You are on page 1of 9

Environmental Pollution 199 (2015) 130e138

Contents lists available at ScienceDirect

Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

A novel eld transplantation technique reveals intra-specic metalinduced oxidative responses in strains of Ectocarpus siliculosus with
different pollution histories
ez a, b, c, Alberto Gonza
lez d, Rodrigo A. Contreras d, A. John Moody e,
Claudio A. Sa
d
Alejandra Moenne , Murray T. Brown a, *
a

School of Marine Science & Engineering, Faculty of Science and Environment, Plymouth University, Drake Circus, PL4 8AA, Plymouth, United Kingdom
Departamento de Medio Ambiente, Facultad de Ingeniera, Universidad de Playa Ancha, Casilla 34-V, Valparaso, Chile
~ a #450, Vin
~ a del Mar, Chile
Centro de Estudios Avanzados, Universidad de Playa Ancha, Traslavin
d
Departamento de Biologa, Facultad de Qumica y Biologa, Universidad de Santiago de Chile, Casilla 40 Correo 33, Santiago, Chile
e
School of Biological Sciences, Faculty of Science and Environment, Plymouth University, Drake Circus, PL4 8AA, Plymouth, United Kingdom
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 October 2014
Received in revised form
11 January 2015
Accepted 15 January 2015
Available online

A novel eld transplantation technique, in which seaweed material is incorporated into dialysis tubing,
was used to investigate intra-specic responses to metals in the model brown alga Ectocarpus siliculosus.
Metal accumulation in the two strains was similar, with higher concentrations in material deployed to
the metal-contaminated site (Ventanas, Chile) than the pristine site (Quintay, Chile). However, the
oxidative responses differed. At Ventanas, strain Es147 (from low-polluted site) underwent oxidative
damage whereas Es524 (from highly polluted site) was not affected. Concentrations of reduced ascorbate
(ASC) and reduced glutathione (GSH) were signicantly higher in Es524. Activities of the antioxidant
enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione
reductase (GR) all increased in Es524, whereas only SOD increased in Es147. For the rst time, employing
a eld transplantation technique, we provide unambiguous evidence of inter-population variation of
metal-tolerance in brown algae and establish that antioxidant defences are, in part, responsible.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Brown algae
Metal accumulation
Antioxidant metabolism
Active biomonitoring

1. Introduction
Beyond certain threshold concentrations, essential and nonessential metals can produce detrimental effects in marine algae,
including photo-inhibition and disruption of electron transport
chains in chloroplasts and mitochondria. As a consequence, there is
over-production of reactive oxygen species (ROS) resulting in
oxidative stress (Torres et al., 2008); for example, Cu excess disrupts electron transport chains in chloroplast and mitochondria
generating over-production of the highly oxidizing O
2 , the prelez et al., 2010). However,
cursor of H2O2, a less reactive ROS (Gonza
while exposure to excess metals can be harmful for many macroalgal species, some display inherent metal-tolerance (e.g. Brown
et al., 2012; Ratkevicius et al., 2003), while others have evolved
metal-tolerant ecotypes following long-term exposure to metal
pollution (Contreras et al., 2005; Ritter et al., 2010). For example,
* Corresponding author.
E-mail address: MTBrown@plymouth.ac.uk (M.T. Brown).
http://dx.doi.org/10.1016/j.envpol.2015.01.026
0269-7491/ 2015 Elsevier Ltd. All rights reserved.

Nielsen et al. (2003) observed that growth rates of embryos and


adults of Fucus serratus from a metal polluted location were
signicantly higher than those from a pristine location, when
exposed up to 2 mmol L1 Cu in vitro. Although the mechanisms of
tolerance have not been fully elucidated, there is evidence that the
foundation for inter- and intra-specic variation in tolerance is a
combination of efcient cell exclusion, synthesis of metal-chelating
compounds and activation of reactive oxygen metabolism
lez et al., 2010;
(Contreras et al., 2009; Gledhill et al., 1999; Gonza
Mellado et al., 2012; Pawlik-Skowronska et al., 2007).
Most of what is known about metal-mediated responses in
seaweeds has been derived from experimentation under controlled
conditions in the laboratory, but, despite these data being invaluable, they may not necessarily explain what is happening in the
more complex natural environment. Therefore, if results from eld
investigations reect those from laboratory studies greater
credence can be accorded to the latter. In estuaries and coastal
waters, seaweeds are the main primary producers at the base of
trophic networks and provide habitat for a large diversity of other

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

ez et al., 2012b). Because of their ecological


marine organisms (Sa
importance and metal accumulation capacities brown seaweeds of
the orders Fucales and Laminariales are the most widely used
macroalgae for metal bioaccumulation studies (Brown and
ez et al., 2012a). Typically, assessment of metal
Depledge, 1998; Sa
pollution using seaweeds has involved a passive bio-monitoring
approach, whereby resident species are sampled and analysed for
their metal content (e.g. Pawlik-Skowronska et al., 2007;
ez et al., 2012a). However, intrinsic and
Ratkevicius et al., 2003; Sa
extrinsic factors can inuence the accumulation of some metals and
population responses to metal exposure; the latter may result in
differential tolerance and potentially create uncertainties in the
interpretation of data leading to false environmental diagnoses
(Brown et al., 2012). As an alternative procedure, several authors
have suggested active bio-monitoring as a better option, whereby
individuals of species whose responses to metal-pollution are well
researched are cultured in the laboratory and then transplanted to
locations to be investigated (Brown et al., 2012; Chaphekar, 1991;
ez et al., 2012a). Advantages of this approach include inSa
dividuals from the same population, or ramets of the same individual, can be transplanted into different locations, similar age
individuals can be used, the time period of exposure is known and
monitoring can be carried out even if the species is absent from a
site. Of the active-bio-monitoring efforts so far reported most have
douin et al., 2008; Serisawa et al.,
involved fucoids and kelps (e.g. He
2002), but, their transplantation can be logistically complex and
time-consuming due to their large stature, morphological
complexity and relatively slow growth. Thus, the approach would
benet from improvements to the methodologies and using a
wider range of species that should include morphologically less
complex and faster growing species such as Ectocarpus siliculosus.
E. siliculosus is a small lamentous brown alga that inhabits the
low intertidal and shallow subtidal, growing free-oating or
attached to hard substrate or epiphytic on larger seaweeds
(Charrier et al., 2008). It has a global distribution in temperate
coastal environments, and has been accepted as the model organism for the Phaeophyceae (Charrier et al., 2008). Our recent results

131

from in vitro Cu exposure experiments, of up to 2.4 mmol L1 for


10 d, have established that strains with different pollution histories
display divergent responses that involve cellular exclusion of Cu,
production of metal chelators and antioxidant defences (Roncarati
et al., 2015; S
aez et al., 2015).
A eld transplantation technique has been developed in which
seaweed material is incorporated into dialysis tubing and then
deployed to eld stations with different levels of metal contamination, to investigate the responses of different strains of
E. siliculosus in situ. Metal accumulation, oxidative damage and
antioxidant responses were measured for this purpose. The aims of
our investigation were threefold: a) to evaluate the effectiveness of
the novel methodology and assess its suitability for incorporation
into bio-monitoring programmes, b) identify mechanistic differences in stress responses between two strains of E. siliculosus with
different pollution histories and c) to compare the results obtained
in this eld investigation with those from controlled laboratory
experimentation.

2. Materials and methods


2.1. Transplantation experiment sites
Two different locations in central Chile were chosen for the
transplantation experiments, one polluted and one pristine. Due to
the documented information of metal contamination in the localez et al., 2008; Sa
ez et al., 2012a, 2012b), the Bay of
tion (Gonza
Ventanas (32 440 36.5500 S and 71290 35.7000 W) was chosen as the
contaminated site (Fig. 1A); metal concentrations of up to
68 mg g1 have been recorded in subtidal sediments at this site
ez et al., 2012a). Sources of pollution come from Cu smelting and
(Sa
casting industries and thermoelectric complexes (Neaman et al.,
2009), and from wastewaters of unknown origin delivered via an
ez et al., 2012a, 2012b). The pristine
illegal sewage outfall (Sa
location used was Quintay (3311046.1600 S and 7142018.7300 W)
(Fig. 1A), a site with no history of metal pollution.

Fig. 1. (A) Map of the transplantation sites in central Chile, (B) diagram of the transplantation device, and (C) sample of the transplantation device in the eld.

132

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

To avoid proliferation of non-native E. siliculosus, the two strains


used were native to Chile: Es524 (Culture Collection of Algae and
Protozoa (CCAP) 1310/333), was isolated from Caleta Palito (Cha~ aral, Chile), a coastal site with a long history of high levels of Cu
n
pollution resulting from mining activities and Es147 (CCAP 1310/
340), isolated from Caleta Coloso (Antofagasta, Chile), a harbour
with a recent history of low metal inputs. The strains were cultured
in 10 L polycarbonate bottles with clean autoclaved seawater and
Provasoli nutrients (Provasoli and Carlucci, 1974), and cultured at
16  C, 14/10 of light/dark cycle, 45 mmol photons m2 sec1, with
aeration to avoid CO2 depletion.

200 mL was mixed with 200 mL of 0.5% thiobarbituric acid (TBA) in


10% TCA. Samples were heated at 95  C for 45 min in a water bath
and then cooled to room temperature. Two hundred mL of the
mixture were added to each well and the absorbance measured at
532 nm. Malondialdehyde (MDA; SigmaeAldrich) was used as a
standard.
Concentrations of the pigments chlorophyll a (Chla) and c (Chlc),
and fucoxanthin (fx) were measured using a modied version of
Seely et al. (1972). A biomass of 200 mg was added to a 1.5 mL tube
containing 800 mL of dimethyl sulfoxide (DMSO). After 5 min,
samples were centrifuged at 15,000 g for a few seconds to separate
biomass from supernatant. Supernatant was diluted in a DMSO:
water ratio of 4:1. Pigments were calculated using the equations:

2.3. Transplantation device and eld experiments

Chla A665 =72:5

E. siliculosus is a small lamentous seaweed that grows up to


30 cm (Charrier et al., 2008); the nature of its morphology and
stature would inevitably lead to biomass loss and biofouling (with
bacteria and microalgae) if transplanted without being enclosed.
Thus, 6 g FW thalli of E. siliculosus were enclosed in 76 mm at
width dialysis tubing (SigmaeAldrich, D9402), cellulose membranes permeable to only small molecules and ions, including
metals (De Philippis et al., 2003), and lled with autoclaved
seawater. Because of the composition of the tubing and likelihood
of it being grazed, it was placed inside transparent 2 L plastic
bottles with one hundred 1 mm holes to allow for water exchange
(see Fig. 1B). Devices were deployed to eld sites and placed 2 m
below the lowest tide mark by attaching them to a c. 5 kg rock with
nylon shing line (Fig. 1C). The transplanted material remained in
situ for 10 d after which the seaweeds were blotted dried, weighted
and immediately frozen in liquid nitrogen, and then stored
at 80  C to await analyses.

Chlc A631 A582  0:297A665 =61:8

2.2. Strains and culture

2.4. Metal accumulation


Frozen biomass was freeze-dried for 24 h (30e60 mg) and
digested with 2 mL of HNO3 in a MARS 6 microwave
(Supplementary Table 1). After digestion, the volume of each
sample was adjusted to 10 mL with milli-Q water (18 U). The total
concentrations of Cu, Cd, Al, Fe and Pb were determined using ICPMS (Thermo Scientic, X Series 2). The choice of metals analysed
was based on recently acquired data of high concentrations in
ez
sediments and the kelp Lessonia trabeculata from Ventanas (Sa
et al., 2012a, 2012b). To validate the data, the digestion protocol
was also applied to certied reference material (Ulva lactuca, BCR279) (Supplementary Table 2).
2.5. Measurement of oxidative stress parameters
The concentrations of H2O2 were measured according to Sergiev
et al. (1997) using a plate reader (VersaMax, Molecular Devices,
Sunnyvale, CA, USA). One hundred milligram of frozen biomass
were placed in a 1.5 mL tube to which 1 mL of 10% trichloroacetic
acid (TCA) was added. Glass beads (3 mm) were added to the tubes
and vortexed for 5 min. The homogenate was centrifuged (Sanyo
Hawk 15/05) for 10 min at 21,000 g and supernatant was then
transferred to a new tube. Fifty mL of supernatant were added to
each well, with 150 mL of 50 mM potassium phosphate buffer (pH
7.0) and 100 mL of 1 M KI then added. Absorbance was measured at
390 nm and commercial H2O2 (SigmaeAldrich, G5389) was used as
a standard.
Levels of lipid peroxidation (LPO) were measured according to
Heath and Packer (1968) in a plate reader. The biomass was
extracted as for H2O2 measurements. A supernatant volume of

fx A480  0:722A631 A582  0:297A665  0:049A665


 =130

2.6. Activities of antioxidant enzymes


Protein extracts for determining antioxidant enzyme activities
were prepared as described by Ratkevicius et al. (2003) and, according to that method, proteins were precipitated with ammonium sulphate. Total protein content was determined using bovine
serum albumin (BSA) as standard (Ratkevicius et al., 2003).
Superoxide dismutase (SOD) activity was determined in a plate
reader according to Mishra et al. (1993), with some modications.
Proteins (20 mg) were added to 290 mL of a mixture containing
100 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 11 mM
cytochrome-c, 11 mM xanthine, and 0.002 Units of xanthine oxidase.
Activity of SOD was calculated according to Kuthan et al. (1986).
Catalase (CAT) activity was determined according to Aebi (1984).
The activity was quantied by adding 15 mg of protein extracts to
1 mL of 100 mM potassium phosphate buffer (pH 7) and 16 mM
H2O2. The decrease in absorbance was followed at 240 nm for 30 s
and the activity was calculated using the extinction coefcient
43.1 M cm1.
Ascorbate peroxidase (APX) activity was measured according to
Nakano and Asada (1981). Proteins (15 mg) were added to 100 mM
potassium phosphate buffer (pH 7), containing 0.5 mM ASC and
16 mM H2O2 (Ratkevicius et al., 2003). The decrease in absorbance
at 290 nm was monitored for 30 s and the activity was calculated
with the extinction coefcient of ASC ( 2.8 mM cm1).
The activity of glutathione reductase (GR) was measured in a
plate reader according to Sen Gupta et al. (1993). Proteins (50 mg)
were added to 290 mL of a solution containing 100 mM potassium
phosphate buffer (pH 7), 0.5 mM oxidized glutathione and 0.15 mM
NADPH. The decrease in absorbance was followed at 340 nm for
5 min and the activity was quantied with the extinction coefcient of NADPH ( 6.22 mM cm1).
2.7. Concentrations of antioxidant compounds
The concentrations of reduced ascorbate (ASC) and dehydroascorbate (DHA) were measured according to Benzie and Strain
(1999), using a plate reader. A biomass of 300 mg FW was ground to
powder in a mortar with liquid nitrogen, and mixed with 1.2 mL
0.1 M HCl. Samples were centrifuged at 21,000 g for 10 min at 4  C.
To quantify ASC, 290 mL of tripyridyl triazine (Fe III TPTZ) were

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

133

Table 1
Concentration of metals in the strains of E. siliculosus Es524 and Es147 transplanted to Quintay (pristine site) and Ventanas (polluted site), both located in central Chile, for 10 d.
Concentrations of Cu, Fe, and Al are expressed in mmol g1 dry weight (DW) and Cd and Pb are in nmol g1 DW. Different letters represent signicant differences for each metal
measured (p < 0.05). Errors are 1 SD, n 3.
Strain
Es147
Es147
Es524
Es524

Location
Quintay
Ventanas
Quintay
Ventanas

Cu
0.74
8.1
1.12
9.3

Fe

0.11
0.4 c
0.11 a
0.8 c

0.78
4.4
1.05
5.8

Al

added to each well containing 10 mL of extracts, with the assumption that nothing but ascorbate would react. The absorbance at
593 nm was measured immediately after adding Fe III TPTZ. To
measure total ascorbate, 500 mL of extracts were incubated in the
presence of 5 mL of 100 mM dithiothreitol for 1 h. To stop the reaction, 5 mL (w/v) of N-ethylmaleimide were added. Total ascorbate
was measure as for ASC. Concentrations of DHA were calculated by
subtracting ASC levels from total ascorbate. L-ASC (SigmaeAldrich)
was used as standard.
Glutathione in its reduced (GSH) and oxidized (GSSG) forms was
measured with modications to Queval and Noctor (2007). The
biomass was extracted as for ascorbate, but in this case the supernatant was neutralized with 5 M K2CO3 to a nal pH 6e7. To
measure total GSH, 10 mL of supernatant were added to each well,
and 290 mL mixture containing 0.1 M NaH2PO4 (pH 7.5) and 6 mM
EDTA, 10 mL of 0.34 mM NADPH, 0.4 mM DTNB, and 1 unit of GR
were then added. The change in absorbance was monitored at
412 nm for 5 min, and concentrations were calculated using GSH
(SigmaeAldrich) as standard. To quantify GSSG, 250 mL of neutralized supernatant were incubated for 20 min with 5 mL of 4vinylpyridine and the mixture was centrifuged at 21,000 g for
5 min at 4  C. Levels of GSSG were measured as for total GSH, and
calculated using GSSG (SigmaeAldrich) as standard.
Concentrations of phenolic compounds were measured according to Van-Alstyne (1995). One hundred milligram were mixed
with 5 mL of 80% methanol in distilled water. Glass beads (c.10,
3 mm) were added and the mixture vortexed at 550 rpm for 24 h at
4  C and then centrifuged at 6000 g at 4  C for 10 min. Supernatant
of 12.5 mL was added to 500 mL of 17% Folin-Ciocalteu reagent. After
5 min incubation at room temperature, the solution was made
alkaline with 250 mL of 1 M Na2CO3. Samples were placed in a water
bath at 50  C and incubated for 30 min. The absorbance was
measured at 765 nm using phloroglucinol as standard.
2.8. Statistical analyses
The ShapiroeWilk and Bartlett Tests were performed to assess
requirements of normality and homogeneity of variances, respectively. To study differences in mean values, one-way ANOVA and
post-hoc Tukey test at 95% condence were performed. Statistical
indicators of signicant differences were added to gures and
tables.
3. Results and discussion
3.1. Metal accumulation
The concentrations of Cu, Fe, Al and Pb were signicantly higher
in E147 and Es524 transplanted to the polluted site of Ventanas
than to the pristine location of Quintay (Table 1). At Ventanas, while
the accumulation of Cu and Al was not signicantly different between strains, the accumulation of Fe and Pb was signicantly
higher in Es524 than Es147 (Table 1). Differences in the accumulation of metals between strains may reect cell exclusion capacity

0.09
0.4 c
0.11 a
0.8 b

0.4
8.4
0.7
9.6

Pb

0.05
0.5 c
0.07 a
0.5 c

6.1
52
10.1
78

Cd

0.6
3d
1.4
8c

14.8
6.5
16.6
8.3

2.1
1.0
2.7
1.0

a
b
a
b

and cell wall characteristics that can affect competition between


metals for binding sites; for example, differences in the conformation of alginic acid which is composed by mannuronic (M) and
guluronic acid (G). Changes in M/G ratios have been observed to
mediate the availability of binding sites and thus the total metal
binding capacity of cell walls (Sinnott, 2007). The concentrations of
Cu, Fe, Al and Pb accumulated in Es524 and Es147 at Ventanas were
signicantly lower than those reported from a passive biomonitoring study at the same location using Lessonia trabeculata,
results that are consistent with the shorter period of exposure to
the metals. The data conrms Ventanas as a metal-polluted
location.
In contrast to the above, concentrations of Cd were lower in
algae transplanted to Ventanas than to Quintay (Table 1) and are
ez
consistent with the results from the study on L. trabeculata (Sa
et al., 2012a). Quintay is located in close proximity to one of the
most important upwelling hotspots of the south-western Pacic
coast (Aiken et al., 2008) and it has been well documented that Cd
binds to different biomolecules, and following biological and
oceanographic processing is subsequently transported in nutrientez et al., 2012b).
rich deep waters to coastal areas (see Sa
3.2. Indicators of oxidative damage
The concentrations of H2O2 in Es147 were signicantly higher
than those in Es524 transplanted to the polluted site (Ventanas).
There were no signicant differences between Es524 and Es147
from the pristine site (Quintay) or between Es524 from Ventanas
and Quintay (Fig. 2A). Similarly, the highest levels of lipid peroxidation (LPO) were found in Es147 from Ventanas, while LPO in
Es524 from Ventanas and in both strains from Quintay did not differ
signicantly from one another (Fig. 2B). Thus, Es147 displayed an
oxidative stress condition in the highly polluted environment of
Ventanas whereas no stress response was observed in Es524, the
strain originating from a metal contaminated site. These ndings
are similar to those obtained in a laboratory study using
E. siliculosus strains Es524 and LIA (from a pristine site in N.W.
Scotland) exposed up to 2.4 mmol L1 Cu for 10 d; only LIA displayed
an oxidative stress condition as evidenced by increases in H2O2 and
LPO with increasing Cu exposure (S
aez et al., 2015).
Signicant decreases in total chlorophyll, Chla and Chlc were
found only in strain Es147 transplanted to Ventanas. These results
comply with those from the laboratory study on Cu excess
described above, with a decrease in chlorophyll concentrations
only observed in LIA originating from a pristine site (S
aez et al.,
2015). At elevated concentrations Cu can outcompete Fe for uptake, making Fe less available for the synthesis of chlorophyll and
thus reducing photosynthetic activity (Patsikka et al., 2002). In
addition, Cu can replace Mg in the chlorophyll molecule, reducing
photosynthetic performance (Kuepper et al., 2002). However, since
there were no differences in pigment concentrations of Es524
transplanted to Quintay and Ventanas, it is more likely that Es147
had a lower capacity to buffer ROS excess than Es524 and consequently experienced greater oxidative damage to chlorophyll

134

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

Fig. 2. H2O2 (A) and lipid peroxidation (B) levels in two strains of E. siliculosus transplanted for 10 d to the pristine site Quintay (white bars), and the metal-polluted location
~ aral,
Ventanas (black bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan
northern Chile. Different letters represent signicant difference at 95% condence interval (p < 0.05). Error bars are SD, n 3.

Fig. 3. Total chlorophyll (A), chlorophyll a (B), chlorophyll c (C), and fucoxanthin (D) concentrations in two strains of E. siliculosus transplanted for 10 d to the pristine site Quintay
(white bars), and the metal-polluted location Ventanas (black bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from
~ aral, northern Chile. Different letters represent signicant difference at 95% condence interval (p < 0.05). Error bars are SD, n 3.
a highly Cu-polluted site in Chan

molecules. This hypothesis is also supported by the LPO results that


provide evidence for oxidative damage only in Es147 transplanted
to Ventanas. As for fucoxanthin concentrations, there was a signicant increase in Es524 transplanted to Ventanas (Fig. 3D), a
result that is in agreement with the ndings from the in vitro Cu
ez et al., 2015). The carotenoid fucoxanexposure experiment (Sa
thin is the main accessory light-harvesting pigment in brown algae.
It is very efcient at transferring energy to Chla but may also have a
protective role under high irradiance (Evstigne and Paramono,
1974). A strong antioxidant capacity, better even than b-carotene,
has been recorded for this pigment (Masashi and Kazuo, 2007), but
there is no evidence for any antioxidant role in the metabolism of
brown algae. However, the results obtained in this study and from
our previous laboratory investigation suggest that fuxoxanthin
could be acting as an antioxidant in the chloroplast, providing

protection to chlorophyll from degradation.

3.3. Activities of antioxidant enzymes


The activity of SOD has been recognized as the rst line of
defence against ROS, catalysing the dismutation of O
2 into H2O2
and O2 (Ken et al., 2005). SOD activity was highest in Es147 from
Ventanas (Fig. 4A). Levels in Es524 were lower than in Es147 from
Ventanas, but signicantly higher than in both strains from Quintay. This information suggests that more SOD is needed in O
2
stressed Es147 from Ventanas, which would result in greater production of H2O2. CAT and APX are known to cooperate in detoxifying H2O2 in chloroxygenic organisms, such as brown algae (Collen
and Davison, 2001; Contreras et al., 2009) and vascular plants
(Asada, 1992; Mizuno et al., 1998), including under metal stress. For

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

135

Fig. 4. Activity of antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and glutathione reductase (GR) (D) in two strains of
E. siliculosus transplanted for 10 d to the pristine site Quintay (white bars), and the metal-polluted location Ventanas (black bars), in central Chile. Strain Es147 is from a low metal~ aral, northern Chile. Different letters represent signicant difference at 95%
polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan
condence interval (p < 0.05). Error bars are SD, n 3.

example, Contreras et al. (2009) found that APX and CAT cooperate
by increasing their activities in the brown seaweeds Lessonia
nigrescens and Scytosiphon lomentaria under Cu stress in vitro,
although the extent of the increase differed between the two species. Therefore, to counteract the effects of increased concentrations of H2O2 in Es147 from Ventanas, the activities of CAT and APX
might be expected to increase, but this was not observed (Fig. 4B,
C). In contrast, CAT and APX activities in Es524, although lower than
in Es147 from Ventanas, increased signicantly in the material
transplanted to Ventanas compared to Quintay, and were sufciently active to respond to any increases in H2O2 (Fig. 4B,C). Thus,
as well as inter-species variation in the activities of APX and CAT in
response to metal stress, our results show that activities can be
strain (population)-specic. In this particular case, the higher activities of APX and CAT in Es524 when exposed to elevated metal
concentrations imply a greater ability to respond to increased H2O2
production compared with Es147 (see Fig. 2A).
The enzyme GR is critical in the recycling of glutathione by
catalysing the reduction of GSSG to GSH (Noctor et al., 2012). In situ
studies on resident seaweed species, such as S. lomentaria
(Contreras et al., 2005), and the green seaweed Ulva compressa
(Ratkevicius et al., 2003), have usually found lower levels of GR
activity in individuals collected from metal contaminated locations
than from pristine sites, perhaps indicative of metal-mediated GR
cleavage (Schtzendbel and Polle, 2002). In contrast, we observed
higher levels of GR activity in Es524 from Ventanas than from
Quintay, whereas there were no signicant changes in Es147 between locations. Our data suggest that under metal-mediated
oxidative stress the higher GR activity in Es524 is important for
maintaining sufcient GSH, a powerful antioxidant, a metalchelator, and the precursor of the metal-chelating polypeptide,
phytochelatin (Noctor et al., 2012).

3.4. Concentrations of antioxidant compounds


The behaviour of ascorbate in Es524 and Es147 was similar at
the two test sites. There was an almost 20 fold increase in the total
ascorbate content of both strains transplanted to Ventanas
compared with Quintay (Fig. 5A), indicating that ascorbate was
synthesized in response to metal stress. ASC and DHA levels were
higher in both strains from Ventanas compared with Quintay, and
at Ventanas ASC levels were signicantly higher in Es524 than
Es147 (Fig. 5B, C). Despite the latter result, the marked increases in
DHA in the strains transplanted to Ventanas showed the clearest
pattern; DHA levels in both strains from Quintay were around 40%
of total ascorbate whereas at Ventanas the values were almost 90%.
ez
These results are in general agreement with those reported by Sa
et al. (2015); total ascorbate, ASC and DHA increased concomitantly
with increasing exposure of up to 2.4 mmol L1 Cu for 10 d in two
tolerant strains (Es524 and REP) isolated from polluted locations,
while in the less tolerant strain LIA (from a pristine site) ASC levels
were signicantly lower. Although the results obtained from the
eld and laboratory experiments are broadly similar, the extent of
the increase in DHA differed markedly. Under exposure to Cu in the
laboratory DHA concentrations in LIA (non-tolerant) accounted for
no more than about 60% of the total ascorbate pool and in the two
tolerant strains (Es524 and REP) levels did not exceed 50% of total
ez et al., 2015). These ndings imply that the
ascorbate (Sa
bioavailability of Cu and other metals to E. siliculosus at Ventanas is
greater than in our Cu-exposure laboratory experiments and is
having a distinct impact on ROS over-production which is apparently counteracted by ascorbate metabolism. Similar results, of
marked increases in DHA under metal excess, have been found for
L. nigrescens and S. lomentaria (Contreras et al., 2009, 2005) and U.
compressa (Mellado et al., 2012; Ratkevicius et al., 2003). Thus, at

136

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

Fig. 5. Total ascorbate (A), reduced ascorbate (ASC) (B), dehydroascorbate (DHA) (C), total glutathione (D), reduced glutathione (GSH) (E), oxidized glutathione (GSSG) (F), and total
phenolic compounds (G) concentrations in two strains of E. siliculosus transplanted for 10 d in to pristine site Quintay (white bars), and the metal-polluted location Ventanas (black
~ aral, northern Chile.
bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan
Different letters represent signicant difference at 95% condence interval (p < 0.05). Error bars are SD, n 3.

Ventanas the increased levels of ASC in the more tolerant strain


(Es524) are being subsequently oxidized to DHA in order to buffer
the metal-mediated ROS production. Synthesis of ASC could either
be via the L-galactose pathway, as previously reported by Mellado
et al. (2012) in U. compressa or, alternatively, through the HalliwelleAsada Cycle within which GSH is used as a substrate for DHA
reduction to ASC by the enzyme dehydroascorbate reductase
(DHAR) (Noctor and Foyer, 1998). Since GSH concentrations where
higher in Es524 than Es147 from Ventanas (see below), any increase
in DHAR activity (not measured) would also have contributed to the
higher ASC concentrations in Es524 than Es147 from this location.
Patterns in glutathione metabolism were similar to those
observed for ascorbate, although not as pronounced. Even though
there were higher concentrations of the total glutathione pool in
both strains from Ventanas than Quintay, the increase was signicantly higher in Es524 with about a 2-fold increase (see Fig. 5D).
Similar patterns can be seen for GSH and GSSG (Fig. 5E,F), but the
incremental change was greater for GSSG, suggesting an oxidative
response. At Ventanas, GSSG levels increased approximately 3-fold
in Es524 but only 2-fold in Es147, compared to Quintay. Similar
results have been reported for the brown seaweed S. lomentaria
(Contreras et al., 2005) and also for several vascular plants exposed
to various environmental stressors (Mhamdi et al., 2010; Noctor
et al., 2012). As GR catalyses the reduction of GSSG back to GSH,
the higher GSH and greater GR activity in Es524 than Es147 from
Ventanas can explain, at least in part, better glutathione recycling

in Es524. Furthermore, the increase in GSH in both strains transplanted to Ventanas may have been induced by GSH synthesis
through the pathway involving the activities of the enzymes gglutamylcysteine synthetase (g-GCS) and glutathione synthase
(GS), as observed in E. siliculosus strains Es524 and REP exposed up
to 2.4 mmol L1 Cu (Roncarati et al., 2015). The fact that in both
Es524 and Es147 transplanted to Ventanas, GSH levels increased,
albeit to a greater extent in the former strain, suggests that glutathione recycling is sufcient to maintain GSH at concentrations
capable of counteracting metal-mediated oxidative damage, to
some extent. Taking account of our ndings on the dynamics of
ascorbate and glutathione, together with the changes in the activities of antioxidant enzymes, the information strongly suggests that
the HalliwelleAsada (glutathione-ascorbate) cycle is one of the
main, if not the most important, antioxidant mechanisms in
E. siliculosus and brown seaweeds which allows this ecologically
important group macroalgae to thrive in metal contaminated
coastal environments.
In addition to their antioxidant properties phenolic compounds
have been recognized as effective extra- and intra-cellular metal
chelators and play an important role in metal-stress metabolism of
brown algae (Connan and Stengel, 2011). Concentrations of total
phenolic compounds were signicantly higher in both strains
transplanted to Ventanas than Quintay, with the highest concentrations in Es147 (Fig. 5G). These results are in agreement with the
ez et al. (2015), who found similar increases in total
ndings of Sa

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

phenolic compounds with Cu exposure in three E. siliculosus strains.


These results suggest that while phenolic compounds play a role in
reactive oxygen metabolism and/or as metal chelators in
E. siliculosus under metal exposure, their content is not directly
correlated with the pollution histories of the different strains.

3.5. Activation of the antioxidant metabolism and intra-specic


tolerance to Cu
Of course, other interacting chemical, physical and biological
stress factors (e.g. Heinrich et al., 2012; Rijstenbil et al., 2000), in
addition to metal exposure, can result in the over-production of
ROS and oxidative stress and they may have had some inuence on
our ndings. That notwithstanding, Ventanas and Quintay were
selected as suitable study sites because of their similarities in terms
of bathymetry, oceanography, geomorphology and marine comez et al., 2012a; S
munities (see Sa
aez et al., 2012b). Furthermore,
the correspondence between the results obtained from previous
ez et al., 2015),
laboratory investigations (Roncarati et al., 2015; Sa
and those reported here strongly support the view that the intraspecic differences in ROM and the enhanced antioxidant defences observed in strain Es524 are, indeed, in response to metal
stress.
Other mechanisms also contribute to metal-resistance in
E. siliculosus including exclusion from cells by their binding to cell
walls and intra-cellular sequestration by metal-chelating compounds such as phytochelatins, as found in our recent laboratory
ez et al., 2015). Thus it is likely that
studies (Roncarati et al., 2015; Sa
the tolerance of Es524 to metal pollution at Ventanas is achieved
through a combination of efcient cell exclusion, antioxidant defences and enhanced production of metal-chelators such as phytochelatins. Future eld experiments will address this hypothesis.

3.6. Conclusions
The deployment of E. siliculosus within dialysis tubing to eld
sites for 10 d proved to be very effective, with no loss of material
and very little biofouling of either the tubing or the protective
bottles. The results obtained from the study offer a reliable representation of the metal bioavailability at the two locations, being in
general agreement with the available published data that conrms
lez
Ventanas to be the more metal-contaminated location (Gonza
ez et al., 2012a, 2012b). These reasons, together
et al., 2008; Sa
with its simplicity and cost-effectiveness, make this novel device
and the approach highly suitable for incorporating into biomonitoring programmes for assessing chemical pollution in
coastal waters and estuaries. The methodology would lend itself to
other lamentous and frondose seaweed species and to the
microscopic stages (e.g. gametophytes, germlings) of morphologically more complex ones.
In this study we were able to establish that the responses
recorded in a serious of laboratory studies reect those occurring in
nature. The congruence between eld and laboratory results
strongly imply that the different responses of Es524 and Es147 are
mediated by metal stress and conrm Es524, isolated from a highly
metal-impacted location, as the more tolerant strain. Therefore, for
the rst time employing eld transplantation experiments, we
provide unambiguous evidence for inter-population variation in
metal-tolerance in brown seaweeds. In the case of E. siliculosus
strain Es524 the mechanisms include a strong antioxidant capacity
which is associated with the synthesis of the antioxidants GSH and
ASC, and increased activities of the antioxidant enzymes SOD, APX,
CAT, and GR.

137

Acknowledgements
This study was funded by the Santander Postgraduate Mobility
Support Scholarship 2011e12, awarded at Plymouth University to C.
ez. We thank nancial support for PhD studies to C. A. Sa
ez
A. Sa
from CONICYT Becas Chile Scholarship (72110557). We also
acknowledge Chita Guisado and Roberto Maltrain at Universidad de
Valparaso for providing laboratory facilities for culturing
E. siliculosus and the necessary materials for the transplantation
experiments.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2015.01.026.
References
Aebi, H., 1984. Catalase in vitro. Methods Enzym. 105, 121e130.
Aiken, C.M., Castillo, M.I., Navarrete, S.A., 2008. A simulation of the Chilean Coastal
Current and associated topographic upwelling near Valparaso, Chile. Cont.
Shelf Res. 28, 2371e2381.
Asada, K., 1992. Ascorbate peroxidase e a hydrogen peroxide-scavenging enzyme in
plants. Physiol. Plant. 85, 235e241.
Benzie, F.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological uids and modied version for
simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzym. 299, 15e23.
Brown, M.T., Depledge, M.H., 1998. Determinants of trace metal concentrations in
marine organisms. In: Bebianno, M.J., Langston, W.J. (Eds.), Metabolism of Trace
Metals in Aquatic Organisms. Chapman & Hall, London, pp. 185e217.
Brown, M.T., Newman, J.E., Han, T., 2012. Inter-population comparisons of copper
resistance and accumulation in the red seaweed, Gracilariopsis longissima.
Ecotoxicology 21, 591e600.
Chaphekar, S.B., 1991. An overview on bioindicators. J. Environ. Biol. 12, 163e168.
Charrier, B., Coelho, S.M., Le Bail, A., Tonon, T., Michel, G., Potin, P., Kloareg, B.,
Boyen, C., Peters, A.F., Cock, J.M., 2008. Development and physiology of the
brown alga Ectocarpus siliculosus: two centuries of research. New Phytol. 177,
319e332.
Collen, J., Davison, I.R., 2001. Seasonality and thermal acclimation of reactive oxygen
metabolism in Fucus vesiculosus (Phaeophyceae). J. Phycol. 37, 474e481.
Connan, S., Stengel, D.B., 2011. Impacts of ambient salinity and copper on brown
algae: 2. Interactive effects on phenolic pool and assessment of metal binding
capacity of phlorotannin. Aquat. Toxicol. 104, 1e13.
Contreras, L., Mella, D., Moenne, A., Correa, J.A., 2009. Differential responses to
copper-induced oxidative stress in the marine macroalgae Lessonia nigrescens
and Scytosiphon lomentaria (Phaeophyceae). Aquat. Toxicol. 94, 94e102.
Contreras, L., Moenne, A., Correa, J.A., 2005. Antioxidant responses in Scytosiphon
lomentaria (phaeophyceae) inhabiting copper-enriched coastal environments.
J. Phycol. 41, 1184e1195.
De Philippis, R., Paperi, R., Sili, C., Vincenzini, M., 2003. Assessment of the metal
removal capability of two capsulated cyanobacteria, Cyanospira capsulata and
Nostoc PCC7936. J. Appl. Phycol. 15, 155e160.
Evstigne, V.B., Paramono, L.I., 1974. Investigation of characteristics of fucoxanthin
with respect to elucidation of its role in photosynthesis e isolation, purication
and adsorption spectrum. Biokhimiya 39, 394e400.
Gledhill, M., Nimmo, M., Hill, S.J., Brown, M.T., 1999. The release of coppercomplexing ligands by the brown alga Fucus vesiculosus (Phaeophyceae) in
response to increasing total copper levels. J. Phycol. 35, 501e509.
lez, A., Vera, J., Castro, J., Dennett, G., Mellado, M., Morales, B., Correa, J.A.,
Gonza
Moenne, A., 2010. Co-occurring increases of calcium and organellar reactive
oxygen species determine differential activation of antioxidant and defense
enzymes in Ulva compressa (Chlorophyta) exposed to copper excess. Plant Cell
Environ. 33, 1627e1640.
lez, I., Muena, V., Cisternas, M., Neaman, A., 2008. Copper accumulation in a
Gonza
plant community affected by mining contamination in Puchuncavi valley,
central Chile. Rev. Chil. Hist. Nat. 81, 279e291.
Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I. Kinetics
and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophysics 125,
189e198.
douin, L., Bustamante, P., Fichez, R., Warnau, M., 2008. The tropical brown alga
He
Lobophora variegata as a bioindicator of mining contamination in the New
Caledonia lagoon: a eld transplantation study. Mar. Environ. Res. 66, 438e444.
Heinrich, S., Valentin, K., Frickenhaus, S., John, U., Wiencke, C., 2012. Transcriptomic
analysis of acclimation to temperature and light stress in Saccharina latissima
(Phaeophyceae). PLoS ONE 7.
Ken, C.F., Hsiung, T.M., Huang, Z.X., Juang, R.H., Lin, C.T., 2005. Characterization of
Fe/Mn e superoxide dismutase from diatom Thallassiosira weissogii: cloning,
expression, and property. J. Agric. Food Chem. 53, 1470e1474.

138

ez et al. / Environmental Pollution 199 (2015) 130e138


C.A. Sa

Kuepper, H., Setlik, I., Spiller, M., Kuepper, F.C., Prasil, O., 2002. Heavy metal-induced
inhibition of photosynthesis: targets of in vivo heavy metal chlorophyll formation. J. Phycol. 38, 429e441.
Kuthan, H., Haussmann, H.J., Werringloer, J., 1986. A spectrophotometric assay for
superoxide-dismutase activities in crude tissue fractions. Biochem. J. 237,
175e180.
Masashi, H., Kazuo, M., 2007. Benecial Health Effects of Seaweed Carotenoid,
Fucoxanthin, Marine Nutraceuticals and Functional Foods. CRC Press,
pp. 297e320.
lez, A., Dennett, G., Moenne, A., 2012. CopperMellado, M., Contreras, R.A., Gonza
induced synthesis of ascorbate, glutathione and phytochelatins in the marine
alga Ulva compressa (Chlorophyta). Plant Physiol. Biochem. 51, 102e108.
Mhamdi, A., Hager, J., Chaouch, S., Queval, G., Han, Y., Taconnat, L., Saindrenan, P.,
Gouia, H., Issakidis-Bourguet, E., Renou, J.-P., Noctor, G., 2010. Arabidopsis
glutathione reductase plays a crucial role in leaf responses to intracellular H2O2
and in ensuring appropriate gene expression through both salicylic acid and
jasmonic acid signaling pathways. Plant Physiol. 153, 1144e1160.
Mishra, N.P., Mishra, R.K., Singhal, G.S., 1993. Changes in the activities of antioxidant enzymes during exposure of intact what leaves to strong visible light
at different temperatures in the presence of protein synthesis inhibitors. Plant
Physiol. 102, 903e910.
Mizuno, M., Kamei, M., Tsuchida, H., 1998. Ascorbate peroxidase and catalase
cooperate for protection against hydrogen peroxide generated in potato tubers
during low-temperature storage. Biochem. Mol. Biol. Int. 44, 717e726.
Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867e880.
Neaman, A., Reyes, L., Trolard, F., Bourrie, G., Sauve, S., 2009. Copper mobility in
contaminated soils of the Puchuncavi valley, central Chile. Geoderma 150,
359e366.
Nielsen, H.D., Brownlee, C., Coelho, S.M., Brown, M.T., 2003. Inter-population differences in inherited copper tolerance involve photosynthetic adaptation and
exclusion mechanisms in Fucus serratus. New Phytol. 160, 157e165.
Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen
under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249e279.
Noctor, G., Mhamdi, A., Chaouch, S., Han, Y.I., Neukermans, J., Marquez-Garcia, B.,
Queval, G., Foyer, C.H., 2012. Glutathione in plants: an integrated overview.
Plant Cell Environ. 35, 454e484.
Patsikka, E., Kairavuo, M., Sersen, F., Aro, E.M., Tyystjarvi, E., 2002. Excess copper
predisposes photosystem II to photoinhibition in vivo by outcompeting iron
and causing decrease in leaf chlorophyll. Plant Physiol. 129, 1359e1367.
Pawlik-Skowronska, B., Pirszel, J., Brown, M.T., 2007. Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on
species and metal concentrations of the habitat. Aquat. Toxicol. 83, 190e199.
Provasoli, L., Carlucci, A.F., 1974. Vitamins and growth regulators. In: Stewart, W.D.P.
(Ed.), Algal Physiology and Biochemistry. Blackwell, Oxford, pp. 741e778.
Queval, G., Noctor, G., 2007. A plate reader method for the measurement of NAD,
NADP, glutathione, and ascorbate in tissue extracts: application to redox
proling during Arabidopsis rosette development. Anal. Biochem. 363, 58e69.

Ratkevicius, N., Correa, J.A., Moenne, A., 2003. Copper accumulation, synthesis of
ascorbate and activation of ascorbate peroxidase in Enteromorpha compressa (L.)
Grev. (Chlorophyta) from heavy metal-enriched environments in northern
Chile. Plant Cell Environ. 26, 1599e1608.
Rijstenbil, J.W., Coelho, S.M., Eijsackers, M., 2000. A method for the assessment of
light-induced oxidative stress in embryos of fucoid algae via confocal laserscan
microscopy. Mar. Biol. 137, 763e774.
Ritter, A., Ubertini, M., Romac, S., Gaillard, F., Delage, L., Mann, A., Cock, J.M.,
Tonon, T., Correa, J.A., Potin, P., 2010. Copper stress proteomics highlights local
adaptation of two strains of the model brown alga Ectocarpus siliculosus. Proteomics 10, 2074e2088.
ez, C.A., Greco, M., Gledhill, M., Bitonti, M.B., Brown, M.T., 2015.
Roncarati, F., Sa
Response differences between Ectocarpus siliculosus populations to copper
stress involve cellular exclusion and induction of the phytochelatin synthesis
pathway. Aquat. Toxicol. 159, 167e175.
ez, C.A., Lobos, M.G., Macaya, E., Oliva, D., Quiroz, W., Brown, M.T., 2012a. VariSa
ation in patterns of metal accumulation in thallus parts of Lessonia trabeculata
(Laminariales; Phaeophyceae): implications for biomonitoring. PLoS ONE 7,
e50170. http://dx.doi.org/10.1371/journal.pone.0050170.
ez, C.A., Pe
rez-Matus, A., Lobos, M.G., Oliva, D., Va
squez, J.A., Bravo, M., 2012b.
Sa
Environmental assessment in a shallow subtidal rocky habitat: approach
coupling chemical and ecological tools. Chem. Ecol. 28, 1e15.
ez, C.A., Roncarati, F., Moenne, A., Moody, J.A., Brown, M.T., 2015. Copper-induced
Sa
intra-specic oxidative damage and antioxidant responses in strains of the
brown alga Ectocarpus siliculosus with different pollution histories. Aquat.
Toxicol. 159, 81e89.
Schtzendbel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metalinduced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53,
1351e1365.
Seely, G.R., Duncan, M.J., Vidaver, W.E., 1972. Preparative and analytical extraction of
pigments from brown algae with dimethyl sulfoxide. Mar. Biol. 12, 184e188.
Sen Gupta, A., Webb, R.P., Holaday, A.S., Allen, R.D., 1993. Overexpression of superoxide dismutase protects plants from oxidative stress. Plant Physiol. 103,
1067e1073.
Sergiev, I., Alexieva, V., Karanov, E., 1997. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress
markers in plants. Proc. Bulg. Acad. Sci. 51, 121e124.
Serisawa, Y., Yokohama, Y., Aruga, Y., Tanaka, J., 2002. Growth of Ecklonia cava
(Laminariales, Phaeophyta) sporophytes transplanted to a locality with
different temperature conditions. Phycol. Res. 50, 201e207.
Sinnott, M., 2007. Carbohydrate Chemistry and Biochemistry: Structure and
Mechanism. RSC Publishing, Cambridge.
Torres, M.A., Barros, M.P., Campos, S.C.G., Pinto, E., Rajamani, S., Sayre, R.T.,
Colepicolo, P., 2008. Biochemical biomarkers in algae and marine pollution: a
review. Ecotoxicol. Environ. Saf. 71, 1e15.
Van-Alstyne, K.L., 1995. Comparision of 3 methods for quantifying brown algal
polyphenolic compounds. J. Chem. Ecol. 21, 45e58.