Physiological, structural and ultrastructural impacts of silver nanoparticles on the seagrass Cymodocea nodosa

Journal Pre-proof Physiological, structural and ultrastructural impacts of silver nanoparticles on the seagrass Cymodocea nodosa Zoi Mylona, Emmanuel Panteris, Michael Moustakas, Theodoros Kevrekidis, Paraskevi Malea PII:

S0045-6535(20)30259-9

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126066

Reference:

CHEM 126066

To appear in:

ECSN

Received Date: 7 November 2019 Revised Date:

23 January 2020

Accepted Date: 29 January 2020

Please cite this article as: Mylona, Z., Panteris, E., Moustakas, M., Kevrekidis, T., Malea, P., Physiological, structural and ultrastructural impacts of silver nanoparticles on the seagrass Cymodocea nodosa, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126066. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Zoi Mylona: methodology, formal analysis, investigation, writing original draft, visualization. Emmanuel Panteris: conceptualization, methodology, investigation, resources, writing review & editing. Michael Moustakas: methodology, resources, writing review & editing. Theodoros Kevrekidis: conceptualization, methodology, formal analysis, writing original draft, writing review & editing. Paraskevi Malea: conceptualization, methodology, formal analysis, investigation, resources, writing original draft, writing review & editing, visualization, supervision, project administration. .

1 2

Physiological, structural and ultrastructural impacts of silver nanoparticles on

3

the seagrass Cymodocea nodosa

4

Zoi Mylonaa, Emmanuel Panterisa, Michael Moustakasa, Theodoros Kevrekidisb,

5

Paraskevi Maleaa* a

6 7 8 9

Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece

b

Laboratory of Environmental Research and Education, Democritus University of Thrace, Nea Hili, GR-68100 Alexandroupolis, Greece

10 11 12 13 14

* Corresponding author: [email protected] (Paraskevi Malea)

15 16 17 18 19 20 21 22 23 24 25 26 1

27

ABSTRACT

28

Silver nanoparticles (AgNPs) are an emerging contaminant, currently considered to be a

29

significant potential risk to the coastal environment. To further test potential risk, and to determine

30

effect concentrations and sensitive response parameters, toxic effects of environmentally relevant

31

AgNP concentrations on the seagrass Cymodocea nodosa were evaluated. Alterations of the

32

cytoskeleton, endoplasmic reticulum, ultrastructure, photosystem II function, oxidative stress

33

markers, cell viability, and leaf, rhizome and root elongation in C. nodosa exposed to AgNP

34

concentrations (0.0002-0.2 mg L-1) under laboratory conditions for 8 days were examined. An

35

increase in H2O2 level, indicating oxidative stress, occurred after the 4th day even at 0.0002 mg L-1.

36

Increased antioxidant enzyme activity, potentially contributing to H2O2 level decline at the end of

37

the experiment, and reduced protein content were also observed. Actin filaments started to

38

diminish on the 6th day at 0.02 mg L-1; microtubule, endoplasmic reticulum, chloroplast and

39

mitochondria disturbance appeared after 8 days at 0.02 mg L-1, while toxic effects were generally

40

more acute at 0.2 mg L-1. A dose-dependent leaf elongation inhibition was also observed; as for

41

juvenile leaves, toxicity index increased from 2.8 to 40.7% with concentration. Hydrogen peroxide

42

(H2O2) overproduction and actin filament disruption appeared to be the most sensitive response

43

parameters, and thus could be utilized as early warning indicators of risk to seagrass meadows. A

44

risk quotient of 1.33 was calculated, confirming previous findings, that AgNPs may pose a

45

significant risk to the coastal environment.

46

Keywords: marine plant, toxicity, cellular effect, physiological response, biomarker, risk

47

assessment

48 49

1. Introduction

50

Seagrasses grow submerged and rooted in shallow coastal waters throughout the world.

51

These angiosperms form extensive beds, which play a key multifunctional role. Seagrasses are

52

important primary producers, supply organic food to a variety of food webs, stabilize the substrate

2

53

and serve as habitats and nurseries for numerous species, including commercially important fish

54

and shellfish (Costanza et al., 1997). However, a global trend of regional seagrass declines, caused

55

by a combination of natural and anthropogenic disturbances, has been documented. Anthropogenic

56

inputs of phytotoxic chemicals are suspected to contribute to seagrass declines (Lewis and

57

Devereux, 2009). However, laboratory toxicity results are mainly available for relatively few

58

seagrass species and chemical classes (e.g. Lewis and Devereux, 2009; Diepeus et al., 2017;

59

Buapet et al., 2019; Mochida et al., 2019), restricting a realistic evaluation of the role of

60

anthropogenic chemicals in seagrass declines. Therefore, additional toxicity information for

61

conventional chemical contaminants and, even more for, chemicals of emerging concern is needed

62

to evaluate potential risks posed to seagrass meadows.

63

Silver nanoparticles (AgNPs) are a pollutant of emerging environmental concern. AgNPs are

64

increasingly utilized in many applications and consumer products, exploiting their distinctive

65

physical and chemical characteristics, as well as their antibacterial and antifungal properties

66

(Fabrega et al., 2011). The extensive application of AgNPs leads to their release in the

67

environment during production, use and after disposal; AgNPs enter the environment directly or

68

indirectly via a technical system and inevitably end up in the coastal environment (Fabrega et al.,

69

2011).

70

The increasing AgNP use has raised concerns about the environmental and human safety,

71

since AgNPs are associated with cellular disturbances (Vale et al., 2016). The ecotoxicological

72

literature suggests that AgNPs can affect higher plants at the molecular, cellular, physiological and

73

morphological level; however, in most of the studies dealing with AgNP impacts on terrestrial

74

higher plants, high, not environmentally relevant AgNP concentrations were used (> 0.2 - 5000 mg

75

L-1; see review in Rastogi et al., 2017, and Yan and Chen, 2019). Notably, a number of studies

76

have focused on the toxicity of AgNPs to freshwater higher plants (exposure concentrations: 0.005

77

- 1000 mg L-1; e.g. see review in Thwala et al., 2016; Zou et al., 2016; Dewez et al., 2018; Pereira

78

et al., 2018; Minogiannis et al., 2019), indicating adverse effects even at relatively low

3

79

concentrations (0.005 mg L-1; Gubbins et al., 2011; Minogiannis et al., 2019). The phytotoxicity of

80

AgNPs to a seagrass species (Halophila stipulacea) has been also recently examined, indicating

81

actin filament (AF) damage and oxidative stress induction at concentrations as low as 0.0002 mg

82

L-1; in addition, it was assessed that a risk posed by AgNPs cannot be excluded, and thus further

83

testing is required (Mylona et al., 2020). Considering the above, as well as that AgNP effects may

84

be species-specific (Yan and Chen, 2019), research concerning interactions between seagrasses

85

and AgNPs needs to be extended to several other seagrass species.

86

The present study aims to provide information on AgNP toxicity to seagrasses assessing

87

alterations in several structural, biochemical and physiological traits of the little Neptune grass

88

Cymodocea nodosa (Ucria) Aschers. (Cymodoceaceae) upon exposure to environmentally relevant

89

AgNP concentrations. Toxicity information would allow us to derive effect concentrations, to

90

determine sensitive response parameters as early warning indicators of risk and to assess potential

91

risks. C. nodosa is along with Posidonia oceanica the most important and widespread seagrasses

92

in the Mediterranean Sea. P. oceanica meadows are considered one of the climax communities of

93

the Mediterranean coastal area (Procaccini et al., 2003), while C. nodosa is the most common

94

seagrass species in shallow sheltered to semi-exposed Mediterranean sub-tidal environments

95

(Orlando-Bonaca et al., 2015). In particular, C. nodosa is considered as a species with great

96

phenotypic plasticity and a high ability to adapt to environmental variability, and thus to colonize

97

new substrates (Malea and Zikidou, 2011). The utilization of C. nodosa as a biomonitor of

98

environmental quality through the use of biomarkers has been previously recommended; toxicity

99

data are available mainly for trace metals, while response parameters more commonly monitored

100

in toxicity tests include the cytoskeleton, photosynthetic activity, cell viability and growth (Malea

101

et al., 2013a, 2013b, 2014, 2019; Portillo et al., 2014; Moustakas et al., 2016, 2017; Llagostera et

102

al., 2016; Adamakis et al., 2018).

103

We investigated potential alterations in (a) the cytoskeleton (AFs and microtubules, MTs),

104

endoplasmic reticulum (ER) and ultrastructure in leaf cells, (b) photosystem II functioning and

4

105

oxidative stress parameters in leaves, (c) cell viability and elongation of leaves, rhizomes and roots

106

of C. nodosa exposed under laboratory conditions to AgNP concentrations ranging from 0.0002 to

107

0.2 mg L-1 for 8 days. In addition, toxic effect levels and the relative sensitivity of the response

108

parameters were determined, and the risk posed by the AgNPs to the coastal environment was

109

assessed.

110 111

2. Materials and methods

112

2.1. Seagrass collection

113

Cymodocea nodosa was collected from Epanomi site, Thermaikos Gulf, Northern Aegean

114

Sea, Mediterranean Sea (40º24'23.62"N, 22º53'44.38" E) during July 2017 from 1 m depth with a

115

20 cm diameter acrylic corer, which penetrated to a depth of 30 cm. At the sampling site, this

116

seagrass forms a monospecific meadow, displaying in mid-summer a leaf density of about 5000

117

leaves per m2. Plants were rinsed in seawater at the sampling site and transported to the laboratory

118

in plastic containers with seawater.

119

2.2. Treatments

120

C. nodosa plants, consisting of plagiotropic rhizomes with the corresponding roots, orthotropic

121

rhizomes and leaf shoots were placed for 24 h under laboratory conditions in constantly aerated

122

aquaria containing seawater to equilibrate. A leaf shoot usually consists of 3 adult, 1 intermediate,

123

and 1-2 juvenile leaves (Malea et al., 2014).

124

A stock suspension of 20 mg L-1 of AgNPs (primary particle size 20-40 nm, purity 99.9% on

125

metal basis, Alfa Aesar, Taufkirchen, Germany) was prepared in Milli-Q water and ultrasonicated

126

for 3 min with a microtip probe (VibraCell 400 W, Sonics & Materials Inc., USA). Plants were

127

incubated in glass-made aquaria containing AgNPs at concentrations 0.0002, 0.002, 0.02 and 0.2 mg

128

L-1 in filtered (0.45 µm Whatman GF/C) seawater, and filtered seawater with no added AgNPs

129

(control). The seawater was collected from the sampling site, and had salinity 34.9 psu, pH 7.0,

130

dissolved O2 8.34 mg L-1 and dissolved silver concentration 0.0047 µg L-1. The experiments were

5

131

conducted under 16 h day / 8 h night regime at an ambient temperature of 22 ± 1oC / 18 ± 1oC day /

132

night, respectively, with photon flux density of 120 ± 20 µmol m-2 s-1. The aquaria were constantly

133

aerated using pumps. The suspensions in the aquaria were changed every day. The experiment was

134

conducted in triplicate (three aquaria per concentration). Plant samples were randomly removed after

135

2, 4, 6 and 8 days.

136

2.3. AgNPs characterization

137

Particles’ primary size and morphology were examined in ultrasonicated stock suspension

138

(20 mg L-1; number of particles: 551) by Transmission Electron Microscopy (TEM, Jeol JEM

139

1010). Hydrodynamic size was assessed by dynamic light scattering (DLS) analysis and surface

140

charge by zeta (ζ) potential measurements (three repetitions) using Nano ZS Zetasizer (Malvern

141

Instrument, Worcestershire, UK).

142

2.4. Imaging of cell structural components

143

AF, MT and ER effects were examined in meristematic and differentiating interface leaf

144

cells in six juvenile leaves per cell component, concentration and day (two leaves per aquarium).

145

Leaves were cut into ca. 2 mm x 2 mm pieces; three subsamples of leaf pieces were examined for

146

each staining. TRICT-phalloidin was used to stain AFs according to Adamakis et al. (2018). MT

147

and ER immunostaining was performed according to Malea et al. (2013a, b) and Zachariadis et al.

148

(2001), respectively, after modifications (see Mylona et al., 2020). A Zeiss LSM780 Confocal

149

Laser Scanning Microscope (CLSM) was used for observation and ZEN2011 software for image

150

acquisition. Observations were conducted on about 20-30 cells per image in at least three randomly

151

selected images. The effects on AFs were estimated, compared to the control, investigating z-series

152

projections with Image J software, and expressed by an arbitrary scale of 1 to 3. The number of

153

cortical MTs per cell was determined through z-series projections, following image interpretation

154

and manual measurements based on an imaginary line, vertical to MTs, processed in Image J

155

software; MTs were counted, and measurements were expressed as percentages (%) of the number

156

of MTs per cell in relation to the average MT number per cell in the control.

6

157

Ultrastructural observations were conducted by Transmission Electron Microscopy (TEM)

158

(Panteris et al., 2018) in three intermediate leaf blades (one leaf per aquarium), derived from the

159

shoots used for cytoskeleton examination. Ultrathin sections were examined by a JEOL JEM 1011

160

TEM (Ltd., Tokyo, Japan) equipped with a Gatan ES500W digital camera and micrographs were

161

obtained with 3.11.2 software.

162

2.5. Imaging of hydrogen peroxide production

163

Intermediate leaf blades from the control and 0.0002-0.02 mg L-1 treatments (three leaves per

164

aquarium) were incubated with 2’, 7’-dichlorofluorescein diacetate (DCF-DA, Sigma) in dimethyl

165

sulfoxide (DMSO) (Moustakas et al., 2017), and then, were observed under a Zeis AxioImager Z.2

166

fluorescence microscope equipped with an MRc5 Axiocam. Images were acquired and the

167

fluorescence intensity of the cells was measured using Image J software. The corrected total cell

168

fluorescence (CTCF) was calculated (CTCF: integrated density – (area of selected cell * mean

169

fluorescence of background readings), about eighty CTCF values (three areas per leaf part * three

170

parts (tip, middle and basal part) * nine leaves) were obtained per concentration and day, and the

171

ratio of mean CTCF in AgNP treated leaves to mean CTCF in the control was calculated.

172

2.6. Assays for antioxidant enzymes, and protein and malondialdehyde content

173

Antioxidant enzymes activity, total protein content and content of malondialdehyde (MDA),

174

which is an end product of lipid peroxidation were assayed on the 6th-8th days at 0.0002-0.02 mg

175

L-1 and in the control to assess various levels of the oxidative stress response. Six intermediate leaf

176

blades per concentration, day and technique were used (two leaves per aquarium). Extracts were

177

prepared by homogenization of leaf material (0.1 g wet wt) in liquid nitrogen with phosphate

178

buffer (pH 7.8); total protein content (mg g-1 wet wt) was determined under Coomassie Brilliant

179

Dye (G-250) reaction (Bradford, 1976); superoxide dismutase (SOD) activity (U mg-1 protein) was

180

detected based on the inhibition rate of nitro blue tetrazolium (NBT) to photochemical decline

181

(Beyer and Fridovich, 1987); ascorbate peroxidase (APX) activity (U mg-1 protein) was assayed as

182

proposed by Nakano and Asada (1981). MDA content (nmol g-1 wet wt) was assayed according to

7

183

Heath and Packer (1968) protocol; leaf homogenization (0.1 g wet wt) was carried out in liquid

184

nitrogen with the addition of trichloroacetic acid, followed by centrifugation. Analyses were done

185

in triplicate.

186

2.7. Chlorophyll fluorescence analysis

187

Chlorophyll fluorescence was estimated at room temperature using a pulse modulation

188

chlorophyll fluorometer (JUNIOR-PAM, Walz, Germany) (Wu, 2016). Six intermediate leaf

189

blades (two leaves per aquarium) per concentration and day from the control and 0.0002-0.02 mg

190

L-1 treatments were dark adapted for 10 min and measured with an actinic light (AL) intensity of

191

120 µmοl photons m-2 s-1. The following parameters were measured: effective quantum yield of

192

photochemical energy conversion in PSII (ΦPSII), quantum yield of regulated non-photochemical

193

energy loss in PSII (ΦNPQ), quantum yield of non-regulated energy loss in PSII (ΦNO), relative PSII

194

electron transport rate (ETR), non-photochemical quenching (NPQ), photochemical quenching (qP)

195

and maximum quantum yield of PSII photochemistry (Fv/Fm) (Malea et al., 2019).

196

2.8. Evans Blue staining

197

Untreated and AgNP treated juvenile leaves and intermediate leaf blades from six shoots per

198

concentration and day (two shoots per aquarium), and the corresponding orthotropic rhizomes and

199

plagiotropic rhizome roots were incubated at 0.25% aqueous Evans Blue staining (e.g. Malea et al.,

200

2014). The occurrence of dead epidermal leaf cells was observed under a Zeiss AxioImager Z.2

201

light microscope; observations were made in three areas at each leaf part (tip, middle and basal,

202

about 3000 cells per area), and measurements were expressed as percentages (%) of dead cells.

203

Same compartments were pooled per concentration and day. Three subsamples per

204

compartment were analyzed. In particular, juvenile leaf segments of 1.2 cm length, intermediate

205

leaf blade segments of 2 cm length, rhizome segments of 0.5 cm length and root segments of 1.5

206

cm from the tip, middle and basal parts of the plant compartments were excised. The dye of

207

compartment segments was extracted and the optical density (OD) of extracts was measured with a

208

spectrophotometer (PharmaSpec UV-1700; Shimadzu, Tokyo, Japan) (Mylona et al., 2020).

8

209

2.9. Seagrass compartment elongation

210

Plagiotropic rhizomes marked by tagging at the beginning of the experiment in the control

211

and at AgNP treatments (at least six rhizomes per concentration - two rhizomes per aquarium); a

212

small hole was also made in their leaves with a needle just above the sheath of the oldest adult leaf

213

(Pérez and Romero, 1994). Leaf elongation was measured as the length of newly formed leaf

214

segments (distance between the hole and the leaf base) (Adamakis et al., 2018). Elongation of the

215

corresponding orthotropic rhizomes and roots was also measured. Toxicity Index (TI, %) was

216

calculated as follows: TI= [(EC-ET) / EC] * 100, where: EC= elongation in the control, ET=

217

elongation at AgNP treatment.

218

2.10. Data analysis

219

Non-parametric tests were used, as primary analysis on raw and log-transformed data

220

indicated unequal variances and severe violation of the normality assumption. Mann-Whitney U-

221

test was applied to determine significant differences in variables between AgNP treatments, and

222

between AgNP treatments and the control. Kruskal-Wallis Analysis of Variance was performed to

223

determine significant differences in variables among treatments.

224

2.11. Risk assessment

225

The risk posed by AgNPs was assessed considering a direct release of sewage treatment

226

plant (STP) effluents into a coastal area as a worst-case scenario. The risk quotient (RQ) was

227

calculated according to the European approach (ECB, 2003), and taking also into account an initial

228

dilution factor (DF) of 10, since discharges to a coastal area are subject to a notable dilution, but

229

initial dilution may only occur on calm days in low tidal range areas (ECB, 2003).

230

The predicted environmental concentration (PEC, mean: 2.65ng L-1) provided by the

231

advanced model of Sun et al. (2016) for AgNPs in STP effluents in the EU in 2014 was used. As

232

for the predicted no effect concentration (PNEC), the no observed effect concentration (NOEC,

233

Crane and Newman, 2000) can be used (ECB, 2003); if the lowest observed effect concentration

234

(LOEC) is the lowest tested concentration and thereby, a definitive NOEC cannot be provided, the

9

235

LOEC could be used as a starting point (Gubbins et al., 2011). The NOEC, or alternatively the

236

LOEC, was divided by an assessment factor of 1000 to calculate the PNEC; the highest assessment

237

factor proposed for deriving PNEC for seawater for long-term studies was applied due to the

238

scarcity of available data (ECB, 2003; ECHA, 2008).

239 240

Thereby, RQ= (PEC / DF) / PNEC. An RQ value >1 indicates that a risk cannot be excluded and thus, a refinement of the risk assessment is recommended (ECB, 2003).

241 242

3. Results

243

3.1. Silver nanoparticle characterization

244

TEM analysis of AgNPs in stock suspension showed that primary particles had a uniform

245

spherical shape and a diameter of 12.30-212.64 nm (mean ± SD: 35.01 ± 12.68 nm) (Appendix A).

246

DLS measurements demonstrated zeta potential and hydrodynamic diameter values of -2.58 ± 0.88

247

mV and 874.3 nm, respectively, indicating a high propensity for aggregation, and a polydispersity

248

index value of 0.605, indicating that smaller and larger aggregate sizes were present.

249

3.2 Effects on structural cell components

250

AFs in meristematic and differentiating interphase cells of C. nodosa juvenile leaves were

251

affected by AgNPs in a concentration- and time-dependent manner (Table 1). F-actin was firstly

252

affected at 0.02 mg L-1 (6th day), exhibiting signs of diminishing (Fig. 1.A2 cf 1.A1, 1F cf 1E). At

253

0.2 mg L-1, only some thick AF bundles persisted in meristematic cells (Fig. 1.A3), while in

254

differentiating cells more AF bundles were observed (Fig. 1.A7). At 0.2 mg L-1, F-actin

255

disappeared totally in the meristematic zone after 6 days (Fig. 1.A4), while in the differentiating

256

zone AFs appeared diminished, yet still present (Fig. 1.A8).

257

ER was evenly distributed in untreated juvenile leaves (Fig. 1.B1). AgNPs affected ER in a

258

concentration- and time-dependent manner. In particular, aggregation effects were observed at

259

0.02 mg L-1 on the 8th day (Fig. 1.B2). Aggregation was intensified at 0.2 mg L-1, even from the 6th

260

day (Fig. 1.B3).

10

261

In untreated cells cortical MTs were abundant (Fig. 1C1, C4). The effects of AgNPs on MTs

262

in meristematic and differentiating cells of juvenile leaves were concentration-dependent (Table 1).

263

At 0.02 mg L-1, cortical MTs appeared significantly diminished, compared to the control (Mann-

264

Whitney U-test, p< 0.001) and fragmented (Fig. 1C2, C5) on the 8th day, while at 0.2 mg L-1 they

265

were more severely affected (Fig. 1C3, C6). At the above concentrations, no dividing cells were

266

detected, in opposite to the control and the lower concentrations (Fig. 1C2).

267

Ultrastructural investigation of intermediate leaves after 8 days of AgNP treatment revealed

268

structural defects in chloroplasts and mitochondria (Fig. 2C-F), compared to the control (Fig. 2A,

269

B). At 0.02mg L-1, the frets in chloroplasts appeared swollen (Fig. 2C) and mitochondria were

270

devoid of cristae at their central area (Fig. 2D). At 0.2 mg L-1, both frets and grana appeared

271

further swollen (Fig. 2E, F), while the plastid envelope locally consisted of only one membrane

272

(Fig. 2E). At the above concentration, the ptDNA compartments were remarkably expanded in the

273

stroma (Fig. 2E, F), while mitochondria were similar in appearance to those observed at 0.02 mg

274

L-1.

275

3.3. Changes in oxidative stress parameters

276

H2O2 level, expressed as the ratio (r) of mean CTCF in AgNP-treated leaves to mean CTCF

277

in the control, increased after the 2nd day at 0.0002-0.02 mg L-1, and peaked (r= 64:1 at 0.0002 mg

278

L-1, 368:1 at 0.002 mg L-1, and 79:1 at 0.02 mg L-1) on the 4th or 6th day; this increase was followed

279

by a decrease on, or up to the 8th day (Fig. 3, Appendix B). Mean CTCF values on the 4th, 6th and

280

8th days at all three AgNP treatments were significantly higher than those in the control (Mann-

281

Whitney U-test, p< 0.01 or 0.001), except for that on the 8th day at 0.002 mg L-1 (p> 0.05).

282

Antioxidant enzymes activity on the 6th day was generally higher compared to the control

283

(Fig. 3). In particular, SOD activity on the 6th day at 0.002 mg L-1 and APX activity on the 6th day

284

at 0.0002-0.02 mg L-1 were significantly higher than those in the control (Mann-Whitney U-test,

285

p< 0.05). Total protein content on the 6th and 8th days at 0.0002 and 0.002 mg L-1 and MDA

11

286

concentration on the 8th day at 0.0002 and 0.02 mg L-1 were significantly lower, compared to the

287

control (Mann-Whitney U-test, p< 0.05) (Fig. 3).

288

3.4. Changes in chlorophyll fluorescence parameters

289

Changes in the three excitation energy fluxes at PSII, namely, ΦPSII, ΦNPQ, and ΦNO during

290

the incubation period in the control and at 0.0002-0.02 mg L-1 are given in Appendix C. Similarly,

291

changes in NPQ, that reflects heat dissipation of excitation energy in the antenna system, ETR, qp,

292

that represents the fraction of open PSII reaction centers, and Fv/Fm are also given in Appendix C.

293

The values of these parameters during the incubation period did not significantly differ compared

294

to the control at all three AgNP treatments (Mann-Whitney U-test, p> 0.05).

295

3.5. Cell death

296

Mean values of the OD of extracts from leaves, orthotropic rhizomes and roots over the

297

incubation period showed a non-significant increasing trend with increasing concentration

298

(Kruskal-Wallis Chi-squared; χ2: 0.52-4.43; df: 4, n: 20, p> 0.05), indicating no marked cell death

299

upon AgNP exposure. In particular, mean OD at 0.2 mg L-1 was over 117%, 140%, 146% and

300

128 % to the control’s OD as for juvenile leaves, intermediate leaf blades, orthotropic rhizomes

301

and roots, respectively (Appendix D).

302

In accordance to the above, a few epidermal leaf cells were observed to die upon AgNP

303

treatment, in contrast to the control, where no positive Evans Blue staining was observed. In

304

particular, epidermal leaf cells stained blue were observed only at 0.2 mg L-1 from the 4th day

305

onward; their percentages in juvenile and intermediate leaves were < 1.2%.

306

3.6. Effects on plant compartment elongation

307

Leaf length (mean ± SE) at the beginning of the experiment was 3.91 (± 0.49), 14.49 (± 1.38)

308

and 18.88 (± 0.93) cm as for juvenile, intermediate and adult leaves, respectively, while

309

orthotropic rhizome and root length were 1.76 (± 0.37) and 9.47 (± 1.26) cm, respectively.

310

As for juvenile leaves, mean elongation over the incubation period significantly decreased

311

with increasing exposure concentration (Kruskal-Wallis Chi-squared; χ2: 13.006, df: 4, n: 128, p<

12

312

0.05) (Fig. 4); in particular, leaf elongation at 0.2 mg L-1 was significantly lower than that in the

313

control and at 0.0002 mg L-1 (Mann-Whitney U-test, p< 0.01). The average toxicity index over the

314

incubation period varied from 2.8% at 0.0002 mg L-1 to 40.7% at 0.2 mg L-1.

315

On the other hand, mean intermediate leaf, adult leaf and orthotropic rhizome elongation

316

over the incubation period showed no significant variation with concentration (Kruskal-Wallis

317

Chi-squared; χ2: 3.225, 3.747 and 11.876, respectively; df: 4; n: 58, 167 and 116 respectively; p>

318

0.05). No measurable root elongation was observed.

319

3.7. Risk assessment

320

The LOEC for examined oxidative stress parameters was found to be the lowest tested

321

concentration (0.0002 mg L-1). Thus, this LOEC value was used for determining the PNEC (=

322

0.0002 mg L-1 / 1000= 0.2 ng L-1). Based on this PNEC value and on PEC mean value for STP

323

effluents (2.65 ng L-1), a RQ of 1.33 was calculated.

324 325

4. Discussion

326

AgNP phytotoxicity to C. nodosa was evaluated. AgNPs appeared to be able to affect

327

structural and physiological traits of this seagrass species even at environmentally relevant

328

concentrations. Toxic effects may be mainly due to AgNPs and AgNP aggregates adsorbed to

329

seagrass surfaces and internalized into the seagrass body, and not to Ag dissolved from AgNPs in

330

the culture medium, since free chlorine ions form complexes with Ag+ in seawater regulating Ag+

331

availability (Sendra et al., 2017). Internalized AgNPs may have led to toxic effects indirectly by

332

generating reactive oxygen species (ROS) and inducing oxidative stress, or directly by reacting

333

with cell components and interfering with their function (see also Yan and Chen, 2019). During

334

AgNP uptake, Ag+ may have been released from AgNPs; released Ag+ may have also induced toxic

335

effects by generating ROS, and by binding to cell components and modifying their activities (see

336

also Yan and Chen, 2019).

13

337

The induction of ROS overproduction in C. nodosa leaves after AgNP exposure is indicated

338

by the elevated H2O2 levels observed on early time even at the lowest concentration. The data on

339

enzymatic antioxidants (SOD and APX) activity suggest that antioxidant defense mechanisms

340

were stimulated to eliminate ROS toxic effects; this activation potentially contributed to the

341

decrease in H2O2 levels at the end of the experiment. However, despite antioxidant enzymes up-

342

regulation, inhibition of protein biosynthesis or acceleration of protein decomposition due to

343

oxidation possibly occurred, as indicated by the observed reduction in protein content (Jiang et al.,

344

2014). On the other hand, the fact that MDA content showed no significant increase indicates no

345

marked lipid peroxidation (Zou et al., 2016).

346

A similar time-variation pattern of H2O2 level was also observed in the seagrass H.

347

stipulacea exposed to 0.0002-0.02 mg L-1 of AgNPs for 8 days; in addition, an up-regulation in

348

SOD activity was also observed, but a decrease in APX activity, an increase in MDA content and a

349

non-significant alteration in protein content as well (Mylona et al., 2020), indicating some

350

variability in responses to oxidative stress. AgNPs at concentrations of 0.05-10 mg L-1 have been

351

reported to cause ROS overproduction and to induce alterations in other oxidative stress

352

parameters in freshwater plants (e.g. Oukarroum et al., 2013; Jiang et al., 2014; Zou et al., 2016;

353

Pereira et al., 2018).

354

AgNP-induced ROS overproduction possibly contributed to the observed cell structural and

355

ultrastructural disturbances. Similarly to H. stipulacea (Mylona et al., 2020), in C. nodosa F-actin

356

appeared to be more vulnerable to AgNPs than MTs, being also consistent with the effects of

357

bisphenol A on the same seagrass (Adamakis et al., 2018). Although this does not elucidate the

358

mechanism, via which AgNPs affect F-actin, it strongly excludes any correlation between the two

359

cytoskeletal components in their disruption. In contrast, ER susceptibility can be readily associated

360

with F-actin disruption, since ER integrity and distribution is associated with AFs (see Mylona et

361

al., 2020). The increased F-actin stability in differentiating cells, compared to meristematic cells,

14

362

could be attributed to the significance of cytoplasmic streaming (see Volkmann and Baluska,

363

1999) for the larger differentiating cells in comparison to the small meristematic ones.

364

Although in H. stipulacea mitochondria appeared not affected by AgNPs at concentrations of

365

0.0002- 0.2 mg L-1 (Mylona et al., 2020), in C. nodosa mitochondrial cristae appeared diminished,

366

which agrees with the oxidative nature of AgNP toxicity. Furthermore, fret thylakoid swelling

367

reveals that, while in H. stipulacea chloroplasts remained unaffected, in C. nodosa they are

368

sensitive to a certain degree. It appears thus that ultrastructural susceptibility to AgNPs may be

369

species-specific. AgNP effects on freshwater plants at the ultrastructural level have been also

370

reported, particularly on chloroplast structure of Spirodela polyrhiza, but at a higher concentration

371

than those applied in the present study (10 mg L-1; Jiang et al., 2014).

372

The observed dose-dependent inhibition of leaf elongation, being a potential result of AgNP-

373

induced oxidative stress, AgNP-specific effects and / or Ag+-specific effects on seagrass cell

374

structure and function (Yan and Chen, 2019), indicates an increasing deterioration of the global

375

state of C. nodosa with increasing AgNPs concentrations. As MTs and AFs have an essential role

376

in higher plant growth and morphogenesis (Mathur et al., 1999; Hasezawa and Kumagai, 2002),

377

the observed AF and MT disruption may have contributed to leaf elongation inhibition. Cell

378

division cessation in the meristematic leaf areas, at the highest concentrations, also deprived the

379

leaves from new cell production, potentially contributing to elongation impairment. The above

380

findings are consistent with those recently reported for the effects of AgNPs on H. stipulacea leaf

381

elongation; particularly, in this species, the toxicity index increased from 1.1 to 32.2% with

382

exposure concentration (0.0002- 0.2 mg L-1), and a marked elongation impairment occurred at 0.2

383

mg L-1 (Mylona et al., 2020). Inhibition of freshwater plants’ growth upon exposure to AgNPs at

384

concentrations even as low as 0.005 mg L-1 has been also reported (Gubbins et al., 2011;

385

Minogiannis et al., 2019).

386

On the other hand, no inhibition of PS II function was observed in C. nodosa at AgNPs

387

concentrations up to 0.02 mg L-1, despite a ROS overproduction and a chloroplast impairment. A

15

388

deterioration of photosynthetic activity after exposure for 24 h, 48 h or 7 days to higher AgNP

389

concentrations (1 – 10 mg L-1), compared to those applied in the present study, has been previously

390

observed in freshwater plants (Jiang et al., 2012; Zou et al., 2016; Dewez et al., 2018). In addition,

391

no marked reduction in cellular viability was observed in the present study; on the contrary, a

392

marked reduction in cellular viability in young leaves of H. stipulacea treated with 0.02-0.2 mg L-1

393

of AgNPs for 4-6 days has been reported (Mylona et al., 2020). AgNPs have been reported to

394

induce cell death, as a potential result of excess ROS, in a freshwater plant, particularly Lemna

395

gibba at concentrations of 0.1-10 mg L-1 (Oukarroum et al., 2013).

396

Alterations in H2O2 level, occurring with the concomitant changes in antioxidant enzymes

397

activities and protein content, appear to be an early AgNP-induced stress marker in C. nodosa. AF

398

damages in meristematic and differentiating leaf cells of this seagrass species also appear to be a

399

reliable and relatively sensitive indicator of AgNPs stress, while ER, MTs, chloroplasts,

400

mitochondria and juvenile leaf elongation impairment appear to be comparatively less sensitive

401

biomarkers. The above findings, along with those of Mylona et al. (2020), particularly that AF

402

disturbance in differentiating leaf cells of H. stipulacea and elevated H2O2 levels occur at early

403

time after exposure to 0.0002 mg L-1 of AgNPs, suggest that these response parameters could be

404

utilized as early warning indicators of risk posed by AgNPs to seagrass meadows. The observed

405

response differences between C. nodosa and H. stipulacea indicate some interspecific variation in

406

biological traits influencing the AgNP-seagrass interaction.

407

The lowest effect concentration observed in C. nodosa and H. stipulacea (0.0002 mg L-1; see

408

also Mylona et al., 2020) is lower, compared to the lowest concentrations at which effects of

409

AgNPs on freshwater higher plants under laboratory conditions have been reported. In particular,

410

in toxicity tests for AgNPs and freshwater plants, effect levels have been ≥ 0.005 mg L-1; in these

411

tests, subcellular, photosynthetic and growth effects have been mainly examined (see review in

412

Thwala et al., 2016; Zou et al., 2016; Dewez et al., 2018; Pereira et al., 2018; Minogiannis et al.,

413

2019). Elevated ROS production and antioxidant defense mechanisms activation, impairment of

16

414

cell ultrasrtucture, photosynthetic efficiency and growth, and cell death after exposure to AgNPs

415

have been also observed in terrestrial plants, but at concentrations generally higher (≥ 0.01 mg L-1;

416

e.g. Panda et al., 2011; Qian et al., 2013; Sosan et al., 2016; Vinković et al., 2017; review in Yan

417

and Chen, 2019) than those in the present study. The variation in effect levels may be due to

418

differences in exposure concentrations, but also in nanoparticle physico-chemical properties, plant

419

species morphology and physiology, response parameters sensitivity, culture media charactreristics

420

(e.g. ionic strength, pH) and experimental techniques (e.g. Sendra et al., 2017; Yan and Chen,

421

2019).

422

The occurrence of toxic effects of AgNPs on C. nodosa at exposure to concentration as low

423

as 0.0002 mg L-1, also reported for H. stipulacea (Mylona et al. 2020), suggests that AgNPs may

424

pose threat to seagrasses. The fact that a RQ value >1 was calculated suggests that AgNPs may

425

pose a significant potential risk to the coastal environment (ECB, 2003). This finding is supported

426

by Mylona et al. (2020), who also determined a RQ>1 for AgNPs, based on toxicological data on

427

H. stipulacea. In this context, the fact that models providing PECs do not consider AgNP fate

428

processes, and that validation of modeled concentrations is currently lacking (Bundschuh et al.,

429

2018) should be taken into account. On the other hand, the fact that AgNP concentrations in

430

untreated wastewater from a wastewater treatment plant were detected to be as high as 1.9 µg L-1

431

(Hoque et al., 2012) further supports our findings. These findings are consistent with those of

432

Gubbins et al. (2011), who examining AgNP phytotoxicity to the freshwater plant Lemna minor,

433

also determined a RQ> 1. The above also reinforce the findings of Fabrega et al. (2011) and Vale

434

et al. (2016), whο, reviewing the relevant ecotoxicological literature, noted that AgNPs may pose a

435

high risk to aquatic biota.

436 437

Conclusions

438

The present study represents one of the first attempts to assess AgNP effects on seagrasses.

439

Our results indicate that AgNPs can induce oxidative stress in C. nodosa and impair several

17

440

structural and physiological traits at environmentally relevant concentrations, posing a significant

441

potential risk to the coastal environment; changes in oxidative stress markers and AF damage in C.

442

nodosa leaves can be utilized as early warning indicators of AgNP stress. Considering also the

443

previously reported data, the above findings can potentially apply to other seagrass species. These

444

findings can be utilized in the overall effort of protection and management of the seagrass

445

meadows.

446

Acknowledgments Authors are grateful to Assoc. Prof. D. Fatouros (School of Pharmacy, AUTH

447

Greece) and Prof. T. Kechagias (School of Physics, AUTH, Greece) for their support with AgNP

448

characterization. Thanks are due to the anonymous referees for thoughtful critiques and comments.

449 450

References

451

Adamakis, I.-D.S., Malea, P., Panteris, E., 2018. The effects of Bisphenol A on the seagrass

452

Cymodocea nodosa: Leaf elongation impairment and cytoskeleton disturbance. Ecotoxicol.

453

Environ. Saf. 157, 431-440.

454 455 456 457

Beyer, W.F.J.R., Fridovich, I., 1987. Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions. Anal. Biochem. 161, 559-566. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

458

Buapet, P. Mohammadi, N.S., Pernice, M., Kumar, M., Kuzhiumparambil, U., Ralph, P.J., 2019.

459

Excess copper promotes photoinhibition and modulates the expression of antioxidant-related

460

genes in Zostera muelleri. Aquat. Toxicol. 207, 91-100.

461

Bundschuh, M., Filser, J., Lόderwald, S., McKee, M.S., Metreveli, G., Scaumann, G.E., Schulz,

462

R., Wagner, S., 2018. Nanoparticles in the environment: where do we come from, where do

463

we go to? Environ. Sci. Eur. 30, 6. doi: 10.1186/s12302-018-0132-6

464

Costanza, R., d'Arge, R., de Groot, R., Faber, S., Grasso, M., Hannon, B., Limburg, K., Naeem, S.,

18

465

O'Neill, R.V., Paruelo, J., Raskin, R.G., Sutton, P., van den Belt, M., 1997. The Value of the

466

World's Ecosystem Services and Natural Capital. Nature 387, 253-260.

467 468

Crane, M., Newman, M.C., 2000. What level of effect is a no observed effect? Environ. Toxicol. Chem. 19, 516-519.

469

Dewez, D. Goltsev, V., Kalaji H., Qukarroum A., 2018. Inhibitory effects of silver nanoparticles

470

on photosystem II performance in Lemna gibba probed by chlorophyll fluorescence. Current

471

Plant Biol. 16, 15-21.

472

Diepeus, N.J., Buffan-Dubau, E., Budzinski, H., Kallerhoff, J., Merlina, G., Silvestre, J., Auby, I.,

473

Tapie, N., Elger, A., 2017. Toxicity effects of an environmental realistic herbicide mixture

474

on the seagrass Zostera noltei. Environ. Pollut. 222, 393-403.

475 476

ECB, 2003. Technical Guidance Document on Risk assessment, European Chemicals Bureau, Institute for Health and Consumer Protection, Part II, European Commission, Dublin.

477

ECHA, 2008. Guidance on information requirements and chemical safety assessment. Chapter

478

R.10: Characterisation of dose (concentration)-response for environment. Guidance for the

479

implementation of REACH. European Chemicals Agency, 2008, pp.65.

480 481 482 483 484 485 486 487

Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S., Lead, J.R., 2011. Silver nanoparticles: Behaviour and effects in the aquatic environment. Environ. Int. 37, 517-531. Gubbins, E.J., Batty, L.C., Lead, J.R., 2011. Phytotoxicity of silver nanoparticles to Lemna minor L. Environ. Pollut. 159, 1551-1559. Hasezawa, S., Kumagai, F., 2002. Dynamic changes and the role of the cytoskeleton during the cell cycle in higher plant cells. Int. Rev. Cytol. 214, 161-91. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189-198.

488

Hoque, M.E., Khosravi, K., Newman, K, Metcalfe, C.D., 2012. Detection and characterization of

489

silver nanoparticles in aqueous matrices using asymmetric-flow fractionation with

490

inductively coupled plasma mass spectrometry J. Chromatography A 123, 109-115.

19

491

Jiang, H.-S., Li, M., Chang, F.-Y., Li, W., Yin, L.-Y., 2012. Physiological analysis of silver

492

nanoparticles and Ag NO3 toxicity to Spirodela polyrhiza. Environ. Toxicol. Chem. 31(8),

493

1880-1886.

494

Jiang, H.-S., Qiu, X.-N., Li, G.-B., Li, W., Yin, L.-Y., 2014. Silver nanoparticles induced

495

accumulation of reactive oxygen species and alteration of antioxidant systems in the aquatic

496

plant Spirodela polyrhiza. Environ. Toxicol. Chem. 33, 1394-1405.

497 498

Lewis, M.A., Devereux, R., 2009. Nonnutrient anthropogenic chemicals in seagrass ecosystems: Fate and effects. Environ. Toxicol. Chem. 28, 644-661.

499

Llagostera, I., Cervantes, D., Sanmartí, N., Romero, J., Pérez, M., 2016. Effects of Copper

500

Exposure on Photosynthesis and Growth of the Seagrass Cymodocea nodosa: An

501

Experimental Assessment. Bull. Environ. Contamin. Toxicol. 97, 374-379.

502 503

Malea, P., Adamakis, I.-D.S., Kevrekidis, T., 2013a. Microtubule integrity and cell viability under metal (Cu, Ni, Cr) stress in the seagrass Cymodocea nodosa. Chemosphere 93, 1035-1042.

504

Malea, P., Adamakis, I.-D.S., Kevrekidis, T., 2013b. Kinetics of cadmium accumulation and its

505

effects on microtubule integrity and cell viability in the seagrass Cymodocea nodosa. Aquat.

506

Toxicol. 144-145, 257-264.

507

Malea, P., Adamakis, I.-D.S., Kevrekidis, T., 2014. Effects of lead uptake on microtubule

508

cytoskeleton organization and cell viability in the seagrass Cymodocea nodosa. Ecotoxicol.

509

Environ. Saf. 104, 175-181.

510

Malea, P., Charitonidou, Κ., Sperdouli, I., Mylona, Z., Moustakas, M., 2019. Zinc uptake,

511

photosynthetic efficiency and oxidative stress in the seagrass Cymodocea nodosa exposed to

512

ZnO nanoparticles. Materials 12, 2101. doi:10.3390/ma12132101

513 514

Malea, P., Zikidou, C., 2011. Temporal variation in biomass partitioning of the seagrass Cymodocea nodosa at the Gulf of Thessaloniki, Greece. J. Biol. Res. 5, 75-90.

20

515

Mathur, J., Spielhofer, P., Kost, B., Chua, N., 1999. The actin cytoskeleton is required to elaborate

516

and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana.

517

Development 126, 5559-5568.

518

Minogiannis, P., Valenti, M., Kalantzi, O.-I. , Biskos, G., 2019. Toxicity of pure silver

519

nanoparticles produced by spark ablation on the aquatic plant Lemna minor. J. Aerosol Sci.

520

128, 17-21.

521

Mochida, K., Hano, T., Onduka, T., Ito, K., Yoshida, G., 2019. Physiological responses of eelgrass

522

(Zostera marina) to ambient stresses such as herbicide, insufficient light, and high water

523

temperature. Aquat. Toxicol. 208, 20-28.

524

Moustakas, M., Malea, P., Haritonidou, K., Sperdouli, I., 2017. Copper bioaccumulation,

525

photosystem II functioning, and oxidative stress in the seagrass Cymodocea nodosa exposed

526

to copper oxide nanoparticles. Environ. Sci. Pollut. Res. Int. 24, 16007-16018.

527

Moustakas, M. Malea, P., Zafeirakoglou, A., Sperdouli, I., 2016. Photochemical changes and

528

oxidative damage in the aquatic macrophyte Cymodocea nodosa exposed to paraquat-

529

induced oxidative stress. Pestic. Biochem. Physiol. 126, 28-34.

530

Mylona, Z., Panteris, E., Kevrekidis, T., Malea, P. 2020. Silver nanoparticle toxicity effect on the

531

seagrass Halophila stipulacea. Ecotoxicol. Environ. Saf. 189, 109925.

532

https://doi.org/10.1016/j.ecoenv.2019.109925

533 534

Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867-880.

535

Orlando-Bonaca, M., Mavrič, B., Francé, Grego, M., 2015. A new index (MediSkew) for the

536

assessment of the Cymodocea nodosa (Ucria) Ascherson meadow’s status. Mar. Environ. Res.

537

110, 132-141.

538

Oukarroum, A., Barhoumi, L., Pirastru, L., Dewez, D., 2013. Silver nanoparticle toxicity effect on

539

growth and cellular viability of the aquatic plant Lemna gibba. Environ. Toxicol. Chem. 32,

540

902-907.

21

541

Panda, Kamal K., Achary, Mohan M., Krishnaven, R., Padhi, Bijaya K., Sarangi, Sachindra N.,

542

Sahu, Surendra N., Panda, Brahma, B., 2011. In vitro biosynthesis and genotoxicity bioassay

543

of silver nanoparticles using plants. Toxicology in Vitro 25, 1097–1105.

544

Panteris, E., Diannelidis, B.-E., Adamakis, I.-D.S., 2018. Cortical microtubule orientation in

545

Arabidopsis thaliana root meristematic zone depends on cell division and requires severing

546

by katanin. J. Biol. Res. 25, 12. doi: 10.1186/s40709-018-0082-6

547

Pereira, S.P.P., Jesus, F., Aguiar, S., de Oliveira, R., Fernandes, M., Ranville, J., Nogueira, A.J.A.,

548

2018. Phytotoxicity of silver nanoparticles to Lemna minor: Surface coating and exposure

549

period-related effects. Sci. Total Environ. 618, 1389-1399.

550 551

Pιrez, M., Romero J., 1994. Growth dynamics, production and nutrient status of the seagrass Cymodocea nodosa in a Mediterranean semi-estuarine environment. Mar. Ecol. 15, 51-64.

552

Portillo, E., Ruiz, de la Rosa, M., Louzara, G., Ruiz, J.M., Marín-Guirao, L., Quesada, J.,

553

González, J.C., Roque, F., González, N., Mendoza, H., 2014. Assessment of the abiotic and

554

biotic effects of sodium metabisulphite pulses discharged from desalination plant chemical

555

treatments on seagrass (Cymodocea nodosa) habitats in the Canary Islands. Mar Pollut Bull.

556

80, 222-233.

557

Procaccini, G., Buia, M.C., Gambi, M.C., Perez, M., Pergent, G., Pergent-Martini, C., Romero, J.,

558

2003. The seagrasses of the western Mediterranean, in: Green, E.P., Short, F. (Eds.). World

559

Atlas of Seagrasses. University of California Press, London, England, pp. 48-58.

560

Qian, H., Peng, X., Han, X., Ren, J., Sun, L., Fu, Z., 2013. Comparison of the toxicity of silver

561

nanoparticles and silver ions on the growth of terrestrial plant model Arabidopsis thaliana. J.

562

Environ. Sci. 25, 1947–1956.

563

Rastogi, A., Zivcak, M., Sytar, O., Kalaji, H.M., He, X., Mbarki, S., Brestic, M., 2017. Impact of

564

metal and metal oxide nanoparticles on plant: A critical review. Frontiers in Chemistry 7,

565

doi: 10.3389/fchem.2017.00078.

22

566

Sendra, M., Yeste, M.P., Gatica, J.M., Moreno-Garrido, I., Blasco, J., 2017. Direct and indirect

567

effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas

568

reinhardtii and Phaeodactylum tricornutum). Chemosphere 179, 279-289.

569

Sosan, A., Svistunenko, D., Straltsova, D., Tsiurkina, K, Smolich, I., Lawson, T., Subramaniam, S.,

570

Golovko, V., Anderson, D., Sokolik, A., Colbeck, I., Demidchik, V., 2016. Engineered silver

571

nanoparticles are sensed at the plasma membrane and dramatically modify the physiology of

572

Arabidopsis thaliana plants. Plant J. 85, 245–257.

573

Sun, T.Y., Bornhöft, N., Hungerbόhler, K., Nowack, B., 2016. Dynamic probalistictic modeling of

574

environmental emissions of engineered nanomaterials. Environ. Sci. Technol. 50, 4701-

575

4711.

576

Thwala, M., Klaine, S.J., Musee, N., 2016. Interactions of metal-based engineered nanoparticles

577

with aquatic higher plants: A review of the state of current knowledge. Environ Toxicol.

578

Chem. 35, 1677-1694.

579 580

Yan, A., Chen, Z., 2019. Impacts of silver nanoparticles on plants: A focus on the phytotoxicity and underlying mechanism. Int. J. Mol. Sci. 20, 1003. doi: 10.3390/ijms20051003

581

Vale, G., Mehennaoui, K., Cambier, S., Libralato, G., Jomini, S., Domingos, R.F., 2016.

582

Manufactured nanoparticles in the aquatic environment-biochemical responses on freshwater

583

organisms: A critical overview. Aquat. Toxicol. 170, 162-174.

584

Vinković , T. Novák, O., Strnad, M., Goessler, W., Jurašin, D.D., Paradikovič, Vinković Vrček, I.,

585

2017. Cytokinin response in pepper plants (Capsicum annuum L.) exposed to silver

586

nanoparticles. Environ. Res. 156, 10-18.

587 588

Volkmann, D., Baluška, F., 1999. Actin cytoskeleton in plants: From transport networks to signaling networks. Microsc. Res. Techniq. 47, 135-154.

589

Wu, Η. 2016. Effect of Different Light Qualities on Growth, Pigment Content, Chlorophyll

590

Fluorescence, and Antioxidant Enzyme Activity in the Red Alga Pyropia haitanensis

591

(Bangiales, Rhodophyta). BioMed Res. Intern. ID7383918.

23

592 593 594 595 596

http://dx.doi.org/10.1155/2016/7383918 Zachariadis, M., Quader, H., Galatis, B., Apostolakos, P., 2001. Endoplasmic reticulum preprophase band in dividing root-tip cells of Pinus brutia. Planta 213, 824-827. Zou, X., Li, P., Huang, Q., Zhang, H., 2016. The different response mechanisms of Wolffia globose: Light-induced silver nanoparticle toxicity. Aquat. Toxicol. 176, 97-105.

597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617

24

618

Figure captions

619

Figure 1. Single CLSM sections of interphase Cymodocea nodosa juvenile leaf cells, either

620

untreated (A1, A5) or treated with AgNPs (A2-A4, A6-A8) after TRITC-phalloidin staining. A1-

621

A4. Meristematic cells: AFs gradually diminish, in a concentration- and time-dependent pattern.

622

A5-A8. Differentiating cells: At 0.02 mg L-1 AFs diminish (A6), while at 0.2 mg L-1, they appear

623

highly bundled on the 4th day (A7), diminishing on the 6th day (A8). B1-B3: ER at maximum

624

intensity projection of CLSM sections in untreated and AgNP-treated leaf cells. B1. ER is evenly

625

distributed in the control. B2. ER aggregations on the 8th day at 0.02 mg L-1, further intensified at

626

0.2 mg L-1 (B3). (C1-C6): Single CLSM sections after tubulin immunostaining, in meristematic

627

and differentiating leaf cells. Densely arranged cortical MTs and dividing cells (pointed out by

628

arroheads in C1) can be observed in control meristematic cells (C1). In differentiating cells, MTs

629

are numerous, yet unaligned (C4). After treatment with AgNPs in both meristematic (C2, C3) and

630

differentiating cells (C5, C6), MTs are fragmented and diminished (C2, C5). The above resulted

631

from at least three sample images and were observed at single, cortical, central CLSM sections and

632

projections. Scale bars: 10 µm.

633

Figure 2. TEM micrographs of Cymodocea nodosa mature leaf cells on the 8th day: A, B. In

634

untreated (control) epidermal cell, chloroplasts (A) are limited by an intact plastid envelope (arrow

635

in A). Mitochondria with typical cristae (B) are present in typically dense cytoplasm (double arrow

636

in B). C, D. At 0.02 mg L-1, frets of chloroplasts (C) appear swollen (diamond crosses in C). At

637

some areas, the chloroplast envelope consists of a single membrane (arrowhead in C). Under the

638

same treatment, mitochondria (D) exhibit translucent matrix and swollen cristae. E, F. At 0.2 mg

639

L-1, grana stacks in the chloroplasts are swollen (diamond crosses in E and F), apart from the frets,

640

while the chloroplast envelope consists of a single membrane (arrowhead in E) and the ptDNA

641

compartments (asterisks in E and F) are considerably expanded in the stroma. Ch: Chloroplast, M:

642

Mitochondrion. Scale bars: 1µm.

25

643

Figure 3. A. Changes in internal cell H2O2 level in Cymodocea nodosa leaves exposed for 2, 4, 6

644

and 8 days to 0.0002 - 0.02 mg L-1 AgNP concentrations, expressed as the ratio of mean corrected

645

total cell fluorescence (CTCF) in AgNP treated leaves to that in the control; mean CTCF values

646

were derived from about eighty values (three areas per leaf part*three parts (tip, middle and basal

647

part)*nine leaves). B. Changes in SOD and APX activity, and total protein and MDA content in C.

648

nodosa leaves exposed for 6 and 8 days to the control and 0.0002-0.02 mg L-1 AgNP

649

concentrations; mean±SE from three subsamples; different letters express significantly different

650

values between AgNP treatments and the control and between AgNP treatments (Mann–Whitney

651

U–test; p<0.05).

652

Figure 4. Time course of elongation (in cm) of Cymodocea nodosa juvenile, intermediate and

653

adult leaves, and orthotropic rhizomes after exposure to 0.0002-0.2 mg L-1 AgNP concentrations;

654

mean ±SE from at least 6 shoots and the corresponding orthotropic rhizomes.

655

Appendix A. Transmission Electron Microscope (TEM) image (A.1) and size distribution (A.2) of

656

AgNPs in stock suspension (20 mg L-1); scale bar: 100 nm. See also Mylona et al. (in press).

657

Appendix B. Representative images at ZEISS Axioimager Ζ.2 microscope of hydrogen peroxide

658

(H2O2) production, after 2', 7'-dichlorofluorescein diacetate (DCF-DA) staining in Cymodocea

659

nodosa leaves exposed to 0.0002-0.02 mg L-1 AgNP concentrations; the corrected total cell

660

fluorescence (CTCF) values are mentioned upon the images. Scale bar: 200 µm.

661

Appendix C. Changes in chlorophyll fluorescence parameters in Cymodocea nodosa leaves

662

exposed for 2, 4, 6 and 8 days to the control and 0.0002-0.02 mg L-1 AgNP concentrations:

663

Effective quantum yield of photochemical energy conversion in PSII (ΦPSII), quantum yield for

664

dissipation by down-regulation in PSII (regulated heat dissipation, a loss process serving for

665

protection) (ΦNPQ), quantum yield of non-regulated energy dissipated in PSII (non-regulated heat

666

dissipation, a loss process due to PSII inactivity) (ΦNO), non-photochemical fluorescence

667

quenching (NPQ), relative PSII electron transport rate (ETR), relative reduction state of QA,

668

reflecting the fraction of open PSII reaction centers (qP), and maximum quantum yield of PSII

26

669

photochemistry (Fv/Fm). Mean±SE based on six leaves. The same letters express no significantly

670

different values (Mann-Whitney U-test, p>0.05).

671

Appendix D. Optical density (OD) of extracts from untreated (control) and treated with 0.0002-0.2

672

mg L-1 AgNPs Cymodocea nodosa compartments stained with Evans Blue; mean±SE over the

673

incubation period.

27

Table 1. Time course of silver nanoparticle (Ag NP) effects on (a) actin filaments (AFs) and (b) cortical microtubules (MTs) in interphase meristematic (mc) and differentiating (dc) leaf cells of Cymodocea nodosa. AF Ag NP concentrations (mg L-1) Control 0.0002 0.002 0.02 0.2

mc 1 1 1 1 1

dc 1 1 1 1 1

mc 1 1 1 1 2

Control 0.0002 0.002 0.02 0.2

100 100 100 100 100

100 100 100 100 100

100 100 100 100 100

2 day

4 day

6 day dc 1 1 1 1 2

mc 1 1 1 3a 3b

8 day dc 1 1 1 3a 3a

mc 1 1 1 3a 3b

dc 1 1 1 3a 3a

100 100 100 100 100

100 100 100 77.87±1.55 64.27±1.54

100 100 100 73.12±1.36 71.71±2.30

MT integrity (%) 100 100 100 100 100

100 100 100 100 100

Effects on AFs are classified on a scale of 1 to 3; 1: unaffected AFs; 2: extensive AF bundling/fine filament disappearance; 3a: AF decrease/depolymerization; 3b: AF total disappearance. Effects on MTs are expressed as percentages (%) of the number of microtubules per cell in relation to the control; mean±SE from about 90 meristematic and 50 differentiating cells.

CTCF ratio

400 300

A

0.0002 mg L-1 0.0002 0.002 mg L-1 0.002 0.02 mg L-1 0.02

200 100

0 2 day

4 day

6 day

10

200

8 a ab

a

100

a a

bc

c

a

50

APX activity (U mg-1 protein)

SOD activity (U mg-1 protein)

150

0

B

a b

a

a

a

c

a

a

2

6 day

8 day

100

75

80

a a

b

b

b

b

25

MDA concentration (nmol g-1 wet wt)

a

a

Total protein concentration (mg g-1 wet wt)

4

8 day

100

50

control control 0.0002 0.0002mg mgL¯¹L-1 0.002 mg 0.002 mgL¯¹L-1 0.02 0.02mg mgL¯¹L-1

6

0 6 day

8 day

60

ab ab

b

a

a

a c

40 20 0

0 6 day

8 day

6 day

8 day

b

Juvenile leaf elongation (cm)

A 3

2

1

Intermediate leaf elongation (cm)

4

4

B

3

2

1

0

0 0.0002

0.002

0.02

1

C

0.8

0.6

0.4

0.2

0

Control

0.0002

0.002

0.02

Concentrations (mg

L-1)

Control

0.2

0.2

Orthotropic rhizome elongation (cm)

Control

Adult leaf elongation (cm)

0-2 days 0-4 0-6 0-8

0.0002

0.002

0.02

0.2

0.2

D 0.15

0.1

0.05

0

Control

0.0002

0.002

0.02

Concentrations (mg

L-1)

0.2

Highlights - AgNPs at environmentally relevant concentrations cause toxic effects on seagrasses - AgNPs induce oxidative stress in Cymodocea nodosa at low concentrations (0.2 µgL-1) - AgNPs impair cytoskeleton, endoplasmic reticulum, cell ultrastructure, leaf growth - H2O2 level and actin filament damage are early warning indicators of AgNP stress - AgNPs may pose a significant potential risk to the coastal environment

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: