Chen Liang

Haibo Li

Yi Dong

Yuan Zhao

Zhencheng Tao

Chaolun Li

Wuchang Zhang
email: wuchangzhang@qdio.ac.cn

Gérald Grégori

Gerald Gregori

Planktonic ciliates in different water masses in open waters near Prydz Bay (East Antarctica) during austral summer, with an emphasis on tintinnid assemblages

Keywords: Planktonic ciliates, Abundance, Tintinnid, Water mass, Prydz Bay, Antarctica

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Marine planktonic ciliates constitute a major, ubiquitous, and diverse group of protozoa. These organisms range from 5 to 200 µm in length and can be divided into tintinnids and aloricate ciliates, which belong to the subclasses Oligotrichia and Choreotrichia, respectively, in the class Spirotrichea [START_REF] Lynn | Ciliated protozoa: characterization, classification, and guide to the literature, 3rd edn[END_REF]. As one of the main components of microzooplankton, ciliates are a trophic link between the microbial food web and the traditional food chain. Planktonic ciliates feed on nanoplankton [START_REF] Heinbokel | Studies on the functional role of tintinnids in the Southern California Bight. III. Grazing impact of natural assemblages[END_REF][START_REF] Capriulo | Abundance, species composition and feeding impact of tintinnid micro-zooplankton in central Long Island Sound[END_REF][START_REF] Verity | Abundance, community composition, size distribution, and production rates of tintinnids in Narragansett Bay, Rhode Island[END_REF]) and picoplankton [START_REF] Bernard | The role of picoplankton (cyanobacteria and plastidic picoflagellates) in the diet of tintinnids[END_REF], while they are preyed upon by mesoplankton and fish larvae [START_REF] Azam | The ecological role of water column microbes in the sea[END_REF]Lavalpeuto et al. 1986;Pierce andTurner 1992, 1994). Thus, they play an important role in the transfer of energy and material through the entire pelagic food web [START_REF] Beers | The species distribution of some naturally-occurring quaternary ammonium compounds[END_REF][START_REF] Pierce | Ecology of planktonic ciliates in marine food webs[END_REF].

The various genera of tintinnids have been divided into several biogeographical types (cosmopolitan, neritic, warmwater, boreal, and austral) according to their global occurrence [START_REF] Pierce | Global biogeography of marine tintinnids[END_REF]Dolan et al. 2013a). Therefore, different biogeographical areas can be characterized by different tintinnid assemblages [START_REF] Li | Boreal tintinnid assemblage in the Northwest Pacific and its connection with the Japan Sea in Summer 2014[END_REF]). In the austral assemblage, [START_REF] Dolan | Southern Ocean biogeography of tintinnid ciliates of the marine plankton[END_REF] divided the austral species (40°S-78°S) into Southern Ocean endemic species (only found south of 40°S) and widespread species (those with a geographic range extending into the Southern Ocean). In Antarctic waters, ciliate abundance was observed in different habitats (oceanic areas, transitional areas, polynyas, edges of glaciers, and edges of sea ice) in the Amundsen Sea (e.g., [START_REF] Jiang | Spatial patterns in pelagic ciliate community responses to various habitats in the Amundsen Sea (Antarctica)[END_REF][START_REF] Jiang | Pelagic ciliate communities within the Amundsen Sea polynya and adjacent sea ice zone, Antarctica[END_REF][START_REF] Jiang | An approach to bioassess pelagic ciliate biodiversity at different taxonomic resolutions in response to various habitats in the Amundsen Sea (Antarctica)[END_REF] and the Weddell Sea (e.g., [START_REF] Heinbokel | Patterns of tintinnina abundance and reproduction near the edge of seasonal pack-ice in the Weddell Sea, November 1983[END_REF][START_REF] Garrison | Protozooplankton in the Weddell Sea, Antarctica: abundance and distribution in the ice-edge zone[END_REF][START_REF] Garrison | Plankton assemblages in the ice edge zone of the Weddell Sea during the austral winter[END_REF][START_REF] Gowing | Abundance and feeding ecology of larger protozooplankton in the ice edge zone of the Weddell and Scotia Seas during the austral winter[END_REF]. These investigations generally evaluated ciliate assemblages at a horizontal level while providing very little vertical distribution information. Plankton distribution has been found to be closely related to water masses [START_REF] Longhurst | Ecological geography of the sea, 2nd edn[END_REF], and previous studies have provided evidence of different phytoplankton community structures in various water masses (e.g., [START_REF] Stoecker | Changes in the sea-ice brine community during the spring-summer transition, McMurdo Sound, Antarctica. I. Photosynthetic protist[END_REF][START_REF] Mura | Water-mass influences on summer Antarctic phytoplankton biomass and community structure[END_REF]Zhang et al. 2012). Although water masses are well defined in the Southern Ocean [START_REF] Tomczak | Regional oceanography: an introduction, 1st edn[END_REF], no studies have investigated the relationship between ciliates and different water masses in this ocean to date.

There are several vertically divided water masses in waters near Prydz Bay (East Antarctica) during austral summer; namely, Summer Surface Water (SSW), Winter Water (WW), Circumpolar Deep Water (CDW), Antarctic Bottom Water (ABW) and Antarctic Shelf Water (ASW) [START_REF] Smith | Water masses and circulation in the region of Prydz Bay, Antarctica[END_REF][START_REF] Middleton | Thermohaline structure and mixing in the region of Prydz Bay, Antarctica[END_REF][START_REF] Le | An analysis on water masses and thermohaline structures in the region of Prydz Bay, Antarctica[END_REF][START_REF] Vaz | Physical oceanography of the Prydz Bay region of Antarctic waters[END_REF][START_REF] Williams | Antarctic bottom water from the Adélie and George V Land coast, East Antarctica[END_REF][START_REF] Shi | Progress of Chinese research in physical oceanography of the Southern Ocean[END_REF]. Moreover, upwelling occurs sporadically in Prydz Bay [START_REF] Lin | An analysis on the upwelling of the modified circumpolar deep water over the shelf region of Prydz Bay in the summer of 2013[END_REF]. Bathymetrically, there is a basin known as the Amery Depression located at a depth of about 700 m seaward of the Amery Ice Shelf that shoals gently to outer shelf banks at a depth of around 200 m [START_REF] Gao | Advances of oceanographic study on Prydz Bay and adjacent region, Antarctica[END_REF]. Few studies have investigated ciliate assemblage in Prydz Bay and its surrounding waters. Planktonic ciliates were investigated at 0 and 5 m below sea ice at two stations very close (3 km) to shore [START_REF] Paterson | Sea ice microbial dynamics over an annual ice cycle in Prydz Bay, Antarctica[END_REF]. In addition, ciliate abundances were determined at a depth of 15 m in Prydz Bay, 5 km offshore from the Australian Antarctic station of Davis [START_REF] Davidson | Protist abundance and carbon concentration during a Phaeocystis-dominated bloom at an Antarctic coastal site[END_REF], but there is no record of tintinnid species in this area. However, there have been studies of phytoplankton, zooplankton, bacteria, and viruses in Prydz Bay and its adjacent areas without considering water masses (e.g., [START_REF] Zhu | convallaria; (13) Cymatocylis antarctica Online Resource 2 Cluster analysis using group-average linkage based on the Bray-Curtis similarity matrix of fourth root transformed tintinnid abundances in all sampling points. (group I) tintinnid species in Pattern 1; (group II) tintinnid species in Pattern 2; (group III) tintinnid species in Pattern 3; (group IV) tintinnid species with no distribution pattern Online Resource 3 Vertical scatter distribution of typical tintinnid species in three patterns. (a) Pattern 1: tintinnid mainly occurred in SSW; (b) Pattern 2: tintinnid mainly occurred in WW[END_REF][START_REF] Waters | Summer microbial ecology off East Antarctica (80-150°E): protistan community structure and bacterial abundance[END_REF][START_REF] Thomson | Distribution and abundance of marine microbes in the Southern Ocean between 30 and 80 E[END_REF][START_REF] Liang | Distribution of marine viruses and their potential hosts in Prydz Bay and adjacent Southern Ocean, Antarctic[END_REF]. Moreover, Zhang et al. (2012) used flow cytometry to show that the SSC (side scatter, optical parameter) signal of picophytoplankton increased horizontally from out of the bay to a coastal station without considering the vertical division of water masses in Prydz Bay.

The notion that different water masses contain different indigenous species of plankton is the base of plankton biogeography [START_REF] Longhurst | Ecological geography of the sea, 2nd edn[END_REF][START_REF] Priede | Biogeography of the oceans: a review of development of knowledge of currents, fronts and regional boundaries from sailing ships in the sixteenth century to satellite remote sensing[END_REF], as well as the base of expatriate species (Angel 1993;[START_REF] Kobari | Seasonal appearance of expatriated boreal copepods in the Oyashio-Kuroshio mixed region[END_REF], stray species [START_REF] Dolan | Southern Ocean biogeography of tintinnid ciliates of the marine plankton[END_REF]) and indicator species (e.g., [START_REF] Kato | Tintinnid ciliates as indicator species of different water masses in the western North Pacific Polar Front[END_REF]. Most of the biogeographical studies have been conducted in the horizontal division of water masses, however, we hypothesized that different ciliates have different preferred water masses in the vertical direction in Antarctic waters. Therefore, we compared the ciliate assemblages in different vertically divided water masses in open waters near Prydz Bay during austral summer. Our specific objectives were to compare (1) the contributions of aloricate ciliates and tintinnids to total abundance, (2) proportions of different-sized aloricate ciliates in total aloricate ciliate abundance, and (3) tintinnid species distributions in different water masses.

Materials and methods

Sampling was conducted at 34 stations in four South-North transects (P2, P3, P4, P5) in open waters near Prydz Bay (65°28′-73°3′E, 65°-68°S) (Fig. 1) onboard the R. V. "Xuelong", during the late austral summer (3-27 February) of 2015. The study area was outside of Amery Depression and free of ice. The minimum and maximum depths of the stations were 102 m (St. P2-9) and 3991 m (St. P5-2), respectively. At each station, vertical profiles of temperature, salinity and in vivo Chlorophyll a (Chl a) fluorescence were determined from the sea surface to a depth of 300 m or 2 m above the bottom when the water depth was < 300 m using the SBE911-conductivity-temperature-depth (CTD) unit. Water masses were determined according to temperature and salinity [START_REF] Smith | Water masses and circulation in the region of Prydz Bay, Antarctica[END_REF][START_REF] Middleton | Thermohaline structure and mixing in the region of Prydz Bay, Antarctica[END_REF][START_REF] Le | An analysis on water masses and thermohaline structures in the region of Prydz Bay, Antarctica[END_REF][START_REF] Vaz | Physical oceanography of the Prydz Bay region of Antarctic waters[END_REF][START_REF] Williams | Antarctic bottom water from the Adélie and George V Land coast, East Antarctica[END_REF][START_REF] Shi | Progress of Chinese research in physical oceanography of the Southern Ocean[END_REF].

Water was sampled using 10-L Niskin bottles on a rosette CTD carrousel. Seawater samples of 1 L were collected from each sampling point (surface, 25, 50, 75, 100, 150, 200, 300 m at each station), then fixed with Lugol's solution (1% final concentration, vv -1 ). A total of 264 water samples were collected.

In the laboratory, each water sample was concentrated to about 100 mL by gently siphoning out supernatant water after allowing it to settle for at least 48 h. The settling and siphoning processes were repeated to concentrate each sample to a final volume of ~ 50 mL. The concentrated sample was then allowed to settle in an Utermöhl counting chamber for at least 24 h, after which it was examined using an Olympus IX 71 inverted microscope (×100 or ×400).

For each sample, the entire concentrated sample was counted. A maximum of 25 mL of the concentrated sample was examined in each count; therefore, at least two counts were made to complete sample analysis. A smaller volume was examined for each count when the microscopic view was blurred because of the high concentration of phytoplankton. The species present in low abundance (especially Antarctic endemic species of tintinnids) were counted completely in every count to ensure accuracy. Highly abundant species were not counted if a high number was obtained in a previous count. Because mechanic and chemical disturbances associated with collection and fixation procedures could provoke detachment of the protoplasma from the loricae [START_REF] Paranjape | Cultivation of marine pelagic protozoa[END_REF]Alder 1999), empty loricae of tintinnid species were counted as living cells in this study. In addition, some loricae might be empty when they were sampled [START_REF] Kato | Tintinnid ciliates as indicator species of different water masses in the western North Pacific Polar Front[END_REF][START_REF] Dolan | Observations of apparent lorica variability in Salpingacantha (Ciliophora: Tintinnida) in the Northern Pacific and Arctic Oceans[END_REF], which might have resulted in overestimated numbers. For each species, sizes (e.g., length or width, according to shape) of the cell (aloricate ciliate) or lorica (tintinnid, especially length and oral diameter) were measured for at least (if possible) 20 individuals in the entire study area. According to lorica morphology and size, tintinnids were identified to the species level according to references [START_REF] Laackmann | Die Tintinnodeen der deutschen Südpolar-Expedition 1901-1903[END_REF]Kofoid andCampbell 1929, 1939;[START_REF] Hada | The protozoan plankton of the Antarctic and subantarctic seas[END_REF][START_REF] Boltovskoy | Intraspecific variability in Antarctic tintinnids: the Cymatocylis affinis/convallaria species group[END_REF]Alder 1999;Zhang et al. 2012;Dolan et al. 2013b;[START_REF] Kim | Morphological and ribosomal DNA-based characterization of six Antarctic ciliate morphospecies from the Amundsen Sea with phylogenetic analyses[END_REF]. Ciliate volumes were then estimated using appropriate geometric shapes (cone, ball, and cylinder). Tintinnid carbon biomass was estimated using the equation: C = lorica volume (µm 3 ) × 0.053 + 444.5 [START_REF] Verity | Relationships between lorica volume, carbon, nitrogen, and ATP content of tintinnids in Narragansett Bay[END_REF]. The conversion factor of carbon biomass for aloricate ciliates used in this study was 0.19 pg C µm -3 as defined by [START_REF] Putt | An experimentally determined carbon: volume ratio for marine "Oligotrichous" ciliates from estuarine and coastal waters[END_REF]. Southern Ocean endemic species were defined according to [START_REF] Dolan | Southern Ocean biogeography of tintinnid ciliates of the marine plankton[END_REF].

Data analysis

The occurrence frequency of each tintinnid species was calculated by dividing the total number of sampling points by the number of sampling points where this species occurred. Principal component analysis (PCA) was conducted using Canoco for Windows 4.5 software. For PCA, environmental variables included seawater temperature, salinity, Chl a and depth, while biological variables consisted of the abundance of 13 tintinnid species. Cluster analysis was conducted using the Primer 5 software. Group-average linkage based on the Bray-Curtis similarity matrix of the fourth root transformed tintinnid abundances was utilized.

Results

Hydrography and water mass division

The water column temperature (Fig. 2) showed obvious sandwich structures, with warm water (T > -1.5 °C) lying on and below a cold water (T < -1.5 °C) layer. We defined the cold-water belt (T < -1.5 °C) in the middle as WW, the warm water upon WW in the upper 50 m as SSW, and the warm water below WW as CDW. Upwelling occurred in transect P3 (Sts. 6, 7), P4 . The upwelling in transects P4 and P5 were stronger, with -1.2 °C outcropping to the surface, while the upwelling was comparatively weak in Transect P3 with a temperature of only -0.8 °C outcropping to the surface. WW lied between 50 and 100 m on the offshore side of the transects, whereas it occupied the water column down to 300 m on the coastal side.

The salinity was less than 34 in the upper 50 m (SSW) at most stations, while WW and CDW exhibited higher salinity (> 34) (Fig. 2). The position of salinity 34 isohaline was similar to that of the -1.5 °C isothermal underlying SSW. The in vivo Chlorophyll a (Chl a) decreased with depth, and the highest values (> 3 mg m -3 ) were limited to coastal sites in the upper 50 m of SSW, with a maximum value of 19.18 mg m -3 being observed at 4.5 m at St. P5-9. According to the spatial distribution of the concentration of Chl a, SSW was divided into an area with Chl a concentration > 3 mg m -3 near the shelf and another area with concentration < 3 mg m -3 in the north. The horizontal division of the two sectors overlapped with that of the upwelling, with the exception of transect P2, for which upwelling was not detected in SSW.

Ciliate abundance and biomass in different water masses

Ciliate abundance and biomass decreased from SSW to CDW (Table 1, Figs. 3,4,5a). High ciliate abundance (> 1000 ind. L -1 ) and biomass (> 4 µg C L -1 ) occurred in SSW. The aloricate ciliate proportion accounted for > 57% of the total ciliate abundance in all water masses. Average abundances of aloricate ciliates and total ciliates in SSW Chl a > 3 (SSW, Chl a > 3 mg m -3 ) were less than in SSW Chl a < 3 , but their average biomasses were greater than in this fraction of water (Table 1, Fig. 5b). The proportion of tintinnids to the total ciliate abundance in SSW Chl a > 3 (38.2%) was much higher than in any other water masses (< 13%), with CDW having the lowest tintinnid proportion (2.2%, Fig. 5b). In addition, SSW Chl a > 3 contained larger sized aloricate ciliates, with those in the 10-20 µm size class compromising < 15% of the total aloricate ciliate abundance, while this value was more than 39% in other water masses (Fig. 5c).

Thirteen tintinnid species belonging to five genera were identified (Table 2, Fig. 6), and the occurrence frequencies and abundances of different tintinnids varied greatly. Generally, tintinnids with a higher occurrence frequency also had a higher abundance (both average abundance and maximum abundance, Online Resource 1). Salpingella sp. was the most abundant and frequent species, while Amphorides laackmanni was the least abundant (Table 2, Fig. 5d, Online Resource 1). Tintinnid total abundance in different water masses was different, with SSW Chl a > 3 having the highest average tintinnid total abundance (209 ind. L -1 ) among all water masses (Fig. 5d, Table 1). The average abundance of all tintinnid species in CDW was low (< 0.9 ind. L -1 ) (Fig. 5d, Table 2).

There were seven Southern Ocean endemic tintinnid species (Codonellopsis gaussi, Laackmanniella naviculaefera, Cymatocylis affinis/convallaria, C. antarctica, C. affinis/convallaria forma cristallina, C. affinis/convallaria forma drygalskii and C. vanhoeffeni). Laackmanniella naviculaefera and C. affinis/convallaria forma cristallina were the most abundant endemic species in WW (Table 2). The average abundance of endemic species in SSW Chl a > 3 was highest (Fig. 5d), and the Southern Ocean endemic species abundance in WW (> 30%) was much higher than in other water masses (Fig. 5e).

Tintinnid species in different water masses

Cluster analysis divided all tintinnid species into four groups according to their distribution (Online Resource 2). Species in groups I, II and III had higher occurrence frequencies, making it possible to study their distribution pattern. Three main patterns corresponding to the three groups were identified according to their distribution characteristics (Figs. 7,8,9). Pattern 1 was defined by tintinnids that mainly occurred in SSW. These organisms were distributed in the surface or subsurface layer of SSW, but disappeared in the deep water. Tintinnids with this pattern were identified as Salpingella sp. and C. gaussi. The maximum abundance of Salpingella sp. was observed in the upper 25 m, while that of C. gaussi was found in the upper 50 m (Fig. 7, Online Resource 3). Pattern 2 was defined as tintinnids that mainly occurred in WW. These species (Salpingella costata, S. faurei, C. affinis/convallaria forma drygalskii and C. vanhoeffeni) did not exist in the upper 25 m and mainly occurred in the subsurface and stretched into deep water in WW in the coastal area, with a decreasing trend to the north (Fig. 8, Online Resource 3). Finally, L. naviculaefera and C. affinis/convallaria forma cristallina formed Pattern 3. These organisms inhabited both SSW Chl a > 3 and WW, and were distributed from the surface of SSW Chl a > 3 to deep water in WW (Fig. 9, Online Resource 3). These two species were the most abundant Southern Ocean endemic species in WW (Table 2). Species in group IV (Codonellopsis glacialis, C. affinis/convallaria, C. antarctica, Salpingella acuminata, and A. laackmanni) occurred too rarely (occurrence frequency < 8%, Table 2) to show any obvious occurrence pattern (Online Resource 4).

Principal component analysis of the 264 samples and 13 tintinnid species was conducted to examine the contribution of the environmental variables to tintinnid species occurrence (Fig. 10). Two principal components discriminated the environmental conditions in three water masses. These components explained a large proportion (68.1%) of the variance, as well as 44.7 and 23.4% of the tintinnid species variation, respectively (Fig. 10). The first principal component was closely related to seawater depth, Chl a, Salpingella sp., C. gaussi and C. glacialis. These three tintinnid species had a strong positive correlation with Chl a but a strong negative correlation with depth. Chl a had the greatest influence on C. gaussi and C. glacialis. The second principal component was closely related to temperature, S. costata, S. faurei, C. affinis/convallaria forma drygalskii and C. vanhoeffeni, C. affinis/convallaria forma cristallina and L. naviculaefera. These species had a strong negative correlation with temperature (Fig. 10). Different tintinnid groups had different temperatures and Chl a preferences: those showing Pattern 1 occurring in warm water in the surface and those showing Pattern 2 being present in cold water. Tintinnid species in Pattern 3 occurred in cold water and warm water with high Chl a levels (Online Resource 5).

Discontinuity of ciliate distribution

Isolines of low abundance of aloricate ciliates, tintinnids, and total ciliates showed upwelling in the corresponding horizontal position of the upwelling. The total ciliate abundance isoline of 200 ind. L -1 upwelled in transects P3, P4 and P5, and even reached the surface in SSW in transect P5. The aloricate ciliate isoline of 200 ind. L -1 was distributed in generally the same pattern as the total ciliate assemblage. The tintinnid abundance isoline of 40 ind. L -1 reached the surface in SSW and defined areas of low abundance in surface water for all transects (Fig. 3). According to the tintinnid distribution (Figs. 7,8,9), there was an obvious low-abundance division area that split the tintinnid abundance distribution into two parts. The Salpingella sp. abundance distribution in Transect P3 could easily be split into two parts by the 5

Table 1

Variations of abundance ± SE (ind. L -1 ) and biomass ± SE (µg C L -1 ) of ciliates in different water masses ind. L -1 isoline, while the 60 ind. L -1 isoline split Transect P4 and P5 into two parts (Fig. 7). In the case of tintinnids with Patterns 2 and 3 (C. affinis/convallaria forma drygalskii, C. vanhoeffeni, S. costata, S. faurei, L. naviculaefera and C. affinis/convallaria forma cristallina), their distribution area was also divided by low abundance isolines, and their abundance was higher on the coastal side of the cold-water upwelling (Figs. 8,9).

SSW

Discussion

Ciliate abundance and biomass in different water masses

Data concerning ciliate abundance and biomass are scarce in open waters near Prydz Bay. Paterson and Laybourn-Parry (2012) reported a maximum ciliate abundance of 3000 ind. Red isotherm of -1.5 °C and green Chl a isoline of 3 mg m -3 were attached L -1 in the sea ice area 3 km offshore in April. In addition, higher ciliate abundance of 1.6 × 10 5 ind. L -1 was recorded at depth of 15 m at a site 5 km offshore from the Australian Antarctic station of Davis in January [START_REF] Davidson | Protist abundance and carbon concentration during a Phaeocystis-dominated bloom at an Antarctic coastal site[END_REF]. The close proximity of these stations to the shoreline might explain their higher abundance. The average ciliate abundance (301 ind. L -1 ) and biomass (1.6 µg C L -1 ) in this study were higher than during the same season in the Bellingshausen and Amundsen seas, where there were average ciliate abundance of 139 ind. L -1 and a biomass of 0.34 µg C L -1 , respectively [START_REF] Wickham | Ciliate distributions and forcing factors in the Amundsen and Bellingshausen Seas (Antarctic)[END_REF]). The tintinnid biomass ranged from 0.02 µg C L -1 under the sea-ice to 1.3 µg C L -1 in the open water column in the Weddell Sea [START_REF] Buck | Abundance and distribution of tintinnid ciliates in an ice edge zone during the austral autumn[END_REF].

The proportion of tintinnid abundance to total ciliate abundance has been shown to be < 20% in oceanic waters [START_REF] Suzuki | Standing crops and vertical distribution of four groups of marine planktonic ciliates in relation to phytoplankton chlorophyll a[END_REF][START_REF] Gómez | Trends on the distribution of ciliates in the open Pacific Ocean[END_REF][START_REF] Sohrin | Full-depth profiles of prokaryotes, heterotrophic nanoflagellates, and ciliates along a transect from the equatorial to the subarctic central Pacific Ocean[END_REF][START_REF] Yu | Differences in the vertical distribution and response to freshwater discharge between aloricate ciliates and tintinnids in the East China Sea[END_REF]. [START_REF] Yu | Differences in the vertical distribution and response to freshwater discharge between aloricate ciliates and tintinnids in the East China Sea[END_REF] found that tintinnid abundance was < 10% in stations deeper than 80 m. In the present study, the proportion of tintinnids in SSW Chl a < 3 (SSW, in vivo Chlorophyll a < 3 mg m -3 ) and WW was close to that of oceanic water. A higher tintinnid proportion was found in SSW Chl a > 3 than in other water masses. This phenomenon was consistent with the idea that tintinnids may be Blue line: upwelling range at the 0-m depth. SSW: Summer Surface Water; WW: Winter Water; CDW: Circumpolar Deep Water. Red isotherm of -1.5 °C and green Chl a isoline of 3 mg m -3 were attached more favored than aloricate ciliates under eutrophic conditions in oceanic waters [START_REF] Gómez | Trends on the distribution of ciliates in the open Pacific Ocean[END_REF]. [START_REF] Suzuki | Standing crops and vertical distribution of four groups of marine planktonic ciliates in relation to phytoplankton chlorophyll a[END_REF] also found that tintinnids were adapted to high Chl a concentrations.

The size class of aloricate ciliates has rarely been investigated. In our study, the percentages of different size class aloricate ciliates in different layers of a low Chl a concentration area was roughly the same as in the tropical Western Pacific [START_REF] Wang | Vertical distribution of planktonic ciliates in tropical Western Pacific[END_REF]. However, the size class of aloricate ciliates became larger in the higher Chl a concentration area. There were no similar results in other studies.

Tintinnid species composition

This study was the first to investigate tintinnid species communities in open waters near Prydz Bay. A neritic assemblage has also been described in Antarctic waters (Dolan et al. 2013a). However, neritic genera (Favella, Helicostomella, Leprotintinnus, Metacylis, Stenosemella, Stylicauda, Tintinnidium, and Tintinnopsis) (Dolan et al. 2013a) were not found in the present study. These findings indicate that we did not identify the southernmost limit of the austral assemblage. The tintinnid assemblage might extend further south until it meets the neritic assemblage. However, there has been only one investigation [START_REF] Yu | Differences in the vertical distribution and response to freshwater discharge between aloricate ciliates and tintinnids in the East China Sea[END_REF] of the expansion of neritic species to offshore areas. That study showed neritic species expanded from the coastal line to the 80-m (distance from bottom) isoline in the East China Sea. If this phenomenon is similar in the Antarctic, neritic species would be found in waters shallower than 80 m. However, the shallowest depth in our study was 102 m; therefore, it is assumed that neritic species did not occur in our study area. Thus, further studies should be conducted in nearshore areas to reveal the mixing between austral assemblage and neritic assemblages in Antarctic waters. [START_REF] Dolan | Southern Ocean biogeography of tintinnid ciliates of the marine plankton[END_REF] divided tintinnid species reported from locations between 40°S and 78°S into Southern Ocean endemic species (32 species) and widespread species (161 species). In this study, we found seven Southern Ocean endemic species which was only a small part of the total endemic species list. Most endemic species did not emerge in our survey area, which was likely because their abundance was below the detection limit or they inhabited other areas of Antarctic waters. Dolan et al. (2013b) suggested that several species in the genus Cymatocylis were different morphotypes of Cymatocylis affinis/convallaria. [START_REF] Kim | Morphological and ribosomal DNA-based characterization of six Antarctic ciliate morphospecies from the Amundsen Sea with phylogenetic analyses[END_REF] Therefore, we considered C. drygalskii, C. cristallina and C. convallaria to be polymorphic forms and combined them under the name C. affinis/convallaria. However, this was not done for C. antarctica and C. vanhoeffeni because of their very different morphotypes. In addition, L. naviculaefera and C. gaussi were treated as different species because they showed distinct morphologies in our study.

In this study, C. affinis/convallaria forma cristallina was the dominant morphotype, and C. affinis/convallaria forma affinis and convallaria occurred occasionally in Prydz Bay. However, C. affinis/convallaria forma convallaria was dominant in Admiralty Bay (King George Island) in austral summer, occupying up to 90% of the total tintinnid proportion, while C. affinis/convallaria forma cristallina did not appear [START_REF] Wasik | Annual cycle of tintinnids in Admiralty Bay with an emphasis on seasonal variability in Cymatocylis affinis/convallaria lorica morphology[END_REF]. This distinct difference might reflect geographic differences of tintinnid occurrence.

In the present study, Salpingella sp. was the most abundant species, which was consistent with the results of previous studies. For instance, the maximum abundance of Salpingella sp. reached 1300 ind. L -1 at a nearshore site in Signy Island [START_REF] Leakey | The annual cycle of planktonic ciliates in nearshore waters at Signy Island, Antarctica[END_REF]) and 380 ind. L -1 in the ice-edge zone of the Weddell Sea (Buck et al.1992).

Based on pictures and the size of lorica observed in previous studies [START_REF] Heinbokel | Ciliates and nanoplankton in Arthur Harbor, December 1984 and[END_REF][START_REF] Buck | Abundance and distribution of tintinnid ciliates in an ice edge zone during the austral autumn[END_REF]), the Salpingella sp. observed in the present study may be the same as that of Salpingella sp. #1 observed in the Weddell Sea [START_REF] Buck | Abundance and distribution of tintinnid ciliates in an ice edge zone during the austral autumn[END_REF].

All Southern Ocean endemic tintinnid species except for C. gaussi were limited to the onshore side of the transects from 65°S to 67.51°S in the present study. Similarly, C. affinis/convallaria forma drygalskii was defined as most abundant in the ice-covered coastal zone in the Bellingshausen Sea (Alder and Boltovskoy 1991), while it was dominant in the Amundsen Sea, where it tended to be distributed near the shore [START_REF] Jiang | Spatial patterns in pelagic ciliate community responses to various habitats in the Amundsen Sea (Antarctica)[END_REF]. In another study, C. affinis/convallaria forma drygalskii was almost entirely restricted to the region between 74°S and the Filchner Iceshelf [START_REF] Boltovskoy | Summer Weddell sea microplankton: assemblage structure, distribution and abundance, with special emphasis on the Tintinnina[END_REF].

Indigenous tintinnid species in water masses determined their vertical distribution

Because some tintinnid species had higher abundance in a certain water mass than in other water masses, we took them In each water mass are displayed the average abundance ± SE (ind. L -1 ) of each tintinnid species with its maximum value (ind. L -1 ) in parenthesis as well as its occurrence frequency (%) OF occurrence frequency of each tintinnid species calculated by dividing the total number of sampling points by the number of sampling points where this species occurred, SSW Chl a < 3 Summer Surface Water with Chl a < 3 mg m -3 , sampling points n = 51, SSW Chl a > 3 Summer Surface Water with Chl a > 3 mg m Although previous studies of tintinnids in Antarctic waters did not define any tintinnid species-water mass relationship, some studies have investigated their vertical distribution (e.g., [START_REF] Boltovskoy | Microzooplankton and tintinnid species-specific assemblage structures: patterns of distribution and year-to-year variations in the Weddell Sea (Antarctica)[END_REF][START_REF] Kivi | Late winter microbial communities in the western Weddell Sea (Antarctica)[END_REF][START_REF] Christaki | Microbial food web structure in a naturally ironfertilized area in the Southern Ocean (Kerguelen Plateau)[END_REF]. Since water masses were vertically divided in our study area, we were able to compare our results with these previous studies. In our study, Salpingella sp. and C. gaussi mainly inhabited SSW, which was consistent with the results of previous studies that showed they were present in higher abundance in surface waters. For example, the abundance of Salpingella sp. was higher in the upper 50 m than in deeper waters in the ice-edge zone of the Weddell Sea [START_REF] Buck | Abundance and distribution of tintinnid ciliates in an ice edge zone during the austral autumn[END_REF]. C. gaussi (maximum 40.3 ind. L -1 ) mainly existed in the upper 50 m of the ice-edge zone in the Weddell Sea [START_REF] Boltovskoy | Microzooplankton and tintinnid species-specific assemblage structures: patterns of distribution and year-to-year variations in the Weddell Sea (Antarctica)[END_REF].

Four species (S. fauri, S. costata, C. affinis/convallaria forma drygalskii and C. vanhoeffeni) were mainly found in WW in this study. No specific vertical distribution pattern of these species was reported in previous studies. [START_REF] Boltovskoy | Microzooplankton and tintinnid species-specific assemblage structures: patterns of distribution and year-to-year variations in the Weddell Sea (Antarctica)[END_REF] reported that Salpingella sp. was mainly distributed in deep water (> 100 m) in the Weddell Sea. This species distribution pattern was similar to that of S. fauri and S. costata observed in the present study. Because there was no concrete species information about Salpingella sp. reported in [START_REF] Boltovskoy | Microzooplankton and tintinnid species-specific assemblage structures: patterns of distribution and year-to-year variations in the Weddell Sea (Antarctica)[END_REF], we suggest that the Salpingella sp. observed in their study might be the S. fauri or S. costata found in the present study.

In the present study, C. affinis/convallaria forma cristallina and L. naviculaefera were found in high abundance in SSW Chl a > 3 and WW. L. naviculaefera is a surface species for which the maximum abundance (131 ind. L -1 ) was observed at 25 m in our study. This species has previously been reported to be present in maximum abundance (40.2 ind. L -1 ) at 50 m in the Weddell Sea [START_REF] Boltovskoy | Microzooplankton and tintinnid species-specific assemblage structures: patterns of distribution and year-to-year variations in the Weddell Sea (Antarctica)[END_REF]. To the best of our knowledge, there is no vertical distribution data for C. affinis/convallaria forma cristallina in the available literature. Other species like A. laackmanni and C. antarctic with no concrete pattern were rare in our study and we did not find any previous distribution data regarding these species.

Influences of upwelling on ciliate distribution

Another characteristic of the distribution pattern is the influence of upwelling. Although this upwelling has long been recognized [START_REF] Middleton | Thermohaline structure and mixing in the region of Prydz Bay, Antarctica[END_REF], its influence on plankton distribution has not been investigated to date. The combined effects of the coast and upwelling could be responsible for Chl a intensification in surface waters at the coastal ends of the transects. Upwelling occurred in the same location in which low abundance of surface ciliates and some tintinnid species occurred. Because deep water had low ciliate abundance, we propose that this upwelling was responsible for the low abundance areas. L. naviculaefera and C. affinis/convallaria forma cristallina occurred in WW, and were the most abundant Southern Ocean endemic species in WW. Therefore, these two species might be more adaptive than other endemic species in WW. We speculate that these two species might be the original inhabitants in WW. When WW was brought to the surface by upwelling, these two species were able to survive and prosper, while others perished. Because the upwelling moved both upward and coastward, these two species occurred in SSW Chl a > 3 but not in SSW Chl a < 3 .

Upwelling in Prydz Bay is a part of the "Antarctic divergence" which generally occurs at 60°S-65°S, although it shows great variations with time [START_REF] Mann | Dynamics of marine ecosystems: biological-physical interactions in the oceans[END_REF][START_REF] Lin | An analysis on the upwelling of the modified circumpolar deep water over the shelf region of Prydz Bay in the summer of 2013[END_REF]. Thus, the influence of upwelling on ciliate distribution in our study might have occurred in other Antarctic waters in which upwelling was found.

Prediction of spatial and temporal variations of austral tintinnid assemblage

Because different water masses inhabited different indigenous assemblages, we predicted tintinnid assemblages in other areas and times according to the spatial and temporal variations of water masses in the Southern Ocean. The sandwich structures of SSW, WW, and CDW stretched from waters near Prydz Bay northward until Antarctic Circumpolar Current (ACC) mixed the water layers. Therefore, we speculate that the distributions of austral tintinnid assemblages were similar in waters from our study area to those of ACC.

According to data recorded at the Chinese Antarctic Station of Zhongshan, air temperature was the highest in January (http://polar .china re.gov.cn/meteo /). Our study was conducted at the end of austral summer, when WW was at its minimum range. As time went on, SSW gradually disappeared and was replaced by WW; thus, WW occupied more of the surface water. Therefore, in winter, the abundance of the dominant species, Salpingella sp. We do not have species information regarding aloricate ciliates; however, the aloricate assemblage might utilize the same mechanism as tintinnid species because of their close phylogenetic relationship. Although there have been no reports on other plankton differences in different water masses in the Antarctic, this mechanism might also exist in phytoplankton and zooplankton.

In summary, the results of this study supported our hypothesis that different water masses had ciliate assemblages with different characteristics and indigenous tintinnids. Tintinnid vertical distribution could be determined by whether they were indigenous to water masses. Our results will help predict the spatial and temporal variations in assemblages of ciliates and other plankton according to the dynamics of water masses in Antarctic waters.

Fig. 1

 1 Fig. 1 Sampling stations in open waters near Prydz Bay during the late austral summer. Red star Chinese Antarctic station of Zhongshan; Red circle Australian Antarctic station of Davis

Fig. 2

 2 Fig. 2 Vertical distributions of temperature (T, °C), salinity (S), in vivo Chlorophyll a (Chl a) fluorescence along the transects (P2, P3, P4, P5) in open waters near Prydz Bay. Dot symbols indicate both the depths at which ciliates were sampled and the water masses where

Fig. 3

 3 Fig. 3 Vertical distributions of abundance (ind. L -1 ) of aloricate ciliate, tintinnid, and total ciliate along the transects (P2, P3, P4, P5) in open waters near Prydz Bay. Dot symbols indicate both the depths at which ciliates were sampled and the water masses where samples were collected (purple circle SSW; red diamond WW; green square

Fig. 4

 4 Fig. 4 Vertical distributions of biomass (µg C L -1 ) of aloricate, tintinnid, and total ciliate along the transects (P2, P3, P4, P5) in open waters near Prydz Bay. Dot symbols indicate both the depths at which ciliates were sampled and the water masses where samples were col-

  classified C. drygalskii and C. convallaria as C. affinis, and C. gaussi as Laackmanniella (L. naviculaefera = L. prolongata).

Fig. 5

 5 Fig. 5 Abundance of ciliate (a) and proportions of aloricate ciliate and tintinnid (b), proportions of different-sized aloricate ciliate in total aloricate ciliate abundance (c), abundances (d) and proportions (e) of different tintinnid species in different water masses. Southern

Fig. 6

 6 Fig. 6 Photos of some tintinnid species in open waters near Prydz Bay. a Laackmanniella naviculaefera, b Salpingella sp., c S. faurei, d Codonellopsis glacialis, e C. gaussi, f Cymatocylis affinis/conval-

Fig. 7

 7 Fig. 7 Vertical distribution of abundance (ind. L -1 ) of tintinnid species (Pattern 1) in open waters near Prydz Bay. Dot symbols indicate both the depth at which ciliates were sampled and the water masses where samples were collected (purple circle SSW; red diamond WW;

Fig. 8

 8 Fig. 8 Vertical distribution of abundance (ind. L -1 ) of tintinnid species (Pattern 2) in open waters near Prydz Bay. Dot symbols indicate both the depth at which ciliates were sampled and the water masses where samples were collected (purple circle SSW; red diamond WW;

Fig. 9

 9 Fig. 9 Vertical distribution of abundance (ind. L -1 ) of tintinnid species (Pattern 3) in open waters near Prydz Bay. Dot symbols indicate both the depth at which ciliates were sampled and the water masses where samples were collected (purple circle SSW; red diamond WW;

Table 2

 2 Tintinnid species' distribution in different water masses in open waters near Prydz Bay

  -3 , sampling points n = 27, WW Winter Water, sampling points n = 119, CDW Circumpolar Deep Water, sampling points n = 67, PB Prydz Bay, sampling points n = 264

	Species	Water masses
		SSW Chl a < 3		SSW Chl a > 3		WW		CDW		PB
		Abundance	OF	Abundance	OF	Abundance	OF	Abundance	OF	OF
	Codonellopsis gaussi 0.53 ± 1.78 (9)	7.23 2.76 ± 5.06 (18)	9.52 0.58 ± 2.75 (27)	4.52 0.05 ± 0.37 (3)	2.94 11.36
	C. glacialis	0.22 ± 0.93 (6)	4.82 1.75 ± 5.06 (24)	5.95 0.41 ± 2.99 (32)	3.01 0.00	0.00 7.20
	Cymatocylis affinis/	1.06 ± 3.00 (12)	8.43 6.75 ± 12.31 (63)	17.86 1.54 ± 3.06 (16)	12.95 0.01 ± 0.12 (1)	2.94 25.00
	convallaria forma
	cristallina
	C. affinis/convallaria 0.08 ± 0.45 (3)	2.41 0.11 ± 0.42 (2)	2.38 0.02 ± 0.13 (1)	0.60 0.01 ± 0.12 (1)	2.94 2.65
	C. affinis/convallaria	0.45 ± 1.43 (6)	6.02 0.41 ± 0.94 (3)	5.95 0.92 ± 1.92 (10)	9.94 0.01 ± 0.12 (1)	2.94 16.67
	forma drygalskii
	C. vanhoeffeni	0.20 ± 1.40 (10)	1.20 0.30 ± 1.55 (8)	1.19 0.55 ± 1.42 (8)	7.23 0.00	0.00 9.85
	C. antarctica	0.00	0.00 0.00	0.00 0.11 ± 0.69 (6)	1.20 0.03 ± 0.24 (2)	2.94 1.89
	Laackmanniella	4.97 ± 13.00 (72)	16.87 21.17 ± 28.88 (131)	25.00 3.34 ± 5.35 (32)	19.58 0.10 ± 0.55 (4)	8.82 39.02
	naviculaefera
	Salpingella acuminata 0.00	0.00 0.00	0.00 0.17 ± 0.73 (6)	3.01 0.19 ± 0.74 (4) 17.65 6.06
	S. costata	1.43 ± 8.99 (64)	3.61 0.00	0.00 3.81 ± 18.71 (201) 13.55 0.42 ± 1.69 (10) 20.59 20.83
	S. faurei	0.59 ± 2.42 (16)	6.02 0.45 ± 1.83 (9)	2.38 2.91 ± 9.99 (79)	10.54 0.29 ± 1.88 (15) 11.76 17.42
	Salpingella sp.	55.51 ± 80.26 (346) 43.37 174.98 ± 255.15	28.57 3.88 ± 11.30 (91) 12.05 0.87 ± 4.58 (36) 23.53 40.91
				(1014)
	Amphorides laack-	0.00	0.00 0.34 ± 0.87 (5)	1.19 0.55 ± 3.61 (33)	1.81 0.01 ± 0.12 (1)	2.94 3.03
	manni

to be indigenous species of this water mass. All the indigenous species form indigenous assemblages of this certain water mass. In this study, SSW indigenous assemblage was composed of Salpingella sp. and C. gaussi, WW indigenous assemblage included S. costata, S. faurei, C. affinis/convallaria forma drygalskii and C. vanhoeffeni, and there was no indigenous assemblage in CDW. The tintinnid assemblage in one certain water mass was a mixture of indigenous

Acknowledgements

We thank the 31st CHINARE Antarctic expedition for providing logistical support and environmental data. We thank Jeremy Kamen, MSc, from Liwen Bianji, Edanz Group China (www. liwen bianj i.cn/ac), for editing the English text of a previous version of this manuscript. This study was supported by the National Natural Science Foundation of China (NSFC 41576164); Chinese Polar Environment Comprehensive Investigation & Assessment Programmes (CHINARE2015-01-06 and CHINARE2015-01-05); the Scientific and Technological Innovation Project by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02); and the CNRS-NSFC Joint Research Projects Program (NSFC 41711530149).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical standards All procedures involving animals were performed in accordance with the ethical standards of the institutions at which the studies were conducted

Alder VA (1999) Tintinnoinea. In: Boltovskoy D (ed) South Atlantic zooplankton. Backhuys, Leiden, pp 321-384 Alder VA, Boltovskoy D (1991)


A Nouhi
email: nouhi@univ-tln.fr

H Hajjoul

R Redon

J P Gagné

S Mounier

Time-resolved Laser Fluorescence Spectroscopy of Organic Ligands by Europium : Fluorescence Quenching and Lifetime Properties

Keywords: Fluorescence quenching, Europium (III), Natural Organic Matter, Time-resolved laser-induced fluorescence spectroscopy, Parallel Factor Analysis, Lifetime Analysis

Time-resolved Laser Fluorescence Spectroscopy (TRLFS) has proved its usefulness in the fields of biophysics, life science and geochemistry to characterize the fluorescence probe molecule with its chemical environment. The purpose of this study is to demonstrate the applicability of this powerful technique combined with Steady-State (S-S) measurements. A multi-mode factor analysis, in particular CP/PARAFAC, was used to analyze the interaction between Europium (Eu) and Humic substances (HSs) extracted from Saint Lawrence Estuary in Canada. The Saint Lawrence system is a semi-enclosed water stream with connections to the Atlantic Ocean and is an excellent natural laboratory.

CP/PARAFAC applied to fluorescence S-S data allows introspecting ligandsmetal interactions and the one-site 1:1 modeling gives information about the stability constants. From the spectral signatures and decay lifetimes data given by TRLFS, one can deduce the fluorescence quenching which modifies the fluorescence and discuss its mechanisms. Results indicated a relatively strong binding ability between europium and humic substances samples (Log K value varies from 3.38 to 5.08 at pH 7.00). Using the Stern-Volmer plot, it has been concluded that static and dynamic quenching takes places in the case of salicylic acid and europium interaction while for HSs interaction only a static quenching

Introduction

Improved insight on the interactions between natural dissolved organic matter with lanthanides and actinides has been widely studied [START_REF] Kim | Complexation of americium (III) with humic acid[END_REF]; [START_REF] Moulin | On the use of laser-induced time-resolved spectrofluorometry for interaction studies between organic matter and actinides: application to curium[END_REF]; [START_REF] Kim | Complexation of trivalent actinide ions (Am 3+ , Cm 3+ ) with humic acid: a comparison of different experimental methods[END_REF]; [START_REF] Panak | A study of ternary complexes of Cm (III) with humic acid and hydroxide or carbonate in neutral pH range by time-resolved laser fluorescence spectroscopy[END_REF]; [START_REF] Czerwinski | Complexation of trivalent actinide ions (Am 3+ , Cm 3+ ) with humic acid: the effect of ionic strength[END_REF]; [START_REF] Tiseanu | Time-resolved fluorescence spectroscopy of fulvic acid and fulvic acid complexed with Eu 3+ : A comparative study[END_REF]; [START_REF] Morgenstern | The formation of mixed-hydroxo complexes of Cm (III) and Am III) with humic acid in the neutral pH range[END_REF]; [START_REF] Monsallier | Spectroscopic study (TRLFS and EXAFS) of the kinetics of An (III)/Ln (III) humate interaction[END_REF]; [START_REF] Schmeide | Plutonium (III) complexation by humic substances studied by X-ray absorption fine structure spectroscopy[END_REF]; [START_REF] Brevet | Spectral and temporal luminescent properties of Eu (III) in humic substance solutions from different origins[END_REF]; [START_REF] Jain | Spectroscopic investigation on europium complexation with humic acid and its model compounds[END_REF]; [START_REF] Lukman | Speciation of Eu 3+ bound to humic substances by time-resolved laser fluorescence spectroscopy (TRLFS) and parallel factor analysis (PARAFAC)[END_REF]; [START_REF] Pacold | Direct observation of 4f intrashell excitation in luminescent eu complexes by time-resolved X-ray absorption near edge spectroscopy[END_REF]. Indeed, it is of paramount importance to better understand transport and toxicity pathways of metal ions as well as creating efficient remediation techniques for contaminated environments [START_REF] Hiraide | Heavy metals complexed with humic substances in fresh water[END_REF]; [START_REF] Dube | Adsorption and migration of heavy metals in soil[END_REF]; [START_REF] Choppin | Actinide speciation in the environment[END_REF]; [START_REF] Sachs | Influence of humic acids on the actinide migration in the environment: suitable humic acid model substances and their application in studies with uranium-A review[END_REF]; [START_REF] Cobelo-García | COST action TD1407: Network on technology-critical elements (NOTICE)from environmental processes to human health threats[END_REF]. Various spectroscopic techniques are used for these purposes.

Fluorescence spectroscopy in S-S measurements provides quantitative and qualitative information. However, more data such as fluorescence lifetime measurements could be gathered using TRLFS. In fact, TRLFS is a convenient technique when dealing with different elements in the same sample because it can offer the spectral and temporal resolution together with its high sensitivity. The discriminating capability of TRLFS relies on the fact that the different chemical species between organic matter and a fluorescent metal ion can have different decay lifetimes and spectral shapes which is adequate for the study of such a complex system [START_REF] Collins | Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides[END_REF]. The greatest advantages of TRLFS are its relatively low detection limit and good discriminating capability for different chemical species.

An assessment of the influence of HSs on the mobility of Eu in the environment is necessary and has to include knowledge of stability constants for the formation of Ln(III)-HSs complexes. In this work, complexation of Eu 3+ by Humic Acids (HA) and Fulvic Acids (FA) from St. Lawrence Estuary (Canada) was first studied by fluorescence quenching using Excitation Emission Matrices of fluorescence (EEMs) with a large range of europium concentration. Metal additions were performed using logarithmic increments to cover a wide range of concentration from 2.2×10 -7 to 1.5×10 -3 M. The obtained data was treated by CP/PARAFAC to extract the different fluorescent components using a MATLAB R home-made program [START_REF] Bro | tutorial and applications[END_REF]; [START_REF] Luciani | A simple correction method of inner filter effects affecting FEEM and its application to the PARAFAC decomposition[END_REF]. Then a fitting based on the onesite model (1 : 1) [START_REF] Ryan | Copper (II) complexing capacities of natural waters by fluorescence quenching[END_REF] was done on the CP/PARAFAC components to determine their stability constants. TRLFS is a complementary technique to determine the composition of the mixtures by measuring the fluorescence lifetimes and see its variation with the metal addition. Fluorescence properties of Eu(III) with Salicylic Acid (SA) and HSs complexes were studied from Eu(III) site [START_REF] Irving | The formation constants of europium (III)-salicylate complexes and their extraction into isoamyl alcohol[END_REF]; [START_REF] Hasegawa | Complexation of lanthanoid (III) with substituted benzoic or phenylacetic acids and extraction of these acids[END_REF] 2017) and also from the ligand site, but only for SA [START_REF] Aoyagi | Fluorescence characteristics of complex formation of Europium (III)-Salicylate[END_REF]. Analysis of the literature has shown that data of humic substances complexes with Eu(III) from the ligand site is still lacking. As a matter of fact, it raises some interesting questions that have not been discussed before. Therefore, with this study, we wanted to investigate how these interactions occur focusing on the HSs fluorescence emission. Analysis of fluorescence lifetimes and Stern-Volmer plots [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF] will give reliable information on the quenching process that takes place. This line of study is important to settle a general relationship between organic matter properties from St Lawrence Estuary and static/dynamic quenching contributions.

Experimental setup

Sample preparation

The complexation of Eu(III) by SA was first studied in order to calibrate our assays and compare our results with literature [START_REF] Aoyagi | Fluorescence characteristics of complex formation of Europium (III)-Salicylate[END_REF]. Indeed, the compositions of HSs are complex and partly unknown. The HSs are composed of a complex mixture of organic and organomineral compounds and can differ depending on its type. Therefore, SA provides a carboxyl and hydroxyl group that mimic the important functionalities of HSs [START_REF] Stevenson | Humus chemistry: genesis, composition, reactions[END_REF] and form strong complexes with Eu(III) [START_REF] Kuke | Intramolecular deactivation processes in complexes of salicylic acid or glycolic acid with Eu (III)[END_REF].

Salicylic acid (NormaPur R , Prolabo) was used as received. Its aqueous solution was prepared at a concentration of 5 × 10 -4 M with UHQ water. HA and FA were obtained from the St. Lawrence Estuary [START_REF] Tremblay | Organic matter distribution and reactivity in the waters of a large estuarine system[END_REF] following the IHSS protocol [START_REF] Swift | Method for extraction of IHSS soil fulvic and humic acids[END_REF].

Europium standard (1,000 mg.L -1 in 2% HNO 3 , SPEX CertiPrep) was used as a quencher without further purification.

Twelve solutions with different concentrations of Eu(III) were prepared by mixing the stock solutions in a 1 cm quartz cell. The final concentrations of SA, HA and FA were kept constant at respectively 5 × 10 -5 M, 11 and 16 mg of matter per liter. Eu(III) was added using logarithmic increments of concentrations from 2.2×10 -7 M to 1.5×10 -3 M. Every sample was allowed to equilibrate for at least 15 min and no precipitation was observed at higher europium concentration. The pH of the samples was adjusted to 7.00 ± 0.09 using 1 mL of HEPES buffer (0.1 M). HEPES was used to maintain the pH constant and avoid pH adjustment for each sample. Preliminary measurements without metal additions were done to verify that there is no interaction between the fluorescent ligands and the HEPES buffer. The results did not show any variation of the fluorescence intensity. Thus, no interaction between the HEPES buffer and ligands was observed. A total volume of 3 mL was achieved by adding UHQ water.

Necessary pH adjustments of metal solutions were done with NaOH (1M) and HCl (1 M) and the changes in the volume were taken into account.

Fluorescence and lifetime measurements

Two series of experiments were done on the prepared samples. In order to perform S-S measurements, a spectrophotometer (HITACHI F4500) equipped with a Xenon lamp (450 W) and connected to a computer was used. Twelve individual EEMs gathered from S-S measurements of SA, HA, and FA were collected in fluorescence quenching experiments. The excitation wavelength range was from 250 nm to 450 nm with a 10 nm slit and 5 nm excitation step. The corresponding emission wavelengths were collected at 5 nm step between 300 nm and 700 nm and 10nm slit. The scan speed was set at 2,400 nm.min -1 and the photomultiplier tension was at 700 V.

TRLFS data was carried out using a pulsed Nd:YAG laser (Quanta-Ray INDI, Spectra Physics) and performed at room temperature (294 K). The Nd:YAG source provides an excitation with a pulse width of 5 ns and a repetition rate of 20 Hz. The excitation source for TRLFS was the third harmonic generation (355 nm) and the fluorescence emission was collected at 90 degrees through a bundle made of 16 optical fibers of 200 µm into the entrance slit of a Czerny-Turner spectrograph (TRIAX 180, Horiba Jobin-Yvon). The latter has three UV-VIS gratings (300 g/mm, 900 g/mm and 2,400 g/mm). The spectral resolutions were respectively 0.27 nm, 0.09 nm and 0.034 nm. The second grating was used for these experiments.

The fluorescence signal was detected by an intensified couple charged device camera (ICCD 300V, Horiba Jobin-Yvon). For the time-resolved measurements, the initial timing of the fluorescence signal was varied by changing the delay of the trigger through a pulse generator (DG535, Stanford Research System). In order to increase the accuracy of the measurements, each laser pulse was corrected in energy fluctuation by splitting the laser beam through a pyroelectric sensor (PE10, Ophir Electronics). The power of pulse was constant within around 3-5 µJ pulse during the acquisition. Seven hundred spectra were accumulated at each time step of 0.2 ns and a time delay of 200 ns with a 35 ns gate width. These parameters were set to measure the fluorescence focusing on the ligand fluorescence emission.

Every mixture was allowed to equilibrate for at least 10 min and bubbled by a nitrogen flux to avoid photobleaching.

Data Treatment

For S-S data treatment, CP/PARAFAC algorithm was used to determine the contribution of components to the EEMs by separating independent sources after elimination of scattering phenomena [START_REF] Bro | tutorial and applications[END_REF]. The first step of decomposition consists of cleaning all EEMs from the diffusion signals (Rayleigh and Raman) by cutting the Rayleigh diffusion bands (20 nm) and applying the Zepp procedure [START_REF] Zepp | Dissolved organic fluorophores in southeastern US coastal waters: correction method for eliminating rayleigh and raman scattering peaks in excitation-emission matrices[END_REF]. No inner effect was encountered because chosen concentrations were in the linear zone of fluorescence. Dilution curves were done to determine these value (data not shown). Thus, no numerical correction was applied to the EEMs. The estimation of the number of components is particularly critical because users have to test a range of model to evaluate the best number of components. In this study, it was defined by evaluating the COR-CONDIA score. Decomposition investigation was done from 1 to 4 components and the number of component giving a CORCONDIA score higher than 60% was selected as optimal model. Iterations and number of runs were implemented until the algorithm converged.

The optimization between experimental and calculated fluorescence using onesite model with one ligand [START_REF] Ryan | Copper (II) complexing capacities of natural waters by fluorescence quenching[END_REF] was executed by evaluating the bias errors. The bias is equal to the sum of the square of the subtraction between the experimental fluorescence intensity and the calculated fluorescence intensity.

Data obtained with TRLFS were analyzed using a home-made MATLAB R program. The evaluation of the best fitting of the integrated fluorescence intensity was done by time deconvolution to determine fluorescence amplitudes and fluorescence lifetimes. Supposing a system excited by an infinitely sharp pulse of light (δ-function) and assuming the response to be linear, the measured fluorescence decay is given by the convolution integral :

y(n) = h(n) ⊗ IRF (1)
where h(n) is the intrinsic fluorescence decay and IRF is the Instrument Response Function. Experimentally, the IRF is gathered by measuring only the solvent diffusion.

The estimation of h(n) was achieved by time deconvolution in order to obtain the amplitudes of each fluorophore and lifetimes. Assuming a multi-exponential decay as follow :

h(n) = i α i exp -t/τi (2)
The amplitudes α i and the lifetimes τ i can be considered to be adjustable parameters. They are calculated employing Simplex algorithm minimizing the error of the equation :

[y(n) -y (n)] = ε(n) (3)
It is known that quenching can occur by different mechanisms. Dynamic quenching occurs when the excited-state fluorophore is deactivated upon contact with other molecules in solutions due to physical collision [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]. In this case, the fluorophore returned to the ground-state during a diffusive encounter with the quencher. The molecules are not chemically modified in the process and can be shown by the Stern-Volmer equation 4.

I 0 I Q = 1 + K SV [Q] = 1 + k q τ Q [Q] ( 4
)
where I 0 and I Q are the fluorescence intensities in the absence and presence of quencher respectively, τ Q is the fluorescence lifetime in the presence of metal, K SV is the Stern-Volmer quenching constant and k q is the bimolecular quenching constant.

If dynamic quenching takes place, the intensity of fluorescence and lifetime follows the relationship:

I 0 I Q = τ 0 τ Q (5)
In this expression τ 0 is the fluorescence lifetime in the absence of quencher. It is noteworthy that dynamic quenching is a time-dependent process.

Aside from collisional quenching, fluorescence quenching can occur due to a complex formation. Fluorophores can form non-fluorescent complexes with quenchers [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]. This process is referred to as static quenching since it occurs at ground-state and does not rely on diffusion or molecular collisions.

The dependence of the fluorescence intensity upon quencher concentration is given by the following expression

I 0 I Q = 1 + K S [Q] (6)
where K S is the association constant of the complex.

Note that the I 0 /I Q on [Q] is linear, which is identical to dynamic quenching.

Hence, the measurement of fluorescence lifetimes is the most ultimate method to distinguish static and dynamic quenching.

Results & Discussions

Steady-State and fluorescence quenching results

The CP/PARAFAC decomposition of SA-Eu 3+ quenching experiment shows that two components are obtained in this interaction (Fig. 1). The first component (λ ex /λ em )=295/410 nm shows a fluorescence intensity and is obviously the excitation/emission wavelength of SA Tauler et al. (1998) whereas, the second component does not show any fluorescence intensity variation (Fig. 2. A) nor fluorescence peak. Thus, the peak could be a noise factor and was not considered in the following discussion.

The relative contributions of each components of SA-Eu 3+ interaction are shown in Fig. 2. A. indicating the quenching of SA depending on the Eu 3+ concentration. The corresponding conditional stability constant (K value) for the first component is reported in Table 1 and was estimated as Log K SA = 3.38 based on the 1 : 1 complexation assumption between metal and ligand. This value is higher than Log K=2.08 ± 0.02 obtained by [START_REF] Aoyagi | Fluorescence characteristics of complex formation of Europium (III)-Salicylate[END_REF] at pH 4 from the slope of the intensity SternVolmer plot and higher than Log K=2.02±0.05 obtained by [START_REF] Hasegawa | Complexation of lanthanoid (III) with substituted benzoic or phenylacetic acids and extraction of these acids[END_REF] at pH 6 from the glass electrode method.

The stability constant of SA-Eu 3+ found in this study is consistent considering that the stability constant should increase when pH increases.

As for the extracted humic substances, the presence of abundant aromatic structures in organic matter provides reasonably good fluorescence characteristics. CP/PARAFAC decomposition of the EEMs obtained in the case of HA (1996).

The relative contribution of this component to the EEMs during the quenching experiment is shown in Fig. 2.B. and the corresponding stability constant is Log K HA,C1 = 4.43 (Table 1). The second component (λ ex /λ em )=270/505 nm shows a longer emission wavelength characteristic of a higher conjugated structure usually found in soils. This component behaves similarly to component 1 (Fig. 2005) reported a stability constant at pH 5 equal to 6.4±0.2 for FA-Eu(III) interaction using synchronous fluorescence spectroscopy. This value is higher than our stability constant. This data is in good agreement with an other published work : Log K=6.00±0. 10 Bidoglio et al. (1991). Furthermore, [START_REF] Lead | Europium binding by fulvic acids[END_REF] reported only one stability constant at a pH between 2.5 and 3.5 : Log K=2.36±0.13 much like [START_REF] Wei | Evaluation of the interactions between water extractable soil organic matter and metal cations (Cu (II), Eu (III)) using excitation-emission matrix combined with parallel factor analysis[END_REF] :

Log K=5.84. The values of the stability constants found in the literature are higher due to the fact that the fulvic acids studied are mainly derived from soils or rivers and may have greater complexing capacities than the fulvic acids used in this study.

TRLFS results

As mentioned above in section 2.3, the quenching process cannot be assessed using steady-state measurements. Therefore, fluorescence lifetime measurements using TRLFS are the most ultimate method to distinguish whether static, dynamic or both quenching processes occur based on Stern-Volmer plots [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF].

After S-S quenching experiments, eight samples out of twelve from the quenching curves were selected for lifetime measurement, because of the time required for each measure. The first sample does not contain any concentration of Eu 3+

while the seven other samples contain an increasing volume of it at a concentration varying from 2.2 × 10 -5 M to 2.2 × 10 -3 M.

For the interaction between SA-Eu 3+ , the decay was found to be monoexponential. The average lifetime τ a recorded was 2.97±0.2 ns. In Fig. 3.A (2006). On the other hand, [START_REF] Aoyagi | Fluorescence characteristics of complex formation of Europium (III)-Salicylate[END_REF] found a fluorescence lifetime equal to 2.02±0.01 ns and a static quenching occurring for SA-Eu 3+ interaction.

This discrepancy could be due to the pH value as they worked at pH 4.

For HA-Eu 3+ interaction, CP/PARAFAC decomposition gives three components while the time deconvolution suggests a bi-exponential decay and two lifetimes denoted by exponent indexes τ A and τ B . One very short, indicated by τ A 0 /τ A Q in Fig. 3. B and the second one, specified by τ B 0 /τ B Q , longer than the first one (11.33 ns). The two fluorescence lifetimes turned out to be independent on total concentration of Eu 3+ . This suggests a static quenching. The dependence of I 0 /I Q of component 1 and component 2 on [Eu 3+ ] is therefore non-linear.

In the case of static and dynamic quenching, the characteristic feature of the Stern-Volmer plot is an upward curvature, concave towards the y-axis. The concave pattern of the Stern-Volmer plot is interpreted as the manifestation of fluorophores that are not accessible for quenching [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]. Thus, the data cannot be accurately fitted using the linear Stern-Volmer equation 4 in this situation. However, [START_REF] Reiller | Bi-exponential decay of Eu (III) complexed by suwannee river humic substances: spectroscopic evidence of two different excited species[END_REF] quenching. The previous study ( Tiseanu et al. (1998)) also reported a static quenching in the case of FA-Eu(III) interaction.

Conclusion

In this work, the interactions of salicylic acid, humic acid and fulvic acid with Eu 3+ were studied by steady-state and time-resolved laser-induced fluorescence spectroscopy focusing on the ligand fluorescence emission using a nanosecond pulsed laser. Lifetime analysis using Stern-Volmer plot showed that part of dynamic quenching process or both static and dynamic quenching contributed to the global quenching of fluorescence in the case of salicylic acid-Eu 3+ interaction. Data obtained is not exactly in agreement with literature [START_REF] Aoyagi | Fluorescence characteristics of complex formation of Europium (III)-Salicylate[END_REF] since they found a static quenching in the case of SA-Eu(III) interaction. This discrepancy with SA should be investigated at different pH to define precisely the quenching process dependence on pH. Static quenching process took place for humic acid and fulvic acid extracted from St. Lawrence Estuary. [START_REF] Tiseanu | Time-resolved fluorescence spectroscopy of fulvic acid and fulvic acid complexed with Eu 3+ : A comparative study[END_REF] also found a static quenching in the case of FA-Eu(III) interaction, which is in a good agreement with our study. The stability constants of all the interactions studied were calculated using the one-site 1 : 1 model and are in concordance with literature for SA and HA. For FA, we found constants slightly lower than those in the literature which may be due to the origin of FA. It is noteworthy that TRLFS successfully discriminates the fluorescence quenching process that takes place and gives original lifetimes of HSs and constant of complexation with Eu(III). Further application can be extended to quantitatively analyze other lanthanides and actinides.

  ; Tiseanu et al. (1998); Hasegawa et al. (1990); Toraishi et al. (2005); Brevet et al. (2009); Jain et al. (2009); Kuke et al. (2010); Lukman et al. (2012); Saito et al. (

  2.B) and has the higher stability constant (Log K HA,C2 = 5.08). The third component (λ ex /λ em )=250/395 nm is more relative to the protein-like structures and presents a weaker quenching effect. This means that the related fluorescent chemical functions are less affected by europium or less complexing. This is demonstrated by the lowest stability constant value Log K HA,C3 = 3.63. In comparison,[START_REF] Wei | Evaluation of the interactions between water extractable soil organic matter and metal cations (Cu (II), Eu (III)) using excitation-emission matrix combined with parallel factor analysis[END_REF] reported two stability constants using synchronous fluorescence spectroscopy. Their constants at pH 6 are higher than found in our study : Log K 1 =5.51 and Log K 2 =5.60. However, the value reported by Hahn et al. (2017) using ultrafiltration and CE-ICP-MS at pH=5.00±0.05 seems to be also higher than ours : Log K=6.36±0.02 and Log K=6.1±0.1. This work clearly shows that several fluorescent sites complex the europium in this particular HA from St. Lawrence estuary. More investigation should be done to compare the data, in particular for the pH conditions.For the FA, two components were found by CP/PARAFAC showing an interesting fluorescence quenching (Fig.2. C.) The first component (Fig.1. C) located at (λ ex /λ em )=310/415 nm is generally another marine humic-like component and is designated as peak M[START_REF] Coble | Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy[END_REF]. The second component (λ ex /λ em )=265/475 nm is larger and is designated as peak A[START_REF] Coble | Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy[END_REF] with a slight red shift in emission. The corresponding fluorescence intensities are given in Fig. 2. C. showing a homogeneous and similar behavior of both components. This is demonstrated by the values of the stability constants respectively Log K F A,C1 = 4.42 and Log K F A,C2 = 4.43 of the first and second component. The study of Chung et al. (

Figure 1 :

 1 Figure 1: Components obtained for A) Salicylic acid-Eu 3+ B) Humic acid-Eu 3+ and C) Fulvic acid-Eu 3+ interaction after CP/PARAFAC decomposition

  observed a bi-exponential decay for the luminescence of Eu(III)-HSs complexes and reported a deactivation of at least two different excited-states from Eu(III) site. The reported values of fluorescence lifetime cannot be compared to our study since they measured the fluorescence lifetime values from Eu(III) site.The intensity ratios of the fulvic acid with Eu 3+ in Fig.3. C. seem to be linear except the penultimate point which could be attributed to an error in the measurement. According to[START_REF] Tiseanu | Time-resolved fluorescence spectroscopy of fulvic acid and fulvic acid complexed with Eu 3+ : A comparative study[END_REF], the existence of a tri-modal decay time distribution with time values centered around 0.7, 3 and 11 ns was

Figure 3 :

 3 Figure 3: Stern-Volmer plot showing the intensities ratio and the fluorescence lifetimes ratio of A) Salicylic acid-Eu 3+ , B) Humic acid-Eu 3+ and C) Fulvic acid-Eu 3+ interactions

Table 1 :

 1 Stability constants and average fluorescence lifetimes of SA and HSs complexed with 3+ suggests the presence of three components (Fig. 1.B). The first component in the UV range is located at (λ ex /λ em )=270/445 nm, and has been designated as peak A. It was attributed to humic substances Coble

	Eu 3+
		Log K SA Log K .,C1 Log K .,C2 Log K .,C3 τ A a ns τ B a ns
	SA	3.38	-	-	-	2.97	-
	HA	-	4.43	5.08	3.63	0.61	11.33
	FA	-	4.42	4.43	-	0.73	12.54
	interaction with Eu