimation (Prasad et al., 2001; Drost et al., 2007; Appenroth et al., 2010; Biswas et al., 2010). Lemnaceae are small, freshwater hydrophytes that are commonly used for regulatory toxicity testing of chemicals (OECD, 2000; ISO, 2001; Brain and Solomon, 2007). The suitability of Lemnaceae for toxicity testing relates to growth characteristics such as the small size, rapid reproduction and ease of culturing (cf. Landolt, 1986). Uptake and distribution of chemicals by Lemnaceae depends on anatomical and morphological characteristics, including the extent of contact with the liquid growth medium (i.e. floating versus submerged species). Lemna minor, a floating species, is reported to be sensitive to a range of toxins (Lahive et al., 2010), and this species is commonly used for ecotoxicological testing. Biological impacts of chemicals can be easily quantified as altered biomass growth rates, frond and colony numbers, chlorophyll content or chlorophyll-a fluorescence (Appenroth et al., 2010; Lahive et al., 2010). Colony disintegration can be readily observed in stressed cultures, and this has been hypothesised to be a survival strategy that prevents toxin transfer from mature to younger fronds (Li and Xiong, 2004a,b). Several studies have shown that alumina nanoparticles can enhance the electron transfer efficiency of isolated photosynthetic reaction centres (Govorov and Carmeli, 2007; Nadtochenko et al., 2008). The importance of this finding in terms of the growth and development of the intact plant has not been determined. In this study, the hypothesis was tested that aluminium oxide (alumina, Al2 O3 ) nanoparticles can enhance the growth of L. minor. This was done by quantifying the effects of engineered alumina nanoparticles on growth, morphology and photosynthesis of L. minor. This study addresses a gap in our knowledge about the biological impacts of engineered nanoparticles (Handy et al., 2008). 2. Materials and methods 2.1. Lemnaceae culturing Cultures of L. minor were maintained at the School of Biological, Earth and Environmental Sciences, University College Cork, Ireland. L. minor was originally sourced from a pond in Blarney, Co. Cork, Ireland. Autotrophic cultures were kept under aseptic, optimal growth conditions on half-strength Hutner’s medium (Brain and Solomon, 2007; Biswas et al., 2010) in magenta vessels with

Fig. 2. Effects of alumina nanoparticles on 7-day biomass accumulation by L. minor cultures. At the start of the experiment, alumina nanoparticles were added to halfstrength Hutner’s medium, together with four equally sized L. minor colonies. The initial fresh weight was 14 mg. Standard deviations are shown (n = 4). ANOVA (post hoc Tukey) showed a highly significant effect of alumina nanoparticle treatment (P < 0.01). Points marked with * or ** are significantly different from the control (P < 0.05, P < 0.01, respectively). The concentration of nanoparticles is given on a half-logarithmic scale.

vented lids. The initial pH of the growth medium is 4.6–4.7. The light regime for culturing and for all toxicity tests was 16 hr light: 8 hr darkness at a light intensity of 50 ␮mol m−2 s−2 (cool white fluorescent lamps) and a temperature of 22 ± 2 ◦ C. These conditions facilitate an 8-fold increase in frond numbers over 7 days as stipulated by OECD (2000), although temperature and light levels are slightly less than recommended. 2.2. Alumina exposure A stock suspension of 10 g/L alumina nanoparticles (Al2 O3 , Alfa Aesar, No44757, Heysham, UK) was prepared in 70% ethanol, and this stock suspension was used to make up all exposure concentrations less than 30 mg/L alumina. For higher concentrations, alumina suspended in a small volume of ethanol, was directly added to the medium. The final ethanol concentration never exceeded 0.1% to avoid plant toxicity. Ethanol (0.1%) was also added to untreated, control cultures. A final volume of 200 mL half-strength Hutner’s medium was used for all experiments. The pH of all aluminananoparticle treatments was set at 5.3 in all but the “exposure range” finding experiment (Fig. 1), using HCl. Effects of alumina nanoparticles on endpoints were scored after 7 days. To study potential, indirect effects of alumina nanoparticles (especially particle dissolution) on L. minor growth, particles were incubated for either an hour, or for 7 days in half-strength Hutner’s medium, after which particles were removed from the medium by ultracentrifugation (Sorval RC-5B Refrigerated Superspeed centrifuge, Dupont, USA) for 20 min at 12.500 g, followed by decanting of the supernatant into a new container. This supernatant was subsequently used for a 7 day L. minor growth assay. The biological effects of molecular aluminium sulphate were studied using Al2 (SO4 )3 (Sigma–Aldrich, Dorset, UK) in concentrations ranging from 0.003 mM to 1 mM. Free aluminium concentrations are likely to have been slightly lower due to presence of a chelator (2.9 mg/L EDTA) in Hutner medium. The pH was set at 5.3. Effects of aluminium sulphate on endpoints were scored after 7 days. The biological effects of bulk alumina were studied using Al2 O3 (Sigma–Aldrich, Dorset, UK) with an average size of approximately 5–10 ␮m (micro-particle), and in concentrations ranging from 1 mg/L to 1000 mg/L. Bulk alumina did not affect the pH of the medium, hence no adjustment was required in these experiments. Endpoints were scored after 7 days.

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500

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Alumina concentration (mg/L) Fig. 3. Effects of nano-alumina on L. minor, (A) biomass; (B), root length; (C), frond number; (D), colony number; and (E) total surface area. All parameters were measured after 7 days growth. The initial fresh weight was 16 mg which comprised 4 colonies each with three fronds. Standard errors of the mean are shown (n = 5). ANOVA (post hoc Tukey) showed significant effects of alumina nanoparticle treatment on biomass (P < 0.05), root length (P < 0.01), and frond number (P < 0.01). Points marked with * or ** are significantly different from the control (P < 0.05, P < 0.01, respectively).

2.3. Lemna minor growth assay For growth assays, four similarly sized colonies of L. minor each with three fronds, were transferred to a magenta vessel containing 200 mL medium. A minimum of four replicates was analysed for each alumina exposure concentration. Magentas were kept in growth rooms (conditions as described for “Lemnaceae culturing”) and after 7 days endpoints were measured to assess the biological impact of added alumina. In each series of experiments, controls were cultures not exposed to alumina nanoparticles, bulk alumina or aluminium sulphate, but that were in all other aspects treated similarly. 2.4. Endpoints quantified The biomass (fresh weight) of four freshly harvested L. minor colonies was measured at 0.1 mg sensitivity (Ohaus Explorer, Switzerland) on day 1, and the mean of five replicates served as the initial biomass for the calculation of biomass increases. Accumulated biomass was again measured after 7 days of growth. Morphological parameters such as frond and colony number, length of roots and total surface area of the culture were quantified on digital photographs using ImageJ software (NIH, USA). Chlorophyll-a fluorescence was measured using a modulated imaging fluorometer equipped with ImagingWin software (PAM, Effeltrich, Germany). Three colonies were randomly selected from each replicate and surface water was removed using absorbent

paper. Plants were then dark-adapted for 15–20 min. A low intensity, measuring light (<1 ␮mol/m2 /s1 ) was used to determine Fo, the minimum fluorescence obtained in the dark adapted state. Maximum fluorescence (Fm) was determined for the dark adapted state by applying a saturating pulse of white light. The variable fluorescence (Fv) was calculated as the difference between the maximum fluorescence and the minimum fluorescence. The maximum quantum efficiency of photosystem II (PSII) was calculated as: Fv/Fm = (Fm − Fo)/Fm. Plants were exposed for 15 min to an actinic light intensity of 145 ␮mol/m2 /s1 in order to determine steady state fluorescence parameters. The steady state quantum yield of photosystem II (Y(II)), and coefficients of photochemical quenching (Qp ) and non-photochemical quenching (Qn ) were calculated as Y(II) = (Fm − F)/Fm , Qp = (Fm − F)/(Fm − Fo ) and Qn = (Fm − Fm )/(Fm − Fo ) (Maxwell and Johnson, 2000; Vassilev, 2002), whereby Fm was the maximum fluorescence yield under light adapted conditions, Fo was the minimum fluorescence yield under light adapted conditions, and F was the fluorescence intensity under actinic light. Areas of Interest, i.e. areas within colonies where chlorophyll-a fluorescence was measured, were selected in the centre of mature fronds. 2.5. Physico-chemical characterisation nano-alumina Aluminium oxide nanoparticles (Al2 O3 ) were characterised by an average nominal size of 20 nm, APS power, an SA of 200 m2 /g and a formula weight of 101,96 g (Alfa Aesar, Ward

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Fig. 4. Effects of ammonium sulphate on L. minor, (A) biomass accumulation and (B) root elongation, and of bulk alumina on (C) biomass accumulation and (D) root elongation. All parameters were measured after 7 days growth. The initial fresh weight was 16 mg. Standard errors of the mean are shown (n = 4). ANOVA (post hoc Tukey) showed a highly significant effect of aluminium sulphate on biomass (P < 0.01) and root length (P < 0.01), and a significant effect of bulk alumina on root elongation (P < 0.05). Points marked with ** are significantly different from the control (P < 0.01).

Hill, MA, USA). Alumina nanoparticle primary size was further investigated using Transmission Electron Microscopy (TEM). TEM images were recorded using a Jeol JEM-2011 Transmission Electron Microscope equipped with a Gatan DualVision 600 CCD. Samples were prepared by drop casting a 2.5 ␮L aliquot of the alumina nanoparticle stock solution onto a 300 mesh carbon-coated copper grid, which was allowed to evaporate under ambient conditions. Average alumina nanoparticle diameters were determined by quantifying the dimensions of approximately 200 nanoparticles (located in different regions of the grid) on TEM images, using ImageJ software (NIH, USA). The size distribution, number and mean hydrodynamic diameter of the alumina particles were further characterised using a NanoSight LM20 system with a laser output of 30 mW at 650 nm (NanoSightTM , NanoSight Ltd., Wiltshire, SP4 7RT, UK). Mean square displacements of single particles were determined by tracking the scattered light using Nanoparticle Tracking Analysis (NTA) technology. Measurements were carried out at particle concentrations of 10 mg/L and 1 g/L in half-strength Hutner’s medium and results were the means of quintuplicate runs. All data were subsequently analysed using the instrument software (NanoSightTM) version 1.5. Zeta potential was measured on a Zetasizer Nano ZS ZEN3600 (Malvern Instruments Ltd., Malvern, UK) operating with a He–Ne laser at a wavelength of 633 nm using back scattered light. Measurements were carried out on the stock solution with particle

concentrations of 10 mg/L after sonication of the solution for 30 min. Zeta potential was also measured in half-strength Hutner’s medium with particles concentrations of 10 mg/L. Results were the means of quintuplicate runs. 2.6. Statistical analysis Dose–response curves were constructed and standard errors were calculated. The response of each endpoint to a particular alumina concentration was compared to that of others using ANOVA, post hoc Tukey test (P < 0.05) using SPSS (PASW version 17, IBM, NY, USA) and/or Minitab (version 12.21, PA, USA). 3. Results 3.1. Physico-chemical characterisation of nano-alumina particles Alumina (Al2 O3 ) nanoparticles were characterised by an average nominal size of 20 nm, an SA of 200 m2 /g and a formula weight of 101.96 g (Alfa Aesar, Ward Hill, MA, USA). Average alumina nanoparticle diameter was determined by quantifying the dimensions of approximately 200 nanoparticles on TEM images. A representative TEM image of alumina nanoparticles (Fig. 1A) shows the range of particle sizes and morphologies observed (Table 1). A log-normal distribution of the particles sizes was observed, with an average size of 9.01 nm and a standard deviation of 3.2 nm (median size 8.38 nm). The size distribution, number and mean hydrody40

A

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1 hour incubation

Fig. 5. Effects of the removal of alumina nanoparticles on the enhancement of L. minor growth. Particles were incubated for either 1 h or 1 week in half strength Hutner’s medium, removed by ultracentrifugation, and the medium was subsequently tested for growth promoting ativities. Control medium was treated identical to the other media, except that no nano-particles were ever added. Effects on (A) biomass accumulation and (B) root length on day 7. The initial fresh weight was 16 mg. Standard errors of the mean are shown (n = 4). ANOVA (post hoc Tukey) revealed no statistically significant effects.

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namic diameter of the particles were further characterised using Nanoparticle Tracking Analysis (NTA) technology. Measurements were carried out at particle concentrations of 10 mg/L and 1 g/L in half-strength Hutner’s medium. Analysing a freshly made (ca. 1 h) suspension of particles in medium, we observed a multimodal distribution in particle size at the two concentrations tested (Fig. 1C and D). The main peaks in particle size were approximately 20, 40, 80, 100, 200 and 275 nm for 10 mg/L, and 20, 60, 80 and 120 nm for a 1 g/L suspension of alumina nanoparticles. No changes in particle size were observed during the course of the experiment. The data indicate various degrees of aggregation of the Alumina nanoparticles although most of the particles had a hydrodynamic diameter lower than 200 nm (Fig. 1C and D). At a concentration of 10 mg/L, the mean hydrodynamic diameter of these particles was 165 ± 38 nm (average ± SD) and the mean particle number was 2.75 ± 1.4 × 107 particles/mL. At a concentration of 1 g/L, the mean hydrodynamic diameter was 189 ± 38 nm (average ± SD) and the mean particle number was 7.06 ± 2.54 × 107 particles/mL. Zeta potential was 5.3 mV in half-strength Hutner’s medium at a particle concentration of 10 mg/L (Table 1). 3.2. Alumina nanoparticles enhance biomass accumulation by L. minor L. minor colonies were exposed for 7 days to alumina nanoparticles. Concentrations ranged from 0 to 10 g/L (Fig. 2). Exposure to such concentrations of alumina nanoparticles did not cause any macroscopic damage, and neither frond mortality nor colony disintegration was observed. Instead, a significant increase in accumulated biomass with increasing alumina concentrations was noted. Highest accumulated biomass was measured at 0.3 g/L alumina (Fig. 2). At this concentration, accumulated 7-day biomass was some 2.4-fold higher compared to the control raised in the absence of nano-alumina. Significant increases in 7-day biomass accumulation could be measured at nano-alumina concentrations of 100 mg/L (P < 0.05), 0.3 g/L (P < 0.01), 1 g/L (P < 0.01) and 3 g/L (P < 0.05)(Post Hoc Tukey), compared to the untreated control. Exposure to alumina concentrations greater than 0.3 g/L caused a decrease in the enhancement of biomass production (Fig. 2). The addition of alumina nanoparticles caused an increase in the pH of the Lemnaceae medium from pH 4.7 in the standard halfstrength Hutner’s medium to 5.8 after addition of 1 g/L alumina. To exclude the possibility that the increase in biomass seen in nanoalumina treated samples (Fig. 2) was caused by this increase in pH, the pH of all alumina treatments and their untreated controls was set at pH 5.3. In the absence of variations in pH, the addition of 1 g/L alumina increased fresh weight biomass accumulation of L. minor by >65% (P < 0.015)(Fig. 3A). Therefore, it was concluded that alumina nanoparticles can enhance biomass accumulation in the absence of changes in the pH of the liquid medium. 3.3. Alumina nanoparticles alter the morphology of L. minor colonies Apart from the increase in biomass, the most notable consequence of exposure to alumina nanoparticles was an increase in root length (Fig. 3B). In the absence of variations in pH, 1 g/L alumina increased significantly (>150%) the root length of L. minor (P < 0.01). Exposure to a concentration of just 10 mg/L alumina already increased root length in L. minor by 80% (P < 0.01). To further characterise the alumina induced increase in growth in L. minor, frond, colony and total-surface area were quantified. A small but significant (P < 0.01) increase in the number of fronds (24%) was observed in the cultures treated with 1 g/L alumina (Fig. 3C). In contrast, the number of colonies did not change significantly in response to nano-alumina exposure (Fig. 3D), suggesting that the

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number of fronds per colony was slightly increased at the higher concentration of alumina nanoparticles. Also, the total surface area covered by all fronds in each experimental unit remained unaltered irrespective of alumina treatment (Fig. 3E). 3.4. Alumina mediated enhancement of growth is “nano-specific” A pertinent question is whether the changes in biomass production and root elongation are caused by alumina nanoparticles, or rather by aluminium ions that may be released due to the potential dissolution of the particles. To address this question, fronds were exposed to a range of ionic aluminium concentrations (0–1 mM) to test the potential effects of aluminium on the growth of L. minor. It was found that aluminium-sulphate inhibits both biomass accumulation and root elongation (Fig. 4A and B). Concentrations as low as 0.03 mM Al2 (SO4 )3 significantly inhibited both biomass accumulation and root elongation (P < 0.01). The inhibition of growth and the onset of toxicity symptoms, such as the decrease in chlorophyll content and colony disintegration, are consistent with aluminium toxicity. The addition of 0.03 mM Al2 (SO4 )3 , increased the SO4 concentration in the medium by just 4.9% and this is unlikely to have caused toxicity. Thus, the impact of ionic Al2 (SO4 )3 on L. minor is noticeably different from that by alumina nanoparticles. To further exclude the possibility that ionic aluminium, released due to dissolution of nano-alumina particles, caused the observed growth stimulation, we quantified the biological effects of medium from which alumina nanoparticles had been removed. Alumina nanoparticles were incubated for either 1 h, or 7 days in halfstrength Hutner’s growth medium, after which particles were removed using ultracentrifugation. This medium was used for a 7day analysis of L. minor growth. The control comprised centrifuged medium that had not been in contact with alumina nanoparticles. Plants raised on centrifuged medium were similar with respect to biomass accumulation and root growth (Fig. 5A and B), irrespective of whether the medium had earlier contained alumina nanoparticles. Therefore, it is unlikely that any ion leaked from the alumina particles causes the observed growth stimulation. The alumina nano-specificity of the observed biological effects was further ascertained by exposing plants to bulk aluminiumoxide. These particles were characterised using TEM, showing irregularly shaped particles with sizes ranging from 3 to 10 ␮m. These micrometer sized particles did not significantly affect biomass accumulation (Fig. 4C). A small increase in root length was observed in cultures exposed to the highest concentration of bulk alumina (Fig. 4D). 3.5. Alumina mediated enhancement of photosynthetic electron transfer L. minor was grown indoors on optimised medium (pH and nutrients). A potentially limiting factor for L. minor growth under indoor conditions is the lack of sufficiently high levels of photosynthetic active radiation (PAR). Alumina nanoparticles stimulated biomass accumulation at low light levels of PAR, suggesting increased light capture and/or photosynthetic efficiency. To investigate this, effects of alumina nanoparticles on photosynthetic efficiency were quantified. On average, our data did not show an alumina effect on the maximal quantum yield of photosystem II (Fv/Fm) in dark adapted fronds (Fig. 6A). However, under steady state photosynthetic conditions, the quantum yield of photosystem II, Y(II) was significantly (P < 0.05) increased in L. minor fronds treated with alumina nanoparticles (Fig. 6A). The level of photochemical quenching (Qp ) was not significantly increased (Fig. 6B), and the non-photochemical quenching (Qn ) was not affected by alumina nanoparticles (Fig. 6C). These data suggest that

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(Rout et al., 2001). Removal of alumina particles from the medium through ultracentrifugation showed that the nanoparticles themselves were responsible for the biological effect (Fig. 5A and B). Therefore, it is concluded that the growth enhancement by alumina does not involve aluminium ions produced through particle dissolution, but that enhancement depends on the direct contact between particle and plant. Bulk alumina had no effect on biomass accumulation (Fig. 4A and B), thus the growth enhancement by alumina nanoparticles is “nano-specific”. The latter conclusion is in agreement with work by Gao et al. (2008), who showed that titanium oxide nanoparticles stimulated growth of spinach, but that bulk titanium oxide had no significant effect. In our experiments, no evidence for alumina nanoparticle induced toxicity was found. However, a decreasing growth enhancement at alumina concentrations greater than 1 g/L was noted, and it is possible that alumina nanoparticles can cause some damage to L. minor at very high concentrations. Toxic, growth-inhibiting properties of aluminananoparticles towards a range of plant species have previously been reported (Lin and Xing, 2007; Yang and Watts, 2005). 4.2. Mechanisms underlying altered growth of L. minor exposed to nano-alumina

Fig. 6. Effects of alumina nano-particles on chlorophyll a fluorescence parameters in L. minor measured after 7 days exposure. Shown are the steady state quantum yield of PSII (Y(II)); photochemical quenching (Qp ); and non-photochemical quenching (Qn ). Standard errors of the mean are shown (n = 5). Nanoparticles have a statistically significant effect on Y(II) (P < 0.05).

alumina nanoparticles increased photosynthetic electron transfer efficiency. 4. Discussion 4.1. Alumina nanoparticles enhance growth of L. minor Alumina nanoparticles increased growth of L. minor, and this could be observed as enhanced biomass accumulation, morphological adjustments and increased photosynthetic efficiency (Figs. 2, 3 and 6). Previously, it was found that carbon nanotubes enhance seed germination and growth of tomato plants, an effect that was linked to water uptake (Khodakovskaya et al., 2009). Using metal-nanoparticles, Gao et al. (2008) showed that titanium oxide increased biomass accumulation in spinach by 60%. The recent discovery of the growth promoting effects of titanium oxide nanoparticles extends work from the 1930s which showed that titanium-salts can enhance plant growth (cf. Hruby´ et al., 2002). The beneficial effects of titanium-salts involve upregulation of a variety of stress acclimation mechanisms, and have been hypothesised to constitute a hormetic response, similar to that induced by a wide variety of stressors (Kovács et al., 2009). The relationship, if any, between the ionic titanium enhanced growth and the apparently similar enhancement by titanium oxide nanoparticles has not yet been established. However, the apparent similarity in response emphasises the importance of considering particle dissolution in nano-biological studies. Ionic aluminium was found to be toxic to L. minor (Fig. 4A and B) and this is in agreement with an extensive body of literature on the effects of aluminium on plants

A striking, alumina nanoparticle induced morphological change is the increase in root length in L. minor (Fig. 4B). Previously, alumina nanoparticles were found to enhance root elongation in Arabidopsis thaliana seedlings (Lee et al., 2009), although inhibitory effects were reported for a whole range of other species (Yang and Watts, 2005; Lin and Xing, 2007). There are many reports in the literature with respect to altered plant root development, especially in response to nutrient deficiencies. For example, phosphate starvation induces alterations in the spatial configuration of root systems, including increased lateral root formation and decreased elongation of the primary (López-Bucio et al., 2002). In contrast, nitrate deficiency stimulates primary root elongation, but does not affect lateral root formation (Linkohr et al., 2002). These contrasting responses have been interpreted in terms of their functional role in reaching out for plant nutrients (Linkohr et al., 2002). A possible explanation for the alumina nanoparticle induced increase in root length is that the alumina particles bind plant nutrients, decreasing their bioavailability, and thus inducing a “nutrient-shortage” response. However, this is considered unlikely as alumina nanoparticles strongly enhance biomass accumulation (Fig. 2), arguing against any nutrient shortage. Moreover, we also note that Lemnaceae absorb substantial amounts of nutrients directly through the lower surface of the floating fronds, thus limiting the role of roots in nutrient uptake (Ice and Couch, 1987). Recently, it was recognised that a broad range of non-optimal environmental conditions can induce generic, “stress induced morphogenic responses” (SIMRs), such as altered root elongation. SIMRs comprise a redistribution of growth (Potters et al., 2007). The mechanisms underlying these responses appear to be conserved across a broad range of environmental conditions. Key components of the SIMR control mechanism are Reactive Oxygen Species (ROS) and the phytohormone auxin (Potters et al., 2007). Several studies have reported nanoparticle induced changes in ROS production (Nel et al., 2006; Hao et al., 2009), and ROS have also been shown to play a regulatory role in frond division in Lemnaceae (Moschopoulou et al., 2007). A ROS-mediated effect on cell physiology would not even require cellular uptake of the nanoparticles. Effects of auxin and other phytohormones on Lemnaceae development are well established. Exposure to low concentrations of auxins causes development of longer roots, while auxin also controls frond size in Lemnaceae (cf. Landolt, 1986). Thus, it is possible that the altered morphology of nano-alumina exposed fronds is the result of interference with SIMR-signalling cascades, implying that alumina

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nanoparticles can act as a morphogen. It is, perhaps, not immediately obvious how redistribution of growth can lead to accelerated growth, i.e. enhanced biomass accumulation. One possibility is that alumina nanoparticles act to redistribute growth activity in favour of more frond surface area, such that overall colony photon capture and hence primary productivity is increased under the relatively low, indoor light levels. Consistent with this possibility, alumina nanoparticles increased the number of fronds (Fig. 3C). However, the larger number of fronds did not result in an increase in total “light capturing” surface area (Fig. 3E). Thus, while it is possible that alumina nanoparticles act as morphogens in L. minor, it appears less likely that this morphogenic effect underlies the observed enhancement of biomass accumulation in L. minor grown indoors under light limiting conditions. Measurements of an increased photosynthetic quantum yield of photosystem II under steady state conditions suggest another possible explanation for the enhanced biomass accumulation (Fig. 2). The increase in the steady state quantum yield of photosystem II (Fig. 6), together with the non-significant increase in photochemical quenching (Qp ), are indicative of an increased photosynthetic electron flow efficiency under steady state conditions (Vassilev, 2002), in alumina nanoparticle treated plants. Alumina nanoparticles increase the quantum yield of photosystem II, but not the maximal quantum yield of photosystem II (Fv/Fm), perhaps suggesting that the alumina nanoparticle effect is not directly on PSII. Artificial structures composed of a photosynthetic system and various metal nanoparticles also display strong enhancements of photosynthetic efficiency, and this is caused by parallel increases in light absorption by chlorophylls and energy transfer from chlorophylls to nanoparticles (Govorov and Carmeli, 2007; Nadtochenko et al., 2008; Mingyu et al., 2007). By analogy, it could be argued that an increase in photosynthesis in alumina nanoparticle treated fronds is caused by increased efficiencies in the early reactions of photosynthesis. Whether increases in light capture per sé can explain the observed changes in chlorophyll a fluorescence parameters measured in this study, remains to be seen. Exposure of leaves to increased light (as a proxy for increased light capture) typically results in a decreasing steady state quantum yield of photosystem II, and in lower photochemical quenching. However, our data do not reveal any decrease in these parameters (Fig. 6). An alternative hypothesis to explain increased photosynthetic quantum yield centres on increased Rubisco activase activity, which was reported following treatment of spinach with titanium oxide nanoparticles (Gao et al., 2008). Rubisco activase activity is light dependent through its response to the ADP/ATP ratio in the stroma, and this enzyme plays a dominant and critical role in controlling Rubisco activity, and hence the rate of the Calvin-Benson (dark-reactions) cycle of photosynthesis. However, it remains to be established whether the enhancement of Rubisco activase activity by titanium oxide nanoparticles is the primary nanoparticle effect, or rather a secondary consequence of accelerated photosynthetic yield.

4.3. Conclusion The alumina nanoparticle mediated growth enhancement constitutes a “response potential”. Exposure of L. minor for 7 days to a constantly high level of just one type of nanoparticle, under otherwise optimised growth conditions, is not realistic from an environmental perspective. Further work is required to explore the environmental relevance of the identified growth response. However, the advantage of controlled laboratory experiments as used in this study is that they enable basic studies on the fundamental mechanisms underlying alumina nanoparticle mediated growth stimulation.

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