The evaluation of waste, surface and ground water quality using the Allium test procedure

Genetic Toxicology

ELSEVIER

Mutation Research 368 (1996) 171 - 179

The evaluation of waste, surface and ground water quality using the Allium test procedure Vesna Smaka-Kincl

a,*,

Peter Stegnar b, Milan Lovka c, Mihael J. Toman d

a Public Sert~ices Administration, Department of Em:ironmental Protection, Slovenska 40, 62000 Maribor, SIocenia b lnstitut 'Jo~efStefan ', Jamot:a 39, 61000 Ljubljana, Slovenia c Institute for Biology, Karlocgka 19, 61000 Ljubljana, Slot,enia d UnieersiO' of Ljubljana, Department of Biology, Ve~na pot 111, 61000 Ljubljana, Slovenia

Received 28 October 1994; revised 2 May 1995; accepted 28 November 1995

Abstract

The bulbs of Allium cepa were grown in test liquids of various pollution levels as follows: undiluted industrial and municipal waste water; biological treatment plant output water; water from the Drava river upstream and downstream of the city of Maribor; and non-chlorinated drinking water as a negative control test. The paper presents the response of the Allium cepa genetic material to the presence of potential cytotoxic and genotoxic substances in test liquids and the suitability of the Allium cepa testing procedure as a method for short-term determination of water pollution level. The suitability of the Allium test procedure as a system for environmental monitoring is presented. The influence of water pollution on macroscopic and cytologic parameters of the common onion by application of the biological testing method was examined. The macroscopic parameter was inhibition of root growth. The cytological parameters were: aberrant cells in metaphase and anaphase, index of micronuclei appearance and inhibition of cell division. The possibility of categorization the different polluted test liquids into quality classes is presented according to the influence of the test liquids on macroscopic and cytologic parameters. Test liquids are divided into 8 quality classes: the first class is the least polluted surface waters, the second and the third classes are more polluted surface waters, the fourth and the fifth classes are biological treatment plant output waters, the sixth till the eighth quality classes are untreated waste waters. The most polluted test liquids (untreated industrial and municipal waste waters) caused sublethal and even lethal effects. The most polluted tested liquids cause the inhibition of root growth over 50% (even up to 74%), decrease of mitotic index over 36% (even up to 66%), increase of presence of interphase cells with micronuclei over 3% and increase of presence of aberrant cells for more than 10 times in comparison to control test. Keywords." Allium test; Bioassay; Cytotoxicity; Genotoxicity; Drinking water; River water; Waste water

1. I n t r o d u c t i o n

* Corresponding author. Tel.: +38 (6) 6220080; Fax: +38 (6) 62224815.

0165-1218/96/$15.00 © 1996 Elsevier Science B.V. PII S0165- 12 1 8(96)00009-2

H i g h e r plants are very suitable testing systems for m o n i t o r i n g the presence o f m u t a g e n i c substances in the e n v i r o n m e n t as well as for the study o f harmful

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effects of a particular mutagen in known concentrations (Ruiz et al., 1992). Plant testers are relatively inexpensive and can easily be stored and handled. Due to the size of their chromosomes, higher plants are suitable for cytological analysis and they show good correlation with other bio-testing systems (Fiskesj6, 1985). Pollution level or quality of natural surface or underground water and of waste waters is determined by physical/chemical, saprobiological, radiological and recently also by cytogenetical and genotoxical analyses. Cytogenetical tests are suitable for identification of harmful effects of particular known substances in various concentrations over different exposure times and for evaluation of their influence on organisms (Al-Sabti and Kurelec, 1985; A1-Sabti, 1989; Abdou et al., 1989; Arrigoni et al., 1989; Kak and Kaul, 1989; Kumar and Sinha, 1989; Rao, 1989; Chauhan and Sunderaraman, 1990; EI-Khodary et al., 1990; Panda et al., 1990; Singh et al., 1990; Kumar et al., 1991; De-Serres, 1992). These tests also provide comprehensive data on harmful effects on testing organisms and are commonly used for bio-monitoring the pollution extent as well as for evaluation of combined effects of all toxic and mutagenic substances on organisms in natural environments (Degrassi and Rizzoni, 1982; Al-Sabti and Kurelec, 1985; Dixit and Nerle, 1985; Fiskesj6, 1988; De Marco et al., 1988; A1-Sabti, 1989). Biological tests for toxicity and genotoxicity are indispensable for the evaluation of reactions of living organisms to the complex environmental pollution and for an indication of potential synergistic effects of various pollutants, while physical and chemical analyses provide nothing but determinations of presence and concentrations of different pollutants. 2. Materials and methods The experiments were carried out on test liquids from 7 different sampling sites once a month from October 1st 1989 until July 1st 1990, overall 10 times in each of the sampling sites listed below. 1. The river Drava near Mariborski otok: this sampling site is located upstream of the city of Maribor where, according to the data from the State Hydrometeorologic Council, the water pollution level is classified as II to I I / I I I .

2. The river Drava at Dravski dvor: this sampling site is located downstream of the city of Maribor at the electric power plant supply duct, which conducts approximately 80% of the total Drava river water. The city of Maribor is not yet provided with a sewage treatment plant, so the river Drava at this site contains practically all industrial and urban effluents from the city and the water pollution level is classified I I l / I I . 3. The inlet to the sewage treatment plant in Race, where the water contains urban waste waters from a population of some 500 people as well as industrial waste waters from chemical and meatprocessing plants. 4. The outlet of the sewage treatment plant in Race. 5. The pumping station of the municipal waste water trunk sewer in Melje, containing, in spite of its name, both urban and industrial effluents from chemical, textile, metal- and food-processing facilities. 6. The pumping station of the industrial waste water trunk sewer in Melje, which, like sampling site 5, contains a mixture of urban and industrial effluents. 7. Non-chlorinated water obtained directly from the drinking water well in Vrbanski plato; the water corresponds to the relevant regulations for use as drinking water and was used in the experiments as a negative control liquid. As the Maribor industrial plants mainly do not provide pretreatment of waste water and Maribor does not have a central treatment plant, there are oscillations in test liquid quality. Because of the oscillations in quality of test liquids (Bla~eka, 1991; Krajnc, 1991) 8-h average samples were taken from sampling sites 1-6. Samples were taken by a peristaltic pump made by Polo, Maribor. The pump was adjusted to gather 5 1 of test liquid in a period of 8 h. For bioindication tests, the bulbs of the common onion (Allium cepa L.) were used. They were all purchased from the same farmer and were stored in a refrigerator at +4°C. Only bulbs of good condition and of approximately the same weight ( 4 - 7 g) were used in experiments. For each test, 10 bulbs were prepared. All dry scales were peeled off and the

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ROOT L E N G H T (cm)

8

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Drava upstr.

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Drava downs|r. ~7~

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1 Race ou|lel

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Munic.sewer

Fig. 1. Root length after 7 days of growth in different test liquids repeated once a month for a period of 10 months.

% MITOTIC INDEX 8

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Fig. 2. Mitotic index values of root tip meristems from different test liquids repeated once a month for a period of 10 months.

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bulbs were placed directly into the test liquid, where they were grown for 7 days in the dark at a temperature of 19-21°C. During the first 3 days, the test liquids were replaced every day, while later on, test jars were only topped up with small quantities of the test liquids. Tests were always initiated and finished between 07.00 and 08.00 h, as the highest mitosis frequency in the onion is recorded between 06.00 and 09.00 h (Sharma, 1983). After 7 days of growth, the length of the roots was measured and root tips fixed in a 3:1 mixture of ethanol and glacial acetic acid. Fixed root tips were kept in the refrigerator at - 2 5 ° C for at least 1 h and then hydrolysed in 1 N HC1 at 60°C for 3 min, rinsed in tap water and stained in Schiff's reagent following Feulgen's method. Microscopic slides were prepared by squashing the root tips in acetocarmine. Cells divisions and cytogenetical abnormalities were observed and photographed under a Jena Carl Zeiss research microscope. For evaluation of harmful effects of test liquids on meristematic cells, microscopic slides of 4 squashed root tips were examined once a month for 10 months from each of the 7

sampling sites. So for each sampling site, 8000 cells (2000 cells in each of the 4 root tips) were analysed 10 times. Meristematic cells were observed in order to determine the proportion of dividing meristematic cells (expressed as mitotic index), the number of interphase cells with micronuclei, aberrant metaphase and anaphase cells. In order to find out the relationships between the water pollution levels of test liquids and the amount of observed cytogenetical abnormalities Student's t-test and the statistical significance of variations between arithmetic means of all observed parameters were determined using the SPSS PSC + statistics program.

3. Results After 7 days of growth in the negative control the average lengths of roots were between 6.2 and 7.7 cm (average 6.7 cm). Fig. 1. shows root length after 7 days growth in different test liquids repeated once a month in period of ten months.

ABERRANT METAPHASES (%) 7O 60 50 40 30 20 10 0

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Fig. 3. Percentage of aberrant metaphase cells in root tip meristems from different test liquids repeated once a month for a period of 10 months.

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ABERRANT ANAPHASES (%) 60 50 40 30 20 10 0 1

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REPLICATION Neg.con.

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Ind.sewer

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Orava upstr. I Race outlet

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Drava downstr.~'~

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Fig. 4. Percentage of aberrant anaphase cells in root tip meristems from different test liquids repeated once a month for a period of 10 months.

INTERPHASE CELLS WITH MICRONUCLEI (%) 0.1

0.08 0.06 0.04

0.02

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Fig. 5. Percentage of interphase cells with micronuclei in root tip meristems from different test liquids repeated once a month for a period of l0 months.

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176

The mitotic index in meristems of roots grown in negative control ranged from 6.34 to 7.54% (average 7.06%). Fig. 2. shows the values of mitotic index in root meristems which grew in different test liquids, repeated once a month for a period of 10 months. The influence of various genotoxic substances from single test liquids was determined by analysing the types and frequencies of aberrant metaphase and anaphase cells and by determining the existence of micronuclei in interphase cells. The most common cytogenetical aberrations in metaphase cells were chromosome stickiness, clumping, chromosome fragments and c-metaphase. In the anaphase cells disrupt and multipolar spindles, chromosome laggards and fragments were found in various frequencies. Aberrant mitotic cells were counted and their ratio was expressed as a percentage of all cells in the same stage of cell division. The percentage of aberrant metaphase cells is shown in Fig. 3 and the percentage of aberrant anaphase cells in Fig. 4. In root meristems of the negative control, the percentage of aberrant metaphase cells ranged from 0.9 to 5.7% (average 2.4%), while the percentage of aberrant anaphase cells was between 0 and 4.9% (average 1.9%). The percentage of interphase cells with micronuclei in meristems of roots growing in negative control ranged from 0 to 0.001%. Fig. 5. shows the percentage of interphase cells with micronuclei which grew in different test liquids during the period of 10 months. Statistical significance of variations between arithmetic means of individual examined parameters was determined by the Student t-test. Selected

threshold of statistical significance was p < 0.05. Results of the negative control were compared with the influence of other test liquids on observed parameters and are shown in Table 1. The largest difference in root length after 7 days growth ( p < 0.001) was found in tests performed in water from both trunk sewer lines at Melje (sampling sites 5 and 6) and in water from the inlet as well as the outlet of the sewage treatment plant in Race (sampling sites 3 and 4). The statistical significance of variations in the length of roots after 7 days growth was also indicated in water from the Drava river at Mariborski otok (significance level p < 0.05) and at Dravski dvor (significance level p < 0.01) (sampling sites 1 and 2). The mitotic index of root tips meristems indicates statistical significance of differences for all tested liquids. In plants growing in water from the Drava river at Mariborski otok (sampling site 1) the relevance level was p <0.01, while in all other test liquids the relevance level of p < 0.001 was determined. The proportion of aberrant metaphase and anaphase cells also indicates different levels of statistical significance of variations for all test liquids. The highest level of statistical significance of p < 0.001 was determined for the amount of aberrant metaphase cells in root tips from the municipal as well as the industrial waste water trunk sewer in Melje (sampling sites 5 and 6) and from the Drava river water at Dravski dvor (sampling site 2). The same level of statistical relevance was also determined for the amount of aberrant anaphase cells in root tips from the municipal waste water trunk at

Table 1 Results of toxicological and cytogenetical analyses in Allium cepa exposed to different water samples (test liquids from different sampling sites) Sample no.

Control 1 2 3 4 5 6

Root length

Mitotic index

Aberrant metaphase cells

Aberrant anaphase cells

Micronucleated cells

(cm)

(%)

(%)

(%)

(%)

2.4 _+ 1.40 6.1_+ 3.70 b 7.4_+ 2.11 ~ 19.7__ 15.94 b 19.8+15.83 b 13.0-t- 4.79 c 10.7_+ 4.05 c

1.9 _+ 7.1_+ 8.3_+ 24.5-t16.1_+ 15.9+ 13.1_+

6.8 _+ 0.46 6.3+0.43 6.1+0.31 1.8_+0.87 4.1+1.22 1.7+0.85 3.2+1.38

p<0.05;hp<0.01;c

a h c c c c

7.1 _+ 0.40 5.9_0.83 5.1+1.19 2.4+0.93 3.5+0.85 3.5+0.98 4.5+0.85

p<0.001.

h ~ c ~ ~ ~

1.91 5.52 5.71 12.0l 6.73 7.91 8.26

~' b ~ ~ ~ h

0.003 +_ 0.01 0.011+0.01 0.008+0.01 0.042+0.03 0.036+0.01 0.035+0.01 0.022+0.02

" ~ ~ b

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Melje (sampling site 5) and from the inlet as well as from the outlet of the sewage treatment plant in Race (sampling sites 3 and 4). The statistical significance of differences between the occurrence of micronuclei in the interphase cells of root tip meristems were indicated at a significance level of p < 0.001 for tests performed in water from the municipal waste water trunk at Melje (sampling site 5) and from the inlet as well as from the outlet of the sewage treatment plant in Race (sampling sites 3 and 4), at a significance level of p < 0.01 for the industrial waste water trunk at Melje (sampling site 6), while occurrence of micronuclei in other sampling sites was of no statistical significance. Despite their advantages in detecting the influences of complex mixture of pollutants, in most countries cytogenetic tests have not yet been included in the official legislation regarding water quality determination, mainly due to the fact that there are no standard testing methods. For standardisation of cytogenetic test procedure and explanations of the results from various test organisms, a GHP (genotoxic harmful potential) unit has been provisionally suggested (Hantage, 1992). Classification of test liquids in our case was built up according to presence of cytotoxic and genotoxic substances and their influence on the onion. Considering the influence of cytotoxic and genotoxic substances, classification of the test liquids was carried out and 8 classes were formed. Table 2 shows our suggested classification of tested liquids using the GHP-values, determined by the types and amount of cytogenetic effects of test liquids on the bioindicator organism used in our experiments ( A l l i u m cepa). According to this suggested classification, water samples in our experiments can be determined as described below. The value GHP 1 was determined for the least polluted surface water from Drava river upstream of Maribor city (sampling site 1), which, compared with negative control test, caused very weak cytotoxic and genotoxic effects. The value GHP 2 was determined for more polluted surface water from Drava river downstream of Maribor city (sampling site 2), which, compared with negative control test, caused weak cytotoxic and genotoxic effects. The value GHP 5 was determined for biological

Table 2 Suggested classification of tested liquids using the GHP values, determined by the types and amount of cytogenetic effects of test liquids GHP Length Mitotic Increasefactors of: value of roots i n d e x Aberrant Cells with (%) (%) metaphase+ micronuclei anaphase cells l 2 3 4 5 6 7 8

91-98 81-90 71-80 61-70 51-60 41-50 31-40 21-30

84-95 70-83 62-69 54-61 49-55 45-48 40-44 30-39

2.5- 3.5 3.6- 4.0 4.1- 5.1 5.2- 6.9 7.0- 8.4 8.5- 9.0 9.1- 9.5 9.6-11.0

2.5- 3.0 3. l - 3.5 3.6- 6.0 6.1- 7.1 7.2- 8.0 8.1- 9.0 9.1-10.0 10.1-12.0

treated waste water from the outlet of the sewage treatment plant in Race (sampling site 4), which, compared with negative control test, caused strong, even sublethal cytotoxic and genotoxic effects. The values GHP 6 - 8 were determined for untreated waste waters (sampling sites 3, 5 and 6) which compared with negative control test caused strong cytotoxic and genotoxic, even lethal effects.

4. Discussion The Allium test has often been used for the determination of cytotoxic a n d / o r genotoxic effects of various substances (Grant, 1982). It is considered to be a standard procedure for quick testing and detection of toxicity and pollution levels in the environment. Toxic effects may be evaluated by analysing macroscopic (root growth decrease) as well as cytologic parameters (types and frequencies of aberrations and disturbed cell divisions). Results of the Allium test may indicate the presence of certain cytotoxic, genotoxic or mutagenic substances in the environment, which represent direct or indirect risks for all living organisms. The toxicity of the test liquids was indicated by a decrease in root growth. According to the root growth decrease, the samples of untreated waste water showed highest toxicity. These waters may be considered as highly toxic. Less toxic and only weak sublethal effects were expressed in the biological

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treatment plant treated waste water. Water samples from Drava fiver expressed only weak toxic effects. The fact that the root growth decrease over 45% strongly indicates the presence of toxic substances (Fiskesji3, 1985) having sublethal effects on plants (Hidalgo et al., 1989; Wierzbicka, 1988; Antonsiewicz, 1990). After 7 days' growth, the parameter root length was shown as a reliable indicator of toxicity, even for the least polluted test liquids. This parameter is sensitive enough to monitor the pollution level of slightly poquted surface waters. In samples from sampling sites 1-6, different degrees of mitotic index decrease were determined, which indicates the presence of cytotoxic substances in waters. As expected, the highest decrease was found in root tip meristems of plants growing in untreated waste water. A high cytotoxicity level of these waters is also indicated by the development of deformed interphase nuclei. Inhibition of mitotic activities is often used for tracing cytotoxic substances (Linnainmaa et al., 1978). The cytotoxicity level can be determined by the decreased rate of the mitotic index. A mitotic index decrease below 22% of the negative control causes lethal effects on test organisms (Antonsiewicz, 1990), while a decrease below 50% usually has sublethal effects (Panda and Sahu, 1985) and is called cytotoxic limit value (Sharma, 1983). In our studies, the mitotic index decrease in onion root meristem was found to be a reliable means for quick determination of the presence of cytotoxic substances in the environment, for monitoring the cytotoxic pollution level in the natural environments and for evaluations of water pollution levels. This parameter is sensitive enough to also be used for monitoring the pollution levels of slightly polluted surface waters. Statistical analysis of the results showed that the percentage of aberrant metaphase as well as anaphase cells for all test liquids expressed a statistically significant difference varying only in relevance levels. The increase of percentage of aberrant metaphase and anaphase cells in onion root tip meristems indicates genotoxic effects of test liquids. The highest percentage of aberrant cells appeared in the roots, which grew in the most polluted test liquids, that was untreated waste water.

Waste waters, before as well as after treatment procedure, do cause a statistically significant increase in the amount of interphase micronuclei, while this is not the case in slightly polluted surface waters. The results of the Student t-test show that parameter frequency of interphase cells with micronuclei is not sensitive enough for the evaluation of water quality and for discrimination between drinking water quality and surface waters classified as quality groups II-III. The test liquids were classified into 8 quality classes according to their polluted level concerning GHP values. This classification would be more representative with possibility of introducing test batteries which include different bioindicator organisms. In that case, the differences between metabolisms of various bioindicator organisms can be decreased. The Allium test with A l l i u m cepa is one of the short-term biological tests which can identify influence of law-concentration of cytotoxic and genotoxic substances. This research has confirmed that differently polluted surface and waste waters have harmful effects on test materials, in our case, A l l i u m cepa. The possibility of using root tips as a system of monitoring the toxic and mutagenic effects of test liquids is shown. The rate of growth of A l l i u m cepa in differently polluted waters could provide evidence of the inhibition of root growth, decreased percentage of cell division and increased number of aberrant cells. The parameter of interphase cells with micronuclei was proved to be reliable only in cases of more polluted test liquids, e.g., waste waters.

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