Solution culture protocols to study trace metal phytotoxicity
Pax Blamey, Peter Kopittke, Colin Asher, Bernhard Wehr, Neal Menzies
The University of Queensland, Brisbane Qld, Australia
Many trace metals are essential for plant growth while others have no proven benefit – but all are toxic above a certain limit. Plant physiologists are interested in determining the concentrations present in the root environment that result in normal growth, those that cause deficiencies or toxicities, and the factors that influence trace metal uptake by plants.
Terminology and equations
Trace metals: natural components of the environment generally present at low concentrations in soils and aquatic systems (exclude Ca, Mg, K, or Na).
Numerous solution culture techniques have been used to study the phytotoxic effects of trace metals. Underlying all techniques is the need to expose plants to toxic but appropriate concentrations in solution. Unfortunately, this has not always been considered, and innumerable results in the published scientific literature do not relate to levels found to be toxic in soils or in aquatic systems – surely an important aim of many solution culture experiments.
Trace metals must be in solution to impact directly on plant growth. These elements arise from the parent material through weathering or from other sources (e.g. atmosphere, dust, fertilizers), some of which are immediately soluble or have to undergo weathering over time (Figure 1). Once in solution, the elements are present mostly as ions which are either positively charged (cations) or negatively charged (anions). An exception and though not a trace metal, boron (B) is present mostly as uncharged molecules of boric acid (H3BO3). The cations and anions in the soil solution are taken up by microbes, plant roots and soil fauna or are fixed to varying degrees to clays or organic matter. As cations and anions are removed from solution, they are replaced with varying speed by those fixed to minerals and organic matter – the important property of a soil’s buffering capacity.
Figure 1. Simplified relationships of elements present as minerals in a soil and added as dust, in rain and as fertilizer or from plant and animal residues, as cations and anions in the soil solution, present in the soil organic fraction or absorbed by plants, and lost below the root zone by leaching.
In recent years, improved techniques to sample the soil solution have provided more information of the concentrations of cations and anions in the soil solution (Table 1). These concentrations vary during the course of the seasons due to changes in biological activity, but the soil solution is surprisingly dilute in non-saline soils with an ionic strength of about 5 mM. Also important is the low concentration of phosphorus (P) found in the soil solution (< ca. 5 μM), increasing to < ca. 50 μM soon after P fertilization.
Table 1. Calculated ionic strength and concentrations of nutrients in the soil solution of a Krasnozem (Oxisol), Queensland, Australia, Hoagland’s No. 2 solution, and a dilute nutrient solution.
|Soil solution a (unfertilized) ( M)||Hoagland’s No. 2 b solution ( M)||Dilute nutrient c solution ( M)|
|Ionic strength||4 900||26 000||2 700|
|NO3– – N||1 740||14 000||450|
|NH4+ – N||320||1 000||150|
a Surface soil of a highly-weathered Krasnozem (oxisol), Queensland, Australia (Menzies and Bell 1988)
b Hoagland and Arnon (1950)
c Wheeler et al. (1993)
Some common solution culture techniques including Hoagland’s solution (Table 1) (Hoagland and Arnon 1950) or its modifications were developed to grow plants in conveniently small volumes of solution. Hoagland and Arnon (1950) emphasized that there is no -magic’ involved in growing plants in nutrient solutions and listed solutions that might prove useful among many others previously developed. Such solutions are not appropriate for scientific research, however, if the aim is to study the function of roots in soils because the “elements … may be several orders of magnitude higher than those found in the soil around plant roots” (Taiz and Zeiger 2006). Shaff et al. (2010) have provided examples of problems that arise when using high ionic strength solutions. These problems are especially pertinent in solution culture studies of Al toxicity, but apply also to toxicities of other trace metals both because of precipitation and competition for sorption by the roots.
For example, Shaff et al. (2010) demonstrated that for Yoshida’s rice nutrient solution containing 1.3 mM Al, most of the Al precipitates as Al2(SO4)3 whilst the Fe precipitates as FePO4.
Asher and Edwards (1983) and Parker and Norvell (1999) have provided summaries of nutrient solution culture techniques that approximate the soil solution and overcome some of the problems associated with the use of high nutrient concentrations. These techniques, however, introduce problems of maintaining solution pH and concentrations of elements without the buffering capacity of the soil. This may be done in various ways, such as using large volumes of solution, periodically replacing the solution, or adding nutrients removed by the plants. Wheeler et al. (1993), for example, have demonstrated good wheat growth in nutrient solutions approximating those of the soil solution (Table 1). Asher and Cowie (1970) provided information on maintaining nutrient concentrations using Programmed Nutrient Addition based on plant growth rate; this was later computerized by Asher and Blamey (1987) and Blair and Taylor (2004) used a controlled delivery system to add specified volumes of nutrient stock solution.
Ranges of values
Of the many trace metals in the environment, aluminium (Al) causes the most widespread and serious problem because of soil acidification. Von Uexküll and Mutert (1995) estimated that acid soils occupy approximately 30% or 3950 m ha of the world’s ice free land area. Acidification increases the solubility of most trace metals; with Al being the most common metal in mineral soils (ca. 8 %) it is the most likely to become toxic as soils acidify. Of the remaining phytotoxic trace metals, the most important include lead (Pb), mercury (Hg), copper (Cu), cadmium (Cd), arsenic (As), cobalt (Co), nickel (Ni), zinc, (Zn), and manganese (Mn).
A review of the scientific literature from 1975 to 2009 by Kopittke et al. (2010) revealed a great range in concentrations that are toxic. Reasons include differences in (i) inherent toxicity among trace metals, (ii) among plant species in sensitivity, and (iii) and experimental techniques. . Indeed, it is often the aim of phytotoxicity studies is to identify genotypic differences in sensitivity. For example, Edwards and Asher (1982) found that across 13 crop and pasture species, the external Mn concentration needed to reduce dry mass by 10% varied from 1.4 μM in maize (Zea mays L.) and wheat (Triticum aestivum L.) to 65 μM in sunflower (Helianthus annuus L.). By discarding information from studies that used inappropriate experimental techniques, the median toxic concentration ranged from 0.30 M Pb to 46 M Mn.
For a diagramatic summary of the literature relating to concentrations of nine trace metals that reduced growth of plants in solution culture, see Figure 1 of Kopittke et al. 2010.
Because many trace metals form relatively insoluble compounds, there is a need to measure the concentrations of the soluble trace metal to which the plants are exposed (Kopittke et al. 2010). For example, Blamey et al. (2010) reported that approximately 80-90% of the silver (Ag) added to nutrient solutions had precipitated within 48 h. Similarly, lead (Pb) is known to form highly insoluble phosphates that may precipitate from solution (Kopittke et al. 2008). Such findings stress the importance of measuring the concentrations of trace metals in solution.
Health, safety and hazardous waste disposal considerations
Many trace metals are highly toxic to human health, and great care is needed in the conduct of solution culture experiments and in the disposal of these hazardous wastes.
Safe use of hazardous chemicals starts with Material Safety Data Sheets (MSDS) which ensures that the product is used as intended and forms the basis of risk assessment. Institutions must make sure that MSDS are available to employees, and employees must comply with institutional guidelines in the use of hazardous chemicals. Trace metal salts and solutions should be locked storage.
When being used, care is needed in preventing any contamination, including by dust and by solutions. The MSDS provide information on procedures needed should spillage occur. Personal protection, including use of laboratory coats and safety glasses, must be used at all times.
Disposal of trace metal solutions requires special consideration given that trace metals are not degraded by microbes. Salts and solutions at high concentration need to be disposed of as stipulated by each institution; low level concentrations may often be diluted further before disposal via the sewage system. Increasing the solution pH or adding soluble phosphates can decrease further some trace metals’ biological availability.
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