Surface analytical methods are applied to examine the environmental status of seawaters. The present overview emphasizes advantages of combining surface analytical methods, applied to a hazardous situation in the Adriatic Sea, such as monitoring of the first aggregation phases of dissolved organic matter in order to potentially predict the massive mucilage formation and testing of oil spill cleanup. Such an approach, based on fast and direct characterization of organic matter and its high-resolution visualization, sets a continuous-scale description of organic matter from micro- to nanometre scales. Electrochemical method of chronoamperometry at the dropping mercury electrode meets the requirements for monitoring purposes due to the simple and fast analysis of a large number of natural seawater samples enabling simultaneous differentiation of organic constituents. In contrast, atomic force microscopy allows direct visualization of biotic and abiotic particles and provides an insight into structural organization of marine organic matter at micro- and nanometre scales. In the future, merging data at different spatial scales, taking into account experimental input on micrometre scale, observations on metre scale and modelling on kilometre scale, will be important for developing sophisticated technological platforms for knowledge transfer, reports and maps applicable for the marine environmental protection and management of the coastal area, especially for tourism, fishery and cruiser trafficking.
Macroaggregation phenomena in the northern
Adriatic:
Different human activities introducing substances and energy in marine
environment can have serious environmental threats with wide ranging impacts
and possibly long lasting consequences. The impact of human activities
(land-based and marine) on coastal and marine ecosystems, and how to manage
them, demonstrates the need for a monitoring study of the environmental
status of seawaters, which is the main goal of the Marine Directive. Here, we
address the major challenges faced by the Adriatic Sea: risks and threats of
oil pollution and mucilage formation, where eutrophication most probably
contributes to the frequency of mucilage appearance (Fonda-Umani et al.,
1989). The Adriatic Sea is a semi-closed, narrow basin with a northern shallow
zone and is thus very sensitive to environmental changes. According to the
Croatian Hydrocarbon Agency (
Organic matter in the water column has a highly reactive nature; it
continuously undergoes biotic and abiotic transformations and consequently
exhibits high variation in time and space, with a tendency to accumulate at
the interfaces. Biogeochemical transformation of organic matter is a rather
complex process, in which chemical composition of the seawater plays an
important role. The fate of marine organic matter depends not only on the inorganic
constituents but also on biota, influencing its physicochemical
properties, distribution, stability and bioavailability of metal–organic
complexes. Organic matter in the marine environment is classified as
dissolved (DOM), consisting of truly dissolved and colloidal fractions, and
particulate based on the traditional oceanographic routines. DOM can
be produced by phytoplankton, macrophytes and marine plants through primary
production (Carlson, 2002), and 10 % of it can self-assemble to form
nano-, micro- or even macrogels (Verdugo et al., 2004; Verdugo, 2012). Because of
the great complexity and heterogeneous composition of marine organic matter,
its characterization remains a challenge. In the following, organic matter characterization is summarized briefly
based on the measurement techniques.
Characterization of DOM in terms of its composition and molecular structure
has advanced substantially in the last decade, mainly due to the
development of advanced instrumental analytical methods and the
corresponding procedures: for example, Fourier transform ion cyclotron, mass
spectrometry, homo- and hetero-correlated multidimensional nuclear magnetic
resonance spectroscopy, excitation emission matrix fluorimetry with
parallel factor analysis for UV–fluorescence spectroscopy, and
advances made in sample preparation (i.e. desalting/extraction techniques;
Nebbioso and Piccolo, 2013; Roth et al., 2015; Kujawinski et al., 2016;
Mopper et al., 2007). In addition, development of particle counters
(electronic, electrochemical), histological staining and sophisticated
microscopical methods in the last decades helped establish new particle
classes, predominantly non-living, which had remained undetected due to
their small size, transparency and susceptibility to degradation
(Žutić and Svetličić, 2000). These particle classes have
shown large abundance and reactivity, consequently having a crucial role in
aquatic environments, particularly in the cycling of nutrients, aggregation
of organic matter and the food web (Koike et al., 1990; Wells and Goldberg 1994;
Long and Azam, 1996). The abundance of non-living particles greatly exceeds
the abundance of living microorganisms, algae and bacteria, and viruses.
Major classes of non-living organic particles in euphotic layer are (i) colloidal
particles (5–200 nm) collected by ultracentrifugation and observed
under transmission electron microscopy (Wells and Goldberg, 1991, 1993;
Kepkay, 1994; Leppard et al., 1997); (ii) submicrometre particles
(0.4–1
While relatively broad scientific literature is available on the application of different techniques for characterization of marine organic matter, most of the methods include relatively expensive equipment and/or complex sample preparation procedures that effect the state of the fragile fractions of organic matter. It is now generally agreed that organic matter of a given chemical composition can occur in a large range of forms under the influence of dynamic aquatic conditions: monomer molecules, polymer chains, globules, vesicles and complex networks such as gels (Žutić and Svetličić, 2000). All of these will undoubtedly affect organic matter's persistence, stability, aggregation behaviour and bioavailability. Little is known about the distribution of environmental particles due not only to the difficulties in sampling, sample handling, insufficient sensitivity and selectivity of analytical methods but also to the heterogeneity of natural samples (Buffle and van Leeuwen, 1992). For example, the seawater sample needs to be unperturbed in order to preserve the heterogeneous distribution and primary structure of organic particles. High heterogeneity makes both the analysis and data interpretation challenging. Therefore, both development of methodological approaches and additional scientific understanding for assessing the environmental status of seawater are required. The aim of this overview is to emphasize advantages of a combined methodological approach applied to investigating hazardous situations in the Adriatic Sea, such as monitoring the first aggregation phases of dissolved organic matter in order to potentially predict the massive mucilage formation and testing of oil spill cleanup. Surface analytical methods, namely chronoamperometry at the dropping mercury electrode (DME) and atomic force microscopy (AFM), applied to monitor organic matter dynamics and oil pollution in the marine environment are based on fast, direct characterization and visualization from micro- to nanometre scale. We underline the importance that sample pretreatment is not required in order to capture the physical structure and organization of organic matter as closely as it exists in the natural system. Often, sample pretreatments such as filtration, centrifugation, staining and/or fixation introduce artefacts. The electrochemical approach is quickly compared to other methods, with sample characterization taking only a couple of minutes, making it suitable for analysis of a large number of natural seawater samples and thus applicable for monitoring purposes. In contrast, AFM can be applied to reveal structural organization of organic matter on the molecular level and to obtain three-dimensional (3-D) images of fine, fragile and soft structures. These data can be correlated to the electrochemical ones in order to shed more light on the biotic and abiotic transformations of organic matter at micro- and nanoscales (Ciglenečki and Svetličić, 2015). The advent of AFM introduced the possibility to directly explore these processes at a scale that determines the fate of organic matter and its interactions at the interfaces.
The electrochemical method used here is chronoamperometry of dissolved
oxygen at a charged DME (Barradas and Kimmerle, 1966; Zvonarić et al.,
1973). Chronoamperometry at a mercury electrode is an important and
convenient tool for the in situ single particle analysis in an aqueous
electrolyte solution. Mercury, as a liquid substrate, has several very
unique advantages: it is atomically smooth, fluid and chemically inert with
a large set of interfacial data in various aqueous electrolyte solutions,
necessary for the analysis of the amperometric signal of an organic
particle. Fluidity of mercury perfectly mimics interactions with other fluid
interfaces in contact (e.g. biological membranes, vesicles, bubbles).
Electrochemical measurement is performed by immersing the DME directly into
the electrochemical vessel containing a fresh seawater aliquot of 15 mL. The
mercury electrode has a surface area of only 4.7 mm
Illustration of different classes of organic constituents in seawater and their electrochemical signals at the electrode: biopolymer and small colloids, surface-active particles and gel particles (adapted from Svetličić et al., 2006).
Basic principle of electrochemical adhesion-based detection: interaction of oil droplet with the charged electrode interface (adopted from Svetličić et al., 2001).
Basic principle of atomic force microscopy.
In order to access more quantitative information from the amperometric signal of an organic particle, the measurement has to be performed in a previously deaerated solution under nitrogen purging for a few minutes so as to remove redox reaction of dissolved oxygen. In this way, it is possible to retrieve information about: organic particle diameter, particle surface area at the interface, number of molecules in the monolayer, surface charge of the particle, critical interfacial tensions of adhesion and kinetic parameters of the adhesion process through analysis of the amperometric signal using a reaction kinetics model and the corresponding methodology (Ivošević DeNardis et al., 2012, 2015).
AFM is based on a relatively simple principle: it involves raster scanning of a sharp and hard tip (probe) located at the free end of a flexible cantilever. The tip is scanned over the surface of a sample, sensing the interaction forces between the tip and sample (Fig. 4). The sample is mounted on a piezoelectric scanner, which allows three-dimensional positioning with subnanometre accuracy. Interaction between the tip and the surface of the sample leads to cantilever bending, which is measured by laser light reflected from the cantilever to a position sensitive photodetector. As changes in cantilever deflection result in variation of the distance between the tip and sample, a constant distance is re-established with a feedback loop between the sample–tip positioning system and a computer-controlled piezoelectric scanner. Registered values of cantilever deflection are electronically converted into a pseudo 3-D image of the sample. As a result, AFM gives real 3-D images of the sample with a vertical resolution of 0.1 nm and lateral resolution of 1 nm. The main advantages of AFM over conventional light or electron microscopy in studies of marine organic matter include (i) 3-D high-resolution imaging of different seawater constituents (biotic and abiotic) connecting micrometric and nanometric dimensions; (ii) samples being non-destructively imaged in the air and under near-natural aqueous conditions; (iii) nanomechanical mapping (i.e. Young's modulus, deformation, hydrophobicity, adhesion); and (iv) determination of intra- and inter-molecular forces in heterogeneous molecular assemblies (i.e. Pletikapić et al., 2014).
Annual variation of concentrations of surface-active particles (SAP), gel microparticles (GeP) and surfactant activity determined by electrochemical analysis of natural seawater samples taken at sampling station SJ 105 in the northern Adriatic.
AFM topographic images of marine gel representing
networks with
After a dramatic mucilage event in the northern Adriatic in the summer of
1997, which had negative impact on the fishery and tourism, the monitoring
programme “Systematic Study of the Adriatic Sea as a Base for
Sustainable Development of the Republic of Croatia” was launched in 1998.
Monitoring of organic matter dynamics was carried out to examine the
environmental status in the northern Adriatic and to predict mucilage event.
We focus on naturally occurring, non-living and micrometre-sized
surface-active particles. This fraction mainly involves soft, hydrophobic,
hydrophilic and reactive particles susceptible to degradation.
Surface-active particles are described as vesicle or micelle-like structures
formed by self-assembly of organic matter, primarily of lipid,
polysaccharide and proteinaceous components deriving “mostly” from excreted cells and/or from their decomposed products (Žutić and
Svetličić, 2000). They have an affinity to accumulate at the
interfaces, and they can be measured directly electrochemically on the
single particle level based on their interfacial properties, structural
organization and hydrophobic–hydrophilic character. We introduced a direct
electrochemical method to probe the state of marine organic matter without
perturbing its original heterogeneous distribution. Figure 5 shows
distribution of SAP (hydrophobic), gel microparticles (GeP) (hydrophilic) and surfactant activity
measured over a period of 10 years at sampling station SJ 105.
This particular sampling station was selected because it is distant from the
coastline and thus the influence of direct input of inland freshwater is
minimized (Fig. 1b). SAP concentrations ranged from 5
AFM as a high-resolution imaging technique that requires only easy sample
preparation has a huge potential in investigating marine organic matter,
particularly in elucidating the structure, transformations and dynamics of
organic matter in the marine environment. Of all forms of organic matter,
marine fibrils and fibrillar networks were the most studied form. For
example, Santschi et al. (1998) were the first to image individual
fibrillar polysaccharides in marine macromolecular organic matter. From
there on, AFM has been applied to study both (i) the networks secreted by
marine bacteria and algae (Nishino et al., 2004; Svetličić et al.,
2005, 2013; Malfatti and Azam, 2009; Malfatti et al., 2010; Urbani et al., 2012; Bosak et al., 2012; Villacorte et al., 2015b) and (ii) marine
fibrils (Mišić Radić et al., 2011; Svetličić et al.,
2011) forming giant gel macroaggregates (Vollenweider and Rinaldi, 1999;
Giani et al., 2005a, b), including their self-assembly and molecular
structure at different levels of association (Mišić Radić et
al., 2011; Pletikapić et al., 2014). Application of AFM imaging for
monitoring by itself is often not suitable. This is due to the large number
of samples required in the monitoring studies, making AFM analysis, if used
alone, a time-consuming process. It is therefore recommended to analyse
seawater samples first with rapid and “bulk” techniques (i.e.
chronoamperometry) in order to identify samples of interest. For AFM
imaging, the drop deposition method modified for marine samples and imaging
in air was found to be most adequate for visualization of macromolecular
organic matter organization (Pletikapić et al., 2011; Mišić
Radić et al., 2011, for more detail). Typically, AFM
measurements of marine organic samples are performed at room temperature and
50–60 % of relative humidity, which leaves samples with a small hydration
layer adhering to the substrate, helping to maintain the native structure
(Balnois and Wilkinson, 2002). It was found that direct deposition of a drop
of seawater (5
Large marine vesicle of 1.5
Sampling station of Rt Murva (
In addition, gel network formation was visualized at station SJ 107 in
August 2010, when concentrations of micrometre-sized GeP and SAP were low
(0.2 and 2.3
However, despite this, AFM is not yet exploited up to its full potential. Significant advances in the field are expected by integrating AFM into versatile hybrid devices that would combine two or three complementary techniques in one instrument, allowing a more detailed and comprehensive analysis of marine samples. While simultaneous AFM imaging and mechanical mapping (stiffness, friction, dissipation and/or adhesion) is already showing its potential by increasing the number of studies conducted on biological samples (Dufrêne et al., 2013, and references therein), including marine samples (Francius et al., 2008; Pletikapić et al., 2012a), of particular interest for investigation of marine organic matter is coupling AFM with different optical, spectroscopic and/or interfacial techniques (Moreno and Toca-Herrera, 2009). In line with the topic covered in this paper, integration of mercury as a substrate in the AFM set-up is drawing particular attention (Schon et al., 2013). An AFM cantilever has been developed using a mercury fountain pen probe allowing simultaneous probing of mechanical and electrical properties, for instance in biological membrane research. In addition, a hybrid AFM-optical (fluorescence) microscope (Kassies et al., 2005; Geisse et al., 2009) would be extremely useful for deepening the research on different classes of organic particles traditionally detected by staining and microscopic analysis. An additional step forward in the field is expected by combining AFM and IR to provide simultaneous correlation of topographical (organizational) and chemical data (Dazzi et al., 2012; Amenabar et al., 2013; Kulik et al., 2014). Alternatively, one could use chemically functionalized tips and, by simultaneously performing imaging and force curve acquisition, generate chemical maps (Blanchette et al., 2008). Such studies would allow relating molecular organization of marine organic matter with chemical information on the micro- and nanometre scales, which would undoubtedly widen the spectrum of information about samples of interest.
Surface analytical methods can also be applied for testing of the
environmental status of seawater after oil spill cleanup. We detected,
characterized and visualized the presence of dispersed oil droplets in the
seawater due to the accidental sinking of a ship and oil spill in the Bay of Kotor (south Adriatic sea, Montenegro). The ship sank on 11 October
2013 at Rt Murva (15 m depth) in the Bay of Kotor (Fig. 8;
Ivošević DeNardis et al., 2014). The ship had an overall length of
32.7 m and weighed 325 tons, carrying oil and diesel oil (type D2).
One month after the mechanical removal of oil spill, dispersed oil droplets
were still present in the whole water column. Characterized oil droplets
were in the continuum size range from micro to nano. Smaller oil droplets
tend to accumulate at the halocline, while larger rise to the surface where
they may coalesce. The highest droplet concentration of 2.0
Surface analytical methods are applied for hazardous situation in the
Adriatic Sea such as monitoring the first aggregation phases of dissolved
organic matter in order to potentially predict the massive mucilage
formation and testing of oil spill cleanup. The monitoring study conducted
in the northern Adriatic enabled investigation of the environmental status
of seawaters based on the spatiotemporal distribution of organic matter. A
few months prior to the mucilage event, promoted by certain meteorological
and hydrodynamics conditions, the concentration of accumulated SAP in the
halocline reached 1
Corresponding data sets referring to this paper can be accessed upon request to authors.
This work was supported by the Croatian Ministry of Science, Education and Sports through the projects (i) Surface Forces on Atomic Scale Applied in Marine Science and Nanotechnology, (ii) National Monitoring Programme (Project Jadran), Systematic Research of the Adriatic Sea as a Base for Sustainable Development of the Republic of Croatia, and (iii) Croatia–Montenegro bilateral scientific cooperation “Impact Assessment and Determination of Organic Pollutants in the Waters” (project leaders: Zoran Kljajić and NID). The authors thank V. Svetličić and V. Žutić for coordinating the organic microparticle study in the framework of Project Jadran. We thank many coworkers who contributed to the activities described in this paper. This review is dedicated to the memory of the late Zoran Kljajić of the Institute of Marine Biology, Kotor, Montenegro. Edited by: A. Olita Reviewed by: A. Podestà and M. G. Giani