Using Tradescantia Micronucleus Assay

Chemosphere 209 (2018) 1e6

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Clastogenecity evaluation of water of Lake Sevan (Armenia) using Tradescantia micronucleus assay

E.A. Aghajanyan a, R.E. Avalyan a, A.E. Simonyan a, A.L. Atoyants a, B.K. Gabrielyan b, * R.M. Aroutiounian a, A. Khosrovyan c, a Laboratory of General and Molecular Genetics, RI “Biology”, Faculty of Biology Yerevan State University, 8, Charents Str, 0025 Yerevan, Armenia b Scientiﬁc Center of Zoology and Hydroecology, National Academy of Sciences of Armenia, 7 Paruyr Sevak, 0014, Yerevan, Armenia c UNESCO UNITWIN/WiCop, Physical Chemistry Department, Faculty of Marine and Environmental Sciences, University of Cadiz, Polígono Río San Pedro S/n, Puerto Real 11510, Cadiz, Spain highlights

We tested clastogenecity of the water of a natural lake using Tradescantia assay. Clastogenic effects in pollen microspores might be linked to Co and Ni ions. A comparison was made with the results of another Tradescantia-based test. Occurrence of micronuclei in microspores coincided with that of dwarf stamen hair. article info abstract

Article history: The clastogenic effects of water samples in seven locations of Lake Sevan (Armenia) with the application Received 15 February 2018 of Trad-MCN (micronuclei) bioassay using Tradescantia (clone 02) were investigated. A signiﬁcant in- Accepted 24 May 2018 crease in the frequency of micronuclei in tetrads of pollen microspores and tetrads with micronuclei Available online 24 May 2018 exposed to the test samples compared to the control has been revealed. A multivariate analysis indicated Handling Editor: Jian-Ying Hu linkage between the frequencies of occurrence of micronuclei in the cells and Ni and Co ions. The results were compared with the endpoints of another Tradescantia-based test system (stamen hair mutation Keywords: test) performed on the same water samples and generation of the plant: occurrences of micronuclei in Tradescantia (clone 02) sporogenic cells coincided with that of non-surviving stamen hair. Genotoxicity © 2018 Elsevier Ltd. All rights reserved. Micronuclei Water pollution Pollen microspores

1. Introduction determination of low concentrations. Therefore, in parallel with the chemical analysis of water it is essential to evaluate its toxicity The aquatic environment is the recipient of different contami- using appropriate biological responses. It was shown that plant nants, a large proportion of which could have genotoxic/carcino- bioassays are sensitive systems to detect the genotoxic and clas- genic potential (Claxton et al., 1998). Many hazardous substances togenic effects of environmental pollutants (Majer et al., 2005). could occur in concentrations beyond their detection limit, despite Among them Tradescantia bioassays Tradescantia micronucleus substantial improvements in methodologies and techniques for (Trad-MCN) and Tradescantia stamen hair mutation (Trad-SHM) are widely used for evaluation of genotoxicity and clastogenecity of natural reservoirs contaminated with heavy metals (Ma, 1999;

* Corresponding author. UNESCO UNITWIN/WiCop, Physical Chemistry Depart- Majer et al., 2002; Steinkellner et al., 1998), organic compounds ment, Faculty of Marine and Environmental Sciences, University of Cadiz. Polígono (Kim et al., 2003), agrochemicals (Mohammed and Ma, 1999; Río San Pedro s/n, Puerto Real 11510, Cadiz, Spain. Rodrigues et al., 1998) and also for mixtures of pollutants (Duan E-mail addresses: [email protected] (E.A. Aghajanyan), [email protected] et al., 1999). Both assays are considered as valuable systems for (R.E. Avalyan), [email protected] (A.E. Simonyan), [email protected] revealing and monitoring a wide range of toxic agents in soil, air (A.L. Atoyants), [email protected] (B.K. Gabrielyan), [email protected] (R.M. Aroutiounian), [email protected] (A. Khosrovyan). and water due to the simplicity of their procedure and high https://doi.org/10.1016/j.chemosphere.2018.05.142 0045-6535/© 2018 Elsevier Ltd. All rights reserved. 2 E.A. Aghajanyan et al. / Chemosphere 209 (2018) 1e6 sensitivity (Aroutiounian, 2006; Batalha, 1999; Cesniene et al., Water samples were collected during Spring 2016 from seven 2010; Garcia et al., 2011; Misík et al., 2011, 2016; Simonyan et al., sites of Lake Sevan, Armenia: Artanish, Karchaghbyur, Tsapatakh, 2016; Thewes et al., 2011). An increase in the level of micronuclei Noradus, Litchk, Martuni and Masrik (Fig. 1). The samples were frequency in Trad-MCN test was found in microspore tetrads of collected by hand from approximately 30 cm depth from three Tradescantia treated with municipal wastewater, as well as nearby locations per sampling site in 1 L pre-cleaned plastic bottles groundwaters contaminated with industrial wastewater (Monarca per location. Samples were stored at þ4 C until delivery to the et al., 2000; Haider et al., 2002; Biscardi et al., 2003). laboratory; tests were initiated next day. More detailed description Lake Sevan is one the largest freshwaters in the world, playing a of the sites can be found in Avalyan et al. (2017). unique role in the economic development of Armenia. The lake is fed by 28 rivers that drain residential, agricultural and industrial 2.2. Trad-MCN bioassay areas. Despite this, only hydrochemical (nutrients, metals, metal- loids) and hydrobiological (quantitative and qualitative analysis of Tradescantia (clone 02) used for this study was obtained from the food web) monitoring is regularly conducted in the lake's basin. the greenhouse of Yerevan State University. It is an interspecific The aim of this study was to assess the clastogenic potential of hybrid T. occidentales and T. ohiensis (Ma et al., 1994), having a water from Lake Sevan by Trad-MCN bioassay. A comparison was multitude of sprouts where inflorescences develop. The test is also conducted with the results of Tradescantia stamen hair muta- based on the scoring of MCN frequency in pollen mother cells at tions test (Trad-SHM) performed in the earlier work (Avalyan et al., disturbances in the process of microsporogenesis (Ma et al., 1994; 2017) with the same water samples and the generation of Trades- Misík et al., 2011). Formation of MCN is related to chromosomal cantia (clone 02). instability and is indicative of genomic damage. The contents of all bottles with sampled water (totally, 3 L) per 2. Material and methods site were mixed together and the sub-samples of 150 mL were taken for testing. The tests were conducted at room temperature, 2.1. Study area and sampling according to Klumpp et al. (2004). Five-seven young inflorescences of Tradescantia were dipped into the sub-samples. The duration of Lake Sevan, one of the largest and most exploited alpine lim- treatment was 24 h (18/6 day/night cycle). The inflorescences were nosystems in the world. Anthropogenic pressures on the lake removed and fixed in Carnoy's solution (3:1 ethanol-glacial acetic include water extraction since the 50s (which has resulted in the acid). Preparation of the tetrads and the scoring of the micronuclei water level dropping by around 20 m and the drying out of the were carried out as described by Ma et al. (1994). The following test spawning grounds of two-subspecies of endemic trout), watershed criteria were used: the number of tetrads with micronuclei (Tetr/ development (which resulted in an increased organic input from MCN) and the number of micronuclei per 100 tetrads (MCN/tetr). domestic and industrial effluents), and overfishing (which resulted For each water sample 3000 tetrads were analyzed. Tap water was in the depletion of the commercial stock). The lake has turned from used as a control and test vessels were set in triplicate. an oligotrophic “trout” water body into a mesotrophic “carp” reservoir. The problem of ineffective use and pollution of the 2.3. Chemical and statistical analysis ecosystem is urgent and an improvement of the ecological situation in the lake basin is extremely important (Hovhanissyan, 1994; Lake Water samples were analyzed for chemical composition by a Sevan, 2016; Matishov et al., 2016). certified laboratory, following standard methods (APHA, 1998).

Artanish

Tsapatakh Noradus

Masrik Litchk

Martuni Karchaghbyur

Fig. 1. Location of sampling areas in Lake Sevan, Armenia: Artanish, Tsapatakh, Masrik, Karchaghbyur, Martuni, Litchk, Noradus. E.A. Aghajanyan et al. / Chemosphere 209 (2018) 1e6 3

Several elements were measured (total concentrations): Al, Ni, Zn, a,b). The maximum manifestation of the clastogenic effects by As, Cu, Fe, Cr, Co, Mo, Cd, Sn, Pb, Mg, Mn, Na, Ca, K. pH level at all both criteria was observed in water samples Masrik, Martuni, Tsa- sites varied from 8.6 to 8.7. Electrical conductivity varied from 650 patakh, Lichk. These results are in good agreement with the gene to 846 mS/cm: Artanish e 650, Karchaghbyur e 690, Noradus e 846, mutations in the somatic cells of Tradescantia detected by the Trad- Masrik e 733, Tsapatakh e 699, Litchk e 752, Martuni e 840. SHM test (Avalyan et al., 2017). However, higher clastogenic re- Electrical conductivity of the tap water control was less than sponses were revealed in Masrik site by Trad-MCN, while the Trad- 200 mS/cm, pH 8.3. SHM test detected a higher number of somatic mutations in Nor- Chemical data were compared to the legislated water quality adus and Litchk (recessive mutation events, RME), Litchk and Tsa- standards for aquaculture (from the Environmental Impact Moni- patakh (white mutation events, WME) and morphological changes toring Center of the Ministry of Nature Protection of Armenia, (dwarf or non-surviving stamen hair, NS) in Masrik. On the con- www.armmonitoring.am), USEPA aquatic life guidelines and WHO trary, both tests similarly identified the samples with a smaller drinking-water quality standards (Table 1). The details of chemical number of clastogenic and genotoxic responses - Artanish and analysis were published by Avalyan et al. (2017). Karchaghbyur. Significant differences between the endpoints, observed in the It should be noted that both criteria of the Trad-MCN test samples and the control were revealed by the independent t-test characterized the samples in a similar manner: higher number of with the help of STATGRAPHICS Centurion16.2 statistical program clastogenic effects in Masrik, followed by almost same-level re- (StatPoint Technologies, Inc. USA; Warrenton, VA). The data are sponses in other stations and with the lowest in Artanish (Fig. 2a b). expressed as mean ± standard error of mean (SEM). Normality and The previous Trad-SHM test revealed a similar distribution of NS homogeneity of variances were checked prior to the analysis to responses across the stations (Avalyan et al., 2017). conform with the assumptions of the test. The multivariate analysis indicated a possible linkage between Principal component analysis (PCA) with Varimax as the NS, WME, Tetr/MCN and Ni and Co with relevance to Masrik and extraction method was selected as a multivariate analysis method Noradus (Fig. 3a and b). Co ions were also associated with all the to reveal linkages among the endpoints of the bioassay and biological responses in Tsapatakh, Noradus, Litchk, Masrik. How- contaminant levels. PCA test also allowed to identify associations ever, in Tsapatakh and Litchk, none of the other metal ions (Al, As, K, between data and sampling locations. For interpreting linkages, Mg, Ca, Cu, Cr and Fe) could be connected to the biological effects loadings more than 0.4 (by absolute value) were considered and for (Fig. 3c). Increasing levels of Ni from anthropogenic sources in the associations of parameters with the sampling locations positive different environments can cause deleterious effects in living or- factor scores were used. ganisms (Nickel Institute, 2011). In several studies with application of Trad-MCN (Misík et al., 2011; Pedro-Escher et al., 2014) and other 3. Results and discussion plant objects (Kieling-Rubio et al., 2012) the toxicity of Ni among other metal contaminants was expected. Opposite results, where Ni One can see that concentrations of many ions (Al, Mg, Cr, Cu, V) did not increase the micronuclei frequency in the pollen tetrads of exceeded standard levels in almost every station, except for Masrik Tradescantia plants are also known (Fargasova et al., 2015). To the and Martini where only V and Cr levels were higher than the best of our knowledge, the toxicity of Co and Ni to aquatic organ- standards (Table 1). Clastogenic effects in the generative sphere of isms received little attention, possibly because of difficulties in Tradescantia showed a significant increase in the frequency of the revealing the mechanisms of toxicity especially in plants (e.g., occurrence of MCN in tetrads and the number of tetrads with Bhalerao et al., 2015). However, in our earlier research these two micronuclei in all studied samples compared to the control (Fig. 2 metals were also related to the mutation events in the stamen hair

Table 1 Total concentrations of chemicals in different parts of Lake Sevan and control, national water quality standards for aquaculture, USEPA aquatic life criteria for continuous concentrations and WHO drinking-water quality standards. Marked area show levels exceeding standards.

Artanish Karchaghbyur Noradus Masrik Tsapatakh Litchk Martuni Control (tap) National water Aquatic life criteria Guidelines for water quality standards for continuous drinking water for aquaculture concentration quaity (WHO) (since 1990) (USEPA)

mg L 1 Na 47.80 72.90 48.60 40.60 61.80 37.90 8.47 8.47 120.00 ee Mg 41.70 58.37 41.90 36.10 52.80 30.10 5.80 5.83 40.00 ee Al 0.05 0.10 0.07 0.04 0.09 0.09 0.00 0.00 0.04 0.09 0.10 p 0.25 0.07 0.07 0.04 0.22 0.06 0.00 0.00 3.50 e K 14.80 19.40 15.06 13.60 20.20 12.30 1.96 1.97 50.00 ee Ca 18.40 25.10 18.90 21.60 23.90 16.30 12.90 12.89 180.00 ee mgL 1 V 5 8 5 5 7 7 5 23 1.00 e b Cr 20 30 20 20 20 20 7 7 1.00 74/11a 50 Mn 0.7 1 0.2 0.2 0.7 1 1 1.1 10.00 e 400.00 Fe 5 460 70 60 190 70 70 8.6 500.00 1000.00 e Co 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.00 10.00 ee Ni 2 3 2 3 3 1 1 1.7 10.00 7.85 70 Cu 1 3 2 1 2 0.6 1 1.1 1.00 e 2000.00 Zn 0.7 7 0.7 0.2 0.6 0.3 0.5 64 10.00 17.80 e As 8 9 8 9 10 8 4 3.8 50.00 150.00 10 Mo 5 7 5 5 5 2 4 2.3 500.00 e 70.00 Pb 0.00 0.1 0.04 0.03 0.00 0.2 0.5 0.1 100.00 0.21 10.00

- Standards not available. a Cr(III)/Cr(VI). b Depends on geographic region (typically, 1e6 mg L-1)(WHO, 2000). 4 E.A. Aghajanyan et al. / Chemosphere 209 (2018) 1e6

Fig. 2. Frequency of clastogenic effects in the sporogenic cells of Tradescantia (clone 02) exposed to water samples from different zones of Lake Sevan basin. a e frequency of tetrads *** with micronuclei, b e micronuclei in tetrads. p < 0,001 - signiﬁcant differences from the control. of Tradescantia in Tsapatakh, Artanish and Karchaghbyur samples Alternatively, a presence of other agents not measured in this work (Avalyan et al., 2017), suggesting that the exposure pathway cannot be excluded. employed in our studies (inﬂorescences submerged into water) Importantly, the levels of many metals in water (e.g., Ni, Co, could provide important information for understanding the effects Mn, Fe, As, Mo, Pb) did not exceed national regulatory standards. of dissolved metals. Further research on the uptake of ionic Ni and However, effects may be observed at concentrations far below Co (including their combination) via different exposure pathways those predicted to be safe by regulatory frameworks (Liess et al., may elucidate their toxicity and mechanisms. 2016). Furthermore, Tradescantia test systems are known for their Further, in Masrik and Martuni samples, V and Cr concentrations high performance at low concentrations and the present results exceeded the standards (Table 1). However, the results of PCA did have also shown this. Moreover, the toxicity of a mixture depends not associate biological responses with V neither in these nor any not only on the exposure concentration of each mixture constit- other sample, although highest occurrences of micronuclei (Tetr/ uent and its ratio but also on the means of the toxicants to act MCN and MCN/tetr) in microspores of Tradescantia were found in jointly (Altenburger et al., 2004). A chemical mixture of metals the above-mentioned samples. This may suggest the high sensi- appears more toxic than individual metals if their action is syn- tivity of the test to V and Cr in general or to their particular ergistic. On the other hand, the antagonistic effects of some chemical forms. Knasmuller et al. (1998) reported clear dose-effect metals in a mixture diminish the uptake of individual metals responses obtained from the Tradescantia polen MCN test when (Barata et al., 2002; Panda and Panda, 2002) e.g., by competing for exposed to Cr6þ and Ni2þ (in contrast to Cr3þ), although the the same uptake sites at the cell surface (Bervoets and Blust, concentrations tested were much higher than those in our study. 2000; Qu et al., 2013). E.A. Aghajanyan et al. / Chemosphere 209 (2018) 1e6 5

inﬂorescences by the same agents in the water, assuming the collective effect of more than individual metals, even at low concentration.

4. Conclusion

In this work, we intended to identify toxicants in complex mixtures by linking exposure information to biological effects. According to the results of our studies, a marked increase in the level of clastogenicity in the water samples of Lake Sevan has been detected. This points out to the presence of toxic agents that are able to inﬂuence the processes occurring in the generative sphere of Tradescantia. It also indicates the effectiveness of the application of Trad-MCN bioassay for the assessment of clastogenicity effects of natural waters, especially at low level of metal contaminants. Tetr/ a) MCN and MCN/tetr endpoints similarly identiﬁed the impact of the lake's water on microspores of the Tradescantia. When compared to the results of Trad-SHM test, it showed a similar pattern to NS occurrences across the sites. Both tests demonstrate high sensitivity to environmental agents in the lake that are able to cause mutations in somatic and clastogenecity in sporagenic cells of Tradescantia (clone 02).

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