HE Shan,LIU Hongbing,YANG Xue,LI Chunxia,and GUAN Huashi

Key Laboratory of Marine Drugs,Ministry of Education, Marine Drugs and Food Institute,Ocean University of China, Qingdao 266003, P.R.China

1 Introduction

Deep-sea water (DSW) generally refers to sea water from a depth of more than 200 m,where sunlight can not reach and photosynthesis can not take place (Tsuchiyaet al.,2002).Besides,water at those depths is free of the pollutants and pathogen that can taint surface-sea water(SSW).For these reasons,DSW has many properties that SSW has no,such as purity,low temperature stability,and a wealth of mineral components (Suzuki,2000).Researchers have confirmed the safety of DSW and showed that DSW is safe as drinking water,while SSW has some subacute toxicity (Tsuchiyaet al.,2004).Besides,DSW has been demonstrated to manifest diverse beneficial health effects such as inhibition of atherosclerosis,hypertension,obesity,ostaeoporosis,atopic eczma/dermatitis syndrome,fatigue and cancer (Yoshiokaet al.,2003;Hataguchiet al.,2005; Kozukaet al.,2006; Katsudaet al.,2008; Maehiraet al.,2008; Hwanget al.,2009; Wanget al.,2009).Although the development and utilization of DSW has entered into the commercialization stage in Japan,Korea and America,there is no related research in China.

As a major developing country with a long coastline,China should fully utilize the marine resources for the sustainable development.Additionally,water resource shortage and water pollution are becoming more and more serious in China.Thus,from the perspectives of economy and treatment of disease,the study on DSW has both scientific significance and application values.

Through retrieving information from different literatures on DSW,we found that the pumping depth of DSW has not been very precisely prescribed.For example,in Korea,the depth is 1100 m (Hwanget al.,2009),while in Japan,DSW is pumped up from about 300 m (Tsuchiyaet al.,2004),344 m (Katsudaet al.,2008),374 m (Miyamuraet al.,2004),or 612 m (Machiraet al.,2008).Hence the question is raised as to DSW from what depth DSW should be pumped up from in the South China Sea.Although 200 m is the limiting depth of sunlight-penetration in seawater theoretically,there is a transition layer between SSW and DSW.In deep ocean,the chemical composition of sea water is relatively stable and does not change with depth,sea site and season (Wangersky,1974;Bada and Lee,1977).

The chemical ingredients of seawater are composed of inorganic elements and organic components.Inductively coupled plasma mass spectrometry (ICP-MS) was generally considered as a interference-free technique for analysis of trace elements in natural water samples (Zhuet al.,2006),which allows multi-element analysis,wide linear dynamic range and high sensitivity.It could provide useful information when utilized together with chemometrics methods such as hierarchical cluster analysis (HCA) and principal component analysis (PCA) (Giacominoet al.,2011).Dissolved organic matter (DOM) in seawater is one of the largest organic carbon reservoirs on the earth.However,the major fraction of DOM is still chemically uncharacterized due to the polydisperse and highly complex chemical nature of DOM (Kochet al.,2008).The content of DOM is generally represented as levels of dissolved organic carbon,which could be analyzed by methods of wet chemical oxidation and high-tem- perature catalytic oxidation (Sharpet al.,1995).Ultra performance liquid chromatography-mass spectrometry (UPLC-MS)combined with PCA are powerful for analyzing complex mixtures (Massonet al.,2010),but they have not been used in the analysis of DOM in seawater.

In this study,the pumping depth of DSW in the South China Sea was determined on the basis of chemical ingredients.Seawater was pumped up from 150,200,300,500 and 1000 m.These depths were chosen based on information from different researchers who have accepted 150m as SSW,1000 m as DSW,and 200 (Tsuchiyaet al.,2002),300 (Qian,1978),and 500 m (Wangersky,1974;Bada and Lee,1977) as boundaries between SSW and DSW.The analyses of inorganic elements and dissolved organic matter (DOM) were performed by ICP-MS and UPLC-MS respectively.The raw data were used for hierarchical cluster analysis (HCA) and principal component analysis (PCA).The depth of seawater which has similar chemical composition to that of 1000 m was regarded as the pumping depth of DSW.

2 Materials and Methods

2.1 Collection and Pre-Treatment of Seawater

Seawater was pumped up in the area 500 km away from the coastline of Shantou (120?30′15′E,20?59′57′′N,the South China Sea),and from the depths of 150,200,300,500 and 1000 m using CTD water sampler (Sea-Bird Electronics Inc.,Bellevue,Washington,USA).After pumped up,the seawater was immediately filtered through a microfilter with a pore size of 0.45 μm (Labbase Instrumentation Co.,Ltd,China) to remove the phytoplankton and marine microorganisms,and stored in a refrigerator for further analysis.

2.2 Chemical Composition Analysis of Seawater

2.2.1 Inorganic elements analysis of seawater ICP-MS analysis of inorganic elements

A total of 52 elements were determined: Li,B,F,Na,Mg,Al,Cl,K,Ca,V,Cr,Mn,Fe,Co,Ni,Zn,Ga,As,Se,Br,Rb,Sr,Y,Mo,Ag,Cd,Sb,I,Cs,Ba,La,Ce,Pr,Nd,Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm,Yb,Lu,W,Hg,Pb,Bi,Th,U,SO42?and NO3?.Metal element analysis was carried out using inductively coupled plasma mass spectrum(ICP-MS,Agilent 7500a).The emission intensity measurements were conducted under the following conditions:RF Power 1200 W,nebulizer flow 15 L min-1,and auxiliary gas 1.0 L min-1.Nonmetals elements such as F,Cl,Br,I,SO42?and NO3?were analyzed by Ion Chromatography(Dionex ICS5000) under the following conditions: column,TSK gel Super IC-AZ (150 mm×4.6 mm i.d.); mobile phase,3.8 mmol L-1NaHCO3with adding with 3.0 mmol L-1Na2CO3; flow rate,0.8 mL min-1; injection volume,30μL; column temperature,40℃.

Data analysis

The data sets were used as an input for SPSS Program,version 16.0 (SPSS Inc.,Chicago,USA) to perform HCA and PCA.Euclidian distances and average linkage were used for HCA (Ma and Dai,2010).

2.2.2 DOM analysis of seawater Extraction of DOM

DOM was extracted as described by Kochet al.(2008)and Dittmaret al.(2008).Seawater samples (5 L) were pre- fi ltered with 0.45 μm fi lter membrane.The fi ltrate was acidi fi ed with hydrochloric acid to pH 2 and pumped through a cartridge filled with sorbent (PPL,10 g,Varian)at a flow rate not exceeding 40 mL min-1.The cartridges were previously rinsed with methanol and acidi fi ed ultrapure water (with HCl to pH 2) (Milli-Q system,Millipore).Before elution of DOM,the cartridge was rinsed with 2 volumes of ultrapure water for complete removal of salt,and DOM was eluted with 10 mL methanol (Burdick & Jackson,Honeywell) at a flow rate not exceeding 40 mL min-1.DOM was dried by evaporation,and then redissolved in 1 mL methanol and stored in the sealed ampoules at ?20℃ in the dark for further analysis.

UPLC-MS analysis of DOM

The ultra-performance liquid chromatograph-mass spectrum (UPLC-MS) system consisting of an ACQUITYTM UPLC BEH C18 column (1.7 μm,50 mm×2.1 mm i.d.;Waters Corp.,Milford,MA,USA) and a SynaptTMhighresolution mass spectrometer (Waters Corp.,Milford,MA,USA) was employed for analysis.The column temperature was maintained at 35℃.DOM extracts were chromatographed by UPLC using a gradient mobile phase consisting of 0.1% formic acid in water as solvent A and acetonitrile as solvent B.The gradient conditions of the mobile phase were 0 min 10% B,20.0 min 100% B.The flow rate was 0.40 mL min-1.The injection volume was 3 μL.

For operation in MS mode,ionization was achieved using electron spraying ionization with positive and negative modes.The electrospray source parameters were fixed as: capillary voltage 3 kV,cone voltage 35 V,source temperature 100℃ and desolvation temperature 350℃.The cone and desolvation gas flows were 50 and 800 L h-1respectively.For mass accuracy,leucine enkephalin (MW=555.62 Da) (200 pg μL-1in acetonitrile/water 50:50) was used as a lock mass.Full scan data were collected from 100 to 1000 m/z over a period of 20 min.MassLynx software (Waters Corp.,Milford,USA) was used for system control and data acquisition.The experiments were carried out in triplicate.

Data analysis

The raw data were analyzed using MarkerLynx Appli-cations Manager version 4.1 (Waters Corp.,Milford,USA).Peak list data obtained by MarkerLynx,including the peak number (RT-m/z pair),sample name,and normalized ion intensity were used for PCA.

3 Results and Discussion

3.1 Difference of Mineral Ingredients in Seawater from Different Depths

A total of 52 elements were investigated to determine the difference among seawater from the five depths.The contents of the examined elements are presented in Table 1.

Table 1 Content of mineral ingredients in seawater samples from five depths

Notes: a) in μg L-1; b) in mg L-1.

PCA is a projection method,and dimension reduction of the data can be achieved using a smaller number of principal components than original variables (Skrbi?et al.,2005).Four principal components (PCs) are listed in Table 2,which could account for 100% of the total variance in the data.The first PC (PC1) explained 64.16% of the total variance in the data.The contents of V,Cr,Mn,Zn and Se were positively correlated with PC1,while the contents of Hg and Pb were negatively correlated with PC1.Therefore,V,Cr,Mn,Zn,Se,Hg,and Pb were regarded as the characteristic elements of seawater.The distribution of these characteristic elements in seawater from the five depths is shown in Fig.1.Among these elements,contents of V,Cr,Mn,Zn and Se increased with the increase of depth,meanwhile the levels of poisonous elements,i.e.,Hg and Pb,decreased sharply.

Table 2 PCA for microelements in seawater samples from five depths: varimax rotated principal component loadings

Fig.1 Distribution of characteristic elements in seawater samples from five depths.

HCA is a convenient tool to compare samples and visualize their differences.It was performed for investigating similarities between seawater samples from different depths,based on the levels of all determined elements.The horizontal line of the branches in Fig.2 represented the degree of distance; the vertical lines indicated the distance values.The shorter distance between two samples demonstrated the higher similarity.The distance and similarity between samples was represented in a cluster diagram.As shown in Fig.2,seawater from 500 m and 1000 m shared higher similarity than other samples,which suggested they were very similar in inorganic components.

Fig.2 HCA of inorganic components in seawater samples from five depths (1,150 m; 2,200 m; 3,300 m; 4,500 m;5,1000 m).

3.2 Difference of DOM in Seawater from Different Depths

The base peak intensity (BPI) chromatograms of DOM in seawater from different depths are shown in Fig.3.Although some differences could be visually noted among the five samples,more subtle changes could be found using a pattern recognition approach,such as PCA.

Scores plots from PCA were used to assess the degree of similarity between samples (Massonet al.,2010).PCA scores plot for the DOM in seawater from different depths analyzed in ESI+and ESI?modes are shown in Fig.4.Replicate injections of the same samples clustered tightly,illustrating the stability of the UPLC-MS platform throughout the whole run (Massonet al.,2011).Samples of 150,200 and 300 m were separated from samples of 500 m and 1000 m,while the latter two were tightly clustered.This result suggested that seawater from 500 m and 1000 m shared higher similarity in organic component than other samples.

Taken together,for both inorganic and organic components,seawater from 500 m and 1000 m shared higher similarity than other samples,so seawater from depths more than 500 m has relatively stable chemical composition,and could be used in the development of DSW in the South China Sea.

As the chemical components of DSW may be influenced by ocean current and submarine topography (Wangerskyet al.,1974),more samples need to be collected and their chemical components should be compared in the future studies.Additionally,it has been reported that DSW could prevent the hyperlipidemia and arteriosclerosis in animals(Miyamuraet al.,2004).This means DSW may be distinct from SSW in its pharmacological effect,which needs to be confirmed by further study.

Fig.3 BPI chromatograms of DOM in seawater samples from five depths in ESI+ mode.

Fig.4 PCA scores plot of DOM in seawater samples from five depths.A.ESI+ mode; B.ESI? mode.

Acknowledgements

This research was supported by Special Fund for Marine Scientific Research in the Public Interest (2010-05024).The authors gratefully thank Professor Chen Yongxing and the crew in research vessel ‘Dong Fang Hong 2’ for their generous assistance.

Bada,J.L.,and Lee,C.,1977.Decomposition and alteration of organic compounds dissolved in seawater.Marine Chemistry,5 (4): 523-534.

Dittmar,T.,Koch,B.,Hertkorn,N.,and Kattner,G.,2008.A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater.Limnology and Oceanography:Methods,102 (6): 230-235.

Giacomino,A.,Abollino,O.,Malandrino,M.,and Mentasti,E.,2011.The role of chemometrics in single and sequential extraction assays: A review.Part II.Cluster analysis,multiple linear regression,mixture resolution,experimental design and other techniques.Analytica Chimica Acta,688 (2): 122-139.

Hwang,H.S.,Kim,H.A.,Lee,S.H.,and Yun,J.W.,2009.Anti-obesity and antidiabetic effects of deep sea water on ob/ob mice.Marine Biotechnology,11 (4): 531-539.

Hataguchi,Y.,Tai,H.,Nakajima,H.,and Kimata,H.,2005.Drinking deep-sea water restores mineral imbalance in atopic eczma/dermatitis syndrome.European Journal of Clinical Nutrition,59 (3): 1093-1096.

Koch,B.P.,Ludwichowski,K.U.,Kattner,G.,Dittmar,T.,and Witt,M.,2008.Advanced characterization of marine dissolved organic matter by combining reversed-phase liquid chromatography and FT-ICR-MS.Marine Chemistry,111 (6):233-241.

Kozuka,Y.,and Tuji,A.,2006.Anticancer drug using deep sea water.US 2006/02634422 A1.2006-11-23.

Katsuda,S.I.,Yasukawa,T.,Nakagawa,K.,Miyake,M.,and Yamasaki,M.,2008.Deep-sea water improves cardiovascular hemodynamics in Kurosawa and Kusanagi-Hyper-Clesterolemic (KHC) rabbits.Biological and Pharmaceutical Bulletin,31 (1): 38-46.

Machira,F.,Iinuma,Y.,Eguchi,Y.,Miyagi,I.,and Teruya,S.,2008.Effects of soluble silicon compound and deep-sea water on biochemical and mechanical properties of bone and the related gene expression in mice.Journal of Bone and Mineral Metabolism,26 (2): 446-451.

Masson,P.,Alves,A.C.,Ebbels,T.M.D.,Nicholson,J.K.,and Want,E.J.,2010.Optimization and evaluation of metabolite extraction protocols for untargeted metabolic pro fi ling of liver samples by UPLC-MS.Analytical Chemistry, 82 (18):7779-7786.

Masson,P.,Spagou,K.,Nicholson,J.K.,and Want,E.J.,2011.Technical and biological variation in UPLC-MS-based untargeted metabolic profiling of liver extracts: Application in an experimental toxicity study on galactosamine.Analytical Chemistry,83 (3): 1116-1123.

Wangersky,P.J.,1974.Particulate organic carbon: sampling variability.Limnology and Oceanography,19 (5): 980-984.

Miyamura,M.,Yoshioka,S.,Hamada,A.,and Takuma,D.,2004.Difference between deep seawater and surface seawater in the preventive effect of atherosclerosis.Biological and Pharmaceutical Bulletin,27 (1): 1784-1790.

Qian,Z.G.,1978.Chemical constituent of dissolved organic matter in seawater.Marine Science,6 (4): 21-34.

Skrbi?,B.,Durisi?-Mladenovi?,N.,and Cvejanov,J.,2005.Principal component analysis of trace elements in Serbian wheat.Journal of Agriculture and Food Chemisty,53 (6):2171-2175.

Sharp,J.H.,Benner,R.,Bennett,L.,and Tupas,L.M.,1995.Analyses of dissolved organic carbon in seawater: the JGOFS EqPac methods comparison.Marine Chemistry,48 (2): 91-108.

Suzuki,H.,2000.Characteristics,utilization,and functionality of deep-sea water.Food Research,7 (3): 37-41.

Tsuchiya,Y.,Nankamura,K.,Sekikawa,H.,and Kawamura,H.,2002.Subacute effects of deep-sea water from the Japan Sea on blood examination values in mice.Environmental Health and Preventive Medicine,7 (4): 189-196.

Tsuchiya,Y.,Watanabe,A.,Fujisawa,N.,Kaneko,T.,Ishizu,T.,Fujimoto,T.,and Nakamura,K.,2004.Effects of desalted deep seawater on hematologic and blood chemical values in mice.The Tohoku Journal of Experimental Medicine,203 (3):175-182.

Wang,S.T.,Hwang,D.F.,and Chen,R.H.,2009.Effect of deep sea water on the exercise-induced fatigue of rats.Journal of Food and Drug Analysis,17 (2): 133-143.

Wangersky P.J.,1974.Particulate organic carbon: sampling variability.Limnology and Oceanography,19 (5): 980-984.

Yoshioka,S.,Hamada,A.,Cui,T.,and Yamamoto,S.,2003.Pharmacological activity of deep-sea water: examination of hyperlipemia prevention and medical treatment effect.Biological and Pharmaceutical Bulletin,26 (11): 1552-1559.

Zhu,Y.B.,Itoh,A.,Fujimori,E.,Umemura,T.,and Haraguchi,H.,2006.Determination of rare earth elements in seawater by ICP-MS after preconcentration with a chelating resin-packed minicolumn.Journal of Alloys and Compounds, 408: 985-988.