This article was downloaded by: [Dalhousie University] On: 13 July 2014, At: 22:59 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Bioremediation of an Iron-Rich Mine Effluent by Lemna minor a
S. Teixeira , M. N. Vieira
b c
a
, J. Espinha Marques & R. Pereira
b d
a
Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences , University of Porto , Porto , Portugal b
Department of Biology, Faculty of Sciences , University of Porto , Porto , Portugal c
CIIMAR - Interdisciplinary Centre of Marine and Environmental Research , University of Porto , Porto , Portugal d
Click for updates
CESAM – Centre of Environmental and Marine Studies , University of Aveiro , Aveiro , Portugal Accepted author version posted online: 12 Aug 2013.Published online: 10 Mar 2014.
To cite this article: S. Teixeira , M. N. Vieira , J. Espinha Marques & R. Pereira (2014) Bioremediation of an Iron-Rich Mine Effluent by Lemna minor , International Journal of Phytoremediation, 16:12, 1228-1240, DOI: 10.1080/15226514.2013.821454 To link to this article: http://dx.doi.org/10.1080/15226514.2013.821454
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Dalhousie University] at 22:59 13 July 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
International Journal of Phytoremediation, 16:1228–1240, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.821454
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY LEMNA MINOR S. Teixeira,1 M. N. Vieira,2,3 J. Espinha Marques,1 and R. Pereira2,4
Downloaded by [Dalhousie University] at 22:59 13 July 2014
1
Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences, University of Porto, Porto, Portugal 2 Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal 3 CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal 4 CESAM – Centre of Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal Contamination of water resources by mine effluents is a serious environmental problem. In a old coal mine, in the north of Portugal (S˜ao Pedro da Cova, Gondomar), forty years after the activity has ended, a neutral mine drainage, rich in iron (FE) it stills being produced and it is continuously released in local streams (Ribeiro de Murta e Rio Ferreira) and in surrounding lands. The species Lemna minor has been shown to be a good model for ecotoxicological studies and it also has the capacity to bioaccumulate metals. The work aimed test the potential of the species L. minor to remediate this mine effluent, through the bioaccumulation of Fe, under greenhouse experiments and, at the same time, evaluate the time required to the maximum removal of Fe. The results have shown that L. minor was able to grow and develop in the Fe-rich effluent and bioaccumulating this element. Throughout the 21 days of testing it was found that there was a meaningful increase in the biomass of L. minor both in the contaminated and in the non-contaminated waters. It was also found that bioaccumulation of Fe (iron) occurred mainly during the first 7 days of testing. It was found that L. minor has potential for the bioremediation of effluents rich in iron. KEY WORDS: neutral mine drainage, phytoremediation, iron, Lemna minor
INTRODUCTION Mining activity deeply alters soils properties and generates large amounts of wastes, usually accumulated in piles. Such waste accumulations represent a serious environmental problem that affects soil and sediments, groundwater and surface waters, living beings and ecosystems (Babbou-Abdelmalek et al. 2011).
Address correspondence to Sara Teixeira, Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences, University of Porto, Rua do Campo Alegre 4169-007 Porto, Portugal. E-mail: sararmteixeira@ gmail.com 1228
Downloaded by [Dalhousie University] at 22:59 13 July 2014
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY L. MINOR
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The solid wastes are usually present during mine exploitation or in abandoned mines, exposed to atmospheric conditions, where apart from acid mine drainage they can also give rise to neutral mining drainage (NMD) (Babbou-Abdelmalek et al. 2011). This type of drainage can occur in coal or metallic mines due to: i) low concentration of sulphides; ii) the presence of monosulphides instead of pyrite; iii) the grain size of pyrite; iv) the acid neutralization by natural waters with high alkalinity or by the presence of mineral carbonates or silicates; v) groundwater circulation without direct contact with the sulfides or, vi) the lack of contact of sulfides with oxygen (Mayo et al. 2000; Dold and Fontbot´e 2002; Desbartats and Dirom 2007). The contamination of groundwater in mining areas is an important problem because it affects an essential source of drinking and irrigation water. Besides, since in the hydrological systems groundwater circulation influences surface water circulation, for example, by providing stream baseflow, this type of contamination may be widely spread in the environment (e.g., Fetter 1999, 2001; Kløve et al. 2011; Keesstra et al. 2012). In the last few decades, the use of plants and algae as agents for the removal of pollutants from water has awakening the interest of the scientific community, (e.g., Kara 2004) and has been subject of several studies aiming to assess the real bioaccumulation capabilities of different plant species (Khellaf and Zerdaoui 2009; Kara et al. 2003; Alvarado et al. 2008; Hossain et al. 2011; Rahman and Hasegawa 2011). Macrophytes are plants present in almost all types of aquatic habitats (Hossain et al. 2011; Rahman and Hasegawa 2011; Mudgal et al. 2010; Kheir et al. 2007). They are responsible by biomass and oxygen production, the fixation and stabilization of sediments, the provision of habitat and refuge for other living beings, the removal of toxic substances and nutrients from water and sediments, among others services (Mudgal et al. 2010). From the standpoint of environmental remediation, is their ability to detoxify or remove toxic substances from their medium that makes macrophytes a group of plants with so much interest. Some species of macrophytes have been often tested to remove nutrients (Kiran et al. 1991; Fang et al. 2007; Maltby et al. 2010), metals (e.g., Kara 2004; Mkandawire and Dudel 2005; Demirezen et al. 2007; Hou et al. 2007; Alvarado et al. 2008; Mishra and Tripathi 2008) and organic pollutants, including emerging substances (e.g., Olette et al. 2008; Dosnon-Ollete et al. 2009; Reinhold et al. 2010) from aquatic systems. Within others the floating aquatic hyperaccumulating species, are of particular interest as they accumulate contaminants with all the body (Rahman and Hasegawa 2011). Lemna minor Linn´e (1753) is a good example of macrophytes, with great abilities for water remediation (Mkandawire and Dudel 2007). This is a kind of small floating monocotyledoneous macrophyte belonging to the family of Lemnaceae (Maltby et al. 2010). They are common and abundant in many slow flowing and lentic nutrient-rich water bodies, both in tropical and temperate regions (Mkandawire and Dudel 2007; Maltby et al. 2010). Due to their small size, rapid growth rate, vegetative reproduction, ease of cultivation and adaption to different environmental conditions and sensitivity to numerous pollutants, Lemna species were also considered an appropriate model for ecotoxicological studies (Wang et al. 1987) and good ecological indicators (B¨oc¨uk et al. 2013). Despite entering into nonsense with their reported sensitivity (e.g., Drost et al. 2007), Lemna species, have proved a major role in the extraction and accumulation of metals from the water (Kara et al. 2003) adapting well to adverse conditions in terms of contamination. Some studies indicate that L. minor can accumulate high concentrations of several metals and metalloids, namely nickel, copper, cadmium, zinc, manganese, boron, uranium and arsenic (Jain et al. 1988; Mkandawire and Dudel 2005; Demirezen et al. 2007; Hou et al. 2007; Alvarado et al.
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2008; Megateli et al. 2009; Uysal and Taner 2010; B¨oc¨uk et al. 2013). Other authors also admit that L. minor might be used for the removal and accumulation of other metal such as lead, iron and mercury, as it is a plant with a great hiperaccumulation potential (Khellaf and Zerdaoui 2009). The aim of the present work was to test the ability of Lemna minor to remove iron from an neutral iron-rich effluent which is contributing for the contamination of a small stream (Ribeiro de Murta) and of a main river downstream (Rio Ferreira). The time required for maximum bioaccumulation/biosorption was also tested. MATERIAL AND METHODS
Downloaded by [Dalhousie University] at 22:59 13 July 2014
Origin of the Effluent The exploitation of coal (anthracites) in the Douro Coalfield (N Portugal) began around 1795, lasted for nearly two centuries and took place in several sites including the S. Pedro da Cova Mine (e.g., Lemos de Sousa and Wagner 1983), where the underground mining ceased in the early 1970’s. For decades, mine effluents with high iron concentration have been drained by galleries emerging approximately 1300 m to SE of the main mining area and released in local streams (Ribeiro de Murta and Rio Ferreira) and in the surrounding soils. Presently, the drainage is neutral most likely due to acid neutralization by alkaline thermomineral waters. Water Sampling and Analysis Water sampling was carried out in Ribeiro de Murta, S˜ao Pedro de Cova (Porto, Portugal). Five distinct sampling points were chosen, namely: point 1 located upstream of the mine galleries in Ribeiro de Murta; point 2 and 3 immediately after the discharges of the effluent from the mine galleries; point 4 located downstream of the mine galleries and point 5 in the confluence of Ribeiro de Murta and Rio Ferreira. Surface water samples were collected in plastic bottles properly decontaminated by acid washing. The water to be used in the phytoremediation assay with L. minor, for the determination of nutrients and for the R was frozen at –20◦ C. The water samples for the determination toxicity test with Microtox of iron content were acidified with nitric acid 65% (v/v), Suprapur, Merck. Water pH, electrical conductivity (mS/ cm), temperature (◦ C), total dissolved solids – TDS (mg/L) and dissolved oxygen (mg O2 / L) were measured in situ using a multiparametric probe HI98129 from Hanna Instruments. Total content of iron in water samples was determined by Atomic Absorption Spectrometry, with flame atomization by the method described in Reis et al. (2012), and the results are expressed in mg/L. The concentration of nitrates, nitrites, phosphates and ammonia present in the water of the five sampling points were measured using a photometer C200 from Hanna Instruments, following the methods proposed by the supplier and approved by R 1978). the Environmental Protection Agency (EPA) (Hanna Instruments R Toxicity Assay with Microtox
The toxicity of the water samples collected at different sampling points in the stream R assay. During this assay, the bacteria and in the river was evaluated with the Microtox Vibrio fischeri is exposed for 5, 15, and 30 min to different concentrations of the water samples collected (15 ± 1◦ C) in accordance with the 81.9% Basic Test protocol, in a 500 Microtox Toxicity Analyzer (Azur Environmental 1998) At the end of the assays the value
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY L. MINOR
1231
of highest percentage of effect (% Effect), for each sample, was recorded since it was not possible to calculate EC20 or EC50 values and respective confidence intervals. Plant Material - Lemna minor
Downloaded by [Dalhousie University] at 22:59 13 July 2014
L. minor fronds used in the phytoremediation experiments were collected in a greenhouse of the botanical garden of Porto. The macrophyte fronds were brought to the laboratory in a plastic box filled with water from the pond, to which they were adapted and where they were developing healthy. A sample of the same water was collected, in the starting day of the experiment, to be used as control in the assay, since no contamination was expected in this water. In the laboratory, the fronds of L. minor were visually inspected and carefully selected for the assay. Phytoremediation Assay In the phytoremediation assay were considered six treatments: i) four treatments with contaminated water from points 2 and 3; these points were tested up to concentrations of 100% and 50%, since it was feared that growth of macrophytes was inhibited in the undiluted samples; ii) water from point 1 (non-contaminated) which basically acted as field control, and water from the botanical garden pond (additional control). For each treatment and exposure period (7, 14, and 21 days) were prepared three replicates, in a total of 54. Each replicate (prepared in propylene vessels with 5.5 cm diameter and 7.3 cm height) contained 40 mL of the corresponding water sample and 60 L. minor fronds randomly assigned. The test was carried out in the greenhouse of Botanical Garden of Porto, where the macrophyte were maintained under conditions of light and temperature to which they were adapted. L. minor Growth At the beginning of the assay, three sets of 60 L. minor fronds were randomly separated from the initial pool of fronds, to estimate the dry weight of the fronds added to each treatment. The three sets of fronds were placed in an oven in aluminum foil, at 70◦ C, for 36 hours, till weight stabilization, and thereafter weight to the nearest 0.001 g. An average dry weight of 6.7 ± 0.8 mg of L. minor fronds was initially added to each treatment. At the end of each test period (T7, T14, and T21), the fronds of L. minor from each replicate were collected for dry weight determination, by the methodology described above. Iron Total Content of Fronds After dry weight determination, the dried material of each replicate was digested for subsequent determination of iron total content, in the fronds, grown during different exposure periods. To this end, 1 mL of nitric acid was added to each replicate which were left at room temperature, for 24 hours. Afterwards, 250 μL of hydrogen peroxide R ) were added to each tube, followed by heating at 60◦ C, for 8 hours. (Suprapur Merck Once cooled, the samples were centrifuged at 4000 rpm, for 10 minutes, and the supernatant was decanted. This step was required to remove some white fibers that subsisted after the wet digestion. The samples were then diluted with deionized water till a volume of 10 mL
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and analyzed by ICP-MS (Thermo X-Series Quadrupole ICP-MS, Thermo Scientific). For quality control of the wet digestion process, two sample blanks were prepared following the procedure described above, but without sample addition. The bioconcentration factors (BCF) (Rezvani and Zaefarian 2011) for fronds growing in non-diluted water from sites 2 and 3, were calculated as the ratio of the concentration of iron in the L. minor dry biomass and the concentration in the water.
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Statistical Analysis Fronds biomass (dry weight) and iron total content data recorded during the phytoremediation assay were subjected to statistical analysis, using the software IBM SPSS Statistics V.20. Initially a two-way ANOVA test was carried out to assess the effect of exposure time and type of water in both endpoints. Thereafter, one-way ANOVAs were performed to test the effect of each factor separately in order to verify if there were differences over time in terms of biomass and Fe bioaccumulated between macrophytes exposed to different types of water, within each exposure period. When a significant interaction between both factors (exposure time X type of water) was recorded in the previous twoway ANOVA, one-way ANOVAs and Tukey multicomparison tests were performed with correction for simple main effects (Zar 2010). The analysis of variance was performed, even when the assumption of the homogeneity of variances was not respected, since this analysis was considered robust when ni for groups are equal (Zar 2010). RESULTS AND DISCUSSION Water Physical and Chemical Parameters In Table 1 are shown the results for the assessment of physical-chemical parameters of water samples collected near the mine, and in the pond of Botanical Garden of Porto. Table 2 displays the Maximum Recommended Values (MRV) and Maximum Permissible Values (MAV) set by Decree-Law 236/98, of 1st of August, for water for irrigation purposes (Annex XVI) (MA 1998). Table 1 Physico-chemical parameters determined for water samples collected in the S. Pedro da Cova mine and in the pond of the Botanical Garden of Porto.
Parameter
Point 1
Point 2
Point 3
Point 4
Point 5
Botanical Garden
pH Electrical Conductivity (μS/cm) Total dissolved solids (mg/L) Dissolved oxygen (saturation%) Nitrate (mg/L NO3 –) Ammonia (mg/L NH3 ) Phosphate (mg/L PO4 –) DL: 0.241 μmol/L Nitrite (mg/L NO2 –) DL: 0.268 μmol/L Iron (mg/L)
7.49 70 30 78.04 0.001 0.001 0.12 BDL
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Bioremediation of an Iron-Rich Mine Effluent by Lemna minor a
S. Teixeira , M. N. Vieira
b c
a
, J. Espinha Marques & R. Pereira
b d
a
Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences , University of Porto , Porto , Portugal b
Department of Biology, Faculty of Sciences , University of Porto , Porto , Portugal c
CIIMAR - Interdisciplinary Centre of Marine and Environmental Research , University of Porto , Porto , Portugal d
Click for updates
CESAM – Centre of Environmental and Marine Studies , University of Aveiro , Aveiro , Portugal Accepted author version posted online: 12 Aug 2013.Published online: 10 Mar 2014.
To cite this article: S. Teixeira , M. N. Vieira , J. Espinha Marques & R. Pereira (2014) Bioremediation of an Iron-Rich Mine Effluent by Lemna minor , International Journal of Phytoremediation, 16:12, 1228-1240, DOI: 10.1080/15226514.2013.821454 To link to this article: http://dx.doi.org/10.1080/15226514.2013.821454
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Dalhousie University] at 22:59 13 July 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
International Journal of Phytoremediation, 16:1228–1240, 2014 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.821454
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY LEMNA MINOR S. Teixeira,1 M. N. Vieira,2,3 J. Espinha Marques,1 and R. Pereira2,4
Downloaded by [Dalhousie University] at 22:59 13 July 2014
1
Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences, University of Porto, Porto, Portugal 2 Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal 3 CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Portugal 4 CESAM – Centre of Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal Contamination of water resources by mine effluents is a serious environmental problem. In a old coal mine, in the north of Portugal (S˜ao Pedro da Cova, Gondomar), forty years after the activity has ended, a neutral mine drainage, rich in iron (FE) it stills being produced and it is continuously released in local streams (Ribeiro de Murta e Rio Ferreira) and in surrounding lands. The species Lemna minor has been shown to be a good model for ecotoxicological studies and it also has the capacity to bioaccumulate metals. The work aimed test the potential of the species L. minor to remediate this mine effluent, through the bioaccumulation of Fe, under greenhouse experiments and, at the same time, evaluate the time required to the maximum removal of Fe. The results have shown that L. minor was able to grow and develop in the Fe-rich effluent and bioaccumulating this element. Throughout the 21 days of testing it was found that there was a meaningful increase in the biomass of L. minor both in the contaminated and in the non-contaminated waters. It was also found that bioaccumulation of Fe (iron) occurred mainly during the first 7 days of testing. It was found that L. minor has potential for the bioremediation of effluents rich in iron. KEY WORDS: neutral mine drainage, phytoremediation, iron, Lemna minor
INTRODUCTION Mining activity deeply alters soils properties and generates large amounts of wastes, usually accumulated in piles. Such waste accumulations represent a serious environmental problem that affects soil and sediments, groundwater and surface waters, living beings and ecosystems (Babbou-Abdelmalek et al. 2011).
Address correspondence to Sara Teixeira, Department of Geosciences, Environment and Spatial Planning, Faculty of Sciences, University of Porto, Rua do Campo Alegre 4169-007 Porto, Portugal. E-mail: sararmteixeira@ gmail.com 1228
Downloaded by [Dalhousie University] at 22:59 13 July 2014
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY L. MINOR
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The solid wastes are usually present during mine exploitation or in abandoned mines, exposed to atmospheric conditions, where apart from acid mine drainage they can also give rise to neutral mining drainage (NMD) (Babbou-Abdelmalek et al. 2011). This type of drainage can occur in coal or metallic mines due to: i) low concentration of sulphides; ii) the presence of monosulphides instead of pyrite; iii) the grain size of pyrite; iv) the acid neutralization by natural waters with high alkalinity or by the presence of mineral carbonates or silicates; v) groundwater circulation without direct contact with the sulfides or, vi) the lack of contact of sulfides with oxygen (Mayo et al. 2000; Dold and Fontbot´e 2002; Desbartats and Dirom 2007). The contamination of groundwater in mining areas is an important problem because it affects an essential source of drinking and irrigation water. Besides, since in the hydrological systems groundwater circulation influences surface water circulation, for example, by providing stream baseflow, this type of contamination may be widely spread in the environment (e.g., Fetter 1999, 2001; Kløve et al. 2011; Keesstra et al. 2012). In the last few decades, the use of plants and algae as agents for the removal of pollutants from water has awakening the interest of the scientific community, (e.g., Kara 2004) and has been subject of several studies aiming to assess the real bioaccumulation capabilities of different plant species (Khellaf and Zerdaoui 2009; Kara et al. 2003; Alvarado et al. 2008; Hossain et al. 2011; Rahman and Hasegawa 2011). Macrophytes are plants present in almost all types of aquatic habitats (Hossain et al. 2011; Rahman and Hasegawa 2011; Mudgal et al. 2010; Kheir et al. 2007). They are responsible by biomass and oxygen production, the fixation and stabilization of sediments, the provision of habitat and refuge for other living beings, the removal of toxic substances and nutrients from water and sediments, among others services (Mudgal et al. 2010). From the standpoint of environmental remediation, is their ability to detoxify or remove toxic substances from their medium that makes macrophytes a group of plants with so much interest. Some species of macrophytes have been often tested to remove nutrients (Kiran et al. 1991; Fang et al. 2007; Maltby et al. 2010), metals (e.g., Kara 2004; Mkandawire and Dudel 2005; Demirezen et al. 2007; Hou et al. 2007; Alvarado et al. 2008; Mishra and Tripathi 2008) and organic pollutants, including emerging substances (e.g., Olette et al. 2008; Dosnon-Ollete et al. 2009; Reinhold et al. 2010) from aquatic systems. Within others the floating aquatic hyperaccumulating species, are of particular interest as they accumulate contaminants with all the body (Rahman and Hasegawa 2011). Lemna minor Linn´e (1753) is a good example of macrophytes, with great abilities for water remediation (Mkandawire and Dudel 2007). This is a kind of small floating monocotyledoneous macrophyte belonging to the family of Lemnaceae (Maltby et al. 2010). They are common and abundant in many slow flowing and lentic nutrient-rich water bodies, both in tropical and temperate regions (Mkandawire and Dudel 2007; Maltby et al. 2010). Due to their small size, rapid growth rate, vegetative reproduction, ease of cultivation and adaption to different environmental conditions and sensitivity to numerous pollutants, Lemna species were also considered an appropriate model for ecotoxicological studies (Wang et al. 1987) and good ecological indicators (B¨oc¨uk et al. 2013). Despite entering into nonsense with their reported sensitivity (e.g., Drost et al. 2007), Lemna species, have proved a major role in the extraction and accumulation of metals from the water (Kara et al. 2003) adapting well to adverse conditions in terms of contamination. Some studies indicate that L. minor can accumulate high concentrations of several metals and metalloids, namely nickel, copper, cadmium, zinc, manganese, boron, uranium and arsenic (Jain et al. 1988; Mkandawire and Dudel 2005; Demirezen et al. 2007; Hou et al. 2007; Alvarado et al.
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2008; Megateli et al. 2009; Uysal and Taner 2010; B¨oc¨uk et al. 2013). Other authors also admit that L. minor might be used for the removal and accumulation of other metal such as lead, iron and mercury, as it is a plant with a great hiperaccumulation potential (Khellaf and Zerdaoui 2009). The aim of the present work was to test the ability of Lemna minor to remove iron from an neutral iron-rich effluent which is contributing for the contamination of a small stream (Ribeiro de Murta) and of a main river downstream (Rio Ferreira). The time required for maximum bioaccumulation/biosorption was also tested. MATERIAL AND METHODS
Downloaded by [Dalhousie University] at 22:59 13 July 2014
Origin of the Effluent The exploitation of coal (anthracites) in the Douro Coalfield (N Portugal) began around 1795, lasted for nearly two centuries and took place in several sites including the S. Pedro da Cova Mine (e.g., Lemos de Sousa and Wagner 1983), where the underground mining ceased in the early 1970’s. For decades, mine effluents with high iron concentration have been drained by galleries emerging approximately 1300 m to SE of the main mining area and released in local streams (Ribeiro de Murta and Rio Ferreira) and in the surrounding soils. Presently, the drainage is neutral most likely due to acid neutralization by alkaline thermomineral waters. Water Sampling and Analysis Water sampling was carried out in Ribeiro de Murta, S˜ao Pedro de Cova (Porto, Portugal). Five distinct sampling points were chosen, namely: point 1 located upstream of the mine galleries in Ribeiro de Murta; point 2 and 3 immediately after the discharges of the effluent from the mine galleries; point 4 located downstream of the mine galleries and point 5 in the confluence of Ribeiro de Murta and Rio Ferreira. Surface water samples were collected in plastic bottles properly decontaminated by acid washing. The water to be used in the phytoremediation assay with L. minor, for the determination of nutrients and for the R was frozen at –20◦ C. The water samples for the determination toxicity test with Microtox of iron content were acidified with nitric acid 65% (v/v), Suprapur, Merck. Water pH, electrical conductivity (mS/ cm), temperature (◦ C), total dissolved solids – TDS (mg/L) and dissolved oxygen (mg O2 / L) were measured in situ using a multiparametric probe HI98129 from Hanna Instruments. Total content of iron in water samples was determined by Atomic Absorption Spectrometry, with flame atomization by the method described in Reis et al. (2012), and the results are expressed in mg/L. The concentration of nitrates, nitrites, phosphates and ammonia present in the water of the five sampling points were measured using a photometer C200 from Hanna Instruments, following the methods proposed by the supplier and approved by R 1978). the Environmental Protection Agency (EPA) (Hanna Instruments R Toxicity Assay with Microtox
The toxicity of the water samples collected at different sampling points in the stream R assay. During this assay, the bacteria and in the river was evaluated with the Microtox Vibrio fischeri is exposed for 5, 15, and 30 min to different concentrations of the water samples collected (15 ± 1◦ C) in accordance with the 81.9% Basic Test protocol, in a 500 Microtox Toxicity Analyzer (Azur Environmental 1998) At the end of the assays the value
BIOREMEDIATION OF AN IRON-RICH MINE EFFLUENT BY L. MINOR
1231
of highest percentage of effect (% Effect), for each sample, was recorded since it was not possible to calculate EC20 or EC50 values and respective confidence intervals. Plant Material - Lemna minor
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L. minor fronds used in the phytoremediation experiments were collected in a greenhouse of the botanical garden of Porto. The macrophyte fronds were brought to the laboratory in a plastic box filled with water from the pond, to which they were adapted and where they were developing healthy. A sample of the same water was collected, in the starting day of the experiment, to be used as control in the assay, since no contamination was expected in this water. In the laboratory, the fronds of L. minor were visually inspected and carefully selected for the assay. Phytoremediation Assay In the phytoremediation assay were considered six treatments: i) four treatments with contaminated water from points 2 and 3; these points were tested up to concentrations of 100% and 50%, since it was feared that growth of macrophytes was inhibited in the undiluted samples; ii) water from point 1 (non-contaminated) which basically acted as field control, and water from the botanical garden pond (additional control). For each treatment and exposure period (7, 14, and 21 days) were prepared three replicates, in a total of 54. Each replicate (prepared in propylene vessels with 5.5 cm diameter and 7.3 cm height) contained 40 mL of the corresponding water sample and 60 L. minor fronds randomly assigned. The test was carried out in the greenhouse of Botanical Garden of Porto, where the macrophyte were maintained under conditions of light and temperature to which they were adapted. L. minor Growth At the beginning of the assay, three sets of 60 L. minor fronds were randomly separated from the initial pool of fronds, to estimate the dry weight of the fronds added to each treatment. The three sets of fronds were placed in an oven in aluminum foil, at 70◦ C, for 36 hours, till weight stabilization, and thereafter weight to the nearest 0.001 g. An average dry weight of 6.7 ± 0.8 mg of L. minor fronds was initially added to each treatment. At the end of each test period (T7, T14, and T21), the fronds of L. minor from each replicate were collected for dry weight determination, by the methodology described above. Iron Total Content of Fronds After dry weight determination, the dried material of each replicate was digested for subsequent determination of iron total content, in the fronds, grown during different exposure periods. To this end, 1 mL of nitric acid was added to each replicate which were left at room temperature, for 24 hours. Afterwards, 250 μL of hydrogen peroxide R ) were added to each tube, followed by heating at 60◦ C, for 8 hours. (Suprapur Merck Once cooled, the samples were centrifuged at 4000 rpm, for 10 minutes, and the supernatant was decanted. This step was required to remove some white fibers that subsisted after the wet digestion. The samples were then diluted with deionized water till a volume of 10 mL
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and analyzed by ICP-MS (Thermo X-Series Quadrupole ICP-MS, Thermo Scientific). For quality control of the wet digestion process, two sample blanks were prepared following the procedure described above, but without sample addition. The bioconcentration factors (BCF) (Rezvani and Zaefarian 2011) for fronds growing in non-diluted water from sites 2 and 3, were calculated as the ratio of the concentration of iron in the L. minor dry biomass and the concentration in the water.
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Statistical Analysis Fronds biomass (dry weight) and iron total content data recorded during the phytoremediation assay were subjected to statistical analysis, using the software IBM SPSS Statistics V.20. Initially a two-way ANOVA test was carried out to assess the effect of exposure time and type of water in both endpoints. Thereafter, one-way ANOVAs were performed to test the effect of each factor separately in order to verify if there were differences over time in terms of biomass and Fe bioaccumulated between macrophytes exposed to different types of water, within each exposure period. When a significant interaction between both factors (exposure time X type of water) was recorded in the previous twoway ANOVA, one-way ANOVAs and Tukey multicomparison tests were performed with correction for simple main effects (Zar 2010). The analysis of variance was performed, even when the assumption of the homogeneity of variances was not respected, since this analysis was considered robust when ni for groups are equal (Zar 2010). RESULTS AND DISCUSSION Water Physical and Chemical Parameters In Table 1 are shown the results for the assessment of physical-chemical parameters of water samples collected near the mine, and in the pond of Botanical Garden of Porto. Table 2 displays the Maximum Recommended Values (MRV) and Maximum Permissible Values (MAV) set by Decree-Law 236/98, of 1st of August, for water for irrigation purposes (Annex XVI) (MA 1998). Table 1 Physico-chemical parameters determined for water samples collected in the S. Pedro da Cova mine and in the pond of the Botanical Garden of Porto.
Parameter
Point 1
Point 2
Point 3
Point 4
Point 5
Botanical Garden
pH Electrical Conductivity (μS/cm) Total dissolved solids (mg/L) Dissolved oxygen (saturation%) Nitrate (mg/L NO3 –) Ammonia (mg/L NH3 ) Phosphate (mg/L PO4 –) DL: 0.241 μmol/L Nitrite (mg/L NO2 –) DL: 0.268 μmol/L Iron (mg/L)
7.49 70 30 78.04 0.001 0.001 0.12 BDL
