Temperature-dependent toxicities of nano zinc oxide

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http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, Early Online: 1–12 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/17435390.2013.848949

ORIGINAL ARTICLE

Temperature-dependent toxicities of nano zinc oxide to marine diatom, amphipod and fish in relation to its aggregation size and ion dissolution Stella W. Y. Wong and Kenneth M. Y. Leung

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The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China

Abstract

Keywords

This study, for the first time, concurrently investigated the influence of seawater temperature, exposure concentration and time on the aggregation size and ion dissolution of nano zinc oxides (nZnO) in seawater, and the interacting effect of temperature and waterborne exposure of nZnO to the marine diatom Skeletonema costatum, amphipod Melita longidactyla and fish Oryzias melastigma, respectively. Our results showed that aggregate size was jointly affected by seawater temperature, nZnO concentration and exposure time. Among the three factors, the concentration of nZnO was the most important and followed by exposure time, whereas temperature was less important as reflected by their F values in the three-way analysis of variance (concentration: F3, 300 ¼ 247.305; time: F2, 300 ¼ 20.923 and temperature: F4, 300 ¼ 4.107; All p values 50.001). The aggregate size generally increased with increasing nZnO concentration and exposure time. The release of Zn ions from nZnO was significantly influenced by seawater temperature and exposure time; the ion dissolution rate generally increased with decreasing temperature and increasing exposure time. Growth inhibition of diatoms increased with increasing temperature, while temperature and nZnO had an interactional effect on their photosynthesis. For the amphipod, mortality was positively correlated with temperature. Fish larvae growth rate was only affected by temperature but not nZnO, while the two factors interactively modulated the expression of heat shock and metallothionein proteins. Evidently, temperature can influence aggregate size and ion dissolution and thus toxicity of nZnO to the marine organisms in a species-specific manner.

Chlorophyll a fluorescence, heat shock protein, metallothionein, nanomaterial, toxicity

Introduction The US Environmental Protection Agency has recently published a report on the evaluation of vulnerability of aquatic ecosystems exposed to multiple stressors, in particular, interactions of global climate change with anthropogenic influences (USEPA, 2011). The potential releases of engineered nanomaterials (NMs) into the aquatic environment have received much concern from governments and scientists, and their ultimate behavior and fate may be determined by various physicochemical conditions including temperature, pH and salinity. Several in vitro projects concerning the cellular uptake of NMs at different temperatures have been undertaken over the recent decade (Arsianti et al., 2010; Bejjani et al., 2005). However, as the purpose of most cellular researches was to evaluate the role of NMs as drug delivery carriers, the temperatures used for comparison were normally 4  C (where energy-dependent endocytosis ceases almost completely and cell membrane becomes too rigid for passive diffusion) and 37  C (normothermia) (Arsianti et al., 2010). Moreover, Rispoli et al. (2010) reported that higher temperatures led to decreased

Correspondence: Professor Kenneth M. Y. Leung, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. Tel: +852 22990607. Fax: +852 25176082. E-mail: [email protected]

History Received 15 November 2012 Revised 16 September 2013 Accepted 23 September 2013 Published online 13 November 2013

aggregate size of nano zero-valent copper in nutrient broth medium, and thus aggravated its toxicity toward Escherichia coli. On the other hand, in vivo experiments with aquatic plants and animals are relatively rare, with only two documented in vivo studies. Tao et al. (2009) observed that mortality and development of the freshwater flea Daphnia magna exposed to 0.2 mg L1 of fullerene (C60) was temperature-independent. Oukarroum et al. (2012) also observed that temperature had an influence on the aggregate formation and zeta potential of nano silver, which subsequently affected their inhibitory effect on the freshwater microalgae Chlorella vulgaris and Dunaliella tertiolecta. Our laboratory has previously documented a study comparing the toxicity of nano zinc oxide (nZnO), bulk ZnO and Zn ion to five marine organisms at room temperature (25  C; Wong et al., 2010); while nanoparticulate effects were observed, the toxicity of nZnO to the marine organisms was mainly attributed to dissolved Zn ions. This study, for the first time, was designed to concurrently examine the influence of temperature on the ion dissolution and size aggregation of nZnO in seawater and the interacting effect of temperature and waterborne exposure of nZnO to three selected marine organisms, namely, the diatom Skeletonema costatum, amphipod Melita longidactyla and fish Oryzias melastigma. These species represent the primary producer, primary consumer and secondary consumer, respectively. Our experimental results showed that temperature can significantly influence aggregate size and ion dissolution and thus toxicity of nZnO, although the interacting effect of temperature and nZnO is also highly species dependent.

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S. W. Y. Wong & K. M. Y. Leung

Materials and methods Waterborne exposure preparation

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A series of 96-h toxicity tests was undertaken with S. costatum, M. longidactyla and O. melastigma, through waterborne exposure to nZnO. Test endpoints included chlorophyll fluorescence for the diatom, mortality for the amphipod and growth and protein expressions for the fish larvae. The maximum predicted environmental concentration of nZnO found in the literature was found to be 0.76 mg L1 in the UK (assuming full market penetration of nZnO-containing products; Boxall et al., 2007), and the exposure range employed in this study (0.1–16 mg L1) would cover this scenario. License for the Animals (Control of Experiments) Ordinance was obtained from the Department of Health, the Government of Hong Kong Special Administrative Region, prior to experimentation. Chemical preparation and characterization nZnO powder (average primary particle size, 20 nm) was purchased from Nanostructured & Amorphous Materials Inc. (Los Alamos, NM). Zinc Pure AS calibration standard (1000 mg L1 dissolved in 2% HNO3) was obtained from PerkinElmer (Waltham, MA). All glasswares used in the study were acid-washed in 10% HNO3, rinsed with MilliQ water and autoclaved before use. The morphology of nZnO has been described in Wong et al. (2010). To evaluate the aggregate size distribution, six replicates, each replicate consisting of 15 mL of nZnO suspensions (at 10, 20, 50 or 100 mg L1) dispersed in 0.22-mm filtered artificial seawater (FASW; salinity, 30  0.5%; pH, 8.0  0.1; sea salt: Tropic Marin, Wartenberg, Germany), were shaken inside an orbital shaker/mixer chamber ( 200 rpm; set at 10, 15, 25, 28 or 30  C; model 3528-1; Lab-Line Instruments Inc., Melrose Park, IL) for 7 days. During both characterization and exposure studies, temperatures, salinities and pH of all test solutions were monitored frequently to ensure that their values remained relatively constant. The aggregation size distribution was quantified by dynamic light scattering technique (DLS; Delsa Nano C, Beckman Coulter Inc., Fullerton, CA). The measurement with DLS was conducted at days 1, 4 and 7, and samples were not shaken for 30 min prior to start of the measurement to simulate the exposure schematics. Preliminary trials showed that measurements at concentrations below 10 mg nZnO L1 was not viable with the DLS machine. To evaluate the dissolved metal ion concentrations, 15 mL of 100 mg L1 of nZnO suspensions were dispersed in FASW, shaken inside the orbital shaker/mixer chamber ( 200 rpm; set at 10, 15, 25, 28 or 30  C) for 7 days. Sampling took place at days 1, 3, 5 and 7. At each sampling time point, four replicates were withdrawn and filtered through a 0.02-mm syringe filter with alumina-based membrane (Whatman, Kent, UK). The samples were not centrifuged prior to filtering, since this step would increase the variability of the amount of released Zn2þ. The Zn2þ concentration in the filtrate was then measured by inductively coupled plasma optical emission spectrometry (PE Optima 8300, Perkin-Elmer, Branchburg, NJ). The detection limit for Zn was 1.2 mg L1. The method of standard additions was utilized to account for matrix interference (Perkin-Elmer, 1982). Briefly, each replicate was divided into three samples (labeled as A, B and C), each consisting of 4 mL of filtrate. Sample B was spiked with 2 mL of 4 mg Zn L1 standard, while sample C was spiked with 4 mL of the same standard. Sixty-five percent HNO3 and MilliQ water were then added to samples A, B and C for digestion to make up to 8 mL of analyte solution consisting of 2% HNO3. The samples were then wrapped in aluminum foil and stored at 4  C until analysis.

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For exposure studies, stock solutions of 100 mg nZnO L1 were produced by dispersing 0.025 g of the nZnO powder in 250 mL FASW. The nZnO suspensions were stirred incessantly with a magnetic stirrer ( 200 rpm) at the test temperatures for at least 7 days before experimentation. Growth inhibition test for the diatom and chlorophyll a fluorescence measurements In 2009, sea surface temperatures (SSTs) in Hong Kong ranged from 13.8 to 33.2  C (EPD, 2009), and thus the temperature treatments chosen for this study closely approximated this range, which was assumed to be the thermal tolerance range of the test organisms (Bao et al., 2008). The marine diatom S. costatum (strain code: CCMP 1332; Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow, AR) was acclimated to the test conditions (f/2 þ Si medium; 16 h light: 8 h dark cycle with light intensity between 1000 and 1500 Lux (Lux/FC light meter TM201, Tenmars, Taiwan)) utilizing different temperatures (15, 25 and 28  C) for two sub-cultured batch generations. In areas which receive intense solar radiation and relatively little wind action, SST can undergo diurnal variations up to 6  C (Beggs, 2010); therefore, the set temperatures were achieved for the initial batch by increasing or decreasing from 25  C in a stepwise manner of 1  C every 4 h. Subcultures attempted at 10  C and 30  C were unsuccessful as the diatom cells died, and thereafter abolished. Initial algal concentration of 105 cells mL1 was exposed to different concentrations (i.e. 0, 0.5, 1, 2, 4, 8 and 16 mg L1) of nZnO (three replicates per treatment) dispersed in 6 mL of autoclaved f/2 þ Si medium in autoclaved glass vials (capped with autoclaved rubber lids). The glass vials were then placed inside thermostatically controlled water baths fitted with a cooler or heater and shaken manually once every 12 h. Chlorophyll fluorescence was recorded at the start and end of experiment (i.e. 96 h), with the aid of WATER-PAM fluorometer (Heinz Walz GmbH, Effeltrich, Germany), and combining the instructions and terms used by Roha´cˇek & Barta´k (1999) and Maxwell & Johnson (2000; Supplementary Table S1). Live algae instead of chlorophyll extracts were used for the fluorescent measurements. Briefly, the glass vial was covered with black paper for at least 20 min, then 3 mL of sample was aliquoted into a quartz cuvette and the fluorescence (Fo) was measured using a weak measuring light (0.15 mmol photons m2 s1). The fluorescence of the darkadapted chlorophyll was then excited to a maximum state (Fm) by activating a saturating pulse of white light (43000 mmol photons m2 s1) for 0.8 s. To obtain light-adapted chlorophyll, actinic radiation (1000 mmol photons m2 s1) was applied continuously until the fluorescence reached a steady level (Fs) – this was usually achieved within 5 min. The saturating pulse was once again triggered and the corresponding maximum fluorescence 0 ). The fluorescence yield (Fo0 ) was subseyield recorded (Fm 0 quently estimated from an equation based on Fo, Fm and Fm (Supplementary Table S1; Oxborough & Baker, 1997). Pilot tests using the maximum test concentration of nZnO (16 mg L1) indicated that the nanoparticles did not introduce shading effects or interferences on the fluorescence signals. Acute mortality tests for the amphipod The gammarid amphipod M. longidactyla was harvested from the outdoor culture tanks at the Swire Institute of Marine Science, which was supplied with a constant flow of seawater from the Cape d’Aguilar Marine Reserve, Hong Kong (22 130 N, 114 120 E). The average whole animal size collected was 5 mm in length. The animals were kept under the laboratory conditions

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DOI: 10.3109/17435390.2013.848949

for at least 1 week, fed with the seaweed Ulva lactuca during this acclimation period. Temperature was adjusted in a similar stepwise manner as the procedures described above for the diatom exposure experiment, using the thermostat-installed water baths. Once the designated temperature (10, 15, 25 or 30  C) had been reached, animals were acclimated at the constant conditions for at least 24 h prior to start of chemical exposure. The total time taken for temperature modification followed by acclimation was the same across all treatments ( 4 d). None of the amphipods survived at 35  C, and this treatment was thus terminated. The animals were unfed during both temperature adjustment and chemical exposure periods. Each treatment consisted of three replicates, each being a 250mL glass beaker containing 10 M. longidactyla individuals in 200 mL of test solution (0.22-mm FASW; 16 h light: 8 h dark cycle). The test concentrations consisted of 0, 0.1, 0.5, 2.5 and 10 mg nZnO L1. Two nylon mesh strips (30 mm  10 mm) were added to each beaker to provide a substrate for the amphipods. The exposure was semi-static (i.e. each test solution was changed at 48 h) and lasted for 96 h. Mortality was monitored daily, and dead animals were removed. Control mortality was below 20% in accordance with OECD guidelines for testing of chemicals. Growth rate and protein stress responses in the fish Preliminary results showed that for the marine medaka fish, O. melastigma fish larvae less than 24 h old, mortality did not occur at concentration as high as 100 mg nZnO L1. Therefore, sublethal effects were investigated instead, and the nZnO concentration used for the treatment group was 10 mg L1. The exposure conditions and containers were similar to those employed for M. longidactyla with five replicates of 10 larvae (524 h old) for the growth rate experiment and three replicates of 10 larvae (524 h old) for the protein expression investigation. Growth in medaka larvae 4–13 day post-hatch has been demonstrated as a more sensitive endpoint to chemical exposures than mortality, and 7-day was deemed as minimum time required for an accurate appraisal of pollutant toxicity (Manning et al., 2001). In this study, fish larvae 524 h old were gradually acclimated to the test temperatures, as described afore for the algae and amphipod, and dosed with chemicals when they were 4 days old. The fish were fed daily, and half the solution was changed following feeding. Photos of the larvae were taken at the start of experiment and the end of day 7 using a dissecting microscope equipped with camera, and total length (TL) was measured from the images using the Image J version 1.44 (NIH, Washington, DC). For each tank, the average growth rate (mm per day) was calculated based on the difference between mean initial TL and mean final TL over the 7-day of experimentation. Control mortality was below 10% in accordance with OECD guidelines for testing of chemicals, and exceedance of the criteria at 35  C led to abandon of the data at this temperature treatment. For protein stress responses, fish were harvested at 48 and 96 h after the start of chemical exposure. The steps and reagents involved in tissue homogenization, protein extraction and quantification and Western blot analysis followed those described in Wong et al. (2010). For Western blot analysis, equal loading of protein was further ensured by visualization with 0.1% Ponceau S in 1% acetic acid after the protein sample was completely transferred onto a nitrocellulose membrane. Primary antibodies used were rabbit anti-cod polyclonal MT (Cat. No. M04406201-500; Biosense Laboratories AS, Bergen,

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Norway), mouse anti-HSP70 monoclonal IgG1 (Cat. No. MA3007; Affinity BioReagents, Golden, CO) and rat anti-HSP90 monoclonal 16F1 (Cat. No. ADI-SPA-835; Stressgen Biotechnologies Corp., Victoria, Canada) with corresponding secondary antibodies of peroxidase-goat anti-rabbit IgG, peroxidase-goat anti-mouse IgG and peroxidase-rabbit anti-mouse IgG (all purchased from H þ L, Zymed Laboratories, South San Francisco, CA). Statistical analysis Data analysis was performed with Graphpad Prism version 5.00 (Graphpad Software, San Diego, CA) and SPSS version 17 (SPSS Inc., Chicago, IL). Three-way analysis of variance (ANOVA) was used to test the significance of seawater temperature, nZnO concentration and exposure time, and their interactions on influencing the mean hydrodynamic diameters (DH), while twoway ANOVA was used to test the significance of seawater temperature and exposure time and their interaction on influencing the dissolved Zn2þ ion concentrations. To determine the IC50 (concentration inhibiting 50% of algal fluorescence) and LC50 (concentration causing lethality to 50% of amphipods), data were fitted to a sigmoidal log (dose)-response curve (variable slope) with a four-parameter logistic equation. Statistical difference was implied if the 95% confidence intervals (CI) of the IC50s or LC50s did not overlap. Two-way ANOVA using temperature and exposure concentration of nZnO as fixed factors was used to test for equality of the means of each chlorophyll fluorescence parameter. For Western blot analysis, results were represented as fold changes relative to the control. Mean differences in MT, HSP70 and HSP90 protein inhibition/induction among different temperature treatments were compared using one-way ANOVA, while Spearman’s rank correlation test was used to compare the expression patterns of different proteins at each time point. For all ANOVAs conducted, Tukey’s post-hoc test was also carried out for factors that were flagged as having significant effect (p50.01 was chosen as the significant level due to unequal variances between treatments).

Results Aggregation size and ion dissolution of nZnO at various temperatures There was no overall interaction between seawater temperature, exposure time and exposure concentration of nZnO on the DH of nZnO (F24, 300 ¼ 1.014, p40.05); however, temperature significantly interacted with time and concentration separately to affect DH (Figure 1; temperature  concentration: F12, 300 ¼ 3.236, p50.001; F8, 300 ¼ 4.194, p50.001; Supplementary Table S2). Among the three factors, the concentration of nZnO was the most important and followed by exposure time, whereas temperature was less important as reflected by their F values in the ANOVA results (concentration: F3, 300 ¼ 247.305; time: F2, 300 ¼ 20.923 and temperature: F4, 300 ¼ 4.107; all p values 50.001; Table S2). The aggregate size generally increased with increasing nZnO concentration and exposure time (Figure 1). Although there was a significant interaction between temperature and concentration/ time, the corresponding F values were small, and the pattern of the aggregate size across different temperatures was somewhat unclear (Figure 1). The release of Zn2þ ion from nZnO generally increased with decreasing seawater temperature and increasing exposure time (Figure 2; temperature: F4, 60 ¼ 135.702; time: F3, 60 ¼ 15.964; Table S3), although there was a significant yet weak interacting effect of the two factors (F12, 60 ¼ 2.068, p ¼ 0.033).

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Figure 1. Hydrodynamic diameter (DH) of nZnO aggregations at different seawater temperatures (10, 15, 25, 28 and 30  C), exposure time (days 1, 4 and 7) and nZnO concentrations (10, 20, 50 and 100 mg L1; n ¼ 6; mean  2SD).

According to the F values, seawater temperature was the key factor leading the change of ion dissolution of nZnO though the process was time dependent. Obviously, the Zn ion concentration was considerably higher at 10  C and 15  C than the other higher temperatures (Figure 2). As the results of the characterization studies illustrated that the DH values under most temperature regimes were reaching a plateau by day 7 (Figure 1), and the stock solution was stirred for at least 7 days prior to being used in exposure studies, in the following sections, the toxicity test results would be discussed based on the DH of nZnO obtained on day 7 (Table 1) where applicable. Based on both literature review and results of our preliminary experiment (data not shown), at room temperature, the ion dissolution of nZnO in seawater at maximum equilibrium is 70–100% at 0.1–1 mg nZnO L1 (Fairbairn et al., 2011; Miller et al., 2010), and at above 10 mg nZnO L1, the dissolution is concentration-independent (Peng et al., 2011). Assuming a similar concentration, trends are observed for different temperature regimes, a Monte Carlo method was employed to generate nZnO concentration-dissolution curves under different temperature scenarios. One hundred random values were generated based on the mean and standard deviation obtained from literature and the day 7 dissolved Zn concentration from this study (as provided by DLS), and non-linear regression curves were fitted to the simulated dataset (Figure 3). In the following sections, the toxicity test results would be evaluated based on the dissolved Zn content as calculated from the equations of these regression curves where applicable.

Temperature-dependent toxicities of nZnO toward algae and amphipod The toxicity of nZnO toward the algae and amphipod displayed similar patterns across temperature groups in this study (Figures 4 and 5). The median inhibition concentration (IC50) of nZnO on the fluorescence of the diatom S. costatum (Table 2) was significantly lower at 28  C as compared to that observed at 15 and 25  C, indicating higher toxicity at 28  C. Similarly, mean 96 h-LC50 value of nZnO on the amphipod M. longidactyla decreased from 410 to 8.33 mg L1 (Table 3) when temperature rose from 10 to 15  C (Table 3). The values were significantly lower at 25  C (0.04 mg L1) and 30  C (0.1 mg L1), which were not significantly different based on their 95% CIs (Table 3). It was clear that the amphipod were more susceptible to nZnO toxicity at higher temperatures. When expressed in terms of dissolved Zn concentration, the results remained the same for M. longidactyla (Table 3), but the results were slightly different for S. costatum as a significant difference between the treatment groups at 15 and 25  C was observed (Table 2). Temperature and nZnO were shown to have an interactional effect on all inspected chlorophyll parameters (Figure 6; Supplementary Table S4). Increasing nZnO concentrations generally led to reduction in photochemical quenching (i.e. decreased PO, 2 and qp values; Figures 6a–c), whereas the patterns of non-photochemical energy dissipation (NPQ and qo) varied under different temperature regimes (Figures 6e and f). There was no distinctive pattern for PO, 2 and qp of the control under different temperatures, except for noticeable reduction of PO and

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DOI: 10.3109/17435390.2013.848949

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Figure 2. Release rates of 0.02 -mm filterable Zn2þ from 100 mg L1 of nZnO in filtered artificial seawater (FASW) at different temperatures (10, 15, 25, 28 and 30  C). Bars with the same letter are statistically indifferent (i.e. p40.01, Tukey’s post-hoc comparison test). Table 1. Physicochemical characteristics of nZnO under different temperatures (10, 15, 25, 28 and 30  C) and concentrations (10, 20, 50 and 100 mg L1) at day 7. Hydrodynamic diameter (DH; mm; Mean  SD) 

Temperature ( C) 10 15 25 28 30

10 mg nZnO L

1

0.52  0.22 0.52  0.31 0.64  0.27 0.51  0.19 0.50  0.17

20 mg nZnO L1

50 mg nZnO L1

100 mg nZnO L1

0.71  0.14 1.1  0.35 0.76  0.23 0.79  0.17 0.76  0.24

1.3  0.41 1.2  0.17 0.93  0.13 1.0  0.14 0.92  0.22

1.3  0.12 1.4  0.17 1.2  0.14 1.3  0.18 1.4  0.10

2 at 28  C (Figures 6a and b). W tended to increase with nZnO concentration, and temperature did not have conspicuous effect on the control except for 28  C, where W was elevated compared to the other temperatures (Figure 6d). For the lowest temperature treatment (15  C), both NPQ and qo tended to increase at lower nZnO concentrations, followed by a decline at higher chemical levels (Figures 6e and f). However, at the highest test temperature (28  C), NPQ and qo decreased with increasing nZnO concentration, and no intermediate elevation was observed (Figures 6e and f). Temperature-dependent toxicity of nZnO toward fish In this study, temperature rather than nZnO had a significant effect on the growth rate of the marine medaka fish O. melastigma, while

there was no significant interaction between the two factors (twoway ANOVA: Ftemperature ¼ 60.525, p50.001; Ftreatment ¼ 0.466, p40.05; Ftemperature*treatment ¼ 0.741, p40.05; Figure 7). Due to mass mortality at day 4, results for 35  C were not shown. O. melastigma larvae exposed to 10 mg nZnO L1 displayed similar HSP70 (Figure 8a) and HSP90 (Figure 8b) expressions across the different temperature treatments regardless of the exposure time (Spearman’s rank correlation test for 48 h:  ¼ 0.837, p50.01; and for 96 h:  ¼ 0.753, p50.05; Figure 9). Based on the Western blot images (Figures 8a,b), it is apparent that the HSP production increased at sub-optimal (15  C) and supra-optimal (30  C) temperatures. Except for 25  C at 48 h, nZnO generally inhibited the HSP expressions. At 15  C, both HSPs were suppressed by the chemical treatment at 48 h, followed by a return to the control level at 96 h. At 25  C, the expressions

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Figure 3. Simulated ion dissolution rates of zinc ions (Zn2þ) from different concentrations of nZnO in filtered artificial seawater (FASW) at different temperatures (10, 15, 25, 28 and 30  C) and at day 7 of the exposure. Nonlinear regression curves were fitted to the five temperature treatments as follows: for 10  C: ½Zn2þ  ¼ 3:6262  ð1  e0:2544 ½nZno Þ, adjusted R2 ¼ 0.9903, p50.0001; for 15  C: ½Zn2þ  ¼ 1:5991 ð1  e0:5532 ½nZno Þ, adjusted R2 ¼ 0.9579, p50.0001; for 25  C: ½Zn2þ  ¼ 1:1767  ð1  e0:8199 ½nZno Þ, adjusted R2 ¼ 0.8503, p50.0001; for 28  C: ½Zn2þ  ¼ 1:1008  ð1  e1:0224 ½nZno Þ, adjusted R2 ¼ 0.9354, p50.0001; for 30  C: ½Zn2þ  ¼ 0:9702  ð1  e1:0916 ½nZno Þ, adjusted R2 ¼ 0.7900, p50.0001.

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Figure 5. 48-h and 96-h concentration-response relationships of nZnO toward Melita longidactyla at different temperatures (10, 15, 25 and 30  C) (n ¼ 3; mean  2 SD). Table 2. 96-h IC50 values (95% confidence interval (CI)) of nZnO toward Skeletonema costatum at different temperatures (15, 25 and 28  C) based on the measured fluorescence level relative to the controls. Temperature ( C) 15 25 28

96-h IC50 (mg nZnO L1)

96-h IC50 (mg dissolved Zn L1)

17 (11–27)a 12 (10–14)a 3.0 (2.8–3.3)b

1.82 (1.64–2.03)a 1.18 (1.17–1.18)b 1.01 (0.99–1.03)c

Values with the same letter denote overlapping of 95% CI (i.e. no significant difference, p40.05). Figure 4. 96-h concentration–response relationships of nZnO toward Skeletonema costatum at different temperatures (15, 25 and 28  C) based on the measured fluorescence level relative to the controls (n ¼ 3; mean  2 SD).

of HSPs were increased relative to the control at 48 h, but were depressed by 96 h. At 30  C, the HSPs were inhibited throughout the exposure period. The two MT isoforms (Figure 8c) expression patterns across all temperature treatments were also comparable between the two time points (48 h:  ¼ 0.704, p50.05 and 96 h:  ¼ 0.912, p50.01; Figure 9). A reversal of the HSP trend seemed to be happening with MTs at the lower temperatures: at 15  C, MT-I was increased at both 48 and 96 h, while MT-II was increased at 48 h only. At 25  C, both MTs were depressed by 96 h. At 30  C, expressions of MTs were suppressed at 48 h but returned to the control level at 96 h.

Discussion Aggregation kinetics of various NMs have been previously discussed in various texts and are generally described by integration of two factors – aggregation rate (which is dependent

on collision rate) of particles and attachment efficiency (defined as the probability of irreversible attachment between two collided colloidal particles; Quik et al., 2011). NM aggregation determination is theoretically affected by temperature of the suspending medium (Petosa et al., 2010), initial primary particle concentration and time (Chen et al., 2006). The results from the present study in general support this hypothesis, although no clear pattern on the aggregate size could be distinguished between different temperatures (Figure 1). Intriguingly, it was found that the release of Zn2þ ion from nZnO increased with decreasing temperature (Figure 2). Similar results for bulk ZnO had been yielded in another study, where the solubility of Zn decreased from around 3.5 mg L1 at 15  C to less than 1 mg L1 at 35  C (Yebra et al., 2006). Zn2þ release from nZnO in seawater is achieved via the following equilibrium: ZnOðsÞ þ H2 O þ 2Cl $ 0:5ZnCl2 4  þ 0:5ZnðOHÞ þ 0:5OH 3

ð1Þ

It is hypothesized that at lower temperatures, Zn2þ tends to accumulate in the solution as the activation energy of the reverse reaction is theoretically higher than that required for

Temperature-dependent toxicities of nano zinc oxide

DOI: 10.3109/17435390.2013.848949

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Table 3. 48-h and 96-h LC50 values (95% confidence interval (CI)) of nZnO toward Melita longidactyla at different temperatures (10, 15, 25 and 30  C).

Temperature (C) 10 15 25 30

48-h LC50 (mg nZnO L1)

48-h LC50 (mg dissolved Zn L1)

96-h LC50 (mg nZnO L1)

96-h LC50 (mg dissolved Zn L1)

410 410 0.11 (0.020–0.76) 0.32 (0.14–0.74)

43.3 41.6 0.0883 (0.016–0.269) 0.257 (0.112–0.552)

410a 8.33 (2.55–27.23)b 0.04 (0.01–0.17)c 0.1 (0.08–0.13)c

43.3a 1.651 (1.190–2.291)b 0.032 (0.008–0.087)c 0.080 (0.064–0.104)c

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Values with the same letter denote overlapping of 95% CI.

Figure 6. Interactions of nZnO concentration (0, 0.5, 1, 2, 4, 8 and 16 mg L1) and temperature (15, 20, 25 and 28  C) on various chlorophyll a fluorescence parameters ((a) PO, (b) 2, (c) qp, (d) W, (e) NPQ and (f) qo) in the diatom Skeletonema costatum (n ¼ 3; 2 SD). The description of each parameter is given in Supplementary Table S1 and the statistics are given in Supplementary Table S4.

ZnO dissolution process (Yebra et al., 2006), hence accounting for the observed phenomenon. Ion solubility has been cited many times as a main contributor toward nZnO adverse effects on aquatic organisms (Aruoja et al., 2009; Franklin et al., 2007; Wong et al., 2010). Following this line of reasoning, toxicity of nZnO would be expected to be highest at the lowest temperature, where Zn2þ bioavailability was optimized. However, our observed toxicities of nZnO to the

algae and amphipod, which generally increased with increasing temperature, did not support this postulation. Conceivably, the nZnO toxicity to marine organisms is highly species-specific given that different species often have different avoidance behavior and different modes for toxicant uptake and its elimination. For instance, most of the amphipods were in dormant stage at the lowest temperature (data not shown), and thus their uptake of nZnO and Zn2þ was possibly greatly reduced as

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8

S. W. Y. Wong & K. M. Y. Leung

Figure 7. Average growth rates (mm day1) of medaka fish larvae Oryzias melastigma under exposure to the control and 10 mg nZnO L1 at different temperatures (15, 25 and 30  C; n ¼ 5, 2 SD) for 7 days (twoway ANOVA: Ftemperature ¼ 60.525, p50.001; Ftreatment ¼ 0.466, p40.05; Ftemperature*treatment ¼ 0.741, p40.05). Bars with the same letter are statistically indifferent (i.e. p40.01, Tukey’s post-hoc comparison test).

compared to those that stayed active at higher temperatures, even though the Zn2þ abundance in solution was relatively greater. Dormancy under certain stress conditions (such as high toxicant levels and low temperatures) has been reported for the copepod Tigriopus japonicus (Bao et al., 2008; Kwok & Leung, 2005) and is associated with an extremely low metabolic rate, which in turn decreases chemical uptake, bioaccumulation and toxicity. As temperature increased, metabolic rate also increased, and thus M. longidactyla became more vulnerable at high temperatures due to narrowed aerobic scope and elevated energy demand (Po¨rtner, 2002). Toxicity of nZnO toward M. longidactyla did not increase further between 25 and 30  C, presumably because Zn2þ ion release might become a limiting factor at 30  C (Table 3). In addition, aggregation might have played a role in the toxicity of nZnO at higher concentrations, as DH increased with nZnO concentration (Table 1), and amphipods might have ingested aggregated nZnO that had settled on the bottom of the container. It has been shown that this factor contributes toward the toxicity of nZnO in D. magna, as animals exposed to nZnO and Zn2þ displayed dissimilar gene expression profiles (Poynton et al., 2011). For the diatom S. costatum, lower IC50 value was also obtained at 28  C as compared to that observed at 15 and 25  C (Table 2). The elevated toxicity at 28  C might be attributable to the increase uptake of nZnO and Zn ions in the diatom under such a high temperature, and a possibly thermal stress condition. It is important to note that S. costatum cannot survive at 30  C (based on the current results) and thus 28  C is very close to their upper thermal limit presenting a thermal challenging condition to the diatom. In another study on cadmium (Cd) stress to the marine diatom Thalassiosira nordenskioeldii, maximum sensitivity was also induced at the highest test temperature of 30.5  C (Wang & Wang, 2008). These authors explained that such a result was attributed to enhanced cellular Cd accumulation and weakened detoxification ability of cells due, in part, to nitrogen- and glutathione-deficiency (Wang & Wang, 2008). Nitrate assimilation in S. costatum is optimized at 15  C but falls with increasing temperature due to reduction of nitrate reductase activity (Gao et al., 2000). With suppression of normal physiological function at high temperature, the diatom is expected to be more susceptible to the toxicity brought by nZnO and associated Zn2þ. As for the chlorophyll a fluorescence parameters in S. costatum, dwindling PO, 2 and qp values at higher nZnO

Nanotoxicology, Early Online: 1–12

concentrations indicate hindered electron transport from photosystem II (PSII) to photosystem I, and there are multiple potential causes, which have been succinctly summarized in Smith et al. (2005). Briefly, this may be due to (1) a smaller pool of available electron transport constituents (e.g. plastoquinone), (2) competitive conversion to heat energy either directly from PSII antennae or within PSII reaction centers (RCs) and (3) reduced RCs as a result of phosphorylation and relocation of light-harvesting complexes away from PSII. The nanoparticles of nZnO may exert its own effect through reactive oxygen species generation, damaging the thylakoid membranes and RC protein structures, as had been demonstrated for nCuO in Chlamydomonas reinhardtii (Saison et al., 2010). Rashid et al. (1994) also suggested that Zn2þ can disrupt the function of oxygen evolving complex (OEC) within the PSII by non-competitive displacement of certain inorganic cofactors (Ca2þ and Mn2þ) at their binding sites, and thus inhibit water oxidation processes. This mechanism was affirmed by the mounting W values, often associated with Mn-deficiency in the OEC (Kriedemann et al., 1985) at higher nZnO concentrations in this study (Figure 6d). Similar trends have been observed for the variations of NPQ and qo in algae exposed to other chemicals such as copper (Juneau et al., 2002), isoproturon (Fai et al., 2007) and zinc (Mateos-Naranjo et al., 2008). At lower nZnO concentrations, it is anticipated that increase in heat dissipation is complementary to partial inhibition of electron transport in PSII, and such response is considered as ‘‘protective photoinhibition’’ (El-Sabaawi & Harrison, 2006). However, as nZnO concentration was elevated, damage to protein configurations was so extensive such that NPQ also becomes inhibited. At 25 and 28  C, NPQ did not increase even at lower nZnO concentrations, again suggesting a reduction in pigments, which might be involved in photoprotection. NPQ is dependent on both presence of trans-thylakoid membrane pH gradient (DpH) and activities of xanthophyll cycle, which converts monoepoxide diadinoxanthin into de-epoxidized diatoxanthin (Lavaud et al., 2002). Studies on temperature-dependence of diadinoxanthin cycle, as compared to its violaxanthin counterpart, are despairingly lacking, and therefore no solid conclusion can be drawn at present about the effect of thermal stress on NPQ-regulating mechanisms. Based on literature review, Zn2þ may contribute toward the observed decrease in fluorescence (which should be positively correlated with chlorophyll a concentration) in S. costatum by suppressing protochlorophyll reduction to chlorophyll, which is mediated by protochlorophyllide reductase activities (De Filippis & Pallaghy, 1976; Miao et al., 2005). Overall, the role of Zn ion in nZnO toxicity remains intangible, as the physiology of the diatom may overshadow Zn2þ bioavailability, and toxicity of nZnO toward the diatom cannot be predicted based on the concentration of dissolved Zn alone (Table 3). While at lower concentrations, most of the nZnO would have dissolved, at higher concentrations the formation of larger aggregates (Table 1) might have entrapped the algal cells, hence limiting their nutrient intake and inhibiting their growth. Such effects of NM aggregation on the toxicity of nZnO toward algae have been assessed in Aruoja et al. (2009). Although Zn is known to be an essential trace element for fish, it does not appear to affect their growth unless present in relatively high concentrations (Zhao et al., 2011). The current results also concur with Kwok (2009), whereby 5 mg nZnO L1 did not induce growth change in the marine medaka fish O. melastigma at day 7, although growth inhibition was observed at day 14. In contrary, another study, which exposed the rainbow trout to nTiO2 for 8 weeks showed no impact on growth performance (Ramsden et al., 2009). It is possible that growth may be temporarily stalled during early stages of

DOI: 10.3109/17435390.2013.848949

Temperature-dependent toxicities of nano zinc oxide

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Figure 8. Examples of Western blots of HSP70 ((a) 68–73 kDa), HSP90 ((b) 85–90 kDa), MT-I ((c) 14–15 kDa; upper band) and MT-II ((c) 6–7 kDa; lower band) in the fish larvae Oryzias melastigma after exposure to 0 (left lane) and 10 mg nZnO L1 (right lane) for 48 and 96 h under various temperatures.

exposure, while long-term acclimation to chemicals will eventually lead to compensation for any deleterious effects (De Schamphelaere & Janssen, 2004). From these controversial reports, it can be deduced that caution should be exercised when using fish growth as a biomarker, as exposure time can have major implications on its turnout and subsequent interpretations. Hence, chronic toxicity tests covering a full life cycle are more preferable if time and resources allow. HSPs are noted for their involvement in growth at high temperatures, and also protection against diseases such as atrophy and myopathy at low temperatures (Roberts et al., 2010). At 15  C, as solubility of nZnO was elevated and thus more Zn ion was bioavailable, HSP might have been up-regulated preceding 48 h, and as they became over-expressed their expression was repressed – hence the down-regulation observed at 48 h, followed by return to normal level (Figure 9). At 25  C, this pattern was repeated, but as ion release rate

was slower, the response also became lagged. At 30  C, HSP70 was persistently suppressed throughout chemical exposure period, which might be associated with intensive cellular injury and debilitated restoration of cellular integrity (Ruete et al., 2008). A plausible theory regarding the expression patterns of MTs is that metabolism kinetics may overcome the solubility kinetics, since MT synthesis rate is found to be positively correlated with temperature and exposure period to metal ions (Leung et al., 2000). Therefore in spite of the relatively high solubility of nZnO at 15  C, MT-I was not up-regulated in the fish at 48 h and only augmented later on at 96 h. At 25  C, MT-II was not significantly increased at 48 h, but the overall mechanism should have proceeded at a faster rate than 15  C since it was already downregulated by 96 h. At 30  C, the response was possibly so quick that negative feedback might have already achieved by 48 h and basal levels accomplished by 96 h. In summary, nZnO was found

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S. W. Y. Wong & K. M. Y. Leung

Nanotoxicology, Early Online: 1–12

Figure 9. The relative expression levels of HSP70, HSP90, MT-I and MT-II in medaka fish Oryzias melastigma after exposure to 0 and 10 mg nZnO L1 at different temperatures for 48 and 96 h. Bars with the same letter are statistically indifferent (i.e. p40.01, Tukey’s post-hoc comparison test).

to have very low toxicity to the fish larvae in terms of growth inhibition while their growth rate increased with increasing temperature. The combined effects of nZnO and temperature on protein expression of HSPs and MT in the fish were rather complex as their syntheses were highly temporally and thermally dependent.

Conclusions The results of our experiments suggested that the aggregation size of nZnO increased with increasing exposure concentration of nZnO and time of exposure, while temperature had a relatively minor influence on its aggregate size. Clearly, the ion dissolution rate of nZnO decreased with increasing temperature, and such a process was also time dependent. It is important to note that the equilibrium concentration presented in this study is not per se and is likely a function of the size of the test container; nonetheless, the relationship between temperature and aggregation formation and release of metal ions from nano metal oxides deserves further investigation, as they are shown in this study to be significantly correlated, and this in turn might affect the toxicity of nZnO under different temperature regimes. Overall, the temperature-dependent toxicities of nZnO varied among the three testing species, as the physiology, avoidance behavior, chemical uptake and elimination strategies are highly species-specific (or taxa-specific). For the diatom,

the possible increased thermal stress, elevated chemical uptake and decreased toxicant assimilation rates at high temperatures probably concomitantly led to an increased toxicity. Dormancy may explain the relatively low toxicity of nZnO on the amphipod at low temperatures, thus also demonstrating that temperature-dependent animal physiology and behavior can alter the toxicity of nano-materials. The growth rate of the medaka fish was unaffected by nZnO in this study, while HSPs and MTs production were shown to be regulated by interactions between temperature and exposure duration. The results of all these three sets of experiments in tandem highlighted the importance of temperature as an influential factor modulating the aggregation size, ion dissolution and toxicity of nZnO, and prudence should be given concerning the thermal conditions under which similar nano metal oxides are released into the marine environment.

Acknowledgements The authors would like to thank Dr. Priscilla Leung for her advice on the Western blot analysis, Dr. Yunwei Dong, Xiamen University, for his help in the identification of the amphipod and Dr. X.Y. Li, Civil Engineering Department, HKU for allowing us to use the laser diffractometry instrument. The authors also thank the anonymous reviewers for their valuable comments on this manuscript and Ms. Helen Leung and Ms. Cecily Law for their technical support throughout the project.

DOI: 10.3109/17435390.2013.848949

Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This research is funded by Research Grants Council of the Hong Kong Special Administrative Region Government via a General Research Fund (HKU 703511). Stella Wong was partially supported by the Area of Excellence Scheme under the University Grants Committee of the Hong Kong Special Administration Region, China (Project No. AoE/P-04/ 2004).

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Supplementary Materials Supplementary Table S1. Terminology valid for chlorophyll fluorescence and calculated fluorescence parameters used in this experiment (adapted from Kriedemann et al. (1985), Maxwell and Johnson (2000), Oxborough and Baker (1997), Roháček and Barták (1999), and Roháček (2002)) Basic terms used in chlorophyll fluorescence measurement

Definition

Fo

Minimum fluorescence measured on a DAC with open PSII reaction centres (RCs) and minimized non-photochemical processes in thylakoid membrane

Fm

Maximum fluorescence measured on a DAC with closed PSII RCs and minimized nonphotochemical processes in thylakoid membrane

FS

Steady-state fluorescence on a LAC induced by actinic radiation, and when there is no change in external conditions

Fm’

Maximum fluorescence measured on a LAC with closed PSII RCs and optimized active non-photochemical processes in thylakoid membrane

Table S1 (Cont’d) Basic terms used in chlorophyll fluorescence measurement

Definition

Fo’

Minimum fluorescence measured on a LAC with open PSII RCs and optimized active non-photochemical processes in thylakoid membrane. Can be estimated using the equation

Calculated fluorescence parameters

Equation

Significance

Photochemical quenching parameters ΦPo (maximum quantum yield of PS II photochemistry)



Intrinsic or potential quantum efficiency when all PSII RCs are open

Φ2 (effective quantum yield of photochemical energy conversion in PSII or Genty parameter)



Fraction of total absorbed light energy by PSIIassociated chlorophyll that is used in photochemistry; used in quantification of electron transport rate



Discerns manganese deficiency

ΦW (efficiency of water-splitting apparatus)

Table S1 (Cont’d) Calculated fluorescence parameters Photochemical quenching parameters

Equation

Significance



Measures the photochemical capacity of PSII in LAC; corresponds to the proportion of open or oxidized PSII RCs at steady state

qp (photochemical quenching of variable fluorescence) Non-photochemical quenching parameters NPQ (non-photochemical fluorescence quenching)

Correlated with sum of rate constants of nonphotochemical dissipation pathways (fluorescence, heat, spillover of PSII excitation energy to PSI) within thylakoid membranes; also reflects zeaxanthin formation

qo (relative change of minimum fluorescence)

Linked with pH-gradient induced mechanisms that adjust electron flow, disruption of PSII RC activities, de-epoxidation (i.e. oxygen removal) of violaxanthin, and changes in pigment-protein complex configurations

Supplementary Table S2. Results of the three-way ANOVA on the effects of nZnO concentration (10, 20, 50 and 100 mg L-1), temperature (10, 15, 25, 28 and 30°C) and time (Day 1, 4 and 7) on the hydrodynamic diameter (DH) of nZnO (n = 6) Numbers in bold indicate that the factor has a significant effect on the parameter. Source

Type III Sum of Squares

df

Mean Square

F

p

Temperature

0.581

4

0.145

4.107

< 0.010

Time

1.480

2

0.740

20.923

< 0.001

Concentration

26.244

3

8.748

247.305

< 0.001

Temperature * Time

1.187

8

0.148

4.194

< 0.001

Temperature * Concentration

1.374

12

0.114

3.236

< 0.001

Time * Concentration

0.200

6

0.033

0.943

0.465

Temperature * Time * Concentration

0.861

24

0.036

1.014

0.447

Error

10.612

300

0.035

Supplementary Table S3. Results of the two-way ANOVA on the effects of temperature (10, 15, 25, 28 and 30°C) and time (Day 1, 3, 5 and 7) on the ion dissolution of nZnO (n = 4). Numbers in bold indicate that the factor has a significant effect on the parameter. Source

Type III Sum of Squares

df

Mean Square

F

P

Time

3.782

3

1.261

15.964

< 0.001

Temperature

42.863

4

10.716

135.702

< 0.001

Time * Temperature

1.959

12

0.163

2.068

0.033

Error

4.738

60

0.079

Supplementary Table S4. Results of the two-way ANOVA on the effects of nZnO concentration (0, 0.5, 1, 2, 4, 8 and 16 mg L-1) and temperature (15, 20, 25 and 28 °C) on various chlorophyll fluorescence parameters (ΦPO, Φ2, ΦW, qp, NPQ, qo) of Skeletonema costatum (n = 3). Numbers in bold indicate that the factor has a significant effect on the parameter. Chlorophyll fluorescence parameter

Source

df

Sum of squares

F

p

ΦPO

Temperature

3

2.004

293.833

< 0.001

Concentration

6

0.502

36.790

< 0.001

Temperature*Concentration

18

0.151

3.682

< 0.001

Temperature

3

0.211

83.096

< 0.001

Concentration

6

0.140

27.521

< 0.001

Temperature*Concentration

18

0.043

2.796

< 0.010

Temperature

3

1720.985

132.224

< 0.001

Concentration

6

1147.552

44.083

< 0.001

Temperature*Concentration

18

2268.131

29.044

< 0.001

Temperature

3

0.102

8.196

< 0.001

Concentration

6

0.602

24.238

< 0.001

Temperature*Concentration

18

0.238

3.198

< 0.001

Temperature

3

1.482

75.575

< 0.001

Concentration

6

0.426

10.862

< 0.001

Temperature*Concentration

18

0.960

8.163

< 0.001

Temperature

3

0.143

46.145

< 0.001

Concentration

6

0.041

6.666

< 0.001

Temperature*Concentration

18

0.252

13.521

< 0.001

Φ2

ΦW

qp

NPQ

qo

Cited references: 1. Kriedemann PF, Graham RD, Wiskich JT. 1985. Photosynthetic dysfunction and in vivo chlorophyll a fluorescence from manganese-deficient wheat leaves. Aust J Agric Res 36: 157 – 169. 2. Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence – a practical guide. J Exp Bot 51: 659 – 668. 3. Oxborough K, Baker NR. 1997. Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components – calculation of qP and Fv’/Fm’ without measuring Fo’. Photosynth Res 54: 135 – 142. 4. Roháček K. 2002. Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships. Photosynthetica 40: 13 – 29. 5. Roháček K, Barták M. 1999. Technique of the modulated chlorophyll fluorescence: basic concepts, useful parameters, and some applications. Photosynthetica 37: 339 – 363.

 

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