Marine Pollution Bulletin 64 (2012) 20392046

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Comparative efciency of macroalgal extracts and booster biocides

as antifouling agents to control growth of three diatom species
Alla Silkina a,,1, Alexandra Bazes a,2, Jean-Luc Mouget b, Nathalie Bourgougnon a
a
Laboratoire de Biotechnologie et Chimie Marines (LBCM), Universit de Bretagne-Sud (UBS), Universit Europenne de Bretagne (UEB), Centre de Recherche Saint Maud,
56321 Lorient Cedex, France
b
Ecophysiologie et Mtabolisme des Microalgues (EA 2160, Mer, Molcules, Sant), Facult des Sciences et Techniques, Universit du Maine, Av. O. Messiaen,
72085 Le Mans Cedex 9, France

a r t i c l e

i n f o

Keywords:
Antifouling
Ceramium botryocarpum
Sargassum muticum
Booster biocides
Diatoms
EC50

a b s t r a c t
The application of booster biocides Diuron, Tolyluanid and Copper thiocyanate inbantifouling paints,
used to prevent development of biofouling, needs to be monitored before assessing their impacts on
the environment. An alternative approach aims to propose eco-friendly and effective antifoulants isolated
from marine organisms such as seaweeds. In this study, the effects of booster biocides and the ethanol
and dichloromethane extracts from a brown (Sargassum muticum) and a red alga (Ceramium botryocarpum)
have been compared by algal growth inhibition tests of marine diatoms. The most efcient extracts were
ethanol fraction of S. muticum and C. botryocarpum extracts with growth EC50 = 4.74 and 5.3 lg mL1
respectively, with reversible diatom growth effect. The booster biocides are more efcient
EC50 = 0.52 lg mL1, but are highly toxic. Results validate the use of macroalgal extracts as non toxic antifouling compounds, and they represent valuable environmentally friendly alternatives in comparison with
currently used biocides.
2012 Published by Elsevier Ltd.

1. Introduction
Marine biofouling, can be dened as the growth of unwanted
organisms on the surface of articial structures immersed in water
(Yebra et al., 2004; Buma et al., 2009). Biofouling causes important
problems to governments and industry with severe economic consequences particularly on vessels due to increased fuel consumption and reduced inter docking periods (Bellas, 2007, 2008; ICS
and ISF, 2009). In addition, ecological implications of biofouling
include increased carbon emission and potential dispersion of
invasive alien species (Bellas, 2006; Floerl et al., 2009).
Antifouling paints have been widely used to prevent and control
biofouling (Bao et al., 2011) and therefore their economic and
ecological relevance is questionable. For example, tributyltin
(TBT)-based coatings, introduced in the early 70s, are the most
effective at reducing biofouling due to their high toxicity to fouling
Corresponding author. Tel.: +44 (0) 1792295446; fax: +44 (0) 1792295447.
E-mail addresses: alla.silkina@gmail.com, a.silkina@swansea.ac.uk (A. Silkina),
alexbazes@yahoo.fr (A. Bazes), jean-luc.mouget@univ-lemans.fr (J.-L. Mouget),
nathalie.bourgougnon@univ-ubs.fr (N. Bourgougnon).
1
Present address: Algal Biotechnology for Wales Knowledge Transfer Centre,
Centre for Sustainable Aquatic Research, Swansea University, Singleton Park, Swansea
SA2 8PP, UK.
2
Present address: Institut des Sciences de la Vie, Laboratoire de Biochimie
cellulaire, nutritionnelle et toxicologique, Universit catholique de Louvain, Croix
du Sud 5, boite 3, 1348 Louvain la Neuve, Belgique, Belgium.
0025-326X/$ - see front matter 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.marpolbul.2012.06.028

organisms. However these compounds were prohibited as antifoulants in 2008 (IMO Resolution A. 895 21, 25/11/1999). As a result
an intense effort has been devoted to the development of alternative biocides. Several booster biocides (e.g. Irgarol 1051, Diuron,
Copper thiocyanate and Tolyluanid) have thus been approved
for use and replaced TBT in antifouling products. However, recently
some of those booster biocides have also been banned or their use
regulated in Europe because of their environmental persistence
and toxicity to non-target organisms (e.g. Dafforn et al., 2011), thus
highlighting the need to perform adequate risk assessment procedures for antifouling biocides.
The recent restrictions on the use of traditional toxic antifouling
paints, also led to a growing need for new alternative compounds
that ensure satisfactory performance without polluting the marine
ecosystem (Hellio et al., 2009; Dafforn et al., 2011). Since many
sessile marine organisms have developed efcient defense mechanisms against microbial epibionts, there is an increasing interest in
such organisms as a source of naturally released antifouling substances (Hellio et al., 2001; Bazes et al., 2006, 2009; Marechal
and Hellio, 2009; Dafforn et al., 2011). By producing secondary
metabolites, macroalgae carry signicantly less fouling organisms
on their thallium, compared to co-occurring biolms on inanimate
substrata (Dubber and Harder, 2008; Hellio et al., 2009; Ioannou
et al., 2009). Use of these secondary metabolites as alternatives
to toxic antifouling compounds is a new eld of biotechnology.

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A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

The antifouling activity of marine macroalgal compounds is

generally assayed using extracts in various organic solvents (Liao
et al., 2003). Previous works have shown that ethanol/water and
ethanol/dichloromethane extracts from Sargassum muticum (Heterokonta, Sargassaceae) and Ceramium botryocarpum (Rhodophyta,
Ceramiaceae) presented antifouling properties against representative marine organisms such as marine bacteria, phytoplankton,
and spores of macroalgae (Bazes et al., 2006, 2009; Silkina et al.,
2009). In particular, crude extracts of S. muticum and C. botryocarpum displayed clear antifouling properties against two diatom species, Fragilaria pinnata and Cylindrotheca closterium. The macroalgal
compounds affected the growth and the pigment content of the
diatoms. The mechanism by which the extracts act as antifouling
agents relies on the alteration of the photosynthetic apparatus, as
evidenced by pigment changes and PS II inhibition (Silkina et al.,
2009). The efciency of the macroalgal extracts is lower than that
of Diuron, but their inuence is reversible, hence they represent an
environmentally acceptable alternative to toxic products for the
control of fouling organisms (Bazes et al., 2006, 2009; Silkina
et al., 2009). In these previous works, F. pinnata and C. closterium
were selected because diatoms constitute a major component of
primary producers in most aquatic ecosystems. In particular, diatoms constitute 50% of the total oceanic production and they present special interest for ecotoxicological studies of antifouling
compounds in the marine environment (Falciatore and Bowler,
2002; Molino and Wetherbee, 2008).
Considering the need for environmentally friendly antifouling
agents and the general lack of information about the activity of
compounds extracted from marine organisms, the rst objective
of the present work was to assess the potential of macroalgal
crude extracts from S. muticum and C. botryocarpum as natural
biocides. The second objective of this study was to determine
their efciency in comparison with chemical antifouling agents,
by estimating the potential toxicity of some of the most frequently used booster biocides, Diuron, Copper thiocyanate and
Tolyluanid. Bioassays were conducted with three diatom species:
F. pinnata and C. closterium, which are well-known fouling species
(Gatidou and Thomaidis, 2007 Silkina et al., 2009; Arajo et al.,
2010), and Thalassiosira pseudonana, a non-target common species
of marine phytoplankton (Porsbring et al., 2009). From the biological responses and the sensitivity of these diatoms exposed to the
different biocides, the antifouling capacities of the tested compounds are discussed, as well as their potential impact to the
aquatic environment.

2. Materials and methods

2.1. Test organisms
The pennate marine diatoms F. pinnata (Fragilariophyceae), C.
closterium (Nitzschiaceae) and the centric marine diatom T. pseudonana (Thalassiosiraceae) were obtained from the Algae Culture Collection of the University of Caen (France). The diatoms were grown
in simplied seawater-based culture medium (Guillard and Ryther,
1972) with Guillards F/2 (Sigma), in sterile conditions at 18 C. The
simplied seawater was made with NaCl (30 g L1), MgCl2
(10.2 g L1) and KCl (0.74 g L1) and sterilized before use. Guillards
F/2 was added after sterilization and the culture medium was
stored at 4 C until use. Strains were maintained in 250 mL Erlenmeyer asks under controlled illumination (100 lmol photons m2 s1 provided by cool-white uorescent lamps with a
16 h:8 h light:dark cycle). Regular dilutions with fresh medium ensured that exponential growth was maintained prior to testing. Cultures of F. pinnata, C. closterium and T. pseudonana were inoculated
with ca. 67  105, 37  104 and 75  105 cell mL1, respectively.

2.2. Natural antifouling extracts

The natural extracts were prepared from two species of macroalgae: S. muticum (Heterokonta, Fucaleacea) collected in Locmariaquer (48440 N, 3590 W, France); C. botryocarpum (Rhodophyta,
Ceramileacea) cultivated in raceways by the Innovalg Company
(Bouin, France). For this study, two types of extracts with different
polarity were tested, namely extract A (ethanol/water) and extract
B (ethanol/dichloromethane). The detail extraction step was performed as previously (Hellio et al., 2001; Bazes et al., 2006; Silkina
et al., 2009).
2.3. Synthetic booster biocides
Diuron (N-[3,4-dichlorophenyl]-N,N-dimethylurea), Copper
thiocyanate (Thiocyanic acid, copper (1+) salt) and Tolyluanide
(N-([Dichlorouoromethyl]Thio)-N0 ,N0 -Dimethyl-N-P-Tolylsulfamide) (Nautix, France (purity 97.6%)) demonstrated a low solubility
in water, therefore an organic solvent was used for the preparation
of stock and working solutions. Among the different water-miscible
solvents proposed in standardized protocols (OECD, 1981), dimethyl
sulfoxide (DMSO) was the least toxic for diatoms (Okumura et al.,
2001), and was used as the carrier solvent for Diuron, Copper thiocyanate and Tolyluanid in the bioassays. Stock and working solutions
were prepared in acetone-rinsed glassware. Biocides were rst dissolved in dimethyl sulphoxide (DMSO; 99%, BDH Ltd., England), then
further diluted with autoclaved deionised water to give a stock solution of 10 mg mL1 and kept in the dark at room temperature to prevent photodegradation. The stock solution was further diluted with
the culture medium to achieve the target concentrations for toxicity
tests. The nal DMSO concentration in experimental vessels never
exceeded 0.1% (v/v), a concentration lower by one order of magnitude than the NOEC (no observable effect concentration) values
(11,000 ppm) observed for DMSO in two diatom species, Skeletonema costatum and Chaetoceros calcitrans (Okumura et al., 2001).
Furthermore, the toxic effect of DMSO alone towards F. pinnata
was examined in a preliminary experiment at 200 and 1000 ppm,
to estimate the optimum solvent volume which did not affect the
growth of this diatom. Experiments were run in triplicate, and none
of the tested concentrations was toxic towards F. pinnata.
2.4. Algal growth inhibition tests
The diatoms F. pinnata, C. closterium and T. pseudonana were subjected to standard 72 h algal growth inhibition tests, performed in
250 mL Erlenmeyer asks lled with media prepared according
ISO standards 10253 for marine diatoms (ISO, 1995). Dilution water
used for media preparation was of ultrapure quality, produced from
distilled water by means of Milli-Q apparatus (Millipore, Bedford,
MA, USA). Macroalgal extracts and booster biocides were added
into nutrient solutions in volumes appropriate to achieve the correct nal concentration, determined in preliminary experiment
for each substance and each tested diatom (Bazes et al., 2006). Each
strain was exposed to 0.1, 1, 25 and 50 lg mL1 of Diuron, Copper
thiocyanate, Tolyluanid and macroalgal extracts, along with a control with DMSO (200 ppm). All the experiments were performed in
triplicate. Every 24 h the algal density was quantied by using light
microscope (Olympus BH2) and a Malassez haemocytometer.
Growth rate was calculated daily as:

l lnN2 =N1 =t2  t1

where N1 and N2 are cell concentrations, at times t1 and t2,

respectively.
Furthermore, the optical density at 750 nm (OD750, measured as
percent transmission in a Shimadzu UV 160 spectrophotometer)
was used as a proxy for biomass estimation, and the chlorophyll

A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

a (Chl a) content was determined from spectrophotometric measurements in a UV visible spectrophotometer of 90% acetone extracts using the equation described in Sawant et al. (1995):

Chl a lg L1 11:47  OD664  0:4  OD630

From growth rates, biomass estimations and Chl a contents recorded in the different series of test solutions, the concentrations
causing 50% inhibition of growth rate, biomass and Chl a content
were determined at 72 h and expressed as EC50 values (OECD,
2006). The acute inhibition response obtained in these bioassays
was plotted against the tested compound concentrations to obtain
the doseresponse curves. In order to enable direct comparison of
dose responses for three different endpoints (raw data expressed
in different units), all responses were expressed as deviation from
the controls (% inhibition). Unless specied, relative changes in
growth rate, biomass and Chl a content from times t0t72 resulting
from the inhibition by biocides, were calculated as follows, according to Magnusson et al. (2008):

Biomass inhibition : OD750 t72 treatment

 OD750 t0 control
 100=OD750 t 72 control
 OD750 t0 control

Growth rate inhibition : lnN72 =N0 =t 72  t 0 treatment

 100=lnN 72 =N0 =t 72  t0 control

Chl a inhibition : chl at 72 treatment  chl at0 control

 100=chl at72 control  chl at 0 control

Previous work (Silkina et al., 2009) was identied the EC50 of Diuron
at 0.09 0.01 lg mL1, and the concentration of 1 lg mL1 was inhibit the pigments ratio at 1.6 times for Chl a/Chl c, at 1.3 times for
fucoxanthin/Chl a, and at 3 times for diatoxanthin/Chl a.
2.5. Reversible toxicity of biocides
Aliquots of an actively growing culture of each strain were
grown for 72 h in medium containing effective concentrations
(25 lg mL1) of the booster biocides or algal extracts. This cultivation time was presented in Fig. 2 by (A). After this, the algal cells
were then recovered by gentle centrifugation (2000g, 10 min,
20 C, Beckman Avanti J30I) to separate the cells from the medium
containing the booster biocides and the macroalgal extracts. The
pellets containing the algal cells were then re-suspended in a fresh
plain medium (without biocides) for another 96 h, in Fig. 2 this
part represented by (B). The algal growth was assessed daily
(Sawant et al., 1995) and all the experiments were carried out in
triplicate (Bazes et al., 2006; Silkina et al., 2009).
2.6. Statistic analyses
The 72 h EC50, i.e. the effective concentration reducing the
growth rate by 50%, was calculated using linear interpolation.
The data were tested for normality and homogeneity of variance,
and Dunnetts multiple comparison test was used to determine
which treatments differed signicantly from controls (one-tailed
test, p = 0.05). If data were pooled to gain a single EC50 value based
on multiple tests, the Bonferroni t-test or the Wilcoxon rank sum
test was used to compare which treatments differed signicantly
from controls, because the numbers of replicates in each treatment
were unequal (e.g. more replicates for the controls). Statistical
computations were performed using standard software packages
(XLSTAT version 2001).

2041

3. Results
The three test organisms consistently exhibited an exponential
phase of growth in all controls from t24 to t72h (F. pinnata
l = 0.83 0.05, C. closterium l = 0.62 0.03, T. pseudonana l =
0.75 0.03 [means SE, n = 20]) throughout the experiments, and
the cell counts and OD750 values remained a valid representative
for cell density without any adjustments to the initial equations.
In the absence of tested compounds, the Chl a cell content in the
cultures during exponential growth (from t24 to t72h), was
5.76 0.01 lg Chl a 106 cells1 for F. pinnata, 2.63 0.01 for C. closterium, and 3.36 0.01 for T. pseudonana (means SE, n = 3).
3.1. Impacts of extracts and biocides on biomass, growth rate and Chl a
content
The effects of different concentrations of the natural compounds (A and B extracts of S. muticum and C. botryocarpum), and
of booster biocides (Diuron, Copper thiocyanate, Tolyluanid) on
the growth of F. pinnata, C. closterium and T. pseudonana grown in
a batch mode were studied over a 72 h period. In comparison with
control data (% inhibition) the acute inhibition response obtained
in these bioassays was plotted against the tested compound concentrations to obtain the doseresponse curves. The doseresponse
curves for the different compounds exhibited very similar shapes
and slopes for each endpoint and for the three diatom species,
but different threshold concentrations, in function of the response
studied (results for F. pinnata shown in Fig. 1). The concentrations
of antifouling compounds which inhibited 50% of the growth rate
(l), biomass or Chl a content, were consistent for each of the compounds and species tested (Table 1). The responses of the diatoms
exposed to the different booster biocides showed that Chl a was
the most sensitive parameter (Table 1). In contrast, biomass estimated by OD750 measurements was the least sensitive parameter,
with an EC50 for the booster biocides signicantly higher
(p P 0.005) than for growth rate and Chl a content for all microalgae, a pattern also observed in the presence of the natural antifoulants. Contrary to the booster biocides, the growth rate was the
most sensitive parameter in diatoms exposed to the macroalgal extracts (Table 1).
Whatever the diatom tested, booster biocides were one/two
orders of magnitude more efcient than natural macroalgal extracts. Diuron (average EC50 for the three diatom species and
parameters, 0.069 lg mL1) was the most efcient antifouling
compound, 510 more effective than Copper thiocyanate and Tolyluanid. Regarding the natural extracts from macroalgae, A extracts of S. muticum and of C. botryocarpum were 24 times more
efcient than B extracts (Table 1).
3.2. Sensitivity of the tested diatoms
The three diatoms F. pinnata, C. closterium and T. pseudonana
were highly sensitive to the tested compounds especially booster
biocides (Table 1). The lowest differences between species were
observed for Diuron, the most efcient antifouling compound.
For most parameters (l, Chl a content, OD750) and antifoulants
tested (particularly for booster biocides and A extracts of macroalgae), F. pinnata was the most sensitive and T. pseudonana the
least sensitive diatom. This difference in sensitivity in function of
the species was also observed with EC10 values (data not shown).
The data of the Chl a content have a linear correlation (R2 P 90)
with biomass for the three studied species. The Chl a cellular content inhibition for the three tested diatoms (Fig. 2) conrmed than
between these studied species the highest sensitivity corresponded to F. pinnata for the booster biocides and A extracts of

2042

A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

100

100

Relative value

80

100

100

80

80

80

60

60

60

40

40

40

40

20

20

20

20

Biomass
Growth rate
Chl a content

60

0
0.01

10

25

50
-1

25

100
Biomass
Growth rate
Chl a content

80

Relative value

10

50

A extract C. botryocarpum (g mL-1)

A extract S. muticum (g mL )

100

0
0.01

0
0.01

10

25

B extract S. muticum (g mL )

100

80
60

60

40

40

40

20

20

20

10

25

50

Diuron (g mL-1)

0
0.01

10

25

50

B extract C. botryocarpum (g mL-1)

80

60

0
0.01

50
-1

0
0.01

10

25

0
0.01

50

10

25

50

Tolylfluanid (g mL-1)

Copper Thiocyanate (g mL-1)

Fig. 1. F. pinnata 72 h growth rate, biomass and Chl a content per cell response to (A) A extract S. muticum, (B) A extract C. botryocarpum, (C) B extract S. muticum, (D) B extract
C. botryocarpum, (E) Diuron, (F) Copper thiocyanate, (G) Tolyluanid.

Fig. 2. Efciency (in %) of Copper thiocyanate and Tolyluanid (EC50) in comparison with Diuron, as more efcient biocide (A), and efciency (in %) of A extract C.
botryocarpum, B extract S. muticum, B extract C. botryocarpum in comparison with A extract S. muticum (B), as more efcient extract.

macroalgae inuence. The lowest sensitivity was for T. pseudonana.

Whatever the booster biocide or macroalgal extract, the Chl a cellular content was already affected after the rst 24 h exposure,
with the exception of Diuron, for which an increase was even observed for F. pinnata at t = 24 h.

cells did not exhibit any growth and the cultures died. Regarding
the diatoms exposed to the macroalgal extracts, the growth was
also inhibited, but to a lower extent, being 2540% of the control.
However, the same cells exhibited almost normal growth when
transferred to an extract-free fresh medium (0.3 d1, which represents 7080% of the controls).

3.3. Reversibility of the antifouling activity

4. Discussion
The toxicity of the booster biocides and macroalgal extracts
and their persistency are shown in Fig. 3. The graphs represent
the evolution with time of the mean cell concentration of diatom
cultures exposed for 72 h to 25 lg mL1 of each product, a concentration previously demonstrated to be effective (Silkina et al.,
2009). The cells were then transferred to a fresh biocide- or extract-free medium for another 96 h period of growth (Fig. 3). For
all three diatom species, the growth rate in presence of the booster
biocides, was strongly inhibited as compared to the controls, by 5
times with Diuron and by 2.32.5 with Tolyluanid and Copper
Thiocyanate. After their transfer into a biocide-free medium, the

4.1. Natural antifouling defenses in marine organisms

The fouling of an aquatic organism surface, results in signicant
negative consequences, such as decreased growth and reproduction,
or increased diseases (e.g. Ganti et al., 2006). To reduce these biological costs, some marine plants and animals have developed physical,
mechanical or chemical defenses against the settlement and growth
of fouling organisms on their surfaces (Chambers et al., 2006; Fusetani, 2004, 2011). One of the best known mechanisms to prevent
fouling, results from the production and accumulation of secondary

Chl a content (lg Chl a 106 cells1).

Growth rate (l).
Biomass (OD750).
a

Biomass

0.097 0.001c
0.770 0.003c
0.870 0.004c
11.240 0.048c
13.200 0.056c
19.430 0.083c
35.200 0.156c
0.058 0.001a
0.470 0.002a
0.623 0.003a
8.563 0.050b
9.788 0.041b
14.433 0.061b
24.847 0.110b
0.062 0.001b
0.520 0.002b
0.690 0.003b
5.546 0.023a
6.513 0.028a
9.587 0.041a
16.368 0.074a

Chl a content

0.092 0.001c
0.610 0.002c
0.820 0.003c
9.150 0.039c
10.870 0.046c
17.960 0.077c
32.600 0.140c
0.051 0.001a
0.390 0.001a
0.540 0.002a
5.984 0.017b
7.986 0.034b
13.263 0.056b
23.345 0.102b

4.2. Toxicity of booster biocides: the diatom proof

0.087 0.001b
0.490 0.002c
0.720 0.003c
8.240 0.035c
8.700 0.037c
18.160 0.078c
29.900 0.128c
0.050 0.001a
0.306 0.001a
0.530 0.002a
6.137 0.025b
6.257 0.027b
13.439 0.057b
21.975 0.094b
0.053 0.001a
0.350 0.001b
0.670 0.002b
4.166 0.017a
4.293 0.018a
8.961 0.038a
14.753 0.063a
Diuron
Copper thiocyanate
Tolyluanid
A S. muticum
A C. botryocarpum
B S. muticum
B C. botryocarpum

2043

metabolites at the organisms surface (Fusetani, 2011). Extensive literature exists on this subject and identies numerous marine organisms which produce natural antifoulants. Compounds responsible
for antifouling activities have been extracted mainly from macroalgae (Bazes et al., 2006; Hellio et al., 2000, 2009) and sponges
(Dobretsov et al., 2007), but also from many other organisms (e.g.
Maida et al., 2006; Daoud et al., 2011). This strategy pathway is under demanding development. However, the causal link between
their ecological role and their activity in antifouling assays is in most
cases tenuous (Steinberg and de Nys, 2002; Raveendran et al., 2008;
Chambers et al., 2011).
The two macroalgal species used in our experiments, S. muticum
and C. botryocarpum, represent a rich source of allelochemicals and
secondary metabolites, able to exert a high level of inhibition
against colonizing microorganisms. The ethanolic and dichloromethane extracts of S. muticum and C. botryocarpum, have already
shown antifouling properties which inhibit the development of
marine bacteria, phytoplankton and spores of Ulva sp. at concentrations between 25 and 150 lg mL1 (Hellio et al., 2000, 2001; Stiger et al., 2004; Bazes et al., 2006, 2009).

0.0588 0.001b
0. 430 0.001b
0.710 0.003b
4.515 0.019a
5.363 0.023a
8.862 0.038a
16.086 0.069a

T. pseudonana

Biomass

Chl a content
C. closterium

Biomass
Chl a content
F. pinnata
EC50 (SE)

Table 1
For each diatom species, letters next to EC50 values indicate signicant differences between parameters: (Bonferroni t-test, p < 0.05 ). For each parameter, EC50 values in bold (the lowest) and italic (the highest) indicate signicant
differences when comparing different diatom species.

A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

Booster biocides have been in use since 1990s to replace TBT in

antifouling paints. Their toxicological effects on algal growth and
their respective EC50 values have been well documented (e.g. Ma
et al., 2004, 2006; Koutsaftis and Aoyama, 2006), as well as the
lower sensitivity of growth in comparison with other biological
parameters such as the PSII quantum yield (Macedo et al., 2008).
Despite this, assessment of the risks associated with their use is
still urgently needed (Voulvoulis et al., 2002). For example, toxicity
of Diuron has long been known generally under <0.7 lg L1 (e.g.
Giacomazzi and Cochet, 2004), the environmental impacts of Copper thiocyanate (10 lg mL1) (Reitsema, 2008) and the concentration of Tolyluanid in Swedish surface water was 0.2 lg mL1
(Trneman and Johansson, 2009) but limited information is available about their inhibition effect for target and non target diatoms
(Yebra et al., 2004).
In the present study, F. pinnata was the most sensitive species;
Diuron the most powerful booster biocide; and Chl a content the
most altered parameters tested. In the literature, effective Diuron
concentrations for different microalgal species ranged from 0.004
to 0.090 lg mL1 (Ma et al., 2004, 2006; Koutsaftis and Aoyama,
2006). These differences (one order magnitude) in EC50 reect distinct specic responses and acclimation capacities to the biocides,
and are in accordance with EC50 values (inhibition of the Chl a content) measured in our work, which varied from 0.048 to
0.058 lg mL1. This result is also in agreement with Bengtson Nash
et al. (2005) study. Diuron was the most powerful phytotoxicant
tested by the mode of action of preventing electron transfer from
QA to QB and oxidative stress in PSII. (e.g. Rutherford and Krieger-Liszkay, 2001), the species-dependent response was observed
on the low exposure (1 lg L1). The similar Diuron alteration of
F. pinnata pigment biosynthesis was studied by Silkina et al.,
2009, and conrmed by present results (Fig. 2)
The Copper thiocyanate treatment in our study showed a EC50
(Chl a content) of 0.300.47 lg mL1, depending on the species, a
result in agreement with van Wezel and van Vlaardingen (2004),
who measured EC50 of 0.97 lg mL1 on a microalgal microcosm
in a marine environment. The presence of the ion Cu2+ increases
the toxicity of this biocide (Koutsaftis and Aoyama, 2006) and for
marine biota is toxic from 1 lg mL1 (Omae, 2003).
Tolyluanid is a biocide often used in agriculture as a fungicide,
but it is toxic against a large number of species, including various
microalgae and the microcrustacean Daphnia sp. (Fernndez-Alba
et al., 2002). In the present study, Tolyluanid presented EC50

500

400

300

200

100

II

0
0

24

48

72

96

120 144 168

300

200

100

II

0
0

24

48

Time (hours)

72

96

120 144 168

Time (hours)

Biomass (cell concentration x 105cell mL-1)

A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

Biomass (cell concentration x 105cell mL-1)

Biomass (cell concentration x 105cell mL-1)

2044

500

400

300

200

100

II

0
0

24

48

72

96

120 144 168

Time (hours)

Fig. 3. Growth curves of F. pinnata (A), C. closterium (B), T. pseudonana (C) subjected to 25 lg mL1 of the A and B extracts of S .muticum and C. botryocarpum or synthetic
biocides (I) before and (II) after transferring them to an extract or biocide-free medium.

values (growth rate inhibition) between 0.67 and 0.71 lg mL1, in

accordance with a previous studies (Bayer CropScience, 2003),
where EC50 (growth inhibition) was 1 lg mL1 for the green alga
Desmodesmus subspicatus. In diatoms, as in other photosynthetic
organisms, exposure to Copper thiocyanate and Tolyluanid, results in direct or indirect light-induced oxidative stress (Geoffroy
et al., 2003, 2004), due to the disconnection of Chl molecules from
their energy transfer proteins, which leads to the production of Chl
triplet state able to induce the formation of reactive oxygen species
(ROS) (Rutherford and Krieger-Liszkay, 2001). This was conrmed
by our results.
4.3. Can macroalgal extracts prevent fouling by diatoms efciently?
The marine diatoms used in the present work, F. pinnata, C. closterium and T. pseudonana present the bioindicators in ecotoxicological studies to assess the physico-chemical quality of aquatic
environments (Rao, 1994). In accordance with previous works
(Bazes et al., 2006; Silkina et al., 2009), and present results conrm
the sensitivity of these diatoms exposed to the macroalgal extracts,
and as a matter of fact, the potential of macroalgal metabolites as
sources of antifouling agents. Concerning the antifouling activity of
macroalgal extracts, elucidation of the mode of action is not
straight forward. In contrast with booster biocides, the addition
of macroalgal extracts mostly inhibited the growth rate in present
study. The fact that growth, which is an integrated biological response to environmental constraints, proved to be the most sensitive parameter for macroalgal extracts, could reect the existence
of different modes of action, due to various bioactive molecules
present in the extracts. Regarding the present work, the inhibition
of algal growth by studied macroalgal extracts is clear but understanding the mechanisms underlying the diatoms response is
not obvious, as the information which can be inferred from diatom
cell characteristics, such as, production of extracellular polysaccharide (EPS), which could shield cells from toxins (Underwood and
Paterson, 2003). According to Urbani et al. (2005), T. pseudonana
produced more EPS in comparison with C. closterium and F. pinnata.
Indeed, Urbani et al. (2005) showed that the production of EPS per
unit cell was higher in T. pseudonana (28.4 lmol C 106 cells) than
in C. closterium (2.56 lmol C 106 cells). For F. pinnata, the EPS production was 0.99 lmol C 106 cells (Renaud et al., 1999) thus presents much smaller production than for T. pseudonana and C.
closterium, the protection by EPS no extend for this species.
Although some components from macroalgal crude extract have
been shown to alter the photosynthetic apparatus activity, but by
a mechanism not explained yet (Silkina et al., 2009).

zIt needs to be highlighted, that the natural extracts efciency

was lower by at least one order of magnitude than that of booster
biocides. This difference in efciency observed in the present study,
could be explained by the compounds from macroalgae being crude
extracts. Indeed, extracts of macroalgae contain many different
molecules produced from their metabolic activities, some of which
eventually having antifouling activity, for example fatty acid, as
palmitic acid (Bazes et al., 2009). The identication of active compounds, the elucidation of their mode of action, and the assessment
of possible interactions (synergistic as well as antagonistic effects)
between these secondary metabolites, represent a challenge before
being able to provide standardized environmentally friendly antifouling coatings, produced from marine organisms.
4.4. Environmental relevance of macroalgal antifouling compounds
The data about the environmental occurrence, fate, toxicity and
persistence of these booster biocides is limited, as well as on their
potential risks for marine ecosystems due to their increasing use.
In the context of water framework directives, the environmental
risk assessment of biocidal products is required, especially considering ecotoxicological studies, such as acute and chronic toxicity,
persistence and bioaccumulation (Koutsaftis and Aoyama, 2006;
Zhou et al., 2007).
The concentrations of Tolyluanid and Copper thiocyanate,
including no-effect concentration for a wide variety of aquatic
organisms, are reported to be higher than 100 ng L1 (Bellas,
2006; Thomas and Brooks, 2010). The concentration of Diuron provides inhibition for the biota, specially for algae is 10 ng L1 (Jones,
2004). Our results showed that fouling and non-target diatoms
were highly sensitive to booster biocides at concentrations which
are environmentally relevant: e.g. growth and Chl a content were
reduced by 10% at concentrations as low as 1.01.4, 0.971.4,
and 6.211.4 lg L1, for Diuron, Tolyluanid and Copper thiocyanate, respectively. The environmental detected concentrations of
Diuron were >8 lg L1 (Magnusson et al., 2008). Average concentrations of 6.72 ng L1 for Tolyluanid were measured by Mukherjee et al. (2009) in the marine environment and of 0.91.4 lg L1
for Copper thiocyanate in open marina, by Boxall et al. (2000). Even
low concentration of these compounds in the water may exacerbate the inhibitory effect on microalgal communities (Magnusson
et al., 2008). Inhibition of electron transport by booster biocides
at low concentrations likely decreases growth rates and biomass
of critical primary producers in estuarine habitats (Magnusson
et al., 2008; Fai et al., 2009). Reductions in microalgal biomass
may in turn change the composition of the microorganism commu-

A. Silkina et al. / Marine Pollution Bulletin 64 (2012) 20392046

nity, thus altering food availability and quality for benthic feeders
(Arrhenius et al., 2004). Changes in pigment and protein contents
may also change the nutritional value of the microalgae (Magnusson et al., 2008), thus affecting the food chain. Indeed, the toxicity
of the booster biocides Diuron, Copper thiocyanate and Tolyluanid towards diatom populations can induce changes in the pigment composition and in the structure of phytoplankton, as
observed by others (Advisory Committee on Pesticides, 2000;
Perschbacher and Ludwig, 2004; Fa et al., 2007, 2010).
In contrast with the toxic stable effects of the booster biocides,
the most striking specicity in the mode of action of the macroalgal extracts is their non toxic and non residual effect, as evidenced
by the reversible toxicity tests. The pattern of diatom growth rates
exposed to the extracts showed that these extracts were not toxic.
This in turn suggests that the diatoms exposed to effective concentrations of the extracts were alive, but unable to grow. The macroalgal crude extracts represent a valuable alternative source of
active compounds from marine organisms, which could be used
in the formulation of environmentally friendly antifouling
coatings, due to their absence of toxicity (Bazes et al., 2006,
2009; Silkina et al., 2009).
5. Conclusion
The present study has met two objectives. First, it extends our
knowledge of the toxicological effects on growth rate, Chl a content
and biomass production in diatoms of two widely used, but poorly
studied biocides, Copper thiocyanate and Tolyluanid. The presence of these chemical compounds in antifouling paints results in
environmental risk for primary producers in marine ecosystems.
Second, this study conrms the antifouling activity of extracts from
S. muticum and C. botryocarpum observed on different diatom species, representing fouling organisms and non-target species. Hence
the deleterious effects of booster biocides justify their replacement by natural compounds devoid of high toxic effect on biota,
and macroalgae appear good resources to provide such biological
biocides. These good substitutes prevent fouling by inhibiting the
development of diatoms, but are not toxic to fouling agents, nor
to non-target organisms. Nevertheless incorporation of those natural compounds in antifouling paints will require purication and
identication of the active metabolites from the crude extracts.
Such identication of natural products is often difcult, but it is
paramount because it would instigate the development of ecologically desirable, highly specic, biological algaecides.
Acknowledgement
The authors would like to acknowledge Jacob Scolding provided
language editing support.
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