Journal of Environmental Accounting and Management

Volume 6 Issue 4 December 2018

ISSN 2325-6192 (print) ISSN 2325-6206 (online) CN 10-1358/X

Journal of Environmental Accounting and Management

ISEE International Society for Environmental Ecology

Journal of Environmental Accounting and Management Editors Zhifeng Yang School of Environment Beijing Normal University Beijing 100875, China Fax: +86 10 58800397 Email: [email protected]

Sergio Ulgiati Department of Science and Technology Parthenope University of Napoli Centro Direzionale, Isola C4. 80143 Napoli, Italy Fax: +39 081 547 6515 Email: [email protected]

Associate Editors Mark T. Brown (System analysis) Department of Environmental Engineering Sciences University of Florida. Gainesville, FL, USA Fax: 352-392-3624 Email: [email protected]

Olga Kordas (Urban system/Smart cities) Department of Sustainable development, Environmental science and Engineering KTH Royal Institute of Technology Teknikringen 34, S - 100 44 Stockholm, Sweden Email: [email protected]

Hans Schnitzer (Energy system analysis) Institute for Process Engineering Graz University of Technology Graz, Austria Fax: 43(0)316-873/7469 Email: [email protected]

Biagio Giannetti (Cleaner production) Paulista University Laboratório de Produção e Meio Ambiente (LaProMA) Sao Paulo, Brazil Email: [email protected]

Antonino Marvuglia (Computational Sustainability) Luxembourg Institute of Science and Technology Environmental Research & Innovation Department 5 avenue des hauts fourneaux L-4362 Esch sur Alzette - Luxembourg Email: [email protected]

Walter A. Pengue (Agriculture/Water management) Universidad Nacional de General Sarmiento Gepama Fadu UBA Buenos Aires, Argentina Emails: [email protected]

Salvatore Arico Division of Ecological Sciences UNESCO, Paris, France Fax: 33 1 4568 5804 Email: [email protected]

Francesco Cherubini Dept. of Energy and Process Engineering Norwegian Univ. of Science and Technology Trondheim, Norway Email: [email protected]

Pier Paolo Franzese Department of Science and Technology Parthenope University of Napoli Centro Direzionale, Isola C4. 80143 Napoli, Italy Email: [email protected]

Feni Agostinho Paulista University Laboratório de Produção e Meio Ambiente (LaProMA) Rua Dr. Bacelar, 1212, CEP 04026-002, São Paulo, Brasil Email: [email protected]

Liang Dong Institute of Environmental Sciences Leiden University P.O. Box 9518 2333 RA Leiden, the Netherlands Email: [email protected]

Andrea Genovese Logistics and Supply Chain Management Research Centre Management School, The University of Sheffield Room B.063, Conduit Road, Sheffield S10 1FL, UK. E-mail: [email protected]

Bhavik Bakshi Department of Chemical Engineering The Ohio State University Columbus Ohio 43210, USA Fax: (614)292-3769 Email: [email protected]

Stefano Dumontet Department of Environmental Sciences Parthenope University of Napoli Centro Direzionale, Isola C4. 80143 Napoli, Italy Email: [email protected]

Dabo Guan School of International Development University of East Anglia, St Edmund's College, Cambridge Email: [email protected]

Enrico Benetto Luxembourg Institute of Science and Technology ERIN - Environmental Research & Innovation Department 41, rue du Brill, L-4422 Belvaux Email: [email protected]

Juan Jose Cabello Eras Energy Department Universidad de la Costa Colombia Email: [email protected]

Helmut Haberl Institute of Social Ecology Alpen Adria Universität Vienna, Austria Email: [email protected]

Editorial Board

Federico M. Butera Dept. of Building & Environment Science & Technology (BEST) Politecnico di Milano, Milano, Italy Fax: 02-2399-5151 Email: [email protected] Guoqian Chen Dept.of Mechanics and Engineering Science Peking University Beijing, China Email: [email protected]

Brian D. Fath Department of Biological Sciences Towson University Towson, MD 21252, USA Fax: 410-704-2405 Email: [email protected] Matthias Finkbeiner Department of Sustainable Engineering Techincal University Berlin, Germany Fax +49 (0)30 314-21720 Email: [email protected]

Gordon Huang Dept. of Environmental Systems Eng. University of Regina Regina, Saskatchewan, Canada Fax: (306) 585-4855, Email: [email protected] Wesley W. Ingwersen Sustainable Technology Division National Risk Management Research Laboratory, US EPA Cincinnati, OH 45268, USA Email:[email protected]

Continued on the back materials

Journal of Environmental Accounting and Management Volume 6, Issue 4, December 2018

Editors Zhifeng Yang Sergio Ulgiati

L&H Scientific Publishing, LLC, USA

Publication Information Journal of Environmental Accounting and Management (ISSN 2325-6192 (print), eISSN 2325-6206 (online), CN 10-1358/X) is published quarterly (March, June, September, and December) by L & H Scientific Publishing, LLC, P.O. Box 99, Glen Carbon, IL62034, USA. Subscription prices are available upon request from the publisher or from this journal website. Subscriptions are accepted on a prepaid basis only and entered on a calendar year basis. Issues are sent by standard mail (Surface in North America, air delivery outside North America). Priority rates are available upon request. Claims for missing issues should be made within six months of the date of dispatch.

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Printed in USA on acid-free paper.

Journal of Environmental Accounting and Management 6(4) (2018) 291-294

Volume 1 Issue 1 March 2013

ISSN 2325-6192 (print) ISSN 2325-6206 (online)


Journal of Environmental Accounting and Management Journal homepage: https://lhscientificpublishing.com/Journals/JEAM-Default.aspx

Environmental Quality and Management Pier Paolo Franzese1†, Giulia Maisto2 , Anna De Marco2 , Carmen Arena2 , Elvira Buonocore1 1 2

Department of Science and Technology, Parthenope University of Naples, Italy Department of Biology, University of Naples “Federico II”, Italy

Submission Info Communicated by Sergio Ulgiati Received 20 October 2018 Accepted 23 October 2018 Available online 1 January 2019 Keywords Environmental quality Environmental management Environmental assessment Environmental accounting

Abstract This volume gathers theoretical, methodological, and applied papers exploring different issues related to environmental quality and management. Human economy depends on healthy ecosystems capable of ensuring a life support system delivering ecosystem services vital for human well-being. Integrated environmental assessment and environmental accounting are essential research areas to assess the quality of environmental matrices while supporting managers and policy makers with scientifically sound information.

©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction The massive exploitation of natural resources by humans is strongly affecting biodiversity and the functionality of natural ecosystems (Cardinale et al., 2012; MA, 2005; TEEB, 2010). The lack of understanding about the dependence of human economy upon natural resources has generated critical environmental problems, among which chemical pollution, eutrophication, soil erosion, biodiversity loss, water crisis, and climate change (Folke et al., 2011; Meadows et al., 2004; Rockström et al., 2009). Over the past few decades, efforts have been done to explore the link between environmental quality and human well-being. Indeed, healthy ecosystems are capable of maintaining their structures and functions, ensuring the generation and maintenance of natural capital stocks and ecosystem services flows (Costanza et al., 1997, 2014; Häyhä and Franzese, 2014). The increased awareness on the interdependency between natural and human economy has led to the development of nature conservation actions and sustainable management schemes, also boosting natural capital and ecosystem services assessment (Franzese et al., 2015, 2017; Häyhä et al., 2015; Nikodinoska et al., 2018; Picone et al., 2017; Vassallo et al., 2017). Environmental monitoring and natural resource accounting represent key tools to perform integrated environmental assessment focusing on the quality of environmental matrices (air, soil, and water) and the resilience of natural ecosystems (Folke et al., 2004). † Corresponding

author. Email address: [email protected] ISSN 2325-6192, eISSN 2325-6206/$-see front materials © 2018 L&H Scientific Publishing, LLC. All rights reserved. DOI:10.5890/JEAM.2018.12.001

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The design of sustainable management strategies requires the integration of environmental, economic, and social aspects (Franzese et al., 2008, 2014; Russo et al., 2014; Ulgiati et al., 2010; Viglia et al., 2013). These aspects can be investigated through multicriteria assessment frameworks capable of supporting local managers, policy-makers, and other stakeholders (Buonocore et al., 2012, 2014; Nikodinoska et al., 2017).

2 Goal of this special issue The main goal of this special issue is to present a set of articles exploring different aspects related to the issue of environmental quality and management. In particular, this special issue gathers theoretical, methodological, and applied papers exploring the following research areas: a) characterization and functionality of aquatic and terrestrial ecosystems, b) relationships between chemical alterations and biological components of ecological communities, c) chemical, biological, and ecotoxicological indicators to assess environmental quality, d) spatial analysis and environmental accounting tools to assess anthropogenic impacts and ecosystem resilience, e) renewable energy options and their implications on economy and environment.

3 Papers presented in the special issue This special issue collects papers presented at the X Biennal International Workshop Advances in Energy Studies (BIWAES) and the XXVII Congress of the Italian Society of Ecology (S.It.E.), summarized as follows. Esposito et al. (2018) evaluate heavy metals concentration in leaves of Q. ilex L. collected in two municipalities located in Southern Italy. Figlioli et al. (2018) assess the effects of increasing Mn concentrations on the uptake of a set of micro and macro nutrients in Cistus salvifolius L. grown in hydroponic culture. Amitrano et al. (2018) analyze specific physiological traits of mung bean (Vigna radiata) seedlings grown at different light quality regimes to assess the best light treatment promoting photosynthesis and plant development. Romanucci et al. (2018) test the effects on five selected plant species of phytotoxic extracts added in subsurface drip irrigation system. Sapio (2018) discusses the effects of regional redistribution of renewable energy subsidies and shows that regions characterised by high energy demand and low renewable energy endowments disproportionately contribute to the green energy budget. Schiavo et al. (2018) assess the toxicity of virgin plastic pellet leachates on organisms of different trophic levels, spanning form prokaryotes to eukaryotes, in freshwater environment. Marzaioli et al. (2018) investigate the effect of biochar amendment on soil quality and crop yield in a greenhouse environment of an organic farm located in Southern Italy. Appolloni et al. (2018) perform an integrated landscape-seascape spatial analysis of the Gulf of Naples (Southern Italy) to detect differences in the spatial patterns of ecosystem patches between landscape and seascape and to compare the effects of anthropogenic impact in terms of spatial heterogeneity. Buonocore et al. (2018) use bibliometric network analysis to investigate the global scientific literature on “natural capital”, exploring the relationships among authors, countries, journals, and main keywords on the subject.

4 Concluding remarks The Guest Editors hope that this volume will boost the interdisciplinary knowledge on different aspects regarding the issue of environmental quality and management.


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Acknowledgement A special thank is due to all the Reviewers who contributed their time and valuable effort. Without their work and scientific support this special issue would not have been possible.

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6(4), 312-322. Meadows, D.H., Randers, J., and Meadows, D.L. (2004), Limits to Growth. The 30-Year Update. Chelsea Green Publishing, USA. Nikodinoska, N., Buonocore, E., Paletto, A., and Franzese, P.P. (2017), Wood-based bioenergy value chain in mountain urban districts: An integrated environmental accounting framework, Applied Energy, 186, 197-210. Nikodinoska, N., Paletto, A., Pastorella, F., Granvik, M., and Franzese P.P. (2018), Assessing, valuing and mapping ecosystem services at city level: The case of Uppsala (Sweden), Ecological Modelling, 368, 411-424. Picone, F., Buonocore, E., D’Agostaro, R., Donati, S., Chemello, R., and Franzese P.P. (2017), Integrating natural capital assessment and marine spatial planning: A case study in the Mediterranean sea, Ecological Modelling, 361, 1-13. ˚ Chapin, F.S., Lambin, E.F., Lenton, T.M., Scheffer, M., Folke, C., Rockström, J., Steffen, W., Noone, K., Persson, A., Schellnhuber, H.J., Nykvist, B., de Wit, C.A., Hughes, T., van der Leeuw, S., Rodhe, H., Sörlin, S., Snyder, P.K., Costanza, R., Svedin, U., Falkenmark, M., Karlberg, L., Corell, R.W., Fabry, V.J., Hansen, J., Walker, B., Liverman, D., Richardson, K., Crutzen, P., and Foley, J.A. (2009), A safe operating space for humanity, Nature, 461, 472-475. Romanucci, V., Ladhari, A., De Tommaso, G., De Marco, A., Di Marino, C., Di Fabio, G., and Zarrelli, A. (2018), Phytotoxic extracts as possible additive in subsurface irrigation drip for organic agriculture, Journal of Environmental Accounting and Management, 6(4), 332-340. Russo, T., Buonocore, E., and Franzese, P.P. (2014), The Urban Metabolism of the City of Uppsala (Sweden), Journal of Environmental Accounting and Management 2(1), 1-12. Sapio, A. (2018), Regional redistribution effects of renewable energy subsidies, Journal of Environmental Accounting and Management, 6(4), 323-331. Schiavo, S., Oliviero, M., Romano, V., Dumontet, S., and Manzo, S. (2018), Ecotoxicological assessment of virgin plastic pellet leachates in freshwater matrices, Journal of Environmental Accounting and Management, 6(4), 341-349. TEEB (2010), The Economics of Ecosystems and Biodiversity: Mainstreaming the Economics of Nature: A Synthesis of the Approach, Conclusions and Recommendations of TEEB. Ulgiati, S., Ascione, M., Bargigli, S., Cherubini, F., Federici, M., Franzese, P.P., Raugei, M., Viglia, S., and Zucaro, A. (2010), Multi-method and multi-scale analysis of energy and resource conversion and use. In: Barbir, F. and Ulgiati, S. (Eds.), Energy Options Impacts on Regional Security. NATO Science for Peace and Security Series – C: Environmental Security, pp. 1-36, Springer. (ISBN: 978-90-481-9567-1; ISSN 1874-6519). Vassallo, P., Paoli, C., Buonocore, E., Franzese, P.P., Russo, G.F., and Povero, P. (2017), Assessing the value of natural capital in marine protected areas: A biophysical and trophodynamic environmental accounting model, Ecological Modelling, 355, 12-17. Viglia, S., Nienartowicz, A., Kunz, M., and Franzese, P.P. (2013), Integrating environmental accounting, life cycle and ecosystem services assessment, Journal of Environmental Accounting and Management, 1(4), 307-320.






Light Fertilization Affects Growth and Photosynthesis in Mung Bean (Vigna radiata) Plants Chiara Amitrano1, Ermenegilda Vitale2 , Veronica De Micco1 , Carmen Arena2† 1 2

Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, Portici (NA), Italy Department of Biology, University of Naples Federico II, 80125 Naples, Italy Submission Info Communicated by Pier Paolo Franzese Received 17 April 2018 Accepted 30 September 2018 Available online 1 January 2019 Keywords Mung bean Light quality Photosynthesis Crop production Proteins

Abstract In a climate change scenario, the optimization of growth conditions for food crop species plays a key role for the sustainability of cultivation. Agrotechnologies need to be improved to set up the best conditions to maximize plant development, production and resource use efficiency in growth chambers and greenhouses. The manipulation of light quality during plant growth may be used as a powerful mean to obtain specific functional traits. This practice may be useful to improve plant growth, also avoiding the use of large doses of chemical fertilizers, which may compromise the environment and human health. In our study, we analyzed specific physiological traits of mung bean (Vigna radiata) seed-lings grown at different light quality regimes (W-White, R-Red and RB-Red-Blue light), to assess the best light treatment in promoting plant development and photosynthesis. Plant growth was monitored measuring stem and root elongation, dry biomass and total leaf area. The integrity of the photosynthetic machinery was monitored through fluorescence an emission measurements and content of photosynthetic pigments and total proteins. Our results showed that the growth under R wavelengths promoted stem elongation compared to W and RB. This light treatment was also responsible for the highest production of total chlorophylls. Photochemistry was not affected by the different light qualities. RB light induced a compact architecture of plants and the highest amount of proteins. Overall results indicate that different light quality regimes can be applied during the cultivation to consciously modify plant growth and development. Thus, it will be fundamental to optimize and choose opportunely not only the intensity but also the spectral composition of light to maximize the productivity of a specific crop in quantitative and qualitative terms. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction The optimization of production processes in food crops is a fundamental issue in the perspective of the increasing demand in quantity and quality of food (Lairon, 2010). † Corresponding


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Vigna radiata (L.) R. Wilczek also known as mung bean, is an important legume crop in a large number of countries (Lakhanpaul et al., 2000). The seeds and sprouts of mung bean have been consumed as common food in China for more than 2000 years; they are used in fresh salads in India, Bangladesh, South East Asia and western countries providing the main source of protein (240 g kg−1 ) for a large proportion of vegetarians as well as abundant dietary fiber, carbohydrates (630 g kg−1 ) and a vast range of bioactive phytochemicals with antioxidant, antimicrobial, anti-inflammatory and antitumor activity (Kanatt et al., 2011; Nair et al., 2013; Tang et al., 2014). Mung bean can be considered an excellent example of functional food because it presents a high digestibility and it is rich in proteins (about 20% - 24% of the total composition of mung bean) and essential amino acids (Kudre et al., 2013; Tang et al., 2014). In this species, seeds germination and formation of sprouts are processes strongly influenced by light that represents the main driver for plant development, flower and seed production (Singh et al., 2015). In fact, it is well known that some useful metabolites are synthesized during the initial stage of germination (El-Adawy et al., 2003). Beside germination, light quality and intensity affect numerous other processes, such as photo-morphogenesis, biomass accumulation, photosynthetic efficiency, flowering and phytochemical synthesis. In particular light quality and intensity modify the signaling cascade of specific photoreceptors (phytochromes, cryptochromes and phytotropins), thus changing the expression of a high number of genes (Yorio et al., 2001; Massa et al., 2008; Li and Kubota, 2009; Lin et al., 2013; Olle and Virˇsil˙e 2013; Arena et al., 2016). Within the whole visible light spectrum, pure red wavelengths strongly affect the vegetative growth, photosynthetic apparatus development, photosynthetic process, morphogenesis, flowering and budding; pure blue wavelengths play a fundamental role in the regulation of vegetative growth and photosynthesis through the control of chlorophyll biosynthesis, stomata opening and photo-morphogenesis (Urbonaviˇci¯ut˙e, et al., 2007; Chen et al., 2014; Singh et al., 2015). Plants have a considerable physiological and morphological plasticity in adapting to different light qualities (Barreiro et al., 1992; Arena et al., 2016). In particular blue light combined with red wavelengths positively influences plant growth, tissue nutritional value, chlorophyll ratio and number of seeds in vegetables (Yanagi et al., 1996; Goins et al., 1997; Li et al., 2012). It is not possible to delineate major common responses for all crops because there are species-specific reactions to light quality and intensity (Li, 2012; Singh et al., 2015). The modulation of light quality is more and more recognized as an efficient mean to optimize not only plant development but also nutritional quality of edible organs. At present, many greenhouses are equipped with artificial light provided by LED technology, which in combination with solar light as the main light source guarantee an optimal illumination during plant growth (Bian et al., 2015). A proper lighting during plant development is a valuable resource, especially in regions where solar radiation is weak or photoperiod is not sufficient to guarantee optimal growth (Opdam et al., 2005). Cultivation under controlled conditions plays an important role in the production of vegetables: the main advantage provided by the indoor production is the control of the growth environment in terms of light (quality and quantity), temperature, humidity, CO2 concentration, water and nutrients (Gary, 2003; Gruda, 2005). For light optimization, the LEDs technology used in artificial systems is a suitable device easily integrated into digital control systems to modulate both the intensity or spectral composition during the whole plant life cycle (Yeh and Chung, 2009). The manipulation of LED lighting represents a useful mean to realize appropriate light fertilization protocols in order to satisfy specific light requirements for each species. This is a fundamental requisite to obtain high photosynthetic rates and biomass production without the addition of high doses of chemicals. An appropriate light fertilization is “eco-friendly” because allows minimizing the use of fertilizers to obtain high crop yield, thus reducing the impact on the environment. For example, the modulation of light quality regulates the nitrate concentration in plants: red light stimulates the nitrate reductase activity decreasing the nitrate concentration in plants, whereas blue light increases the overall nitrogen concentration, reducing the plant demand for this macronutrient (Deng et al., 2000; Lillo and Appenroth, 2001; Ohashi-Kaneko et al., 2006). The selection of a proper light spectrum in growth chambers or in greenhouses in combination with the most suitable environmental parameters (T◦ , RH%, CO2 concentration) is promising to achieve high yield in a short time. In this study we applied specific “light-fertilization” treatments to improve the growth and quality of mung bean (Vigna radiata L.) seedlings. To this purpose, we analyzed plant


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development, photosynthesis, biomass partitioning, chlorophyll and total protein production, providing useful information for the design of light systems for the cultivation under controlled conditions.

2 Materials and methods 2.1

Plant material and experimental design

Seeds of Vigna radiata (L.) R. Wilczek, commonly known as mung bean, were incubated in Petri dishes, lined with three layers of filter paper, at 22◦ C in the dark for two days. Afterward, germinated seeds were incubated in a growth chamber under controlled conditions of temperature (22-23 ◦ C), relative humidity (60-65%) and photoperiod (12 h), under three different regimes of light, equivalent for intensity (photosynthetic photon flux density, PPFD, 140-200 µ mol photons m−2 s−1 ), but different for quality: white light (W) with a spectrum from 380-750 nm, pure red light (R) with a maximum intensity at 660 ± 5 nm, and a mixture of red (R: 66%) and blue (B: 33%) light (RB). The white light was provided by two types of Phosphor-Coated LEDs for white light (5000K-6500K). 2.2

Plant growth

After a week, at the emergence of the first two true leaves, 10 seedlings for each light treatment were transplanted into pots filled with commercial soil and incubated for four weeks, under the three conditions reported above. During the growth period, seedlings were regularly watered to reintegrate water lost by evapotranspiration. Plant height, stem and root length, root/shoot ratio, leaf number, leaf area and plant dry weight were recorded at the end of the experiment. The leaf area (surface of the lamina) was quantified on digital images taken by means of a digital camera and analyzed through “ImageJ” software (Rasband, W.S., U.S. NIH, Bethesda, Maryland, USA, 1997-2012). 2.3

Fluorescence “a” emission

Chlorophyll a fluorescence emission was measured using a pulse amplitude modulated fluorometer (JuniorPAM, Walz, Germany). Light fast-kinetic response curves (LFRCs) were performed in mung bean seedlings exposed to different light treatments in order to evaluate the photosynthetic capacity at Photochemical Photon Flux Densities (PPFD) ranging from 0 to 1500 µ mole photons m−2 s−1 . For each irradiance level, photochemical parameters, in the dark and in the light, were calculated as reported in Arena et al. (2005). On 30 min dark-adapted leaves, the background fluorescence signal, F0, was induced by a weak light of 0.5 µ mol photons m−2 s−1 at 0.6 kHz frequency. Maximal fluorescence in the dark-adapted state, Fm, was obtained by applying a 1 s saturating light pulse (8,000 µ mol photons m−2 s−1 ) at 20 kHz frequency. The photochemical quenching qP, which indicates the proportion on PSII reaction centers that are open, was calculated according to Genty et al. (1989). The non-photochemical quenching (qN), the fraction of absorbed light energy not utilized in photochemistry and dissipated as heat was calculated according to Van Kooten and Snel (1990). 2.4

Content of chlorophylls and proteins

After fluorescence measurements, 5 leaves were collected from 3 plants per each treatment for the determination of photosynthetic pigment content. The content of chlorophylls a and b, the relative a/b ratio, and total carotenoids (x+c) were determined. Photosynthetic pigments were extracted in ice-cold 100% acetone, quantified and expressed in µ g cm−2 as reported in Arena et al. (2014). For protein extraction, 0.3 g of leaves from seedlings per each light treatment were powdered with liquid nitrogen. Proteins from the cytoplasmic fraction, were extracted from the powder according to Wang et al. (2006) and Sorrentino et al. (2018). Proteins were estimated by the method of Bradford (1976) based on the

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colorimetric assay with Coomassie blue R-250, using the Bio-Rad Protein Assay (Bio-Rad). A standard curve with bovine serum albumin (Sigma) was used to calculate protein concentrations in the extracts, expressed in mol [BSA] g−1 FW. 2.5

Data elaboration

All data were analyzed by one-way analysis of variance (ANOVA) through the Sigma-Stat 3.5 software (Jandel Scientific, San Rafael, CA, USA). The Holm-Sidak test was applied for all multiple comparison tests with a significance level of p < 0.05. Data are reported as mean values ± standard error (n = 5). 3 Results and discussion 3.1

Effect of light quality on plant growth

V. radiata seedlings grown under R light showed a significantly higher values (P < 0.05) of plant height and leaf area than those grown under W and RB regimes (Fig. 1a, e). These results are in agreement with other studies where the stem elongation in Chrysanthemum (Kim et al., 2004c) and grape (Puspa et al., 2008) was stimulated by red light. The addition of blue to red wavelengths determined significant reduction (P < 0.05) in stem elongation and in root/shoot ratio, accompanied by an increase in leaf number compared to the other light treatments (Fig. 1a, c, d). The high root/shoot ratio is due to the significant increase in root length recorded in RB seedlings compared to W and R ones (Fig. 1b). These results are in agreement with previous studies which reported an increment in shoot length under 100% of red light (Hoenecke et al., 1992; Miyashita et al., 1997; Brown et al., 1995; Goins et al., 1997; Okamoto et al., 1997). Although both red and blue light can mediate stem elongation (Huche-Thelier et al., 2016), many studies have shown that blue light was more effective than red light in suppressing shoot elongation in a range of plant species (Hoenecke et al., 1992; Brown et al., 1995; Kong et al., 2012). Generally, different plant species show different responses to specific light receipts (Goins et al., 1997): for some crops, such as lettuce (Lactuca sativa L.), spinach (Spinacia oleracea L.) and radish (Raphanus sativas L.), the growth under pure Red LEDs was not sufficient to achieve a normal development root/shoot ratio and a minimum blue light was necessary to guarantee this balancing (Bula et al., 1991; Yorio et al., 2001). The growth under RB wavelengths induced a more compact structure characterized by the short stem elongation and the higher number of smaller leaves (at least compared to the R seedlings). In these plants, the blue light component added to the red one likely promoted the partitioning of photosynthates to roots, conversely to R plants where the total absence of blue determined the unbalancing of photosynthetic products towards the shoot. The increasing in root length under RB light and the rise of stem elongation under R light were responsible of the higher values (P < 0.05) of total dry weight in RB and R seedlings compared to W seedlings (Fig. 1f). The higher partitioning of photosynthates towards the epigeal biomass in R compared to W plants as well as the increased leaf area expansion may be likely due to the need by photosynthetic apparatus to harvest as much light as possible, since the pure R light does not represent a favorable condition for plant growth. When blue light is added to the red wavelengths, the stomatal opening is promoted and thus gas exchanges are improved, resulting in increased production of biomass in many species (Sharkey and Raschke, 1981; Goins et al., 1997; Zeiger and Zhu, 1998). Okamoto et al. (1997) reported that the stem elongation in lettuce seedlings decreases significantly with the increase of blue wavelengths. It is well demonstrated that small variation within blue light component can determine significant changes in stem growth (Brown et al., 1995). The synergistic interaction of the blue and red lights receptors (cryptochromes and phytochromes) may account for such responses (Kim et al., 2004 a,b,c). Also in our experiment, even if the light intensity was the same during plant growth for all light treatments, the different light quality may have stimulated specific responses, mediated by diverse photoreceptors, that make seedling able to optimize their growth according to the specific light environment.


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Fig. 1 Plant height (a), root length (b), root/shoot ratio (c), leaf number (d), leaf area (e), and dry weight (f) of Vigna radiata L. seedlings grown under the three different light quality treatments: White (W), Red (R) and Red-Blue (RB). Each value represents the mean ± SE (n = 5). Different letters indicate significant differences between light treatments (P < 0.05).

3.2

Effect of light quality on photochemistry

The photosynthetic efficiency of seedlings subjected to the different light-growth regimes was analyzed in vivo by fluorescence emission measurements. Light fast-kinetic response curves have been performed to assess if the capability of photosynthetic apparatus in converting the light energy to photosystems may be affected by light quality. Ft showed an increase according to the increasing irradiation in all seedlings, irrespective of the specific light quality treatment. However, in W seedlings the Ft values were significantly higher (P < 0.01) than in R and RB seedlings not only at growth irradiance of 200 µ mol photons m−2 s−1 (see the bar plots inside the graph) but in the whole range of PPFD tested (Fig. 2a). On the other hand, the photochemical quenching, qP, exhibited a progressive reduction as irradiation increased. At growth irradiance, qP was higher (P < 0.05) in R and RB than in W seedlings (Fig. 2b). The decline in photochemical quenching with photon flux density increase was accompanied by the rise of non-photochemical quenching (qN) (Fig. 3c). RB seedlings showed the highest values (P < 0.01) of qN compared to W and R in the whole range of PPFD, including the plant growth irradiance (Fig. 2c). The progressive increase of Ft together with lower qP indicated a loss of photosynthetic efficiency in W compared to R and RB seedlings. It has been demonstrated that the reduction of photochemical reactions leads to the depletion of PSII reaction center function (Maxwell et al., 1994; Wang et al., 2009), resulting in decline of plant productivity. Generally, the decrease in photochemistry is balanced by the rise of non-photochemical quenching that acts as compensation mechanism to avoid an over excitation of photosynthetic electron transport chain (Maxwell and Johnson 2000). This is not the case or V. radiata plants exposed to white light, in which the lowest values of qN indicates a reduced capacity to dissipate thermally the excess of light energy to photosystems. On the other hand, the opposite common behavior of R and RB seedlings (higher qP values) confirms the positive role of R and B wavelengths on light reactions of photosynthesis. In previous studies, highest values of photochemical quenching were found in tomato seedlings grown under RB condition compared to white and pure red light (Liu

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Fig. 2 Fast kinetics light-response curves (from 125 to 1500 µ mol photons m−2 s−1 ) of Ft - basal fluorescence in the light (a), qP-photochemical quenching (b), qN - non-photochemical quenching (c) of Vigna radiata L. seedlings grown under three different light quality regimens: white (W), Red (R) and Red-Blue (RB). The bar plot diagram enclose in each fast kinetics light-response curve represents the value of the parameter at growth irradiance in chamber of 200 µ mol photons m−2 s−1 . Each point is the mean ± SE (n=5). Different letters indicate significant differences among light treatments (P < 0.05).

et al., 2011). Conversely to other species, such as Cucumis sativus and Acacia mangium (Wang et al., 2009; Yu and Ong, 2003), the growth under pure red did not induce a decline of qP compared to white light in V. radiata seedlings, highlighting the species-specific response to light quality. In the specific case of V. radiata seedlings, the growth under RB wavelengths stimulates the thermal dissipation mechanisms not reducing the photochemical reactions. This could be a favorable trait because it makes RB seedlings stronger against photoinhibitory damage risks. It is well known that an increase of qN may occur as photoprotective process against light-induced damages (Hong et al., 1999; Maxwell and Johnson, 2000).


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Table 1 Content of Chl a and b, chlorophyll a/b ratio, content of total carotenoids (x+c) in Vigna radiata L. seedlings under the three different light quality treatments: White (W), Red (R) and Red-Blue (RB). Each value represents the mean ± SE (n = 5). Different letters indicate significant differences between light treatments (P < 0.05). Chl a [µ g cm−2 ]

Chl b [µ g cm−2 ]

White (W)

16.7 ± 0.52 a

Red (R )

15.41 ± 1.01 a

Red-Blue (RB)

6.62 ± 1.57 b

Light

x+c[µ g cm−2 ]

Chl a/b ratio

11.42 ± 0.61 a

2.85 ± 0.37 a

1.46 ± 0.13 a

10.05 ± 0.47 a

4.04 ± 0.98 b

1.46 ± 0.05 a

4.98 ± 1.02 b

2.15 ± 0.11 c

1.32 ± 0.01 b

Fig. 3 Total protein content in Vigna radiata L. plants grown under the three different light quality treatments: White (W), Red (R) and Red-Blue (RB). Each value represents the mean ± SE (n = 5) and is referred to a BSA (albumin serum bovine) standard curve, at the wavelength of 595nm. Different letters indicate significantly different values (P < 0.05).

3.3

Effect of light quality on pigments and total proteins

The different light quality treatments during growth significantly affected the content of photosynthetic pigments in V. radiata seedlings (Table 1). Compared to W and R, RB seedlings showed lower values (P < 0.05) of chlorophyll a and b and total carotenoid content as well as Chl a/b ratio. Compared to W, seedlings developed under pure R exhibited similar values for chlorophyll content and chlorophyll a/b ratio and higher (P < 0.01) total carotenoid (x+c) content (Table 1). The higher amount of chlorophylls and carotenoids in R than RB plants together with the higher values of leaf area and stem elongation, compared to W seedlings, may be interpreted as a strategy to optimize the light harvesting by photosynthetic apparatus under limiting light conditions. Our hypothesis is in agreement with data reporting that an increase in photosynthetic pigment synthesis is often associated with an increase in leaf lamina expansion to improve light interception (Kubota et al., 2012). In W plants the high pigment content was not accompanied by an increase in stem elongation and leaf area, probably because the mix of different wavelengths is more suitable to guarantee the right amount of light to photosystems reducing the need for allocating biomass in above-ground organs. The lowest (P < 0.05) chlorophyll a/b ratio in RB seedlings is indicator of a different acclimation to light in these plants when the red wavelengths are supplemented with blue. This parameter may be considered as a “bioassay for the light environment of a plant” (Dale and Causton, 1992). In our experiment the significant reduction of the chlorophyll a/b ratio under RB treatment compared to W and R, indicating a simultaneous decrease of both chlorophyll a and b; this may represent a signal that the light at leaf lamina is not limiting for photochemical reactions in RB seedlings. Regarding the protein content, results from the Bradford assay indicated that the highest amount was found in RB seedlings which showed significantly higher values (p < 0.05) than R and W seedlings (Fig. 3). This is in agreement with previous experiments showing that the blue wavelengths promote protein synthesis and accumulation (Zhang et al., 2010; Li et al., 2012). The stimulatory effect on proteins played by RB conditions makes this light regime interesting to induce a valuable trait in V. radiate seedlings.

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4 Conclusions The different light quality significantly influenced specific traits in V. radiata seedlings. The increasing in root growth under RB light, and the rise in stem elongation under R light, lead to higher values of total dry weight in RB and R seedlings compared to W seedlings. Moreover, the development of plants under the mix of RB lights, induced a more compact structure, very helpful in the sight of its cultivation in growth chambers when volume availability is a constrain. When blue wavelengths are mixed with R ones, not only the capability of photosynthetic apparatus to use the light energy is enhanced but also the thermal dissipation mechanisms are promoted in V. radiata. From the eco-physiological point of view, this trait is important to give these plants an intrinsic resistance against photoinhibition risks. Finally, compared to W and R treatments, RB light regime enhances the content of total proteins. This outcome could be very important from a nutraceutical point of view. However, further analyses are needed, also considering light quality effects on edible organs also in other species. The overall results indicate that it is possible to use specific light fertilization treatments as a mean to drive crop growth and development, thus obtaining desirable traits in plants. The application of different light spectral composition may complement and/or reduce the utilization of other growth-promoting factors. However, these outcomes seem to be highly depending on the species and this aspect has to be adequately considered.

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Capture Rate of Selected Heavy Metals In Q. Ilex L. Leaves Collected At Two Sites With Different Land Uses F. Esposito†, V. Memoli, S. C. Panico, A. De Marco, G. Maisto Dipartimento di Biologia, Universit degli Studi di Napoli Federico II, Via Cinthia, 80126 Napoli, Italy Submission Info Communicated by Pier Paolo Franzese Received 17 April 2018 Accepted 30 September 2018 Available online 1 January 2019 Keywords Metal capture rate Urban area Industrial area Leaf deposit

Abstract Anthropization causes an increase of pollutants in the atmosphere that, in turn, leads to a decline of air quality. Leaves from selected tree species are useful tools to evaluate air quality as they intercept air deposition and accumulate, through stomata, pollutants in gaseous form or in fine particulate. However, leaf morphology and biochemical characteristics may be negatively affected by air pollution. The aims of the study were: i) to evaluate the concentrations of Cd, Cr, Cu, Ni and Pb in leaves and deposit on them in specimens of Q. ilex L., widely used as biomonitor; ii) to estimate the relationships between metal accumulation and morphological leaf traits (length, width, petiole length, leaf area) in two municipalities: Pomigliano (ME) and Naples (UE), respectively, characterized by mixed (urban and industrial) and urban environments. At both site typologies, the investigated metals, with the exception of Cd, were accumulated in leaf deposits, as their concentrations were higher in unwashed than washed. The comparison of the metal concentrations in deposits on leaves collected at the two site typologies highlighted that for Pb values were statistically different with concentrations higher at ME. Instead, the leaves widely differed for metal composition, with statistically higher values of Cd at UE. All the metal concentrations exceeded the chemical fingerprint, in particular Pb and Cd respectively in mixed and urban environments. Besides, the metal capture rate, an estimation of the adsorbed or captured heavy metals on the leaf surface respect to the total concentration, showed statistically lower values for Cu and Ni in leaves collected at ME, suggesting the consistent presence of fine particulate. Finally, metal accumulation in leaves collected at ME was linked to leaf morphology as leaf traits showed values lower than in leaves collected at UE. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction Heavy metals are the main contaminants in human impacted areas (Gu et al., 2016). In fact, they are emitted to the atmosphere by industrial, agricultural and urban activities. For instance, paintings, exhausted batteries, industrial wastes and vehicular emission are considered the main emission sources for Pb; Cr is produced by † Corresponding


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different agricultural and industrial activities as well as, by lubricants, which are antiwear protectants for vehicles; Cd, Ni and Cu derive by oils, pneumatics and old car remains (Nagajyoti et al., 2010; Malizia et al., 2012; Iodice et al., 2016). Heavy metals are released to the atmosphere in gaseous and liquid forms or as particulate matter of different sizes, depending on the prevailing combustion or manufacturing processes (Sawidis et al., 2011). As they cause a decrease of air quality, their concentrations should be under some threshold values in order to preserve environment quality and human health (Du et al., 2013). For these reasons, monitoring programs of air quality at brief- and long-term are required. In the last decades, the degree of inorganic and organic pollution has been evaluated through selected plants (Maisto et al., 2004; De Nicola et al., 2014, 2017), as their leaves can intercept air particulate and accumulate pollutants. Besides, the use of evergreen species provides many advantages in biomonitoring surveys for the high spatial and temporal resolution due to the excellent availability of plants and low sampling costs (Sawidis et al., 2011). In the Mediterranean Region, Quercus ilex L. is a widespread evergreen species often also used as ornamental plant in urban area and with well-proven capability to monitor air quality (Alfani et al., 2000; Maisto et al., 2013; Ugolini et al., 2013). However, metal uptake and accumulation can cause changes in leaf structure, morphology and physiology (Shi and Cai, 2009; Arena et al., 2014, 2017). The aims of this research were: i) to evaluate the concentrations of Cd, Cr, Cu, Ni and Pb in unwashed and washed leaf tissue of Q. ilex leaves collected at two municipalities, respectively, characterized by mixed (urban and industrial, Pomigliano D’Arco) and urban environments (Naples); ii) to estimate the relationships between metal accumulation and morphological leaf traits (length, width, petiole length, area).


Study area and sampling

The samplings were carried out on March 2017 at three sites inside each area distinguished by different emissions in atmosphere: the municipality of Naples (total area: 6.2 km2 ; geographical coordinates of the sites: 40◦ 49’21.99”N - 14◦ 11’39.00”E; 40◦ 50’1.09”N - 14◦ 11’47.19”E; 40◦ 50’11.51”N - 14◦ 10’59.15”E) and the municipality of Pomigliano d’Arco (total area: 11.44 km2 ; geographical coordinates of the sites: 40◦ 54’44.42”N - 14◦ 23’52.25”E; 40◦ 54’49.50”N - 14 ˚ 23’50.70”E; 40◦ 54’14.97”N - 14◦ 23’57.24”E) in the surrounding of Naples. They were characterized by urban (UE) and mixed (urban and industrial, ME) environments, respectively. At each site, Q. ilex L. branches located 2-4 m above the ground, from the outer part of the canopies and from the four sides of the tree (N, S, E, W), were cut by pruning shears of eight trees. In order to obtain a homogeneous sample, 70-90 two-year-old leaves were taken by hand from the branches, taking care to minimise contact with the leaf surface. A composite sample of all leaves from the eight trees was subsequently divided into three groups for each site. 2.2

Heavy metal analyses in leaf samples

One group of leaves (15 g) was pre-treated for the direct measurement (unwashed leaves, UL) of element concentrations as below described. The other group of leaves (15 g) was washed by 4 consecutive shakings (each lasting 20 min) in distilled water (1:1.5 = w:v) on a table shaker, in order to mechanically remove the airborne particulate from the leaf surface (washed leaves, WL). The UL samples provide information about the element concentrations in the leaf tissue and air particulate deposited on leaf surface; WL samples provide information about the element concentrations hold in leaf tissue and fine un-removed particulate after water washing. The samples were oven dried (75 ◦ C), homogenised through pulverization (Fritsch pulverisette) and the powder used to prepare 3 replicates. A full description of the method used for the heavy metal analyses in leaf samples appears in Alfani et al. (2000). Briefly, the element concentrations in leaves were measured after digestion with a mixture of HF (50%) and HNO3 (65%) at a ratio of 1:2 (v:v) in a microwave oven (Milestone mls 1200 -


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Microwave Laboratory Systems) on oven-dried leaves (75 ◦ C, until constant weight) and pulverized samples. The concentrations of Cd, Cr, Cu, Ni and Pb were measured by atomic absorption spectrometry (SpectrAA 220, Varian) via graphite furnace. In order to ascertain the accuracy of the method employed, a concurrent analysis of reference materials was carried out (BCR No. 62 from the Community Bureau of Reference from the Commission of the European Communities): the recovery ranged from 86 to 99%, depending on each analysed element. 2.3

Capture rate

To separate the contributions of the adsorbed and captured heavy metals, the capture rate (CR) was used. The CR was calculated using the following equation (Liang et al., 2017): CR = (

CUL −CW L ) × 100 CUL

(1)

where CUL and CW L are the concentrations of the metals in unwashed and washed leaves, respectively. 2.4

Leaf functional traits

Expanded leaves, without damages at the lamina were chosen for determination of area, length, width, and petiole length. The length, width, petiole length and leaf area were determined using the program Image J 1.45 (Image Analysis Software). For each site, three replicates on ten leaves were performed. 2.5

Statistical analyses

To assess the normality of the distribution of the data set, the Shapiro-Wilk test was performed. The two-way analysis of variance (ANOVA), followed by the post hoc test of Holm-Sidak, was performed in order to evaluate the differences in leaf heavy metal concentrations according to different treatments (UL and WL), site typologies (UE and ME) and their interactions. A paired t-test was performed in order to evaluate the differences among the site in capture rate and the leaf traits. Spearman test, as the data showed a non-normal distribution, was performed in order to evaluate the relationships between the leaf chemical composition and leaf traits. The statistical assays, performed by Systat SigmaPlot 12.2 software (Jandel Scientific, USA), were considered statistically significant for P < 0.05.

3 Results The element contents in unwashed (UL) and washed (WL) leaves are showed in Fig 1. In the holm oak leaves, at both sites, Cu was the most abundant element, with mean values approximately of 21.8 and 13.5 µ g g−1 d.w., respectively, in UL and WL; instead, Cd was the less abundant with mean values of 0.07 and 0.1 µ g g−1 d.w. in UL and WL, respectively (Fig. 1). The comparisons between heavy metal concentrations in UL and WL highlighted that, with the exception of Cd, higher values were detected in UL (Fig. 1) with values statistically significant for Cr and Cu at both sites, for Ni at UE and Pb at ME (Fig. 1). Besides, Cd concentrations in WL were statistically higher than those in UL at UE (Fig. 1). The concentrations of Cr, Cu and Ni and Pb in UL and WL leaves did not statistically differ between the sites. Instead, Cd concentrations in UL and WL were statistically higher at UE (Fig. 1). The highest values of capture rate (CR) were observed for Ni at UE, whereas the lowest were observed for Cu at ME (Table 1). The CRs for Cr and Pb were similar at both sites, whereas those for Cu and Ni were statistically higher at UE compared to ME, the mixed environment (Table 1).

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Fig. 1 Mean values (± s.e.) of metal concentrations in Q. ilex L. unwashed (UL, oblique pattern bars) and water washed (WL, empty bars) leaves collected at sites characterized by mixed, ME (grey), and urban, UE (white), environments. Asterisks indicate statistically significant differences (two-way analysis of variance, ANOVA, P < 0.05) between leaf treatments inside each site typology; different letters indicate statistically significant differences (two-way analysis of variance, ANOVA, P < 0.05) inside the same leaf treatment between the site typologies.

Table 1 Mean values of capture rate (± s.e.) of Cr, Cu, Ni and Pb calculated for Q. ilex L. leaves collected at sites characterized by mixed (ME) and urban (UE) environments. Different letters indicate statistically significant differences (Paired t-test, P < 0.05) between the site typologies. Cr ME UE

Cu

Ni

Pb

64.0a

25.7b

61.1b

61.5a

(±2.06)

(±5.21)

(±9.61)

(±10.5)

63.8a

46.1a

89.7a

52.1a

(±2.59)

(±4.73)

(±1.16)

(±7.50)

Table 2 Coefficients of Spearman’s correlations performed between metal concentrations in washed leaves and leaf traits. Asterisks indicate the levels of significance (*P < 0.05; **P < 0.01).

Leaf length

Cr

Cu

Ni

Pb

-0.155

-0.622*

-0.481

-0.664*

Leaf width

-0.198

-0.650*

-0.580*

-0.664*

Leaf area

-0.113

-0.777**

-0.608**

-0.806**

Petiole length

-0.057

-0.777**

-0.735**

-0.763**

Morphological traits of leaves collected at ME had significantly lower values than those of leaves collected at UE (Fig 2). An overall evaluation highlighted negative relations between element concentrations in WL and leaf traits with statistically significant values found for Cr, Cu and Ni (Table 2). By contrast, no statistically significant relations were found between element concentrations in UL and leaf traits.


309

Fig. 2 Box plot of values of leaf length and width, petiole length and leaf area of Q. ilex L. leaves collected at sites characterized by mixed, ME (grey), and urban, UE (white), environment. The boxes indicate the 25th and 75th percentiles, the continuous lines indicate the median values and the upper and lower whiskers indicate, respectively, the maximum and minimum values of the dataset. The asterisks indicate statistically significant differences (Paired t-test, P < 0.05) in the leaf traits between the site typologies.

4 Discussion The different content of elements in unwashed and washed leaves at the investigated sites suggest different associations of elements to air particulate of various sizes likely depending on prevalent source emission. In particular, Pb, Cr and Cu seem mainly to be present, at both the sites, in soluble forms (Alfani et al., 2000; Morselli et al., 2003; Tomaˇsević et al., 2005; Sawidis et al., 2011), as water washing reduced the metal content, likely by removing a fraction of the particulate matter deposited on leaf surfaces. For Ni a different behaviour was observed; in fact, at ME the washing reduced the metal of about 50%, while at UE it dropped near to zero, suggesting that in this site Ni was prevalently associated to washable PM. In addition, Ni at ME and Pb at UE would seem to be scarcely present in soluble forms but mainly in the fine particulate matter; moreover, it can be supposed that PM could be trapped in leaf cuticular layer (Deljanin et al., 2016) or among leaf hairs. A different composition of particulate matter in the two site typologies cannot be excluded. In fact, the capture rate (CR) of Cu and Ni was statistically higher for leaves collected at UE than at ME, although the concentrations of these metals in WL leaves did not statistically differ between the sites. Likely, at the area with typical urban environment, Cu and Ni were mainly associated to coarse particles, deposited on leaf surface, as they can be easily removed by water washing. Conversely, at the area with mixed environment, Cu and Ni could be bound to fine or ultrafine particles that can accumulate in cuticular waxes (Deljanin et al., 2016) or enter in leaf tissue through stomata (Song et al., 2015). This hypothesis is corroborated by the assumption that industries represent the third most important contributor for PM2.5 in atmosphere (Belis et al., 2013). Cadmium showed behaviour completely different from those observed for the other investigated metals. In fact, the higher concentrations measured in WL than in UL suggest the scarce presence of Cd in the deposit on the leaf surface that does not allow highlighting differences between the two subsamples of leaves (WL and UL). Alternatively, water washing could not be a recommendable practice to remove this element from leaves (Ugolini et al., 2013). In addition, it is widely reported that Cd has high water-solubility and mobility in soils and therefore it can be easily uptaken by roots (De Nicola et al., 2008) and sometimes absorbed in place of Zn

310


(Baldantoni et al., 2014). Anyway, likely this pathway scarcely occurred in the investigated areas as Cd in leaves would seem to be mainly associated to small air particles, trapped in leaf cuticular layer or among leaf hairs. As the metal concentrations exceeded the chemical fingerprint (Bargagli, 1998), it can be supposed that the investigated sites were metal polluted. In particular, Cd at UE showed values 2-fold higher than the chemical fingerprint, and Pb showed values 6- and 4-fold higher, respectively, at ME and UE. Although the water washing drastically decreased the concentrations of Cd and Pb, these metals still exceeded the chemical fingerprint (Bargagli, 1998), suggesting accumulation of these elements in leaves. According to leaf morphology, it can be supposed that ME more than UE presented an environment with stress conditions, as narrower, shorter and smaller leaves were observed as compared to the leaves collected at UE, where also shorter leaf petioles were observed (Seyyednejad et al., 2011). This supposition is corroborated by the negative correlations between leaf morphological traits and heavy metal concentrations. A reduction of leaf size may be a plant mechanism to survive under environmental pressures; in fact, plants exposed to heavy metals for long period showed inhibition of metabolic activity, causing growth decrease and slower development (Molas, 1997; Ambo-Rappe et al., 2011). This is in agreement with Arena et al. (2014) who reported a reduction in leaf lamina expansion for holm oak specimens grown in polluted area as compared to the unpolluted one.

5 Conclusion Metal pollution likely occurred at the investigated sites as the metal concentrations in Q.ilex L. leaves exceeded the chemical fingerprint, although different chemical forms of the metals can be supposed. In particular, Pb, Cr and Cu seem mainly to be present, at both the sites, in soluble forms. Instead, Cd could be present in gaseous forms, as it would seem to be directly uptaken by leaves, being absent in leaf deposit. Besides, at the area with typical urban environment, Cu and Ni were mainly associated to coarse particles deposited on leaf surface; conversely, at the area with mixed environment, Cu and Ni could be bound to fine or ultrafine particles. Finally, in mixed environment, metal accumulation in leaves and leaf morphology showed a negative relation, as the investigated leaf traits showed values lower than in the leaves collected at the area with typical urban environment.

Acknowledgments The research was funded by MonAir Project (Monitoraggio dell’aria del Comune di Pomigliano d’Arco, NA) and by the Department of Biology of the University of Naples Federico II (Italy).

References Ambo-Rappe, R., Lajus, D.L., and Schreider, M.J. (2011), Heavy metal impact on growth and leaf asymmetry of seagrass, Halophila ovalis, Journal of Environmental Chemistry and Ecotoxicology, 3, 149-159. Alfani, A., Baldantoni, D., Maisto, G., Bartoli, G., and Virzo De Santo, A. (2000), Temporal and spatial variation in C, N, S and trace element contents in the leaves of Quercus ilex within the urban area of Naples, Environmental Pollution, 109, 119-129. Arena, C., De Maio, A., De Nicola, F., Santorufo, L., Vitale, L., and Maisto, G. (2014), Assessment of eco-physiological performance of Quercus ilex L. leaves in urban area by an integrated approach, Water, Air & Soil Pollution, 225, 18241835. Arena, C., Santorufo, L., Cataletto, P.R., Memoli, V., Scudiero, R., and Maisto, G. (2017), Eco-physiological and antioxidant responses of holm oak (Quercus ilex L.) leaves to Cd and Pb, Water, Air, & Soil Pollution, 228, 459-471. Baldantoni, D., Cicatelli, A., Bellino, A., and Castiglione, S. (2014), Different behaviours in phytoremediation capacity of two heavy metal tolerant poplar clones in relation to iron and other trace elements, Journal of Environmental


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Management, 146, 94-99. Bargagli, R. (1998), Trace elements in terrestrial plants. (ed R. Bargagli), Springer-Verlag, Berlin. Belis, C.A., Karagulian, F.B., Larsen, R., and Hopke, P.K. (2013), Critical review and meta-analysis of ambient particulate matter source apportionment using receptor models in Europe, Atmospheric Environment, 69, 94-108. De Nicola, F., Maisto, G., Prati, M.V., and Alfani, A. (2008), Leaf accumulation of trace elements and polycyclic aromatic hydrocarbons (PAHs) in Quercus ilex L, Environmental Pollution, 153, 376-383. De Nicola, F., Alfani, A., and Maisto, G. (2014), Polycyclic aromatic hydrocarbon contamination in an urban area assessed by Q. ilex leaves and soil, Environmental Science and Pollution Research, 21(12), 7616-7623. De Nicola, F., Baldantoni, D, Maisto, G., and Alfani, A. (2017), Heavy metal and polycyclic aromatic hydrocarbon concentrations in Quercus ilex L. leaves fit a priori subdivision in site typologies based on human management, Environmental Science and Pollution Research, 24, 11911-11918. Deljanin, I., Antanasijević, D., Bjelajac, A., Aniˇcić Uroˇsević, M., Nikolić, M., Perić-Grujić, A., and Ristić, M. (2016), Chemometrics in biomonitoring: distribution and correlation of trace elements in tree leaves, Science of the Total Environment, 545-546, 361-371. Du, Y., Gao, B., Zhou, H., Ju, X., Hao, H., and Yin, S. (2013), Health risk assessment of heavy metals in road dusts in urban parks of Beijing, China, Procedia Environmental Sciences, 18, 299-309. Gu, Y.G., Gao, Y.P., and Lin, Q. (2016), Contamination, bioaccessibility and human health risk of heavy metals in exposedlawn soils from 28 urban parks in southern China’s largest city, Guangzhou, Applied Geochemistry, 67, 52-58. Iodice, P., Adamo, P., Capozzi, F., Di Palma, A., Senatore, A., Spagnuolo, V., and Giordano, S. (2016), Air pollution monitoring using emission inventories combined with the moss bag approach, Science of The Total environment, 541, 1410-1419. Liang, J., Fang, H.L., Zhang, T.L., Wang, X.X., and Liu. Y.D. (2017), Heavy metal in leaves of twelve plant species from seven different areas in Shanghai, China Urban Forestry & Urban Greening, 27, 390-398. Maisto, G., Alfani, A., Baldantoni, D., De Marco, A., and Virzo De Santo, A. (2004), Trace metals in the soil and in Quercus ilex L. leaves at anthropic and remote sites of the Campania Region of Italy, Geoderma, 122, 269-279. Maisto, G., Baldantoni, D., De Marco, A., Alfani, A., and Virzo De Santo, A. (2013), Ranges of nutrient concentrations in Quercus ilex leaves at natural and urban sites, Journal of Plant Nutrition and Soil Science, 176, 801-808. Molas, J. (1997), Changes in morphological and anatomical structure of cabbage (Brassica oleracea L.) outer leaves and in ultrastructure of their chloroplasts caused by an in vitro excess of nickel, Photosynthetica, 34, 513-522. Malizia, D., Giuliano, A., Ortaggi, G., and Masotti, A. (2012), Common plants as alternative analytical tools to monitor heavy metals in soil, Chemistry Central Journal, 6(2), 1-10. Morselli, L., Olivieri, P., Brusori, B., and Passarini, F. (2003), Soluble and insoluble fractions of heavy metals in wet and dry atmospheric depositions in Bologna, Italy, Environmental Pollution, 124(3), 457-469. Nagajyoti, P.C., Lee, K.D., and Sreekanth, T.V.M. (2010), Heavy metals, occurrence and toxicity for plants: a review, Environmental Chemistry Letters, 8 199-216. Sawidis, T., Breuste, J., Mitrovic, M., Pavlovic, P., and Tsigaridas, K. (2011), Trees as bioindicator of heavy metal pollution in three European cities, Environmental Pollution, 159(12), 3560-3570. Seyyednejad, S.M., Niknejad, M., and Koochak, H. (2011), A review of some different effects of air pollution on plants, Journal of Environmental Science, 10, 302-309. Shi, G. and Cai, Q. (2009), Leaf plasticity in peanut (Arachis hypogaea L.) in response to heavy metal stress, Environmental and Experimental Botany, 67, 112-117. Song, Y., Maher, B.A., Li, F., Wang, X., Sun, X., and Zhang, H. (2015), Particulate matter deposited on leaf of five evergreen species in Beijing, China: Source identification and size distribution, Atmospheric Environment, 105, 53-60. Tomaˇsević, M., Vukmirović, Z., Rajˇsić, S., Tasić, M., and Stevanović, B. (2005), Characterization of trace metal particles deposited on some deciduous tree leaves in an urban area, Chemosphere, 61(6), 753-760. Ugolini, F., Tognetti, R., Raschi, A., and Bacci, L. (2013), Quercus ilex L. as bioaccumulator for heavy metals in urban areas: Effectiveness of leaf washing with distilled water and considerations on the trees distance from traffic, Urban Forestry & Urban Greening, 12, 576-584.

Journal of Environmental Accounting and Management 6(4) (2018) 313-324 Volume 1 Issue 1 March 2013




Impact of Biochar Amendment on Soil Quality and Crop Yield in a Greenhouse Environment Rossana Marzaioli1†, Elio Coppola1 , Paola Iovieno2, Alfonso Pentangelo2 , Catello Pane2 , Flora Angela Rutigliano1 1

Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università degli Studi della Campania “Luigi Vanvitelli”, Caserta 81100, Italy 2 Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria – Centro di ricerca Orticoltura e Florovivaismo, Pontecagnano 84091, Italy

Submission Info Communicated by Sergio Ulgiati Received 17 April 2018 Accepted 30 September 2018 Available online 1 January 2019 Keywords Biochar Greenhouse agriculture Crop yield Microbial biomass Microbial activity Rhizoctonia solani Suppressiveness

Abstract Greenhouse agriculture, a widespread practice in the Mediterranean basin, is prone to impoverishment in soil organic carbon because of crop removal and high decomposition rate. Open-field experimentation has shown that the addition of biochar, a product of thermochemical conversion of biomass, under a limited concentration of oxygen, increases the soil organic C pool, enhances crop productivity and improves C terrestrial sink. The present study investigates the effect of biochar amendment in a greenhouse environment in a Southern Italy organic farm. Two doses (10 or 20 t ha−1 ) of biochar from conifer pruning wastes were applied immediately before planting 1-week old plants of pepper (Capsicum annuum L.). Plant growth and crop yield were evaluated six months later, at the end of cultivation, in biochar-treated and in control (without biochar) plots. Soil samples were collected in the same plots immediately after biochar addition and six months later and were analyzed for the following parameters: bulk density, water-holding capacity, pH, electrical conductivity, organic carbon, mineral nitrogen, total microbial biomass and fungal mycelium contents, soil respiration, nitrogen mineralization, potential nitrification, soil suppressiveness to Rhizoctonia solani. A single biochar application caused no apparent damage to the crop; on the other hand, no improvement was observed in crop yield or soil suppressiveness to R. solani. In contrast, the single char application positively affected soil respiration, nitrogen mineralization and potential nitrification. These preliminary results suggest that soil amendment with biochar is a potentially useful practice in greenhouse agriculture, yet further experimentation is necessary to assess optimal amounts for better crop productivity and soil quality. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

† Corresponding


314

Rossana Marzaioli et al. / Journal of Environmental Accounting and Management 6(4) (2018) 313–324

1 Introduction Greenhouse cultivation is a growing agricultural sector, with about 2 million hectares employed in the world and over 400,000 ha in the Mediterranean Basin, mainly Spain, Italy, France, Greece, and Turkey (ScarasciaMugnozza et al., 2011). Greenhouse cultivation in Italy currently covers an area of about 42,000 ha, mainly in Sicily, Campania, Lazio, Veneto and Liguria Regions (ISTAT, 2010). Of these, 37,000 ha are employed for the production of vegetables and fruit, the rest for cultivation of ornamental plants and flowers (ISTAT, 2010). The expansion of this agricultural sector depends on several advantages offered by greenhouse cultivation. Controlled microclimatic conditions, relatively low building costs and high crop yields throughout the year (also thanks to pest control) afford farmers high net earnings (Lamont, 2005). On the other hand, this type of cultivation entails a steady decline in soil quality due to organic matter loss; this depends on high decomposition rate, due to optimal temperature and water conditions, and continuous removal of crop residues (Bonanomi et al., 2011). The reduction in organic matter content negatively impacts on other soil properties, such as waterholding capacity, cation-exchange capacity, nutrient content (Karlen et al., 1997) and, consequently, on soil microbial growth and activity (Bonanomi et al., 2014). An increase in soil salinity is also frequent because of unbalance between upward movement of soil water (transporting salts to the soil surface), caused by high evapotranspiration, and water input by irrigation (Chen et al., 2004). Maintaining and/or improving the pool of soil organic matter is crucial for sustainable agriculture (Vanlauwe et al., 2010), as this parameter affects fundamental soil services such as nutrient cycling and carbon sequestration (Adhikari and Hartemink, 2016). The application of organic amendments, such as compost, is a reliable and effective practice to increase soil organic carbon and consequently soil fertility and crop yield (Smith et al., 1997). However, accelerated decomposition in greenhouse conditions demands high doses and repeated applications of organic amendments, and, consequently, increased costs and carbon dioxide emissions (Kaur et al., 2008). A particularly stable organic amendment is carbonized material known as “biochar”, obtained by thermochemical conversion of biomass (such as plant wastes) in an oxygen-limited environment (IBI, 2013). Biochar addition to soil has been reported to produce increased plant growth and higher yields (Asai et al., 2009; Vaccari et al., 2011, 2015). Moreover, biochar was found to improve plant resistance to pathogens by stimulating antagonistic microflora and/or introducing biochar-associated organic antifungal compounds (De Corato et al., 2015). Biochar improves long-term soil fertility by increasing the pH and enhancing the water-holding capacity, bulk density and micro-aeration, as well as the cation-exchange capacity, nutrient retention, and organic-matter adsorption (Lehmann et al., 2006; Laird et al., 2010). Biochar addition may also positively affect the soil microbial community, which is fundamental for the soil functioning and ecosystem service provisioning (Nannipieri et al., 2012; Schmidt et al., 2014). Because of its molecular structure dominated by aromatic C, biochar is much more resistant to microbial decomposition (from 1,000 to 10,000 years; Warnock et al., 2007) than uncharred organic matter (Baldock and Smernik, 2002), therefore biochar employment for soil amendment is considered a promising way of increasing the terrestrial C sink. Biochar treatment also reduces emission of greenhouse gas N2 O (Castaldi et al., 2011; Xu et al., 2014). Moreover, biochar production from organic wastes for use in agriculture is an effective strategy for waste disposal, with positive effects on economy and environment (Raveendran et al., 1995; Nik-Azar et al., 1997). Biochar application has been reported to have either positive or negative effects on soil microbial community, depending of its characteristics. An increase in total microbial biomass due to biochar treatment probably depends on the release of organic and inorganic nutrients (Lehmann et al., 2011). Moreover, the microporous structure of biochar is thought to provide microbes with a favorable habitat (Thies and Rillig, 2009) and a refuge from predators (Warnock et al., 2007). On the other hand, the high pH and the abundance of aromatic compounds of biochar could negatively affect the soil microbial community, in particular the fungal populations (Warnock et al., 2010). The effects of biochar application in open-field crops have been investigated quite extensively (Steiner et al., 2008; Asai et al., 2009; Vaccari et al., 2011; Rutigliano et al., 2014); several studies have been conducted on the


315

use of biochar as a substrate for pot plants (Baronti et al., 2010; 2014; Deenik et al. 2010; Gartler et al., 2013; Smider and Singh, 2014; Xu et al., 2014), but as far as we are aware there is no report on the effects of in-situ biochar addition to greenhouse soil. The Italian legislation (D.L.vo 75/2010, as modified in 2015) has recently included biochar in the list of amendments approved in agriculture. In contrast to the considerable body of information on the use biochar amendment in open field, as of yet the general effects and dosage application of biochar in greenhouse environment have not been directly tested. The aim of the present study was i) to verify the absence of negative effects of biochar on crops and soil and ii) to assess benefits from biochar addition on crop yield and soil quality in a greenhouse environment. In combination with chemical and physical soil properties, we investigated microbial biomass and activity because the soil microflora is a highly dynamic soil component that reacts to change of environmental conditions long before chemical parameters, such as organic C or total N content (Powlson et al., 1987; Jørgensen and Emmerling, 2006).

2 Material and methods 2.1

Study area

The study was carried out in 2016 in a large greenhouse belonging to an organic farm located in Eboli (Southern Italy). The soil present in the greenhouse is classified as Vertic Calcisols, with clay texture (Scotti et al., 2016). Soil physical and chemical properties were reported by Scotti et al. (2016). Before the greenhouse was built, in 2009, the area used to be an orchard. Until 2014, the greenhouse has been employed for cultivation of cabbage and lettuce in winter, melon and watermelon in summer. Before annual cultivation cycle (generally in June), the soil was amended with a compost produced in the same farm from cultural residues (as described by Scotti et al., 2016), worked up to 50 cm depth and solarized. Soil solarization was realized bringing soil to field capacity with 90 m3 ha−1 of water and covering it with a plastic film of polyethylene from June to all August. Soil temperature reached 60-65 ◦ C in the first 10 cm of depth. Sub-alkaline well water (pH = 7.7-8.0) was used for irrigation. In line with organic farming rules, no pesticides were applied to the crops. The greenhouse had not been in use for two years before the present study. 2.2

Experimental design

The biochar experiment was carried out following an experimental design with randomized blocks, defined by three tunnels, each 40 m in length and 2.4 m in width. Each tunnel was divided into three 10-m-long plots that were treated with biochar in amounts equivalent to 10 t ha−1 (B10), 20 t ha−1 (B20), 0 t ha−1 (control). A strip of about 2.5 m was left between neighboring plots to avoid interference between treatments. Before biochar treatment (15 April 2016), the soil underwent milling; sprinkler irrigation (5 minutes) was applied immediately after biochar addition, followed by new mild vertical milling to incorporate the biochar in the soil. The following day (16 April 2016), one-week-old pepper seedlings (Capsicum annuum L., Kaptur F1 variety from Monsanto vegetable seeds) from a nursery were planted in the experimental plots. During pepper cultivation, 12 treatments with the organic liquid fertilizer BIO ENERGY (Biolchim) were applied by fertigation every ten days at rate of 30 L ha−1 . As reported in a previous study carried out in the same farm (Scotti et al., 2016), the routinely employment of compost fertilization and fertigation by farmers resulted in a mean soil N content of about 1.3 g kg−1 dry soil. The biochar applied (obtained from Agrindustria s.n.c.; Cuneo, Italy) was produced by gasification (1000◦ C), in low oxygen concentration, from spruce wood (2-8 cm) of Northwestern Italy (Bedussi et al., 2015). Characteristics of the resulting biochar (ø ≤4.4 mm) and measurement methods are reported in Table 1.

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2.3


Sampling and analyses

Plant growth was determined by measuring the mean height (cm) of 20 randomly-collected plants from each plot (60 plants for treatment). Harvesting of berries was performed six times in the period July-October, and data were cumulated. Crop yield was evaluated by determining the average number of berries for plant, average weight (g) of a single berry, and total weight (kg) of berries for square meter, assayed on the whole plot ( = 50 plants; 150 plants for treatment). Two soil samplings were carried out: the first immediately after biochar addition and before transplanting the pepper seedlings (time t1h ; 15/04/2016); the second soil sampling was performed immediately after crop collection (14/10/2016; time t6M ). Each time, two soil replicates were collected (15 cm depth) from each plot, thus obtaining 6 replicates for treatment, for a total of 36 samples (2 replicates x 3 plots with the same treatment×3 treatments×2 sampling times). Immediately after sampling, the samples were transferred to the laboratory and sieved (sieve mesh: 2 mm) to remove coarser fragments (roots, stones, etc.). Each sample was then separated into two aliquots; the first was air dried to constant weight for determination of organic carbon content, pH and electrical conductivity, the second was stored at 4 ◦ C for determination of water content, NH+ 4− N and NO3 -N content, microbial biomass, fungal mycelium, potential respiration, nitrogen mineralization and potential nitrification, as well as suppressiveness against Rhizoctonia, a plant pathogen (see below). In addition, an undisturbed soil core (15 cm depth) was collected each time from each plot to assess water-holding capacity and bulk density. Soil water content and water-holding capacity were determined gravimetrically (Allen et al., 1989); bulk density was assayed on the basis of dry weight and volume of soil cores (USDA-NRCS, 2004); total organic C (Corg ) was determined by wet digestion in 0.1667 M of K2 Cr2 O7 (Walkey and Black, 1934; Sleutel et al., 2007); pH and electrical conductivity (EC) were measured (USDA-NRCS, 2004) in 1:2.5 and 1:2 soil/water suspensions, respectively. Microbial biomass was evaluated with the chloroform fumigation-extraction method (Vance et al., 1987): organic carbon extracted with 0.5 M K2 SO4 from fumigated and non-fumigated soil samples was determined by chemical digestion with 0.4 N K2 Cr2 O7 at 160 ◦ C, followed by titration with 0.04 N iron (II) sulphate. Microbial biomass, expressed as microbial carbon (Cmic ), was calculated from the difference between organic carbon of fumigated and non-fumigated soil samples according to the equation by Vance et al. (1987). Fungal mycelium was assayed with the membrane filter technique (Sundman and Sivelä, 1978), evaluating the length of hyphae by the intersection method (Olson, 1950). Potential soil respiration was determined by gas chromatography (Kieft et al., 1998, modified). Soil was pre-incubated in 50-ml vials for 3 days in standard conditions (25 ◦ C and 55% water-holding capacity) in order to eliminate abiotic CO2 . After pre-incubation, the vials were flushed with CO2 -free air for 3 minutes, sealed and incubated in standard conditions for 1 h (Anderson and Domsch, 1978). CO2 evolution was quantified by gas chromatography. N mineralization and potential nitrification were measured as described by Castaldi et al. (2009) on soil samples incubated aerobically for 21 days (60% water-holding capacity, 25 ◦ C, in the dark), with no substrate or with addition of ammonium sulphate (100 mg N g−1 dry soil) to determine net N mineralization and potential nitrification, respectively. After incubation, inorganic nitrogen extracted with 0.5 M K2 SO4 (1:5 soil: extractant ratio) was measured using + selective electrodes for NO− 3 and NH4 (Castaldi et al., 2009). Soil natural suppressiveness against Rhizoctonia solani damping-off on the host plant Raphanus sativus was assessed in microcosm experiments on soil samples from treated plots. Five tray cells per treatment were filled with infected 5-mm sieved soil, with control cells fully filled with healthy uninfected soil. Pathogen inoculum preparation, disease incidence calculation and data elaboration were carried-out as described by Pane et al. (2013). The design included three replications (each with five cells) and the experiment was repeated twice. 2.4

Statistical analysis

Before applying parametric tests, a normality test (Kolmogorov-Smirnov) was performed (SigmaPlot12). The parameters were normalized by log10 transformation when not normally distributed. One-Way ANOVA was


317

Table 1 Mean values (±uncertainties) 1 of physical and chemical characteristics of non-dusty biochar utilized in the present study. Parameters

Reference values

(Methods)

(D.L.vo 75/2010 mod. 2015) ≥ 20, ≤30 (*3) > 30, ≤60 (*2) > 60 (*1)

Organic C (%) (DM 21/12/2000; G.U. n. 21 26/01/2001) Electrical conductivity (mS m−1 ) (DM 17/06/2002; G.U. 220/2002) Water content (%) (DM 24/03/1986) Ash (%) (UNI EN 13039:2012) H/C (molar) pHH2O (DM 17/06/2002; G.U. 220/2002) Elemental analysis (UNI EN 15407:2011) H (%) N (%) O (%) S (%) (as SO3 ; UNI EN 15309:2007)

54.1 (±5.8)2 1065.5 (±19.0)

≤ 1000

8.5 (±0.5)3

≥ 20 (only for dusty material)

0.11 3

> 40, ≤60 (*3) ≥ 10, ≤40 (*2) < 10 (*1) ≤ 0.7

11.52 (±0.34)

4-12

0.48 (±0.08)2 < 0.502

-

13.9 (±1.7)2

-

(±0.32)2

-

7.04 (±0.36)4

1.39

1 uncertainty was calculated with a probability level = 0.95, K = 2; 2 Chelab S.r.l.

from data provided by Chelab

S.r.l; 4 Bedussi

(Mérieux NutriSciences company); 3 calculated et al., 2015; *1, *2, *3: quality classes.

used and integrated, when required, by Student-Newman-Keuls test, to evaluate the significance (P 0 for at least one period t. From this, one can derive the A3 parameter α∗ that guarantees a balanced green budget:

α∗ =

T ∑t=1 ∑z max{0, π − pt }Rzt . T ∑t=1 ∑z Dzt

This is increasing in renewables and decreasing in demand.

(22)

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Alessandro Sapio / Journal of Environmental Accounting and Management 6(4) (2018) 325–333

In other words, when RE generation increases in a zone, keeping the green budget balanced requires a higher α parameter: ∂ α ∗ π − pt + ∑z pt Rzt = ≥0 (23) T ∂ Rst ∑t=1 ∑z Dzt The above results can find an interesting interpretation in light of the merit order effect when markets are integrated. As shown by Ketterer (2014) among others, market integration is one factor weakening the merit order effect pt . Thus, without market integration (that is, with zonal separation), the numerator of the expression for α ∗ would be only π − pst + pst Rst , but pst would be higher (due to a stronger merit order effect). As an implication, the integration of market zones under certain conditions implies a higher unit cost of RE support. This could be seen as a hidden cost of market integration. Since market integration pushes wholesale prices down, more public resources are needed in order to support the green energy sources. On the other hand, integration also neutralises the merit order effect of renewables. Therefore, if this effect is strong enough, the subsidy burden could be relieved by integration.

6 Conclusion The proposed model of RE subsidies shows that the region with higher income and lower RE endowments (the North in Italy) contributes disproportionately to the “green energy budget”. Then, the redistributive effect is emphasised by a support regime that ties the subsidies to wholesale electricity prices, because of the merit order effect associated to renewables, which justifies higher subsidies to RE generators by depressing the electricity price. When regions are physically integrated, one region’s increase in RE production affects the net contribution of the other region as well, through its effect on the national electricity price. Despite such externalities, the North is still contributing more than the South, and any new RE installation in the South implies a burdenshifting effect. However, this pattern - North subsidising the South - is blurred if RE ownership is distributed across zones. Finally, considering a balanced budget rule shows that in the market-based subsidy regime, the unit burden on taxpayers is made heavier by market integration, because it mitigates the national electricity price and hence, more financial resources are absorbed by the subsidy system. These results are relevant with respect to the on-going debate on the decentralization of regional energy policies during the green economy transition. As one learns from the literature on climate change negotiations, international action against climate change involves solving a coordination problem among sovereign entities. This is a challenge nearly as hard even within countries whenever energy policies are decentralized, as with regional energy plans in Italy. Indirect evidence of a need for regional coordination in energy policy actions comes from empirical works. In Sapio (2015), one argues that congestion induced by renewables is not accounted for in local authorization procedures for new RE plants. Gianfreda et al. (2016) propose a similar argument based on a sample of European countries. In light of the results illustrated in this paper, coordination among regions should be aimed at preserving redistribution only if equitable. One way to accomplish this goal could entail differentiated subsidy parameters. Indeed, because of heterogeneity in locations and environmental preferences there may be little reason for uniform support means, as claimed by Lin (2016) based on evidence of spatial misallocation of wind farms in the US. Future research will extend the model to account for dynamic and stochastic relationships. Following Figueiredo et al. (2016), a new extension of the model should account for positive correlation between wind chill and electric heating demand. Due to such correlation, higher subsidies accruing to a region could be partly offset by a higher gross contribution to the green energy budget. Common components in RE generation dynamics across regions would also be interesting to explore, as burden-shifting effects may be blurred in those cases. Secondly, the redistributive patterns studied in this paper should be assessed in light of the environmental benefits implied by intensified investments in green technologies. Even if a region ends up as a net contributor to the green energy budget, its citizens may support incentives to renewables because of non-monetary benefits,

Alessandro Sapio / Journal of Environmental Accounting and Management 6(4) (2018) 325–333

333

such as access to clear air and health safety. A final insight for future research is empirical in nature and involves building a dataset including prices and demand (from GME, the wholesale electricity market operator), tariffs from ARERA (Energy Authority), transmission capacity and renewables from Terna, as well as ownership data.

Acknowledgment The author would like to thank 2 anonymous references, Antoine Mandel, and the audiences in Milan (1st symposium of the Italian Association of Energy Economists, December 2nd 2016) and Turin (seminar at the Department of Economics and Statistics, February 1st 2017) for their useful comments and suggestions. Financial support by Parthenope University of Naples, Bando di sostegno alla ricerca individuale per il triennio 2015-2017, annualita’ 2016, is gratefully acknowledged. Any remaining error is solely the author’s responsibility.

References Anaya, K.L. and Pollitt, M.G. (2015), Integrating distributed generation: Regulation and trends in three leading countries, Energy Policy, 85, 475-486. Borenstein, S. (2012), The private and public economics of renewable electricity generation, The Journal of Economic Perspectives, 26(1), 67-92. Caneppele, S, Riccardi, M., and Standridge, P. (2013), Green energy and black economy: mafia investments in the wind power sector in Italy, Crime, Law and Social Change, 59(3), 319-339. Carmona, R. and Coulon, M. (2014), A survey of commodity markets and structural models for electricity prices, Quantitative Energy Finance Springer New York, 41-83. Clò, S., Cataldi, A., and Zoppoli, P. (2015), The merit-order effect in the Italian power market: The impact of solar and wind generation on national wholesale electricity prices, Energy Policy, 77, 79-88. Figueiredo, N.C., Patra Pereira da, S., and Derek, B. (2016), Weather and market specificities in the regional transmission of renewable energy price effects, Energy, 114, 188-200. Fumagalli, E. (2016), Energy investment: The many lives of energy storage, Nature Energy, 1, 16096. Gianfreda, A, Parisio, L., and Pelagatti, M. (2016), Revisiting long-run relations in power markets with high RES penetration, Energy Policy, 94, 432-445. Joskow, P.L. (2011), Comparing the costs of intermittent and dispatchable electricity generating technologies, The American Economic Review, 101(3), 238-241. Kemp, R. (2010), Eco-Innovation: definition, measurement and open research issues, Economia Politica, 27(3), 397-420. Ketterer, J.C. (2014) The impact of wind power generation on the electricity price in Germany, Energy Economics, 44, 270-280. Lin, Y. (2016), Where does the wind blow? Green preferences and spatial misallocation in renewable energy sector, CEP Discussion, Paper No 1424. Rodriguez, M.C., Hascic, I., Johnstone, N., Silva, J., and Ferey, A. (2015 Sep 1), Renewable Energy Policies and Private Sector Investment: Evidence from Financial Microdata, Environmental and Resource Economics, 62(1), 163-188. Rogers, E. (1983), Diffusion of innovations, New York: Free Press. Sapio, A. (2015), The effects of renewables in space and time: A regime switching model of the Italian power price, Energy Policy, 85, 487-499. Sapio, A. and Spagnolo, N. (2016), Price regimes in an energy island: Tacit collusion vs. cost and network explanations, Energy Economics, 55, 157-172. Sensfuss, F., Ragwitz, M., and Genoese, M. (2008), The meritorder effect: A detailed analysis of the price effect of renewable electricity generation on spot market prices in Germany, Energy Policy, 36(8), 3086-3094.






Phytotoxic Extracts as Possible Additive in Subsurface Irrigation Drip for Organic Agriculture V. Romanucci1 , A. Ladhari1 , G. De Tommaso1, A. De Marco2 , C. Di Marino1 , G. Di Fabio1 , A. Zarrelli1† 1 2

Department of Chemical Sciences, University of Naples, Via Cintia 4, 80126, Italy Department of Biology, University of Naples, Via Cintia 4, 80126, Italy Submission Info Communicated by Pier Paolo Franzese Received 17 April 2018 Accepted 1 October 2018 Available online 1 January 2019 Keywords Hydroalcoholic extracts Lactuca sativa Lycopersicon esculentum Allium cepa Phytotoxicity Anti-radical activity

Abstract The subsurface drip irrigation (SDI) system is a micro-irrigation technique applied below the soil surface through drip lines buried at a depth depending on the characteristics of the soil and on the plants to be irrigated. SDI distributes precise amounts of water directly to the root area, with the possibility of leaving the soil surface dry and less subject to weeds. This system reduces the use of water, herbicides, and environmental pollution. Furthermore, SDI allows the use of urban wastewater, advantageous from the environmental point of view since it reduces the consumption of ground water and energy costs required for its pumping. In addition, it reduces the use of chemical fertilizers through the enhancement of organic fertilizer content in the waste. However, there are issues related to the use of SDI systems, such as the elimination or reduction of roots that wrap the dripper thus blocking the water flow. It has been hypothesized that it would be useful to add a pure or blended phytotoxic mixture to plastic during the production of drippers, whose herbicidal action dissolves gradually with the passage of water. Five species of plants have been selected in this study: Vetch villosa, Brassica juncea, Secale cereale, Juncus effusus, and Vallisneria natans. The phytotoxicity has been tested in vivo on Lactuca sativa, Lycopersicon esculentum, and Allium cepa. The plants showed the same behavior but the aerial biomass of V. natans resulted the most active ones. The phytotoxicity of the hydroalcoholic extract of each plant was evaluated on the same test organisms, with peak inhibitions up to 60, 70, and 80% at concentrations ranging from 10−4 to 10−7 M. In general, the most active hydroalcoholic infusion was that of V. villosa. Finally, after some chromatographic steps and LC/GC-MS analyses, the most abundant metabolites of the hydroalcoholic extracts were identified. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction The recent forums promoted by international organizations have defined the third millennium as that of the thirst for water (G20 Agriculture Ministers’ Action Plan, 2017). It is estimated that in the world there is only 0.3% of † Corresponding


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V. Romanucci et al. / Journal of Environmental Accounting and Management 6(4) (2018) 335–343

renewable freshwater with a very differentiated availability, while the promises of genetic engineering of more productive plants will serve little if there is not enough water to feed them. The continuous increase in population and the need to improve the standard of living feed and exacerbate the conflict between different competing uses of freshwater resources: civil, industrial, and agricultural. The agricultural activities are the largest user of the water resource (on average two thirds of global consumption) and efforts are being made to find solutions to the growing water scarcity. Nowadays, irrigation of fruit and vegetable crops in modern agriculture is increasingly carried out with subsurface drip irrigation (SDI) systems (Camp, 1988). The SDI system is a micro-irrigation technique applied below the soil surface through drip lines buried at a depth depending on the characteristics of the soil and the plants to be irrigated. SDI distributes precise amounts of water directly to the root area, with the possibility of leaving the soil surface dry and less subject to weeds. In the last decades all the experimental tests have demonstrated the validity of the SDI in terms of greater productivity of the crops, water saving due to the lack of evaporation loss, and facilitating the mechanization of the various cultivation operations. The genesis of this technique is to be found in soil aridity increased by global climate change pushing employees in the primary sector to resort to systems allowing water savings. SDI systems reduce the use of water, herbicides, and environmental pollution. Furthermore, SDI allows the use of urban wastewater, advantageous from the environmental point of view since it reduces the consumption of ground water and energy costs required for its pumping. In addition, it reduces the use of chemical fertilizers through the enhancement of organic fertilizer content in the waste (Selvaratnam et al., 2016). The current knowledge on the advantages and mechanisms of operation of micro-irrigation, integrated with new agronomic and technological studies, has made possible the widespread diffusion of sub-irrigation, which has proved to be applicable to an increasing number of crops. The main obstacle to the diffusion of this technique in the last decades is due to the intrusion of root hairs that, looking for water and nutrients, are able to penetrate from the emission points by clogging the delivery devices. In this study, it has been hypothesized that it would be useful to add a pure or blended phytotoxic mixture to plastic during the production of drippers, whose herbicidal action dissolves gradually with the passage of water. The specific aims of the study were: 1) identify from the selected Mediterranean plants the most phytotoxic hydroalcoholic extract on germination and seedling growth of target species; 2) evaluate the insertion of the most active extract or one of its components in the production cycle of drippers; 3) evaluate the possible release of the extract or of its pure component to the passage of water. Five species of plants were selected in this study: Vetch villosa Roth. (Ercoli et al., 2007), Brassica juncea L. (Ercoli et al., 2007), Secale cereale L. (Ercoli et al., 2007), Juncus effusus L. (Ervin et al., 2000) and Vallisneria natans L. (Wu et al., 2000). The phytotoxicity was tested in vivo on Lactuca sativa, Lycopersicon esculentum, and Allium cepa.


Plant material

In this study, V. villosa, B. juncea, S. cereale, J. effusus and V. natans, five species from Mediterranean plant communities, were collected during May 2015, near the city of Naples (Southern Italy). These plants were easily available in large quantities and from geographically accessible locations. 2.2

Extraction and isolation

Each selected plant (roots + aerial parts) was air dried and grinded to fine powder (200 g), and then macerated with methanol/water 1:9 (v/v; 2 L) for 3 days. The prepared extracts were filtered and stored at 4 ◦ C until their use (DellaGreca et al., 2007).


337

Fig. 1 Separation procedures of compounds 1-4 of V. villosa.

2.3

In vitro phytotoxicity bioassay

The phytotoxicity of the crude hydroalcoholic extracts and the 1:1 and 1:3 dilutions was tested in vitro on Lactuca sativa L., Lycopersicon esculentum L., and Allium cepa L. (Macias et al., 2000; Ladhari et al., 2013). The seeds of the target species were surface sterilized with sodium hypochlorite solution (0.4%, v/v) for 3 min and soaked in sterile distilled water for 30 min. The test solutions (10−4 M) were prepared using 10 mM MES buffer (2-N-morpholino-ethanesulfonic acid, pH 6). Then, 25 sterilized seeds of target species were separately placed on the filter paper (Whatman No. 1) in 5 cm Petri dishes. After adding the seeds and 4.5 mL of the test solution, the Petri dishes were sealed with Parafilm® to ensure closed-system models. The seeds were placed in a growth chamber (KBW Binder 240) at 25◦ C, in the dark (5 days for L. sativa and L. esculentum, and 7 days for A. cepa). After growth, the plants were frozen at -20 ◦ C to avoid subsequent growth until measurement. Effects of the hydroalcoholic extracts from the 5 selected plants on the germination and growth of L. sativa, L. esculentum and A. cepa were tested. Treatments were arranged in a completely randomized design with three replications and data were transformed to percent of control for analysis. 2.4

Isolation of the phytotoxic compounds

The hydroalcoholic extract of V. villosa, the most active among those tested, was suspended in water and partitioned between ethyl acetate (EA) and water (W) (Fig. 1). The EA fraction was fractionated into neutral and acidic fractions using an aqueous 2N NaOH solution. The neutral fraction was washed with water and concentrated in vacuo (EAN fraction) while the acidic fraction was first acidified with aqueous 2N HCl to neutral pH and then extracted with ethyl acetate (EAA fraction), according to Fig. 1. The neutral fraction was subjected to silica gel column chromatography using gradient solvent systems. The fractions were injected into the GC-MS, and the structure of compound 1 (Fig. 1) was

338


measured for comparison with the reported data in the literature and in the database. A volume equal to one tenth of the W fraction was partially lyophilized (320 mg) and filtered using a Sep-Pak RP-18 by eluting with water, methanol and acetonitrile (WW, WM, WA, respectively). The fractions were dissolved in 0.8 mL of MeOH, filtered through a 0.45 mm filter and injected into the LC-MS, identifying compound 2 from the WW fraction and compounds 3 and 4 from WM fraction (Fig. 1). 2.5 2.5.1

Identification of the phytotoxic compounds GC-MS/MS and LC-MS/MS analysis

Approximately 1 mg of EAN fraction was dissolved in 0.8 mL of acetone and subjected to a triple quadrupole gas chromatograph mass spectrometer (GC-MS). The analyses were performed with the following temperature program: 40 ◦ C for 5 min, 220 ◦ C at 5 ◦ C/min, and 220 ◦ C for 10 min, and the injection volume was 1 µ L (DellaGreca et al., 2011; Zarrelli et al., 2011). Approximately 1 mg of WW and WM fractions were dissolved in 0.8 mL of acetone and subjected to LCMS analysis. The most appropriate precursor ion, daughter ion, cone voltage, and collision energy were adjusted according to each analysis. 2.6 2.6.1

Spectrophotometric determination of cyanamide Preparation of the standard cyanamide solution

Standard solution of cyanamide (2.5 mM as final concentration) was obtained dissolving pure cyanamide in 0.1 M HCl, freshly prepared and the pH was brought to a final value of 7.0. The calibration line obtained allowed to determine a molar extinction coefficient of 2950 cm−1 M−1 , in good agreement with the values reported in the literature (Yoshifumi et al., 2009; Nieman et al., 1976). 2.7

Statistical analysis

The phytotoxic essays were conducted in a complete randomized design with three replications and a two-way ANOVA was performed to analyze treatment differences followed by the post hoc test of Tukey. The statistical assays, performed by using the Systat SigmaPlot 12.2 software (Jandel Scientific, USA), were considered statistically significant for P < 0.05.

3 Results and discussions 3.1

Phytotoxic evaluation of plant extracts

Our first objective was to identify easily accessible terrestrial or aquatic plants from the Mediterranean area and evaluate their phytotoxicity on test organisms normally used for this purpose (DellaGreca et al., 2004, 2011). Five plants were selected: V. villosa, B. juncea, S. cereale, J. effusus, and V. natans, infused with methanol/water 1:9 for three days at room temperature. The phytotoxicity of the obtained extracts and the corresponding diluted 1:1 and 1:3 was measured on the two dicotyledonous e` L. sativa and L. esculentum and on the monocotyledonous A. cepa (Table 1). Among the five aqueous infusions, V. villosa was the most active regardless of dilution degree and in relation to both germination and elongation of the root and hypocotyl (Table 1). In particular, the undiluted extract of V. villosa showed an anti-germination activity higher than 70% on all three test organisms, while the 1:1 and 1:3 diluted extracts showed an activity of 45-50% and ∼30% respectively. The other extracts showed a generally lower activity of 9-13% and, in particular, that of J. effusus was the least active (Table 1), especially on L. sativa (-25%).

A) Germination L. sativa

L. esculentum

A. cepa

Plant

Extract

1:1

1:3

Extract

1:1

1:3

Extract

1:1

1:3

V. villosa

-76.7±1.1A

-44.8±1.2A

-28.1±0.7A

-70.0±1.8

-44.5±0.9A

-27.2±1.1A

-77.8±1.1A

-50.2±0.8A

-31.8±1.5A

B. juncea

-68.1±0.9B

-40.0±0.8A

-13.1±0.4B

-65.7±1.3

-33.2±0.8B

-9.9±0.8B

-63.8±0.8B

-31.8±0.5B

-15.6±1.0B

S. cereale

-64.3±1.6B

-31.8±0.8B

-17.8±0.8C

-64.9±1.2

-27.8±1.0B

-10.8±0.8B

-64.9±1.2B

-34.1±0.9B

-11.0±0.8B

J. effusus

-52.2±0.8C

-21.4±1.2C

-12.2±0.5B

-63.2±0.8

-33.2±1.1B

-15.2±1.1BC

-72.0±2.1A

-24.8±1.0C

-18.3±1.1B

V. natans

-64.3±1.0B

-33.1±1.1B

-18.3±0.6C

-65.2±1.1

-29.1±0.8B

-16.9±0.7C

-60.3±1.5B

-21.9±0.6C

-8.9±0.8B

B) Root elongation L. sativa

L. esculentum

A. cepa

Plant

Extract

1:1

1:3

Extract

1:1

1:3

Extract

1:1

1:3

V. villosa

-80.7±2.1A

-58.2±0.9A

-35.3±0.8A

-85.2±2.1A

-49.1±1.2A

-33.2±1.0A

-88.9±2.3A

-60.7±1.8A

-40.2±1.1A

B. juncea

-64.9±1.8B

-45.1±0.8B

-18.1±0.5B

-70.2±1.6B

-47.8±1.2A

-20.4±0.8B

-71.2±2.1B

-44.9±1.3B

-20.8±0.8B

S. cereale

-71.2±1.3C

-47.6±1.0B

-24.8±0.6C

-74.9±1.2AB

-48.4±1.3A

-21.3±0.8B

-71.7±1.6B

-40.1±1.1B

-26.7±0.8B

J. effusus

-60.3±1.0B

-35.4±0.7C

-25.1±0.6C

-71.2±2.2B

-30.6±0.6B

-20.4±0.6B

-57.8±1.2C

-24.6±0.8C

-5.2±0.4C

V. natans

-73.2±1.5C

-38.7±0.8C

-27.6±0.8C

-70.3±1.8B

-37.5±1.0C

-10.8±0.5C

-80.2±2.0A

-22.9±0.6C

-31.1±1.1B

C) Shoot elongation L. sativa

L. esculentum

Plant

Extract

1:1

1:3

Extract

1:1

V. villosa

-88.1±2.2A

-58.8±1.2A

-28.2±1.0A

-91.2±2.5A

-60.9±1.3A

B. juncea

-85.2±1.8A

-45.2±1.1B

-18.1±1.1B

-93.1±2.2A

-45.2±1.0B

S. cereale

-69.8±1.6B

-25.1±0.9C

-7.3±0.5C

-66.8±1.2B

-20.8±0.8C

J. effusus

-73.8±1.8B

-28.2±0.6C

-9.1±0.4C

-71.4±1.2B

-28.3±1.1C

V. natans

-68.1±1.5B

-23.3±0.5C

-11.0±0.8C

-77.7±1.5B

-31.0±1.2C

A. cepa 1:3

Extract

1:1

1:3

-34.7±1.2

A

-87.4±1.6A

-60.1±1.1A

-31.4±1.0 A

-20.2±0.9

B

-87.7±1.5A

-37.7±0.8B

-18.2±1.0B

-6.3±0.4 C

-68.9±1.2B

-20.6±0.8 C

-7.4±0.6C

-9.1±0.6 C

-71.3±1.2B

-21.8±0.9 C

-9.3±0.6C

-7.3±0.6C

-65.8±1.4B

-19.2±0.7C

-8.2±0.7C


Table 1 Effect (mean ± SD) of the hydroalcoholic extracts of V. villosa, B. juncea, S. cereale, J. effusus and V. natans, on the germination (A), root elongation (B) and shoot elongation (C) of L. sativa, L. esculentum and A. cepa, for the extracts as such, diluted 1:1 and diluted 1:3, respectively. Positive percentages represent stimulation while negative values represent inhibitions. The three extracts concentrations showed always statistically different effects. Different uppercase letters indicate a significant difference among the five types of extracts (extracts of V. villosa, B. juncea, S. cereale, J. effusus and V. natans). Statistically differences were performed by two-way ANOVA (P < 0.05).

339

340


Regarding the root elongation, the undiluted hydroalcoholic extract V. villosa was able to inhibit it up to about 90%, while those diluted 1:1 and 1:3 showed an inhibition of 60% and slightly less than 40%, respectively. The other extracts were less active about 9-21% and, among these, the less active was once again the extract of J. effusus, especially on A. cepa (30-35%) (Table 1). A slightly different situation in the evaluation of shoot elongation was found (Table 1). In this case there were two extracts that were far more active than the others, namely V. villosa and B. juncea, with inhibition peaks higher than 85% for the undiluted extract. The other three were generally less active than 18-20%. However, the diluted 1:1 extract of V. villosa was 15% more active than B. juncea extract and 35% more than the others, while the diluted 1:3 extract showed an almost double activity of B. juncea extract and 2-5 times more than others (Table 1). 3.2

Isolation of major components of V. villosa extract of and their phytotoxic evaluation

Considering that the extract of V. villosa was the most active, it was decided to continue the investigation by evaluating its chemical composition, at least in the commercially and economically most abundant accessible components. The whole extract showed a composition strongly depending on the extraction conditions and so hardly reproducible. Moreover, its use in the production of the dippers was advised. The hydroalcoholic extract was partitioned between ethyl acetate and water (EA and W, respectively; Fig. 1). In turn, the EA fraction was fractionated into neutral (EAN) and acidic (EAA) fractions that were then subjected to silica gel column chromatography using gradient solvent systems. The obtained fractions were injected into the GC-MS identifying the structure of compound 2-Methyl-1,4-benzoquinone (1). Only part of fraction W was reduced in volume and filtered on a Sep-Pak RP-18 by eluting with water, methanol and acetonitrile (WW, WM and WA, respectively). The fractions were dissolved in 0.8 mL of MeOH, filtered through a 0.45 mm filter and injected into the LC-MS identifying 2,6-Dimethyl-1,4-benzoquinone (2) from the fraction WW and Cyanamide (3) and Calcium cyanamide (4) from fraction WM (Fig. 1). The four identified compounds were tested to determine their phytotoxicity on the three test organisms already considered, at four different concentrations between 10−4 and 10−7 M (Table 2) (D’Abrosca et al., 2005; Fiorentino et al., 2007; Cutillo et al., 2004; DellaGreca et al., 2003). The phytotoxicity assays were carried out according to the previous protocol. In particular, the quantities necessary for the preparation of the test solution (10−4 M) were taken from the compounds and subsequent concentrations (10−5 -10−7 M) were prepared by dilution: all compounds were dissolved in MES in the ratio of 5 L per mL of buffer. The compounds tested were active and, generally, more than 4-hydroxybenzoic acid (HBA), the herbicide used as a reference, at all concentrations considered (DellaGreca et al., 2007). At the highest concentration (10−4 M), compounds 1-4 were able to inhibit the germination of the three test organisms with percentages between 70 and 85%, at least 0-25% more than the reference herbicide (Table 2). Compound 3 was the most active, with a percentage of germination inhibition that rarely fell below 70% even at the concentration of 10-7 M, in particular with an activity around 80% on A. cepa, regardless of concentration tested. Its calcium salt (4) was slightly less active but still more than the compounds 1 and 2 (Table 2). Compound 3 was the most active also in inhibiting root elongation, never below 50% until the concentration of 10−7 M, and with peaks up to 90% at the highest concentration on L. esculentum. Compounds 1, 2 and 4 showed slightly lower activities, similar among them but the first two slightly more active than compound 4 (Table 2). The same trend was found also for the shoot elongation, the most active were cyanamide (3) and its calcium salt (4). Compounds 1 and 2 were much active at the two highest concentrations, but less than com-pounds 3 and 4, and much less at the lower concentrations. Often, at the concentration of 10−7 M compounds 1 and 2 were even slightly stimulating the shoot elongation (Table 2).

Table 2 Effect (mean ± SD) of the compounds 1-4 on the germination (A), root elongation (B) and shoot elongation (C) of L. sativa, L. esculentum and A. cepa. at four different concentrations between 10−4 and 10−7 M. Positive percentages represent stimulation while negative values represent inhibitions. Different lowercase letters indicate a significant difference among the four concentrations. Different uppercase letters indicate a significant difference among different compounds. Statistically differences were performed by two-way ANOVA (PPS>PE. PP toxicity could be related to the presence of solvents (methanol, oil, cyclohexane) employed for its production, whereas PS toxicity was probably due to the depolymerization, occurring in water, followed by styrene release, while the mild toxic effects of PE and its temporary bio stimulation could be attributable to the thermoregulatory additives present in the polyethylene resins. Our results highlighted that also the virgin plastic pellets could be responsible of toxic effects that should not be neglected. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction The global consumption of plastic materials is nowadays steadily increasing, with more than 240 million tons of plastic produced annually. Geyer et al. (2017) estimated that the cumulative amount of plastic waste, from 1950 to 2017, totals around 6 billion of metric tons. The same authors estimate that this value will increase up to 25 billion of metric tons by 2050. The low recycling rates of plastic products, together with their persistence in the environment, ends up in accumulation of plastic debris and plastic microparticles in both terrestrial and marine systems (Barnes et al., 2009). † Corresponding


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S. Schiavo et al. / Journal of Environmental Accounting and Management 6(4) (2018) 345–353

The plastic polymers are usually not regarded as hazardous materials, because their large molecular size and their insolubility in water, that make them not bio-available and biochemically inert (da Costa, 2018). However, several low molecular weight and non-polymeric molecules present in plastic products are known as hazardous. They are either weakly bound or not bound at all to the polymeric macromolecules and may be released from the plastic product (Crompton, 2007; OECD, 2004). These include residual monomers, oligomers, low molecularweight fragments of catalyst remnants, polymerization solvents, and a wide range of additives (Crompton, 2007). Release from plastic products of hazardous substances as phthalates, brominated flame retardants, bisphenol A, formaldehyde, acetaldehyde, 4-nonylphenol, and many volatile organic compounds has been already reported by several authors (Tønning et al., 2010; Kim et al., 2006; Brede et al., 2003; Mutsuga et al., 2006; Fernandes et al., 2008; Henneuse-Boxus and Pacary, 2003). The study of the impact of plastic residues on different environmental matrices needs a multidisciplinary approach integrating chemical analysis (Wagner et al., 2014; Syberg et al., 2015) and ecotoxicological tests. Generally, the toxicity study on plastic polymers found in the environment also include the exposure of test organisms to leachates of virgin plastic pellets, supposed to be free from any additives and/or residual monomers, in order to separate the effects derived for virgin plastic itself from those due to chemicals used during product manufacturing and/or sorbed from the environment. An important work in this field was authored by Lithner et al. (2009), who studied the toxicity of leachates of 32 different plastic products founding acute toxicity in Daphnia magna exerted by leachates of plasticized PVC and polyurethane. Similarly, Wagner and Oehlmann (2009) cultivated mud-snails in polyethylene terephthalate (PET) mineral water bottles founding evident endocrine disrupting effects, if compared to those cultivated in borosilicate Erlenmeyer flasks. However, the absence of any leachable compounds in virgin plastic is still a question under debate. Scientific literature dealing with toxicity tests on plastic leachatesis relatively scant, despite theseare recognised as dangerous pollutant of high ecotoxicological concern (Hermabessiere et al., 2017). In this line, this study is aimed at studying the acute and chronic adverse effects of leachates, coming from different virgin plastic polymers, on test organisms belonging to several species and pertaining to different trophic levels. The test organisms used here were: a) the bacteria Aliivibrio fischeri (acute toxicity), b) the plants Sorghum saccharatum, Lepidium sativum, and Sinapis alba (sub-chronic toxicity) and Vicia faba (chronic toxicity) c) the crustacean Daphnia magna (acute and chronic toxicity). Moreover, in order to identify the possible ecotoxicological risk for the freshwater biota stemming of the investigated plastic leachates, the toxicity test battery integrated index (TBI) (Manzo et al., 2008) was used.

2 Material and methods 2.1

Plastic samples

Toxicity tests have been performed using the leachates of three different polymers: polypropylene (PP), polyethylene (PE), and polystyrene (PS) pellet. 2.2

Samples preparation and leaching

The test is conducted according to Italian and European Standard procedure (UNI EN 12457-2: 2004) by using pure water (18MΩ resistivity) as leaching solution. Each sample (PE, PP, PS) was shaken for 24h at room temperature, with a liquid-to-solid ratio of 10 (L/S=10). At the end of the leaching process, the solid was removed through decantation followed by a filtration on qualitative filter papers Whatman Grade 1 (11 μ m porosity) to remove plastic fragments, and the water phase was tested for its toxicity. 2.3

Acute toxicity • Aliivibrio fischeri


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The Microtox® tests were performed according to the supplier’s protocol (Modern Water, New Castle, DE). Lyophilized bacteria were rehydrated with the reconstitution solution supplied by the manufacturer prior to perform the test. Luminescence inhibition of polymers leachates (PE, PP, PS) was assessed for 5, 15 and 30 min of exposure according to the 81.9% Basic Test Protocol (screening test) (Azur Environment Ltd, 1998). The sample toxicity was evaluated measuring the decrease of the intensity of light produced by the luminescent bacteria. A control group was also set up without leachates. The negative control was the Microtox® diluent (NaCl 2%). The luminescence decrease was evaluated after 5, 15 and 30 min of exposure by using a Microbics Model 500 Toxicity Analyzer, according to the manufacturer’s instructions (Modern Water, New Caste, DE). The results were expressed as luminescence inhibition percentage with respect to the control using the Abbot’s formula and as EC50 calculated using ICp method (USEPA, 1993) • Daphnia magna The D. magna immobilization test was performed according to OECD (2004). Four 2.5 folds serial dilutions were prepared in order to reach the leachate concentration of 100%, 75%, 50% and 25%. Ten individuals, aged less than 24 h, were transferred in Petri dishes and exposed to 10 mL of each leachate dilutions at 20 ±2◦ C. After 24 and 48 h the immobilization and mortality of D. magna individuals were recorded. Tests were performed in triplicate. Results were reported as percentage of the effect respect to the control (Abbott, 1925). 2.4

Subchronic toxicity on plants

Germination and root elongation tests were carried out on S. alba, S. saccharatum and L. sativum according to Italian official protocol (UNICHIM 10780/2003). Ten seeds were placed in Petri dishes, lined with filtered paper imbibed with non-diluted PE, PP, PS leachates. Five replicates for each polymer leachates were prepared. Deionized water was used as negative control. The Petri dishes were incubated in darkness at 25 ±2◦ C in closed plastic bags in order to avoid evaporation. After 72 h, the number of germinated seeds (n) and the root length (L) were measured and the germination index (GI) and the root elongation inhibition (REi) were calculated as follows: %Gi = [(Gc/Gk) ∗ (Lc/Lk)] ∗ 100 (1) % Root elongation inhibition (REi) = (Lk − Lc)/Lk) ∗ 100

(2)

where: Gc = mean of germinated seeds in samples; Gk = mean of germinated seeds in the control; Lc = mean of root length in samples; Lk = mean the root length in the control. 2.5

Chronic toxicity • Daphnia magna

The 21-day life experiments were performed with D. magna, according to OECD 202 (OECD, 2008), on 2 dilutions (50% and 75%) of each leachate. All test solutions were prepared and stabilized at 20◦ C one day before the onset of the experiment. Twenty neonates (< 24h old) were exposed in 50 ml of the sample suspensions in glass vessels. Daphnids were fed twice a week with Raphidocelis subcapitata and Saccaromyces cerevisiae (1.5 10 elevated to 5 cells mL−1 ) in conjunction with the renewal of the test medium. All experiments were performed under a controlled light cycle (16 h of light: 8 h of dark). Survival and number of neonates were recorded daily. A negative (standard water) and positive control (K2 Cr2 O7 ) were performed simultaneously. Results were expressed in terms of mortality and reproduction inhibition (Ri). • Vicia faba The chronic test with V. faba was carried out using a modified Monteiro et al. (2009) protocol. Briefly, germinated V. faba seeds were cultivated in a hydroponic solution (Hoagland and Amon, 1950) supplemented with

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polymers leachates in a concentration of 50%. For each polymer 5 replicates (3 plantules for each replicate) were provided. The experiment was performed under controlled condition: light cycle, 16 h of light: 8 h of dark), temperature of 25◦ C during the day and 16◦ during the night. After 21 days, the following parameters were evaluated: fresh weight, fresh weight of aerial parts, dry weight of aerial parts, fresh weight roots, dry weight roots, leaves number, roots length, length of aerial parts. 2.6

Data integration

For each polymer leachate, the results of the different tests were integrated using the Toxicity test Battery integrated Index (TBI) (Manzo et al, 2008). For each polymer leachate, the effects on the chosen endpoints were expressed as percentage and classified according to Ispra (2011). To calculate the TBI, the percentage of the effect (%E) on each endpoint was corrected to obtain the Score test Endpoint (SEi) using the following formula: SEi = %E(M ∗ S)SCF

(3)

where: SCF (Statistical Correction Factor) = Student t-test differences between samples and control. The values of 0, 1, 2, 3 and 4 were attributed to SCF, corresponding to no effect (p > 0.05), biostimulation (p < 0.05), high biostimulation (p < 0.01), toxicity (p < 0.05), and high toxicity (p < 0.01), respectively; Matrix (M) was set as 2 for samples leachates; Severity (S) was set as 2 for bioluminescence, 3 for root elongation, germination and plants growth, 4 for reproduction and 5 for mortality. SEi is expressed in a 0–100 scale relative to test battery utilized as follows: %SEi = SEi(

%Em ) SE max

(4)

where: %Em = maximum effect percentage observed corresponding to the maximum MS obtained, and SEmax is the maximum Score test Endpoint calculated. The Toxicity test Battery integrated Index (TBI) is calculated according to the following formula %T BI = (

∑ %SEi ) N

(5)

where: N = number of endpoints. The ecotoxicological risk is defined as follows: not significant (TBI ≤ 5%), medium (5< TBI ≤ 20%), high (20 < TBI ≤ 50%), very high (TBI >50%). 3 Results 3.1

Plants

The results of the phytoxicity tests are reported in table 1. PE and PS polymer leachates showed no toxic effects on tested plants. PP leachate resulted non-toxic for L. sativum and S. alba, but exerted a negative effect on germination and root elongation of S. saccharatum. The IG % values highlighted a biostimulating effect on S. saccharatum and S. alba in the case of PE leachate. A slight biostimulation on root elongation was obtained also in the case of PS leachate for all the tested plants. On the contrary, the chronic test carried out with V. faba (Fig. 1) showed severe negative effects on different endpoints. A significant toxicity was recorded as visible damages, such as necrosis and chlorosis, during the test. Among the 3 tested leachates, PS showed the highest adverse effects with values surpassing the 50% for all considered endpoints. In particular, the more pronounced negative effect was recorded upon the root elongation, the most sensitive endpoint among those considered here.


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Table 1 Germination Index (GI) and Root Elongation Inhibition (REI) for S. saccharatum, S. alba L. sativum exposed to PE, PP and PS leachates. Polymers

S. sacchratum

S. alba

L. sativum

GI %

REI%

GI %

REI%

GI %

REI%

PE

101.4

28.6

125.7

14.4

90.7

5.3

PP

41.8

56.6

91.8

7.9

83.5

16.5

PS

70.5

29

92.8

-5.6

97.3

2.7

c

PE

PP

PS

fresh weight 0 fresh weight aerial parts

leaves number 1

roots lenght

dry weight aerial parts

2

length aerial parts

fresh weight roots dry weight roots

Fig. 1 Evaluation of different endpoint for V. faba chronically exposed to PE, PP and PS leachates.

% effect

100

50

0 PE

PP 5'

15'

PS

30'

Fig. 2 Inhibition of bioluminescence (% effect) for A.fischeri exposed to PE, PP, PS (5, 15 and 30 min).

3.2

Bacteria

Toxic effects registered on A. fischeri exposed to plastic leachates are reported in figure 2. To the best of our knowledge, no data are available in literature on the effects of exposition of A. fischeri to plastic leachates. These leachates inhibited the bio-luminescence of A. fischeri below the threshold of 25%. Among the 3 leachates, PS

350


100

% effect

50

0 PE

PP

PS

-50

-100

-150 Mortality

Reproduction

Fig. 3 Mortality and reproduction (% effect) for D. magna exposed to PE, PP, PS at the end of exposure time (21 th day).

exerted the highest effects. Similar results were recorded at 5, 15 and 30 min of exposure. Despite the low effect recorded, the inhibition of bioluminescence seems to be the most sensitive test among the acute assays carried out in this work. 3.3

Cladocera

Although the D. magna 48 h acute exposure test showed no mortality, adverse effects became evident during the 21 days of exposure to leachates. In Figure 3, the effects of PE, PS and PP leachates in terms of surviving and reproduction rate (number of nauplii/number of individual) were reported . D. magna survival was significantly affected by PP and PS leachates, as the recorded rates of mortality were around 69% for PS and slightly below 50% for PP. The first dead individuals were recorded after 4 days for PP, 7 days for PS and 11 days for PE. The negative effect increased with time for PP and PS, whereas PE leachate showed a very low mortality rate (around 7%) (Fig. 4a). The reproduction of D. magna (11th day) was affected by the exposure to PP leachate, while no effect was noticeable up to 2 weeks for PS and PE. Starting from the 3rd week, an increasing inhibition trend was more evident for PS and PE exposure. PS reached almost the 100% of the effect (Fig. 4b). For PE, instead, an enhancement of reproduction rate was observed starting from the 14th day. This increased along the exposure time with a final 98% increment of reproduction rate respect the control (Fig. 4). 3.4

Data integration

The data analysed through the TBI approach allowed to rank the toxicity of leachates as follow PP > PS > PE. The PP leachate showed a high ecotoxicological risk (TBI=12.4%), according to the battery of test organism used here, whereas PS showed a moderate risk (TBI=8.4%) and PE a negligible one (TBI= 4%).

4 Discussion The toxic effect of the leachate of virgin plastic pellets observed here could be due to residues of chemicals used in the polymer production and non-intentionally added substances (impurities, polymerisation by products, breakdown products), catalysts, solvents, and additives easily leaching from virgin plastic materials. Leachates often exert higher toxic effects than virgin plastic themselves, suggesting that some compounds became more


351

Mortality PP

100%

PS

PE

A

% effect

50%

0% 0

4

7

11

14

18

21

Days -50%

Reproduction

B

100%

% effect

50% 0% -50% -100% -150% 0

4

7

11

14

18

21

Days PP

PS

PE

Fig. 4 Mortality (A) and reproduction (B) (% effect) for D. magna exposed to PE, PP, PS at different time of exposure.

bioavailable during leaching (Teuten et al., 2009). In addition, Browne et al. (2013) and Nobre et al. (2015) observed that additives used in the production of different plastics are more toxic than pollutants adsorbed on plastic polymers. In this study, the TBI highest toxicity was evidenced for PP virgin plastic and could be related to the presence of solvents (methanol, oil, cyclohexane) used in polypropylene production (Harding et al. 2007). The moderate toxicity exerted by PS was probably due to its depolymerization, occurring in water, followed by styrene release, whose ecotoxicity has been well documented (Gibbs et al., 1997; Thaysen et al., 2018). According to Murphy (2001), PE mild toxicity effects and its bio stimulation effect on D. magna reproduction rate could be due to the effect of some additives present in the polyethylene resins with known estrogenic potential (Yang et al., 2011). As expected, single biological responses to the complex chemical mixtures present in leachates varied with the polymer concerned and the species specific susceptibility of the exposed model organism. In our experiments, we evidenced relevant differences and sometimes contrasting effects among the exposed organisms. Actually, anthropogenic stressors can differ markedly in their effects on species diversity (Petrin et al., 2008). Taking into account that the variability of the responses of tested organisms is a common frame in ecotoxicological studies, the use a data integration algorithm was of great help in classifying the risk posed by such chemicals to the

352


investigated ecosystem. In the recent past, the “worst-case” assumption was considered adequate to assess the ecotoxicological risk stemming from the presence of molecule with xenobiotic characteristics in the environment (Calow and Forbes, 2003). In the last decades, it became evident that this approach does not satisfactorily assess the environmental risk connected to the exposure to chemicals (Grenni et al., 2018). The development of indices, able to combine the responses to different tests into a toxicity hazard score (Hartwell, 1997), allows to translate the test results to the effects exerted by xenobiotic compounds on complex natural systems in which many individuals and species live and thrive.

5 Conclusion Virgin plastic pellets of different polymers are often used in particle toxicity studies as reference materials. However, a release of bioavailable additives and unknown substances could occur during the exposure in aquatic matrices, provoking adverse effects upon the test organisms especially when they are chronically exposed. We assessed that PP, PS and PE virgin plastic pellets leachates exerted several and diverse toxic effects upon the battery of selected test organisms: bacteria (A. fischeri), plants (V. faba, S. saccharatum, S. alba, L. sativum) and cladocera (D. magna). In particular, D. magna showed the highest susceptibility in the chronic exposure and the A. fischeri in the acute one. Therefore, the selected tests, in particular the chronic assays, proved to be a suitable tool for assessing the toxicity of different plastic polymers. In addition, the TBI approach, adopted here to integrate the toxicity data, was able to classify the toxicity of the investigated leachates as follows: PP > PS > PE. Overall, considering the assessed toxicity related with the virgin polymers, the direct loss of virgin plastic pellets into the environment during manufacturing and transport should be also monitored. The known heterogeneity of plastic properties was mirrored in our results, which showed a highly variable and material-dependent leachates toxicity to the organisms tested here. However, due to industry policies of privacy we have no additional information about the type and the concentration of plastic additives used in the virgin granules studied here. All this makes a more accurate discussion difficult.

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Uptake of Micro and Macronutrients in Relation to Increasing Mn Concentrations in Cistus salvifolius L. Grown in Hydroponic Cultures F. Figlioli1, V. Memoli1, G. Maisto1, V. Spagnuolo1†, S. Giordano1 , E. O. Leidi2 , S. Rossini Oliva3 1

Dipartimento di Biologia, Università Federico II, Via Cinthia 4, 80126 Napoli, Italy IRNAS-CSIC, Av R. Mercedes 10, 41012 Seville, Spain IRNAS-CSIC, Av R. Mercedes 10, 41012 Seville, Spain 3 Dpto. Biolog´ıa Vegetal y Ecolog´ıa, Avda. Reina Mercedes, 41080 Sevilla, Spain

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Submission Info Communicated by Pier Paolo Franzese Received 17 April 2018 Accepted 1 October 2018 Available online 1 January 2019 Keywords Manganese Plant metal stress Plant mineral uptake SEM-XS ray microanalysis

Abstract Mining and smelting activities can alter the ecosystem degrading vegetation and landscape, causing loss of soil fertility and changes in hydrology and microclimate. The mining area of Rio Tinto is one of the largest metallic sulfide deposits in the world, extending to southern Portugal and the Rio Tinto region (Huelva, SW Spain). Soils, characterized by low pH, are strongly impoverished in macro- and micronutrients essential to the plant metabolism and contain very high concentrations of As, Cu, Fe, Mn, and Pb. The aim of this study was to evaluate the effects of increasing Mn concentrations (0, 50, 100, 200, and 300 mg /L) on the uptake of a set of micro and macro nutrients in Cistus salvifolius L., a species native of the Rio Tinto region. The plants, grown in hydroponic culture, were analyzed by AAS for elemental content and by SEM-XS ray microanalysis for element localization. The results of this study showed a stunted growth and ultrastructural alterations in the root of C. salvifolius, with the most evident damages occurring at the highest Mn concentration. Chemical analyses confirm that the higher the concentration in culture medium, the higher the uptake of Mn in plant tissue; both lower and higher Mn concentrations influence the absorption of other essential nutrients, as Fe, Zn, K, and Mg. The visible state of stress observed in plants grown with addition of 300 ppm Mn may therefore be due to such variations in the absorption of micronutrients and/or to the Mn itself. Future studies should focus on possible synergistic and antagonistic activities of Mn versus other essential elements for proper plant development. ©2018 L&H Scientific Publishing, LLC. All rights reserved.

1 Introduction Mining and smelting activities induce landscape degradation, vegetation disruption, soil fertility loss, and hydrological and microclimate changes, deeply disturbing the ecosystems (Runolfsson and Arnalds, 2004). † Corresponding


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Dipartimento di Biologia et al. / Journal of Environmental Accounting and Management 6(4) (2018) 355–363

The abandoned mining area of Rio Tinto is included in the Iberian Pyrite Belt (IPB) one of the largest metallic sulfide deposits in the world, extending in Southern Portugal and the Rio Tinto district (Huelva, SW Spain). The exploitation of this region has been going on for about 5000 years, producing a total of about 1600 million metric tons of waste material (Davis et al., 2000); the area is currently occupied by open pits, tailing deposits and other mining wastes. As a result of those activities and intensive deforestation, natural landscape disruption and vegetation lack occur at large areas of the region. The average mineralogical composition found in the area consists of pyrite (83.1%), sphalerite (5.4%), galena (2.1%), chalcopyrite (1.4%), and arsenopyrite (0.9%), the remaining fraction (7.1%) corresponds to unproductive minerals (Almodovar et al., 1998). The area has a Mediterranean climate, with annual rainfall from 600 to 800 mm and mean annual temperature of 18 ◦ C. Summers are very hot and dry and rainfall occur mostly during autumn and winter. Usually, soils developing on mine wastes are spontaneously colonized by pioneer plant species, which provide an important contribution for land recolonization and natural rehabilitation (Anawar et al., 2013). These plants tolerate the increased availability of potentially toxic metals in the soils through genetically-based mechanisms which allow either to prevent root metal uptake and translocation or detoxification and compartmentalization of absorbed metals. The tolerance in these plants is not metal specific, varying among different genotypes (ecotypes, physiotypes or races) of the same species (Bargagli, 1998). Several families of vascular plants, such as Caryophyllaceae, Cyperaceae and Ericacaeae, include species with morphological and physiological plasticity able to evolve tolerance mechanisms and, to a certain extent, to modulate their evolution according to substrate characteristics (Rossini-Oliva et al., 2016). A number of studies (Kidd et al., 2004; Freitas et al., 2004; Santos et al., 2009; de la Fuente et al., 2010; Abreu et al., 2012; Jiménez et al., 2011) show that several Cistus species (e.g. C. salvifolius L., C. monspeliensis L., C. albidus L., C. crispus L., C. populifolius L. and C. ladanifer L.) are able to survive in very hostile habitats. Manganese is an essential element for plant growth, activating some enzymes involved in citric acid cycle (tricarboxylic acid cycle); besides, a central role of manganese cluster complexes in oxidation of water to oxygen has been reported (Dharmendra et al., 2013). However, its excess, especially in acidic soils, can affect plant survival disturbing physiological functions and mineral uptake (Kochian et al., 2004; Ducic and Polle, 2005). Manganese in plants participates in the structure of photosynthetic proteins and enzymes. Its deficit is dangerous for chloroplasts because it affects the water-splitting system of photosystem II (PSII), which provides the necessary electrons for photosynthesis (Buchanan et al., 2000). However, its excess seems also to be particularly damaging to the photosynthetic apparatus (Mukhopadhyay and Sharma, 1991). Toxic Mn levels fall in the range of 1000-12000 mg kg−1 , depending on the plant species; some species have been found with Mn contents in the range 1000-5000 mg kg−1 both on soils with Mn concentrations higher than 1% and on soils with lower Mn concentrations. Ultramafic soils may have 1000-5000 mg kg−1 , which is not regarded as strongly abnormal. Manganese toxicity is favored in acid soils (Pendias and Pendias, 1992); with low pH values, the amount of exchangeable Mn – mainly Mn2+ – increases in the soil solution. In the present study the effect of increasing Mn concentrations on the uptake of selected micro and macronutrients, such as Fe, Zn, K, Mg, and Mn itself, was evaluated in Cistus salvifolius, a plant native of the Rio Tinto region. The aim of the experiment was to assess if mineral nutrition is affected by Mn concentrations in these plants, naturally growing in soil enriched in Mn. To this aim, the plants were cultured in hydroponic medium and analyzed by AAS for their element content; in addition, SEM microscopy X-ray spectrometer equipped was applied to follow Mn distribution in plant tissues and reveal eventual anatomical alterations possibly related to Mn stress.


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Plant material

Cistus salvifolius L. is a shrub belonging to the Cistaceae family, typical of Mediterranean scrub. It is a shrubby plant not exceeding 50-60 cm height (Farley and McNeilly, 2000). Combined with other bushes, it can spread several meters and form impenetrable clusters. It is resistant to prolonged drought conditions, and not demanding about soil pH. It grows well in neutral, slightly limestone or slightly sandy soils. The plant prefers a sunny exposure, but it also suits partially shady conditions, so it can also be found in open-wood forests; it is also well adapted to areas exposed to environmental disturbances such as recurrent fires and mine contamination. 2.2

Experimental design and plant culture

The seeds of C. salvifolius were treated in a stove at 40 ◦ C for 24 hours to activate seed germination, and subsequently seeded on humid paper in the dark. The plantlets were then transferred and grown in hydroponic solution with a modified Hoagland solution (Hoagland and Arnon, 1950; modified according to Rossini-Oliva et al., 2016). After obtaining seedlings of at least 5 cm long, the samples were exposed to different Mn concentrations for a period of 15 days. We tested the effect of 4 different Mn concentrations added to the culture medium in the form of MnSO4 to obtain final concentrations of 50 ppm, 100 ppm, 200 ppm, 300 ppm; Mnuntreated plants were also cultured in the same conditions and used as control. The environmental conditions provided were: 16/8 hrs light/darkness; ambient temperature between 22 and 26 ◦ C, and light intensity 150-200 µ E m−2 s−1 . At the end of the growth period the samples were collected and prepared for the chemical and SEM-EDS analyses. 2.3

Chemical analysis

To determine the total concentration of the metals and their site of accumulation in the plant, samples were split in shoot and root, oven dried at 70 ◦ C and pulverized in a miller (Retsch S 100) equipped with an agate pocket. An aliquot of 250 mg of each sample were digested with HF (50%) and HNO3 (65%) at a ratio of 1:2, in a microwave oven (Milestone mls 1200-Microwave Laboratory Systems). To avoid the risk of contamination, blank samples (mineralization solutions without plant samples) were also analysed. Elemental concentrations were measured by AAS (SpectrAA-Varian) with graphite furnace and calculated considering the values of the blanks; the measurements were performed for each sample in triplicate; in addition, the mean value and standard error were calculated. A standard reference material (CTA-OTL 1, tobacco leaves) was analysed in parallel and used to calculate recover percentages and for analytical quality control. The content of Fe, K, Mg, Mn and Zn in the standard reference material showed recovery percentages in the range 86-109%, indicating a good accuracy of the chemical analysis. 2.4

SEM-EDS analysis

For SEM-EDS analysis, small pieces (1-3 mm) of leaves and roots of both control and Mn-treated plants of Cistus salvifolius were fixed in glutaraldehyde at 2% in phosphate buffer 65 mM at pH 7.2 for 2 hours. Samples were then dehydrated in 10 ml of alcohol at increasing concentrations (30, 50, 70, 90 and 100%) for a time of 15 min for each concentration except 100% of alcohol left to act for 1 hour. After dehydration, the samples were dried in a stove at 40 ◦ C for 1 hour, mounted on aluminum stubs and coated with a thin carbon layer to make conductive their surface. Samples were observed under a scanning electron microscope (SEM; JEOL JSM 5310). The samples were also analyzed by energy-dispersive X-ray spectroscopy (EDX; Oxford INCA).

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Table 1 Element concentrations (µ g/g d.w.) in the shoots (mean ± SE, n = 3) of C. salvifolius. Different letters indicate significant differences (P