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Aug 22, 2017 - Landfill Leachate Effects on Germination and Seedling Growth of Hemp Cultivars (Cannabis Sativa L.) Magdalena Daria Vaverková1 · Jan ...

Waste Biomass Valor DOI 10.1007/s12649-017-0058-z


Landfill Leachate Effects on Germination and Seedling Growth of Hemp Cultivars (Cannabis Sativa L.) Magdalena Daria Vaverková1 · Jan Zloch1 · Dana Adamcová1 · Maja Radziemska2 · Tomáš Vyhnánek3 · Václav Trojan3 · Jan Winkler3 · Biljana Đorđević3 · Jakub Elbl4 · Martin Brtnický4 

Received: 7 June 2017 / Accepted: 22 August 2017 © Springer Science+Business Media B.V. 2017

Abstract  Landfill leachate is one of the major sources of pollutions discharged into the environment. It is composed from a complex mixture of chemicals and handling typically involves treatment either on-site or at a wastewater treatment plants but phytoremediation is a promising method. The aim of this work was to evaluate the potential of agronomic plant species with high annual biomass yield (Cannabis sativa L.) for toxicity removal from landfill leachate. Raw leachate collected from the pond of untreated leachate at sanitary landfill in Czech Republic was used in the study. The hemp cultivation experiments were performed in the beginning of 2017 under laboratory conditions using three hemp cultivars

registered in the European Union: Tiborszállási (Hungary), Bialobrzeska (Poland) and Monoica (Hungary). The seeds were used for modified standard mustard germination test. The germination of hemp cultivars was tested using the hydroponics medium supplemented with leachate 25, 50, 75, 90 and 100%. The control seeds were growing on untreated nutrient medium under the same condition. The nature of germination of seeds was studied. Based on the obtained results, it can be concluded that the tested samples of leachate were toxic for hemp cultivars (C. sativa L.). Growth inhibition (%) at the studied samples ranged from −6.48 to 75.78%.

* Magdalena Daria Vaverková [email protected] 1

Department of Applied and Landscape Ecology, Faculty of AgriSciences, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic


Department of Environmental Improvement, Faculty of Civil and Environmental Engineering, Warsaw University of Life Sciences, Nowoursynowska 159, 02‑776 Warsaw, Poland


Department of Plant Biology, Faculty of AgriSciences, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic


Department of Geology and Pedology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 1/1665, 613 00 Brno, Czech Republic



Waste Biomass Valor

Graphical Abstract 

Keywords  Phytotoxicity · Toxicity · Landfill leachate · Cannabis sativa L.

Introduction Heavy metals are known as a major source of contamination discharged by various human activities. These heavy metals and persistent organic pollutants (POPs) are harmful to animals as well as humans due to its tendency to accumulate in the food chain [1–3]. The leachate commonly contains large amounts of organic matter, ammonium, heavy metals, and chlorinated organic and inorganic salts, which cause a great threat to soil–water environment in the vicinity of a landfill site [4]. Landfill leachate is one of major sources of heavy metals discharged into the environment, and this has continued to attract public concern due to potential deleterious impact of heavy metals on human health and environment [5, 6]. Therefore, heavy metals removal from landfill leachates is a growing area of research [5, 7, 8]. Leachate handling typically involves treatment either on-site or at a wastewater treatment plants [9–12] but phytoremediation is a promising method. Phytoremediation is a process that uses living green plants for the in situ


risk reduction of contaminated soil, sludge, sediments and groundwater through contaminant removal, degradation or containment [13, 14]. Phytoremediation covers a wide range of pollutants like inorganic chemicals including heavy metals and metalloids, many organic substances including persistent organic pollutants and radioactive elements. Phytoremediation has gained much popularity over the last 20 years and has been considered as an acceptable technique in many countries due to its cost-effectiveness compared to traditional practices [15]. The plants used in phytoremediation are generally annual herbs which don’t have any economic value, but do have a very high extraction potential, namely hyper accumulators [16]. The phytoextraction efficiency of a plant is dependent on the heavy metal contents in the biomass and the biomass production [17]. Some agricultural crops (e.g., Brassica napus L., Brassica juncea L., Cannabis sativa L., Helianthus annuus L., Phaseolus vulgaris L., Sinapis alba L., and Zea mays L.) were found to be effective in removing metals under model conditions but their evaluation in field conditions is missing [18–24]. Hemp (C. sativa L.) is an annual crop with a 6000-year cultivation history. Hemp fiber, seeds and raw materials are used in the textile, oil, paper-making, automotive,

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construction, bio-fuel, functional food, cosmetics, personal care and pharmaceutical industries [25, 26]. It also could phytoremediate environment polluted by heavy metals [16, 26–28]. Hemp is suited to various climates around the world, but it was prohibited in the last century in many countries because it is the raw material source of tetrahydrocannabinol (THC), which is a unique secondary metabolite used as a narcotic [25]. In recent years, hemp cultivation has been permitted to recover in an increasing number of countries because of its wide use, positive effects on the environment, high caloric value, and high yield, low cultivar input [29]. Based on previous research, we conducted a laboratory experiment to compare the response to leachate at different levels exposure at the seed soaking and germination stages and evaluate the phytotoxicity effect of leachate on the three hemp cultivars. This paper aims to assess the potential of agronomic plant species with high annual biomass yield (C. sativa L.) for phytoremediation of heavy metals from landfill leachate. Here, we explore the following issues: (a) the phytotoxicity of the landfill leachate, (b) landfill leachate effects on germination and seedling growth of hemp cultivars and, to find out the answers to these issues, we carried out a laboratory trial on raw leachate collected from the pond of untreated leachate at a sanitary landfill in Czech Republic (CR).

Materials and Methods Site Description The present study was conducted in the Kuchyňky landfill (49.2490778N, 17.3121181E), which is located in a triangular space delimited by main roads connecting the villages of Zdounky, Nětčice and Troubky-Zdislavice (Fig. 1). It is a sanitary landfill incorporated with multilayer composite bottom liner, leachate and landfill gas collection system, and a final cover system. The landfill has a leachate recirculation system, which operates mainly in the summer period (May–September). In terms of maintenance, the landfill is classified in the S-category—other waste, sub-category S-OO3. The designed area of the landfill is 70,700 m2 in five stages with a total volume of 907,000 m3, i.e., ca. 1,000,000 ­103 kg of waste. The facility receives waste (category of other waste) from a catchments area with the population of ca. 75,000 residents. The annually deposited amount of waste is ca. 40,000 103 kg of which 50% are from the communal sphere [30, 31]. Leachate Sampling Raw leachate collected from the pond of untreated leachate was used in the study. The landfill is still under operating and producing young leachate which is mixed together with old (mature) leachate. Two samples (0.5 L sample) of landfill leachate were collected in pre-sterile bottles and brought to the laboratory directly after sampling. Sampling bottles had been previously treated for 24 h in 5 mol/L ­HNO3 and then rinsed with ultrapure water. The samples were preserved

Fig. 1  Schematic diagram showing the landfill and aerial photograph (inset) of the landfill site


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Table 1  Raw landfill leachate characteristics—leachate heavy metals concentrations Heavy metal



Cd Cr Ni Pb Zn Hg LDO pH EC NH4+

mg/L mg/L mg/L mg/L mg/L mg/L mg/L – mS/cm mg/L

0.003 ± 0.02 0.78 ± 0.43 0.11 ± 0.25 0.03 ± 0.03 0.04 ± 0.26 0.001 ± 0.00 5.03 ± 0.11 8.03 ± 2.33 10.33 ± 0.5 292.0 ± 13.19

LDO dissolved oxygen, EC electrical conductivity, Values are mean (n = 3) and ±standard deviation

at a temperature of 4 °C ± 1 until analysis. Some characteristics of the landfill leachate are presented in Table 1. Leachate samples were analyzed for pH, electric conductivity (EC), dissolved oxygen (LDO) (Multi-Parameter Meter HQ30d Portable) and a series of heavy metals. The leachate samples were analyzed for the content of heavy metals (Cd, Cr, Ni, Pb, Zn, and Hg) in the Analytical laboratory at the Department of Chemistry and Biochemistry, Faculty of AgriSciences, Mendel University in Brno. Plant Material and Phytotoxicity Test The hemp cultivation experiments were organized in 2017 under laboratory conditions using three hemp cultivars approved for commercial use by the European Union (EU): Tiborszállási (Hungary), Bialobrzeska (Poland) and Monoica (Hungary). Seeds of hemp (C. sativa L.) cv. Tiborszállási and Bialobrzeska, were obtained from SEMO Inc. Smržice (CR), cv. Monoica were obtained from Agritec Ltd. Šumperk (CR). The seeds were used for modified standard mustard germination test. Seeds were washed ten times in sterile deionized water. Each leachate sample was diluted to give final leachate concentrations of 25, 50, 75, 90 and 100%. Each concentration of the dilution series was tested with three replicate samples. The test organisms were exposed to the landfill leachate solutions for a total of 72 h. The seeds of hemp (Cannabis sativa L.) were germinated in Petri dishes (Fig. 2) with a 14 cm diameter on a layer of filter paper at the bottom (sterilized at 121 °C, 25 min, 1.033 kg/cm2). The hydroponic solution (distilled water with the following chemical ingredients (mg/L): Ca(NO3)2 0.8, ­KH2PO4 0.2, ­KNO3 0.2, ­MgSO4.7 ­H2O 0.2, KCl 0.2, ­FeSO4 0.01, pH 5.2) with tested liquid (landfill leachate) was added into each dish, and 15 healthy looking seeds were evenly


Fig. 2  Petri dish with seeds of C. sativa L.

spread onto the surface of the filter paper. The control seeds were growing on an untreated nutrient medium on the same condition. The Petri dishes were covered by a glass cap to prevent loss due to evaporation and were located in the dark thermostat Ecocell (t = 24 °C, air humidity 80%). After 72 h, at the end of the test period germination percentage, and longest root seedlings were determined. Calculations and Data Analysis The analyses and the length measurements were performed using the Image Tool 3.0 for Windows (UTHSCSA, San Antonio, USA). The bioassays were performed in three replicates. The percent of root growth inhibition (RI) were calculated with the formula (Eq. 1): (1) where A—means root length in the control; B—means root length in the test [32].

RI = A − B∕A × 100

Results The root growth inhibition of crop plants was tested using the hydroponics medium supplemented with landfill leachate 25, 50, 75, 90 and 100%. The root length of germinated seeds was compared with the control. Results are expressed as mean ± standard deviation. Figure 3 presents the effect of the landfill leachate (concentration 100%—sample 100% leachate Bialobrzeska (LB), 90%— sample 90% LB, 75%— samples 75% LB, 50%—samples 50% LB and 25%—samples 25% LB) on the inhibition of seed germination and root growth as related to the test C. sativa L.—Bialobrzeska. The growth inhibition (%) for sample 25% LB was 212.89%, sample 50% LB was 23.34%, sample 75% LB was 37.63%, sample 90% LB was 75.78% and sample 100% LB was 73.87%. With increasing landfill leachate concentrations, the

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Fig. 3  Growth inhibition of C. sativa L.—Bialobrzeska. Different letters indicate statistical significance at p