JPR Advance Access originally published online on October 11, 2004
Journal of Plankton Research 2004 26(12):1369-1377; doi:10.1093/plankt/fbh155
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Journal of Plankton Research Vol. 26 No. 12 © Oxford University Press 2004; all rights reserved
HORIZONS |
How to assess toxin ingestion and post-ingestion partitioning in zooplankton?
1 School of Marine Science and Technology, University of Newcastle upon Tyne, Ridley Building, Claremont Road, Newcastle upon Tyne NE1 7RU, UK, 2 Environment Canada, National Water Research Institute, Burlington, Ontario, Canada L7R 4A6 and 3 University of Calgary, Division of Ecology, Department of Biological Sciences, Calgary, Alberta, Canada T2N 1N4
* Corresponding Author: swatson{at}ucalgary.ca; sue.watson{at}ec.gc.ca
Received August 17, 2004; accepted in principle August 25, 2004; accepted for publication October 6, 2004; published online October, 2004
ABSTRACT
Algal toxins can have severe impacts on marine and aquatic ecosystems and often bioaccumulate through the food chain. One of the major obstacles facing research in this area is the lack of standardized and realistic techniques with which to probe algal–grazer interactions. Here we present a synopsis on current and innovative techniques which can be used to monitor the ambient concentration and fate of algal toxins in grazers and determine their eventual target within the grazers’ tissues. We focus on a newly recognized class of oxylipin toxins (volatile polyunsaturated aldehydes and oxo-acids), which have been identified from lipid-rich microalgae. While some workers have reported that these biotoxins have negative effects on fecundity and recruitment of metazoan grazers, this mechanism is currently under debate as a result of conflicting evidence. We argue that the use of a variety of non-standard and often invalid or unrealistic assays has confounded our ability to scrutinize this mechanism systematically.
INTRODUCTION
The efficiency of energy transfer from primary producers to higher trophic levels will ultimately determine aquatic ecosystem productivity. Abiotic and biotic factors which modify this energy transfer (e.g. temperature, light, nutrients, anthropogenic contaminants, predation, disease, etc.) have long been the subject of extensive research. This has resulted in the development of mechanistic and empirical models to predict productivity at individual food web or ecosystem levels and provided the basis for the management, remediation and/or commercial exploitation of these resources (Hansen et al., 2003
; Kantha, 2004). However, many of these models have limited success as a result of considerable unexplained variance, and more recently, the potentially significant role of chemical ecology in moderating energy transfer has been recognized (Watson and Cruz-Rivera, 2003).
Algae produce a huge range of secondary metabolites (Carmichael, 1997; Hay and Kubanek, 2002; Cembella, 2003; Watson, 2003). For most of these compounds, it is still not known whether they function as semiochemicals (i.e. are bioactive) or simply represent metabolic waste, but an increasing number are now recognized to act as potential pheromones, kairomones or allelogens (Cembella, 2003; Watson, 2003). Research in this area faces two major problems. It is often extremely difficult to identify or measure these chemicals, many of which are released at ultra trace levels in response to specific environmental cues (Pohnert, 2002; Pohnert and Boland, 2002; Pohnert et al., 2002). It is also difficult to demonstrate their effects on other biota, particularly where these are delayed or indirect (Gross, 2003).
Many algae produce toxic metabolites which deter grazing (Shaw et al., 1995), cause grazer mortality (Blom et al., 2001; Granéli and Johansson, 2003) or limit grazer reproductive fitness (Ianora et al., 2003; Miralto et al., 2003), and eventually, this affects ecosystem productivity. However, the complexity of food web interactions can obscure these allelogenic effects, particularly since grazer mortality and fecundity can be affected by numerous other related or unrelated factors. In an early review of the subject, Willis (1985) outlined a number of general requirements that should be fulfilled to conclusively demonstrate allelopathy. We propose that these criteria can be modified and applied to algal–grazer chemical interactions as a standard means of demonstrating these effects. Specifically, the following must be established: (i) an inhibitory effect on the grazer (growth, feeding, reproduction, etc.), (ii) the production of an algal toxin, which can be isolated and (preferably) chemically identified, (iii) a mode of toxin transfer from the algal cell to the target animal, (iv) a means of toxin introduction and/or storage by the target animal and (v) that the observed inhibitory pattern cannot be explained solely by physical or alternative biotic factors.
To fulfil these requirements, which cover molecular processes through to feeding ecology, it is necessary to integrate interactions at organism, tissue and molecular levels. This requires the development of new and innovative techniques. Concepts discussed in this article are applicable to any algal toxin. Here we focus on microalgal-derived polyunsaturated fatty acid (PUFA) derivatives (oxylipins) and their effects on invertebrate grazers, a chemical interaction that is currently the subject of considerable debate.
The highly cytotoxic C7–C10 unsaturated aldehydes (2,4-heptadienal, 2,4-octadienal, 2,4- and 2,6-nonadienal, 2,4-decadienal and 2,4,7-decatrienal) are produced during the enzymatic oxidative catabolism of cell PUFAs. Enzyme action is typically initiated by the loss of cell membrane integrity, for example, during senescence, cell death or physical disruption by grazers (Jüttner, 1995, 2001). This process has long been identified in plant tissues as a primary wound response and as a primary cause of rancid off flavours in PUFA-rich food (e.g. grains, oils and nuts) and drinking water (Galliard, 1978). More recent work has identified these aldehydes among marine and freshwater microalgae with high cell lipid content: diatoms (Jüttner, 1995, 2001; Miralto et al., 1999; d’Ippolito et al., 2002a; Pohnert et al., 2002), synurophytes, chrysophytes, cryptophytes, dinoflagellates (Watson et al., 2001; Watson and Satchwill, 2003; Watson, 2003) and prymnesiophytes (Hansen et al., 2004), and a number of these studies have recognized the potential semiochemical role of these compounds. Recently, field-collected phytoplankton samples dominated by Thalassiosira rotula were identified as producing oxylipins at a concentration of 47.7 fmol cell–1, which was much higher than the cultured strain commonly used in laboratory feeding assays (Wichard et al., in press). However, there are pronounced inter- and intra-specific differences in oxylipin production which may reflect the presence or absence of genes coding for catabolic enzymes and/or the effects of algal physiological status (Pohnert et al., 2002; Watson and Satchwill, 2003). Some authors have proposed that these oxylipins can regulate populations of metazoan grazers (notably calanoid copepods) by inhibiting embryogenesis, hatching and post-embryonic fitness (Miralto et al., 1999; Ianora et al., 2004). Laboratory investigations have described a range of symptoms and pathologies associated with ingestion or exposure to oxylipin-producing algae and purified toxins, ranging from decreased egg production (Ianora et al., 1996), abortion (Chaudron et al., 1996), teratogenesis (Tosti et al., 2003; Lewis et al., 2004), cellular apoptosis and necrosis (Miralto et al., 1999; Poulet et al., 2003; Romano et al., 2003) and fertilization failure (Caldwell et al., 2002, 2004; Tosti et al., 2003). The oxylipins with Michael acceptor properties, i.e. with at least one double bond conjugated to the aldehyde functionality, are broadly cytotoxic, creating a highly active nonspecific chemical reactivity towards nucleophilic biomolecules (Adolph et al., 2004).
In addition to variation in production of oxylipins by algae, there is also marked variation in the responses of grazers to feeding on toxic species (Ban et al., 1997). Most studies have failed to account for the potential of grazers to detoxify oxylipins after ingestion. Detoxification often involves breaking a toxic molecule into smaller components; however, the detoxification products may be more toxic than the initial compounds (Kashian, 2004). This raises a number of important questions: (i) Do oxylipins accumulate within metazoan tissues? (ii) Where and in what form do they accumulate? And, (iii) are modified oxylipins more or less biologically active? It has been further suggested that these oxylipins are concentrated in the metazoan oocytes during vitellogenesis, a hypothesis that has remained untested (Poulet et al., 1994). Toxic properties of these oxylipins have been under investigation in a number of laboratories and have been the subject of a recent international workshop (Paffenhöfer et al., in press); however, several authors have questioned the ecological relevance of the protocols employed in these assays and the applicability of laboratory observations to a field situation (Jónasdóttir et al., 1998; Irigoien et al., 2002). Sensitive and practical analytical techniques have now been developed which allow algal oxylipin concentrations to be determined from the field (Ianora et al., 2004; Wichard et al., in press) and should be used in all future studies concerning oxylipin effects on grazers. A study similar in geographical scope to that of Irigoien et al. (Irigoien et al., 2002) but with a distinct chemistry emphasis would provide a major contribution to the field, not least in providing more information on realistic toxic concentrations to use in a laboratory context.
The key problem facing this research has been the lack of an effective method that can deliver these compounds into the body of small aquatic animals via a normal route of exposure (e.g. the gut) and then follow their eventual fate and effects. In this article, we first review and evaluate the existing protocols and then suggest new approaches to toxin delivery to improve both the ecological relevance and the accuracy of future laboratory-based studies. We also suggest methods which can be used to rigorously test the ‘accumulation via vitellogenesis’ hypothesis first posed by Poulet and coworkers (Poulet et al., 1994).
The majority of laboratory studies which have assayed oxylipins have used direct aqueous immersion exposure (Miralto et al., 1999; Caldwell et al., 2002, 2003; d’Ippolito et al., 2002a; Adolph et al., 2003; Ceballos and Ianora, 2003; Romano et al., 2003; Tosti et al., 2003), whereby adults, larvae or embryos are incubated with toxin solutions. The ecological relevance of this approach is highly questionable. Since these oxylipins are only produced upon PUFA breakdown and are highly cytotoxic, they are not produced intracellularly in healthy cells but primarily released upon cell damage and/or death. They are highly labile and breakdown rapidly upon release, occurring typically at ppb levels or less in natural surface waters, even following the decay of a large bloom, while concentrations used in direct immersion assays have been an order of magnitude higher, in the order of 1 ppm (Watson, 2003). In fact, under natural conditions, exposure to such toxins is likely to be highly localized, primarily via the grazer’s digestive system as opposed to an indiscriminate whole organism exposure.
Direct feeding on toxic species
The traditional approach to the study of ingesting toxic algae has used direct grazing experiments (Chaudron et al., 1996; Dutz, 1998; Lincoln et al., 2001; Turner et al., 2001, 2002). This is an effective and ecologically relevant method for toxin delivery and assessment as it closely imitates natural feeding conditions. There are, however, several drawbacks to this approach. In research with toxins that affect reproductive output, there is always the confounding issue of nutritionally linked effects (Jónasdóttir et al., 1998). The relationship between nutritional condition and reproductive fitness necessitates that all such experiments be verified by nutritional analysis, both of the algal diet and the grazer. Equally, such experiments must consider feeding rates and efficiency and the acceptability of any given algal species to the grazer. Differences in feeding modes among grazers will potentially result in different levels of toxin transfer from a given diet and are an important consideration. Prey-specific ingestion or avoidance can vary considerably among closely related animal taxa and age classes, as well as between functionally distinct groups such as raptorial copepods and filter-feeding cladocerans (Turner et al., 2002). A number of grazing-based models based on mixed diets have been proposed to differentiate between a toxic diet and a nutritionally inadequate diet (Jónasdóttir et al., 1998; Turner et al., 2001; Colin and Dam, 2002). While this may identify a diet as toxic, it will not provide information on the fate of the toxins, which is a fundamental theme of this paper. To determine the response of a grazer to a known concentration of ingested toxin, there must be parallel chemical analysis of the diet, as algal production of lipid-based (and other) toxins varies with age, growth conditions and resource availability (Watson and Satchwill, 2003). A substantial benefit of using alternative delivery vehicles such as microencapsulated diets is that nutritive quality control and the concentration of toxins could be precisely managed and manipulated independent of the variable physiological condition of algal cultures. Importantly, a standard toxin-laced diet can be distributed and tested independently by any investigator with results directly comparable to those of other laboratories/studies.
Use of algal cells as transfer vehicles
Intact algal cells have been experimentally enriched with specific compounds and fed to zooplankton. This approach was first developed by von Elert (von Elert, 2002) in nutritional studies, using fatty-acid-enriched cyanobacteria and diatoms fed to Daphnia. A similar approach has been used more recently by Ianora et al. (Ianora et al., 2004) in toxicological studies of unsaturated aldehydes whereby decadienal was adsorbed onto the surface of a non–oxylipin-producing dinoflagellate and fed to marine copepods. This approach has considerable potential as a means of delivering a known quantity of toxin under semi-natural conditions as the efficiency of adsorption and aldehyde longevity can be calculated, thus allowing the quantities of aldehydes ingested to be determined (Ianora et al., 2004). Such enrichment experiments must be rigorously designed to verify quantitatively the efficiency of both the compound enrichment and delivery (i.e. prey acceptability) for the grazers.
Nutritional studies have used complex lipid emulsions to investigate PUFA limitation in zooplankton (DeMott and Mûller-Navarra, 1997; Sundbom and Vrede, 1997; Weers and Gulati, 1997; Boersma et al., 2001; Park et al., 2003). Other approaches have utilized carriers such as gum arabic-coated lipid microcapsules (Goulden et al., 1998) or bovine serum albumin (Von Elert and Wolffrom, 2001) to deliver fatty acids. Liposomes have been used extensively as chemical or pharmaceutical delivery agents over the past 30 years (Bally et al., 1988) and, more recently, have been applied in feeding experiments. These artificial vesicles have a basic membrane structure of phospholipids and cholesterol which resembles that of living cells and is readily bioavailable to grazers such as Artemia nauplii (McEvoy et al., 1996; Ozkizilcik and Chu, 1994), Daphnia (Ravet et al., 2003) and filter-feeding molluscs (Heras et al., 1994). Liposomes are therefore well suited to serve as carriers in experimental diets; both the vesicle and membrane can be modified with lipophilic and other molecules for use in toxicity studies. A major benefit of lipid emulsions and liposomes is that they are relatively easy to make and manipulate within the laboratory (Philippot et al., 1983) and may also allow for parallel testing of the combined effects of varied nutritional content and toxins on invertebrate reproductive fitness. Liposomes would also permit combinations of oxylipins to be tested, thereby reflecting the diverse oxylipin species and concentrations produced by algae. A disadvantage of this approach is that liposomes of >6 µm diameter are unstable over multiday experiments (Ravet et al., 2003); however, for shorter-term incubations, liposomes would provide an effective carrier vehicle.
Microparticulate diets were developed primarily as larval aquaculture feeds (López-Alvarado et al., 1994; Jones, 1998; Langdon, 2003) and have been successfully used in nutritional studies with calanoid copepods (Kumlu, 1997). This approach has been used to identify the key factors controlling crustacean larval ingestion, digestion, energetic requirements and nutrition and to deliver bioactive compounds (hormones, amino acids and vitamins) to larvae (Yúfera et al., 2003). The most effective of these is protein microencapsulation. This has been used to determine the toxicity of diesel oil to mussels (Strømgren and Nielsen, 1991) and permethrin to Daphnia (Sibley and Kaushik, 1991). Importantly, microencapsulation has proven to be effective at delivering volatile chemicals such as 2-ethyl-1-hexanol and benzyl alcohol to target organisms in food form (Arneson et al., 1995; Goubet et al., 2001). An added benefit of microparticulate diets is that the nutritional content can also be experimentally manipulated, allowing parallel testing of the effects of nutrition on invertebrate reproduction.
In summary, there is a clear need to systematically assess the most suitable toxin delivery vehicle(s), which should be simple, robust and verifiable. The most promising delivery agents to date are liposomes and microparticulates, but this area of research would benefit greatly from the continued development of innovative new approaches. In the interests of comparison and continuity with previously published studies, future investigations should also include aqueous immersion exposure to provide a benchmark for toxicity levels until an alternative standard protocol is developed and sufficient data is generated.
QUANTIFYING TOXIN INGESTION AND MAPPING TOXIN PARTITIONING WITHIN TISSUES
In any toxicological study, it is important to discriminate the target tissues. Thus, to test the ‘accumulation via vitellogenesis’ hypothesis, we must characterize the transport and fate of toxic oxylipins. This is a difficult problem, as many of the techniques available have been developed for much larger animals than zooplankton. Scaling techniques, e.g. whole-organism autoradiography down to the size of a copepod, as such represent a tantalizing challenge. With this in mind and with respect to the broader implications of oxylipin cytotoxicity (e.g. human health), it may be more pertinent to initially apply the following techniques to biological models other than zooplankton. The application of alternative models has yielded valuable information concerning the activity of these molecules, e.g. in tunicates (Tosti et al., 2003), oysters and yeast (Adolph et al., 2004). We suggest that physically larger animals such as filter-feeding bivalves be used, which have the added benefit of being ecologically relevant, as planktonic microalgae represent a major dietary component. Equally, there already exists considerable literature on the interactions between bivalves and other phycotoxins such as saxitoxin and domoic acid. Mussels in particular can also be applied in both marine (Mytilus sp.) and freshwater (Dreissena sp.) situations. We suggest the filter-feeding planktonic crustaceans Daphnia magma and adult Artemia salina as intermediate model species. This will allow techniques to be refined prior to application to what would inevitably be the more difficult task of working with copepods and other small zooplankton.
Gas chromatography mass spectrometric analysis of biological samples
Gas chromatography mass spectrometry (GC–MS) has been an essential analytical tool in the identification of volatile products from biological samples (Jüttner, 2001; d’Ippolito et al., 2002a; Pohnert et al., 2002). A number of extraction techniques, both solvent based and solvent-less, have been developed for analysis of volatile compounds from microalgae (Miralto et al., 1999; Watson et al., 1999; Pohnert, 2000; d’Ippolito et al., 2002b; Pohnert et al., 2002). The same principals can be applied to determining the presence, distribution and quantity of oxylipins in grazers’ tissues. This approach would offer the potential for routine and economical sampling of field material to complement both field-based studies on grazer reproductive success and in situ oxylipin production by algae. Initial attempts to develop this approach have shown some promise (Fig. 1) (Caldwell, 2004), although polyunsaturated aldehydes were not detected. This may indicate that the aldehydes are rapidly transformed into other compounds such as non-volatile adducts or bound to biological molecules because of their high reactivity, in which case direct gas chromatography mass spectrometric analysis of grazer tissues would prove an ineffective measure. In any of these studies, it is extremely important to note that oxylipins occur naturally within some invertebrate species (Zeeck and Hardege, 1990). It is therefore necessary to eliminate the possibility that these compounds are produced by the grazer itself, or as an artefact in the sample. This can be accomplished by prescreening with starved specimens of the target animals, thereby ensuring that algal-derived oxylipins are not present in the gut, or by denaturing the grazer’s lipoxygenase enzymes prior to analysis. Detection of algal-derived labile oxylipins in grazer tissues could be enhanced by applying derivatizing agents such as dimethyl disulfide (Ho et al., 1996), (carbetoxyethylidene)-triphenylphosphorane (d’Ippolito et al., 2002b) or pentafluorobenzylhydroxylamine (Ianora et al., 2004).
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Stable isotopes of an element maintain the same chemical characteristics and therefore react in the same way. There are a wide range of isotopically labelled compounds available, and in many cases, those not yet available can be custom synthesized. In fact, custom synthesis allows the number of labelled atoms and their position within the molecule to be specified, which is a valuable property when seeking to study the potential catabolic products of the original molecule. For example, 13C-labelled oxylipins can be fed to grazers during vitellogenesis and the isotope enrichment can be determined in resultant oocytes and larvae. Adult tissues including gonads, spawned oocytes and larval stages can by analysed by GC–MS alone, or coupled with isotope ratio mass spectrometry (IR-MS), an approach that is used widely for the study of chemical metabolism. This would quantitatively determine 13C enrichment in each fraction, enabling the fate and possible end products of oxylipin ingestion to be traced. A protocol has been developed to incorporate isotopic markers into algal-derived oxylipins (Pohnert et al., in press), therefore making their application in tracer studies highly feasible. The major drawback of this approach is the initial costs associated with compound synthesis. Background noise is likely to be relatively unimportant because of the enriched nature of the samples.
Radiolabelled compounds have been used to study polychlorinated biphenyl (PCB) toxin bioaccumulation in marine invertebrates (Danis et al., 2003) and have also been used in previous studies of the fate of algal biotoxins in surface waters (Hyenstrand et al., 2003). Whole-body autoradiography (WBA) was developed in the 1950s, enabling a detailed and complete visualization of the distribution of an administered compound throughout the entire body. However, the procedure was complex and time consuming and contained a number of inherent weaknesses including a narrow dynamic range and poor sensitivity to the key isotopes, 14C and 3H. Storage-phosphor autoradiography (SPA), which produces a quantifiable luminescent reaction when exposed to a laser beam (Kanekal et al., 1995), was an improvement on this approach, but as with WBA, SPA has a number of weaknesses that do not make it a suitable technique for tiny samples (Walker et al., 2001). Dissection autoradiography is a semi-quantitative technique to determine the distribution of a radioisotope throughout an entire animal (Walker et al., 2001). It is significantly less labour intensive that WBA or SPA; however, sensitivity is decreased. The use of radioisotopes therefore appears to be a less feasible approach than using stable isotopes because of lack of sensitivity for small organisms. Furthermore, this approach is particularly inappropriate as a means of tracing the fate of oxylipins in animal tissues because of the rapid metabolism of oxylipins. Therefore, tissue uptake of a radioisotope may not reflect any direct links with these compounds.
Chemical and fluorescent signatures
Fluorescent approaches have been developed to assess exposure of aquatic invertebrates to toxicants (Leweke et al., 1996). However, in parallel, it must also be clearly demonstrated that attaching a probe to a toxin does not affect its biological activity. The confocal laser scanning microscope (CLSM) utilizes fluorescent probes and has been applied to study factors affecting the reproductive and developmental biology of Crustacea (Zupo and Buttino, 2001; Buttino et al., 2003, 2004; Poulet et al., 2003; Romano et al., 2003). A significant benefit with this technique is the generation of three-dimensional images. When coupled with specific fluorescent probes, the CLSM represents a powerful tool to image and map molecular interactions from the subcellular to organism scale. A number of fluorescent probes have been developed that have allowed unparalleled visualization and semi-quantification when bound to a specific compound (Chang and Lu, 2000). If such a probe could be coupled with a toxin, the CLSM would represent an ideal tool to visually and potentially quantitatively map the fate of a toxin at cellular, tissue and organism scales. For example, the oxylipin 2E,4E-decadienal is known to form adducts with 2'-deoxyadenosine and DNA in mammalian cells by epoxidation and subsequent addition to the N2 amino group (Carvalho et al., 1998, 2000; Loureiro et al., 2000). The formation and measurement techniques for DNA etheno adducts have been reviewed by Martinez et al. (Martinez et al., 2003). The adducts are highly fluorescent and can be isolated by reverse-phase high-performance liquid chromatography and may therefore represent a potential fluorescent biomarker for oxylipin exposure. This approach is promising and could be verified by an isotope tracer technique. However, it is as yet highly specific to interactions of decadienal with specific nucleotides; adducts with, for example, proteins would not be observed as fluorescently active products. An alternative microscopy technique is optical projection tomography (OPT), which is more capable of mapping larger specimens than the CLSM (Sharpe, 2003). However, specimens must be fixed for visualization with CLSM and OPT. Selective plane illumination microscopy (SPIM) is a novel technique which can visualize live specimens (Huisken et al., 2004). SPIM has two further advantages over its rivals, namely it is less complicated to build than a confocal microscope and has greater penetrative depth; however, SPIM is not yet commercially available.
If our understanding of the interactions between toxic microalgae and their grazers is to develop, there needs to be a truly multidisciplinary collaboration between biologists, chemists and technologists to develop and implement reliable and verifiable methods. The techniques should also be scalable; in other words, they should be able to move from initial laboratory-based development to mesocosm experiments and eventually be applied to direct field studies. A number of powerful techniques are available to map the location of toxins within zooplankton. The approach with the most immediate utility is the application of stable isotopes; however, with the continuing rapid development of microscopy and imaging technologies, the need for spectroscopy may be superseded by quantitative fluorescent microscopy. Advances are currently being made with regard to the biochemistry, biosynthesis and molecular biology of toxic oxylipins from algae (T. Wichard, personal communication). Once specific primers, antibodies and molecular probes are available, genetic techniques such as knockout genes and protein biochemistry may be applied to study the effects of oxylipins on grazers. However, before molecular and genetic techniques can be applied with confidence, numerous obstacles with regard to analytical chemistry must be overcome. The combination of controlled toxin delivery methods with high-resolution analytical chemistry/microscopy will help clear the current controversy surrounding the effects of oxylipins on invertebrate reproduction. We recommend that stable isotope-labelled oxylipins transferred in microencapsulated diets or liposomes be fed to mussels, daphnids and adult Artemia sp. before application to the more logistically challenging copepods. With this approach, nutrition and toxin content can be carefully manipulated and the biological fate of the toxins can be accurately determined. Points 1–3 of the modified Willis requirements have already been fulfilled (see above); the experimental approaches described provide a means to critically examine point 4.
ACKNOWLEDGEMENTS
We are grateful for financial support from the Natural Sciences and Engineering Research Council of Canada, Environment Canada, the Ontario Water Works Research Consortium and the University of Newcastle upon Tyne. We also acknowledge the benefits of lively discussions with the participants of the Colloquium on Diatom–Copepod Interactions, Ischia, 2002.
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