Aquatic Toxicology 150 (2014) 220–228

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Physiological plasticity of Dictyota kunthii (Phaeophyceae) to copper

excess
C. Sordet a,∗ , L. Contreras-Porcia b , C. Lovazzano b , S. Goulitquer c , S. Andrade a ,
P. Potin d , J.A. Correa a
a
Pontificia Universidad Católica de Chile, Departamento Ecología, Facultad de Ciencias Biológicas, Santiago, Chile
b
Universidad Andres Bello, Departamento de Ecología y Biodiversidad, Facultad de Ecología y Recursos Naturales, República 470, Santiago, Chile
c
Plate-forme MetaboMER, CNRS & UPMC, FR2424, Station Biologique, 29688 Roscoff, France
d
Université Pierre et Marie Curie - Paris 6, UMR 7139 CNRS, Marine Plants and Biomolecules, Station Biologique, 29688 Roscoff, France

a r t i c l e i n f o a b s t r a c t

Article history: The brown alga Dictyota kunthii is one of the dominant species in the coastal areas of northern Chile
Received 28 October 2013 affected by copper enrichment due to accumulated mining wastes. To assess its physiological plasticity
Received in revised form 21 February 2014 in handling copper-mediated oxidative stress, 4-days copper exposure (ca. 100 ␮g/L) experiments were
Accepted 24 February 2014
conducted with individuals from a copper impacted area and compared with the responses of plants
Available online 4 March 2014
from a non-impacted site. Several biochemical parameters were then evaluated and compared between
populations. Results showed that individuals from the copper-impacted population normally displayed
Keywords:
higher levels of copper content and antioxidant enzymes activity (catalase (CAT), ascorbate peroxidase
Seaweed
Copper stress
(AP), dehydroascorbate reductase (DHAR), glutathione peroxidase (GP) and peroxiredoxins (PRX)). After
Cu complexing compounds copper exposure, antioxidant enzyme activity increased significantly in plants from the two selected
Exudates sites. In addition, we found that copper-mediated oxidative stress was associated with a reduction of
Copper accumulation glutathione reductase (GR) activity. Moreover, metabolic profiling of extracellular metabolites from both
populations showed a significant change after plants were exposed to copper excess in comparison with
controls, strongly suggesting a copper-induced release of metabolites. The copper-binding capacity of
those exudates was determined by anodic stripping voltammetry (ASV) and revealed an increased ligand
capacity of the medium with plants exposed to copper excess. Results indicated that D. kunthii, regardless
their origin, counteracts copper excess by various mechanisms, including metal accumulation, activation
of CAT, AP, DHAR, GP and PRX, and an induced release of Cu binding compounds. Thus, plasticity in
copper tolerance in D. kunthii seems constitutive, and the occurrence of a copper-tolerant ecotype seems
unlikely.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction wastes accumulated in some coastal areas in Chile continue to

Copper is usually present at low levels in the oceans. How-
ever, in coastal waters it may be present at much higher levels,
mostly due to anthropogenic inputs from industries, agriculture
or mining (Castilla and Nealler, 1978; Bryan and Langston, 1992;
Correa et al., 1999). Despite improvements in mining practices,
Abbreviations: AP, ascorbate peroxidase; ASV, anodic stripping voltammetry;
CAT, catalase; DHAR, dehydroascorbate reductase; GP, glutathione peroxidase; GR,
glutathione reductase; ICP–MS, inductively coupled plasma mass spectrometry; IS,
impacted site; LPX, lipoperoxides; NIS, non-impacted site; PRX, peroxiredoxin; ROS,
reactive oxygen species.
∗ Corresponding author. Tel.: +56 2 2354 2642.
E-mail address: camille.sordet@yahoo.fr (C. Sordet).
be responsible for some major sources of focal pollution, from
2400 ␮g/L at the discharge point in the past to 20–25 ␮g/L in the
present (Castilla, 1996; Correa et al., 1999). Copper enrichment of
the water in those areas is accompanied by a reduction in algal
diversity and abundance (Correa et al., 1999) and a replacement by
opportunistic copper tolerant species like Scytosiphon lomentaria,
Scytosiphon tenellus (Phaeophyceae) (Medina et al., 2005), Ulva
compressa (Chlorophyceae) (Castilla, 1996) and more recently Dic-
tyota kunthii (Phaeophyceae) (Andrade et al., 2006). Indeed, algae
are directly affected by copper excess, which impacts distinct cel-
lular functions such as photosynthesis, fatty acid metabolism and
enzymes activity. Copper, in excess, as a redox active metal, can
initiate via the Haber–Weiss reaction, the formation and accumu-
lation of highly toxic reactive oxygen species (ROS) and thus cause
oxidative damage (Gallego et al., 1996; Cuypers et al., 1999). This

http://dx.doi.org/10.1016/j.aquatox.2014.02.018
0166-445X/© 2014 Elsevier B.V. All rights reserved.
 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228
oxidative damage of membrane lipids, nucleic acids and proteins
can then lead to cellular death (Halliwell and Gutteridge, 1984; Aust
et al., 1985; Goldstein and Czapski, 1986).
The ability to counteract oxidative stress to survive under excess
copper exposure depends mainly on the antioxidant capacity of
algae. Different antioxidative mechanisms, in particular a com-
plex machinery of specialized antioxidant enzymes, are involved
in the inactivation of ROS. Hydrogen peroxide (H2 O2 ), resulting
from the decomposition of superoxide anions O2 •− , can be detox-
ified by ascorbate peroxidase (AP) via the oxidation of ascorbate
in the Halliwell–Asada cycle, by glutathione peroxidase (GP) via
the oxidation of glutathione in the glutathione cycle or by cata-
lase (CAT). This ascorbate–glutathione pathway plays a key role in
the antioxidant capacity of plants (Noctor and Foyer, 1998; Asada,
1999) and macroalgae (Ratkevicius et al., 2003; Contreras et al.,
2005; Wu and Lee, 2008). Other enzymes in this pathway, such
as dehydroascorbate reductase (DHAR) and glutathione reductase
(GR), catalyze the regeneration of ascorbate and glutathione after
AP and GP activity.
In algae, copper excess has been correlated with higher antiox-
idant enzyme activity (Ratkevicius et al., 2003; Contreras et al.,
2005; Contreras et al., 2009; Contreras et al., 2007; Wu et al.,
2009; Lovazzano et al., 2013). Other strategies, however, have also
evolved in plants and algae to counteract the effects of copper
excess. For example, there is some evidence showing that organic
ligands are released by macroalgae in response to copper excess
(Gledhill et al., 1999; Vasconcelos and Leal, 2001; Andrade et al.,
2010). Such ligands could modulate metal speciation by estab-
lishing stable complexes and thus reducing metal bioavailability
to macroalgae or enhance uptake of metal in a non-toxic form
(Gledhill et al., 1997). Release of metal binding compounds, either
to chelate metals outside the cells or to act as metal carriers, may
represent another mechanism for metal tolerance in macroalgae
(Gledhill et al., 1997).
Previous studies demonstrated that U. compressa (Correa et al.,
1996; Ratkevicius et al., 2003) and S. lomentaria (Contreras et al.,
2005; Contreras et al., 2009), both growing in the same copper-
enriched environment in Chañaral bay, are highly tolerant to copper
excess due to powerful antioxidant defenses. For D. kunthii, which
co-exists with U. compressa and S. lomentaria in Chañaral bay, no
information is yet available on the mechanisms that allow it to live
under copper excess.
Tolerance to metals can be the result of (i) a high physiological
plasticity, as demonstrated in S. lomentaria that, by quickly chang-
ing the activity of its antioxidative enzymes (i.e. within hours),
can tolerate a broad range of copper levels including those in the
Chañaral bay area (Contreras et al., 2005) or (ii) local adaptation (i.e.
by natural selection of tolerant individuals, with narrower plastic-
ity) to areas with long lasting natural or anthropogenic high metal
levels, as suggested for Ectocarpus siliculosus (Russell and Morris,
1970). Our previous studies suggested that copper tolerance in U.
compressa and S. lomentaria is a constitutive trait and clearly related
to a great physiological plasticity, rather than genetic adaptation to
a copper enriched local condition (Correa et al., 1996; Contreras
et al., 2005).
In this study, we aimed at identifying the mechanisms that may
be involved in copper tolerance in D. kunthii (Phaeophyceae), as
its recent colonization around Chañaral bay strongly suggests this
alga must buffer the effects of high levels of copper still present in
the area. D. kunthii individuals exposed for 4 days to copper excess
(ca. 100 ␮g/L) under laboratory conditions were used to measure
antioxidant enzymes activity, Cu accumulation and release of metal
complexing ligands to the medium. We also aimed at finding evi-
dence to support that copper tolerance is the result of several
complementary mechanisms and not a single process. To address
these issues, differences in tolerance between two populations
221
of D. kunthii, one from Chañaral bay (Impacted Site, IS; Correa
et al., 2006) and the other from Cachagua (Non-Impacted Site, NIS;
Contreras et al., 2005) were included in the study. Results provided
information on the physiological plasticity of D. kunthii, a feature
required to cope with copper excess, which supports the idea that
local adaptation to metal excess in Chañaral bay is unlikely.
2. Materials and methods
2.1. Algal sampling
Individuals of D. kunthii were collected in two sites from cen-
tral and northern Chile: Cachagua (32◦ 34 S, 71◦ 28 W) and Punta
Achurra (26◦ 18 S, 70◦ 39 W). The two sites were selected because of
their contrasting history of mining-related impact. Punta Achurra
(26◦ 18 S, 70◦ 39 W; impacted site (IS)), located in Chañaral bay in
northern Chile, has received wastes discharges from the copper
mine in El Salvador for more than 60 years, which has resulted in
the persistent enrichment of the seawater with copper (Ratkevicius
et al., 2003; Medina et al., 2005). The coast around Chañaral
provides an ideal research area, with no other anthropogenic or
natural forms of contamination apart of copper from mining wastes
(Stauber et al., 2005). Cachagua, considered as a non-impacted site
(NIS), is a small village located in central Chile and far away from
any effect of copper mining. Cachagua displays normal levels of
copper in the water, ranging from <1 to 5 ␮g/L and displays normal
biological diversity at the rocky intertidal zone (Ratkevicius et al.,
2003; Contreras et al., 2005; Medina et al., 2005). After collection,
algae were transported to the laboratory in a cooler at 5–7 ◦ C, where
individuals were rinsed with filtered seawater and stored at 12 ◦ C,
12:12 h photoperiod and 40–50 ␮mol m−2 s−1 irradiance for a 36 h
acclimation period.
2.2. Laboratory experiments
Thirty grams of fresh algal tissue were placed in 1 L of 0.2 ␮m fil-
tered and nutrient enriched (2 mM NaNO3 and 0.1 mM NaH2 PO4 ),
coastal seawater from central Chile, supplemented with a nominal
concentration of 100 ␮g/L copper added as CuCl2 (Titrisol, Merck).
Preliminary tests showed that 100 ␮g/L consistently triggered a sig-
nificant and rapid stress response within 4 days. Control fronds
placed in filtered and nutrient enriched seawater without copper
addition were also included. Triplicates for each treatment were
incubated at 12 ◦ C, 12:12 h photoperiod and 40–50 ␮mol m−2 s−1
irradiance. Seawater was changed daily. Algal samples (n = 3)
were collected at the beginning and after 96 h to determine
lipoperoxide production and specific activity of antioxidant
enzymes.
2.3. Copper accumulation
To determine copper content, algal samples (n = 4) were col-
lected at 0, 24, 48, 72 and 96 h and dried at 50 ◦ C. Approximately
0.2 g (dry weight, dw) of algal material were digested in 7 mL of con-
centrated HNO3 and 1 mL H2 O2 (30%) at 200 ◦ C for 20 min in a high
pressure microwave (Milestone Ethos 1) and enough Nano-pure
distilled water to complete 50 mL. Samples were then analyzed
for total Cu by ICP–MS, in a Thermo Scientific XSERIES 2. Detec-
tion limits were 0.03 ␮g of metal g−1 dw. Analytical grade reagents
were used to prepare relevant blanks and calibration curves. Sam-
ples of Lessonia nigrescens with a known copper content were used
to check the accuracy of the digestion and analytic procedures
during determination of total metals in D. kunthii. Copper accumu-
lation in the samples was determined by subtracting basal copper
222 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228
concentrations to copper content value measured after 24, 48, 72
and 96 h of exposure to copper excess.
2.4. Detection of lipoperoxides (LPX)
LPX levels were determined as thio-barbituric acid reactive
species (T-BARS) according to Ratkevicius et al. (2003). Briefly, algal
tissue (ca. 0.5 g dw) was frozen in liquid nitrogen and homogenized
with a pestle in a mortar. Three to 5 mL (w/v) of trichloroacetic acid
were added during homogenization and the homogenate was then
centrifuged for 20 min at 7400 × g. LPX were detected by addition
of 100 ␮L of the clear homogenate to a reaction mixture contain-
ing 0.5% thiobarbituric acid (in 20% trichloroacetic acid) in a final
volume of 1 mL. The reaction mixture was incubated at 100 ◦ C for
30 min and the absorbance was measured at 512 nm. The extinc-
tion coefficient of the synthesized adduct (ε = 155 mM−1 cm−1 ) was
used to determine the amount of lipoperoxides.
2.5. Preparation of protein extracts and detection of antioxidant
enzymes activities
Algal tissue (5 g fresh weight) was frozen in liquid nitro-
gen and homogenized in a prechilled mortar using a pestle.
A total of 15 mL of 0.1 M phosphate buffer pH 7, containing
5 mM 2-mercaptoethanol was added during homogenization. The
homogenate was allowed to thaw at room temperature, filtered
through Miracloth paper (Calbiochem) and centrifuged at 7400 × g
for 20 min at 4 ◦ C. Proteins were precipitated by addition of 0.5 g
of ammonium sulfate/mL of extract during 2.5 h at 4 ◦ C. The pro-
tein pellet was dissolved in 0.5 mL of 0.1 M phosphate buffer
pH 7. Protein concentration was determined by a bicinchoninic
acid assay, using BSA as standard. Protein extracts were stored at
−80 ◦ C. Ascorbate peroxidase (AP), catalase (CAT), dehydroascor-
bate reductase (DHAR), glutathione reductase (GR), and glutathione
peroxidase (GP) activities were determined according to Contreras
et al. (2005). The AP reaction mixture contained 0.1 M phosphate
buffer pH 7.0, 400 ␮M ascorbate (ASC) and 16 mM H2 O2 . After the
addition of ASC, its consumption was measured at 290 nm for 1 min,
and the activity was calculated using the extinction coefficient of
ASC (ε = 2.8 mM−1 cm−1 ). The CAT reaction mixture contained 0.1 M
phosphate buffer pH 7.0, and 14 mM H2 O2 . After the addition of
H2 O2 , its consumption was measured at 240 nm for 1 min, and
the activity was calculated using the extinction coefficient of H2 O2
(ε = 39.4 mM−1 cm−1 ). For DHAR activity, the reaction mixture con-
tained 0.1 M phosphate buffer pH 7.0, 2 mM reduced glutathione
(GSH) and 300 ␮M dehydroascorbate (DHA). After the addition of
DHA, its reduction to ASC was monitored at 265 nm for 1 min,
and the activity was calculated using the extinction coefficient of
ASC (ε = 2.8 mM−1 cm−1 ). The GR reaction mixture contained 0.1 M
phosphate buffer pH 7.0, 500 ␮M oxidized glutathione (GSSG) and
90 ␮M NADPH. After the addition of NADPH, its oxidation was mon-
itored at 340 nm for 1 min, and the activity was calculated using the
extinction coefficient of NADPH (ε = 6.2 mM−1 cm−1 ). The GP reac-
tion mixture contained 0.1 M phosphate buffer pH 7.0, 200 ␮M GSH,
10 mM H2 O2 , 90 ␮M NADPH, and 1 U of GR (Sigma-Aldrich). After
the addition of NADPH, its oxidation was monitored at 340 nm for
1 min, and the activity was calculated using the extinction coeffi-
cient of NADPH (ε = 6.2 mM−1 cm−1 ). Peroxiredoxin activity using
DTT as reducing agent (PRX/DTT) was determined according to
Lovazzano et al. (2013). For that, 50–100 ␮g of protein extract were
pre-incubated with dithiotreitol (DTT) 0.2 mM in phosphate buffer
0.1 M pH 7.0 for 30 min at 37 ◦ C. Reaction was initiated by adding
50 ␮M H2 O2 or t-butyl peroxide (t-BOOH) to the protein extract,
and incubated for 30 min at 37 ◦ C. Reaction was stopped adding
trichloroacetic acid (10% final concentration) and centrifuged at
18,700 × g for 10 min to precipitate the proteins. Seven hundred
␮L aliquot of the supernatant with the remaining peroxide was
mixed with 200 ␮L of (NH4 )2 Fe(SO4 ) 10 mM and 100 ␮L of KSCN
2.5 M, forming a red-colored complex. Peroxides concentration was
spectrophotometrically measured at 480 nm. The peroxide quan-
tification was performed using a calibration curve with known
quantities of H2 O2 prepared in a range from 1 ␮M to 100 ␮M.
2.6. Profiling of metabolites released
In order to minimize background signals that could hide exu-
dates signals during the mass spectrometry analysis, metabolic
profiling experiments were conducted in a nutrient enriched (2 mM
NaNO3 and 0.1 mM NaH2 PO4 ) artificial seawater (Instant Ocean®
Sea Salt), which provides reduced risk of chemical and biolog-
ical contamination. Exudates extracts were obtained by Solid
Phase Extraction (SPE). Triplicates of 600 mL culture medium
from both conditions were slowly passed through EASY car-
tridges (Chromabond 3 mL, 200 mg, Macherey-Nagel, Germany)
after 24, 48, 72 and 96 h exposure. A sand cartridge (empty
Chromabond cartridge filled with ∼3 mL of cleaned and acidified
sea sand) was then mounted inline before the EASY® cartridge
as filter. After washing with deionized water, the EASY® car-
tridges were air-dried and then eluted in glass vials with 2 mL
methanol, followed with 2 mL methanol/acetone (v/v 1:1). Car-
tridge enrichments by SPE have the advantage of extracting
a broader spectrum of metabolites. This fast chromatographic
method, coupled with mass spectrometry (MS) analysis, enables
the generation of large data sets with high throughput in reasonable
time.
Sample fingerprinting was performed on a Dionex Ulti-
mate 3000 RSLC system with an autosampler, a tertiary
pump and coupled to a LTQ-OrbitrapTM hybrid mass spectrom-
eter (Thermo Fisher Scientific, Bremen, Germany). Chromato-
graphic separation was done on an Acclaim 120 C18 column
(100 mm × 2.1 mm × 2.2 ␮m particle size, Dionex). Mobile phase
consisted in water containing 0.1% acetic acid (A) and acetoni-
trile containing 0.1% acetic acid (B). The elution gradient (A:B,
v/v) was as follow: 80:20 from 0 to 5 min; 95:5 at 15 min and
hold for 10 min; 80:20 at 26 min and hold for 4 min. The injected
volume was 5 ␮L, the flow rate was 0.25 mL/min and the temper-
ature of the column was maintained at 20 ◦ C. The U-HPLC (ultra
high-performance liquid chromatography) column was connected
without splitting to the electrospray interface operating in posi-
tive ion mode. In the positive ion mode, the electrospray voltage
was set to 3.5 kV, the capillary voltage to 45 V, and the tube lens
offset to 130 V. The sheath and auxiliary gas flows (both nitrogen)
were set to 5 arbitrary units (a.u.), and the drying gas temperature
was set to 300 ◦ C. Same parameters were used in the negative ion
mode. Mass spectra were recorded from 50 m/z up to 1000 m/z at
a resolution of 30 000 (FWHM at m/z 400) acquired in the centroid
mode.
Following their acquisition by Xcalibur® software (Thermo
Fisher Scientific), metabolomic fingerprints were deconvoluted to
allow the conversion of the three-dimensional raw data (m/z, reten-
tion time, ion current) to time- and mass-aligned chromatographic
peaks with associated peak areas. Massmatrix File Conversion was
used to convert the original Xcalibur data files (*.raw) to a more
exchangeable format (*.mzXML). Data processing was then per-
formed using the open-source XCMS software. XCMS parameters
for the R language were implemented in an automated script. Cent-
Wave was used for the peak picking. The interval of m/z value was
set to 0.1, the signal to noise ratio threshold was set to 10, the
group band-width was set to 10 and the minimum fraction was
set to 0.75. Statistical analysis were performed with SIMCA 13.0
(Umetrics, Sweden).
 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228 223

2.7. Copper complexing capacity

Copper complexing capacity was determined in artificial seawa-

ter enriched with SPE extracts used for the profiling of metabolites
released. Extracts presenting clear separation of copper treatment
and control in exudates profiles were used. And because of the
similarity of exudates profiles, extracts T2 and T3 for IS popula-
tion were pooled and used as IS sample, such as T1, T2 and T3
for NIS population as NIS sample. 130 ␮L of samples were diluted
in 20 mL of artificial seawater before measurement. To check the
accuracy of the analysis, a 10−6 M EDTA solution in artificial sea-
water was used as a positive control. Measurements were done
using a voltammetric analyzer 797 VA computrace (Metrohm) with
a thin mercury film (TMF) deposited in a rotating glassy-carbon disk
as working electrode (RGCDE), a double junction Ag/sat AgCl ref-
erence electrode with salt bridge (3 M KCl), and a platinum wire
counter electrode. Samples were previously deoxygenated with
nitrogen. The voltammetric scans were performed with the square
wave modulation using the following parameters: frequency 50 Hz;
amplitude 20 mV; electrode rotation 3000 rpm; deposition poten-
tial −0.65 V (vs. Ag/AgCl); deposition time 60 s; equilibration time Fig. 1. Changes of Cu content in Dictyota kunthii from non-impacted (NIS) and
impacted (IS) sites. Values of accumulated copper were determined by subtracting
5 s; scan range −0.65 to 0.0 V. Samples were titrated with an
basal Cu concentrations to copper levels measured after 24, 48, 72 and 96 h of cop-
increasing amount of ionic copper and equilibrate for 5 min with per exposure. Bars represent mean values of four replicates ± SD; dw: dry weight.
natural ligands present in the sample. Measurements of the cop- Letters on the bars indicate results of Tukey tests: means with the same letter are
per oxidation peak height at each copper addition were recorded. not significantly different at P = 0.05.
Titration curves were constructed by plotting each copper oxida-
tion peak height vs. total copper concentration (CuT). ASV-labile
copper concentration (Cu ) was calculated dividing the peak height accumulated copper in material from IS, was 5 (24 h; t = 22.68;
value by the slope of the curve. Concentrations of organically P < 0.001), 4 (48 h; t = 16.53; P < 0.001), 4 (72 h; t = 15.23; P < 0.001)
complexed copper (CuL) were calculated by difference of con- and 2,5 times (96 h; t = 9.86; P < 0.001) higher than concentration in
centrations between the total copper (CuT) and the ASV-labile material from NIS (Fig. 1). At the end of the trial, mean concentra-
copper (Cu ). Titration curves were linearly transformed by plot- tion of accumulated copper was 115 ± 11 ␮g/g dw for IS population
ting Cu /CuL vs. Cu (Sueur et al., 1982). Copper complexing organic and 44 ± 6 ␮g/g dw for the NIS population
ligand concentrations (L) and conditional stability constants (log K )
were calculated from the slope and intercept of the linear least
squares regression of the linearization plot. 3.2. Lipoperoxide (LPX) content

No significant differences (t =1.58 ; P = 0.62) in the basal level of

2.8. Statistical analyses LPX was observed between individuals from NIS (147 ± 7 nmol/g
dw) and those from the IS (131 ± 6 nmol/g dw). Moreover, lipoper-
The significance of the differences in all parameters tested were oxide levels in both populations remained unchanged after 96 h of
determined by one and two-way analysis of variance (ANOVA) exposure to copper excess (all cases P > 0.05) (Fig. 2).
followed by Tukey’s multiple comparisons tests (t) using treat-
ment as a fixed effects factor. Prior to the statistical analyses, data
were checked for variance homogeneity using Levene and Bartlett
tests and for normal distribution using Kolmogorov–Smirnov and
Anderson–Darling tests (Zar, 2010). Differences between mean val-
ues were considered to be significant at a probability of 5% (P < 0.05)
(Zar, 2010).
PCA was performed with SIMCA 13.0 (Umetrics, Sweden) and
realized using the Eigenvalue similarity level at 0.05 and the confi-
dence level on parameters at 95%.

3. Results

3.1. Copper accumulation

The results showed differences in the copper content of plants

from both populations (i.e. Cachagua, non-impacted site (NIS) and
Punta Achurra, impacted site (IS)). Individuals from IS had a basal
concentration 60 times higher (181 ± 4 ␮g/g dry weight (dw)) than
material from NIS (2.8 ± 0.2 ␮g/g dw). Moreover, algal tissue from
both populations exposed during 4 days to copper excess (100 ␮g/L Fig. 2. Lipoperoxides levels in Dictyota kunthii from non-impacted (NIS) and
impacted (IS) sites exposed for 96 h to control seawater (gray bars) and to cop-
of CuCl2 ) showed a progressive copper accumulation and cop-
per (100 ␮g/L) enriched seawater (black bars). Basal value is represented by white
per content reached 47 ± 6 ␮g/g in individuals from the NIS and bars. Lipoperoxides are expressed in nanomoles per gram of dry weight tissue (dw).
297 ± 12 ␮g/g dw in individuals from the IS. Concentration of Bars represent mean values of three replicates ± SD.
224 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228

Table 1 increased (CAT: 7-fold, t = −4.59, P < 0.05; AP: 4-fold, t = −8.29,
Mean (n = 3) of basal activities of antioxidant enzymes in Dictyota kunthii from non-
P < 0.01; GP: 16-fold, t = −5.37, P < 0.01) and significantly higher
impacted site (NIS) and copper impacted site (IS).
than plants under control conditions. GR activity, by the contrary,
Antioxidant enzymes Dictyota kunthii Dictyota kunthii was depressed by copper excess, althought the difference with the
NIS IS
controls was not significant (t = 1.67; P = 0.573). In individuals from
(Specific activitya ) (Specific activitya )
IS, specific activities of enzymes coupled in the Halliwell–Asada
CAT 5 ± 2 33 ± 11* cycle were 4 (t = −6.66; P < 0.05) and 2 times higher (t = −4.21;
AP 266 ± 40 2688 ± 514*
P = 0.01) for AP and DHAR, respectively. Specific activity of CAT, GP
DHAR 702 ± 122 1723 ± 375*
PRX 9 ± 2 16 ± 1* and PRX were 2-fold (t = −4.09; P = 0.01), 4-fold (t = −10.75; P < 0.05)
GR 119 ± 16 5 ± 5* and 1.5 fold (t = −4.96; P < 0.05) higher respectively. GR specific
GP 6 ± 5 52 ± 17* activity was reduced after 4 days of copper exposure, as in indi-
a
Specific activity expressed in nmol min−1 mg−1 protein. viduals from the NIS, even though differences were not significant
*
Significant (P < 0.05) differences among values determined in both populations. (Fig. 3, t = −2.66; P = 0.155).

3.3. Antioxidant enzymes activities 3.4. Profiling of released metabolites

CAT, AP, DHAR, PRX and GP basal activities in individuals from
IS were significantly higher (all cases P < 0.05) than those from the
NIS (Table 1). Contrarily, basal activity of GR was significantly lower
(t = 6.67; P < 0.001) in individuals from IS than NIS (Table 1).
In the laboratory, specific activities of CAT, AP, DHAR, PRX and
GP in individuals from both sites exposed to copper excess were sig-
nificantly higher than controls (i.e. culture medium without copper
addition) (Fig. 3). Specifically, individuals from the NIS after 4 days
of copper treatment displayed CAT, AP and GP activities strongly
Metabolites were extracted daily from the culture media to
characterize the profiling of compounds released by D. kun-
thii under copper excess. Metabolites profiles were complex
and Principal Component Analyses (PCA) were used to compare
the intensity of mass/retention time pairs between all chro-
matograms. The differences between profiles were visualized using
a scores plot of medium extracts. The resulting plots for NIS
extracts showed clear separation of copper treatment and con-
trols, after 24 h (T1), 48 h (T2) and 72 h (T3) (Fig. 4), but no

Fig. 3. Activities of the antioxidant enzymes CAT, AP, DHAR, PRX, GR and GP in Dictyota kunthii from non-impacted (NIS) and impacted (IS) sites exposed for 96 h to control
seawater (gray bars) and to copper-enriched (100 ␮g/L) seawater (black bars). Bars represent mean values of three replicates ± SD. Letters on the bars indicate results of
Tukey tests: means with the same letter are not significantly different at P = 0.05.
 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228 225

Fig. 4. PCA score plots for U-HPLC-orbitrap data of released metabolites from Dictyota kunthii from non-impacted (NIS) and impacted (IS) sites extracted after 24 h (T1), 48 h
(T2), 72 h (T3) or 96 h (T4) of incubation in a copper enriched (0) or control medium ().

obvious separation for T4. Score plots for IS extracts showed
a separation between copper treatment and control after 48 h
(T2) and after 72 h (T3) but no obvious separation for T1 or
T4.
3.5. Copper complexing capacity
Our results revealed that in presence of copper excess, D.
kunthii released significantly more Cu complexing ligands to the
media than plants from culture media with no copper addi-
tion (Fig. 5). The maximum concentration of Cu-binding ligands,
obtained in exudate extracts of individuals from NIS, was 4.5
times higher than controls (t = −9.24; P < 0.005). However, plants
source (i.e. NIS or IS) had no significant effect (t = 1.55; P = 0.64)
on the mean Cu complexing capacity. Finally, the conditional Cu
complex stabilities, determined by log K , were not markedly dif-
ferent: log K ranged from 8.44 ± 0.4 and 8.06 ± 0.3 in NIS and
IS culture controls respectively, to 8.36 ± 0.8 and 8.42 ± 0.2 in
NIS and IS culture exudates of algae exposed to copper excess,
respectively.
4. Discussion
The coastal area of Chañaral bay, receiving the discharges of cop-
per mine remains as an environment stressful to macroalgae as a
result of persistently high levels of total dissolved copper. These
elevated concentration of copper in the water of Punta Achurra (IS)
is reflected in an increased copper accumulation in D. kunthii, reach-
ing levels almost 60 times higher than in individuals from Cachagua
(NIS). Copper levels higher than normal were reported in the tissues
of S. lomentaria and U. compressa growing in the same impacted-site
(Ratkevicius et al., 2003; Contreras et al., 2005; Andrade et al., 2006)
or from other copper contaminated places around the world, such
as Gracilariopsis longissima, Porphyra spp. or Enteromorpha spp.;
(Vasconcelos and Leal, 2001; Brown et al., 2012). In algae, tolerance
can not always be predicted on the basis of the content of metal in
the algal tissues, as some tolerant species such as Ectocarpus silicu-
losus or Chlorella vulgaris have evolved exclusion mechanisms (Hall,
2002). However, copper tolerance in D. kunthii, whether or not it
inhabits a copper enriched environment, seems to be related to
copper accumulation.
226 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228
Fig. 5. Production of complexing ligands (nM) by Dictyota kunthii from non-
impacted (NIS) and impacted (IS) sites. Extracts collected at 24 h intervals during
4 days of copper treatment (black bars) or control (gray bars), were tested for cop-
per complexing capacity by ASV. Letters on the bars indicate results of Tukey tests:
means with the same letter are not significantly different at P = 0.05.
Accumulation of high levels of copper in the tissues is known
to induce oxidative stress which generally triggers abnormal
increases in oxidized by-products in vascular plants and algae
(Pinto et al., 2003; Ratkevicius et al., 2003; Contreras et al., 2005;
Weckx and Clijsters, 2006). However, when D. kunthii from IS and
NIS were compared, no differences in lipoperoxide levels were
detected, in spite that basal levels of copper in the tissues from
IS were significantly higher (i.e. 60 times) than those in plants from
NIS. The absence of an effect on lipid peroxidation could result from
attenuation of their production, enhanced degradation or both.
In vascular plants facing abiotic stress, PRXs have the capacity to
reduce intracellular lipoperoxide levels by buffering ROS or by act-
ing like molecular chaperones (Mowla et al., 2002; Kim et al., 2010).
In Scytosiphon gracilis, Lovazzano et al. (2013) demonstrated that
inhibition of thiol-dependant peroxidase activity (PRX belongs to
TDP family) was correlated with an accumulation of LPX. This find-
ing is consistent with our observations on D. kunthii, where PRX
activity was much higher (almost twice) in individuals from IS
than in those from NIS. As in S. gracilis, copper-induced activation
of PRX in D. kunthii likely controls intracellular lipoperoxide lev-
els and maintains membrane stability under conditions of copper
stress.
This study showed that D. kunthii from IS and NIS responded
to copper excess by increasing the activity of various antioxidant
enzymes. CAT, AP and GP showed the highest increases and demon-
strated the activation of the Halliwell–Asada and glutahione cycles,
two main pathways to detoxify ROS in plants and algae (Noctor and
Foyer, 1998; Collén and Davison, 1999; Contreras et al., 2005, 2009).
As expected, an increase in AP was accompanied by an increase
in DHAR that allowed regeneration of ascorbate. In contrast, GR
activity declined when individuals were exposed to copper excess.
The low GR activity in D. kunthii was consistent with previous
results obtained in both Cu-tolerant and Cu-sensitive algal species
exposed to copper stress. The response of GR has been suggested to
result from the direct action of copper on cysteine residues in the
enzyme’s catalytic site (Contreras et al., 2005; Contreras et al., 2009)
or be a consequence of using catalase activity to attenuate the toxic
effects of ROS (Foyer and Mullineaux, 1994). It is also important to
highlight the physiological plasticity of the antioxidant response
in D. kunthii, revealed by the responses of plants from IS whose
antioxidant enzymatic activity returned to normality after 4 days
in absence of copper excess (control conditions).
Lower levels of enzyme activity after 4 days in absence of copper
excess, in spite of a persistently high level of accumulated copper,
suggests that D. kunthii from IS accumulates copper in a form within
the cell, that may avoid the toxic effects of the metal. To cope with
metal toxicity, hyperaccumulator plants have developed mecha-
nisms by which metal is sensed and immediately complexed and
inactivated by intracellular and extracellular metal binding com-
pounds (Hall, 2002). Exudation of metal chelating compounds to
the rhizosphere play an important role in metal sequestration as
they can enhance metal uptake and likely help carrying metals
into plants tissues (Hall, 2002; Sheoran et al., 2010). In buckwheat,
for example, oxalic acid is specifically secreted in response to Al
stress, and non-toxic Al-oxalate complexes are accumulated in
the leaves, preventing the binding of Al to cellular components
(Ma et al., 1997). Similarly, in macro and microalgae, addition
of ligands in the culture medium to form complexes with heavy
metals promotes their uptake (Gledhill et al., 1997; Vasconcelos
and Leal, 2008; Aristilde et al., 2012). There is also some evidence
which shows that macroalgae release compounds with the capac-
ity to bind copper (Sueur et al., 1982; Vasconcelos et al., 2002)
in response to metal excess (Gledhill et al., 1999; Andrade et al.,
2010). However, little is known about the influence of copper on
the exudation process and the role of those exudates. Based on
the mechanisms employed by higher plants and microalgae, the
hypothesis of exudation of Cu ligands was considered a feasible
mechanism for copper tolerance of D. kunthii. Our results demon-
strated that copper excess triggers exudation of compounds by D.
kunthii from both NIS and IS. This direct effect of copper was simul-
taneously accompanied by an increased concentration of Cu ligands
in the medium, just as described for other macroalgae exposed to
copper excess (Gledhill et al., 1999; Andrade et al., 2010). Con-
ditional stability constants of ligands produced by D. kunthii (i.e.
8–8.4) are within the range of those reported for ligands released
by other seaweeds (i.e. 7–13; Gledhill et al., 1997). Thus, Cu binding
molecules produced by D. kunthii can be considered weak lig-
ands (log K = 7–11), similar to those produced by Fucus vesiculosus
(Gledhill et al., 1999) and Lessonia spicata (Andrade et al., 2010).
Those types of ligands are involved in the formation of a tran-
sient ternary complex with metal transporters at the cell surface
and have been suggested to play a key role in the bioavaibility
and uptake of metals in phytoplankton (Sunda and Huntsman,
1998; Aristilde et al., 2012). D. kunthii from IS exhibited higher Cu
uptake capacity than plants from NIS, which could be explained
by an increased density of Cu transporters on the cell surface, a
higher affinity for the metal or both, as an adaptation to a pro-
longed exposure to a copper enriched environment, as reported
for Zn transporters in phytoplankton (Sunda and Huntsman, 1992,
1998; Aristilde et al., 2012). Higher Cu uptake capacity of D. kunthii
from IS and copper induced exudation of Cu complexing ligands are
consistent with the hypothesized development of transient ternary
complexes with metal transporters to uptake copper, but the exact
role of those compounds in metal uptake in general, and Cu tol-
erance in particular, remain uncertain waiting for their chemical
characterization.
Despite differences in copper pollution history between Cach-
agua (NIS) and Punta Achurra (IS), results of copper exposure under
laboratory conditions of plants from the two sites failed to show
interpopulation differences. Occurrence of ecotypes with differ-
ent levels of copper tolerance have been described for some algae
(Russell and Morris, 1970; Hall, 1981) but most frequently consti-
tutive tolerance has been reported (Correa et al., 1996; Contreras
et al., 2005; Brown et al., 2012). Thus, current available infor-
mation on ecotypic differenciation after a persistant exposure to
copper enriched environments is not conclusive, suggesting that
ecotype development is a complex process and seems to vary from
species to species. In our case, presence of D. kunthii in IS seems
 C. Sordet et al. / Aquatic Toxicology 150 (2014) 220–228
more related to physiological plasticity giving the ability to tolerate
copper excess, via copper accumulation, activation of antioxidant
enzymatic responses and release of Cu binding compounds, than to
the occurrence of a copper tolerant ecotype.
5. Conclusion
Copper accumulation, the activation of an antioxidant system
and the release of Cu binding compounds in response to copper
excess, suggest that there is not a single process that can account
for copper tolerance in D. kunthii. Exposure to in vitro copper excess
revealed similar responses in both populations (NIS and IS) of the
alga, which helps to support the hypothesis that copper tolerance
in this species is a constitutive trait, and that evolution to a copper-
tolerant ecotype seems unlikely.
Acknowledgements
This work was supported by funding from research grants
FONDECYT 3110034 to CS, FONDAP 1501-0001 (CONICYT) to
the Center for Advanced Studies in Ecology & Biodiversity
(CASEB), program 7 to JC, FONDECYT 1120117 to LC, IDEALG
Grants ANR-10-BTBR-04-02 and 04-04 “Investissements d’avenir,
Biotechnologies-Bioressources” and the European Regional Devel-
opment Fund (ERDF)-FEDER 33765, Région Bretagne.
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