**1. Introduction**

246 Radioisotopes – Applications in Physical Sciences

Vázquez A., (2001), Vertical profile determination of gamma emitting radionuclides with

*Environmental Engineering,* Veracruz University, Mexico, 2001, pp 23-32

major concentration in Caribbean Sea and Gulf of Mexico, M. Sc. Thesis,

The chemical speciation of trace metals in seawater is of critical importance to studies in marine biogeochemistry; as such information is essential for interpreting and understanding the chemical reactivity of trace metals in the environment. Foremost in this respect are studies into the role that chemical speciation plays in determining the biological availability (bioavailability) or toxicity of metals to organisms. Research on this topic over the last 30 years has clearly shown that open ocean productivity can be directly limited by iron. Other studies have revealed more subtle effects, such as co-limitation or limitation/toxicity affecting only some phytoplankton species, can occur with other trace metals and lead to controls on the composition of the phytoplankton community. Thus studies addressing chemical speciation in seawater are of relevance to the entire marine ecosystem.

Work on chemical speciation draws on skills and expertise from a diverse range of fields including; analytical chemistry, environmental chemistry, toxicology, geochemistry, genomics, proteomics, biological oceanography, physical oceanography and chemical oceanography. A tool common to all of these fields is the use of radioisotopes to examine the transfer or exchange between chemical species at environmentally relevant concentrations, which would be impossible with conventional analytical techniques. In this role radiotracers have been invaluable in the development of several key discoveries in Chemical and Biological Oceanography:


Oceanographic field research requires the ability to work on a moving ship in the ocean and if this was not difficult enough, work on trace metals necessitates the use of ultraclean techniques to avoid the ubiquitous contamination from the ship itself. Combining this with the normal precautions and safe working environment needed for using radioisotopes can present researchers with a formidable challenge. However despite these problems radiotracers have always been a useful tool for marine scientists, both on land

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 249

Element Concentration Inorganic Speciation Distribution Al 0.3 – 40 nM Al(OH)4-, Al(OH)3 Scavenged Ti 6 – 250 pM TiO(OH)2 Scavenged

Cr 3 – 5 nM CrO42-, Cr3+ Nutrient Mn 0.08 – 5 nM Mn2+ Scavenged Fe 0.02 – 2 nM Fe(OH)2+, Fe(OH)3 Nutrient Co 4 – 300 pM Co2+,Co3+ Nutrient Ni 2 – 12 nM Ni2+ Nutrient Cu 0.5 – 4.5 nM Cu2+ Nutrient Zn 0.05 – 9 nM Zn2+ Nutrient

Mo 105 nM MoO42- Conservative Cd 1 – 1000 pM CdCl2 Nutrient Table 1. Dissolved concentrations of bio-active trace metals in seawater (Bruland and Lohan,

Traditionally chemical oceanographers have made a simple distinction between particulate and dissolved forms by separation via filtration (0.2 µm or 0.4 µm). More recently with the application of ultrafiltration techniques the dissolved fraction has been further divided into soluble (passes through a 1-200 kDa ultrafilter) and colloidal (difference between dissolved and soluble). Particulate forms include metals located intracellularly, or adsorbed extracellularly to biogenic particles or metals that form the matrix of minerals or are

An important concept in the development of the field of chemical oceanography was that of "Oceanographic consistency" (Boyle and Edmond, 1975), by which data for dissolved metals

1. *Conservative type distribution* - Metals showing this behaviour have concentrations that maintain a relatively constant ratio to salinity, have long oceanic residence times

2. *Nutrient type distribution* – Concentrations are lowest in surface waters and increase with depth and are often strongly correlated to the distribution of the macronutrients (N, P and Si), indicating that these metals are assimilated by plankton in the euphotic zone

3. *Scavenged type distribution* – Typical of trace metals that are adsorbed to particles (scavenged) and have oceanic residence times (100-1000 y) less than the mixing time of the ocean. Highest concentrations are found nearest the sources of these elements to the

2. Have correlations with other elements that share the same controlling mechanisms. Application of this approach has resulted in a reliable test for analytical data and led to the determination of vertical profiles for all the natural elements of the periodic table (Nozaki, 1997). Based on the shape of the vertical profile of each trace metal they can be grouped into

three distinct groups reflecting their chemical behaviour in seawater:


2- Nutrient

V 30 – 36 nM HVO4

Se 0.5 – 2.3 nM SeO42-, SeO3

2003; Donat and Bruland, 1995; Nozaki, 1997).

adsorbed to them.

(> 105 years).

ocean.

had to meet the following criteria: 1. Form smooth vertical profiles.

and remineralized at depth.

and sea, as they allow the direct quantification of rates or fluxes and the identification of transformation pathways and mechanisms related to biogeochemical processes in the ocean. In recent years the development of extremely sensitive analytical techniques to determine the concentration of stable elements in seawater (ICP-MS) and phytoplankton (e.g. Nanosims, Synchtron XRF), coupled with the problems of using radioisotopes, has seen a general decline in their use compared to the genesis of trace metal marine biogeochemistry in the 1970's and 1980's. However in recent years a number of new questions have emerged where radioisotopes once again can provide crucial data and this has seen a mini-renaissance in their use.

The aim of this article is to provide a short overview on the previous use of radioisotopes in marine biogeochemistry (Section 2), where they have been applied directly in studies where chemical speciation is directly addressed. For the purposes of this article we only consider studies that utilize radioactive isotopes to directly assess the chemical speciation of trace metals in seawater or the use of chemical speciation techniques to determine the kinetics of exchange between different chemical species and their uptake by phytoplankton.

Research in marine biogeochemistry continues to develop rapidly and radioisotopes will continue to have a role as tools for enhancing our understanding of key processes involving trace metals in the ocean. New areas of research include the impacts of global warming, ocean acidification and ocean deoxygenation, all of which will impact on trace metal speciation and bioavailability. For this work radiotracers still have an important role to play and will continue to be utilised in major ocean-going international programs such as GEOTRACES and SOLAS and in laboratory work. As despite recent developments in analytical techniques, some important processes are still more easily followed by the use of radiotracers. In this context, here we also provide data from new applications (Section 3) of radioisotopes to current problems in this growing field.

#### **1.1 Trace metals in seawater**

In the following section we provide a short overview of trace metal chemistry in seawater and for more information we refer the reader to other review articles on this topic (Bruland and Lohan, 2003; Donat and Bruland, 1995). Other review articles examine the role of trace metals in biological cycles in the ocean (Bruland et al., 1991; Hunter et al., 1997; Morel et al., 2003).

**Note on the units used in this section:** It is common practice for oceanographers to report concentrations on both the molality (moles per kg of seawater) and molarity (moles per liter of seawater) scales, conversion between the two scales is easily accomplished when the density of the seawater is known (easily calculated from the salinity and temperature). In this text we report concentrations on the molarity scale (symbol M, units of mol L-1) using the following standard SI abbreviations: µM (1x 10-6 M), nM (1x 10-9 M), pM (1x 10-12 M), fM (1x 10-15 M) and aM (1x 10-18 M). Radiochemical activities are given in Becquerels (Bq).

#### **1.1.1 Concentrations and distributions of trace metals in seawater**

Trace metals are found in seawater over a wide range of concentrations stretching from µmol kg-1 to amol kg-1 (Bruland and Lohan, 2003) and can exist in a variety of physical and chemical forms (Section 1.2). Table 1 (below) lists the typical concentrations in seawater found for the bio-active trace metals considered in this work.

and sea, as they allow the direct quantification of rates or fluxes and the identification of transformation pathways and mechanisms related to biogeochemical processes in the ocean. In recent years the development of extremely sensitive analytical techniques to determine the concentration of stable elements in seawater (ICP-MS) and phytoplankton (e.g. Nanosims, Synchtron XRF), coupled with the problems of using radioisotopes, has seen a general decline in their use compared to the genesis of trace metal marine biogeochemistry in the 1970's and 1980's. However in recent years a number of new questions have emerged where radioisotopes once again can provide crucial data and this

The aim of this article is to provide a short overview on the previous use of radioisotopes in marine biogeochemistry (Section 2), where they have been applied directly in studies where chemical speciation is directly addressed. For the purposes of this article we only consider studies that utilize radioactive isotopes to directly assess the chemical speciation of trace metals in seawater or the use of chemical speciation techniques to determine the kinetics of exchange between different chemical species and their uptake by

Research in marine biogeochemistry continues to develop rapidly and radioisotopes will continue to have a role as tools for enhancing our understanding of key processes involving trace metals in the ocean. New areas of research include the impacts of global warming, ocean acidification and ocean deoxygenation, all of which will impact on trace metal speciation and bioavailability. For this work radiotracers still have an important role to play and will continue to be utilised in major ocean-going international programs such as GEOTRACES and SOLAS and in laboratory work. As despite recent developments in analytical techniques, some important processes are still more easily followed by the use of radiotracers. In this context, here we also provide data from new applications (Section 3) of

In the following section we provide a short overview of trace metal chemistry in seawater and for more information we refer the reader to other review articles on this topic (Bruland and Lohan, 2003; Donat and Bruland, 1995). Other review articles examine the role of trace metals in biological cycles in the ocean (Bruland et al., 1991; Hunter et al., 1997; Morel et al., 2003). **Note on the units used in this section:** It is common practice for oceanographers to report concentrations on both the molality (moles per kg of seawater) and molarity (moles per liter of seawater) scales, conversion between the two scales is easily accomplished when the density of the seawater is known (easily calculated from the salinity and temperature). In this text we report concentrations on the molarity scale (symbol M, units of mol L-1) using the following standard SI abbreviations: µM (1x 10-6 M), nM (1x 10-9 M), pM (1x 10-12 M), fM (1x 10-15 M) and aM (1x 10-18 M). Radiochemical activities are given in Becquerels

Trace metals are found in seawater over a wide range of concentrations stretching from µmol kg-1 to amol kg-1 (Bruland and Lohan, 2003) and can exist in a variety of physical and chemical forms (Section 1.2). Table 1 (below) lists the typical concentrations in seawater

has seen a mini-renaissance in their use.

radioisotopes to current problems in this growing field.

**1.1.1 Concentrations and distributions of trace metals in seawater** 

found for the bio-active trace metals considered in this work.

**1.1 Trace metals in seawater** 

phytoplankton.

(Bq).


Table 1. Dissolved concentrations of bio-active trace metals in seawater (Bruland and Lohan, 2003; Donat and Bruland, 1995; Nozaki, 1997).

Traditionally chemical oceanographers have made a simple distinction between particulate and dissolved forms by separation via filtration (0.2 µm or 0.4 µm). More recently with the application of ultrafiltration techniques the dissolved fraction has been further divided into soluble (passes through a 1-200 kDa ultrafilter) and colloidal (difference between dissolved and soluble). Particulate forms include metals located intracellularly, or adsorbed extracellularly to biogenic particles or metals that form the matrix of minerals or are adsorbed to them.

An important concept in the development of the field of chemical oceanography was that of "Oceanographic consistency" (Boyle and Edmond, 1975), by which data for dissolved metals had to meet the following criteria:

1. Form smooth vertical profiles.

2. Have correlations with other elements that share the same controlling mechanisms.

Application of this approach has resulted in a reliable test for analytical data and led to the determination of vertical profiles for all the natural elements of the periodic table (Nozaki, 1997). Based on the shape of the vertical profile of each trace metal they can be grouped into three distinct groups reflecting their chemical behaviour in seawater:


Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 251

rapid reduction rates and slower oxidation rates leads to significant concentrations of the lower oxidation states of some metals in ambient seawater. For more on this and the impact of sulfide on metal speciation see general reviews on this subject (Cutter, 1992; Emerson and

There are a number of trace metal radioisotopes that are commonly used for speciation work


to 60Ni β(318.7), (1173, 1332)

to 63Cu LSC (66.9)

to 64Zn (511)

to 99mTc LSC (739, 778)

beta decay, - gamma counting and LSC – liquid

Isotope Half-life Mode of decay Detection Method (keV) 48V 15.98 d EC to 48Ti (983, 1312) 51Cr 27.7 d EC to 51V (320)

55Fe 2.73 y EC to 55Mn LSC (5.9) 57Co 271.8 d EC to 57Fe (122.1) 58Co 70.88 d EC to 58Fe (810.8) 59Fe 44.51 d - to 59Co (1099,1292)


65Zn 243.8 d EC to 65Cu (1115) 67Cu 2.58 d - to 67Zn (185) 75Se 119.78 d EC to 75As (136,265)

109Cd 462 d EC to 109Ag (22) Table 2. Commonly used radioisotopes for trace metal speciation work in seawater (Data complied from sources mentioned in the text). Abbreviations used: d – days, h – hours, y –

Most of the radioisotopes listed above are routinely available commercially and many can be obtained as high specific activity carrier free solutions. See the later sections for more

Typically speciation work in marine biogeochemistry has utilized radioisotopes for two types of experiment: (i) Biological uptake under conditions of chemical equilibrium. (ii)

Radioisotopes have been extremely important in improving our understanding of the links between chemical speciation and bioavailability of trace metals to phytoplankton and bacteria in the ocean. The genesis of this field began with the application of trace metal

**1.4 Typical applications of radioisotopes to trace metal speciation in seawater** 

**1.3 Commonly used radioisotopes for trace metal seawater speciation studies** 

54Mn 312.2 d EC to 54Cr

60Co 5.271 y -

63Ni 100 y -

99Mo 2.7476 d -

details regarding experiments involving the individual metals.

Kinetics of transformation of a known species in seawater.

**1.4.1 Uptake of trace metals by phytoplankton** 

years, EC – electron capture, -

scintillation counting.

64Cu 12.7 h EC to 64Ni

Huested, 1991; Morse et al., 1987).

and they are listed in Table2 below.

### **1.2 Trace metal speciation in seawater**

Studies on trace metal speciation in seawater are concerned with determining the concentrations and processes affecting individual chemical species. Operationally this typically requires the application of specific techniques for the determination of analytically distinguishable chemical species or the application of thermodynamic or kinetic models to predict the behaviour of different species in seawater. Over the years the term 'speciation' began to be used for a number of different uses so to avoid confusion the International Union for Pure and Applied Chemistry (IUPAC) has published guidelines or recommendations for the definition of speciation analysis (Templeton et al., 2000):


#### **1.2.1 Inorganic speciation**

The inorganic speciation of trace metals in seawater (Table 1) is reasonably well described due to the extensive work performed by physical chemists in simple salt solutions. For the more complex media that is seawater, the use of Pitzer equations (Pitzer, 1973) is required but for many species in seawater this data is still missing. The reader is referred to a number of review chapters that cover the inorganic speciation of trace metals in more detail (Byrne et al., 1988; Turner et al., 1981). In particular recent reviews (Byrne, 2010; Millero et al., 2009) have focused on those elements whose inorganic speciation is dominated by hydroxide and/or carbonate species which are sensitive to decreases in pH and increasing CO2 concentrations due to anthropogenic inputs.

#### **1.2.2 Organic speciation**

Many trace metals have been found to be strongly complexed by organic ligands in seawater, most notably iron (Gledhill and van den Berg, 1994) and copper (Coale and Bruland, 1988). However very little is known about these metal-organic complexes though it appears that they are produced by organisms in response to metal stress (Croot et al., 2000). Only a few of these ligands have been isolated and the chemical structures determined; iron complexing siderophores (Martinez et al., 2001) and heavy metal sequestering thiol complexes such as phytochelatins (Ahner et al., 1994). For a general overview of organic speciation in seawater see Hirose (2006). A recent paper by Vraspir and Butler (2009) provides a summary of the current information on trace metal binding ligands that have been isolated and identified in seawater.

#### **1.2.3 Redox speciation – Importance of kinetics**

For many trace metals there are major differences in the reactivity, bioavailability and toxicity between redox species. A critical factor here is the role of kinetics and/or oxygen concentrations in maintaining thermodynamically unstable redox species in solution where

Studies on trace metal speciation in seawater are concerned with determining the concentrations and processes affecting individual chemical species. Operationally this typically requires the application of specific techniques for the determination of analytically distinguishable chemical species or the application of thermodynamic or kinetic models to predict the behaviour of different species in seawater. Over the years the term 'speciation' began to be used for a number of different uses so to avoid confusion the International Union for Pure and Applied Chemistry (IUPAC) has published guidelines or

i. *Chemical species*. Chemical elements: specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure ii. *Speciation analysis.* Analytical chemistry: analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample iii. *Speciation of an element; speciation*. Distribution of an element amongst defined chemical

iv. *Fractionation.* Process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity)

The inorganic speciation of trace metals in seawater (Table 1) is reasonably well described due to the extensive work performed by physical chemists in simple salt solutions. For the more complex media that is seawater, the use of Pitzer equations (Pitzer, 1973) is required but for many species in seawater this data is still missing. The reader is referred to a number of review chapters that cover the inorganic speciation of trace metals in more detail (Byrne et al., 1988; Turner et al., 1981). In particular recent reviews (Byrne, 2010; Millero et al., 2009) have focused on those elements whose inorganic speciation is dominated by hydroxide and/or carbonate species which are sensitive to decreases in pH and increasing CO2

Many trace metals have been found to be strongly complexed by organic ligands in seawater, most notably iron (Gledhill and van den Berg, 1994) and copper (Coale and Bruland, 1988). However very little is known about these metal-organic complexes though it appears that they are produced by organisms in response to metal stress (Croot et al., 2000). Only a few of these ligands have been isolated and the chemical structures determined; iron complexing siderophores (Martinez et al., 2001) and heavy metal sequestering thiol complexes such as phytochelatins (Ahner et al., 1994). For a general overview of organic speciation in seawater see Hirose (2006). A recent paper by Vraspir and Butler (2009) provides a summary of the current information on trace metal binding ligands that have

For many trace metals there are major differences in the reactivity, bioavailability and toxicity between redox species. A critical factor here is the role of kinetics and/or oxygen concentrations in maintaining thermodynamically unstable redox species in solution where

recommendations for the definition of speciation analysis (Templeton et al., 2000):

**1.2 Trace metal speciation in seawater** 

species in a system

**1.2.1 Inorganic speciation** 

**1.2.2 Organic speciation** 

concentrations due to anthropogenic inputs.

been isolated and identified in seawater.

**1.2.3 Redox speciation – Importance of kinetics** 

properties.

rapid reduction rates and slower oxidation rates leads to significant concentrations of the lower oxidation states of some metals in ambient seawater. For more on this and the impact of sulfide on metal speciation see general reviews on this subject (Cutter, 1992; Emerson and Huested, 1991; Morse et al., 1987).

#### **1.3 Commonly used radioisotopes for trace metal seawater speciation studies**

There are a number of trace metal radioisotopes that are commonly used for speciation work and they are listed in Table2 below.


Table 2. Commonly used radioisotopes for trace metal speciation work in seawater (Data complied from sources mentioned in the text). Abbreviations used: d – days, h – hours, y – years, EC – electron capture, beta decay, - gamma counting and LSC – liquid scintillation counting.

Most of the radioisotopes listed above are routinely available commercially and many can be obtained as high specific activity carrier free solutions. See the later sections for more details regarding experiments involving the individual metals.

#### **1.4 Typical applications of radioisotopes to trace metal speciation in seawater**

Typically speciation work in marine biogeochemistry has utilized radioisotopes for two types of experiment: (i) Biological uptake under conditions of chemical equilibrium. (ii) Kinetics of transformation of a known species in seawater.

#### **1.4.1 Uptake of trace metals by phytoplankton**

Radioisotopes have been extremely important in improving our understanding of the links between chemical speciation and bioavailability of trace metals to phytoplankton and bacteria in the ocean. The genesis of this field began with the application of trace metal

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 253

Work on iron biogeochemistry has greatly benefited from the easy availability of both 55Fe

The solubility of iron in seawater is a controlling factor in its distribution in the ocean and information on this topic has been achieved predominantly through the use of radioisotope experiments. Initial work (Kuma et al., 1992) focused on the determination of the rate of dissolution as a function of pH, as measured using a dialysis tube (1kDa), of amorphous ferric oxides formed upon addition of 59Fe(III) to seawater. This approach was then adapted to determine solubility directly in seawater samples by simple syringe filtration with a 0.025 µm filter (Millipore MF) of a seawater solution that had been amended with 100 nM of radiolabelled Fe (Kuma et al., 1996). This technique has subsequently been applied to a range of oceanic environments; coastal Japan (Kuma et al., 1998b), the Pacific Ocean (Kuma et al., 1998a) and the Indian Ocean (Kuma et al., 1996). Liu and Millero (2002) used the same approach but employed a 0.02 µm Anotop filter to measure iron solubility in UV irradiated seawater as a function of temperature and salinity. Field studies using the Anotop filter and 55Fe have been reported from the Mauritanian upwelling (Schlosser and Croot, 2009). Ultrafiltration (Vivaflow 50) has also been applied to studies of the effects of different ligands on iron solubility in seawater (Schlosser and Croot, 2008). The kinetics of iron hydroxide formation was determined using 55Fe and ion-pair solvent extraction of chelated

Iron radioisotopes have proven extremely useful for examining the exchange kinetics between different iron species in seawater. Hudson et al. (1992) utilised 59Fe in combination with ion-pair solvent extraction of iron chelated by sulfoxine (8-hydroxyquinoline-5 sulfonate). Using this approach they measured the rate at which the inorganic Fe(III) hydroxide species at seawater pH (referred to as Fe') are complexed by EDTA and the natural terrestrial siderophore desferrioxamine B (DFO-B). Another approach to measuring Fe' in UV irradiated seawater was developed by Sunda and Huntsman (2003) using solid phase extraction with EDTA on C18 Sep-Paks, by where the Fe' was retained on the column. The phenomena of colloidal pumping, where iron initially in the colloidal size range is transformed into particles has also been investigated in seawater using 59Fe (Honeyman and

**2.1.3 Iron uptake by phytoplankton and regeneration by zooplankton grazing** 

The use of radioisotopes to determine the rate of iron uptake by phytoplankton in trace metal buffered media is the best example there is for the advantages that this approach has over stable isotopes. The literature abounds with several key studies from Morel's group that shaped the direction of marine research on iron; the availability of Fe(II) and Fe(III) to diatoms (Anderson and Morel, 1980), iron colloids (Rich and Morel, 1990), the ability to separate intracellular from extracellular iron (Hudson and Morel, 1989) and the importance of kinetics (Hudson and Morel, 1990). Later work by Sunda and colleagues showed the differences in iron requirements between coastal and oceanic species (Sunda and Huntsman, 1995; Sunda et al., 1991) and the relationship between iron, light and cell size (Sunda and

and 59Fe for tracer studies and no other trace metal has been so widely studied.

**2.1.1 Solubility of iron in seawater** 

iron (Pham et al., 2006).

Santschi, 1991).

Huntsman, 1997).

**2.1.2 Kinetics of exchange between different iron species** 

buffers utilizing aminocarboxylate ligands; Nitrilotriacetic acid (NTA), Ethylenediaminetetraacetic acid (EDTA) and Diethylenetriaminepentaacetic acid (DTPA). A well characterized seawater media, AQUIL, was developed for use in trace metal uptake experiments (Price et al., 1989). New analytical tools were also required to determine the intracellular metal content from that simply adsorbed (Hudson and Morel, 1989) and this allowed the determination of metal quotas for cells (metal to carbon ratio, or metal per cell). Theoretical developments occurred simultaneously with new important paradigms and hypotheses that could be tested based on thermodynamic equilibrium between species; The Free ion association model (FIAM), see review by Campbell (1995) and later the Biotic Ligand Model (BLM) (Di Toro et al., 2001). The recognition that for some metals the system is not in equilibrium, due to slow exchange reactions (Hering and Morel, 1989; Hering and Morel, 1990), saw the use of pulse-chase experiments where a radio-isotope is added as a known species and its uptake followed over time. These new approaches led to important concepts with regard to the kinetic limitations (Hudson and Morel, 1993) on uptake by phytoplankton and how this can impact phytoplankton physiology (e.g. cell size, number of transport ligands).

Applications of the FIAM and BLM to experiments with natural seawater and phytoplankton communities are more complex as the chemical species which are bioavailable are mostly unknown. However if the added radio-isotope is in isotopic equilibrium natural uptake rates and metal quotas can be determined.

#### **1.4.2 Kinetics of exchange between trace metal species**

Experiments investigating the kinetics of exchange between chemical species in seawater have also been applied using radioisotopes. This has typically been done in a pulse-chase fashion utilizing an analytical detection method that was capable of determining the chemical species of interest. These experiments have not always been at the lab bench scale, as past experiments have been performed under controlled conditions in mesocosms, including sediments, and using multiple tracers (Li et al., 1984; Santschi et al., 1987; Santschi et al., 1980).

### **2. Present state of the art**

In the following sections we review the current and previous use of radioisotopes in seawater speciation studies for bio relevant trace metals.

#### **2.1 Iron (Fe)**

Our understanding of the marine biogeochemistry of iron (Fe) has developed rapidly over the last 30 years. The thermodynamically favoured redox form of Fe in seawater, Fe(III), is only weakly soluble in seawater (Millero, 1998). The reduced form, Fe(II), is found in oxic waters as a transient species, primarily generated by photochemical processes (Croot et al., 2008), and existing at extremely low concentrations (picomolar or less) because of rapid oxidation by O2 and H2O2 in warm surface waters. The oxidation of Fe(II) to the less soluble Fe(III) species, leads to the formation of colloidal oxyhydroxide (Kuma et al., 1996) species which coagulate and form particulate iron (Johnson et al., 1997). Dissolved iron is strongly organically complexed throughout the water column (Boye et al., 2001). Iron is an essential element for all life and is a limiting nutrient in many parts of the global ocean as has been so clearly demonstrated in the mesoscale iron enrichment experiments (de Baar et al., 2005). Work on iron biogeochemistry has greatly benefited from the easy availability of both 55Fe and 59Fe for tracer studies and no other trace metal has been so widely studied.

#### **2.1.1 Solubility of iron in seawater**

252 Radioisotopes – Applications in Physical Sciences

buffers utilizing aminocarboxylate ligands; Nitrilotriacetic acid (NTA), Ethylenediaminetetraacetic acid (EDTA) and Diethylenetriaminepentaacetic acid (DTPA). A well characterized seawater media, AQUIL, was developed for use in trace metal uptake experiments (Price et al., 1989). New analytical tools were also required to determine the intracellular metal content from that simply adsorbed (Hudson and Morel, 1989) and this allowed the determination of metal quotas for cells (metal to carbon ratio, or metal per cell). Theoretical developments occurred simultaneously with new important paradigms and hypotheses that could be tested based on thermodynamic equilibrium between species; The Free ion association model (FIAM), see review by Campbell (1995) and later the Biotic Ligand Model (BLM) (Di Toro et al., 2001). The recognition that for some metals the system is not in equilibrium, due to slow exchange reactions (Hering and Morel, 1989; Hering and Morel, 1990), saw the use of pulse-chase experiments where a radio-isotope is added as a known species and its uptake followed over time. These new approaches led to important concepts with regard to the kinetic limitations (Hudson and Morel, 1993) on uptake by phytoplankton and how this can impact phytoplankton physiology (e.g. cell size, number of

Applications of the FIAM and BLM to experiments with natural seawater and phytoplankton communities are more complex as the chemical species which are bioavailable are mostly unknown. However if the added radio-isotope is in isotopic

Experiments investigating the kinetics of exchange between chemical species in seawater have also been applied using radioisotopes. This has typically been done in a pulse-chase fashion utilizing an analytical detection method that was capable of determining the chemical species of interest. These experiments have not always been at the lab bench scale, as past experiments have been performed under controlled conditions in mesocosms, including sediments, and

In the following sections we review the current and previous use of radioisotopes in

Our understanding of the marine biogeochemistry of iron (Fe) has developed rapidly over the last 30 years. The thermodynamically favoured redox form of Fe in seawater, Fe(III), is only weakly soluble in seawater (Millero, 1998). The reduced form, Fe(II), is found in oxic waters as a transient species, primarily generated by photochemical processes (Croot et al., 2008), and existing at extremely low concentrations (picomolar or less) because of rapid oxidation by O2 and H2O2 in warm surface waters. The oxidation of Fe(II) to the less soluble Fe(III) species, leads to the formation of colloidal oxyhydroxide (Kuma et al., 1996) species which coagulate and form particulate iron (Johnson et al., 1997). Dissolved iron is strongly organically complexed throughout the water column (Boye et al., 2001). Iron is an essential element for all life and is a limiting nutrient in many parts of the global ocean as has been so clearly demonstrated in the mesoscale iron enrichment experiments (de Baar et al., 2005).

equilibrium natural uptake rates and metal quotas can be determined.

using multiple tracers (Li et al., 1984; Santschi et al., 1987; Santschi et al., 1980).

**1.4.2 Kinetics of exchange between trace metal species** 

seawater speciation studies for bio relevant trace metals.

transport ligands).

**2. Present state of the art** 

**2.1 Iron (Fe)** 

The solubility of iron in seawater is a controlling factor in its distribution in the ocean and information on this topic has been achieved predominantly through the use of radioisotope experiments. Initial work (Kuma et al., 1992) focused on the determination of the rate of dissolution as a function of pH, as measured using a dialysis tube (1kDa), of amorphous ferric oxides formed upon addition of 59Fe(III) to seawater. This approach was then adapted to determine solubility directly in seawater samples by simple syringe filtration with a 0.025 µm filter (Millipore MF) of a seawater solution that had been amended with 100 nM of radiolabelled Fe (Kuma et al., 1996). This technique has subsequently been applied to a range of oceanic environments; coastal Japan (Kuma et al., 1998b), the Pacific Ocean (Kuma et al., 1998a) and the Indian Ocean (Kuma et al., 1996). Liu and Millero (2002) used the same approach but employed a 0.02 µm Anotop filter to measure iron solubility in UV irradiated seawater as a function of temperature and salinity. Field studies using the Anotop filter and 55Fe have been reported from the Mauritanian upwelling (Schlosser and Croot, 2009). Ultrafiltration (Vivaflow 50) has also been applied to studies of the effects of different ligands on iron solubility in seawater (Schlosser and Croot, 2008). The kinetics of iron hydroxide formation was determined using 55Fe and ion-pair solvent extraction of chelated iron (Pham et al., 2006).

#### **2.1.2 Kinetics of exchange between different iron species**

Iron radioisotopes have proven extremely useful for examining the exchange kinetics between different iron species in seawater. Hudson et al. (1992) utilised 59Fe in combination with ion-pair solvent extraction of iron chelated by sulfoxine (8-hydroxyquinoline-5 sulfonate). Using this approach they measured the rate at which the inorganic Fe(III) hydroxide species at seawater pH (referred to as Fe') are complexed by EDTA and the natural terrestrial siderophore desferrioxamine B (DFO-B). Another approach to measuring Fe' in UV irradiated seawater was developed by Sunda and Huntsman (2003) using solid phase extraction with EDTA on C18 Sep-Paks, by where the Fe' was retained on the column. The phenomena of colloidal pumping, where iron initially in the colloidal size range is transformed into particles has also been investigated in seawater using 59Fe (Honeyman and Santschi, 1991).

#### **2.1.3 Iron uptake by phytoplankton and regeneration by zooplankton grazing**

The use of radioisotopes to determine the rate of iron uptake by phytoplankton in trace metal buffered media is the best example there is for the advantages that this approach has over stable isotopes. The literature abounds with several key studies from Morel's group that shaped the direction of marine research on iron; the availability of Fe(II) and Fe(III) to diatoms (Anderson and Morel, 1980), iron colloids (Rich and Morel, 1990), the ability to separate intracellular from extracellular iron (Hudson and Morel, 1989) and the importance of kinetics (Hudson and Morel, 1990). Later work by Sunda and colleagues showed the differences in iron requirements between coastal and oceanic species (Sunda and Huntsman, 1995; Sunda et al., 1991) and the relationship between iron, light and cell size (Sunda and Huntsman, 1997).

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 255

Huntsman (1988) found that in the Sargasso Sea that Mn oxidation was inhibited by sunlight, consistent with photoinhibition of manganese oxidizing bacteria. Moffett (1997) confirmed this for the Sargasso Sea but found in the Equatorial Pacific that phytoplankton uptake of Mn may be more important. Similar Mn oxidation studies have been performed in the Eastern Caribbean (Waite and Szymczak, 1993) and in hydrothermal plumes (Mandernack and Tebo, 1993). A number of field studies by Moffett and co-workers have sought to link bacterial Mn

The dissolution of 54MnO2 in seawater has been extensively investigated and found to be strongly related to the presence of H2O2 formed by the photoreduction of O2 by dissolved organic matter (Sunda et al., 1983). Photoreduction of MnO2 in shallow sediments has also been observed (McCubbin and Leonard, 1996). Laboratory studies have also investigated the

The speciation of Copper (Cu) in seawater is dominated by organic complexation (Coale and Bruland, 1988) by ligands which are believed to be produced by phytoplankton in response to Cu stress (Croot et al., 2000). While Cu(II) is the thermodynamically favoured redox state in oxygenated seawater there is growing evidence that Cu(I) may also be significant. Radiotracer studies into Cu chemistry however are limited by the short half-lives

Initial studies on Cu uptake by phytoplankton used 64Cu and were focused on pulse chase experiments with NTA buffers and lipophilic 64Cu complexes that could pass directly through the phytoplankton cell wall (Croot et al., 1999). Later work showed the existence of an efflux system for Cu from the Cu stressed cells of the cyanobacteria *Synechococcus* (Croot et al., 2003). Recent works on the uptake of Cu by phytoplankton have utilized the longer lived isotope 67Cu to obtain important information on the uptake kinetics of Cu by diatoms (Guo et al., 2010), determined cellular Cu quotas for different phytoplankton types (Quigg et al., 2006) and showed the dependence of Cu on Fe uptake (Maldonado et al., 2006) and in turn the role of Fe in determining the cellular quota for Cu (Annett et al., 2008). However the most exciting application so far has been the first reported use of 67Cu for work performed using natural phytoplankton assemblages from the North Pacific (Semeniuk et al., 2009).

Zinc (Zn) is a required metal for bacteria and phytoplankton in the ocean as it serves as a metal cofactor for many important processes (Vallee and Auld, 1993). Most notably Zn is utilized for both nucleic acid transcription and repair proteins (Anton et al., 2007) in the enzyme alkaline phosphatise (Shaked et al., 2006) and for the uptake of CO2 via the enzyme Carbonic Anhydrase (CA) (Morel et al., 1994). The strong requirement for Zn by phytoplankton results in low concentrations in surface waters and a nutrient like profile in the ocean (Table 1). In most surface waters Zn is strongly organically complexed (Bruland, 1989), however in deep waters and in surface waters of the Southern Ocean inorganic

The use of 65Zn was central to the first speciation studies of Zn uptake by phytoplankton performed on cyanobacteria (Fisher, 1985) and diatoms (Sunda and Huntsman, 1992).

oxidation to the oxidation of Co (Moffett and Ho, 1996) and Ce (Moffett, 1994).

impact of humic acids on the photoreduction of MnO2 (Spokes and Liss, 1995).

of the two isotopes available 64Cu (t½ = 12.7 hours) and 67Cu (t½ = 2.58 days).

**2.2.3 Mn photoreduction** 

**2.3 Copper (Cu)** 

**2.4 Zinc (Zn)** 

complexes can dominate (Baars and Croot, 2011).

There have also been a number of studies examining the role of zooplankton grazing in transforming iron contained in phytoplankton back into the dissolved phase (Hutchins and Bruland, 1994). A dual tracer (55Fe and 59Fe) approach has also been used to study the fate of intracellular and extracellular iron in diatoms when grazed by copepods (Hutchins et al., 1999). The direct remineralisation of colloidal iron by protozoan grazers has also been observed (Barbeau et al., 1996). Other trophic transfer mechanisms investigated include the transfer of bacterial iron to the dissolved phase by ciliates (Vogel and Fisher, 2009) and remineralisation via viral lysis (Poorvin et al., 2004).

#### **2.1.4 Iron redox speciation**

Somewhat surprisingly there have been very few studies examining iron redox processes in seawater using radioisotopes. Though in part this is most likely due to the short life-time of this species in ambient seawater and the application of chemiluminescence techniques to detect pM Fe(II) (Croot and Laan, 2002). The photoreduction of 59Fe-EDTA has been used as a model system by both Hudson et al. (1992) and Sunda and Huntsman (2003). Photoreduction of natural iron complexes in the Southern Ocean as been shown to be strongly related to UV-B (Rijkenberg et al., 2005). The biological reduction of iron by phytoplankton has also been investigated by Shaked et al. (2004) who used Ferrozine as an Fe(II) chelator and then retained the complex on C18 Sep-Paks.

#### **2.2 Manganese (Mn)**

Manganese (Mn) is a redox sensitive element which is important to phytoplankton due to its involvement in photosynthesis through photosystem II in converting H2O to O2 (Falkowski and Raven, 1997). Mn is also utilized in superoxide dismutases (Peers and Price, 2004). Mn has a scavenged type profile (Landing and Bruland, 1987) and a secondary Mn maximum occurs in the oxygen minimum zone (Johnson et al., 1996). Mn(IV) is the thermodynamically favoured form in seawater but is strongly hydrolysed forming particulate MnO2. Mn(II) is weakly hydrolysed in seawater and does not form strong organic complexes in seawater and is slowly oxidized to particulate Mn(III) and Mn(IV) under seawater conditions (von Langen et al., 1997).

#### **2.2.1 Mn uptake by phytoplankton**

The uptake of Mn by phytoplankton has been investigated for a number of species by using 54Mn. In a series of now classical laboratory studies by Sunda and Huntsman (1983; 1996) the interactive effects between Mn and Cu, Zn and Cd in phytoplankton were investigated and showed clearly the competition for uptake by these elements for the same transport ligands in the diatom species tested. Other work has shown that the 54Mn taken up by phytoplankton can be recycled back into the dissolved phase through the action of zooplankton grazing by copepods (Hutchins and Bruland, 1994).

#### **2.2.2 Mn oxidation**

The oxidation of dissolved Mn(II) in seawater to particulate manganese oxides has been studied extensively in the field via the use of 54Mn (Emerson et al., 1982) and taking advantage of the differences in the solubilities of the different Mn redox states. Initial studies focused on the role of oxygen in the bacterially mediated oxidation of Mn(II) in suboxic zones (Tebo and Emerson, 1985). Work in oxygenated surface waters by Sunda and Huntsman (1988) found that in the Sargasso Sea that Mn oxidation was inhibited by sunlight, consistent with photoinhibition of manganese oxidizing bacteria. Moffett (1997) confirmed this for the Sargasso Sea but found in the Equatorial Pacific that phytoplankton uptake of Mn may be more important. Similar Mn oxidation studies have been performed in the Eastern Caribbean (Waite and Szymczak, 1993) and in hydrothermal plumes (Mandernack and Tebo, 1993). A number of field studies by Moffett and co-workers have sought to link bacterial Mn oxidation to the oxidation of Co (Moffett and Ho, 1996) and Ce (Moffett, 1994).

### **2.2.3 Mn photoreduction**

The dissolution of 54MnO2 in seawater has been extensively investigated and found to be strongly related to the presence of H2O2 formed by the photoreduction of O2 by dissolved organic matter (Sunda et al., 1983). Photoreduction of MnO2 in shallow sediments has also been observed (McCubbin and Leonard, 1996). Laboratory studies have also investigated the impact of humic acids on the photoreduction of MnO2 (Spokes and Liss, 1995).

#### **2.3 Copper (Cu)**

254 Radioisotopes – Applications in Physical Sciences

There have also been a number of studies examining the role of zooplankton grazing in transforming iron contained in phytoplankton back into the dissolved phase (Hutchins and Bruland, 1994). A dual tracer (55Fe and 59Fe) approach has also been used to study the fate of intracellular and extracellular iron in diatoms when grazed by copepods (Hutchins et al., 1999). The direct remineralisation of colloidal iron by protozoan grazers has also been observed (Barbeau et al., 1996). Other trophic transfer mechanisms investigated include the transfer of bacterial iron to the dissolved phase by ciliates (Vogel and Fisher, 2009) and

Somewhat surprisingly there have been very few studies examining iron redox processes in seawater using radioisotopes. Though in part this is most likely due to the short life-time of this species in ambient seawater and the application of chemiluminescence techniques to detect pM Fe(II) (Croot and Laan, 2002). The photoreduction of 59Fe-EDTA has been used as a model system by both Hudson et al. (1992) and Sunda and Huntsman (2003). Photoreduction of natural iron complexes in the Southern Ocean as been shown to be strongly related to UV-B (Rijkenberg et al., 2005). The biological reduction of iron by phytoplankton has also been investigated by Shaked et al. (2004) who used Ferrozine as an

Manganese (Mn) is a redox sensitive element which is important to phytoplankton due to its involvement in photosynthesis through photosystem II in converting H2O to O2 (Falkowski and Raven, 1997). Mn is also utilized in superoxide dismutases (Peers and Price, 2004). Mn has a scavenged type profile (Landing and Bruland, 1987) and a secondary Mn maximum occurs in the oxygen minimum zone (Johnson et al., 1996). Mn(IV) is the thermodynamically favoured form in seawater but is strongly hydrolysed forming particulate MnO2. Mn(II) is weakly hydrolysed in seawater and does not form strong organic complexes in seawater and is slowly oxidized to particulate Mn(III) and Mn(IV) under seawater conditions (von

The uptake of Mn by phytoplankton has been investigated for a number of species by using 54Mn. In a series of now classical laboratory studies by Sunda and Huntsman (1983; 1996) the interactive effects between Mn and Cu, Zn and Cd in phytoplankton were investigated and showed clearly the competition for uptake by these elements for the same transport ligands in the diatom species tested. Other work has shown that the 54Mn taken up by phytoplankton can be recycled back into the dissolved phase through the action of

The oxidation of dissolved Mn(II) in seawater to particulate manganese oxides has been studied extensively in the field via the use of 54Mn (Emerson et al., 1982) and taking advantage of the differences in the solubilities of the different Mn redox states. Initial studies focused on the role of oxygen in the bacterially mediated oxidation of Mn(II) in suboxic zones (Tebo and Emerson, 1985). Work in oxygenated surface waters by Sunda and

remineralisation via viral lysis (Poorvin et al., 2004).

Fe(II) chelator and then retained the complex on C18 Sep-Paks.

zooplankton grazing by copepods (Hutchins and Bruland, 1994).

**2.1.4 Iron redox speciation** 

**2.2 Manganese (Mn)** 

Langen et al., 1997).

**2.2.2 Mn oxidation** 

**2.2.1 Mn uptake by phytoplankton** 

The speciation of Copper (Cu) in seawater is dominated by organic complexation (Coale and Bruland, 1988) by ligands which are believed to be produced by phytoplankton in response to Cu stress (Croot et al., 2000). While Cu(II) is the thermodynamically favoured redox state in oxygenated seawater there is growing evidence that Cu(I) may also be significant. Radiotracer studies into Cu chemistry however are limited by the short half-lives of the two isotopes available 64Cu (t½ = 12.7 hours) and 67Cu (t½ = 2.58 days).

Initial studies on Cu uptake by phytoplankton used 64Cu and were focused on pulse chase experiments with NTA buffers and lipophilic 64Cu complexes that could pass directly through the phytoplankton cell wall (Croot et al., 1999). Later work showed the existence of an efflux system for Cu from the Cu stressed cells of the cyanobacteria *Synechococcus* (Croot et al., 2003). Recent works on the uptake of Cu by phytoplankton have utilized the longer lived isotope 67Cu to obtain important information on the uptake kinetics of Cu by diatoms (Guo et al., 2010), determined cellular Cu quotas for different phytoplankton types (Quigg et al., 2006) and showed the dependence of Cu on Fe uptake (Maldonado et al., 2006) and in turn the role of Fe in determining the cellular quota for Cu (Annett et al., 2008). However the most exciting application so far has been the first reported use of 67Cu for work performed using natural phytoplankton assemblages from the North Pacific (Semeniuk et al., 2009).

#### **2.4 Zinc (Zn)**

Zinc (Zn) is a required metal for bacteria and phytoplankton in the ocean as it serves as a metal cofactor for many important processes (Vallee and Auld, 1993). Most notably Zn is utilized for both nucleic acid transcription and repair proteins (Anton et al., 2007) in the enzyme alkaline phosphatise (Shaked et al., 2006) and for the uptake of CO2 via the enzyme Carbonic Anhydrase (CA) (Morel et al., 1994). The strong requirement for Zn by phytoplankton results in low concentrations in surface waters and a nutrient like profile in the ocean (Table 1). In most surface waters Zn is strongly organically complexed (Bruland, 1989), however in deep waters and in surface waters of the Southern Ocean inorganic complexes can dominate (Baars and Croot, 2011).

The use of 65Zn was central to the first speciation studies of Zn uptake by phytoplankton performed on cyanobacteria (Fisher, 1985) and diatoms (Sunda and Huntsman, 1992).

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 257

*weissflogii*. Later Dupont and co-workers investigated the uptake of 63Ni by the globally important cyanobacteria *Synechococcus* in a laboratory study (Dupont et al., 2008) and made field measurements of 63Ni uptake by natural phytoplankton assemblages (Dupont et al., 2010). They showed that Ni was a required element for many strains of *Synechococcus* and by comparison to the available genomic data most likely all strains of *Procholorococcus*. This was due to the use of a Ni containing superoxide dismutase and in the enzyme for urease uptake. Importantly they also observed that isotopic equilibrium was not established between the added radiotracer and the natural pools of Ni within 24 hours indicating the

Selenium (Se) is found in very low concentrations in seawater (< 1 nM) and its chemistry is under kinetic redox control (Cutter, 1992) with the oxyanions Selenate (SeO42-) (thermodynamically favoured in oxygenated seawater and selenite (SeO32-), both showing nutrient like profiles in the ocean. In surface waters enhanced concentrations of organic

The radioisotope 75Se has been used in a number of studies to elucidate the biogeochemistry of Se in phytoplankton cells. A key early finding was the identification of the pathway of uptake of selenite into the diatom *Thalassiosira pseudonana* and its conversion into the Se containing enzyme glutathione peroxidase (Price and Harrison, 1988). Later studies using the coccolithophorid *Emiliania huxleyi* as a model organism (Obata et al., 2004) , have shown in more detail the steps involved into the uptake of selenite by the cells, and identified a pool of low molecular weight compounds which are used to store Se before incorporation into specific seleno-proteins in the cell. The interspecies differences in selenite uptake and accumulation have also been assessed (Vandermeulen and Foda, 1988), and the release of Se contained in phytoplankton cells by zooplankton grazing or phytoplankton decomposition

Molybdenum (Mo) is almost conservative (105 nM) in oxygenated seawater (Collier, 1985)

reduced to form Mo-sulfides (Erickson and Helz, 2000) and is rapidly precipitated from the water column (Helz et al., 2004). Molybdenum is an essential element for phytoplankton which is utilized as a co-factor in a number of different enzymes (Kisker et al., 1997) and in particular in nitrogenase and nitrate reductase which catalyze the reduction of N2 and nitrate to bioavailable N in the ocean (Mendel, 2005). While Mo has been shown to be a limiting nutrient for freshwater organisms the evidence of Mo limitation in marine organisms is unclear. The use of 99Mo in seawater studies requires special sample handling so that the daughter product 99Tc does not interfere for this reason it has been almost used exclusively in nitrogen fixation studies where the focus has been on the potential for sulfate inhibition of Mo uptake leading to Mo limitation of nitrogen fixation (Marino et al., 2003).

The marine biogeochemistry of Cadmium (Cd) has already yielded a number of surprises as it was long thought to be simply only toxic to organisms making its nutrient like profile in seawater and tight coupling with phosphate difficult to understand. However the

2-), under reducing conditions Mo is

selenide (operationally defined) is typically present (Cutter and Bruland, 1984).

slow exchange kinetics of Ni in seawater (Hudson and Morel, 1993).

**2.6.2 Selenium (Se)** 

(Lee and Fisher, 1992).

**2.6.4 Cadmium (Cd)** 

**2.6.3 Molybdenum (Mo)** 

where it is present as the oxyanion molybdate (MoO4

Studies into 65Zn uptake by bacteria (Vogel and Fisher, 2010) found a much lower uptake of Zn than Cd. 65Zn has also been used in assessing the release of Zn from phytoplankton (Hutchins and Bruland, 1994) and bacteria (Vogel and Fisher, 2009) during zooplankton grazing.

#### **2.5 Cobalt (Co)**

Cobalt (Co) is present in seawater at very low concentrations (< 100 pM) and can exist as either inert Co(III) complexes or more labile Co(II) organic species (Saito and Moffett, 2002). Despite the range of Co isotopes available (Table 2) there have been relatively few studies examining the seawater speciation of Co. Work by Nolan et al. (1992) utilised a dual tracer approach where the uptake of 57Co-cobalamine was compared to that of 60Co-Co(II) and found that the cobalamine was taken up significantly faster and retained for longer in phytoplankton. 57Co was also used to show that Co could replace Zn in the enzyme carbonic anhydrase in some phytoplankton (Yee and Morel, 1996). An early finding with 57Co was that the oxidation of Co(II) to Co(III) in solution (Lee and Fisher, 1993) may be mediated by the same bacteria responsible for Mn oxidation (see section 2.3.2). Though new data (Murray et al., 2007) suggests no Co(II) oxidation occurs in the complete absence of Mn(II) and that the mechanism by which bacteria oxidize Co(II) is through the production of the reactive nano-particulate Mn oxide. Co has also been found to be released back to the dissolved phase from grazed and decomposing phytoplankton (Lee and Fisher, 1994).

#### **2.6 Speciation studies with other trace metals**

Studies into the biogeochemical cycles of other elements in seawater that are strongly hydrolysed and thus analogous to iron are limited by the lack of suitable radiotracers. There are no Aluminium (Al) radioisotopes suitable for use in trace metal speciation studies, as the majority of them have half-lives shorter than 10 minutes. The long lived isotope 26Al (710,000 y) has found application in paleo applications (Lal et al., 2006). Similarly there are also no seawater studies on Titanium (Ti) biogeochemistry with radioisotopes due to the short half-lives of 45Ti (3.08 h), 51Ti (5.76 min) and 52Ti (1.7 min). The longest lived Ti isotope, 44Ti (43.96 y) is difficult to produce and it is not yet available commercially.

Vanadium (V) exists in oxygenated seawater as the inorganic vanadate (VO4 3-) species and while a useable radio-isotope exists, 48V (Table 2), it has so far been only applied to a few studies in marine systems, most notably examining the uptake of vanadate by ascidians who accumulate high concentrations of vanadium in their blood (Michibata et al., 1991). Chromium (Cr) exists in seawater in two redox states as either chromate (CrO42-) or the reduced form Cr(III). There have only been a limited number of studies using either 48Cr or 51Cr (Table 2) and most have focused on the uptake of CrO4 2- (Wang and Dei, 2001b) or colloidal Cr (Wang and Guo, 2000) by phytoplankton.

#### **2.6.1 Nickel (Ni)**

In seawater Nickel (Ni) shows a nutrient like behaviour and is present in surface waters at nM concentrations (Table 1). While some studies have shown organic complexation of Ni in seawater such work is complicated by the slow exchange kinetics for Ni(II) in seawater. A number of important advances in our understanding of Ni biogeochemistry in the ocean have come about through the use of 63Ni. Firstly Price and Morel (1991) observed that Ni was required for growth on urea, a Ni containing enzyme, by the diatom *Thalassiosira*  *weissflogii*. Later Dupont and co-workers investigated the uptake of 63Ni by the globally important cyanobacteria *Synechococcus* in a laboratory study (Dupont et al., 2008) and made field measurements of 63Ni uptake by natural phytoplankton assemblages (Dupont et al., 2010). They showed that Ni was a required element for many strains of *Synechococcus* and by comparison to the available genomic data most likely all strains of *Procholorococcus*. This was due to the use of a Ni containing superoxide dismutase and in the enzyme for urease uptake. Importantly they also observed that isotopic equilibrium was not established between the added radiotracer and the natural pools of Ni within 24 hours indicating the slow exchange kinetics of Ni in seawater (Hudson and Morel, 1993).

#### **2.6.2 Selenium (Se)**

256 Radioisotopes – Applications in Physical Sciences

Studies into 65Zn uptake by bacteria (Vogel and Fisher, 2010) found a much lower uptake of Zn than Cd. 65Zn has also been used in assessing the release of Zn from phytoplankton (Hutchins and Bruland, 1994) and bacteria (Vogel and Fisher, 2009) during zooplankton

Cobalt (Co) is present in seawater at very low concentrations (< 100 pM) and can exist as either inert Co(III) complexes or more labile Co(II) organic species (Saito and Moffett, 2002). Despite the range of Co isotopes available (Table 2) there have been relatively few studies examining the seawater speciation of Co. Work by Nolan et al. (1992) utilised a dual tracer approach where the uptake of 57Co-cobalamine was compared to that of 60Co-Co(II) and found that the cobalamine was taken up significantly faster and retained for longer in phytoplankton. 57Co was also used to show that Co could replace Zn in the enzyme carbonic anhydrase in some phytoplankton (Yee and Morel, 1996). An early finding with 57Co was that the oxidation of Co(II) to Co(III) in solution (Lee and Fisher, 1993) may be mediated by the same bacteria responsible for Mn oxidation (see section 2.3.2). Though new data (Murray et al., 2007) suggests no Co(II) oxidation occurs in the complete absence of Mn(II) and that the mechanism by which bacteria oxidize Co(II) is through the production of the reactive nano-particulate Mn oxide. Co has also been found to be released back to the dissolved

Studies into the biogeochemical cycles of other elements in seawater that are strongly hydrolysed and thus analogous to iron are limited by the lack of suitable radiotracers. There are no Aluminium (Al) radioisotopes suitable for use in trace metal speciation studies, as the majority of them have half-lives shorter than 10 minutes. The long lived isotope 26Al (710,000 y) has found application in paleo applications (Lal et al., 2006). Similarly there are also no seawater studies on Titanium (Ti) biogeochemistry with radioisotopes due to the short half-lives of 45Ti (3.08 h), 51Ti (5.76 min) and 52Ti (1.7 min). The longest lived Ti isotope,

Vanadium (V) exists in oxygenated seawater as the inorganic vanadate (VO43-) species and while a useable radio-isotope exists, 48V (Table 2), it has so far been only applied to a few studies in marine systems, most notably examining the uptake of vanadate by ascidians who accumulate high concentrations of vanadium in their blood (Michibata et al., 1991). Chromium (Cr) exists in seawater in two redox states as either chromate (CrO42-) or the reduced form Cr(III). There have only been a limited number of studies using either 48Cr or 51Cr (Table 2) and most have focused on the uptake of CrO42- (Wang and Dei, 2001b) or

In seawater Nickel (Ni) shows a nutrient like behaviour and is present in surface waters at nM concentrations (Table 1). While some studies have shown organic complexation of Ni in seawater such work is complicated by the slow exchange kinetics for Ni(II) in seawater. A number of important advances in our understanding of Ni biogeochemistry in the ocean have come about through the use of 63Ni. Firstly Price and Morel (1991) observed that Ni was required for growth on urea, a Ni containing enzyme, by the diatom *Thalassiosira* 

phase from grazed and decomposing phytoplankton (Lee and Fisher, 1994).

44Ti (43.96 y) is difficult to produce and it is not yet available commercially.

**2.6 Speciation studies with other trace metals** 

colloidal Cr (Wang and Guo, 2000) by phytoplankton.

**2.6.1 Nickel (Ni)** 

grazing.

**2.5 Cobalt (Co)** 

Selenium (Se) is found in very low concentrations in seawater (< 1 nM) and its chemistry is under kinetic redox control (Cutter, 1992) with the oxyanions Selenate (SeO42-) (thermodynamically favoured in oxygenated seawater and selenite (SeO32-), both showing nutrient like profiles in the ocean. In surface waters enhanced concentrations of organic selenide (operationally defined) is typically present (Cutter and Bruland, 1984).

The radioisotope 75Se has been used in a number of studies to elucidate the biogeochemistry of Se in phytoplankton cells. A key early finding was the identification of the pathway of uptake of selenite into the diatom *Thalassiosira pseudonana* and its conversion into the Se containing enzyme glutathione peroxidase (Price and Harrison, 1988). Later studies using the coccolithophorid *Emiliania huxleyi* as a model organism (Obata et al., 2004) , have shown in more detail the steps involved into the uptake of selenite by the cells, and identified a pool of low molecular weight compounds which are used to store Se before incorporation into specific seleno-proteins in the cell. The interspecies differences in selenite uptake and accumulation have also been assessed (Vandermeulen and Foda, 1988), and the release of Se contained in phytoplankton cells by zooplankton grazing or phytoplankton decomposition (Lee and Fisher, 1992).

#### **2.6.3 Molybdenum (Mo)**

Molybdenum (Mo) is almost conservative (105 nM) in oxygenated seawater (Collier, 1985) where it is present as the oxyanion molybdate (MoO42-), under reducing conditions Mo is reduced to form Mo-sulfides (Erickson and Helz, 2000) and is rapidly precipitated from the water column (Helz et al., 2004). Molybdenum is an essential element for phytoplankton which is utilized as a co-factor in a number of different enzymes (Kisker et al., 1997) and in particular in nitrogenase and nitrate reductase which catalyze the reduction of N2 and nitrate to bioavailable N in the ocean (Mendel, 2005). While Mo has been shown to be a limiting nutrient for freshwater organisms the evidence of Mo limitation in marine organisms is unclear. The use of 99Mo in seawater studies requires special sample handling so that the daughter product 99Tc does not interfere for this reason it has been almost used exclusively in nitrogen fixation studies where the focus has been on the potential for sulfate inhibition of Mo uptake leading to Mo limitation of nitrogen fixation (Marino et al., 2003).

#### **2.6.4 Cadmium (Cd)**

The marine biogeochemistry of Cadmium (Cd) has already yielded a number of surprises as it was long thought to be simply only toxic to organisms making its nutrient like profile in seawater and tight coupling with phosphate difficult to understand. However the

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 259

*<sup>K</sup>*Fe'L = [FeL]

where *K*Fe'L is the conditional stability constant with respect to Fe under these specific conditions (in this case pH 8.0 seawater). To convert *K*Fe'L to *K*FeL, the conditional stability constant for FeL with respect to free Fe3+ , the relationship between Fe and Fe3+ , Fe' =

Upon addition of the competing ligand TAC, a new equilibrium is established between

[FeT] = [Fe] + [FeLi] + [Fe(TAC)2] (4)

Fe(TAC)2 = [Fe(TAC)2]

Titrations performed using CLE-ACSV yield the fraction of Fe complexed by TAC at different Fe concentrations. This fraction is related to the side reaction coefficient by the

*<sup>K</sup>i* L*i* + 2[TAC]2 =

side-reaction coefficient for the naturally occurring ligands, and 2[TAC]2 the side-reaction coefficient for TAC complexes, which was determined previously (Croot and Johansson, 2000). The side-reaction coefficient of Fe (Fe) for all naturally occurring ligands (including

Data in this study were analyzed with a single ligand model that was a nonlinear fit to a Langmuir adsorption isotherm (Gerringa et al., 1995). The single ligand model is derived from equation 3, where [LT] = [L] + [FeL]. Rearranging Eq. 3, 4 and 8 yields a reciprocal

[Fe] = *K*[LT]

We solved Eq. 9 for *K* and [L] by nonlinear regression analysis (Levenberg, 1944; Marquardt, 1963) with Fe as the independent variable and [FeL]/[Fe] as the dependent variable using a purpose built program running LabviewTM . For the case of multiple ligands a more correct

1 + *K*[Fe]

 = <sup>1</sup> 1 + *Ki* L*<sup>i</sup>*

[Fe]/[Fe3+], can be used (e.g. *K*FeL = Fe' *K*Fe'L).

The complexation of Fe by TAC can be described as:

following relationship (all relative to Fe):

Langmuir isotherm:

form of the equation is:

[Fe(TAC)2 ]

Fe'(TAC)2 = [Fe(TAC)2]

[FeT] = 2[TAC]2 1 + 

The side reaction coefficient for Fe(TAC)2 with respect to Fe is then denoted by:

As [TAC] >> [FeT] for this method, the assumption [TAC] = [TACT] can be used.

*Ki* is the conditional stability constant, L*i* the concentration of the *i*th natural ligand,

inorganic ligands) is related to the concentration of [Fe] by the relationship:

[Fe] [FeT] - [Fe(TAC)2]

[FeL ]

TAC, the natural organic ligands and iron:

[Fe][L] (3)

[Fe][TAC]2 (5)

Fe'(TAC)2 (7)

(8)

(9)

o is the

[Fe] = Fe(TAC)2 [TAC]2 (6)

Fe'(TAC)2

 1 + o +

finding that Cd could replace Zn in the enzyme carbonic anhydrase showed for the first time a biological function for Cd (Lane and Morel, 2000) and changed biogeochemists view of this element. Cd appears to be weakly complexed by organic ligands in surface waters with inorganic species dominating in deeper waters (Bruland, 1992). The radioisotope 109Cd is frequently used for studies investigating the uptake of Cd by phytoplankton (Sunda and Huntsman, 1996). A particular focus has been with regard to bio-dilution effects and the role that growth limitation may have on their Cd content, either through iron limitation (Sunda and Huntsman, 2000), macronutrient limitation (Wang and Dei, 2001a) or temperature and irradiance uptake (Miao and Wang, 2004). Other studies have investigated the release of Cd from phytoplankton by cellular efflux mechanisms (Lee et al., 1995) or from zooplankton grazing/phytoplankton decomposition (Xu et al., 2001). The uptake of 109Cd has also been followed in natural phytoplankton communities in the English Channel (Dixon et al., 2006).

#### **3. Potential new applications for iron speciation in seawater**

In this section we outline some new applications using 55Fe for determining thermodynamic and kinetic information on iron speciation in seawater. Each of the outlined methods has been evaluated during shipboard trials at sea.

#### **3.1 Organic speciation of iron in seawater**

Iron organic species are important to the biogeochemical cycling of iron in the ocean as they may determine the bioavailability of iron to phytoplankton and increase the solubility of iron in seawater. Currently most techniques to determine Fe speciation in seawater use voltammetry(Croot and Johansson, 2000; Gledhill and van den Berg, 1994; Rue and Bruland, 1995). In the following we have adapted the chemistry of an existing voltammetric method (Croot and Johansson, 2000) for use with a radiotracer.

#### **3.1.1 Theory: Competitive Ligand Exchange (CLE) – 55Fe TAC**

The theory behind the CLE approach was introduced by Ruzic (1982), van den Berg (1982). A brief outline of the theory for determining dissolved iron speciation is presented below, a fully treatment can be found in Croot and Johansson (2000).

For dissolved iron in ambient seawater, a mass balance can be constructed:

$$\left[\text{Fe}\_{\text{I}}\right] = \left[\text{Fe}^{\prime}\right] + \left[\text{FeL}\_{i}\right] \tag{1}$$

where [Fe] represents the sum of all the inorganic species (predominantly Fe(OH)x(3-x)+ ) and [FeLi] represents the organically bound iron with Li being classes of natural organic ligands. The speciation of Fe(II) is not considered, as in most cases the long equilibration times used in these experiments should have seen the oxidation of any Fe(II) present. Reactions between one class of the natural ligands and Fe can be expressed as:

$$\text{Fe}' + \text{L}' \rightarrow \text{FeL} \tag{2a}$$

$$\text{FeL} \rightarrow \text{Fe}^{\prime} + \text{L}^{\prime} \tag{2b}$$

where L is the Fe-binding ligand not already bound to Fe(III). The equilibrium expression is then:

$$K'\_{\rm Fe'L} = \frac{[\rm FeL]}{[\rm Fe'][L']} \tag{3}$$

where *K*Fe'L is the conditional stability constant with respect to Fe under these specific conditions (in this case pH 8.0 seawater). To convert *K*Fe'L to *K*FeL, the conditional stability constant for FeL with respect to free Fe3+ , the relationship between Fe and Fe3+ , Fe' = [Fe]/[Fe3+], can be used (e.g. *K*FeL = Fe' *K*Fe'L).

Upon addition of the competing ligand TAC, a new equilibrium is established between TAC, the natural organic ligands and iron:

$$\begin{bmatrix} \text{Fe}\_{\text{I}} \end{bmatrix} = \begin{bmatrix} \text{Fe}^{\prime} \end{bmatrix} + \begin{bmatrix} \text{FeL}\_{\text{i}} \end{bmatrix} + \begin{bmatrix} \text{Fe}(\text{TAC})\_{\text{2}} \end{bmatrix} \tag{4}$$

The complexation of Fe by TAC can be described as:

258 Radioisotopes – Applications in Physical Sciences

finding that Cd could replace Zn in the enzyme carbonic anhydrase showed for the first time a biological function for Cd (Lane and Morel, 2000) and changed biogeochemists view of this element. Cd appears to be weakly complexed by organic ligands in surface waters with inorganic species dominating in deeper waters (Bruland, 1992). The radioisotope 109Cd is frequently used for studies investigating the uptake of Cd by phytoplankton (Sunda and Huntsman, 1996). A particular focus has been with regard to bio-dilution effects and the role that growth limitation may have on their Cd content, either through iron limitation (Sunda and Huntsman, 2000), macronutrient limitation (Wang and Dei, 2001a) or temperature and irradiance uptake (Miao and Wang, 2004). Other studies have investigated the release of Cd from phytoplankton by cellular efflux mechanisms (Lee et al., 1995) or from zooplankton grazing/phytoplankton decomposition (Xu et al., 2001). The uptake of 109Cd has also been followed in natural phytoplankton

In this section we outline some new applications using 55Fe for determining thermodynamic and kinetic information on iron speciation in seawater. Each of the outlined methods has

Iron organic species are important to the biogeochemical cycling of iron in the ocean as they may determine the bioavailability of iron to phytoplankton and increase the solubility of iron in seawater. Currently most techniques to determine Fe speciation in seawater use voltammetry(Croot and Johansson, 2000; Gledhill and van den Berg, 1994; Rue and Bruland, 1995). In the following we have adapted the chemistry of an existing voltammetric method

The theory behind the CLE approach was introduced by Ruzic (1982), van den Berg (1982). A brief outline of the theory for determining dissolved iron speciation is presented below, a

 [FeT] = [Fe] + [FeLi] (1) where [Fe] represents the sum of all the inorganic species (predominantly Fe(OH)x(3-x)+ ) and [FeLi] represents the organically bound iron with Li being classes of natural organic ligands. The speciation of Fe(II) is not considered, as in most cases the long equilibration times used in these experiments should have seen the oxidation of any Fe(II) present. Reactions

Fe + L → FeL (2a)

 FeL → Fe + L (2b) where L is the Fe-binding ligand not already bound to Fe(III). The equilibrium expression is

communities in the English Channel (Dixon et al., 2006).

been evaluated during shipboard trials at sea.

**3.1 Organic speciation of iron in seawater** 

then:

(Croot and Johansson, 2000) for use with a radiotracer.

**3.1.1 Theory: Competitive Ligand Exchange (CLE) – 55Fe TAC** 

For dissolved iron in ambient seawater, a mass balance can be constructed:

between one class of the natural ligands and Fe can be expressed as:

fully treatment can be found in Croot and Johansson (2000).

**3. Potential new applications for iron speciation in seawater** 

$$\mathcal{J}\_{\text{Fe(TAC)2}} = \frac{[\text{Fe(TAC)}\_2]}{[\text{Fe'}][\text{TAC'}]^2} \tag{5}$$

The side reaction coefficient for Fe(TAC)2 with respect to Fe is then denoted by:

$$\text{L}\_{\text{Fe}(\text{TAC})2} = \frac{\text{[Fe(TAC)}\_2\text{]}}{\text{[Fe']}} = \beta' \text{ }\_{\text{Fe(TAC)2}}\text{[TAC']^2} \tag{6}$$

As [TAC] >> [FeT] for this method, the assumption [TAC] = [TACT] can be used. Titrations performed using CLE-ACSV yield the fraction of Fe complexed by TAC at different Fe concentrations. This fraction is related to the side reaction coefficient by the following relationship (all relative to Fe):

$$\frac{[\text{Fe(TAC)}\_2]}{[\text{Fe}\_\text{I}]} = \frac{\pounds\,^\prime\_2[\text{TAC}]^2}{1 + 2\text{K}^\prime\_i\,\text{L}\_i + \pounds^\prime\_2[\text{TAC}]^2} = \frac{\mathcal{O}\text{Fe}(\text{TAC})2}{1 + \alpha\_\text{o} + \alpha\_\text{Fe}(\text{TAC})2} \tag{7}$$

*Ki* is the conditional stability constant, L*i* the concentration of the *i*th natural ligand, o is the side-reaction coefficient for the naturally occurring ligands, and 2[TAC]2 the side-reaction coefficient for TAC complexes, which was determined previously (Croot and Johansson, 2000). The side-reaction coefficient of Fe (Fe) for all naturally occurring ligands (including inorganic ligands) is related to the concentration of [Fe] by the relationship:

$$\frac{\text{[Fe'}]}{\text{[Fe}\_{\text{I}}\text{]} - \text{[Fe(TAC)}\_{\text{2}}\text{]}} = \frac{1}{1 + \Sigma K\_i \text{ L'}\_i} \tag{8}$$

Data in this study were analyzed with a single ligand model that was a nonlinear fit to a Langmuir adsorption isotherm (Gerringa et al., 1995). The single ligand model is derived from equation 3, where [LT] = [L] + [FeL]. Rearranging Eq. 3, 4 and 8 yields a reciprocal Langmuir isotherm:

$$\frac{\text{[FeL]}}{\text{[Fe']}} = \frac{K[\text{L}\_\text{T}]}{1 + K[\text{Fe'}]} \tag{9}$$

We solved Eq. 9 for *K* and [L] by nonlinear regression analysis (Levenberg, 1944; Marquardt, 1963) with Fe as the independent variable and [FeL]/[Fe] as the dependent variable using a purpose built program running LabviewTM . For the case of multiple ligands a more correct form of the equation is:

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 261

using electrochemical techniques (Croot et al., 2004). The data shown here provides initial confirmation that this approach can be applied to measuring iron speciation in seawater and could potentially be less labour intensive and time consuming than the current

Fig. 1. (left) Recovery of Fe(TAC)2 as a function of the total iron in solution (natural iron plus added radiotracer). (right) The concentration of organic iron (FeL) measured in the samples as a function of the total iron in solution (natural iron plus added radiotracer). FeL(Titration) refers to the FeL determined by difference from the measured 55Fe-TAC concentrations and FeL(C18) is the directly measured concentration of the seawater filtrate

Fig. 2. (left) Van den berg/Ruzic fit to the data shown in Figure 1. (right) Non-linear fit

electrochemical method.

that passed through the C18 Sep-Pak.

(equation 9) to the data shown in Figure 1.

$$\frac{\Sigma[\text{FeL}\_i]}{[\text{Fe}']} = \Sigma K'\_i \text{ L}\_{i(i \ge 1)} + \frac{[\text{L}\_1]K'\_1}{1 + K'\_1[\text{Fe}']} \tag{10}$$

 K*i* L*<sup>i</sup>*(*<sup>i</sup>* > 1) is the side-reaction coefficient for the weaker ligands, and K1 and L1 represent K and L in Eq. 7.

#### **3.1.2 Methodology: Competitive Ligand Exchange (CLE) – 55Fe TAC**

The seawater samples analyzed here were collected by the snorkel sampling system on the Polarstern (Schüßler and Kremling, 1993) from the Atlantic sector of the Southern Ocean during Polarstern expedition ANTXXIII-9 ((1) March 9, 2007 at 61 55.67' S, 72 43.23' E (2) March 19, 2007 at 61 58.51' S, 82 50.00' E).

A 0.01 M stock solution of TAC 2-(2-Thiazolylazo)-p-cresol; Aldrich was prepared in HPLC grade methanol, when not in use the stock solution is kept refrigerated. A 1.0 M stock buffer of EPPS (N-(2-hydroxyethyl)piperazine-N'-2-propanesulfonic acid; pKa 8.00; SigmaUltra) was prepared in 1 M NH4OH (Fluka, TraceSelect). Ultrapure (R > 18 M cm-1) deionized water (denoted UP water) was produced using a combined systems consisting of a Millipore Elix 3 and Synergy point of use system. All equipment used was trace metal clean and performed under a class 100 laminar airflow bench (AirClean Ssystems). Waters C18 Sep-Pak cartridges (holdup volume 1 mL) were pre-cleaned using 5 mL of quartz-distilled methanol (Q-MeOH) and 5 mL of UP water.

In this work the 55Fe (Hartmann Analytics, Braunschweig, Germany) had a specific activity of 157.6 MBq/mg Fe, a total activity of 75 MBq and was dissolved in 0.51 mL of 0.1 M HCl. The 55Fe stock solution was diluted to form working stock solutions with UP water and acidified with quartz-distilled HCl (Q-HCl) to a pH < 2 to prevent precipitation of the iron.

Subsamples (20 mL) of seawater were pipetted into a series of 12 Teflon bottles (60 mL) and 100 µL of 1M EPPS added. Iron (55Fe) was added to all but two of the bottles, yielding concentrations from 0 to 12 nM. The added Fe was allowed to equilibrate with the natural ligands for one hour at laboratory temperature. At the end of this equilibration period, 20 µL of 10 mM TAC was added and then left to equilibrate for 24 hours. At the end of this time the complete sample was pumped through the C18 column, followed by 5 mL of UP water and the filtrate (including UP water rinse) collected for counting. The 55Fe TAC complex retained on the column was recovered by a 5 mL rinse with Q-MeOH.

The activity of the samples was quantified using a liquid scintillation counter (Packard Tri-Carb 2900TR) with the scintillation cocktail Lumagel Plus (Lumac LSC). The efficiency of the instrument was obtained by quench curve calibration measurements. Separate quench curves were obtained for samples with seawater or methanol/TAC.

#### **3.1.3 Example: Competitive Ligand Exchange (CLE) – 55Fe TAC**

The results of the ligand titration are shown in Figure 1 below and are analogous to similar titrations using electrochemical detection (Croot and Johansson, 2000). The 55Fe-TAC complex is efficiently retained by the C18 column (Baliza et al., 2009). Comparison of the FeL concentration determined by difference between the measured 55Fe-TAC concentration and that which directly passed through the C18 column indicates that either an appreciable amount of the FeL was hydrophobic and retained on the C18 column after the methanol rinse (see section 3.3). Non-linear fitting of the data (Figure 2) to equation 9 gave log *K* = 21.23 ± 0.08 and LT = 1.50 ± 0.07, values consistent with other data from the Southern Ocean

[Fe] = *K*i L*<sup>i</sup>*(*<sup>i</sup>* > 1) + [L1]*K*<sup>1</sup>

K*i* L*<sup>i</sup>*(*<sup>i</sup>* > 1) is the side-reaction coefficient for the weaker ligands, and K1 and L1 represent K

The seawater samples analyzed here were collected by the snorkel sampling system on the Polarstern (Schüßler and Kremling, 1993) from the Atlantic sector of the Southern Ocean during Polarstern expedition ANTXXIII-9 ((1) March 9, 2007 at 61 55.67' S, 72 43.23' E

A 0.01 M stock solution of TAC 2-(2-Thiazolylazo)-p-cresol; Aldrich was prepared in HPLC grade methanol, when not in use the stock solution is kept refrigerated. A 1.0 M stock buffer of EPPS (N-(2-hydroxyethyl)piperazine-N'-2-propanesulfonic acid; pKa 8.00; SigmaUltra) was prepared in 1 M NH4OH (Fluka, TraceSelect). Ultrapure (R > 18 M cm-1) deionized water (denoted UP water) was produced using a combined systems consisting of a Millipore Elix 3 and Synergy point of use system. All equipment used was trace metal clean and performed under a class 100 laminar airflow bench (AirClean Ssystems). Waters C18 Sep-Pak cartridges (holdup volume 1 mL) were pre-cleaned using 5 mL of quartz-distilled

In this work the 55Fe (Hartmann Analytics, Braunschweig, Germany) had a specific activity of 157.6 MBq/mg Fe, a total activity of 75 MBq and was dissolved in 0.51 mL of 0.1 M HCl. The 55Fe stock solution was diluted to form working stock solutions with UP water and acidified with quartz-distilled HCl (Q-HCl) to a pH < 2 to prevent precipitation

Subsamples (20 mL) of seawater were pipetted into a series of 12 Teflon bottles (60 mL) and 100 µL of 1M EPPS added. Iron (55Fe) was added to all but two of the bottles, yielding concentrations from 0 to 12 nM. The added Fe was allowed to equilibrate with the natural ligands for one hour at laboratory temperature. At the end of this equilibration period, 20 µL of 10 mM TAC was added and then left to equilibrate for 24 hours. At the end of this time the complete sample was pumped through the C18 column, followed by 5 mL of UP water and the filtrate (including UP water rinse) collected for counting. The 55Fe TAC complex

The activity of the samples was quantified using a liquid scintillation counter (Packard Tri-Carb 2900TR) with the scintillation cocktail Lumagel Plus (Lumac LSC). The efficiency of the instrument was obtained by quench curve calibration measurements. Separate quench

The results of the ligand titration are shown in Figure 1 below and are analogous to similar titrations using electrochemical detection (Croot and Johansson, 2000). The 55Fe-TAC complex is efficiently retained by the C18 column (Baliza et al., 2009). Comparison of the FeL concentration determined by difference between the measured 55Fe-TAC concentration and that which directly passed through the C18 column indicates that either an appreciable amount of the FeL was hydrophobic and retained on the C18 column after the methanol rinse (see section 3.3). Non-linear fitting of the data (Figure 2) to equation 9 gave log *K* = 21.23 ± 0.08 and LT = 1.50 ± 0.07, values consistent with other data from the Southern Ocean

retained on the column was recovered by a 5 mL rinse with Q-MeOH.

curves were obtained for samples with seawater or methanol/TAC.

**3.1.3 Example: Competitive Ligand Exchange (CLE) – 55Fe TAC** 

1 + *K*1[Fe]

(10)

[FeL*i*]

(2) March 19, 2007 at 61 58.51' S, 82 50.00' E).

methanol (Q-MeOH) and 5 mL of UP water.

**3.1.2 Methodology: Competitive Ligand Exchange (CLE) – 55Fe TAC** 

and L in Eq. 7.

of the iron.

using electrochemical techniques (Croot et al., 2004). The data shown here provides initial confirmation that this approach can be applied to measuring iron speciation in seawater and could potentially be less labour intensive and time consuming than the current electrochemical method.

Fig. 1. (left) Recovery of Fe(TAC)2 as a function of the total iron in solution (natural iron plus added radiotracer). (right) The concentration of organic iron (FeL) measured in the samples as a function of the total iron in solution (natural iron plus added radiotracer). FeL(Titration) refers to the FeL determined by difference from the measured 55Fe-TAC concentrations and FeL(C18) is the directly measured concentration of the seawater filtrate that passed through the C18 Sep-Pak.

Fig. 2. (left) Van den berg/Ruzic fit to the data shown in Figure 1. (right) Non-linear fit (equation 9) to the data shown in Figure 1.

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 263

Fig. 3. Comparison of ultrafiltration methods using water collected by GO-FLO and

over 95% of the Fe is found in the > 0.025 µm fraction (Okumura et al., 2004).

**3.2.3 Methodology – Dissociation kinetics of weak iron-organic complexes** 

The following approach is based on the assumption that the observed decrease in soluble iron with time is due to the exchange of Fe between the weak organic ligands and the colloidal phase which does not pass through the filter. Support for this assumption lies partly in the findings that inorganic iron colloids formed from oversaturation of the solution will be formed very rapidly (Nowostawska et al., 2008) and be considerably larger (Hove et al., 2007) than the cutoff of the Millipore MF filter (25 nm) or Anotop (20 nm – though see 3.2.1 above). An earlier study by Okumura confirms that in the absence of a strong chelator

Thus the formation and dissociation of Fe complexes can be described by equations 2a and 2b from section 3.1.1. We now further assume that the ligands can be divided into two groups; a strong ligand (LS) that is practically inert to dissociation and a weaker ligand (LW) that at equilibrium is not able to keep iron in solution. Thus the soluble Fe concentration can be described by the following equation as a function of time, assuming that the formation of

 Fesol = FeLS+FeLW(e-kt) (11) where Fesol is the measured solubility of iron, FeLS is the concentration of the strong ligand and FeLW is the concentration of the weaker ligands which at thermodynamic equilibrium do not prevent the precipitation of iron from solution, *k* is the observed dissociation rate of

Filtered (0.2 µm) seawater samples were obtained from throughout the water column using GO-FLO sampling bottles on a trace metal clean line at two stations in the Tropical Atlantic

amended with 20 nM 55Fe

**3.2.2 Dissociation kinetics of weak complexes** 

both weak and strong complexes is equally rapid.

the weaker iron organic complexes.

#### **3.1.4 Issues: Competitive Ligand Exchange (CLE) – 55Fe TAC**

If this method is to be used more routinely there are a number of issues that would need to be addressed further. A critical factor in the interpretation of the data is whether the C18 column also retains hydrophobic organic Fe in addition to the 55Fe TAC complex as this could result in the retention of 55Fe not bound to TAC and lead to an underestimation of LT if these complexes are removed by the MeOH rinse used to elute the 55Fe TAC complex. We examine the issue of natural Fe hydrophobic organic complexes in more detail in section 3.3. Additionally this method relies on an accurate measurement of the dissolved iron concentration in the seawater and this needs to be taken into account for the addition of 55Fe, as the ratio of 55Fe to stable iron increases with each subsequent addition of 55Fe. This variation in the overall specific activity of the solution could have an impact on the time required to establish isotopic equilibrium between radiotracer and ambient iron. In the present case it is assumed that the 24 hour equilibration time used was sufficient given that TAC most likely reacts with natural iron ligands via an adjunctive mechanism (Hering and Morel, 1990), though this is not yet confirmed. Finally the use of a high specific activity 55Fe source is essential if low level (< nM) work is performed.

#### **3.2 Dissociation kinetics of weak iron binding complexes**

The earlier work on iron solubility in seawater by Kuma and co-workers (1992, 1993) assumed that the decrease in the concentration of soluble iron with time was due to the aging of meta-stable iron colloids and reduction in their solubility. However an alternative explanation is also possible as subsequent research has suggested that much of the added iron is initially complexed by weaker iron binding ligands (Gerringa et al., 2007) that slowly dissociate over time resulting in the loss of soluble iron from solution. In the following we adapt an existing radiotracer protocol for iron solubility measurements to determine the kinetics of the dissociation of the weak iron binding complexes in seawater.

#### **3.2.1 Filtration and size exclusion**

Recently we became aware of a potential problem when comparing between different filters. In comparing an ultrafiltration system (Vivaflow 50) and the Anotop (Whatman) syringe filters (Figure 3) we found that the Anotop retained far more 55Fe than the ultrafilters. Further measurements comparing the 0.02 µm Anotop filters with another type of ultrafiltration membranes (5, 10, 30, and 100 kDa) also found that the Anotop filters have a much smaller molecular weight cut off (< 5 kDa) than 20 nm (C. Schlosser, unpublished data). It seems likely then that the aluminium oxide matrix of the Anotop filter may also interact and adsorb some inorganic and organically complexed Fe species. Our finding agrees with an earlier study by Chen et al. (2004) which reported that they had observed that the Anotop filters were considerably different from its rated pore size of 0.02 µm (or 2000 kDa) as they found by using fluorescein tagged macromolecular compounds that it had an actual cutoff of 3 kDa. Our own initial work with 0.025 µm Millipore MF filters suggest that these have a cutoff more in keeping with their stated poresize based on comparison with ultrafiltration. These results contrast with an ICP-MS study reporting on the existence of colloidal Fe in the ocean (Wu et al., 2001) which found apparently good agreement between the Millipore MF 0.025 µm filters and the Anotop 0.02 µm syringe filters. More work is needed urgently to address and understand the differences between ultrafiltration systems and how this effects our interpretation of the natural system being investigated.

If this method is to be used more routinely there are a number of issues that would need to be addressed further. A critical factor in the interpretation of the data is whether the C18 column also retains hydrophobic organic Fe in addition to the 55Fe TAC complex as this could result in the retention of 55Fe not bound to TAC and lead to an underestimation of LT if these complexes are removed by the MeOH rinse used to elute the 55Fe TAC complex. We examine the issue of natural Fe hydrophobic organic complexes in more detail in section 3.3. Additionally this method relies on an accurate measurement of the dissolved iron concentration in the seawater and this needs to be taken into account for the addition of 55Fe, as the ratio of 55Fe to stable iron increases with each subsequent addition of 55Fe. This variation in the overall specific activity of the solution could have an impact on the time required to establish isotopic equilibrium between radiotracer and ambient iron. In the present case it is assumed that the 24 hour equilibration time used was sufficient given that TAC most likely reacts with natural iron ligands via an adjunctive mechanism (Hering and Morel, 1990), though this is not yet confirmed. Finally the use of a high specific activity 55Fe

The earlier work on iron solubility in seawater by Kuma and co-workers (1992, 1993) assumed that the decrease in the concentration of soluble iron with time was due to the aging of meta-stable iron colloids and reduction in their solubility. However an alternative explanation is also possible as subsequent research has suggested that much of the added iron is initially complexed by weaker iron binding ligands (Gerringa et al., 2007) that slowly dissociate over time resulting in the loss of soluble iron from solution. In the following we adapt an existing radiotracer protocol for iron solubility measurements to determine the

Recently we became aware of a potential problem when comparing between different filters. In comparing an ultrafiltration system (Vivaflow 50) and the Anotop (Whatman) syringe filters (Figure 3) we found that the Anotop retained far more 55Fe than the ultrafilters. Further measurements comparing the 0.02 µm Anotop filters with another type of ultrafiltration membranes (5, 10, 30, and 100 kDa) also found that the Anotop filters have a much smaller molecular weight cut off (< 5 kDa) than 20 nm (C. Schlosser, unpublished data). It seems likely then that the aluminium oxide matrix of the Anotop filter may also interact and adsorb some inorganic and organically complexed Fe species. Our finding agrees with an earlier study by Chen et al. (2004) which reported that they had observed that the Anotop filters were considerably different from its rated pore size of 0.02 µm (or 2000 kDa) as they found by using fluorescein tagged macromolecular compounds that it had an actual cutoff of 3 kDa. Our own initial work with 0.025 µm Millipore MF filters suggest that these have a cutoff more in keeping with their stated poresize based on comparison with ultrafiltration. These results contrast with an ICP-MS study reporting on the existence of colloidal Fe in the ocean (Wu et al., 2001) which found apparently good agreement between the Millipore MF 0.025 µm filters and the Anotop 0.02 µm syringe filters. More work is needed urgently to address and understand the differences between ultrafiltration systems and how this effects our

**3.1.4 Issues: Competitive Ligand Exchange (CLE) – 55Fe TAC** 

source is essential if low level (< nM) work is performed.

**3.2.1 Filtration and size exclusion** 

interpretation of the natural system being investigated.

**3.2 Dissociation kinetics of weak iron binding complexes** 

kinetics of the dissociation of the weak iron binding complexes in seawater.

Fig. 3. Comparison of ultrafiltration methods using water collected by GO-FLO and amended with 20 nM 55Fe

#### **3.2.2 Dissociation kinetics of weak complexes**

The following approach is based on the assumption that the observed decrease in soluble iron with time is due to the exchange of Fe between the weak organic ligands and the colloidal phase which does not pass through the filter. Support for this assumption lies partly in the findings that inorganic iron colloids formed from oversaturation of the solution will be formed very rapidly (Nowostawska et al., 2008) and be considerably larger (Hove et al., 2007) than the cutoff of the Millipore MF filter (25 nm) or Anotop (20 nm – though see 3.2.1 above). An earlier study by Okumura confirms that in the absence of a strong chelator over 95% of the Fe is found in the > 0.025 µm fraction (Okumura et al., 2004).

Thus the formation and dissociation of Fe complexes can be described by equations 2a and 2b from section 3.1.1. We now further assume that the ligands can be divided into two groups; a strong ligand (LS) that is practically inert to dissociation and a weaker ligand (LW) that at equilibrium is not able to keep iron in solution. Thus the soluble Fe concentration can be described by the following equation as a function of time, assuming that the formation of both weak and strong complexes is equally rapid.

$$\text{Fe}\_{\text{sol}} = \text{FeL}\_{\text{S}} + \text{FeL}\_{\text{W}}(\text{e}^{\text{alt}}) \tag{11}$$

where Fesol is the measured solubility of iron, FeLS is the concentration of the strong ligand and FeLW is the concentration of the weaker ligands which at thermodynamic equilibrium do not prevent the precipitation of iron from solution, *k* is the observed dissociation rate of the weaker iron organic complexes.

#### **3.2.3 Methodology – Dissociation kinetics of weak iron-organic complexes**

Filtered (0.2 µm) seawater samples were obtained from throughout the water column using GO-FLO sampling bottles on a trace metal clean line at two stations in the Tropical Atlantic

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 265

Fig. 5. (left) Vertical profile of iron solubility after 24 hours at TENATSO (ANTXXVI-4,

Fig. 6. (left) Vertical profile of dissociation rate (*k*) for FeLW at TENATSO. (right) Vertical profile of marine humic fluorescence (arbitrary units, 320 nm excitation, 420 nm emission). In earlier studies in the Pacific, iron solubility in intermediate and deep waters has been found to be highly correlated to the fluorescence of marine humic substances (Tani et al., 2003). The humic fluorescence profile at the TENATSO station is shown in figure 6 and is clearly poorly correlated with LS at this location. However at station (S283) in the Equatorial

S287). (right) Chlorophyll fluorescence (arbitary units) at TENATSO.

during the Polarstern expedition ANTXXVI-4. All sample handling was performed in a clean room container. In this work the 55Fe (Perkin Elmer) had a specific activity of 1985.42 MBq/mg Fe, a total activity of 75 MBq and a concentration of 1466.79 MBq/mL. The 55Fe stock solution was diluted as described in section 3.1.2.

Seawater (200 mL) from different depths was transferred into Teflon FEP bottles (1 L) and an aliquot of 55Fe was added to the bottles to give an addition of 21 nM. Subsamples (20 mL) for filtration were taken after 3, 6, 24 and 48 hours and were filtered through 47 mm 0.025 µm Millipore MF filters using an all Teflon filtration unit (Savillex), the filtrate was collected in a Teflon vial. All experiments were performed at 23° C. The activity of the samples was quantified as described in section 3.1.2.

#### **3.2.4 Example – Dissociation kinetics of weak iron-organic complexes**

Samples for the kinetic experiments were obtained from vertical profiles at two stations in the Tropical Atlantic; (i) S283 - April 28, 2010 at 01 46.62' N, 23 00.18' W in the Equatorial Atlantic and (ii) S287 - May 4, 2010 at 17 34.98' N, 24 15.18' W, the TENATSO time series site (for more information on TENATSO see Heller and Croot (2011)). Figure 4 below shows example results for the time dependent decrease in soluble iron from two different depths from the TENATSO site.

In all samples soluble Fe was initially high (10-13 nM) and declined rapidly over the first 24 hours with only small or no changes in the concentration over a further 24 hours. This indicates that the equilibrium value for LS was reached typically within 24 hours and that the weaker LW ligands had dissociated within the same timeframe. Previous studies have indicated that this equilibrium is established over timescales ranging from 1-2 weeks for studies using 0.45 µm pre-filtration (Kuma et al., 1996; Liu and Millero, 2002) to less than 24 hours when 0.2 µm pre-filtration was used (Chen et al., 2004). This highlights the role of colloidal matter/ligands in the time it takes to reach equilibrium. All data was fit to equation 11 using least squares regression.

during the Polarstern expedition ANTXXVI-4. All sample handling was performed in a clean room container. In this work the 55Fe (Perkin Elmer) had a specific activity of 1985.42 MBq/mg Fe, a total activity of 75 MBq and a concentration of 1466.79 MBq/mL. The 55Fe

Seawater (200 mL) from different depths was transferred into Teflon FEP bottles (1 L) and an aliquot of 55Fe was added to the bottles to give an addition of 21 nM. Subsamples (20 mL) for filtration were taken after 3, 6, 24 and 48 hours and were filtered through 47 mm 0.025 µm Millipore MF filters using an all Teflon filtration unit (Savillex), the filtrate was collected in a Teflon vial. All experiments were performed at 23° C. The activity of the samples was

Samples for the kinetic experiments were obtained from vertical profiles at two stations in the Tropical Atlantic; (i) S283 - April 28, 2010 at 01 46.62' N, 23 00.18' W in the Equatorial Atlantic and (ii) S287 - May 4, 2010 at 17 34.98' N, 24 15.18' W, the TENATSO time series site (for more information on TENATSO see Heller and Croot (2011)). Figure 4 below shows example results for the time dependent decrease in soluble iron from two different depths

Fig. 4. Change in the concentration of Soluble Fe (0.025 µm Millipore MF) over 48 hours after the addition of 21 nM 55Fe to water samples from the TENATSO station in the Eastern Tropical Atlantic. (left) 20 m depth. (right) 400 m depth. Error estimates are the result of duplicate measurements and correspond to the 95% confidence interval. The least squares fit

In all samples soluble Fe was initially high (10-13 nM) and declined rapidly over the first 24 hours with only small or no changes in the concentration over a further 24 hours. This indicates that the equilibrium value for LS was reached typically within 24 hours and that the weaker LW ligands had dissociated within the same timeframe. Previous studies have indicated that this equilibrium is established over timescales ranging from 1-2 weeks for studies using 0.45 µm pre-filtration (Kuma et al., 1996; Liu and Millero, 2002) to less than 24 hours when 0.2 µm pre-filtration was used (Chen et al., 2004). This highlights the role of colloidal matter/ligands in the time it takes to reach equilibrium. All data was fit to

**3.2.4 Example – Dissociation kinetics of weak iron-organic complexes** 

stock solution was diluted as described in section 3.1.2.

quantified as described in section 3.1.2.

to equation 11 are also shown (solid line).

equation 11 using least squares regression.

from the TENATSO site.

Fig. 5. (left) Vertical profile of iron solubility after 24 hours at TENATSO (ANTXXVI-4, S287). (right) Chlorophyll fluorescence (arbitary units) at TENATSO.

Fig. 6. (left) Vertical profile of dissociation rate (*k*) for FeLW at TENATSO. (right) Vertical profile of marine humic fluorescence (arbitrary units, 320 nm excitation, 420 nm emission).

In earlier studies in the Pacific, iron solubility in intermediate and deep waters has been found to be highly correlated to the fluorescence of marine humic substances (Tani et al., 2003). The humic fluorescence profile at the TENATSO station is shown in figure 6 and is clearly poorly correlated with LS at this location. However at station (S283) in the Equatorial

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 267

More work is clearly needed on this subject and the method outlined here should be a key

Fig. 8. Correlation between humic fluorescence and Fe solubility (after 24 hours) for samples

As noted early in section 3.1 information on hydrophobic Fe complexes is important for the interpretation of methods using C18 columns to recovery the Fe from solution. Such information is also useful for assessing the scavenging behaviour of iron organic complexes to particles in seawater. Hydrophobic Fe complexes are known to exist as many siderophores possess a hydrophobic tail which facilitates the uptake of iron by the phytoplankton (Martinez et al., 2000). A number of siderophore complexes are quantitatively retained by C18 columns including the terrestrial siderophore desferrioxamine B and its Fe chelate, ferrioxiamine B (Gower et al., 1989). This has lead to the development of extraction techniques for siderophores from seawater using C18 solid phase extraction (Freeman and Boyer, 1992). Other dissolved organic matter is also retained by this approach (Mopper et al., 2007) including marine humic complexes, though

There have been a number of studies that have utilized C18 or similar substrates to trap organic complexes using solid phase extraction techniques (Mackey, 1983). Previous work combining C18 solid phase extraction with radioisotopes in seawater has utilized 64Cu, finding that there is a significant but variable concentration of hydrophobic Cu complexes

The description of the seawater sampling, sample handling and 55Fe standard preparation are the same as described in section 3.2.3. In these experiments, 20 mL of the seawater samples described in section 3.2.3 were pumped through Waters C18 Sep-Paks (cleaned as

from Station 283 in the Tropical Atlantic (Polarstern ANTXXVI-4).

recoveries are highest when the sample is acidified (Amador et al., 1990).

**3.3.1 Methodology – Hydrophobic organic Fe complexes** 

**3.3 Hydrophobic organic Fe complexes** 

(Croot et al., 2003).

contribution to this.

Atlantic (Figure 7) we did observe a strong correlation between LS and humic fluorescence (Figure 8).

The vertical distribution of iron solubility after 24 hours at TENATSO is shown in figure 5 and shows a generally increasing trend with depth with a small local maximum in the surface waters in the vicinity of the chlorophyll maximum consistent with a biological source for LS. Values for LW showed no systematic variation in the water column and ranged from 8-10 nM. The estimated dissociation rate, *k*, for LW ranged from 2 x 10-5 to 8 x 10-5 s-1 similar to voltammetric observations of weak iron binding ligands (Gerringa et al., 2007).

Fig. 7. (left) Vertical profile of Iron solubility after 24 hours at S283 in the Tropical Atlantic (ANTXXVI-4). (right) Marine Humic fluorescence (arbitrary units, 320 nm excitation, 420 nm emission).at the same location.

At S283 LW showed no consistent pattern over the depth range examined with values from 6-10 nM. Estimated rates for the dissociation of the iron complexes from the weak ligands, *k*, ranged from 0.4 – 4.4 x 10-5 and also showed no discernable pattern with depth. The correlation between humic fluorescence and LS suggests that in this case the ligands were mostly derived by the same process inferred for the production of marine humics, the remineralisation of organic matter by microbial action. It furthermore suggests the photochemical destruction of the ligands in near surface waters at both S283 and TENATSO. The differences in the profiles between S283 and TENATSO may be related to a greater production of iron binding ligands by phytoplankton or bacteria at TENATSO. This may be in response to the greater dust flux this site receives as it is lies directly under the path of the Saharan dust plume (Heller and Croot, 2011).

Our approach here clearly provides important information with regard to the kinetics of processes relevant to dust deposition to the ocean (Baker and Croot, 2010) and highlights the role that weaker ligands may play in solubilising iron from aerosols and allowing phytoplankton a critical few extra hours were it is still soluble and potentially bioavailable.

Atlantic (Figure 7) we did observe a strong correlation between LS and humic fluorescence

The vertical distribution of iron solubility after 24 hours at TENATSO is shown in figure 5 and shows a generally increasing trend with depth with a small local maximum in the surface waters in the vicinity of the chlorophyll maximum consistent with a biological source for LS. Values for LW showed no systematic variation in the water column and ranged from 8-10 nM. The estimated dissociation rate, *k*, for LW ranged from 2 x 10-5 to 8 x 10-5 s-1 similar to voltammetric observations of weak iron binding ligands (Gerringa et al., 2007).

Fig. 7. (left) Vertical profile of Iron solubility after 24 hours at S283 in the Tropical Atlantic (ANTXXVI-4). (right) Marine Humic fluorescence (arbitrary units, 320 nm excitation, 420 nm

At S283 LW showed no consistent pattern over the depth range examined with values from 6-10 nM. Estimated rates for the dissociation of the iron complexes from the weak ligands, *k*, ranged from 0.4 – 4.4 x 10-5 and also showed no discernable pattern with depth. The correlation between humic fluorescence and LS suggests that in this case the ligands were mostly derived by the same process inferred for the production of marine humics, the remineralisation of organic matter by microbial action. It furthermore suggests the photochemical destruction of the ligands in near surface waters at both S283 and TENATSO. The differences in the profiles between S283 and TENATSO may be related to a greater production of iron binding ligands by phytoplankton or bacteria at TENATSO. This may be in response to the greater dust flux this site receives as it is lies directly under the path of the

Our approach here clearly provides important information with regard to the kinetics of processes relevant to dust deposition to the ocean (Baker and Croot, 2010) and highlights the role that weaker ligands may play in solubilising iron from aerosols and allowing phytoplankton a critical few extra hours were it is still soluble and potentially bioavailable.

(Figure 8).

emission).at the same location.

Saharan dust plume (Heller and Croot, 2011).

More work is clearly needed on this subject and the method outlined here should be a key contribution to this.

Fig. 8. Correlation between humic fluorescence and Fe solubility (after 24 hours) for samples from Station 283 in the Tropical Atlantic (Polarstern ANTXXVI-4).

#### **3.3 Hydrophobic organic Fe complexes**

As noted early in section 3.1 information on hydrophobic Fe complexes is important for the interpretation of methods using C18 columns to recovery the Fe from solution. Such information is also useful for assessing the scavenging behaviour of iron organic complexes to particles in seawater. Hydrophobic Fe complexes are known to exist as many siderophores possess a hydrophobic tail which facilitates the uptake of iron by the phytoplankton (Martinez et al., 2000). A number of siderophore complexes are quantitatively retained by C18 columns including the terrestrial siderophore desferrioxamine B and its Fe chelate, ferrioxiamine B (Gower et al., 1989). This has lead to the development of extraction techniques for siderophores from seawater using C18 solid phase extraction (Freeman and Boyer, 1992). Other dissolved organic matter is also retained by this approach (Mopper et al., 2007) including marine humic complexes, though recoveries are highest when the sample is acidified (Amador et al., 1990).

There have been a number of studies that have utilized C18 or similar substrates to trap organic complexes using solid phase extraction techniques (Mackey, 1983). Previous work combining C18 solid phase extraction with radioisotopes in seawater has utilized 64Cu, finding that there is a significant but variable concentration of hydrophobic Cu complexes (Croot et al., 2003).

#### **3.3.1 Methodology – Hydrophobic organic Fe complexes**

The description of the seawater sampling, sample handling and 55Fe standard preparation are the same as described in section 3.2.3. In these experiments, 20 mL of the seawater samples described in section 3.2.3 were pumped through Waters C18 Sep-Paks (cleaned as

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 269

Fig. 10. Data from Station 283 (Polarstern expedition ANTXXVI-4. (left) Hydrophobic iron recovered by C18 Sep-Pak (t=24 hours). (right) percentage of iron passing through 0.025 µm

Thus at S283 it suggests that in waters below 200 m the Fe complexation was dominated by hydrophilic humic complexes while nearer to the surface complexation was by ligands that were more hydrophobic in nature. This is the first data on iron complexation in the ocean to show information on the hydrophobic nature of complexes in the water column. This data also points to the role that hydrophobic Fe complexes may play in the biogeochemical cycling of iron in the ocean. The chemistry of these complexes is a rapidly developing field as shown in a recent review article on metallosurfactants (Owen and Butler, 2011) and their

The data we present above is for Fe retained on a C18 column and eluted with methanol. Previous work has shown that the retention of the Fe complex under similar conditions to those employed here varies between different iron species and that the methanol elution may not remove all the Fe that was trapped (Freeman and Boyer, 1992). Other studies have used nitric acid instead of methanol to recover the Fe retained on the C18 column (Abbasse et al., 2002). In the present study we did not attempt a nitric acid rinse due to potential problems with this acid in the scintillation cocktail. Further work is needed to develop a

Similarly colloidal inorganic iron has been found to be almost quantitatively trapped on C18 Sep-Paks (Sunda and Huntsman, 2003), in the present case it is unlikely that there is significant concentrations of this form of iron as outlined early in section 3.2. It would be even more unlikely that colloidal inorganic iron was formed in the 55Fe TAC experiment as this solution would be under saturated with respect to iron precipitation and the TAC is likely to dissolve any amorphous iron colloids quite rapidly (Croot and Johansson, 2000).

filter that is hydrophobic.

role in bioinorganic processes.

**3.3.3 Issues: Hydrophobic organic Fe complexes** 

more complete protocol that includes a complete mass balance.

described in section 3.1.2), rinsed with 5 mL UP water and then the 55Fe retained on the C18 column was eluted with 5 mL Q-MeOH. The activity of the MeOH samples was quantified as described in section 3.1.2. Samples were taken after 3, 6, 24 and 48 hours after the addition of the 55Fe.

#### **3.3.2 Example – Hydrophobic organic Fe complexes**

Seawater for this experiment was obtained from 4 depths at S283 (see description in section 3.2.4) and run as described above. The results from two of the kinetic runs are shown in Figure 9 below. In the sample from 40 m there was a clear decrease with time in the concentration of the hydrophobic Fe trapped by the C18 column. This was similar to the decrease in Fe solubility for the same sample (data not shown) suggesting that for this sample a significant portion of the weak organic ligands (section 3.2) were hydrophobic in nature. Contrastingly samples from deeper in the water column showed little variation with time (Figure 9) indicating that the bulk of the hydrophobic component here were stronger iron binding ligands.

Fig. 9. Hydrophobic organic Fe complexes at S283 in the Equatorial Atlantic. Samples were obtained from 40 m depth (circles) and 400 m depth (triangles). The 95% confidence intervals for the data are represented as error bars.

The vertical distribution of hydrophobic Fe complexes is shown in Figure 10 and indicates a maximum in near surface waters with lower concentrations in deep waters suggesting a biological source. Comparison with the iron solubility data from section 3.2 indicates that the percentage of hydrophobic Fe that was in the soluble phase was high in surface waters and decreased rapidly to be only ~10 % below 200 m (Figure 10).

described in section 3.1.2), rinsed with 5 mL UP water and then the 55Fe retained on the C18 column was eluted with 5 mL Q-MeOH. The activity of the MeOH samples was quantified as described in section 3.1.2. Samples were taken after 3, 6, 24 and 48 hours after the

Seawater for this experiment was obtained from 4 depths at S283 (see description in section 3.2.4) and run as described above. The results from two of the kinetic runs are shown in Figure 9 below. In the sample from 40 m there was a clear decrease with time in the concentration of the hydrophobic Fe trapped by the C18 column. This was similar to the decrease in Fe solubility for the same sample (data not shown) suggesting that for this sample a significant portion of the weak organic ligands (section 3.2) were hydrophobic in nature. Contrastingly samples from deeper in the water column showed little variation with time (Figure 9) indicating that the bulk of the hydrophobic component here were stronger

Fig. 9. Hydrophobic organic Fe complexes at S283 in the Equatorial Atlantic. Samples were obtained from 40 m depth (circles) and 400 m depth (triangles). The 95% confidence

The vertical distribution of hydrophobic Fe complexes is shown in Figure 10 and indicates a maximum in near surface waters with lower concentrations in deep waters suggesting a biological source. Comparison with the iron solubility data from section 3.2 indicates that the percentage of hydrophobic Fe that was in the soluble phase was high in surface waters

intervals for the data are represented as error bars.

and decreased rapidly to be only ~10 % below 200 m (Figure 10).

addition of the 55Fe.

iron binding ligands.

**3.3.2 Example – Hydrophobic organic Fe complexes** 

Fig. 10. Data from Station 283 (Polarstern expedition ANTXXVI-4. (left) Hydrophobic iron recovered by C18 Sep-Pak (t=24 hours). (right) percentage of iron passing through 0.025 µm filter that is hydrophobic.

Thus at S283 it suggests that in waters below 200 m the Fe complexation was dominated by hydrophilic humic complexes while nearer to the surface complexation was by ligands that were more hydrophobic in nature. This is the first data on iron complexation in the ocean to show information on the hydrophobic nature of complexes in the water column. This data also points to the role that hydrophobic Fe complexes may play in the biogeochemical cycling of iron in the ocean. The chemistry of these complexes is a rapidly developing field as shown in a recent review article on metallosurfactants (Owen and Butler, 2011) and their role in bioinorganic processes.

#### **3.3.3 Issues: Hydrophobic organic Fe complexes**

The data we present above is for Fe retained on a C18 column and eluted with methanol. Previous work has shown that the retention of the Fe complex under similar conditions to those employed here varies between different iron species and that the methanol elution may not remove all the Fe that was trapped (Freeman and Boyer, 1992). Other studies have used nitric acid instead of methanol to recover the Fe retained on the C18 column (Abbasse et al., 2002). In the present study we did not attempt a nitric acid rinse due to potential problems with this acid in the scintillation cocktail. Further work is needed to develop a more complete protocol that includes a complete mass balance.

Similarly colloidal inorganic iron has been found to be almost quantitatively trapped on C18 Sep-Paks (Sunda and Huntsman, 2003), in the present case it is unlikely that there is significant concentrations of this form of iron as outlined early in section 3.2. It would be even more unlikely that colloidal inorganic iron was formed in the 55Fe TAC experiment as this solution would be under saturated with respect to iron precipitation and the TAC is likely to dissolve any amorphous iron colloids quite rapidly (Croot and Johansson, 2000).

Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 271

Baars, O. and Croot, P.L., 2011. The speciation of dissolved zinc in the Atlantic sector of the

Baker, A.R. and Croot, P.L., 2010. Atmospheric and marine controls on aerosol iron

Baliza, P.X., Ferreira, S.L.C. and Teixeira, L.S.G., 2009. Application of pyridylazo and

Barbeau, K., Moffett, J.W., Caron, D.A., Croot, P.L. and Erdner, D.L., 1996. Role of protozoan grazing in relieving iron limitation of phytoplankton. Nature, 380: 61-64. Boye, M. et al., 2001. Organic complexation of iron in the Southern Ocean. Deep Sea

Boyle, E. and Edmond, J.M., 1975. Copper in surface waters south of New Zealand. Nature,

Bruland, K.W., 1989. Complexation of zinc by natural organic ligands in the central North

Bruland, K.W., 1992. Complexation of cadmium by natural organic ligands in the central

Bruland, K.W., Donat, J.R. and Hutchins, D.A., 1991. Interactive influences of bioactive trace

Bruland, K.W. and Lohan, M.C., 2003. Controls of Trace Metals in Seawater. In: D.H.

Byrne, R.H., 2010. Comparative carbonate and hydroxide complexation of cations in

Byrne, R.H., Kump, L.R. and Cantrell, K.J., 1988. The Influence of Temperature and pH on

Campbell, P.G.C., 1995. Interactions between Trace Metals and Aquatic Organisms: A

Chen, M., Wang, W.-X. and Guo, L., 2004. Phase partitioning and solubility of iron in natural

Coale, K.H. and Bruland, K.W., 1988. Copper complexation in the northeast Pacific.

Collier, R.W., 1985. Molybdenum in the Northeast Pacific Ocean. Limnology and

Croot, P.L., Andersson, K., Öztürk, M. and Turner, D., 2004. The Distribution and Speciation

Croot, P.L. et al., 2008. Regeneration of Fe(II) during EIFeX and SOFeX. Geophysical

Research Letters, 35(19): L19606,doi:10.1029/2008GL035063.

metals on biological production in oceanic waters. Limnology and Oceanography,

Heinrich and K.T. Karl (Editors), Treatise on Geochemistry. Pergamon, Oxford, pp.

Critique of the Free-ion Activity Model. In: A. Tessier and D.R. Turner (Editors), Metal Speciation and Bioavailability in Aquatic Systems. IUPAC Series on Analytical and Physical Chemistry of Environmental Systems. John Wiley & Sons,

seawater controlled by dissolved organic matter. Global Biogeochemical Cycles, 18:

of Iron along 6° E, in the Southern Ocean. Deep-Sea Research II, 51(22-24): 2857-

solubility in seawater. Marine Chemistry, 120: 4-13.

Pacific. Limnology and Oceanography, 34: 269-285.

North Pacific. Limnology and Oceanography, 37: 1008-1017.

seawater. Geochimica et Cosmochimica Acta, 74(15): 4312-4321.

Trace Metal Speciation in Seawater. Marine Chemistry, 25: 163-181.

Press, Corrected Proof.

of metals. Talanta, 79(1): 2-9.

Research, 48: 1477-1497.

253: 107-109.

36: 1555-1577.

Chichester, pp. 45-102.

doi:10.1029/2003GB002160.

Oceanography, 30: 1351-1354.

Limnology and Oceanography, 33: 1084-1101.

23-47.

2879.

Southern Ocean. Deep Sea Research Part II: Topical Studies in Oceanography, In

thiazolylazo reagents in flow injection preconcentration systems for determination

#### **4. Conclusions and future prospects**

Radioisotopes are a vital tool for trace metal marine biogeochemists as they allow pulse chase experiments for rapid assessment of the kinetics of processes under natural conditions. The applications that radioisotopes can be applied to in seawater speciation studies is strongly linked to, and drives, new analytical developments in techniques to distinguish between individual chemical species. In the present work we have outlined new approaches for examining Fe organic speciation in seawater, but these methods could easily be applied to other trace metal radioisotopes also. New research is needed urgently for assessing the current status and the potential for change in oceanic systems due to global warming, ocean acidification and ocean deoxygenation. In this context we encourage future research into the use of trace metal radioisotopes for determining the changes in speciation and kinetic reactivity for oceanic redox processes in oxygen minimum zones. As this is a critical area of research that needs to be developed over the next decade in order to better assess the impact ocean deoxygenation may play on trace metal redox cycles in the ocean.

### **5. Acknowledgments**

This work is a contribution of the Collaborative Research Centre 754 "Climate - Biogeochemistry Interactions in the Tropical Ocean" (www.sfb754.de), which is supported by the German Research Association. Financial support for this work has come from the DFG through SFB754 "Climate – Biogeochemistry Interactions in the Tropical Ocean" and research grants to PLC (CR145/5, CR145/9 and CR145/18). The technical support of Uwe Rabsch, Kerstin Nachtigall and Peter Streu is gratefully acknowledged.

#### **6. References**


Radioisotopes are a vital tool for trace metal marine biogeochemists as they allow pulse chase experiments for rapid assessment of the kinetics of processes under natural conditions. The applications that radioisotopes can be applied to in seawater speciation studies is strongly linked to, and drives, new analytical developments in techniques to distinguish between individual chemical species. In the present work we have outlined new approaches for examining Fe organic speciation in seawater, but these methods could easily be applied to other trace metal radioisotopes also. New research is needed urgently for assessing the current status and the potential for change in oceanic systems due to global warming, ocean acidification and ocean deoxygenation. In this context we encourage future research into the use of trace metal radioisotopes for determining the changes in speciation and kinetic reactivity for oceanic redox processes in oxygen minimum zones. As this is a critical area of research that needs to be developed over the next decade in order to better assess the impact ocean deoxygenation may play on trace metal redox cycles in the ocean.

This work is a contribution of the Collaborative Research Centre 754 "Climate - Biogeochemistry Interactions in the Tropical Ocean" (www.sfb754.de), which is supported by the German Research Association. Financial support for this work has come from the DFG through SFB754 "Climate – Biogeochemistry Interactions in the Tropical Ocean" and research grants to PLC (CR145/5, CR145/9 and CR145/18). The technical support of Uwe

Abbasse, G., Ouddane, B. and Fischer, J.C., 2002. Determination of total and labile fraction of

Ahner, B.A., Price, N.M. and Morel, F.M.M., 1994. Phytochelatin production by marine

Amador, J., Milne, P.J., Moore, C.A. and Zika, R.G., 1990. Extraction of chromophoric humic

Anderson, M.A. and Morel, F.M., 1980. Uptake of Fe(II) by a diatom in oxic culture medium.

Annett, A.L., Lapi, S., Ruth, T.J. and Maldonado, M.T., 2008. The effects of Cu and Fe

Anton, M. et al., 2007. Identification and comparitive Genomic Analysis of Signalling and

metals in seawater using solid phase extraction and inductively coupled plasma atomic emission spectrometry (ICP-AES). Journal of Analytical Atomic

phytoplankton at low free metal ion concentrations: Laboratory studies and filed data from Massachusetts Bay. Proceedings of the National Academy of Sciences, 91:

availability on the growth and Cu:C ratios of marine diatoms. Limnology and

Regulatory Components in the diatom *Thalassiosira Psuedonana*. Journal of

Rabsch, Kerstin Nachtigall and Peter Streu is gratefully acknowledged.

substances from seawater. Marine Chemistry, 29: 1-17.

Spectrometry, 17(10): 1354-1358.

Marine Biology Letters, 1: 263-268.

Oceanography, 53(6): 2451-2461.

Phycology, 43(3): 585-604.

**4. Conclusions and future prospects** 

**5. Acknowledgments** 

**6. References** 

8433-8436.


Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 273

Gerringa, L.J.A. et al., 2007. Kinetic study reveals weak Fe-binding ligand, which affects the solubility of Fe in the Scheldt estuary. Marine Chemistry, 103: 30-45. Gledhill, M. and van den Berg, C.M.G., 1994. Determination of complexation of iron(III)

Gower, J.D., Healing, G. and Green, C.J., 1989. Determination of desferrioxamine-available

Guo, J. et al., 2010. Copper-uptake kinetics of coastal and oceanic diatoms. Journal of

Heller, M.I. and Croot, P.L., 2011. Superoxide decay as a probe for speciation changes

Hering, J.G. and Morel, F.M.M., 1990. Kinetics of trace metal complexation: ligand exchange

Hirose, K., 2006. Chemical speciation of trace metals in seawater: a review. Analytical

Honeyman, B.D. and Santschi, P.H., 1991. Coupling Adsorption and Particle Aggregation:

Hove, M., van Hille, R.P. and Lewis, A.E., 2007. Iron solids formed from oxidation precipitation of ferrous sulfate solutions. AIChE Journal, 53(10): 2569-2577. Hudson, R.J.M., Covault, D.T. and Morel, F.M.M., 1992. Investigations of iron coordination

extraction of amphiphilic iron complexes. Marine Chemistry, 38: 209-235. Hudson, R.J.M. and Morel, F.M.M., 1989. Distinguishing between extra- and intracellular iron in marine phytoplankton. Limnology and Oceanography, 34: 1113-1120. Hudson, R.J.M. and Morel, F.M.M., 1990. Iron transport in marine phytoplankton: Kinetics

Hudson, R.J.M. and Morel, F.M.M., 1993. Trace metal transport by marine microorganisms: implications of metal coordination kinetics. Deep-Sea Research, 40: 129-150. Hunter, K.A., Kim, J.P. and Croot , P.L., 1997. Biological roles of trace metals in natural

Hutchins, D.A. and Bruland, K.W., 1994. Grazer-mediated regeneration and assimilation of

Hutchins, D.A., Wang, W.-X., Schmidt, M.A. and Fisher, N.S., 1999. Dual-labeling

waters. Environmental Monitoring and Assessment, 44: 103-147.

communities. Aquatic Microbial Ecology, 19: 129-138.

Laboratory Studies of "Colloidal Pumping" Using 59Fe-Labeled Hematite.

and redox reactions in seawater using 59Fe radiometry and ion-pair solvent

of cellular and medium coordination reactions. Limnology and Oceanography, 35:

Fe, Zn and Mn from planktonic prey. Marine Ecology Progress Series, 110: 259-269.

techniques for trace metal biogeochemical investigations in aquatic plankton

reactions. Environmental Science and Technology, 24: 242-252.

Environmental Science and Technology, 25: 1739-1747.

voltammetry. Marine Chemistry, 47: 41-54.

Biochemistry, 180(1): 126-130.

Phycology, 46(6): 1218-1228.

et Cosmochimica Acta, 53: 611-618.

Sciences, 22(8): 1055-1063.

1002-1020.

131-142.

applied to Cu speciation data in the estuarine environment. Marine Chemistry, 48:

with natural organic complexing ligands in seawater using cathodic stripping

iron in biological tissues by high-pressure liquid chromatography. Analytical

during dust dissolution in Tropical Atlantic surface waters near Cape Verde. Marine Chemistry, In Press, Corrected Proof. doi:10.1016/j.marchem.2011.03.006. Helz, G.R., Vorlicek, T.P. and Kahn, M.D., 2004. Molybdenum scavenging by iron monosulfide. Environmental Science & Technology, 38(16): 4263-4268. Hering, J.G. and Morel, F.M.M., 1989. Slow coordination reactions in seawater. Geochimica


Croot, P.L. and Johansson, M., 2000. Determination of iron speciation by cathodic stripping

Croot, P.L., Karlson, B., van Elteren, J.T. and Kroon, J.J., 1999. Uptake of 64Cu-Oxine by Marine Phytoplankton. Environmental Science and Technology, 33(20): 3615-3621. Croot, P.L., Karlson, B., van Elteren, J.T. and Kroon, J.J., 2003. Uptake and efflux of 64Cu by

Croot, P.L. and Laan, P., 2002. Continuous shipboard determination of Fe(II) in Polar waters

Croot, P.L., Moffett, J.W. and Brand, L., 2000. Production of extracellular Cu complexing

Cutter, G.A., 1992. Kinetic controls on metalloid speciation in seawater. Marine Chemistry,

Cutter, G.A. and Bruland, K.W., 1984. The marine biogeochemistry of selenium: A re-

de Baar, H.J.W. et al., 2005. Synthesis of 8 Iron Fertilization Experiments: From the Iron Age

Di Toro, D.M. et al., 2001. Biotic ligand model of the acute toxicity of metals. 1. Technical

Dixon, J.L. et al., 2006. Cadmium uptake by marine micro-organisms in the English Channel

Dupont, C.L., Barbeau, K. and Palenik, B., 2008. Ni uptake and limitation in marine Synechococcus strains. Applied And Environmental Microbiology, 74(1): 23-31. Dupont, C.L., Buck, K.N., Palenik, B. and Barbeau, K., 2010. Nickel utilization in

Emerson, S. et al., 1982. Environmental oxidation rate of manganese(II): bacterial catalysis.

Emerson, S.R. and Huested, S.S., 1991. Ocean anoxia and the concentrations of molybdenum

Erickson, B.E. and Helz, G.R., 2000. Molybdenum(VI) speciation in sulfidic waters: Stability

Fisher, N., 1985. Accumulation of metals by marine picoplankton. Marine Biology, 87: 137-

Freeman, R.A. and Boyer, G.L., 1992. Solid phase extraction techniques for the isolation of

Gerringa, L.J.A., Herman, P.M.J. and Poortvliet, T.C.W., 1995. Comparison of the linear van

and vanadium in seawater. Marine Chemistry, 34(3-4): 177-196.

Falkowski, P.G. and Raven, J.A., 1997. Aquatic Photosynthesis. Blackwell Scientific.

Basis. Environmental Toxicology and Chemistry, 20(10): 2383-2396.

evaluation. Limnology and Ocenography, 29: 1179-1192.

and Celtic Sea. Aquatic Microbial Ecology, 44(1): 31-43. Donat, J.R. and Bruland, K.W., 1995. Trace Elements in the Oceans, pp. 247-281.

Part I: Oceanographic Research Papers, 57(4): 553-566.

Geochimica et Cosmochimica Acta, 46: 1073-1079.

(TAC). Electroanalysis, 12(8): 565-576.

Oceanography, 48: 179-188.

Oceanography, 45: 619-627.

doi:10.1029/2004JC002601.

Acta, 466: 261-273.

40(1-2): 65-80.

1158.

142.

2276.

voltammetry in seawater using the competing ligand 2-(2-Thiazolylazo)-p-cresol

the marine cyanobacterium *Synechococcus* (WH7803). Limnology and

using flow injection analysis with chemiluminescence detection. Analytica Chimica

ligands by eucaryotic phytoplankton in response to Cu stress. Limnology and

to the Age of Enlightenment. Journal of Geophysical Research, 110: C09S16,

phytoplankton assemblages from contrasting oceanic regimes. Deep Sea Research

and lability of thiomolybdates. Geochimica Et Cosmochimica Acta, 64(7): 1149-

siderophores from aquatic environments. Journal of Plant Nutrition, 15(10): 2263 -

den Berg/Ruzic transformation and a non-linear fit of the Langmuir isotherm

applied to Cu speciation data in the estuarine environment. Marine Chemistry, 48: 131-142.


Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 275

Maldonado, M.T. et al., 2006. Copper-dependent iron transport in coastal and oceanic

Mandernack, K.W. and Tebo, B.M., 1993. Manganese scavenging and oxidation at

Marino, R., Howarth, R.W., Chan, F., Cole, J.J. and Likens, G.E., 2003. Sulfate inhibition of

seawater conditions: a non-reversible effect. Hydrobiologia, 500(1): 277-293. Marquardt, D.W., 1963. An Algorithm for Least-Squares Estimation of Nonlinear

Martinez, J.S., Haygood, M.G. and Butler, A., 2001. Identification of a natural

Martinez, J.S. et al., 2000. Self-Assembling Amphiphilic Siderophores from Marine Bacteria.

McCubbin, D. and Leonard, K.S., 1996. Photochemical dissolution of radionuclides from

Mendel, R.R., 2005. Molybdenum: biological activity and metabolism. Dalton

Miao, A.J. and Wang, W.X., 2004. Relationships between cell-specific growth rate and

Michibata, H. et al., 1991. Uptake of V-48 Labeled Vanadium by Subpopulations of Blood-Cells in the Ascidian, Ascidia-Gemmata. Zoological Science, 8(3): 447-452. Millero, F.J., 1998. Solubility of Fe(III) in seawater. Earth and Planetary Science Letters, 154:

Millero, F.J., Woosley, R., Ditrolio, B. and Waters, J., 2009. Effect of Ocean Acidification on

Moffett, J.W., 1994. The relationship between cerium and manganese oxidation in the

Moffett, J.W., 1997. The importance of microbial Mn oxidation in the upper ocean: a

Moffett, J.W. and Ho, J., 1996. Oxidation of cobalt and manganese in seawater via a common microbially catalysed pathway. Geochimica et Cosmochimica Acta, 60: 3415-3424. Mopper, K., Stubbins, A., Ritchie, J.D., Bialk, H.M. and Hatcher, P.G., 2007. Advanced

Morel, F.M.M., Milligan, A.J. and Saito, M.A., 2003. Marine Bioinorganic Chemistry: The

Morse, J.W., Millero, F.J., Cornwell, J.C. and Rickard, D., 1987. The chemistry of the

comparison of the Sargasso Sea and equatorial Pacific. Deep-Sea Research I, 44:

Instrumental Approaches for Characterization of Marine Dissolved Organic Matter: Extraction Techniques, Mass Spectrometry, and Nuclear Magnetic Resonance

Role of Trace Metals in the Oceanic Cycles of Major Nutrients. In: D.H. Heinrich and K.T. Karl (Editors), Treatise on Geochemistry. Pergamon, Oxford, pp. 113-143. Morel, F.M.M. et al., 1994. Zinc and carbon co-limitation of marine phytoplankton. Nature,

hydrogen sulfide and iron sulfide systems in natural waters. Earth-Science

the Speciation of Metals in Seawater. Oceanography, 22(4): 72-85.

marine environment. Limnology and Oceanography, 39: 1309-1318.

Spectroscopy. Chemical Reviews, 107(2): 419-442.

uptake rate of cadmium and zinc by a coastal diatom. Marine Ecology-Progress

Parameters. SIAM Journal on Applied Mathematics, 11(2): 431-441.

marine sediment. Marine Chemistry, 55(3-4): 399-408.

hydrothermal vents and in vent plumes. Geochimica et Cosmochimica Acta, 57(16):

molybdenum-dependent nitrogen fixation by planktonic cyanobacteria under

desferrioxamine siderophore produced by a marine bacterium. Limnology and

diatoms. Limnology And Oceanography, 51(4): 1729-1743.

3907.

Oceanography, 46: 420-424.

Transactions(21): 3404-3409.

Science, 287: 1245-1247.

Series, 275: 103-113.

323-329.

1277-1291.

369: 740-742.

Reviews, 24(1): 1-42.


Johnson, K.S., Coale, K.H., Berelson, W.M. and Gordon, R.M., 1996. On the formation of the

Johnson, K.S., Gordon, R.M. and Coale, K.H., 1997. What controls dissolved iron

Kisker, C., Schindelin, H. and Rees, D.C., 1997. Molybdenum-cofactor-containing enzymes: Structure and mechanism. Annual Review of Biochemistry, 66: 233-267. Kuma, K., Katsumoto, A., Kawakami, H., Takatori, F. and Matsunaga, K., 1998a. Spatial

Kuma, K., Katsumoto, A., Nishioka, J. and Matsunaga, K., 1998b. Size-fractionated iron

Kuma, K., Nakabayashi, S., Suzuki, Y. and Matsunaga, K., 1992. Dissolution rate and

Kuma, K., Nishioka, J. and Matsunaga, K., 1996. Controls on iron(III) hydroxide solubility in

Lal, D. et al., 2006. Paleo-ocean chemistry records in marine opal: Implications for fluxes of

Landing, W.M. and Bruland, K.W., 1987. The contrasting biogeochemistry of iron and manganese in the Pacific Ocean. Geochimica et Cosmochimica Acta, 51: 29-43. Lane, T.W. and Morel, F.M.M., 2000. A biological function for cadmium in marine diatoms.

Lee, B.-G. and Fisher, N.S., 1994. Effects of sinking and zooplankton grazing on the release of elements from planktonic debris. Marine Ecology Progress Series, 110: 272-281. Lee, B.G. and Fisher, N.S., 1992. Decomposition and release of elements from zooplankton

Lee, B.G. and Fisher, N.S., 1993. Microbially Mediated Cobalt Oxidation In Seawater

Lee, J.G., Roberts, S.B. and Morel, F.M.M., 1995. Cadmium: A nutrient for the marine diatom Thalassiosira weissflogii. Limnology and Oceanography, 40: 1056-1063. Levenberg, K., 1944. A Method for the Solution of Certain Non-Linear Problems in Least

Li, Y.-H., Burkhardt, L., Buchholtz, M., O'Hara, P. and Santschi, P.H., 1984. Partition of

Liu, X. and Millero, F.J., 2002. The solubility of iron in seawater. Marine Chemistry, 77: 43-

Mackey, D.J., 1983. Metal-Organic Complexes in Seawater - an Investigation of Naturally

concentrations in the world ocean? Marine Chemistry, 57: 137-161.

Pacific Ocean. Deep-Sea Research, 45: 91-113.

Estuarine Coastal and Shelf Science, 47(3): 275-283.

Geochimica Et Cosmochimica Acta, 70(13): 3275-3289.

debris. Marine Ecology Progress Series, 88(2-3): 117-128.

Squares. The Quarterly of Applied Mathematics, 2: 164-168.

Cosmochimica Acta, 48(10): 2011-2019.

Chemistry, 13: 169-180.

60(8): 1291-1299.

143.

Oceanography, 41: 396-407.

97(9): 4627-4631.

1602.

54.

manganese maximum in the oxygen minimum. Geochimica Et Cosmochimica Acta,

variability of Fe(III) hydroxide solubility in the water column of the northern North

concentrations and Fe(III) hydroxide solubilities in various coastal waters.

solubility of colloidal hydrous ferric oxide in seawater. Marine Chemistry, 38: 133-

seawater: The influence of pH and natural organic chelators. Limnology and

trace elements, cosmogenic nuclides (Be-10 and Al-26), and biological productivity.

Proceedings of the National Academy of Sciences of the United States of America,

Revealed By Radiotracer Experiments. Limnology And Oceanography, 38(8): 1593-

radiotracers between suspended particles and seawater. Geochimica et

Occurring Complexes of Cu, Zn, Fe, Mg, Ni, Cr, Mn and Cd using High-Performance Liquid Chromatography with Atomic Fluorescence Detection. Marine


Utilizing Radioisotopes for Trace Metal Speciation Measurements in Seawater 277

Santschi, P.H. et al., 1987. Relative mobility of radioactive trace elements across the

Santschi, P.H., Li, Y.H. and Carson, S.R., 1980. The fate of trace metals in Narragansett Bay,

Schlosser, C. and Croot, P., 2009. Controls on seawater Fe(III) solubility in the Mauritanian upwelling zone. Geophys. Res. Lett., 36: L18606, doi:10.1029/2009GL038963. Schlosser, C. and Croot, P.L., 2008. Application of cross-flow filtration for determining the

Schüßler, U. and Kremling, K., 1993. A pumping system for underway sampling of

Semeniuk, D.M. et al., 2009. Plankton copper requirements and uptake in the subarctic

Shaked, Y., Kustka, A.B., Morel, F.M.M. and Erel, Y., 2004. Simultaneous determination of

Shaked, Y., Xu, Y., Leblanc, K. and Morel, F.M.M., 2006. Zinc availability and alkaline

Spokes, L.J. and Liss, P.S., 1995. Photochemically induced redox reactions in seawater, I.

Sunda, W. and Huntsman, S., 2003. Effect of pH, light, and temperature on Fe-EDTA chelation and Fe hydrolysis in seawater. Marine Chemistry, 84(3-4): 35-47. Sunda, W.G. and Huntsman, S.A., 1983. Effect of competitive interactions between

Sunda, W.G. and Huntsman, S.A., 1992. Feedback interactions between zinc and phytoplankton in seawater. Limnology and Oceanography, 37: 25-40. Sunda, W.G. and Huntsman, S.A., 1995. Iron uptake and growth limitation in oceanic and

Sunda, W.G. and Huntsman, S.A., 1996. Antagonisms between cadmium and zinc toxicity and

Sunda, W.G. and Huntsman, S.A., 1997. Interrelated influence of iron, light and cell size on

Sunda, W.G. and Huntsman, S.A., 2000. Effect of Zn, Mn, and Fe on Cd accumulation in

Sunda, W.G., Huntsman, S.A. and Harvey, G.R., 1983. Photoreduction of manganese oxides in

ocean. Limnology and Oceanography, 51(1): 299-309.

coastal phytoplankton. Marine Chemistry, 50: 189-206.

marine phytoplankton growth. Nature, 390: 389-392.

Cations. Marine Chemistry, 49: 201-213.

Research Papers, 35(8): 1297-1317.

45(7): 1501-1516.

Journal Of Marine Research, 45(4): 1007-1048.

Marine Science, 10(6): 635-654.

Methods, 6: 630-642.

40: 257-266.

56(7): 1130-1142.

Methods, 2: 137-145.

sediment-water interface in the MERL model ecosystems of Narragansett Bay.

Rhode Island: Radiotracer experiments in microcosms. Estuarine and Coastal

solubility of iron species in open ocean seawater. Limnology and Oceanography:

dissolved and particulate trace elements in near-surface waters. Deep Sea Research,

Northeast Pacific Ocean. Deep-Sea Research Part I-Oceanographic Research Papers,

iron reduction and uptake by phytoplankton. Limnology and Oceanography:

phosphatase activity in *Emiliania huxleyi*: Implications for Zn-P co-limitation in the

manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira. Limnology and Oceanography, 28: 924-934. Sunda, W.G. and Huntsman, S.A., 1988. Effect Of Sunlight On Redox Cycles Of Manganese

In The Southwestern Sargasso Sea. Deep-Sea Research Part A-Oceanographic

manganese limitation in a coastal diatom. Limnology and Oceanography, 41: 373-387.

phytoplankton: Implications for oceanic Cd cycling. Limnology and Oceanography,

seawater and its geochemical and biological implications. Nature, 301(5897): 234-236.


Murray, K.J., Webb, S.M., Bargar, J.R. and Tebo, B.M., 2007. Indirect Oxidation of Co(II) in

Nolan, C.V., Fowler, S.W. and Teyssie, J.-L., 1992. Cobalt speciation and bioavailabilty in

Nowostawska, U., Kim, J.P. and Hunter, K.A., 2008. Aggregation of riverine colloidal iron in

Nozaki, Y., 1997. A Fresh Look at Element Distribution in the North Pacific. EOS, 78(21):

Obata, T., Araie, H. and Shiraiwa, Y., 2004. Bioconcentration Mechanism of Selenium by a Coccolithophorid, Emiliania huxleyi. Plant and Cell Physiology, 45(10): 1434-1441. Okumura, C., Hasegawa, H., Mizumoto, H., Maki, T. and Ueda, K., 2004. Size fractionation

Owen, T. and Butler, A., 2011. Metallosurfactants of bioinorganic interest: Coordinationinduced self assembly. Coordination Chemistry Reviews, 255(7-8): 678-687. Peers, G. and Price, N.M., 2004. A role for manganese in superoxide dismutases and growth of iron-deficient diatoms. Limnology and Oceanography, 49: 1774-1783. Pham, A.N., Rose, A.L., Feitz, A.J. and Waite, T.D., 2006. Kinetics of Fe(III) precipitation in

Pitzer, K.S., 1973. Thermodynamics of Electrolytes. I. Theoretical Basis and General

Poorvin, L., Rinta-Kanto, J.M., Hutchins, D.A. and Wilhelm, S.W., 2004. Viral release of iron and its bioavailabiltiy to marine plankton. Limnology and Oceanography, 49: 1734-1741. Price, N.M. et al., 1989. Preparation and Chemistry of the Artificial Algal Culture Medium

Price, N.M. and Harrison, P.J., 1988. Specific Selenium-Containing Macromolecules in the Marine Diatom Thalassiosira pseudonana. Plant Physiology, 86: 192-199. Price, N.M. and Morel, F.M.M., 1991. Colimitation of phytoplankton growth by nickel and

Quigg, A., Reinfelder, J.R. and Fisher, N.S., 2006. Copper uptake kinetics in diverse marine

Rich, H.W. and Morel, F.M.M., 1990. Availability of well-defined iron colloids to the marine diatom Thalassiosira weissflogii. Limnology and Oceanography, 35: 652-662. Rijkenberg, M.J.A. et al., 2005. The influence of UV irradiation on the photoreduction of iron

Rue, E.L. and Bruland, K.W., 1995. Complexation of Iron(III) by Natural Organic Ligands in

Ruzic, I., 1982. Theoretical Aspects of the Direct Titration of Natural Waters and its Information Yield for Trace Metal Speciation. Analytica Chimica Acta, 140: 99-113. Saito, M.A. and Moffett, J.W., 2002. Temporal and spatial variability of cobalt in the Atlantic

the Central North Pacific as Determined by a New Competitive Ligand Equilibration/Adsorptive Cathodic Stripping Voltammetric Method. Marine

Equations. The Journal of Physical Chemistry, 77: 268-277.

nitrogen. Limnology and Oceanography, 36: 1071-1077.

in the Southern Ocean. Marine Chemistry 93: 119-129.

Ocean. Geochimica et Cosmochimica Acta, 66: 1943-1953.

phytoplankton. Limnology and Oceanography, 51(2): 893-899.

Aquil. Biological Oceanography, 6: 443-461.

marine organisms. Marine Ecology Progress Series, 88: 105-116.

Appl Environ Microbiol., 73(21): 6905-6909.

Bunseki Kagaku, 53(11): 1215-1221.

110(3-4): 205.

Acta, 70(3): 640-650.

Chemistry, 50: 117-138.

221-223.

the Presence of the Marine Mn(II)-Oxidizing Bacterium Bacillus sp. Strain SG-1

estuaries: A new kinetic study using stopped-flow mixing. Marine Chemistry,

of iron compounds in phytoplankton cultures in the presence of chelating ligands.

aqueous solutions at pH 6.0-9.5 and 25 degrees C. Geochimica Et Cosmochimica


**14** 

*Sphagnum* **Plants** 

*Agricultural Sciences,* 

*1,2Sweden 3Ukraine* 

**Cesium (137Cs and 133Cs), Potassium and** 

**Rubidium in Macromycete Fungi and** 

Mykhailo Vinichuk1, 3, Anders Dahlberg2 and Klas Rosén1

*2Department of Forest Mycology and Pathology, Swedish University of* 

*3Department of Ecology, Zhytomyr State Technological University,* 

*1Department of Soil and Environment, Swedish University of Agricultural Sciences,* 

**1.1 Cesium (137Cs and 133Cs), potassium and rubidium in macromycete fungi** 

Radiocesium (137Cs) released in the environment as result of nuclear weapons tests in the 1950s and 1960s, and later due to the Chernobyl accident in 1986, is still a critical fission product because of its long half-life of 30 years and its high fission yield. The study of the cesium radioisotope 137Cs is important, as production and emission rates are much higher than other radioisotopes. This chapter comprises results obtained in several experiments in Swedish forest ecosystems and aims to discuss the behavior of cesium isotopes (137Cs and 133Cs) and their counterparts potassium (K) and rubidium (Rb) in the "soil-fungi-plants transfer" system. The chapter consists of two parts: one mainly dealing with 137Cs, 133Cs, K and Rb in forest soil and macromycete fungi, and the other with the same isotopes in

The bioavailability of radionuclides controls the ultimate exposure of living organisms and the ambient environment to these contaminants. Consequently, conceptually and methodologically, the understanding of bioavailability of radionuclides is a key issue in the field of radioecology. Soil-fungi-plants transfer is the first step by which 137Cs enters food

The availability of radionuclides (137Cs in particular) in soils of different ecosystems is to a large extent regulated by various vascular plants and fungal species. Thus, the behavior of 137Cs in forest ecosystems differs substantially from other ecosystems, foremost due to the abundance of fungal mycelia in soil, which contribute to the persistence of the Chernobyl radiocesium in the upper horizons of forest soils (Vinichuk & Johanson, 2003). Both saprotrophic and mycorrhizal fungi have key roles in nutrient and carbon cycling processes in forest soils. The mycelium of soil fungi has a central role in breaking down organic matter

**1. Introduction** 

chains.

separate segments of *Sphagnum* plants.

**1.1.1 The role of fungi in 137Cs transfer in the forest** 

