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Development of an innovative, completely automated antifouling test system for professional examinations of marine coatings

Final Report Summary - IATS (Development of an innovative, completely automated antifouling test system for professional examinations of marine coatings)

Executive Summary:
Prevention of fouling on ships, bridges, pillars, platforms, buoys and all others staff made from wood, polymers or steel seems to be a significant problem nowadays. Fouling cause decreasing of efficiency of ships transport because it influence on sailing speed and fuel consumption. Scientists all over the world try to develop coatings that will prevent of fouling on all elements immersed in sea or fresh water. Development of new coating formulation takes about 5 years now, beginning from R&D study to the date of launching to the marked of new product. Taking into account the marine market development (few percent per year) and the continuous change of low requirements connected to the environmental issues (these requirements directly influence on coating formulations) this time distance seems to be too long. Formulation time distances is depended not only on formulation scientific problems but also on testing of developed coatings formulations. Nowadays the only available in Europe and common used all over the World method is testing in natural conditions. Ocean immersion testing is generally considered the "gold standard" for evaluating the performance of marine coatings. Antifouling tests are carried out in different regions of the world (different seas, oceans, rivers) to check performance in different biological and chemical environmental conditions. Coatings are typically prepared on large raft panels and immersed in the ocean to evaluate the accumulation and adhesion strength of fouling organisms over time. The way of testing and the big amount of places where the new formulation must be tested cause very high price of new coating formulation development. Just antifouling tests cost about 100 000 euro for 5 samples per one year. The high price and the long time duration from beginning of formulation development till the new product launching cause too long payback time for SME that exist on the market. That is the reason why just big companies are able to develop new coating formulation and the marked is not available for SMEs. Significant decrease of antifouling test performance will cause large decrease of new coating formulations development time and cost, will let SMEs to enter to the market and finally will decrease the antifouling coatings prices. Basing on the previous study of RTD project partners - project's idea was to develop, prepare and implement of an innovative, completely automated antifouling test system for professional examinations of marine coatings. The developed test system makes it possible to perform tests in laboratory conditions and gives results in a much shorter time than experiments carried out in natural conditions. We shall undertake to decrease the test time by a minimum 500% (in reality, the results are seen after 10 days). The appearance of fouling is measured automatically by the image analyzer. The test system allows one to detect the appearance of fouling even if it is not visible to the naked eye. The developed system makes it possible to observe also the differences in the rate of fouling on different samples. The tests is able to be carried out automatically and on a continuous basis (24 hours a day) without supervision. The test data are collected and stored on a continuous basis by the database and this data are available on-line for all the network users. Selected species are so characteristic as to ensure that during laboratory tests the worst conditions for coatings that exist in natural conditions are replicated. The project evaluation let us to construct relatively cheap, available for SME antifouling test set that let European SME to enter to the huge marine coatings market and become a rival company for market potentates.

Project Context and Objectives:
Ships, bridges, pillars, platforms, buoys and all others staff made from wood, polymers or steel which are immersed in the sea or fresh water are exposed to the biofilm fouling and corrosion. That is the reason that development of coating with good antifouling properties makes a high interest of scientists and companies that produce coatings.
Fouling that appears on immersed in water elements cause not only problems with repainting those elements every few years but also cause decreasing of efficiency of ships transport because it influence on sailing speed and fuel consumption.
Antifouling solutions can cause some fuel savings what is a very important factor nowadays when the savings of mineral oil sources play a crucial role. In the same way antifouling coatings influence on transport efficiency by reducing the speed decreasing closed by biofilm. So the antifouling coatings formulation development seems to play one of the crucial role in efficiency of marine transport.
The antifouling solution are still expensive and the marked is completely controlled by big companies. The problem nowadays is long time duration and very high cost of coatings formulation development. Development of new coating formulation takes about 5 years now, beginning from R&D study to the date of launching to the marked of new product. Taking into account the marine market development and the continuous change of low requirements connected to the environmental issues (these requirements directly influence on coating formulations) this time distance seems to be too long.
The high price of development study and the long time duration from beginning of formulation development till the new product launching cause too long payback time for SME that exist on the market. That is the reason why just big companies are able to develop new coating formulations and the marked is not available for SMEs.
Formulation time distances is depended not only on scientific problems but also and mainly on testing time duration of developed coatings formulations. Nowadays the only available in Europe and common used all over the World test method is coatings examination in natural conditions. Ocean immersion testing is generally considered the “gold standard” for evaluating the performance of marine coatings. Antifouling tests are carried out in different regions of the world (different seas, oceans, rivers.) to check performance in different biological and chemical environmental conditions.
Coatings are typically prepared on large raft panels and immersed in the ocean to evaluate the accumulation and adhesion strength of fouling organisms over time. In the figures bellow are shown two different coating samples after two years exposure in natural sea water conditions.
The way and places of antifouling test examination cause very high cost of those tests. Antifouling test of just 5 samples of coatings cost about 100 000 euro per year; and the standard amount of tested samples is about 50 per year or more. After failing the two years duration antifouling tests the formulation is reformulated and the sample must be tested for two years again.
That is the reason why just big companies with big R&D budget are able to develop new coating formulations and the marked is not available for SMEs.
The new test system is going to be produced by European SME and is dedicated for the European SMEs. Developed in the project test system will contribute to the increasing significance of SME in a market that is dominated by potentates.

Scientific and technological objectives
The overall technical objective was to develop, prepare and implement of an innovative, completely automated antifouling test system for professional examinations of marine coatings. The developed test system makes it possible to perform antifouling tests in laboratory conditions and gives results in a much shorter time than experiments carried out in natural conditions. In addition, specific technological objectives were:
1) To develop biological technology by using of fresh water species that let to achieve results comparable to the tests performed in natural conditions;
2) To decrease test time by a minimum 500%;
3) To develop technology that will let to detect the appearance of fouling even if it is not visible to the naked eye;
4) To develop technology that let tests to be carried out automatically and on a continuous basis (24 hours a day and simulation of changes time day – intensity on light) without supervision;
5) To develop technology that let test data to be collected and stored on a continuous basis by the database and this data will be available on-line for all the network users;
Background technological objectives were:
6) To develop simply in use, cheap and user friendly test system for fast antifouling testing.

The scientific objective of the project involve the extension to current knowledge of :
• mechanism of fouling and corrosion of coatings
• relation between fresh and sea water species and the correlation of fouling
• accelerate of time duration of developing of new coatings formulations and faster launching of new products to the market
• decreasing of price of antifouling tests and open the market for SME
Scientific base of project
The processes of surface fouling have few stages: molecular fouling (conditioning film), microfouling (by bacteria and later by microalgae and fungi), macrofouling (by macroalgae and invertebrates).
There were selected some of the freshwater species having desirable properties. These species in freshwater create the periphyton assemblage. This community is composed of a complex mixture of algae, cyanobacteria, heterotrophic microbes, and detritus that is attached to submerged surfaces. Hydrobiologist diverified few types periphyton: Epilithon (community which includes the attached microorganisms found on the rocks and stones of the aquatic environment); Epixylon (refers to the microorganisms found on the fallen woods in water bodies); Epipsammon (community of microorganisms associated with sand grains); Epipelon (microorganisms inhabiting detritus and fine sediment surface are referred to as epipelon) and Epiphyton (community inhabiting submerged surface of macrophytes).
Species for fauling assays will be searched among these periphytonic algae and animals. We want to verify some taxons.
Diatoms (Bacillariophyta). Generally, diatoms are good candidates for testing. They are scurvy, able to movement and are able to adhere (to sick) better than double-sided sticky tape. The observed motility rates of individual cells of 0.1 to 0.3 mm /s.
Bluegreens (Cyanobacteriaceae). These mat-forming Cyanobacteria are slowly motile, presumably moving in response to oxygen tension, carbon dioxide concentration, and light. Some of these species are very promising,
Greens, (Chlorophyceae). Despite of typical planktonic species there are a lot of sessile species suitable in bioassays.
Protists (Protozoa) are an animal member of fouling community. There are colonial peritriches (Carchesium), some suctorians (Stentor, Podophrya fixa). They have a preference for stones or fragments of detritus as settling sites. All have developed efficient mechanisms for trapping pass in food, an essential attribute in non-motile organisms. All of these sessile ciliates (peritrichs) feed on non-attached bacterial species. For some species of protists, Amarat et al. (1999) successfully used Principal Component Analysis (PCA) technique for species identification. A three-dimensional representation was generated and several protozoa (Opercularia, Colpidium, Tetrahymena, Prorodon, Glaucoma and Trachelophyllum) species could be positively identified. This programme was created to semi-automatically analyse protozoal digitised images.

Project Results:
The whole concept of the project was divided for two parts: biological and mechanical. In the biological part there were species isolated and tested and tests methods developed. In the mechanical part there were designed and constructed mechanical parts of IATS tester including analytical software.


1. Culture of a collection of pure test species.
Out of all isolated algal and cyanobacterial strains (see WP1 report) 25 was preselected for further investigation. The main criterion for the strain selection was a satisfactory growth in laboratory conditions. Although some of the species comprised a frequent component of biofilms (e.g. the rhodophyte Audouinella), we were not able to maintain the algal culture for a prolonged time period, which is necessary for the purpose of the IATS project. Exact determination of all selected strains was not possible, based solely on morphological characters, seen in the light microscope (genus name of the species is followed by sp. - e.g. Leptolyngbya sp.). Sequencing of selected molecular markers (mostly used markers are 16S rDNA for Cyanobacteria and 18S rDNA for eukaryotic algae) has to be employed for exact characterization of the strains. Cyanobacterial strains were represented by Phormidium molle (SAG 26.99) Leptolyngbya sp. (HR5), Calothrix sp. (Probe 11 Klon 5_1) and by a marine strain Pseudanabaena lonchoides (Zurek). Nine isolates of diatoms (Stramenopiles, Ochrophytes) originated from freshwater biofilmes - Achnanthidium minutissimum (SAG RJ D410), Achnanthidium sp. (EH1 Klon 1), Navicula sp.1 (Lovosice), Navicula sp.2 (Lovosice), Navicula sp.3 (Lovosice), Gomphocymbella sp. (Lovosice), Nitzschia sp. (Lovosice), Achnanthidium sp. (EH1 Klon 3), Achnanthidium sp. (FH3 Kl. 2A), Nitzschia sp. (Probe 8 Klon 11). Two diatom strains (Achnanthes sp. KATZ 09425 and Navicula perminuta Zurek ) were isolated from PPG rafts in Kats (Nederland), where samples of coatings are tested in natural seawater. Diatoms represent an important part of both freshwater and marine biofilms, the cells are capable of producing extracellular polysaccharides (EPS) that may ease their attachment to the solid surfaces. However, long term maintaining of diatom strains may be problematic. Each diatom cell is enclosed in a unique cell wall made of silica (frustule), which is composed of two overlapping parts (reminding the box and its lit). Reproduction among these organisms is primarily asexual, with each daughter cell receiving one of the parental frustule parts. This one is used as the larger part, into which second, smaller part is constructed. This form of division results in a size reduction of the population up to about one-third their maximum size. In order to restore the size of the population, sexual reproduction must occur. However, in the cultures that resulted from a single cell, captured by a glass micropipette, sexual reproduction may not happened, as the produced gametes (sexual cells) are either male or female, but not both. Only one strain of the Xanthophyceae – class related to diatoms (Stramenopiles, Ochrophytes), Tribonema sp. Lovosice was obtained. Frequent isolates belonged to green algae of Trebouxiophyceae (Heterochlorella luteoviridis, Chlorella sp. Lovosice, Diplosphaera sp. Lovosice, Sphaerochlamydella sp. Lovos.), Chlorophyceae (Stigeoclonium sp. Lovosice, Stigeoclonium sp. EX07 K01, Chlorococcum sp. Zurek 04, Chlorococcum sp. Lovosice, Ankistrodesmus sp. V.H.S 5). The filamentous green alga Klebsormidium sp.Lovosice belongs to the evolutionary linage that gave rise to vascular plants. The strains selected for further experiments and tests are listed in Table 1.

Table 1. The list of strains used in growth experiments and/or in anti-fouling tests. Information concerning the selected optimal medium, isolation and optimal culture conditions (where investigated) is provided. Taxonomical groups of algae are differentiated by color boxes. SAG = Sammlung von Algenkulturen Gottingen (SAG Culture Collection), CUNI – Charles University in Prague, INNOPOL – Innowacja Polska, UGOE – University of Goettingen, BrM – Brakish water medium, BBM - Bold's basal medium, WC – WC culture medium. – See Annex 1

For the long term maintenance the strains were kept as a liquid cultures in triplicates (50 ml Erlenmayer flask covered with aluminum foil) and/or on agar slants and subcultured regularly. The strains were placed in a special refrigerator (conditioned for 17 ºC), with continuous illumination of 40µmol m-2 • s-1 provided by 18W cool fluorescent tubes (Philips TLD 18W/33).

2. Investigation of optimal growth conditions for test species

- to investigate the optimal growth conditions of isolated cyanobacteria and algae (temperature, irradiance)
- to suggest time of cultivation

Before the algal strain was used for the growth experiments, sufficient volume of exponentially growing dense culture had to be obtained. Cells were transferred from the long-term maintenance suboptimal conditions to about 0.5 liter of the fresh medium and kept at 22 ºC for ca.14 days, until the sufficient biomass was produced. Cultivation in crossed gradients of temperature and light was performed in a Labio unit (Czech Republic). The Labio unit is composed of the thick metal desk, where the right side is cooled while the left side is warmed – providing a continuous gradient of temperature; for details see Kvíderová & Lukavský (2001). Diluted, exponentially growing, culture was pipetted to 96 (for diatoms) or 9-wells culture plates. Cells were cultivated for 9-15 days under continuous illumination (daylight fluorescent tubes Osram Dulux L). The strains were cultivated in 25 combinations of five temperature levels (5°C, 10°C, 15°C, 20°C, 25°C) and five irradiance levels (42, 60, 86, 127, 165 μmol.m-2.s-1) with three replicates in each combination of factors. Biomass was estimated as percentage cover or by using an image analysis software (NAJA; Hauer & Jirka, 2007) in three day intervals. 2D graphs were obtained by Sigmaplot software.

Survey of optimal growing conditions (temperature and light intensity) of strains investigated in a cross gradient of temperature and light is provided in Table 1 (See Annex 1).

Cyanobacterium Phormidium molle SAG 26.99 has relatively broad temperature range as it grows in the whole temperature gradient, however it has narrow irradiance optimum of 42 μmol.m-2.s-1 (Fig. 2). At lower temperature P. molle is able to sustain a higher irradiance level (up to about 100 μmol.m-2.s-1) production of secondary carotenoids is induced to protect the cells from higher light intensity, consequently the whole biomass converts to the red color. Optimal conditions: 15 ºC, 42 μmol.m-2.s-1.

Figure 1. Assessment of optimal growing conditions in Tribonema sp. (Lovosice). Inserted figures show the amount of biomass produced in a triplicate of wells under the conditions tracked by an arrow. See Annex 1

Nine strains of diatoms were investigated. The growth optimum of Achnanthidium sp. (EH1 Klon 1) is shifted towards higher temperatures with a broad tolerance to light intensity levels. At lower temperatures (10-15 ºC) the optimal conditions lie at the lower light intensity levels (Fig. 3a). Optimal conditions: 20 ºC, 42-60 μmol.m-2.s-1.
Nitzschia sp. (Probe 8 Klon 11) provides very similar pattern of optimal growth conditions compared to Achnanthidium sp. (EH1 Klon 1) with a broad tolerance to high light intensity (Fig. 3b). At lower temperatures (10-15 ºC) the optimal conditions lie at the lower to medium light intensity levels (up to 100 μmol.m-2.s-1). Optimal conditions: 20 ºC, 86-127 μmol.m-2.s-1.
In Nitzschia sp. (Lovosice) two optima were revealed. A dense culture was provided at lower temperature (15 ºC) and light intensity of 60 μmol.m-2.s-1 however even higher biomass was obtained in a combination of higher temperature (25 ºC) and low irradiance (Fig. 3c). Optimal conditions: 25 ºC, 42 μmol.m-2.s-1.
Navicula sp.1 (Lovosice) represents a diatom strain growing in a narrow range of optimal conditions provided by high temperature combined with high light intensity (Fig. 3d). Optimal conditions: 25 ºC, 127 μmol.m-2.s-1.
Navicula sp.2 (Lovosice) is growing in a narrow range of optimal conditions. Optimum is shifted to higher temperature combined with low light intensity (Fig. 3e). Optimal conditions: 25 ºC, 60 μmol.m-2.s-1.
Navicula sp.3 (Lovosice) still has a relatively restricted range of optimal conditions. The shift towards higher temperatures has to be compensated by higher irradiance in order to sustain optimal growth (Fig. 3f). Optimal conditions: 20 ºC, 86 μmol.m-2.s-1.
Gomphocymbella sp. (Lovosice) represents a strain with extremely broad temperature tolerance (within the whole gradient of investigated temperature: 5-25 ºC) and optimum in lower to medium light intensity (Fig. 4a). Optimal conditions: 20-25 ºC, 42-60 μmol.m-2.s-1.
Achnanthidium sp. (EH1 Klon 3) growth optimum is shifted towards higher temperatures with higher light intensity levels. At lower temperatures (10-15 ºC) the optimal conditions lie at the lower light intensity levels (Fig. 4b). Optimal conditions: 20 ºC, 140-160 μmol.m-2.s-1. Achnanthidium sp. (FH3 Klon 2A) has an optimum growth in a relatively narrow range of temperature combined with a wide range of light intensity (Fig. 4c). At 20 ºC compared to 15 ºC the optimum is shifted towards higher light intensity. Optimal conditions: 15 ºC, 60 μmol.m-2. s-1.

Figure 2. Phormidium molle SAG 26.99 - a graph illustrating biomass amount at the end of the cultivation period (9 days) on the crossed gradients. Maximum amount of biomass indicated in red, minimum in blue color. See Annex 1.
Figure 3. Graph illustrating biomass amount at the end of the cultivation period (15 days) on the crossed gradients in diatoms. a) Achnanthidium sp. (EH1 Klon 1. b) Nitzschia sp. Probe 8 Klon 11. c) Nitzschia sp. Lovosice. d) Navicula sp.1 Lovosice. e) Navicula sp.2 Lovosice. f) Navicula sp.3 Lovosice. Maximum amount of biomass indicated in red, minimum in blue color. See Annex 1.
Figure 4. Graph illustrating biomass amount at the end of the cultivation period (15 days) on the crossed gradients in diatoms. a) Gomphocymbella sp. Lovosice. b) Achnanthidium sp. EH1 Klon 3. c) Achnanthidium sp. FH3 Kl. 2A. See Annex 1.

Maximum amount of biomass indicated in red, minimum in blue color.
Tribonema sp. Lovosice provides on optimum growth in combination of low temperature and low irradiance, while at higher temperature, medium light intensity supports best conditions for growth (Fig. 1). Optimal conditions: 20-25 ºC, 60 μmol.m-2.s-1.

Green algae
Seven strains belonging to three different classes and two different evolutionary lineages (Trebouxiophyceae, Chlorophyceae – chlorophyte lineage and Klebsormidiophyceae – streptophyte lineage) were investigated.
Chlorella sp. (Lovosice) is the strain with broad irradiance optimum (except for the lowest values) and relatively broad temperature optimum (Fig. 5a). Chlorella sp. is not inhibited even at the maximum tested level of light intensity (165 μmol.m-2.s-1). Optimal conditions: 15-20 ºC, 86-126 μmol.m-2.s-1.
Sphaerochlamydella sp. (Lovosice) has its growth optimum in combination of higher temperature and medium irradiance. The shift towards higher temperature has to be compensated by higher irradiance in order to sustain optimal growth (Fig. 5b). Optimal conditions: 20 ºC, 86 μmol.m-2. s-1.
Stigeoclonium sp. (Lovosice) has a broad range of optimal temperature (the whole tested gradient 5-25 ºC), however this strain is able to grow only at low to medium levels of light intensity. The growth is completely inhibited at higher light intensity levels (Fig. 5c). Optimal conditions: 15-25 ºC, 42-60 μmol.m-2.s-1.
Chlorococcum sp. (Zurek 04) represents the strain with the broadest growth conditions investigated so far. Except for very high levels of light intensity, Chlorococcum sp. Zurek 04 is able to grow sufficiently in a whole gradient of combined variables (Fig. 5 d). Optimal conditions: 5-15 ºC, 42-60 μmol.m-2.s-1.
Chlorococcum sp. (Lovosice) has an optimum for growth shifted towards higher temperature and low to medium irradiance (Fig. 5e). Optimal conditions: 25 ºC, 42 μmol.m-2.s-1.
Ankistrodesmus sp. (V.H.S) represents a strain with a narrow temperature range accompanied by a wide irradiance range (Fig. 5f). Optimal conditions: 20 ºC, 86 μmol.m-2.s-1.
Klebsormidium sp. (Lovosice) is able to grow optimally in en extremely low range of studied conditions. Klebsormidium sp. (Lovosice) is able to sustain only lowest light intensity (Fig. 6). Optimal conditions: 20-25 ºC, 42 μmol.m-2.s-1.

Figure 5. Graph illustrating biomass amount at the end of the cultivation period (9 days) on the crossed gradients in green algae. a) Chlorella sp. Lovosice. b) Sphaerochlamydella sp. Lovosice. c) Stigeoclonium sp. Lovosice. d) Chlorococcum sp. Zurek 04. e) Chlorococcum sp. Lovosice f) Ankistrodesmus sp. V.H.S. Maximum amount of biomass indicated in red, minimum in blue color. See Annex 1
Figure 6. Graph illustrating biomass amount at the end of the cultivation period (9 days) on the crossed gradients in a streptophyta alga Klebsormidium sp. (Lovosice). Maximum amount of biomass indicated in red, minimum in blue color. See Annex 1.

Algal growth in a culture is determined by number of various abiotic factors, from which temperature, pH, intensity and quality of light and nutrient supply are thought to play the prime role. Eighteen strains were investigated in a crossed gradient of light and temperature and the strain-specific optimal conditions were revealed.
Although the optimal conditions are strain-specific, some generalizations within diatoms and green algae may be concluded (see Fig. 8). Most of the diatom strains grow optimally at temperature of 20 ºC and light intensity of 86 μmol.m-2.s-1. Several strains are able to sustain optimal growth in a wide range of light intensity (e.g. Achnanthidium sp. EH1 Klon 1, Nitzschia sp. Probe 8 Klon 11 or Achnanthidium sp. FH3 Klon 2A), however none of the strains is able to grow optimally in a wide range of temperature. Green algal strains (including xanthophyt Tribonema), on the other hand, provide more diverse response. We have recorded strains with a wide range of optimal temperature (e.g. Chlorococcum sp. Zurek 04, Stigeoclonium sp. Lovosice), but also with wide light intensity optima (e.g. Chlorella sp. Lovosice, Ankistrodesmus sp. V.H.S). Most of the green algal strains grow optimally at temperature of 20 ºC and light intensity of 60 μmol.m-2.s-1.
Duration of a cultivation experiment depends on inoculum density (cell concentration at the beginning of cultivation), duration of an adaptation phase (a culture has to be adapted to the new conditions) and resource limitation (nutrients, light – the cells shade each other). The amount of biomass was estimated in three-day intervals and for this type of experimental design a 9-day and 15-day cultivation period was optimal for green algae (including cyanobacterium Phormidium and xanthophyte Tribonema) and diatoms, respectively.

Figure 7. The summary of the optimal growth conditions in diatoms and green algae. The image arose by projection of the growth optimum ranges of diatoms (green algae) into a single 2D diagram. See Anex 1.

3. Investigation of optimal culture medium for test species.
To keep the operation of the IATS test system as easy as possible, we selected two basic inorganic culture media, a Bold's basal medium (BBM; Bischoff & Bold 1963) for all strains except diatoms and WC medium (Guillard & Lorenzen 1972) for freshwater diatoms. Brackish water medium with 1% Silicate was used to perform anti-fouling tests on marine strains in order to compare fouling properties of marine vs. freshwater species. Recipes for all culture media used are listed in a Table 2. Investigated algal and cyanobacterial strains and media used for their cultivation are listed in Table 1. BBM medium is also available commercially as mixed stock solution (Sigma-Aldrich Company, B5282). The stock solution is sterilized and contains all vitamins, 2 ml of stock solution diluted into 1000ml of distilled sterilized water provide standardize BBM medium ready for use. This can ease the supply of the medium to the users of proposed IATS test system.

Table 2. The culture media used for maintenance, growing experiments and anti-fouling tests. BBM = Bold's basal medium. See Annex 1.

A special interest is in elaborating procedure of testing the toxic samples in reasonable time.
In order to do it, the special procedure was tested. We obtained two kinds of toxic samples prom PPG named as AF1 and AF2. PPG said us the chemical constitution of them. Paints contains 30 and 50 weight % cuprous oxide and between the 1 and 5 weight % of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one.
This compound is also known as DCOIT; 4,5-Dichloro-2-n-octyl-3(2H)-isothiazolone; 4,5-Dichloro-2-n-octyl-isothiazolin-3-one; 4,5-Dichloro-2-octyl-3(2H)-isothiazolone; Duracide L; Isothiazolone, 4,5-dichloro-2-octyl-; Kathon 287T; Kathon 930; Sea Nine-211. Molecular Formula is C11H17Cl2NOS. Melting point 44°C. Compound is used as Fungicide, algaecide applied in marine paint, coating and plastic. Is distributed by PPG Coatings Europe BV, Hempel UK Ltd, Jotun Paints (Europe) Ltd.
We studied literature in this matter. Settlement and subsequent development on surfaces require some form of resistance to or protection from the incorporated biocides. The minimum leaching rate necessary to prevent animal and plant fouling on cuprous and mercuric oxide paints was calculated as 10 μg cm−2 day−1 and 2 μg cm–2 day –-1 respectively (Harris, 1946; Anon, 1952). Different parameters are used for effect of Cu estimation: survival time, spawning per female, root length, respiration rate, respiration, reproduction, phosphatase activity, nutrient uptake, nitrogen fixation, mortality, life time, ingestion rate, hatching, immoblization, growth rate, egg production, dry weight, diversity, deformations, de-flagellation, filtration rate and so on.

Conception of diminishing toxicity.
To decrease and accelerate leaching rate toxic, paints were washed in distilled water in room temperature and in 50-60 oC during almost half a year. Samples were exposed in 600 ml bakers. Water was changed every 5 days at the beginning of experiment later every 10 days and then, every 14-18 days. After paint exposition water divided into two subsamples. The first one was analysed for Cu concentration and second one of 250 ml was inoculated by few ml of dense culture of was Scenedesmus acutus. Prior inoculation basic solution of medium Jaworski was added. During 10 days number of cells was counted by Coulter Counter for obtaining the growth curve. All growth bioassays were negative during 180 days in both temperature variants of leaching.

4. Laboratory anti-fouling tests on selected freshwater species
- to perform extensive laboratory anti-fouling tests, where the marine coating samples will be submerged and exposed to selected freshwater species

Anti-fouling tests on freshwater species were conducted at the University of Goettingen (UGOE) and at the Charles University (CUNI). 11 strains were investigated (one cyanobacterium, four diatom strains and six green algal strains). Phormidium molle SAG 26.99 and Chlorococcum sp. (Zurek 04) were tested in both UGOE and CUNI laboratories. Exponentially growing dense cultures served as inoculum. Experiments were conducted in triplicates. Methodologies used in both laboratories were congruent, however differed in some details.
UGOE: the strains were grown in 100ml Erlenmeyer flasks covered by an aluminium cap under the optimal conditions (inferred in growing experiments) in a conditioned cultivation chamber (Fig. 13). Coating paint samples were placed horizontally and submersed to the algal culture. To prevent sedimentation of the cells, cultures were agitated by horizontal shaker. Fouling properties of algae on a coating paint sample, a glass bead and/or a piece of silicon were compared. Ability of algae to grow in suspension was also recorded in order to distinguish strains, that grow in the medium, however do not foul on the coating paint samples. Duration of the anti-fouling tests was: 30 days. Ca. 200 anti-fouling tests were conducted (different combinations of coating paint samples and tested algal (cyanobacterial) strains).
CUNI: the strains were grown in 100 ml beakers covered by an aluminium foil under the optimal conditions (inferred in growing experiments) in a conditioned cultivation room. Cultures were agitated by a magnetic stirrer. Ability of algae to grow in the culture medium and simultaneously their ability to foul on a coating paint sample were assessed. Duration of the anti-fouling tests was 30 days. 67 anti-fouling tests were conducted.
To evaluate fouling properties of tested algal strain and its ability to grow in the medium, a four-degree scale was introduced: 0 – no growth, 1 – poor growth, 2 – good growth, 3 – very good growth. The degree of algal growth was evaluated either visually or by chlorophyll fluorescence in small diatom species (Nikon Eclipse 80i) after brief rinsing in distilled water.
18 different types of coating paint samples were provided by PPG. (coating specification codes: 0, 1 (=A4), 3G, 5, 81, 82 (=A2), 83, 84, 85 (=A5), CT, C1 (=A3), N2 (=A1), IC-13, IC-14, IC-17F, IC-6, IC-7, IC-8).

Results and conclusions
The results of anti-fouling tests on freshwater strains are summarized in Table 3. Anti-fouling tests on Phormidium molle SAG 26.99 conducted in UGOE and CUNI laboratories provided highly congruent results, however we do not have enough data on Chlorococcum sp. (Zurek 04) to compare the anti-fouling test results.
18 tested PPG coating paint samples were divided to three categories according their anti-fouling properties:
- very good anti-fouling properties (coating specification codes: 0, 81, 82 (=A2), 83, 84, 85 (=A5), CT, C1 (=A3), N2 (=A1), IC-13, IC-14, IC-17 (=F), IC-6, IC-7, IC-8).
- medium anti-fouling properties (coating specification codes: 1=A4, 3G)
- poor anti-fouling properties (coating specification code: 5)

We are aware of the fact, that some coating paint samples were not sufficiently tested, we were not supply enough pieces to conduct all anti-fouling tests and some of the coating samples were no longer available. However, the aim of this part was to find algal (cyanobacterial) strains with good fouling abilities, mimicking natural communities in marine environments, and this aim was met.

The two new series of coating paint samples were released by PPG recently (A1-A6, J1-J8). While A1-A5 were already tested previously (see Tab. 4), A6 and J1-J8 are new. There are several advantages listed by PPG:
- The series contains commercial products, so the correlation with ships in practice is possible.
- The series contain both toxic and none toxic formulations. The series contain stepwise increased amount of active material.
- PPG has these materials available on request.

We have already started testing of the new series using both freshwater and marine strains. Since both series have been exposed on the Kats rafts since July 2011, we do not have a source of correlation, yet. The results are listed in Table 4. As some samples from series J are toxic, we obtained no algal growth even in a culture medium. The new methodology, how to overcome this problem, is tested including extensive rinsing in distilled water and the use of the active carbon for toxic metal (Cu, Zn) remedition.

Table 3. The results of anti-fouling tests on freshwater strains. Ability of algae to grow in the culture medium (=culture) and simultaneously their ability to foul on a coating paint sample (=coat) were assessed. Four-degree scale was used: 0 – no growth (green), 1 – poor growth (yellow), 2 – good growth (orange), 3 – very good growth (red). See Annex 1.
Table 4. The results of anti-fouling tests provided by UGOE. The new coating series are marked in a grey colour. See Annex 1.

5. Laboratory anti-fouling tests on seawater species
- to perform laboratory anti-fouling tests, where the marine coating samples will be submerged and exposed to selected marine species

Anti-fouling tests on marine species were conducted at the University of Goettingen (Achnanthes sp. KATS 09425) and by INNOPOL (Navicula perminuta Zurek and aquarium experiments). The methodology used by UGOE was the same as described above for freshwater strains anti-fouling tests. Achnanthes sp. KATS 09425 was cultivated in seewater medium (Brackish water medium, see Table 2).
INNOPOL: Paint coating samples were exposed to a culture of Navicula perminuta (strain Zurek) in 600 ml volume beakers. Cells were cultivated in artificial sea water enriched by compounds from f/2 medium or BG11 (http://www.ccap. media/media.htm). Cultures were not agitated and samples were placed horizontally. Coating samples were observed, photographed by a classic digital camera, by a microscopic camera or by a fluorcamera (Fig. 14) and ability of algae to foul on a sample was assessed. Simultaneously grow in the culture medium was recorded.

Table 5. The results of anti-fouling tests on marine strains. Ability of algae to grow in the culture medium (=culture) and simultaneously their ability to foul on a coating paint sample (=coat) were assessed. Four-degree scale was used: 0 – no growth (green), 1 – poor growth (yellow), 2 – good growth (orange), 3 – very good growth (red). See Annex 1.

Results and conclusions
The results of anti-fouling tests on Achnanthes sp. (KATS 09425) and Navicula perminuta (Zurek) are listed in Table 5. Performance of different coating paint samples in these two cultures is compared to data obtained in natural marine localities in the next chapter (Task 2.6.)
Results on two new series of coating paint samples (A1-A6 and J1-J6) are summarized in Table 6. Similarly as in freshwater algae tests, the toxic samples totally prevented the growth of algae even in the culture (e.g. J1), although some samples were evidently less toxic (J5 and J8). Pseudanabaena lonchoides in an aquarium coped better with toxicity of some samples due to a higher volume of medium.

Table 6. Results of anti-fouling tests on marine strains in a 20 l aquarium and 150 ml beakers. Four-degree scale was used: 0 – no growth, 1 – poor growth, 2 – good growth, 3 – very good growth. See Annex 1.

6. Comparison of results of freshwater and seawater tests with marine coating data
- to compare the results of anti-fouling laboratory tests with PPG data based on long-term investigation of submerged samples inthe seawater rafts
- to select the best strains for the IATS test technology

Long-term testing of 18 coating paint samples was performed by PPG in a different marine regions and at varying lengths of time.
Anti-fouling properties of the coating paint samples were assessed and samples were divided into three categories, in the same way as for the purpose of the laboratory anti-fouling experiments (see page 16).
- very good coating with very good anti-fouling properties, the fouling appears very slow or does not appear at all (coating specification codes: 0, 81, 82 (=A2), 83, 84, 85 (=A5), CT, C1 (=A3), N2 (=A1), IC-13, IC-14, IC-17 (=F), IC-6, IC-7, IC-8).
- medium quality coating with medium anti-fouling properties, the slight fouling appears, the process of fouling is relatively slow (coating specification codes: 1=A4, 3G)
- bad coating with poor anti-fouling properties, the fouling appears very fast (coating specification code: 5)

To compare the freshwater and seawater anti-fouling tests with marine coating data the table similar to Table 3 and 5, was introduced (Table 7) containing all tested strains, both freshwater (12 strains) and marine (two strains). Samples of coating paints in the left column are colored with respect to their anti-fouling properties: bright green = very good anti-fouling properties (corresponds to category (1) described previously); ping = medium anti-fouling properties (corresponds to category (2) described previously); orange = poor anti-fouling properties (corresponds to category (3) described previously). Numbers 0-3, in the columns of algal strains, indicate the ability of algae to grow in the culture medium (=culture) and simultaneously their ability to foul on a coating paint sample (=coat). The colour of the cell indicate, whether the performance of the coating pain sample in marine environment (according to PPG data) and in the laboratory algal culture is the same. Dark green = same performance (or pass the comparison), while red = different performance (or failed in comparison). Laboratory experiments, where no algal grow was recorded, were excluded from analysis. To provide an example: the coating pain sample 5 performed poor anti-fouling properties in both Achnanthidium minutissimum (SAG RJ D410) culture and marine environment, results are congruent in both environments – the cells are colored in green. On the other hand, the coating pain sample N2 performed good anti-fouling properties in marine environment, but poor anti-fouling properties in a culture of Achnanthidium minutissimum (SAG RJ D410), results are incongruent - the cells are colored in red. The last row of the table summarizes the numbers of congruence and incongruence results (P/F) for the tested strains.

Table 7. Comparison of results of freshwater and seawater tests with marine coating data. See the text for detailed explanation of the table.7. See Annex 1.

Fig 9 (left) represents relative ratios of pass/fail in tested freshwater strains. In four strains Navicula sp.3 (Lovosice), Chlorococcum sp. (Zurek 04), Stigeoclonium sp. (Lovosice) and Klebsormidium sp. (Lovosice) all tested coating paint samples (100%) performed the same anti-fouling properties as in marine environment. However, in Navicula sp.3 (Lovosice), Chlorococcum sp. (Zurek 04) and Klebsormidium sp. (Lovosice) only four coating paint samples were tested, so the results may rise by change. More coating paint samples should be tested in these algal cultures, in order to obtain confidential results. Eight coating paint samples were tested in Stigeoclonium sp. (Lovosice) culture and all performed in the same way as in marine environment.
Figure 9. Congruency of the results of laboratory experiments conducted on freshwater strains with the data obtained in real marine environment a) relative ratios of pass/fail in tested freshwater strains. b) absolute ratios of pass/fail in tested freshwater strains. See text for details. See Annex1.

Stigeoclonium sp. (EX07 K01), Phormidium molle (SAG 26.99) Stigeoclonium sp. (Lovosice) and Chlorella sp. (Lovosice) are the best candidates to be used for IATS technology on the base of absolute number of congruent experiments. In Stigeoclonium sp. (EX07 K01) eleven out of 15 tested coating paint samples performed congruent results, in Phormidium molle (SAG 26.99) nine out of 12 tested, in Stigeoclonium sp. (Lovosice) eight out of eight tested and in Chlorella sp. (Lovosice) seven out of eight.
Fig. 10 shows the position of the marine isolates (Achnanthes sp. KATS 09425 and Navicula perminuta Zurek) among the freshwater ones. Both marine strains were isolated from biofilms fouling samples exposed to seawater in one of the PPG testing station in Kats (Nederlands). In Achnanthes sp. KATS 09425 seven out of 12 tested coating paint samples performed congruent results with marine tests and in Navicula perminuta (Zurek) three out of six tests were congruent. Several freshwater strains provided more congruent results on both relative and absolute scales. Diatoms in a marine biofilm probably interact with other organisms (chemical communication, release of micronutrients and vitamins ect.) and may not be so successful fouling agens being isolated to a monoclonal culture.

Figure 10. Congruency of the results of laboratory experiments conducted on freshwater and marine strains with the data obtained in real marine environment (marine strains are highlighted). See Annex 1.
Table 8. Species selected for IATS test technology accompanied by optimal growth conditions (temperature and light intensity) and the type of selected culture medium. See Annex 1.

The automated antifouling test system will cover the temperature range from 10 to 25 °C and irradiance range from 30 to 150 μmol.m-2.s-1.

Optimal test species selected according to before mentioned laboratory tests:

Chlorococcum sp. (INNOPOL)
- Medium: BBM
- Temperature: 10–15 °C
- Illumination: 60 mmol•m-2•s-1

Stigeoclonium sp. EX 07 (SAG)
- Medium: BBM
- Temperature: 20–25 °C
- Illumination: 40–60 mmol•m-2•s-1

Phormidium molle (SAG)
- Medium: BG 11
- Temperature: 15 °C
- Illumination: 40 mmol•m-2•s-1

Chlorella sp. (CUNI)
- Medium: BBM
- Temperature: 15–20 °C
- Illumination: 100–160 mmol•m-2•s-1

Navicula sp. 2 (CUNI)
- Medium: WC
- Temperature: 25 °C
- Illumination: 60 mmol•m-2•s-1

Cultures should be unpacked immediately after receipt and stored at 15-18°C under low light intensity (north window, no direct sun light, or weak white fluorescent light). Screw caps or vessels should be loosened but not removed. Further maintenance or multiplication of cultures requires sterile transfer into new culture media.
Culture media
To approximately 900 ml of distilled H2O add the listed components in the order specified while stirring continuously. Bring total volume to 1000 ml with distilled H2O. Cover and autoclave medium. Allow to cool then store at refrigerator temperature.


Mechanical construction levels of the IATS tester.

The system is composed of IV separate levels of mechanical construction:

Fig. 11 IATS prototype tester. Front view. See Annex 1
Fig. 12 IATS prototype tester. Mechanical construction levels. See Annex 1

Level I
- XYZ robot manipulator system
This level is located at the top and constitutes the mechanical part of the tester (Fig. 11,12 See annex 1). It’s main task is to manipulate the sample in the XYZ space of the tester that will allow for the following activities: placing the sample at specified location in the XY space of the tester, rotating the sample by 180 degrees, raising and lowering the robotic arm along the Z-axis of the tester’s system of coordinates. The XYZ robot manipulator system was realized based on industrial control engineering components developed by company B&R.

The following components were employed for Level I:
- 4 step motors with lead screws exercising the feed mechanism of the manipulator in XYZ directions in the system of coordinates of the tester made by company B&R,
- pneumatic gripping device, designed and developed for the purpose of this project, allowing for closing and opening of the sample in the grip,
- rotary pneumatic actuator allowing for rotating the grip by 360 degrees,
- 6 motion limit sensors for specific XYZ directions, 2 for each direction.

Fig.13. Level I. XYZ robot’s manipulator unit. Robotic arm. See Annex 1.
Fig.14. Level I. XYZ robot’s manipulator unit. Step motors for the X-axis (master, slave). See Annex 1.
Fig.15. Level I. Rotary pneumatic actuator of the XYZ robot. See Annex 1.
Fig.16. Level I. XYZ robot’s manipulator unit. Robot’s pneumatic grip. See Annex 1.

Technical specifications of manipulator robot.
- Electric voltage: 230 VAC
- Max. power: 700 W
- Control voltage: 24 VDC
- Temperature: 0 C 55 C
- Compressed air pressure: 6 bar

- Control unit - The device is composed of a control and executive units. The control unit comprises the following: PLC controller located in the control box +SS1, and components responsible for:
- Work safety - Communication with the operator (PC, switches, control lamps)
- Power supply - Transferring input signals from the unit to the controller. Transferring output signals from the controller to device mechanisms

PLC controller
The system is controlled by means of the PLC controller developed by the B&R Company. The controller analyzes current input signals from the sensors and operator’s requests sent from the PC or through switches, and develops output control signals.

Safety unit
The safety unit consists of an emergency stop switch, safety switch for disabling doors, and a transmitter switch. It is responsible for disabling mobile mechanical device elements in situations considered by the employee as hazardous for people and the system. Turning the “emergency stop” switch on whenever someone trespasses the working area or opens the door when it is not allowed disables the mechanical elements.

Control box +SS1- The control box +SS1 consists of:
- Main power switch
- Emergency switch
- Transmitters of the emergency stop unit
- Fuses
- Service power plug
- 24V DC power supply adaptor
- programmable controller made B&R
- step motor controllers
- 27VDC power supply adaptors for the step motor
- signal lamps
- control switches
- terminal strips and wiring ducts

Executive unit -The device is composed of the following:
- step motors controlling the robot in directions: X, Y, Z,
- rotary actuator
- pneumatic grip

Description of communication of the PLC controller with the PC.
Communication between the controller and the PC is possible through the Modus RTU (RS232) protocol.
The number of the reactor and command controlling the robot (number from 1 to 12), for previously transmitted reactor’s number, are sent to the controller. Later, the impulse “start work step” is sent, which transmits the information to the controller. The controller returns the information containing the number of received sample, work step undertaken, or number of error provided the step could not be completed. After the work step is finished, an informative signal is sent to notify whether the step was successful. During the process, a confirmation containing the information of selected reactor and number of work step in progress is sent to the computer. The controller also transmits the information of current alerts and robot’s state.

Level II – devices for environment simulation, analysis, and initial preparation of the IATS prototype.
The following devices are located at Level II of the system:

This device serves the purpose of creating and maintaining proper environmental conditions for growing algae, which are placed in a solution inside the Bioreactor. The device is composed of the following two parts:
- Docking station, which serves the purpose of protecting the Bioreactor against mechanical damages. It is also a reference point for setting up an unambiguous position for the Bioreactor in the IATS tester’s XYZ system of coordinates. The main task of the docking station is also to provide a sufficient amount of light and heat to the Bioreactor. The light is brought into the Bioreactor by light-emitting diodes (LED), which are placed on both sides of the docking station. The bottom part of the station is made of stainless steel and placed on a plate heat exchanger that allows for exchange of heat between the reactor and the docking station within the range of temperatures from 15 to 24 degrees Celsius.
- Bioreactor, made of stainless steel, which sidewalls are made of toughened glass. Transparent walls allow for the visual assessment of tests’ progress, determination of the level of contamination of the bioreactor with organic material, and for the provision of essential amount of light to the solution during the tests (Fig. 17).

Fig.17. Level II. Bioreactor in the docking station. View from the side. The cover contains a mechanism tightening the grip. See Annex 1.

The top part of the Bioreactor’s chamber is protected by a cover that contains the following:
-Outlet for supplying air directly into the Bioreactor. Compressed air is pumped into the Bioreactor through an L shaped pipe made of stainless steel, which is located inside the Bioreactor. This process of pumping air into the reactor results in emerging air bubbles in the solution, which by flying up to the top cause movement in the solution and provide essential dosage of oxygen to the bio-solution.
- Outlet for outflowing excessive amounts of air and gases coming from the photosynthesis processes.
- Outlet for supplying the bio-solution (automatic process)
- Outlet for automatic measurement of temperature. Profiled hole is adjusted to the size of a temperature sensor.
Moreover, the top of the cover also contains the following: grips allowing for lifting the reactor’s container from the docking station, profiled crevice allowing for manual or automatic placement of the sample with a specially designed mechanism tightening the sample’s grip to the bioreactor’s surface.

Detox Chamber
This device consists of a water container, and a valve for inflowing and outflowing water. The device is protected with a cover carved with a row of crevices allowing for a safe placement of the sample. The process of rinsing out toxic substances is based on cyclical washing the samples placed in the chamber containing water. The frequency of washing the samples is managed by the IATS computer by means of the high-level ‘IATS Management’ software that controls the parameters of rinsing intensity, inflows and outflows of water to and from the chamber’s container.
The chamber is protected against water overflows by a liquid level sensor, which is mounted on the opposite side of where the valve for inflowing and outflowing water is placed. The container is entirely made of Teflon. The Chamber may be dismantled and rinsed off if necessary. In order to do so, there is no need for any special equipment or tools. The waste is drained to a separate utilization system outside the IATS.

Fig.18. Level II. Detox Chamber. See Annex 1.

Washing Chamber
The main task of this device involves rinsing the sample off with clear water in order to remove the excess of biomaterial from the surface of the sample. The structure and the way this machine operates is analogical to the Detox Chamber – the only difference is its size. The rinsing process is based on dipping the sample in clear water solution, which is pumped into the container. A sensor for controlling the level of liquid in the chamber that is located on the opposite side of the water valve protects the container from being overflown with water. Similarly to the Detox Chamber, the waste is drained out to a separate utilization system. There is a possibility of dismantling the chamber container for cleaning if it requires washing

Fig.19. Level II. Washing Chamber. See Annex 1.

The main idea behind antifouling tests is the determination whether organisms attach to the sample or not. The IATS tester prototype allows for a hands-free realization of this idea. Here, the phenomenon of luminescence of chlorophyll is applied. The FluorCam device was especially designed for this purpose. Its unique construction allows for both chlorophyll fluorescence and GFP imaging. The appropriate emission filter is used for GFP and chlorophyll detection. FluorCam is an innovative construction with red-orange flashing LED panels (620 nm) and blue actinic/saturating pulse LED panels (455 nm). The LED panels provide uniform irradiance over an area of 3.5 x 3.5 cm – suitable for imaging of seedlings, small plants, native or detached leaf segments, etc.
The FluorCam is supplied with a high-resolution CCD camera, which is specifically intended for detection of weak signals demanding long integration times. Such setup retains most of the functionalities for chlorophyll fluorescence kinetics measurement. The sample after being taken out from the Bioreactor is subject to rinsing off in the Washing Chamber. Then, it is placed in front of the FluorCam, where it is exposed to light and photographed. The picture is analyzed by means of the FluorCam high-level software. As a result, the user receives an information on the number of points, pixels, which glow in the dark representing the chlorophyll level. This test is then automatically repeated with frequency selected by the user.
Continuous analysis of the trend representing the growth of glowing field serves as a basis for determining whether the sample is or isn’t resistant. The FluorCam device is controlled directly by the central computer for the prototype system via USB port and FluorCam high-level software installed on the computer. The main condition for proper operation of the FluorCam device is complete darkness inside the IATS prototype.

Fig.21. The principle of operation of the FluorCam device. IATS. See Annex 1.

Level II control unit is based on the use of a PLC controller made by OMRON and placed in the control box, which is located at Level IV on the right. The box contains components responsible for: communication with operator (PC, switches, control lamps), power supply, transmission of signals between the controller and the object, transmission of signals coming out of the controller and directed to the Bioreactor, Washing Chamber, and the Detox Chamber (Fig. 22).

Fig. 22 Arrangement of the elements of the control box. See Annex 1.

The controller processes current input signals from the sensors and operator’s requests coming from the PC by means of the ‘IATS Management’ software, and works out control signals for managing: inflows and outflows of water in the Detox and Washing Chambers, and airing and lighting (LED) in the Bioreactor. The control box also contains the following elements: main power switch, fuses, service power outlet, 24V DC power supply adaptor, signal lamps, terminal strips, and wiring ducts. Communication between the controller and computer is possible by means of the TCP/IP protocol.

Level III and IV of the mechanical aspect of realization of the IATS project.
Level III, plate heat exchanger was built in form of a plate carved with parallel placed ducts connected with small pipes. The flow of warm or cold water through the heat exchanger at the Level IV allows for maintaining uniform temperature in the IATS prototype (within the range of temperatures of 15-24 oC).

Fig. 23 Level IV. IATS prototype. Compressor. See Annex .
Fig. 24 Level IV. From the left: IATS computer, heat exchanger. See Annex 1.

Level IV. PLC controller boxes are located at Level IV, for controlling the XYZ robot’s manipulator (Level 1), and managing processes and devices of Level II such as the Bioreactor, Washing Chamber, and Detox Chamber. Level IV was designed to protect electronic devices against water spills. Therefore, it is separated from Level III with a tabletop and all cables are sealed. Below the tabletop the main IATS computer is placed with Windows 7 x64 Pro and ‘IATS Management’ software. A compressor for the pneumatic motor of Level I is also located at Level IV, along with a water and air pump, water valves, and waste draining installation.

IATS control units – communication with IATS Management software
The following separate control units are singled out in the IATS tester prototype (Fig 25):

Fig. 25. IATS control scheme. See Annex 1.

- Process of controlling the XYZ robot’s manipulator and grip – Level I. The control unit for this process was realized using the PLC controller developed by B&R.
- Process of controlling Bioreator’s parameters, processes in the Washing Chamber, and Detox Chamber. The control unit for this process was realized using the PLC controller developed by OMRON.
- Process of controlling FluorCam and analysis of images, which was realized by means of the PC using the high-level FluorCam software.

All processes are controlled by a superior software - ‘IATS Management’, installed on the IATS prototype’s PC, to which access is possible through a touch screen built in the tester’s casing. The software managing the PLC controllers was developed based on programming tools dedicated to specific drivers offered by producers within the scope of the Embedded C language technical support.
In order to be able to quickly reprogram the XYZ robot’s manipulator a user graphical interface was developed.

Fig. 26. IATS Managament High-Level Software. Main screen. See Annex 1.
Fig. 27. Graphical interface allowing for quick programming XYZ robot’s movements. See Annex 1,.

A test of the sample in the IATS unit is based on the following operational stages:

Description of the testing process:
1. Preparation of the tester’s mechanical unit to analysis. This stage involves resetting the tester’s location to initial values 0,0,0, in the XYZ manipulator’s system of coordinates. The homing procedure is realized after each finished measurement session.
2. Shifting of the XYZ robot’s manipulator to the Bioreactor’s position. This stage involves moving the manipulator’s arm directly above the selected Bioreactor along the X- and Y-axis.
3. Lowering the manipulator’s arm and placement of the sample in the grip. This stage involves moving the manipulator’s arm along the Z-axis to its bottom position, gripping the sample, shifting the sample to the top position, and placing it above the Washing Chamber.
4. Rinsing the sample with clear water. This stage focuses on rinsing the sample with clear water by dipping it in the container. By slowly taking out the sample out of the water, we are sure that biological material that is not attached to the sample was rinsed off. After being put in the top position, the sample is placed in the position of the FluorCam camera.
5. Taking a picture of the biological material attached to the sample. This stage involves placing the sample near the FluorCam camera’s lens, and taking a picture of both sides of the sample. The reference side is photographed by rotating the sample by 180 degrees. Ready pictures are automatically sent to the computer, registered in the tester’s database, and subject to analysis by means of the FuorCam software in order to determine the fluorescent surface of the biological material.

IATS Management Software.

IATS program, which oversees the entire system, is an application which runs under the control of the Windows 7 operating system and libraries Framework. NET 4.0. It is using database software: Microsoft SQL Server 2008 R2 Express (Instance name: SQLEXPRESS).

IATS system works with the subsystem FluorCam designed for imaging of chlorophyll fluorescence emitted by living algae and cyanobacterial strains fouling on the paints inserted to the reactors. FluorCam Software should be running during the tester operation.
The main window contains of: Main, Results, History, Options, Detoxic Chamber, Help tabs. The basic tab 'Main' contains the image of 9 reactors and their current status. A green arrow (under reactor number) means that the measurements is in progress (the schedule shows running analysis). Red symbol under the reactor means that analysis was completed.
At the bottom of the main screen there is a box with a log of the program, which contains the activities that have been carried out by the program and information about all system errors. On the right side of the window contains information about the temperature and bubbling during the experiment, and the buttons for starting/stopping, setting a new experiment and import, export all
schedules for all reactors. In this tab user can start new schedules (clear all schedules), set schedules for reactors and start/stop/continue analysis (measurements).

System configuration.
When you first run the application it must be configured by selecting the 'Options' tab on the main window. This window allow:
Measurements configuration (Part 1)
- set the temperature in the reactors during the experiment (global temperature),
- set the default lighting in reactors (default lighting),
- default the analysis threshold F (equation 1). This value can be changed for any reactor in the reactor window. If analysis value F exceeds this treshold the analysis for the reactor are not continued (default analysis treshold),
- default minimal time between two operations during the measurements. If this value is 10, this means that if the previous operation (eg. analysis) began after 40 minutes, the next can begin soon after 50 minutes (Default minimal time between two operation),

Area MAX value (see Equation 1) is given by the FluorCam software for sample completely covered by algae

F=100 Area/〖Area〗_MAX ,% (1)
F - fraction of the sample surface covered by algae,
Area - area of sample covered by algae that is reflected as area where fluorescence signal is emitted (It is calculated by Fluorcam),
Area MAX - amount of light reflected by the sample as a whole covered by algae.

Devices configuration (Part 2)
The next three values determine whether the program is working with real subsystems that manage succession: a robot responsible for the movement of samples (manipulator), the camera to perform photos (photocamera), environmental control subsystems in the chambers (chambers control). During testing system values can be set to 'Yes' (Virtual), but during the operation of the system, these values must be set to 'No',
Local IP address of the machine running the system and the IP server address of the PLC. These values are used to communicate with the reactors (lighting, bubbling, detoxic reactor and the temperature in the system). LocalIP must be in this same subnetwork. For example:
PLC IP: local IP: Subnetwork mask:
COM port is used to determine the serial port, after which the system communicates with the robot responsible for the movement of samples.

Devices testing (Part 3)
Additionally you can view test panel. Using controls in this panel you can control and test different parts of the machine: photo camera, mechanical arm, subsystem of light and water.
To show admin panel press 'show admin panel' and enter password: pa

Setting up the experiment.
The configuration of the experiment should be started by resetting all reactors by pressing the 'Clear Schedule' button on the first tab of the main window.
During setting up of the new experiment the experiment program (schedule) should be set for each reactor separately. The configuration of each reactor is accomplished by clicking on the picture of the reactor in the first tab of the main window. This causes the appearance of a new window.
During reactor schedule configuration there should be set a number and name of the sample and the threshold of the analysis. If analysis result exceeds this threshold the analysis for the reactor is not continued.
The first tab of the window "Reactor" contains also graph, which will be complemented with the data from the analysis of a sample in this reactor (according with equation 1). The second tab 'Schedule' is used to define actions that can be performed on the sample during the experiment.
During the experiment, in the reactor it is allowed to:
- change the bubbling for all reactors, 0 (Off) or 100% (On)
- change lighting in the reactor in the range of 0-100%
- perform analysis of samples, consisting of removal of the sample from the reactor and take a picture, which after analysis tells you how much of the sample is covered with algae.
Change of the lighting and bubbling require a parameter. Change of the operation of the analysis is parameter less. Each of these activities requires time definition, during which it should be performed (number of days, hours and minutes after the start of the experiment).
At the bottom of this tab, there are buttons to ‘Add’ and ‘Delete’ actions on the schedule, and to ‘Import’ and ‘Export’ the entire schedule (to copy the schedule analysis of one reactor to another).
The last tab in the window of the reactor is an 'Analysis'.
In this tab it possible to see two photos for each analysis (base and reference photo), time of the analysis, and the value that specifies how much of the sample is covered with algae. This value is calculated according to equation 1.
In the 'Analysis' tab it is possible to ‘Print’ the results of the experiment (for the reactor).

Running the experiment.
The experiment can be initiated by pressing the 'Start analysis' button. Experiment can be terminated by pressing the 'Stop analysis' button. An additional option is possibility to terminate the measurement for a single reactor by clicking an icon symbolizing the current state (green arrow) of the reactor. It is located below the reactor number.
During the experiment, we can see the results of the analyzes for individual reactors in three ways:
- press the 'Show reactors charts' displays thumbnails chart instead of scheme of reactors in the main window.
- In the second tab ‘Results’ there is chart with results of analysis for all reactors. There is possible to show or hide data for each reactor and there is also possibility to observe the trend lines for each reactor (Trend describes a 3rd degree polynomial). The first number in the description of the graph line is the number of samples, while the second number is the number of the reactor in which the sample is located. There is also possible to print chart or export results to Excel file format. It is possible to show table with results of analyses.
- Clicking on the specific reactor scheme opens the reactor, where the first tab is shown a graph of the results of the analyzes.

History of performed experiments.
History of performed experiments (the time of the experiment, the end state, value of last analysis) is shown in the 'History' tab.
There is possible to save (to PDF format) report of the one selected sample, save (to excel format) results of measurements for many selected samples or save (to PDF format) history of measurements.

Detoxic Chamber
Defined actions to be performed on the detoxic chamber is related the 'Detoxic chamber' tab.
By clicking on the diagram of the reactor appears the window. There is possibility to specify the time of filling with water and draining the water from the detoxic chamber.
Defining the schedule of detoxic reactor it is completely independent of the measurements. Available actions in detoxic chamber are emptying and filling the chamber and additionally refill the chamber.
The filling chamber is set to the closed circulation of water in the chamber, where the water pump is operated for 5 minutes, and then remains inactive for a time t = 'action parameter' - 5 min). Then the pump starts to work again for 5 minutes. This procedure is repeated until the next action in the schedule.

Potential Impact:
Market potential

The primary direct markets for the antifouling test system (IATS) are the marine and protective coatings market. Therefore, the market size, segmentation analysis and the SMEs economic benefit estimations are based on evaluating markets in this sectors. Consortium have also described the future end-users and companies which will indirectly benefit from the project’s results. It would be the following sectors: Marine, Infrastructure, Power, Offshore, Oil and Gas, Chemical Processing, Transportation – Rail and Intermodal. Due to it the rapid growth one or more of them will be also an advantage for the protective coatings development.

Marine coatings
Marine coatings systems are applied to ships and structures in both the sea and fresh water environments. They serve the dual purpose of protecting again deterioration and keeping ships looking good. The global marine coatings industry is poised to sail forward on the growing demand for reduced fuel consumption in cargo and cruise ships. Increasingly stringent environmental regulations are boosting the prospects of high-value, eco-friendly coatings. Innovation has been the only constant, with all companies striving to offer eco-friendly products.
New analysis from Frost & Sullivan finds that the market earned revenues of $5,030.1 million in 2011 and estimates this to reach $10,216.3 million in 2018. The research covers anti-corrosive, anti-fouling and foul-release coatings.
"The need to lower fuel consumption is a strong market driver and antifouling coatings applied to ships’ hulls offer one way to combat emissions and reduce fuel consumption, explained Frost & Sullivan Research Director. "Foul-release technology, which also results in substantial fuel savings, is particularly useful for large cargo ships, which consume a lot of fuel."
Marine coatings manufacturers are generally conservative in adopting new practices. However, increasingly stringent environmental legislation, paralleled by customer preference for more eco-friendly products, is pushing innovation in the market.
Until the beginning of 2014, new ship building activities are expected to fall. This will lead to declining volume demands in the near-term. Consolidation of the shipping management companies will increase their buying power, while placing additional pressure on marine coatings prices.
The global marine coatings market is heavily consolidated, with 80% of the market owned by five companies AkzoNobel, through its International Paint business, Chugoku Marine Paints Hempel’s Marine Paints, Jotun and PPG.
Aggressive marketing, improved customer service – as sales are mostly direct – high performance products and strategic alliances in regions like China will help improve market share. As the market is dominated by a few large companies and barriers to entry are high, smaller companies should focus on offering differentiated products and superior customer service.
Protective coatings
The global protective coatings sector is larger than marine, standing at just over 1.85 million tonnes (in 2007). Growth of this market is expected across all regions for the medium-term (although uniform growth is not anticipated) .
The global protective coatings market is also highly fragmented – most of it (about 62% of value market) is shared by minor manufacturers. Rest of the market (38%) belongs to nine main coatings companies: PPG, AkzoNobel, Jotun, RPM Painting, S&W Painting, Kansai Paint, Chugoku Marine Paints, Hempel’s Marine Paints and KCC Paints.
New analysis finds that the market earned revenues of EUR 876.2 million in 2008 and estimates this to reach EUR 944.6 million by 2015 . This research covers the following application sectors: oil and gas, power generation, infrastructure, commercial architecture, water and wastewater and the manufacturing industries. There are currently more than 360 major players in the protective coatings market in Europe. In 2008, unit shipments for the European protective coatings market stood at 118.4 million liters. By 2015, unit shipments may well reach 121.1 million liters, growing at a Compound Annual Growth Rate (CAGR) of 1.1 percent. The protective coatings market will be buoyed by investments in energy facilities, power generation plants, and infrastructure projects. The current economic downturn is restraining the market, with limited growth projected for several quarters in some applications, growth prospects are set to improve in the near term.
In terms of both volume and revenue, the highly mature Western European market is experiencing limited growth in comparison with its rapidly growing Eastern European counterpart. Volumes in the Western European protective coatings market fell by 5 to 7 percent in January 2009, resulting in intensifying competition as coating companies vied for declining market share. In Eastern Europe, the protective coatings market shows more promise, both in terms of volume and revenue. Coating companies in Eastern Europe will see revenue growth thanks to robust market growth in the region.

The European shipbuilding industry is a dynamic and competitive sector both in the EU and on a global scale. It has great importance from both an economic and a social perspective, and also involves other areas including transport, security, research and the environment. The EU promotes its development and addresses competitiveness issues the sector is facing.
Shipbuilding is an important and strategic industry in a number of EU Member States. Shipyards often play a significant role for the regional industrial infrastructure and, with regard to military shipbuilding, for national security interests. The European shipbuilding industry is the global leader in the construction of complex vessels such as cruise ships, ferries, mega-yachts and dredgers. It also has a strong position in the building of submarines and other naval vessels. Equally, the European marine equipment industry is world leader for a wide range of products from propulsion systems, large diesel engines, environmental and safety systems to cargo handling and electronics.
There are around 150 large shipyards in Europe, with around 40 of them active in the global market for large sea-going commercial vessels. Around 120,000 people are directly employed by shipyards (civil and naval, new building and repair) in the European Union. With a market share of around 15% in volume terms, Europe is still vying (with South Korea) for global leadership in terms of the value of civilian ships produced (15 billion Euros in 2007).

Offshore wind turbines.
The market for offshore wind turbines has to be seen separated from the onshore market. Different turbine sizes, the harsh offshore environment together with limited access for service and maintenance require specially designed offshore wind turbines.
During 2011, work was carried out on 15 offshore wind farms in Europe:
• two full-scale farms were fully completed and grid connected;
• three experimental floating concepts were tested, one of which full-scale and grid connected;
• five wind farms were partially completed and grid connected;
• one further turbine was installed at a pre-existing near-shore test site;
• in four other projects, offshore work has begun, but no turbines were connected during 2011.
With over 750 MW grid connected in British waters during 2011, 87% of new capacity was added in the United Kingdom. 108 MW were added in Germany (13%), a 3.6 MW turbine grid connected in Denmark. Three experimental floating turbines were also installed in Norway, Sweden and Portugal. The latter being the only full-scale, grid connected model (2 MW) of the three and the first full-scale offshore turbine in southern European waters.
Today only five manufacturers offer offshore wind turbines, two of them, Bard and Multibrid are specialized offshore wind turbine manufacturers whereas Vestas, Siemens and Repower offer on- and offshore wind turbines.
Siemens installed and grid connected 200 turbines in 2011: 85% of all offshore turbines installed and connected during the year. REpower was in second place with 22 (9%), followed by BARD (12 turbines) and Vestas (one turbine).
The average capacity of offshore wind turbines grid connected during 2011 was 3.6 MW.
A total of 1,371 offshore turbines are now installed and grid connected in European waters totaling 3,812.6 MW spread across 53 wind farms in 10 countries. The offshore wind capacity installed by the end of 2011 will produce, in a normal wind year, 14 TWh of electricity, enough to cover 0.4% of the EU’s total consumption. In 2010, Thanet, a 300 MW project in the UK, was the largest offshore wind farm completed and fully grid connected in the world. During 2011 over 380 MW were installed at Greater Gabbard, also in the UK. Once completed, Greater Gabbard’s total capacity will be 504 MW. However, construction has also started on the first phase of the London Array project. Once completed, it will be 630 MW. The UK is by far the largest market with 2,094 MW installed, representing over half of all installed offshore wind capacity in Europe. Denmark follows with 857 MW (23%), then the Netherlands (247 MW, 6%), Germany (200 MW, 5%), Belgium (195, 5%), Sweden (164, 4%), Finland (26 MW in near-shore projects) and Ireland 25 MW. Norway and Portugal both have a full-scale floating turbine (2.3 MW and 2 MW respectively).

Partners Economic Benefit and Joint Investment Pay Back in Months.
All SME partners in this project expect to become the initial players in the supply chain able to deliver the product and license manufacturing technology on global basis. The predicted business benefits for each SME contributor, within the first five years after the end of the RTD work is quantified in the table below.
The benefits for SME contributors are derived from 2 sources:
- IATS tester sales,
- IATS maintenance.
The profits figures are divided between the partners in accordance to their contribution to the project and the role they will play in supply chain. The projected payback time for each partner is between 18 and 24 months, although it is difficult to predict it accurately because the market uptake is likely to be exponential, starting slowly at the introduction stage and ramping up as the demand increases. The total potential profit for all SMEs partners amount to €4,35 mln.
IATS sales analysis assumes that the manufacturing cost target is €71,000 for the one entire test system (based at the breakdown costs shown in the table above). Consortium partners agreed to reach the final sales price in the range on the €150,000. Comparing to the only similar solutions from the USA, this price seems to be very reasonable and attractive for the potential end users. Those assumption had been confirmed by the potential future end user – one of the project Partners – PPG company.
More than 360 major players in the protective coatings market in Europe, approximately 1,000 all over the world, a numerous laboratories and research organizations which could be interests in the IATS product give a quite promising market outlook. All the sales predictions bases on the total IATS system sale during the five post project years at the level of 50 testers, what is less than 5% of all potential end users. It have to be mentioned that the biggest protective coatings manufacturers have more than one company’s laboratory and can potentially buy more than one piece of the final product. According to the business practice the first years’ sales will be a bit lower that in the following years.

IATS sales graph represents the total increased revenue per annum, before any capital investment and depreciation is taken into account. Within 5 years after the end of the RTD work, IATS would reach 0,2% of the protective coatings market at the year 2015. Protective coatings markets were precisely discussed in the Market Potential paragraph. The total income from IATS is expected to reach approximately €7,75 mln.
The estimated yearly sales of IATS has been shown in the table below. This is seen as a long term sustainable level given the total market opportunity available.

The total potential profit for SMEs partners from IATS system sales and its maintenance amounts to €4,35 mln within 5 year post-project. Annually profit shows the graph next by. All SME partners will reap from selling the final product and all necessary components. It should be emphasized that all predictions were assumed on safe and realistic basis what makes them more than likely to attain.
Total €7.752.000 sales during 5 years will allow to create 19 new work places mostly in the companies which will take part in the project. Calculations based on €80.000 Euros new sales pa turnover providing 1 new job and are explained more detailed in paragraph Working Conditions& Employment in the next subsections.

Exploitation foreground and IPR protection.
The ownership of the results of the project will be agreed in detail between the SME partners when drawing up the final agreement concerning IPR protection. All intellectual property created under this project will reside with the SME partners and be exploited by them in the form of ownership, patents, or through license agreements. In short, the default regime applies, whereby the rights to the foreground developed by the RTD Performers are passed on to the SMEs in the consortium. Even though PPG is a potential end-user in the project, it has not expressed interest in disseminating or using the results generated by the project, and so shall be strictly considered a potential purchaser of the commercial version of the IATS and will not have any rights to any part of the IATS.
The second aspect of the IATS IPR regime is that ownership (or being a patent holder, or co-owner) of a particular elements of IATS will be assigned to the SME that is likely to manufacture the said elements. According to the plans, ECEL has the manufacturing potential to deliver most if not all of the automatic and mechanical elements of the IATS (excepting the photoluminescence chamber), which means that it will have ownership over the entire concept (jointly with PSI and EDESIGN), as well as over the methodology for testing the anti-fouling properties. PSI will have the priority in terms of the photoluminescence chamber, especially since this is one of the few components in the project which can technically be adapted to a different laboratory setting and application and PSI main expertise is in these types of laboratory equipment. EDESIGN will be the owner of the process control hardware design and the process control software, because it has the necessary fabricating equipment and previous experience developing control hardware/software solutions for its clients. EDESIGN will make available these elements of IATS to the main integrator – EC Electronics – on the basis of a license agreement, which will made available on a royalty free basis. The initial goal of the IATS SME group during the commercialization phase will be to position themselves as the effective and preferred suppliers in this product category.

The commercial route envisaged for the exploitation of the results will be via consortium members, as well as outside business partners.

Product market strategy and post-project activities necessary for market entry.
After the project is concluded there will be a strong need to take actions necessary for market entry and to persuade potential buyers that the IATS is a great opportunity for them. The system will be promoted by SMEs to the protective coatings producers, taking into consideration especially the marine coatings manufacturers. It has been decided that the biological laboratories, universities and research public or private organizations would be interested in the developed product. ECEL will be the leader of the this activities and take care of contacting the potential buyers. All the SME companies, due to their contacts with the biological research organizations will take care of popularized the idea across, not only Europe, but also at the other continents, especially in the North America and Asia where the most powerful protective manufacturers have their headquarters.
ECEL, EDESIGN and PSI will focus also on IATS promotion in their home countries. It has been foreseen to put the scientific articles in the branch journals and magazines. There is expected advertising in different journals related to marine business and by sending information to potentially interested companies. Afterwards the marketing campaign will be broaden in order to meet potential buyers in the whole Europe.
Issues connected with service and technical assistance in different countries was agreed between interested parties. Those obligations will be held by Photon Systems Instruments (Czech Republic).
Currently there are held business negotiations with the first client of IATS solution – PPG Coatings Europe BV from Netherlands.

Contribution to community societal objectives.
Improving Levels of Skills
The proposed development will increase the effectiveness of the whole process and the desirability of the job by up-skilling the current staff and systems.
• Increased demand for production staff and technicians, generated by the additional equipment that will be necessary to produce the new products, will create an estimated 19 additional medium skilled jobs. While initially this will lead to a price premium being paid for those already qualified, the lack of a suitably skilled work force will very quickly stimulate workers to acquire new skills.
• Cross-sectoral skills transfer between RTD Performers, measuring instruments, specialized electronic systems design for control, monitoring and diagnostics fields, electronic hardware and software manufacturers and end user partners will take place within the consortium. This will provide opportunities for designers, production and marketing staff to gain an understanding and appreciation of complimentary production processes which will catalyse new ways of thinking and solving problems.
As the technology finds new market opportunities in other sectors and territories, both inside and outside of the European Union, the partners will be required to undertake technology transfer programmes, to train new licensees in aspects of the technological implementation and biological aspects of our Innovative Anti-Fouling Test System. These third party firms will initially be recruited from areas of the Union and Accession countries which we cannot service directly from one location. Additionally all the end-users which will buy the new test system are going to get a brief training how to use properly the Anti-fouling test system. Hence, significant levels of pan-European technology transfer training will take place as the technology proliferates.

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