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Hans Elwing, Ph.D. Professor in Biotechnology

Dept. of Cell and Molecular Biology/Interface Biophysics

Göteborg University, Sweden

The Barnacle

- our target

Typical Biofouling on a sailingboat

In Sweden (Three summer month)

Copper paint

Barnacle

Green algae

The Barnacle

1. A Crustacean

2. Present in all marine

environment

3. Difficult to remove

4. Resistant to toxinsBarnacle sex_WMV V9.wmv

1. Adult

2 Planktonic -

form3. Cyprid-larvae

4. Established cyprid

The life cycle of BarnacleHans Elwing, Interface Biotechn, GU

photo courtesy International Paint Ltd)

Barnacle may dominate Marine Biofouling in Sweden

Sacrifying anod

Propellers are favourites ...

Foto: Per Jonsson Foto: Jill Key

Drev is another favorite ...

Propeller

Traditional

erosive anti-

fouling paint

Biocid in coating Biocid dissolved in water

From L.D. Chambers et al ”Modern approaches to marine antifouling coatings” Surf and Coat Tech 2006, 201, 6, 3642-3652

Layer thickness

Time

Erosive Paints

+ Inexpensive

+ Long experience

- Toxic

Gradual erosion (controlled release)

Pine rosin

Important

Binder

Typical Biocides used in anti-fouling formulations

Compound

Copper [Cu(I)O, CuSCN, Cu(s)]Cybutryne (Irgarol)DCOIT (Sea-Nine)Dichlofluanid TolylfluanidCopper pyrithioneZinc pyrithioneZineb

NewTralopyril (Echonea)Medetomidine

Katemin (Medetomidin)

Inhibits colonization of Cyprid larvae

www.i-tech.se

Inventors: Hans Elwing Lena

Mårtensson and Mia Dahlström

PCT Filed: January 25, 2000 PCT

No.: PCT/SE00/00149 PCT Pub.

Patent: Inhibition of marine biofouling of surfaces

Mia Dahlström Fil. Dr. 2004

Lena Lindal

*Very low acute toxicity

*Induce a swimming reflexon cyprid larvae

Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisusAuthor(s): Dahlstrom M, Martensson LGE, Jonsson PR, et al.Conference Information: International Symposium on Marine Biofouling, JUL 07-09, 1999 UNIV PLYMOUTH, PLYMOUTH, ENGLANDSource: BIOFOULING Volume: 16 Issue: 2-4 Pages: 191-+ Published: 2000

+ 17 other publications

PUBLICATIONS Medetomidine antifouling

1. Title: Bioaccumulation of the new antifoulant medetomidine in marine organismsAuthor(s): Hilvarsson, A; Ohlauson, C; Blanck, H, et al.Source: MARINE ENVIRONMENTAL RESEARCH Volume: 68 Issue: 1 Pages: 19-24 Published: 2009

2. Title: Adsorption of antifouling booster biocides on metal oxide nanoparticles: Effect of different metal oxides and solventsAuthor(s): Shtylova, L; Fant, C; Handa, P, et al.Source: PROGRESS IN ORGANIC COATINGS Volume: 64 Issue: 1 Pages: 20-26 Published: 2009

3. Title: Melanophore sensitivity and pigmentation in fish exposed to medetomidineAuthor(s): Lennquist, A; Martensson, L; Forlin, LSource: MARINE ENVIRONMENTAL RESEARCH Volume: 66 Issue: 1 Pages: 73-73 Published: 2008

4. Title: Effects of medetomidine on hepatic EROD activity in three species of fishAuthor(s): Lennquist, A; Celander, MC; Forlin, LSource: ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY Volume: 69 Issue: 1 Pages: 74-79 Published: 2008

5. Title: Medetomidine as a candidate antifoulant: Sublethal effects on juvenile turbot (Psetta maxima L.)Author(s): Hilvarsson, A; Halldorsson, HP; Granmo, ASource: AQUATIC TOXICOLOGY Volume: 83 Issue: 3 Pages: 238-246 Published: JUL 20 2007

6. Title: Effects of a candidate antifouling compound (medetomidine) on pheromone induced mate search in the amphipod Corophium volutatorAuthor(s): Krang, AS; Dahstrom, MSource: MARINE POLLUTION BULLETIN Volume: 52 Issue: 12 Pages: 1776-1783 Published: DEC 2006

7. Title: Alteration of CYP1A activity in fish as a response to medetomidine exposureAuthor(s): Lennquist, A; Forlin, LSource: MARINE ENVIRONMENTAL RESEARCH Volume: 62 Pages: S72-S73 Supplement: S Published: 2006

8. Title: Antifouling agent release from marine coatings-ion pair formation/dissolution for controlled releaseAuthor(s): Handa, P; Fant, C; Nyden, MSource: PROGRESS IN ORGANIC COATINGS Volume: 57 Issue: 4 Pages: 376-382 Published: DEC 1 2006

9. Title: Complexation chemistry for tuning release from polymer coatingsAuthor(s): Fant, C; Handa, P; Nyden, MSource: JOURNAL OF PHYSICAL CHEMISTRY B Volume: 110 Issue: 43 Pages: 21808-21815 Published: NOV 2 2006

10. Title: Effects of medetomidine, a novel antifouling agent, on the burrowing bivalve Abra nitida (Muller)Author(s): Bellas, J; Hilvarsson, A; Birgersson, G, et al.Source: CHEMOSPHERE Volume: 65 Issue: 4 Pages: 575-582 Published: OCT 2006

PUBLICATIONS Medetomidine antifouling

13Title: Interaction between medetomidine and alkyd resins: NMR and FTIR investigation of antifouling marine paint model systemsAuthor(s): Shtykova, L; Ostrovskii, D; Jacobsson, P, et al.Source: JOURNAL OF APPLIED POLYMER SCIENCE Volume: 99 Issue: 5 Pages: 2797-2809 Published: MAR 5 2006

12. Title: Sublethal effects of a new antifouling candidate on lumpfish (Cyclopterus lumpus L.) and Atlantic cod (Gadus morhua L.) larvaeAuthor(s): Bellas, J; Hilvarsson, A; Granmo, ASource: BIOFOULING Volume: 21 Issue: 3-4 Pages: 207-216 Published: 2005

13. Title: Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus Author(s): Dahlstrom, M; Lindgren, F; Berntsson, K, et al.Source: JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-COMPARATIVE EXPERIMENTAL BIOLOGY Volume: 303A Issue: 7 Pages: 551-562 Published: JUL 1 2005

14. Title: NMR diffusometry and FTIR in the study of the interaction between antifouling agent and binder in marine paintsAuthor(s): Shtykova, LS; Ostrovskii, D; Handa, P, et al.Source: PROGRESS IN ORGANIC COATINGS Volume: 51 Issue: 2 Pages: 125-133 Published: NOV 2004

15. Title: Impact of polymer surface affinity of novel antifouling agentsAuthor(s): Dahlstrom, M; Jonsson, PR; Lausmaa, J, et al.Source: BIOTECHNOLOGY AND BIOENGINEERING Volume: 86 Issue: 1 Pages: 1-8 Published: APR 5 2004

16. Title: Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisusAuthor(s): Dahlstrom, M; Martensson, LGE; Jonsson, PR, et al.Conference Information: International Symposium on Marine Biofouling, Date: JUL 07-09, 1999 UNIV PLYMOUTH PLYMOUTH ENGLAND Source: BIOFOULING Volume: 16 Issue: 2-4 Pages: 191-+ Published: 2000

New solutions ...

Streptomyces avermitilis - a Bacteria living in soil

Produce Avermectin

A macrocyclic lactone

A large and water insoluble

Molecule

Many variants such as

ivermectin, selamectin,doramectin and abamectin.

Avermectin – A solution from Nature

*Very low toxicity (harmless) to Humans.

*High acute toxicity to crustacean and most insects.

Avermectin – mode of action

Glutamate – gated chloride channel

Cl-

Avermectin

Increase of Chloride permeability

Cell – membraneNerve or muscle

Loss of nervousfunction

Source of Avermectin

Contains Ivemectin 10 mg/mlGlycerol formal 40%Propylene glycol 60% Co-solvents

1. Pure Ivermectin

2. Ivermectin for vetrinary use (Injection)

Field experiment

Avermectin additive - three month

Toxicity levels EC50 (LD50) of Avermectin in water solution.

Adult Barnacle (B.improvisus)..................................... 1 ng/mlCyprid larvae (B.improvisus)....................................... 1 ng/mlNauplie (B. Improvisus).......................................below 1 ng/ml

Artemia salina (brine shrimp) ...............................below 1ng/ml

Toxicity levels EC50 (LD50) of Avermectin-containing coatings

Metod. Avermectin (Ivermectin) was added in colofonium based paintformulations and was painted on small panels. Elution (soaking) of panels was made at lab with stirring seawater for several weeks. Toxicity of elution waterwas tested on Artemia.

Experiment Toxic levels (1/2 dilution step)

Expected resultA 10 % erosion of the paint 10 µg Avermectin/ml Dilution about1/1.000.000 to reach non-toxic levels!

Petri-dish with painted panel

ArtemiaTox test

Toxicity levels EC50 (LD50) of Avermectin-containing coatings

Metod. Avermectin (Ivermectin) was added in colofonium based paintformulations and was painted on small panels. Elution (soaking) of panels was made at lab with stirring seawater for several weeks. Toxicity of elution waterwas tested with Artemia.

Experiment Toxic levels (1/2 dilution step)

Real resultPure coating ..................................................................... 0 - 1/4Avermectin (1 %) containing coating .............................. 0 – 1/32 Very lowAvermectin (1%) + 10 % Cosolvent................................. 0 - !/32

Conclusion. The anti-barnacle effect of Avermectin-coatings are not related toemission (leaching) of Bioactive avermectin

Petri-dish with painted panel

Atremia

Soft807-3

Hard807-1

Front Backside

Penetration effects of Barnacle in soft (and very hard) polymers. No Avermectin added.

Photo: Mats Hulander

No paintat side

See penetration !

No penetration !

See paintat side !

Control sideAvermectin side

Hard Coating

Soft Coating

Barnacle

No Barnacle !

Field experiment – Hard and soft coatings

Avermectin – Post settlement inhibition (PSI) on soft

polymers

Small barnacleCoating

Support

Without AvermectinAdult Barnacle

With Avermectin

Coating

A AAADetachment

Penetration

AVery little emission As judged by Artemiaassay

Wikstrom, SA; Pavia, H Chemical settlement inhibition versus post-settlement mortality as an explanation for differential fouling of two congeneric seaweedsOECOLOGIA Volume: 138 Issue: 2 Pages: 223-230 Published: JAN 2004

Natural Phenomena !

Influence of Cosolvent in Ivomec

Inhibition of Barnacle growth

with respect to control

%

Control Experiment

Field panel

Low toxic colofonium basedPaint (xylol)

Three summer month

Synergisticeffect !

n=6

One explanation to the cosolvent synergistic effect

Metod: Flourescent Avermectin and flourescence mircroscopColofoniumbased formulation dissolved in Xylol

Avermectin_10% CosolventAvermectin -No Cosolvent

Increased dispersionIncreased Bio-avaliability

Solvent incompatibility, not cosolvent

Boat experiments

Boat Association at the westcoast of Sweden

Avermectin boat-experiment -08

Three types of ”Biocidal” free paints were used.

SoftSemi - soft Propeller (semi-soft)

Avermectin was added into this paints (0.1%)

Very Hard(Copper)

Semi-soft anti-fouling paint. Four month in Swedish water. Photo. Mats Hulander Båtägare: Mauritz Palm.

AvermectinSurface

Control

Surface

Soft anti-fouling paint . Four month in Swedish water. Photo and Boat owner: Jesper Eriksson

AvermectinSurface

ControlSurface

Motor Boat (Volvo Penta) Four month in Swedish water. Photo Lars-Erik Ohlsson, Volvo Penta.

AvermectinSurface

ControlSurface

Copper paintTF + Avermectin

TC Nano

Propeller. Boatowner and och Photo: Hans Andersson

Very hard anti-fouling paint . Four month in Swedish water. Photo and Boat owner: Jan Rydström

With 0.1 % Avermectin No Avermectin

Barnacles

No effect of Avermectin on Barnacle Colonisation.

The (PSI) Avermectin project + Inexpensive- 0.1 Euro /l paint

+ Apperant low non-target effects+ Applicable in most soft paints+ Previous marine use (Salmon farming)+Low concentration (0.001 %)+ Replaces 30 tonnes/y of copper i Sweden

Conclusions

Next Step

Use of non-eroding polymers (very low emission). Quantification of emission (HPLC)May replace DDT in many countries (eg. China)Isolation of more PSI-substances

Yong salmon infested with sea-lice

Avermectin (Emamectin benzoate)

Is used as food additive for salmon

for sea-lice protection. No adverse

ecotoxicological effect

are reported

About 25 kg Avermectin is used

annually in Norway. Corresponds

to 2 million boats for similar risk

Avermectin in salmon farming

Conclusion – The cosolvent project

Application to other Biosides (eg Boroside, sea-nine)Dispersion analysis with TOF-Sims

+ Inexpensive+ Little registration efforts

The research group

University of GothenburgSP, Borås, SwedenSwedish Research Counsil

Emiliano Pinori Ph. D. Student (Marine antifouling)

Mats Hulander, Ph. D. Student (Biomaterials)

Anders Lundgren Ph.D. Student (Biosensors)

Mia Dahlstrom, Ph. D. (Marine Biofouling)

Mattias Berglin Ph. D. Associate Professor

David Wessling Diploma worker

Marinord- A research programme

Thats all folks !