A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis

Trends in Analytical Chemistry, Vol. 30, No. 2, 2011 Trends

A European perspective on progressin moving away from the mousebioassay for marine-toxin analysisKatrina Campbell, Natalia Vilarino, Luis M. Botana, Christopher T. Elliott

This review considers the ethical and technical problems currently associated with employing mouse bioassays for marine-toxin

analysis and the challenges and the difficulties that alternative methods must overcome before being deemed applicable for

implementation into a regulatory monitoring regime. We discuss proposed alternative methods, classified as functional, imm-

unological and analytical, for well-established European toxins as well as emerging toxins in European waters, highlighting their

advantages and disadvantages. We also consider emerging tools and technologies for future toxin analysis.

Even though regulatory bodies have recently recommended analytical methods for a number of toxins, there is still scope for

functional and immunological methods in rapid screening and detecting emerging toxins. Future developments foreseen in the

analysis of marine biotoxins are multiplex-based analysis, miniaturization and portability for on-site testing. However, the

longstanding lack of reference materials and standards continues to pose a severe limitation on progress in development,

validation and therefore implementation of any alternative method based on the criteria stipulated by European Union legislation.

ª 2010 Elsevier Ltd. All rights reserved.

Keywords: Analytical method; Antibody; Biosensor; Functional assay; Immunology; Marine toxin; Mouse bioassay; On-site testing; Rapid screening;

Receptor

Katrina Campbell*,

Christopher T. Elliott

Institute of Agri-Food and Land

Use, School of Biological

Sciences, Queen�s University

Belfast, BT9 5AG, UK

Natalia Vilarino,

Luis M. Botana

Departamento de

Farmacologıa, Facultad de

Veterinaria, USC, 27002 Lugo,

Spain

*Corresponding author.

Tel.: +44 (0)28 9097 6796;

E-mail: katrina.campbell@

qub.ac.uk

0165-9936/$ - see front matter ª 2010

1. Introduction

Marine biotoxins are naturally-occurringpoisonous substances synthesized bymicroscopic toxin-producing algae or theirassociated bacteria, though normally innon-harmful quantities. However, a com-bination of increased temperatures, sun-light and nutrient-rich waters is believedto cause rapid algal reproduction andthereby lead to potentially ‘‘harmful algalblooms’’. Worldwide, increasing occur-rences of toxic blooms are thought to belinked to climate change, increased oceaneutrophication and commercial shipping[1]. These toxins transfer through thetrophic chain into shellfish and fish. Mol-luscan shellfish are bivalve-filter feedersand ingest the algae, whereupon toxinsmay increase to levels that are potentiallylethal to humans or other consumers (e.g.,marine mammals and birds). Hence, asthis has major implications for publichealth, seafood destined for human con-sumption is routinely monitored by regu-latory bodies worldwide and is deemed fitfor consumption based on regulatory lim-

Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.10.01

its and methods established to preventacute poisoning [2–7]. The monitoring ofmarine toxins is vital to the aquacultureindustry, as these toxins may cause sub-stantial ecological damage and economiclosses through frequent or prolongedcontamination and closure of harvestingsites [8].

Marine biotoxins detected worldwide,but particularly in European waters, wereoriginally classified based on their acutesymptomatic effect in humans followingintoxification. The three main groupsmonitored in the European Union (EU)are:� Paralytic Shellfish Poisoning (PSP) tox-

ins;� Diarrheic Shellfish Poisoning (DSP) tox-

ins; and,� Amnesic Shellfish Poisoning (ASP).

However, as alternative detection meth-ods are considered, classification isbeginning to focus more on chemicalstructures and properties of the toxins. DSPtoxins have in recent times become knownas lipophilic toxins incorporating oka-daic acid, dinophysistoxins, azaspiracids,

0 239

mailto:katrina.campbell@�qub.ac.uk

mailto:katrina.campbell@�qub.ac.uk

http://dx.doi.org/10.1016/j.trac.2010.10.010

Trends Trends in Analytical Chemistry, Vol. 30, No. 2, 2011

pectenotoxins and yessotoxins with the last two notproved to cause diarrheic symptoms following intoxica-tion. For each of these three main toxin groups and sub-groups, the occurrence of the toxins, their chemicalcharacteristics, toxicokinetic evaluations, human-expo-sure assessments and detailed review of potential methodsof analysis have in recent years been published by theEuropean Food Safety Authority (EFSA) as scientificopinions [9–14]. The diversity of the numerous analoguesor natural enzymatic metabolites of marine biotoxins hasbeen described [15]. Fig. 1 highlights the structure of theparent or reference toxin within each group and an indi-cation of the number of relative analogues or naturalenzymatic metabolites. Table 1 lists the producers of thetoxin, mechanism of action and effects in humans inaddition to the current European Union (EU) referencemethods of analysis and regulatory limits in shellfish meatapplied in the monitoring regimes.

Currently, EU regulations stipulate that the referencemethods for the detection of marine biotoxins are twodistinct animal bioassays based on the hydrophilic [16]and lipophilic [17] solvents used for the extractionprocedure. The detection of domoic acid is an excep-tion where the reference method is high-performanceliquid chromatography with ultraviolet detection

(a) PSP toxins (> 30 analogues) [85]

N

N

R1

NH

HN

R3

+H2N

R4

R

N

R1 R2 R3 R4: OCONH2 R

H H H Saxitoxin (STX)

H H OSO3- Gonyautoxin (GTX) 2

H OSO3- H GTX 3

OH H H Neosaxitoxin (NEO)

OH H OSO3- GTX 1

OH OSO3- H GTX 4

Carbamate toxins

Figure 1. Chemical structure of the parent/reference toxin(s) for

240 http://www.elsevier.com/locate/trac

(HPLC-UV) [3,5]. HPLC with fluorimetric detection(HPLC-FLD) for saxitoxin and analogues [4] and anenzyme-linked immunosorbent assay (ELISA) for do-moic acid [5] are officially accepted as screeningmethods but the reference methods for these toxins arethe aforementioned.

However, this review also includes prospectiveemerging toxins to European waters [e.g., cyclic imines,palytoxin, tetrodotoxin, maitotoxin, ciguatoxins andneurotoxin-poisoning brevetoxins (Fig. 2)], as theiroccurrence could have severe implications with regardsto seafood safety [18]. In addition, as the shellfish tradeexpands globally with increased exports and imports toand from regions of the world where these toxins areprevalent, effective monitoring methods will need to bein place within the EU. Although EFSA has publishedscientific opinions for emerging toxins (Table 2) [19–22],with the exception of tetrodotoxin, these toxins are notspecified by the current EU regulations. At present, theirdetection is coincidental, as some co-extract with DSP orPSP toxins using the specified sample-preparation pro-tocols for the EU-approved animal bioassays. However,in many other regions of the world, animal bioassays arethe method of choice for monitoring these phycotoxinsin various seafoods. This review discusses the problems

NH2+

OH

2

OH

-Sulfocarbamoyl toxins

Decarbamoyl (dc) toxins

Deoxydecarbamoyl (do) toxins

4: OCONHSO3- R4: OH R4: H

B1 (GTX 5) dc-STX do-STX

C1 dc-GTX 2 do-GTX 2

C2 dc-GTX 3 do-GTX 3

B2 (GTX 6) dc-NEO

C3 dc-GTX 1

C4 dc-GTX 4

marine biotoxin groups regulated by the European Union.

(ii) Azaspiracid (~20 analogues) [88,89]

Azaspiracid-1

(iii) Pectenotoxin (~13 analogues) [90]

Pectenotoxin-2

(iv) Yessotoxin (~36 analogues) [91]

(b) DSP / Lipophilic toxins

(i) Okadaic acid and dinophysistoxins (DTXs) (>10 analogues and esters) [86,87]

O

O

CH3

CH3

O

O

O

CH2

OH

OR6 CH3

O

O

O

OR2

R3

H3C OHR1O

R4 R5

Toxin analogue R1 R2 R3 R4 R5 R6

Okadaic Acid H H CH3 H H H

DTX-1 H H CH3 CH3 H H

DTX-2 H H H H CH3 H

DTX-3

(Acylated forms of OA, DTX-1 and DTX-2)

H Fatty acid H / CH3 H / CH3 H / CH3 H

Fig 1. (continued)


associated with animal bioassays and the progress anddifficulties in finding alternative methods.

2. The problems with bioassays

The animal bioassays for marine biotoxins, discussed ina previous review [23], are widely considered to be

antiquated, cruel methods that involve injecting shellfishextract into a mouse or rat and observing if the animallives or dies over a defined time frame as well as theresulting symptoms. At least two animals, and oftenthree, are used per sample tested and are sacrificed at theend of the test, irrespective of the toxicity outcome. Themajor drawbacks of these assays are that there isgrowing recognition that they are both unethical, due to

http://www.elsevier.com/locate/trac 241

(c) ASP toxins – Domoic acid (~10 analogues) [92]

Fig 1. (continued)


the suffering and the sacrifice of laboratory animals, andare technically inadequate.

A conflict of interest between two EU Directives, 91/492 for public health control of marine biotoxinsimplementing the animal bioassays [24] and 86/609 onthe protection of laboratory animals [25], has in morerecent years increased the pressure within Europeancountries to move away from animal bioassays. From atechnical perspective, the methods are costly to performwith the animals requiring housing and restricted toweight conditions. Furthermore, they lack sensitivitywith limits of detection approaching the regulatorylimits, suffer from non-specificity (whereby toxins cannotbe identified or individually quantified) and are prone toinaccuracies in detection [26–28].

If the recent EFSA opinion on marine biotoxins is tobe implemented [29] and lower regulatory limits areestablished, these bioassays will not be fit for purpose.Conversely, these bioassay methods have been appliedfairly effectively for over 70 years for PSP toxins and30 years for DSP toxins in protecting the consumer,even though they lack adequate validation by today�sstandards. The aquaculture industry is generallyreluctant to change. More sensitive assays may trans-late to a greater number or longer unnecessary closuresof harvesting sites. However, there is increasing con-cern that the effects of toxin consumption at sub-reg-ulatory levels over more chronic, long-term exposurewere not taken into account when the risks were beingdefined. Irrespective, with increasing toxic occurrencesand new toxins presenting in established risk areas andwell-known toxins occurring in new locations, thesebioassays are becoming unsustainable, due in part tothe large numbers of mice required and the perfor-mance of the procedures.

There is a worldwide trend towards expansion of theshellfish industries, so the frequency of sampling andtesting is rising, exacerbated by the increasing numbersof short-term blooms, which increase the potential forlarge-scale shellfish poisoning episodes (e.g., those thatoccurred over the past decade, particularly for DSPtoxins) [30]. These episodes have an immense detri-mental effect on the industry.


3. Challenges facing alternative methods

In order to protect consumer health, the recent EFSAopinion on marine biotoxins [29] recommends, with theexception of yessotoxin, the implementation of substan-tially lower regulatory limits for toxin contents in sea-food destined for human consumption. For regulatoryauthorities to be in a position to comply with the EFSAopinion, although not a draft for legislation, access tosuitable alternative detection methods will therefore beessential.

These new methods must be reliable and have ade-quate sensitivity in order to detect the presence of thetoxins and analogues at not only the current regulatorylevels but also lower levels that are likely to result fromthe recommendations of EFSA and other regulatorybodies worldwide. Alternative methods with improvedsensitivity, compared to the mouse bioassay, have beendeveloped to replace the bioassays over the past30 years, but a series of drawbacks and limitations haveprevented their full implementation as referencemethods in routine-monitoring programs. In order toprotect the health of the consumer and the aquacultureindustry, regulatory authorities have establishedstringent performance criteria that must be realized byan alternative method before the mouse bioassay is dis-continued. The most challenging requirement is thatintra-laboratory and inter-laboratory validation must beperformed to internationally-recognized standards [3].The complexity of toxin analogues and their limitedavailability as standard material, particularly fromcompetitive commercial sources, not only restrictsmethod development but also proves validation to bepractically impossible to such accredited levels.

Standards for marine toxins are generally producedfollowing their extraction and purification from large-scale culturing of toxic algal or from contaminatedshellfish material harvested from regions following anoutbreak [31]. Specialist laboratories able to performsuch work are rare worldwide, as they are expensive toestablish and to maintain. Explicit, dedicated expertise inchemical separation and purification techniques is re-quired in addition to a regular supply of contaminated

Table 1. Predominant toxins covered by European Union legislation, including action and effect and regulatory methods employed

Toxin group Reference toxin (number

of analogues)

Algal species derived

from

Action and effect

in humans

Current EU

Regulatory limits

Regulation (EC) No.

853/2004 (lg/kg of

shellfish meat)

Current EU reference

monitoring method

Limit of detection

(LOD)/Limit of

quantification (LOQ)

[29]

Standardized method

Paralytic

shellfish

poisoning

toxins

[12]

Saxitoxin

(>30 analogues) [85]

Alexandrium species

Gymnodinium species

Pyrodinium species

Blockage of site 1 of

the voltage-gated

sodium channel

causing

cardiorespiratory

failure and death

800 STX Eq Mouse bioassay with 0.1

M HCL (15 min)

Regulation (EC) No 2074/

2005

LOD: 370 lg STX Eq/

kg

AOAC method 959.08

HPLC-FLD (Lawrence

method)

(For screening purposes)


2006

amending (EC) No 2074/

2005

LOQ: 10–80 lg STX

Eq/kg for individual

analogues

AOAC method 2005.06

Diarrheic

shellfish

poisoning

toxins

[9–11,13]

Okadaic acid and

dinophysistoxins (>10

analogues)[86,87]

Dinophysis species

Prorocentrum lima

Inhibit protein

phosphatases by

binding to PP1 and

PP2a receptor sites

causing diarrhea.

160 OA Eq Mouse Bioassay or Rat

Bioassay with acetone

extraction (24 h)

Regulation (EC) 2074/2005

Unknown for each

toxin. These bioassays

are incapable of

detecting these toxins

at their current

regulatory limit with

100% certainty.

For okadaic acid the

probability of

detection at the

regulatory limit is as

low as 40%.

No

Pectenotoxin-2

(�13 analogues) [90]

Dinophysis species In vitro disruption of

actin cytoskeleton and

diarrheic effects are in

dispute

160 OA Eq

Azaspiracid-1

(�20 analogues)

[88,89]

Azadinium spinosum Action is still

unknown but causes

diarrhea and

neurotoxic effects

160 AZA Eq

Yessotoxin


Protoceratium

reticulatum

Lingulodinium

polyedrum

Gonyaulax spinifera

Action not fully

known but interacts

with

phosphodiesterase

enzymes and diarrheic

effects are being

questioned

1000 YTX Eq

Amnesic

shellfish

poisoning

toxins [14]

Domoic acid


Pseudo-nitzschia

species

Chondria armata

Interacts with kainite

receptors causing

neurological damage,

memory loss and

death

20 000 DA Eq HPLC-based methods


2007


2005

LOD: 0.2–1 mg DA/kg

LOQ: 1–2.5 mg DA/kg

AOAC method 991.26

CEN method 14176

Antibody-based

methods(ELISA)

(For screening purposes)

Regulation (EC) No1244/

2007


2005

LOD: 0.003 mg DA/kg

LOQ: 0.01 mg DA/kg

AOAC method 2006.02

LC-FLD, Liquid chromatography-fluorescence detection; HPLC, High-performance liquid chromatography; Eq, Equivalents; ELISA, Enzyme-linked immunosorbent assay; CEN, European Committee for Standardization.

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material. Currently, the only source of certified referencestandards and material is the Institute for Marine Bio-sciences, National Research Council of Canada. Anumber of research groups have shown that the organicsynthesis of structurally-complex toxins is feasible [32–38]. Yet only small amounts of toxin have been syn-thesized using this approach, as it is time consuming andexpensive. The production of each toxin generally re-quires a team of dedicated, prominent, organic chemiststo perform many reactions, sometimes more than 20,and, at each reaction step, the product must be purifiedand characterized before proceeding. The large-scaleproduction and commercialization of marine-biotoxinstandards is difficult by any measure. A further compli-cation lies in the Chemical Weapons Convention, theinternational legislation pertaining to biological weapons,which is particularly pertinent to the PSP toxins [39], soonly those researchers with sufficient access to toxinscan meet the EU requirements for validation, and thus,to date, progress is slow in moving away completely fromthe mouse bioassay. A Europe-wide initiative to developbetter supply sources would be welcomed by both reg-ulatory authorities and researchers to advance methoddevelopment and validation.

The drive for the next generation of methods is focusedon the design of procedures that require low toxin con-sumption, are cost effective, rapid and sufficiently high-throughput to provide a more efficient turn-around timein the monitoring regime to inform the industry prior toharvesting and distributing shellfish into the market.Alternative tests are not only being designed for moni-toring laboratories but are being targeted at the industryto be used in end-product or field testing as risk-assess-ment tools for hazard analysis and critical control points(HACCP). This movement towards self regulation opensup other topics for debate, particularly on what is re-quired in relation to sample design and representation,quality control and quality assurance. The cost of suchprograms could be prohibitive, if based on large samplenumbers.

4. Current alternative methods

Published alternative methods for each toxin may beclassed into biological functional assays, biochemicalimmunological assays and chemical analytical methods,as outlined in Table 3. The EFSA opinions, available formost toxin groups, provide a concise review of the cur-rent, most promising alternative methods of analysisdeveloped for each toxin group listing their advantagesand disadvantages [9–14,19–22,29].

Monitoring programs typically cover a range of mar-ine toxins, as contamination incidents can often involveseveral toxins from within a group or different groupswith varying toxicities. The ideal method for seafood


analysis would be to detect all toxins based on their oraltoxicity in humans as the ultimate functional assay.However, for immunological or analytical methods,varying toxicities within a group pose problems foraccurate quantification of toxic potential. These alter-native methods utilize structural and chemical propertiesof molecules as opposed to their toxicity. The overalltoxicity of a sample for a particular group of toxins mustthen be calculated using toxicity equivalence factors(TEFs). TEFs compare the toxicity of different analoguesor toxins within a group to the toxicity of a parent orreference compound. However, application of TEFs re-quires discrimination or resolution of individual toxinanalogues by the detection method used.

4.1. Functional assaysReviews focusing on functional assays in marine-bio-toxin detection have been published in recent years[23,40]. Functional assays are traditionally defined asdetection methods that use the mechanism of action ofthe toxin for quantification, which, in turn, relates to thetoxicity of the compound. These assays are receptor orcell based and the most prominent assays for each toxinare reported in Table 3.

Okadaic acid and dinophysistoxins have been identi-fied as protein-phosphatase-inhibiting marine toxins.Enzyme-inhibition assays have been developed wherebythe inhibition is detected using radioisotopes, absorbanceor fluorescence (FL) [41]. This FL-based inhibition assayis claimed to be a useful rapid-screening method due toits high sensitivity, and data generated have been shownto be in good agreement with the mouse bioassay andchromatographic techniques. An electrochemical-basedassay has also been developed for this toxin-enzymeinteraction [42]. Phosphodiesterase enhancement hasbeen exploited for the detection of yessotoxins and ana-logues using surface-plasmon resonance (SPR) [43],resonant-mirror biosensors [44] or microplate assayswith FL detection [45].

The saxiphilin receptor isolated from the bull frog wasused to produce an assay for PSP toxins [46] and areceptor-based assay for domoic acid has been developedbased on the kainite receptor, but, as interference maybecaused by glutamate, this method requires a pre-treat-ment step with glutamate decarboxylase to improveselectivity and sensitivity [47]. An FL-polarization assaythat uses the nicotinic-acetylcholine receptor from Tor-pedo membranes has been shown to be capable ofdetecting the cyclic imines, gymnodimine and spirolides[48].

Receptors can display high affinities and sensitivitiesand provide a response proportional to total toxin tox-icity. However, the receptors may not be specific to aparticular toxin group, so toxin identification is neverunequivocally possible with a functional assay, onlydetection of toxic activity. The isolation and the

Figure 2. Chemical structure of the parent toxins for emerging marine biotoxins in European Union waters.


retention of the stability of receptors are also majorlimiting factors in the broad application of these assays.However, crude extracts or cell lines abundant inreceptors may be used but there are more complex bio-analytical systems that have been employed in two

different assay formats. The toxicity present in a sampleis either derived from end-point assays that use thesurvival rate of the cell or FL assays that are sensitive tochanges in membrane potential when toxin is present.Cell-morphology-based assays (e.g., those used for DSP,


Table 2. Potential emerging toxins in European waters, including action and effect in humans and current status of European Union monitoring

Toxin Derived from 1Bacterium2Coral 3Algae

Action and effect in humans Current status within EUmonitoring program

Tetrodotoxin[99] Shewanella alga species1

Pseudoalteromonastetradonis1

Pseudomonas species1

Vibrio species1

Blockage of site 1 of thevoltage-gated sodiumchannel causing cardio-respiratory failure and death

There are no EUregulatory limits set forthese particular toxinsToxins may present inmouse bioassay for PSPbased on theirhydrophilic solubility inextraction solventsLOD and LOQ for theseindividual toxins withthis assay are unknown

Palytoxin and ostreocins [19,93,94] Palythoa species2

Ostreopsis siamensis3In vitro binds to the sodiumpotassium ATPase.Causes vomiting, diarrhea,respiratory distress and death

Maitotoxin [100] Gambierdiscus toxicus3 Activates calcium-cationchannels causingneurological, gastrointestinaland cardiac symptoms anddeath

Cyclic imines[21,95]

Gymnodimine Gymnodinium species3

Karenia species3Interacts with nicotinicacetylcholine receptorsEffects in humans have notbeen reported.In mice, followingintraperitoneal injection,these toxins (gymnodimineand spirolides) cause similareffects to those caused byDSP toxins, thereby causinga positive response in theDSP assay

There are no EUregulatory limits set forthese particular toxinsToxins may present inmouse bioassay or ratbioassay for DSP toxinsbased on their lipophilicsolubility in extractionsolvents.LOD and LOQ for theseindividual toxins withthis assay are unknown

Spirolides Alexandrium ostenfeldii 3

PinnatoxinsPteriatoxinsProrocentrolides Prorocentrum species3

Spiroprorocentrimines Prorocentrum species3

Ciguatoxins(responsible for ciguatera fish poisoning)[20,98]

Gambierdiscus toxicus3 Activates the voltage-gatedsodium channels at site 5,causing neurological,gastrointestinal and cardiacsymptoms

For ciguatoxins,Regulation No. 853/2004 states fisheryproducts containingtoxins must not bemarketed but analyticalmethods are notspecified. Mousebioassay applied byBanner et al. [101] andvalidated by Lewis andSellin [102] is used.

Neurotoxic shellfish poisoning toxins:[22,96,97]Brevetoxin ABrevetoxin B

Karenia species3

Chattonella3Activates the voltage-gatedsodium channels at site 5,causing neurological,gastrointestinal and cardiacsymptoms

For brevetoxins, theAmerican Public Healthassociation method usesthe mouse bioassay withdiethyl-ether extractionwith the limit being anydetectable level/100 gshellfish [103].


azaspiracids and palytoxin) employ cell lines (e.g., neu-roblastoma cells, rat hepatocytes, human epidermoidcarcinoma cells or intestinal epithelial cell lines). Whenthese cells are exposed to DSP toxins, morphologicalchanges can be detected by microscopy, spectropho-tometry or FL measurements of the F-actin levels [41].

Neuroblastoma-based assays depend on the binding ofPSP toxins, brevetoxin and ciguatoxin to voltage-sensi-tive sodium channels in neuroblastoma nerve cells. Toenhance the specificity, ouabain and veratridine, which


inhibit the sodium release from the cell, and whichslightly open the sodium channels, respectively, areusually added to the culture prior to the experiment. Thetoxins bind to the receptors with an affinity proportionalto their toxic potency. Tetrodotoxin (TTX) and PSPtoxins block the sodium channels, promoting cell sur-vival, whereas brevetoxin and ciguatoxin open them,promoting cell death. The effect of the toxins on thesodium channels has been monitored by microscopy,spectrophotometry and FL [23]. A frog-bladder mem-

Table 3. Prospective alternative methods to the mouse bioassay for each toxin

PSP toxins Molecular weight ofreference toxin

Analytical method Functional bindingassays

Immunologicalmethods

Saxitoxin andanalogues[12,104]

372 HPLC-FLD post column oxidationAOAC approved [71]\\

Sodium-channelreceptor-binding assayIAEA Support\[49]

Radioimmunoassay[115]

HPLC-FLD pre column oxidation [72] Saxiphilin receptor-binding assay\[46]

Immunological (Jellet) [57]

HPLC-MS [105]HILIC-MS[106–108]

Neuroblastoma assay\

(Sodium-channelreceptor) [111,112]

ELISA\

[53,116,117]

Capillary electrophoresis [109] Electrophysiologicalassay [113]

SPR Biosensor[63]

Chemosensors [110] Fluorimetric assays [114] SPR coupled to HILIC [65](Biacore)

DSP toxins Molecular weight ofreference toxin



Okadaic acid andDTXs[9]

805 HPLC-FLD[118–120]HPLC-MS\\

[121–124]

Protein phosphatase-2a-assays with absorbance[125] or fluorescence[126]\

F-Actin cell-basedfluorimetric microplateassay [127]Cell morphology [128]

ELISA\

[53,117,129]SPR biosensor [62]Quartz crystalmicrobalance biosensor[130]Electrochemicalbiosensor [131,132]

Pectenotoxin-2 andanalogues[13]

859 HPLC-FLD/UVD[118–120]HPLC-MS\\

[133]

F-Actin cell-basedfluorimetric microplateassay[127]

ELISA [13]

Azaspiracid-1 andanalogues[10]

841 HPLC-MS\\

[121,124,133]Cell morphology [128]

Yessotoxin andanalogues [11]

1186 HPLC-FLD [134]HPLC-MS\\

[121,124,133]Capillary electrophoresis[135,136]

E-cadherinfragmentation [137]Phosphodiesteraseenhancement, using SPR[43] or resonant mirrorbiosensors [44,138] ormicroplate assay[45,139]

ELISA\

[53,140]

ASP toxins Molecular weight ofreference toxin



Domoic Acid[14] 311 HPLC-UVD\\

[141]HPLC-MS [121]Thin layer chromatography [142]

Kainic-acid receptor-binding assay\ [47]

ELISA\

[53,117][[143](Biosense)]SPR Biosensor [61]

Emerging toxins Molecular weightoftoxin


Immunological methods

Tetrodotoxin 319 HPLCHPLC-MS[144]

Frog-bladder assay(Sodium-channel receptor)[145]Neuroblastoma assay(Sodium-channel receptor)[111,112]

Screen-printedelectrodes [59]SPR [64]ELISA [146]

Palytoxin [19] 2677 HPLC-MS[147]

Cell-based assays[148–150]

ELISA[56]

Maitotoxin 3422 Neuroblastoma assay[151]

(continued on next page)



Table 3 (continued)

Emerging toxins Molecular weightoftoxin


Immunological methods

Cyclic Imines [21] 507–711 HPLC-MS \\[123,133] Fluorescent polarizationassay [152]

Ciguatoxin [20,98,153] 1110 HPLC-MS[154]

Fluorimetric assay[155] Neuroblastoma assay

(Sodium-channel receptor)[156]

Radioimmunoassay[157]ELISA[54]Lateral flowimmunoassay[158,159]

Brevetoxin-1Brevetoxin-2[22]

866894

HPLC-MS[160,161]

Sodium-channel receptor-binding assay\ [162]Fluorimetric assay[163]Neuroblastoma assay(Sodium-channel receptor)[164]

Radioimmunoassay[163]ELISA\

[164,165]

\Expert consultation by the FAO/IOC/WHO considered alternative methods.\\Expert consultation by the FAO/IOC/WHO considered and recommended alternative methods.


brane, rich in sodium channels, was used to cover asodium electrode and integrated into a flow cell to form atissue biosensor. The toxins could be detected by moni-toring the transport of sodium ions and the toxicity levelsof the PSP toxins correlated with the mouse bioassay.For TTX, concentrations one order of magnitude belowthe limit of detection of the bioassay could be detected[41]. A single-laboratory validation study has beenconducted for the microplate receptor-binding assay forPSP toxins in shellfish using titrated saxitoxin [49].Unfortunately, this radioligand is in extremely limitedsupply and very costly to produce. Although sodium-channel assays are more sensitive than the mouse bio-assay, they are not cost effective for routine screening.The source of the sodium channels (i.e. rat brains) is alsoa disadvantage from an animal-welfare perspective, but,in relation to this topic, at least the sodium-channelassay has shown that the numbers of animals requiredto be sacrificed for analytical purposes can be reduced.

A drawback to the functional assay is that, for all thetoxins, corresponding specific receptor targets have notyet been clearly identified and the stability of receptorsreduces their ability in being automated on sensor sys-tems (e.g., SPR).

4.2. Immunological (biochemical) methodsImmunology-based assays depend on the ability of aspecific antibody to distinguish the three-dimensionalstructure of a specific toxin from other molecules. Anti-bodies have the advantages of high specificity and sen-sitivity, the latter allowing for sample dilution thatreduces matrix effects from shellfish samples. However,an antibody is raised to a particular toxin in the groupand the specificity of the antibody is unlikely to corre-


spond to the inherent toxicity of individual toxins,depending on their structural differences. There is atrade-off in usefulness between highly-specific antibod-ies, able to detect a given toxin at low levels, and lessspecific antibodies, able to detect all the members of atoxic family. Hence, broad cross-reactivity profiles of anantibody may be an advantage or a limitation, depend-ing on the purpose of the test.

Immunological methods developed for marine biotox-ins fall into four main categories:(1) ELISA;(2) radioimmunoassay;(3) lateral flow tests; and,(4) immunosensors.

These immunological methods, particularly rapid orhigh-throughput tests, can be effective monitoring tools.The term ‘‘rapid test’’, used for promoting a method,usually refers to a procedure much faster than therespective reference methods. However, rapid methodsshould have a number of other common features (i.e. themethod should be simple, easy to use and capable oftesting marine biotoxins in the field as well as the labo-ratory). An alternative quantitative confirmatory test isgenerally required to reopen shellfish-growing areasclosed due to high toxin levels.

4.2.1. ELISA. ELISAs for marine-biotoxin analysis havebeen available for more than 25 years. Both direct andindirect competitive inhibition formats have beeninvestigated with assays developed for DSP, PSP, ASP,NSP, ciguatoxins, TTX and palytoxin [50–56], some ofwhich are available commercially. The integration offour assays for DSP, PSP, ASP and NSP toxins into asingle screening format has been demonstrated [53].


Their limited success as commercial products has beenmost frequently linked to lack of toxins for standards,inadequate specificity to all the major toxins within aparticular group and a disproportionate estimation oftotal toxicity due to cross reactivity. However, with theuse of highly-specific antibodies, these methods have theability to be fully quantitative or semi-quantitative forindividual toxins. ELISAs are generally regarded as highthroughput with low sample-volume requirements andoften fewer sample extract clean-up procedures com-pared to analytical methods (e.g., HPLC or LC-MS),making them simple and portable for the detection ofmarine biotoxins in the field.

4.2.2. Radioimmunoassays. Radioimmunoassays havebeen developed for saxitoxin, okadaic acid, brevetoxins,and ciguatoxins, but are now considered defunct, due tohazards associated with use and disposal of radioactivity.

4.2.3. Lateral flow tests. The lateral flow test or strip testhas been considered a feasible screening option forphycotoxins over the past decade, although one com-pany (Jellet Rapid Testing (JRT) Limited) has monopo-lized the market [57]. The JRT assay for PSP toxins wasaccepted as a regulatory screening method in the USAfor making precautionary closures of shellfish-harvestingsites upon contamination. The benefits of lateral flowtests are that they are user-friendly, very rapid, havelong-term stability under various storage conditions, andare suitable for testing for marine biotoxins in the field asan HACCP tool. However, at best, the technology canprovide only semi-quantitative results; for any positivesamples, and the exact marine-biotoxin concentrationmust be elucidated by a reference method. It is likely thatother commercial lateral flow devices will enter themarket as the amount of testing increases to providemarket opportunities.

4.2.4. Immunosensors. An immunosensor is a devicethat uses specific biochemical reactions mediated byantibodies to detect chemical compounds, usually byelectrical, thermal or optical signals. Substantial researchin using immunosensors for marine-biotoxin analysis hasbeen conducted with systems {e.g., quartz crystalmicrobalance, electrochemical and SPR biosensors, withthe last of these appearing extremely promising[41,58,59]}. A review on bioaffinity-detection systemssummarizes the principles of these methodologies andother upcoming surface-based technologies [60].

SPR is widely recognized as being the leading tech-nology for label-free toxin detection, and methods havenow been developed and undergone single-laboratoryvalidation for domoic acid, okadaic acid, DTXs and PSPtoxins, with results for naturally-contaminated shellfishsamples in good agreement with the current regulatorymethods for these toxins [61–63]. An SPR method for

TTX has also been developed but not yet validated [64].An advantage of SPR is that it can be linked to MS, sothat samples screened by SPR and found to contain toxincan be identified and quantified using MS [65].

Currently, the mouse bioassay for DSP toxins underEU regulations can detect four structurally-differenttoxin groups as a single test. Recent concerns raised bymonitoring laboratories are that to perform this taskwith immunological assays will require four separatetests rather than one that would result in more time-consuming, costly analysis. Research programs aretherefore now focusing on multiplex formats forachieving a single test for marine biotoxins.

As part of the EU 7th Framework Program Conffidence(www.Conffidence.eu), research is pursuing develop-ment of a multiplex SPR method for the simultaneousanalysis of four key toxins: saxitoxin, neosaxitoxin,okadaic acid and domoic acid. This prototype system hasthe potential to analyze up to a total of 16 toxins on amicroarray-sensor chip.

Another particularly exciting aspect to immunosen-sors is their budding potential for miniaturization andportability, which make them interesting prospects foron-site monitoring. Electrochemical sensors were ini-tially shown to be more suitable for this application, buta portable SPR sensor has been developed for detectingdomoic acid [66]. These immunology-based systemsshould be thought of as bioanalytical tools for the pre-liminary screening of toxins in a sample. If the sample isconsidered suspicious, complementary analytical tech-niques should be applied in tandem to provide anaccurate toxin determination and quantification.

4.2.5. Immunoaffinity column clean up. Immunologicalsample-preparation methods can be used as options fortoxin extraction and concentration from complicatedmatrices [67]. Immunoaffinity columns have been as-sessed for sample clean up in marine-biotoxin analysis[68–70]. This technique for toxin capture displays thesame advantages and disadvantages as an immunology-based detection method.

4.3. Analytical methodsOptions for analytical methods for marine-toxin detec-tion are dominated by LC, with FL, UV or MS detection.LC has proved a valuable tool for toxin detection, but,when the compound of interest does not contain achromophore for FL or UV detection, the toxins must firstbe derivatized pre-column or post-column, as in the caseof PSP toxins [71,72]. However, it is well establishedthat methods (e.g., LC-FL) can be subject to artifacts(e.g., reported imposter peaks caused by compounds thatbehave in a chromatographically identical manner to atoxin, including its FL properties) [73].

Coupled LC tandem MS methods (LC-MS/MS) are re-ported to be a powerful combination for the detection


http://www.Conffidence.eu


and quantification of toxins in both plankton and shell-fish at trace levels, the chemical characterization of newtoxins, and the traceability of toxin production byplankton and the investigation of toxin metabolism inshellfish [74,75].

LC-MS methods have been constructed for all knowntoxin groups and the emphasis is now on developingsimple single-sample preparations for all toxins. How-ever, these methods are relatively expensive and, to date,their routine application, in particular sample prepara-tion, is generally laborious, often with multiple extrac-tion steps, boiling or freezing and/or solid-phaseextraction clean up being required for samples. Theanalysis and the interpretation of the results are difficultfor determination and quantification, requiring trained,experienced personnel. The use of TEFs based on mouse-toxicity data for each toxin with the same mechanism ofaction increases the difficulty in quantification, espe-cially when standards are unavailable. A choice ofsuitable marker compounds can ease the analyticalburden, but positive samples will generally require moredetailed follow-up analyses. Nevertheless, LC-MS meth-ods are unequivocal in their confirmatory applicationdue to the identification based on the unique fragmen-tation pattern of a given toxin. Of the methods that theexpert consultation of FAO/IOC/WHO considered as op-tions (Table 3), LC-UV, LC-FL, and LC-MS were recom-mended for domoic acid, saxitoxin and lipophilic toxinsrespectively [6,30,75].

Even though the FAO/IOC/WHO has recommended theanalytical methods as options for certain toxins, thisleaves potential gaps in the analysis of emerging toxins inEurope and elsewhere. For example, recently the first caseof tetrodotoxin poisoning was observed in Spain [76], andshellfish samples tested negative using both the Lawrence[71] and Oshima [72] HPLC methods for PSP toxins.

If the mouse bioassay is removed completely, then it isimperative that all emerging toxins identified withinEuropean waters become part of the EU legislation interms of recommended methods of analysis and estab-lished regulatory limits. However, it is difficult orimpossible to predict which toxin(s) will emerge asproblems or threats to human health in a given region,thereby allowing legislation to be established; assaysbased on detecting toxic activity without the need forstructural information are thus still valuable in this re-gard. Analytical methods such as LC-based techniqueswill allow identification and/or detection of only thosetoxins for which standards are available.

5. Emerging tools and technologies

In addition to the multiplexing immunosensors, otheremerging tools and technologies have potential forapplications in marine-toxin analysis. Research on the


synthesis of cloned receptors would prove beneficial inboth functional assays and as more stable alternativebinders to antibodies for sensor technologies. For otherbinding molecules, there is the progression of recombinantantibody technology, which was recently illustrated witha palytoxin-detection assay [77], and the potentialdevelopment of generic aptamers for toxin groups.

Research on chemosensors based on photoinducedelectron transfer and designed as FL-recognitionmolecules for the toxins have shown some potential forsaxitoxin determination [41]. Advances in the use ofmolecularly-imprinted polymers as synthetic receptorsfor solid-phase extraction, and chromatographic matri-ces for separations and biosensor devices may have fu-ture roles [41,78]. Other antibody-based detectiondevices, in particular flow cytometry [79], {e.g., luminexxMAP technology [80–82]} are showing promise interms of versatility and reproducibility in other fields andmay have potential for high-speed, high-throughput,multi-toxin detection. As complementary tools for toxindetection in seafood, microarray technologies [83,84],which may be employed for detecting toxic algal species,are also evolving as early-warning tools for predictingtoxic outbreaks.

6. Conclusions

The FAO/IOC/WHO has recently recommended solelyanalytical methods as alternative reference methods tothe mouse bioassay. However, there is immense scope forfunctional and immunological assays when monitoringfor marine toxins and in particular emerging toxins. Themajor application for such assays will be on-site, rapid,end-product testing. Successful multiplex immunosen-sors have the potential to provide high-throughput toxinanalysis for multiple-toxin groups. However, with theimminent removal of the mouse bioassay, resources foralternative method implementation are still required, inaddition to substantial changes to the legislation, tocover all toxins current and emerging not only for re-gional testing but for import and export of seafood pro-duce.

Future developments foreseen in the analysis ofmarine biotoxins are multiplex-based analysis, minia-turization and portability for on-site testing. The lack ofreference materials and standards that has prevailedover the years has been a severe limitation to progressand this factor may slow advancement still.

AcknowledgementsThis work was funded by the European Commission aspart of the 6th Framework Program Integrated ProjectBioCop (Contract FOOD-CT-2004-06988) and the 7thFramework Program Conffidence (Contract 211326).


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A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis

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