Molecular Mechanisms in the Phytoremediation of Heavy Metals from ...

Molecular Mechanisms in the Phytoremediation of Heavy Metals from Coastal Waters

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Subrata Trivedi and Abid Ali Ansari

19.1

Heavy Metal Pollution in Coastal Areas: An Introduction

The rapid pace of industrialization and urbanization of coastal areas has created several pollution-related problems in which heavy metal pollution is a major area of concern. Coastal areas are one of the most important places for human inhabitation (McKinley et al. 2011). Major world cities like Los Angeles, San Francisco, New York, Tokyo, Osaka, Beijing, Singapore, Hong Kong, Sidney, Mumbai, Dubai, etc. are located along the coastal regions around the world. With rapid urbanization and industrialization, heavy metals are continuously carried to the estuarine and coastal areas from upstream of tributaries and from the industrial and sewage discharge. Major part of the anthropogenic metal load in coastal areas has a terrestrial source, mainly from mining and industrial activities along major rivers and estuaries (Mitra et al. 1995; Ridgway et al. 2003; Caeiro et al. 2005; Usero et al. 2005; Sundaray et al. 2011). Water pollution by heavy metals is a global issue that needs to be addressed properly. The coastal areas where the terrestrial and marine ecosystems converge are of great significance because a large number of plants and animals, both marine and estuarine, thrive in this dynamic and fragile habitat. Heavy metal contamination could affect the water quality and bioaccumulation of metals in aquatic organisms, resulting in potential long-term implication on the health of humans and also the ecosystem (Fernandes et al. 2007; Abdel-Baki et al. 2011; Trivedi et al. 1995; Mitra et al. 1996). Pollution by heavy metals is a very serious problem due to

S. Trivedi, M.Sc., M.Phil., Ph.D. (*) • A.A. Ansari, Ph.D. Department of Biology, Faculty of Science, University of Tabuk, Tabuk 71491, Saudi Arabia

their toxicity and ability to accumulate in the biota (Islam and Tanaka 2004). In many countries drinking water is produced and then supplied to different locations by desalination of seawater. The presence of high level of metals in seawater, especially coastal waters, poses severe problem for such activities. Some heavy metals which are essential components in metabolism may become toxic when present in high concentration. Some of these heavy metals, like Hg, Cd, Pb, As, and Se, are not essential for most of the plants, since they do not perform any known physiological function. Other heavy metals like Zn, Co, Cu, Fe, Mn, Mo, and Ni are considered as essential elements because they are required for normal growth and metabolism of plants. These latter elements can easily lead to poisoning at higher concentrations. Heavy metal toxicity may result from alterations of numerous physiological processes caused at cellular or molecular level by inactivating enzymes. They may block functional groups of metabolically important molecules, displace or substitute essential elements, and also disrupt membrane integrity. Heavy metal poisoning commonly results in the enhanced production of reactive oxygen species (ROS) due to interference with electron transport activities, particularly in the chloroplast membranes. Increase in ROS has several negative consequences like exposure of cells to oxidative stress leading to lipid peroxidation, biological macromolecule deterioration, ion leakage, membrane dismantling, and DNA-strand cleavage.

19.1.1 Phytoremediation and Types of Phytoremediation (Definitions) Bioremediation is one of the most applicable methods for the management of environmental contaminants by biological mechanisms (including microorganisms) in soil and water. Plant-based bioremediation technologies are collectively termed as phytoremediation. Thus, phytoremediation refers

A.A. Ansari et al. (eds.), Phytoremediation: Management of Environmental Contaminants, Volume 2, DOI 10.1007/978-3-319-10969-5_19, © Springer International Publishing Switzerland 2015

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S. Trivedi and A.A. Ansari

to the use of the plants to clean up contaminated soil and water. Phytoremediation is also called green remediation, agro-remediation, botano-remediation, or vegetative remediation. Different techniques used in phytoremediation are:

19.1.1.1 Phytoextraction Phytoextraction refers to the uptake of contaminants by plant roots and translocation within the plants. Contaminants are generally removed by harvesting the plants. It is one of the best methods to remove contaminants from soil, sediment, and sludge. 19.1.1.2 Rhizofiltration In rhizofiltration, plants, both terrestrial and aquatic, are used to absorb and concentrate contaminants including heavy metals from polluted aqueous sources in their roots. 19.1.1.3

Phytostabilization

Phytostabilization refers to the use of plants to reduce the mobility or bioavailability of pollutants in the environment, thus preventing their migration to groundwater or their entry into food chains.

19.1.1.4 Phytovolatilization In this method plants are used in the uptake of contaminants from soil and waste water, transforming them into volatilized compound and then transpiring into the atmosphere.

19.2

Phytoremediation of Heavy Metals from Coastal Waters (General)

It is reported that a common water hyacinth Eichhornia crassipes may serve as a phytoremediation tool for cleaning up of several metals from coastal areas. In a study at the coastal area of Ondo State in Nigeria, enrichment factor (EF) and translocation factor (TF) were evaluated for ten metals, namely, As, Cd, Cu, Cr, Fe, Mn, Ni, Pb, V, and Zn. Heavy metal accumulation was observed in water as well as in the roots and shoots of Eichhornia crassipes. The results indicated that Eichhornia crassipes was able to accumulate high levels of Cr, Cd, Pb, and As both in the roots and shoots (Agunbiade et al. 2009). Phytoremediation using halophytes could be a possible bioremediation technique to control heavy metal pollution in coastal environments. Phytoremediation using halophytes is being applied to recover polluted coastal lagoons (Madejón et al. 2006; Lone et al. 2008).

19.2.1 Phytoremediation of Cadmium Cadmium is not essential for plant growth and is very toxic to many organisms. In humans, cadmium causes several health problems such as damage to the kidneys and lung tis-

sues, emphysema, and carcinogenesis. It is also an endocrine disruptor and interferes with calcium regulation in biological systems (Degraeve (1981), Salem et al. (2000), and Awofolu (2005)). Cadmium is used in industrial operations to prevent corrosion of machinery. Plants show Cd toxicity like chlorosis, reddish veins and petioles, curled leaves, severe reduction in growth of roots and tops, and a number of tillers. Echinochloa polystachya, a fast-growing perennial grass, is able to accumulate high levels of cadmium and is a good candidate for Cd phytoextraction. A study conducted in Saudi Arabia showed that the translocation factor (TF) was high for Cd in Calotropis procera (Badr et al. 2012). Azolla pinnata accumulates 740 mg kg−1 Cd (Rai 2008) and Thlaspi caerulescens and Solanum photeinocarpum accumulate 263 and 158 mg kg−1 Cd, respectively (Lombi et al. 2001; Zhang et al. 2011). Hyperaccumulation of cadmium in Arabidopsis halleri (Cosio and Keller 2004; Kupper et al. 2000) and Brassica juncea (Salt et al. 1997) has also been reported.

19.2.2 Phytoremediation of Arsenic It is reported that over 137 million people in more than 70 countries are affected by arsenic poisoning from drinking water (Arsenic in drinking water seen as threat, “USAToday. com,” August 30, 2007). Arsenic poisoning or arsenicosis is caused by the ingestion, absorption, or inhalation of high levels of arsenic. Initial symptoms of arsenic poisoning include headaches, confusion, diarrhea, and drowsiness. With increased poisoning, convulsions and changes in fingernail pigmentation (leukonychia striata) occur. In acute arsenic poisoning severe diarrhea, vomiting, blood in the urine, hair loss, cramping muscles, stomach pain, and more convulsions take place. Arsenic poisoning also affects the skin, lungs, kidneys, and liver (Test ID: ASU. Arsenic, 24 h, Urine, Clinical Information, Mayo Medical Laboratories Catalog, Mayo Clinic. Retrieved 2012-09-25). Arsenic is related to heart disease and cancer (Smith et al. 1992; Chiou et al. 1997). Cancers related to As in drinking water are reported in Taiwan, Argentina, Chile, Bangladesh, and India (WHO 2001). Arsenic pollution is also linked to chronic lower respiratory diseases and diabetes (NavasAcien et al. 2008; Kile and Christiani 2008). Chronic exposure to arsenic may result in deficiency of vitamin A and night blindness (Hsueh et al. 1998). Arsenic poisoning ultimately results in coma and death. As (as arsenate) is an analogue of phosphate and interferes with essential cellular processes such as oxidative phosphorylation and ATP synthesis (Tripathi et al. 2007). Hence, removal of arsenic from water is of prime importance. Some marine algae, which are constantly exposed to arsenate in seawater, have the biochemical capability to convert arsenate to harmless organo-arsenic compounds intercellularly (Edmonds and Francesconi 1987). Pteris vittata

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(Chinese brake fern) can accumulate up to 95 % of the arsenic from soil in its fronds (Ma et al. 2001; Zhang et al. 2011). This hyperaccumulator fern species can be used in phytoremediation of arsenic (Wilkins and Salter 2003). Some properties like high biomass, fast growth, versatility and hardiness, extensive root system, high As accumulation in fronds, perennial, resistance to disease and pests, diverse ecological niches, and mycorrhizal associations of P. vittata make it an ideal candidate for phytoremediation of arsenic. The plant is reported to accumulate approximately 1,000 mg kg−1 As (Baldwin and Butcher 2007). Another study suggested that the level of As in P. vittata is 8,331 mg kg−1 (Kalve et al. 2011). Besides P. vittata four other species belonging to the genus Pteris, namely, P. biaurita, P. cretica, P. quadriaurita, and P. ryukyuensis, also accumulate high levels of arsenic, i.e., ~2,000, ~1,800, ~2,900, and 3,647 mg kg−1, respectively (Srivastava et al. 2006). In recent years much progress has been made in understanding As tolerance and its hyperaccumulation by P. vittata. However, more research on P. vittata is needed and certain technical barriers are to be overcome. Corrigiola telephiifolia, which accumulates 2,110 mg kg−1 As, is also a known hyperaccumulator species for arsenic (Garcia-Salgado et al. 2012). In seawater, marine algae can transform arsenate into nonvolatile methylated arsenic compounds (methanearsonic and dimethylarsinic acids). It is a beneficial step to the primary producers and also to the higher trophic levels, since nonvolatile methylated arsenic is much less toxic to marine invertebrates.

19.2.3 Phytoremediation of Lead Lead is an extremely toxic heavy metal, which can cause severe problems in children such as impaired development, reduced intelligence, short-term memory loss, learning disabilities, and coordination problems. High level of lead causes renal failure and increased risk for development of cardiovascular disease (Salem et al. 2000; Padmavathiamma and Li 2007; Wuana and Okieimen 2011; Iqbal 2012). Several plant species have been reported as important hyperaccumulators of lead. A leguminous shrub Sesbania drummondii and several Brassica species can accumulate significant amounts of lead in their roots (Blaylock et al. 1997; Sahi et al. 2002; Wong et al. 2001). Piptatherum miliaceum accumulate lead directly correlating to soil concentrations and do not show any symptoms of toxicity for 3 weeks. Sesbania drummondii can tolerate lead levels up to 1,500 mg L−1 and accumulate 40 g kg−1 shoot dry weight (Sahi et al. 2002). Lead is relatively insoluble because most of the lead is accumulated in the stems and not in the leaves (Kumar et al. 1995). The main problem for the phytoremediation of lead is its extremely low solubility (Huang et al.

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1997). The aquatic weed, Eichhornia crassipes, has phytoremediation potential for removal of lead from effluent (Sukumaran 2013).

19.2.4 Phytoremediation of Copper Copper is an essential element and enzyme cofactor for oxidases like cytochrome oxidase, superoxide dismutase, and tyrosinases but some plants and animals can accumulate even toxic levels of copper. High levels of copper can cause brain and kidney damage, liver cirrhosis and chronic anemia, and stomach and intestinal irritation (Salem et al. 2000; Wuana and Okieimen 2011). Salix nigra can accumulate more cadmium and copper but more studies are necessary to determine the feasibility of this species for phytoremediation (Kuzovkina et al. 2004). Eichhornia crassipes is estimated to accumulate high levels of copper and could be potentially used for phytoremediation (Liao and Chang 2004). Aquatic weed, Typha latifolia, can prominently remove copper, cadmium, and arsenic from effluent (Sukumaran 2013). Eleocharis acicularis which hyperaccumulates Cu at 20,200 mg kg−1 may be considered as a potential phytoremediation species for removal of copper (Sakakibara et al. 2011).

19.2.5 Phytoremediation of Chromium High level of chromium causes cancer and extensive hair loss (Salem et al. 2000). Crotalaria juncea and Crotalaria dactylon can remediate Cr and Cu (Saraswat and Rai 2009). Tumbleweeds Salsola kali (Gardea-Torresday et al. 2005) and Gynura pseudochina are known to be Cr hyperaccumulators (Mongkhonsin et al. 2011). Pteris vittata is a potential chromium phytoremediation species which accumulates 20,675 mg kg−1 Cr (Kalve et al. 2011). The species is also considered as phytoremediation tool for arsenic management.

19.2.6 Phytoremediation of Manganese Manganese has been reported for its negative effects on the respiratory and nervous system. Symptoms of manganese poisoning are hallucinations, forgetfulness, and nerve damage. Higher levels of manganese can also cause Parkinson disease, lung embolism, bronchitis, and impotency. Manganese can cause both toxicity and deficiency symptoms in plants. Chengiopanax sciadophylloides is a Mn hyperaccumulator species and ZIP family transporter genes have been isolated from this species (Mizuno et al. 2008 ). Austromyrtus bidwillii (Bidwell et al. 2002), Phytolacca

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americana (Pollard et al. 2009), and Maytenus founieri (Fernando et al. 2008) are capable of Mn hyperaccumulation. Schima superba is capable of hyperaccumulating Mn to a level of 62,412.3 mg kg−1 (Yang et al. 2008).

19.2.7 Phytoremediation of Nickel Nickel can cause allergic dermatitis known as nickel itch. Inhalation of Ni can cause cancer of the lungs, nose, throat, and stomach. Ni is hematotoxic, immunotoxic, neurotoxic, genotoxic, reproductive toxic, pulmonary toxic, nephrotoxic, and hepatotoxic metal. Furthermore it causes hair loss (Salem et al. 2000; Khan et al. 2007; Das et al. 2008). Ni-hyperaccumulating plants are Berkheya coddii (Robinson et al. 1997; Moradi et al. 2010), Sebertia acuminate (Jaffre et al. 1976; Perrier 2004), Phidiasia lindavii (Reeves et al. 1999), and Bornmuellera kiyakii (Reeves et al. 2009). Isatis pinnatiloba can accumulate Ni to a level of 1,441 mg kg−1 (Altinozlu et al. 2012). Five species belonging to the genus Alyssum, namely, Alyssum bertolonii, A. caricum, A. pterocarpum, A. murale, and A. corsicum, hyperaccumulate Ni with levels of 10,900, 12,500, 13,500, 15,000, and 18,100 mg kg−1, respectively (Li et al. 2003). A different study showed that Alyssum murale hyperaccumulates 4,730–20,100 mg kg−1 and Alyssum markgrafii accumulates 19,100 mg kg−1 Ni, respectively (Bani et al. 2010). Alyssum serpyllifolium accumulates 10,000 mg kg−1 Ni (Prasad 2005). Hence it is evident that all the seven different Alyssum species are capable of hyperaccumulating Ni and can be considered as a potential candidate for phytoremediation of nickel. After phytoremediation, plant biomass containing accumulated heavy metals can be combusted to get energy and the remaining ash is called bio-ore which can be processed for the recovery or extraction of the heavy metals. Phytomining is commercially used for Ni and it is less expensive compared to conventional extraction methods. Using Alyssum murale and Alyssum corsicum, we may grow biomass containing 400 kg Ni ha−1 with production costs of $250–500 ha−1. Considering Ni price of $40 kg−1 (in 2006, Ni metal was trading on the London Metal Exchange at more than $40 kg−1), Ni phytomining is considered a highly profitable agricultural technology (crop value = $16,000 ha−1) for Ni-contaminated soils (Chaney et al. 2007). This bio-based mining would be more attractive since it would be helpful in limiting environmental pollution (Siddiqui et al. 2009).

19.2.8 Phytoremediation of Vanadium Vanadium is used mainly to produce certain alloys, and V2O5 is used as a catalyst in manufacturing sulfuric acid and maleic anhydride and in making ceramics. The blood cells of

S. Trivedi and A.A. Ansari

some marine animals like ascidians hyperaccumulate vanadium which is 107 times higher than the vanadium found in seawater (Trivedi et al. 2003). In humans the acute effects of vanadium are irritation of the lungs, throat, eyes, and nasal cavities. Very little information is available on the phytoremediation of this metal. In seawater, many marine algae accumulate vanadium which is utilized in the functioning of vanadium-dependent haloperoxidases. The levels of vanadium in sediments, roots, stems, and leaves of a mangrove species Avicennia marina have been reported from Mtoni, Msimbazi, and Mbweni mangrove ecosystems (Mremi and Machiwa 2003). Among mushrooms, Amanita muscaria concentrates vanadium to levels of 100 times (2 mmol kg−1 dry weight) than those found in other mushrooms and higher plants.

19.2.9 Phytoremediation of Zinc Overdosage of zinc can cause dizziness and fatigue (Hess and Schmid 2002). The unicellular green alga, Dunaliella salina, showed high tendency for zinc accumulation and is a candidate for phytoremediation of zinc (Magda 2008). Studies conducted in China have identified Sedum alfredii as hyperaccumulator for Zn and Cd, and it has been intensively investigated by various researchers in their studies conducted in hydroponics and/or the uncontaminated and contaminated soils. Thlaspi caerulescens (Kupper and Kochian 2010), Arabis gemmifera, A. paniculata (Kubota and Takenaka 2003; Tang et al. 2009), Arabidopsis halleri (Zhao et al. 2000), and Picris divaricata (Du et al. 2011) also have the capability to hyperaccumulate zinc. Eleocharis acicularis which accumulates 11,200 mg kg−1 Zn is a potential candidate for zinc phytoremediation (Sakakibara et al. 2011).

19.2.10 Phytoremediation of Metals by Mangroves Located between marine and terrestrial environments, mangroves are transitional coastal ecosystems which are found mostly in the tropical and subtropical regions. In these regions, about 75 % of the coastline and nearly 18 million hectares are occupied by mangrove forests (Kathiresan and Qasim 2005). There are more than 14.5 million hectares of mangrove forests in the Indo-Pacific region (6.9 million), Africa (3.5 million), and the Americas (4.1 million) Sahoo and Dhal (2009). The mangrove ecosystems are of great ecological and economic significance. They serve as buffer zone and provide primary protection against storm surges and coastal erosion. Besides this, they are also an important nursery for various marine and estuarine faunas including fish, crustaceans, etc. The global economic value (USD) of mangrove habitat is estimated as 181 billion (Alongi 2002).

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One excellent example of mangrove ecosystem is Sunderbans which is located in India and Bangladesh. The Sunderbans is the single largest block of tidal halophytic mangrove forest listed in the UNESCO world heritage list (http://whc.unesco.org/en/list) which is regarded as a biodiversity hot spot. This region is also regarded as the world’s largest natural nursery where a large number of marine and estuarine species come to breed and the juveniles stay back to exploit its rich natural resources (Trivedi et al. 2007). Several researches on heavy metal accumulation by different floras and faunas in this region have been reported (Mitra et al. 1994a, b, 1995, 1996; Trivedi et al. 1995). A comparative study on the heavy metal accumulation by different mangrove plants in this area was conducted (Mitra et al. 1994c). Mangroves act as sink or buffer and remove/immobilize metals before reaching the nearby aquatic ecosystems like the estuaries and creeks. Due to high proportion of fine clays, organic matter and low pH, mangrove mud effectively sequester metals, often immobilized as sulfides in anaerobic sediments. Mangroves are considered to be tolerant and significantly adaptive to the presence of heavy metals (Chiu and Chou 1991; Walsh et al. 1979). A concentration of trace metals is reported for at least 33 mangrove species (Lewis et al. 2011). The widely distributed fast-growing mangrove plant Rhizophora mucronata has the potential for metal phytoremediation (Pahalawattaarachchi et al. 2009). Sonneratia caseolaris, a mangrove species belonging to family Sonneratiaceae, is found near the banks of tidal rivers in brackish water and provides essential congregating place for fireflies. The fermented juices of this mangrove species have the ability to arrest hemorrhage and the half-ripe fruit is used to treat coughs (Perry 1980). Recent studies showed that Sonneratia caseolaris possess the capacity to take up selected heavy metals through its roots and store certain heavy metals in its leaves without any sign of injury, thereby suggesting the potential of Sonneratia caseolaris as a phytoremediation species (Nazli and Hashim 2010). Bioaccumulation of heavy metals by certain mangrove species reveals that these plants can act as bio-purifier or biofilter (Zaman et al. 2013). The concentration of heavy metals in different parts of mangrove plants may be efficiently used for water quality monitoring program (Mitra et al. 2004). Different mangroves can be used for phytoextraction, phytostabilization, rhizofiltration, and phytovolatilization. Table 19.1 shows examples of trace metal bioaccumulation in mangrove tissue.

19.2.11

Molecular Mechanisms of Metal Phytoremediation

Heavy metal ions that are incorporated to the tissues of living organisms can bind to macromolecules like protein. In order to understand the mechanism of accumulation of the metal, it is important to search for the metal-binding proteins.

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Since these proteins are in turn encoded by specific genes, the searching and subsequent analysis of those genes provides important clues for the hyperaccumulation of the metals. The analysis of the expressed sequence tags (ESTs) plays an important role in elucidating the metal hyperaccumulation sites. It is noted that the frequency of ESTs for a gene encoding metal-binding protein in each developmental stage and in the adult tissue roughly reflects the level of mRNA expression. If the cDNA encoding the metal-binding protein is available, then the recombinant metal-binding protein can be produced in the laboratory. In this case, the cDNA encoding the metal-binding protein is ligated to a cloning vector by the process of genetic engineering. The recombinant DNA is then transformed using suitable competent cells. These bacterial cells when grown in proper condition in appropriate culture medium produce the metal-binding protein of interest. During the process of isolation of metal-binding protein, sometimes it is difficult to purify the protein from the mixture of several proteins. This situation often arises with the newly discovered metal-binding proteins or their genes which are not well characterized. In such a situation, it is a good idea to prepare fusion proteins. For example, vanadium-binding protein (CiVanabin5) was ligated to maltose-binding protein (MBP) to produce fusion protein CiVanabin5-MBP. This fusion protein was then subjected to amylose resin column chromatography. The fusion protein CiVanabin5-MBP was eluted from the column using a buffer containing maltose. Subsequently, the junction between the CiVanabin5 and MBP was cut, and metal-binding assay was conducted using immobilized metal ion affinity chromatography or IMAC (Trivedi et al. 2003). There has been significant progress in determining the molecular basis for metal accumulation, which provides a strong scientific basis to outline several strategies for phytoremediation of metals. Biotechnological approaches are now used to produce improved plant varieties for enhancing natural hyperaccumulation of heavy metals. After mobilization, metals first bind to the cell wall, which is an ion exchanger of comparatively low selectivity. Subsequently, transport systems and intracellular high-affinity binding sites then mediate and help the uptake across the plasma membrane through secondary transporters such as channel proteins and/or H+-coupled carrier proteins (Chaney et al. 2007). In recent years several membrane transporter gene families have been identified and characterized by heterologous complementation screens and sequencing of ESTs and plant genome studies. Many cation transporters have been identified in recent years, most of which are Zn-regulated transporter (ZRT), Fe-regulated transporter (IRT), natural resistance-associated macrophage proteins (NRAMP), Al-activated malate transporter (ALMT), cation diffusion facilitator (CDF), P-type ATPase (heavy metal associated), yellow stripe-like (YSL),

Avicennia marina Acanthus ilicifolius Ceriops decandra Avicennia officinalis Rhizophora apiculata Rhizophora mucronata Excoecaria agallocha Bruguiera cylindrica Ceriops decandra Aegiceras corniculatum Acanthus ilicifolius Avicennia marina

Avicennia marina Aegiceras corniculatum Hibiscus tiliaceus Excoecaria agallocha Bruguiera gymnorrhiza Not reported Rhizophora mangle (mg/g) Rhizophora mangle Many species (review)

Species Laguncularia racemosa Avicennia marina Rhizophora mangle 14 species including Rhizophora conjugata Acanthus ilicifolius Bruguiera caryophylloides Carapa moluccensis Scyphiphora hydrophyllacea Excoecaria agallocha Avicennia alba Avicennia marina Avicennia marina Avicennia marina R RM MC R

R MC R

SV MC M R SV

R

R

Location Panama Australia Brazil West Malaysia

China Australia

Australia

Puerto Rico Brazil China

India

India

Australia

Aerial roots

Leaf Roots Leaf Young leaves Old leaves Wood Bark Fruit Leaves Leaves Standing crop Leaves Roots Leaves Stems Roots Leaves

Tissue Leaves Leaves Fruits Leaves BD-12

Cu 2.3–5.0 2.9–24.8

BD

2–12

8.1–95.1

0.01–0.30 1.8–13.8 101 9 BD 18.3 3.6–12.2 4.3–20.5 6.6–21.4 6.1–4.8