The most important advantage of the Allium test is that it is a “low budget” method, which besides being fast and easy to handle also gives reliable results. The duration of the genotoxicity test is three to four weeks, including initial toxicity testing, scoring of aberrations, and statistics. It can be described briefly as follows:
Week 1: A 96-h root growth inhibition test is carried out in order to determine the toxicity level of the test chemical or environmental sample, and EC50 is determined by the dose-response relationship by interpolation.
Week 2: The 48-h genotoxicity test is carried out with 3-4 concentrations below the EC50, and the root tip cells are prepared for microscopic analysis.
Week 3: Chromosome aberrations are scored in anaphase and telophase cells.
Week 4: Calculations, statistics and reporting.
MATERIALS AND CHEMICALS
The test organism
The common onion, Allium cepa (Stuttgarter Rie- sen) is used. In Denmark, onions can be obtained from Dahnfeldt, Odense. The onions are sown in spring and harvested in late summer. The bulbs should be 15-22 mm in size and weigh 2-4 g. However, onions of other sizes and sorts can be used. If kept dry at 10-15 °C, the onions can be used within a year after harvest. The onions need to rest for about two to three months before they are able to grow roots fast enough for the assay. The yellow shallows and the dry bottom plate inside the root primordia are carefully removed prior to the test.
Glassware
Figure 1 shows some special equipment used for the Allium test. The glass tubes (Wallin-glass) are bottomless, and a 70-mm ruler is mounted on the side of the glass and then used to measure the length of the root bundle. The beakers are made of polycarbonate and are disposable. This equipment is produced at our own laboratory, but the assay can also be carried out using normal test tubes in a rack and an ordinary ruler for the measurement of the root length. However, the advantage of this special equipment compared to the test tubes is that it is easier to change the test solutions in the beaker. A hole in the cover makes it also possible to aerate the solution in the beaker, which is more complicated if six test tubes are used. One disadvantage of using one beaker instead of six test tubes is that the volume of test solution is about twice as large as for test tubes.
Chemicals
Tap water of good quality is used for negative control and for dilution of chemicals. Good quality means, for example, that the water is not containing any chlorine compounds and that the water pipes are not made of copper. If the quality of the tap water is poor, it is recommended to use synthetic fresh water made of Millipore water containing
MgSO4 60 mg/l, NaHCO3 96 mg/l, KCl 4 mg/l and CaSO4 60 mg/l (CaSO4 which should be dissolved by heating and stirring before it is mixed with the other salts). If the test chemicals are not water- soluble, DMSO, acetone or ethanol can be used as a solvent. Methyl methanesulfonate (MMS) can be used as positive control. Fixation and maceration is carried out using a solution of 9 parts of 45% acetic acid and 1 part of 1 M HCl. The chromosomes are stained with 2% orcein in 45% acetic acid.
Microscope and photo equipment
A light microscope (e. g., Dialux from Leitz) is used with an oil immersion objective and 500D magnification. For discussion of aberrations, it is useful to install a video camera on the microscope and transfer the pictures to a computer.
METHODS
Prior to the Allium test the pH of an environmental sample (e. g., wastewater) should be adjusted to about 7 with 1 M HCl or NaOH. The test is carried out at room temperature and the onions should be kept away from direct sunlight during the experiment.
Root growth inhibition test
The toxicity assay is performed as a 96-h semi-static exposure test, and 6-9 concentrations of the test chemical or complex mixture are used. Every 24 h the test solutions are replaced by fresh solutions. The test solutions are kept cold but should have reached room temperature before use. At the termination of exposure, one onion (out of six) with the poorest growth is discharged and the length of the root bundle is measured for the rest five onions. The effect of growth inhibition is shown on a graph with the log concentration against the root length expressed as percent of control (Fig. 4). The EC50 can be calculated with a computer programme or found by a simple interpolation.
Genotoxicity assay
The genotoxicity assay is carried out with four sample concentrations. They can, for example, be composed of the EC50 as the highest concentration followed by 50%, 25% and 10% of the EC50. Tap water or synthetic fresh water can be used as a negative control, and if DMSO or other solvents are used, a solvent control should be included in the assay. MMS, 10 mg/l, is used as positive control, but maleic hydrazide, 5 mg/l, can also be used. Six onions are exposed to each concentration. For the first 24 h, the onions are grown in tap water or synthetic fresh water, whereafter they are exposed to the test chemicals for 48 h, which is close to two cell cycles. As for the toxicity test, the test solution is changed after 24 h. The onion with the poorest growth is excluded for every concentration, and the remaining five onions are prepared for microscopy.
For every onion one slide is made using the following procedure: 5 root tips at a length of 10 mm are cut off and placed in a test tube with 2 ml acetic acid - hydrochloric acid solution and heated for 5 min at 50 °C. Thus, the root cells will become fixed and macerated. Thereafter, the roots are placed on a slide and the terminal root tips (1-2 mm) are cut off and used for further preparation. The rest of the material is removed from the slide and the excess of liquid is sucked up by a piece of blotting paper. Two drops of fresh filtrated 2% orcein solution are added and mixed with the root tips by stirring and knocking with a blunt stick of stainless steel (or something alike). In the final phase, a cover slip is placed on the root cells and allowed to absorb stain for 5-10 min. Afterwards, the cells are squashed by placing to layers of blotting paper on the cover glass and pressing slightly down with the thumb. The cover slip is fixed carefully to the slide with nail varnish. The slides can be kept fresh for 2 months in a freezer.
Microscopic examination
All slides are coded and examined blind. The microscopic analysis includes the mitotic index and scoring of chromosome aberrations in anaphase and telophase cells. The mitotic index, MI is found by counting all stages of mitotic cells out of 1000 cells. It is recommended only to score chromosome aberrations if MI is above 10, otherwise there will be too few anaphase and telophase cells for the analysis. The slides are examined from right to left, up and down, and the first 100 anaphase and telophase cells are scored for aberrations.
Two classical categories of aberrations, fragments and bridges, are scored. These are the most frequent aberrations and indicate that the test chemical or complex mixture is clastogenic. Very often fragments are seen together with bridges and these cells can be scored as a specific category. Figure 2 shows how chromatid breakage and rejoining can result in these aberrations. Categories as vagrant and laggards indicating interaction with the spindle are also scored. Other less frequent aberrations such as multipolarity, c-mitotic anaphases, polyploidy and pulverised chromosomes, are also registered. Different kinds of aberrations are shown in Fig. 3.
The control level from exposure to tap water or synthe tic freshwater is about 1% of aberrant cells, and for the positive control with MMS, 10 mg/l, or maleic hydrazide, 5 mg/l, it is about 25% of aberrant cells. If the toxicity is not too high, it is possible to score 100 anaphase and telophase cells per slide. With five onions per beaker it gives 500 cells per concentration. However, if the mitotic index is very low, it can be impossible to find 100 cells per onion.
Statistics
As the distribution of aberrant cells is binomial, we used the χ2-test for statistical calculations. The calculations were carried out on the assumption that the five onions made one sample, and each concentration was tested against the control sample.
RESULTS AND DISCUSSION
The results from a 96-h root growth inhibition test of maleic hydrazide are shown in Fig. 4. The sigmoid graph is a typical dose-response curve for this kind of toxic effects. However, if the chemical or environmental sample has a low acute toxicity, it can be difficult to obtain a usable graph for determination of EC50. From earlier studies (Nielsen, 1994; Rank et al., 1994; Rank et al., 1998), EC50 values from testing chemicals, wastewater and wastewater sludge are shown (Table 1).
Table 2 shows the corresponding results from negative and positive controls. When tap water was used as negative control (Rank et al., 1997), 0.2% and 1.4% of aberrant cells were found, and for synthetic freshwater (Rank et al., 2002) the values were 0.4% and 1.6%. The present results for MMS, 10 mg/l, varied from 18.4% to 28.6%, but in other studies we have seen levels lower than 10% of aberrant cells. The explanation for these variations is that MMS degrades very fast, even if it is kept cold, and therefore it is recommended not to use a batch for more than half a year after it is opened.
In Table 2 one can see that MMS, 10 mg/l, decreased the mitotic index (9–21%) compared tothe controls. Further, a big variation (42–65) can be seen for the MI of the controls. Therefore, it could be questioned if the MI should be used as a quantitative measure of toxicity, and in the Allium anaphase-telophase chromosome aberration assay the MI is only used for evaluation purposes to see if there will be mitotic cells enough for the analysis of chromosome aberrations. As pointed out earlier, it has been found that with an index below 10 there will normally be too few anaphase and telophase cells to score at the slide.
The Allium anaphase-telophase chromosome aberration assay was developed as a modification of the Allium test described by Fiskesjo (1985) to make a simpler and faster assay for detection of the ge- notoxicity of chemicals and environmental samples. Fortunately, the test system has been found useful for many different studies. Odeigah et al. (1997) used the Allium test to show the genotoxicity of wastewater from an oil field, and Monarca et al. (2000) investigated urban wastewater disinfected by different chemicals and showed a positive response in the Allium test when peracetic acid was used as a disinfectant. The Allium test also showed good results when aqueous extracts from lead-contaminated soils before and after remediation were examined for genotoxic effects (Chang et al., 1997). Further, in a soil study using the Allium test, Kovalchuk et al. (1998) found a strong, significant correlation of chromosome aberrations with 137Cs activity in soils contaminated by the Chernobyl accident.
In conclusion, the Allium cepa anaphase- telophase chromosome aberration assay is useful for many types of environmental samples and can be recommended as a tool for monitoring the genoto- xic effects and thereby contributing to environmental risk assessment m eans, which are becoming ever more important.
References
1. Chang L. W., Meier J. R., Smith M. K. Application of plant and earthworm bioassays to evaluate remediation of a lead-contaminated soil. Archives of Environmental Contamination and Toxicology. 1997. Vol. 32. P. 166-171.
2. Claxton L. D, Houk V. S., Hughes T. J. Genotoxicity of industrial wastes and effluents. Mutation Research. 1998. Vol. 410. P. 237-243.
3. Fiskesjo G. The Allium test as a standard in the environmental monitoring. Hereditas. 1985. Vol. 102. P. 99-112.
4. Fiskesjo G. The Allium test - an alternative in environmental studies: The relative toxicity of metal ions. Mutation Research. 1988. Vol. 197. P. 243-269.
5. Grant W. F., Lee H. G., Logan D. M., Salomone M. F. The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment. Mutation Research. 1992. Vol. 270. P. 53-64.
6. Kihlman B. A. Root tips for studying the effects of chemicals on chromosomes. In: A. Hollaender (ed.). Chemical Mutagens. Plenum Press, New York, 1971. P. 449-514.
7. Kovalchuk O., Kovalchuk I., Arkhipov A., Telyuk P., Hohn B., Kovalchuk L. The Allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the Ukraine contaminated by the Chernobyl accident. Mutation Research. 1998. Vol. 415. P. 47-57.
8. Monarca S., Feretti D., Collivignarelli C., Guzzella L., Zerbini I., Bertanza G., Pedrazzani R. The influence of different disinfectants on mutagenicity and toxicity of urban wastewater. Water Research. 2000. Vol. 34. No. 17. P. 4261-4269.
9. Nielsen M. H., Rank J. Screening of toxicity and ge- notoxicity in wastewater using the Allium test. Here- ditas. 1994. Vol. 121. P. 249-254.
10. Odeigah P. G. C., Nurudeen O., Aund O. O. Geno- toxicity of oil field wastewater in Nigeria. Hereditas. 1997. Vol. 126. P. 161-167.
11.Rank J., Nielsen M. H. A modified Allium test as a tool in the screening of genotoxicity of complex mixtures. Hereditas. 1993. Vol. 118. P. 49-53.
12.Rank J., Nielsen M. H. Evaluation of the Allium anap- hase-telophase test in relation to genotoxicity screening of industrial wastewater. Mutation Research. 1994. Vol. 312. P. 17-24.
13.Rank J., Nielsen M. H. Allium cepa anaphase-telo- phase root tip chromosome aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azi- de, and ethyl methansulfonate. Mutation Research.
1997. Vol. 390. P. 121-127.
14.Rank J., Nielsen M. H. Genotoxicity testing of wastewater sludge using the Allium cepa anaphase-telop- hase chromosome aberration assay. Mutation Research.
1998. Vol. 418. P. 113-119.
15.Rank J., Lopez L. C., Nielsen M. H., Moretton J. A two-laboratory study of the Allium cepa anaphase-te- lophase chromosome assay comparing genotoxicity of maleic hydrazide, acridine and DEHP. Hereditas. 2002. Vol. 136. P. 13-18.
Jette Rank
ALLIUM ANAFAZINIy-TELOFAZINIy CHROMOSOME ABERACIjy TYRIMO METODAS
Santrauka
Svoguno Allium anafaziniq-telofaziniq chromosomq abe- racijq tyrimo metodas buvo sukurtas greitam cheminiq me- dziagq ir aplinkos pavyzdziq genotoksikologiniam jvertini- mui. Pries genotoksiskumo tyrimq atliekamas 96 val. truk- mes sakneliq augimo inhibicijos testas. Chromosomq abe- racijos nustatomos sakneles paveikus tiriamj medziaga 48 val. Klastogeniskumo rodikliai yra atsirandantys chro- mosomq tiltai ir fragmentai, o atsiliekancios ir pasimetu- sios chromosomos rodo aneugeninj poveikj. Metodas yra greitas bei patikimas ir gali buti taikomas nutekamiesiems vandenims, upes vandenims, uzterstoms dirvoms ir sude- tingiems misiniams tirti.
Raklazodziai: Allium cepa, genotoksiskumas, chromo- somq aberacijos, anafaze, telofaze
Jette Rank
ALLIUM ANAFAZINIy-TELOFAZINIy CHROMOSOME ABERACIjy TYRIMO METODAS
Santrauka
Svoguno Allium anafaziniq-telofaziniq chromosomq abe- racijq tyrimo metodas buvo sukurtas greitam cheminiq me- dziagq ir aplinkos pavyzdziq genotoksikologiniam jvertini- mui. Pries genotoksiskumo tyrimq atliekamas 96 val. truk- mes sakneliq augimo inhibicijos testas. Chromosomq abe- racijos nustatomos sakneles paveikus tiriamj medziaga 48 val. Klastogeniskumo rodikliai yra atsirandantys chro- mosomq tiltai ir fragmentai, o atsiliekancios ir pasimetu- sios chromosomos rodo aneugeninj poveikj. Metodas yra greitas bei patikimas ir gali buti taikomas nutekamiesiems vandenims, upes vandenims, uzterstoms dirvoms ir sude- tingiems misiniams tirti.
Raklazodziai: Allium cepa, genotoksiskumas, chromo- somq aberacijos, anafaze, telofaze