TRANSFORMATION OF PARALYTIC SHELLFISH TOXINS IN BENTHIC CLAM, POLYMESODA SIMILIS FED WITH TOXIC DINOFLAGELLATE, ALEXANDRIUM MINUTUM (DINOPHYCEAE)

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1 TRANSFORMATION OF PARALYTIC SHELLFISH TOXINS IN BENTHIC CLAM, POLYMESODA SIMILIS FED WITH TOXIC DINOFLAGELLATE, ALEXANDRIUM MINUTUM (DINOPHYCEAE) ABSTRACT P. T. Lim 1, P. S. Daniel Ngilek 1, C. P., Leaw 2, O. Bojo 1, and G. Usup 2 1 Department of Aquatic Science, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak 2 Marine Science Program, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Corresponding author: ptlim@frst.unimas.my; Paralytic shellfish poisoning (PSP) is a type of shellfish poisoning due to the contamination of microalgae derived- toxins. Thousands of poisoning cases have been reported in Malaysia due to three species of marine dinoflagellate, viz. Pyrodnium bahamense var. compressum, Alexandrium tamiyavanichii and A. minutum. In the present study, biotransformation study of paralytic shellfish toxins was carried out in benthic clam, Polymesoda similis fed with cultured cells of A. minutum. Toxin analysis was carried out using high performance liquid chromatography with post column derivatization with authentic toxins as reference. Toxin composition observed in the initial clam samples (8 hrs after feeding) was identical to the A. minutum s, with GTX4 as the predominant toxin (up to 86%). However, GTX1 increased dramatically accompanied by the decrease of GTX4 after 24 hrs due to the epimerization process of GTX4 to GTX1. Biotransformation of toxin was observed after 96 hrs with the decrease of GTX1 and an increase in GTX2 (24-34%) and GTX3 (16-22%). However, toxicity remained unchanged for six days (samples 4-6) clearly indicated the low toxin depuration rate in P. similis. Toxins that accumulated in P. similis and its composition also reaffirmed our earlier finding that A. minutum was the causative organism of the PSP event in Tumpat, Kelantan in year Keywords: Paralytic shellfish poisoning (PSP), biotransformation, benthic clams, Polymesoda similis, Alexandrium minutum INTRODUCTION Paralytic shellfish Poisoning (PSP) is a type of shellfish poisoning caused by the consumption of toxins contaminated shellfish that derived from several species of marine microalgae. PSP has been seriously affecting shellfish industries in many countries (Fernandez et al. 2003) and caused public health concern, particularly in countries where shellfish is an important source of protein. In Malaysian waters, PSP was first reported in the east coast of Sabah following blooms of Pyrodinium bahamense (Roy, 1977). Since then, thousands of cases have been reported but only confined to the west coast of Sabah. In recent years, PSP have been increasingly reported in the Peninsula Malaysia, with several sporadic incidences on both South China Sea and Malacca Strait (Usup et al., 2002). The dinoflagellate, Alexandrium tamiyavanichii was suspected as the culprit for the event in Sebatu, Malacca where three persons were hospitalized with no fatality. In September 2001, six people were poisoned including one fatality was reported in Tumpat, Kelantan after consuming the benthic clam, Polymesoda similis or locally known as lokan (Lim et al. 2004). The causative organism responsible for the event was identified as Alexandrium minutum (Lim et al. 2004) based on the dinoflagellate cultures established and wild specimens collected from location where the incidence occurred. PSP have been reported in many species of shellfish such as Argopecten irradians, Cardium edule, Mya arenaria, Mytilus edulis, Pecten maximus, Saxidomus gigantus and Spisula solidae (Shumway et al. 1990; Emsholm et al. 1995). The toxin accumulation and depuration rates differed greatly depending on the types of shellfish. However, little is known about the toxin accumulation and depuration of paralytic shellfish toxins (PSTs) in the benthic clam, P. similis. This species is commonly found in muddy intertidal zone of swamp and mangrove forest and consumed as delicacy among the local folk. Knowledge on the accumulation and depuration of PSTs in the clam is 197

2 important in shellfish monitoring where outbreaks occurred. In our present study, a post-column derivatization high performance liquid chromatography (HPLC) method was applied to evaluate the toxin accumulation and depuration of PSTs in P. similis fed with toxic Alexandrium minutum. The main objective of this study is to determine the biotransformation of PSTs in P. similis and to verify that A. minutum as the causative organism of PSP event in September 2001 in Tumpat, Kelantan. MATERIALS AND METHODS Alexandrium minutum Culture The clonal culture of dinoflagellate, Alexandrium minutum was established from plankton haul samples in Tumpat, Kelantan (Figure 1). The culture was grown in ES-DK medium (Kokinos & Anderson, 1995) contained 380µM of nitrate and 12µM of phosphate, and addition of 8mL/L soil extract. The culture was maintained at 26 ± 0.5 o C with a 12:12 hour light dark cycle. Salinity of medium was adjusted to 15 PSU with distilled waters. Three liters of exponential phase culture were harvested by sieving through 20μm mesh size sieve and centrifuged at 2000 g for 10 mins. Cell density was determined by microscopic cell count before fed to the clams. Figure 1. Map showing location of Geting River, Tumpat, Kelantan where clonal culture of Alexandrium minutum (inset) was isolated during the blooms event in September Feeding Experiments For feeding experiments, the benthic clam, Polymesoda similis collected from the local peat swamp were used in this study. All the clams used were properly cleaned with seawater to remove sediment and epiphytes attached. P. similis was kept in seawater with salinity adjusted to 15 PSU which similar to the salinity of location where the clam was collected. The experiment was carried out in a plastic tray containing 2L seawater, supplemented with approximately cells of A. minutum. Feedings were done at 0 hr, 24 hr, 72 hr, 96 hr and 144 hr over the experiment period. Total of eight pieces of clams were randomly taken for toxin analysis (before feeding, 8 hrs, 24 hrs, 96 hrs, 168 hrs, 240 hrs and 312 hrs after feeding). The clam samples were kept in -20 o C before toxin extraction and analysis. Toxin Extraction From Alexandrium Minutum And Polymesoda Similis Toxin extraction of A. minutum was carried out as described earlier (Lim and Ogata, 2005; Lim et al., 2006). Late exponential phase culture of A. minutum was harvested by centrifugation. Toxin was extracted in 0.05 M acetic acid (AcOH) using ultra-sonication homogenizer in ice waters. Toxin extracts were kept in -20 o C until further analysis. For clams, the samples were dissected, rinsed, weighted and added with equal volume of 1 N of HCl according to AOAC method. The mixture was 198

3 homogenized using blender until it became slurry. The samples were then centrifuged at 6000 g for 10 mins to separate clam tissue from the toxin extract. The supernatant was carefully removed and purified using ISOLUTE C-18 SPE columns according to manufacturer instruction (Waters, Massachusetts, USA). Toxin Analysis High performance liquid chromatography (HPLC) analysis was carried out using a Shimadzu VP HPLC system with isocratic, post-column derivatization method of Oshima (Oshima, 1995) with minor modification. The samples were run through a Genesis or Apex C18 column (4.6 mm i.d. 15 cm, 120Å, 4 μm). The chromatographic conditions were as follows. The column mobile phase contained 2 mm heptanesulfonate in 30 mm ammonium phosphate buffer, ph 7.1. The post-column oxidizing reagent was 7 mm periodic acid in 80 mm sodium phosphate buffer, ph 9.0, while the acidifier was 0.5 M acetic acid. Flow rates for the mobile phase were 0.8 ml min -1, and 0.4 ml min -1 for each post-column reagent. Detection wavelengths were set at 330 nm excitation and 390 nm emission. Toxins identification and quantification were determined by comparison with authentic toxin standards. RESULTS AND DISCUSSION Filter feeders such as bivalves that fed on the toxic algae during the blooms ingested and accumulated the toxins. Several studies have been carried out on accumulation and depuration of paralytic shellfish toxins (PSTs) in shellfishes. However, those studies were mainly conducted on bivalves from temperate waters. Thus far, no PSTs accumulation study was conducted on benthic clams such as Polymesoda similis. This is the bivalve species that caused poisoning with a fatality after consuming the clams in Tumpat, Kelantan in September In our present study, considerable high amount of toxins was detected in the clam tissues with μg STXeq. 100g -1 tissue (>400 MU 100g -1 tissue) following eight hours of toxic cells exposure (Figure 2, Table 1). This indicated a fast PSTs accumulation rate in the clam, P. similis. The toxins accumulated in the clams also exceeded the permissible level of 400 MU 100g -1 tissue (80μg PSTs 100g -1 tissue) in commercial shellfish. However, the total toxin contents did not increase further even after several feedings. The clams accumulated between 38 to 68 μg of PSTs per 100g clam tissue. It is well known that the capacity to accumulate ingested PSTs in bivalves vary significantly among species. Bougrier et al. (2001) found that king scallops only accumulated 8% of the total toxins contained in the ingested algae cells. On the other hand mussels such as Mytilus edulis or M. californianus showed high accumulation of 74-96% (Bricelj and Shumway, 1998) μg STX eq. 100g Time after feeding with A. minutum (hr) Figure 2. Total toxin contents found in the clams (μg STXeq. 100g -1 tissue) throughout the experiment. 199

4 Table 1. Toxin composition of benthic clam, Polymesoda similis fed with toxic A. minutum Clam Samples Collection time of clams PSTs (% STXeq.) after feeding (hrs) a GTX1 GTX2 GTX3 GTX4 dcgtx3 STXs Total toxin MU b 100g -1 tissue (μg STXeq. 100g -1 ) * * Trace (50.05) * * Trace (37.91) * Trace (55.71) Trace (56.34) Trace (68.38) Trace (64.71) a Collection time of clams for toxin analysis (hours) after feeding; b MU= mouse unit: One mouse unit is toxin that required to kill a 20g mice in 15 min when injected intra-peritoneally; * Toxin congener was not detected in the samples. Toxin composition observed in the initial clam samples (8 hrs exposure) was identical to the A. minutum s, with GTX4 as the predominant toxin (up to 86%) (Figure 3). However, GTX1 increased dramatically accompanied by the drastic decrease of GTX4 after 24 hrs of exposure. This was probably due to the epimerization process of GTX4 to GTX1 and the structural characteristics of the toxin itself. Epimerization of 11-hydroxysulfate toxins occurred in all shellfish, with beta-epimers (such as GTX4) gradually transformed into chemically more stable alpha-epimers (such as GTX1) (Oshima et al., 1995). Our clonal culture of A. minutum used in this study produced GTX4 and GTX1 as dominant toxin congeners (up to 97% mole). Other toxin congeners, GTX2, GTX3 and STX were only found in trace amount. Our result also showed that toxins ingested at the initial stages (before 96 hrs exposure) did not undergoing any chemical change. Transformation of toxins was observed after 96 hrs of toxic cells exposure, with the 2-fold decrease of GTX1 and increases in GTX2 and GTX3 (Figure 3). Natural reductants (such as glutathione and cysteine) are commonly found in shellfish which facilitated the conversion of N-OH group toxins (GTX1, GTX4, and neostx) to N-H group toxins (GTX2, GTX3, and STX) (Arakawa et al., 1984). Similar transformation was also observed in scallop, Patinopecten yessoensis (Oshima et al., 1995). The species has been shown to retain toxins for several months and a change in toxin profile was observed during the depuration process (Oshima et al., 1995). Most saxitoxin derivatives undergo transformation in the bivalve organisms which they accumulated. This was evident from the variation in toxin profiles of bivalves and the toxic algae found in the surrounding waters (Schantz et al. 1975). The transformation in bivalve was also species dependent and affected by environmental conditions (Fernandez et al. 2003). The variation in stability and the existence of interconversions can cause discrepancies in toxin analysis. According to Shimizu (1987) the stability of saxitoxin derivatives are in order of STX > neostx > GTX2,3 > GTX 1,4, with STX as the most stable form. The carbamate toxins (STX, neostx, GTX1 4) are times more toxic than the N-sulfocarbamoyl derivatives (GTX5, 6 and C toxins) (Cembella et al. 1993). Furthermore, the specific toxicity of each toxin congeners may also caused drastic change in total toxicity without any intake or losses of toxin. Increases in GTX3 and GTX2 after toxic cells exposure period also strongly supported our initial toxin analysis of contaminated clams collected after two weeks of the Tumpat event where GTX3 and GTX2 were found in high proportion. Toxicity of the clams (sample 4 to 6) remained unchanged after toxic cells exposure period for six days clearly indicated the low toxin depuration rate in the clam, P. similis (Figure 3). Saxitoxins 200

5 % STX eq. 100 GTX GTX4 GTX2 GTX Time after feeding with A. minutum (hr) Figure 3. Changes of toxin compositions in benthic clam, Polymesoda similis after feeding with toxic A. minutum. Arrows indicate time of feeding (0 hr, 24 hr, 72 hr, 96 hr and 144 hr). persist in shellfish for varying periods, depending on the shellfish and the tissue involved. Shellfish that clear the toxins very quickly are toxic only during period of actual blooms (Taylor, 1988). Mussels could become highly toxic within a few hours to a few days of the onset of a toxic algal bloom, but lose their toxicity rapidly. Generally, clams and oysters do not become as toxic as mussels. However, they require more time to accumulate high level of toxins and longer period to cleanse themselves. The Alaska butter clam (Saxidomus giganteus) is never safe to consume as it retains toxins for years (Schantz, 1973; Schantz, 1984; Shumway, 1990). Unfortunately, the depuration rate of P. similis in this study was not able to determine due to the short duration opted. In this study, the toxin composition found in the clam, P. similis after exposed to toxic A. minutum also reaffirmed our earlier finding that A. minutum was the causative organism for the PSP event in Tumpat, Kelantan in year The toxin composition and profile were identical to the contaminated clams collected from the area after the poisoning event (Lim et al. 2004). Absence of other PSTs producing dinoflagellates, such as Pyrodnium bahamense and A. tamiyavanichii in the plankton samples (Usup et al., 2002) also supported our conclusion. Our result clearly showed that the benthic clam, P. similis is able to accumulate PSTs in a rapid manner on the onset of a toxic algae bloom and could take longer period to depurate the toxins. The public should be advised to avoid consumption of the clams during toxic algal blooms. ACKNOWLEDGEMENTS The study was financially supported by the Universiti Malaysia Sarawak (UNIMAS) short term research grant to P.T. Lim. P. S. Daniel Ngilek was supported by Zamalah graduate studentship from UNIMAS. REFERENCES Bricelj, M. & Shumway, S Paralytic shellfish toxins in bivalve mollusk: occurrence, transfer kinetic and biotransformation. Rev. Fish. Sci. 6: Bougrier, S., Lassus, P., Beliaeff, B., Bardouil, M., Masselin, P., Truquet, P., matignon, F., Mornet, F. Le Baut, C Feeding behavior of individuals and groups of king scallops (Pecten maximus) contaminated experimentally with PSP toxins and detoxified. In. Hallegraeff, G., Backburn, S., Bolch, C., Lewis, R. (Eds), Harmful Algal Blooms IOC UNESCO, pp Cembella, A., Shumway, S., Lewis, N., Anatomical distribution and spatio-temporal variation in paralytic shellfish toxin composition in two bivalve species from the gulf of Maine. J. Shellfish Res. 12:

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