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Effects of lead accumulation on the Azolla caroliniana–Anabaena association
Ecotoxicology and Environmental Safety 102 (2014) 100–104
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of lead accumulation on the Azolla caroliniana–Anabaena association Anne E. Roberts a,b, Charles W. Boylen a,b, Sandra A. Nierzwicki-Bauer a,b,n a b
Darrin Fresh Water Institute, Rensselaer Polytechnic Institute, 5060 Lakeshore Drive, Bolton Landing, NY 12814, United States Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, United States
art ic l e i nf o
a b s t r a c t
Article history: Received 22 September 2013 Received in revised form 11 January 2014 Accepted 13 January 2014 Available online 5 February 2014
The effect of lead accumulation on photopigment production, mineral nutrition, and Anabaena vegetative cell size and heterocyst formation in Azolla caroliniana was investigated. Plants were exposed to 0, 1, 5, 10, and 20 mg L 1 lead acetate for ten days. Lead accumulation increased when plants were treated with higher lead concentrations. Results revealed a statistically significant decline in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids in 5, 10, and 20 mg Pb L 1 treatment groups as compared to plants with 0 or 1 mg Pb L 1 treatments. No statistically significant change in anthocyanin production was observed. Calcium, magnesium, and zinc concentrations in plants decreased in increasing treatment groups, whereas sodium and potassium concentrations increased. Nitrogen and carbon were also found to decrease in plant tissue. Anabaena vegetative cells decreased in size and heterocyst frequency declined rapidly in a Pb dose-dependent manner. These results indicate that, while A. caroliniana removes lead from aqueous solution, the heavy metal causes physiological and biochemical changes by impairing photosynthesis, changing mineral nutrition, and impeding the growth and formation of heterocysts of the symbiotic cyanobacteria that live within leaf cavities of the fronds. & 2014 Published by Elsevier Inc.
Keywords: Azolla Lead accumulation Toxicology Symbiosis Phytoremediation
1. Introduction The heavy metal lead (Pb) is a hazardous and persistent environmental pollutant. Pb is used in many industrial and mining processes and is highly toxic to humans when ingested or inhaled (Duruibe et al., 2007). While Pb has no known biological function in aquatic plants, many plants have the ability to sequester large amounts of the metal via bioaccumulation (Pourrut et al., 2011). Known as phytoremediation, this process is an emerging tool used to extract metals from the environment (Environmental Protection Agency (EPA), 2000). Phytoremediation is an efficient and cost effective method to decontaminate environments. However, in order to optimize these systems, knowledge of how Pb affects plant physiology must be obtained prior to system design (PilonSmits, 2005). Lead has been shown to interfere with photosynthesis and change elemental makeup in many plants, ultimately leading to chlorosis and stunted growth (Sengar et al., 2008). Toxicological response to heavy metals varies amongst plant species (Pourrut et al., 2011); therefore, any plant under consideration for Pb removal should be evaluated prior to its use. A leading candidate for Pb removal from aquatic systems is the water fern, Azolla (Sood et al., 2012). Azolla is a small floating n Corresponding author at: Darrin Fresh Water Institute, Rensselaer Polytechnic Institute, 5060 Lakeshore Drive, Bolton Landing, NY 12814, United States. E-mail addresses: [email protected] (A.E. Roberts), [email protected] (C.W. Boylen), [email protected] (S.A. Nierzwicki-Bauer).
0147-6513/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ecoenv.2014.01.019
macrophyte with a nearly global distribution. Azolla can be found on the surfaces of rice paddies, lakes, ponds, and marshes, and can tolerate a wide range of environmental conditions. Under ideal growth conditions, Azolla has doubling rates of approximately 2–4 days. This rapid production of biomass over a short period of time makes Azolla an ideal candidate for use in phytoremediation systems. As the fern floats on the water surface, it is easy to dispose of when the plant becomes saturated with a pollutant. Because the weight of the plant is 90–94% water, once dried, biomass volume is reduced dramatically, allowing for convenient transport and disposal (Pabby et al., 2004; Scharpenseel and Knuth, 1985). Azolla been shown to successfully extract Pb directly from environmental sites, such as a coalmine effluent and a flyash pond (Bharti and Banerjee, 2012; Pandey, 2012). Many studies have monitored Azolla's biomass and Pb accumulation overtime and all indicate that the plant is effective for the removal of Pb from aquatic solutions (Gaumat et al., 2008; Guar et al., 1994; Jafari et al., 2010; Jain et al., 1990; Rakhshaee et al., 2006; Stepniewska et al., 2005). Many of these studies have also investigated ways in which to increase Pb accumulation by Azolla through chemically treating the fern or its environment (Khorsravi et al., 2005; Taghi ganji et al., 2005; Rakhshaee et al., 2006). Only a few studies have looked in depth at the toxicological effects of Pb on Azolla. Gaumat et al. (2008) studied photosynthetic changes in Azolla pinnata, and Oren Benaroya et al. (2004) included research on the impact of mineral nutrient uptake by Azolla filiculoides. Each species of Azolla
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may exhibit differences in tolerance to Pb toxicity; therefore, before Azolla caroliniana can be fully exploited for phytoremediation purposes, a thorough investigation on how the plant is affected by Pb has been conducted. To date, no study has examined the toxicology effects of Pb on the fern's nitrogen-fixing symbiotic partner, Anabaena azollae, which lives in the leaf cavities. A. azollae can provide the total nitrogen demand of the fern, allowing the fern to thrive in nitrogen-free environments (Peters and Meeks, 1989). As the health of A. azollae is critical to this symbiosis, it is vital to understand if the symbiont is affected by Pb. If the fern is used for Pb remediation in a nitrogen-limiting environment and the N-production of A. azollae is affected by Pb, Azolla may not perform to its full capacity. Most uptake studies performed on Azolla for Pb accumulation have used lead nitrate (Oren Benaroya et al., 2004; Gaur et al., 1994; Khorsravi et al., 2005; Jain et al., 1990; Rakhshaee et al., 2006; Stepniewska et al., 2005; Taghi Ganji et al., 2005), which may shield the effects of Pb toxicity on the symbiosis by the addition of nitrate. To eliminate this complication, lead acetate was used as the Pb source in this study. The aim of this study was to determine what effect Pb accumulation has on photopigment production, mineral nutrition, and Anabaena heterocyst formation in A. caroliniana grown in N-free medium. The results of this research will help guide the efficient use of Azolla in Pb removal from aqueous environments.
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2.4. Pigment analysis After 10 days, whole plants were collected from each treatment group and weighed. Plants were ground in a small glass homogenizer in chilled 80% acetone. Samples were centrifuged for 2 min at 5000 rpm. The supernatant was transferred to borosilicate tubes and read in a Spectronic Genesys 5 spectrophotometer at absorbances of 470, 646, and 663 nm. Total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid concentrations were estimated according to the equations by Lichtenthaler and Wellburn (1983). Anthocyanin content was estimated as described by Sims and Gamon (2002). All pigment analyses were performed under low light conditions. 2.5. Anabaena preparation and cell analysis One plant was selected from each replicate and cyanobacteria were exuded using the “gentle roller” method (Peters and Mayne, 1974). Squash buffer (Tris: EDTA, 50:50 v/v, pH¼ 8) was collected and wet mounts were prepared from 1 mL of solution. Slides were observed using a 10 ocular and a 40 objective (400 ) using an inverted Nikon microscope. Vegetative cells and heterocysts were counted in filaments found in 10 random frames for each sample, 200 cells per sample. Heterocyst frequency per filament was determined. Vegetative cell area was measured using SPOT software 4.7 where 468 sensor pixels¼ 50 mm. Vegetative cells were measured in five fields of view and photographed at 400 . 2.6. Statistical analysis Significant differences between treatments for each analyte were determined at p r 0.05 using the statistical software SigmaPlot 11.0© using a one-way ANOVA, followed by a Bonferroni post hoc analysis. All data are expressed as mean 7 standard error (SE).
2. Materials and methods
3. Results 2.1. Azolla cultivation A. caroliniana (strain 3001) was obtained from the Azolla Germplasm Collection at the International Rice Research Institute (Manila, Philippines). Plants were cultured in a nitrogen free IRRI nutrient solution: 20 mg L 1 NaH2PO4, 40 mg L 1 K2SO4, 40 mg L 1 CaCl2, 40 mg L 1 MgSO4, 0.5 mg L 1 FeSO4 EDTA, 0.5 MnCl2, 0.15 mg L 1 NaMoO4, 0.2 mg L 1H3BO3, 0.01 mg L 1 ZnSO4, 0.01 mg L 1 CuSO4, and 0.01 mg L 1 CoCl2, buffered to pH 5.5 70.2 with 1 M NaOH (Watanabe et al., 1992). Plants were maintained in a plant growth chamber in 250 mL glass beakers under a 16/8 h light/dark cycle at a temperature of 25 1C/22 1C with cool white fluorescent lights at 45 μmol m 2 s 1.
2.2. Experimental design Approximately 2 g fresh weight of A. caroliniana was transferred to beakers containing 100 mL of 0, 1, 5, 10, or 20 mg L 1 Pb uptake solution, pH 6.5 7 0.2. These concentrations were selected for the results of this study to be comparable with previously performed research (Oren Benaroya et al., 2004; Gaumat et al., 2008; Jafari et al., 2010; Jain et al., 1990; Rakhshaee et al., 2006). Pb uptake solution was prepared in IRRI media using a stock lead acetate solution. Six replicates were prepared for each treatment. Experiments were performed in a random block design under the growth conditions described above. To negate changes in Pb concentration due to evaporation, a static renewal test was employed. On days 2, 4, 6, and 8 Pb uptake solution was completely replenished, and 0.1 g of plant material was removed for pigment analysis. On day 10, the plants were removed from the medium, washed thoroughly with distilled water, and separated for subsequent metal, nutrient, and pigment analysis. Plants were also collected for microscopic examination.
3.1. Pb accumulation in A. caroliniana Lead accumulation occurred in all treatments, excluding the control which contained no addition of Pb (Fig. 1). As Pb treatments increased, plants accumulated higher concentrations of the metal. It was observed that plants in the 10 and 20 mg Pb L 1 treatment groups appeared smaller and slightly yellow in color compared to the control following a 10-day incubation period. Higher Pb concentration treatments also resulted in root abscission. 3.2. Changes mineral nutrition Mineral nutrient content in A. caroliniana changed under Pb stress (Table 1A). Levels of Ca in the plants decreased with
2.3. Metal and nutrient analysis Plants were oven dried at 80 1C for approximately 48 h or until constant weight remained (Schor-Fumbarov et al., 2005; Gaur et al., 1994). A small amount of dried plant material from each treatment was removed to analyze total nitrogen and carbon content using a CE-Elantech CNH analyzer. Prior to digestion in 2 mL concentrated nitric acid, the weight of the plant was recorded. Digests were performed similar to the method of Guar et al. (1994). Briefly, digests were placed in a heat block until a clear solution of approximately 0.5 mL remained. Digests were brought to a standard volume with deionized water for a sample concentration with 1% nitric acid. Uptake solution and digested plant material were tested for Pb, Ca, Mg, Zn, Na, and K concentrations using a Perkin-Elmer Atomic Absorption Spectrophotometer.
Fig. 1. Pb accumulation in plants after 10 days incubation in differing lead concentrations: 0, 1, 5, 10, and 20 mg Pb L 1. Mean values are expressed as bars7 S.E. (n ¼6).
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Table 1 (A) The effect of Pb treatment on the mineral composition of Ca, Na, K, Mg, Zn, and N in A. caroliniana. (B) The impact of Pb on carotenoid and anthocyanin content in A. caroliniana. Mean values are expressed 7 S.E. (n¼6). Small letters indicate significant differences between treatments at p r0.05. A. Mineral nutrient content Pb Treatment (mg L 1) 0 1 5 10 20
Ca (mg g d.w. 1)
Na (mg g d.w. 1)
K (mg g d.w. 1)
Mg (mg g d.w. 1)
Zn (mg g d.w. 1)
N (mg g d.w. 1)
6.6 7 0.42a 5.3 7 0.79a/b 4.3 7 0.44a/b 3.6 7 0.49b 3.6 7 0.41b
8.2 7 0.82a 20.17 2.51a/b 23.2 7 3.51b 20.0 7 3.37a/b 27.17 3.23b
14.8 7 1.00a 43.5 7 6.16b 57.5 7 7.67b 42.6 7 6.57b 56.4 7 6.07b
1.2 7 0.12 1.17 0.20 0.84 7 0.08 0.86 7 0.19 0.89 7 0.05
0.137 0.03 0.107 0.02 0.08 7 0.01 0.077 0.01 0.077 0.01
4.0 7 0.07a 3.9 7 0.11a/b 3.7 7 0.14a/b 3.7 7 0.11a/b 3.5 7 0.08b
B. Carotenoid and anthocyanin content Pb Treatment (mg L 1)
Carotenoids (mg g f.w. 1)
0 1 5 10 20
26.7 7 2.9a 26.6 7 2.3a 14.3 7 1.2b 13.17 1.6b 11.7 7 1.4b
Anthocyanins (ABS529–0.24(ABS650) g f.w. 1) 1.9 7 0.18 2.7 7 0.54 1.9 7 0.08 1.9 7 0.23 1.6 7 0.19
Significance analyzed within each treatment. a is significantly different than b. a/b is not significantly different from a or b.
Fig. 2. The effect of Pb on total chlorophyll (white bars), chlorophyll a (gray bars), chlorophyll b (light gray bars), and carbon content (black circles) in A. caroliniana. Bars represent mean values7 S.E. (n ¼6). Significant differences (pr 0.05) in total chlorophyll between Pb treatments are indicated by the small letters a and b. The number of stars (* or **) indicate a significant different (pr 0.05) in chlorophyll a between Pb treatments. Significant differences (p r 0.05) in chlorophyll b between Pb treatments are indicated by large letters (A, B, or C). Labels that contain the same letter indicate similarity.
Fig. 3. The area of vegetative cells measured from each Pb treatment. The total number of vegetative cells measured from each treatment group are as follows: 852, 546, 601, 466, 584 for 0, 1, 5, 10, and 20 mg Pb L 1 respectively. Bars represent mean values7 S.E. Small letters indicate significant differences between given treatments at p r 0.05. Labels that contain the same letter indicate similarity.
increasing Pb concentrations. Significant decreases in Ca were found in the two highest treatment groups, 10 and 20 mg Pb L 1. A decrease in Mg and Zn was also observed in plant tissue; however, these changes were not statistically significant. Interestingly, levels of both K and Na were found to increase in plants with higher Pb concentrations. All plants exposed to Pb were found to have a significant change in K concentrations, whereas, a significant increase in Na was only seen in plants grown in the 5 and 20 mg Pb L 1 treatments. Total N and C were found to decrease (Table 1A and Fig. 2). Plants in the highest Pb concentration treatment group had a statistically significant decrease in N.
highest Pb treatments (Fig. 2). This trend was also observed for chlorophyll a concentrations. The lowest metal treatment, 1 mg Pb L 1, had little impact on photopigment production. Chlorophyll b had a significant decrease only in the 5 mg Pb L 1 treatment group as compared to the control. Carotenoids were found to decrease significantly in the three highest Pb concentration treatments (Table 1B). There was no net change in anthocyanin production regardless of the Pb treatment concentration (Table 1B).
3.3. Changes in photopigments
Pb accumulation had a significant impact on the size of vegetative cells within Anabena filaments as well as the percentage of heterocysts in filaments (Figs. 3 and 4). Vegetative cell area decreased significantly in 5 mg Pb L 1 as compared to the control and the lowest Pb treatment. Vegetative cells continued to decline
Pb accumulation in A. caroliniana affected the production of total chlorophyll, chlorophyll a, and chlorophyll b (Fig. 2). Total chlorophyll was found to decrease significantly only in the three
3.4. Changes in vegetative cell size and heterocyst frequency
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Fig. 4. The effect of Pb accumulation on Anabaena heterocyst frequency. Bars represent mean values of average heterocyst frequency calculated for each plant 7 S.E. (n ¼6). Small letters indicate significant differences between treatments for heterocyst frequency at p r 0.05. Labels that contain the same letter indicate similarity.
in size in the 10 and 20 mg Pb L 1 treatments. The number of heterocysts present within filaments decreased as Pb accumulation increased. Plant treatments at 1 and 5 mg Pb L 1 concentrations had significantly fewer heterocysts than the controls. Plants in treatments 10 and 20 mg Pb L 1 had significantly fewer heterocysts than the control and 1, and 5 mg Pb L 1 treatments. There were approximately 50% fewer heterocysts in plants from the two highest treatment concentrations.
4. Discussion Azolla has been shown repeatedly to be an excellent candidate for metal remediation (Sood et al., 2012). As demonstrated in this study, the fern is able to accumulate Pb; however, the fern and its symbiont are not immune to metal toxicity. While previous uptake studies have shown the effect of Pb on the biomass of Azolla (Gaur et al., 1994; Khosravi et al., 2005; Rakhshaee et al., 2006; Stepniewska et al., 2005), only two studies have noted more specific biochemical and physiological changes (Oren Benaroya et al., 2004; Gaumat et al., 2008). The results of this study support some of the previous findings, but the studies used different Azolla species and different forms of Pb; therefore, comparisons must be made with this in mind. Oren Benaroya et al. (2004) found Pb to accumulate in the vacuoles and in the cell walls of Azolla but Pb was not localized in the cyanobacterial symbiont. The results of the current study indicate that the symbiont is negatively affected by the presence of Pb through diminished cell growth and reduced production of heterocysts (Figs. 3 and 4). As the nitrogen-fixing cyanobacteria were the only source of nitrogen for Azolla in this study, the reduced number of heterocysts may have led to the N deficiency (Table 1A). This postulation is supported by a study performed using the free-living cyanobacteria, Nostoc muscorum, which showed that Pb induced a loss of heterocysts formation and also inhibited nitrogenase reductase (Rai et al., 1990). Loss of heterocyst formation leads to senescence in Azolla if the fern is not provided an additional source of N (Forni et al., 1991). With the reduction of symbionts containing heterocysts, adding additional nitrate to the solution may be a way to keep the fern healthier longer in order to maximize its efficiency during Pb remediation.
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The decrease in total chlorophyll and decline in total carbon found in this study supports the previous findings of Gaumat et al. (2008), which showed a reduction of chlorophyll in A. pinnata after incubation with Pb. This study confirms that Pb exposure to Azolla causes an interruption of photosynthesis. Carotenoids have been suggested as potential protective mechanisms in Azolla against oxidative stress caused by arsenic exposure (Sánchez-Viveros et al., 2011), but this study found Pb was deleterious to carotenoid levels in Azolla. Carotenoid levels may increase in plants shortly after response to metal stress to help combat oxidative stress; however, studies have shown that decreased carotenoid content is a common response after many days of continued exposure to metal stress (Leblebici and Aksoy, 2011; Rastgoo and Alemzadeh, 2011; Shu et al., 2012; Wang et al., 2011). Likewise, anthocyanin production in Azolla may also increase shortly after exposure to metals in response to oxidative stress (Dai et al., 2012). Over time, anthocyanin production may diminish due to loss of other macronutrients vital to the plant, such as Zn (Hajiboland and Amirazad, 2010). As this study measured these photopigments after a 10-day period, any immediate increases would not have been observed. Pb may also not cause a strong change in anthocyanin synthesis in Azolla as compared to changes caused by Cd. Heavy metal stress has been shown to interfere with nutrient uptake in plants (Sengar et al., 2008). Lead induced nutrient changes appear to vary widely in plants (Cao et al., 2004; Kibria et al., 2009; Wang et al., 2011). Changes in mineral nutrition may occur from blockage of root absorption, a decrease in translocation processes, or by competition between ions, impairing critical physiological processes (Pourrut et al., 2011). The results of this study showed a slightly different response in mineral nutrient uptake to Pb than previously reported by Oren Benaroya et al. (2004). The latter study found an increase in Na and Ca and a decrease Mg. The current study showed an increase in Na and K, but a decrease in both Ca and Mg. This difference may be due to the difference in lead salts added but may also be a species specific response as A. filiculoides was used in the previous study and A. caroliniana in the current study. Relationships to the elements that changed under Pb stress in Azolla with known uptake pathways or stress responses should be studied in the future to determine what metabolic processes are specifically changed as a result of Pb toxicity.
5. Conclusions While A. caroliniana is capable of accumulating an abundance of the heavy metal Pb, the effects of the metal are deleterious to the health of the fern and its cyanobacterial symbiont. Chlorophyll and carotenoid levels in the plant were found to decrease, and changes in vital nutrients Ca, Mg, Zn, N, and C were found to decrease or for Na and K, increase. Likewise, the health of the nitrogen-fixing cyanobacterial symbionts appeared diminished through size of vegetative cells and a reduction in heterocyst frequency, likely contributing to the reduction of the health of the fern. By more accurately monitoring the health of the plant caused by the decline of the symbiotic nitrogen-fixing cyanobacteria which had been masked in previous studies using lead nitrate, a more complete understanding of the impact of Pb on the Azolla– Anabaena association has been elucidated. These results strengthen the assertion that a variety of Azolla species are potentially useful candidates for Pb remediation. These results also indicate that Azolla plants may need to be removed and replenished after 10 days when exposed to higher concentrations of Pb due to the negative impacts of these higher Pb concentrations on the plant's health.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of lead accumulation on the Azolla caroliniana–Anabaena association Anne E. Roberts a,b, Charles W. Boylen a,b, Sandra A. Nierzwicki-Bauer a,b,n a b
Darrin Fresh Water Institute, Rensselaer Polytechnic Institute, 5060 Lakeshore Drive, Bolton Landing, NY 12814, United States Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, United States
art ic l e i nf o
a b s t r a c t
Article history: Received 22 September 2013 Received in revised form 11 January 2014 Accepted 13 January 2014 Available online 5 February 2014
The effect of lead accumulation on photopigment production, mineral nutrition, and Anabaena vegetative cell size and heterocyst formation in Azolla caroliniana was investigated. Plants were exposed to 0, 1, 5, 10, and 20 mg L 1 lead acetate for ten days. Lead accumulation increased when plants were treated with higher lead concentrations. Results revealed a statistically significant decline in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids in 5, 10, and 20 mg Pb L 1 treatment groups as compared to plants with 0 or 1 mg Pb L 1 treatments. No statistically significant change in anthocyanin production was observed. Calcium, magnesium, and zinc concentrations in plants decreased in increasing treatment groups, whereas sodium and potassium concentrations increased. Nitrogen and carbon were also found to decrease in plant tissue. Anabaena vegetative cells decreased in size and heterocyst frequency declined rapidly in a Pb dose-dependent manner. These results indicate that, while A. caroliniana removes lead from aqueous solution, the heavy metal causes physiological and biochemical changes by impairing photosynthesis, changing mineral nutrition, and impeding the growth and formation of heterocysts of the symbiotic cyanobacteria that live within leaf cavities of the fronds. & 2014 Published by Elsevier Inc.
Keywords: Azolla Lead accumulation Toxicology Symbiosis Phytoremediation
1. Introduction The heavy metal lead (Pb) is a hazardous and persistent environmental pollutant. Pb is used in many industrial and mining processes and is highly toxic to humans when ingested or inhaled (Duruibe et al., 2007). While Pb has no known biological function in aquatic plants, many plants have the ability to sequester large amounts of the metal via bioaccumulation (Pourrut et al., 2011). Known as phytoremediation, this process is an emerging tool used to extract metals from the environment (Environmental Protection Agency (EPA), 2000). Phytoremediation is an efficient and cost effective method to decontaminate environments. However, in order to optimize these systems, knowledge of how Pb affects plant physiology must be obtained prior to system design (PilonSmits, 2005). Lead has been shown to interfere with photosynthesis and change elemental makeup in many plants, ultimately leading to chlorosis and stunted growth (Sengar et al., 2008). Toxicological response to heavy metals varies amongst plant species (Pourrut et al., 2011); therefore, any plant under consideration for Pb removal should be evaluated prior to its use. A leading candidate for Pb removal from aquatic systems is the water fern, Azolla (Sood et al., 2012). Azolla is a small floating n Corresponding author at: Darrin Fresh Water Institute, Rensselaer Polytechnic Institute, 5060 Lakeshore Drive, Bolton Landing, NY 12814, United States. E-mail addresses: [email protected] (A.E. Roberts), [email protected] (C.W. Boylen), [email protected] (S.A. Nierzwicki-Bauer).
0147-6513/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ecoenv.2014.01.019
macrophyte with a nearly global distribution. Azolla can be found on the surfaces of rice paddies, lakes, ponds, and marshes, and can tolerate a wide range of environmental conditions. Under ideal growth conditions, Azolla has doubling rates of approximately 2–4 days. This rapid production of biomass over a short period of time makes Azolla an ideal candidate for use in phytoremediation systems. As the fern floats on the water surface, it is easy to dispose of when the plant becomes saturated with a pollutant. Because the weight of the plant is 90–94% water, once dried, biomass volume is reduced dramatically, allowing for convenient transport and disposal (Pabby et al., 2004; Scharpenseel and Knuth, 1985). Azolla been shown to successfully extract Pb directly from environmental sites, such as a coalmine effluent and a flyash pond (Bharti and Banerjee, 2012; Pandey, 2012). Many studies have monitored Azolla's biomass and Pb accumulation overtime and all indicate that the plant is effective for the removal of Pb from aquatic solutions (Gaumat et al., 2008; Guar et al., 1994; Jafari et al., 2010; Jain et al., 1990; Rakhshaee et al., 2006; Stepniewska et al., 2005). Many of these studies have also investigated ways in which to increase Pb accumulation by Azolla through chemically treating the fern or its environment (Khorsravi et al., 2005; Taghi ganji et al., 2005; Rakhshaee et al., 2006). Only a few studies have looked in depth at the toxicological effects of Pb on Azolla. Gaumat et al. (2008) studied photosynthetic changes in Azolla pinnata, and Oren Benaroya et al. (2004) included research on the impact of mineral nutrient uptake by Azolla filiculoides. Each species of Azolla
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may exhibit differences in tolerance to Pb toxicity; therefore, before Azolla caroliniana can be fully exploited for phytoremediation purposes, a thorough investigation on how the plant is affected by Pb has been conducted. To date, no study has examined the toxicology effects of Pb on the fern's nitrogen-fixing symbiotic partner, Anabaena azollae, which lives in the leaf cavities. A. azollae can provide the total nitrogen demand of the fern, allowing the fern to thrive in nitrogen-free environments (Peters and Meeks, 1989). As the health of A. azollae is critical to this symbiosis, it is vital to understand if the symbiont is affected by Pb. If the fern is used for Pb remediation in a nitrogen-limiting environment and the N-production of A. azollae is affected by Pb, Azolla may not perform to its full capacity. Most uptake studies performed on Azolla for Pb accumulation have used lead nitrate (Oren Benaroya et al., 2004; Gaur et al., 1994; Khorsravi et al., 2005; Jain et al., 1990; Rakhshaee et al., 2006; Stepniewska et al., 2005; Taghi Ganji et al., 2005), which may shield the effects of Pb toxicity on the symbiosis by the addition of nitrate. To eliminate this complication, lead acetate was used as the Pb source in this study. The aim of this study was to determine what effect Pb accumulation has on photopigment production, mineral nutrition, and Anabaena heterocyst formation in A. caroliniana grown in N-free medium. The results of this research will help guide the efficient use of Azolla in Pb removal from aqueous environments.
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2.4. Pigment analysis After 10 days, whole plants were collected from each treatment group and weighed. Plants were ground in a small glass homogenizer in chilled 80% acetone. Samples were centrifuged for 2 min at 5000 rpm. The supernatant was transferred to borosilicate tubes and read in a Spectronic Genesys 5 spectrophotometer at absorbances of 470, 646, and 663 nm. Total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid concentrations were estimated according to the equations by Lichtenthaler and Wellburn (1983). Anthocyanin content was estimated as described by Sims and Gamon (2002). All pigment analyses were performed under low light conditions. 2.5. Anabaena preparation and cell analysis One plant was selected from each replicate and cyanobacteria were exuded using the “gentle roller” method (Peters and Mayne, 1974). Squash buffer (Tris: EDTA, 50:50 v/v, pH¼ 8) was collected and wet mounts were prepared from 1 mL of solution. Slides were observed using a 10 ocular and a 40 objective (400 ) using an inverted Nikon microscope. Vegetative cells and heterocysts were counted in filaments found in 10 random frames for each sample, 200 cells per sample. Heterocyst frequency per filament was determined. Vegetative cell area was measured using SPOT software 4.7 where 468 sensor pixels¼ 50 mm. Vegetative cells were measured in five fields of view and photographed at 400 . 2.6. Statistical analysis Significant differences between treatments for each analyte were determined at p r 0.05 using the statistical software SigmaPlot 11.0© using a one-way ANOVA, followed by a Bonferroni post hoc analysis. All data are expressed as mean 7 standard error (SE).
2. Materials and methods
3. Results 2.1. Azolla cultivation A. caroliniana (strain 3001) was obtained from the Azolla Germplasm Collection at the International Rice Research Institute (Manila, Philippines). Plants were cultured in a nitrogen free IRRI nutrient solution: 20 mg L 1 NaH2PO4, 40 mg L 1 K2SO4, 40 mg L 1 CaCl2, 40 mg L 1 MgSO4, 0.5 mg L 1 FeSO4 EDTA, 0.5 MnCl2, 0.15 mg L 1 NaMoO4, 0.2 mg L 1H3BO3, 0.01 mg L 1 ZnSO4, 0.01 mg L 1 CuSO4, and 0.01 mg L 1 CoCl2, buffered to pH 5.5 70.2 with 1 M NaOH (Watanabe et al., 1992). Plants were maintained in a plant growth chamber in 250 mL glass beakers under a 16/8 h light/dark cycle at a temperature of 25 1C/22 1C with cool white fluorescent lights at 45 μmol m 2 s 1.
2.2. Experimental design Approximately 2 g fresh weight of A. caroliniana was transferred to beakers containing 100 mL of 0, 1, 5, 10, or 20 mg L 1 Pb uptake solution, pH 6.5 7 0.2. These concentrations were selected for the results of this study to be comparable with previously performed research (Oren Benaroya et al., 2004; Gaumat et al., 2008; Jafari et al., 2010; Jain et al., 1990; Rakhshaee et al., 2006). Pb uptake solution was prepared in IRRI media using a stock lead acetate solution. Six replicates were prepared for each treatment. Experiments were performed in a random block design under the growth conditions described above. To negate changes in Pb concentration due to evaporation, a static renewal test was employed. On days 2, 4, 6, and 8 Pb uptake solution was completely replenished, and 0.1 g of plant material was removed for pigment analysis. On day 10, the plants were removed from the medium, washed thoroughly with distilled water, and separated for subsequent metal, nutrient, and pigment analysis. Plants were also collected for microscopic examination.
3.1. Pb accumulation in A. caroliniana Lead accumulation occurred in all treatments, excluding the control which contained no addition of Pb (Fig. 1). As Pb treatments increased, plants accumulated higher concentrations of the metal. It was observed that plants in the 10 and 20 mg Pb L 1 treatment groups appeared smaller and slightly yellow in color compared to the control following a 10-day incubation period. Higher Pb concentration treatments also resulted in root abscission. 3.2. Changes mineral nutrition Mineral nutrient content in A. caroliniana changed under Pb stress (Table 1A). Levels of Ca in the plants decreased with
2.3. Metal and nutrient analysis Plants were oven dried at 80 1C for approximately 48 h or until constant weight remained (Schor-Fumbarov et al., 2005; Gaur et al., 1994). A small amount of dried plant material from each treatment was removed to analyze total nitrogen and carbon content using a CE-Elantech CNH analyzer. Prior to digestion in 2 mL concentrated nitric acid, the weight of the plant was recorded. Digests were performed similar to the method of Guar et al. (1994). Briefly, digests were placed in a heat block until a clear solution of approximately 0.5 mL remained. Digests were brought to a standard volume with deionized water for a sample concentration with 1% nitric acid. Uptake solution and digested plant material were tested for Pb, Ca, Mg, Zn, Na, and K concentrations using a Perkin-Elmer Atomic Absorption Spectrophotometer.
Fig. 1. Pb accumulation in plants after 10 days incubation in differing lead concentrations: 0, 1, 5, 10, and 20 mg Pb L 1. Mean values are expressed as bars7 S.E. (n ¼6).
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Table 1 (A) The effect of Pb treatment on the mineral composition of Ca, Na, K, Mg, Zn, and N in A. caroliniana. (B) The impact of Pb on carotenoid and anthocyanin content in A. caroliniana. Mean values are expressed 7 S.E. (n¼6). Small letters indicate significant differences between treatments at p r0.05. A. Mineral nutrient content Pb Treatment (mg L 1) 0 1 5 10 20
Ca (mg g d.w. 1)
Na (mg g d.w. 1)
K (mg g d.w. 1)
Mg (mg g d.w. 1)
Zn (mg g d.w. 1)
N (mg g d.w. 1)
6.6 7 0.42a 5.3 7 0.79a/b 4.3 7 0.44a/b 3.6 7 0.49b 3.6 7 0.41b
8.2 7 0.82a 20.17 2.51a/b 23.2 7 3.51b 20.0 7 3.37a/b 27.17 3.23b
14.8 7 1.00a 43.5 7 6.16b 57.5 7 7.67b 42.6 7 6.57b 56.4 7 6.07b
1.2 7 0.12 1.17 0.20 0.84 7 0.08 0.86 7 0.19 0.89 7 0.05
0.137 0.03 0.107 0.02 0.08 7 0.01 0.077 0.01 0.077 0.01
4.0 7 0.07a 3.9 7 0.11a/b 3.7 7 0.14a/b 3.7 7 0.11a/b 3.5 7 0.08b
B. Carotenoid and anthocyanin content Pb Treatment (mg L 1)
Carotenoids (mg g f.w. 1)
0 1 5 10 20
26.7 7 2.9a 26.6 7 2.3a 14.3 7 1.2b 13.17 1.6b 11.7 7 1.4b
Anthocyanins (ABS529–0.24(ABS650) g f.w. 1) 1.9 7 0.18 2.7 7 0.54 1.9 7 0.08 1.9 7 0.23 1.6 7 0.19
Significance analyzed within each treatment. a is significantly different than b. a/b is not significantly different from a or b.
Fig. 2. The effect of Pb on total chlorophyll (white bars), chlorophyll a (gray bars), chlorophyll b (light gray bars), and carbon content (black circles) in A. caroliniana. Bars represent mean values7 S.E. (n ¼6). Significant differences (pr 0.05) in total chlorophyll between Pb treatments are indicated by the small letters a and b. The number of stars (* or **) indicate a significant different (pr 0.05) in chlorophyll a between Pb treatments. Significant differences (p r 0.05) in chlorophyll b between Pb treatments are indicated by large letters (A, B, or C). Labels that contain the same letter indicate similarity.
Fig. 3. The area of vegetative cells measured from each Pb treatment. The total number of vegetative cells measured from each treatment group are as follows: 852, 546, 601, 466, 584 for 0, 1, 5, 10, and 20 mg Pb L 1 respectively. Bars represent mean values7 S.E. Small letters indicate significant differences between given treatments at p r 0.05. Labels that contain the same letter indicate similarity.
increasing Pb concentrations. Significant decreases in Ca were found in the two highest treatment groups, 10 and 20 mg Pb L 1. A decrease in Mg and Zn was also observed in plant tissue; however, these changes were not statistically significant. Interestingly, levels of both K and Na were found to increase in plants with higher Pb concentrations. All plants exposed to Pb were found to have a significant change in K concentrations, whereas, a significant increase in Na was only seen in plants grown in the 5 and 20 mg Pb L 1 treatments. Total N and C were found to decrease (Table 1A and Fig. 2). Plants in the highest Pb concentration treatment group had a statistically significant decrease in N.
highest Pb treatments (Fig. 2). This trend was also observed for chlorophyll a concentrations. The lowest metal treatment, 1 mg Pb L 1, had little impact on photopigment production. Chlorophyll b had a significant decrease only in the 5 mg Pb L 1 treatment group as compared to the control. Carotenoids were found to decrease significantly in the three highest Pb concentration treatments (Table 1B). There was no net change in anthocyanin production regardless of the Pb treatment concentration (Table 1B).
3.3. Changes in photopigments
Pb accumulation had a significant impact on the size of vegetative cells within Anabena filaments as well as the percentage of heterocysts in filaments (Figs. 3 and 4). Vegetative cell area decreased significantly in 5 mg Pb L 1 as compared to the control and the lowest Pb treatment. Vegetative cells continued to decline
Pb accumulation in A. caroliniana affected the production of total chlorophyll, chlorophyll a, and chlorophyll b (Fig. 2). Total chlorophyll was found to decrease significantly only in the three
3.4. Changes in vegetative cell size and heterocyst frequency
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Fig. 4. The effect of Pb accumulation on Anabaena heterocyst frequency. Bars represent mean values of average heterocyst frequency calculated for each plant 7 S.E. (n ¼6). Small letters indicate significant differences between treatments for heterocyst frequency at p r 0.05. Labels that contain the same letter indicate similarity.
in size in the 10 and 20 mg Pb L 1 treatments. The number of heterocysts present within filaments decreased as Pb accumulation increased. Plant treatments at 1 and 5 mg Pb L 1 concentrations had significantly fewer heterocysts than the controls. Plants in treatments 10 and 20 mg Pb L 1 had significantly fewer heterocysts than the control and 1, and 5 mg Pb L 1 treatments. There were approximately 50% fewer heterocysts in plants from the two highest treatment concentrations.
4. Discussion Azolla has been shown repeatedly to be an excellent candidate for metal remediation (Sood et al., 2012). As demonstrated in this study, the fern is able to accumulate Pb; however, the fern and its symbiont are not immune to metal toxicity. While previous uptake studies have shown the effect of Pb on the biomass of Azolla (Gaur et al., 1994; Khosravi et al., 2005; Rakhshaee et al., 2006; Stepniewska et al., 2005), only two studies have noted more specific biochemical and physiological changes (Oren Benaroya et al., 2004; Gaumat et al., 2008). The results of this study support some of the previous findings, but the studies used different Azolla species and different forms of Pb; therefore, comparisons must be made with this in mind. Oren Benaroya et al. (2004) found Pb to accumulate in the vacuoles and in the cell walls of Azolla but Pb was not localized in the cyanobacterial symbiont. The results of the current study indicate that the symbiont is negatively affected by the presence of Pb through diminished cell growth and reduced production of heterocysts (Figs. 3 and 4). As the nitrogen-fixing cyanobacteria were the only source of nitrogen for Azolla in this study, the reduced number of heterocysts may have led to the N deficiency (Table 1A). This postulation is supported by a study performed using the free-living cyanobacteria, Nostoc muscorum, which showed that Pb induced a loss of heterocysts formation and also inhibited nitrogenase reductase (Rai et al., 1990). Loss of heterocyst formation leads to senescence in Azolla if the fern is not provided an additional source of N (Forni et al., 1991). With the reduction of symbionts containing heterocysts, adding additional nitrate to the solution may be a way to keep the fern healthier longer in order to maximize its efficiency during Pb remediation.
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The decrease in total chlorophyll and decline in total carbon found in this study supports the previous findings of Gaumat et al. (2008), which showed a reduction of chlorophyll in A. pinnata after incubation with Pb. This study confirms that Pb exposure to Azolla causes an interruption of photosynthesis. Carotenoids have been suggested as potential protective mechanisms in Azolla against oxidative stress caused by arsenic exposure (Sánchez-Viveros et al., 2011), but this study found Pb was deleterious to carotenoid levels in Azolla. Carotenoid levels may increase in plants shortly after response to metal stress to help combat oxidative stress; however, studies have shown that decreased carotenoid content is a common response after many days of continued exposure to metal stress (Leblebici and Aksoy, 2011; Rastgoo and Alemzadeh, 2011; Shu et al., 2012; Wang et al., 2011). Likewise, anthocyanin production in Azolla may also increase shortly after exposure to metals in response to oxidative stress (Dai et al., 2012). Over time, anthocyanin production may diminish due to loss of other macronutrients vital to the plant, such as Zn (Hajiboland and Amirazad, 2010). As this study measured these photopigments after a 10-day period, any immediate increases would not have been observed. Pb may also not cause a strong change in anthocyanin synthesis in Azolla as compared to changes caused by Cd. Heavy metal stress has been shown to interfere with nutrient uptake in plants (Sengar et al., 2008). Lead induced nutrient changes appear to vary widely in plants (Cao et al., 2004; Kibria et al., 2009; Wang et al., 2011). Changes in mineral nutrition may occur from blockage of root absorption, a decrease in translocation processes, or by competition between ions, impairing critical physiological processes (Pourrut et al., 2011). The results of this study showed a slightly different response in mineral nutrient uptake to Pb than previously reported by Oren Benaroya et al. (2004). The latter study found an increase in Na and Ca and a decrease Mg. The current study showed an increase in Na and K, but a decrease in both Ca and Mg. This difference may be due to the difference in lead salts added but may also be a species specific response as A. filiculoides was used in the previous study and A. caroliniana in the current study. Relationships to the elements that changed under Pb stress in Azolla with known uptake pathways or stress responses should be studied in the future to determine what metabolic processes are specifically changed as a result of Pb toxicity.
5. Conclusions While A. caroliniana is capable of accumulating an abundance of the heavy metal Pb, the effects of the metal are deleterious to the health of the fern and its cyanobacterial symbiont. Chlorophyll and carotenoid levels in the plant were found to decrease, and changes in vital nutrients Ca, Mg, Zn, N, and C were found to decrease or for Na and K, increase. Likewise, the health of the nitrogen-fixing cyanobacterial symbionts appeared diminished through size of vegetative cells and a reduction in heterocyst frequency, likely contributing to the reduction of the health of the fern. By more accurately monitoring the health of the plant caused by the decline of the symbiotic nitrogen-fixing cyanobacteria which had been masked in previous studies using lead nitrate, a more complete understanding of the impact of Pb on the Azolla– Anabaena association has been elucidated. These results strengthen the assertion that a variety of Azolla species are potentially useful candidates for Pb remediation. These results also indicate that Azolla plants may need to be removed and replenished after 10 days when exposed to higher concentrations of Pb due to the negative impacts of these higher Pb concentrations on the plant's health.
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