5.1.5 Aquatic biotoxins (Hans Henrik Huss)

The possible presence of natural toxins in fish and shellfish has been known for a long time. Most of these toxins are produced by species of naturally occurring marine algae (phytoplankton). There are over 4,000 species of marine algae, but only 70-80 species (~2%) are known to produce toxins (Scoging, 1998).

A proportion of the toxic phytoplankton has a red-brown pigmentation, giving rise to the naming of algal blooms as "red tides". However, it should be emphasised that not all coloured algae are toxic, and incidence of poisoning have occurred in the absence of red tides. Visible red tides may contain from 20 000 to > 50 000 algal cells per ml. Concentrations as low as 200 cells/ml may produce toxic shellfish. During a bloom, bivalves can accumulate sufficient toxin to cause human illness after filter feeding for only 24h (Scoging, 1998) (see Figure 5.14).

Molluscan shellfish are filter feeders and continually pump water through their gills where particulate matters is removed and ingested. Mussels ingest food particles of any type of 2 to 90 mm in size with a rate of ingestion dependent on water temperature and environment. Optimally, they can filter 2.5 l/h extracting 98% of the available algae. Consequently, any toxin associated with the phytoplankton ingested can rapidly accumulate and hence become concentrated in the bivalve mollusc. The consumption of these toxic shellfish by humans causes illness with symptoms ranging from mild diarrhoea and vomiting to memory loss, paralysis and death.

Toxins associated with phytoplankton are known as phycotoxins. These toxins have been responsible for incidents of wide-scale death of sea-life and are increasingly responsible for human intoxication. There are a number of different seafood poisoning syndromes associated with toxic marine algae and these include paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP) and azaspiracid shellfish poisoning (AZP). There are also different types of food poisoning associated with finfish and these include ciguatera poisoning and puffer fish poisoning. Consumption of raw molluscan shellfish poses well-known risks of food poisoning, however, intoxication from finfish is not so well known. Most of the algal toxins associated with seafood poisoning are heat stable and are not inactivated by cooking. It is also not possible to visually distinguish toxic from non-toxic fish and shellfish. Many countries rely on biotoxin monitoring programmes to protect public health and close harvesting areas when toxic algal blooms or toxic shellfish are detected. In non-industrialized countries, particularly in rural areas, monitoring for harmful algal blooms does not routinely occur and death due to "red tide toxins" commonly occurs.

Figure 5.14 Generalized pathways of human intoxication with molluscan shellfish toxins via filter feeding bivalves and carnivorous and scavenging gastropods. (from Anderson et al., 2001).

The toxins are accumulated in the digestive gland of the shellfish (hepatopancreas) and do not affect the shellfish themselves. The shellfish may reduce toxicity in clean water, but depuration times vary greatly according to the bivalve specie involved, the pumping activity of the bivalve and the hygrographic conditions.

Fish may also consume toxic algae and cause disease in humans (ciguatera). Also, there are toxins in some fish species that do not involve marine algae (puffer fish poisoning), see Table 5.26.

Paralytic shellfish poisoning (PSP)

a) The disease and epidemiological aspects

Intoxication after consumption of shellfish is a syndrome that has been known for centuries, the most common being PSP. It is caused by a group of toxins (saxitoxins and derivates) produced by dinoflagellates of the genera Alexandrium, Gymnodium and Pyrodinium.

Table 5.26 Marine biotoxins and the associated poisonings.

Symptoms of PSP initially involve numbness and a burning or tingling sensation of the lips and tongue that spread to the face and fingertips. This leads to a general lack of muscle coordination in the arms, legs and neck. Severe cases of PSP have resulted in respiratory paralysis and death. There are an estimated 1 600 annual cases of PSP world-wide, approximately 300 of these will be fatal (Scoging, 1998). Mortality rates in outbreaks of PSP have reached 40%. There is an extreme variation in sensitivity to the toxin, but intoxication has followed oral intake of 144 µg to 1,660 µg per person with fatalities occurring at levels of 300 µg to 12 400 µg PSP per person (van Egmond et al., 1993).

b) Prevalence in fish and fishery products

PSP is the most widespread shellfish poisoning and outbreaks are occurring worldwide as shown in Figure 5.15.

Blooms of toxic algae - and outbreaks of PSP - occur regularly throughout Europe, and the EU-monitoring programmes regularly detect high toxin levels (van Egmond et al., 1993). The dinoflagellates bloom as a function of water temperature, light, salinity, presence of nutrients and other environmental conditions. Blooms of toxic algae have recently become more prevalent, and many experts believe coastal pollution and shipping practices have contributed to this expansion (Anderson 1994). Water temperature must be (5-8°C for blooms to occur. If temperature decreases to below +4°C, the dinoflagellates will survive as cysts buried in the upper layer of the sediments.

Shellfish that have fed on toxic dinoflagellates retain the toxin for varying periods of time depending on the shellfish. Some clear the toxin very quickly and are only toxic during the actual bloom. Others retain the toxin for a long time, even years (Schantz, 1984).

c) Stability of toxin

The toxic compounds are water-soluble and heat stable. A 5-minute cook will reduce toxicity by only 30% and increasing this to 20 min. will only effect a 40% denaturation (Scoging, 1998).

Figure 5.15 World distribution of outbreaks of paralytic shellfish poisoning (black spots) and ciguatera (shaded area). See Huss (1994) for references.

Diarrheic shellfish poisoning (DSP)

Thousands of cases of gastrointestinal disorders caused by DSP have been reported in Europe, Japan, South East Asia, North- and South-America (Sechet et al., 1990). The causative dinoflagellates, which produce the toxins are within the genera Dinophysis and Prorocentrum. These dinoflagellates are widespread, which means that this illness could also occur in any other parts of the world. A great number of toxins has been identified including okadaic acid (OA) and associated toxins (DTX 1-4). Levels producing diarrhoea in adults are estimated at ³ 40 µg for OA and ³ 35 µg for DTX 1 (Scoging, 1998).

Onset of disease is within half an hour to a few hours following consumption of shellfish, which have been feeding on toxic algae. Symptoms are gastrointestinal disorder (diarrhoea, vomiting, abdominal pain) and victims recover within 3-4 days with or without treatment. No fatalities have ever been observed.

The toxins are heat stable and survive normal cooking.

Neurotoxic shellfish poisoning (NSP)

The occurrence of NSP has historically been limited to the west coast of Florida, where blooms of the dinoflagellate Gymnodinium breve occurs regularly offshore and is carried inshore by wind and current conditions. However, also shellfish harvested on the southern Atlantic coast may be toxic and there have been reports of outbreaks of NSP in New Zealand.

The responsible toxins are a family of brevetoxins. The toxins are extraordinarily stable (survive heat up to 300°C) and the oral LD₅₀ value in rats being in the order of 520-6 600 µg/kg (Llewellyn 2001). Pathogenic dose for human is in the order of 42-72 mouse units (MU).

Typical symptoms of NSP are tingling in the face, throat and digits, dizziness, fever, chills, muscle pains, abdominal pains, nausea, vomiting, headache and reduced heart rate. There have been no recorded human deaths from NSP, but the toxin is fatal to fish and can cause massive fish kill.

Amnesic shellfish poisoning (ASP)

ASP is the only shellfish poison produced by a diatom. Disease was first identified in Canada in 1987, where more than 100 people became ill often consuming contaminated shellfish (Todd, 1993). The disease was named after one of the more curious symptoms, which was loss of short-term memory. Other symptoms include nausea, vomiting, diarrhoea, headache and neurological effects including dizziness, disorientation and confusion. In severe cases seizures followed by coma and death may occur. The short-term memory loss seems to be permanent in surviving victims.

Outbreaks have so far been confined to Canada and the USA, although the responsible algae has been found in many other areas.

The causative agent is domoic acid. In the Canadian 1987 outbreak, human toxicity occurred at 1-5 mg/kg (Todd, 1993).

Ciguatera fish poisoning (CFP)

CFP is one of the most common food-borne illnesses related to finfish consumption. Its true incidence is not known, but it has been estimated that 10 000-50 000 people a year suffer from this disease. It is caused by consumption of fish that have become toxic by feeding on toxic dinoflagellates or toxic herbivore fish. The principal source is the benthic dinoflagellates Gambierdicus toxicus, which is found primarily in the tropics where it lives in association with macro algae, usually attached to dead corals. More than 400 species of fish are known to be vectors of ciguatoxins. Toxins can be detected in the gut, liver and muscle tissue by means of mouse assay. Some fish may be able to clear the toxins from their systems (Taylor, 1988). The toxic fish may be found in tropical and subtropical Pacific and Indian Ocean regions and in the tropical Caribbean as shown in Figure 5.15.

Ciguatoxins arise from bio-transformation in the fish of precursor toxins produced in the dinoflagellates and it causes disease when present in ³ 1 ppb (0.1 µg/kg) in the flesh of the fish (Lehane and Lewis, 2000).

Clinical symptoms vary widely but are characterized by gastrointestinal, neurological and cardiovascular disturbances often within 10 min but also up to 24 h after ingestion of toxic fish. The initial gastrointestinal symptoms are similar to any other food poisoning (abdominal pain, nausea, vomiting, diarrhea). The neurological symptoms most often encountered are tingling and numbness in the mouth, hand and feet, muscle cramping and weakness, temperature reversal, superficial hyperesthasia with a sensation of burning. Headache, vertigo, stiffness, convulsions, hallucinations, transient blindness, salivation, perspiration are symptoms that may occur. A slow, irregular pulse and law arterial pressure may follow. Cardiovascular disorders usually disappear within 48-72 h while neurological effects may persist for weeks, even years in severe cases. Death from CFP is rare (<1% worldwide).

Puffer fish (Tetrodotoxic) poisoning (PFP)

Tetrodotoxin (TTX) is one of the most potent non-proteinacous toxins known and responsible for numerous fish poisonings. The toxin is named after the order Tetraodontidae (common names: puffer fish, balloon fish, globe fish, fugu, toad fish, blow fish), since many of these fish often carry the toxin. Apart from Tetraodontidae toxin has been found in goby, blue-ringed octopus, various gastropods, newts and houseshoe crab.

PFP has frequently occurred in Japan, where these fish are a traditional food. Nearly 300 cases (nearly 500 patients) were recorded in the 10-year period 1987-1996 with an average mortality rate of 6.6% (Yoshikawa-Ebesu et al., 2001). Sporadic cases of PFP are seen in other Asian and Pacific countries incl. USA. Symptoms of PFP occur within minutes and rarely more than 6 h after ingestion of toxic fish. Nausea and vomiting may or may not occur, but the most common symptoms are tingling or pricking sensation and dizziness. Disease may progress to muscle and respiratory paralysis. Where death occurs it is usually within 6 h and sometimes as rapidly as 20 min following toxin ingestion. Persons who have not died within 24 h generally recover completely.

The distribution of the toxin in the fish is mainly in the ovaries (eggs), liver and skin. The muscle tissue is normally free of toxin. The origin of the toxin has historically been much debated (Figure 5.16). The question has been whether it is endogenous or exogenous. It is now assumed that TTX in fish comes directly from its feed. The toxin is produced by bacteria, absorbed on or precipitated with plankton, transmitted to TTX-bearing animals such as small gastropods, starfish, flatworks etc. and from here transmitted to fish and large gastropods. Fish, except those processing tetrodotoxin such as puffers and tropical goby, do not accumulate tetrodotoxin even where toxin-containing diets are fed to them at sub-lethal doses (Yoshikawa-Ebesu et al., 2001).

TTX is a potent toxin with a LD₅₀ of 2 mg for man. The minimum dose necessary to cause symptoms has been estimated to 0.2 mg (Yoshikawa-Ebesu et al., 2001).

Control and prevention of natural toxins

Figure 5.16 Assumed mechanism of toxification of tetrodotoxin-bearing animals (after Yoshikawa-Ebesu et al., 2001).

Natural toxins are very heat stable. Normal household cooking (e.g. boiling, steaming, frying) has no or very little effect on toxin levels. Also a heat treatment of 70°C in 20 min was insufficient to reduce the toxin level significantly and even after retorting (120°C for 60 min) some toxicity remained (Nagashima et al., 1991). The normal industrial canning process may significantly decrease the toxin levels present in shellfish, but it is only sufficient when the initial toxin level is relatively low. Thus in the European Union it is acceptable to utilise bivalve molluscs when the initial level of contamination with PSP exceeds the limit of 80 µg/100 g laid down in Council Directive 91/492/EEC but is below 300 µg/100 g (EC, 1996). However, the molluscs have to undergo the following operations sequentially:

1. Preliminary cleaning in fresh water for a minimum of two minutes at a temperature of 20°C, plus or minus 2°C

2. Pre-cooking in fresh water for a minimum of three minutes at a temperature of 95°C, plus or minus 5°C

3. The separation of flesh and shells

4. Second cleaning in running fresh water for a minimum of 30 seconds at a temperature of 20°C, plus or minus 2°C

5. Cooking in fresh water for a minimum of nine minutes at a temperature of 98°C, plus or minus 3°C

6. Cooling in running cold fresh water for approximately 90 seconds

7. The separation of the edible parts (foot) from the non-edible parts (gills, viscera and mantle) mechanically with water pressure

8. Conditioning in containers closed hermetically in a non-acidified liquid medium

9. Sterilisation in autoclave at a minimum temperature of 116°C for a time calculated according to the dimension of the containers used but which cannot be lower than 15 minutes.

A new chemical method for decontamination of PSP toxins in shellfish was recently developed by Lagos et al. (2001). The method involves one or two alkaline treatment (pH (9) followed by boiling and washing. The method was reported to yield 99% decontamination.

Detection of natural toxins is mainly based on mouse-bioassays, while analytical methods may be used for confirmatory analysis of toxic compounds. Only in one case (analysis for domoic acid) is an analytical method - high-performance liquid chromatography (HPLC) - approved as a certified method.

Mouse-bioassays are cheap to carry out, but it is to their disadvantage that they involve live animals, a practice, which has become increasingly unpopular, and that they require experienced personnel and careful standardisation of assay conditions. Also bioassays are less sensitive and less precise than analytical methods. For an overview of present and emerging technologies in detecting natural toxins see Kitts (2001), Price and Tom (1999) or Anderson et al. (2001).

The regulatory tolerances established for natural toxins by FDA (1998) and others are listed in Table 5.27.

Table 5.27 Monitoring of biotoxins

1. PD = Pathogenic dose for humans
2. LD = Lethal dose for humans.

The primary preventive tool for intoxications with natural toxins is the monitoring of toxin levels in algae in the harvesting areas (see Chapter 9). Based on the presence of toxins, waters can be classified and harvesting of shellfish forbidden if levels of toxin are too high. Other elements of a control programme will include (FDA, 1998):

- a requirement that containers of in-shell molluscan shellfish bear a tag that identifies the type and quality of shellfish, harvester, harvest location and date of harvest

- a requirement that molluscan shellfish harvesters be licensed

- a requirement that processes that chuck molluscan shellfish or ship, repack the chucked product be certified

- a requirement that containers of chucked shellfish bear a label with the processor's name, address and certification number.

Depuration and ozonation are not effective and are not used in reducing toxins in shellfish (Anderson et al., 2001).