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Investigation of the efficiency of the lipophilic biotoxin MBA extraction method to remove a model hydrophilic biotoxin (dc-saxitoxin)

Project Code: B16008

31/10/2005

Integrin Advanced Biosystems Ltd
Mckenzie, J ; Ward, C

Background

Since 2001 some shellfish extracts from England, Wales and Northern Ireland have demonstrated a fast and unusual form of toxicity in the mouse bioassay for lipophilic toxins (DSP-MBA). This *atypical response* in the bioassay is characterised by the rapid death (within minutes) of injected mice. The cause of the atypical response in the DSP-MBA is unknown

The DSP-MBA involves homogenisation of shellfish flesh, extraction of the homogenate using solvents. Acetone is used first which is then evaporated to leave an aqueous extract, which is then extracted with ether. The ether extract is washed with water and evaporated to leave a residue, which is suspended in an aqueous solution before administration to mice.

The Shellfish Industry have suggested that the atypical response is an artefact of the methodology, focusing their attention on the presence of solvents in the extract injected into the mouse and the possibility of carry-over of water-soluble interfering factors into the injected extract. The DSP MBA is designed to remove water-soluble toxins as these are a known source of interference in the assay. In France, the presence of low levels of water soluble PSP biotoxins have been found to cause rapid death of mice in the bioassay for lipophilic biotoxins used at this time, which simply involved extraction of the shellfish with acetone and evaporation of the acetone before suspension in Tween-60.

In November 2003, a standardised extraction protocol (UK-NRL DSP1) was introduced, which included measures to control solvent levels. Although atypical responses in the DSP-MBA initially ceased after the introduction of the UK-NRL DSP1, some samples in the Spring of 2004 showed atypical responses. The available data on solvent levels in the final extract from the recent atypical responses in the DSP MBA, suggest that residual levels of solvents are too low to be the direct cause of these mouse deaths.

The UK-NRL DSP1 has a smaller backwash volume (2 x 5 ml) compared with those used in similar protocols elsewhere in Europe ( e.g. 20 ml in France). It may be the volume of water used is inadequate to reduce the potential contamination of the final extract with PSP toxins to levels that will not interfere with the MBA.

Rationale and Objectives

The purpose of the study was to measure the clearance of a model hydrophilic PSP toxin (dc-saxitoxin) from shellfish extract being prepared using UK-NRL DSP1 where the wash step is omitted then study the effects of different wash volumes. Dc-saxitoxin was chosen as the model compound because a controlled reference material (CRM) (mussel flesh) containing this compound is readily available. The chemical structures of PSP toxins suggests that dc-saxitoxin will be the least water-loving of the many PSP biotoxins (saxitoxin, neosaxitoxin and gonyautoxins 1-6). Thus, it can be concluded that a wash step that effectively reduces the carry-over of dc-saxitoxin in the final extract will also be applicable to other PSP toxins. An additional experiment was introduced to look at the effect of pre-saturating the ether with water.

Approach

Dc-saxitoxin CRM was mixed with mussel homogenate to produce an effective concentration of 40 µg/100 g of dc-saxitoxin (half the regulatory limit). This was then extracted using the solvent extraction steps defined by UK NRL DSP1. Instead of injecting the extract into mice, the amount of dc-saxitoxin in the extract was measured using a chemical detection method (ELISA) which can measure much lower levels of PSP toxins than the mouse assay. No mice were used in the experiments. Several experiments were run using different modifications of the backwash volume and a further experiment was done comparing the effect of using *dry* ether and ether that had been pre-saturated with water before use.

Results

When no backwash was used, approximately 12% of the dc-saxitoxin was carried over into the final extract. This was far in excess of the amount necessary to kill mice. When a 2x 5ml backwash was used the amount present in the final extract fell to about 1% of the original amount. Slightly less was carried over with 2 x 20 ml washes and using 50 ml washes resulted in there being no detectable dc-saxitoxin in the final extracts. These results both demonstrate the necessity for the backwash step and the desirability of using sufficiently large volumes of water. Under the conditions used, both the 2 x 5 ml and 2 x 20 ml backwash steps were inadequate to reduce the levels of dc-saxitoxin below detectable levels. In one extract where 2 x 100 ml volume of backwash water was used a low amount of dc-saxitoxin was detected. This suggests that factors other than the backwash volume can influence the result.

When pre-saturated ether was used in the extraction, the amount of dc-saxitoxin left in the extract was further reduced (almost by half).

What it means and why it is Important

The experiments undertaken were not an attempt to investigate the possibility that PSP toxins are the cause of the atypical response. Instead, they were designed to challenge the methodology using experimentally contaminated materials to see how effective the methodology was in removing these water-soluble toxins. As the design of the extraction procedure is meant to remove possible interference from water soluble toxins in the DSP assay, this is an important point.

Without any backwash step, the dc-saxitoxin was present in amounts sufficient to kill mice. The backwash step is thus an important and necessary component of the assay. Increasing the volume of the backwash resulted in increasingly smaller amounts of dc-saxitoxin appearing in the extract and thus a reduced possibility that any hydrophilic toxin present in the sample would interfere with the assay. The dc-saxitoxin was reduced below detectable limits when 2 x 50 ml washes were used meaning that volumes less than this may be inadequate to prevent possible interference by hydrophilic toxins breaking through into the final extract. However, the reduction caused by use of pre-saturated ether shows that other measures may also be useful in reducing the possibility of water soluble toxins interfering with the assay.

The methodology used in the UK is currently being revised. This short piece of work has shown the critical need to use sufficient volumes of backwash water and will be of use in setting limits for the revised methodology.

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