THE ROLE OF SALT MARSH PLANTS AND ...

In: Handbook of Environmental Research Editor: Aurel Edelstein and Dagmar Bär

ISBN: 978-1-60741-492-6 © 2009 Nova Science Publishers, Inc.

Chapter 19

THE ROLE OF SALT MARSH PLANTS AND MICROORGANISMS IN SEDIMENT METAL BIOGEOCHEMISTRY Bernardo Duarte* 1 and Isabel Caçador1 1

Centro de Oceanografia Faculdade de Ciências, Universidade de Lisboa, Campo Grande 1749-016 Lisboa.

ABSTRACT Salt marshes were often installed in the proximity of cities and industrialized areas. In these cases, flooding transports large quantities of contaminants in both dissolved and suspended particulate forms to the salt marsh areas. Anthropogenic metals are incorporated in the sediments, decreasing their availability in the water column. Vascular plants in salt marshes are determinant to the dynamics of the estuarine ecosystem and strongly influence the processes of accumulation and retention of heavy metals in these areas. The utilization of wetlands as filters has gained great interest in the past decades. However, the role that plants play in the filter function of wetlands in relation to metals is still a matter of investigation. When the metal contaminants enter in the salt marsh they spread along with the tides and periodic floods and interact with soil and the biotic community. Salt marsh plants are known to accumulate large amounts of metals in their aerial and belowground organs but also for their ability to phytostabilize these contaminants in the rhizosediment, playing an important role in the this ecosystem autoremediative processes and biogeochemistry. When we consider the possible toxic effects of metals to the marsh ecosystem and also to human health, the total amount of metal is not as important as the chemical form that is present, responsible for the bioavailability to the plant uptake and consequently to the introduction in the food web. Activity of plant roots and associated microbes can alter physical and chemical properties of the sediment, influencing geochemical fractionation of metals and thus availability to the plant. Organic matter pools are known to be highly efficient sinks of heavy metals, constituting an important fraction of metal species, *

Portugal, [email protected], [email protected]

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Bernardo Duarte and Isabel Caçador present in salt marsh sediments. Organic matter cycles are supported by microbial enzymatic degradation mechanisms and consequently affect the organic bound metals. Along with this, microbial driven sulphidisation is also an important factor when considering speciation processes. Without ignoring physic-chemical factors, as pH, Eh and oxygen profiles, microbial speciation processes assume a very important role, unconsidered in most studies. In this chapter it will be discussed the influence of vascular plants and microbial activity in salt marsh sediments and its consequences in metal chemical speciation.

GENERAL INTRODUCTION Tagus estuary is located near a very densely populated and industrialized area, majorly occupied by Lisbon metropolitan area, in the north side, and by the industrial and urban zones of Barreiro, Seixal and Montijo cities in the south side. This geographical display makes this estuary subjected to discharges from industries and effluents of activity sources. Particularly in the south side there are large salt marsh areas with very rich vegetation. There is an evident zonation of these species distribution from the upper marsh areas to the lower marsh and mudflats (Caçador et al., 2007). In the upper marsh one of the more abundant specie is Halimione portulacoides (L.) Aellen (Chenopodiaceae) that coexists with Sarcocornia species, while the lower marsh is mostly dominated by Spartina maritima Loisel (Poaceae). The large submersion periods and the physical-chemical characteristics of these two distinct areas, along with the competitive interspecific relations are the major factors that contribute to this zonation. Also the different plant coverage verified in the upper and lower marsh is also an important factor that will influence sediment characteristic such as contaminate retention and sediment redox state (Caçador et al., 2009).Due to the large submersion periods that these salt marsh areas are subjected, this kind of sediment is often waterlogged and shows low levels of oxygen, being adverse to plant growth (Richert et al., 2000). However salt marsh plants are well known for pumping oxygen from the atmosphere to the sediment, turning the redox conditions of the root zone oxidative (Ludemann et al., 2000). All these sediment – plant interactions are very important to considerer when considering contaminant retention. Tidal flooding of the salt marsh supplies considerable amounts of heavy metals from nearby urban and industrialized areas, which tend to accumulate in sediments and plant tissues. These metals retained in the sediment present various forms (Tessier, 1979) depending on the bounds they establish with the different sediment components. This is also a dynamic process very influenced by the sediment and external factors (hydrodynamics, weather and seasonal variation) but also by the vegetation that colonizes the area (Reboreda and Caçador, 2007). In soils and sediments, elements (metallic and non-metallic) exist in several different forms and associated with various components of distinct natures. These physical-chemical forms of the elements are essential to understand their behavior in the environment (mobility, pathways, bioavailability). These physical-chemical forms are considered as elemental species, formed by bound establishment of the elements with the sediment matrix constituents. Although there isn’t an established and standard definition for metal speciation, it is widely accepted to be the identification and quantification of the different, defined species/forms, in which an element occur in a determined matrix (Tack and Verloo, 1995). There are also evidences that microbial activity can greatly influence metal speciation, throughout interactions with metal ligands (Gadd, 2001 and 2004; Tabak et al., 2005). These transformations include reactions of

The Role of Salt Marsh Plants and Microorganisms in Sediment Metal Biogeochemistry 3 metal precipitation by metallic sulfides and redox reactions with changes on the metal specie and associations (Hullebusch et al., 2005). Caçador and col. (2000) showed a strong seasonal variation of plant biomass in these ecosystems together with a variation in metal concentrations in plant tissues, indicating a possible similar variation in the metal biogeochemistry.

HEAVY METAL SPECIATION Heavy metal inputs reaches salt marshes throughout tidal flooding and are retained in the sediments in various forms (Tessier, 1979), depending on the bounds they establish with the sediment components. There is a large variability in this process depending not only on the sediment characteristics but also on external factors (hydrodynamics, weather, seasonal variations and plant coverage) as it was seen in previous studies (Reboreda and Caçador, 2007; Duarte et al., 2008). The bibliography points out several evidences that microbial interactions with metals greatly influence their speciation (Gadd, 2001 and 2004; Tabak et al., 2005; Duarte et al., 2008). At this point this interaction between extracellular enzymes, organic matter and metals will be abordaded as an essential, but not unique, speciation mechanism. These transformations include metal precipitation reactions by metallic sulfides and redox reactions causing changes on the metal species and its associations (Hullebusch et al., 2005). Heavy metal speciation is very variable being dependent on both on salt marsh to salt marsh and on plant coverage (Reboreda and Caçador, 2007 and 2008). Several sequential extraction procedures have been developed throughout the years in order to assess metal speciation in different types of soils and sediments (Tessier, 1979; Miller et al., 1986; Keller and Verdi, 1994; European Comission, 1999). These different chemical protocols are based in selective extractants of increasing strength, that extract metals bounded to different sediment components (for example, oxides, salts, organic matter, sulphides, etc). Nowadays there are two procedures widely referenced: the Bureau of Certification and Reference (BCR) protocol and the Tessier protocol. These protocols are very similar, although the Tessier scheme was specifically developed for sediments, while the BCR protocol was developed initially for soils. The Tessier scheme applies a serie of extractants that will separate heavy metals in X fractions: exchangeable/available fraction, carbonate bound fraction, Fe and Mn oxides bound fraction, organic bound fraction and the residual fraction. Recently some adaptations of this method were developed to fulfill specific proposes of some studies, focusing fractions while discarding others, like the described in Hullebush et al., 2005. In this protocol the Fe and Mn oxides fraction is not extracted being lately solved in the residual extraction step. Salt marsh sediments are often very organic, a fact that can be an obstacle to the BCR procedure, due to the employing of high temperatures in the extraction of the organic bound metals with hydrogen peroxide, which leads to a very violent reaction and sample losses. The method proposed by Hullebush and colleagues (2005) was found to be the more adequate procedure for this study and for the type of samples (very organic rhizosediments). Metal chemical speciation becomes an important issue to aboard, not only in ecological risk assessment but also in phytoremediation processes. For these last it is important to consider this mechanisms to take advantage of them in the bioavailabilty modifications involved during this process. Overlooking the organic-bound fraction of all metals and the organic matter content and humic acids content in the sediment, a similar seasonal pattern is observed between these

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Bernardo Duarte and Isabel Caçador

components (Figure 2). This similarity indicates that a depletion of the organic-bound fraction of metals is driven by a decrease in the organic matter content in the sediment, and not by desorption of these metals from the organic matter.

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Figure 1. Metal speciation in the rhizosediments of H. portulacoides, along the studied period (from Duarte et al., 2008).

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The Role of Salt Marsh Plants and Microorganisms in Sediment Metal Biogeochemistry 5

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Figure 2. Comparasion between organic matter content (LOI), humic acids and the total heavy metals present in the organic bound fraction (from Duarte et al., 2008).

As it was previously referred, microbial enzymes will have its prime effect on the organic fraction of the sediments, where there is a large amount of heavy metals bounded. To better understand this mechanism the organic fractions of the metals should be compared with the activity of the sediment compounds and microbial activities. These enzymes exhibit different patterns of activity throughout the seasons, with peaks of activity in different periods, leading to a differential degradation of the organic components of the sediment. The enzymatic activity is greatly affected by medium conditions, in this case the pH, Eh and salinity of the sediment. From Spring to Autumn an increase of the organic matter content in the sediments colonized by H. portulacoides can be observed, which is in agreement with the increase of root biomass found by Caçador et al. (2000). The enzymatic analysis indicates that there are two different periods of organic matter cycling during the year. High protein degradation activities were also evident. During Spring and Summer high β-N-acetylglucosaminidase and phenol oxidase activities are also observed. β-N-acetylglucosaminidase degrades chitin and the release of this enzyme is associated with the ecdysis process. As for phenol oxidase, it catalyzes the degradation of recalcitrant phenolics materials, such as lignin (Freeman et al., 2004). Both enzymes degrade large structural polymers of animals and plants. As previously mentioned, chitin exoskeletons are known to be able to accumulate toxic metals (Bergey and Weis, 2007). There are also reports (Sousa et al., 2008) that lignin from H. portulacoides can accumulate small amounts of heavy metals. As chitin is a protein, its degradation is due not only to β-N-acetylglucosaminidase but also to protease, explaining the simultaneous high activity of both this enzymes. Merging the peaks of activity of these two enzymes with the organic-bound fraction of metals found, it is noticeable that, along with this degradation process, there is also a high depletion of the organic-bound fraction of metals, indicating that these metals are probably bound to chitin like proteins. Phenol oxidase shows its maximum activity during Summer and a total inhibition in the seasons hereafter. This inhibition is due to the obligatory need of molecular oxygen (Freeman et al., 2004) for the activity of this enzyme, scarce in the rhizosediments during the cold seasons, as indicated by the negative redox values verified during these periods. Together with a decrease in the activity of

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protease and β-N-acetylglucosaminidase there was a peak of phenolic degradation in Summer. This mechanism keeps the organic-bound metal fractions in low values, compared with Autumn, when all enzymes were found to be inactive. This indicates a degradation of plant residues, releasing the percentage of metals associated to phenolics. Whilst in Spring and Winter an increase of the percentage of metals in the residual fraction together with a depletion of metals in the organic bound fraction could be observed, the same didn’t happen in Summer. This is caused to the peaks of sulphatase activity detected in Spring and Winter. Some authors (Hullebusch et al., 2005) point out that high sulphatase activity can lead to the conversion of the sulphate produced by this enzyme to sulphides, by sulphate reducing bacteria. Sulfides can chemically reduce metals into a stable form for extended periods of time (Tabak et al., 2005), increasing therefore the metal concentrations in the residual fraction. This is also found when comparing the amount of metals in the more available form with sulphatase activity. This analysis indicated what it seemed to be a second major period of organic matter depletion, most significantly observed in Winter. In this season all enzymes except sulphatase, peroxidase and protease are inactive or inexistent. Extracellular peroxidase is known to be produced mostly by ligninolytic white fungi in order to degrade plant litter (Johnsen and Jacobsen, 2008). In the presence of hydrogen peroxide, this enzyme catalyzes the degradation of ligninocellulosic litter. Due to this mechanism peroxidase can operate even when the Eh values are low (as it was verified in Winter) without the need of molecular oxygen contrarily to phenol oxidase. Previous studies showed that, in salt marshes saprophytic fungi are majorly present in leaves and stems as epiphytes (Castro and Freitas, 2000). As previously stated, low Eh values prevailing in Winter, most probably do not allow the activity of phenol oxidase, being the peroxidase activity the principal source of organic matter recycling during Winter. In this season there are great inputs of plant litter due to leaf and stem senescence. Metals retained metals in these decaying parts, become available for peroxidasic activity, the principal source of organic matter recycling during this period. The degradation of structural polymers releases phenolic substances of lower molecular weight, which are accumulated in the sediment due to the absence of phenol oxidase activity. Together with this intense activity there is also a high protease activity, due to the degradation of lignin and associated components. This renders protein content more accessible for protease degradation. The breakdown of these bounds releases metals in the surrounding environment, and as verified in Spring, the high sulphatase activity detected could be responsible for the depletion of the labile metal fraction contrarily to which would be expected. These modifications in metal speciation due to EEA are important to be considered within the entire ecosystem. Organic matter is known to be an important sink of heavy metals, by the strong ligations that metals establish with organic compounds. The hydrolysis and breakdown of this organic compounds lead to the release of these metals to the surrounding medium, and consequently change their speciation. These changes are also important to be considered. Metals that were previously bound to organic matter suffer after its breakdown changes in their availability and also in their mobility, affecting therefore the community. This availability can be increased if these metals don’t establish stable connections with any other sediment components, and meaning that can then be uptaken by plants and consequently enter the food chain.

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The Role of Salt Marsh Plants and Microorganisms in Sediment Metal Biogeochemistry 7

Figure 3. Influence of extracellular enzymatic activities of Protease, Peroxidase, Fenol Oxidase and βN-acetylglucosaminidase (line) on total organic bound metals (bars). From Duarte et al., 2008.

CONCLUSION Salt march sediments are often very rich in organic matter pools, well known to be highly efficient sinks of heavy metals, constituting an important fraction of metal species present in salt marsh sediments (organic bound fraction). Organic matter cycles are supported by microbial enzymatic degradation mechanisms and consequently affect the organic bound metals. In addition, microbial driven sulphidisation is also an important factor when considering speciation processes. As previously referred this metallic speciation process is affected by a large number of environmental conditions (tide, sediment physic-chemistry, plant coverage, fauna and microbes).Throughout this study it became very evident that it is essential to consider microbial EEA as a key factor in not only affecting organic matter biogeochemical recycling, but also heavy metal speciation models. Without detriment of physical and chemical factors, such as pH, Eh and oxygen profiles, microbial speciation processes assume a very important role in metal speciation, which has been largely neglected so far by most studies. In conclusion, these sediment characteristics influence metal speciation not only directly throughout chemical processes but also indirectly by its effect in the activity of several extracellular enzymes. It becomes therefore clear

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that the role of the EEAs here presented is important enough to be taken into account in the future for metal availabity studies. Applying this knowledge of metal mobility/availability it is possible to improve several remediation techniques such as, in situ bioremediation projects, as well as with batch reactors or constructed wetlands and water treating plants or phytoremediation processes. 0,8

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Figure 4. Influence of extracellular enzymatic activites of Sulphatase (line) on total exchangeable metals (bars). From Duarte et al., 2008

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