Ecotoxicology and Environmental Safety 95 (2013) 27–32
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Phytotoxicity assessment of a methanolic coal dust extract in Lemna minor Nadia Coronado-Posada, Maria Cabarcas-Montalvo, Jesus Olivero-Verbel n Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia
art ic l e i nf o
a b s t r a c t
Article history: Received 2 December 2012 Received in revised form 10 April 2013 Accepted 2 May 2013 Available online 30 May 2013
Coal mining generates negative effects on environment, human health, hydrodynamics of mining areas and biodiversity. However, the impacts of this activity are less known in plants. Lemna minor is one of the most commonly used plants in aquatic toxicity tests due to its ubiquitous distribution in ponds and lakes, culture conditions and the free-floating habitat that exposes it to hydrophobic as well as dissolved compounds. The goal of this research was to evaluate the effects of a methanolic coal dust extract on L. minor. Macrophytes were exposed to six different concentrations of coal extract (from 7.81 to 250 mg/L) for 5 days, following the OECD test guideline 221. The coal extract had a half inhibitory concentration (IC50) of 99.66 (184.95–54.59) mg/L for the number of fronds. Several signs of toxicity such as chlorosis, reduction in the size of the fronds, abscission of fronds and roots, and the presence of necrotic tissues were observed at concentrations lower than the IC50. Preliminary Gas Chromatography-Mass Spectrometry analysis of the coal dust extract revealed the presence of several compounds, including, among others, alkanes, carboxylic acids and polycyclic aromatic hydrocarbons (PAHs), these lasts, may be responsible for some of the observed effects. These results demonstrated that coal dust has phytotoxic effects and should not be considered as an inert material. & 2013 Elsevier Inc. All rights reserved.
Keywords: Coal mining Growth inhibition Lemna minor Phytotoxicity
1. Introduction Coal is a combustible sedimentary rock and a versatile fossil fuel made of a complex and heterogeneous mixture of mostly organic matter (Alpern and Lemos de Sousa, 2002; Vassilev and Vassileva, 2009), and some inorganic materials. The International Energy Agency (IEA, 2010) reported coal as the second most used energy source in the world after oil. It is employed for electric power generation and other industrial processes, such as steel and cement production, as well as a liquid fuel (Miller, 2011). It has been estimated there are over 847 billion tonnes of coal reserves worldwide, available in almost every country. In Latin America, Colombia has the largest coal reserves, ranking first in the list of coal-producing countries (BP, 2012), being the USA and Europe the major destinations. According to the Colombian Mining and Energy Planning Unit (UPME, 2009), coal reserves are mainly located on the Atlantic Coast in the departments of La Guajira, Cesar, and Córdoba (Renzoni, 2006). Although coal mining is one of the core industries that contributes to the economic development of a region, it deteriorates the
n Correspondence to: Environmental and Computational Chemistry Group, School of Pharmaceutical Sciences, University of Cartagena, Campus of Zaragocilla Cartagena, Colombia. Fax: +57 5 6698323. E-mail addresses:
[email protected],
[email protected] (J. Olivero-Verbel).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.05.001
environment (Miller, 2005). The impact involves the whole coal fuel cycle: mining, transport, storage, combustion and conversion. Coal mining activity moves large quantities of pollutants such as dust particles, organic and inorganic compounds, trace elements, including arsenic (As), lead (Pb), cadmium (Cd) and mercury (Hg), causing a wide range of respiratory diseases and other human health problems (Finkelman, 2004; Miller, 2005). It is also a recognized source of polynuclear aromatic hydrocarbons (PAHs). These lipophilic substances can be absorbed through diet or occupational exposure, being further metabolized into highly reactive molecular initiators of carcinogenesis (Skupiska et al., 2004), nephropathy (Orem et al., 2006), immune system disorders, and inflammatory responses (Goulaouic et al., 2008). The release of pollutants from burning coal and waste banks presents potential environmental and human health hazards, especially when environmental controls are inefficient (Finkelman, 2004). The particles derived directly from the combustion of coal or its ashes have shown genotoxic effects in human lymphocytes (Kleinjans et al., 1989), transformation of human cells into tumor cells (Wu et al., 1990), and even participation in the reduction of photosynthetic activity in plants (Naidoo and Chirkoot, 2004). During coal mining and transport from mines to ports, tiny mineral particles are released by friction, forming the coal dust. In ports, the release of these particles during loading results in their accumulation on beaches. Long-time and intensive mining has caused spatio-temporal cumulative effects on soils, sediments and
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water, promoting ground subsidence, river hydrodynamics and land use changes, soil and air pollution and ecosystem evolution (León et al., 2007). This means deterioration of environmental quality in the affected areas, as well as the devaluation of land and infrastructure of tourism services. Coal dust extracts have also been reported to be cytotoxic and mutagenic in mammals (Ulker et al., 2008), and studies in coal mining areas have shown that organisms that live in these environments have greater DNA damage than those from reference sites (Cabarcas-Montalvo et al., 2012). However, there is a lack of research evaluating the phytotoxic potential of coal on aquatic plants. One of the widely accepted models for toxicity testing is the floating plant L. minor (Lemnaceae), commonly known as duckweed. The small size, simplicity of structure, asexual reproduction and short generation time are characteristics that make it a suitable species for laboratory tests (Wang, 1986, 1990). L. minor is recognized as a bioindicator of ecological relevance for the detection and monitoring of pollution (Garnczarska and Ratajczak, 2000). In the present study, specimens of L. minor were exposed to coal dust extracts prepared from samples collected at mines in the department of Cesar, Colombia, in order to assess toxic effects on the plant. The observed toxicity was then explained based on the analytical characterization of the coal extract.
2. Materials and methods 2.1. Duckweed sample Young plants of L. minor were collected from natural freshwater ponds, with no apparent sources of pollution, located within the urban zone of the city of Cartagena, and cultured in the lab for several weeks before used for the assays. The macrophytes were washed with tap water and healthy specimens selected for assays. The stock culture was maintained in the laboratory of Ecotoxicology at the University of Cartagena in a 4 L fishbowl. Plants were conditioned with culture medium, at temperature of 30 7 1 1C, and a 12:12 h light/dark cycle (Nauman et al., 2006).
during 5 consecutive days, with replenishment of culture media every 2.5 days, corresponding to a semi-static test system. Each experiment was run in triplicate, and a maximum DMSO concentration of 0.25 mg/L was used in the treatments and control during the experiment. This concentration had no measurable adverse effects on L. minor. 2.5. Evaluation of the effect of the methanolic coal dust extract solutions on L. minor The experiments performed to determine the phytotoxicity of coal extracts included daily measurements of the number of live/dead colonies, fronds and roots, at different tested concentrations. The occurrences of chlorosis and necrosis were recorded, and the last day of exposure, pictures were taken with a Nikon SMZ745T Stereoscopic Microscope. For the test to be valid, the doubling time (Td) of frond number in the control must be less than 2.5 days (60 h). In this work, Td was calculated using the formula Td¼(ln2/μ)nd (OECD TG 221, 2006), where μ is the average specific growth and d is the total number of days utilized for the experiment. This value was obtained for each test concentration and control, based on the number of fronds, utilizing the equation: μi−j ¼[ln(Nj)−ln(Ni)]/(Δt), where μi−j is the average specific growth rate from time i to j; Ni and Nj are the number of fronds observed in the test or control vessel at time i and j, respectively; and Δt is the time lapsed from the start of the exposure period to the end of the experiment. The concentration-response curve for the inhibition of growth rate (Ir) was obtained for each test concentration according to the formula: %Ir ¼ [(μC−μT)/μC] 100, where % Ir is the average percent inhibition of growth rate; μC is the mean value for μ in the control, and μT is the mean value for μ in the treatment group (OECD TG 221, 2006). 2.6. Gas chromatography-mass spectrometry (GC–MS) analysis The methanolic coal dust extract was analyzed by gas chromatography coupled to mass spectrometry (GC–MS). The chromatographic data were obtained with a gas chromatograph, GC 7890C (Agilent Technologies) equipped with a mass selective detector MSD 5975C (electron impact ionization, EI, 70 eV; Agilent Technologies), split/splitless injector, and an MSChemStation data system, which included the spectral libraries Wiley and NIST 2008. A capillary column HP-5 MS of 30 m 250 μm 0.25 μm coated with 5% phenyl methyl siloxane was used. The GC oven temperature was programmed from 45 1C (5 min) to 250 1C (5 min) at 5 1C/ min. The temperatures of the injection port and transfer line were set at 250 and 2850 1C, respectively. Helium (99.9990%, Cryogas, Cartagena, Colombia) was used as carrier gas, at 50 mL/min of constant flow. Mass spectra and reconstructed (total) ion chromatograms were obtained by automatic scanning at 3.85 scan/s. Chromatographic peaks were checked for homogeneity and identified with the aid of the mass chromatograms for the characteristic fragment ions, and the help of the peak purity function of the MS-Chemstation software.
2.2. Culture medium for experiments with L. minor 2.7. Statistical analysis The culture medium used in the experiments was described by Appenroth et al. (1996). It consisted of a solution containing 3.46 mM KNO3, 0.66 mM KH2PO4, 72 mM K2HPO4, 0.41 mM MgSO4, 1.25 mM Ca(NO3)2, 1.94 mM H3BO3, 0.63 mM ZnSO4, 0.18 mM Na2MoO4, 0.91 mM MnCl2, 2.81 mM FeCl3, and 4.03 mM EDTA-Na2. The nutrient solution was sterilized (121 1C, 20 min) and stored in a cool, dark place, adjusting the pH with NaOH (1 M) or HCl (1 M) to 6.5 (OECD TG 221, 2006; ISO DIS 20079, 2004).
The assumptions of normality, and equal variance were evaluated. ANOVA and Dunnett's post-test were used to compare the means of examined variables in the different groups or time exposures. The concentration of dust coal extract that produced 50% inhibition (IC50) of any particular variable was calculated by probit analysis (Finney, 1971). A P-valueo 0.05 was set as statistically significant.
2.3. Preparation of methanolic coal dust extract
3. Results
The coal samples were obtained from La Loma coal mine (10123′58′′ N, 75130′9′′ W) in the Department of Cesar, Colombia, the second largest in the country. The sample was grounded in a mortar, and sifted through a mesh screen (40/200) to get a particle size of ∼75 μm. For the preparation of the extract, 25 g of coal dust were used for Soxhlet extraction with 150 mL HPLC grade methanol for 12 h. The solvent was removed by rotary evaporation. A total of 20 different dust coal extractions were performed, producing around 10 g of a partially dried extract, which was subsequently freeze-dried. The extract was then dissolved in dimethyl sulfoxide (DMSO), sonicated for 18 hours, and then filtered through Whatman paper no. 40.
The specific growth rate (μ) and the doubling time (Td) of fronds in the control were 3.3570.32 day−1 and 1.03 days, respectively. These values are within the validity criteria according to the OECD guideline 221 (OECD TG 221, 2006). Time/concentration growth curves generated for number of colonies, fronds and roots of L. minor per vessel are presented in Fig. 1. After the first and second days of exposure, plants showed no significant changes in the evaluated parameters. Data for colonies showed statistical differences compared to control from day 3 for 125 mg/L and 250 mg/L concentrations (Fig. 1A). In the case of fronds, statistical differences were detected since day 3 for the three greatest concentrations (62.5, 125, 250 mg/L; Fig. 1B). However, compared to the control, on day 5 all the evaluated concentrations showed a statistically significant decrease in this parameter. For root number, changes were observed starting on day 3 for all tested concentrations (Fig. 1C). Taken together, these data revealed after the third day, methanolic coal dust extract solutions impacted the growth of the plants, in terms of number of colonies,
2.4. Macrophyte exposure to coal dust extract Methanolic coal dust extract solutions were prepared in culture medium, the same day of testing, at concentrations of 7.81, 15.62, 31.25, 62.5, 125 and 250 mg/L, adjusting the pH to 6.5 7 0.05. In this paper, the term “frond” is used in the sense of Ashby et al. (1949) and it refers to a single leaf from which root and younger (or older) attached appendages have been dissected away. Colony, on the other hand, is a frond with its root and pocket containing developing fronds. Healthy colonies formed by 2 or 3 visible fronds were transferred from the culture to test containers (75 mL glass vessels). Care was taken to avoid any physical injure to the plants. Each container had a total of 2 plants exposed to treatment or vehicle control (DMSO)
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Fig. 1. Effect of methanolic coal dust extract solutions in the growth of (A) colonies, (B) roots and (C) fronds of Lemna minor evaluated over a period of 5 days. Symbols on top of the graphs indicate statistical differences between particular tested concentrations and control.
Fig. 2. Percentage inhibition of Lemna minor growth exposed to methanolic coal dust extract solutions.
growth rate, with an IC50 of 99.66 mg/L (184.95 mg/L−54.59 mg/L; R2 ¼0.93) (Fig. 2). Morphological effects under examined conditions were also evaluated, including chlorosis, necrosis, and abscission of fronds and roots (Fig. 3). Chlorosis was the predominant feature, coupled with the abscission of the roots, and tissue necrosis. The 250 mg/L extract coal concentration resulted in a particular response. Under this conditions, it seems all the normal processes of the plant were halted, as 100% of the tissues were necrotized. However, the fronds maintained the same size and the roots remained attached to their colonies. It should be noted that during the last exposure days, the solutions turned murky, slightly brown, viscous, foul-smelling, and with algae in the necrotic fronds. The relative chemical composition of the examined sample of coal dust extract is presented in Fig. 4. The analysis revealed the presence of 142 different chemicals, including alkanes, alkyl and aromatic carboxylic acids, esters, and PAHs, among other compounds.
4. Discussion fronds and roots, compared to those obtained with the control samples (Fig. 1). However, among evaluated parameters, the roots of L. minor showed an increased sensitivity to the concentrations used in the experiment. The tolerance of the duckweed to coal dust solutions was determined by plotting a concentration/Ir response curve (Fig. 2). It shows a clear concentration-dependent inhibition of the frond
The results presented here demonstrate the methanolic coal extract causes phytotoxicity in L. minor. Aquatic plants exposed to methanolic coal dust extract solutions experienced toxic effects including chlorosis, decrease in the size of the fronds, abscission of fronds and roots, and tissue necrosis. The most characteristic phytotoxicity sign found in exposed plants was the presence of
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Fig. 3. Colonies of Lemna minor in the evaluated groups. (A) Control, (B) 7.81 mg/L, (C) 15.62 mg/L, (D) 31.25 mg/L, (E) 62.5 mg/L, (F) 125 mg/L, and (G) 250 mg/L. Some observed findings are evidenced in pictures.
Fig. 4. Chemical composition of the methanolic extract of coal dust. GC–MS analysis was performed as described in Materials and Methods. The relative distribution of all compounds (Panel A) or PAHs (Panel B) present in the chromatogram are depicted as pie graphs. Compounds identified to a match quality of 90% or greater against library spectra (Wiley and NIST 2008) were assumed to be reliable identifications. These chemicals were the following: 1. 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester. 2. 3-(2-Thiazol-5-Yl-Benzoimidazol-1-Yl)-Propionitrile. 3. Benzoic acid. 4. Docosanoic acid. 5. Eicosane. 6. Eicosanoic acid. 7. Heptadecane. 8. Heptadecanoic acid. 9. Hexacosane. 10. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester. 11. Octadecanoic acid. 12. Oxirane, hexadecyl. CB80H90. Compounds with match quality lower than 90% but greater than 80%. CB80. Compounds with match quality lower than 80%. In Panel B, identified compounds were: I. 1H-Cycloprop[e]azulene, decahydro-1,1,4,7-tetramethyl. II. Naphthalene, 1,6-dimethyl-4-(1-methylethyl). III. Other PAHs.
chlorotic leaves. This condition evidences photosynthesis impairment, leading to necrosis (Hess, 2000). In fact, any significant alteration in chlorophyll concentration may lead to marked effects
on the entire plant metabolism (Hamid et al., 2009). Coal extract exposure promotes a decrease in photosynthetic activity, linked to reduced plant growth, leaf area, and chlorophyll content. Several
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studies have reported an inverse correlation between the level of peroxidation and the rate of growth (Siegel and Galston, 1967; Bacon et al., 1997; Lin and Kao, 2001), suggesting a possible role for oxidative species on growth inhibition. Preliminary GC–MS analysis of the extract showed the presence of different types of hydrocarbons, mainly alkanes, and carboxylic acids, as well as PAHs (14.7%). These compounds act as ligands of the aryl hydrocarbon receptor (AhR); a ligand-activated transcription factor that regulates the transcription of a wide range of genes, including some drug metabolizing enzymes, several genes involved in cell proliferation, apoptosis and cell growth (phenomena evaluated) (Abel and Haarmann-Stemmann, 2010). These chemicals are also well recognized tumor initiators, as they can covalently bind to DNA, leading to adduct formation, which may end up in mutations and cell damage. Therefore, it is likely that the relatively high percentage of PAHs in the mixture may be responsible for the phytotoxicity effects detected in this assay. Since coal is formed from ancient plant material, it may also contain heavy metals. However, in this study, it is likely that these chemicals were not present in the methanolic extract, as it mainly removes organic compounds. Accordingly, observed effects may solely derive from the exposure to organic chemicals. The mechanism of abscission of fronds and roots in L. minor exposed to coal extracts can be interpreted as a response to stress experienced by the plant in contact with phytotoxic agents, whose action leads to separation and disintegration of the colony in isolated units with little chance of survival. Such frond abscission has been observed in the presence of known toxic compounds (Slovin, 1997; Li and Xiong, 2004). The presence of aromatic hydrocarbons and other substances with known toxic effects in the methanolic coal dust extract solutions may be associated with signs of toxicity found in the assay. In nature, it may be quite difficult to find concentrations of coal extract components as high as those used in this study, however, the results of the observed effects are a projection that must be taken into account in environmental impact studies of areas surrounding the mines. The coal dust and/or its components may reach aquatic ecosystems by leaching processes occurring in the mines, as well as by the release of material that is stored in the coal ports, or by the action of ocean currents on deposits in areas of low water, creating a potential risk to the environment and human health (National Research Council, 2006). In summary, these results revealed the occurrence of toxicity signs behavior and growth when plants were exposed to coal dust extracts. L. minor represents a microaquatic species that appears to be sensitive to these metabolites, and consequently it may be useful for measuring phytotoxic effects on plant growth.
5. Conclusions Obtained data evidenced that even low concentrations of methanolic coal extract produce phytotoxic effects verified by inhibition of growth based on colonies, fronds and roots number as well as decrease of chlorophylls content in L. minor. Also it was confirmed the relevance of duckweed as a sensitive indicator species of the phytotoxic potential of complex solutions such as those of coal dust extract.
Acknowledgments The authors wish to thank the University of Cartagena and Colciencias (Colombia) for their financial support (Grant 110749326186). Technical expertise from Nerlys Pajaro is also appreciated. Nadia Coronado-Posada is sponsored by the Virginia
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Gutierrez de Pineda Young Investigator Program of Colciencias University of Cartagena, Colombia (2012-2013).
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