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Preliminary test of arsenic and mercury uptake by Poa annua
e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
available at www.sciencedirect.com
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Review
Preliminary test of arsenic and mercury uptake by Poa annua Elena Comino ∗ , Adriano Fiorucci, Stefania Menegatti, Cecilia Marocco Politecnico di Torino, Land, Environment and Geo-Engineering Department, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
a r t i c l e
i n f o
a b s t r a c t
Article history:
Arsenic (As) and mercury (Hg) are among the most dangerous heavy metals to humans and
Received 28 February 2008
the environment because of their toxicity towards all living organisms and their related
Received in revised form
accumulation capability. It is known that some plant species are able to detoxify water and
16 September 2008
soil from some pollutants. In this paper we strive to investigate how a common plant species
Accepted 28 September 2008
is able to accumulate these metals. In this research we considered Poa annua, a plant species easily growing in Italy and deeply involved in the food chain, to understand problems related to its use as fodder
Keywords:
for wild and farm animals (i.e. cattle) and suitability to be used for phytoremediation
Arsenic
purposes. Hydroponic experiments were set up; P. annua was seeded in different substrates: gravel
Mercury
and zeolite, alone and mixed at different percentage.
Zeolite
For each metal three different levels of contamination were chosen, for As 0.25, 0.5 and
Gravel Constructed wetland
5 mg L−1 , for Hg 0.1, 0.2 and 2 mg L−1 . No substantial difference in metal absorption among
Accumulation
plant samples watered with different As and Hg concentrations, was observed during the
Poa annua
testing phase.
Rhizofiltration
Nevertheless, results show that concentrations of As and Hg accumulated in P. annua increase with the increasing contamination exposure. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Although metal concentrations in domestic wastewater are usually low and do not constitute an immediate risk, metals are persistent elements and do continue to accumulate in the sediments and plants of the constructed wetlands (CWs) during their operational lifetime. The major processes responsible for metal removal in CWs (Kadlec and Knight, 1996) are binding to sediments and soils, precipitation as insoluble salts and uptake by plants and bacteria. The use of heavy metals hyperaccumulating plant species has been already proposed by many authors (Baker and Brooks, 1989; Baker et al., 2000; Manios et al., 2003; Reeves,
∗
Corresponding author. Tel.: +39 0115647647; fax: +39 0115647699. E-mail address: [email protected] (E. Comino). 0925-8574/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2008.09.017
1992). In this paper we strive to investigate the phytoextraction ability of Poa annua (chosen as a crop plant). Several studies have been carried out about P. vittata arsenic traslocation capability, including Ma et al. (2001) who reported 5000 mg kg−1 accumulated in P. vittata aboveground biomass under normal conditions in a soil with an average of 184 mg kg−1 in 20 weeks Objectives of this study are: • Compare arsenic and mercury uptake by P. annua in different arsenic and mercury contaminated substrates. • Estimate P. annua phytoextraction capability for very low As and Hg concentration in water to evaluate if a phytoex-
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e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
traction process can be applied for final treatment when concentrations are extremely low.
1.1.
Toxic metals and toxicity mechanisms
Metals play an integral role in the life processes: (1) Some metals, such as calcium, chromium, copper, iron, potassium, magnesium, manganese and sodium, are essential serve as nutrients and finally are used for redoxprocesses. (2) They stabilize molecules through electrostatic interactions. (3) They are components of various enzymes. (4) They regulate osmotic pressure (Bruins et al., 2000). Other metals have no biological role (e.g. arsenic and mercury). They are nonessential (Bruins et al., 2000) and potentially toxic to organisms. Toxicity of nonessential metals occurs through the displacement of essential metals from their native binding sites and thus inhibits the activity of sensitive enzymes (Nies, 1999). In addition, at high levels, both essential and nonessential metals can: (1) (2) (3) (4)
Damage cell membranes. Alter enzyme specificity. Disrupt cellular functions. Damage the structure of DNA (Bruins et al., 2000).
Arsenic (As) occurs naturally in a wide range of minerals. The common valence states of arsenic in nature include −3, 0, +3 and +5 (Jain and Ali, 2000). In soils, the most often encountered arsenic forms are inorganic As(III) (arsenite) and As(V) (arsenate) (Balasoiu et al., 2001). In addition, As(V) and As(III) can be volatilized to arsine (AsH3 ). Contribution to environmental pollution is due through man’s use of arsenic containing insecticides, herbicides, fungicides, pesticides and wood preservatives and through mining and burning of coal. The impacts of arsenic on biological systems depend on concentration and vary from organism to organism. In general, the toxicity of arsenic is dependent on its oxidation state: trivalent arsenic forms, that correspond to both arsenite and arsine, are approximately 100 times more toxic than the pentavalent derivatives (Cervantes et al., 1994). According to Gao and Burau (1997), soil parameters and properties such as pH, temperature, organic matter content and texture affect the arsine evolution rate as well the presence of certain elements and ions, such as antimony (Andrewes et al., 2000—inhibition), phosphate (Huysmans and Frankenberger, 1991—inhibition), and molybdate (Oremland et al., 2000—enhancement). Arsenic accumulation by aquatic macrophytes facilitates the entry its into the food chain. Plants are the starting point of most food chains. Humans may be directly affected if plants, such as watercress and mint, are consumed, or indirectly when they consume species that have high As levels due to contamination of their food chain. Mercury is present in numerous chemical forms. Mainly it occurs naturally in the environment as mercuric sulfide. Ele-
mental mercury itself is toxic and cannot be broken down into less hazardous compounds. Elemental or inorganic forms can be transformed into organic (especially methylated) forms by biological systems. Not only are these methylated mercury compounds toxic, but highly bioaccumulative as well. The increase in mercury results in relatively high levels of mercury in fish consumed by humans as it rises in the aquatic food chain. Hg in soils can be readily transferred up the food chain from plants to herbivores and carnivores (Gnamus et al., 2000). Plant uptake of Hg is affected by selenite and selenate. Shanker et al. (1996) reported that Se (as selenate) in the growth solution (0.5–6 g/ml) could effectively inhibit Hg uptake by tomato. Arsenate is chemically similar to selenate, and therefore may influence plant uptake of Hg. Hg, on the other hand may influence plant uptake of As. Meharg and Jardine (2003) recently reported that Hg treatment could substantially inhibit the uptake of both arsenite and arsenate by rice seedlings.
2.
Materials and methods
The best technique for the evaluation of heavy metals accumulated in plants is Zheljazkov and Nielson protocols described below as resulted from a thorough bibliographic research (Huang et al., 2000; Jørgensen, 1993). In the present study we used Zheljazkov and Nielson protocols approach in order to extract metals from P. annua. The protocols indicate use of more than 1 g of substance. In our experiment we analyze a plant with a low biomass production, then we decided to test the possibility of using the protocol but with only 1 g of substance. The aim of the experiment is to study the capacity of P. annua to phytoextract heavy metals (As, Hg). Concentrations are 1/10, 1, 10 times standard limit concentrations.
2.1.
Materials
First in order to carry out experiments we consider interaction between the seeded P. annua and the initial concentrations. Gravel and zeolite were chosen as substrates to be used for P. annua germination and growth. Fig. 1 shows the grain distribution curves of the gravel and zeolite (Violante, 2000). The gravel used for tests of sieving and hydraulic conductivity is 3–8 mm, commonly used in wetlands. The same procedure was applied for zeolite (1–5 mm) taking care not to destroy the breakable grains during the sieving operations. These substrates are capable of supporting plant growth and have the ability to anchor plant root systems. Moreover, the substrates are chemically stable and have some hydraulic conductivity to enable soakage of effluent into the substrate. One of the most important parameters to be considered in CW design is the hydraulic conductivity (K). Hydraulic conductivity values for the used gravel and zeolite have been evaluated (Fig. 2) using a self-constructed tank built up according to the Darcy’s law. The test starts with filling the tank until reaching the upper controlled level of the tank surface discharge points. The back-
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e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
Fig. 1 – Size grain distribution curves of the gravel (a) and zeolite (b) used in our study.
ground tap for the system was open in order to reach the conditions of stability. The input excess water will be removed from two surface discharge points, and afterwards, by the exhaust grid. The flow flowing from the tap will stabilize around a value dependent on the cross section and on the different load per-
meability. Therefore the flow, the cross section A and the pressure drop i, were measured. Once we stabilized the flow from the tap, we measured the permeability. The permeability was calculated as:K = Q/At × i: Kgravel = 3.13 × 10−3 m s−1 ,
Kzeolite = 3.56 × 10−3 m s−1
Fig. 2 – Hydraulic conductivity values calculated for gravel and zeolite used in our study.
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e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
Table 1 – Heavy metal concentrations used for the laboratory trials. Metal As Hg a
b
CI (mg L−1 ) 0.25 0.0025
CIIa (mg L−1 ) 0.5 0.005
CIII (mg L−1 )
Cb (mg L−1 )
5 0.05
0.02 0.001
Wastewater superficial bodies discharge standard limits according to Dlgs 152/99. Wastewater discharge after treatment system according to DM 185/2003.
H3 BO3 , 0.1 M (NH4 )6 Mo7 O24 × 24H2 O, 0.2 M ZnSO4 × 7H2 O, 0.15 M CuSO4 × 5H2 O) in deionised water to avoid any inhibition caused by metals. After this germination period plants were grown for 3 weeks indoors, but under a natural day/night regime, moistened every 3 days with Rorison’s solution (B = blank) and treatment solutions, at different concentrations, dissolved in Rorison’s nutrient solution. The metals chosen for research are not covered by the Rorison’s solution, so as not to compromise the concentration of metallic detected in P. annua.
2.2. The substrates were used both individually and mixed, five mix have been performed, subdivided as percentage of weight: (1) (2) (3) (4) (5)
100% gravel. 75% gravel and 25% zeolite. 50% gravel and 50% zeolite. 25% gravel and 75% zeolite. 100% zeolite.
Three different concentrations have been considered for each set. The reference concentration (CII) has been chosen based on Dlgs 152/1999 and represents the standard limit for discharging in superficial water bodies, CI is half the standard limit concentration, while CIII has been chosen ten times the reference concentration. C# is the concentration according to new legislative set of rules for wastewater reuse with a restrictive approach, according to Ministerial decree DM 185/03. No presence of As and Hg was identified using mineralizator DK6/42 at the beginning of research in the digestion of gravel and zeolite.As and Hg concentrations have been compared according to Italian legislation as described above (Table 1).
2.1.1.
P. annua
In this research we were interested in considering a natural plant species easily growing in Italy and involved in the food chain to understand problems related to its use as fodder for wild and farm animals (i.e. cattle). P. annua is a valuable meadow and pasture grass spread throughout Europe and especially in the central United States, characterized by tall stalks and slender bright green leaves. P. annua is also known as Kentucky bluegrass. It favours moist conditions, including reservoir shores (Hoffman et al., 1980). It can withstand flooding (Schalitz, 1977) with subsequent freezing, ice and snow (Beard, 1964). The interaction of the effects of soil texture and climate on P. annua is demonstrated by its high frequency on dry sites, on shallow neutral soils overlying gravel (Maycock and Guzikowa, 1983) and low frequency on sandy soils. The experiment was conducted in 8 cm diameter and 15 cm tall plastic containers in three replicates in hydroponic conditions. Around 100 seeds of P. annua were placed on the surface for the different substrates and watered, during the overall germination period of 18 days, with Rorison’s solution (200 M Ca(NO3 )2 × 4H2 O, 100 M MgSO4 × 7H2 O, 100 M K2 HPO4 × 3H2 O, 5 M Fe-chelate, 1 M MnSO4 × 4H2 O, 5 M
Analytical methods
The plants collected were washed with tap water to remove sand and dirt, and then rinsed thoroughly with distilled water. Stainless steel scissors were used to cut the plants leaves, stems and roots. The material was milled in a micro-hammer cutter and sieved through a 1.5-mm sieve to ensure uniform distribution of metals in the sample. After homogenisation, the plant samples were placed in clean paper bags. Each plant sample was dried at 65 ◦ C for 48 h before digestion to analyze heavy metals. They were put separately into labelled polythene bags preserved in desiccators for drying. The drying of collected material is important because it protects the plant material from microbial decomposition and also ensures a constant reference value by determining the dry weight in contrast to fresh weight, which is difficult to quantify (Markert, 1993). Digestion was performed using mineralizator DK6/42 produced by Velp Scientifica, this heating block, designed for wet digestion, can hold up to 6 tubes of 42 mm diameter. The nitric acid digestion was chosen among wet digestion methods, this procedure is the most efficient method to extract metals (Zheljazkov and Nielson, 1996). 1 g of plant material was placed in a 300mL digestion tube and 10 mL of concentrated HNO3 was added a 0.5-g sample was used. When sample mass was not enough, because not all the P. annua dried samples get 1 g. The sample was then heated for 45 min at 90 ◦ C Then the temperature was increased to 180 ◦ C at which the sample boiled for 6 h, until a clear solution was obtained and the volume was reduced to about 5–10 mL. After cooling, the interior walls of the tube were washed down with a low distilled water and the tube was swirled throughout the digestion to keep the wall clean and prevent the loss of the sample. The solution was filtered with cellulose acetate filter paper (45 um) mounted on a vacuum pump. The crucible was well rinsed with distilled water and transferred to the 50 or 100 mL volumetric flask, and was then made up to the mark with distilled water: for sample weight around 0.5 g, the resultant residue was diluted to 50 mL volumetric flasks mark with distilled water. In order to be stored, they were then transferred into a 50or 100-mL plastic bottle (it depends on the sample’s mass). Arsenic concentrations were determined by Electrothermal Atomic Absorption Spectrometry (APHA, 3113 B method, 1998) while determination of mercury was performed by ColdVapour Atomic Absorption Spectrometric Method (APHA, 3112 B method, 1998).
e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
3.
Results
About 50% of P. annua plants germinated 1 week after it was seeded and at the end of the entire period the germination pergentage increased to 90%. At first sight none of the adopted heavy metals levels caused roots or leaves alteration visible to the naked eye, but it is true the contamination levels were slight. Within the study period of 21 days, symptoms of As and Hg toxicity were in general absent. In this study both arsenic concentration, as ppm (mg L−1 ), and Hg concentration, as ppb, were determined for each sample and an average calculated from the three replicates. P. annua can not be considered an hyperaccumulating plant species as concentration in the plant samples was up to 290 mg kg−1 d.w. (dried weight of the collected material) for As and 0.04 mg kg−1 d.w. for Hg: according to Brooks et al. (1977)
347
As hyperaccumulating plants have been defined as plants that accumulate greater than 1000 mg kg−1 . The most significant data in Fig. 3a and b show that, for every substrate type, there is no As or Hg in samples grown in blanks, instead the highest figure concerning As absorption match with the C3 concentration (5 mg L−1 ): at lower environmental concentrations (0.5 mg L−1 ) corresponds very low – two order of magnitude – accumulation of arsenic in plant (2 mg kg−1 d.w.). Instead, mercury concentrations in plants increase proportionally from initial concentration to 0.05 mg L−1 (Fig. 3b). This means P. annua is sensible to Hg and its uptake occurs even in presence of very low initial concentration. P. annua accumulation reaches the maximum value at the 100% gravel substrate and the minimum one with 100% zeolite, this highlights the main characteristic of gravel to have a zero absorption coefficient that is indeed maximum for the zeolite. Where gravel and zeolite were mixed, the accumula-
Fig. 3 – (a) Comparison between As added in Rorison’s solution and As accumulation in plants and (b) comparison between Hg added in Rorison’s solution and Hg accumulation in plants.
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tion trend changed with the different concentration of zeolite. It creates two different trends having no reference to any mathematic function, although it is not clear how the trend concentration has got an edge in correspondence to the mixed substrate of 25% gravel–75% zeolite (see also Fig. 4). To understand the P. annua efficiency in extracting these metals from the environment in which the plant is growing, we compared the concentration of both As and Hg in the plant samples in relation to the concentration in water, in this way we could also standardize P. annua adsorption ability toward different metals. For this purpose the bioaccumulation coefficient (BAC) was considered (Brooks and Robinson, 1998):
BAC =
Meplant Mewater
L kg−1
where [Meplant ] is the metal concentration in plant = mg kg−1 d.m., [Mewater ] is the metal concentration in water = mg L−1 .
The bioaccumulation coefficient histogram (Fig. 4) shows how As and Hg uptake varies and depends on the inlet concentration plotted on the horizontal axes. P. annua has a maximum BAC of 57 L kg−1 for arsenic at concentration of 5 mg L−1 in water, although the trend how P. annua accumulates is not directly proportional to the initial concentration. Indeed this species seems to be effective in removing only medium high concentration of As. Concerning mercury, the most significant data are the decreasing of the bioaccumulation coefficient with an increase of the Hg concentration in water, in this case the maximum value is about 2020 L kg−1 , in correspondence with the lowest initial metal concentration in solution. Mercury can be adsorbed at very low concentrations, but its bioaccumulation tendency decreases with higher Hg concentration in water. From Fig. 3a and b it results that concentrations in the plant tissues do not give important results as the concentration in the dissolved phase was very low, but
Fig. 4 – Bioaccumulation coefficient for the tested heavy metals: (a) As concentration that corresponds to 0.25 mg L−1 = CI; 0.5 mg L−1 = CII; 5 mg L−1 = CIII and (b) Hg concentration that corresponds to 0.0025 mg L−1 = CI; 0.005 mg L−1 = CII; 0.05 mg L−1 = CIII.
e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 343–350
from the BAC evaluation we can assume P. annua has got a good capacity for mercury extraction although mercury uptake values are very low. From these preliminary results, P. annua extracted both arsenic and mercury, even if concentrations are low, but the bioaccumulation coefficient plot shows As is accumulated more at higher environmental concentration while Hg can be adsorbed starting from very low concentration values and P. annua mercury bioaccumulation decreases with an increase of the inlet concentrations.
4.
Discussion and conclusion
Results demonstrated that Mineralizator DK6/42 was found to be appropriate for analysing plants. This technique homogenisation does not need because digestion smoulder every plant tissues. Moreover results showed that water polluted with As and/or Hg can be absorbed and accumulated by P. annua. Is very important that this contaminated plant not be used as fodder because can carry heavy metals (As and Hg) in the food chain, as shown in Cubadda et al. (2005). P. annua accumulates metals even in condition of very low concentrations. Analyzing 1 g of P. annua it is possible evaluate metals’ presence, even if Zheljazkov and Nielson protocol suggest to use more than 1 g of substance. In particular, mercury uptake occurs even with very low initial concentrations in water, instead in case of low arsenic pollution this plant species seem not accumulating As very much. Indeed bioaccumulation is higher when metals are in more bio-available forms in the environment. The measurement of the absorption of the vegetation to tolerate high metal concentrations may also vary between laboratory trials, field samply and monitoring due to increased stresses and more complexities in this environment. Especially pH, temperature and organic matter should be evaluated. Further to Du et al. (2005) studies demonstrating that Oryza sativa root arsenic concentration decreased significantly with increasing external Hg concentration: the present research recommends that additional investigation should take into account also the interactions between Hg and arsenate uptake. The comparison among metal uptake in different contaminated substrates shows how metals concentrations diminish if the substrate is 100% zeolite compared to 100% gravel medium (Fig. 3a). Even if the adsorption mechanisms’s zeolites are still not clear, the low plant uptake in the case of natural zeolite may be explained by low As bioavailability due to retention of inorganic arsenic species (Kumar et al., 2007). Follow up research activities will develop the following issues: (1) Evaluation of heavy metals in roots and not only the air plant entering in the food chain. This will introduce rhizofiltration and phytoremediation comparing the results from this experiment with those of Ma et al. (2001), who showed P. vittata could hyperaccumulate As. It is clear that the plant species tested in
349
this experiment have no specialized As-uptake mechanism. However in phytoremediation, roots plants’ are very important. Roots have a high capacity to extract metals even if are present in low concentrations. According to discussion above, we may assume that P. annua can be also used for rhizofiltration treatment because it is suitable to remediate extracted groundwater, surface water, and wastewater with low of concentration contaminants. Indeed an advantage associated with rhizofiltration is that contaminants do not have to be traslocated to the shoots. Thus, species other than hyperaccumulators may be used (Henry, 2000) for this purpose. This application can integrate and improve the performance of existing wastewater treatments, thanks to P. annua ability in recovering metals even in very low concentration. This technique can be used for final treatment when concentrations are already very low. (2) The relationship between heavy metals absorbed and stabilized in aerial parts or in roots of plants and heavy metals that remain in the substrates. (3) The adsorption behave of different rates of gravel and zeolite mix.
references
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Cubadda, F., Raggi, A., Marconi, E., 2005. Effects of processing on five selected metals in the durum wheat food chain. Microchem. J. 79, 97–102. Du, X., Zhu, Y.G., Liu, W.J., Zhao, X.s., 2005. Uptake of mercury (Hg) by seedlings of rice (Oryza sativa L.) grown in solution culture and interactions with arsenate uptake. Environ. Exp. Bot. 54, 1–7. Gao, S., Burau, R.G., 1997. Environmental factors affecting rates of arsine evolution from and mineralization of arsenicals in soil. J. Environ. Qual. 26, 753–763. Gnamus, A., Byrne, A.R., Horvat, M., 2000. Mercury in the soil–plant–deer–predator food chain of a temperate forest in Slovenia. Environ. Sci. Technol. 34, 3337–3345. Henry, J.R., 2000. Overview of the Phytoremediation of Lead and Mercury. U.S. Environmental Protection Agency Technology Innovation office, Washington, DC. Hoffman, G.R., Hodges, M.B., Stanley, L.D., 1980. Germination of plant species common to reservoir shores in the northern Great Plains. Bull. Torr. Bot. Club. 107, 506–513. Huang, Y., Zhao, Z., Xu, M., Gao, Y., 2000. Biological approaches for disposing and reusing chemical wastewater. Ecol. Eng. 16, 281–292. Huysmans, K.D., Frankenberger, W.T., 1991. Evolution of trimethylarsine by a Penicillium sp. isolated from agricultural evaporation pond water. Sci. Tot. Environ. 105, 13–28. Kadlec, R.H., Knight, R.L., 1996. Treatment Wetlands. CRC, Boca Raton, FL. Kumar, P., Jadhav, P.D., Rayalu, S.S., Devotta, S., 2007. Surface-modified zeolite-A for sequestration of arsenic and chromium anions. Curr. Sci. 92 (5). Jain, C.K., Ali, I., 2000. Arsenic: occurrence, toxicity and speciation. Water Res. 34, 4304–4312. Jørgensen, S.E., 1993. Removal of heavy metals from compost and soil by ecotechnological methods. Ecol. Eng. 2, 89–100. Ma, L.Q., Komar, K.M., Tu, C., Zhang, W., Cai, Y., 2001. A fern that hyperaccumulates arsenic. Nature, 409–579.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ecoleng
Review
Preliminary test of arsenic and mercury uptake by Poa annua Elena Comino ∗ , Adriano Fiorucci, Stefania Menegatti, Cecilia Marocco Politecnico di Torino, Land, Environment and Geo-Engineering Department, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
a r t i c l e
i n f o
a b s t r a c t
Article history:
Arsenic (As) and mercury (Hg) are among the most dangerous heavy metals to humans and
Received 28 February 2008
the environment because of their toxicity towards all living organisms and their related
Received in revised form
accumulation capability. It is known that some plant species are able to detoxify water and
16 September 2008
soil from some pollutants. In this paper we strive to investigate how a common plant species
Accepted 28 September 2008
is able to accumulate these metals. In this research we considered Poa annua, a plant species easily growing in Italy and deeply involved in the food chain, to understand problems related to its use as fodder
Keywords:
for wild and farm animals (i.e. cattle) and suitability to be used for phytoremediation
Arsenic
purposes. Hydroponic experiments were set up; P. annua was seeded in different substrates: gravel
Mercury
and zeolite, alone and mixed at different percentage.
Zeolite
For each metal three different levels of contamination were chosen, for As 0.25, 0.5 and
Gravel Constructed wetland
5 mg L−1 , for Hg 0.1, 0.2 and 2 mg L−1 . No substantial difference in metal absorption among
Accumulation
plant samples watered with different As and Hg concentrations, was observed during the
Poa annua
testing phase.
Rhizofiltration
Nevertheless, results show that concentrations of As and Hg accumulated in P. annua increase with the increasing contamination exposure. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Although metal concentrations in domestic wastewater are usually low and do not constitute an immediate risk, metals are persistent elements and do continue to accumulate in the sediments and plants of the constructed wetlands (CWs) during their operational lifetime. The major processes responsible for metal removal in CWs (Kadlec and Knight, 1996) are binding to sediments and soils, precipitation as insoluble salts and uptake by plants and bacteria. The use of heavy metals hyperaccumulating plant species has been already proposed by many authors (Baker and Brooks, 1989; Baker et al., 2000; Manios et al., 2003; Reeves,
∗
Corresponding author. Tel.: +39 0115647647; fax: +39 0115647699. E-mail address: [email protected] (E. Comino). 0925-8574/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ecoleng.2008.09.017
1992). In this paper we strive to investigate the phytoextraction ability of Poa annua (chosen as a crop plant). Several studies have been carried out about P. vittata arsenic traslocation capability, including Ma et al. (2001) who reported 5000 mg kg−1 accumulated in P. vittata aboveground biomass under normal conditions in a soil with an average of 184 mg kg−1 in 20 weeks Objectives of this study are: • Compare arsenic and mercury uptake by P. annua in different arsenic and mercury contaminated substrates. • Estimate P. annua phytoextraction capability for very low As and Hg concentration in water to evaluate if a phytoex-
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traction process can be applied for final treatment when concentrations are extremely low.
1.1.
Toxic metals and toxicity mechanisms
Metals play an integral role in the life processes: (1) Some metals, such as calcium, chromium, copper, iron, potassium, magnesium, manganese and sodium, are essential serve as nutrients and finally are used for redoxprocesses. (2) They stabilize molecules through electrostatic interactions. (3) They are components of various enzymes. (4) They regulate osmotic pressure (Bruins et al., 2000). Other metals have no biological role (e.g. arsenic and mercury). They are nonessential (Bruins et al., 2000) and potentially toxic to organisms. Toxicity of nonessential metals occurs through the displacement of essential metals from their native binding sites and thus inhibits the activity of sensitive enzymes (Nies, 1999). In addition, at high levels, both essential and nonessential metals can: (1) (2) (3) (4)
Damage cell membranes. Alter enzyme specificity. Disrupt cellular functions. Damage the structure of DNA (Bruins et al., 2000).
Arsenic (As) occurs naturally in a wide range of minerals. The common valence states of arsenic in nature include −3, 0, +3 and +5 (Jain and Ali, 2000). In soils, the most often encountered arsenic forms are inorganic As(III) (arsenite) and As(V) (arsenate) (Balasoiu et al., 2001). In addition, As(V) and As(III) can be volatilized to arsine (AsH3 ). Contribution to environmental pollution is due through man’s use of arsenic containing insecticides, herbicides, fungicides, pesticides and wood preservatives and through mining and burning of coal. The impacts of arsenic on biological systems depend on concentration and vary from organism to organism. In general, the toxicity of arsenic is dependent on its oxidation state: trivalent arsenic forms, that correspond to both arsenite and arsine, are approximately 100 times more toxic than the pentavalent derivatives (Cervantes et al., 1994). According to Gao and Burau (1997), soil parameters and properties such as pH, temperature, organic matter content and texture affect the arsine evolution rate as well the presence of certain elements and ions, such as antimony (Andrewes et al., 2000—inhibition), phosphate (Huysmans and Frankenberger, 1991—inhibition), and molybdate (Oremland et al., 2000—enhancement). Arsenic accumulation by aquatic macrophytes facilitates the entry its into the food chain. Plants are the starting point of most food chains. Humans may be directly affected if plants, such as watercress and mint, are consumed, or indirectly when they consume species that have high As levels due to contamination of their food chain. Mercury is present in numerous chemical forms. Mainly it occurs naturally in the environment as mercuric sulfide. Ele-
mental mercury itself is toxic and cannot be broken down into less hazardous compounds. Elemental or inorganic forms can be transformed into organic (especially methylated) forms by biological systems. Not only are these methylated mercury compounds toxic, but highly bioaccumulative as well. The increase in mercury results in relatively high levels of mercury in fish consumed by humans as it rises in the aquatic food chain. Hg in soils can be readily transferred up the food chain from plants to herbivores and carnivores (Gnamus et al., 2000). Plant uptake of Hg is affected by selenite and selenate. Shanker et al. (1996) reported that Se (as selenate) in the growth solution (0.5–6 g/ml) could effectively inhibit Hg uptake by tomato. Arsenate is chemically similar to selenate, and therefore may influence plant uptake of Hg. Hg, on the other hand may influence plant uptake of As. Meharg and Jardine (2003) recently reported that Hg treatment could substantially inhibit the uptake of both arsenite and arsenate by rice seedlings.
2.
Materials and methods
The best technique for the evaluation of heavy metals accumulated in plants is Zheljazkov and Nielson protocols described below as resulted from a thorough bibliographic research (Huang et al., 2000; Jørgensen, 1993). In the present study we used Zheljazkov and Nielson protocols approach in order to extract metals from P. annua. The protocols indicate use of more than 1 g of substance. In our experiment we analyze a plant with a low biomass production, then we decided to test the possibility of using the protocol but with only 1 g of substance. The aim of the experiment is to study the capacity of P. annua to phytoextract heavy metals (As, Hg). Concentrations are 1/10, 1, 10 times standard limit concentrations.
2.1.
Materials
First in order to carry out experiments we consider interaction between the seeded P. annua and the initial concentrations. Gravel and zeolite were chosen as substrates to be used for P. annua germination and growth. Fig. 1 shows the grain distribution curves of the gravel and zeolite (Violante, 2000). The gravel used for tests of sieving and hydraulic conductivity is 3–8 mm, commonly used in wetlands. The same procedure was applied for zeolite (1–5 mm) taking care not to destroy the breakable grains during the sieving operations. These substrates are capable of supporting plant growth and have the ability to anchor plant root systems. Moreover, the substrates are chemically stable and have some hydraulic conductivity to enable soakage of effluent into the substrate. One of the most important parameters to be considered in CW design is the hydraulic conductivity (K). Hydraulic conductivity values for the used gravel and zeolite have been evaluated (Fig. 2) using a self-constructed tank built up according to the Darcy’s law. The test starts with filling the tank until reaching the upper controlled level of the tank surface discharge points. The back-
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Fig. 1 – Size grain distribution curves of the gravel (a) and zeolite (b) used in our study.
ground tap for the system was open in order to reach the conditions of stability. The input excess water will be removed from two surface discharge points, and afterwards, by the exhaust grid. The flow flowing from the tap will stabilize around a value dependent on the cross section and on the different load per-
meability. Therefore the flow, the cross section A and the pressure drop i, were measured. Once we stabilized the flow from the tap, we measured the permeability. The permeability was calculated as:K = Q/At × i: Kgravel = 3.13 × 10−3 m s−1 ,
Kzeolite = 3.56 × 10−3 m s−1
Fig. 2 – Hydraulic conductivity values calculated for gravel and zeolite used in our study.
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Table 1 – Heavy metal concentrations used for the laboratory trials. Metal As Hg a
b
CI (mg L−1 ) 0.25 0.0025
CIIa (mg L−1 ) 0.5 0.005
CIII (mg L−1 )
Cb (mg L−1 )
5 0.05
0.02 0.001
Wastewater superficial bodies discharge standard limits according to Dlgs 152/99. Wastewater discharge after treatment system according to DM 185/2003.
H3 BO3 , 0.1 M (NH4 )6 Mo7 O24 × 24H2 O, 0.2 M ZnSO4 × 7H2 O, 0.15 M CuSO4 × 5H2 O) in deionised water to avoid any inhibition caused by metals. After this germination period plants were grown for 3 weeks indoors, but under a natural day/night regime, moistened every 3 days with Rorison’s solution (B = blank) and treatment solutions, at different concentrations, dissolved in Rorison’s nutrient solution. The metals chosen for research are not covered by the Rorison’s solution, so as not to compromise the concentration of metallic detected in P. annua.
2.2. The substrates were used both individually and mixed, five mix have been performed, subdivided as percentage of weight: (1) (2) (3) (4) (5)
100% gravel. 75% gravel and 25% zeolite. 50% gravel and 50% zeolite. 25% gravel and 75% zeolite. 100% zeolite.
Three different concentrations have been considered for each set. The reference concentration (CII) has been chosen based on Dlgs 152/1999 and represents the standard limit for discharging in superficial water bodies, CI is half the standard limit concentration, while CIII has been chosen ten times the reference concentration. C# is the concentration according to new legislative set of rules for wastewater reuse with a restrictive approach, according to Ministerial decree DM 185/03. No presence of As and Hg was identified using mineralizator DK6/42 at the beginning of research in the digestion of gravel and zeolite.As and Hg concentrations have been compared according to Italian legislation as described above (Table 1).
2.1.1.
P. annua
In this research we were interested in considering a natural plant species easily growing in Italy and involved in the food chain to understand problems related to its use as fodder for wild and farm animals (i.e. cattle). P. annua is a valuable meadow and pasture grass spread throughout Europe and especially in the central United States, characterized by tall stalks and slender bright green leaves. P. annua is also known as Kentucky bluegrass. It favours moist conditions, including reservoir shores (Hoffman et al., 1980). It can withstand flooding (Schalitz, 1977) with subsequent freezing, ice and snow (Beard, 1964). The interaction of the effects of soil texture and climate on P. annua is demonstrated by its high frequency on dry sites, on shallow neutral soils overlying gravel (Maycock and Guzikowa, 1983) and low frequency on sandy soils. The experiment was conducted in 8 cm diameter and 15 cm tall plastic containers in three replicates in hydroponic conditions. Around 100 seeds of P. annua were placed on the surface for the different substrates and watered, during the overall germination period of 18 days, with Rorison’s solution (200 M Ca(NO3 )2 × 4H2 O, 100 M MgSO4 × 7H2 O, 100 M K2 HPO4 × 3H2 O, 5 M Fe-chelate, 1 M MnSO4 × 4H2 O, 5 M
Analytical methods
The plants collected were washed with tap water to remove sand and dirt, and then rinsed thoroughly with distilled water. Stainless steel scissors were used to cut the plants leaves, stems and roots. The material was milled in a micro-hammer cutter and sieved through a 1.5-mm sieve to ensure uniform distribution of metals in the sample. After homogenisation, the plant samples were placed in clean paper bags. Each plant sample was dried at 65 ◦ C for 48 h before digestion to analyze heavy metals. They were put separately into labelled polythene bags preserved in desiccators for drying. The drying of collected material is important because it protects the plant material from microbial decomposition and also ensures a constant reference value by determining the dry weight in contrast to fresh weight, which is difficult to quantify (Markert, 1993). Digestion was performed using mineralizator DK6/42 produced by Velp Scientifica, this heating block, designed for wet digestion, can hold up to 6 tubes of 42 mm diameter. The nitric acid digestion was chosen among wet digestion methods, this procedure is the most efficient method to extract metals (Zheljazkov and Nielson, 1996). 1 g of plant material was placed in a 300mL digestion tube and 10 mL of concentrated HNO3 was added a 0.5-g sample was used. When sample mass was not enough, because not all the P. annua dried samples get 1 g. The sample was then heated for 45 min at 90 ◦ C Then the temperature was increased to 180 ◦ C at which the sample boiled for 6 h, until a clear solution was obtained and the volume was reduced to about 5–10 mL. After cooling, the interior walls of the tube were washed down with a low distilled water and the tube was swirled throughout the digestion to keep the wall clean and prevent the loss of the sample. The solution was filtered with cellulose acetate filter paper (45 um) mounted on a vacuum pump. The crucible was well rinsed with distilled water and transferred to the 50 or 100 mL volumetric flask, and was then made up to the mark with distilled water: for sample weight around 0.5 g, the resultant residue was diluted to 50 mL volumetric flasks mark with distilled water. In order to be stored, they were then transferred into a 50or 100-mL plastic bottle (it depends on the sample’s mass). Arsenic concentrations were determined by Electrothermal Atomic Absorption Spectrometry (APHA, 3113 B method, 1998) while determination of mercury was performed by ColdVapour Atomic Absorption Spectrometric Method (APHA, 3112 B method, 1998).
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3.
Results
About 50% of P. annua plants germinated 1 week after it was seeded and at the end of the entire period the germination pergentage increased to 90%. At first sight none of the adopted heavy metals levels caused roots or leaves alteration visible to the naked eye, but it is true the contamination levels were slight. Within the study period of 21 days, symptoms of As and Hg toxicity were in general absent. In this study both arsenic concentration, as ppm (mg L−1 ), and Hg concentration, as ppb, were determined for each sample and an average calculated from the three replicates. P. annua can not be considered an hyperaccumulating plant species as concentration in the plant samples was up to 290 mg kg−1 d.w. (dried weight of the collected material) for As and 0.04 mg kg−1 d.w. for Hg: according to Brooks et al. (1977)
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As hyperaccumulating plants have been defined as plants that accumulate greater than 1000 mg kg−1 . The most significant data in Fig. 3a and b show that, for every substrate type, there is no As or Hg in samples grown in blanks, instead the highest figure concerning As absorption match with the C3 concentration (5 mg L−1 ): at lower environmental concentrations (0.5 mg L−1 ) corresponds very low – two order of magnitude – accumulation of arsenic in plant (2 mg kg−1 d.w.). Instead, mercury concentrations in plants increase proportionally from initial concentration to 0.05 mg L−1 (Fig. 3b). This means P. annua is sensible to Hg and its uptake occurs even in presence of very low initial concentration. P. annua accumulation reaches the maximum value at the 100% gravel substrate and the minimum one with 100% zeolite, this highlights the main characteristic of gravel to have a zero absorption coefficient that is indeed maximum for the zeolite. Where gravel and zeolite were mixed, the accumula-
Fig. 3 – (a) Comparison between As added in Rorison’s solution and As accumulation in plants and (b) comparison between Hg added in Rorison’s solution and Hg accumulation in plants.
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tion trend changed with the different concentration of zeolite. It creates two different trends having no reference to any mathematic function, although it is not clear how the trend concentration has got an edge in correspondence to the mixed substrate of 25% gravel–75% zeolite (see also Fig. 4). To understand the P. annua efficiency in extracting these metals from the environment in which the plant is growing, we compared the concentration of both As and Hg in the plant samples in relation to the concentration in water, in this way we could also standardize P. annua adsorption ability toward different metals. For this purpose the bioaccumulation coefficient (BAC) was considered (Brooks and Robinson, 1998):
BAC =
Meplant Mewater
L kg−1
where [Meplant ] is the metal concentration in plant = mg kg−1 d.m., [Mewater ] is the metal concentration in water = mg L−1 .
The bioaccumulation coefficient histogram (Fig. 4) shows how As and Hg uptake varies and depends on the inlet concentration plotted on the horizontal axes. P. annua has a maximum BAC of 57 L kg−1 for arsenic at concentration of 5 mg L−1 in water, although the trend how P. annua accumulates is not directly proportional to the initial concentration. Indeed this species seems to be effective in removing only medium high concentration of As. Concerning mercury, the most significant data are the decreasing of the bioaccumulation coefficient with an increase of the Hg concentration in water, in this case the maximum value is about 2020 L kg−1 , in correspondence with the lowest initial metal concentration in solution. Mercury can be adsorbed at very low concentrations, but its bioaccumulation tendency decreases with higher Hg concentration in water. From Fig. 3a and b it results that concentrations in the plant tissues do not give important results as the concentration in the dissolved phase was very low, but
Fig. 4 – Bioaccumulation coefficient for the tested heavy metals: (a) As concentration that corresponds to 0.25 mg L−1 = CI; 0.5 mg L−1 = CII; 5 mg L−1 = CIII and (b) Hg concentration that corresponds to 0.0025 mg L−1 = CI; 0.005 mg L−1 = CII; 0.05 mg L−1 = CIII.
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from the BAC evaluation we can assume P. annua has got a good capacity for mercury extraction although mercury uptake values are very low. From these preliminary results, P. annua extracted both arsenic and mercury, even if concentrations are low, but the bioaccumulation coefficient plot shows As is accumulated more at higher environmental concentration while Hg can be adsorbed starting from very low concentration values and P. annua mercury bioaccumulation decreases with an increase of the inlet concentrations.
4.
Discussion and conclusion
Results demonstrated that Mineralizator DK6/42 was found to be appropriate for analysing plants. This technique homogenisation does not need because digestion smoulder every plant tissues. Moreover results showed that water polluted with As and/or Hg can be absorbed and accumulated by P. annua. Is very important that this contaminated plant not be used as fodder because can carry heavy metals (As and Hg) in the food chain, as shown in Cubadda et al. (2005). P. annua accumulates metals even in condition of very low concentrations. Analyzing 1 g of P. annua it is possible evaluate metals’ presence, even if Zheljazkov and Nielson protocol suggest to use more than 1 g of substance. In particular, mercury uptake occurs even with very low initial concentrations in water, instead in case of low arsenic pollution this plant species seem not accumulating As very much. Indeed bioaccumulation is higher when metals are in more bio-available forms in the environment. The measurement of the absorption of the vegetation to tolerate high metal concentrations may also vary between laboratory trials, field samply and monitoring due to increased stresses and more complexities in this environment. Especially pH, temperature and organic matter should be evaluated. Further to Du et al. (2005) studies demonstrating that Oryza sativa root arsenic concentration decreased significantly with increasing external Hg concentration: the present research recommends that additional investigation should take into account also the interactions between Hg and arsenate uptake. The comparison among metal uptake in different contaminated substrates shows how metals concentrations diminish if the substrate is 100% zeolite compared to 100% gravel medium (Fig. 3a). Even if the adsorption mechanisms’s zeolites are still not clear, the low plant uptake in the case of natural zeolite may be explained by low As bioavailability due to retention of inorganic arsenic species (Kumar et al., 2007). Follow up research activities will develop the following issues: (1) Evaluation of heavy metals in roots and not only the air plant entering in the food chain. This will introduce rhizofiltration and phytoremediation comparing the results from this experiment with those of Ma et al. (2001), who showed P. vittata could hyperaccumulate As. It is clear that the plant species tested in
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this experiment have no specialized As-uptake mechanism. However in phytoremediation, roots plants’ are very important. Roots have a high capacity to extract metals even if are present in low concentrations. According to discussion above, we may assume that P. annua can be also used for rhizofiltration treatment because it is suitable to remediate extracted groundwater, surface water, and wastewater with low of concentration contaminants. Indeed an advantage associated with rhizofiltration is that contaminants do not have to be traslocated to the shoots. Thus, species other than hyperaccumulators may be used (Henry, 2000) for this purpose. This application can integrate and improve the performance of existing wastewater treatments, thanks to P. annua ability in recovering metals even in very low concentration. This technique can be used for final treatment when concentrations are already very low. (2) The relationship between heavy metals absorbed and stabilized in aerial parts or in roots of plants and heavy metals that remain in the substrates. (3) The adsorption behave of different rates of gravel and zeolite mix.
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