Advances and pitfalls in developing natural products as antifoulants in Brazil.
Advances and pitfalls in developing natural products as antifoulants in Brazil.
Ricardo Coutinho
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Advances and pitfalls in developing
Natural products as antifoulants in
Brazil
Ricardo Coutinho, Ph.D
Division of Biofouling
Biofouling,, Department of Ocenography
Ocenography,, Instituto
de Estudos do Mar Almirante Paulo Moreira, Brazil
Gothenburg.. 19.11.2009
Gothenburg
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SUMMARY
The beginning
The development
Problems and alternatives
Study case
Future perspective
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The beginning
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-The use of natural products as antifoulants
has been an active research area for the last
30 years. Nevertheless, little advances has
been achieve in order to produce an
antifouling paint with a natural substance in
their matrix.
-Research in natural products in Brazil started
in 1995 in the Brazilian Navy and Fluminense
Federal University with emphasis in the field
and laboratory test of antifouling (AF) activity
extracted.
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Methodology
Field tests – In 1995, Mark Hay and Willian Fenical went to
Arraial do Cabo,Rio de Janeiro, Brazil to make a survey in the
species that had natural products used to defense against
herbivore. Several experiments were done in the field using
differents methods.
-Some of the methods were adapted to test antifouling products
from marine organisms.
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Extract of Marine organisms
were incorporated into Phytogel,
Phytogel,
place in plastic dish and exposed
to the field. The extract was
liberated slowly, as it presumably
occurs in live organisms
organisms..
bóia
superfície
distorcedor
P
R
O
F
U
N
D
I
D
A
D
E
Suporte de alumínio
T1
Peso
T2
T3
C
T4
C - Controle
T - Tratamentos
T5
1,0 m
T6
Peso
fundo
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Rafts trials where
used in theses
tests.
Cabo Frio Island
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Laboratory Test – Based on Da Gama et. al. 2002jj
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First Test
Laurencia
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Bioincrustação ao longo das semanas
Controle
L. obtusa
100
120
100
60
Controle
Laurencia sp1
40
20
Incrustação (%)
80
Incrustação (%)
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Bioincrustação ao longo das semanas
80
60
40
20
0
0
1
2
3
4
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Tempo (semanas)
Cromatografia em camada delgada das espécies estudadas de
Laurencia,, eluída duas vezes em clorofórmio, visualizada e revelada com
Laurencia
sulfato cérico - T = todas;
todas; L.o.= Laurencia obtusa;
obtusa; L.f.= L. filiformis
filiformis;; L.a.=
L. arbuscula
arbuscula;; L.s.= L. scoparia
scoparia;; L. sp
sp2
2= Laurencia sp
sp2
2; L. sp
sp1
1= Laurencia
sp1
sp1; L.al
al..= L. aldingensis
aldingensis..
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3
4
Tempo (semanas)
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Behavior of exploration and
seleciotion of the substrate
by Perna perna and controls.
Bioensaio com Perna perna
Atividade Antiincrustante (% Controle)
Perna perna
100
L. obtusa*
L. arbuscula*
L. aldingensis*
L. scoparia*
L. filiformis*
Laurencia sp2*
Laurencia sp1*
80
N = 10 réplicas
60
* p < 0,05
40
20
0
Espécies Estudadas
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The development
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Surface elatol concentrations did not inhibit herbivory
by sea urchins,settlement of barnacle larvae,
or mussel attachment, but thallus did.
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Sudatti. et. al.2008
Corps in cerise (*) and many small
and large vesicles (arrows) inside
the cortical cells are seen. Scale bar,
6 um.
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Salgado et.al.2008
Fig. 1. Differential interferential optical microscopy images of living cortical, medullar and tricoblast cells of Laurencia obtusa. (a)
Transverse section view of cortical and medullar cells. Note the presence of two CC (*) in a cortical cell; (b) image of tricoblast cells,
where it can be also observed the presence of CC. Note the presence of connections between the CC and the cell periphery (arrows);
(c and d) superficial view of cortical cells showing elongated structures connecting CC to cell periphery (bars = 10 lm).
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Fig. 5. Optical microscopy images (DIC, fluorescence and overlaid images) of semisemi-thin transversal sections. (a) A DIC
image where it can be observed the CC (CC), the cell walls (CW) and the chloroplasts (C). (b) An image revealing the
typical autoauto-fluorescence observed in these cells. (c) An overlaid image composed by DIC and by fluorescence images,
respectively. Note the autoauto-fluorescence emission in CC and also in many vesicles distributed along the i ntrantra-cellular
space and in chloroplast (bars = 10 lm).
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Fig. 6. Optical microscopy images of a cortical cell obtained using video-microscopy analysis. Note, in the sequence
‘a–f’, the vesicles displacement (arrow and arrow heads) migrating from CC in direction to the cell periphery. *,
chloroplast. (bar = 3 lm).
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Fig. 9. TriTri-dimensional reconstruction of the tubular membranous connections. (a
(a–
–c) TEM images of sections used in the 3
3--D
reconstruction process where is possible observe the tubular membranous connections (arrows), a CC region (*) and a vesicle inside
the connections (arrow head); (d and e) 3-D reconstruction. Cell wall is represented in blue color; the CC is red color; the membrane of
tubular connections in yellow color; and transported vesicle in orange color (bars = 400 nm).
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Antifouling activity of natural products from
Brazilian Seaweeds
-In
a recent work, we tested crude organic extracts from 51 populations
comprising 42 species of macroalgae from eight locations along the
Brazilian coast against a relevant fouling organism in laboratory
bioassays, the brown mussel Perna perna.
Da Gama et. al. 2008
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Despite the unbalanced number of macroalgae tested among different
localities, there seems to be no latitudinal trend of increased
antifouling activity towards lower latitudes, where fouling pressure is
presumed to be higher.
There was a clear phylogenetic pattern in antifouling
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activity, with red macroalgae having the highest
proportion (55%) of active species (moderate or strong
fouling Inhibition), followed by brown macroalgae (14%).
Green seaweeds never exhibited strong AF activity
These results suggest that research efforts should be
focused on tropical red macroalgae in the quest for new
antifoulants
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Problems and alternatives
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Problem : Synthesis of Elatol
Alternatives :
-Comercial Analogs already with permission to use.
-Synthesis of others compounds :
-Easy synthesis
-Enough material to incorporated in a paint
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Problem : Low investiments in research (in Brazil).
Alternatives : Join projects with Universities, Federal and
State agency and paint companies.
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Study case
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Objectives
To test all the process from synthesis to the incorporation
In the paint matrix and to test in the field.
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The synthesis
Our biocide were synthesized in the Federal University of
Rio de Janeiro, based on a chemistry structure similar a
substance produced by a sponge and incorporated in a
paint matrix
matrix..
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Incorporation in the Paint’s
Matrix
The experimental paint was made in a join project withe
The International Paint Company (Akzo Nobel) located in
Niterói, Rio de Janeiro.
The Rio de Janeiro State University (UERJ) also
participated in the laboratoy test of the paint.
paint.
Field tests
Guanabara Bay, Rio de
Janeiro
Cabo Frio Island
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Treatments
Were tested three coating (3 repliqueted each):
-Main biocide ( Copper) substitute by our “natural” biocide.
-Commercial paint
-Our “natural” biocide used as accessory biocide together
with copper.
-Control (without antifouling paint)
The experiments lasting by 1 (one) year.
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List of species presents in the plates
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Sinopse taxonômica
Reino Plantae
Divisão Chlorophyta
Classe Chlorophyceae
Ordem Ulvales
Família Ulvaceae
Divisão Phaeophyta
Tipo morfo-funcional/ Grupos
Foliácea
Filamentosa parda
Classe Phaeophyceae
Ordem Ectocarpales
Família Ectocarpaceae
Divisão Rhodophyta
Ordem Ceramiales
Família Ceramiaceae
Filamentosa Vermelhas
Reino Animalia
Filo Cnidaria
Classe Hydrozoa
Ordem Hydroida
Família Campanulariidae
Obelia dichotoma (Linnaeus, 1758)
Classe Anthozoa
Ordem Actiniaria
Família Actiniidae
Filo Annelida
Classe Polychaeta
Ordem Canalipalpata
Família Serpulidae
Filo Arthropoda
Subclasse Cirripedia
Ordem Thoracica
Família Balanidae
Megabalanus tintinnabulum (Linnaeus, 1758)
Balanus trigonus
Balanus improvisus
Filo Ectoprocta
Classe Gymnolemata
Ordem Cheilostomata
Família Bugulidae
Bugula neritina (Linnaeus, 1758)
Outros briozoários arborescentes
Família Schizoporellidae
Schizoporella errata (Johnston, 1847)
Filo Chordata
Classe Ascidiacea
Ordem Stolidobranchia
Familia Styelidae
Styela plicata (Lesueur, 1823)
Anêmonas
Serpulídeos
Balanídeos
Briozoários incrustantes
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Treatment 1 –”natural biocide” (4 months)
Revestimento 1 - Matriz com Biocida Natural
100%
90%
Porcentagem de cobertura
80%
70%
60%
50%
40%
30%
20%
10%
0%
1a
2a
3a
4a
5a
6a
8a
Sem anas
a
Biofilme
Obelia dichotoma
Balanídeos
Filamentosas Pardas
Bugula neritina
Styela plicata
Serpulídeos
Tubos mucosos
Espaço vazio
b
c
12a
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Treatment 2- Commercial paint
Revestimento 2 - Matriz Comercial
100%
Porcentagem de cobertura
80%
60%
40%
20%
0%
1a
2a
3a
4a
5a
6a
8a
Sem anas
Biofilme
Obelia dichotoma
Balanídeos
Filamentosas Pardas
Bugula neritina
Styela plicata
Serpulídeos
Tubos mucosos
Espaço vazio
12a
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Treatment 3 – “natural biocide as accessory
Revestimento 3 - Matriz com Biocida Natural de Acessório
100%
Porcentagem de cobertura
80%
60%
40%
20%
0%
1a
2a
3a
4a
5a
6a
8a
Semanas
Biofilme
Obelia dichotoma
Balanídeos
Filamentosas Pardas
Bugula neritina
Styela plicata
Serpulídeos
Tubos mucosos
Espaço vazio
12a
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Control
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Controle
100%
Porcentagem de cobertura
80%
60%
40%
20%
0%
1a
2a
3a
4a
5a
6a
8a
Sem anas
Biofilme
Obelia dichotoma
Balanídeos
Filamentosas Pardas
Bugula neritina
Styela plicata
Serpulídeos
Tubos mucosos
Espaço vazio
12a
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treat.. 1
treat
treat. 2
treat. 3
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control
May
June
Jully
August
October
November
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Future Perspectives
• New synthesis
•Biocide incorporation in paint matrix
•Investiments by companies
•Field tests (plates and ships)
•Toxicity tests
•Patents
•Registration
•Production
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Advances and pitfalls in developing natural products as antifoulants in Brazil.
Ricardodo Mar Almirante Paulo Moreira, Brazil
INSTITUTO DE ESTUDOS DO MAR
ALMIRANTE PAULO MOREIRA
Arraial do Cabo, Rio de Janeiro
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Acknowledgements
Hans Elwing
Renato Crespo Pereira - Fluminense Federal University (UFF)
Bernardo da Gama – Fluminense Federal University (UFF)
Daniela Sudatti – Fluminense Federal University (UFF)
Leonardo Salgado – Botanic Gardem-Rio de Janeiro (JB)
Gilberto Amado Filho-Botanica Garden-Rio de Janeiro (JB)
Claudio Lopes – Rio de Janeiro Federal University (UFRJ)
Rosangela Lopes -Rio de Janeiro Federal University of Rio(UFRJ)
Aricelso Limaverde –Rio de Janeiro State University (UERJ)
5 post-doc, 19 Ph.Ds, 23 MSc.
Akzo Nobel
Sponsor : Brazilian Navy, CNPq, FINEP, FAPERJ
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Thanks
Questions ?
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