Research

Joint research of UJEP, ZU, NULP
Cooperation with liasoning institution Zagreb University and end-users from village St.Helena, Croatia

On May 2-9th, 2017 prof. Valentina Pidlisnyuk did a mission trip to Croatia. The goals of the mission were to discuss on-going research on M.xgiganteus growing at the contaminated land and to enrich the deposit Mxgiganteus plantation located at village St.Helena with partners from Agronomy Department at Zagreb University and potential end-uses of the phytotechnology.

On May, 3rd prof. Pidlisniuk met with Dr. Zeljka Zgorelec, ZU. Results of 2 years research on M.giganteus growing at the soil artificially contaminated by heavy metals and 2 years experiment with soil contaminated by metals taken from Bakar, Croatia were discussed. The design of training 2 scheduled at UJEP for November, 7-9 was observed and participation of ZU in this event was discussed. Dr.Zgorelec agreed to do lecturing on quality ensuring of soils and plants analysis. Prof. Pidlisnyuk met with Iva Hrelja, student from the Master program “Agroecology”, Antonija Jonjic and Ana Lijovic, students from Master program in “Organic farming and agrobusiness” and shared results of NATO SPS G4687 project.

During the visit prof.Pidlisnyuk updated on-going 3rd year semi-field experiment with M.xgigateus at the soil taken from Bakar and did sampling.

On May 4-5 the planting of additional deposit plantation at village st.Helena was done. The farmer organization passed the site for UJEP research on cost-sharing base and assisted in field plugging and planting process. The consultation with potential end-users- local farmers regarding planting of energetic crops at the Northern Croatia was done and questions about treatment of M.xgiganteus plantation against weeds was developed. The field was plugged, prepared for planting and 230 rhizomes of M.xgiganteus were planted. Currently the deposit plantation calculated 545 rhizomes. On May, 7 the fence from decorative bushes were planted in order to mark plantation.

On May, 8 prof. Pidlisnyuk made a field trip to Bakar, Rijeka region for additional sampling of metal- contaminated soil. Cost –sharing for that trip was done by potential end-users from village St.Helena.

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Planting of M.xgiganteus at the deposit field
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Meeting at the Agricultural Department, Zagreb University
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Updating the 3rd year experiment
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Field plugging
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Preparation of the field
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Assistance from the local end-users
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Consultation with local farmers
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Sampling at Bakar, Croatia

National University Lvivska Polytechnika (NULP)

Establishing field plots at Dolyna, Ukraine

On April 3-5, 2017 the new research plots were established in Dolyna, Ivano-Frankivsk region, Ukraine. The place is former military site used as base of soviet army rockets and possess constantly a negative effect to the health of Dolyna habitats. The local authority is strongly aware of implementing the proposed phytotechnology for cleaning the site and production of biomass. Mayor of the city Volodymir Garazd participated in Training 1 in Lviv on October, 6-7, 2016 and initiated and promoted the establishing of that research Being involved in EU project on alternative energy and energy saving cities is implementing in Ukraine by Association “Energy Efficient Cities of Ukraine he proposed to use the site in Dolyna as demonstration one for governmental officials and professionals interested in alternative energy, including those involved in the EU project.

The establishing was jointly done by NULP and NULES in cooperation with new project’s partners from Ivan Franko Lviv National University and local administration of Dolyna city. Consequently, Co-Director Dr.Pavlo Shapoval, NULP led the establishing at the place assisted by PhD student Vytaliy Stadnik, NULP and PhD Student Artem Medkov, NULES. The design of the plots and scientific assistance was done by Dr.Volodymir Kvak, NULES and Dr. Lubov Byn’o, IFNUL. The local governmental officials: Ruslana Havrulkiv (Deputy Mayor of Dolyna city) and Andrij Yanishevskiy (Head of the Dolyna Soil Station) assisted in paper work and supplying the technical equipment for planting, which was a cost-sharing from the local authority.

Two plots (one - at the contaminated land and another - at the control land) were established with the size of 15,4 x22,1 m each, altogether 480 rhizomes were planted. Half of the M.x giganteus rhizomes were treated by PGRs “Stimpo” in order to increase the biomass quality and quantity.



Research at UJEP, Czech Republic
Monitoring mission to the research sites at Czech Republic

On August, 17-18 prof. Pidlisnyuk and assoc.prof. Trogl made a control mission to the research sites in Mimon (contaminated and control plots) and deposit site in Chomutov.

The both plots in Mimon show the good shooting rate of Miscanthus xgiganteus about 60%, and plantations are not much littering by weeds. The plantations were established in June because of delay in permission for planting and overcame the extremely hot temperature waves were in Czech Republic in July and August.

On August 18 prof. Pidlisnyuk and assoc.prof. Trogl visited the deposit site planted at the Agricultural research station in Chomutov and met with Ing.Honzuk, head of department at the unit. The necessary work on maintaining plantation and cleaning it from weeds was discussed, also ways of treatment the MxG plantation by PGRs in 2018 was overviewed.

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discussion the joint research on PGRs
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First year plantation of MxG at Mimon (contaminated soil)
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Former airport Hradcany, Mimon
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Meeting at Chomutov agricultural station prof.Pidlisnyuk, assoc.prof.Trogl, Ing.Honzik
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monitoring of the control site,Mimon
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monitoring of the deposit site, Chomutov
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Mxg at the contaminated site, Mimon

New experiment with miscanthus growing at the oil-contaminated soil at UJEP

On July, 19 the new experiment was established at UJEP with growing miscanthus at the artificially oil-contaminated soil. The idea run from the fact that during sampling in spring 2017 one locality was found at the research site in Mimon contaminated by oil products. The source of the contamination was caused by previous release of oil-products into the soil by soviet troops while living airport in 90th. Such contaminated localities were cleaned up in 1996-2006 during implementation of projects supported by Czech Ministry of the Environment and other governmental agencies based at oxidation of oil-products inside the soil depth. However, the locality found is still remained contaminated.

The experiment has been designed in a way, that miscanthus seedling first produced at the regular agricultural soil during 2 month and further re-planted to the pots. The clean soil for the experiment was artificially contaminated by oil in the different proportions referred to the real concentration of contaminants at Mimon site. One seedling of miscanthus was planted in each pot which has 11 kg of differently contaminated soil.

The miscanthus growth will be monitored during the vegetation season and content of oil concentration will be measured.

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Dr. Trogl and Erasmus student Gosterislioglu during soil samping in Mimon
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Former military site at Mimon
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Former storage of oil-products at Mimon
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Planting of miscanthus seedling by PhD student Diana Nebeska
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Soil sampling at the research site in Mimon on April 25

Field experiment in Hradčany airport – Mimoň, Czech Republic

Two experimental fields were settled in Hradčany former military airport near city Mimoň on June 1 2017. One locality is slightly contaminated with residuals of organic compounds as consequence of activities of Soviet army and the second one is control without hydrocarbon contamination.

Main goals of the experiment are:

  • Research of biomass production on military affected soil
  • Research of phytoremediation process
  • Research of influence of plant growth regulator on phytotechnology

Both localities were prepared by ploughing and 92 rhizomes were planted into each locality, half of them was treated by Stimpo plant growth regulator.

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Members of UJEP team planting Miscanthus
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Miscanthus rhizomes
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Planting at control field
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Preparation of field

Semi-field experiment at UJEP, Czech Republic

First year experiment was founded at UJEP starting from May, 2016. The design of the experiment was developed during meeting of Co-Directors in Kyiv, Ukraine in Aril and is unified for teams from UJEP, NULES and IBPP. The experiment consists of two main directions: research of phytoremediation process and research of biomass production.

The contaminated soil was taken from former military site, located at Mimon, North Bohemia. The site used to be a place for soviet air-force location Hradcany. The negotiation was done at the Ministry of Defense of Czech Republic and branchof Military Forest at Mimon for receiving an access to the site for sampling. The soil was taken from the site and analyzed for contamination by heavy metals and residues of organic substances.

The control soil was taken from the agricultural land located in 3 km from the site. The contaminated and control soils were mixed in different proportion, put in pots. 2 Miscanthusxgiganteus rhizomes were planted in each pot.

The monitoring of biomass growing is providing constantly and was finished as leaves become deep yellow and dry, on November, 16, 2016.

During visit to UJEP prof. Octaviana Trijillo, Northern Arizona University and University of Arizona ( USA) on August,1st the peculiarities of phytotechnologies developed were discussed and decision was proposed to present the approach with using second generation crops for phytoremediation at the Department of Soil, Water and Environmental Sciences University of Arizona. Prof. Trijillo was presented the semi-field research on phytotechnology at UJEP.

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Using microbiology indicators: phospholipid fatty acid and enzymes for assessment changing in military contaminated soil’s ecosystem during application of phytotechnology with Miscanthus x giganteus

The efficiently of the proposed approach has to be assessed and constantly monitored in order to develop an advanced phytotechnology. The progress may be assessed by three main approaches:

  • assess of phytoremediation efficiently by analyzing translocation factors
  • assess the physiological parameters during phytotechnology application: i.e. plant growth, volume of biomass, photosynthetic pigments, and antioxidant enzymes
  • assess the microbial indicators of soil ecosystem during process of phytotechnology application.

There is a luck of data regarding impact to microbial community of rhizospheres, its dynamic and role in the process of phytoremediation. In particular it is necessary to have that data during growing of second generation biofuel crop Miscanthusxgiganteus at the military contaminated soils taking into account the need for assessing such phytotechnology.

The main goal of the research is to explore the microbial community in a contaminated soil close to Miscanthus x giganteus rhizospheres and in the treated soil, it’s changing during vegetation seasons and to calculate microbiological indicators in order to improve the overall process of phytotechnology with Miscanthus x giganteus phytotechnoly to be applied for the former military sites in-situ.

The research is going on with the soils taken from the military sites at Sliac, Slovakia; Mimon, Czech Republic and with the abandoned soil from Bakar, Croatia.

Research at NULES
On-going research at Kurakhove

Yellow blue trap installed for monitoring of aerial-flying insects of M.x g.

On July 6-8, 2017 Dr.Volodymur Kvak (NULES) visited the experimental sites in Kurakhovе, Donetsk region. Research site is located at the place of former military activities and was appeared in 2015 during temporary occupation of that territory by Russian Military troops. Control site is located at the agricultural field in suburb. Research and control sites were established with assistance of the State Enterprise "Center for Certification and Examination of Seeds and Planting Material" (CCESPM), potential end-user of the phytotechnology. Dr. Kvak inspected established plots of M.xg., installed the insect traps and took soil samples for analysis.

It was observed that Mxg planted in autumn have shown better establishment results than those planted in spring. Due to the long spring drought (two months without precipitation), plants are currently in stress state and some already dried up.

Dr. Kvak had a meeting with CCESPM representatives Mykola Bilyi and Elvira Paschenko, potential end-users of the phytotechnology. They considered the idea to increase drought tolerance of Mxg by frequent watering and using amendments.

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M.x g. experimental site with evidence of drought stress
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Meeting with potential end-users
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Sampling at the research site

NULES field research at Kurakhove, Eastern Ukraine

Field research on growing Miscanthusxgigateus at the former military land contaminated by heavy metals is going on in Kurachhove site (Donetzka region). Soil is heavily polluted by heavy metals due to the former antiterrorist operation was there in 2015 year and industrial activities.
The research is focused on testing the impact of time planting and treatment of rhizomes by PGRs. To the biomass production. In addition, growing process of different varieties of Miscanthus x giganteus is monitoring. The plants planted in the fall 2016 were hibernated successfully. Spring planting in 2017 also gave a satisfactory results of seedling appearance and establishment. Since the site is located in the zone where precipitation is limited the special attention is given to the water availability to the crop.

Along with NULES team members of the Donetsk’ branch of State Enterprise on certification and expertise of seeds and plant materials have been assisted in research voluntarily.

Manual removal of weeds at the research site, May 2017.

Enrichment of research at NULES

Semi-field and field experiment on growing Miscanthus x giganteus at the contaminated military soils taken from military sites in Ukraine have been continuing at the Laboratory of Phytotechnologies and Biocontrol, NULES. The second year experiment with miscanthus growing at the soil taken from Kurakhovo was updated and is currently monitored. The miscanthus deposit field established in 2016 was enriched. Since 2017 the research objectives were extended to testing study effect of PGRs to plant’s better adaptation to stress factors while growing crop at the contaminated soils. The research in laboratory at NULES is complementary to experiment carrying out at UJEP. For helping with this new field of research a new young researcher Mgr. Artem Medkov joint NULES NATO team recently: he started PhD study in this topic under supervising of assoc. prof. Tatyana Stefanovska. Artem has an experience in effectiveness of PGRs while applied to the different crops including energy plants when they were grown at the agricultural soil. The topic of his dissertation thesis is focused at peculiarities of biomass quality and quantity when Miscanthusx gigateus is treated by PGRs ( application was done to rhizomes and will be done to leaves). That research field is within G4687 objective on improving the biomass quality.

Master student Van Shuay has started research with supervising of assoc. professor Tatyana Stefanovska. His thesis will be focused at herbivorous diversity at Miscanthus x giganteus growing at the soil taken from Dolyna (former military site).

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Assoc. professor Ludmila Kava checks miscanthus plants after hibernation
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Students from Agricultural Entomology Class carry out laboratory stidy at miscanthus deposit plot
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Assoc.professor Tatyana Stefnovska and Master student Van Shuay are sampling entomophauna at the pot experiment
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Master student Van Shuay is studying method of PGR ‘s application
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PhD student Artem Medkov is setting up the laboratory trial with soil from Dolyna site

Research at NULES is led by assoc.prof. Stefanovska and focused at the following main fields:
  • Developing indicators for estimation the efficiency of the phytotechnology with miscanthus biomass production using nematodes and other soil organisms. The research is going on in cooperation with liasoning istitutions: WMU and WUELS, Poland.
  • Phytotechnology experiment in lab with the military contaminated soil from Kamenetz-Podilsky. In cooperation with NULP and UJEP.
  • Erection and observation of field plot at Kurakhovo, Donetsk region, Eastern Ukraine
  • Impact of different factors to the quality of miscanthus biomass when growing the plant at the military contaminated soil. Research is going on in cooperation with IPBB and UJEP with assistance of firm Agrotech.
  • Erection and observation of the Demonstration site in Dolyna, Ivano-Frankivsk oblast. In cooperation with end-users from Dolyna local authority and Lviv Ivan Franko National University.

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Biomass experiment monitoring ( from left to right): PPD Dr.Tetyana Stefanovska, PhD Artem Medkow, Dr. Ludmila Kava, prof. Valentina Pidlisnyuk
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Erection of field plot in Kurakhove, Donetst oblast
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Dr.Volodymir Kvak establishing biomass experiment
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Students majoring in Plant Protection during laboratory research with M.xgiganteus
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Erection of M.xgiganteus plantation at the research field of NULES. Dr. Volodymir Kvak- at the right side
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PPD Tetyana Stefanovska leads the laboratory research with M.xgiganteus
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Mrg. Svitlava Yaschuk (at the back) plans the design of field research in Kurakhove, Eastern Ukraine

Research at Kansas State University and Kansas Agricultural Experimental Station, USA
A greenhouse pot experiment was established in 2017 at the Department of Agronomy, Kansas State University

A greenhouse pot experiment was established  in 2017 at the Department of Agronomy, Kansas State University in order to research the growing of Miscanthusxgiganteus at the Pb contaminated soil modelled  contaminated land at  the military site in Ft-Riley, Kansas.

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About twenty days before first cutting, June 4, 2017
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Before first cutting, June 25, 2017
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Before first cutting, June 25, 2017
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One month after planting Mxg at the contaminated soil, April 18, 2017
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Soil preparation for the Lab experiment, PhD student Zafir Alasmary, March 22, 2017
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Ten days after planting Mxg at the contaminated soil , April 5, 2017
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Twenty days after first cutting, July 12, 2017

The small-plot field study at Fort Riley, Kansas, U.S.A.

The small-plot field study initiated in spring 2016 at Fort Riley, Kansas, U.S.A., is nicely progressing in 2017.

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2016 small-plot study. Picture was taken on April 9, 2017
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2016 small-plot study. Picture was taken on June 7, 2017
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A biosolids-amended plot, 2016 small-plot study. Picture was taken on June 7, 2017
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A biosolids-amended plot, 2016 small-plot study. Picture was taken on May 2, 2017
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No-tillage control plot, 2016 small-plot study. Picture was taken on May 2, 2017
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Triple superphosphate-treated plot, 2016 small-plot study. Picture was taken on May 2, 2017

A research field site has been established at Fort Riley, Kansas to grow Miscanthus x giganteus on lead contaminated soil. The first planting was in spring 2015; additional treatments were planted in spring 2016. The growth of miscanthus has been good in 2015 and 2016 thanks in large part to favorable rainfall amount and distribution.

The small-plot field study initiated in spring 2016 at Fort Riley, Kansas, U.S.A., consists of 5 treatments selected to evaluate how different establishment methods and soil amendments affect soil and plant lead content and stability. The experimental design is a randomized complete block with four replications. The site soil was contaminated with lead (about 1000 mg Pb kg-1 of soil), and soil pH was near neutral. The use of phosphorus to reduce lead bioavailability has been proposed as an effective in situ stabilization option for lead contaminated soils. Also, to minimize absorption of lead by plants maintain soil pH levels above 6.5 to 7.0 is preferred. Therefore, based on soil characteristics, we chose two sources of phosphorus as our soil treatments/amendments. Those were triple superphosphate (TSP, inorganic phosphorus fertilizer applied at 5:3 Pb:P molar ratio) and class B biosolids (organic source of phosphorus applied at 45 Mg/ha). The biosolids material was obtained from a nearby water treatment facility (Topeka, Kansas).

Table 1. Miscanthus establishment treatments.

Treatments: 
1ControlLeave existing vegetation in place with no amendments
2Miscanthus No-tillKill existing vegetation, plant miscanthus directly into undisturbed soil, no amendments
3Miscanthus TillKill existing vegetation, till to depth of 3 to 4 inches before planting miscanthus, no amendments
4Miscanthus Till + TSPKill existing vegetation, incorporate P fertilizer to depth of 3 to 4 inches to influence lead chemistry in surface soil before planting miscanthus
5Miscanthus Till + BiosolidsKill existing vegetation, incorporate Compost to depth of 3 to 4 inches to influence lead chemistry in surface soil before planting miscanthus

Table 2. Selected properties of the Fort Riley Soil

Soil propertiesTest site
pH (1:10 soil: water)6.85
CEC, cmol+ kg-119.5
Sand, silt, and clay, %11.3,  59.8,  28.9
Mehlich III-P, mg kg-1408.25
Ext. K, mg kg-1589.45

Table 3. Selected soil properties before amendments.

TREATMENTMehlich-3 extractable P*Extractable KAvailable NpH
NH4-NN03-N
…………………. (mg kg-1) ……………………. 
Control50.6 ± 3.49*516.7 ± 15.46.9 ± 1.141.9 ± 0.766.9 ± 0.07
Miscanthus No-till43.6 ± 2.39540.2 ± 7.76.0 ± 0.561.3 ± 0.376.7 ± 0.10
Miscanthus Till41.1 ± 7.26539.2 ± 14.85.8 ± 0.491.7 ± 0.536.8 ± 0.07
Miscanthus Till + TSP32.9 ± 7.94553 ± 19.55.4 ± 0.271.1 ±0.286.8 ± 0.06
Miscanthus Till + Biosolids43.4 ± 10.02523 ± 17.45.8 ± 0.171.4 ± 0.466.8 ± 0.08
Miscanthus Till + Biosolids1.68  ± 0.071.51  ± 0.110.50  ± 0.030.69 ± 0.011.35 ±0.10

*Values are means of four replicates±standard error.
** Dry matter content as a fraction of FW
A greenhouse experiment will be initiated in March 2017 to evaluate phosphorus source and rate effects on soil lead bioavailability and soil quality.

IPBB, Kazakhstan

Research on microbial population and mezophauna in the military soil, exploring soil characteristics: metal bioavailability, organic content, microbial population.

Experiment was provided at IPBB with military soil taken from village Maili, Balkhash district, Almaty region, Kazakhstan. Soil samples were taken at the depths of 0-20 and 20-60 cm, control samples were taken at the distance of 1 km from the village, totally, 20 soil samples were researched. Determination of heavy metal ions in the contaminated and control soil samples were carried out by inductively coupled plasma mass spectrometry (ICP-MS) using Agilent 7500 series instrument. Concentrations of organochlorine pesticides in the soils were determined by gas chromatography with mass spectrometric detection 7890A/5973N (Agilent, USA) equipped with Combi-PAL (CTC Analytics AG, Switzerland) auto sampler. It was established that research soil was saline, sandy, and pH of aqueous extract was 8.27-9.89. The soil was contaminated with heavy metals of 1st, 2nd and 3rd class of danger and organochlorine pesticides, particularly, DDT metabolites, γ HCH, heptachlor. The residual concentrations of POP pesticides in the research soil were below MACs (maximum allowable concentrations). However, detected residues of g-HCH, 2-4-DDD, 4,4’-DDD in soil was the evidence of danger to environment and humans. A possible reason of detected POP pesticides was likely related to agricultural activities. The proof was the presence of pesticides’ residues in the control soil samples. Determination the total content of heavy metals in the research soil showed that Pb concentration exceeded MAC up to 29 times, As - up to 5 times, Zn - up to 11 times, Ba - up to 1200 times, Sr - up to 22 times, Cu - up to 13 times.

The high concentrations of contaminants and presence of organochlorine pesticides in the soil may cause imbalance of physical, chemical and biological processes in it. Therefore, biochemical activity and phytotoxicity of the soil were assessed. Enzymatic activity indicators have good prospects for effective use in evaluation and monitoring of environmental conditions of soil, and allow characterizing modern conditions of soil life and predicting dynamic processes and trends in it and determining effect on soil anthropogenic factors. To identify the anthropogenic influence on fertility, biological activity of the contaminated soil: urease and cellulolytic activity were studied. The urease activity is one of the most important indicators of soil biological activity, it is regarded as a measure of self-purification capacity of soil, and the cellulolytic activity is a measure of intensity of destructive processes.

For analysis of soil biology, control and contaminated soils with the total level of contamination Zc 53.2-71.7 mg/kg were researched. A rapid method was used as methodological techniques for determination biochemical activity of soils. The essence of the method is that velocity of decomposition of organic nitrogen-containing compound (urea) and changes in ambient air pH for 1.5-2.0 units due to emission of ammonia are recorded (in hours). During the decomposition of urea, formation of volatile alkali – ammonia actively occurs, as a consequence, the air above soil gradually becomes alkaline. Colour of test strip varies in accordance with changes in pH of the medium. Time quantity at increasing alkalinity of vapour in equilibrium with soil in presence of urea was considered, as an indicator of urease activity.
Cellulolytic activity of soil was evaluated by application method. Flax was used as an applicator. The intensity of tissue destruction process was evaluated by the loss of weight. Express method for assessing the biological activity of the soil using flax applicator allows for a short time to determine the status of the biological activity of soil contaminated with anthropogenic pollutants.

The slight increase of urease activity in contaminated soil was found which indicated a decrease in resistance of enzyme to heavy metal ions. After 24 hours, the pH value of the soil reached 9, and pH of the soil around the plant was 10. The obtained results proofed that microorganisms which could degrade toxic compounds were less active in contaminated area, i.e., self-cleaning capacity of soil was weak.

In contrast with contaminated soil, a high level of urease activity after 24 hours was revealed in control samples, the pH reached 12, what characterizes self-cleaning ability of a potential soil. Reduction in biological activity of soil contaminated with xenobiotics, and cellulolytic activity was reduced relative to control up to 14% (30% control) was observed indicating toxicity of heavy metals. They inhibit soil microflora. It is known that slow process of cellulose decomposition inhibits a flow of organic components for reproduction of humus.

These results reiterate that microorganisms are not able to remove harmful heavy metals from soil, so biotech remediation methods are ineffective due to the slow degradation by microorganisms, contaminated soil does not have a self-cleaning and destructive capacity. Results confirmed the necessity to apply the phytotechnology approach for restoration of the military contaminated soils.

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Joint research


UJEP, ZU, NULP

UJEP, NULES, IPBB