Application of marine biotechnology in the production of natural biocides for testing on environmentally innocuous antifouling coatings.

Abstract The widely recognized biofouling phenomenon has many negative consequences for artificial structures that are in contact with seawater in the form of structural defects and additional expenses for maritime companies due to cleaning and prevention processes. After having analyzed the serious environmental problems caused by an indiscriminate use of highly toxic biocides coming from organic derivatives of tin compounds and the controlled emissions of volatile organic compounds (VOC) to the atmosphere, the evolving technology of antifouling paintins (further mandated by current environmental standards) aims to develop environmentally innocuous water-based coverings in which extracts of the very same marine world are used as biocide compounds. Water-based coatings are being developed that use low-toxic elements and natural biocides, where bacteria is isolated from surfaces immersed in the marine environment, creating a promising source of natural antifouling compounds. The result is a new environmentally friendly antifouling coating that is able to mitigate the problem of biofouling without affecting the surrounding medium, and which may be applied on any artificial structure in contact with seawater.

Key words Marine biotechnology, Biofouling, Antifouling coatings, Marine natural products

Introduction

Any artificial structure in contact with seawater is rapidly coated by a microbiological biofilm, which serves as a base on which macroorganisms will grow. This phenomenon, known as biofouling, causes structural problems and its mitigation involves a severe economic outlay for maritime industries.

Biofouling can be defined as "the undesirable phenomenon of adherence and accumulation of biotic deposits on a submerged artificial surface or in contact with seawater" This accumulation or incrustation consists of a film composed of microorganisms affixed to a polymeric matrix created by themselves (biofilm), where inorganic particles (salts and/or corrosive products) may arrive and be retained as a consequence of other types of fouling that develop in the course of the process. This bofilm (microbial biofouling or micro-fouling) can begin the accumulation of macroorganisms (macrobial biofouling or macrofouling). (1)

Biofouling is made up of hundreds of species, such as tubicolous bacteria, protozoan, seaweed, mollusks, bryozoans, cirripeds, polychaetes, ascidians, hydrozoans, and so on. These organisms adhere themselves to the substrate, developing a fast growth rate and great reproductive potential. The strategy of the different families is to obtain the resources that the ecosystem offers, avoiding the competition among them by differentiating the periods of colonization in such a way that the incrustation begins with the settling of the phytobenthonic organisms during the springtime, then continuing with the adhesion of zoobenthonic organisms.

Biofouling, therefore, accelerates the processes of corrosion of manmade materials (2) (3) and causes break downs in the performance of the structures. (4) This damage takes place on movable and stationary structures such as boats, petroliferous or gas platforms, oceanographic investigation implements, thermal energy conversion plants, and subaqueous wounding equipment. It also damages martitime cultivation facilities (aquariums, cages, conduits, and pumps), as well as their cultivated organisms. (5)

In ships, biofouling increases the friction between the hull and the water, which results in an increase in fuel consumption (up to 40-50% with low-density biofouling) and a decrease in speed and maneuverability. The hull of a ship unprotected by antifouling systems can accumulate up to 150 kg of biological incrustation per square meter after 6 months in the sea. A long tanker with 40,000 [m.sup.2] of underwater hull supposes an increase in weight of six metric tons of biological incrustations, (6) which can lead to enormous economic losses.

To avoid economic loss, as well as an accelerated deterioration of the artificial structures in contact with seawater. different types of protection have been used over time. Among them are the copper coatings that were introduced by the Phoenicians and continued to be successfully used on wooden ships until the 18th century. When iron ships were first built, paints widely known as patents, in which the copper sulfate acted as a biocide principle, began to be manufactured. In I960, the use of paints composed of copper, mercury, arsenic, organic derivatives of tin (organic compounds of tin like tributyltin (TBT) and trifeniltina (TPT)) spread widely, but eventually they proved to be a real risk for the marine ecosystem.

In the 1970s, (here was continued use of antifouling paints based on the biocide performance of the organic derivatives of tin, especially tributyltin (TBT). As a result, most of the navigation ships covered their hulls with these types of paints, which turned out to be effective and economic. (7)

During the 1980s. we discovered the negative consequences of using TBT in antifouling paint on the marine ecosystem, especially in areas of low water interchange by tidal influence, such as bays or estuaries, where it is especially detrimental to populations of some invertebrates, mollusks, crustaceans, and fish, with serious malformations detected in some species. (7) As a result of these findings, several countries introduced controls to limit the use of TBT in antifouling paints used on small Stips, In 1982. France prohibited the use of TBT in ships with a length shorter than 25 m. Japan, the United Kingdom, the United States, Norway, Australia, New Zealand, and other countries soon followed.

At the beginning of the 1990s, an International Maritime Organization (IMO) resolution recommended that governments ban the use of tributyltin in ships less than 25 m long and imposed restrictions in the leaching process of tributyltin that require it to be less than 4 [micro]g/[cm.sup.2] per day. The use of antifoulings that contain TBT was forbidden in countries such as Japan, New Zealand, and Australia.

In 1997, Japan prohibited the production of anti-fouling paints with TBT. Since January 1, 2003, using organotin compounds as part of a ship's antifouling system has been prohibited around the world. January % 2008 has been set as the designated date for these compounds to completely disappear from all ships' surfaces.

Natural products from marine organisms can be used as replacements for the chemicals commonly used in antifouling coatings. (8) Many sessile marine animals arc not affected by biofouling and produce metabolites that demonstrate antifouling properties, presumably as a means of protection from colonization by foul ins; organisms, or as a strategy for space in highly competitive environments. Some seaweed species can also interfere with bacterial colonization on their surfaces by the release of antifouling compounds. (10) Recent studies have also highlighted the important role that bacteria plays in colonizing the surfaces of marine organisms. Bacteria growing on the surfaces of the larvae of some crustaceans produce antimicrobial compounds that protect the developing larvae from fungal infection. (11) Biofilms of the bacterium. Pseudoaheromonas tunicata, isolated from the surface of a tunicate showed antifouling activity against Balanus amphrite and Ciona intestinails larvae. (12) Bacteria isolated from the surface of seaweeds have also been shown to release compounds that repel other fouling bacteria, suggesting that they may protect the seaweed from fouling by other organisms. (13)

Thus, taking the Bay of Santander as the area of study, a chemical antifouling treatment has been developed using natural extracts as biocides. The result is a coating capable of controlling the problem of biofouling that can be applied on any artificial structure in contact with seawater without negatively affecting the surrounding environment.

The Santander bay (http://es, wikipedia.org/wiki/Bahia_de_Santander) is a 22.5 [km.sup.2]estuary located on the Cantabrian coast in northern Spain. Various rivers and streams flow into the bay supplying it with large amounts of rain water and open up to the sea through the Sardinero inlet, which allows for a continual renovation of water due to its tidal flows. The meteorological conditions make this location natural paradise with a temperate climate and plenty of rain.

Following a scrupulously designed work plan. and a method contrasted with previous experiments of a similar nature. (14) the research group tested the paints on polyvinyl chloride (PVC) test pieces in natural environmental conditions, as well as in the laboratory under different hydodynamics and light conditions, in two phases of experimentation. The result obtained were satisfactory.

Materials and methods

preliminary work

Collection of seaweed and marine animals and isolation of epiphytic bacteria

The most representative seaweed was Fucus seraratus, Fucus spiralis, Ulva lactuca, Laminaria Sp., Chondrus crispus, Palmaria palmate and Delesseria sanguine, all of which were collected and the bacteria from their surfaces isolated on a variety of media. Bacteria were also isolated from the surfaces of a number of sponges (Haliclona viscosa, Halichondria bowerbanki, Myxilla incrustans, Halichondria panacea, and Pachymatisma johnstonia), a featherstar (Antedon bifida), and two compound tunicates (Botryllus schlosseri and an unidentified specie). These different surfaces increased the range of bacteria isolated. To date, over 650 strains have been isolated from surfaces. There is ongoing isolation of bacteria from seaweeds in an effort to collect bacteria throughout the year, as bacterial strains found on seaweed in different seasons are known to differ. By continuing the collection, therefore, a wider range of strains should be obtained. Bacteria are being isolated at a variety of temperatures, also to increase the numbger of strains that are isolated.

Each of the bacterial strains isolated from seaweed or invertebrate surfaces undergo preliminary screening. Antibiotic assays against nine fouling bacteri were carried out using either a standard disk diffusion assay (15) or a well assay. For the well assay, 10 mm diameter wells were cut in marine agar (MA) plates that had been freshly swabbed with a lawn of each fouling straing. To these wells, 100 [micro]L of the cell-free supernatant were added. The plates were incubated overnight at 28 [degrees]C. Strains giving inhibition zones greater than 11 mm were considered to be antibiotic producers. Forty-two (6.5%) of the 650 isolates exhibited antibacterial activity against at least one of the fouling strains.

Of the 42 antibacterial compound-producing isolates, strains FS55 (Bacillus pumilus isolated from Fucus serratus (Brown alga)), NUDMB50-11 (Pseudomonas sp. isolated from Archidoris pseudoargaus (Nudibranch)), and II-111-5 (Bacillus subtitils isolated from Palmaria palmata (Red alga)) exhibited antibacterial activity against more than two marine fouling bacteria. These three isolates were grown in MA, with the supernatants separated from the cells, and then the supernatant put through an XAD-16 column. The XAD-16 column is a polystyrene organic matrix that removes all the organic material from the supernatant, allowing the salt water in the medium to run out the bottom. The organic compounds were then eluted from the column using methanol. The methanol fraction was rotationally evaporated to remove the methanol and any remaining water was removed by freeze drying. The extract was redissolved in methanol to further purify the crude extract before it was incorporated into resin. (16)

NUDMB50-11 was grown in 5 L of SM 9 medium at 28[degrees]C with shaking for 5 days. The culture was centrifuged (2500 rev per [min.sup.-1], 1 h) and the chloroform extract (2.0g) was subjected to silica gel column chromatography and eluted with a chloroform methanol gradient (0~100% methanol). Fractions with similar patterns on TLC (silica gel preparative (chloroform/methanol)) were combined and 15 fractions were obtained. Further silica gel column chromatography and crystallization, combined with a comparison of their 2D NMR spectral data with literature values, were used for identification of the chemical structures of these metabolites. (16)

Incorporation of bacterial extracts into paints

Supernatant and cell extracts from FS55, NUDMB50-11, and II-111-5 cultures were dissolved in minimal volumes of either methanol or dichloromethane. The solutions were mixed with a water-based paint resin [Revacryl 380 (Harlaw Chemical Company, Ltd.)]. This resin is an acrylic copolymer mixed with water with a 50% solids content, white in color, with a particle size of 0.1 micras and a PH of 8.

The concentration of the cell extracts was 28 mg [mL.sup.-1] while that of the supernatant was 20 mg [mL.sup-1]. Therefore, each bacterial extract incorporated into the resin in a specific concentration created a different paint, tested along with a copper thyocyanate (40%) as its positive control, and without an antifouling compound (100% resin) as a negative control. All paints were applied over aluminum strips with a 25 micra thick layer when dry and a drying time of 48 h at room temperature. The strips were pushed into agar that was previously inoculated with lawns of fouling bacteria. The paints were tested against six of the fouling strains of bacteria that were isolated from a rock and a glass slide. Studies have shown that the bacteria found on inanimate and animate objects are different, and because we want the compounds to be effective on an inanimate object it was important to test the paint against bacterial strains isolated from inanimate surfaces. The plates were left to incubate at room temperature. After 4 days any clear areas in the bacterial lawn were traced and these areas measured using image analysis. This process was repeated after 7 days. There was some variation in the length of time for an effect to be seen with different paints. however after 7 more days there were no changes in the areas of bacteria cleared by the paint. In addition to testing the bacterial extract paints, controls containing methanol and dichloromethane were tested. (16)

Since the results showed that NUDMB50-11 supernatant extract was the best antimicrobial extract when incorporated into the paint, a further laboratory test of this paint was carried out in which the concentration was varied by 50-25-10-5-1%, and with a positive and negative control. The paint was put onto aluminum strips and suspended in coarsely filtered seawater using plastic coated wire. The strips were left for 2 weeks before being removed and any microbes on their surfaces being fixed with glutaradehyde. The microbes were then stained using the DNS- specific fluorochrome DAPI, and examined under a fluorescence microscope. There were few bacteria attached to the strips with bacterial extract, however the presence of some flagellates was noted. These may have been attracted to the build-up of organic material on the surface of the strip. The negative control strips colonized 75% of the surface, while the positive controls and the paints with a percentage of a supernatant extract of either 50% or 25% occupied 5% of the surface and 10-20% on the other paints.

The settling laboratory tests were carried out either with animal or vegetable organisms. Barnacles (Balanus improvisus) are used as animal organisms because they are considered a type of species that severely contributes to biofouling worldwide. Because of their ubiquity and their basic life cycle, the Ulva lactuca was also used as a vegetable organism.

Field tests

The fields tests are carried out in natural and laboratory controlled conditions. The testing period took place between March and October (spring summer) because this time period is the most favorable for the settling and development of biofouling. Abiotic factors, such as the photoperiod (a mean value of 14 h), seawater temperature (annual mean value of 14-15[degrees]C, while summer temperatures are 19-20[degrees]C), the dissolved [O.sub.2] levels (annual mean value of 9.2 mg per L with summer highs of 9.8 mg per L) have maximal values that contribute to the start and development of the phytobenthic and zoobenthic organisms' life cycle.

For tests in natural conditions, a floating punt is used. The test samples are fixed to the shell sides of the punt at a distance of 15 cm below the water's surface. The zone of the entrance to the Gamazo dry-dock in the Bay of Santander was chosen as the sampling site due to its geographical proximity to the laboratory at the Nautical Technical School. Further-more, this zone is limited and protected by the Port Authorities, it is far away from the navigation channel, is protected from the prevailing winds, and has good water renovation.

In the biofouling laboratory, two tanks with a capacity of 2000 L were installed, and these received the seawater directly from the bay. This allowed us to test the samples in conditions of static and dynamic flow. In the static flow test conditions, the samples remained in natural temperature conditions (+2[degrees] C over the sampling point in the bay) and photoperiod (14 h), with a daily water renovation of 25%, to guarantee the supply of nourishment necessary for the normal development of the vital cycle of the biofilm. In the dynamic flow test, a stirring device is incorporated and the samples remain in variable conditions of temperature (up to 32[degrees]C) and the photoperiod (up to 24 h) so that will be exposed to the most favorable conditions for the settling and growth of biofouling. To supply the laboratory with solar light, we used actinic light because its light spectrum is very similar to that of the sun, placing a florescent light in each of the tanks with a 636 W light and an illumination of 2000 lux.

Each of the formulated paints were applied to standardized test samples of PVC measuring 200 x 100 x 4 [mm.sup.3 5]. Each test samples was evaluated twice in the different test conditions.

Periodically, the physical and chemical parameters of the seawater (pH, salinity, dissolved oxygen, temperature, hardness, chloride, calcium, alkalinity, and density) were analyzed, and the test samples were removed from the water to analyze the settling of the biofouling qualitatively and quantitatively by means of weighing, analyzing, and taking photographs of the treated surface. Concurrently with this biological analysis, a physical analysis of the sample was undertaken to check the process of paint lixiviation by checking the thickness of the paint layer and the appearence of physical defects (osmosis, cracks, and adherence) in the paint film.

First phase of experimentation

In this phase, eight different paints in water base with white (paints 1-5), red (paint 6), blue (paint 7), and green (paint 8) colors were manufactured with the compositions shown in Table 1. One uncoated panel is submerged to compare results. The participating company, Pinturas Ferroluz SA, was in charge of the production process and the supply of raw materials.

Table 1: Composition of tested coatings in the first phase of
experimentation of the field tests

Paint no.     [white box] 1  [white box] 2  [white box] 3  [white box] 4

Polyamide          1.22           1.35           1.22           1.26
resin 360

Revacryl          22.88          27.49          24.88          25.63
380

Water             36.58          33.69          36.59          37.68

Cuprous            -             17.52          15.85          16.33
thiocyanate

Tophelex           5.36           5.93           5.36           5.53

BHA                 -             -              -              0.50

NUDMB50-11         0.49           -              0.49           -

Titanium          28.54          14.02          12.68          13.07
dioxide

Red iron
oxide

Blue
luconyl

Green chrome
oxide

Isopropyl          2.93                          2.93           -
alcohol

Total            100            100            100            100

Paint no.     [white box] 5  [red box] 6  [blue box] 7  [green box]8

Polyamide
resin 360

Revacryl        28.84            26.97       29.28          26.97
380

Water           19.03            19.31       12.40          19.31

Cuprous         36.05            33.14       36.21          33.14
thiocyanate

Tophelex         6.59             6.71        6.70           6.71

BHA

NUDMB50-11

Titanium         9.49
dioxide

Red iron                         13.87
oxide

Blue                                         15.41
luconyl

Green chrome                                                13.87
oxide

Isopropyl
alcohol

Total          100              100         100            100

We were particularly interested in how the paint compositions developed, the use of water as a volatile vehicle, and the utilization of an acrylic resin as a base. With respect to the antifouling additives, it is important to underline the use of cuprous thiocyanate (CuSCN), Tophelex, BHA, and NUDMB50-11. For pigments we used titanium dioxide, red iron oxide, blue luconyl, and green chrome oxide.

CuSCN is a white powder used as a antifouling agent because of its low toxicity. Tophelex is a new bioactive compound that has been developed based entirely on extracts from edible plants by Ecosearch (International) Ltd. (www.biopartneruk.com/Partner Listing/Ecosearch (International) Ltd.). BHA (butylated hydroxyanisole) ([C.sub.11] [H.sub.16] [O.sub.2]) is a mixture of two isomers, 2-tertiary-butyl-4-hydrocy-anisole and 3-tertiary-butyl-4-hydroxy-anisole, that is effective as an antioxidant agent. NUDMB50-11 is a marine bacteria extract obtained in some preliminary work of this investigation.

Titanium dioxide pigment (Ti[O.sub.2]) is a white powder with high opacity, brilliant whiteness, excellent covering power and resistance to color change. These properties have made it a valuable pigment and opacifier for a broad range of applications in paints. Red iron oxide ([Fe.sub.2][P.sub.3]) is an extraordinary old, widespread, and versatile family of relatively dull but extremely permanent, nontoxic pigments. Blue Luconyl [R] (Cu phthalocyanine, beta) is a blue, stable, and nontoxic pigment. Green chrome oxide comes as a crystal powder with a good covering strength, high temperature resistance, and sunlight fastness, and is of very excellent quality and color firmness.

These water-based paints of high solid content are applied in two layers of 40 [mu]m each--one in dry with an interval of 24h, a tact drying time of 30 min, and a total drying time of 4h. The solid content in Paints 1, 2, 3, and 4 are [+ or -]65% in weight, while Paints 5, 6, 7, and 8 are [+ or -]80% in weight, with a surface coverage of 12-13 L/[m.sup.2] per dry layer of 40 [mu]m. After 3 months packaged, they present a perfect aspect with a smooth consistency.

Second phase of experimentation

After analyzing the results of the first phase of experimentation, we decided to attempt to reduce the settlement of weed fouling on the paint in a second phase of the experiment. The original paint was based on an acrylic, water-based resin as the main binder. This resin is highly seawater resistant and reduces the solubility of the paint. Two new paints were prepared, taking into account the results from Phase 1:

--For Paint A we used Paint 3 from the first phase of the testing as a reference, reducing the acrylic resin binder by 10% to increase the water penetration and increasing the cuprous thiocyanate by 25% by increasing the percentage of NUDMB50-11 to 4%.

--For Paint B, we did the same as for Paint A but reduced the cuprous thiocyanate by 25% and compensated for it with the presence of 3% BHA.

--One uncoated panel was used as a control.

The composition of this phase's paints is seen in Table 2, with a solid content of 60% of its weight, and a surface coverage of 12-13 L/[m.sup.2] per dry layer of 40 [mu]m.

Table 2: Composition of tested coatings in the second phase of
experimentation of the field tests

     Paint no.        [white box] A  [white box] B

Revacryl 380               22.50        22.50
Water                      40.12        40.12
Cuprous thiocyanate        11.88         8.91
Tophelex                    5.36         5.36
BHA                         -            2.97
NUDMB50-11                  3.96         3.96
Titanium dioxide           16.18        16.18
Total                      100         100

Toxicity test

To evaluate the acute toxicity of the substances employed in the elaboration of the antifouling coatings, PARCOM standarized tests were used.

The PARCOM toxicity standarized tests (18) were carried out with fish. In our case, we used young brill or turbot (Scophthalmus maximus) that were 4-6 cm in length in two 200 L conic tanks. After a conditioning period of 12 days in the laboratory, the fish were exposed to the substance we wished to evaluate for a period of 4 days. During the 16 days that the tests lasted, the fish were fed according to the test indications. Daily measurements of dissolved oxygen, photoperiod, temperature, pH, and salinity were recorded.

Results

In this section, it is important to differentiate between the results of the preliminary tests carried out in the laboratory and the results of the field tests carried out with the definitive paints manufactured in the first and second phases of experimentation.

Preliminary work

Over 650 isolates of marine epibiotic bacteria from different sources were screened. Three of the most active were used to produce extracts that were incorporated into antifoulding coatings. One of the most active isolates was a Pseudomonas sp., strain NUDMB50-11. Figure 1 shows the area covered by algal spores in Petri dishes treated with tested antifouling pints. The areas are expressed as a percentage of the negative control with vertical lines equaling 95% confidence limits. NUD S represents NUDMB50-11 supernatant extract paint, FS55 S represents FS55 supernatant extract paint, and 111-5 C represents II-111-5 cell extract paint.

[FIGURE 1 OMITTED]

All the resin paint-extract preparations were active against fouling. The cell and supernatant extracts of FS55 were not significantly more active than the controls of resin alone or resin with methanol. All NUDMB50-11 extracts were significantly more active and extracts had a pronounced effect in inhibiting the growth of the other strains. Almost all of the coatings were more active than the commercial antifouling paint. The NUDMB50-11 extract was active against the strain before incorporation into the paint base.

In an effort to compare the bacterial extracts used in each paint, the results were calculated to measure the cleared zone obtained per mg of pure extract. The NUDMB50-11 and methanol-soluble cell extract and supernatant extract were the most active, being significantly better than all of the other extracts. The NUDMB50-11 water-soluble extract was more effective than that of FS55.

The paints exhibiting antifouling activity in the antibacterial assays described above were used in barnacle settlement assays. Two of the paints showed a significant decrease in the number of settled barnacles in comparison with the control. The paints of interest were those formulated from the NUDMB50-11 supernatant. All other paints showed no significant antisettlement activity. Figure 2 shows the settling frequency of barnacle larvae in Petri dishes treated with antifouling paints. Settling frequency is expressed as a percentage of the negative controls, with vertical lines equaling 95% confidence limits.

[FIGURE 2 OMITTED]

Of the paints described above, the paint containing NUDMB50-11 supernatant showed a significant inhibition in the settling frequency and the growth rate of Ulva lactuca compared to the control. Figure 3 shows the settling frequency of algal spores in Petri dishes treated with antifouling paints. Settling frequency is expressed as a percentage of the negative controls, with vertical lines equaling 95% confidence limits.

[FIGURE 3 OMITTED]

Field tests

First phase of experimentation

TESTS IN NATURAL CONDITIONS: Paint 1 (0% cuprous thiocyanate, 5.4% Tophelex, and 0.49% NUDMB50-11) and Paint 2 (17.5% cuprous thiocyanate, 5.9% Tophelex, and 0% NUDMB50-11) showed a more severe settling of seaweeds than other paints, being the accumulated biomass at the end of the phase of experimentation of 24 and 26 g, respectively. Paint 3 (15.8% cuprous thiocyanate, 5.4% Tophelex, and 0.49% NUDMB50-11) and Paint 4 (16.3% cuprous thiocyanate, 5.5% Tophelex, and 0.5% BHA) showed the best results with 14 and 12 g. of accumulated biomass, respectively. Paints 5, 6, 7, and 8 (33-36% cuprous thiocyanate and 6.6-6.7% Tophelex, in different colors) showed similar results, with 16-18 g of accumulated biomass. The uncoated panel contained approximately 120 g of crustacean, seaweeds and companion fauna at the end of the test period. Figure 4 shows a comparison of the weight of the test samples treated with the formulated paints in this phase of experimentation after a period of seawater immersion of 8 months.

[FIGURE 4 OMITTED]

It is important to point out that all test samples were colonized by seaweeds, mainly enteromorpha and chaetomorpha from the first month of their stay in the water, without the establishment of any animal species during the testing period, with the accumulation of the biomass on the test samples treated differently relative to the percentage of the biocide used, without any influence from the paint's pigmentation.

Figures 5 and 6 show an illustration of the test panels treated with paints of this phase of experimentation after 1 and 7 months in seawater. In viewing the illustrations, we must note that even though the treated surface is covered by phytobenthic organisms (especially enteromorpha and chaetomorpha), we cannot detect the presence of any zoobenthic settlement, which suggests the control of the most aggressive biofouling organisms on artificial structures in contact with seawater. We must also note the possibility of reducing the amount of Cuprous thiocyanate by adding other biocides like NUDMB50-11 or BHA, for which we would reformulate the paints in the second testing phase in an attempt to exert greater control over the seaweed settlement and to decrease its levels.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

The physical analysis did not show deteriorations in the paints (harmful phenomena, blisters forming, cracks).

TEST IN LABORATORY CONDITIONS: The test results obtained in natural conditions can be extended to the results recorded either in conditions of static or dynamic flow. In conditions of dynamic flow, a larger accumulation of biomass is recorded due to the conditions of temperature, photoperiod, and supply of nourishment experienced by the treated test samples. In conditions of static flow, however, there was less biomass accumulation on the treated test samples. Quantitatively, there is a difference of 5 g of accumulated biomass between the dynamic and static flow conditions, estimated from the difference in the results obtained from natural conditions with a decrease of 25% in accumulated biomass, and the appearance in both cases of chaetomorpha and enteromorpha as the phytobenthic species.

Second phase of experimentation

TESTS IN NATURAL CONDITIONS: The uncoated panel contained appropriately 70g of crustaceans seaweeds, and companion fauna at the end of the second test period. Paints A and B contained no crustaceans. Paint B contained approximately 6 g of weed fouling. It is worth nothing that paint B showed a maximum concentration of weed fouling in July and subsequently showed a very significant reduction in weed content (from 10 to 6 g) in October.

Evolution in weight of the test samples treated with the formulated paints in the second phase of experimentation of the field tests after a period of seawater immersion of 7 months can be seen in Fig. 7. Figure 8 provides an illustration of the uncoated and test panels treated with paints of this phase of experimentation after 4 or 5 months in seawater.

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

TESTS IN LABORATORY CONDITIONS: Like in the first testing phase, the results obtained in the test with natural conditions can be extended to the results recorded either in conditions of static or dynamic flow. In dynamic flow conditions, Paint A reaches an accumulated biomass of 9 g and paint B reaches 3 g, while in the static flow these values are 4 and 1 g, respectively. When there is an increase in water temperature to 25-28[degree]C, and a photoperiod of 24 h, the biomass increases by 50%. In no case is there a settling of zoobenthic organisms, while the chaetomorpha and the enteromorpha are the most significant phytobenthic species.

Toxicity test

The test begins with the period of acclimatization (12 days) of the 200 individuals of Scophthalmus maximus lodged in groups of 25 units in as many compartments as there are paints to test. The initial conditions of the seawater in tanks were a temperature of 15[degrees]C, 7.8 mg per L dissolved oxygen, 8.0 pH, 35.3 salinity, and a photoperiod of 16 h. The load of fish in each one of the compartments also did not surpass the limit of 1 g of fish per L imposed by the PARCOM. The following results can be emphasized:

- Mortality during the test period (96 h) was 0%. The test was satisfactory.

- Abnormal responses from the fish were not observed.

- The fish did not suffer external injuries that indicate some type of pathology.

Analysis and discussion of results

Preliminary work

The first stage of biofilm development is the adsorption of organic macromolecules in the process of forming a conditioning film. (19) This is closely followed by the attachment of bacteria and other unicellular organisms. It has been suggested that effective inhibition of biofilm formation at this early stage would lead to a surface that lacks the necessary characteristics to permit the settlement of larvae of macrofoulers. (20) The development of a paint with antibacterial activity, therefore, may disrupt the early stages of biofilm development and provide an effective antifouling coating for the protection of marine structures. In addition, the use of a water-based paint, Revacryl 380, could overcome the adverse environmental and health impacts of solvent-based paints.

Species of Bacillus and Pseudomonas were the most common bacteria isolated in the present study, which showed good antifouling activity. This may reflect the culture methods used rather than serve as an indication of species diversity in the natural surface films. Three compounds exhibiting antifouling activity were isolated and characterized from a culture of the most active strain, NUDMB50-11. All compounds were reported to be secondary metabolites of Pseudomonas and their antimicrobial activities were documented. (21) Pyolipic acid has also been reported to exhibit mycoplasmacidal and antiviral activities in vitro, (22) and was reported as a biosurfactant with the potential for application in combating marine oil pollution. (23) However, this is the first report describing the antifouling activity of these compounds. The compounds represent three diverse classes of molecules with different physical properties and antimicrobial activities. This range of physical properties and activities may, when the extract is incorporated into a paint, provide active components that are released at different rates from the painted surface and have differential activities against a ramie of target organisms.(16)

Clare (8) reviewed natural antifouling products from marine sources and identified a number of compounds with antifouling activities. The vast majority of these, however, were classified as anlifoulants on the basis of a single assay. The antifouling activity of one specific bacterium has, however, been studied in some detail: Pseudoahevomomis lunicatu. isolated from the surface of the tunicate Ciona intestinalis, produced at least five extra-cellular compounds that inhibited the settlement or development of a range of surface-colonizing species.(12) The compounds, which have not been identified, inhibit the settlement of invertebrates and algal spores, stunt the growth of bacteria and fungi, and retard surface colonization by diatoms.

Field tests

It is evident that the strategies used by the different families to obtain the resources offered by the ecosystem are directed at avoiding competition between themselves by means of the differentiation of the colonization periods. The incrustation starts in the spring with the settling of the seaweeds, followed in the month of June with the colonization stage of the mytilidos. Later, during the summer months, animal settlings such as cirripeds and serpullids are found. (24)

With regard to the annual evolution of the different organisms, it is important to emphasize the seasonal condition of the phytobenthonic organisms, which is why a great number of these disappear at the end of the summer.(14) The same does not occur with zoobenthonic species, which extend their incrustation period in time and develop a large biomass during their growth.

In Santander Bay, Garcia (25) tested an auto-polishing antifouling with percentages by mass of active ingredients of 38.55% cuprous oxide, 4.39% tributytin methacrylate and 1.17% Bis(tributyltin) oxide. Trueba (14) researched the "study of copper oxinate and tetrachloroisophthalonitrile action used as active substances in scale preventer patents with vinly and chlorinated rubber bases applied over artificial framings placed at the Santander Bay." Both authors were able to control the process of biofouling adhesion. The accumulated biomass on the test panels at the end of the experimentation were practically negligible, as were the coat test panels of this investigation. For this reason, we can say that the obtained results were satisfactory (see Fig. 9).

[FIGURE 9 OMITTED]

In the same way, the results obtained by Arias et al. (26) in the Mediterranean Sea can be extrapolated, considering the different environmental conditions that exist between this sea and the Cantabrian. Of these results, the action of tetrachloroisophthalnitrile. (14) organic zinc compounds, (26) and the NUDMB50-11, Tophelex, and BHA used in this investigation we deduce that it must be accompanied by the algaecide action of compounds derived from copper in order to obtain total control on the adhesion of biofouling in the coated surfaces. We can also say that the obtained results are more influenced by the concentration and type of biocides used in the paint's composition than the product bases of the paints.

Conclusions

The strategy adopted in this study has identified a number of surface-associated bacteria that are capable or producing metabolites that, when incorporated into paint, retain their antifouling activity. Three metabolites exhibiting antifouling activity were purified and identified. Broad spectrum antifouling activity was achieved by combinations of these simple antimicrobial compounds.

Analysis of the data after a period of seawater immersion of 8 months indicated that the NUDMB50-11 compound and the Tophelex compound could be used to reduce the cuprous thiocynate compound by 50% without loss inperformance against crustaceans, but no improvement was noted against seaweed settlement.

The paints that were tested are fully effective against the settling of the animal organisms that cause biofouling. This signifies that they provide total protection from those organisms that are most harmful for any artificial structure in contact with the seawater.

The conditions achieved in the biofouling laboratory are extensible to those in the Bay of Santander, as the results obtained are very similar to those recorded in natural conditions.

The work will continue with the aim of isolating and researching new bacterial strains. Further research into the creation of paints will be carried out and those selected for trial will be tested against microbes, barnacles, and seaweeds. Identification of some of the active bacteria strains will be attempted.

The paints produced are fully ecological, are composed of natural substances, and are water based, which suggests a potential advance in pertinent prevailing legislation with regard to eliminating compounds that are toxic to the organisms located around the treated surface.

This work demonstrates the potential of marine bacteria in the production of antifouling coatings based on biodegradable natural products rather than the toxic compounds in current use.

Participating entities

This project (REN2001-0519) is an international collaboration among:

--Department of Sciences and Techniques of Navigation and Shipbuilding. Cantabria University. Spain.

--Department of Biological Sciences. Heriot-Watt University. Edinburgh. Scotland.

--Ecosearch (International), Ltd. Scotland.

--Santander Port Authority. Spain.

--Ferroluz Paints. S. A. Cantabria. Spain.

--We would also like to thank the Spanish Ministry of Education and Science for financial support of Project REN2001-0519.

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E. Eguia, A. Trueba

Department of Sciences and Techniques of Navigation and Shipbuilding, Cantabria University, C/ Gamazo, 1, Santander 39004, Spain

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