Bioassay standard

Pica-Granados, Y., Trujillo, G.D. and Hernandez, H.S. (2000) Bioassay standardization for water quality monitoring in Mexico, Environmental Toxicology 15 (4), 322-330. 

Figure 8.2. Molybdenum bioassay standard series. Arranged left to right are A. niger (M) cultures grown with nil, 0.0001, 0.0002, 0.001, 0.002, 0.005, 0.01, 0.02, and 0.05 ig Mo per 50mL of culture solution.

Naphthalene Acetic Acid and Naphthalene Acetamide. Naphthalene acetic acid [26445-01-2] (38) is historicaHy one of the first plant growth regulators. Reports concerning its activity in crops and plants have been a subject in much of the eady Hterature (57). Consequently, it has been used as a starting matedal for other compounds, eg, vide infra Sevin. Naphthaleneacetamide [31093-43-3] (39) has been used as a standard matedal to evaluate abscission pnor to 1953 and its effect on apple drop was reported in 1953 (58). The substance is used as an internal standard in the abscission bioassay (59). 

Specifications for hGH products are defined by the governmental licensing authorities, eg, the U.S. Pood and Dmg Administration. Draft monographs for hGH have been prepared by both the EnitedStates Pharmacopia and the European Pharmacopeia commissions and should be formally adopted by 1995. These specifications are suitable for biosynthetic hGH. The much less purified pituitary-derived hGH has virtually disappeared from commercial production. An international reference standard for pituitary-derived hGH (lot 80/505) has been used for caUbration, particularly for bioassay purposes. A highly purified biosynthetic hGH standard (lot 88/624) has been prepared and should be formally adopted by 1995, or before. 

Maximum acceptable concentiation figures expressed as fractions of 96 h for most sensitive species in given area. The 96 h is that concentration of a substance which kills 50% of the test species within 96 h under standard bioassay conditions. 

A listing of compounds evaluated in the laboratory as cockroach repeUents summarizes 872 synthetic compounds out of 901 bioassayed from 1953 to 1974 (43). EenchoHc acid [512-77-6] (3-isopropyl-1-methylcyclopentanecarboxyHc acid) has been used as a standard repeUent in tests conducted by placing 20 cockroaches in a glass crystallizing dish without food and water and offering them a choice of two cardboard shelters, one of which was treated with 1 or 2 mL of a 1% solution of the candidate in acetone. Counts were made daily for seven days. 

The impact that a silver compound has in water is a function of the free or weaMy complexed silver ion concentration generated by that compound, not the total silver concentration (3—5,27,40—42). In a standardized, acute aquatic bioassay, fathead minnows were exposed to various concentrations of silver compounds for a 96-h period and the concentration of total silver lethal to half of the exposed population (96-h LC q) deterrnined. For silver nitrate, the value obtained was 16 )-lg/L. For silver sulfide and silver thiosulfate complexes, the values were >240 and >280 mg/L, respectively, the highest concentrations tested (27). 

One unit of bacitracin USP is defined as the activity given by 26 micrograms of the dried FDA master standard. The USP requirement for commercial bacitracin is a potency of at least 40 units per milligram in a bioassay with Micrococcusflavus or Sarcina lutea. Pure bacitracin A has a potency of about 100 units per milligram. Standards for animal feed grades of bacitracin are available (71,81,82) as are analytical methods (67). 

Toxicity Test (Bioassay). Organisms representative of those to be protected are exposed to the test water under rigorously controlled conditions, usually in a laboratory environment. In this test the organisms, normally fish, are exposed for a standard time period in aquaria to various dilutions of waste or river water while some physiological parameter is carefully monitored to determine fish response. Behavior is also observed. 

Methods. As discussed in the previous chapter, a number of approaches have been used to assess the presence of potentially toxic trace elements in water. The approaches used in this assessment include comparative media evaluation, a human health and aquatic life guidelines assessment, a mass balance evaluation, probability plots, and toxicity bioassays. Concentrations of trace elements were determined by atomic absorption spectrometry according to standard methods (21,22) by the Oregon State Department of Environmental Quality and the U.S. Geological Survey. 

Toxicity Bioassay. Ninety-six hour acute toxicity tests were conducted on the effluent streams of major industries. A static renewal procedure was used in which waste waters of various dilutions were renewed at 24 hour intervals over a 96 hour period. Rainbow trout was used as the test organism. Tests were conducted at 13°C in 20 liter aquaria according to standard procedures (22), Results are summarized in Table 8. Chemical and toxicity test results indicate that the trace element quantities identified in Table 8 are not acutely toxic under the prevailing conditions and unlikely to pose an acute threat to aquatic life. In this case a chronic toxicity assessment would require additional research. 

The mouse bioassay for PSP, described in its original form by Sommer in 1937 (29), involves i.p. injection of a test solution, typically 1 mL, into a mouse weighing 17-23 g, and observing the time from injection to death. From the death time and mouse weight, the number of mouse units is obtained by reference to a standard table 1 mouse unit is defined as the amount of toxin that will kill a 20-g mouse in 15 min (77). The sensitivity of the mouse population used is calibrated using reference standard saxitoxin (70). In practice, the concentration of the test solution is adjusted to result in death times of approximately 6 min. Once the correct dilution has been established, 5 mice will generally provide a result differing by less than 20% from the true value at the 95% confidence level. The use of this method for the various saxitoxins and indeterminate mixtures of them would appear... 

Mouse Bioassay. The mouse is the traditional animal of choice for detecting biological activity due to STX and TTX. Mice receive an intraperitoneal injection of sample and are observed for symptoms of intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity of STX and TTX in numerous samples. For the standard STX assay, one mouse unit is defined as that quantity of STX injected i.p. in 1 ml solution that will... 

Given the problems associated with using standard animal bioassays and doses above the level where hormetic effects might be observed (which... 

Improved Methods for Collection, Bioassay, Isolation, and Characterization of Compounds. Techniques used to characterize natural products are evolving rapidly as more sophisticated instrumentation is developed. Plant physiologists and chemists should work closely together on this aspect, since rapid and reproducable bioassays are essential at each step. There is no standard technique that will work effectively for every compound. Briefly, isolation of a compound involves extraction or collection in a appropriate solvent or adsorbant. Commonly used extraction solvents for plants are water or aqueous methanol in which either dried or live plant parts are soaked. After extracting the material for varying lengths of time, the exuded material is filtered or centrifuged before bioassay. Soil extraction is more difficult, since certain solvents (e.g. bases) may produce artifacts. 

Several chemical-assay methods (15,23,50) for tetraethyl pyrophosphate were recently developed and applied by seven collaborating laboratories to samples of representative commercial products and to a sample of purified tetraethyl pyrophosphate which served as a common standard. Concordant results, which correlated well with bioassay results,... 

CTAs are possible in vitro alternatives to the standard approach for the assessment of carcinogenicity (the 2-year bioassay in rodents), which have been shown to be a multistage process able to model the most important stages of in vivo carcinogenesis [50]. CTAs are faster and more economic than in vivo assay and they could be a valid and useful screening tool for chemicals. 

The potential for a compound to induce carcinogenicity is a crucial consideration when establishing hazard and risk assessment of chemicals and pharmaceuticals in humans [53]. To date, the standard approach to assess carcinogenicity at a regulatory level is the 2-year bioassay in rodents. According to the recent REACH... 

All bioassays are comparative in nature, requiring parallel assay of a standard preparation against which the sample will be compared. Internationally accepted standard preparations of most biopharmaceuticals are available from organizations such as the World Health Organization (WHO) or the United States Pharmacopeia. 

Ecotoxicity assessment of water samples was carried out, in parallel to chemical analysis, using three standardized bioassays based on the micro-crustacean Daphnia magna, the algae Pseudokirchneriella subcapitata, and the bioluminescent bacteria Vibrio fischeri. 

For standardised instrumental analytical methods, i.e. biomarkers, biosensors and bioassays, there are well-established standard protocols on the national level, e.g. under Association Francaise de Normalisation (AFNOR), British Standard Institute (BSI), DIN (German Organisation for Standardisation), etc., and all those standards are formed by ISO-Working Groups and by validation studies into ISO - and CEN - Standards. Normal accredited and well-qualified laboratories should be able to perform the monitoring. 

The ISO protocol for the biochemical response EROD (ISO 23893-2/AWI) as a recent example of a bioanalytical (biomarker) [49,50] method standardised under ISO for fish needs harmonisation with the other test systems and between the laboratories (users) before implementation. Use of biomarkers (biochemical responses) in multi-arrays for environmental monitoring according to Hansen et al. [50] is complementary to chemical analysis since they can alert for the presence of ecotoxic compounds. Bringing into the WFD, the effect-related approaches concerning bioassays and biomarkers are only relevant in the context of the QN of environmental relevant substances and the good chemical status. But it is rather difficult to transfer the monitored biochemical responses or biomarkers into an operational effect-related standard. They serve as the basis for environmental protection against hazardous substances. In relation to... 

Since persistence in sediments is longer than that in the water column, the relevant toxicity studies are those that consider longer term, chronic exposures. A number of standard tests have been developed for assessing sediment toxicity and the bioassay of field collected sediments (e.g., [16-24]). The most commonly tested freshwater species are arthropods, including the amphipod shrimp // azteca and chironomid midge larvae, both Chironomus dilutus (formerly C. tentans) and C. riparius. Water-only studies have demonstrated that II. azteca are particularly sensitive to SPs (see Sect. 3) and in the published literature, this is the most commonly tested species for assessing the sediment toxicity of SPs. 

Invertebrate species have been widely used in toxicity studies of pesticides [61]. Zooplankton play a key role in the food chain because they occupy a central position. Therefore, their responses to natural and anthropogenic stresses are intimately linked with other food predator organisms. The most widely accepted bioassays employ species such as Ceriodaphnia dubia, Daphnia magna, Artemia salina, or Thamnocephalus platyurus [62-64]. D. magna has been used for many years as a standard aquatic test species and formally endorsed by the major international organizations such as the EEC, OECD, and ASTM [65-67]. Its choice is mainly because it represents the zooplankton community and is a species of worldwide occurrence. In addition, it has a greater sensitivity to toxicants, particularly pesticides, compared with other aquatic species [61,68] (Table 1). 

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