General bioassay procedures

Finally, in a number of instances bioassays were carried out in a greenhouse (Hall et al. 1982, 1983) or in growth chambers (PPFD of 240-650 timol/m /s Waters and 

2 Plant-Plant Allelopathic Interactions. Phase I The Laboratory 

Blum 1987 Blum and Rebbeck 1989 Klein and Blum 1990b Holappa and Blum 1991 Lehman and Blum 1997, 1999a, b Shafer et al. 1998) where materials and methods described above were modified to meet specific experimental objectives. For details see references cited. 

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The mouse bioassay, an indirect assay, historically has been used to evaluate shellfish toxicity (especially for PSP). Other bioassay procedures have been developed but not generally applied for regulatory purposes (Schantz et al., 1958). The mouse bioassay involves intraperitoneal (i.p.)... 

Since the 1950s, the Canadian-United States Conference on Shellfish Toxicology endorsed a mouse bioassay that was based on the use of purified toxins and has historically been the most universally applied technique for examining shellfish (especially for PSP) other bioassay procedures have been developed but not generally applied. The intraperitoneal minimal lethal dose of the toxin for the mouse was 9 pg kg body weight. The intravenous minimal lethal dose for the rabbit was 3-4pgkg body weight. The minimal lethal dose of the toxin for humans is estimated to be between 1 and 4 mg. 

A cell-based bioassay procedure generally includes cell growth in suspension or adherent cultures for several days/weeks. In a typical assay, cells are activated/inhibited with the therapeutic protein of interest in a multiwell assay... 

The more classical approach to assess the presence of marine biotoxins in seafood is the in vivo mouse bioassay. It is based on the administration of suspicious extracted shellfish samples to mice, the evaluation of the lethal dose and the toxicity calculation according to reference dose response curves, established with reference material. It provides an indication about the overall toxicity of the sample, as it is not able to differentiate among individual toxins. This is a laborious and time-consuming procedure the accuracy is poor, it is nonspecific and generally not acceptably robust. Moreover, the mouse bioassay suffers from ethical implications and it is in conflict with the EU Directive 86/609 on the Protection of Laboratory Animals. Despite the drawbacks, this bioassay is still the method of reference for almost all types of marine toxins, and is the official method for PSP toxins. 

Many questions remain as to the most appropriate extraction or concentration procedures. Nevertheless, residues from similar water sources have now been obtained by using a variety of techniques. The patterns of mutagenic activity obtained from these residues are sufficiently similar so that it is clear the mutagens are generally not artifacts of concentration. Chemical assays and bioassays of these mixtures typically reveal that among the thousands of largely unknown compounds present at low concentrations, there is a diverse minority of mutagens. 

The general procedure for seedling bioassay was as follows 25 seeds of each species were placed per dish, excepting Hordeum vulgare (10 seeds per dish), with 5 mL of the test solution, and incubated in the dark at 25°C. Four replicates for each concentration were set up. Germination and growth time varied for each plant species Lepidium sativum, 3 days Lactuca sativa and Hordeum vulgare, 5 days and Allium cepa, 7 days. 

In practice, animal bioassay data are generally the primary data used in risk assessments. Animal studies are well-controlled with known exposures, and they are carried out with detailed, careful clinical and pathological examinations. The use of laboratory animals to determine potential toxic effects in humans is a necessary and accepted procedure. It is a recognized fact that effects in laboratory animals are usually similar to those observed in humans at comparable dose levels. Exceptions are primarily attributable to differences in the pharmacokinetics and metabolism of the xenobiotics. 

Detailed information on procedures for the collection, handling and preservation of aqueous and sediment samples is given in a series of ASTM (1994a,b) and ISO (1990, 1991, 1992a,b, 1994, 1995, 1998a,b, 1999, 2000) documents. General issues for the collection, storage and preparation of environmental samples for bioassays that need to be considered are summarised below ... 

Bioassays for BT toxin are sensitive because the test animals function as a concentrator and a detector simultaneously. When exposed to very low concentrations, the toxin is accumulated as the animal feeds over a period of days. Compared to immunoassays, bioassays for the BT toxins are generally 50-100 times more sensitive (Table II). As with other residue procedures, sample work-up can be employed to extend the detection limits of an enzyme immunoassay and to remove interfering materials at the same time. Such work up will be essential if immunoassays are to be as sensitive as bioassays using filter feeding organisms. 

This chapter provides an outline of the general approach used in the author s laboratory to purify marine natural products. An outline is given in Fig. 1. In our approach, all separations are carried out on a small scale until pure compounds are reproducibly isolated. A variety of purification methods are used to ensure isolation of all compounds of interest, and all fractions are characterized by either bioassay, NMR, HPLC, and/or TLC. Our overall procedure includes the following steps ... 

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