Trees under polluted environment

Trees under polluted environment Research collection

Snejana Dineva

2017

Contents

Deciduous Tree Species Under Polluted Air Environment and Leaf Blade Modifications..................................................................... 3

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria) ...................................................... 39

Ecotoxicology Assessment Model of Plant-Soil Complex Treated with Radomir Metal Industries Waste Water ................................ 59

Effects of Chronic Ionizing Radiation And Interactions With Other Environmental and Climatic Factors on Plant Growth And Development .............................................................................. 96

2

Deciduous tree species under polluted air environment and leaf blade modifications Snejana Boycheva Dineva1

Abstract The air pollutants such as gaseous, aerosols and dust effect directly and indirectly on plants leaves with or without visible damages. The leaf is a part of the plant that first and most obviously exhibit visible symptoms of injury and that can serve as bioindicator for tolerance of the species. Nevertheless, even without visible injures, biochemical, physiological and cellular changes can take place to mitigate stress and the ample used of the plant internal resources. Typically under ambient air pollution tree plants adapt through leaf blade modifications. Evaluation of leaf alterations in plants exposed to air pollution is an important task for biological monitoring and mitigation strategies. The aim of study is to evaluate leaf blade morphology and structure amendments under industrial air pollution for deciduous trees most applied in urban planting: Acer campestre L, Acer negundo L, Acer saccharinum L, Acer tataricum L, Acer negundo L., Fraxinus americana L, Morus alba L, Platanus acerifolia Willd. Key words: Acer campestre L, Acer negundo L, Acer saccharinum L, Acer tataricum L, Acer negundo L., Fraxinus

1

Trakia University - Stara Zagora; Faculty of Techniques and Technology; http://tk.unisz.bg, Address: Yambol 8602, "Gr. Ignatiev" str. №38, Bulgaria, e-mail: [email protected]

3

americana L, Morus alba L, Platanus acerifolia Willd., leave blades, polluted environment

Introduction Air pollution causes marvellous negative impact on human health, plants and animals populations (Dhaliwal, 2012; Kapoor, 2017). The pollutants with great direct influence are particulate matter, SO2 and O3 (https://www.forestry.gov.uk

/fr/urgc-7edhqh).

Trees

can

capture

significant quantities of health-damaging particles improving local air quality. Plants as attached organisms are unable to avoid hostile environment and are continuously exposed to contaminated conditions (Kumar et al., 2015). The air contamination as atmospheric gases, aerosol particles and precipitations, influence directly on the leaves or needles and bark of trees (Balouet & Chalot, 2015). Therefore, the leaf is the part of the plant that first and most obviously displays visible symptoms of injury from air pollution (Shrivastava & Prakash, 2016). The ambient air pollution initiated complicate leaf lamina modifications that are species-dependent and correlated to protective or adaptive mechanism of plants (Wuytack et al., 2011; Noor et al., 2014). Plants with short life cycle can adapt to external extreme factors through generations, while the long-lived tree species expressed adequate adaptation based on their genetic potential. The study of air pollution in terms of trees may include parts of the tree, individual trees, or entire forests. Nonetheless, the leaf laminas reflect changes in environmental conditions for a relatively short time and are considered as a proper tool for investigation. The

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investigations of leaf blades are mainly used in assessing the destruction of forests, where air pollution is considered a major disturbing factor. Tree leaves are sensitive and highly exposed to air pollution thus can be used to assess the air quality of urban environment. The contaminants from the air primarily leave a chemical signature only in the leaves, needles, and bark, and are not transported into the xylem (Balouet & Chalot, 2015). The application of tree leaves for the assessment of air contamination is highly dependent on the morphological and anatomical parameters of leaves (Baranyai & Posta, 2015). Evaluation of leaf alterations in plants on exposure to pollution is an important step to screen and isolate tolerant plants from sensitive ones (Rai, 2016). The sensitive species are commonly used as bioindicators while the tolerant once for air pollution management. In the last decades many morphological, anatomical, physiological, and biochemical studies have been done to determine the reactions of tree species toward air pollution (Gupta & Ghouse, 1988; Jahan & Iqbal, 1992; Kurteva & Stambolieva, 2007; Pourkhabbaz et al., 2010; Assadi et al., 2011; Wuytack et al., 2011; Thambavani & Maheswari, 2012; Appalasamy et al., 2017; Gupta et al., 2015; 2016; Uka et al., 2017). The aim of investigation is to provide information about the leaf morphology and structure amendments of some deciduous tree species: Acer campestre L, Acer negundo L, Acer saccharinum L, Acer tataricum L, Acer negundo L, Fraxinus americana L, Morus alba L, Platanus acerifolia Willd, developed under polluted environment.

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Importance of deciduous tree species for mitigation of air pollution Pollution caused by particles with aerodynamic diameters less than 2.5μm (PM2.5) is a major environmental problem. According to the WHO (2016b) and EAI analysis, Bulgaria is on the second place of dead’s cases in the World (fig. 1), causing from impure air conditions and on the first place in EU (EEA Report No 28/2016). Planting more trees has been suggested as an unconventional approach to dismiss the problem (Yang et al., 2015). Trees with pronounced adapt ability are used for planting and mitigating the adverse air conditions. According to Beckett et al. (2000), the best choices for pollution-control through implementing plants are conifers trees. Among the broad-leaved species most effective at capturing particles are those with rough leaf surfaces (Beckett et al., 2000). The management of air quality includes reduction of emission at source level, alteration of pollutants to a less harmful compounds and sequestration. However, application of ornamental trees to improve air quality may consider as a well alternative approach (Kapoor, 2017). Green plants create vast positive effect on urban microclimate by reducing air and noise pollution (Beckett et al., 2000; Freiman et al., 2006; Nowak et al., 2006; Bealey et al., 2007; Jim and Chen, 2007; McDonald et al., 2007; Kumar et al., 2013; Stratu et al., 2016).

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Figure 1. The human cost - premature deaths attributable to PM 2.5 exposure

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According to Kapoor (2017), through air quality management by planting plants can be achieved the next goals:  improving the air quality;  supporting and implementing the developmental works;  creating good working conditions;  providing good healthy condition;  enriching the aesthetic value of a site;  reducing other types of environmental pollutions;  ensuring safe and unhazardous journey;  promoting green-belt development;  developing awareness in environment management among the peoples. Many plants are well known for improvement of air quality in addition to their economic importance (table 1). Commonly species with more complexes stem structure and smaller leaves have greater relative deposition velocities. The choice of tree species, a planting design and location from pollution source are critical in defining the effectiveness of refining air by trees (Freer-Smith et al., 2004). The main characteristics used to evaluate tree species’ adaptation to urban environments are: tolerance of poor soil and drought; resistance to pest and disease; tolerance of SO2, NO2 and O3. However, stress tolerance of trees is perhaps the first selection criterion of decision making when the trees are chosen for planting in polluted areas (Grote et al., 2016). Among the top ten most frequently occurring tree species in urban areas, estimated from 328 cities observations, Platanus acerifolia (Aiton) Willd take third position; Acer saccharinum L. – fifth; Acer negundo L. – sixth; and Morus alba L. – tenth, (Yang et al., 2015).

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Platanus acerifolia Willd is considered as less sensitive species to SO2 and fluorides (Mathy P., 1987) and very tolerant to pollution (Gregorová et al., 2010). Fraxinus americana L. has been reported as sensitive to ozone (Oswalt & Clatterbuck, 2005).

Table 1. Economic importance of tree species, place of origin (endemism), and fragility (Stefanov and Ganchev, 1958; Delkov, 1988; Prokopiev, 1978) Tree Species

Importance 1

Acer campestre L.

2

X

1

2

Fragility 3

X

Acer negundo L. Acer tataricum L.

3

Origin

X

+ X

X

X

Fraxinus americana L.

X

+ +

X

+

Morus alba L.

X

X

+

Platanus acerifolia Willd.

X

X

++

Table 1 Legend: Importance of the tree species:

Origin of the tree species:

1) primary economic importance;

1) autochthonous;

2) secondary economic

2) endemic to the Balkan Peninsula;

importance;

3) introduced from other parts of the

3) lowest economic importance

world.

(widely distributed in the parks). Acer saccharinum L. is recommended for designing the plant park and green areas (Gyenov et al., 1995). Silver maple is given as SO2 tolerant species (Davis and Gerhold, 1976; Appleton et al., 2014) and intermediate

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to fluorides (Mathy P., 1987). Silver maple is a tree species with great capacity effectively to remove PM2.5 from urban air (Yang et al., 2015). Acer negundo L effectively remove PM2.5 from urban air (Yang et al., 2015) and it is suitable to be applied for the monitoring of urban dust (Baranyai & Posta, 2015). Boxelder has been reported as resistant to polluting elements in the atmosphere (Oswalt & Clatterbuck, 2005; Stratu et al., 2016) and intermediate to O3 and SO2 (Oswalt & Clatterbuck, 2005). Morus alba L is good carbon sink plants, with high absorption to air pollutants such as chlorine, hydrogen fluoride, and sulphur dioxide (Lu & Jiang, 2003). In many studies has been recommended for green belt development in industrially polluted regions having aesthetic value and ability to reduce air pollution (Orwa et al., 2009; Jian et al., 2012; Gupta et al., 2016). Acer campestre L is a relatively sensitive to high O3 levels (Calatayud et al, 2007), reported as a tolerant to SO2, and intermediate to hydrogen fluoride (Trees for Polluted Air, 1973). It is suitable for planting in industrial area based on its high APTI (Air pollution Tolerance index) value that range between 30 up to 100 (Kapoor, 2017). Field maple has been recognized as ideal for the interception of pollutant particles (Beckett et al., 2000) and for building green belts (Gyenov et al., 1995). Acer tataricum L. is assessed as high resistant species (Ilkun 1971; Antipov 1974; 1975). Nevertheless, there are some reports that refereed Tatarian maple as less resistant or sensitive one (Krasinskii 1950; Tolonnikova 1970). These dissensions are due to the lack of uniform method and criteria for assessment in addition to the different conditions of the environment in which plant genotype has been expressed.

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Characteristic of the polluted region The examined tree species were developed on territory of metallurgical plant "Kremikovtzi" (42°47’N; 23°30’E), that was the largest metalworking factory in Sofia’s valley, stranded on 17 km northeast from Sofia. MP "Kremikovtzi" was a “hot spot” source of SO2, NxOx, Pb, As, Zn, Cu etc. and dust. The heat power stations of “Kremikovtzi” and the metallurgic plant have been emitted 90% of the total pollution amount in Sofia district (Tzekova et al, 2004). Factors determining the extent of tree destruction by pollution on trees are: type and concentration of contaminants; distance from the source of contamination; duration of exposure; weather conditions. Other important factors are the topography of the region, soil moisture and nutrients, maturity of plant tissue, time of year (season) and the type of plants. Metal smelting plants, release mainly SO2, H2S, NO2, and HF into the atmosphere. The main pollutant in Kremikovtzi vicinity is the sulfur dioxide, and during the investigation period the amount of the sulfur dioxide in the observed region was 0.5mg/м3. According to Kapoor (2017) air pollutants affect the plants even in very low concentration from 0.1 to 55 ppm. The experimental data suggest that 1.0-3.3 μg/m3 sulphate particle concentrations that correspond to 150–500 mmol/litre of sulphate in mist are suitable thresholds for trees in areas where cloud cover occurs for more than 10% of the time and visible foliar lesions on leaf surface structure didn’t appears(Cape, 1993). The landscapes, air currents and movements influence pollutant concentration, chemical structure, and the duration of tree exposure to pollutants (Appleton et al., 2014). For the Kremikovtzi region is typical strong winds with speed over 12-14 m/s during spring and autumn, from the West-North-western direction (figure 2). The largest percentage quiet

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time for Kremikovtzi area (42%) leads to longer retention of pollutants in the ground layer of the atmosphere to the area around the plant (Stoyanov, 2011). The plants are more damaged by atmosphere pollutants during cooler months when the stomata are open, than during the warmer months when the stomata control are enhanced to store water needed, and to reduce transpiration (Lange et al., 1989). Water deficiency of the soil, temperature extremes, humidity and light often change the plant response to air pollutants (Edward & Chappelka, 2004). On the figure 3 are given weather conditions for the period of study.

Figure 2. Wind rose, metallurgical plant Kremikovtzi (Stoyanov, 2011)

The seasonal rainfall variations are ample with average 566 mm annual rainfall. The lowest precipitations take place in winter while spring brings most rainfall (Fig. 3). Snow cover remains on average for 43 days (Curchod, 2005).

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Figure 3. Climate: monthly averages, Sofia region (SCOPES, 2004)

The serious environmental problem in the region is the soil heavy-metal contamination. More, than three decades of unrestricted environmental pollution with heavy metals have resulted in a contamination of hundreds of hectares of originally very rich soil to values up to ten times the permissible levels for Cd, Pb, Mn and As (Curchod, 2005). The control trees were growing in Vitosha National Park (42°30’N; 23°15’E), near to the lift station of Dragalevtzi. That area has relatively clean air and similar environmental conditions.

Plant material and methods The study examined morphological and anatomical changes of leaf blade structure under industrial pollution. The trees were with similar age, uniform height and growth form, approximately 15 years old, and sun

13

exposure. The plant material was collected July, from the south side of the crown at 160-200 cm high, randomly. The middle parts of the leaf blades were cut and fixed in 90% ethanol – 90 cm3, ice acetic acid – 5 cm3 and formalin – 5 cm3. Standard histological techniques and light microscopy were used to examine the anatomical characteristics of the leaf blades. The cross-sections of the leaf lamina were prepared and observed under light microscopy. The measurements were repeated 30 times per one parameter. All measurements were carried out with the objective lens 40 and eyepiece 8X and documented with photographs X 200. The coefficient of palisadness (К %) was measured: К (%) = [Mp /М].100 where: Mp – the length of palisade tissue; М – the length of mesophyll tissue. Cell size and thickness of the layers were assessed statistically with ttest.

Common changes of leaf blades under air pollution stress Morphological modifications of lamina The most common and most damaging pollutants in polluted areas are O3 and SO2 that cause more injury to woody plants than all other air pollutants combined do (Davis & Gerhold, 1976). Nitrogen oxides are predominantly responsible for acid precipitation and ground level O3 formation (Kapoor, 2017). SO2 has a reducing properties and lead to chlorotic or brownish red colour of leaves (Kapoor, 2017). Acute sulphur dioxide damage causes severe leaf scorch, usually on upper interveinal leaf surfaces. Moisture in the air or on leaf surfaces may combine with sulphur

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dioxide and form sulfuric acid that causes leaf scorch, spotting, defoliation. The morphology of plant lamina is very important to determine plant respond to the air pollution (Rahul & Jain, 2014). The use of plants as bioindicators of environmental stress represents an easy and inexpensive way of monitoring air pollution. Foliar injury is an earlier obvious response, and it is the most used sign of plant response to air contamination (Davis & Wilhour, 1976; Edward & Chappelka, 2004). The suitable criteria for assessing injury produced by air pollution to forest tree included degree of chlorosis and necrosis (Donald & Raymond, 1976). In sensitive deciduous tree species the foliar injuries are over 50%; in moderately tolerant species normally less than 50% but more than 20%, and in tolerant ones are less than 20% (Nikolaevskiy et al., 1971). According to the above criteria, registered foliar injuries, the tree species can be listed in the following order depending on percent injury from the total surface: Morus alba L. = Acer campestre L. > Fraxinus americana L. > Acer tataricum L. > Platanus acerifolia Willd > Acer saccharinum L. > Acer negundo L. The field surveillance of trees from Kremikovtzi region showed that the most tolerant deciduous tree species under observation were Morus alba L. and Acer campestre L. The leaf blades of white mulberry and field maple from polluted area were in good state, well developed, without any sign of chlorosis and necrosis to the end of vegetation period, and without significant reduction of leaf blade surfaces (table 4). All other tree species expressed to the different degree some injuries on their lamina surfaces. Mulberry was classified as a first-class resistant tree species against SO2 pollution and high resistant to chlorine pollution, and under such conditions the leaves remained undamaged or impaired less than 20% of the total leaf area (Lu et al., 2004). In Poland, Morus alba L. growing near to copper

15

smelter also has been registered as tolerant species with leaf injuries less than Acer negundo L. (33 μm) > Morus alba L (12.74 μm) > Acer saccharinum L. (11.4 μm) > Platanus acerifolia Willd. (8.34 μm) > Acer campestre L (2.44 μm) > Acer tataricum L (- 8.21 μm).

17

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In some species have been registered increasing the lamina thickness in observed trees from polluted area. With a great extent Fraxinus americana L and Acer negundo L increased their lamina thickness under air pollution, leave blades from Kremikovtzi were 250.31 ± 5.13 μm and 137.5 ± 7.46 μm, while in the control were with value 181.39 ± 2.74 μm and 104.5 ± 2.34 μm, at significance p Acer negundo L.** > Acer tataricum L.*** > Platanus acerifolia Willd.* > Morus alba L.* > Acer campestre L.* > Acer saccharinum L.* (table 5).

Registered modifications of abaxial cuticle The alterations of lower cuticle layer didn’t show common pattern and were different for the observed tree species (table 5). Acer negundo L and Acer saccharinum L have the same value of that parameter in both samples. Fraxinus americana L. > Platanus acerifolia Willd. > Acer tataricum L significantly increase the abaxial cuticle layer (table 5), while Morus alba L and Acer campestre L decreased the thickness of lower cuticle, but only for mulberry the difference of mean was significant (table 5). The thick cuticle is a xeromorphic sign and in plants subjected to gas impact is considered as a mechanism for protection (Ilkun, 1970; Ninova, 1970). The thicker cuticle assists troubled transpiration of leaves in deciduous tree plants growing under industrial polluted air (Caput, 1978). In the deciduous tree plants subjected to gas impact as a mechanism of protection often has been noticed deposition of thicker cuticule layer (Ninova, 1970; Ilkun 1970; Ilkun, 1971).

Changes of epidermis Registered modifications of adaxial epidermis The registered modifications of adaxial epidermis are not identical. Two species, Acer saccharinum L and Fraxinus americana L, under polluted air

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pressure diminished the sizes of adaxial epidermis cells with value 1.25 μm at significant level p Acer campestre L. > Acer negundo L. (table 6). Morus alba L and Acer tataricum L didn’t have significant alterations of the upper epidermis layer. Moreover, White mulberry impressed with the large size of adaxial epidermis cells around 22.5 μm to 25 μm in high for the leave blades for the both regions. The flow of modifications in the tree species from enlargement to diminution of that trait value is: Platanus acerifolia Willd. (3.58***) > Acer campestre L. (2.35**) > Acer negundo L. (2.085**) > Morus alba L. (0.7) > Acer tataricum L. (-0.875) > Fraxinus americana L. (-1.25*) > Acer saccharinum L. (-1.25*).

Registered modifications of abaxial epidermis Platanus acerifolia Willd and Fraxinus americana L. increase the lower epidermis layer. Acer saccharinum L. didn’t alter that trait. Other tree species diminished significantly the sizes of lower epidermis cells, under polluted conditions (table 6), as: Acer negundo L. (-2.02**) > Morus alba L. (-1.64**) > Acer campestre L. (-1.22**) > Acer tataricum L. (-0.825*).

Changes of mesophyll Modifications of palisade mesophyll Under polluted air environment have been registered significant expansion of lamina palisade, in following order: Fraxinus americana L. > Acer negundo L. > Platanus acerifolia Willd. > Acer saccharinum L. > Morus alba L. > Acer tataricum L. > Acer campestre L. (table 7).

22

23

Moreover under adverse conditions the palisade mesophyll in Acer negundo L were double striated, while in the control was represented by one layer cells with the mean value only 26,75 μm (σ = 1,03). The palisade mesophyll of Fraxinus americana L. consists of two layers of long-shaped flat cells that under adverse air circumstances significantly grew in height (table 7).

Modifications of spongy mesophyll On leave blades cross sections of tree species growing in the vicinity of Kremikovtzi has been observed modifications of spongy parenchyma that can be describe as reducing the air cavities to full resemblance to palisade mesophyll. Reducing the intercellular air spaces lowered the velocity of gaseous pollutants uptake and water loss, which is regarded as important trait that enable the tolerant genotypes to avoid or to oppose the adverse effects (Giacomo et al., 2010). The changes of spongy mesophyll thickness were inconsistent from enlargement to decrease: Fraxinus americana L. > Acer negundo L. > Acer saccharinum L. > Morus alba L. > Acer campestre L. > Acer tataricum L. > Platanus acerifolia Willd. (table 7).

Coefficient of palisadness (K%) Four from the observed species increased the coefficient of palisadness under polluted circumstances: Platanus acerifolia Willd (with 11.09%) > Acer negundo L. (with 10% > Fraxinus americana L (with 6%) > Acer tataricum L (with 5%). The escalation of palisadness coefficient (K%) is a sign of good resistance of the tree to the type of pollution (Nikolaevskiy, 1979).

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25

Acer saccharinum L., Morus alba L and Acer campestre L preserved their coefficient of palisadness, for Acer saccharinum L 65%; for Morus alba L 41% - 42% for threatened trees and for Acer campestre L. 45% under polluted air and 43.48% for control.

Lamina modifications as biomarkers for air pollution The anatomical and morphological alterations that plants established under environmental stresses and limitations depended on their ecological plasticity, i.e. genetic ability to adapt and tolerate those conditions. Clarifying the alterations of organisms to industrial air pollution stress can be useful as bioindicators for the development of suitable monitoring and mitigation strategies (Rai et al, 2011). A statistically significant increase in the thickness of the palisade and spongy mesophyll, thickness of a lamina and upper epidermis tissue were determined as the adaptive changes of tree species toward gas impurity with correlations of the complex index of air pollution (Neverova et al., 2005). According to the foliar injuries, the tolerance of tree species reduced from Morus alba L. = Acer campestre L. > Fraxinus americana L. > Acer tataricum L. > Platanus acerifolia Willd > Acer saccharinum L. > Acer negundo L., and their capacity to cope with air pollution. According to the internal lamina modification Acer campestre L was detached to the air conditions and preserved the internal functional anatomy (table 8). This well correlated with the visual observations on lamina surfaces that were without chlorosis and necrotic spots. Usually when leaf surface features are affected, but not the internal functional anatomy, it is accepted as a tolerant response that tree can cope with pollution (Dickinson et al., 1991; Pourkhabbaz et al., 2010).

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Table 8. Comparison of internal lamina changes developed under polluted air Tree species

LT

K%

a

b

c

d

e

f

Morus alba L.

+

N

+

N

+

+

-

-

Acer campestre L

N

N

+

+

N

N

-

N

Fraxinus americana L.

+

+

+

-

+

+

+

+

Acer tataricum L.

-

+

+

N

N

-

-

+

Platanus acerifolia Willd.

+

+

+

+

+

-

+

+

Acer saccharinum L.

+

N

+

-

+

+

N

N

Acer negundo L.

+

+

+

+

+

+

-

N

(+) – significantly increased; (N) – non significant changes; (-) – significantly reduced Legend: LT - thickness of lamina; (K%) - coefficient of palisadness; a – adaxial cuticle layer; b – adaxial epidermis; c – palisade mesophyll; d – spongy mesophyll; e – abaxial epidermis; f – abaxial cuticle layer

In all observed species the common thickness of lamina, the thickness of adaxial cuticle layer, the thickness of palisade mesophyll and the coefficient of palisadness express undoubted trend to increase under air polluted conditions.

Acknowledgements I would like to express personal appreciation to Dr. Lilia Drazheva and Assoc. Prof. Krasimira Uzunova – Ex Deputy Dean of Academic Affairs at the Faculty of Biology, Sofia University, for their assistance in the process of investigation. The article includes results from a research project funded by the Faculty of Techniques and Technology – Yambol, Trakia University - Stara Zagora.

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ECOLOGIA BALKANICA 2012, Vol. 4, Issue 2 December 2012 pp. 51-60

Ecotoxicology Assessment of Waste Water Emitted From Radomir Metal Industries (Bulgaria) Мariana I. Lyubenova1, Snejana B. Dineva2, Irina B. Karadjova3 1 - Department Ecology and EP, University of Sofia, Faculty of Biology, 8 D. Tzankov Blvd., 1164 Sofia, BULGARIA 2 - Technical College of Yambol, University of St. Zagora, Gr.Ignatiev Str. 38, 8602Yambol, BULGARIA 3 - Department of General and Inorganic Chemistry, University of Sofia, Faculty of Chemistry, 1 J. Bourchier Blvd., 1164 Sofia, BULGARIA * Corresponding author: [email protected]

Abstract The purpose of this article is to evaluate the toxicological effect of wastewater emitted from „Radomir Metal Industries". It has been registered that the range of 50% mortality (LC50) of great water flea (Daphnia magna St.) is limited between 75% and 80% effluent.

The

data

mortality

rate-effluent

dilution

for

Pseudorasbora parva well correlated with linear regression, R2=0.86. LC50 is reported from exposure to raw sewage (100%).

39

Results indicate that even when individual concentrations of toxic metals are within the permissible limits the effluent remains toxic for the hydrobiota perhaps due to the combine effect of the contaminants. Keywords: ecotoxicology, heavy metals, effluent, D. magna St., P. parva Temminck & Schlegels.

Introduction Metals are perceived as pollutants but they are also naturally existing substances in the environment. Elements like Cd, Hg, Pb, Ni are accepted as priority pollutants for aquatic environment. Most metals do not undergo microbial or chemical degradation and are toxic and their total concentration in soils persists for a long time after their introduction (ADRIANO, 2003; KIRPICHTCH-IKOVA et al., 2006). Their content in aquatic environment is very divers, and it was well known that neither total nor dissolved total metal concentrations are good predictors for their bioavailability and toxicity (NEWMAN, 2008). Many chemical mixtures, with con-centrations of individual chemicals consi-dered as nontoxic, are often presented in aquatic systems. However, it is reckoned that such chemical mixtures where indivi-dual constituents are present at low, nontoxic concentrations may trigger toxicity due to additive or synergistic effects

among

the

constituents

(RAJAPAKSE

et

al.,

2002;

MONTVYDIENE & MARCIULIONIENE, 2004). The surface water environment is under the influence of continuous industrial pressure. It has long been recognized that the formation of organic and inorganic metal complexes and metal sorption to particulate material reduces metal bioavailability and toxicity in the water compartment (LUCK et al., 2008; SAEEDI et al., 2004). Bio-assessment

40

can be used as a tool to detect the presence of hazardous chemicals in the environment evaluating the effects of mixtures with the combined effects that can be expressed as synergism, additivity and antagonism demonstrating bioavailability of contaminants to different species (LOUREIRO et al., 2006; LANDIS et al., 2011; LYUBENOVA & KALCHEV, 2011; LYUBENOVA et al., 2012). However assessment of water quality in the presence of mixture of pollutants by using single– species biotest may be insufficient for a more biologically complex system; there-fore, organisms of different phylogenetic levels and ontogenesis have to

be

involved

in

these

investigations

(MONTVYDIENE

&

MARCIULIONIENE, 2004). The purpose of article is to evaluate the toxicological effect of wastewater emanated from Radomir Metal Industries".

Material and Methods Study area. Bulgarian metal casting plant Radomir Metals is based the southwestern town of Radomir, Pernik district. The plant has been constructing on 1 800 000 m2 area and situated at about 50 кm south-west of the capital of the Republic of Bulgaria - Sofia, on the road Е79 (Coordinates: 42°31'10"N 22°59'12"E). The purification facilities fully meet the requirements of the European Standards for the quality of the air and water (http://radomirmetal industries.com/en/application/index). „Radomir Metal Industries" has a complex permit № 145-NO/2008 on within the scope of paragraph 2.2. Annex 4 of the Law on Environmental Protection. Under the complex permit "Radomir Metal Industries has established annual emission standards presented in Table 1. It was reported (PASKALEV, 2001), that there are some strongly polluted sections in Struma River – after the towns of Pernik, Kjustendil,

41

and Dupnitza. Sources of pollution are the industrial wastewater of the towns of Radomir and Pernik, as well as domestic sewage of some of the bigger settlements (the town of Blagoevgrad and others). In the area of enterprise "Radomir Metal Industries" soil and water monitoring at several different points are conducted. According to the data of Basin Directorate West Region for wastewater, even those emitted from the production are in the emission rate.

TABLE 1. Direct annual pollutant emission standards Pollutants

Mixed stream wastewater (industrial and domestic-faecal) kg / year

Total nitrogen

709.40

Total phosphorous

77.45

Arsenic and its compounds

1.54

Cadmium and its compounds

0.15

Chromium and its

1.54

compounds Copper and its compounds

8.52

Mercury and its compounds

0.15

Nickel and its compounds

0.42

Lead and its compounds

1.54

Zinc and its compounds

15.02

Phenols

4.64

Common organic carbon

6954.5

Chlorides

-

Cyanides

0.57

42

"Radomir Metal Industries” has owns water treatment plant. Purification is a mechanical, biological, as is done drying and stabilization of sludge. Receiver of purified water was the Struma River (II - Second category receiving water). Along the outlet wastewater to the water treatment point, canal water is intensively used for irrigation. Industrial wastewater is discharged to the water purification plant through a channel running through arable land. In this regard, research in this work is done to establish whether there is compliance with established standards. Characteristics of the test – objects. The biological tests evaluated the toxicity of wastewater, polluted air, soil, sediment, etc. or a particular pollutant using standard test organisms. The latter are exposed to different concentrations of the substance and report mortality or change in morphology and physiology of organisms. In determining the toxicity we followed standard protocols in order to have comparability of results. For the purpose of study are conducted tests with test-objects: Daphnia magna (ISO 6341/1996, 10); Pseudorasbora parva (ISO 7346/1:1996, 10). The aims of bioassays are to determine the substance concentration or dilution of water waste, in which occurred 50% mortality (LC50) or change in the appropriate indicator in the test-organisms for determined time. In assessing the toxicity of the effluent of “Radomir Metal Industries” are using change in mortality of D. magna (ISO 6341/1996) and P. parva (ISO 7346/ 1:1996) respectively, at a constant temperature 20±2°C. By pretesting a series of concentrations (in %) for the final testing are defined: 100, 95, 90, 85, 75 и 70 where the D. magna mortality percentage is rendered in account. Used for testing fish of species P. parva are from one and the same generation and with approximately the same dimensions (length ± 5 mm). The sample has been acclimatized in aquaria (1 l aerated water for a fish) and normally fed two weeks until the test beginning. The

43

fish is not fed and the water is not aerated during the test. For the final testing the following dilutions are used: 20%, 40%, 60%, 80%, 100% included smallest lethal and highest not lethal dilution, defined by the pretesting, for 48 h in a two repetitions. The tests are conducted in aquariums with an individual volume of 10 l (with the appropriate dilution of the effluent and the control with a boiled tap water) and with 10 fishes in every aquaria. The dead fishes have been removed two times a day. In 24 h during the test the wastewaters at different dilution have been renewed and the alive fishes have been moved to the new aquariums. LD50 is calculated by graphical interpolation (ISO 6341/1996, ISO 7346/1:1996). The toxicity of Zn (Zn2SO4.7H2O) on P. parva is also been tested using concentrations: 0, 0.001, 0.010, 0.030, 0.040, 0.050 μg.l-1.

Sample analysis Reagents. All solutions were prepared with analytical reagent grade chemicals and ultra-pure water (18 MΩ cm) generated by purifying distilled water with the Milli-QTM PLUS system Nitric acid: Suprapur HNO3 (67% v/v) was purchased from Fluka. The stock standard solutions of Cd, Cu, Cr, Fe, Hg, Ni, Pb and Zn 1000 mg.l-1 were Titrisol, Merck in 2% v/v HNO3 and were used to prepare calibration standards. Sampling. Fish samples (whole fish body) are thoroughly washed with MQ water. The fish specimens were dissected and samples of fish gills are quickly removed and washed again with MQ water. Fish gills were analyzed as obtained without further homogenization. Fish gills (sample amount between 0.1 and 0.3 g) were digested with nitric acid in MW oven (step 1 - 250 W for 3 min; step 2 - 400 W for 3 min, step 3 - 600 W for 3

44

min), solutions cooled, transferred in 5 ml volumetric flask and diluted up to the mark with Milli-Q water. Instrumentation. Determination of Fe and Zn: Flame atomic absorption spectrometric measurements were carried out on a Perkin Elmer Zeeman 1100 B spectrometer with an air/acetylene flame. The instrumental parameters were optimized in order to obtain maximum signal-to-noise ratio. Determination of Cd and Pb in fish gills: Electrothermal atomic absorption spectrometric measurements were carried out on a Perkin Elmer Zeeman 3030 spectrometer with an HGA-600 atomizer. Pyrolytic graphitecoated graphite tubes with integrated platforms were used as atomizers. Pd as (NH4)2PdCl4 was used as modifier for ETAAS measurements of Cd. Pretreatment temperatures used were 500 °C for Cd and Pb and atomization temperatures 1300 °C for Cd and 1900 °C for Pb. Determination of Cd, Cu, Cr, Ni, Pb in water samples: Samples measured by ETAAS under optimized instrumental parameters. Determination of Hg in water samples: Water samples were previously digested with 1 ml HNO3, Mercury content was measured by cold vapour AAS (Varian AA 240 atomic absorption spectrometer equipped with a continuous flow VGA-77 Vapor Generation Accessory) under optimal instrumental parameters. Assessment of water contamination. Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) have been determined for the sewage (ISO 6060:1989). In environmental chemistry, the chemical oxygen demand (COD) test is commonly used to indirectly measure the amount of organic compounds in water. Most applications of COD determine the amount of organic pollutants found in surface water (e.g. lakes and rivers), making COD a useful measure of water quality. It is expressed in

45

milligrams per litre (mg.l-1), which indicates the mass of oxygen consumed per litre of solution. Older references may express the units as parts per million (ppm). Statistics. All obtained results were statistically processed. It has been shown that the resulting averages are representative of performance using ttest. The statistical significance level in this study was defined at p < 0.05.

Results Results of samples from wastewater The table below presents the results of some additional analysis of samples of wastewater and limit concentrations of metals under Regulation №6 (Table 2). The marginal limit rate are defined in the particular circumstances of production taken into account for the manufacture of iron and steel, production of iron and steel castings, cast cars and other nonferrous metals. The results show that the resulting concentrations are within the limits of regulation.

46

TABLE 2. Average values with standard deviation of the analyzed parameters of samples effluent compared to the Marginal limit concentrations Elements

Marginal limite

Acidified

Not acidified

concentrations*,

sample,

sample,

μg. l-1

unfiltered,

unfiltered,

μg l-1

μg l-1

Cd

500

3.2±0.2

2.3±0.2

Cu

500

12.0±2.0

8±1

Cr

500

2.8±0.2

2.1±0.1

Fe

5000

189±14

156±12

Ni

500

4±1

3±1

Pb

200

3.8±0.1

2.1±0.1

Zn

2000

230±15

190±12

Нg

10

103 µGy h−1 sustained for a large fraction of the lifespan (Real et al., 2004). The occurrence of hormesis is becoming broadly discussed, especially in toxicology and radiation biology (Luckey, 1980). Various studies report hormesis effects such as growth stimulation following irradiation with relatively low doses of ionizing radiation (Sax, 1954; Miller, 1987; Marcu et al., 2013). A typical hormetic curve is either U-shaped or has an inverted U-shaped dose–response, depending on the endpoint measured. If the endpoint is growth or longevity, the dose–response would be that of an inverted U-shape; if the endpoint is disease incidence, then the dose– response would be described as U- or J-shaped (Calabrese, 2004). Hormesis is an adaptive response with distinguishing dose-response characteristics that is induced by either direct acting or overcompensationinduced stimulatory processes at low doses. In biological terms, hormesis represents an organismal strategy for optimal resource allocation that ensures homeostasis is maintained (Calabrese & Baldwin, 2002). Study the percentage of cells with chromosome aberrations or micronuclei induced by low doses of acute (dose rate of 47 cGy/min) or chronic (dose rate of 0.01 cGy/min) gamma-irradiation in vitro in Chinese hamster fibroblasts, human lymphocytes, and Vicia faba seeds and seedlings, revealed that the sensitivity of the indicated biological entities to low doses was greater than expected based on linear extrapolation from higher doses. Authors supposed that the induction of DNA repair occurs only after a threshold level of cytogenetic damage and that the higher yield of cytogenetic damage per unit dose at low radiation doses is attributable to an insignificant contribution or the absence of DNA repair processes. The dose-response curves for cytogenetic damage that were obtained were nonlinear when evaluated over the full range of the doses used. At very low

100

doses, the dose-response curves appeared linear, followed by a plateau region at intermediate doses. At high doses the dose response curves again appeared linear with a slope different from that for the low-dose region. There was no statistically significant difference between the yields of cells with micronuclei induced by low doses of acute versus chronic irradiation (Zaichkina et al., 2004). Dose-effect curves on chromosome aberrations in root meristem cells of Pisum sativum plantlets in the dose range of 0-10 Gy also showed non-linear responses with a plateau for doses up to 1 Gy (Zaka et al., 2002). In A. thaliana, Kovalchuk et al. (2007) showed that the genome is regulated differently depending on whether the irradiation was chronic or acute. Growth responses of Arabidopsis thaliana (L.) Heynh, to a gradient of chronic gamma-radiation demonstrated a significant, but nonlinear, response for three variables, number of seedlings emerging, plants flowering, and plant volume. Flowering and plant volume were the most sensitive indicators of radiation exposure. The response of number leaves per plant was not related to daily exposure. LD50 values ranged from 66 R/20 hour day for plant volume to 1231 R/20 hour day for seedling emergence (Daly&Thompson, 1975). Joiner et al. (2001) showed that most cell lines have hyperradiosensitivity to very low radiation doses, which is not predicted by back extrapolation of the cell survival curve from higher doses. Such nonlinear data have led to the recent view that biological effects of ionizing radiation should not be extrapolated from high to low doses based on the LNT model. Many years ago it was shown by Russel (1965) that the mutation yield per unit dose was higher at low doses of radiation than at high doses. Similar results were obtained by studying radiation-induced cytogenetic damage (Luchnik and Sevankaev, 1976; Pohl-Rulling et al., 1983; Lloyd et al., 1988; Zaichkina et al., 1997),

101

transformation (Oftedal, 1990), and cell survival (Joiner, 1994; Joiner et al., 1996). For carcinogens, regulatory agencies accepted that risk is directly proportional to exposure in the low-dose zone and consequently, there is no safe level of exposure, no level is completely harmless. This so-called linear non-threshold (LNT) dose–response model has become the standard model for assessing the health risks of chemical carcinogens and radiation by regulatory agencies in many countries (Calabrese, 2004). The LNT model is in conflict with three other models, the threshold model, which proposes that low doses are harmless; the radiation hormesis model proposes that small doses can be beneficial; the supralinear model suggests that ionizing radiation at very low doses is more harmful per unit dose than radiation at higher doses (Moore, 2002; Tredici, 1987). Currently, radiation protection of the environment and conservation of ecosystem sustainability is of a special concern. Nevertheless, the information on dose-effect relationships at low doses for non-human species is limited despite its importance. The development of a harmonized approach to human and biota protection has been recognized as a challenge for modern radiobiology and radioecology. In this framework, much more information on non-human species response to low level radioactive radiation exposures is needed.

Plant-test models using for carry-on physiology, epigenetics and genetics research Radiation safety standards limiting radiation exposure of man and doses at which radiobiological effects in non-human species were not observed after the Chernobyl accident (Fesenko et al., 2005). A methodological

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approach for a comparative assessment of ionizing radiation based on the use of Radiation Impact Factor (RIF). However, no internationally agreed criteria or policies for protection of the environment from ionizing radiation till now exist. It is difficult to determine or demonstrate whether or not the environment is adequately protected from potential impacts of radiation under different circumstances (ICRP, 2003). In the framework of ICRP a task group has been established aimed at substantiating a representative set of critical species and indicators for estimating radiation effects (Williams, 2003).

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Plant-test modell

Used for monitoring

Arabidopsis gamma radiation; thaliana (L.) chemical Heynh. mutagenesis;

gamma Pinus sylvestris L. radiation

Vicia faba

Allium cepa L.

chemical mutagenesis; chronic and acute gamma radiation gamma radiation

Endpoint

germination rate, survival rate and growth; embryonic test; gene expression; comet assay; enzyme capacity responsible for antioxidative defence mechanisms (SOD, APOD, GLUR, GPOD, SPOD, CAT, ME) cytogenetic alterations in seedling root meristem; enzymatic loci polymorphism; abortive seeds; cytogenetic alterations in coleoptiles of germinated seeds; length of sprouts;

chromosomal aberations; micronuclei;

growth parameters germination rate, survival rate and growth;

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References

McKelvie A.D., 1965; Daly&Thompson, 1975; Abramov et al., 1995; Kim et al. 2014; Kovalchuk et al. 2000; 2007; Vandenhove et al., 2010a, b; 2014;

Geraskin&Volkova, 2014; Geraskin et al., 2010, 2011, 2012; Arkhipov et al. 1994; Kalchenko&Fedotov 2001; Kalchenko et al. 1993a, b; Kovalchuk et al. 2003; Rubanovich&Kalche nko 1994; Shevchenko et al. 1996 Amer et al., 1969; Rank et al., 1994; Ma et al., 2005; Zaichkina et al., 2004; Vaijapurkar et al, 2001; Mohandas&Grant, 1974;

chemical mutagenesis

Phaseolus vulgaris

Pisum sativum

mitotic index and micronuclei %; chromosomal aberrations; chromosome fragmentation; chromosome stickiness and clumping; gamma stem elongation; radiation number of internodes and leaf dry weight; photosynthetic pigment composition; ribulose 1,5bisphosphate carboxylase (Rubisco) activity; glutathione S transferase activity (GST); low doses of germination rate, short-term survival rate; gamma growth (plant size and irradiation weight); reproduction (pod number per plant, seed number per pod); meiotic anomalies (micronuclei); qualitative biochemical traits (seed storage proteins);

Kumari&Vaidyanath, 1989; Grant, 1978; Fiskesjo, 1995; Ma et al. 2005;

Arena et al., 2014

Zaka et al., 2004

Table 1. Mostly used plants as biomonitoring system

Many studies have shown that air, water, soil and food are often contaminated

with

mutagens

and

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carcinogens,

which

increase

environmental carcinogenic hazards. For that reason monitoring of genotoxic compounds in the environment has become an important objective of public health. Plants are used for monitoring the presence of chemical and physical mutagens in polluted habitats. Higher plants provide valuable genetic assay systems for screening and monitoring environmental pollutants (Ecobichon, 1997). The assessments with higher plants confirmed that plant genotoxicity assays are highly sensitive, only with few false negatives in predicting carcinogenicity of test agents (Ennever et al. 1988). There are about 233 plants that have been used in various aspects of mutagenic research (Sherby 1976). Some of them as onion (Allium cepa, 2n=16), Mouse ear cress (Arabidopsis thaliana, 2n=10), Hawks beard (Crepis capillaris, 2n=6), Soybean (Glycine max, 2n=40), Barley (Hordeum vulgare, 2n=14), Spiderwort (Tradescantia clones, 2n=12), Broad bean (Vicia faba, 2n=12) and maize (Zea mays, 2n=20) are the best worked out assays for gene mutation, mitotic and meiotic chromosome aberrations, micronucleus (MNC), sister chromosal exchange (SCE) and the comet assay that evaluates DNA damage (Panda&Panda, 2002). Several numbers of assays have been validated and standardized to stimulate routine use in the detection of environmental mutagens (Grant, 1994). The International Program on Chemical Safety (IPCS) collaborative study on higher plant genetic systems for screening and monitoring environmental pollutants was initiated in 1984. It is a cooperative venture of the United Nations Environment Program, the International Labour Organization and the World Health Organization. Its goal was to develop methodologies for improving the assessment of risks from chemical exposure (Grant&Salamone, 1994; Gopalan, 1999). Under the sponsorship of the IPCS, 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic

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hazards of environmental chemicals (Sandhu et al., 1994). For screening and monitoring environmental pollutants, are choosing the Arabidopsis thaliana white embryo and the Tradescantia stamen hair test for gene mutation assays, while the Vicia faba root tip and Tradescantia micronucleus test were chosen for chromosal aberration (Ecobichon, 1997). Plant bioassays for detection and screening hazardous environment, chemical-induced cytogenetic aberrations and gene mutations existed from many years (Grant, 1994).

Many tests have been recommended to

regulatory authorities, the advantages of these assays to make them ideal for screening potential mutagens and carcinogens are shown on table 2 (Grant, 1994). Table 2. Selection criteria for IPCS Collaborative Study on Higher Plant Genetic Systems 1. 2. 3. 4. 5. 6.

Ease of use. Well-developed methodology Used by a number of investigators A large data base on chemical mutagens Adaptability of protocols to different climatic conditions Ease of distribution of source material

From Grant (1994)

The most generally assays used for studying mutagenicity of various pollutants in plants are based on the detection of chromosomal aberrations in Allium cepa (Fiskesjo, 1995, Ma et al. 2005), Tradescantia (Ichikawa, 1992), Vicia faba plants (Kanaya et al., 1994) or Zea mays (Grant and Owens, 2006). Allium cepa roots chromosomal aberration (AL-RAA) and micronuclei (AL-MCN) tests are widely employed to evaluate the genotoxicity of many chemical compounds and environmental pollutants. These assays are good and sensitive methods for monitoring clastogenic effects (Grant, 1982; Ma et al., 1995). An Allium cepa chromosome

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aberration test that can serve as a rapid screen for toxic effects of chemicals is among them (Grant, 1994м Bolle et al. 2004). The advantages of the Allium cepa test are that it is a fast and inexpensive method, easy to handle, gives reliable results. Due to its sensitivity, the Allium cepa test was the first of nine plant assay systems evaluated by the Gene-Tox. Not only known chemicals but also water-soluble compounds (e.g. salt solutions), heavy metals and complex environmental mixtures are studied by the Allium cepa test: river and lake waters, waters of well, chlorinated drinking water, domestic and industrial wastewaters, leachate of landfill, industrial waste, soil samples and soil extracts have been studied using this test (Fiskesjö, 1985; Cabrera et al., 1999; Monarca et al., 2003). Furthermore, the test can be used to measure also toxicity, studying macroscopic parameters as length of roots, variations in form, colour and consistency of roots, presence of broken root tips, tumors and hooks (Fiskesjö, 1985). Allium cepa was exanimated as test plant model for study ionizing effects on morphological features such as the number of root and length of root formation, and shoot formation but the evidence were not enough confidence to accept them as a biological indicator for lower gamma dose measurement (Vaijapurkar et al., 2001). Another suitable plant for detecting especially different types of hazardous condition in the environment is Tradescantia (Ma et al., 1996). There are two main tests: the stamen hair mutation (Trad-SH) test and the micronucleus assay (Trad-MCN). The first is based on the heterozygosity for flower colour in Tradescantia clones. Clone 4430 is a hybrid of T. hirsutiflora and T. subacaulis reproduced only asexually, through cloning. The visual marker for mutation induction is a phenotypic change in the pigmentation of the stamen cells from the dominant blue colour to recessive pink (Ma et al., 1994a). The Trad-MCN test is based on the

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frequency of micronuclei in tetrad cells induced in male meiotic cells by the tested mutagen (Ma et al., 1994b). These tests may be used under laboratory, or in situ exposure conditions, for monitoring air or water, or for testing radioactive or chemical agents (Gichner&Veleminský, 1999; Knasmuller, 2003; Cebulska–Wasilewska&Plewa, 2003). Among plant based bioassays, the Vicia faba is considered as favorable for evaluating the environmental quality, by DNA damages and abnormalities in cell division. Various chemicals have scored positive in the Vicia faba-based sister chromatide exchange assay (Rank et al. 1994, Ma et al. 2005). The use of V. faba chromosome aberration has been ongoing for decade. Vicia faba seeds (cv. Giza 1) were planted in gamma radiation field and chronically irradiated with gamma-rays (392—2075 r) during the whole life of the plant. Chronic irradiation of Vicia faba plants did not reduce pollen fertility. The percentages of the induced abnormal pollen mother cells (P.M.Cs) as well as the frequency of abnormal P.M.Cs in the different meiotic stages were proportional with the given doses. The main types of chromosome aberrations were anaphase and telophase bridges, fragmentation and lagging chromosomes. The nearest plants to the source showed an inhibition of shoot growth, flower and seed sterility and irregular branching. The most dominant type of anomaly was the presence of micronuclei in the different stages of mitosis and in the resting cells (Amer, 1969). Vicea faba offers many advantages and is ideal for use by scientists in the field of environmental mutagenesis for screening and monitoring of genotoxicity, cytotoxicity and mutagens according to the standard protocols and genetic makeup is similar to other living organisms (Leme&Marin-Morales, 2009; Kristen, 1997). In some systems, e.g. in tests with maize, morphological changes of the pollen are used, or in the case of Arabidopsis thaliana, changes in the color

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of the embryos. Soybean (Glycine max) and tobacco (Nicotiana tabacum), formation of mosaicism which leads to leaf spots varying in their color and morphology; detection of somatic crossing over, chromosome deletions, nondisjunction and point mutations are used (Vig, 1982). A new approach to biomonitoring, which involves transgenic plants is based on the integration into the plant genome of a marker gene of known sequences that will serve as target for mutagenic influences. Essential progress in generation and development of transgenic plants as biomonitors has been made (Lebel et al. 1993; Kovalchuk et al. 1998 and 1999; Ries et al. 2000; Kovalchuk et al. 2001; Li et al. 2006; Boyko et al. 2007; Van der Auwera et al. 2008). Two different transgenic systems were designed to study mutagenic influence via point mutations and homologous recombination events (HR). One of the important advantages of transgenic biosensors is the ability to customize the assay in accordance with monitoring needs. Transgenic plant biomonitors used for the evaluation of genotoxicity are Arabidopsis thaliana and Nicotiana tabacum plants (Kovalchuk and Kovalchuk, 2008). Arabidopsis thaliana (L.) Heynh, (Mouse-ear Cress, or Thale Cress) is currently the most popular plant-test model, with first sequenced genome. Arabidopsis thaliana (L.) Heynh is self-compatible weedy species with a worldwide distribution, often used as a model, because of its small genome, easy growth in lab conditions, and also it is self-fertile. It has proved to be a useful organism for mutation research because of its short life cycle and morphologically distinctive mutants that can be induced. Approximately 1000 mutants were produced in an attempt to look for mutagenic agents giving high mutation rates and offering prospects of mutation specificity (McKelvie A.D., 1965). Mutant of Arabidopsis uvh1, is hypersensitive to both UV-B and UV-C light wavelengths and to ionizing radiation. Uvh1

110

plants showed chlorosis, wilting, and extensive cell death following exposure of leaves to small, acute fluencies of UV-B or UV-C light that did not affect wild-type plants. In addition, irradiation of uvh1 seeds with yrays inhibited the production of the first true leaves at much lower doses than those needed to similarly affect wild-type plants. These hypersensitive mutant phenotypes are due to a single, recessive mutation probably located on chromosome 3. Additional uvh mutants, and five of these mutants are currently being characterized in detail (Greg et al., 1994). Other radiationsensitive mutants of Arabidopsis have recently been described. A UV-Bhypersensitive mutant was isolated using a root bending assay and was shown to have a defect in repair of 6-4 pyo (Britt et al., 1993). Its small stature and short generation time facilitates rapid genetic studies. It grows from far north to equatorial location within a wide climatic and latitudinal range that makes it an excellent model for studying natural variation in adaptive traits. Most examples of heritable epigenetic variation for plants have come from experimental models such as maize (Zea mays L.), Pinus sylvestris L., and Arabidopsis thaliana (L.) Heynh (Richards, 2006; Mousseau et al., 2013). Surveys on genomic consequences of gamma radiation and chemical induced mutagenesis have been widely applied with plant test model Arabidopsis thaliana (L.) Heynh (McKelvie, 1965; Abramov et al, 1995; Kovalchuk et al, 2007). Pisum sativum is determined as radiosensitive plant mentioned in the NATO document AC/25-WP/79 about the effects of radioactive fallout on food and agriculture (Zaka et al, 2004). Pisum sativum has been used for studying all the cytological endpoints that follow treatment of chromosomes by chemical and physical agents. (Grant&Owens, 2002). Detailed descriptions of these assays can be found in Plewa (1982), Sandhu et al. (1989), Grant (1994) and Kanaya et al. (1994), Ma et al. (1994 and

111

1995). The relevance of higher plant genotoxic bioassays has been discussed in detail (Fiskesjo, 1995; Grant, 1994, Grant and Owens 2002). The advantages in utilizing plant systems have been reviewed by many authors (Mann&Story, 1966; Nilan, 1978; Conte et al. 1998). The most serious disadvantage of a plant system for the detection of genetic risks to man is the lack of similarity between vegetative and mammalian metabolism. Pinus sylvestris Scots pine have been widely used as a study organism for estimation the consequences after ionizing radiation, because it is common and widespread in the region near Chernobyl, also these pines are more susceptible to the negative impact of radiation than many other species of trees (Arkhipov et al., 1994; Kalchenko&Fedotov, 2001; Kalchenko

et

al.,

1993a,

b;

Kovalchuk

et

al.,

2003;

Rubanovich&Kalchenko, 1994; Shevchenko et al., 1996). Pinus sylvestris, L. has become one of the primary test objects for ecological and genetic monitoring due to its widespread distribution, similarity of its radiosensitivity to that of humans, reproducibility and sensitivity of the available experimental endpoints (Geraskin et al., 2003). Coniferous plants generally show a high retention capacity and low turnover rate for contaminants taken up by the aerial biomass from the atmosphere, an assessment of cytogenetic anomalies in the intercalar meristem of young needles also appears to be a promising test system. In either case, the damage to the DNA mainly appears as chromosome aberrations at the first mitosis (Geraskin et al., 2003).

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Biological indicators measuring consequences of gamma radiation Ionizing radiations induce morphological, genetical, physiological and biochemical changes, that vary with plant species, irradiation dose and type.

Morphological criterion Typically as morphological parameters for estimation radiosensitivity are used several characters describing plant growth: germination, root test analysis, percentage of plant survival, seedling length and weight, growth reduction or stimulation. The frequently observed symptoms at low dosages are early germination and inhibition at high dosages (Sax, 1963; Luckey, 1980; Sagan, 1987; Planel et al., 1987; Korystov&Narimanov, 1997; Charbaji&Nabulsi, 1999; Kim et al., 2000; Toker et al., 2005; Wi et. al, 2007; Ling et al., 2008; Melki & Marouani, 2009; Borzouei et al., 2010; Wi et al., 2005; Minisi et al, 2013; Chaudhary&Agrawal, 2014), and reduced growth characteristics parallel with increasing the radiation dosages (Dwelle, 1975; Chandorkar&Clark, 1986; Kim et al., 2000; Zaka et al., 2004; Toker et al., 2005; Wi et al., 2007; Kon et al., 2007; Vanhoudt et al., 2014; Chaudhary&Agrawal, 2014). In most cases fluctuations in growth criteria are observed, but with not clear pattern and dose-dependent curve. Treatment of A. thaliana (L.) Heynh seedlings with different gamma radiation doses resulted on variations of root and leaf fresh weights but no dose-dependent growth inhibition have been detected (Vanhoudt et al., 2014). Therefore, authors supposed that those fluctuations are mostly due to biological dissimilarities rather than a distinct radiation effect. Hence, when aiming to construct a dose-response curve, higher total absorbed gamma radiation doses need to be applied on a more sensitive

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developmental stage of the seedlings. The results from investigation low doses irradiated dry and wet seeds of Molucella laevis (L.), at 2.5, 5, 7.5 and 10 Kr, all doses of seeds except 20 Kr had the same plant survival percentage 100% in both seasons. On other hand, the higher doses (12.5, 15, 17.5 and 20 Kr) of wet seeds liner decreased the plant survival percentage with the increase of gamma radiation doses in both seasons (Minisi et al, 2013). The results of the experiments with higher dosage of gamma radiation indicated a pronounce decrease of germination percentage, number of survival plants and plant height (Vaijapurkar et al., 2001; Minisi et al, 2013). Also, wet treatments of radiation caused a simulative effect in most characters. The high doses 12.5 to 17.5 Kr of wet seeds caused some morphological variations. The genetic relationship of the morphological variations can be determined by using RAPD analysis (Minisi et al, 2013). According to the Vaijapurkar et al. (2001), when study ionizing effects on irradiated onion (Allium cepa —red globe—Mathania Desi) concluded that the morphological features such as the number of root and length of root formation, and shoot formation cannot give a confidence to accept them as a biological indicator for lower gamma dose measurement. It can be only used for qualitative measurement for gamma dose evaluation. Analysis showed a relation with delivered gamma-radiation on onions at lower doses, i.e., 50–2000 cGy. The differences in the root numbers and root length of irradiated onions (Allium cepa—red globe—Mathania Desi) at different intervals was extremely significant (P