toxins

TOXINS PSP TOXINS PROFILE ALONG THE CROATIAN ADRIATIC COAST 1Institute

Romana

2 ROJE

and Ivona

1 MARASOVIĆ

of Oceanography and Fisheries, Šetalište Ivana Maštrovića 63, 21000 Split 2Student of Erasmus Mundus Master of Science in Marine Biodiversity and Conservation

[email protected]

www.izor.hr

INTRODUCTION

a)

c) Fig. 1. Geographic location of the sampling stations along the Croatian coast (a). Stations S 1, 2, 3, 4, and 5 are located along West Istrian coast (b) and stations S 6 is in the Mali Ston Bay sites (c).

RESULTS AND DISCUSSION

The purification procedure and High Performance Liquid Chromatography with fluorescence detection (HPLC-FLD) includes a prechromatographic oxidation of the PSP toxins with hydrogen peroxide and periodate were based on Lawrence et al., 2001. The method was used for determination of saxitoxin, decarbamoylsaxitoxin, neosaxitoxin, gonyautoxins 2 and 3, gonyautoxins 1 and 4, decarbamoylneosaxitoxin, gonyautoxin 5 (B 1) decarbamoylgonyautoxins 2 and 3, and Nsulfocarbamoyl-gonyautoxin 2 i 3 (C 1 and C 2) in shellfish (mussels, oysters and scallops) and ascidians. The PSP extracts were analyzed using a Varian ProSTAR 230 HPLC analytical system coupled with a ProStar 363 fluorescence detector with excitation wavelength set to 340 nm and emisssion to 390 nm. Separation of toxin oxidation Fig. 4. Chromatogram of Mix II containing products was carried on reversed-phase C18 STX, dcSTX, GTX 2,3, dcGTX 2,3 and GTX 5 column (Restek, Pinnacle II 250 x 4.6 mm, 5 μm after peroxide oxidation. particle size) protected by a guard cartridge Pinnacle II C18, 20 x 4.0 mm (Varian, SAD). Column temperature was kept at 30°C and run time was set to 15.00 min. Retention times (chromatographic peaks) obtained after periodate and peroxide oxidations of PSP toxins are shown in Table 1. For calculating the concentration of each PSP toxin present in the samples, comparison of the peak areas in chromatograms after HPLC analysis corresponding to those of sample extracts and those of the PSP analytical standards obtained from the National Research Council of Canada (Halifax, Canada) was used (Fig. 3 and 4).

stx

dcstx

Retention time (min) Periodate oxidation Peroxide oxidation

3.1 GTX1,4

4.3 dcGTX2,3 dcGTX2,3

5.0

6.3

6.6

8.6

9.5

10.2

dcNEO dcSTX

NEO dcSTX dcNEO

dcSTX C1,2 dcSTX

dcSTX

GTX1,4 NEO dcGTX2,3 C1,2 dcSTX C1,2 dcGTX2,3

11.4

12.2

GTX1,4 GTX2,3

GTX5

STX NEO dcNEO

GTX2,3

GTX5

STX

dcgtx2,3

gtx5

c1,2

22.Feb.'09.

0

100

200

300

400

500

600

700

800

Toxin concentration (μg STX eq. kg-1)

Fig. 5. Total PSP toxicity (μg STX eq. kg-1) from ascidian at station S 1.

stx

dcstx

gtx2,3

c1,2

06.Apr.'09. 30.Mar.'09. 09.Mar.'09. 19.Dec.'08.

0

100

200

300

400

500

600



stx

dcstx

gtx2,3

gtx5

c1,2

06.Apr.'09. 09.Mar.'09. 22.Feb.'09. 09.Feb.'09. 26.Jan.'09. 29.Dec.'08. 17.Dec.'08.

0

100

200

300

400

500

600

700



gtx2,3

15.Apr'09.

dcstx

16.Mar'09. 12.Mar'09. 09.Mar'09. 02.Mar'09. 23.Feb'09. 16.Feb'09. 14.Feb'09. 04.Feb'09.

S6

S5

0

27.Jan'09. 19.Jan'09.

10

20

30

40

Toxin concentration (μg STX eq. kg-1) 0

200

400

600

800

1000

1200

1400

1600


Table 1. Retention times of PSP toxins after periodate and peroxide oxidations (peaks considered as diagnostic quantitation peaks are underlined).

gtx2,3

09.Mar'09.

Sampling date, station

METHODS

Sampling date

Fig. 2. Microcosmus sulcatus

Sampling date

The PSP contamination affected mussels and ascidians sampled from the West Istrian area and mussels from the south area (Mali Ston Bay) of the Adriatic Sea. Figures 5-9 shows the total PSP toxicity found in shellfish and ascidians at all investigation stations. It is calculated by summing the toxicity contribution of each quantified toxin (μg STX eq. kg-1). STX, dcSTX, GTX 2,3, dcGTX 2,3, GTX 5 and C 1,2 were the toxins determined Mytilus galloprovincialis in ascidian samples from the West Istrian coast at the stations S 1, 2 and 3 during the period between December 2008 and May 2009 (Fig. 5, 6 and 7). The majority of PSP toxin containing samples are from the January to April period, that is in agreement with periodicity found at the Mediterranean coast (Taleb et al., 2001). Mussels were sampled at stations S 4, 5 and 6, and showed STX, dcSTX, GTX 2,3 present during the same period (Fig. 8 and 9). STX and GTX 2,3 were Fig. 3. Chromatogram of Mix I containing NEO, the dominating toxins in terms of concentration dcNEO and GTX 1,4 after periodate oxidation. and toxicity contribution considering all stations. STX had a maximum concentration of 1298.17 μg kg-1, dcSTX reached the maximum concentration of 96.50 μg kg-1, while GTX 2,3 showed the highest concentration of 655.72 μg kg-1. DcGTX 2,3 appeared only twice in concentrations of 348.60 μg kg-1 and 389.18 μg kg-1. GTX 5 was present in the toxin profile with 585.21 μg kg-1 as maximum. C 1,2 was present in the sample 353.85 μg kg-1 as its maximum.

dcstx

06.Apr.'09.

Sampling date

Sampled material

stx

Sampling date

Paralytic Shellfish Poisoning (PSP) is caused by the saxitoxins, potent neurotoxins that cause paralysis and death in consumers of contaminated seafood. Present study is the first attempt to determine PSP toxin types and establish a complete list of PSP toxins for Croatian coastal waters. Shellfish farming is widespread in Croatia along its entire coastline, particularly long tradition on the far south of the Croatian coast (Mali Ston Bay) with a more than one hundred years long tradition of shellfish farming (Fig 1.). Croatian shellfish industry, with its production of 4 000 tons of mussels and 1 million oysters in 2008 is small when compared to the other countries, but it has a high potential for aquaculture expansion within its 31 490 km2 of territorial waters, 5 835 km of coastline and 1 244 islands. There are 25 sampling sites from shellfish breeding and harvesting areas along the Croatian coast of Adriatic Sea included in continuous monitoring of marine biotoxins ASP, DSP and PSP (Fig 1.). From the year 2000 through 2009 sampling frequency per site was once a month from November until May and two times a month from May through October. Istria Peninsula had a weekly sampling frequency from April through November in 2007 and 2008 (Fig. 1b). Starting in 2009, samples were acquired fortnightly from January through March and weekly from April through December, with the exception of sampling sites at Istria Peninsula which had a weekly sampling through the entire year. Included in this study were also 18 samples of ascidian Microcosmus sulcatus, sampled at S 1, 2, and 3 stations at West Istrian coast during the period from December 2008 through May 2009, as there were 5 recorded cases of human poisoning after consumption of fresh sea squirts caught by fisherman nets (Fig. 2).

b)

15.Sep.'08. 27.Oct.'08.

Ivana

1 UJEVIĆ ,

Fig. 9. Total PSP toxicity (μg STX eq. kg-1) from shellfish at station S 5 and 6.

Fig. 8. Total PSP toxicity (μg STX eq. kg-1) from shellfish at station S 4. REFERENCES 1.Lawrence, J.F., Niedzwiadek, B., and Menard, C., 2005. Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection. J. AOAC Int. 88, 1714-1732. 2. Taleb, H., Vale, P., Jaime, E. and Blaghen, M., 2001. Study of paralytic shellfish poisoning toxin profile in shellfish from the Mediterranean shore of Morocco. Toxicon 39, 1855-1861.