Introduction

In response to the expanding stresses on the environment and in the belief that there is no single criterion by which to adequately judge the potential hazard of a given substance (either to the environment or to humans), several biological assay procedures have been developed, proposed, and used to assay toxicant impacts.

In general, there are two main groups of in vitro toxicity-screening tests: (a) the "health effect" tests, and (b) the "ecological effect" tests. "Health effect" toxicity tests are based on the use of subcellular components (e.g., enzymes, DNA, and RNA), isolated cells (e.g., cell cultures, red blood cells), tissue sections, or isolated whole organs. These tests consist in the determination of cell viability (e.g., plating efficiency, colony formation), cell reproduction, or macromolecular biosynthesis. "Ecological effect" tests mainly measure the acute toxicity of chemicals to aquatic organisms representing various trophic levels of the food chain.

These tests use bacteria, algae, zooplankton, ben-thic invertebrates, and fish for the estimation of chemical toxicity in natural and man-modified ecosystems.

In the search for rapid, relatively reproducible and inexpensive tests, bacteria appear to be sensitive sensors of chemical toxicity. This is so because they have relatively short life cycles and quick responses to changes in their environment, where they may be exposed to a wide range of toxic, organic, and inorganic compounds in natural waters, soil, and sewage treatment processes. These characteristics make bacteria suitable for the rapid screening of toxicants in natural waters. Some screening tests are:

a) Mutagenicity test employing microorganisms and viruses, developed and standardized for determining genotoxic chemicals. The most frequently used mutagenicity test is the Salmonella typhimurium reverse mutation assay, which appears to be a reliable indicator of the potential carcinogenicity of organic chemicals. It uses auxotrophic mutants for histidine (his) that have either a normal or an error prone DNA repair capability and, therefore, can detect either base-pair errors or frame-shifts. Exposure to mutagenic agents induces prototrophy, and colonies of such revertants develop in histidine-free media.

b) Assay based on bacterial luminescence using Photobacterium phosphoreum in the Microtox® test. The Microtox® bioassay assesses acute toxicity in aquatic samples. It measures the activity of luminescent bacteria that emit light under normal metabolic conditions. Any stimulation or inhibition of their metabolism affects the intensity of the light output, c) Assays based on the measurement of viability or growth inhibition of specific bacteria (or specific bacteria groups). Sewage microorganisms and bacteria belonging to the genera Pseudomonas, Klebsiella, Aeromonas, or Citrobacter are used for the assays.

Nonetheless, there are specific problems associated with most of these testing protocols (e.g., the choice of test organisms, inocula size, growth media, and substrate concentrations). When using bacterial cultures in toxicology testing, the decision regarding the use of pure or mixed cultures of organisms also poses a problem. Pure cultures entail fewer complications and the results are easier to interpret. If natural mixtures of microbial populations are used, the problem of deciding what sources of populations to use as inocula and how to handle and store them prior to initiating the bioassay must be considered. Pure culture testing eliminates the possibility of interspecies interactions such as synergisms, com-mensalisms, symbiosis, and antagonisms that occur in natural environments and that may be important for biodégradation and adaptation of the biota to the substance under study.

In order to test the toxicant capability of several substances, the viable cell count of a bacterial culture may be determined using nutrient agar, which allows for the growth of a wide variety of microorganisms. The toxicant substances are included in the medium at increasing concentrations, so that the reduction in the number of colony forming units (CFUs) is determined relative to the number grown without the toxicant.

The substances chosen for the present experiment are:

• Sodium azide (NaNj), best known as the chemical found in automobile airbags. It is also used in detonators and other explosives. It also finds use as a chemical preservative in hospitals and laboratories, and in agriculture for pest control because azide anions prevent the function of cytochrome oxidase, an enzyme associated with respiration. Cells die as a result.

Hexavalent chromium, Cr(VI) is a human carcinogen acting upon chronic inhalation exposures. When swallowed, it can upset the gastrointestinal tract and damage the liver and kidneys. Evidence, however, suggests that hexavalent chromium does not cause cancer when ingested, most likely because it is rapidly converted into the triva-lent form, Cr(III) after entering the stomach. Chromium (VI) is a danger to human health, mainly for people who work in the steel and textile industries.

Chromium (III) occurs naturally in many fresh vegetables, fruits, meat, grains, and yeast, and is often added to vitamins as a dietary supplement. It is an essential nutrient for humans, and shortages may cause heart conditions, metabolism disruption, and diabetes. Nevertheless, the uptake of too much Cr(III) can cause health effects (e.g., skin rashes). Chrome green is the green oxide of chromium (III), Q2O3, used in enamel painting and glass staining. Trivalent chromium may cause skin irritation at high doses given parenterally but is not toxic at lower doses given orally.

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