This thesis has been approved by

The Honors Tutorial College and the Department of Chemistry and Biochemistry

______

Dr. Hao Chen Assistant Professor, Chemistry and Biochemistry Thesis Advisor

______

Dr. Lauren McMills Honors Tutorial College Director of Studies, Chemistry

______

Jeremy Webster Dean, Honors Tutorial College

REGULATION AND TESTING FOR MARINE BIOTOXINS

______

A Thesis

Presented to

The Honors Tutorial College

Ohio University

______

In Partial Fulfillment

of the Requirements for Graduation

from the Honors Tutorial College

with the degree of

Bachelor of Science in Chemistry

______

by

Molly C. Semones

August 2010 1

Regulation and Testing for Marine Biotoxins

Molly C. Semones

Department of Chemistry and Biochemistry, Honors Tutorial College, Ohio

University, Athens, Ohio; E-mail: [email protected]

Abstract: The human health threat from marine algal toxins is increasing with expanding human population, increased seaside populations, and the concomitant increase in aquaculture operations and demand for seafood. As humans increase demand for seafood, they create waste and activity that may increase the likelihood of harmful algal blooms (HABs) and phycotoxin production by some of these blooms, notably the production of the causative toxins for ciguatera fish poisoning (CFP), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP) and diarrhretic shellfish poisoning (DSP). The mainstay for regulatory detection of these toxins has long been the mouse bioassay (MBA), with intraperitoneal (i.p.) injection of suspect extracts and subsequent monitoring for symptoms and time of death. The general sentiment in the research community is that there is a need to eliminate, or at least reduce, the use of the mouse bioassay in testing for algal toxins, due to technical limitations of the procedure and its ethical drawbacks.

A number of functional and analytical methods have been developed to this end. This paper reviews the rise of harmful algal blooms, toxin syndromes, historical use of and 2

the subsequent need to find an alternative to the mouse bioassay in detection of algal toxins and obstacles to the development of these alternative methods. The role of large importing countries in this process is then considered; particular attention is paid to the

United States, as there is little discussion of their efforts in the literature.

Keywords: 3Rs; algal toxins; alternative methods; biotoxins; economics; harmful algal blooms; HABs; marine biotoxins; mouse bioassay; MBA; regulation; seafood; shellfish; three Rs; trade

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Table of Contents

1. Introduction 4-14

2. The Mouse Bioassay 14-21

3. The 3Rs in Marine Algal Biotoxin Testing 21-24

4. Applying the 3Rs of Humane Experimental Technique 25-29

5. The National Shellfish Sanitation Program (NSSP), Interstate Shellfish

Sanitation Conference (ISSC) and United States Food and Drug

Administration (FDA) 30-35

6. Alternative Methods 35-37

6.1 Chromatography 37-42

6.2 Immunoassays 43-45

6.3 Receptor Binding Assays 46-47

6.4 Cell Culture Assays 47-48

6.5 Enzyme Inhibition 48-49

6.6 Algae Monitoring Programs 49-50

7. Hurdles to the Implementation of Alternative Techniques 50-61

8. Interplay between Trade and International Adoption of Alternative

Methods 61-70

9. Concluding Remarks 70-71

Acknowledgements 72

References 73-94 4

1. Introduction

Of over 5,000 species of phytoplankton known to date, 300 of these are capable of rapid proliferation and production of harmful algal blooms (HABs) [1, 2].

These blooms, although in some cases beneficial to ecosystem function, can damage marine ecosystems and impact both living marine resources and recreational use of coastal areas, causing considerable damage to human health [3, 4] and economics [5-

7] in the process. Of these 300 species, some 40 produce potent toxins [8], known as phycotoxins, algal toxins, marine biotoxins or simply biotoxins. These algae- produced marine biotoxins transfer through the marine food web and accumulate in seafood commonly consumed by humans. Human consumption of this contaminated seafood—particularly shellfish, which filter-feed on phytoplankton and concentrate the toxins in their flesh—results in illness ranging from nausea and diarrhea to distressing neurological effects and, in extreme intoxications, death [9].

A number of toxins are responsible for these illnesses, and new toxins continue to emerge following persistent investigation. The more recognized of these human health syndromes include neurotoxic shellfish poisoning (NSP, caused by brevetoxins), paralytic shellfish poisoning (PSP, caused by saxitoxins), amnesic shellfish poisoning (ASP, caused by domoic acid and its analogues), diarhettic shellfish poisoning (DSP, caused by okadaic acid and dinophysis toxins), azaspiracid poisoning (AZP, caused by azaspiracids) and ciguatera fish poisoning (CFP, caused by ciguatoxins). The toxins responsible for these intoxications vary in structure, 5

molecular target and lethal dose, as well as the algal species responsible for their production (Figure 1; Tables 1 and 2) [10]. As natural products, they exist as a number of analogues and metabolites of varying potencies, which complicates their detection in programs designed to monitor for marine biotoxins in seafood products

[11]. Transfer of these biotoxins through the marine food web, and subsequent accumulation in fish, shellfish, crustaceans and other seafood products [12-16], results in human exposure through consumption of seafood, illness and, for ASP, PSP and

CFP, the occasional fatality [17-19]. The toxins are odorless and tasteless, typically have no visible affect on contaminated organisms (although they may exhibit sublethal and chronic effects at the population level [20]) and, unlike viral and bacterial pathogens, are relatively unaffected by cooking or industrial processing. In some cases, these processes may actually increase toxin content [21]. While consumption is the predominant mode of exposure, exposure and illness may also occur through drinking or contact with contaminated water as well as, in the case of brevetoxins, inhalation of aerosols containing phytoplankton and toxins broken up by surf action. 6

Figure 1. Representative molecular structures of the six main algal toxins; many analogues and metabolites similar to the structures depicted here exist in situ. Reprinted from [10] with permission from Elsevier.

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Table 1. Marine biotoxins, biological target, method of action, human symptoms of intoxication and animal toxicity. Reprinted from [22] (Table 2, page 1676) with permission from Springer Science+Business Media.

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Table 2. Marine biotoxins, producing species, distribution, common regulatory limits and methods used for biotoxin detection. Reprinted from [22] (Table 1, page 1674) with permission from Springer Science+Business Media. References in table refer to citations in original publication.

9

As consumers become more aware of the health benefits associated with seafood diets high in polyunsaturated fatty acids (PUFAs), demand for seafood is increasing. Growing exports indicate an increase in consumption of seafood not only in the United States of America and the European Union, but also Asian countries such as Japan [23]. Fish, including shellfish, provide 2.9 billion people with at least

15 percent of their average per capita animal protein intake [23]. In areas that import seafood, this increase in consumption is often unaccompanied by a concomitant awareness of the risks associated with consumption of seafood products, including the possibility of marine biotoxin contamination [24-26].

Improved processing, handling, packing and transport of seafood has facilitated international trade [23] and has expanded the threat of marine biotoxin illnesses beyond toxic HAB-prone areas to locations where illnesses caused by these toxins are unfamiliar to both health-conscious consumers [27] and the medical community [28, 29]. Diagnosis of algal biotoxin poisonings is based on self-reported symptoms and a history of consumption of fish or shellfish, and mild symptoms often mimic the generic symptoms of food poisoning, cold or flu. More chronic effects may be diagnosed as psychiatric disorders or chronic fatigue syndrome [29]. Misdiagnosis is thus common and even in endemic areas medical practitioners are frequently unaware of the possibility of marine algal toxins as instigators of illness. In the United

States the Centers for Disease Control and Prevention (CDC) has developed the

Harmful Algal Bloom-Related Illness Surveillance System (HABISS) to track and gain a better understanding of the incidence and distribution of marine biotoxin 10

intoxications within the US [30] (http://www.cdc.gov/hab/surveillance.htm accessed 7

July 2010), of which the CDC estimates that only 2-10 percent of cases are reported

[31].

HABs are expanding in intensity, duration and geographic range [15, 32-35] concurrent with the expansion in seafood production. While the reason for the expansion remains in debate and varies from toxic species to toxic species, progress towards a warmer climate and increasing oceanic carbon dioxide concentrations are expected to favor a number of toxin-producing HAB species over other less detrimental algal species [36]. Current marine biotoxin levels already place substantial economic, sociocultural and health burdens on the global community [35,

37] and, with the expansion of both toxin-producing phytoplankton and seafood demand, the problem will only become more pressing. This makes the accurate and rapid detection of marine biotoxin expressing species and their toxins in seafood increasingly urgent.

Limits have been set for these toxins, and these vary from country to country along with the accepted method used to detect and quantify the toxins in shellfish.

Regulators in many countries stipulate both maximal allowable levels of the toxins in domestic, exported and imported seafood products as well as the appropriate and legally acceptable methods to test for the toxins in seafood products [38]. In the

United States the National Shellfish Sanitation Program (NSSP), administered by the

Interstate Shellfish Sanitation Conference (ISSC) and United States Food and Drug

Administration (FDA), regulates shellfish products to assure consumer safety and 11

approves techniques used to monitor shellfish harvesting waters. The current gold standard method, endorsed by the FDA and ISSC for use in the NSSP for NSP and

PSP toxins, and most commonly used by oversight authorities worldwide, is the mouse bioassay (MBA) [22]. This assay involves intraperitoneal (i.p.) injection of suspected toxic samples, after an extraction step and sans painkillers or sedatives, into live mice, wherein time of death (TOD) and symptoms correlate to the toxic content of the injected sample. When limits are exceeded, regulators close affected shellfish beds to harvesting.

Initially developed for PSP toxins in the 1940s [39], the mouse bioassay has since been adapted to all of the marine biotoxins except ASP. It is perhaps the most straightforward method for detecting algal toxins, requires little specialized equipment, and has, in the past, served as a potent protector of human health in the absence of more sensitive and quantitative techniques. While very few outbreaks have been reported from commercial shellfish resources since the institution of the current regulatory limits and testing with the mouse bioassay (most reports of poisoning originate from recreational or subsistence use or the emergence of new toxins [40, 41]) there are both ethical and technical concerns in the use of the mouse bioassay in testing procedures, as well as concerns that some of these limits may be inadequate for prevention of suspected chronic effects of the toxins. In addition, the mouse bioassay is incapable of determining the exact toxin profile or class involved in a poisoning event on a molecular level. For these reasons, scientists, regulators and the seafood industry have expressed a strong desire to replace the test as a regulatory tool as soon 12

as possible, although the caveat that any replacement should be capable of protection of human health equal to that provided by the mouse bioassay nearly always accompanies this desire when it is expressed [9, 42, 43].

Research needs and the morbidity and mortality associated with consumption of contaminated seafood have driven the development of a number of technologies suitable for the reduction in use, phasing out and eventual replacement of the mouse bioassay, a concept commonly known as the “3R’s” (refinement, reduction, replacement) in animal experimentation [44]. To reach this goal, considerable effort has been and continues to be poured into the development of in vivo and in vitro alternative methods, including chromatography, immunochemistry, and functional techniques. Many of these methods are capable of detection at or below the level of the mouse bioassay, with definitive identification of the toxin classes involved.

Several of the methods have already been validated and adopted for use in regulatory schemes [45-47], and it is hoped in the future that use of the mouse bioassay will be greatly reduced and possibly eliminated through implementation of these alternative methods.

International adoption of these techniques as regulatory tools, however, is influenced by a number of factors, including the biotoxin regulatory policies of large importers of seafood goods that outline legally acceptable methods used to detect and quantify marine biotoxins in imported products. The United States is one such country, the second largest importer of seafood (13,271 US million) behind Japan

(13,971 US million) [23]. As such, it has an influence over the international 13

acceptance of these methods through its policies, in particular those of the NSSP with respect to shellfish. Thus the replacement of the mouse bioassay is tied up with economic and trade concerns as well as the technical and ethical inadequacies of the assay and scientific validity of alternative techniques. Countries wishing to eliminate the mouse bioassay must take into consideration international treaties as well as the

World Trade Organization (WTO)’s policies and the Sanitary and Phytosanitary (SPS)

Agreement, which prohibits use of unnecessarily high health standards as disguised trade barriers.

The regulatory situation with respect to marine biotoxins and efforts to replace the mouse bioassay in Europe [48], another large seafood importer, is extensively covered in the literature, but little information is readily available on similar progress in the United States. This work seeks to gain a better understanding of the system in place for marine biotoxin regulation of seafood in the United States and the United

States’ impact, as a major importer of shellfish products, on the alacrity with which alternatives to the mouse bioassay are adopted internationally. To accomplish this, the advantages and disadvantages of the mouse bioassay and progress towards the 3Rs in marine biotoxin regulatory testing are reviewed, followed by the United States’ policy on laboratory techniques suitable for the detection and quantification of marine biotoxins, as facilitated by the NSSP. Advanced techniques for the replacement of the mouse bioassay are then reviewed, along with several of the major hurdles to their adoption. How this fits in with global food standard harmonization and the possible 14

implications of the United States’ NSSP policies on worldwide marine biotoxin policies is then explored.

2. The Mouse Bioassay

In the majority of countries that regulate marine biotoxins, the mouse bioassay is the official method of detection and determination of toxin content in shellfish products [9, 22]. It was introduced in 1937 [49] for the detection of PSP toxins and has since been adapted to all of the major toxin classes [41, 50-55] with the exception of ASP toxins, for which a high-performance liquid chromatography-ultraviolet detection (HPLC-UVD) method is commonly utilized [56]. The mouse bioassay entails injection of toxic extract from suspected seafood, typically shellfish, into 2-3 mice per sample and monitoring of the mice for time of death (TOD). TOD is then utilized in conjunction with a reference dose response curve to determine sample toxin content. Notably, the bioassay for PSP toxins is the only mouse bioassay to have undergone AOAC validation [57] and the weight of regulatory approval for the rest of the assays rests on their past efficiency in protecting human health in the absence of a more sensitive and specific method of detection. As a functional method, the mouse bioassay accounts for systemic toxicity in a mammalian model and toxin interaction when samples contain multiple toxins. It requires little specialized equipment or personnel to perform and, while the method cannot discriminate between toxins, it gives overall toxicity data, which is essential for the protection of human health [58]. 15

The design of the mouse bioassay and regulatory limits for each of the toxins, however, has been based mainly on incomplete and scant toxicological data obtained from sporadic acute human poisoning events [9]. For some of the toxins, the ideal regulatory limit developed from these events has been adjusted to accommodate the detection limit of the mouse bioassay, as in for the AZP toxins [59] and PSP toxins

[60]. Recently, both the Joint Food and Agricultural Organization of the United

Nations (FAO)/Intergovernmental Oceanographic Commission (IOC)/World Health

Organization (WHO) ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs

[9] and the European Food Safety Authority [42] have expressed the opinion that the common limits for DSP, AZP, PSP and ASP toxins are non-protective for either sensitive or high volume consumers. The Joint Committee determined that there was insufficient data to complete a risk assessment for NSP toxins [9]. Where there is little information on marine biotoxin exposure levels that produce acute symptoms in humans, even less information exists for the development of chronic exposure limits.

Without the necessary toxicological data to complete risk assessments, no tolerable daily intake (TDI) levels for chronic exposure have been developed for any of the marine biotoxins [9]. This is of increasing concern, as a number of the toxins have been linked with possible epidemiological chronic effects in human and animal populations [35, 61-67].

The mouse bioassay for all toxin classes is at the edge of its limit of detection for the current regulatory limits in place for all of the main marine biotoxin classes. If regulatory limits were to be revised to take into account these new recommendations 16

for acute limits and possible future recommendations for chronic TDI, the assay would need to be replaced with a new, more sensitive method of detection and quantification of toxin content; in the case of ASP toxins, where the mouse bioassay has never been considered sensitive enough to protect human health, this has been accomplished with an HPLC-UVD methodology [56] and is successfully integrated into monitoring programs. Additionally, freshwater cyanobacteria are known to produce PSP toxins, and the current established limit for these toxins in drinking water is well below the detection limit of the mouse bioassay [68].

Outside of concerns about the relevancy of the mouse bioassay for toxin limits realistically protective of human health, the assay has long been known for significant variation in results due to mouse gender, strain and weight [69-74] as well as inter- and intralaboratory variation [75]. This makes it necessary for labs to periodically calibrate the assay and determine a conversion factor to use with dose-TOD curves to determine toxicity, involving the use of more animals. Additionally, there is individual variation in mice even within the same strain. In particular, a notable variation in response to toxins in lower weight mice at lower doses of toxin has been observed

[74]. This means that only mice of a certain weight range may be used for the assay, which can be difficult to accommodate.

The mouse bioassay uses a large number of animals with death as the endpoint of the assay, and puts the animals through great pain, distress and suffering before their death. The most severe variation conducted for PSP toxins ideally takes 5-7 minutes, while in the DSP and AZP toxin mouse bioassays the animal may take from 17

5-24 hours to die, depending on the particular toxins surveyed and method chosen.

Beyond the large number of animals as an animal welfare issue, these animals must be raised, cared for, transported and disposed of—one lab in a study conducted by Guy and Griffin (2009) [76] estimated the annual cost of mice for running 4,500 mouse bioassays at $70,000.

Inter- and intralaboratory variation may be due to the handling and treatment of the mice [44], as well as the particular procedure used within the lab. While generally accepted procedures do exist, there is often variation in how the procedures are applied between labs and even within labs from technician to technician [77].

Initially, the mouse bioassay was believed to be ineffective for monitoring AZP toxins

[40], as it was poorly reproducible and the mice appeared less sensitive to the toxin than humans. However, Hess et al (2009) [52] developed a harmonized mouse bioassay procedure that gives good reproducibility and performs comparably to existing mouse bioassays for other toxin classes through eliminating the variability caused by variations in the procedure.

Despite its drawbacks, the mouse bioassay is touted by some for its ability to account for systemic toxicity and screen for new and emerging toxins. This is an example of the high fidelity argument addressed by Russell and Burch [44], under the premise that mice are biologically similar to humans and will thus react to the toxins in a similar manner. While there is a certain amount of truth to this, it has been pointed out that the mouse bioassay has yet to prevent an outbreak due to new toxin classes [76]. This was the case with both ASP toxins in Canadian waters in 1987 [41] 18

and AZP toxins in Irish waters in 1995 [40]. In both incidents, the mouse bioassay was in use monitoring for known toxins but did not catch the appearance of these new toxins, which was essentially left up to a “human bioassay” [76]. In the case of ASP toxins this was due to the lesser sensitivity of mice to the ASP toxins. For AZP toxins it was a consequence of the sample extraction used to prepare the sample for the mouse bioassay. DSP toxins are known to accumulate mainly in shellfish hepatopancreas; AZP toxins distribute throughout whole body shellfish tissue. As a result, since only hepatopancreas was used in extractions, toxicity of AZP toxins in the whole body tissue was not accounted for and led to human intoxications. If the toxin is not extracted in procedures used for routine monitoring in the area, as in the case of

AZP toxins, the mouse bioassay is incapable of acting as a screen for the new toxin. If it is extracted, the mouse model must then be sufficiently sensitive to detect the toxin.

While no new toxin has been detected to date, this conclusion may be challenged soon as currently, in France, a compound is interfering with the mouse bioassay and causing significant closures of oyster beds, with concomitant economic hardships for oyster farmers. If this compound turns out to be toxic to humans, it would be the first successful catch of an emerging toxin by the mouse bioassay. However, there is anecdotal evidence that suggests that the mussels are, indeed, safe to eat [78].

While failing to extract new toxic compounds is of concern, the mouse bioassay has also been known to respond to artifacts of the extraction procedure and compounds that are non-toxic to humans at levels present in shellfish tissue, resulting in false positives. While excessive safety (i.e., closure under these circumstances) 19

results in utmost protection of human health, it imposes economic hardship on growers where no true threat exists. Combes (2003) [79] describes what appears to be a case of procedural artifacts with the DSP mouse bioassay, and Hess (2010) [78] details the closure of shellfish beds in France that may be due to unknown interfering compounds that are non-toxic to humans. Yessotoxins (YTXs), lipophilic marine biotoxins previously considered under the DSP toxin heading but since separated due to their apparent lack of oral toxicity in humans, are 10 times more potent when administered i.p. rather than orally; as a result, where YTXs are present, they cause significant false positives in the mouse bioassay where the general lipophilic mouse bioassay is used

[69, 80, 81]. The fidelity of the mouse bioassay is thus questionable, as human exposure occurs through ingestion and mouse exposure via injection [48, 76]. This method results in the greatest systemic distribution of the toxin, bypasses the important absorption stage and does not account for toxin uptake from the gut—if the toxin can’t reach its target, it is unable to exert its toxicity. Taking this into consideration, the mouse bioassay is less a high fidelity screen for toxins and more a test correlated to toxicity in humans over the years of its implementation.

High zinc [82] and free fatty acid (FFA) [70, 83, 84] levels have been known to cause positive results in the mouse bioassay in the absence of significant toxin content and at levels of zinc and FFA that are non-toxic to humans. In contrast, high salt concentration decreases the apparent toxicity of the mouse bioassay [85], which, to the detriment of human health, leads to false negatives. The solution pH has also been shown to impact assay results [72]. 20

A final issue with the mouse bioassay lies with its strength; the assay gives an overall toxicity value for samples, but is incapable of discerning the exact toxins eliciting the response. Where human and mouse toxicity vary, and there are a number of different toxin classes present that have different regulatory limits, this presents a problem. An excellent example is the mixture of lipophilic toxins, including DSP and

AZA toxins, in European waters. If the presence of a particular toxin that is highly toxic to mice when injected i.p. but marginally or non-toxic to humans when ingested is known (for example, pectenotoxins or YTXs), an alternative extraction can be performed. However, the technician must know or suspect the toxin’s presence, and more animals are required for each sample in order to separately quantify the levels of these toxins. Potentiation may also factor into the toxicity measurement [18], and multiple toxins may influence each other’s actions in the assay. Where human and mouse sensitivity fail to correlate, this is problematic.

The burden of the inaccuracies of the mouse bioassay falls heavily on the producing sector, as, in addition to the factors that cause false negatives and positives, the mouse bioassay has been shown to be ineffective in following the sudden toxification of shellfish and their slow depuration, making it unsuitable for use by producers for planning harvests, and requiring them to use alternative and often expensive end-product testing methods [78]. Exacerbating this, regulatory programs often fail to make their data available in a timely manner. This results in wasted effort harvesting toxic shellfish where the producer does not implement their own testing 21

methods for toxin limits, even in the absence of the inadequacies of the mouse bioassay [78].

As a result, while the mouse bioassay may still have a place in fundamental marine biotoxin research, the seafood industry, scientists and regulators consider the mouse bioassay in need of replacement as soon as possible. It is no longer appropriate in the light of new information on possible subclinical, acute and chronic effects, and, for some, never has been due to animal welfare issues and a lack of validation and sound experimental design [79]. New, more sensitive analytical and biological detection methods, which present possible alternatives to the mouse bioassay, have been developed with the capacity to protect human health more effectively than the long-used mouse bioassay. These are likely to be implemented in the near future once validated and accepted by regulatory bodies.

3. The 3Rs in Marine Algal Biotoxin Testing

Russell and Burch introduced the 3Rs (replacement, reduction and refinement) in 1959 in the publication of The Principles of Humane Experimental Technique [44].

This work was the first to address in depth the effects of animal treatment during experimentation on scientific results, and represented the birth of a field of study recognized to both increase animal welfare standards and, when properly applied, the quality of science produced. 22

Replacement, as used by Russell and Burch, entails replacing conscious, living vertebrates with non-sentient material, and may include the use of invertebrates, tissue cultures or entirely analytical methods that require neither animals nor biological materials garnered from them. Two types of replacement exist in practice—absolute and relative. In absolute replacement, procedures requiring animal usage are eliminated, as seen through the application of chromatography methods to marine biotoxin detection, discussed in Section 6.1. Relative replacement, on the other hand, still requires the use of animals, but these animals are not likely exposed to distress during the procedure, as in the receptor binding assay (RBA) described in Section 6.3, wherein rats are humanely killed to obtain the necessary brain tissue to complete the assay. Besides death, the rats suffer no inhumane consequences of experimentation akin to those endured by mice in the mouse bioassay described in Section 2.

The second R, reduction, entails reducing the animals to the minimal number necessary for an experiment while still maintaining a sufficient number to provide the statistical power required to interpret results. Since the results of experiments that use too few animals are unusable, such experiments are detrimental to animal welfare and scientific concerns as surely as using too many animals. Time saved and personal convenience, or other non-scientific reasons, are not considered “sufficient justification for using more animals than the minimum necessary to obtain meaningful results. Proper statistical design, prior to undertaking the study, and appropriate analysis of the resulting data, may make it possible to obtain results of comparable precision by using fewer animals,” [86]. 23

Refinement, the final R, involves removing as much suffering as possible from the procedure, and consists of everything from the husbandry of the animals to the choice of the final endpoint of the experiment [87].

The first International Symposium on Regulatory Testing and Animal Welfare

(ISRTAW), sponsored by the International Council for Laboratory Animal Science

(ICLAS) and the Canadian Council on Animal Care (CCAC), was held in June of

2001, with the results of the symposium summarized in a supplemental issue of the

Institute for Laboratory Animal Research (ILAR) Journal [88]. The subject of this conference was the impact of the 3Rs on scientific research, and the resulting ILAR issue contains a number of articles detailing the very tangible impact of animal welfare on scientific results. These articles demonstrate that not only does proper use of animals lead to a reduction in animal usage, and concomitant reduction in expense of scientific studies that the public, industry and government must find beneficial, but that the removal of variability caused by issues of animal welfare that unduly stress the animals, beyond that required for the experiment, actually generates more accurate and applicable results.

Balls (2002) [89], in an article discussing future improvements through replacement of animal methods with in vitro methods, succinctly summarizes the impact of the three Rs on scientific endeavors:

Although it is true that this desire [to replace, reduce and refine animal experimentation] is partly driven by animal welfare considerations, the need for better, scientifically more advanced, mechanistically based methods for providing information relevant to human hazard prediction and risk assessment 24

and for protecting the environment is no less compelling. In addition, the alternative methods will also tend to be less expensive to perform, and to have a higher rate of test item throughput. Thus, there are various good reasons for wanting alternatives to animal tests, including scientific, economic, logistical, ethical, legal, and political pressures. A most satisfying aspect of working in this area is that both humans and animals can be afforded great benefits.

The impact of the 3Rs on scientific results, as well as the concept’s pivotal role in furthering animal welfare, makes it an ideal framework for improving regulatory marine biotoxin monitoring. A joint European Centre for the Validation of Alternative

Methods (ECVAM)/DG SANCO Workshop in 2005 held on the Three Rs Approaches in Marine Biotoxin Testing, reported by Hess et al. (2006) [48], used this framework to accomplish a conversation on just such a concept. The same ideas will be applied here. While the technologies currently available to replace, reduce and refine the mouse bioassay may not in some cases yet be ready to replace the time-tested method, they certainly represent advancement in the field of regulating and monitoring for algal toxins. This paper represents a discussion of where regulatory monitoring for marine algal toxins in the food supply chain stands with respect to the 3Rs of Burch and Russell in regards to the use of the mouse bioassay, and the impact large seafood importing countries, such as the United States, can have on the implementation of the

3Rs. 25

4. Applying the 3Rs of Humane Experimental Technique

There are considerable opportunities with the mouse bioassay to implement refinement, reduction and replacement of the procedure [79]. In the European Union, this is actually stipulated by EU Directive 86/609/EEC (Council of the European

Community, 1986) and the Animal (Scientific Procedures) Act (1986), with the requirement to refine, reduce and replace the use of animals used in bioassays and scientific experiments whenever possible. The uniformity of the application of alternative methods varies worldwide; some countries and labs have adopted reduction, replacement and refinement methods, while others have not or have only partially done so. Germany, in particular, relies completely on chromatographic methods and has not conducted a mouse bioassay for the marine biotoxins since the

1980s [90], with no negative impact on human health. Methods utilized in the United

States to implement the 3Rs will be noted, although these seem to be, at least on the official regulatory end, few. This is especially notable in comparison with the regulatory efforts of the EU.

Reduction methods are currently the most commonly utilized, and most typically include screening methods and considering the use of fewer mice for each sample (i.e., 2 instead of 3) or the elimination of titration steps to obtain a more accurate estimate of toxicity when the sample is already known to be highly toxic from preliminary mouse bioassay [91]. An important caveat is that, while reduction is 26

positive, using too few animals to answer the scientific question is detrimental as well

[44]. Notably, if the mouse bioassay were replaced as the confirmatory method, a screening strategy would provide progress towards full replacement of animal tests.

In the United States, the Jellett Rapid Test (JRT) for PSP [45], a rapid lateral flow immunochromatographic assay, has been approved for use as a screening method by the NSSP [92], reducing the number of mice used in PSP toxin monitoring programs in the United States. Oshiro et al. (2006) [93] projected a 30 percent reduction in use of mice for California during the low PSP season when the test was in use. Another study [94] suggested that use of the JRT in UK monitoring programs in

2000 could have resulted in as much as a 61 percent reduction in the use of the mouse bioassay. Functional methods such as the RBA, cell culture assays and enzyme inhibition assays are also under consideration as possible screening tools [95-97], and monitoring programs such as the Olympic Region Harmful Algal Bloom Partnership

(ORHAB) in the United States are aimed towards the development of similar screening methods [98]. These screening methods are particularly sensitive to the need for rapid results that can give a quick “yes or no” answer on whether a particular harvest area should be closed immediately or is safe to harvest, reducing material cost and time commitment. Positive samples initiate immediate closures followed by confirmatory testing methods (i.e., the mouse bioassay where it is still in use, or chromatographic methods where the assay has been replaced), while negative samples eliminate the need to wait for results from distant regulatory laboratories and ease the pressure testing places on growers in planning harvests and preventing recalls (from 27

toxic product reaching the market between the time a product is harvested and the return of the most recent test results) [78, 99]. Such screening methods, while they may be unsuitable for full replacement at this time, or possibly ever, show promise as part of an integrative strategy towards reduction and replacement, providing they do not display false negatives or excessive false positives.

In addition, a well-planned strategy using appropriate scientific methodology and statistical tests can lead to a reduction in animal use; the lack of this in the design of the mouse bioassay is one of Combes’ (2003) [79] major qualms with this method.

Appropriate controls lead to a reduction in variation of the assay, and more reliable results, and so this control over variation is essential to the mouse bioassay. Reducing variability, through reducing the need for excessive calibration, leads to a reduction in animal use.

A number of methods have been developed to aid in the replacement of the mouse bioassays for marine biotoxins. The mouse bioassay is currently considered a confirmatory method wherein a positive mouse bioassay means toxins are present at a quantifiable level. However, the assay is known for its variability and is incapable of discriminating between toxin classes and analogues that may be present in the same sample. The effects are thus of cumulative toxicity, and it is difficult to know whether one or all of the toxins are over regulatory limits, or multiple below with cumulative toxicity causing death. Chromatographic methods such as LC-UVD or FLD and LC-

MS (described in Section 6.1), however, address this problem, and are utilized in several countries’ monitoring programs. These methods are expensive and require 28

specialized personnel and equipment to perform, as well as readily available toxin standards. As in the case of reduction of the mouse bioassay when used as a confirmatory test, functional assays such as the RBA, cytotoxicity assays and enzyme inhibition assays or the sensitive immunoassays could be used as a screen, reserving chromatography for samples that screen positive and leading to significant reduction in expenses and more effective monitoring program. A well-validated functional method would be preferable, as this would have the greatest chance of detecting new and emerging toxins. Theoretically technicians working with chromatographic methods would be familiar with the toxin profile produced by samples, as well, and could test for toxicity when suspicious changes occurred in a toxin profile if chromatographic methods were the only methods in use [76].

Where replacement is not yet feasible, maximal reduction coupled with refinement is the best option [44]. A significant leap forward in general refinement in animal experimentation has been that of the humane endpoint; i.e., choosing an endpoint that causes the animal the least suffering during the progress of the experiment without invalidating the results. The Canadian Council on Animal Care

(1998) and Organization for Economic Cooperation and Development (OECD) [87] published humane endpoint guidelines that have resulted in a general overall reduction in animal use and increased animal welfare. However, while this has considerably impacted on general toxicity testing, little reduction or refinement with respect to humane endpoints has been seen in monitoring for shellfish poisonings [100], as toxicity is calculated from the time of death. While no progress has been made in 29

choosing a humane endpoint for these assays, an anaesthetized mouse model has been developed for the PSP mouse bioassay, where the mouse undergoes anesthesia prior to injection. While this assay takes twice the time for the mouse to die, it is relieved of considerable suffering and distress, and calibration curves developed from this model give a better overall toxicity estimate [101].

In assessing the suitability of the mouse bioassay to monitor for marine biotoxins, the 3Rs represent a major consideration. Regulators, scientists and industry alike have expressed the desire to reach the ultimate goal of replacement as soon as possible without compromising human health and safety. While this has long been the sentiment in the literature, it has yet to be fully accomplished, and a number of factors—scientific, economic, logistical, ethical, legal and political—impact the rate at which this occurs [89]. In the United States, the NSSP regulates interstate commerce in shellfish, and through Memorandums of Understanding has been extended to cover countries that wish to export shellfish products into the United States. As one of the largest seafood importing countries, the US’s policies have a considerable impact on trade partners. 30

5. The National Shellfish Sanitation Program (NSSP), Interstate Shellfish

Sanitation Conference (ISSC) and United States Food and Drug

Administration (FDA)

In the United States, the Food and Drug Administration (FDA) is responsible for setting limits concerning algal biotoxins in seafood and, in conjunction with the

Interstate Shellfish Conference (ISSC), they administer the National Shellfish

Sanitation Program (NSSP), the United State’s joint state-federal-industry cooperative program for regulating interstate commerce in sanitary shellfish products. States may also choose to use the program to regulate intrastate commerce in shellfish products and, where the FDA holds a Memorandum of Understanding (MOU) with foreign governments, those nations also participate in the program and export shellfish products to the United States. The FDA currently holds MOUs specifically for shellfish safety with Australia, Canada, Iceland, Japan, Mexico, New Zealand and the

United Kingdom, as well as a number of more general MOUs with these and other countries on food safety and regulatory practices

(http://www.fda.gov/InternationalPrograms/Agreements/MemorandaofUnderstanding/ default.htm, [accessed 8 July 2010]) [102].

The program is voluntary and reliant on both the states’ adoption of the program and the cooperation of the shellfish industry. Participating states and foreign governments provide a legal framework for enforcing sanitation measures for shellfish 31

products, classify and monitor shellfish growing areas, certify and inspect dealers under their authority and conduct inspections of laboratories responsible for determining the harvesting status of shellfish beds. The FDA, in turn, provides evaluation of state programs and sets various regulatory limits for toxic and deleterious substances in waters or shellfish meat to assure that it is sanitary for human consumption and free from pathogens, biotoxins, or toxic or deleterious substances.

Previously, the FDA also conducted inspections of the majority of state labs; however, due to limited resources and a burgeoning seafood industry, they have encouraged state authorities to employ FDA certified State Shellfish Laboratory Evaluation

Officers (LEOs) to conduct these inspections. Details of the working aspects of the program are found in The Guide for the Control of Molluscan Shellfish [92], which contains the model ordinance that governs the NSSP, as well as a number of supporting documents (http://www.fda.gov/Food/FoodSafety/Product-

SpecificInformation/Seafood/FederalStatePrograms/NationalShellfishSanitationProgra m/default.htm, [accessed 7 July 2010]) [103].

The guidelines are maintained by the Interstate Shellfish Sanitation Conference

(ISSC), whose members consist of representatives from state shellfish control agencies from both producing and non-producing states, federal agencies, industries, academic institutions and foreign governments that have entered into shellfish shipping agreements with the US. Through annual meetings, the ISSC promotes harmonized shellfish sanitation procedures and reviews and amends the guidelines as needed; these amendments are then adopted following FDA approval 32

(http://www.issc.org/about/default.aspx?section=Conference%20Administration,

[accessed 7 July 2010]) [104]. Governing procedures for the body are found in the

ISSC Constitution, Bylaws and Procedures [105].

As part of the framework of the NSSP, all shellfish producing states and MOU countries are required to have and maintain a Marine Biotoxin Contingency Plan; The

Guide for Control of Molluscan Shellfish [92] contains guidance in creating a Marine

Biotoxin Contingency Plan in Section IV. Guidance Documents, Chapter II .02:

Guidance for Developing Marine Biotoxin Contingency Plans. Failure to have a

Marine Biotoxin Contingency plan in place will result in sanctions on the offending authority [92]. This plan “defines administrative procedures, laboratory support, sample collection procedures, and patrol procedures to be implemented on an emergency basis in the event of the occurrence of shellfish toxins,” as well as procedures for emergency recalls and embargoes on contaminated lots and communication with affected authorities [92].

Dealers must incorporate the Biotoxin Contingency Plan into their regular

Hazard and Critical Control Point (HACCP) plan, an integral part of the NSSP model ordinance outlined in the Guide [92]. Under the seafood Hazard Analysis and Critical

Control Point (HACCP) regulation published in the Federal Register on December 18,

1995 (60 FR 65096), seafood processors are required “to conduct an analysis of the potential food safety hazards that are reasonably likely to occur with the seafood products they process and to have and implement written HACCP plans to control any hazards identified in the hazard analysis” and “failure to meet the requirements of the 33

seafood HACCP regulation may cause products to be adulterated under section

402(a)(4) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 342(a)(4))”

(http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDoc uments/Seafood/ucm142704.htm, [accessed 7 July 2010]) [106].

The FDA recognizes the presence of five toxins in US waters (PSP, NSP, ASP,

CFP and DSP) and has issued limits for three of these (PSP - cells/L n/a; 80 μg/100 grams; NSP - 5,000 cells/L or 20 MU (approximate as 80 μg/100 g); ASP - cells/L n/a;

2 mg/100 grams (20 ppm)), along with an advisory to dealers that fish known to be contaminated with CFP toxins should not be placed on the market

(http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDoc uments/Seafood/ucm091782.htm [accessed 7 July 2010]) [107]. States or MOU countries may choose to use cell count data or biotoxin accumulation in shellfish tissue as reason to initiate closures for non-federally regulated biotoxins that occur in their waters. While biotoxins represent a significant threat, and areas subject to frequent marine biotoxin bioaccumulation may be placed under a conditionally approved growing area classification, the NSSP specifically prohibits the designation of growing areas as prohibited unless they are “so highly or frequently affected” that the state is unable to safely regulate the situation [92].

Labs testing for the marine biotoxins are inspected at least once every three years by an FDA certified State Shellfish Laboratory Evaluation Officer (LEO), and must be found to conform or provisionally conform to the NSSP guidelines in order to continue evaluating samples for the NSSP. Detailed guidelines for the inspections, 34

including checklists covering the NSP and PSP toxin mouse bioassays, are found in the NSSP guidelines, and the methods allowed for testing for these toxins are detailed in Section II Model Ordinance, Chapter III Laboratory @.02 Methods C. Biotoxin:

Methods for the analyses of shellfish and shellfish harvest waters shall be: (1) The current AOAC and APHA methods used in the bioassay for paralytic shellfish poisoning toxins: and (2) The current APHA method used in the bioassay for Karenia brevis toxins; or (3) Methods validated for use in the National Shellfish Sanitation Program under Procedure XVI of the Constitution, Bylaws and Procedures of the ISSC and/or cited in the Guidance Documents, Chapter II. Growing Areas .10 Approved National Shellfish Sanitation Program Laboratory Tests [92].

In addition to these methods, the Jellet Rapid Test (JRT) for PSP is allowed under the following conditions, found in Section IV. Guidance Documents Chapter II. Growing

Areas .10 Approved National Shellfish Sanitation Program Laboratory Tests:

Microbiological and Biotoxin Analytical Methods:

i. Method can be used to determine when to perform a mouse bioassay in a previously closed area. ii. A negative result can be substituted for a mouse bioassay to maintain an area in the open status. iii. A positive result shall be used for a precautionary closure [92].

For emerging toxins where no NSSP approved method exists, the ISSC

Executive Board may consider allowing unapproved or unvalidated methods for use in regulating the toxin; within two years, a Single Lab Validation Protocol must be submitted for the method [92]. 35

The ISSC Constitution, Bylaws and Procedures (2008) [105] contain sections on the evaluation and adoption of new test methods in Procedure XVI. Procedure for the Approval of Analytical Methods for the NSSP. Along with scientific validity, the

ISSC takes into consideration the rapidity of the analytical technique or assay for use in field or commercial applications, requirements for skilled staff or specialized instruments or apparatus, cost and the immediate need for the method. Both the ISSC and FDA approve the methods listed in the Guide [92]. Notably, the majority of methods utilized by the NSSP are mouse bioassays, although the JRT was approved for use under specific circumstances in 2004. In order for an alternative method to the mouse bioassay to be widely accepted for use in the United States, then, it would need to pass both ISSC and FDA approval.

6. Alternative Methods

The mouse bioassay provides a quick and dirty estimate of overall sample toxicity, but it has well-recognized drawbacks that, left unsupplemented by other techniques, leaves both researcher and regulator with questions such as which toxin is responsible for a positive mouse bioassay result, whether there are other toxins present and whether there is toxin in a sample that fails to cause acute effects, but may instigate chronic or cumulative effects that researchers and regulators are unaware of.

Researchers, over the years, have developed a suite of chromatographic, immunological and functional techniques to assist in answering these questions where 36

the mouse bioassay has given incomplete or inadequate results. The techniques also typically reduce the ethical implications of animal use as well as the burden of either maintaining a mouse colony or ordering mice through the mail, giving weight to the argument that the most humane technique available makes for the best experiment.

Chromatographic, immunological and functional methods have been adapted to research and regulatory purposes and are variably suited to each—an optimal technique in a research lab, such as the RBA, discussed in Section 6.3, may be less than ideal for regulatory purposes. The same may be said of the reverse. In a similar vein, techniques have been heterogeneously developed with respect to the main marine biotoxin classes, and the method of choice for a given toxin is based on the particular nuances of the toxin and its analogues and the success researchers have had with that toxin and a given technique. Comprehensive toxin review papers include information on the current state of detection methods for the particular toxin reviewed; the recent Toxicon, Vol. 56, Issue 2 [108] is dedicated to the marine biotoxins, and contains more specific information on individual toxins and specialized detection methods.

In general, techniques can be described based on two classification schemes: either structural and activity based, or in vitro and in vivo. Each technique has distinct advantages and disadvantages in terms of specificity, ability to quantify toxin content, time required to execute, specialized staff and knowledge required, technical drawbacks and cost. A number of recent reviews cover the major techniques either in development and/or already implemented in marine biotoxin testing; the reader is 37

referred to these works should they require more technical or specific coverage than that provided in this work [9, 22, 39, 48, 58, 95, 109-115]. The need for methods capable of being used in the field with minimal expertise has resulted in the development of a number of rapid, accurate, and inexpensive detection methods.

Attempts to reach this goal have been addressed by the development of biosensors,

“analytical device[s] incorporating a biorecognition element intimately associated with or integrated within a transducer that converts the response into an electrical signal”

[22]. Several recent works [22, 58] specifically address biosensors, and a number of these methods are based off the principles discussed in Sections 6.1-6.6 and hold promise for rapid, accurate techniques for use in situ.

6.1 Chromatography

Chromatographic methods including gas chromatography (GC), thin-layer chromatography (TLC), liquid chromatography (LC), and liquid chromatography- mass spectrometry (LC-MS), often in combination with spectroscopic methods, are applied in the detection of marine biotoxins. Quilliam (2003) [116] reviews these chromatographic techniques and their applicability in both regulatory and research settings, with particular emphasis on the utility of LC and LC-MS methods.

Coupled with ultraviolet (UVD) or fluorescence (FLD) detection, liquid chromatography was the earliest non-biological method used to study marine biotoxins [39]. As most of the phycotoxin classes lack a natural chromophore, pre- or 38

post-column derivitisation is utilized in conjunction with LC-UVD/FLD to insert a suitable chromophore capable of being detected by these systems. Post-column oxidation requires considerable daily upkeep, and pre-column oxidation, while easily automated, presents complications in interpretation of results, as some toxins give either the same oxidation product despite originating from different parent compounds, or multiple oxidation products originating from the same parent compound.

The advent of electrospray ionization in 1984 [117] allowed the use of mass spectrometry in conjunction with liquid chromatography. In 1989 Quilliam and

Jeffrey [118] applied this technique to marine biotoxins, creating a liquid chromatography method that could be used for detecting marine biotoxins without the need for pre- or post-column derivitisation steps. Methods utilizing LC-MS have since been developed for all known toxins [116]. The ability of LC-MS to assist in structural identification of the toxins has accelerated the understanding of the toxins and their metabolic and environmental fates, with a detection limit capability five times lower than that of the mouse bioassay [39].

LC-UVD/FLD and LC-MS are particularly useful with the complex toxin profiles involved in marine biotoxin detection. Even the smallest toxin family, the

ASP toxins, contains multiple isomers and metabolites. In the case of the more complex families, notably DSP and PSP toxins, many more analogues are present in naturally occurring sample profiles [10]. Small alterations to chemical structure result in changes in toxicity and, although the mouse bioassay provides an integrated 39

response to the relative toxicities of the various toxins, it remains unable to identify specific analogues and its response to specific toxins is not always equivalent to human toxicity. Where multiple toxin classes are present, each with different regulatory limits and modes of action, the bioassay is incapable of discriminating between toxin classes that act on different molecular targets. This means it is unable to determine whether any single toxin class has exceeded its individual regulatory limit. This is the case with lipophilic toxins in European waters, where the basic mouse bioassay for these toxins is incapable of differentiating between DSP, YTX,

PTN, and AZA toxins without modifications to the extraction procedure. Here LC-

MS and LC-UVD/FLD, despite the difficulties associated with both pre- and post- column derivitisation methods, are capable of sensitive separation, identification and quantification of these compounds, providing invaluable information on toxin profiles and protection of consumer health that the conventional mouse bioassay cannot match

[116].

Attempts have been made to develop a multi-toxin LC-MS technique, and in

2005 McNabb et al. [119] conducted a single-laboratory validation and interlaboratory study on a multi-toxin detection method for the amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins and other lipophilic toxins with promising results.

Limitations with developing such a method now lie with developing appropriate sample preparation procedures [116].

A major obstacle to the implementation of chromatographic methods as routine monitoring techniques is the absence of certified reference materials and standards 40

needed for both the calibration of methods and the development of toxic equivalency factors in toxicological studies that would allow the conversion of complex toxin profiles to overall toxicities. Limited certified reference standards are available for a number of marine biotoxins from the European Commission Joint Research Centre’s

Institute for Reference Materials and Measurements (IRMM) and National Research

Council Canada-Institute for Marine Biosciences (NRCC-IMB).

Additionally, equipment set up and maintenance costs for chromatographic techniques are often prohibitive, their operation requires skilled personnel, and many scientists and regulators consider chromatography slower than the mouse bioassay.

The Lawrence AOAC (2001) [46] method has been implemented for detecting PSP in regulatory programs in the UK. However, the method would require NSSP approval in the US in order to become widely accepted in that country, and, according to

Etheridge (2010) [96], is unlikely to be adapted in many laboratories even in the event of NSSP approval due to instrumental cost, time constraints and the need for skilled scientists. A similar situation exists in Canada [76].

Use of one of the following methods in Sections 6.2-6.5 as a screening tool, coupled with chromatographic techniques for confirmation and quantification located at a central laboratory, would likely give the greatest balance between economic considerations and the need to identify toxin profiles [116]. Chromatography methods are also particularly suitable for the identification of new toxins in the case of marine biotoxin-caused outbreaks in conjunction with bioassay directed fractionation (Figure

2) as discussed by Quilliam (2003) [116]. Two methods have been fully validated in 41

interlaboratory collaborative trials; these are the just-mentioned Lawrence PSP AOAC method (2001) [46], recently adopted by the European Union for the detection of PSP toxins, and HPLC-UVD for domoic acid [56]. Hess et al. (2006) [48] provides a review of the chromatographic methods available for the various toxin classes, although all of these except the two already mentioned have yet to undergo successful validation. 42

Figure 2. Flow chart indicating the general principles of the toxin isolation strategy based on the integration of bioassay-directed fractionation with chemical analysis. Reprinted from [116] with Elsevier’s permission. 43

6.2 Immunoassays

Immunoassays are among the most promising techniques for screening purposes. These techniques use the high specificity of antibodies for their associated antigens to quantify toxin content. Antibodies are raised either in animals or cell cultures and used in enzyme-linked immunoassays (ELISAs), lateral flow immunoassays (LFIA) or biosensors based on surface plasma resonance (SPR) [48].

As structural based methods, immunoassays respond to the specific antigen epitope they were raised in response to.

The utility of individual immunotechniques hinges on the antibody’s specificity. The antibody must be capable of cross-reacting with a wide range of toxins within a toxin class, without being so non-specific as to detect non-toxic co- occurring compounds within the matrix [58]. This balancing act limits their utility, as, in order to use immunotechniques for quantification, the cross reactivity of a given antibody must be known to all toxins in the class. Thus, immunoassays have proven easiest to apply for the toxins that have the fewest isomers, such as ASP toxins, where domoic acid is the main isomer responsible for ASP illness [120]. Immunomethods, however, are particularly unsuitable for DSP and other lipophilic toxins, as there is considerable cross-reactivity between the lipophilic toxin classes [48]. A saxitoxin

LFIC method, the Jellett Rapid Test (JRT) for PSP, has been developed and accepted by the NSSP for screening in the United States, but has not yet undergone AOAC 44

validation [45]. Additionally, immunomethods appear particularly promising for brevetoxins [120], and a monoclonal antibody was recently developed for azaspiracid toxins, opening the way for development of immunodetection methods for this toxin class [122].

Like the mouse bioassay, and in contrast to analytical techniques like LC-

UVD/FLD or LC-MS, immunoassays are incapable of distinguishing between toxin cogeners, and their response is typically given in “equivalents” of a toxin standard used to develop the standard curve. This can result in over- or underestimation of toxin content in an area, depending on the predominant toxins in the toxin profile and their respective toxicities in comparison to their affinity for the antibody. However, their high specificity lends them to detecting low levels of toxin in screening programs, which can subsequently be investigated via chromatography methods, discussed in Section 6.1.

The appealing aspect about immunoassays is that they can process a large number of samples, are relatively cheap, require very little equipment to process and minimal training and expertise on the technician’s end [58]. A number of formats have been developed, and the concept has been used to make biosensors. Enzyme- linked immunoassays, whereby toxin endogenous to the sample competes with assay toxin for enzyme-labeled antibodies, are common and available for most of the toxin classes, including okadaic acid, saxitoxin, azaspiracids, brevetoxins, domoic acid, yessotoxin, ciguatoxins, and palytoxins [22]. 45

Figure 3 shows the concept behind the direct competition ELISA for domoic acid, developed by Biosense Laboratories. An integrated system for ASP, NSP and

PSP toxins found in New Zealand has been developed that shows promise as an effective screening tool for multiple toxin classes [39].

Figure 3. The direct competition ELISA developed by Biosense Laboratories. Free DA in the sample competes with DA-conjugated protein coated to a plastic well for binding to anti-domoic acid antibodies. Anti-DA antibodies are conjugated to horseradish peroxidase (HRP). After washing, the wells are incubated with a substrate that reacts with the HRP-antibody conjugate to give a change in color of the solution that is measurable by spectrophotometry at 450 nm, which is inversely proportional to the concentration of DA in the sample. Figure reprinted with permission from Biosense Laboratories from the Biosense ASP ELISA protocol [123].

Radioimmunoassays (RIAs) that incorporate radiolabeled toxins have been developed as well; however, these are mostly restricted to research purposes as they 46

are radioactive and pose both safety and regulatory concerns, which many labs are not equipped to deal with [58].

6.3 Receptor Binding Assays

A number of the toxin classes (CFP, NSP, PSP, ASP) exert their toxicity through binding to known biological receptors within the body. For these toxins, receptor-binding assays (RBAs) have been developed to take advantage of this. RBAs have been developed for CFP, NSP, and PSP toxins using the voltage gated sodium channel [96, 124, 125] and for ASP toxins using the glutamate receptor [126, 127].

Typically, these utilize radio labeled toxin standards in competition with toxins in the sample for the molecular site of action, although some have been developed with chromogenic and fluorescent endpoints. These, however, tend to be less sensitive as the addition of necessary chromophores decreases the affinity of the labeled toxin for the receptor [39, 114].

The RBA relies on the inherent affinity of the toxins for the receptor and, since the receptor utilized is the biological target where the toxins exert their effect, this correlates well with the potential toxicity of the sample in most cases. In contrast with the structural-based immuno- and chromatographic methods, this type of assay is known as a functional assay. Without identifying the toxin profile causing the response, the RBA, much like the mouse bioassay, accounts for the varying toxicity of analogues present in the mixture to yield an overall sample toxicity. 47

Receptors, in contrast to antibodies, are more difficult to work with and have a lower stability than antibodies, and the targets for some toxins, such as AZAs, are still unknown. For these toxins it is not yet possible to develop an RBA. Despite the difficulties of working with receptors and the issue of dealing with radio labeled compounds, the ability to detect overall toxicity makes the RBA attractive, as well as the lack of a requirement for certified reference standards for the various toxin analogues and metabolites [22].

A single laboratory validation of the RBA for the PSP toxins was successful

[97] and an AOAC collaborative study is currently in progress [96].

6.4 Cell Culture Assays

In cell culture assays, toxic sample extract applied to cell cultures induces either cell death or changes in morphology that are correlated to sample toxicity. The cytotoxicity assay for NSP, CFP and PSP toxins is the classical example of this method. Neuroblastoma cells are treated with oubain/vertradine to enhance sodium entry into the cell, cell swelling and eventual lysis. NSP and CFP toxins, which alter the gating of the sodium channel and reduce the voltage required to generate an action potential, accelerate cell death. PSP toxins, which physically bind to and block the pore of the voltage gated sodium channel, increase cell survival. Such assays are automatable [128] which increases their usefulness, and a single rat or mouse brain may yield enough material to run 20 samples [129], reducing animal usage. 48

Assays based on morphological changes and cell death utilizing cells other than neuroblastoma cells have been developed for PSP, ASP, DSP and other lipophilic toxins. However, the cytotoxic response in these assays is more general, typically preventing specific identification of the toxins responsible for cell death [113].

These assays are useful for research, economical and easy to perform, but require the maintenance of tissue cultures and specialized staff and may be time consuming. They may also give confusing results in the presence of toxin mixtures

[39, 58]. Like the bioassay and RBA, however, they are functional methods capable of giving an overall toxicity result that correlates variably with actual toxicity depending on the specific format chosen for the assay [113].

6.5 Enzyme Inhibition

Enzyme inhibition, in particular, has been developed for the DSP toxins.

These toxins exert their effect mainly through the inhibition of protein phosphatases.

Originally the assay was based on the DSP toxins’ capability to inhibit the release of radioactive phosphate from radio labeled phosphorylase by phosphoprotein phosphatase 2A. Development of both spectrophotometric methods utilizing p- nitrophenyl phosphate and fluorimetric methods has since eliminated the need for the use of radioactive material [113].

The sensitivity and correlation of the fluorometric enzyme inhibition assay, along with its ease of use, have led to its consideration by the European Community as 49

a possible replacement for the mouse bioassay for DSP toxins [58]. O’Neill and

Moffat (2000) [130] review the various formats available for the protein phosphatase inhibition assay for DSP toxins.

6.6 Algae Monitoring Programs

Monitoring for known toxic algal species is used both to trigger testing for toxins and, in some cases, for immediate closure at a threshold level of potentially toxic cells. In Florida, the presence of Karenia brevis, which produces the toxins that cause NSP, above 5,000 cells per liter triggers immediate closure of shellfish harvesting waters. Once cell levels have dropped below the 5,000 cells per liter threshold, testing for NSP toxins in shellfish is initiated in order to reopen the waters

[131]. Cell counts may be conducted manually, but this is both tedious and requires considerable taxonomic expertise. Molecular and immunological probes have been developed to bypass this requirement, as well as simple flow-cytometer methods based on harmful algal cells’ optical properties [132].

Toxicity and cell presence do not always correlate since potentially toxic species only produce toxins under species-specific environmental and physiological conditions, and so monitoring for harmful algal species has a tendency to overestimate risk [39]. Additionally, shellfish may remain toxic even after toxic algal species are no longer detectable in routine sampling. Episodes of toxin accumulation in shellfish, however, are generally preceded by an increase in potentially toxic species population. 50

While algal monitoring is not considered as a prospective replacement for the mouse bioassay, it can alert regulators to the need for immediate testing for toxins in shellfish when increased cell counts occur off the normal regulatory schedule for shellfish testing, precautionary closures or, in some cases, the need to monitor for new toxins with the appearance of a known toxin producer that has expanded into new waters.

Combined with environmental monitoring of factors such as wind, water, temperature and salinity that can give indications of when conditions are becoming favorable for a toxic event, integrating algal monitoring gives flexibility in management options and allows for proactive response to toxic blooms in place of reactive responses [9].

7. Hurdles to the Implementation of Alternative Techniques

A number of barriers must be hurdled in order for alternative techniques to be adopted in practice. These include scientific, economic, logistical, legal and political obstacles [89]. Several were addressed in the review of alternative methods in

Sections 6.1-6.6. Of these, the most intractable are the technical scientific issues. In the case of biotoxins, this is a chronic lack of certified reference materials required for the development and implementation of the alternative methods in practice. The lack of these same standards is often cited as a major impediment to the second major obstacle, formal validation of alternative methods. However, if standards were to become available, these validations would become much more feasible, and, while the process of validation is laborious and expensive, the experience of some scientists 51

involved in developing validation procedures has evidenced that scientific validation

“need not be long and costly” and emphasizes that test development and regulatory acceptance, in contrast to the actual formal validation procedure, are truly the rate limiting factors in the adoption of alternative methods [89]. Regulatory acceptance represents a particularly intractable step, as the mouse bioassay has been in use since

1937 for marine biotoxin testing [58]. While it is incredibly variable for toxins such as those in the DSP suite and other lipophilic toxins, and has been known to both inadequately protect human health from these toxins by releasing contaminated shellfish onto the market and causing economic hardships for producers with false positives, the PSP mouse bioassay, in particular, has prevented any reported deaths in shellfish cleared for consumption using the PSP mouse bioassay. Regulators, with such a deadly toxin, have been reluctant to switch from what they consider a reliable method to other methods that may prove as effective in practice, but have not proven themselves through long use, as has the PSP mouse bioassay [76]. In order to overcome these biases regulators and scientists developing alternate methods must communicate clearly the effectiveness and limitations of alternative methods, as well as complete the requisite collaborative multi-laboratory validations. In addition to proving their effectiveness to regulators, the alternatives must also be economically viable and capable of being used, either as stand-alone procedures or in conjunction with other methods in a consolidated strategy, to provide rapid and inexpensive results for the immediate closure of toxic areas in locales far from central laboratories.

Biosensors in this regard, discussed in Section 6, provide promise. 52

Both certified calibrant and matrix reference materials—required for toxicological studies, validation of detection methods and for quality control and calibration of methods in official regulatory labs—are limited in availability, with the demand for these toxins far outstripping the available supply. A reference material

(RM) is defined as “material, sufficiently homogeneous and stable with respect to one or more specified quantities, used for the calibration of a measuring system, or for the assessment of a measurement procedure, or for assigning values and measurement uncertainties to quantities of the same kind for other materials.” A certified reference material (CRM) is a “reference material, accompanied by an authenticated certificate, having for each specified quantity a value, measurement uncertainty, and stated metrological traceability chain,” [133]. RMs may be used for routine work, and some labs produce their own in-house laboratory reference materials (LRMs). Hess (2007)

[134] promoted this practice to prevent the rapid use of CRMs needed for validation and official quality control, since they are in high demand and short supply, and are absolutely required for the development and validation of new, more effective methods of detecting the toxins and calibration in official regulatory labs. This also frees producers to expand the range of available CRMs to new toxins and analogues.

National Research Council Canada (NRCC) and the Japanese Food Research

Laboratory produce the majority of certified marine biotoxin standards, and a number of other organizations produce non-certified reference materials [134].

While it may seem obvious that CRMs should be reserved for essential purposes, the production of standards, certified or not, is a laborious process, and 53

many labs are either incapable of producing LRMs or hesitant to put the effort into it when standards are already available elsewhere, albeit in small quantities [134].

Where synthetic schemes are available, the toxins may be chemically synthesized.

These syntheses, however, tend to be complicated. Where a synthesis does not exist, the toxin must be extracted and purified from either natural phytoplankton blooms and contaminated shellfish tissues or mass-cultured phytoplankton. The sporadic nature of toxic HABs and the use of the non-specific mouse bioassay prevent the routine identification of specific toxins in contaminated shellfish, and many of the toxic phytoplankton are not amiable to culturing. This makes the process of RM and CRM production both technically difficult and opportunistic in most cases [22].

Saxitoxin has been listed as a chemical weapon under the Convention for the

Prohibition of Chemical Weapons, and its trade and transport is strictly regulated.

While saxitoxin CRMs are available, this means they are inaccessible in many countries, especially those that have not signed or ratified the Convention [135].

Formal interlaboratory validation of alternative methods is the second barrier to the adoption of these methods. While many methods come and go informally in the scientific literature without ever undergoing validation, the introduction of a requirement to protect consumers and producers from physical or economic harm requires a formalized validation procedure [136]. Regulatory authorities want to know that the methods they utilize are effective for the purpose they are used for, and so require, in most cases, methods to undergo validation before they are considered for use. The process of validation itself is under revision and constantly evolving [86, 54

136, 137]. When validating any method as a risk management tool, sufficient toxicological data is required in order to create a risk assessment; this is particularly problematic for the marine biotoxins, as epidemiological data on which to base risk assessments is scarce and the standards required for toxicological studies are limited.

When these risks and hazards are not fully understood, as is the case for the marine biotoxins, this leaves some amount of uncertainty as to the appropriate test to which to compare a method to in validation. Traditionally, for marine biotoxin testing, the gold standard method of comparison has been the mouse bioassay integrated, where possible, with epidemiological data from human intoxications. However, perfect correlation with animal tests may not be either possible or desirable [137], and the mouse bioassay has not been formally validated in most cases and certainly has a number of limitations of its own. While few of the alternative techniques have yet been fully validated, the mouse bioassay has only ever been validated for PSP toxins

[57], and its subsequent adaptations to other toxins have never undergone revalidation.

The authority of these versions of the mouse bioassay and regulators’ trust rests not on the weight of scientific trials, but the methods’ long use in regulatory programs for protecting human health [138]. Combes (2003) [79] argues that, had the mouse bioassay been subjected to validation, it would never have been developed due to its incapability to stand up to the rigors of full interlaboratory validation. Despite these arguments, the mouse bioassay is still typically the “gold standard” that any alternative method is validated against [89], and in vitro methods typically come under more scrutiny in validation than corresponding in vivo methods. Thus, while many 55

validations continue to use the mouse bioassay as a comparison, there are those who emphasize that incomplete agreement with in vivo methods should not necessarily prevent validation [89, 136, 137].

Internationally, the International Union of Pure and Applied Chemists

(IUPAC), the International Organization for Standardization (ISO) and the

Association of Analytical Communities (AOAC) International have published harmonized guidelines for conducting collaborative interlaboratory validations as well as single-laboratory validations [139-145], and a number of organizations specifically concerned with the validation and adoption of alternative methods to animal assays, including the European Center for the Validation of Alternative Methods (ECVAM), the Centre for Documentation and Evaluation of Alternatives to Animal Experiments

(ZEBET), and the Interagency Coordinating Committee on the Validation of

Alternative Methods (ICCVAM) are working towards the replacement, reduction and refinement of animal methods.

ISO defines the process of validation as “confirmation through examination of a given item and provision of objective evidence that it fulfils the requirements for a stated intended use,” [133], i.e., a validation is used not only to determine that a particular method works, but its fitness of purpose for a particular application under specified conditions and its ability to accurately measure, with accompanied uncertainty, what it’s intended to measure. If the original bounds of the method are changed (for example, different toxins or different concentrations of those toxins are assessed, in different matrices, as with the marine biotoxin mouse bioassays) the 56

method will need to be revalidated for those bounds [146]. Prior to a full collaborative study, a single-laboratory validation or pre-validation study must be conducted to optimize the method and characterize it with respect to its performance characteristics

(precision, trueness, selectivity/specificity, linearity, operating range, recovery, limit of detection (LOD), limit of quantification (LOQ), sensitivity, ruggedness/robustness, applicability, calibration and traceability) and the analytical system (the purpose and the type of method, the type and the concentration range of analyte(s) being measured, the types of material or matrices for which the method is applied, and a method protocol) involved. Taverniers et al. (2004) [146] describes the process of method validation and quality assurance in depth. Ideally, the concerns of eventual validation are addressed during method development and therefore ease this validation process, although this did not occur with the biotoxin mouse bioassays. The pre-validation study, often in the form of single-laboratory validation, makes formal collaborative interlaboratory validations “much faster, less expensive, and more likely to succeed,”

[89]. AOAC International maintains guidelines for conducting these studies and selecting labs to participate in collaborative studies. The Codex Alimentarius, discussed in Section 8, prefers methods to be validated according to the collaborative

IUPAC/AOAC/ISO harmonized protocol; however, there is an extreme deficiency of interlaboratory validated methods and proficiency testing schemes for phycotoxins, so particular emphasis is placed on single-lab validation and internal quality control [9]. 57

AOAC International maintains a Task Force on Freshwater and Marine Toxins that addresses the global need for improved testing methods for the detection of freshwater and marine biotoxins:

… by focusing efforts, setting priorities, and identifying economic and intellectual resources. The group will establish methods priorities, determine fitness for purpose, identify and review available methodology, recommend methodology for validation, and identify complementary analytical tools. Once appropriate analytical methodology has been identified or developed, the Task Force will identify financial and technical resources necessary to validate the methodology.

In recommending methods for validation, the Task Force considers the need for the method, the priorities of influential outside groups, the likelihood the method will provide a viable alternative to existing animal methods and whether interlaboratory data already exists on the method and the practicality of the method (including whether the standards and techniques involved are “within the budget or expertise limitations of stakeholder laboratories”) for eventual use by all stakeholders

(http://www.aoac.org/marine_toxins/task_force.htm [accessed 7 July 2010]) [147].

Once methods are validated, they face the often-substantial barrier of regulatory acceptance and incorporation into legal requirements. This is assisted, in some cases, by evaluations of the method for its usefulness by organizations such as

ECVAM, ICCVAM and ZEBET. Where validation shows the efficacy of a method under a fixed range of conditions, these organizations evaluate validated methods for practicality in application. Promotion of methods unsuited to the demands of field and regulatory work represents an obvious waste of resources with the many promising 58

methods queued for validation [136]. There are both those that have unrealistic expectations of how quickly alternative methods to in vivo methods can be adopted, and others who may use the process in order to delay or prevent changes to a regulatory system [136]. Schiffelers (2007) [148] points out that a collaborative system involving both in vivo and in vitro methods may be most practicable; how quickly this will be accepted and integrated by regulatory authorities remains to be seen. In some cases, these legislators and regulators may be waiting for a scientific consensus; something that takes a long time to develop in many cases and that may prevent action despite overwhelming evidence [148].

As discussed in Sections 6.1-6.6, each technique has its own strengths and weaknesses when it comes to monitoring applications. Regulators must understand and be convinced of the effectiveness of these techniques before they are willing to adopt them; Hess et al. (2006) [48] recommends the education of regulators in basic biology to facilitate their understanding and the acceptance of functional methods.

Additionally, regulators must be convinced of the efficiency of these methods in protecting human health; very few of the methods have gone through stringent validations, and they must be fiscally viable for both regulatory and industrial use in detecting toxins. Regulators walk a fine line between the benefits of seafood, associated risks and the socioeconomic hardships imposed on producers by preventative closures. In some cases, regulators are unaware of and unfamiliar with existing alternative methods, possibly due to those methods’ constant development and “a lack of resources expended to validate or accept them for regulatory use” [76]. 59

In the US, the ISSC and FDA provide this evaluation for use of methods on shellfish and, if acceptable, incorporate the method into the NSSP. Regulators, once the methods have been validated, must be convinced that the methods are both effective and feasible for use both economically and in field situations.

Despite the validation of several alternative methods, many scientists and regulators do not trust the new alternative methods, and are wary of switching over from what they perceive as an effective management tool [76]. Spielman (2002)

[149], in his discussion of switching to alternatives that promote animal welfare in chemical and pharmaceutical testing, cites the “comfort factor” or “NIH not invented here syndrome” as an impediment to implementation of alternative testing in pharmaceuticals, while Richmond (2002) [150] cites the possibility that “regulatory inertia” may be involved. These same factors are likely involved in the adoption of alternative methods to the mouse bioassay. In all cases, those who discuss the issue have stressed that better communication and transparency between all individuals involved in the process—regulators, scientists, producers and the public— is the solution [76, 148, 149].

A growing emphasis on consumer safety and the idea of zero risk impacts the process of replacing the mouse bioassay with alternative methods, and communications of what is acceptable risk (and what is not) is required to ease the transition between methods [89, 148]. Schiffelers (2007) [148] notes that, while public opinion pushes for animal welfare, consumer safety tends to trump this concern, and those who advocate zero risk are insufficiently aware of the increased 60

and often unnecessary animal testing this creates. Regulators bear heavy responsibility for whatever they release onto the market and so, when consumers are obsessed with any risk being unacceptable, regulators and legislators are understandably hesitant to switch from methods that, while imperfect, may land the blame for any inadequacies of the new method, real or imagined, on their heads [148].

Finally, the development, validation and implementation of alternative methods can be a costly process. While the UK has implemented the Lawrence

AOAC (2005) [151] chromatography method for PSP detection as a primary method in their detection programs [96], this method is, at this time, unlikely to be used in the

US even if the method were NSSP approved due to instrumental cost, time constraints, and the need for skilled scientists. Similar reasons have prevented its adoption in

Canada [76]. Responses from the ethnographic study of regulators and scientists conducted by Guy and Griffin (2009) [76] suggest that government funding, spread over the development and validation of many methods, could impede the acceptance and implementation of those methods already AOAC validated. Notably, the

Lawrence method, developed in Canada, has yet to be adopted in that country, but there are multiple lines of research into many other non-validated methods (Jellett

Rapid Test (JRT), the pre-column HPLC/Lawrence method, post-column HPLC method, receptor binding assay (RBA), HPLC–mass spectrometry (HPLC–MS),

Enzyme-Linked Immunosorbent Assay (ELISA), and Surface Plasmon Resonance

(SPR)). The tendency towards zero risk, discussed previously, combined with delayed results caused by lengthy validation and acceptance time lags in developing alternative 61

methods, may also make legislators and governments hesitant to commit resources to alternative method implementation and acceptance [147]. Also, while legislators and governments are wont to demand scientific certainty, the economic literature on food safety regulations, for the most part, ignores the substantial scientific uncertainty involved in food quality assurance [152].

Reduction in use of and replacement of the mouse bioassay for marine biotoxins faces many obstacles, but, with conscientious development of methods and application of available funds, supported by clear communications about the advantages and disadvantages of each method and sufficient education, is becoming increasingly feasible.

8. Interplay between Trade and International Adoption of Alternative

Methods

Global seafood trade, following a trend in the industry’s expansion, reached

$92 billion in 2007, with 194 countries reporting seafood exports as of 2006. With this expansion the supply chain has become more complex; importation into one country for value-added processing followed by re-exportation to a second country for consumption or further modification is common [23]. This complexity spreads the responsibility of maintaining seafood quality and safety across international borders, where laws and requirements on seafood processing, safety and sale fluctuate [23,

153]. This variation in requirements for seafood processing results in health concerns 62

and unequal trade opportunities between countries [135], leading organizations such as the World Trade Organization (WTO), to advocate international harmonization of seafood standards in order to facilitate free trade. International harmonization of standards is also recognized an efficient method for advancing the 3Rs in regulatory testing [149]. Thus, the high demand for seafood and the link between economics and animal safety places large importing countries in a position to influence the speed and ease with which alternative methods to the mouse bioassay in marine biotoxin testing are adopted. Malik et al. (2010) [154] recognizes that the European Union exerts a huge influence in this regard, and that Japan and other Asian countries are setting increasingly stringent import standards that countries such as the EU, China and US must meet in order to export there. As the second largest importer of seafood worldwide, the US, along with other major importers, is in an influential position over seafood-exporting countries, and especially developing countries, to increase food safety as well as influence the methods utilized to accomplish food safety. In the case of marine biotoxins, this means that major importers have the capability to influence how quickly and effectively alternatives to the mouse bioassay are adopted.

The World Trade Organization (WTO), Organization for Economic

Development and Cooperation (OECD) and the Codex Alimentarius (Codex) work towards harmonization of standards across countries for a variety of food products to increase food safety and, notably, facilitate trade and economic prosperity. The WTO and OECD are primarily concerned with facilitating free trade, whereas the Codex, sponsored by the Food and Agricultural Organization (FAO) and World Health 63

Organization (WHO), shares the trade concern with raising overall global food safety and quality, and is a major player in the development of international food safety standards. The WTO, through the Sanitary and Phytosanitary (SPS) Agreement, encourages the development of basic science-based limits for food safety and animal and plant health standards, but leaves the development of these international standards to groups such as the Codex. Through the SPS agreement, member states are required to avoid the use of food safety standards as disguised trade barriers used to protect domestic markets. Any change in food regulations by member countries, including seafood regulations, must be submitted to the WTO to allow comment by trading partners.

The globalization of food production has complicated the enforcement of SPS standards, and efforts to enforce SPS standards have, in turn, complicated trade [155].

The burden of cumbersome unharmonized regulations ultimately falls on the consumer in the form of higher prices. In 1982 the OECD worked for the harmonization of chemical testing for this reason and, following their lead, the

International Conference on Harmonization (ICH) in 1990 accepted harmonized guidelines for the efficacy and safety testing of drugs and medicine. While this led to a benefit for both producers and consumers, it also reduced the number of animals used for testing [149]. Presumably, similar consumer, producer and animal welfare advantages would stem from parallel harmonization of marine biotoxin standards.

While organizations such as the WTO, OECD and Codex play a large part in the adoption of food safety testing standards and techniques, major importing 64

countries’ willingness to accept international standards and to assist in the development and validation of testing methods impacts the ability of developing countries and large exporters to adopt international standards. Large importers, in conjunction with these international organizations, share influence over safety standards and the methods used to achieve them.

Fernandez (2000) [135] reviews the state of harmonization in the marine biotoxin field as of 2000. An ethnographic survey of Canadian scientists and regulators revealed a common concern that switching to an alternative method without the approval of trading partners could result in trading sanctions, especially as many of these agreements cite Codex methods. The bilateral trade agreement with the US with respect to switching to LC-UVD methods for PSP toxins was specifically cited as problematic; any such change in the Canadian Shellfish Sanitation Program (CSSP) would first have to be approved by the US. Codex has yet to approve suitable replacements for the majority of marine biotoxin mouse bioassays, although they have identified promising methods slated for approval pending full validations [48]. Many international treaties use and reference the Codex’s guidelines in treaty language, and the slow adoption of alternative methods by the Codex could lead to lags in implementation of alternative methods, as countries could be found in violation of treaties and international law. Canada has adopted chromatography methods for detecting DSP toxins, but must test a subset of samples with the DSP mouse bioassay in order to export to the EU [76]. Similarly, while New Zealand “has a strong commitment to the implementation of non-animal based methods” the New Zealand 65

Food Safety Authority, due to exportation, must “ensure compliance with the regulations of importing nations,” [43].

A large gap in seafood quality control lies between developed and developing countries. With the demand for seafood imports high in developed countries and no concomitant rise in domestic supply, developing countries face few import tariffs.

Japan, the US and the EU imported 72 percent of the value of all exported fish products in 2006; of this, 62 percent of the product originated from developing countries. Instead, the major impediment to developing countries exporting seafood products to developed nations is an inability to meet high seafood sanitation requirements of the importing countries [23, 156]. Fisheries management, a daunting task for all countries, is especially so for those with poor infrastructure and funding, leading reform in fisheries management in some instances to be linked with capacity- building assistance incentives through international organizations and bilateral agreements [23]. Their involvement in capacity-building incentives gives developed countries more direct control over the methods and limits used in developing countries.

Richmond (2007) [157] discussed the role of regulation and whether it drives, monitors or manages change. While he and his colleagues came to the conclusion through the evaluation of case studies that regulation does not always drive change, they determined that it has the powerful capability in some situations to do so. The implementation of the HACCP system utilized by major importers is also recognized as a catalyst that introduced significant changes in food sector regulatory policy [156]. 66

This role of importing countries’ regulatory policies places major importers in a position to influence the quality assurance and safety standards of countries that export to them, often times benefiting the exporting country through the resulting improved national seafood safety. While this improved seafood safety capacity is desirable, international agreements have recognized that a balance must be struck between encouraging developing countries to improve food safety and requiring unattainable goals in locales with limited resources [153, 156]. In contrast, limits and methodologies imposed by major importing countries, if restrictive or outdated, may prevent the adoption of more scientifically sound practices, as well as cause economic hardship for exporting countries actively working to update their policies [135].

The discussion of the use of the mouse bioassay and alternative techniques in marine biotoxin testing is particularly active in the EU. The lynchpin for the difference in tenor of discussion of the mouse bioassay in literature within the EU as opposed to outside, and in particular to the US, is presumably due to the possibly conflicting legal requirements of animal welfare and marine biotoxin testing in EU law [48], as well as the predominance of a complex suite of lipophilic toxins in

European waters that the mouse bioassay is particularly technically unsuited to detect.

Dennison (2007) [138] and Hess (2006) [48] discuss the conflict between the two laws, one covering acceptable marine bitoxin testing methods and the other the legal requirement to utilize non-animal methods in experimentation where methods are available and practicable. Possibly, if a similar animal welfare law was present in the

US, the discussion would be much more active there. Additionally, EU law places 67

more weight on the precautionary principle in regulation, whereas the US relies heavily on cost-benefit analysis in developing monitoring and testing regulations for seafood safety [158]. Using the framework utilized by Richmond [156] to discuss the role of regulation in policy change, the “problem stream windows” have aligned to create a period of opportunity for regulatory change within the EU with respect to marine biotoxin testing, leading to a more intense scrutiny and discussion of the mouse bioassay in EU member states.

The impact and legal status of the mouse bioassay in marine biotoxin testing has been scrutinized in the EU [48, 90, 138] and literature is available for Canada as well [76, 101]. Outside of method development and a consensus in the literature on the ethical quandaries and technical limitations of the mouse bioassay, the literature is scarce on US-specific plans for the reduction and elimination of the mouse bioassay from US regulatory testing policy.

Possibly this lack of visible discussion is due to a feeling that there is little need to elaborate a situation so obviously in need of an overhaul, the pervasive feeling in regulatory circles that the mouse bioassay provides adequate protection for the predominant toxin classes in US waters, the idea that the mouse bioassay is capable and adequate to screen for emerging toxins, and/or ever-present fiscal issues. The US may be relying on organizations such as the Codex to prompt change in marine biotoxin regulation policy, and discussion likely appears outside of the regularly published literature. In the case of the discrepancy between discussions in the US and

EU, this may be due to the legal status of animal testing in the EU and current biotoxin 68

monitoring laws and the presence of a complex mixture of lipophilic toxins, including the DSP suite, that are not currently prevalent in US waters and are incapable of being discriminated between in the routine mouse bioassay for lipophilic toxins.

In the US the NSSP and ISSC, discussed in Section 5, work to facilitate trade and uniform harmonization of shellfish safety standards between and within states, as well as with foreign countries that hold MOUs and similar agreements with the FDA.

Acceptance of products between states and countries party to the NSSP helps eliminate the need for expensive and excessive testing. With the growing trade in seafood, need for harmonization has expanded beyond national and to international interactions, seen in the creation of MOUs with foreign countries. With this expansion, such national programs for uniformity must be harmonized with each other at the international level. In the case of biotoxins, this means agreement on both limits and methods of testing for those limits, with which the Codex is intimately involved.

The United States is a member of the WTO and subject to the SPS Agreement.

As such, programs deemed to provide equivalent protection, although not necessarily through the same means, are required by the SPS Agreement to be accepted. This is the case with Canada’s Shellfish Sanitation Program (CSSP), which is considered equivalent to the United States’ NSSP, although not identical [135]. Changes to such programs, such as a switch from the mouse bioassay to an alternative method in testing for marine biotoxins would require evaluation and approval on the part of the importing partner country, a concern revealed in Guy and Griffin’s work (2009) [76].

This could prevent or delay countries from using what they may otherwise consider 69

improved methods for the protection of human health or force the maintenance of a subsidiary regulatory structure for export within a domestic regulatory program.

The US shows little activity in the literature towards implementing alternative methods into regulation. To date the only visible implementation of the 3Rs in US law is reduction in animal usage through the use of the NSSP approved JRT screen for

PSP toxins. Any similar alternative method would first have to be accepted by the

NSSP in order to be used and accepted by the US, and chromatography methods likely face an uphill battle with the fiscal and personnel resources required for their implementation, even following validation [96]. In contrast, the EU, which is the third largest worldwide seafood importer, shows a lively discussion in the literature on options for implementing alternative methods in regulation [48]. Canada and New

Zealand have also taken steps towards eliminating the mouse bioassay in their national programs [38,43]. Since the major impediment to developing countries exporting to developed countries such as the US is the ability to meet seafood sanitary requirements [23], developing countries will likely work towards those importation requirements utilized by countries that they wish to export to. If these countries solely followed US regulation as it currently stands, this would mean adoption and perpetuation of the technically and ethically flawed mouse bioassay.

While the adoption of alternative testing methods into regulatory programs is resource-intensive, opportunities exist to ease the process in the US. The Interagency

Coordinating Committee for the Validation of Alternative Methods (ICCVAM) of the

United States, within the National Institute of Environmental Sciences (NIEHS), has 70

abbreviated procedures for reviewing methods already evaluated by ECVAM [159], as discussed in Section 7. As ECVAM is active in promoting and evaluating alternatives to marine biotoxins, acceptance and use of such abbreviated procedures, or the NSSP developing similar procedures with well-known alternative method evaluating bodies such as ICCVAM, ECVAM and ZEBET, could lead to more rapid adoption of appropriate alternatives to the mouse bioassay and elimination of unnecessary duplications of efforts while minimizing the cost of the validation and evaluation of methods prior to acceptance by regulatory authorities.

The gradual integration of these methods into regulatory systems is becoming a reality with the increasing acceptance of alternative marine biotoxin testing methods by scientists [160]. Large importing countries such as the US, in conjunction with international programs including the WTO and Codex, have the opportunity to be important catalysts in or impediments to the process. So far, outside of methods development, the US seems to be relatively passive in the implementation of new regulatory methods, while countries such as the EU’s member states and New Zealand take the lead in implementing alternative methods.

9. Concluding Remarks

HAB expansion makes marine biotoxin detection in seafood supplies a high priority. The mainstay of detection has long been the mouse bioassay, but, slowly and surely, this is changing. Alternative biological and analytical methods with the 71

capability of replacing, reducing and refining mouse bioassay use for marine biotoxin testing, discussed in Section 6-6.6, are gaining acceptance in the scientific and regulatory communities. LC-MS, in particular, is becoming increasingly accepted as a viable monitoring tool when used in conjunction with suitable screening methods such as ELISA and RBA [160]. The utilization of these methods in regulatory programs would allow, in some cases, both the adoption of more protective safety limits and increased animal welfare, leading to greater protection of human health.

Trade and economic considerations accompany the health and scientific concerns that impact the alacrity of the switch from the mouse bioassay to alternative methods. Major importers such as the US and EU, two out of the three top importing countries [23], have influence over the speed at which the alteration in regulatory structure occurs. The EU has been particularly active in discussing and implementing alternative marine biotoxin testing methods; however, little regulatory change or discussion of change is available for the US. The alacrity or lagging of acceptance of alternative methods to the mouse bioassay by the ISSC, and incorporation of these into the NSSP guidelines, will influence the speed at which these methods are adopted internationally once validated, and so an increased discussion of these plans would be desirable in order to facilitate the timely and least costly approach to adopt more progressive marine biotoxin testing methods. 72

Acknowledgements

Thank you to my family and friends, especially my parents, husband and sister, who supported and put up with me during the process of writing this thesis. Their support, along with that of my Director of Studies, Dr. Lauren McMills, thesis advisor

Dr. Hao Chen, Assistant HTC Dean Jan Hodson and the rest of the HTC College staff, made this work possible. My friend Dan Aldridge has my ever-lasting appreciation and praise for his invaluable assistance editing and nitpicking the final draft of this paper. Special gratitude goes out to the personnel at Interlibrary Loan who fielded my

(many) requests for oceanic-related articles from a land-locked college. 73

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