Assessment of Mutagenicity of Water from Lake Sevan, Armenia with Application of Tradescantia (Clone

Home , Artanish, Karchaghbyur

Mutation Research 800 (2017) 8–13

Contents lists available at ScienceDirect

Mutation Research/Genome instability and disease

journal homepage: www.elsevier.com/locate/molmut

Community address: www.elsevier.com/locate/mutres

Assessment of mutagenicity of water from Lake Sevan, Armenia with

application of Tradescantia (clone 02)

a a b,∗ a a

R.E. Avalyan , E.A. Aghajanyan , A. Khosrovyan , A.L. Atoyants , A.E. Simonyan , a

R.M. Aroutiounian

Laboratory of General and Molecular Genetics, RI “Biology”, Faculty of Biology Yerevan State University, 8, Charents Str., 0025, Yerevan, Armenia

UNESCO UNITWIN/WiCop, Physical Chemistry Department, Faculty of Marine and Environmental Sciences, University of Cadiz. Polígono Río San Pedro s/n,

Puerto Real 11510, Cádiz, Spain

a r t i c l e i n f o a b s t r a c t

Article history: For many decades water resources in Armenia have been affected by anthropogenic activity, therefore,

Received 17 October 2016

a regular bioindication of genotoxic effects of the water bodies is desirable. The genotoxicity of water

Received in revised form 19 February 2017

samples collected from different parts of Lake Sevan were assessed by means of Trad-SHM (stamen hair

Accepted 28 March 2017

mutation) assay using Tradescantia (clone 02). Here we report a signiﬁcant increase in the frequency of

Available online 31 March 2017

somatic mutations and morphological changes in the Tradescantia inﬂorescences exposed to the water

samples compared to the control. The somatic mutations (recessive mutation and white mutation events)

Keywords:

were mostly linked to the concentration of Al, Ni, As, Co and Pb in Artanish, Tsapatakh and Karchaghbyur,

Water pollution

Noradus, Martuni and Litchk, while morphological changes (non-surviving hairs) were related to Co level

Tradescantia (clone 02)

Genotoxicity in Tsapatakh and Karchaghbyur. The results obtained show that Lake Sevan contains substances which

Mutations may cause genotoxicity and teratogenicity in Tradescantia and probably also in aquatic animals. The

Environmental pollution results also show that Trad-SHM assay can be used for monitoring natural resources.

1. Introduction micronucleus (Trad-MCN) and Tradescantia stamen hair mutation

(Trad-SHM) assays [16]. The results revealed genetic alterations in

Current levels of anthropogenic stress on freshwater resources the ﬁsh and plant, statistically attributed to Al, Fe, Cu and Mn lev-

make it practically necessary to periodically evaluate water quality els in the water, which were collected from the sites other than

and the health of hydroecosystems. Lake Sevan, one of the largest those in this study. The results for ﬁsh and Tradescantia were in

Alpine lakes in the world, plays a unique and important role in high agreement.

the economic development of Armenia. However, the basin of the Plants represent important organisms for use in environmental

lake drains 28 tributaries that ﬂow through densely populated monitoring as they are sensitive and reliable indicators of contam-

agricultural and industrial areas. Contaminant inﬂow from these ination of the biosphere. Test-systems based on plant use have

tributaries carrying industrial, agricultural and domestic waste is shown high sensitivity and are recommended for monitoring geno-

an important cause of change in the lake’s ecosystem. In spite toxicity and assessment of carcinogenicity of xenobiotics in the

of this, monitoring of the state of the lake’s ecosystem has been air, water and soil medium [8,12]. Among plants tested, a spe-

restricted to monitoring the chemical content of the water and cial place is occupied by the heterozygote clones of Tradescantia

to biological monitoring. For the ﬁrst time, genotoxicity of the (clones 02 and 4430). They were shown to perform successfully in

lake’s water was assessed by a combination of the water chemical dose-response studies and as biodetectors of chemical mutagens

composition and three bioassays: comet assay in erythrocytes of [14,9,5,7,22]. Tradescantia was used in air monitoring programs

gibel carp (Carassius auratus gibelio) and plant assays: Tradescantia [4], for evaluating toxicity of sewage sludge and liquid samples

[10,15,2] and soil samples [1]. Trad-SHM test is a reliable and sen-

sitive system for monitoring gaseous and liquid mutagens in the

∗ laboratory [12] and in situ [4].

Corresponding author. Tel.: +34 635160070.

The aim of our study was to assess the genotoxicity of water

E-mail addresses: re [email protected] (R.E. Avalyan), [email protected]

from different parts of Lake Sevan, Armenia, Trad-SHM assay with

(E.A. Aghajanyan), [email protected] (A. Khosrovyan), [email protected]

(A.L. Atoyants), [email protected] (A.E. Simonyan), [email protected] Tradescantia (clone 02).

(R.M. Aroutiounian).

http://dx.doi.org/10.1016/j.mrfmmm.2017.03.006

R.E. Avalyan et al. / Mutation Research 800 (2017) 8–13 9

2. Material and methods During the biotest Trad-SHM the following changes have been con-

sidered as indicator test-criteria: change of the cell color from blue

2.1. Sampling sites to pink (recessive mutation events – RME) and appearance of white

cells (undeﬁned white mutation events – WME). Besides somatic

Water samples were collected by hand during Spring 2016 mutations, morphological changes are also taken into account:

at three different times from seven locations in Lake Sevan, under-developed hairs of less than 12 cells (non-surviving hairs –

Armenia (near river discharges and in the bay area) at an approx- NS) and branching hairs (BH). Lastly, changes in the ﬂower structure

imate depth of 30 cm depth in 1 L clean plastic bottles from were taken into account: change in the stamen numbers (normally,

three nearby locations per sampling site (Fig. 1). Samples were 6), fused petals, change in the ﬂower color from blue to pink/white,

◦

stored at +4 C pending delivery to the laboratory and the initi- other possible changes.

ation of tests which were done the next day. According to the Flowers were examined daily for 21 days as they appeared. From

long-term data of the Environmental Impact Monitoring Cen- 8000–14,000 stamen hairs were obtained per sample. The number

ter of the Ministry of Nature Protection of Armenia, Litchk, of mutations per sample and per mutation were calculated per 1000

Karchaghbyur, Artanish and Tsapatakh sites are relatively less hairs, according to standard protocols [13].

contaminated compared to Noradus, Masrik and Martuni sites.

◦ ◦

Litchk (40 11 11,391“N and 45 14 56,327“E) and Karchaghbyur

◦ ◦ 2.4. Chemical and statistical analysis

(40 10 54,225“N and 45 34 53,418“E) sites are located near spawn-

ing rivers of endemic Sevan trout. Both rivers are spring-fed,

Water samples were analyzed for chemical composition by a

relatively short and away from sources of industrial contami-

◦ ◦ certiﬁed laboratory, according to standard methods [3]. Several ele-

nation. The Artanish (40 27 19,303“N and 45 25 12,123“E) site

ments were measured (total concentrations): Al, Ni, Zn, As, Cu, Fe,

is located in the Artanish bay, which is a protected area and

Cr, Co, Mo, Cd, Sn, Pb, Mg, Mn, Na, Ca, K.

a nursing ground for Varicorhinus capoeta and Coregonus lavare-

◦ ◦ Chemical data were compared to the legislated water quality

tus ﬁsh species. Tsapatakh (40 24 34,465“N and 45 27 49,216“E)

◦ standards for aquaculture (from the Environmental Impact Mon-

is another low contaminated site. Noradus (40 24 0,386“N and

◦ ◦ ◦ itoring Center of the Ministry of Nature Protection of Armenia,

45 10 1706“E), Masrik (40 13 25,447“N and 45 38 21,822“E) and

◦ ◦ www.armmonitoring.am), USEPA aquatic life guidelines and WHO

Martuni (40 9 53,285“N and 45 30 21,649“E) sites are located near

drinking-water quality standards.

rivers which discharge untreated industrial or domestic waste to

Principal component analysis (PCA) with Varimax as the extrac-

the lake. Near Masrik, the river is affected by mining activities in its

tion method was selected as a multivariate analysis method to

drainage area, and Martuni and Noradus sites are affected by rivers

observe linkages between mutation events and contaminant lev-

carrying industrial and domestic efﬂuents.

els, and their association to the sampling sites. For interpreting

variables within a principal component, loadings more than 0.4 (by

2.2. Tradescantia

absolute value) were considered.

Signiﬁcant differences between the endpoints, observed in the

Tradescantia (clone 02) is an interspeciﬁc hybrid T.occidentales

samples and the control (p < 0.05, p < 0.01, p < 0.001), were analyzed

and T.ohiensis [13], having a multitude of sprouts where inﬂores-

with the Student t-test (equality of variances is not assumed) by

cences develop. Each inﬂorescence contains a large number of buds.

the statistical program STATGRAPHICS Centurion 16.2 (StatPoint

Normally, the ﬂower emerges every three days, has three blue

Technologies, Inc. USA; Warrenton, VA).

petals that are surrounded with three sepals, a pistil and six sta-

mens. A characteristic feature of the ﬂower is the presence of 30–50

and more hairs (occasionally fewer) on a stamen, and each hair, 3. Results and discussion

in turn, consists of 12–35 somatic cells. The plant is heterozygous

for blue/pink alleles in the ﬂoral parts [13]. Blue ﬂower pigment Genotoxic and morphological effects of the water samples

is dominant while mutant cells are pink. The plant is successfully determined by the Trad-SHM assay are given in Fig. 2(a,b,c) and the

−1

acclimated to the conditions of Armenia, has strong root systems, chemical content of water samples (total concentrations, mg L )

can grow in a variety of soils and is very sensitive to environmental in Table 1. The data represent the mean of three independent

pollutants. experiments. For comparison, national water quality standards for

Tradescantia (clone 02) used for this study was taken from the aquaculture, USEPA aquatic life criteria for continuous concentra-

greenhouse of Yerevan State University. tions and WHO drinking-water quality standards are also given in

Table 1. For metals where toxicity is hardness-dependent (Pb, Ni

2.3. Trad-SHM assay and Zn), a re-calculation was made as suggested by USEPA guide-

lines [18]. While the concentrations of most elements were below

Water samples were investigated for genotoxicity assessment national water quality standards for aquaculture, the concentra-

by means of Trad-SHM assay. Three independent experiments were tions of Mg, Al, V, Cr, and Cu exceeded them in essentially all

carried out with ﬁve replicates per sampling site. The contents of samples. V, Cr, Zn and Cu concentrations in tap water control also

all bottles from each site (totaling 3 L) were mixed together and exceeded the national standards for aquaculture (Table 1). Cd and

150 mL samples taken for testing. The assay has been described Sn levels were almost 100 times below their corresponding stan-

−1

in detail by Pogosyan et al. [15] and Aghajanyan et al. [2]. In brief, dards (5 and 1 ␮g L , not shown). However, when compared to

young inﬂorescences of the plant were submerged in water samples the USEPA aquatic life criteria for continuous effect concentrations,

(3 inﬂorescences per vessel) for 18 h and after a 7-day recovery only Cr (VI) concentration in all sites but Litchk and Martuni might

period, the ﬂower blossoms were examined. Stamens of a ﬂower have exceeded the corresponding criteria, although in our study we

were placed on a slide and stamen hair viewed under a magnifying measured total Cr concentrations. When compared to the available

glass at 10 x for counting blue-to-pink mutations in stamen hairs WHO guidelines for drinking-water quality [20], only Al concentra-

and morphological changes. Tap water was used as control in the tion in Litchk sample exceeded the guideline, while chemical levels

bioassays. at most sites were far below the drinking-water guideline levels

Stamen hairs in Tradescantia ﬂowers, where active cell division (Table 1). In the tap water control, the concentrations of all mea-

takes place, are the most sensitive place for mutations to occur. sured elements were below WHO’s guideline levels. pH level at all

10 R.E. Avalyan et al. / Mutation Research 800 (2017) 8–13

Fig. 1. Location of sampling areas in Lake Sevan, Armenia: Artanish, Tsapatakh, Masrik, Karchaghbyur, Martuni, Litchk, Noradus.

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.

* Cr(III)/Cr(VI).

−1

** Depends on geographic region (typically, 1–6 ␮g L ) [21].

Control with tap water.

- Standards not available.

sites varied from 8.6 to 8.7. Electrical conductivity varied from 650 cal conductivity of the tap water control was less than 200 ␮S/cm,

to 846 ␮S/cm: Artanish – 650, Karchaghbyur – 690, Noradus – 846, pH 8.3.

Masrik – 733, Tsapatakh – 699, Litchk – 752, Martuni – 840. Electri-

R.E. Avalyan et al. / Mutation Research 800 (2017) 8–13 11

a) larly, morphological changes (NS hairs, so called dwarf cells which

** are evidences of potential teratogen effects in Tradescantia ([22])

1.8 ** RME/1000

1.6 were also signiﬁcantly more frequent in all samples, compared to

events 1.4 ** the control (5–30 times), with highest frequency at Masrik (Fig. 2c).

E 1.2 **

Thus, from the results of disturbances in the stamen hairs of the RM

1 *

* plant (RME, WME, NS), it can be suggested that water samples col- of

0.8 *

0.6 lected from different parts of Lake Sevan contained substances that

0.4

were able to cause genotoxic and possibly, teratogen effects.

0.2

The results of this study show that although the levels of

Frequency 0

elements measured did not exceed the drinking-water quality

guidelines set by WHO, somatic mutations and morphological

changes in the plant’s stamen hairs were frequent in all samples.

Sampling sites

Notably, the national water quality standards for aquaculture and

USEPA aquatic life criteria were stricter for some elements, com-

b) pared to the WHO standards for drinking-water, e.g., Al, Cr, Cu. At all

18 sites, V and Cr levels exceeded the national water quality standards

*** WME/1000

16 *** for aquaculture. At Litchk, Tsapatakh, Karchaghbyur and Artanish

14 ***

*** sites that have usually been considered less contaminated, at least events 12 ***

E *** ***

three elements had concentration above the national water quality

WM standards for aquaculture. For example, at Litchk, concentrations of 8 of

6 Al, V, and Cr considerably exceeded the standards: Al by a factor of

three, V and Cr by seven times. At Artanish and Karchaghbyur, Na,

Mg, Al, V, Cr, Ni and As levels also exceeded the standards, in partic- 0

Frequency

ular V ﬁve times and Cr twenty. Hence the high frequency of somatic

mutations occurred in the plant exposed to the waters from less

contaminated sites does not seem unusual, given the high concen-

Sampling sites trations of several chemicals. Alternatively, this may suggest any of

the following: the assay is highly sensitive to particular elements;

the assay is highly sensitive to low concentrations of chemicals;

c) there was an occasional supply of contaminants in those areas by

river discharges or wave/current action at the time of sampling. In

addition, effects may be observed at concentrations far below those ***

NS/1000 predicted to be safe by regulatory frameworks [11].

10 The results of principal component analysis are represented events

8 in two separate graphs to avoid data crowding in a single graph NS

(Fig. 3a,b). Only statistical associations found between the distur-

of ***

6 ***

*** *** bances observed and chemical levels are shown. The percentage of

4 ***

total variance explained by each component is shown near compo-

2 nents. It can be seen that the concentration of Al, P, Ni and As ions

Frequency

0 were statistically linked to the WME events observed in stamen

hair cells of Tradescantia (clone 2) at Artanish, Tsapatakh and Kar-

chaghbyur, while both somatic mutation events (RME and WME)

were linked to Al concentration at Noradus and Litchk (Fig. 3a). The

Sampling sites ions of Co were also related to somatic and morphological distur-

bances at Tsapatakh and Karchaghbyur and Pb levels to somatic

Fig. 2. Frequency of genotoxic and morphological effects of water samples from

disturbances at Noradus, Litchk and Martuni (Fig. 3b). Given that

different zones of Lake Sevan basin in the somatic cells of Tradescantia (clone 02).

both Pb and Co concentrations were far below regulatory values

RME – recessive mutation events; WME – undeﬁned (white) mutation events; NS –

(Table 1), this may assume a likely effect of even low concen-

non-surviving hair (morphological effect). Signiﬁcant differences from the control

are denoted by asterisk, signiﬁcance levels: * p < 0.05, ** p < 0.01, *** p < 0.001. trations of multiple toxicants on plant cells. Toxicological effects

may occur at concentrations far below regulatory levels and syn-

ergistic action of multiple stressors on an organism often exceeds

The mean total number of stamen hairs examined per sampling the effects of individual stressors [6,11,19]. Morphological distur-

site (depending on the number of ﬂowers that blossomed during bances (NS) were mostly related to Tsapatakh and Karchaghbyur

the 21-day count) were as follows: Artanish – 8155, Masrik – 8336, sites (Fig. 3b). Appearance of dwarf NS hairs in Tradescantia is asso-

Karchaghbyur – 8977, Noradus – 9237, Martuni – 13693, Litchk – ciated with teratogenic effects [22] and since NS frequency in all

13738, Tsapatakh – 14420, and control – 10752. water samples was signiﬁcantly higher than in the control, it may

The study of the genotoxicity of the water samples by the Trad- be evidence of the presence of teratogens in the lake, especially at

SHM assay has shown a signiﬁcant increase in the frequency of these sites. Somatic mutations (RME and WME) were closely cor-

somatic mutations (RME and WME) and morphological changes related with each other, which seems to be usual for lower doses

(NS) in all water samples compared to the control. In all samples, the of exposure. Underbrink et al. [17] mentioned that at higher doses

level of RME exceeded that of the control by 3.5–9 times, depend- this relationship changed to one aberrant type predominance.

ing on the sample. The highest frequency of RME occurred in the These results conﬁrm that toxicological effects may be caused

Noradus and Litchk samples (8–9 times) and in the Martuni and even at low levels of contaminants as it has been acknowledged

Tsapatakh samples (6 times) (Fig. 2a). The frequency of WME in all by previous authors. As well, the results conﬁrm the reported high

samples also exceeded that of the control (3.5–4 times), with the sensitivity of Trad-SHM assay to low levels of chemicals.

highest rate in the Litchk and Tsapatakh samples (Fig. 2b). Simi-

12 R.E. Avalyan et al. / Mutation Research 800 (2017) 8–13

Fig. 3. Biplot of component scores and loadings of the sampling sites (blue dots) and selected chemicals and genetic and morphological effects (brown dots) in the coordinates

of the two components. RME – recessive mutation events, WME – undeﬁned mutation events, NS – non survived hairs (morphological changes) in stamen hair cells of

Tradescantia (clone 2). In parentheses, percentage of total variance explained by a component. a,b show associations between genetic mutations and different chemicals.

(For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.).

Moreover, while in all samples the differences in the frequency 4. Conclusion

of mutations and morphological changes were signiﬁcantly higher

than in the control, NS events were mostly connected to Noradus These results indicate that Trad-SHM bioassay of Tradescantia

and Masrik sites and RME and WME with the other sites, accord- (clone 02) can be applied for biotesting of water quality of aquatic

ing to PCA results. This may suggest that different parts of Lake ecosystems, in particular, for genotoxicity testing. Genotoxic effects

Sevan basin are affected by a different contaminant mix, possibly of various waters on Tradescantia (clone 02) suggest that the lake’s

caused by the type of the anthropogenic activity evolved in the basin is contaminated by xenobiotics with the genotoxicity and ter-

area and their cumulative effect is variant. Nevertheless, based on atogenicity potential. Mainly, concentrations of Al, As, Ni, Pb and Co

the signiﬁcantly higher frequency of disturbances observed in all may be the cause of the disturbances observed. It is recommended

samples (Fig. 2) one may assume that river discharges in Lake Sevan to combine the results of Trad-SHM assay with that of other toxicity

supply chemicals that are capable of inducing genotoxicity and ter- assays on aquatic animals to reveal a relationship between them.

atogenicity in Tradescantia (clone 02) and probably in the resident

aquatic organisms. Related to this, one of the important tasks of the

Acknowledgments

biomonitoring of the natural water resources in Armenia is not only

a systematic control over the chemical content of the waters but

This research program (Scientiﬁc Basis of the Integrated Man-

also ecotoxicological assessment of the impact of contamination.

agement of Natural Resources of Lake Sevan, 15RF – 049) was

Our study conﬁrms the usefulness of Trad-SHM assay for genetic

funded by State committee of science (Ministry of Education and

monitoring of the natural water resources and its high sensitivity

Science of Armenia).

to even low concentrations of environmental pollutants. However,

despite the results of Trad-SHM assay having indicated the occur-

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