Toxla~, Vol. 31, No. 3, pp. 237-242, 1993 . Prinmd io (kat l~ .
0041-0101/9316.00 + .00 ~ 1993 lapmo~ 1~ Lod
INFLUENCE OF INITIAL TOXICITY AND EXTRACTION PROCEDURE ON PARALYTIC TOXIN CHANGES IN THE MUSSEL PATRIC[c Lnssvs,' MARTIAL LBDOCrx,2 MICIeàLg BAItDOUIL' and MADELEINE BOIIBC' 'IFREMER, Centre de Nantes, B .P . no. 1049, 44037 Nantes Cedes Ol, Franoe ; and 2CNEVA, Laboratoire Central d'Hy~bne Alimentaire, 43 rVe de Danttig, 75015 Paris, France (Received 19 llfarch 1992; accepted 7 October 1992)
P. LASSVS, M. L»oux, M. BARDOUIL and M. Boxec. Influence of initial toxicity and extraction procedure on paralytic toxin changes in the mussel . Toxtcon 31, 237-242, 1993 .-Farmed mussels were artificially contaminated with a pure culture of an Alextutdrittm tamarense toxic strain (MOG 835), to assess the effect of initial toxicity on paralytic toxin change during the depuration process. As previously observed in mussel, gonyautoxin GT'X2 is eliminated more slowly than other gonyautoxins . A toxic level (1300 ~g PSP per 100 g meat) is required to produce a drastic change in the depuration course, i.e . a `fast' depuration rate followed by a `slow' one. Below this threshold, decontamination becomes slower as the proportion of GTX2 increases over the time course. Although GTX2 is slowly eliminated during the depuration process, it is also formed in increasing quantities during the contamination phase. It remains to be determined whether changes in GTX2/GTX3 ratios are due to chemical or biological transformation. INTRODUCTION
Nus~ROUS studies have investigated the contamination of edible mollusks by paralytic toxins (ÀSARAWA et al., 1985 ; BRICELJ et al., 1990; BFdTLER and LISTON, 1990), but none has considered the influence of initial toxicity on depuration kinetics. Moreover, for reasons of rapid performance and cost efficiency, the techniques of measuring toxicity rates in networks monitoring shellfish safety are generally limited to the mouse test procedure described by the Association of Official Analytical Chemists (AOAC, ANON, 198 . It would thus be desirable to provide shellfish farmers with information concerning the probable length of the depuration period once the animals are placed in a healthy environment and the most suitable methods (high performance liquid chromatography (HPLC) analyses, biological tests] to estimate edibility during decontamination . In an earlier work (LA33US et al., 1989), we studied the contamination/decontamination kinetics of shellfish which had ingested cultures of Alexmedrittm ( = Protogonyttulax) tmrwrense . Decontamination kinetics for mollusks with high rates of contamination (mussels and scallops) proved of interest because (1) residual decontamination was `slow', and (2) elimination differed from that of gonyautoxins (GT'X) 2 and 3, with GTX2 remaining at rather significant concentrations long enough to prevent the shellfish toxin level from dropping below the public health threshold (80 ~eg/100 g of meat). Although 237
P. LASSUS et d.
GTX2 is not among the most virulent agents of paralytic shellfish poisons (PSP), it is one of the most common compounds, together with GTX3, found in the tissues of shellfish contaminated by marine dinoflagellates of the genus dlext~itart (OsHnrA et al., 1978; NtxiUCHi et al., 1981, 1983 ; OrlouE et ul., 1981 ; FR>~t et al., 1989). We suggested that slow GTX2 elimination could be due to biotransformation of the toxins present at the onset of contamination or to a physiological process unfavorable to rapid elimination of this toxin. A similar experimental approach was used in the present study. MATERIALS AND METHODS A toxic strain (MOG835) of Akx~drhon tmnarense (Dinophyceae) was used for a~perimental ahenfleh contamination. Cultures wen performed in 10liter glass bottles fined with continuously aerated sea water (30'/.) flloaed on 0.22 pm membrane (Millipore, Bedford, MA, U.SA.) and enriched with Prowsoli-ES medium (Paovesot.t et d., 195 . The ambient factors was a iemperatttte of 16°C and a right intensity of 2500 f2001ux with a 12 hr right/dark cycle. The same medium and the same procedure wa+e used for non-toxic algae Tetmrtbntt s~ertca (Chlorophycese) . Blue mussels (AlytiGct eduNa) was oonarted in the Bourgnenf Hay and acclimatized to laboratory conditions 1 week before tIm experiment . Then, 10 to IS shellfish were placed in 2 liter of aerated sea water and fed with a daily rate of 0.5 rifer of cailture medium, with the same conditions of temperature and right . The sea water in each container was teneaed daily. A contamination period followed by decontamination in Alearmw6ia~n-free sea water was applied to every shellllsh apa:ies involved in the mcperimeok SheflSah samples were eonected at different stagex of the experiment to perform bioassays and toxin determinations . During conte+~mAhon and decontamination periods, toxicity tests using three male 'Swiss' mice (20 g) wero conducted at least every 3 days on acidified (HCI, 0.1 N) extracts of shellfish meat. Following the AOAC procedure, 1 ml of extract was igjxted i.p. into the mice, and the survival time, fast expressed in mouse units (MU), was then converted into paralytic sheBBeh poison (PSP) concentration rates in meat, i.e . MU x CF - Pg PSP/ml extract, with a eorrectioa factor (CF) - 0.22 and Ng PSP/100g meat = Rg PSP/ml x dilution factor x V, where V ~ volume of extract in ml . After 1 ml of the AOAC addic extract was transferrod into an ultraflltration system (MPS-l, Amicon Corp ., Daavere, MA, U.SA) and centrifuged, 10 kl of dear ultraflltetad solution were igjected into the HPLC system . The HPLC procedure used was that developed and modified by Stnuv~ty and w~.t, (1984, 198 . The paralytic ahellBsh toxins are separated on a divinylbenzene term colmnn (PRP-1, Hamilton and Co, Reno, NV, U.S .A.), with heu~ane a~ heptane sulfonates as ion-pair reagents . This was performed on a HPLC system (655A12LC pump, L-5000 LC controller, Merck, Darmstadt, F.R .G .) equipped with a post~olumn reactor (URS-0Sl ICratoe, Ramsey, NJ, U.S.A.) and a fluorescence detector with a xenon excitation sours (F-1000, Merck, Darmstadt, F.R.G.) set at 340 rim excitation and 400rim emission. The HPLC column wsa maintained at 35°C, and post~olumn reaction was performed with periodic acid in a 1 ml reaction coil maintained at 90°C . Chromatograms were recorded on a dtromato-integrator (D 2000, Merck, Darmstadt, F.R .G.) . RESULTS
Several initial toxicity levels were monitored during decontamination, respectively at 3, 6, 9, 12 and 15 days of contamination, at a daily concentration of 3.2 106 cells/liter - ' in brooding tank water, in order to determine the initial toxic threshold at which a change in the different gonyautoxin ratio could be obtained during decontamination. Overall results are given in Fig. 1 and for each dooontamination series in Fig. 2. Thus, for an initial contamination of only 3 days (320 ~g PSP/100 g of meat), decontamination was already slow and failed to reach the safety threshold in 12 days. The results were the same in the other four trials, GTX2 having a higher concentration than the other toxins at the 12th day of decontamination . These observations were also valid for 6 and 9 days of initial contamination (Fig. 2H and 2C), percentages for GTX1 and GTX2 being higher at the cad of decontamination (9th and 12th days, respectively). For an initial contamination of 12 and 15 days (respectively 1324 and 2371 Rg PSP/100 g of meat), the relative proportion of GTX2 was actually greater than that of the other gonyautoxins (Fig. 2D and E) from the beginning of the decontamination phase. The two-slope decontamination kinetics
Paralytic Toxin in Munel
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Five differont initial toxicity levels were choxn for decontamination monitoring : A, B, C, D, E, or respectively : 320, 89g, 1060, 1324 and 2371 Ng PSP/ 100 g of mussel meat . For each mean toxic level a vertical bar features minimal and maximal values cala>
(`fast' and `slow') previously noted for toxicity levels between 200(1 and 3000 ~g PSP in the scallop (Deans et al., 1989) occurred only after 15 days of contamination in the mussel . In order to check the effects of extraction procedure on toxin profiles, the same contamination/decontamination experiment was subsequently conducted using a 0.1 N hydrochloric acid extraction at room temperature. The results (Table 1) showed a drastic change in the gonyautoxin ratio after 15 days of contamination. GTXl and GTX4 became the major toxins, whereas GTX2 and GTX3 were poorly represented. Nonetheless, after 15 days of decontamination, and despite the total amount of toxin decreased as in the previous experiment, the GTX2 percentage seemed to have increased, whereas highly toxic compounds, STX (saxitoxin) and NeoSTX (neosaxitoxin) were present but in a weak ratio.
Results of the first experiment show that GTX2 is always eliminated more slowly than the other gonysutoxins. Moreover, this experiment indicates that an initial toxicity above 1324 ~g PSP/100 g of meat is required to produce decontamination kinetics initially `fast' and then `slow'. Below this threshold, decontamination becomes slower as the proportion of GTX2 increases over the time course . Finally, it should be noted that there was a gradual increase in the pmportions of GTX2/GTX3 during contamination. Regardless of its initial toxicity, GTX2 was present during decontamination. However, the fact that GTX2 toxicity was proportionally greater fmm the beginning of the decontamination phase (values equal to or greater than 1300 pg PSP/ 100 g of meat) would
P. LASSUS et al. 30
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/ GTX4 ® GTXI
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Days 1FYa . 2. R>3.A17Y8 P~l~rrA~ OF BA(~ (iOiNYAU10x11~i DURatO U®COIiTAI~lAT70N, wrra ~ usr 1® A>t IOOX OF ACCUYULAlt~ l'OXQi Alm A>s ~ PIaSr DAY (O) OP DAY OF lX)NTA~lA170N D~CON'rAYQ4ATlON 1 . Mean results in two series of high-performance liquid chromatography analyses, with a maximum deviation never exceeding IOX of the values (numbs of replicate - 3) . Toxin profiles were etimated every 3 days after 3, 6, 12 and IS days (respectively A, B, C, D, E) of contamination .
Paralytic Toxin in Muasd
TAatB 1. RH<.ATIVB P~ICSPrfA(~S OP OONYAUT07m1S IN 1(pss$. ®CTAACIE AT THE Br1D OF ODNTAI~NATION AND DEOOrrrASm~uTioN rEaioDS. ExTRACnON raoc~uaE wAS rettroa~ wrrH 0.1 N HYDanc~.auc Acm, AT T~eA1vaE
15 days of contamination 15 days of contamination sad 1S days of decontamination
STX, saxitoxin; Neo, nec-saxitoxin; C3TX, gonyautoxin . Staadard erron never exceeded IOY. of the valus (number of replicates a 3).
seem to indicate that this toxin is also formed during the contamination phase in mollusks . Several hypotheses may account for this phenomenon: (1) enzymatic transformation of GTXl and GTX4 into GTX2 and GTX3; (2) a preferential accumulation of GTX2 and GTX3, with release of GTXl and GTX4 from the beginning of the contamination phase; or (3) hydmly~s of GTX8/epi GTX8, the dominant gonysutoxins of the MOG 835 strains, into GTX2 and GTX3. The second experiment aimed to indicate the relative roles of acid extraction and mussel metabolism in hydrolysis of GTX8/epi GTX8, since these weakly toxic compounds are known to produce GTX2 and GTX3 after hydrolysis. It appears that the AOAC procedure certainly affects toxin-profiles and that hydrolysis of GTX8/epi GTX8 in GTX2 and GTX3 probably occurs during extraction process. Nevertheless, even when the integrity of toxin structures is preserved, concentrations of highly toxic compounds increase during decontamination, and, consequently, may affect the overall toxicity of shellfish meat . It is well known (BOYI~t et al., 1986; I3w1.1, and RSICÜARDT, 1984) that heating during the AOAC procedure is detrimental to gonyautoxin chemical stability. Yet this standardized method is used in many national monitoring networks, and it is to be assumed that a depttration process carried out in a shellfish farm could probably be checked by it. It is thus likely that toxin profiles in natural or artificial (nontoxic algal food provided) conditions would resemble those described here, i.e. showing an increase in the relative percentage of GTX2 and a resulting lengthening of depuration kinetics during the `slow' decontamination phase. From our preliminary results, it would effectively appear that the AOAC method (mouse test) systematically produces a lengthening of the decontamination phase, regardless of the initial toxicity level tested. Acid extraction at room temperature should thus prove preferable for monitoring toxin concentrations in shellfish meat, even though it is still likely that the GTX21eve1 increases during decontamination. Application of the I3PLC procedure for daily monitoring of contaminated animals in shellfish farms or depuration stations would be onerous and inappropriate. A simple modification of the AOAC procedure would seem preferable, if restricted to measurements of shellfish depuration . With respect to the safety threshold for human consumption (80 pg STX/100 g of shellfish meat), classic AOAC extraction using the heating process is safer since hydrolysis of gonyautoxins generally increases global sample toxicity . Aabrowkalgenenus-The authoa thank Dr S. HAt .~ . (U.S . FDA, R+ashington DC~ and Dr Y. Os~mtA (Tohoku Univenity, Sendai, Japan) for providing, respectively, the PSP toxin standards and the Akarm~dr6en tanrmrn.Ae toxic stain.
P. LASSUS et d. REFERENCES
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