2.6.1 Depuration
Various attempts have been made at detoxifying shellfish contaminated with paralytic shellfish poisons in an effort to reduce the duration of off market times. The most obvious method is to transfer shellfish to waters free of toxic organisms and allow them to self-depurate. However, transferring large quantities of shellfish is very labour intensive and costly. The rates of detoxification vary considerably between species and some species remain toxic for extended periods of time, for instance up to several months for Crassostrea, Plactopecten, Spisula and others. The time required for elimination in mussel tissue also varies considerably. If the contaminating dinoflagellates have disappeared from the surrounding water there is a gradual decline in the amount of toxin. The toxicity of the mussel Mytilus edulis can decrease by 50 percent in 12 days in dinoflagellate free, salt water with a temperature of 15 to 20° C. The elimination time of PSP toxins from the clam Saxidomus giganteus however is much longer. It takes a year or longer after the exposure to the toxic dinoflagellates to lose the toxins (Mons et al., 1998). The rate of loss varies with season, and low water temperatures apparently retard toxin loss. However, the degree to which temperature affects the uptake and release of toxins is not clearly understood. Further, the rate of detoxification is highly dependent on the site of toxin storage within the animal (toxins in the gastrointestinal tract are eliminated much more readily than toxins bound in tissues) and on initial or peak level of toxicity. Mussels are known to accumulate PSP toxins faster than most other species of shellfish and also eliminate the poisons quickly. While oysters do not accumulate the toxic species as readily as mussels, they take considerably more time to detoxify (Mons et al., 1998).
Detoxification of PSP-toxins using temperature or salinity stress has been tried with marginal success. Instantaneous electrical shock treatments were found to accelerate toxin excretion in scallops. Reduced pH has been tried as a means of detoxifying butter clams but with no success. Chlorination has been used in France; however, this process alters the flavour of the shellfish and thus decreases marketability. However, ozonized seawater can be of value in detoxification of shellfish recently contaminated by the vegetative cell phase of toxic dinoflagellates. In a study during a red tide outbreak, it was shown that ozone treatment of the seawater prevents shellfish from accumulating PSP toxins. This activation could be achieved in a marketable species such as Mya within an economically feasible time frame. On the contrary, ozone is useless in detoxifying cysts or in bivalves that have ingested cysts or have the toxins bound in their tissue over long periods of time. Further, detoxification of algal toxins from shellfish, especially paralytic shellfish poisons, over long periods of time is not economically feasible. Ozone is not recommended as a practical or safe means of eliminating algal toxins from shellfish (Mons et al., 1998).
Cooking has also been proposed as a possible means of detoxifying shellfish contaminated with paralytic shellfish poisons. However, while cooking may reduce levels of toxins it does not eliminate the danger of intoxication. If the initial level of toxicity is low, cooking may effectively reduce toxicity to safe levels. Pan frying seems to be more effective than other methods of cooking. When clams or mussels are steamed or boiled, toxins lost from the tissues are contained in the cooking liquid rendering the fluids extremely toxic (Mons et al., 1998). Boiling of oysters only, for the usual home cooking times (98 °C for 10 minutes), reduced their toxicity by 68 to 81 percent. However, boiling by itself is not sufficient to detoxify extremely high toxic shellfish (Jeong et al., 1999).
Beringuer et al. (1993) studied the effects of operations carried out during the industrial canning process on the contamination of Acanthocardia tuberculatum (Mediterranean cockle) by PSP toxins. The observed effects of boiling and sterilizing averaged over 70.6 to 77.9 percent and 81.8 to 90.9 percent reduction of toxicity, respectively. Takata et al. (1994) investigated the reduction in toxicity of PSP-infected oysters (Crassostrea gigas) by heat treatment. The methods of heat treatment were boiling (at 98 °C for 5 to 60 minutes) and retorting (at 120 °C for 5 to 60 minutes). Boiling at 98 °C resulted in 53 to 88.3 percent detoxification, retorting at 120 °C in 57.4 to 100 percent detoxification. Boiling and retorting for 60 minutes resulted in more detoxification than boiling and retorting for five minutes.
Less detoxification after boiling and autoclaving of PSP-infected oysters was observed. Oysters having 17.4 or 29.8 MU PSP toxins were boiled, canned and autoclaved. The toxicity was reduced by about 20 percent after boiling and by less then 10 percent after autoclaving. The effectiveness of canning as a means of reducing PSP-toxicity levels below quarantine levels is dependent upon the initial levels of toxicity and should be approached with great caution (Mons et al., 1998).
After both the boiled and the smoked canning process of oysters (Crassostrea gigas) contaminated with 185 to 778 mg eq STX/100 g, Jeong et al. (1999) measured a reduction to <80 mg eq STX/100 g. Mole % of toxin components in the shucked oysters was in the order of 25.1% GNTX1, 19.2 mole % GNTX3, 17.2 mole % of GNTX4 and 14.6 mole % of GNTX2. Trace amounts of C1, C2, STX and neoSTX were present. In case of specific toxicity, the major toxins were GNTX1-4. The sum of GNTX1, 2, 3 and 4 was >80 percent of total toxicity. STX and dc-STX were more thermostable than any other toxin component.
The standard canning process (steaming, cooking and sterilization) of pickled mussels and mussels in brine (Mytilus galloprovincialis) resulted in a 50 percent reduction of PSP toxicity in mussel meat. The decrease was not dependent on the toxin levels in the raw mussel meat. Total toxicity reduction did not fully correspond to toxin destruction, which was due to the loss of PSP toxins to cooking water and packing media of the canned product. The detoxification percentages can be affected by changes in the toxin profile due to toxin conversion (Vieites et al., 1999).
Indrasena and Gill (1999) studied the effects of a wide range of pH values (3-7) on the kinetics of thermal destruction for individual PSP toxins in scallop digestive glands. Most of the individual toxins degraded more rapidly when heated at higher temperatures and pH levels for longer times. PSP toxicity decreased rapidly at 130 °C at pH 6-7.
In a later study of Indrasena and Gill (2000a), mixtures of purified and partially purified PSP toxins including C1/2 and B1 toxins, GNTX1-4, neoSTX and STX were heated (90-130 °C) for different times (10 to 120 minutes) at different pHs (3-7) and analysed by LC. C toxins declined rapidly at low pH, and GNTX1/4 toxins decreased at high temperatures and high pH. GNTX 2/3 increased initially at low pH and then declined with subsequent heating, whereas STX increased consistently at pH 3 to 4. The integrated total toxicity declined at pH 6 to 7. The efficacy of thermal destruction was highly dependent on pH, with rapid thermal destruction of carbamate toxins at higher pH. Heating at low pH resulted in conversion of least toxic compounds to highly toxic compounds.
Variations in C-toxins (C1-2), GNTX1-4, STX and neoSTX in scallop digestive glands and a mixture of purified PSP toxins were studied during storage at -35, 5 and 25 °C at different pH levels by Indrasena and Gill (2000b). All toxins were stable at low pH (3-4) and -35 °C. C toxins were the most sensitive followed by GNTX1/4 for changes at all pH levels and at 5 and 25 °C. STX followed by neoSTX were the most stable at -35 and 5 °C, especially at pH 3-4.
With the exception of the methods reported by Berenguer et al. (1993) and Takata et al. (1994) there are hardly useful methods for effectively reducing PSP toxins in contaminated shellfish. Most of the methods tested have been unsafe, too slow or economically unfeasible, or have yielded products unacceptable in appearance and taste. Given the apparent global increase in harmful algal blooms and the continually growing interest in culture of bivalve molluscs, further efforts are needed to develop effective means of detoxifying shellfish contaminated with PSP toxins. Failing the development of any such methods, increased efforts will need to be devoted to monitoring shellfish for the presence of PSP toxins.
Takatani et al. (2003) suggested that microwave heating after pretreatment with salt and alkaline solution might be available for decomposition of PSP toxins in the oyster Crassostrea gigas.
Data on toxicity measured under different conditions for the adductor muscle of highly PSP-infested scallop Patinopecten yessoensis, were reviewed. In the adductor muscle, separated from live or fresh scallop, not any toxicity was observed even though the whole scallop contained levels as high as 2 900 MU/g. On the other hand, the adductor muscle separated from the frozen whole body, showed very small toxicity whose score depended on the different procedures, high especially in slow thawing over many hours. It can be concluded that the adductor muscle of the scallop Patinopecten yessoensis is safe for consumption only when it is prepared from live or fresh scallop with careful removal of toxic viscera, roe and the other organs (Murakami and Noguchi, 2003).
Within the EC Research Programme “Quality of Life and Management of Living Resources”, research is ongoing on an accelerated detoxification system for live marine shellfish, contaminated by PSP toxins. The objective of this project is to determine the effects of micro-algae diet in order to accelerate the detoxification of live shellfish, namely oysters and clams, in a system that overcomes the problems of PSP contamination. The properties of the process are to speed up the shellfish detoxification kinetics with appropriate diets using the effect of non-toxic cell density (Anonymous, 1999a).
2.6.2 Preventive measures
At present the economic feasibility of efficiently detoxifying shellfish on a large scale in artificial systems is not promising. In areas prone to regular outbreaks of toxic algal species, culturists and commercial fishermen alike still depend on monitoring systems to warn of toxic shellfish and plan their activities accordingly. Through combined efforts of an intensive monitoring programme and culture of ‘rapid release’ species (e.g. Mytilus edulis), species known to avoid toxic dinoflagellates (e.g. Mercenaria, most oysters) or scallops, economic loss can be kept to a minimum.
Preventive measurements include regular inspection of seawater bodies in which the shellfish are grown on the possible appearance of toxic dinoflagellates especially in the season that blooms may occur. In addition, the presence of cysts of the dinoflagellates should be explored and the shellfish itself should also be inspected routinely. Therefore, the development of a rapid and reliable method for the detection of Alexandrium species before bloom formation is important (Mons et al., 1998). However, morphological identification continues to be controversial (Sako, 1999). Some of the morphological features may change with varying environmental conditions or growth stage. This necessitates the application of biological, biochemical, immunological and molecular biological techniques. Monoclonal antibody analyses and lectin-binding assays have also been applied. However, these analyses are based on phenotypic characters which may be affected by environmental factors (Adachi et al., 1996). In order to resolve the phenotypic problems, the definition of genetic markers may be useful for classifying the Alexandrium species responsible for the production of PSP toxins. In a number of studies the ribosomal DNA (rDNA) sequences and internal transcribed spacers (ITS) (using DNA probes and whole-cell hybridization) were analysed (Adachi et al., 1996; Sako, 1999).
Haley et al. (1999) reported a stream-lined method for a labour- and resource-intensive protocol for the isolation of A. tamarensis ribosomal DNA (rDNA). This method facilitated the detection of 10-4 ng/ml of A. tamarensis DNA. The kit enabled A. tamarensis to be isolated from the water sources with little signal degradation. This is a valuable technique for the rapid detection of A. tamarensis, even before cell numbers are large enough for morphological identification.
Guzmán et al. (2001) reported that prevention could be strengthened not only through a PSP monitoring programme but also through a strategy of specific training to specific target groups and an appropriate information dissemination system. A strategy applied in southern Chile since 1997 included:
training workshops oriented to diverse groups;
design and dissemination of key information about harmful algal blooms and its effects, oriented to the general community; and
specific activities with primary and high school teachers and students.
In case of contaminated shellfish, measurements have to be taken to prevent consumption and cases of human PSP intoxications should be reported to the health authorities as soon as possible. More information on regulations and monitoring will be presented in Chapter 2.8.
