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Rhizoid Regeneration

Development of New Applications for 'Rhizoid Regeneration Technique' for use in Monitoring Pesticides and Herbicides in Vineyards

Jane Shields developed a 'Rhizoid Regeneration Technique' during post- graduate research at Heriot-Watt University, Edinburgh. The aim of the original research was to develop an effective treatment system for the marine environment using algae. However, there was no effective method for assessment of the contaminants in the water without using marine animals in a LD50.test, (the concentration of a substance required to kill 50% of the organisms). This is a crude measurement and Jane wanted to find an alternative to animal experimentation.

Methods with multicellular algae used dead material or staining, both ineffective and crude. All the available methods focused on the concentrations required to kill a certain type of organism. Her thesis became the development of this technique.

Algae form the first link in many food webs, oxygenate the water and are important in the cycling of dissolved organic and inorganic substances. Toxicants accumulated by algae may be passed up the whole food web, and if stored rather than metabolised may be concentrated and have effects at higher trophic levels.

For this reason the focus was on the development of a sensitive technique that could detect the first signs of stress in algal populations which required the algae to remain alive. Another difficulty to overcome was the variability and steepness of physio-chemical gradients in estuarine water, the continuous and frequently unpredictable oscillations of salinity and dissolved and particulate matter in the water. It was also necessary for the technique to not be affected by reproduction.

The genus, Enteromorpha (Chlorophyta, Ulvales), a green tubular parenchymatous alga was chosen as it has a wide geographical distribution ( both northern and southern hemispheres, well know nutrient requirements, small genetic and phenotypic variation, good taxonomic characterisation of the strains, a high growth rate and ease of handling. The habitat ranges from fresh water to seawater but it is more common in estuarine and coastal habitats.

One of the most precarious stages in the life history of a marine benthic organism is the colonisation of new substrata, for which highly specialised processes of settlement and early growth have developed. In Enteromorpha, a primary rhizoid develops which penetrates a substrate on structures being colonised and other rhizoids soon follow the point of entry. Secondary rhizoid regeneration occurs in nature when the alga is removed from the substrate by either predation or wave action and must reattach.

The technique is based on the secondary rhizoid regeneration process that can be simulated by cutting 5 mm off the basal end of the algal filament which removes the specialised cells that grow rhizoids. This technique has proved to be extremely sensitive as the algal filiments must be in good health in order to regenerate the specialised cells and produce rhizoids. Assessment is based on the number of algal filaments that have regenerated in a given substance or natural waters and the density and number of the rhizoids which is a finer indicator of the degree of stress on the organism.

This technique has been used to assess water quality along an estuary by studying different algal populations located in different parts of an estuary in combination with testing samples along an estuary tested against a base population used as a control, using standard statistical analysis. It has also been shown to be effective in testing particular contaminants at known concentrations, e.g. copper, biocide and salinity.

ITV in France has asked Jane Shields to see if the rhizoid regeneration technique can be developed to monitor pesticides and herbicides. There are hundreds of pesticides and herbicides in use but only a few of these have a test that can analyse it so it is virtually impossible to understand the full impact of these contaminants in the soil or water. Also most are used in mixtures making analysis even more difficult.

We are starting to work on the development of the rhizoid regeneration test for this purpose. Initial funding is expected from ITV.