Article pubs.acs.org/est
Toxin-Producing Ostreopsis cf. ovata are Likely to Bloom Undetected along Coastal Areas Martin Pfannkuchen,*,† Jelena Godrijan,† Daniela Marić Pfannkuchen,† Ljiljana Iveša,† Petar Kružić,‡ Patrizia Ciminiello,§ Carmela Dell’Aversano,§ Emma Dello Iacovo,§ Ernesto Fattorusso,§ Martino Forino,§ Luciana Tartaglione,§ and Margareta Godrijan‡ †
Institute Ruđer Bošković, Center for Marine Research, Giordano Palliaga 5, 52210 Rovinj, Croatia Laboratory for Marine Biology, Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia § Chemistry of Natural Products Department, University of Napoli Federico II, Via Domenico Montesano 49, 80131, Napoli, Italy ‡
S Supporting Information *
ABSTRACT: Mass appearances of the toxic dinoflagellate genus Ostreopsis are known to cause dangerous respiratory symptoms in humans exposed to aerosols. The outbreaks can appear in shallow marine waters of temperate regions around the globe. We followed a massive bloom event on a public beach on the northern Adriatic coast near Rovinj, Croatia. We identified the responsible species and the produced toxins as well as the dynamics of the event with respect to environmental conditions. Ostreopsis cf. ovata appeared in masses from September through October 2010 on a public beach near Rovinj, Croatia but stayed undetected by public health organizations. Respiratory symptoms were observed whenever humans were exposed to substrate samples containing large numbers of Ostreopsis cells. During the mass abundance of O. cf. ovata also exposure to the aerosols on the beach evoked respiratory symptoms in humans. Our measurements showed high cell abundances and high toxin contents with a stable relative contribution of putative Palytoxin and Ovatoxins a-e. Artificial beach structures proved to dramatically reduce settling of the observed Ostreopsis biofilm. Blooms like those reported herein have a high potential to happen undetected with a high potential of affecting the health of coastal human populations. Increased monitoring efforts are therefore required to understand the ecology and toxicology of those bloom events and reduce their negative impact on coastal populations.
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INTRODUCTION Harmful algae blooms (HABs) are caused mostly by unicellular algae. Their highly mobile life style allows them to form mass appearances essentially everywhere and any time, given suitable conditions. This implies severe difficulties in predicting and investigating those events. The importance of a close HAB monitoring becomes more and more evident. Those HABs are affecting human populations in ways like discoloration of the water with subsequent decay on the coastlines, adverse effects on marine resources (from tourism to fisheries), toxin accumulation throughout the food chain, and most severely direct intoxication of humans and animals through contact or aerosols.1−6 The mass appearance of toxic algae is a result of suitable environmental conditions.2,4,7−9 In the last decades the frequency and intensity of mass appearances of toxic algae seems to have increased, albeit it is discussed whether this is a result of changing environments or increased monitoring efforts.10,11 However, the ecologies of HABs are not yet sufficiently understood to propose successful forecast or management scenarios beyond monitoring.11,12 Most toxic algae blooms are discovered after the observation of resulting dramatic symptoms affecting higher animals or humans.13 But © 2012 American Chemical Society
given the increasing number of compounds and toxinproducing species, we must expect a high number of so-far unreported toxic events caused by algae blooms. We therefore do not yet know the full extent to which coastal populations are affected by mass appearances of toxic marine organisms. We herein report on a mass appearance of the dinoflagellate Ostreopsis cf. ovata. O. cf. ovata is known to produce the toxic compounds putative palytoxin and ovatoxins (a-e)14−18 (Figure S1 in the Supporting Information). Palytoxin is the most potent nonprotein toxin known.19 It acts primarily on the Na+/K +-ATPase channel and clinical symptoms range from violent contractions of all muscle types, sperm immobility, to hemorrhagic and ultimately fatal effects. For an exhaustive review of palytoxin toxicity see Ramos and Vasconcelos.16 Uptake routes leading to symptoms range from skin contact over inhalation of aerosols to ingestion of contaminated seafood, with the latter being of reportedly fatal consequences. Received: Revised: Accepted: Published: 5574
January 16, 2012 April 12, 2012 April 24, 2012 April 24, 2012 dx.doi.org/10.1021/es300189h | Environ. Sci. Technol. 2012, 46, 5574−5582
Environmental Science & Technology
Article
Figure 1. (a) Light micrograph of the biofilm (darkfield, bar = 500 μm). (b) Light micrograph of Ostreopsis cf. ovata cells from the biofilm depicted in (a) (differential interference contrast, bar = 50 μm). (c) Fluorescent light micrograph of Calcofluor white stained O. cf. ovata cells showing the characteristic plate structure and pore (po) on the Epitheca (E) and Hypotheca (H). Excitation: 350 nm. Emission: 400 nm −480 nm. Extended focus compilation over a stack of 10 focal layers within 12 nm in z direction after theoretical deconvolution (bar = 20 μm). (d) Scanning electron micrograph of the extracellular matrix produced by and stabilizing the biofilm (bar = 10 μm).
investigations of its geographic distribution independent of reported impacts on human health.30,40−42 Samples from suitable substrates are examined microscopically for the presence of Ostreopsis species and final species determination is traditionally performed with fluorescence microscopy on stained cells and of course electron microscopically by determining the characteristic plate structures.43 To supplement extensive microscopical searches, recently PCR-based essays were developed to detect the species even in very low abundances within environmental samples.44 As a result of the aforementioned methodological improvements and raised interest, mass abundances are also reported for coastal areas in the Indian Ocean and the Atlantic Ocean and the toxin production is confirmed around the world.30,42,43,45−49 Here we report a detailed description of an Ostreopsis mass appearance on a public beach near the city of Rovinj (Croatia). We followed the bloom from September to October 2010. Daily sampling allowed us to closely monitor the development of several abundance peaks as well as the end of the mass appearance. The parallel recording of environmental parameters as well as in situ observations gave further insights into the ecology and bloom dynamics of this toxic dinoflagellate genus on a part of the northern Adriatic coast.
Although the group of toxins was originally described from a tropical soft coral, the most widespread palytoxin producing organisms are dinoflagellates of the genus Ostreopsis J. Schmidt 1901.14,15,20 The risk imposed by palytoxin and ovatoxins and their producing organisms on humans and animals is high and largely underestimated.21 Animal models for toxin detection are tested, but largely the analysis relies on chemical methods such as combinations of liquid chromatography and mass spectrometry (LCMS).18 The chemical structure of the palytoxin family was recently uncovered17,18,22−26 and sensitive LC-MS methods based on selected ion monitoring (SIM) and multiple reaction monitoring (MRM) experiments on triple quadrupole MS25,27 or on high-resolution full MS and MS/MS experiments on linear ion trap-orbitrap FTMS,28,29 or on full MS experiments on time-of-flight (TOF)15 have been reported for their detection. The species of the genus Ostreopsis are known to live mainly attached to benthic substrates. Massive abundances are observed in shallow coastal areas, especially close to the shoreline in only a few meters of depth. The genus is reported from temperate and subtropical regions all around the world, therefore imposing a substantial threat to the world’s population in those regions.30 Blooms with dramatic impacts on human health are so far mainly reported from the coasts around the Mediterranean.25,27,31−39 In the area around Genova, Italy, several hundred persons had to be hospitalized after exposure to aerosols during a bloom of Ostreopsis sp. in summer 2005.33 The rising interest in the genus Ostreopsis, which is able to produce toxic aerosols along shorelines, led to
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MATERIALS AND METHODS The observed bloom took place on a public beach close to the city of Rovinj, Croatia (45°6.018 N, 13°37.704 E, September− October 2010). All underwater work was performed in apnea (snorkeling) in a water depth between one and four meters. 5575
dx.doi.org/10.1021/es300189h | Environ. Sci. Technol. 2012, 46, 5574−5582
Environmental Science & Technology
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Figure 2. Average abundances of O. cf. ovata attached to C. crinita (cells per gram wet weight of substrate) and in the surrounding water as well as environmental conditions at the sampling site (3 substrate samples and 3 replicas each). Error bars show the standard deviation, small crosses and numbers show the maximum observed abundance. The large cross marks the day after the strongest wave impact. The filled circle marks the last storm impact and the end of the bloom. The open circle marks a weaker wind and wave impact, which washed bigger flocks of the biofilm from the substrate and resulted in floating flocks of O. cf. ovata on the water surface.
Water samples of the “surrounding” water were taken in 1.5-L PET bottles. Substrate samples of Cystoseira crinita and hard substrate such as pebbles were taken in 1-L PET containers including the surrounding water. Care was taken to not mechanically disturb the substrate and attached epiphytes during sampling. The samples were immediately transferred to the nearby Center for Marine Research in Rovinj for further processing. For Ostreopsis sp. counting, each specimen of C. crinita was sampled with 1 L of the surrounding water from 1.5 m depth. Cell counts were normalized to the wet weight of the substrate C. crinita. On the base of earlier reported population densities of 5950 g C. crinita per m2 50 we calculated the Ostreopsis cell abundance and total toxin content per square meter by multiplying the normalized cell counts with the population density of C. crinita and by multiplying the Ostreopsis cell abundances with the cellular toxin content respectively. After vigorous shaking, 300 or 600 mL of untreated surrounding water from the substrate samples was filtered on a 0.22-μm glass filter, and the filters were transferred
to a 60% aqueous solution of methanol (Kemika). The samples were stored at −20 °C until they were analyzed by HR LC-MS for the presence of palytoxins and ovatoxins. O. cf. ovata samples for toxin analysis were collected every day in the period when the highest cell concentration was recorded in seawater (September 28−October 7, 2010) and every two days at the end of the HAB (October 15−19, 2010). Cell pellets were provided to the Natural Products Chemistry Department for chemical analysis. Cell numbers are reported in SI Table 2. Pellets were extracted as previously reported.25,27,28 Crude extracts (volume = 10 mL) were analyzed directly by highresolution liquid chromatography−mass spectrometry (HR LCMS) on an Agilent 1100 LC binary system (Palo Alto, CA) coupled to a hybrid linear ion trap LTQ Orbitrap XL Fourier transform MS (FTMS) equipped with an ESI ION MAX source (Thermo-Fisher; San Josè, CA). LC-MS conditions were reported previously.28 For more detailed information see Supporting Information. 5576
dx.doi.org/10.1021/es300189h | Environ. Sci. Technol. 2012, 46, 5574−5582
Environmental Science & Technology
Article
334 306 cells per g wet weight of C. crinita. After October 16 the average abundances fell below 5325 cells per g wet weight of C. crinita and from then on decreased further. Figure 2 shows the abundances of O. cf. ovata observed as attached to the substrate C. crinita and in the surrounding water as well as the abiotic factors measured during the bloom event. A weaker wind and wave impact on October 1, 2010 detached parts of the biofilm, which could afterward be observed floating on the water surface. Shortly after that, the biofilm was restored on the substrate (open circles in Figure 2). Repeatedly higher wind situations coming from the south culminated on October 4 in more than a meter wave heights breaking on the examined beach. This high energy input washed up a lot of sandy sediments and completely removed the observed biofilms from the substrate (upright cross in Figure 2). This event followed the bloom peak abundance with 334 306 cells per g wet weight substrate. During this strong wave impact the number of cells attached to the substrate fell to 58 181 cells per g wet weight substrate. At the same time the number of cells per liter in the surrounding water reached its peak of 42 600. Only 24 h later this number fell again to 1720 cells per liter surrounding water while the number of cells on the substrate rose again. The beginning of the bloom went along with extended rainfalls but only little reduction on salinity around the substrate. The sudden reduction in cells attached to the substrate, directly after the record of highest abundance, was followed by a sudden increase of cells in the surrounding water. This coincided with moderate rainfall and a more pronounced reduction in salinity around the substrate. Nevertheless the following days, with again-raised salinities measured, showed again high numbers of attached Ostreopsis cells with a second maximum of 150 840 cells per g wet weight of C. crinita. A third maximum of 134 665 cells per g wet weight of C. crinita was observed on October 13, 2010 and was followed by an immediate and dramatic decrease of observable Ostreopsis cells on the substrate. This decrease finished in an overall low of Ostreopsis abundance coinciding with another heavy storm impact and heavy (
