The pollen as bioindicator of the genotoxicity of environment

. In recent years the male gametes of plants are used for biotesting in ecotoxicology. High proportion of sterile pollen is the indicator of environmental pollution and the presence of mutagens, which cause genetic disturbances. The viability of pollen of Go rodkov’s rowan ( Sorbus gorodkovii Pojark) and Scots pine ( Pinus sylvestris L.) in the vicinity of environmentally hazard enterprises of the Arctic cities of the Murmansk region have been studied. The following parameters were considered: the number of fertile and sterile pollen and their percentage ratio, the sterility index, and palynotoxic effect of the pollution. High content of the abortive pollen of S. gorodkovii (55 – 65%), the sterility index (2.1 – 2.4) and the palynotoxic effect (38.2 – 50%) indicates the mutagenicity and genotoxicity of the emissions of the Murmansk thermal power plants, that leads to the formation of sterile male gametes. High level of sterility of P. sylvestris pollen (42.5 – 50.7%), the index of sterility (1.4 – 1.7) and the palynotoxic effect (19.1 – 29.8%) indicates, that emissions of the mining and processing plant "Apatit" containes the mutagenic chemicals that have a genotoxic effect on the pine male gametophyte. The pollen of plants can be used for bioindication of environment as the indicator of the impact of gametopathogenic factors.


Introduction
Modern cities have an important role in a global process.Cities are producing 80% of the world's economic output, consume 70% of the world's resources and energy, and thus account near 75% of the anthropogenic carbon emissions [1].One of the goals of "The 2030 Agenda for Sustainable Development", adopted by the United Nations, is the sustainable development and safety of the cities [2].The new innovative methods are used for the monitoring of ecological safety of the urban environment.In ecotoxicology the male gametes of plants are used for biotesting.Studies have shown that pollen is effective in environmental monitoring [3] because it provides very important information of the impact of pollution on the adult plants, because gametes express many genes that correlate with the adaptability of the sporophyte [4].Some of chemical substances have a mutagenic effect on plants, disturbed the process of microgametogenesis, that expressed in decrease of the pollen fertility, which described as the ability to pass genes to the offspring [5].The sterility of the microgametophyte can be considered as the indicator of mutagenic effect of pollutants [6].Many species of plants are good biomonitors and bioindicators of the genotoxicity of polluted air due to the accumulation of pollutants [7].The use of pollen for detecting of mutagenic effects is possible because lethal mutations occur in haploid gametes.It is known that the production of sterile (abortive) pollen by plants increases in the vicinity of industrial enterprises, near the roads with intensive traffic [8].Biotests for mutagenicity of the environment based on the viability of male gametes of plants [9], while higher the content of abortive pollen, higher the toxicity of the environment.The bioindication and biotesting with using of pollen fertility, are carried out in many cities of Russia [8], but they are only few in the urbanized territories of the Arctic [10][11].The purpose of the study is to determine the mutagenicity and genotoxicity of the environment of the industrial cities of the Murmansk region.

Material and methods
The studies were carried out in the cities of Murmansk and Apatity.The city of Murmansk (68°58′ N; 33°05′ E) is large ice-free port of the Russian Arctic with developed infrastructure and industrial facilities.The quality of environment has dramatically deteriorated over the last decade due to the emissions of the thermal power plants operating on fuel oil of low quality, and the dust pollution by the Murmansk Commercial Sea Port.The emissions of the heating plants contain highly mutagenic pollutants (vanadium, benzopyrene, manganese and its compounds, hydrogen sulfide, etc.).The Murmansk Commercial Sea Port is the leader of dry cargo handling in the North-West of Russia, the main of which is coal (93.5%).The composition of coal dust includes silicon dioxide, pyrite, ash, and slag, consisting of oxides of silicon, aluminum, iron (III), calcium, potassium, rare and trace elements (Ge, V, W, Ti, etc.).The Murmansk incineration plant belongs to the I class of environmental hazard and to the I category of negative environmental impact.Specific emissions of the waste incineration plants are super toxicants: dioxins and furans, as well as heavy metals.The Apatity is an industrial city located in the foothills of the Khibiny Mountains (67°34'00" N, 33°24'00" E), in the impact zone of the mining and processing plant "Apatit", one of the world's leading producers of phosphate raw materials for mineral fertilizers.The mining and processing complex includes the mines, the processing plants (ANOF-2 and ANOF-3), the Tailing Dump and several workshops.The wastes of processing of apatite-nepheline ores are stored in the Tailing Dump located on the shore of the lake Imandra in the vicinity of the city of Apatity.The enrichment waste consists of the fraction of finely dispersed nepheline sands, which, under strong winds, turn into an aerosol state, forming dust clouds, and are transported over the long distances.The atmospheric air of the city of Apatity is heavily polluted by dust, which contains toxic compounds.The content of strontium is 4 times higher than background values, copper -2 times, phosphates -10 times, sulfates, and fluorides -3 times [12].
The studies of mutagenicity and genotoxicity of the environment in the city of Murmansk were carried out with use of the pollen of Sorbus gorodkovii Pojark (Gorodkov's rowan), in the city of Apatity -Pinus sylvestris L. (Scots pine).Sorbus gorodkovii is an endemic of the East Fennoscandia, the European hypoarctomontane species, which range is located beyond the Arctic Circle.Gorodkov's rowan forms the basis of green spaces of the city of Murmansk.The pollen grains are three-lobed on the pole and elliptical on the equator.Pinus sylvestris L. (Scots pine) is widely distributed in the cities of the Murmansk region, in the Apatity grows on the outskirts of the city.Pollen grains are two-sacci, heteropolar, bilaterally symmetrical.Sacci have oblong-elliptical shape, sharply separated from the pollen grain, and displaced to the distal side.
In the city of Murmansk, five test sites (figure 1) were laid in the impact zone of environmentally hazardous enterprises: "Roslyakovo Yuzhnoye" -Molodyozhnaya Street,  10 trees were marked on each of the test sites.The male cones of P. sylvestris and flowers of S. gorodkovii with ripe pollen were collected, the samples were fixed in 40% alcohol solution and stored in the refrigerator.Pollen of S. gorodkovii were stained by 1% acetocarmine solution, of P. sylvestris -by Lugol's solution.Cytological analysis of the temporary preparations of pollen grains was carried out by the method of light microscopy (magnification 200 times for P. sylvestris and 400 times for S. gorodkovii).In total, 500 pollen grains were viewed in each sample.Stained pollen was considered as fertile, unstained as sterile.The test of sterility of male gametophyte was used for the screening of the genotoxity of emissions of the industrial enterprises.The following parameters were considered: the number of fertile and sterile pollen grains and their percentage ratio, the sterility index, and palynotoxic effect, according to E. Ibragimova [13].The sterility index (SI) is the value showing how many times the frequency of the induced sterility caused by pollution is higher than the spontaneous sterility (in control).The palynotoxic effect of emissions -PE (%) was calculated using the formula of I. N. Lozanovskaya, modified by E. E. Ibragimova [13] for the analysis of pollen fertility of male gametophyte and was calculated according to the formula: where Fо -the indicator of the value of the spontaneous fertility of pollen of control plants, Fх -the indicator of the value of the induced fertility of pollen of plants under the study.The palynotoxic effect shows by how many percent the fertility level of the test sample (induced fertility) is lower than the pollen fertility of the control (spontaneous fertility) taken as 100%.

Results and discussion
Cytological analysis of the samples of S. gorodkovii pollen in the city of Murmansk showed that the content of fertile pollen grains varies from 35 to 52% (in control -72%) (figure 2).The proportion of sterile pollen is 48-65%, the largest content is in the vicinity of the South thermal power plant (65%), the Central (59%), which operate on the fuel oil, and the coal boiler house "Roslyakovo Yuzhnoye" (55%) (figure 2).This is induced sterility caused by industrial pollution of the environment and indicates the high toxicity of emissions of the Murmansk heating plants.The calculation of the sterility index (SI) showed that the induced sterility of the rowan pollen is two or more times higher than the spontaneous sterility (in the control).The largest sterility indexes are in the vicinity of the South (2.4) and Central thermal power plants (2.1), and the "Roslyakovo Yuznoe" boiler house (2.0).At the sites of the incineration plant and the Murmansk Commercial Sea Port, the sterility index is lower (1.8).The highest indexes of palynotoxic effect of emissions were received in the vicinity of the Murmansk thermal power plants using fuel oil: the South (50%) and the Central (42.3%) (figure 2).At the test site of the coal boiler house "Roslyakovo Yuzhnoye" -38.2%.This indicates a high toxicity of their emissions, which cause a significant decrease of pollen fertility compared to the control.The palynotoxicity effect of the incinerator emissions is lower (25.8%), it does not operate at full capacity.In the impact zone of the Murmansk Commercial Sea Port, the value of PE is 26.4%.The high content of sterile pollen (55-65%), the values of the sterility index (2.1-2.4) and palynotoxic effect (38.2-50%) indicates the mutagenicity and genotoxicity of the emissions of the Murmansk heating plants, which leads to the formation of sterile male gametes of S. gorodkovii.The thermal power plants operated on the fuel oil are the most dangerous for living organisms, including humans.Ash generated by fuel oil combustion usually contains 2-6% of vanadium [14] in the composition of highly toxic divanadium pentoxide (V2O5) [15].The cytological studies of P. sylvestris pollen in the city of Apatity showed that its fertility varies from 49.3 to 57.5%.The proportion of sterile pollen in the samples is 42.5-50.7%.The highest sterility of the pine male gametophyte was found in the northern outskirts of the city (Kozlova Street -50.7%) and near the Tailing Dump (48.4%) (figure 3).The increase of the production of sterile pollen of P. sylvestris in Apatity is induced by emissions of the processing plant ANOF-2 and by dusting of wastes of the Tailing Dump.Calculation of the sterility index (SI) showed that induced sterility of P. sylvestris pollen is 1.4-1.7 times higher than its spontaneous sterility (in the control).The maximum sterility index was found in the Kozlova Street (1.7) and the Tailing Dump (1.6).On the test sites the "Lyubkino swamp" -1.5, the ANOF-2 is 1.4.The largest palynotoxic effect (PE) are on the Kozlova Street (29.8%) and near the Tailings Dump (26.4%), which indicates the greatest toxic effect of pollutants on pollen fertility (figure 3).At the test sites "Lyubkino swamp" and "ANOF-2" palynotoxic effect is lower (21.5 and 18%, respectively) (figure 3).High levels of the sterility of pine pollen, the indexes of sterility and palynotoxic effect indicate that emissions of the Apatit processing plant and the Tailing Dump containes the mutagenic chemicals that have a genotoxic effect on the male gametophyte of P.sylvestris and the process of gamete formation.The emissions of the "Apatit" contains strontium, zinc, phosphorus, calcium, sodium, potassium, aluminum, nitrogen compounds and iron [12].Apatite dust contains some natural radionuclides.It is known that disturbances during microsporogenesis can occur both at early stages, in meiosis, and at later stages -during the division of the generative cell and the formation of spermies, therefore, sterility is associated with disturbances of the processes of sporogenesis and gametogenesis and can be used as indirect indicator of these violations.Cytoplasmic male sterility is expressed in the degeneration of pollen on some stages of development and genetically determined.The pollen fertility is controlled by nuclear and cytoplasmic genes.The pollen sterility can be caused by the action of nuclear genes (genetic male sterility) and cytoplasmic genes.Cytoplasmic male sterility occurs as result of the mutation of mitochondrial genome, leading to anther degeneration or production of abortive pollen [13].

Conclusion
The sterility of pollen caused by industrial pollution is a major environmental problem affecting the productivity of plants.The results of screening of the genotoxicity of pollutants of the industrial enterprises of the cities of the Murmansk region indicates that emissions of the fuel oiled thermal power plants and the Apatit processing plant are highly toxic, cause lethal embryological changes of the male gametes.High proportion of sterile pollen and the palinotoxic effect are indicates the presence of mutagens, which cause genetic disturbances in the process of microsporogenesis of S. gorodkovii and P. sylvestris.The pollen of plants can be used in ecological studies as the indicator of the gametopathogenic environmental factors.For studying of the mutagenicity and genotoxicity of the environment, it is better to use plant species of aboriginal flora that adapted to the regional climate.
170 m from the coal boiler house Roslyakovo Yuzhnoye."Incineration Plant" -Domostroitelnaya Str., 482 m from the plant for thermal treatment of municipal solid waste."Murmansk Port" -950 m from the Murmansk Commercial Sea Port."Central Thermal Power Plant" -Tralovaya Str., 353 m from the Central heating house, operating on the fuel oil."South Thermal Power Plant" -Baumana Str., 930 m from the South heating house, which is the most powerful in the city and the Murmansk region.The control site located in the settlement of Verkhnetulomsky, 83 km from the city of Murmansk in a south-west direction.Four test sites (figure 1) were laid in the city of Apatity: "Tailing Dump" -the forest cenosis, 2 km from the center of the Tailing Dump of ANOF-2."ANOF-2" -the forest cenosis,1 km from the processing plant ANOF-2."Kozlova Street" -the forest area on the northern outskirts of Apatity, 3.8 km from the center of the ANOF-2 and 35 m from the railway."Lyubkino swamp" -the forest park in the south-east part of the city of Apatity, 5.7 km from the center of the ANOF-2.The control site -in the settlement of Verkhnetulomsky, 130 km from the city of Apatity.