White mustard (Sinapis alba) root and shoot growth inhibition test
White mustard (Sinapis alba) is a sensitive test plant. Its growth is inhibited in contact with toxic substances. The toxic effect can be estimated by measuring its root and shoot length. The method was developed to direct contact test applied to solid samples based on HS 21976-17/1988 standard by BME’s Environmental Biology and Biotechnology Group. Direct contact between the soil and the plant root can be achieved without placing filter paper on the soil surface. This way the roots interact more closely with the soil.
5 g solid sample is weighed into 9 mm diameter Petri dish. It is uniformly spread, then wetted to its water holding capacity (approx 3-4 ml distilled water). The germination ability of white mustard seed should be minimum 95%. Germination ability and suitable growth is checked with distilled water. The sensitivity of the seeds is checked with Cu dilution series. The test is run in triplicate. Dilution series are prepared with OECD control soil (10% peat, 20% kaolinite clay min. 30% kaolinite, 70% industrial quartz sand). Distilled water is used as control, or in case of the availability of uncontaminated control material it can be used also, as a control. The samples are kept in thermostate for 72 hours at 20/25 °C, in the dark.
The root and shoot lengths are measured with a ruler. The results of the triplicates are averaged. Root and shoot growth inhibition is expressed in % compared to the root or/and shoot length of the test plants grown on the control soil:
X=(K-M)/K*100, where: X: root or shoot growth inhibition %, K: root or shoot length of plants grown in the control soil, M: root or shoot length of plants grown in the tested soil.
In case of dilution series the inhibition is plotted versus the weighed amount and the ED20 and ED50 values (the amount causing 20 and 50% inhibition) are estimated.
Other factors influencing root growth:
The root length is not always proportional to the inhibition. The root often reacts to the heterogeneously distributed contaminant in the soil with avoidance behaviour, namely developing abnormally extended but very thin roots which can be distinguished from the normally grown roots. For this reason, root length and root growth, as measurement endpoint may be applied only with restrictions in case of soils contaminated by hydrocarbons and other hydrophobic contaminants.
Klebercz Orsolya (2009) noticed that even though during the evaluation of the test soil toxicity was considered proportional to the root and shoot decrease, it did not always reflect the reality confirmed by the weight of the plants. Even if the biomass was similar the physical appearance of the roots was different suggesting unfavourable conditions for their development. The roots of the white mustard react to soil toxicity by intensive growth because the roots try to avoid the toxic soil grains by chemotaxis. Such roots could be easily recognised: they are elongated, thin and long, as if they were escaping to search for suitable soil. Meanwhile the shoots are short, because they do not get enough nutrients. In such cases the shoot/ root ratio is a more adequate indicator which takes into account the chemotaxis and it characterises soil toxicity in a more complex way. Usually, if the shoot/root length ration is < 0.7 then we are faced with „escaping” roots, therefore the root growth inhibition is not proportional with the available concentration of the soil contaminant, soil toxicity could be characterised by the shoot/root length ration.
Gruiz K., Horváth B., Molnár M.: Környezettoxikológia, Műegyetemi kiadó, Budapest, 2001.
Klebercz O.: Toxikus fémekkel szennyezett talaj stabilizációja - Szabadföldi kísérletek, Diplomamunka, BME ABÉT, Budapest, 2009
Feigl, V. Hungarian version of the description of the Sinapis Alba test: https://www.enfo.hu/keptar/3700