Assessment of the use of biomarkers in aquatic plants for theevaluation of environmental quality: application to seagrasses

L. Ferrat a,*, C. Pergent-Martini a, M. Romeo b

a EqEL, University of Corsica, BP 52, 20250 Corte, Franceb R.O.S.E., University of Nice Sophia Antipolis, UMR INRA UNSA 1112, Parc Valrose, BP 71, 06108 Nice, France

Received 17 April 2003; received in revised form 21 May 2003; accepted 7 June 2003

Abstract

The use of aquatic plants as bio-indicators constitutes an irreplaceable tool for investigation in ecological research,

applied to the conservation of littoral ecosystems. Today, studies in both the laboratory and the field have provided

encouraging insights into the capacity of aquatic plants to act as biomonitors of environmental quality, through the use

of biomarkers, and these are reviewed here. Photosynthetic activity, secondary metabolites, heat shock proteins,

enzymes of detoxication, and oxidative stress biomarkers were measured in the case of various stressors, (e.g. light,

thermal, hydric/haline stress, or herbicides, metals, organic contaminants). Most of them seem to be valuable and early

markers of the environmental conditions, as demonstrated by experimentations carried out on Posidonia oceanica .

Nevertheless, none can be in itself a valuable solution, and only a multiparametric approach, including both

‘physiological’ biomarkers, biomarkers of general stress and more specific biomarkers seems to be appreciable in an

ecotoxicological diagnostic.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Aquatic plants; Biomarkers; Oxidative stress; Detoxication; Glutathione S -transferase; Phenolic compounds

1. Introduction

In the early XXth century, researchers proposed

the use of living organisms in parallel with

physico-chemical analyses (that do not provide

either the threshold of sensitivity or the level of

response of organisms to contamination, Amiard

et al., 1998), to evaluate the state of a medium

(Blandin, 1986; Kolwitz and Marson, 1908; Kol-

witz and Marson, 1909 in Amiard et al., 1998).

These organisms are referred to as bio-indicator

species, that is to say ‘species or group of species,

whom, by their presence and/or their abundance,

are representative of one or more properties of the

ecosystem in which they occur’ (Guelorget and

Perthuisot, 1984).These species make it possible to determine,

with precision, the impact and the progression of

anthropic action on biocenosis vitality (Rainbow

and Phillips, 1993). The bio-indicator species must

be sedentary, of ecological importance, widespread

* Corresponding author. Tel.: �/33-495-45-0146; fax: �/33-

495-46-2441.

E-mail address: ferrat@univ-corse.fr (L. Ferrat).

Aquatic Toxicology 65 (2003) 187�/204

www.elsevier.com/locate/aquatox

0166-445X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0166-445X(03)00133-4

and widely studied and sensitive to environmentalvariations (Molfetas and Blandin, 1981).

The use of bio-indicators constitutes an irre-

placeable tool for investigation in ecological re-

search, applied to the conservation of littoral

ecosystems (Rainbow and Phillips, 1993).

Despite of the crucial role of plants (aquatic or

terrestrial) in ecosystems, these organisms have

been underemployed for the diagnosis or predic-tion of the negative consequences of human

activities, although physiological processes, bio-

chemical response and mechanisms of adaptation

or mortality can be used to evaluate the quality of

a medium (Vangronsveld et al., 1998). These

organisms are sedentary, sensitive to environmen-

tal variations and react, as first stages of the food

chain, more rapidly to the presence of pollutantsthan organisms living at higher stages (Lovett

Doust et al., 1994).

2. Aquatic plants as bio-indicators

Belsher (1979) defined the indicator function of

some groups of species, by their behavior under

the impact of pollution (e.g. eutrophication). Herecorded cases of regression (e.g. Phaeophyceae,

Cryptonemiales, Ceramiales and Gigartinales), or

even disappearance within the same group of

species (e.g. Bonnemaisoniales, Gelidiales, Rhody-

meniales), or on the contrary an increase (e.g.

Protoflorideae or Bangiophyceae). In a general

manner, Fucophyceae and Rhodophyceae have

tended to disappear under the impact of pollution(Bellan et al., 1995). Similarly, the massive devel-

opment of chlorophyceae (e.g. Ulva sp. and

Enteromorpha sp.), observed in littoral lagoons

or sheltered bays (e.g. Venice lagoon, Thessaloniki

gulf, California salt marsh, etc.) has led to these

organisms being considered as eutrophication bio-

indicators (Lazaridou et al., 1997; Sfriso and

Marcomini, 1997; Fong et al., 1998). Cystoseira

amentacea (Bory) and Cystoseira mediterranea

(Sauvag.) have also been used as negative sentinel

species for pollution (Bellan-Santini, 1966; Bellan,

1972).

Bianchi and Peirano (1995) demonstrated that

in Liguria, the ratio of magnoliophyta Cymodocea

nodosa (U.) Aschers./Posidonia oceanica (L.) De-lile was correlated with water quality. This ratio

varies from 0.3 in non-polluted waters to 0.8 in

industrialized or urbanized waters. Many authors

have noted a regression of P. oceanica meadows

according to the degree of anthropization. Thus,

given its wide range of distribution throughout the

Mediterranean, P. oceanica would appear to be a

potentially valuable bio-indicator of water quality.(Pergent, 1991) and has been used in bio-monitor-

ing programs for the marine environment (Bou-

douresque et al., 2000).

The use of macrophytes is notable in the bio-

monitoring of trace metal contamination (e.g.

Fucus vesiculosus (L.), Ascophyllum nodosum (L.)

Le Jol., Sargassum sp., Ulva lactuca , see synthesis

in Rainbow and Phillips, 1993; Phillips, 1994). Themechanisms of accumulation of these metals have

been studied under laboratory conditions (e.g.

Padina gymnospora (Kutzing) Vickers and Ulva

lactuca , Amado Filho et al., 1997), and in natural

conditions: e.g. Caulerpa taxifolia (Vahl) C.

Agardh (Gnassia-Barelli et al., 1995); Cystoseira

sp. (Catsiki and Bei, 1992); Fucus vesiculosus

(Ostapczuk et al., 1997); Padina pavonica (Cam-panella et al., 2001); Ulva lactuca (Catsiki and

Papathanassiou, 1993); Ulva rigida C. Agardh

(Favero et al., 1996).

Even if magnoliophyta are less widely used as

bio-indicators, particular attention must be paid to

them with regard to their capacity to accumulate a

wide range of pollutants such as organo-chlorine

compounds (Chabert et al., 1984), artificial radio-nuclids (Florou et al., 1985; Calmet et al., 1991)

and above all heavy metals (Malea and Haritoni-

dis, 1989a).

Several studies, generally in situ, have been

carried out concerning the use of a wide diversity

of magnoliophyta as bio-indicators of metallic

contamination (Table 1).

However, if these species integrate the quality oftheir environment, and constitute a good instru-

ment of investigation, they generally respond

belatedly, thus limiting their utilization for biomo-

nitoring short term (daily to monthly) environ-

mental variations (Rainbow and Phillips, 1993).

This has led researchers to focus on the use of early

symptoms, or biomarkers, defined as cellular,

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204188

molecular and biochemical changes induced by

chemical pollutants, measurable in biological sys-

tems such tissues, cells and biological fluids

(Lagadic et al., 1997; Mc Carthy and Shugart,

1990in Lagadic et al., 1998).

Biomarkers are widely different in their signifi-

cance and terminology (i.e. biomarkers of expo-

sure, of effects, of stress, of alteration. (de

Lafontaine et al., 2000), but in the context of this

paper, we will categorize biomarkers according to

two large types of responses: biomarkers of

exposure and biomarkers of stress.

Biomarker utilization in monitoring programs

for environmental quality is increasingly common

(see synthesis in Amiard et al. (1998)). In contrast

to the simple measurement of contaminants accu-

mulating in tissues, biomarkers can offer a more

complete an biologically more relevant informa-

tion on the potential impact of toxic pollutants on

the health of organisms (van der Oost et al., 1996).

They can provide information on the health of

organisms, and can be used as early warning

signals for general or particular stress (Vangrons-

veld et al., 1998). From the temporal point of view,

they can provide evidence of the dynamics of the

patterns of variation of bio-available pollutantquantities, for the original molecules and degrada-

tion products (Amiard et al., 1998).

Moreover, Lagadic et al. (1997) underlined the

interest in measuring several biomarkers at the

same time in the same organism, which allows a

pertinent approach to evaluate the effects of

pollutants on individuals.

3. Biomarkers in aquatic plants

Today, studies focus on the identification of

stress biomarkers. Aquatic plants, totally or par-

tially submerged, have been studied principally

from lagoon or estuarine ecosystems, under stress

Table 1

Marine plant (particularly magnoliophyta) species employed as bio-indicators of metallic contamination

Species Metals References

Amphibolis Antarctica (Labill.) Sonder and Aschers. ex

Aschers.

Cd, Cu, Fe, Mn, Pb, Zn Harris et al. (1979)

Cymodocea nodosa Cr, Cu, Ni; Al; Ca, Cd,

Cu, Fe, K, Mg, Na, Pb,

Zn

Catsiki and Panayotidis (1993), Malea (1993)

and Malea and Haritonidis (1995)

Cymodocea rotundata Ehrenb. and Hempr. ex Aschers C.

serrulata (R. Brown) Aschers. and Magnus Enhalus

acoroides (L. f) Royle Halodule uninervis (Fors.) Aschers.

H. pinifolia (Miki) den Hartog Halophila ovalis (R. Br.)

Hook. f

Cd, Cu, Pb, Zn Nienhuis (1986)

Halophila stipulacea (Fors.) Aschers. Al; Cd, Cu, Fe, K, Na,

Pb, Zn; Cd; Al

Malea and Haritonidis (1989b), Malea

(1994a,b) and Malea and Haritonidis (1996)

Heterozostera tasmanica (Martins ex Aschers.) den Hartog Cd, Cu, Fe, Mn, Pb,

Zn; Cd

Harris et al. (1979) and Fabris et al. (1982)

Posidonia australis Hook. f Cd, Cu, Mn, Ni, Pb, Zn Ward (1987)

P. oceanica Cd, Cr, Cu, Pb, Zn; Hg;

all

Campanella et al. (2001) and Capiomont et al.

(2000); see synthesis in Pergent-Martini and

Pergent (2000)

Syringodium isoetifolium (Aschers.) Dandy Thalassia hem-

prichii (Ehrenb.) Aschers. Thalassodendron ciliatum

(Forrsk.) den Hartog

Cd, Cu, Pb, Zn Nienhuis (1986)

Zostera marina (L.) Cd; Cd, Cu, Pb, Zn;

Cd, Cu, Cr, Pb, Zn

Faraday and Churchill (1979), Brix et al. (1983)

and Lyngby (1991)

Zostera muelleri Irmisch ex Aschers. Cd, Cu, Fe, Mn, Pb,

Zn; Cu

Harris et al. (1979) and Carter and Eriksen

(1992)

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 189

of various origin (e.g. light, thermal, hydric/halinestress, herbicides, metals, organic contaminants).

Research on biomarkers is frequently carried

out on the entire plant, or only on the leaves (for

magnoliophyta) and there is little information on

the stress response in the different parts of a plant.

However, these responses can be very different,

according to the degree of exposure and the

physiological role of the different parts of theplant (Pflugmacher et al., 1999a).

The principal biomarkers tested are ‘measurable

responses’ that occur in photosynthetic activity,

enzymatic processes of nutrition, secondary meta-

bolite synthesis, oxidative stress and/or detoxica-

tion mechanisms.

3.1. Modification of photosynthetic activity (Table

2)

The abundance and distribution of aquatic

plants is directly correlated with the quantity of

light available. Light is essential to the survival of

endogenous tissues, as they depend on the oxygen

supply from photosynthesis performed by epigen-

ous tissues. Chlorophyll fluorescence measurementis considered as a way to evaluate the biochemical

and physiological state of plants. It is a reliable

technique, easy to carry out, non-destructive and

rapid (Kramer et al., 1987, Walker, 1988 in

Vangronsveld et al., 1998).

Chlorophyll content and chlorophyll fluores-

cence are used to highlight stress due to a single

environmental factor or to a combination ofdifferent environmental factors (Table 2), but

they also constitute potential biomarkers of

anthropogenic stress (Table 2).

3.1.1. Chlorophyll fluorescence

The response of photosynthetic systems from

various plant species has been established for a

number of growth conditions and chemical pro-

ducts, and constitutes a reference base for the useof chlorophyll fluorescence to detect stress and its

effects (Vangronsveld et al., 1998). Chlorophyll a

fluorescence made it possible to detect, after 1 h

exposure (i) in Zostera marina stress due to an

infection by a pathogen (Ralph and Short, 2002),

(ii) in Posidonia australis and Amphibolis antartica

stress caused by a combination of heat anddesiccation (Seddon and Cheshire, 2001), (iii) in

Halophila ovalis a stress induced by herbicides

(Ralph, 2000) or by trace metals (Ralph and

Burchett, 1998). Herbicides (e.g. atrazine,

DCMU) act principally on their binding with the

quinone protein Qb (the second electron acceptor

in the PS II complex), inhibiting electron transport

(Ralph, 2000). Trace metals (e.g. Cu) may act onwater-splitting side of photosystem II and the

electron transport chain (Ralph and Burchett,

1998). Copper and zinc showed greater effects on

photosynthesis than lead and cadmium. The

response to stress was proportional to the metal

concentration and also a function of the time of

exposure. However, Halophila ovalis was highly

tolerant to metals, for the tested concentrations,thus limiting the interest of this species as a bio-

indicator (Ralph and Burchett, 1998).

3.1.2. Photosynthetic pigment concentration

The analysis of photosynthetic pigment concen-

tration generally confirms the results obtained by

chlorophyll fluorescence measurements. Trace me-

tals can substitute for the magnesium ion in the

chlorophyll molecule, leading to the inability tocatch photons and thus to a decrease in the

photosynthetic activity.

Generally, stressed plants increase their carote-

noid concentration to provide protection against

the formation of free radicals. A decrease in total

chlorophyll and in the ratio chlorophyll/carote-

noids are often observed. These variations in

photosynthetic pigments under exposure to tracemetals and herbicides have been observed for

various species (e.g. Halophila ovalis , Ralph and

Burchett, 1998; Ralph, 2000; Salvinia minima ,

planktonic diatoms, Table 2). For Salvinia minima

contamination by Cr reduces all photosynthetic

pigments, even carotenoids (Brent Nichols et al.,

2000). For planktonic diatoms Fargasova (1999)

showed a decrease of chlorophyll a , caused byoxidative stress due to Cu, and a decrease of

chlorophyll c due to Zn (Rijstenbil et al., 1994a).

In conclusion, trace metals may inhibit chlor-

ophyll pigment biosynthesis and enzymes involved

in this process. The same phenomena is observed

with exposure to high irradiance, where photo-

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204190

synthetic pigments (chlorophyll a�/b) decrease

(Yakovleva and Titlyanov, 2001), and this only

in oligotrophic conditions for P. oceanica (Alcov-erro et al., 2001).

3.2. Enzymatic activities related to nutrients

Plant nutrient metabolism can be influenced by

various stressors, so the activity of enzymes

involved in the assimilation of nitrogen (e.g.

glutamine synthetase (GS), nitrate reductase(NR)) and phosphate (alkaline phosphatase, an

enzyme hydrolyzing organic phosphate mono-

esters to inorganic phosphate) can be an interest-

ing biomarker for their capacity to highlight a

nutrient deficiency for plants under stressing

conditions.

In aquatic ecosystems, decreases in irradiance

are frequently observed, from minutes to months,

in estuaries and lagoons. These conditions cancause severe stress. In the magnoliophyta Thalas-

sia testudinum , Kraemer and Denis Hanisak

(2000) reported (i) a decrease of soluble carbohy-

drate content in the leaves with a decrease of light,

(in the same conditions, Peralta et al. (2002)

reported a mobilization of these carbohydrates in

the aboveground parts of Zostera noltii but Ruiz

and Romero (2001) showed a depression in therhizomes of P. oceanica ), (ii) an accumulation of

nitrogenous and phosphorus nutrients during light

exposure and a slow decrease during dark phase,

and (iii) a decrease of GS activity in dark phase,

and a rapid increase when returned to light.

Physiological variations can be measured 24 h

after a variation in light intensity.

Table 2

Changes in photosynthetic activity as biomarkers of stress in aquatic plants

Biomarkers Stress Species Kind of study References

Decrease of chlorophyll fluor-

escence

Infection by L. zosterae Zostera marina In vitro Ralph and Short

(2002)

T8�/desiccation Posidonia australis , Amphi-

bolis antartica

In vitro Seddon and Che-

shire (2001)

Light (P/o/) T8, Cu, triazine Cladophora sp. In situ: Gulf of

Saronikos (Gr)

Hader et al.

(1997)

Different species of Chloro-

phyta, Phaeophyta, Rhodo-

phyta

In situ: Cabo de

Gata (Sp.)

Hader et al.

(1998)

Photosynthetic pigments: de-

crease in ratio chlorophyll/car-

otenoids

Cd, Cu, Zn, Pb, Fe Scenedesmus quadricauda

(Turp.) Breb

In vitro Fargasova (1999)

Ditylum brightwellii (T.

West) Grunow in van

Heurck

Rijstenbil et al.

(1994b)

Phoyosynthetic pigments�/

Chlorophyll fluorescence

Cd, Cu, Pb, Zn Halophila ovalis Ralph and

Burchett (1998)

Salvinia minima (Baker) Brent Nichols et

al. (2000)

Diuron [3-(3?,4?-dichlorophe-

nyl)-1,1-dimethylurea; triazine

Halophila ovalis In vitro Ralph (2000)

Light Halophila ovalis In vitro Ralph and

Burchett (1995)

Light Chondrus crispus Stackh In vitro Yakovleva and

Titlyanov (2001)

Light, T8, salinity Halophila ovalis In vitro Ralph (1999)

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 191

In the case of high irradiance stress, photo-inhibition was demonstrated by a decrease of GS

and NR activity in the freshwater Spirodela

polyrhiza (Schwalbe et al., 1999).

Ralph (1999), demonstrated in Halophila ovalis ,

that the combination of various forms of stress

amplified the sensitivity of the plant to each of

these individual factors.

Alkaline phosphatase activity (APA) was stu-died in P. oceanica and Cymodocea nodosa , under

experimental and natural conditions, for nutritive

stress due to phosphorus deficiency (Perez and

Romero, 1993; Invers et al., 1995). Perez and

Romero (1993) showed that APA is correlated to

phosphorus concentrations in the tissues, and/or

the bio-availability of this element in the medium.

An increase in APA was noted when the plant isgrowing in phosphorus limited medium. Values

were higher in the older parts of the plant, and low

but detectable in the roots (Perez and Romero,

1993). So APA could be a good indicator of

phosphorous deficiency in aquatic plants and an

indicator of the nutritional status in magnolio-

phyta (Hernandez et al., 1999).

3.3. Heat shock proteins

All organisms respond to stress at the cellular

level with the rapid synthesis of a small number of

so-called stress proteins and a simultaneous in-

hibition of normal protein synthesis (Lewis et al.,

2001). The protein synthesized were designated

heat shock proteins (HSPs). The cellular stress

response is induced by a range of stressors includ-ing pollutants (Mc Carthy and Shugart, 1990), and

as primary protective response of cells, is poten-

tially useful in environmental monitoring.

Although some stress proteins are only found in

cells responding to stressors, most are also present

at lower concentrations under normal conditions

(i.e. they are constitutively expressed) and play

essential roles in cellular protein homeostasis byacting as molecular chaperones. This protects cells

allowing them to recover and survive the stress

(Lewis et al., 2001).

Among stress proteins, HSP70 is a popular

choice for biomarker research as this is the most

highly conserved and widely studied of the HSPs

(Ryan and Hightower, 1996). These HSP70 havebeen evaluated as biomarkers of pollution, with

research almost exclusively confined to animals

(see synthesis in Lewis et al., 1998). Studies have

been carried out in Enteromorpha intestinalis

(Lewis et al., 1998, 2001) under exposure to a

variety of stressors. The HSP70 response was

affected by nutrient limiting conditions (reducing

in fact protein metabolism), and copper inducedan increase in HSP70 only under nutrient replete

conditions. Herbicides failed to induce an increase

of HSP70, suggesting that triazines may be weakly

proteotoxic. Lewis et al. (2001) showed that

HSP70 is only induced by stressors that are

strongly proteotoxic. The authors concluded that

HSP70 in E. intestinalis was not useful as a

biomarker for copper and triazine.

3.4. Phenolic compounds

Among secondary metabolites, phenolic com-

pounds have been widely studied (Table 3). These

compounds are of major importance in terms of

protection in plant species despite the fact that

they do not represent a primary function in

physiological processes (Levin, 1971). They corre-spond to a wide range of chemical structures with

at least an aromatic cycle (C6) carrying one or

more hydroxyl groups (Waterman and Mole,

1994).

A large number of these compounds have been

identified and most of them are characteristic of

only one or few species (Ernst and Peterson, 1994;

Waterman and Mole, 1994), and are widelydistributed in terrestrial higher plants, (Bate-

Smith, 1968; Karolewski and Giertych, 1995;

Giertych and Karolewski, 2000), and in aquatic

species (Mc Clure, 1970; Zapata and Mc Millan,

1979; Quakenbush et al., 1986; Pip, 1992). Their

qualitative and quantitative distribution varies

among tissues and is influenced by growth condi-

tions and the physiological state of the plants(Levin, 1971; Macheix, 1996).

Phenolics play an important ecological role and

their synthesis and storage are considered to be

good indicators of stress such as grazing (Ander-

son and Velimirov, 1982; Steinberg, 1984, 1985;

Ragan and Glombitza, 1986), infection by micro-

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204192

organisms (Ragan and Glombitza, 1986; Vergeer

et al., 1995; Vergeer and Develi, 1997) and inter-

and intra-specific competition (Mc Lachlan and

Craigie, 1966). Most of these compounds make a

significant contribution to the antioxidant activity

of plants and have the capacity to bind heavy

metals (Emmons et al., 1999) and are thus of

major importance in the mechanisms of protectionof plants against stress (Swain, 1977).

In Zostera marina , infected by Labyrinthula

zosterae , the concentration of phenolic com-

pounds has been studied (Vergeer and Develi,

1997). The infection caused an increase of a

phenolic compound, the caffeic acid, blocking

the growth of L. zosterae . Under normal growth

conditions, Zostera marina can protect itself,increasing its production of caffeic acid, but this

energetic investment on behalf of defense against

microorganisms did not occur when the plant is

under limited growth conditions.

The hypothesis that phenolic compounds can

act as repellents against herbivores was suggested

at the beginning of the last century (Hunger, 1902

in Schoenwaelder and Wiencke, 2000). It was first

confirmed by various studies (see synthesis in

Schoenwaelder and Wiencke, 2000). Among

them, Steinberg (1984) showed a higher rate of

production of phenolic compounds in the repro-

ductive organs of marine species directly exposed

to grazers. There was a negative correlation

between the total phenolic concentration of planttissues and their palatability for grazers (Anderson

and Velimirov, 1982; Steinberg, 1984), but some

studies have since demonstrated that this correla-

tion is not systematic (Steinberg and Paul, 1990;

Steinberg et al., 1991; Steinberg and Van Altena,

1992).

In addition to these phenolic compounds,

Smolders et al. (2000) measured lower tannin(complex phenolic compounds) concentrations in

submerged than in emerged and floating plants.

They explained these difference by greater expo-

sure to pathogens or to grazers that led these

plants to develop a strong chemical resistance and

so an important energetic investment. Pavia and

Brock (2000) supported in Ascophyllum nodosum

Table 3

Studies realized on phenolic compounds as biomarkers of stress in aquatic plants

Stress Species Kind of study References

Infection by L. zosterae ,

Porter and Muehlstein

Zostera marina In situ: Roscoff, (Fce) Vergeer et al. (1995) and

Vergeer and Develi (1997)

Interspecific competition P. oceanica In situ: Western

Mediterranean (Fr)

Cuny et al. (1995), Agostini

et al. (1998) and Ferrat

(2001)

Hg P. oceanica In situ: Livorno

Mediterranean (It)

Agostini et al. (1998) and

Ferrat (2001)

Cu Ascophyllum nodosum In vitro Toth and Pavia (2000)

Grazing/phenolic com-

pounds concentration

Different species of Chlorophyta, Phaeophyta, Rho-

dophyta

In vitro Anderson and Velimirov

(1982)

Alaria marginata Postels and Ruprecht Steinberg (1984)

Fucus vesiculosus (L.), Halidrys siliquosa (L.), Lyng-

bye Eisenia arborea Areschoug.

Steinberg (1985)

Dictyota spiralis (Montagne), Dictyopteris australis

(Sonder) Askensay, Lobophora variegata , (Lamour-

oux) Womersley Cystoseira trinodis , (P. Forsskal) C.

Agardh Sargassum sp.

Steinberg and Paul (1990)

Different species of Chlorophyta, Phaeophyta, Rho-

dophyta

Steinberg (1985) and

Steinberg and Van Altena

(1992)

Sargassum sp. Steinberg et al. (1991)

Grazing, desiccation,

UVB

Ascophyllum nodosum In vitro Pavia and Brock (2000)

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 193

an induced defense model for the production ofphlorotannins, enhanced by exposure to grazing,

UVB and desiccation.

P. oceanica apparently does not increase phe-

nolics in response to competition from invasive C.

taxifolia . Neither Cuny et al. (1995) nor Agostini

et al. (1998) showed any significant difference for

water-soluble phenolic compounds in a site char-

acterized by strong competition, compared with asite without C. taxifolia . Ferrat (2001) confirmed

that potential stress, generated by competition

with C. taxifolia , had a limited impact on the

production of water-soluble phenolic compounds.

However, an increase in tannin cells in blades from

colonized sites justified reconsideration of the role

of polymerized tannins in the mechanisms of

response to competition.With respect to heavy metals, no modification in

the levels of simple phenolic compounds was

reported in P. oceanica contaminated by mercury

(Agostini et al., 1998), nor in levels of polyphe-

nolics in Ascophyllum nodosum contaminated by

copper (Toth and Pavia, 2000). Two tendencies

were apparent in P. oceanica (Ferrat, 2001). A

decrease of total (simple and complex) or simplephenolic compounds was noted in plants coming

from a contaminated site, in particular in the non-

chlorophyllous basal parts of the leaves (sheaths).

This suggests that complex phenolic compounds

such as flavonoids, act as chelators and antiox-

idants, notably in the sheaths of the plant.

Hydroxycinnamic acid derivates (caffeic acid,

ferulic and p -coumaric acids) seemed to be in-volved to a lesser extent, because of their reduced

antioxidant power compared to the flavonoids.

Beyond their low antioxidant power, these com-

pounds often appeared in the glycosilated form,

further limiting their antioxidant activity (Rice-

Evans et al., 1999; Kahkonen et al., 1999; Ferrat,

2001).

3.5. Oxidative stress biomarkers (Table 4)

Oxidative stress is first characterized by an

oxidative ‘burst’, or a rapid and transient produc-

tion of high quantities of reactive oxygen species

(ROS: e.g. singlet oxygen, superoxide anion,

hydrogen peroxide, hydroxyl and hydroperoxy-

radicals). The production of ROS is a naturalphenomenon (Cossu et al., 1997), triggered by

various external factors (Rijstenbil et al., 1994a),

and generally reduced in plants under normal

conditions of growth (Vangronsveld et al., 1997).

The ‘burst’ can be stimulated directly by various

pollutants (Stegeman et al., 1992; Table 4) or

indirectly by their metabolization (Cossu et al.,

1997). Organic compounds and transition metals(e.g. Cu) were shown to be pro-oxidants and to

accelerate the formation of oxy-radicals in plants

(Salin, 1988), and their excess increased lipid

peroxidation (loss of membrane integrity; Rijsten-

bil et al., 1994a). The oxidative ‘burst’ constituted

an early strategy of defense in plants (Wojtaszek,

1997), in response to various forms of stress

(Kupper et al., 2001).The presence of ROS triggered secondary reac-

tions of defense that prevent the destructive

oxidation of important metabolites (Subhadra et

al., 1991). This antioxidant system was based on

enzymatic (e.g. superoxide dismutase (SOD), per-

oxidase, catalase) and non-enzymatic mechanisms

(e.g. thiol groups, flavones, phytoalexines, hydro-

quinones, vitamins; Cossu et al., 1997; Vangrons-veld et al., 1997). The overproduction of ROS

caused lipid peroxidation only when antioxidant

defenses overlapped (Cossu et al., 1997).

3.5.1. Oxidative burst

The phenomenon of oxidative burst has been

widely studied in the case of the invasion of

terrestrial plants by pathogens, but few studieshave been carried out on the mechanisms of

defense against pathogens in the marine environ-

ment (Potin et al., 1999). The first oxidative burst

in marine species was revealed only in 1994 (Colen

and Pedersen, 1994) in response to mechanical

stress on Eucheuma platycladum . Studies have

been carried out since in the case of an invasion

by a pathogen in Chondrus crispus (Bouarab et al.,1999) and in Gracilaria conferta (Weinberger et al.,

1999).

3.5.2. Lipid peroxidation

Lipid peroxidation leads to the formation of

degradation products such as alkanes and alde-

hydes (e.g. malone dialdehyde). Thiobarbituric

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204194

acid reactive substances (TBARS) concentration is

an interesting biomarker of metal-induced oxida-tive stress. Hamoutene et al. (1995, 1996) and

Vavilin et al. (1998) noted a significant decrease of

TBARS after exposure to trace metals in marine

and freshwater species, respectively. TBARS in-

creased in the foliar sheaths of P. oceanica after 48

h exposure to mercury chloride (HgCl2) at 0.01

and 0.1 mgHg l�1 as compared to the controls

(Ferrat, 2001; Ferrat et al., 2002a). Paradoxically,at higher concentrations of mercury in the medium

(1 mgHg l�1), a significant decrease in TBARS was

noted in contaminated sheaths, compared to the

controls. Non-enzymatic antioxidant molecules

were suggested in counteraction to the oxidative

stress generated by mercury.

3.5.3. Antioxidant enzymes

Catalase accelerates the spontaneous dismuta-tion reaction of hydrogen peroxide. An increase in

its activity has been demonstrated in the presence

of various pollutants (Table 4): e.g. peroxidase,

glutathione reductase and SOD after 24 h expo-

sure to organic pollutants significantly increased in

Lemna minor (Roy et al., 1995).

In Ceratophyllum demersum metal-induced oxi-

dative stress increased antioxydant enzymes suchas peroxidases, catalase and SOD (Rama Devi and

Prasad, 1998). However, in Lemna minor , beyond

a threshold of toxicity, these enzymatic activities

decreased with increasing metal concentrations

(Subhadra et al., 1991). Catalase activity increased

significantly in P. oceanica following 48 h expo-

Table 4

Changes in oxidative stress molecules as biomarkers of stress in aquatic plants (in vitro experiments)

Biomarkers Kind of stress Species References

Generation of oxidative burst

(ROS)

Mechanical stress Eucheuma platycladum (Schmitz) Colen and Pedersen (1994)

Infection by a mi-

croorganism

Chondrus crispus Bouarab et al. (1999)

Laminaria digitata (Hudson) Lamouroux Kupper et al. (2001)

Generation of lipid peroxida-

tion (TBARS)

Infection by a mi-

croorganism

Gracilaria conferta (Schousboe ex Mon-

tagne)

Weinberger et al. (1999) and

Ferrat (2001)

Cu, Zn, Cd P. oceanica Hamoutene et al. (1996)

Chlorella pyrenoidosa Kessler and Huss Vavilin et al. (1998)

Induction of catalase, perox-

idase, SOD; GR activities

HCB Lemna minor (L.) Roy et al. (1995)

Cu, HgCl2,

CH3HgCl

Subhadra et al. (1991)

Ceratophyllum demersum (L.) Rama Devi and Prasad (1998)

HgCl2 P. oceanica Ferrat (2001)

Salinity Hydrilla verticillata (L.f.) Royle, Najas sp. Rout and Shaw (2001)

Decrease in thiol pool (GSH) Cu, Zn, Cd Chlorella pyrenoidosa Vavilin et al. (1998)

Enteromorpha sp. Rijstenbil et al. (1998)

Ceratophyllum demersum Rama Devi and Prasad (1998)

Ditylum brightwellii (T. West) Grunow in

van Heurck, Thalassiosira pseudonana

Rijstenbil et al. (1994a,b)

P. oceanica Hamoutene et al. (1996), Ferrat

(2001) and Ferrat et al. (2003)

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 195

sure to low concentrations of HgCl2 (0.01 mg l�1

and 0.1 mg l�1) as compared to controls (Ferrat,

2001; Ferrat et al., 2002b). In contrast, there was

no significant difference between 1 mg l�1 and

controls, and lipid peroxidation level was low. This

result indicates that other systems of protection

may be involved against oxidative stress (non-

enzymatic induction of phytochelatins).

When freshwater species are grown at high saltconcentrations (Table 4), oxidative stress appar-

ently induces catalase, SOD and peroxidase (Rout

and Shaw, 2001).

3.5.4. Antioxidant molecules

Concerning non-enzymatic mechanisms, re-

search on biomarkers has focused on various

compounds usually involved as intracellular bin-

ders for metals, or involved in their exclusion andtheir detoxication (e.g. vitamin E, thiol groups,

particularly reduced glutathione (GSH) and phy-

tochelatins). GSH and other non-protein-thiol

groups (according to the determination method,

authors consider that the analyzed molecules are

glutathione or non-protein thiol compounds).

Protect against oxy-radicals and pro-oxidant me-

tals (Cu, Fe). When these molecules are not insufficient quantities, antioxidant enzymes (e.g.

SOD) can participate in the elimination of oxy-

radicals (Rijstenbil et al., 1994b).

Glutathione (g L-glutamyl-L-cysteinyl-glycine) is

involved as a co-substrate in conjugation reactions

(with glutathione S -transferase: GST, see below),

and is an important antioxidant in plants (Rij-

stenbil et al., 1994a,b; Hamoutene et al., 1995). Inthe same manner, homoglutathione (g L-glutamyl-

L-cysteinyl-b-alanine), an alanine homologue of

glutathione, can entirely or partially replace glu-

tathione as antioxidant.

Xenobiotics can modify quantities of reduced

(GSH) or oxidized glutathione, indeed, in Cerato-

phyllum demersum , the introduction of metal into

the medium caused a decrease in GSH levelsproportional to its concentration (Rama Devi

and Prasad, 1998).

The comparison of non-protein thiol compound

levels in sites contaminated by Hg, with those

coming from pristine sites in the Mediterranean

showed a significant decrease in reduced glu-

tathione in the foliar tissues (sheaths�/blades) ofP. oceanica from the contaminated sites (Ferrat,

2001; Ferrat et al., 2003). These results are in

agreement with the fact that GSH is involved in

the binding of metals, single or polymerized into

phytochelatins (Gekeler et al., 1989). Moreover,

non-protein thiol levels were higher in blades than

in sheaths (Ferrat, 2001; Ferrat et al., 2003).

These processes appeared to be influenced byenvironmental factors, therefore GSH concentra-

tions cannot be considered as specific indicators of

oxidative stress due to a given pollutant (Rijstenbil

et al., 1998).

3.6. Biomarkers of detoxication (Table 5)

These biomarkers are different according to the

toxic compounds that they can detoxify. Phyto-

chelatins are reported to detoxify heavy metals

whereas phase I and phase II enzymes of bio-

transformation metabolize also organic xenobio-tics.

3.6.1. Phytochelatins

The glutathione molecule above-mentioned isthe precursor of phytochelatins or homophytoche-

latins (metalloisopeptides, (g-Glu-Cys)n-Gly with

n�/2�/8), peptides sequestrating trace metals (Leo-

pold et al., 1999; Schmoger et al., 2000). The

formation of these molecules is achieved by an

enzymatic (g-glutamyl-cysteine synthetase) poly-

merization of glutathione or homoglutathione

(Gekeler et al., 1989). These mechanisms havebeen demonstrated in terrestrial plants (Gekeler et

al., 1989). Moreover, the increase of phytochelatin

levels in macro- and micro-algae is a specific

biomarker of heavy metal bioavailability and

stress caused by these metals. Indeed, Skowronski

et al. (1998) demonstrated phytochelatin synthesis

in response to cadmium uptake in Vaucheria

(xanthophyceae). Concerning micro-algae, experi-mental exposure to trace metals induced acute

stress and a synthesis of phytochelatins in plank-

tonic diatoms (Rijstenbil et al., 1994b), and

Pawlik-Skowronska (2002) demonstrated, in 24

h, the production of Pb-induced phytochelatins in

cells of Stichococcus bacillaris .

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204196

3.6.2. Biotransformation enzymes

The metabolism of xenobiotics proceeds in

plants in three phases (Pflugmacher et al.,

1999b): (i) a phase of functionalization consists

of oxidation of the xenobiotic mostly by the

system of cytochrome P450 monooxygenases, (ii)

a second phase of conjugation facilitates the

binding of the chemical pollutant to an endogen-

ous substrate (glucuronic acid, glutathione, sul-fates). The enzymes involved are generally

transferases (e.g. uridinediphospho-glucuronosyl

transferases, glutathione S -transferases or GSTs,

sulfotransferases). An increase in the enzymatic

activity during the first and second phase indicates

exposure to xenobiotics (Roy et al., 1995), and (iii)

the third phase consists of partitioning and storage

processes in cellular walls or vacuoles.These detoxication mechanisms have been

widely studied in agronomically important terres-

trial plants and in particular in terms of resistance

to herbicides, but there is little information on the

activity of these enzymes in aquatic macrophytes

(Pflugmacher et al., 1999b).

Some studies have been carried out on herbicide

(Tang et al., 1998) and hydrocarbon (Schrenk etal., 1998; Table 5) detoxification in freshwater

species, and on the action of trace metals on

marine species (Hamoutene et al., 1995; Ranvier et

al., 2000; Ferrat, 2001; Ferrat et al., 2002a,c).

3.6.2.1. Phase I enzymes. The system of P450

cytochrome takes an important part in phase I

enzymes. It is responsible for the oxidative meta-

bolism of a wide variety of compounds, includingxenobiotics as well as endogenous compounds (e.g.

fatty acids). These hemoproteins are in fact only

one of the various elements of the electron

transport multienzymatic complex.

The ethoxyresorufin O -deethylase (EROD) ac-

tivity seems to be the most sensitive catalytic

activity that can be used to measure an induction

of cytochrome P450 (Hamoutene et al., 1996). InP. oceanica , these authors observed an inhibition

of cytochrome P450 dependent enzymes in the case

of a metallic contamination, but noted a high

variability of the EROD measurement.

Pflugmacher and Sandermann (1998), Pflugma-

cher et al. (1999a,b) have detected the existence of

cytochrome P450 oxygenases in various marinespecies. Activities of the cytochrome P450 mono-

oxygenases system have been demonstrated for

substrates such as fatty acids (e.g. lauric, palmitic,

stearic acids) but also for xenobiotics like pheno-

barbital (Pflugmacher and Sandermann, 1998) and

polychlorobiphenyls (PCBs) (Pflugmacher et al.,

1999b).

3.6.2.2. Phase II enzymes. GSTs form a super-family of multifunctional enzymes (mostly cyto-

solic), in animal like in vegetal organisms (Pascal

and Scalla, 1999; Plaisance and Gronwald, 1999;

Reade and Cobb, 1999). GSTs exist under homo

or heterodimer forms of around 25 kDa molecular

weight (Marrs, 1996).

GSTs catalyze the conjugation reaction of

reduced glutathione (GSH) with electrophilic sub-strates (Pascal and Scalla, 1999; Plaisance and

Gronwald, 1999; Reade and Cobb, 1999). GSH-

substrate conjugates are more polar and less toxic

(Marrs, 1996).

Plant GSTs have been discovered as part of

herbicide detoxication in terrestrial plants (Shima-

bukuro et al., 1970) and studied for their role in

the selectivity towards these contaminants (La-moureux et al., 1991, Plaisance and Gronwald,

1999). They appear to be responsible for tolerance

to herbicides (Marrs, 1996; Reade and Cobb,

1999), but many authors have demonstrated their

involvement in the detoxication of polycyclic

aromatic hydrocarbons (PAHs), PCBs, heavy

metals (see synthesis in Marrs, 1996).

Various studies have revealed the existence ofGST isozymes in various plants (Pascal and Scalla,

1999), with different degrees of specificity to

herbicides (Timmermann, 1989).

A significant influence of xenobiotic exposure

has been demonstrated on the enzymatic activity

of aquatic species. GST activity was significantly

increased after exposure to PAHs (Schrenk et al.,

1998), HCBs (Roy et al., 1995), and Pflugmacheret al. (1999b) also identified GST and O - and N -

glucosyltransferase activities in various marine

species, in response to contamination by PCBs.

Tang et al. (1998) showed, in freshwater species,

the involvement of GST activity in the metabolism

of atrazine. It seems that the species most tolerant

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 197

Table 5

Induction of biotransformation molecules and enzymes by heavy metals or xenobiotics in aquatic plants

Biomarkers Stress Species Kind of study References

Induction of phytochelatins Heavy metals Ditylum brightwellii , Vaucheria Sticho-

coccus bacillaris

In vitro Rijstenbil et al. (1994a,b), Skowronski

et al. (1998) and Pawlik-Skowronska

(2002)

Induction of phase I enzymes: cyto-

chrome P450 monooxygenases

EROD

Cu P. oceanica In vitro Hamoutene et al. (1996)

Phenobarbital Different species of Chlorophyta,

Phaeophyta, Rhodophyta

In vitro Pflugmacher and Sandermann (1998)

3-Chlorobiphenyl Different species of Chlorophyta,

Phaeophyta, Rhodophyta

In vitro Pflugmacher et al. (1999a,b)

Induction of phase II enzymes:

GSTs

Atrazine Chlamydomonas sp., Chlorella sp., Sce-

nedesmus quadricauda Cyclotella sp.,

Synedra sp.

In vitro Tang et al. (1998)

Cu, HgCl2,

CH3HgCl

P. oceanica In vitro and in situ: Wes-

tern Mediterranean (Fr, It)

Hamoutene et al. (1996), Ranvier et al.

(2000), Ferrat et al. (2002a,c) and

Ferrat (2001)

PAHs Nuphar lutea , Lemna minor , Hippuris

vulgaris , Potamogeton sp.

Eastern European lakes Schrenk et al. (1998)

L.

Ferra

tet

al.

/A

qu

atic

To

xico

log

y6

5(

20

03

)1

87�

/20

41

98

of atrazine are those showing the highest levels ofGST activities (Tang et al., 1998).

Concerning the detoxification of heavy metals,

studies have been carried out with Cd (Hamoutene

et al., 1996), or Hg (Ranvier et al., 2000; Ferrat et

al., 2002a,c), also showing an increase in

GST activity in presence of heavy metal. Experi-

mental contamination of P. oceanica by HgCl2and CH3HgCl as a function of time (144 h),showed that the inorganic form of mercury

preferentially induced GSTs (Ferrat et al.,

2002a). In parallel, there was also an increase of

GST activity in controls as a function of time.

This last observation implied that GSTs are not

specific to contamination by mercury, and re-

sponded as much to stress generated by conserva-

tion in the aquarium as to stress generated bymercury.

Moreover, P. oceanica collected in a contami-

nated and a pristine site of the Western Mediter-

ranean, showed increased GST activity from the

contaminated site. An immunochemical study

performed on these samples made it possible

to determine the presence of A1/A1 isozyme

(with chlorodinitrobenzene affinity, the exogenoussubstrate used for the experiment) only in the

sheaths, compared to the blades. The induction

was higher in the sheaths coming from the con-

taminated site as compared to the pristine one

(Ferrat, 2001).

Both in vitro and in vivo experiments (Ferrat et

al., 2002a,c) have demonstrated that GST activ-

ities were always higher in the sheaths than in theblades. In the light of the results of the immuno-

chemical study (Ferrat et al., 2002a), other GST

isozymes may exist in the blades.

4. Conclusion

In conclusion, the survey presented here shows

the variety of the mechanisms of response that canbe carried out by aquatic plants, in the case of the

alteration of environmental conditions, whether of

natural or anthropogenic origin.

Among the biomarkers mentioned, markers of

photosynthetic activity (chlorophyll fluorescence,

photosynthetic pigments concentration), markers

of oxidative stress (lipid peroxidation, catalase,

GSH) and of detoxication (GST, GSH, phytoche-

latins) seem to offer encouraging possibilities.

They do indeed seem to be representative of the

level of perturbation of the medium and of the

health of the organisms. They could therefore be

used for the early detection of alterations in water

quality. Unfortunately, studies carried out on

HSPs did not still show encouraging results for

their utilization as stress markers.

In addition to the species on which these

mechanisms have been tested, seagrasses and

particularly P. oceanica , have been revealed to be

very sensitive with regard to variations in environ-

mental quality, which is consistent with their use

as ‘classical’ bio-indicator species. Indeed, studies

show that mercury contamination could provoke

measurable damages like membrane degradation

(lipid peroxidation), enzymatic inhibition, but that

the plant could answer by mechanisms of protec-

tion (i) induced directly by mercury like biotrans-

formation enzymes (GST), (ii) induced by oxygen

reactive species (oxidative stress) like antioxidant

enzymes (catalase), antioxidant and metal chela-

tors molecules (GSH, phenolic compounds) very

rapidly. So the study of various biomarkers in this

species offers a good illustration of the multiplicity

of the mechanisms involved and confirms the

importance and the interest of a multiparametric

approach, including both ‘physiological’ biomar-

kers, biomarkers of general stress and more

specific biomarkers. This multiparametric ap-

proach is highly recommended in ecotoxicology.

Moreover, the identification of specific responses

to a particular kind of stress in these plants, given

their extensive range of distribution and their

localization in the littoral zone, could make them

effective tools for the early evaluation of the

quality of this environment that is so exposed to

human activities.

Acknowledgements

The authors would like to thank Dr M. Paul for

proof-reading the English.

L. Ferrat et al. / Aquatic Toxicology 65 (2003) 187�/204 199

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