Comparative Paralytic Shellfish Toxin Profiles in the Strains Of

Baseline / Marine Pollution Bulletin 50 (2005) 208–236 211

Acknowledgment Muir, D., Braune, B., DeMarch, B., Norstrom, R., Wagemann, R., Lockhart, L., Hargrave, B., Bright, D., Addison, R., Payne, J., The authors wish to thank Hector de Haro for the Reimer, K., 1999. Spatial and temporal trends and effects of contaminants in the Canadian Arctic marine ecosystem: a review. sample preparations and the staff of the Natal Sharks Science of the Total Environment 230, 83–144. Board who made the tissue sampling possible. Serrano, R., Fernańdez, M.A., Hernańdez, L.M., Hernańdez, M., Pascual, P., Rabanal, R.M., Gonza´lez, M.J., 1997. Coplanar polychlorinated biphenyl congeners in shark livers from the North- References Western African Atlantic Ocean. Bulletin of Environmental Contamination and Toxicology 58, 150–157. Agency for Toxic Substances and Diseases Registry (ATSDR)/US Storelli, M.M., Marcotrigiano, G.O., 2001. Persistent organochlorine Public Health Service, 1994. Toxicological Profile for 4,40-DDT, residues and toxic evaluation of polychlorinated biphenyls in 4,40-DDE, 4,40-DDD (Update). ATSDR, Atlanta, GA. sharks from the Mediterranean Sea (Italy). Marine Pollution Aguilar, A., 1984. Relationships of DDE/DDT in marine mammals to Bulletin 42, 1323–1329. the chronology of DDT input into the ecosystem. Canadian Swann, R.L., McCall, P.J., Laskowski, D.A., Dishburger, H.J., Journal of Fisheries and Aquatic Science 41, 840–844. 1981. Estimation of soil sorption constants of organic chemicals Cliff, G., Dudley, S.F.J., Davis, B., 1989. Sharks caught in the by high-performance liquid chromatography. In: Aquat. Toxi- protective gill nets off Natal, South Africa. 2. The great white shark col. Hazard Assess., ASTM Special Technical Publication 737, (Carcharodon carcharias). South African Journal of Marine Science pp. 43–48. 8, 131–144. Tren, R., Bate, R., 2004. South AfricaÕs War against Malaria: Lessons Davis, J.A., May, M.D., Greenfield, B.K., Fairey, R., Roberts, C., for the Developing World. Cato Policy Analysis No. 513, Cato Ichikawa, G., Stoelting, M.S., Becker, J.S., Tjeerdema, R.S., 2002. Institute, Washington, DC. Contaminant concentrations in sport fish from San Francisco Bay. Tricas, T.C., McCosker, J.E., 1984. Predatory behavior of the Marine Pollution Bulletin 44, 1117–1129. white shark (Carcharodon carcharias) with notes on its biology. de Kock, A.C., Best, P.B., Cockcroft, V., Bosma, C., 1994. Persistent Proceedings of the California Academy of Sciences 43, 221–238. organochlorine residues in small cetaceans from the East and West Vetter, W., Weichbrodt, M., Scholz, E., Luckas, B., Oelschlager, H., coasts of southern Africa. Science of the Total Environment 154, 1999. Levels of organochlorines (DDT, PCBs, toxaphene, chlor- 153–162. dane, dieldrin, and HCHs) in blubber of South African fur seals Hayteas, D.L., Duffield, D.A., 2000. High levels of PCB and p,p0-DDE (Arctocephalus pusillus pusillus) from Cape Cross/Namibia. Marine found in the blubber of killer whales (Ocinus orca). Marine Pollution Bulletin 38, 830–836. Pollution Bulletin 40, 558–561. Watling, R.J., Watling, H.R., Stanton, R.C., McClurg, T.P., Enge- Howard, P.H., Meylan, W.M., 1997. Prediction of physical properties, lbrecht, E.M., 1982. The distribution and significance of toxic transport, and degradation for environmental fate and exposure metals in sharks from the Natal Coast. South Africa Water Science assessments. In: Chen, F., Schuurmann, G. (Eds.), Quantitative and Technology 14, 21–30. Structure–Activity Relationships in Environmental Sciences—VII, Wintner, S.P., Cliff, G., 1999. Age and growth determination of the Proceedings QSAR96. SETAC Press, Pensacola, FL, pp. 197– white shark, Carcharodon carcharias from the East Coast of South 217. Africa. Fisheries Bulletin 97, 153–169.

Comparative paralytic shellﬁsh toxin proﬁles in the strains of Gymnodinium catenatum Graham from the Gulf of California, Mexico

I. Ga´rate-Liza´rraga a,*, J.J. Bustillos-Guzma´n b, L. Morquecho b, C.J. Band-Schmidt a, R. Alonso-Rodrı´guez b, K. Erler c, B. Luckas c, A. Reyes-Salinas b, D.T. Go´ngora-Gonza´lez b

a Departamento de Plancton y Ecologı´a Marina, Centro Interdisciplinario de Ciencias Marinas (CICIMAR-IPN), Apdo. Postal 592, La Paz, B.C.S. 23000, Me´xico b Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, B.C.S. 23000, Me´xico c Faculty of Biology and Pharmacy, Department of Food Chemistry, Friedrich-Schiller University, Dornburgerstraße 25, 07743 Jena, Germany

Gymnodinium catenatum was described for the first time in the Gulf of California, Mexico by Graham * Corresponding author. Tel.: +52 112 25344/+52 612 1230350x82434; fax: +52 112 25322/+52 612 122 5322. (1943). The known global distribution of this species E-mail addresses: [email protected], [email protected] has increased dramatically in the past few decades, (I. Ga´rate-Liza´rraga). and it is now reported in every continent, its vegetative 212 Baseline / Marine Pollution Bulletin 50 (2005) 208–236 forms having been recorded in Argentina, Alexandria, Australia, Brazil, Cuba, Italy, Japan, Korea, Philip- pines, Portugal, Spain, Singapore, Uruguay, and Vene- zuela (Hallegraeff and Fraga, 1996; Negri et al., 2001; La Barbera-Sańchez and Gamboa-Maruez, 2001; Holmes et al., 2002; Go´mez, 2003; Leal et al., 2003; Park et al., 2004). G. catenatum is the only known gymnodi- niod dinoflagellate that produces paralytic shellfish poisoning (PSP) (Hallegraeff and Fraga, 1996), and up to 20 saxitoxin analogues have been reported (Negri et al., 2001, 2003). G. catenatum is an important compo- nent of the phytoplankton along the Mexican Pacific coastline (Graham, 1943; Mee et al., 1986; Corte´s-Alta- mirano et al., 1999; Figueroa-Torres and Zepeda-Esqui- vel, 2001; Herrera-Galindo, 2002; Ga´rate-Liza´rraga et al., 2004a,b; Morquecho and Lechuga-Deve´ze, 2004; Band-Schmidt et al., 2005). High concentrations have been reported with few human fatalities, and extensive fish mortalities (Mee et al., 1986; Corte´s-Altamirano and Alonso-Rodrı´guez, 1997). Recently, massive mor- tality events of nauplii and adult shrimp related to G. catenatum blooms have been documented along the Fig. 1. Sampling stations ( ) and historical records (d) of distribution Sinaloa shoreline (Alonso-Rodrı´guez and Paéz-Osuna, of Gymnodinium catenatum along the Gulf of California (Graham, ´ 1943; Gilbert and Allen, 1943; Mee et al., 1986; Manrique and 2003; Alonso-Rodrıguez et al., 2004). Molina, 1997; Corte´s-Altamirano and Alonso-Rodrı´guez, 1997; Corte´s- Paralytic shellfish toxin (PST) profiles have been Altamirano et al., 1999; Morquecho and Lechuga-Deve´ze, 2004; determined in several strains of G. catenatum isolated Ga´rate-Liza´rraga et al., 2001; Ga´rate-Liza´rraga et al., 2004a, 2004b; from Bahıá Concepcioń, and nine saxitoxin analogues Alonso-Rodrı´guez et al., 2004; Band-Schmidt et al., 2004; this study). were determined (Band-Schmidt et al., 2005). In wild phytoplankton samples containing G. catenatum from Bahıá Concepcioń, Bahıá de Mazatlań and Bahıáde riched with GSe (Doblin et al., 2000). Vegetative cells La Paz, also nine saxitoxin analogues were determined; were isolated from the enrichment medium with micro- SXT and neoSXT were observed only in the BahıádeLa pipettes using an inverted microscope (Carl Zeiss, Axio- Paz, and Bahıá Concepcioń samples, respectively vert 100). Cells were transferred individually to 24-well (Ga´rate-Liza´rraga et al., 2004a,b). Differences found in plates, previously filled with modified GSe medium the toxin profiles of three G. catenatum populations (Blackburn et al., 1989), and maintained at 21 ± 1 C, led us to compare the toxicity of Mexican strains of L:D, 12:12, and 65 lEm2 s1 light intensity. Culture G. catenatum isolated from three bays of the Gulf of media were prepared using aged Bahıá Concepcioń California in culture conditions to determine if they and Bahıá de La Paz seawater (salinity 35). Both sea- have a typical or similar PST profiles. water and nutrients were sterilized through a 0.22-lm G. catenatum strains were isolated from three embay- filter. Cultures from wells were transferred into 75-mL ments from the Gulf of California (Fig. 1). Four strains culture flasks for experimental and identification from Bahıá Concepcioń (BACO) were isolated from purposes. Nine clonal cultures from vegetative cells vegetative cells collected by vertical tows of a 20 lm and one strain from cyst germination of G. catenatum net during April 2001. A fifth strain was obtained from were established (Table 1). Batch cultures (50 mL) were resting cysts found in sediment samples collected with a grown in tissue culture flasks at 20 ± 1 C with overhead gravity corer (5–20 cm length, 1.3 cm in diameter) dur- illumination of 150 lEm2 s1 (12:12 h L:D cycle) and ing June 2000. The cores were wrapped in aluminum foil harvested during exponential growth phase after inocu- and stored in darkness at 4 C. Strains from Bahıáde lation with G. catenatum strains. Cultures were har- Mazatlań were isolated from water samples taken with vested by filtration through GF/F glass fiber filters and a Van Dorn bottle during a red tide of G. catenatum immediately frozen at 20 C. These filters were used during April 2003. Strains from Bahıá de La Paz were for toxin analysis. also isolated from phytoplankton net (20 lm) samples Paralytic shellfish toxins were extracted by adding collected during January 2003. 2 mL acetic acid (0.03 N) to each G. catenatum contain- Cell concentrates were sieved through a 60-lm mesh ing filter, sonicated at 35 kHz for 60 s in an ice bath, cen- to eliminate larger organisms and to inoculate 250-mL trifuged at 3000 rpm for 5 min, and the supernatant culture container, previously filled with seawater en- filtered with a single-use syringe-filter (0.45 lm). An ali- Baseline / Marine Pollution Bulletin 50 (2005) 208–236 213

Table 1 Cell toxicity (pg STX eq/cell) of 10 strains of G. catenatum isolated from from Bahıá Concepcioń (BACO), Bahıá de Mazatlań (BAMAZ) and Bahıá de La Paz (BAPAZ), Gulf of California Strain Locality Isolation STX neoSTX dcSTX dcGTX2 DcGTX3 B1 B2 C2 Total Average GCPV-1 BAPAZ V – 7.53 1.81 1.19 0.40 0.08 0.18 1.49 12.68 GCPV-2 BAPAZ V – 14.57 5.63 1.21 0.40 0.06 0.03 1.38 23.29 15.12 GCPV-3 BAPAZ V – 1.93 0.48 2.77 0.93 0.04 0.06 3.19 9.40 GCCV-1 BACO V – 4.01 1.08 2.14 0.71 0.03 – 2.34 10.32 GCCV-2 BACO V – 5.69 1.21 3.75 1.25 0.08 – 4.65 16.63 GCCV-3 BACO V – 4.24 0.86 3.73 1.24 0.06 0.05 4.51 14.68 13.30 GCCV-4 BACO V – 6.21 1.39 1.50 0.46 0.06 – 2.03 11.66 GCCQ-1 BACO C – 6.80 1.61 1.67 0.53 0.04 0.18 2.32 13.16 GCMV-1 BAMAZ V 0.19 17.50 0.50 2.31 0.81 0.03 0.41 2.22 23.96 GCMV-2 BAMAZ V – 14.27 0.71 0.87 0.31 – – 0.43 16.60 20.27 V = vegetative; C = cyst; – = not detected. quot (150 lL) of the clarified extract was used for hydrolysis with HCl (1 M). Finally, 10 lL of both extracts (with and without hydrolysis) were injected in the HPLC system. Chromatography was performed as published recently (Hummert et al., 1997; Yu et al., 1998). An ion-pair buffer gradient was used, which was composed of a solution of octanesulfonic acid and ammonia phos- phate at pH 6.9 and acetonitrile to separate PSP-toxins. After post-column oxidation with alkaline periodic acid, the resulting products were detected with a fluorescence detector. Paralytic shellfish toxins were identified by comparing chromatograms obtained from sample ex- Fig. 2. Toxin content (bars, pg PSP/cell) and cell toxicity (lines, tracts with those resulting after injection of standard pgSTXeq/cell) from G. catenatum strains from the Gulf of California. solutions. STX, neoSTX, GTX1, GTX2, GTX3, GTX4, and dcSTX, were purchased from the National These values are higher in comparison to values re- Research Council Canada, Halifax, NS, Canada. Quan- ported by Oshima et al. (1990) and Donker et al. tification of PSP contents was done with the factor re- (1997) who reported values of toxicity between 40 and sponse (peak area/toxin concentration) obtained with 70 pg/cell. The total toxicity on a STXeq-basis of the dif- the injection of known quantities of toxin standards. ferent strains of G. catenatum from three embayments Decarbamoyl toxins, dcGTX2 and dcGTX3, were not the Gulf of California was variable, ranging from 9.40 available, therefore the corresponding carbamoyl toxins to 23.93 pg STXeq/cell (Fig. 2). The lowest toxin con- were used to obtain their respective response factors. centration value was observed for a strain from BACO According with Yu et al. (1998) detection limits for (GCMV-3) and the highest values were found in a strain the method are: 247, 10, 17, 218, 77, 16, and 7 pg for from Bahıá de Mazatlań (GCMV-1) and a strain from GTX1-4, neosaxitoxin, dcSTX, and STX, respectively. Bahıá de la Paz (GCPV-2). Partial contributions to the Other toxin limits are not given because they were not total toxicity for each toxin analogue are summarized determined by us. Toxin composition of the strains stud- in Table 1. No differences in the toxin concentration ied was expressed as the concentration of each toxin (pg/ were observed the strain isolated from cyst and strains cell), cell toxicity (pg STXeq/cell), as well as molar per- obtained from vegetative cells from Bahıá Concepcioń. centages (mol%). In order to compare the derivative The average toxicity from the different strains of G. toxin profiles of the different strains of G. catenatum, catenatum strains in STX equivalents was 15.1 pg we performed a cluster analysis using the Morisita index STXeq/cell for strains from Bahıá de La Paz (BAPAZ); (similarity) (Sneath and Sokal, 1973). Molar percentage 13.3 pg STXeq/cell for strains from Bahıá Concepcioń of each toxin was fed into an agglomerative classifica- (BACO), and 20.3 pg STXeq/cell for strains from Bahıá tion module of a flexible algorithm for the construction de Mazatlań (BAMAZ). These average toxicity are low of dendrograms (De la Cruz-Aguero, 1994). The result- or similar to the cell toxicity reported for Singapore, ing dendrogram grouped the different strains according Uruguayan and Korean isolates (Meńdez and Ferrari, to their toxin similarity in molar percentages. 2002; Holmes et al., 2002; Park et al., 2004). However, Toxin content varied from 36.14 to 183.95 pg/cell for these toxicity values are lower than a previous study the different isolates (Fig. 2) with an average of 90.7 of G. catenatum strains from BACO grown in f/2 (BAPAZ), 129.32 (BACO), and 75.65 pg/cell (BAMAZ). media (26.7 pg STXeq/cell) (Band-Schmidt et al., 214 Baseline / Marine Pollution Bulletin 50 (2005) 208–236

2005). Quantitatively, neoSXT was the more abundant STX, GTX2, GTX3, and B2 were also reported. This toxin, particularly in BAPAZ (17.5 pg STXeq/cell) and suggestion has been partially corroborated in this study. BAMAZ (14.6 pg STXeq/cell) strains. SXT was detected Relatively high proportions of C1-2 and dcGTX2-3 are only in one strain from Bahıá de Mazatlań (GCMV-19) characteristic of BACO strains (Band-Schmidt et al., in very low concentrations (Table 1). Except for the value 2005). However BAMAZ strains differ from BACO of dcSXT (5.6 pg STXeq/cell) for one of the strains from strains by the higher molar percentage of neoSTX. the Bahıá de La Paz (GCPV-2), the rest of the strains Low proportions of SXT and B1 were also detected. presented values under 5 pg STXeq/cell (Table 1). Ga´rate-Liza´rraga et al. (2004b) analyzed PSTs in wild The toxin profile for the different strains resulted G. catenatum samples from Bahıá Concepcioń, and from the presence of three toxins of the decarbamoyl pointed out that the presence of a high molar percentage family: dcSTX, dcGTX2, and dcGTX3; three less potent of neoSTX in BACO material suggests that this toxin toxins, B1, B2, and C2 from the sulfocarbamoyl family; could be a distinctive characteristic of G. catenatum pop- in addition, two carbamate toxins: SXT and neoSXT ulations from this bay. However, in wild phytoplankton were also present (Table 1). The typical HPLC profiles samples of G. catenatum from Bahıá de Mazatlańand of toxin extracts from selected strains from each embay- Bahıá de La Paz neoSTX was not observed. SXT was ment are shown in chromatograms of Fig. 3A–F. This observed in wild phytoplankton samples from this last toxin profile is similar to a previous report (Band- bay (Ga´rate-Liza´rraga et al., 2004a,b). Schmidt et al., 2005) where the analogues dcSTX, On a molar basis, C1, C2, dcSTX, and dcGTX2 were dcGTX2, dcGTX3, C1-2 were suggested to be used as the more important toxins. neoSTX contributed with biomarkers for this population; low proportions of neo- percentages over 11% in two strains from BAPAZ and

8000 20000 10000 3 Intensity( 3 5 3 5 4000 10000 5000 6 4 5 6 4 9 4 0 0 0

25000 80000 7 7 40000 7 20000 (B) 60000 (D) V) 30000 (F) µ 15000 40000 20000 10000 6 8 8

Intensity ( 8 2 20000 2 2 5000 10000 3 5 3 3 5 9 6 6 9 5 9 0 0 0 0 102030400 102030400 10203040 Time (min) Time (min) Time (min)

Fig. 3. Typical HPLC-FD chromatograms of paralytic shellﬁsh toxins in Gymnodinum catenatum strains showing elution of toxins: C-toxins (1), dcGTX2 (2), dcGTX3 (3), B1 (4), neoSTX (5), dcSTX (6), GTX2 (7), GTX3 (8), and STX (9). Before (A, C, E) and after hydrolysis (B, D, F); A–B (BAPAZ strains), C–D (BACO strains), and E–F (BAMAZ strains).

Table 2 Molar percentage of each toxin of different strains of G. catenatum isolated from Bahıá Concepcioń (BACO), Bahıá de Mazatlań (BAMAZ) and Bahıá de La Paz (BAPAZ), Gulf of California Strains Locality STX Neo dcSTX DcGTX2 DcGTX3 B1 B2 C1 C2 GCPV-1 BAPAZ – 12.88 9.72 11.19 3.77 1.81 3.78 42.46 14.39 GCPV-2 BAPAZ – 19.67 23.89 8.98 2.98 1.07 0.51 32.37 10.52 GCPV-3 BAPAZ – 1.99 1.56 15.65 5.25 0.56 0.84 55.63 18.52 Average – 11.51 11.72 11.94 4.00 1.15 1.71 43.49 14.48 GCCV-1 BACO – 5.15 4.37 15.08 5.01 0.57 – 52.84 16.98 GCCV-2 BACO – 3.91 2.61 14.14 4.71 0.69 – 55.95 18.00 GCCV-3 BACO – 3.02 1.93 14.57 4.84 0.52 0.46 56.55 18.11 GCCV-4 BACO – 8.76 6.18 11.65 3.57 1.03 0.05 52.60 16.16 GCCQ-1 BACO – 8.27 6.17 11.19 3.54 0.70 2.78 51.46 15.90 Average – 6.68 4.76 12.47 3.98 0.75 1.09 53.54 16.72 GCMV-1 BAMAZ 0.23 18.16 1.62 13.18 4.60 0.36 5.31 43.59 12.95 GCMV-2 BAMAZ – 42.04 6.59 14.11 4.97 – – 25.06 7.22 Average 0.12 30.10 4.11 13.65 4.79 0.18 2.66 34.32 10.09 Baseline / Marine Pollution Bulletin 50 (2005) 208–236 215 two strains from BAMAZ (Table 2). Less abundant tox- the addition of soil extract and in the micronutrient ins on a molar basis, were B1 + B2 (0.0–5.3%), and composition. Notable differences in toxin profiles of dcGTX3 (<6%). Cluster recognition was performed with G. catenatum in culture have also been observed, 70% of similarity, showing two different groups (Fig. 4). depending on the life history stage (Bravo and Ander- Group A is represented by a strain from Mazatlań son, 1994), and these differences may be a result of the (GCPV-2) and that from Bahıá de La Paz (GCMV-2). expression of enzymes involved in toxin biosynthesis These strains have the lowest and the highest molar per- (Oshima et al., 1993a,b). centage of C1 + C2 and neoSTX, respectively. Likewise Reguera and Oshima (1990) have mentioned that the both strains have low molar percentages (<1.0) of B2. toxin composition does appear not to be a conservative Coincidently, these two strains showed the highest toxic- property of G. catenatum. However, other studies on ity averages. Group B was composed of the rest the G. catenatum toxin profiles have demonstrated a consis- strains, which presented minor differences in toxin com- tency in the toxin profile within populations. Neverthe- position. BACO strains are grouped together; another less, some variations exist between populations (Oshima subgroup is formed by a strain from Bahıá de Mazatlań et al., 1990; Oshima et al., 1993a,b). The PSTs are (GCPV-1) and one from Bahıá de La Paz (GCMV-1). practical and functional biomarkers to differentiate Paralytic toxin profiles from strains from MZT and G. catenatum populations, being a good feature to geo- LAPAZ are closer than those from BACO. This could be graphically distinguish different populations of phyto- explained because Bahıá de Mazatlań and BahıádeLa plankton (Negri et al., 2001). For instance, the strain Paz are open bays, whereas Bahıá Concepcioń is a shal- from Singapore has a unique toxin profile (GTX1, low and protected lagoon. Another explanation could be GTX2, GTX3, GTX4, neoSXT, and SXT). Camino- particular physiological adaptations of populations of Orda´s et al. (2004) reported important differences in G. catenatum. Laboratory studies so far have been per- the toxin profile between the Galicia and Andalucıá formed to determine the effect of temperature, salinity, strains. SXT was observed in the Galicia strains and seawater sources, and culture media, on the vegetative was absent in strains from Andalucıá. Camino-Orda´s growth of clonal cultures of G. catenatum isolated from et al. (2004) report the presence of 13-doSTX in all the Bahıá Concepcioń (Band-Schmidt et al., 2004). This Spanish strains, contrasting with previous studies in strain seems to have evolved physiological and nutritional which no 13-deoxydecarbamoyl toxins (doSTX) were requirements that differ from other strains of this species, observed in G. catenatum strains from Spain (Oshima which could possibly result in a unique toxin profile. et al., 1990; Negri et al., 2001). However, they cannot Band-Schmidt et al. (2005) reported a similar toxin propose the presence of doSXT as a distinctive toxin profile obtained from strains of G. catenatum cultured from the Spanish strains. Park et al. (2004) reported in modified f/2 media. Important differences, such as that strains from the Yellow Sea have predominantly the presence of SXT, B1 toxins, higher molar percent- carbamate toxins, while strains from Sujeongri and ages of neoSXT and the lack of GTX1-3 have also been Chindong in the South Sea contained the N-sulfocarba- observed. These differences could be due to the different moyl toxins, C1-2, as major components including the strains used; however this is unlikely since in the previ- presence of GTX5 and dcSTX in some strains. ous work, 16 strains were analyzed. Differences observed From Bahıá Concepcioń, Band-Schmidt et al. (2005) are probably related to the culture media used (Reguera pointed out that the main toxins are: dcSTX, dcGTX2, and Oshima, 1990). G. catenatum in this study was dcGTX3, C1, and C2; these were always present in growth in GSe medium which differs from f/2 media in strains, suggesting they can be used as biochemical markers. However, the toxins observed consistently in strains from BACO, BAMAZ, and BAPAZ were: neo- SXT, dcSTX, dcGTX2, dcGTX3, B1, C1, and C2, showing the addition of two more saxitoxin analogues of the 5 SXT analogues reported by Band-Schmidt et al. (2005). A distinctive characteristic from the Mexi- can strains of G. catenatum is the low molar percentages of B1-B2 toxins. High molar percentages of B1-B2 toxins have been reported for strains from Portugal and Spain (Donker et al., 1997; Negri et al., 2001; Camino- Orda´s et al., 2004). Besides the similarity in toxin concentration, there were no differences between the toxin profile in the cyst Fig. 4. Similarity dendrogram based on toxin profiles (mol%) for strain (GCCQ-1) and strains obtained from vegetative strains of G. catenatum from Bahıá Concepcioń, Bahıá de Mazatlań cells from Bahıá Concepcioń (GCCV1-4), contrasting and Bahıá de La Paz. with the results reported by Negri et al. (2001). These 216 Baseline / Marine Pollution Bulletin 50 (2005) 208–236 authors found that a number of Australian strains de- and seawater source on the growth of Gymnodinium catenatum rived from the products of wild resting cysts germinated (Dinophyceae) from Bahıá Concepcioń, Gulf of California, Mex- in the laboratory exhibited atypical PST profiles for ico. Journal of Plankton Research 26 (12), 1459–1470. Blackburn, S.I., Hallegraeff, G.M., Bolch, C.J., 1989. Vegetative G. catenatum. Nevertheless, Park et al. (2004) observed reproduction and sexual life cycle of the toxic dinoflagellate the absence of dcSXT in isolates obtained from cyst of Gymnodinium catenatum from Tasmania, Australia. Journal of G. catenatum from the South Sea. This study represents Phycology 25, 577–590. the first comparative study of PSTs profiles produced by Bravo, I., Anderson, D.M., 1994. The effects of temperature, growth strains of G. catenatum from three different bays in the medium and darkness on excystment and growth of the toxic dinoflagellate Gymnodinium catenatum from northwest Spain. Gulf of California: Bahıá Concepcioń, Bahıá de Maz- Journal of Plankton Research 16, 513–525. atlań and Bahıá de La Paz. We found that G. catenatum Camino-Orda´s, M., Fraga, S., Franco, J.M., Orda´s, A., Figueras, A., strains have a large variation in toxin content and PSTs 2004. Toxin and molecular analysis of Gymnodinium catena- profile. These results suggest that the characterization of tum (Dinophyceae) strains from Galicia (NW Spain) and G. catenatum strains using typical toxin profiles is diffi- Andalucıá (S Spain). Journal of Plankton Research 26, 341–349. Corte´s-Altamirano, R., Alonso-Rodrı´guez, R., 1997. Mareas rojas cult and can vary with culture conditions, growth phase, durante 1997 en la bahıá de Mazatlań, Sinaloa, Me´xico. Ciencias strain, etc., In order to have a better understanding of del Mar 15, 31–37. the variations in the toxin profiles of G. catenatum be- Corte´s-Altamirano, R., Nun˜ez-Pasteń, A., Pasteń-Miranda, N., 1999. tween different localities along the Mexican coastline, Abundancia anual de Gymnodinium catenatum Graham dinoflage- toxin profiles will be analyzed during different growth lado to´xico de la costa este del Golfo de California. Ciencias del Mar 35, 51–56. stages as well as varying the nutrient sources and con- Doblin, M.A., Blackburn, S.I., Hallegraeff, G.M., 2000. Intraspecific centrations. Moreover, the study of wild populations is variation in the selenium requirement of different geographic necessary. strains of the toxic dinoflagellate Gymnodinium catenatum. Journal of Plankton Research 22, 421–432. De la Cruz-Aguero, G., 1994. ANACOM. Sistema de ana´lisis de Acknowledgments comunidades. Versioń 3.0. Manual del usuario. Departamento de Pesquerıás y Biologıá Marina, Centro Interdisciplinario de The authors thank the National Council of Science Ciencias del Mar, Instituto Politećnico Nacional, La Paz, B.C.S., Me´xico. and Technology for financial and logistical support Donker, S., Reyero, M.I., Reguera, B., Franco, J.M., 1997. Perfil de (CONACYT grant 144384 to CJB-S, R33598-B, toxinas en seis cepas de Gymnodinium catenatum de Galicia. In: V 33684-V, 37560-V, G37560-V, and CONACYT-DAAD Reunioń Ibe´rica de Fitoplancton To´xico y Biotoxinas, Vigo, program). This research was partially financed by the Espanã, pp. 69–76. DLR (German Center for Air and Airspace), CIBNOR Figueroa-Torres, M.G., Zepeda-Esquivel, M.A., 2001. Mareas rojas del puerto interior, Colima, Me´xico. Scientia Naturae 3, 39–52. institutional research projects (AYCG-8, AYCG-11, Ga´rate-Liza´rraga, I., Bustillos-Guzmań, J.J., Alonso-Rodrı´guez, R., and PC3-1) and Instituto Politećnico Nacional research Luckas, B., 2004a. Comparative paralytic shellfish toxin profiles in projects (CGPI grant 20031093; 20040626; 20040066). two marine bivalves during outbreaks of Gymnodinium catenatum Thanks to the CIBNOR staff for English language edit- (Dinophyceae) in the Gulf of California. Marine Pollution Bulletin ing and to Clara Ramı´rez J. for facilitating literature. 48, 397–402. Ga´rate-Liza´rraga, I., Bustillos-Guzmań, J.J., Erler, K., Mun˜eton- IGL has COFAA and EDI fellowships. JBG, LM and Go´mez, M.S., Luckas, B., Tripp-Quezada, A., 2004b. Paralytic CJB-S are SNI fellow. shellfish toxins in the chocolata clam, Megapitaria squalida (Bivalvia: Veneridae), in Bahıá de La Paz, Gulf of California. Revista de Biologıá Tropical, vol. 52, pp. 133–140. References Ga´rate-Liza´rraga, I., Hernańdez-Orozco, M.L., Band-Schmidt, C.J., Serrano-Casillas, G., 2001. Red tides along the coasts of Baja Alonso-Rodrı´guez, R., Ga´rate-Liza´rraga, I., Luckas, B., Reinhardt, California Sur, Mexico (1984 to 2001). Oceańides 16 (2), 127–134. K., Bustillos-Guzmań, J., 2004. Mortalidad de larvas y camarońen Gilbert, J.Y., Allen, W.E., 1943. The phytoplankton of the Gulf of cultivo en Sinaloa, Me´xico, asociado a mareas rojas de Gymnodi- California obtained by the E.W. Scripps in 1939 and 1940. Journal nium catenatum. In: Resu´menes XII Reunioń Nacional de la of Marine Resources 5, 89–110. Sociedad Mexicana de Planctologıá, A.C. SOMPAC, Nuevo Go´mez, F., 2003. The toxic dinoflagellate Gymnodinium catenatum:an Vallarta, Nayarit, pp. 54–55. invader in the Mediterranean Sea. Acta Botanica Croatia 62, 65– Alonso-Rodrı´guez, R., Paéz-Osuna, F., 2003. Nutrients, phytoplank- 72. ton and harmful algal blooms in shrimp ponds: a review with Graham, H.W., 1943. Gymnodinium catenatum a new dinoflagellate special reference to the situation in the Gulf of California. from the Gulf of California. Transactions of the American Aquaculture 219, 317–336. Microscopical Society 62, 259–261. Band-Schmidt, C.J., Bustillos-Guzmań, J., Ga´rate-Liza´rraga, I., Hallegraeff, G.M., Fraga, S., 1996. Blooms dynamics of the toxic Lechuga-Deve´ze, C.H., Reinhardt, K., Luckas, B., 2005. Profiles Gymnodinium catenatum, with emphasis on Tasmanian and Span- of paralytic shellfish toxin in strains of the dinoflagellate Gymn- ish coastal waters. In: Anderson, D., Cembella, A.D., Hallegraeff, odinium catenatum and the scallop Argopecten ventricosus from G.H. (Eds.), Physiological Ecology of Harmful Algal Blooms. Bahıá Concepcioń, Gulf of California, Mexico. Harmful Algae 48 NATO ASI, Series, pp. 41, 59–80. (1), 21–31. Herrera-Galindo, J.E., 2002. Composicioń, abundancia y distribucioń Band-Schmidt, C.J., Morquecho, L., Lechuga-Deve´ze, C.H., Ander- de los dinoflagelados en la zona cercana a la lıńea de costa y marina son, D.M., 2004. Effects of growth medium, temperature, salinity adyacente al rıó Copalita, en Bahıás de Huatulco, Oaxaca, Baseline / Marine Pollution Bulletin 50 (2005) 208–236 217

Diciembre 1997–Octubre 1998. Master degree thesis. Universidad Blooms 2000. Intergovernmental Oceanographic Commission of del Mar, Puerto A´ ngel, Oaxaca, Me´xico. UNESCO, Paris, pp. 210–213. Holmes, M.J., Bolch, C.J., Green, D.H., Cembella, A.D., Ming-Teo, Negri, A.P., Stirling, D., Blackburn, S., Bolch, C., Burton, I., S.L., 2002. Singapore isolates of the dinoflagellate Gymnodinium Eaglesham, G., Thomas, K., Walter, J., Willis, R., Quilliam, M., catenatum (Dinophyceae) produce a unique profile of paralytic 2003. Three new saxitoxin analogues isolated from the toxic shellfish toxins. Journal of Phycology 38, 96–106. dinoflagellate Gymnodinium catenatum. In: Villalba, A., Reguera, Hummert, C., Ritscher, M., Reinhardt, K., Luckas, B., 1997. Analysis B., Romalde, J.L., Beiras, R. (Eds.), Molluscan Shellfish Safety of the characteristic PSP profiles of Pyrodinium bahamense and Conselleria de Pescae Asuntos Marı´timos a Xunta de Galicia and several strains of Alexandrium by HPLC based on ion-pair Intergovernmental Oceanographic Commision of UNESCO, chromatographic separation, post-column oxidation, and fluores- Spain, pp. 19–28. cence detection. Chromatographia 45, 312–316. Oshima, Y., Blackburn, S.I., Hallegraeff, G.M., 1993a. Comparative La Barbera-Sańchez, A., Gamboa-Maruez, J.J., 2001. Distribution of study on paralytic shellfish toxin profiles of the dinoflagellate Gymnodinium catenatum Graham from shellfish toxicity on the Gymnodinium catenatum from three different countries. Marine coast of Sucre State, Venezuela, from 1989 to 1998. Journal of Biology 116, 471–476. Shellfish Research 20, 1257–1261. Oshima, Y., Itakura, H., Kian-Chuan, L., Yasumoto, T., Blackburn, S., Leal, S.G., Delgado, F., Nodas, 2003. Nuevo registro de microalga Hallegraeff, G.M., 1993b. Toxin production by the dinoflagellate to´xica para aguas cubanas. Revista Investigaciones Marinas 24, Gymnodinium catenatum. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic 155–157. Marine Phytoplankton in the Sea. Elsevier, New York, pp. 907–912. Manrique, F.A., Molina, R.E., 1997. Occurrence of red tides in the Oshima, Y., Sugino, K., Hirota, M., Yasumoto, T., 1990. Comparative Bacochibampo, Guaymas, Sonora, Me´xico. Hidrobiolo´gica 77, studies on paralytic shellfish toxin profile of dinoflagellates and 8184. bivalves. In: Graneli, E., Sundstrom, B., Elder, L., Anderson, D. Mee, L.D., Espinosa, M., Dıáz, G., 1986. Paralytic shellfish poisoning (Eds.), Toxic Marine Phytoplankton. Elsevier, New York, pp. 391– with a Gymnodinium catenatum red tide on the pacific coast of 396. Mexico. Marine Environmental Research 19, 17–92. Park, T.G., Kim, C.H., Oshima, Y., 2004. Paralytic shellfish toxin Meńdez, S., Ferrari, G., 2002. Floraciones algales nocivas en Uruguay: profiles of different geographic populations of Gymnodinium antecedentes, proyectos en curso y revisioń de resultados. In: Sar, catenatum (Dinophyceae) in Korean coastal waters. Phycological E.A., Ferrario, M.E., Reguera, B. (Eds.), Floraciones algales Research 52, 300–305. nocivas en el cono sur americano. Instituto Espanõl de Oceanog- Reguera, B., Oshima, Y., 1990. Response of Gymnodinium caten- rafıá, pp. 269–288. atum to increasing levels of nitrate: growth patterns and Morquecho, L., Lechuga-Deve´ze, C.H., 2004. Seasonal occurrence of toxicity. In: Graneli, E., Sundstrom, B., Elder, L., Anderson, planktonic dinoflagellates and cyst production in relationship to D. (Eds.), Toxic Marine Phytoplankton. Elsevier, New York, environmental variables in subtropical Bahıá Concepcioń, Gulf of pp. 316–319. California. Botanica Marina 47, 313–322. Sneath, P.H., Sokal, R.R., 1973. In: Numerical Taxonomy. W.H. Negri, A.P., Bolch, C.S., Blackburn, S., Dickman, M., Llewellyn, L.E., Freeman and Company, San Francisco, p. 573. Mendez, S., 2001. Paralytic shellfish toxins in Gymnodinium Yu, R.C., Hummert, C., Luckas, B., Qian, P.Y., Zhou, M.J., 1998. catenatum strains from six countries. In: Hallegraeff, G.M., Modified HPLC method for analysis of PSP toxins in algae and Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal shellfish from China. Chromatographia 48, 671–676.

A preliminary report of persistent organochlorine pollutants in the Yellow Sea

J.R. Oh, H.K. Choi, S.H. Hong, U.H. Yim, W.J. Shim, N. Kannan *

South Sea Institute, Marine Environmental Research Laboratory, Korea Ocean Research and Development Institute, 391 Jangmok-ri, Jangmok-myon, Geoje 656830, Republic of Korea

The Yellow Sea is a semi-enclosed, continental shelf, habit the areas that drain into the Yellow Sea (Duda and shallow-sea and one of the 50 Large Marine Ecosystems Sherman, 2002). Large cities near the sea having tens of (LME) of the world. Its area is more than 400,000 km2, millions of inhabitants include Qingdao, Tianjin, Da- with an average water depth of 44 m. China, South and lian, Shanghai, Seoul/Incheon, and Pyongyang-Nampo. North Korea are the 3 countries bordering this LME. Industrial wastewater containing major pollutants from 600 million people, or 10% of the worldÕs population, in- port cities; non-point source contaminants of agricul- tural origin (pesticides); oil discharged from vessels * Corresponding author. Tel.: +82 55 639 8675; fax: +82 55 639 and ports; and oil and oily mixtures from oil exploration 8689. enter this LME from extensive economic development E-mail address: [email protected] (N. Kannan). in the coastal zone. In 1999, the recipient amount of