Marine biotoxins in shellfish – Pectenotoxin group

No abstract available

Pectenotoxin (PTX)-group toxins are a group of polyether-lactone toxins. They have been detected in microalgae and bivalve molluscs in Australia, Japan and New Zealand and in a number of European countries. Their presence in shellfish was discovered due to their acute toxicity in the mouse bioassay after intraperitoneal (i.p.) injections of lipophilic extracts of shellfish. PTX-group toxins are exclusively produced by Dinophysis species. They can be found in filter-feeding bivalve molluscs such as oysters and mussels. To date 15 different analogues have been isolated and characterised. PTX-group toxins are heat stable, but they are easily destroyed under strong basic conditions such as used for hydrolysis of acyl esters of the okadaic acid (OA)-group toxins. PTX-group toxins are also labile under acidic conditions.

PTX-group toxins in shellfish are always accompanied by toxins from the OA-group. This appears to be the basis for grouping PTX-group toxins and OA-group toxins in the European regulation. The Scientific Panel on Contaminants in the Food Chain (CONTAM Panel) concluded that because PTX-group toxins do not share the same mechanism of action as OA-group toxins they should not be included in the regulatory limit for OA-group toxins.

The toxicological database for PTX-group toxins is limited and comprises mostly studies on their acute toxicity in mice. There are no reports on adverse effects in humans associated with PTX-group toxins. The available data on lethality in mice only comprise information following i.p. injection and are not sufficient to establish robust toxicity equivalency factors (TEFs). In order to be prudent the CONTAM Panel proposes a provisional TEF value of 1 to be used for PTX1, PTX2, PTX3, PTX4, PTX6 and PTX11, until more robust data become available. PTX7, PTX 8, PTX 9, PTX2 SA and 7-epi-PTX2 SA are much less toxic and were not assigned TEFs.

Following oral administration PTX-group toxins show low systemic absorption and reported toxicity is mainly restricted to the intestinal tract. No data on the chronic effects of PTX-group toxins in animals are available, therefore the CONTAM Panel could not establish a tolerable daily intake (TDI). In view of the acute toxicity of PTX-group toxins, and due to the lack of observations in humans, the CONTAM Panel decided to establish an acute reference dose (ARfD) based on the available animal data on acute toxicity. 

Although the oral toxicity is not well defined, the CONTAM Panel considered it appropriate to establish an ARfD on the basis of a lowest-observed-adverse-effect-level (LOAEL) of 250 µg/kg body weight (b.w.) for intestinal toxicity of PTX2 observed in mice. Because the effects were mild and reversible, the CONTAM Panel decided to apply a factor of 3 for the extrapolation from a LOAEL to a no-observed-adverse-effect level (NOAEL). The CONTAM Panel established an ARfD of 0.8 µg PTX2 equivalents/kg b.w., based on a LOAEL of 250 µg/kg b.w. and an overall uncertainty factor of 300.

In order to protect against the acute effects of PTX-group toxins, it is important to use a large portion size rather than a long-term average consumption in the health risk assessment of shellfish consumption. Consumption data for shellfish species across the European Union (EU) were limited, therefore the European Food Safety Authority (EFSA) requested the Member States to provide information on consumption of relevant shellfish species. Based on data, provided by five Member States, the CONTAM Panel identified 400 g of shellfish meat as a large portion size to be used in the acute risk assessment of marine biotoxins.

Consumption of a 400 g portion of shellfish meat containing PTX-group toxins at 160 µg/kg shellfish meat (by analogy with the current EU limit for lipophilic toxins of 160 µg OA equivalents/kg shellfish meat) would result in an intake of 64 µg toxin (equivalent to about 1 µg/kg b.w. in a 60 kg adult). This intake is slightly higher than the ARfD of 0.8 µg PTX2 equivalents/kg b.w. (equivalent to 48 µg PTX2 equivalents per portion for a 60 kg adult) and is not considered to constitute a health risk. Based on current consumption and occurrence data, there is a small chance (approximately 0.2 %) to exceed the ARfD of 0.8 μg PTX2 equivalents/kg b.w. when consuming shellfish currently available on the European market. 

The CONTAM Panel concluded that, in order for a 60 kg adult to avoid exceeding the ARfD of 0.8 µg PTX2 equivalents/kg b.w., a 400 g portion of shellfish should not contain more than 48 µg PTX2 equivalents corresponding to 120 µg PTX2 equivalents/kg shellfish meat. 

There is no information on the effects of processing (e.g. cooking, steaming, autoclaving) on the levels of PTX-group toxins in shellfish, but it can be assumed that, as for other lipophilic toxins, water loss during processing may lead to an increase in the concentration of PTX-group toxins in shellfish flesh. 

The mouse bioassay (MBA) and the rat bioassay (RBA) are the officially prescribed reference methods in the EU for the determination of lipophylic toxins, including PTX-group toxins. The CONTAM Panel noted that the MBA has shortcomings e.g. it has an inherent variability, is not quantitative, has an insufficient detection capability, and is not selective for the PTX-group toxins and thus may give both false negative and false positive results. The RBA detects compounds with diarrhetic effects such as OA-group toxins. PTX-group toxins do not have diarrhetic properties, therefore the CONTAM Panel concluded that the RBA is not suitable to detect PTX-group toxins. 

The current EU legislation permits the replacement of the MBA, provided that the alternative methods have been validated according to an internationally recognised protocol. The evidence available at this moment suggests that the methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) have the greatest potential to replace the MBA. These methods also have the possibility for multi-toxin group detection/quantification. At this time however, none of the methods for the determination of toxins from the PTX-group have been validated by interlaboratory studies. The CONTAM Panel noted that, whilst application of single laboratory validation according to recognised international guidelines to demonstrate their fitness-for-purpose can be an impetus for implementation of alternative instrumental analyses of marine biotoxins for regulatory purposes, method performance criteria should be stipulated where possible and validation by interlaboratory trials should be the long-term objective.

Marine biotoxins, pectenotoxin (PTX)-group toxins, shellfish, bivalve molluscs, mammalian biotests, acute reference dose, portion size, methods of analysis, human health, risk assessment