Biofouling, 2003 Vol 19 (Supplement), pp. 161–169

Is the Mussel Test a good Indicator of Antifouling Activity? A Comparison Between Laboratory and Field Assays BERNARDO A P DA GAMAa,b,*, RENATO C PEREIRAa, ANGE´LICA R SOARESc, VALE´RIA L TEIXEIRAa and YOCIE YONESHIGUE-VALENTINb a Departamento de Biologia Marinha, Universidade Federal Fluminense (UFF), P O Box 100 644, CEP 24001-970 Nitero´i-RJ, Brazil; bPrograma de Po´sGraduac¸a˜o em Biotecnologia Vegetal, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro (UFRJ), IIha do Funda˜o CEP 21949-900, Rio de Janeiro-RJ, Brazil; cPrograma de Po´s-Graduac¸a˜o em Quimica Orgaˆnica, Instituto de Quimica, Universidade Federal Fluminense, Nitero´i-RJ, Brazil

(Received 25 June 2002; in final form 9 July 2002)

Current antifouling technologies rely on metal-based paints, but due to their toxicity, an expected worldwide ban of organotin-containing paints is now prompting the quest for safe and effective alternatives. One of these is antifouling coatings whose active components are naturally occurring compounds in marine organisms. A number of laboratory bioassays has been designed to search for antifouling compounds. However, there is no evidence to date that these assays provide results reproducible through ecologically realistic field experiments. Natural concentrations of the extracts from the Brazilian seaweeds Laurencia obtusa and Stypopodium zonale were tested in the laboratory through the ‘mussel test’ and in the field through the ‘phytagel method’ in order to compare the efficiency of these methods in assessing antifouling activity. L. obtusa extract significantly inhibited fouling in both the laboratory and field assays, while S. zonale stimulated fouling in both assays. Major compounds from the extracts were identified. The findings suggest that the ‘mussel test’ is a reliable time and costsaving screening method for antifouling substances, although field assays are more sensitive for detection of their activity spectrum. Keywords: antifouling; Perna perna; brazilian seaweeds; Laurencia obtusa; Stypopodium zonale; marine natural products

INTRODUCTION Fouling, or the process of adsorption, colonization and development of living and non-living materials on an immersed substratum (Clare, 1996) can have a wide range of deleterious effects on man-made structures, and on host organisms or basibionts (Wahl, 1989), being one of the most important problems currently facing marine

technology (Crisp, 1984). The detrimental effects of biofouling (or fouling by organisms such as bacteria, seaweeds and invertebrates) include creation of turbulence on ships’ hulls and in hydro-eletric pipelines with an attendant reduction in speed or power output, respectively, reduction in conductive heat transfer across heat exchanger tubes, biologically induced corrosion of metallic surfaces (Marshall, 1994), and reduction of water flow and the overwhelming of flotation capacity in aquaculture (Lewis, 1994). Antifouling coatings technology is based upon mechanisms in which broad-spectrum biocides, usually toxic metal ions, kill organisms that settle on coatings. Organotin biocides like TBT (tributyltin) are the most effective antifoulants currently available on a commercial scale. Although effective, organotins have become serious pollutants in the marine environment, causing concern for future human health effects, which lead to the recent proposal for a global ban on the use of organotin antifoulants on 1 January 2003 by the Marine Environmental Protection Committee of the International Maritime Organization (MEPC-IMO) (Champ, 2000). Research laboratories worldwide now use bioassays with target fouling organisms to direct purification, identification, and development of new environmentally safe antifoulants. When the outer surfaces of marine organisms living in habitats of intense fouling pressure remain devoid of epibionts, the existence of some natural antifouling defense is suspected. Thus one of the most promising alternatives is offered by

*Corresponding author; fax: þ55-21-27195934; e-mail: [email protected] ISSN 0892-7014 print/ISSN 1029-2454 online q 2003 Taylor & Francis Ltd DOI: 10.1080/0892701031000089534

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the development of antifouling coatings in which the active ingredients are compounds naturally occurring in marine organisms (or their synthetic analogues) and operating as natural anti-settlement agents. The inhibition of fouling from the surfaces of marine organisms has been a theme in the literature for many years (e.g. Sieburth & Conover, 1965; Davis et al., 1989; Clare et al., 1992; Hellio et al., 2001), and a wide array of marine natural products or crude extracts that have some antifouling activity in laboratory bioassays have been isolated from a large number of marine organisms, including marine bacteria, seaweeds, seagrasses, bryozoans, ascidians, cnidarians, and sponges (see Pawlik, 1992; Clare, 1996; Rittschof, 2001 for reviews). However, the role of these compounds or extracts in situ as fouling (or epibiont) deterrents is currently unknown, and have only recently began to be addressed (Henrikson & Pawlik, 1995; 1998; da Gama et al., 2002; Pereira et al., 2002). As Rittschof (2001) pointed out, some antifouling substances are usually narrow spectrum in effectiveness, as prevention of fouling by one kind of organism is routinely supplanted by fouling of another type, and fouling communities are usually composed of thousands of species (Wahl & Mark, 1999). A number of laboratory bioassays has been devised to search for antifouling compounds, often including barnacle larvae (e.g. Rittschof et al., 1986; Maki et al., 1988; Fusetani et al., 1996) or bryozoan larvae assays (e.g. Bryan et al., 1997; Schmitt et al., 1998; Shimizu et al., 2000). Mussels have also been used in several different assays (e.g. Harada et al., 1984; Ina et al., 1989; Goto et al., 1992; Satuito et al., 1993; Davis & Moreno, 1995; Sera et al., 2000; Hellio et al., 2000a) due possibly to a series of advantages, viz. 1) mussels (as well as barnacles) are representative fouling organisms which cause serious problems to ships’ hulls, cooling systems of power plants (Hattori et al., 1998), and aquaculture (Armstrong et al., 1999), 2) although apparently sessile, mussels retain a degree of motility beyond the larval stage, 3) they are able to reattach to surfaces by secreting new byssal threads, 4) they actively select physically and chemically suitable surfaces on which to settle, and 5) unlike barnacle larvae, they can be used soon after collection. Some marine macroalgae are largely free from epibionts, although their surfaces should be particularly susceptible to fouling because they are sessile and restricted to the photic zone, where conditions for fouling growth are optimal (de Nys et al., 1995). Field and laboratory analyses in Brazil have shown that the red alga Laurencia obtusa (Hudson) Lamouroux remains free of epibionts, while the co-occurring brown alga Stypopodium zonale (Lamouroux) Papenfuss is partially covered by fouling plants and animals (da Gama, 2001).

To the authors’ knowledge, this is one of the first studies aimed at comparing laboratory and field antifouling assay methods. The screening for antifouling activity of extracts of L. obtusa and S. zonale through a laboratory antifouling bioassay using the mussel Perna perna (Linnaeus, 1758) is compared with an ecologically relevant field assay with phytagele (Henrikson & Pawlik, 1995; 1998; da Gama et al., 2002; Pereira et al., 2002).

MATERIALS AND METHODS Algal Collection Two seaweed species known to produce secondary metabolites (see Faulkner, 2001 and previous reviews) were chosen for the experiments and collected from two different areas on the Brazilian littoral (Atlantic Ocean), the brown seaweed Stypopodium zonale from the Abrolhos Archipelago, Bahia State (northeastern coast), and the red alga Laurencia obtusa from Cabo Frio Island, Rio de Janeiro State (southeastern coast). Both seaweeds are known to produce diverse natural products exhibiting interesting biological activities such as chemical defense against herbivores (Hay et al., 1987; 1988). The seaweeds were washed in seawater in the laboratory to remove associated organisms.

Extraction Procedures and Identification of Major Metabolites After determination of the volume of fresh material (210 ml to prepare 6 replicates of 35 ml each, see Antifouling Activity in the Field ) by water displacement into a graduated cylinder, the seaweeds were air dried in the dark at room temperature (in order to avoid photolysis and thermal degradation of the metabolites) until a steady dry weight (DW) was obtained (an extract aliquot equivalent to the DW of filterpaper was used in laboratory assays). Each algal species was submitted to exhaustive and successive extraction in a combination of organic solvents (methylene chloride and methanol), in the proportion of 2:1, following standard procedures for natural products chemistry. To increase the effectiveness of the extraction, the seaweeds were submitted to ultrasound for 15 min (Branson model 3210). The solvent was eliminated under reduced pressure and the remainder was weighed to determine the natural (volumetric and DW) concentration of the crude extracts. The L. obtusa crude extract (4.07% of tissue DW) was filtered through a pad (25 mm thick) of silica gel to yield seven fractions. Fraction 2 was applied to preparative thin layer chromatography (TLC) plates to yield a clear oil (0.45% DW) identified as the

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FIGURE 1 Structure of the major secondary metabolites elatol and atomaric acid from Brazilian populations of L. obtusa and S. zonale, respectively.

compound elatol (Figure 1) according to 13C and 1 H-NMR data (Ko¨nig & Wright, 1997). The S. zonale extract (16.52% DW) was shaken with 1N NaOH and subsequently with 1N HCl. The acidic fraction obtained was applied to a vacuum liquid column (VLC, silica gel) and gradient-eluted from hexane to methanol to yield six fractions. Fraction 3 yielded a yellowish oil identified as the meroditerpene atomaric acid (Figure 1) according to 13C and 1 H-NMR data (Wessels et al., 1999). This compound represented 1.65% of the DW of S. zonale. In order to detect the presence of metabolites at the surface of the alga that exhibited antifouling activity (L. obtusa ), fresh specimens were extracted in hexane for 40 s, a time period insufficient to cause cell lysis (de Nys et al., 1998). The resulting extracts were compared by TLC with the extracts used in the experiments. Antifouling Activity Against P. perna Juvenile mussels (P. perna ) were collected during low tide from the rocky coastal area of Itaipu (Nitero´i city, Rio de Janeiro, Brazil) and kept in a 230 l recirculating laboratory aquarium (equipped with biological filtering, protein skimming and activated carbon) at a constant temperature (208C), salinity (ca 35 ‰) and aeration for 12 h. Individuals were then disaggregated by carefully cutting the byssus threads, and divided into size groups according to total shell length, ranging from 0.8 to 4.1 cm in a plastic tray with seawater. Individuals exhibiting substrate exploring behavior (actively exposing their foot and crawling) were selected for experiments. Antifouling activity was measured by the following procedure, being a modification of the method described by Ina et al. (1989) and Goto et al. (1992). Water-resistant filter paper was cut into 9 cm diameter circles and soaked in solvent (control filter). Another 9 cm diameter set of filter papers (treatment filters) was cut in a chess board pattern (1 cm squares) and soaked in a natural concentration of extracts (determined as the extract equivalent to the DW of alga ¼ DW of filter paper) or in a 15 mM solution of CuSO4 (positive control). All filter paper circles were allowed to air dry. Entire filter circles were then placed in the bottom of sterile polystyrene

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Petri dishes, over which treated chess board filters were placed. Dishes were filled with 80 ml of seawater and three mussel specimens (2.0 – 3.0 cm length) were added. In this way, mussels would have the same area of treated (superior, squared) and control (inferior, entire) filter paper to which to attach. Ten replicates of each treatment (blank control, positive control—CuSO4, L. obtusa extract and S. zonale extracts) were used. Experimental dishes were kept in total darkness, as mussels have been shown to produce more byssal threads when held in the dark (Davis & Moreno, 1995). Experiments were allowed to run for 12 h. Mussel activities were recorded immediately after the start of the experiment, after 2 h, and then after 12 h. The activities recorded were substratum exploring behavior, gamete release (as an indicator of possible stress or positive cues) and number of byssal threads attached to each substratum (control or treated filterpaper, shell of another mussel or border of Petri dish). After the 12 h period, all records of attachment were checked, mussels were placed in plastic mesh bags tagged according to treatment, and suspended in a sea aquarium for 24 h to check for possible mortality due to exposure to the test substances. After the trials, filter papers treated with L. obtusa and S. zonale extracts were taken from dishes and allowed to air dry. The filter papers were then reextracted, the solvents evaporated and the residue remaining applied to a TLC plate for comparison with the original crude extracts. Antifouling Activity in the Field The method used was first devised by Henrikson and Pawlik (1995), modified according to da Gama et al. (2002), and has three advantages. First, the organic extracts can be included in the gel at the natural volumetric concentrations found in seaweeds, and the extracts can then slowly diffuse in the water in a manner similar to that occurring in a living organism. Secondly, the extracts are incorporated into the gel, not only on the surface, which helps to keep the physical properties of the gel surface. Thus, the differences observed in larval settlement can only be attributed to the chemical properties of the extracts. Thirdly, settlement of fouling on the experimental gel occurs under natural conditions of flow and diffusion, being exposed to a natural supply of larvae and spores of algae. Algal extracts mixed with phytagele (Sigma Chemical Company) were embedded into sterile polystyrene Petri dishes (treatments). Solvent alone was added to phytagel in the controls. Gel plates were prepared using a mixture of 1.52 g of phytagel and 35 ml of distilled water, heated to boiling point in a microwave oven. The mixture was then vigorously stirred with a glass rod, while adding 0.5 ml of

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methanol (or extract), and poured into a circular mould (Petri dishes) for hardening. The extract added to every 35 ml of gel was equivalent to an extract of 35 ml of fresh seaweed in an attempt to maintain the natural concentrations of metabolites. Gels were kept in circular molds so as to allow extract diffusion through only one side of the experimental plates. This would theoretically reduce diffusion rates by one half relative to Henrikson and Pawlik’s (1995) method, as estimated in previous work (da Gama et al., 2002). It was not possible to include CuSO4 treatment as this substance prevents phytagel from hardening. In the experiment, six 35 ml replicates were prepared for each seaweed extract and for the control. One replicate of each treatment and one control replicate were randomly arranged and fastened to each one of the six rectangular aluminum structures. These represented independent experimental units, eschewing problems of pseudoreplication (see Hulbert, 1984). The structures were then submerged to a depth of 1 m and secured to a swivel (to ensure orientation parallel to water flow) suspended from three flotation rafts moored at Cabo Frio Island (Arraial do Cabo, State of Rio de Janeiro). Settlement of fouling in the field was measured weekly as percentage cover, using a dotgrid method (Foster et al., 1991). A high number of points (235) was used to avoid underestimating rare species and to reduce deviation among replicates (Dethier et al., 1993; da Gama et al., 2002). To prevent the fouling organisms from dying, each structure was kept in a large aluminum tray containing seawater during measurements. The biofilm cover (1st week after immersion) was measured by means of its macroscopic manifestation, i.e. the growth of microorganisms on gels created a conspicuous thin, brownish layer, covering an area of the plate easily estimated. Biofilm samples taken to the laboratory contained mainly bacteria, benthic diatoms and ciliated protists. After the field assay, gels were cut into small pieces, allowed to dry, and re-extracted with the same solvents as used previously. TLC analysis was then performed in order to compare the re-extraction results with the original extracts.

was used for post-hoc comparisons. Differences among treatments were considered significant when p , 0:05 ða ¼ 5%Þ:

RESULTS Antifouling Activity Against P. perna The mussels produced as many as 114 byssal threads per treatment (control trial), and attached to filter paper (treated and control), the Petri dish border, or to the shell of other individuals. As shown in Figure 2a, the L. obtusa extract significantly inhibited byssal thread attachment relative to the control (p , 0:0002; ANOVA and Tukey HSD test), exhibiting the strongest antifouling activity. Similarly, CuSO4 prevented attachment of P. perna ðp ¼ 0:001Þ; but not so effectively (see Figure 2a). In contrast, the S. zonale extract had no significant activity on the total number of byssal threads produced by the mussels. However, as shown in Figure 2b, the mussels showed a significant preference for attachment to filter paper treated with the S. zonale extract ðp , 0:02Þ; indicating that some compound (or compounds) present in this extract may serve as a settlement cue to P. perna. Moreover, the extract of S. zonale considerably stimulated gamete release relative to other treatments, while L. obtusa treatments induced almost no gamete release (Figure 2c). Substratum exploring behavior was always displayed prior to bissus attachment (Figure 2d). As mussels exposed to the L. obtusa extract did not settle, they also did not show this pattern. On treatments containing CuSO4, however, mussels seemed to take some time to detect this substance and stop exploring and attaching to substrata. Mussels from this treatment showed 10% mortality after 24 h, although all individuals from other treatments (including L. obtusa extract) remained alive. TLC analysis of filter paper extracted after the assays showed that all visible spots were still present, including the major metabolites elatol and atomaric acid, found in L. obtusa and S. zonale, respectively. Elatol was also found in the surface extract of L. obtusa.

Statistical Analyses The data from laboratory bioassays were analyzed as the number of attached byssal threads (not as percentages as shown in graphs). Field data were analyzed as the number of dots covered by fouling. Univariate analysis of variance (ANOVA) was performed on all data, as the variances in the different groups of the design were nearly identical (Cochran C statistic and Bartlett test), and the means were not correlated with the variability across the cells of the design (Zar, 1984). The Tukey HSD test

Antifouling Activity in the Field Total cover of fouling on experimental gels through time is shown in Figure 3a. Fouling cover was significantly greater ðp , 0:001Þ on the gels carrying the S. zonale extract after 1 week of immersion. At this time fouling was mainly composed of microorganisms (biofilm). After 2 weeks, the L. obtusa extract presented fouling cover significantly lower than other treatments ðp , 0:01Þ: The same trend continued up to the 7th week, with the L. obtusa

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FIGURE 2 (a) Number of byssal threads (%) attached to different substrata after each experimental trial. (b) Number of byssal threads (%) attached to the indicated substrata (control or treatment filter paper) after trials. (Byssal threads attached to other substrata (mussel shells or Petri dish borders) were not considered in this analysis). Vertical bars ¼ means þ one standard deviation. (c) Gamete release by P. perna expressed as a percentage of the total number of individuals used in each trial (30). (d) Exploring behavior of P. perna (expressed as a percentage of the total number of individuals used in each trial) during two periods of observation (0:00–0:30 h and 2:00–2:30 h) after the beginning of the trial. N ¼ number of replicates. NS ¼ not significant. P value is from one-way ANOVA and Tukey’s HSD test between the control and the indicated treatment.

extract presenting a strong and broad spectrum antifouling activity compared to the control and the plates with S. zonale extract ðp , 0:01Þ: At this time the experiment was terminated, as fouling organisms were predated from the gels by fish. A total of 13 taxa were identified settling and growing on the gels, but total fouling cover was composed mainly of a multi-specific turf of algae and barnacles. The S. zonale extract seemed to attract more fouling species, relative to the controls, while the extract of L. obtusa prevented some species from settling or growing, thereby reducing species richness (Figure 3b). TLC analysis of the gel extracts after the experiments showed that many spots containing polar substances (water-soluble) were lost to the seawater, although the compounds elatol and atomaric acid were still present on L. obtusa and S. zonale treatments, respectively.

DISCUSSION Studies on the antifouling mechanisms utilized by sessile aquatic organisms may provide valuable information for fouling control in marine biotechnology (Hellio et al., 2001). Antifouling agents derived from natural products may be less

environmentally harmful than the current toxins, having less activity against non-target species (Hellio et al., 2000b). Most of the marine phyla adopt the sessile mode of life which characterizes fouling organisms for at least one ontogenetic phase, viz. many bacteria, many protozoa, many diatoms, most macroalgae, all sponges, most cnidarians, many molluscs, some rotifers, most bryozoans and phoronids, many brachiopods and many tube-building polychaetes, some echinoderms, a few crustaceans (e.g. Cirripedia—barnacles), some hemichordates, and all ascidians. For the majority of these species, the algal thalli appear to be little suited for settlement. This may be due to the widespread existence of multilevel antifouling defense systems, encompassing different combinations of mechanical, chemical, associational and/or life history defenses (Wahl & Mark, 1999), developed probably in response to the strong evolutionary disadvantages involved in possessing epibionts. Epibiosis may diminish the light energy and nutrients reaching the basibiont plant, indirectly influencing abundance, distribution, and productivity as well as sexual and vegetative reproduction (Orth & Montfrans, 1984). Additionally, epibionts may attract consumers that otherwise would not feed significantly on the host plant (Karez et al., 2000).

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FIGURE 3 (a) Total cover (%) of fouling on experimental gel plates. Vertical bars ¼ means þ one standard deviation. (*) ¼ Significant differences at p , 0:05 from one-way ANOVA and Tukey’s HSD test between the indicated treatment and the control. (b) Total number of fouling species (richness) on experimental gel plates. N ¼ number of replicates.

Laboratory assays using mussels as model organisms in fouling studies have been reported in literature during the last two decades. These studies either propose new antifouling screening methods using some mussel species (Harada et al., 1984; Ina et al., 1989; Takasawa et al., 1990; 1992; Satuito et al., 1993; Watanabe et al., 1993; Kitajima et al., 1995; Singh & Etoh, 1997; Hellio et al., 2000a; Sera et al., 2000) or describe the antifouling (or fouling-promoting) activity of compounds or extracts of variable nature (Katsuoka et al., 1990; Etoh et al., 1991; Goto et al., 1992; Mizobuchi et al., 1993; 1994; 1996; Shimidzu et al., 1993; Kawamata et al., 1994; Konya et al., 1994; Davis & Moreno, 1995; Dormon et al., 1996; Fusetani et al., 1996; Devi et al., 1998; Hattori et al., 1998; Terada et al., 1999; Cho et al., 2001). On the other hand, papers could not be found dealing with the relationship between laboratory ‘mussel tests’ and field antifouling assays, with the exception of one recent study (Delort et al., 2000), showing that the compound curcumin stimulated both attachment of M. edulis in the laboratory and fouling settlement in short-term field assays. The present data appear to validate the results of these screening programmes, indicating that the laboratory results may be reproduced in ecologically relevant field assays. Settlement trends observed in the laboratory may be more substantial when tested in the field. Studies correlating results of laboratory assays with effects in the field are scarce (Bakus et al., 1985; Rittschof & Costlow, 1989; Delort et al., 2000), but like the present paper, they indicate a relatively good agreement. The present results suggest that the mussel P. perna is an excellent model organism for laboratory antifouling studies, because of its rapid and clear response to test compounds. Although the power of laboratory assays is the rapid, highly sensitive screening of potential antifouling compounds for antifouling effectiveness and toxicity (Rittschof et al., 1992), they have

weaknesses. These include 1) the use of still water, as it is unclear that fouling organisms would respond to similar stimuli under natural flow conditions in the field (Hay et al., 1998); 2) the use of a single fouling organism, making it impossible to determine the activity spectrum of compounds; many new antifoulants are narrow spectrum in activity, therefore prevention of fouling by one kind of organism is routinely supplanted by fouling of another type in the field (see Rittschof, 2001); 3) the concentrations at which compounds or extracts are tested should attempt to reproduce the concentrations potentially experienced by the organisms in nature, if ecological relevance is expected (Jenkins et al., 1998); and 4) the lack of standardized testing (de Nys et al., 1996). Nevertheless, the power of field assays lies in the sensitivity to detect the activity spectrum of compounds or extracts. In this manner, the use of laboratory assays only would not permit the detection of the broad spectrum antifouling activity of the L. obtusa extract. By making collections of L. obtusa around the year it has been observed that this alga becomes fouled only near the end of its growing season, as it approaches senescence (da Gama, unpublished). A similar pattern was observed by Schmitt et al. (1995) with the brown alga Dictyota menstrualis from North Carolina. The increase in fouling cover or epibiosis on Laurencia thalli seems to be related to an observed decrease in elatol levels (da Gama et al., unpublished observations). Phillips and Towers (1982) observed within-thallus variation in bromophenol (lanosol) levels from the red alga Rhodomela larix, relating this variation with epiphyte cover. However, precise concentration measurements would be necessary to support such a statement for L. obtusa. As the authors have identified elatol as the major metabolite in Brazilian L. obtusa, it would be expected that some toxic mechanism is involved, since some authors (de Nys et al., 1996; Ko¨nig & Wright, 1997) describe this sesquiterpene as being

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toxic to some invertebrate larvae (Balanus amphitrite nauplii). Despite that, mussels exposed to the L. obtusa extract remained alive, while 10% of the mussels exposed to a much lower level of CuSO4 died after a 24 h period. Although the above mentioned authors report toxicity to barnacle larvae, they also say that elatol did not inhibit growth of marine bacteria or the green alga Ulva lactuca. Thus it could be concluded that this compound is not as toxic, as in the present work it (mixed in the extract) also did not kill mussel juveniles. As compounds absorbed into filter paper circles were not released (at least completely) into the seawater in the dishes, juvenile mussels could avoid exposure to the L. obtusa extract by refusing to attach (i.e., behaviorally avoiding exposure). However, it seems quite unlikely that mussels could close their shells and stop the filtering and respiration process. In any case, antifouling agents without acute toxicity are generally considered to be nontoxic. However, all compounds that somehow prevent settlement or metamorphosis increase the probability of death of the target organism, because the organism must spend more time in the plankton. An increase in larval time in the water column also increases larval mortality (Rittschof, 2001). Some marine investigators (e.g. Paul, 1992; Schmitt et al., 1995) have noted that secondary metabolites that deter feeding by herbivores could also serve other ecological functions (i.e. they play multiple functional roles). Several authors have ascribed a defensive role against herbivores to elatol (Hay et al., 1987; 1988; Granado & Caballero, 1995). Also, de Nys et al. (1996) and Ko¨nig and Wright (1997) showed that this metabolite (isolated from Australian L. rigida ) inhibited larval settlement of Bugula neritina and Balanus amphitrite in laboratory trials. If elatol were really responsible for the field antifouling activity reported in the present study, it might play a multiple function role in the alga, acting simultaneously as a feeding deterrent against diverse herbivores and as a broad spectrum epibiosis inhibitor. The byssal attachment-promoting activity of the S. zonale extract is also in accordance with the field data. Delort et al. (2000) also found that an attachment stimulant (curcumin) for the mussel M. edulis enhanced fouling in field assays. This activity appears to be reinforced by the elevated rate of gamete release caused by the extract of S. zonale (although this gamete release may also be interpreted as resulting from stress). The identification of settlement promoters may be very useful to aquaculture technology, increasing settling rates of edible molluscs (oysters and mussels) or macroalgae on desired surfaces. Although the screening for new antifouling compounds using laboratory based bioassays can

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be a useful and quite reliable method, the ecological significance of laboratory bioassays appears to be very limited and should be confirmed by subsequent field experiments. Further work is needed to confirm the identity of L. obtusa compounds causing the observed activity, and to examine the precise role of such activity in nature.

Acknowledgements CNPq and FAPERJ supported this research. BAPG and ARS gratefully acknowledge CAPES for providing ScD Fellowships. RCP, VLT, and YYV thank CNPq for their Research Productivity Fellowships. BG Fleury, CEL Ferreira, RC Villac¸a, JHS Miyamoto and TS Ribeiro helped either in field work or in preliminary assays. Comments by FPA Gama improved the manuscript.

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Is the Mussel Test a good Indicator of Antifouling Activity?

1H-NMR data (Wessels et al., 1999). This compound represented 1.65% of the DW of S. zonale. In order to detect the presence of metabolites at the surface of ...

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