Haibo Li

Chaofeng Wang

Chen Liang

Yuan Zhao
email: yuanzhao@qdio.ac.cn

Wuchang Zhang
email: wuchangzhang@qdio.ac.cn

Gérald Grégorie

Tian Xiao

Diversity and distribution of tintinnid ciliates along salinity gradient in the Pearl River Estuary in southern China

Tintinnid community structure in the Pearl River Estuary were investigated 6 times (10

Introduction

Estuaries are important to humans and marine lives. Estuarine environment is characterized by strong interaction of freshwater outflow and seawater intrusion. The mixture of seawater and freshwater present challenges to the physiology of the organisms. Therefore, estuaries support unique communities of organisms that live at the margin of the sea [START_REF] Elliott | The need for definitions in understanding estuaries[END_REF][START_REF] Mclusky | The Estuarine Ecosystem -Ecology, Threats and Management[END_REF]. The variability of environmental features, especially salinity, is the main factor controlling the plankton distribution in an estuary [START_REF] Laprise | Environmental variability as a factor controlling spatial patterns in distribution and species diversity of zooplankton in the St. Lawrence Estuary[END_REF]. Estuarine zooplankton tend to occupy relatively welldefined salinity ranges in relation to their own osmoregulatory abilities [START_REF] Modéran | Zooplankton community structure in a highly turbid environment (Charente estuary, France): spatio-temporal patterns and environmental control[END_REF]. Species composition of both phytoplankton and zooplankton varied along salinity gradient in estuarine areas (Marshall andAlden, 1990, 1993;[START_REF] Sautour | Comparative spring distribution of zooplankton in three macrotidal European estuaries[END_REF][START_REF] Tackx | Zooplankton in the Schelde estuary, Belgium and The Netherlands. Spatial and temporal patterns[END_REF][START_REF] Calliari | Biomass and composition of the phytoplankton in the Río de la Plata: large-scale distribution and relationship with environmental variables during a spring cruise[END_REF][START_REF] Islam | Spatial distribution and trophic ecology of dominant copepods associated with turbidity maximum along the salinity gradient in a highly embayed estuarine system in Ariake Sea, Japan[END_REF][START_REF] Muylaert | Changes in phytoplankton diversity and community composition along the salinity gradient of the Schelde estuary (Belgium/ The Netherlands)[END_REF]. Accordingly, different zooplankton and phytoplankton communities were divided along salinity gradient in the estuarine waters (Marshall andAlden, 1990, 1993;[START_REF] Huang | Species diversity and distribution for phytoplankton of the Pearl River estuary during rainy and dry seasons[END_REF][START_REF] Tan | Seasonal variation in zooplankton[END_REF][START_REF] Muylaert | Changes in phytoplankton diversity and community composition along the salinity gradient of the Schelde estuary (Belgium/ The Netherlands)[END_REF][START_REF] Modéran | Zooplankton community structure in a highly turbid environment (Charente estuary, France): spatio-temporal patterns and environmental control[END_REF].

Tintinnids (Ciliophora: Spirotrichea) are planktonic ciliates with durable lorica [START_REF] Lynn | Ciliated Protozoa: Characterization, Classification, and Guide to the Literature[END_REF], that could be found in marine habitats all over the world. According to tintinnid genera occurrence in the global ocean, tintinnids were divided into five biogeographical patterns 34.1. Our aims were to 1) find out whether there are freshwater or brackish tintinnid species, 2) determine the trends of tintinnid species richness and abundance from the freshwater to seawater, 3) understanding the mixing of the different tintinnid communities. 41,36,26,29 and 30 stations, respectively (Fig. 1). Surfer (Version 12, Golden Software Inc., USA) was used to present the sampling stations.

Materials and methods

Surface

At each station, 80 L of surface water were taken with a bucket while the ship was moving, and gently filtered through a 10 μm meshsize net. The samples (ca. 150 cm 3 ) in the cod end of the net were transferred into bottles and immediately fixed with Lugol's solution (1% final concentration). Surface water temperature (°C) and salinity (measured using the Practical Salinity Scale) were determined using a portable water quality analyzer YSI EC300A (Professional Plus, USA).

In the laboratory, two subsamples (each 25 cm 3 ) from each original (cosmopolitan, neritic, warm-water, boreal, and austral) in genera level [START_REF] Pierce | Global biogeography of marine tintinnids[END_REF][START_REF] Dolan | Diversity and distributions of tintinnid ciliates[END_REF]. Tintinnids mainly occur in marine environments, but also in freshwaters [START_REF] Kofoid | A Conspectus of the Marine Freshwater Ciliata Belonging to the Suborder Tintinnoinea, with Descriptions of New Species Principally from the Agassiz Expedition to the Eastern Tropical Pacific 1904-1905[END_REF][START_REF] Nie | Notes on three new species of fresh-water Tintinnoidea[END_REF][START_REF] Chiang | Notes on the freshwater Tintinnoinea from Jiangsu and Anhui provinces[END_REF][START_REF] Petz | Morphogenesis in some freshwater tintinnids (Ciliophora, Oligotrichida)[END_REF] and brackish waters [START_REF] Sniezek | Nolaclusilis hudsonicus n. sp (Nolaclusiliidae n. fam.) a bilaterally symmetrical tintinnine ciliate from the lower Hudson River Estuary[END_REF][START_REF] Snyder | Nolaclusilis bicornis n. g., n. sp. (Tintinnina, Tintinnidiidae): a tintinnine ciliate with novel lorica and cell morphology from the composition and grazing impact on phytoplankton standing stock in the Pearl River Estuary, China[END_REF][START_REF] Smith | Dartintinnus alderae n. g., n. sp., a brackish water tintinnid (Ciliophora, Spirotrichea) with dual-ended lorica collapsibility[END_REF]. Most studies of tintinnids in brackish waters mainly reported the occurrence salinity in limited sites (Balkis, 2004;Urrutxurtu, 2004). There was no report on those tintinnids distribution variations along salinity gradient around the world. In this case, we are not sure whether those species reported in brackish waters were brackish species or just expatriated from their indigenous areas.

As the l argest e stuary i n southern China, the Pearl River E stuary is a dynamic complex e stuary with 8 river i nlets o n i ts western boundary a nd h as l ots o f f reshwater i nput channels f rom the Pearl River a nd i ts b ranches (Wong e t a l., 2004). The mean a nnual discharge o f the Pearl River i s a bout 1 0,000 m 3 s -1 a nd the discharge is a bout f our times g reater i n the rainy season (April to S eptember) than i n the dry season (October to March) (Dong e t a l., 2004). The mean discharges o f the Pearl River E stuary a re a bout 8000, 1 9,000 and 6000 m 3 s -1 i n March, J une a nd October, respectively (Zheng e t al., 2012). I n the Pearl River E stuary, p hytoplankton was divided into e stuarine, mixed water a nd coastal communities ( Huang e t a l., 2004), the z ooplankton was divided i nto e stuarine, tropical n eritic, warm-temperate n earshore a nd p elagic communities (Tan e t a l., 2004). I n this study, we i nvestigated tintinnid community variation along transects where salinity varied f rom 0 .1 to and 29. respectively. In dry season, temperatures fell in the range of 27.5-28.8 °C and 26.0-28.2 °C at Transects A and B, respectively. In transitional season, temperatures were the lowest, fell in ranges of respectively (Fig. 2).

Salinity preference of tintinnid species

Totally 43 tintinnid species belonging to 15 genera were identified at 6 sampling transects. Tintinnopsis (23 species) was the most diverse genus followed by Codonellopsis (3 species), Tintinnidium (2 species), Leprotintinnus (2 species), Amphorides (2 species), and Eutintinnus (2 species). Other genera each contained one species (Table 1).

In dry season, 32 species belonging to 12 genera occurred (30 species in 11 genera and 26 species in 8 genera at Transects A and B, respectively). In rainy season, 23 species belonging to 8 genera occurred (16 species in 6 genera and 16 species in 4 genera at Transects C and D, respectively). In transitional season, 26 species belonging to 8 genera occurred (19 species in 6 genera and 22 species in 7 genera at Transects C and D, respectively). For individual species, 11 species occurred in all 3 seasons. While 16 species occurred in only 1 season (Table 1).

Salinity ranges were determined for 32 species occurred in ≥5 stations at all transects. These species were divided into freshwater species, brackish species and marine species according to their preferred salinity (salinity with the highest abundance occurred) and abundance variations along salinity gradient (Figs. 3-5 and S1-S3, Table 1 andS1). Eleven species occurred in < 5 stations were occasional species (Fig. S4). We could not assign salinity preference because of their low abundance and occurrence frequencies (Table 1 andS1).

Freshwater species

Three species (Tintinnopsis mayeri, T. urnula and Tintinnidium fluviatile) with the highest abundance in oligohaline (salinity < 3) zone were freshwater species. T. mayeri was the only freshwater species that appeared at all 6 transects in 3 seasons. T. urnula occurred at 5 transects in 3 seasons and T. fluviatile was identified at 3 transects in dry and Fig. 2. Variation of salinity and temperature (°C) at each transect in the Pearl River Estuary. sample were settled in two Utermöhl counting chambers (25 cm 3 ) for at least 24 h. Tintinnid were identified and counted using an inverted microscope (Olympus IX 71) at magnification of 100 × or 400 × , photo of each tintinnid species was taken by a software (Olympus cellSens Standard 1.17) connecting the microscope and computer. Tintinnid species were determined according to lorica morphology and size based on literature (Kofoid andCampbell, 1929, 1939;[START_REF] Hada | The fauna of Akkeshi Bay IV. The pelagic ciliata[END_REF][START_REF] Hada | Studies on the Tintinnoinea from the western tropical Pacific[END_REF][START_REF] Bakker | Tintinnida from coastal waters of the S.W.-Netherlands I. The genus Tintinnopsis stein[END_REF][START_REF] Yoo | Taxonomic studies on tintinnids (Protozoa: Ciliata) in Korean coastal waters. 1. Chinhae Bay[END_REF][START_REF] Yoo | Taxonomical studies on tintinnids (Protozoa: ciliata) in Korean coastal waters. 2. Yongil Bay[END_REF][START_REF] Zhang | An Illustrated Guide to Contemporary Tintinnids in the World[END_REF][START_REF] Zhang | A new tintinnid ciliate (Ciliophora: Spirotrichea) from Yangtze River Estuary, with notes on its habitat[END_REF]. Distributional data were presented as scatter diagrams and bar charts by Grapher (Version 12, Golden Software Inc., USA).

Results

Hydrography

Surface salinity changed greatly along the Pearl River Estuary in each season. In dry season, surface salinity fell in the range of 0.2-32.6 and 0.3-34.1 at Transects A and B, respectively. In rainy season, it fell in the range of 0.1-24.4 and 0.1-12.7 at Transects C and D, respectively. In transitional season, surface salinity ranged in 0.1-32.5 and 0.1-34.3 at Transects E and F, respectively (Fig. 2).

Salinity variation trends along each transect had obvious differences. At Transect A, salinity stayed at 0.2 at the innermost 5 stations and then increased slowly with small fluctuations. At Transect B, salinity increased continuously from upstream to downstream with large fluctuation between Stns. B19 and B28. The salinity fluctuations at Transects A and B were caused by freshwater discharge from branches of the Pearl River. Because of the largest freshwater discharge in rainy season, salinity kept its minimum value (0.1) at upstream stations at both Transects C and D. Salinity increased slower at Transects C and D than the other 4 transects. At Transect E, salinity increased continuously before reaching a plateau at the last three stations. At Transect F, salinity stayed at 0.1 or 0.2 before Stn. F10, then increased rapidly (Fig. 2).

Temperature fluctuations were small (< 3 °C) along each transect. Temperatures were the highest in rainy season, ranged in 28.2-30.8 °C 3

rainy seasons (Fig. 3, Table 1). The highest abundance of T. mayeri (230 ind. L -1 ) was higher than T. urnula (20 ind. L -1 ) and T. fluviatile (9 ind. L -1 ) (Table S1). The highest abundances of T. mayeri, T. urnula and T. fluviatile appeared at salinity of 2.8, 0.9 and 1.6, respectively (Fig. 3). Salinity tolerances of the 3 freshwater species had slight difference. T. fluviatile only appeared in salinity ≤12.7, while both the salinity range of T. mayeri and T. urnula was 0.1-32.5 (Table S1). T. mayeri merely appeared in salinity ≤6.1 at Transects A, B, D and F. At Transects C and E, T. mayeri was identified at high salinity regions and even to 32.5 at Transect E. Maximum abundance of T. mayeri was highest in rainy season, which were 150 and 230 ind. L -1 at Transects C and D, respectively. In dry and transitional seasons, maximum abundance were extremely lower than in rainy season, which were 37, 15, 12 and 5 ind. L -1 at Transects A, B, E and F, respectively (Fig. 3).

Tintinnopsis urnula did not appear at Transect B. It only occurred in salinity ≤4.1 at Transects A, C, D and F. At Transect E, it appeared in salinity ranged in 6.5-32.5. Maximum abundance of T. urnula was the highest (20 ind. L -1 ) at Transect F and the lowest (7 ind. L -1 ) at Transect E (Fig. 3). T. fluviatile did not occur at transitional season. In dry season, it only appeared in salinily ≤2.9 and 5.1 at Transects A and B, respectively. At Transect D, it occurred in salinity 10.3-11.3. Maximum abundance of T. fluviatile was the highest (7 ind. L -1 ) at Transect B and the lowest (3 ind. L -1 ) at Transect D. Maximum abundance of T. fluviatile was 5 ind. L -1 at Transect A (Fig. 3).

Brackish species

Codonellopsis lusitanica, Leprotintinnus nordqvisti, L. simplex, Nolaclusilis bicornis, Tintinnidium sp., Tintinnopsis amoyensis, T. chinglanensis, T. fimbriata, T. nana, T. nucula, T. parva, T. tentaculata, T. tubulosa, and Tintinnopsis sp.1 were defined as brackish species. Salinity ranges of these species were different, but the preferred salinity ranged in 3-23. With the increase of salinity, abundances of most brackish species increased at first, then decreased (Fig. 4).

Tintinnopsis amoyensis and T. fimbriata were the most abundant species, whose highest abundance were 2202 and 1657 ind. L -1 , respectively. Both of them occurred at all transects in 3 seasons. Swarm (abundance > 1000 ind. L -1 ) of T. amoyensis and T. fimbriata occurred at Transect D when salinity ranged in 5.4-9.2 and 3.9-6.1, respectively. At other 5 transects, maximum abundance of T. amoyensis and T. fimbriata were ≤200 ind. L -1 . Salinity range of T. amoyensis and T. fimbriata were 0.2-26.4 and 0.2-24.4, and the preferred salinity of T. amoyensis and T. fimbriata were 5.4 and 3.9, respectively (Fig. 4).

Maximum abundance of N. bicornis, T. nana, T. parva, T. tentaculata, T. tubulosa, and Tintinnopsis sp.1 were in the range of 50-1000 ind. L -1 , which occurred when salinity were 10. 3, 6.6, 7.1, 11.3, 5.4 and 3.9, respectively. T. nana, T. parva and T. tubulosa occurred at all transects in 3 seasons, with salinity ranged in 0.1-19.5. 0.1-30.6 and 0.1-32.6, respectively. T. tentaculata appeared at 5 transects in 3 seasons, with salinity ranged in 1.4-33.8. N. bicornis and Tintinnopsis sp.1 only occurred at Transect D in rainy season, with salinity ranged in 7.8-12.7 and 2.1-12.7, respectively (Fig. 4).

Marine species

Fifteen species with the highest abundance occurred at polyhaline (salinity > 23) regions were considered as marine species: Amphorellopsis acuta, Amphorides amphora, C. morchella, C. ostenfeldi, Dadayiella ganymedes, T. beroidea, T. butschlii, T. estuariensis, T. glans, T. plagiostoma, T. radix, T. schotti, T. tubulosoides, T. tocantinensis and Wangiella dicollaria. Their abundance decreased sharply with salinity decrease from downstream to upstream. They rarely appeared in low salinity (< 15) waters. No marine species was identified at Transect D (Fig. 5, Table S1).

Maximum abundance of W. dicollaria (305 ind. L -1 ) and T. tocantinensis (107 ind. L -1 ) were > 100 ind. L -1 . Salinity ranges of W. dicollaria and T. tocantinensis were 15.3-34.1 and 16.6-34.1, respectively. A. acuta, C. morchella, C. ostenfeldi, D. ganymedes, T. plagiostoma, T. radix and T. tubulosoides had maximum abundance ranged in 20-100 ind. L -1 , with salinity tolerance ranges in 15. 2-34.1, 17.3-34.3, 21.0-34.3, 29.5-34.3, 6.3-34.1, 16.6-34.1 and 11.1-34.1, respectively. Maximum abundances of A. amphora, T. beroidea, T. butschlii, T. estuariensis, T. glans and T. schotti were < 20 ind. L -1 . They occurred in salinity ranges of 21.2-34.3, 17.3-34.1, 29.5-34.1, 16.6-34.1, 14.6-34.3 and 22.5-34.3, respectively (Fig. 5, Table S1).

Amphorellopsis acuta and C. morchella occurred in 3 seasons. But A. acuta appeared at Transects A, B, C, E and F, while C. morchella appeared at Transects B, C and F. T. beroidea, T. glans, T. tubulosoides and W. dicollaria occurred at 4 transects in dry and transitional seasons. A. amphora only appeared at 2 transects in transitional season, while T. estuariensis and T. tocantinensis only appeared at 2 transects in rainy season. T. butschlii, T. plagiostoma, T. radix and T. schotti occurred at 3 Fig. 4. Abundance (ind. L -1 ) variation of brackish tintinnids along salinity gradient at each transect. transects, while C. ostenfeldi and D. ganymedes occurred at 2 transect in different seasons (Fig. 5, Table 1).

Number of tintinnid species variations along salinity gradient

Number of freshwater tintinnid species was ≤2 at each station. Although the number of freshwater tintinnid species was low, the variation trends were quite clear except for Transect E (and partly Transect C) (Fig. 6). Freshwater species disappeared when salinity > 5 at Transects A, B and F. They occurred discontinuously when salinity > 5 at Transects C and D. However, they occurred at high salinity regions at Transect E (Fig. 6).

Number of brackish tintinnid species increased at first, then decreased along salinity gradient at Transects A, B, and F. It was difficult to determine the variation tends at Transects C and D because of the narrower salinity range (Fig. 6). The maximum numbers fell in the range of 5-8 at each transect, occurred when salinity ranged in 5-25 (Fig. 6).

At Transects A and B, number of marine tintinnid species decreased from polyhaline to mesohaline (salinity ranged in 3-23) stations, the maximum number (10 in both Transects A and B) occurred at stations with salinity > 30 (Fig. 6). At Transects E and F, number of marine tintinnid species was ≤6, higher numbers occurred when salinity > 20 and decreased sharply when salinity < 20 (Fig. 6). Number of marine tintinnid species was extremely low at Transect C where the highest salinity was 24.4. No marine species occurred at Transect D (Fig. 6).

Tintinnid species richness ranged in 1-15, 1-13, 1-7, 2-11, 1-7 and 2-8 at Transects A, B, C, D, E and F, respectively. It increased along salinity gradient when salinity < 5 at each transect. Different variation trends of tintinnid species richness were observed when salinity > 5 in different seasons. Tintinnid species richness was high when salinity > 30 at Transects A and B. It stayed stable at mesohaline regions at Transects C, E and F (Fig. 6).

Tintinnid abundance variations along salinity gradient

Abundances of freshwater species were high when salinity was extremely low and decreased sharply with the increase of salinity at each transect except Transect E (Fig. 7). Maximum abundances ranged in 12-230 ind. L -1 at each transect. Maximum abundance was much higher in rainy season (160 and 230 ind. L -1 at Transects C and D, respectively) than in dry season (38 and 22 ind. L -1 at Transects A and B, respectively) and transitional season (12 and 21 ind. L -1 at Transects E and F, respectively) (Fig. 7).

Brackish species abundances increased at first and then decreased along salinity gradient at each transect, high abundance occurred at regions with salinity ranged in 5-20. Maximum abundance was extremely higher at Transect D (2683 ind. L -1 ) than at the other 5 transects (177-376 ind. L -1 ) (Fig. 7).

At polyhaline regions, marine species abundance increased rapidly with the increase of salinity. Maximum abundances were higher in dry season (247-531 ind. L -1 at Transects A and B, respectively) than in transitional season (53-109 ind. L -1 at Transects E and F, respectively) and rainy season (< 20 ind. L -1 at Transect C) (Fig. 7).

Total tintinnid abundances were in the ranges of 4-285, 2-557, 2-235, 10-2693, 1-215 and 1-239 ind. L -1 at Transects A, B, C, D, E Fig. 6. Number of species variation of different type tintinnids along salinity gradient at each transect. Note that total species including freshwater species, brackish species, marine species and occasional species. Fig. 7. Abundance (ind. L -1 ) variation of different type tintinnids along salinity gradient at each transect. Note that total species including freshwater species, brackish species, marine species and occasional species. and F, respectively (Fig. 7). Variation tendency of total tintinnid abundance were similar when salinity was < 15 except at Transect A: along with the increasing salinity, it increased at first before reaching a peak, then decreased to low values (Fig. 7).

Total tintinnid abundances showed bimodal-peak along salinity gradient at 4 transects (Transects A, B, E and F). First abundance peaks occurred when salinity was in the range of 4-11 for all transects, which were contributed by brackish species. The second abundance peaks occurred when salinity > 30 at transects A, B and F, and with salinity about 25 at Transect E. Those peaks were contributed by marine species (Fig. 7).

Tintinnid community variations along salinity gradient

Cumulative data of the 6 transects showed that number of species and abundance of brackish tintinnids had peaks in mesohaline region. Number of species and abundance of marine tintinnids increased with salinity. There were only 3 freshwater species which was too low to show richness trend (Fig. 8). Freshwater species abundance decreased from oligohaline to mesohaline regions. Total species richness was low at oligohaline regions and high at polyhaline regions. Total tintinnid abundance showed peaks at mesohaline and polyhaline regions along salinity gradient (Fig. 8).

Three tintinnid communities (low salinity, brackish and coastal communities) were divided based on the dominance of freshwater, brackish and marine tintinnids (Fig. 9). Low salinity community, dominated by the freshwater species, mainly appeared when salinity was < 2.5 (Fig. 9). At Transects A, B, C and D, the division between low salinity and brackish communities were at the salinity of 2.9, 4.2, 2.0 and 2.4, respectively. The lowest salinity when coastal community first occurred was 29.5, 24.9, 20.1, 19.5 and 21.2 at Transects A, B, C, E and F, respectively (Fig. 9). Therefore, the three communities were generally divided at salinity ranges of 2.0-4.2 and 19.5-29.5, respectively.

The community variations in different season were different: The boundary between different communities were clear cut at Transects A, B, C and D. However, at Transects E and F, low salinity community did not have a clear cut separation with brackish community: freshwater tintinnids bounced back to dominance for several times (Fig. 9).

Discussion

Salinity preference of tintinnid species

Both freshwater and marine organisms could be found at estuaries [START_REF] Mclusky | The Estuarine Ecosystem -Ecology, Threats and Management[END_REF]. There were different opinions on whether true brackish species existed at estuaries. Some reports insisted that brackish species were really marine or freshwater species that can adapt brackish conditions [START_REF] Cognetti | Biodiversity and adaptive mechanisms in brackish water fauna[END_REF][START_REF] Attrill | Ecotone or ecocline: ecological boundaries in estuaries[END_REF]. Most researches agreed that there were true brackish species in mesohaline regions of estuaries [START_REF] Mclusky | The Estuarine Ecosystem -Ecology, Threats and Management[END_REF][START_REF] Elliott | The estuarine quality paradox, environmental homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF][START_REF] Whitfield | Paradigms in estuarine ecology -a review of the Remane diagram with a suggested revised model for estuaries[END_REF]. "(True) brackish species" [START_REF] Barnes | What, if anything, is a brackish fauna?[END_REF][START_REF] Mclusky | The Estuarine Ecosystem -Ecology, Threats and Management[END_REF] and "(true) estuarine species" [START_REF] Carter | Seasonal and spatial distribution of planktonic crustacea in the lower Saint John River, a multibasin estuary in New Brunswick, Canada[END_REF][START_REF] Elliott | The estuarine quality paradox, environmental homeostasis and the difficulty of detecting anthropogenic stress in naturally stressed areas[END_REF] were commonly used in estuarine studies with the same meaning [START_REF] Whitfield | Paradigms in estuarine ecology -a review of the Remane diagram with a suggested revised model for estuaries[END_REF]. We chose to use "brackish species" in our study.

To our knowledge, brackish tintinnid species was first proposed by [START_REF] Bakker | Tintinnida from coastal waters of the S.W.-Netherlands I. The genus Tintinnopsis stein[END_REF]. There were lots of tintinnid taxonomic references about freshwater [START_REF] Kofoid | A Conspectus of the Marine Freshwater Ciliata Belonging to the Suborder Tintinnoinea, with Descriptions of New Species Principally from the Agassiz Expedition to the Eastern Tropical Pacific 1904-1905[END_REF][START_REF] Nie | Notes on three new species of fresh-water Tintinnoidea[END_REF][START_REF] Chiang | Notes on the freshwater Tintinnoinea from Jiangsu and Anhui provinces[END_REF][START_REF] Petz | Morphogenesis in some freshwater tintinnids (Ciliophora, Oligotrichida)[END_REF], brackish [START_REF] Sniezek | Nolaclusilis hudsonicus n. sp (Nolaclusiliidae n. fam.) a bilaterally symmetrical tintinnine ciliate from the lower Hudson River Estuary[END_REF][START_REF] Snyder | Nolaclusilis bicornis n. g., n. sp. (Tintinnina, Tintinnidiidae): a tintinnine ciliate with novel lorica and cell morphology from the composition and grazing impact on phytoplankton standing stock in the Pearl River Estuary, China[END_REF][START_REF] Smith | Dartintinnus alderae n. g., n. sp., a brackish water tintinnid (Ciliophora, Spirotrichea) with dual-ended lorica collapsibility[END_REF] and marine [START_REF] Kofoid | Reports on the scientific results of the expedition to the eastern tropical Pacific, in charge of Alexander Agassiz[END_REF][START_REF] Zhang | An Illustrated Guide to Contemporary Tintinnids in the World[END_REF] species. However, variation of community composition by those tintinnids along salinity gradient in a specific estuary has not been studied before. Based on the intrusion ability from marine to freshwaters in the Lake Shinji-Ohashi River-Lake Nakaumi brackish-water system, tintinnids were divided into high salinity and wide salinity species, while no freshwater or brackish species was divided [START_REF] Godhantaraman | Geographical and seasonal variations in taxonomic composition, abundance and biomass of microzooplankton across a brackish-water lagoonal system of Japan[END_REF].

Freshwater species and marine species may be expatriated to mesohaline areas due to freshwater discharge and oceanic water intrusion, respectively, into estuaries. Those freshwater and marine species might be found in an expatriate state in mesohaline waters [START_REF] Angel | Biodiversity of the pelagic ocean[END_REF]. Brackish species usually have wide salinity tolerance [START_REF] Whitfield | Paradigms in estuarine ecology -a review of the Remane diagram with a suggested revised model for estuaries[END_REF]. Thus, it was inaccurate to determine salinity preference of tintinnid species according to sporadic report of their occurrence. Therefore, it was more suitable to determine whether a tintinnid species is a freshwater, brackish or marine species according to the preferred salinity and abundance variation along salinity gradient.

According to the division standard above, we divided the 32 tintinnid species into freshwater (3 species), brackish (14 species) and Fig. 8. Cumulative number of species and abundance (ind. L -1 ) variation of different type tintinnids along salinity gradient. Note that total species including freshwater species, brackish species, marine species and occasional species. marine (15 species) species (Figs. 345). This was consistent with salinity adaptability of phytoplankton in the Pearl River Estuary that freshwater, half-fresh-water and oceanic phytoplankton species were identified [START_REF] Huang | Species diversity and distribution for phytoplankton of the Pearl River estuary during rainy and dry seasons[END_REF].

Freshwater species

Thirty freshwater tintinnid species have been identified before, and most freshwater species belonging to genera Tintinnopsis (20 species) and Tintinnidium (6 species) [START_REF] Zhang | An Illustrated Guide to Contemporary Tintinnids in the World[END_REF]. In our study, Tintinnidium fluviatile, Tintinnopsis mayeri and T. urnula were identified as freshwater species with preferred salinity values < 3 (Fig. 3). T. fluviatile was considered as a freshwater species in previous studies [START_REF] Chiang | Notes on the freshwater Tintinnoinea from Jiangsu and Anhui provinces[END_REF][START_REF] Zhang | An Illustrated Guide to Contemporary Tintinnids in the World[END_REF]. T. mayeri and T. urnula were not classified as freshwater species before.

Tintinnidium fluviatile occurred in freshwater lakes in Jiangsu Province, China [START_REF] Chiang | Notes on the freshwater Tintinnoinea from Jiangsu and Anhui provinces[END_REF]. It also occurred in the Dock of Woods Hole [START_REF] Gold | Studies on the sizes, shapes, and the development of the lorica of agglutinated tintinnida[END_REF]) and the Río de la Plata estuary [START_REF] Gómez | Seasonal and spatial distribution of the microbenthic communities of the Río de la Plata estuary (Argentina) and possible environmental controls[END_REF]. T. mayeri was found in coastal sites of Jiaozhou Bay without any information of salinity [START_REF] Yin | Primitive investigation of tintinnids in Jiaozhou Bay[END_REF]. However, a 10-year survey in the central Jiaozhou Bay (salinity > 24.6) did not find this species [START_REF] Feng | Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific[END_REF]. In the present study, although T. mayeri appeared when salinity was > 30 in the Pearl River Estuary, the stations T. mayeri appeared were not far from the coast. T. mayeri may discharged to higher salinity areas by river runoff or the branches of the river in expatriate state and die out soon. However, the 10-year survey in Jiaozhou Bay were conducted in the central area of Jiaozhou Bay far from the coast, T. mayeri might not reach those areas even in expatriate state. Our result that T. mayeri was a freshwater species may contribute to explain its presence in [START_REF] Yin | Primitive investigation of tintinnids in Jiaozhou Bay[END_REF] and absence in [START_REF] Feng | Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific[END_REF]. T. urnula mainly occurred at low salinity regions and disappeared at the outermost sites in the Hooghly (Ganges) River Estuary (Rakshit et al., 2016a).

In the present study, we confirmed that freshwater tintinnids can sometimes be brought to high salinity (32.5) regions. However, we did not understand why freshwater tintinnids were expatriated to high salinity in transitional season rather than in rainy season with largest freshwater discharge.

Brackish species

Four brackish species were reported in previous studies: Dartintinnus alderae [START_REF] Smith | Dartintinnus alderae n. g., n. sp., a brackish water tintinnid (Ciliophora, Spirotrichea) with dual-ended lorica collapsibility[END_REF], Nolaclusilis bicornis [START_REF] Snyder | Nolaclusilis bicornis n. g., n. sp. (Tintinnina, Tintinnidiidae): a tintinnine ciliate with novel lorica and cell morphology from the composition and grazing impact on phytoplankton standing stock in the Pearl River Estuary, China[END_REF], N. hudsonicus [START_REF] Sniezek | Nolaclusilis hudsonicus n. sp (Nolaclusiliidae n. fam.) a bilaterally symmetrical tintinnine ciliate from the lower Hudson River Estuary[END_REF] and T. fimbriata [START_REF] Bakker | Tintinnida from coastal waters of the S.W.-Netherlands I. The genus Tintinnopsis stein[END_REF]. In our study, 14 brackish species were identified (Fig. 4). Among them, N. bicornis and T. fimbriata were reported in previous studies. N. bicornis was first identified in oligohaline and mesohaline regions with salinity ranged in 0.7-16.4 in the Chesapeake Bay [START_REF] Snyder | Nolaclusilis bicornis n. g., n. sp. (Tintinnina, Tintinnidiidae): a tintinnine ciliate with novel lorica and cell morphology from the composition and grazing impact on phytoplankton standing stock in the Pearl River Estuary, China[END_REF]. T. fimbriata mainly appeared at mesohaline region in Netherlands coastal waters [START_REF] Bakker | Tintinnida from coastal waters of the S.W.-Netherlands I. The genus Tintinnopsis stein[END_REF] and the Chesterfield Inlet [START_REF] Rogers | Tintinnids of Chesterfield inlet, Northwest Territories[END_REF]. The abundance of T. fimbriata reached 1.08 × 10 6 ind. L -1 at salinity of about 15 and caused a yellowish colouration in the Kiel Bight, Baltic Sea, in March 1999 [START_REF] Agatha | Redescription of the tintinnid ciliate Tintinnopsis fimbriata Meunier, 1919 (Spirotricha, Choreotrichida) from coastal waters of northern Germany[END_REF]. In this study, swarm of T. fimbriata appeared at salinity ranged in 3.9-6.1 and declined both towards the polyhaline and oligohaline waters (Fig. 4).

Other species were not defined as brackish species, although there were reports of some species occurring in brackish waters. T. amoyensis is a common and sometime dominant species at estuarine regions. In the Weihe River mouth of Laizhou Bay, T. amoyensis was the most dominant species with maximum abundance of 6700 ind. L -1 [START_REF] Zhang | Summertime ciliate and copepod nauplii distribution and microzooplankton herbivorous activity in the Laizhou Bay, Bohai Sea, China[END_REF]. T. amoyensis (synonym of T. uruguayensis) was also a Fig. 9. Abundance percentage (%) of different type tintinnids at each station. : Salinity, : freshwater species, : brackish species, : marine species, : occasional species. [START_REF] Khlebovich | Some problems of crustacean taxonomy related to the phenomenon of Horohalinicum[END_REF]. Planktonic protists showed an inverse trend along salinity gradient compared with macrozoobenthos, while the highest species richness occurred in the horohalinicum in the Baltic Sea estuaries (Telesh et al., 2011a,b;2013).

In the present study, 15 marine, 14 brackish and 3 freshwater tintinnid species were identified (Table 1). It was understandable because only 30 freshwater tintinnids while about 900 marine tintinnid species have been reported till now [START_REF] Zhang | An Illustrated Guide to Contemporary Tintinnids in the World[END_REF]. As a result, species richness in mesohaline waters was higher than in oligohaline waters.

Tintinnid species richness were higher in mesohaline than in oligohaline regions in all transects in our study, while the values in polyhaline waters were higher than in mesohaline waters in some transects and lower in other transects (Fig. 6). This was not so consistent with that of planktonic protists in the Baltic Sea estuaries, where the highest species richness occurred in horohalinicum (Telesh et al., 2011a,b;2013). In the Chesapeake Bay with salinity ranged in 9-27, higher numbers of tintinnid species were found in the intermediate stations (salinity ranged in 14-17), low values were found at oligohaline and polyhaline stations [START_REF] Dolan | Estuarine diversity of tintinnids (planktonic ciliates)[END_REF]. This tendency was similar with that of Transects E and F in our study.

Variation of tintinnid abundance along salinity gradient

In our study, two tintinnid abundance peaks appeared at Transects A, B E and F, only one abundance peak appeared at Transects C and D (Fig. 7). This might be because salinity ranges of Transects C and D were the narrowest, if sampled at a larger salinity range, tintinnid abundance may showed bimodal-peak, too.

In the present study, peaks of tintinnid abundance occurred at mesohaline and polyhaline regions along salinity gradient. This abundance distribution pattern was inconsistent with that in the Bay of Biscay, where tintinnids showed a seaward distribution and were restricted to regions with salinity > 25 [START_REF] Urrutxurtu | Seasonal dynamics of ciliated protozoa and their potential food in an eutrophic estuary (Bay of Biscay)[END_REF]. Bimodal-peak of tintinnid abundance along salinity gradient made it difficult to find the correlation between tintinnid abundance and salinity in our study (Fig. 8), which was consistent with that no significant correlation between tintinnid abundance and salinity (ranged in 11.3-33.2) was found in Hiroshima Bay [START_REF] Kamiyama | Seasonal variation in the species composition of tintinnid ciliates in Hiroshima bay, the Seto Inland Sea of Japan[END_REF].

Tintinnid community varations along salinity gradient

According to the abundance percentage variations of freshwater, brackish and marine zooplankton species along salinity gradient, a distinct brackish zooplankton community could be divided between freshwater and marine zooplankton communities [START_REF] Cognetti | Biodiversity and adaptive mechanisms in brackish water fauna[END_REF]. In our study, low salinity, brackish and coastal tintinnid communities were divided using the same method as [START_REF] Cognetti | Biodiversity and adaptive mechanisms in brackish water fauna[END_REF] (Fig. 9).

Tintinnid community division in this study was similar to previous studies of phytoplankton [START_REF] Huang | Species diversity and distribution for phytoplankton of the Pearl River estuary during rainy and dry seasons[END_REF] and zooplankton [START_REF] Tan | Seasonal variation in zooplankton[END_REF] in the Pearl River Estuary. Indeed, estuarine, mixed water and coastal phytoplankton communities [START_REF] Huang | Species diversity and distribution for phytoplankton of the Pearl River estuary during rainy and dry seasons[END_REF], whereas estuarine, tropical neritic, warm-temperate nearshore and pelagic zooplankton communities [START_REF] Tan | Seasonal variation in zooplankton[END_REF] were divided in the Pearl River Estuary. The similar variation tendencies of zooplankton communities were also observed in the St. Lawrence Estuary [START_REF] Laprise | Environmental variability as a factor controlling spatial patterns in distribution and species diversity of zooplankton in the St. Lawrence Estuary[END_REF][START_REF] Winkler | Trophic coupling across the St. Lawrence River estuarine transition zone[END_REF] and upper Ariake Bay of Japan [START_REF] Islam | Spatial and seasonal variations in copepod communities related to turbidity maximum along the Chikugo estuarine gradient in the upper Ariake Bay, Japan[END_REF]. Those results indicated that it was a common phenomenon that plankton varied from low salinity to brackish to coastal communities along salinity gradient in estuarine regions.

In the present study, low salinity tintinnid community was absolutely dominant by 3 freshwater species. Brackish tintinnid community was shaped by several abundant brackish species such as T. tubulosa, T. amoyensis, T. tentaculata, T. fimbriata, T. parva, and T. nana. While coastal tintinnid community was much more complex, it was composed by more species with low abundance . This distribution pattern was consistent with that of zooplankton in the Gironde estuary, where the upstream zone was exclusively dominated by one copepod, major tintinnid species at estuarine environment in southwest coast of India [START_REF] Sivasankar | Loricate ciliates as an indicator of eutrophication status in the estuarine and coastal waters[END_REF]. In this study, swarm of T. amoyensis occurred at salinity 5.4-9.2 (Fig. 4). In our study, swarm of T. amoyensis and T. fimbriata occurred at Transect D caused the extreme high abundance of brackish species at transect D (Figs. 4 and7).

Tintinnopsis tubulosa was considered as a wide salinity species in the Hooghly (Ganges) River Estuary with the highest salinity of 26 (Rakshit et al., 2016a). Leprotintinnus nordqvisti was considered as a high salinity species in a brackish-water system in Japan [START_REF] Godhantaraman | Geographical and seasonal variations in taxonomic composition, abundance and biomass of microzooplankton across a brackish-water lagoonal system of Japan[END_REF]. T. tentaculata, T. parva, and T. nana were mostly found at regions with salinity range of 3.2-14.4 in the Hooghly (Ganges) River Estuary (Rakshit et al., 2016a). T. nucula, T. chinglanensis, L. simplex, and Codonellopsis lusitanica were sporadically reported at coastal or estuarine environments [START_REF] Zhang | Summertime ciliate and copepod nauplii distribution and microzooplankton herbivorous activity in the Laizhou Bay, Bohai Sea, China[END_REF][START_REF] Li | Interaction between neritic and warm water tintinnids in surface waters of East China Sea[END_REF]Rakshit et al., 2016b) without detailed salinity range.

In taxonomical studies, brackish tintinnid species were less frequently targeted in brackish waters, where in turn the most recently discovered species and genera, such as D. alderae [START_REF] Smith | Dartintinnus alderae n. g., n. sp., a brackish water tintinnid (Ciliophora, Spirotrichea) with dual-ended lorica collapsibility[END_REF], N. hudsonicus [START_REF] Sniezek | Nolaclusilis hudsonicus n. sp (Nolaclusiliidae n. fam.) a bilaterally symmetrical tintinnine ciliate from the lower Hudson River Estuary[END_REF] and N. bicornis [START_REF] Snyder | Nolaclusilis bicornis n. g., n. sp. (Tintinnina, Tintinnidiidae): a tintinnine ciliate with novel lorica and cell morphology from the composition and grazing impact on phytoplankton standing stock in the Pearl River Estuary, China[END_REF], have been found. We find that preferred salinity of brackish species were in the range of 3-23, which could be helpful for taxonomist to find more brackish tintinnid species.

Marine species

Most marine tintinnids in our study were common species in coastal and estuarine waters around the world (G odhantaraman and Uye, 2003; [START_REF] Urrutxurtu | Seasonal succession of tintinnids in the Nervion River estuary, Basque Country, Spain[END_REF][START_REF] Li | Interaction between neritic and warm water tintinnids in surface waters of East China Sea[END_REF][START_REF] Feng | Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific[END_REF][START_REF] Sivasankar | Loricate ciliates as an indicator of eutrophication status in the estuarine and coastal waters[END_REF]. Their abundances plummeted sharply with the decrease of salinity in the Pearl River Estuary (Fig. 5).

There were some reports about decreasing abundances of some species from coast to inner part of estuaries. For example, Wangiella dicollaria was restricted at regions with salinity > 24 in the Hooghly (Ganges) River Estuary (Rakshit et al., 2016a). T. berodea only appeared when salinity was > 18 in the Nervion River estuary, its high abundance occurred when salinity was > 25 [START_REF] Urrutxurtu | Seasonal succession of tintinnids in the Nervion River estuary, Basque Country, Spain[END_REF]. T. radix appeared when salinity ranged in 19.7-20 in the Sea of Marmara [START_REF] Balkis | Tintinnids (Protozoa: Ciliophora) of the Buyukcekmece Bay in the sea of Marmara[END_REF] and 29.17-32.89 in Southern Yellow Sea [START_REF] Zhang | Meso-scale spatial distribution of large tintinnids in early summer in southern Yellow Sea[END_REF]. It was the dominant species at all stations in Shenhu Bay of China with salinity ranged in 31-33.6 [START_REF] Wang | Spatial and seasonal variations of large tintinnid ciliates in Shenhu Bay of China[END_REF]. T. estuariensis was a species first recorded in the Changjiang River Estuary with salinity ranged in 3.9-29.8 [START_REF] Zhang | A new tintinnid ciliate (Ciliophora: Spirotrichea) from Yangtze River Estuary, with notes on its habitat[END_REF].

Tintinnid composition variation along salinity gradient

In the present study, different tintinnid species occupied different salinity ranges with drastic abundance variations (Figs. 345). Variations of tintinnid composition along salinity gradient in the present study were consistent with previous studies of phytoplankton and zooplankton. Estuarine plankton community structure was strongly affected by salinity. Species composition and abundance of both phytoplankton and zooplankton varied along salinity gradient (Marshall andAlden, 1990, 1993;[START_REF] Sautour | Comparative spring distribution of zooplankton in three macrotidal European estuaries[END_REF][START_REF] Tackx | Zooplankton in the Schelde estuary, Belgium and The Netherlands. Spatial and temporal patterns[END_REF][START_REF] Calliari | Biomass and composition of the phytoplankton in the Río de la Plata: large-scale distribution and relationship with environmental variables during a spring cruise[END_REF][START_REF] Islam | Spatial distribution and trophic ecology of dominant copepods associated with turbidity maximum along the salinity gradient in a highly embayed estuarine system in Ariake Sea, Japan[END_REF][START_REF] Muylaert | Changes in phytoplankton diversity and community composition along the salinity gradient of the Schelde estuary (Belgium/ The Netherlands)[END_REF]. Correspondingly, communities of zooplankton and phytoplankton varied along salinity in estuaries (Marshall andAlden, 1990, 1993;[START_REF] Huang | Species diversity and distribution for phytoplankton of the Pearl River estuary during rainy and dry seasons[END_REF][START_REF] Tan | Seasonal variation in zooplankton[END_REF][START_REF] Muylaert | Changes in phytoplankton diversity and community composition along the salinity gradient of the Schelde estuary (Belgium/ The Netherlands)[END_REF][START_REF] Modéran | Zooplankton community structure in a highly turbid environment (Charente estuary, France): spatio-temporal patterns and environmental control[END_REF].

Variation of tintinnid species richness along salinity gradient

In estuary regions, number of freshwater and marine macrozoobenthos were higher than brackish species, freshwater and marine macrozoobenthos decreased in diversity with a progression from both upstream freshwater and downstream marine ends, caused the lowest species richness when salinity ranged in 5-8 [START_REF] Remane | Die brackwasserfauna[END_REF][START_REF] Mclusky | The Estuarine Ecosystem -Ecology, Threats and Management[END_REF][START_REF] Whitfield | Paradigms in estuarine ecology -a review of the Remane diagram with a suggested revised model for estuaries[END_REF]. The salinity range of 5-8 was considered as critical salinity zone or horohalinicum (Khlebovich and

Conclusions

In this study, diversity and distribution variations of tintinnid along salinity gradient from 0.1 to 34.1 in the Pearl River Estuary were investigated. We found that freshwater, brackish and marine tintinnid species appeared successively along salinity gradient. Preference salinity of freshwater, brackish and marine tintinnid species ranged in < 3, 3-23 and > 23, respectively. The alternation of freshwater, brackish and marine tintinnid species caused the variation of tintinnid species richness and abundance. Tintinnid species richness was low at oligohaline regions in 3 seasons. Variation tendency of tintinnid species richness along salinity gradient were different in different seasons. Two abundance peaks appeared at mesohaline and polyhaline regions. According to the dominance of freshwater, brackish and marine tintinnid species, low salinity, brackish and coastal communities were divided in the Pearl River Estuary. Future research of tintinnid community variation along large salinity gradient in other estuaries is needed in order to elucidate whether there is different characteristics in other estuaries.

the median zone was co-dominated by 2 copepods, while the downstream zone presented the highest diversity [START_REF] David | Estuarine habitats structure zooplankton communities: implications for the pelagic trophic pathways[END_REF]. The similar pattern was also observed in the Chikugo Estuary [START_REF] Islam | Spatial distribution and trophic ecology of dominant copepods associated with turbidity maximum along the salinity gradient in a highly embayed estuarine system in Ariake Sea, Japan[END_REF] and the Sendai Bay [START_REF] Kazama | Relative importance of physical and biological factors regulating tintinnid populations: a field study with frequent samplings in Sendai Bay, Japan[END_REF]. The similar variation patterns of tintinnid and plankton communities along salinity gradient demonstrated that with the increase of salinity, structure of plankton communities become more complex.

Seasonal variations of tintinnid composition

The waxing and waning of different species within the system was the main reason causing the seasonal changes in tintinnid species compositions [START_REF] Dolan | Diversity and distributions of tintinnid ciliates[END_REF]. The present study showed that 11 in 43 species occurred in 3 seasons, while 16 species only appeared in 1 season. This was consistent with previous studies that most tintinnid species have obvious seasonal occurrence [START_REF] Modigh | Variability and persistence in tintinnid assemblages at a Mediterranean coastal site[END_REF][START_REF] Dolan | Diversity and distributions of tintinnid ciliates[END_REF][START_REF] Feng | Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific[END_REF]. A 4-year study sampled weekly in the Gulf of Naples indicated that none of the 10 most common species occurred even half the time and most occurred less than a third of the time [START_REF] Modigh | Variability and persistence in tintinnid assemblages at a Mediterranean coastal site[END_REF]. A 10-year study sampled monthly at 4 stations in the Jiaozhou Bay showed that 10 species had a year-round distribution, 2 species occurred in 11 months while the other 14 species were found in less than 7 months [START_REF] Feng | Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific[END_REF].

As plenty of studies showed very distinct seasonal changes in the occurrence of individual tintinnid species, we sampled at 3 seasons (dry, rainy and transitional) in order to collect more tintinnid species in the Pearl River Estuary. The purpose of this study was to show tintinnid community variation along salinity gradient. Therefore we did not discuss too much on seasonal variation of tintinnid community in the Pearl River Estuary.

  waters were collected onboard R/V "Nanfeng" in transects approximately along the axis of Pearl River Estuary in dry season (Transect A on 10 October 2014; Transect B on 29 October 2014), rainy season (Transect C on 11 June 2015; Transect D on 30 June 2015) and transitional season (Transect E on 15 March 2017; Transect F on 1 April 2017). All transects had the same innermost station (Stns. A1, B1, C1, D1, E1 and F1) in Xinzhou Wharf. However, they had different lengths and different number of stations: Transects A, B, C, D, E and F had 19,

Fig. 1 .

 1 Fig. 1. Stations and sampling days of each transect in the Pearl River Estuary.

Fig. 3 .

 3 Fig.3. Abundance (ind. L -1 ) variation of freshwater tintinnids along salinity gradient at each transect.

  Maximum abundance of C. lusitanica, L. nordqvisti, L. simplex, Tintinnidium sp., T. chinglanensis and T. nucula were < 25 ind. L -1 . The preferred salinity of the 6 species ranged in 4.6-22.5. Salinity ranges of C. lusitanica, L. nordqvisti, L. simplex, Tintinnidium sp., T. chinglanensis and T. nucula were 0.8-14.2, 9.2-34.1, 11.8-34.1, 6.3-23.6, 8.3-31.4 and 0.2-34.1, respectively. Among them, L. nordqvisti occurred at 4 transects in dry and rainy seasons, Tintinnidium sp. occurred at Transects A, B and D in dry and rainy seasons, L. simplex occurred at Transects B, C and E in 3 seasons, T. nucula occurred at Transects A, B and F in dry and transitional seasons, while C. lusitanica and T. chinglanensis occurred at Transects E and F in transitional season (Fig. 4).

Fig. 5 .

 5 Fig. 5. Abundance (ind. L -1 ) variation of marine tintinnids along salinity gradient at each transect.

Table 1

 1 Species list and their occurrence frequencies (times) at Transects A, B, C, D, E and F. Nineteen, 41, 36, 26, 29 and stations were conducted at Transects A, B, C, D, E and F, respectively.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC Nos. 41576164 and 41706192), National Basic Research Program of China (973 Program No. 2014CB441504), CNRS-NSFC Joint Research Projects Program (No. 41711530149) and 2017-2019 Sino-French Cai Yuanpei Programme. We thank the crew on the R/V "Nanfeng" for their support and assistance during the cruises.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecss.2019.106268.


Patrice Berthod
email: patrice.berthod@centraliens-lille.org

Zohra Himeur
email: himeurzahra@gmail.com

Dependence on the chromium content of the high temperature oxidation behavior of Ta-rich nickel-based cast alloys

Keywords: Cast nickel-based alloys, High tantalum content, Varying chromium content, High temperature oxidation

The high-temperature stability of the primary tantalum carbides is a problem of importance for chromium-rich cast alloys, based on cobalt or nickel. The focus of this study was nickel-based alloys, as these alloys are particularly sensitive to a lack of TaC carbides in the ascast state and by dissolution due to high-temperature exposure. In this work, a possible way for promoting the formation of many TaC carbides by changing from the usual 30wt.% chromium content was investigated. Five alloys with Cr content varying from 10 to 50 wt.% were prepared and then subjected to microstructure characterization and to oxidation tests. In contrast with what was expected, decreasing the Cr content in comparison to the Ni-30Cr-0.4-6Ta reference alloy did not succeed in obtaining more TaC carbides, but instead had the opposite effect. Concerning the high-temperature oxidation behavior at 1127 and 1237°C, loss of resistance was observed only for a Cr content at the lower level of 10 wt.%. It was noticed that a subscale CrTaO 4 developed during oxidation and seemed to promote oxide spallation during cooling.

INTRODUCTION

Chromium is, with aluminum and silicon, one of the three main elements added in rather high quantities in superalloys to allow them resisting oxidation by hot gases in service [START_REF] Kofstad | High Temperature Corrosion[END_REF][START_REF] Young | High Temperature and Corrosion of Metals[END_REF]. In presence of carbon chromium may also play the role of a carbide-former element. In favorable chemical compositions, it allows obtaining Cr 7 C 3 or Cr 23 C 6 , the types of chromium carbides which are usually met in superalloys [START_REF] Sims | The Superalloys[END_REF][START_REF] Bradley | Superalloys: A technical guide[END_REF]. It is commonly considered that 20 wt.%Cr is the low limit for a nickel-based alloy for keeping a chromia-forming behavior on long time when it is exposed at high temperature in oxidant atmosphere [START_REF] Kofstad | High Temperature Corrosion[END_REF][START_REF] Young | High Temperature and Corrosion of Metals[END_REF][START_REF] Birks | Introduction to the High-Temperature Oxidation of Metals[END_REF]. However chromium may present several disadvantages. One already knows the toxicity problems of the Cr VI specie [START_REF] Langard | Biological and Environmental Aspects of Chromium[END_REF] and the possible re-oxidation of the protective chromia scale into gaseous species [START_REF] Young | High Temperature and Corrosion of Metals[END_REF][START_REF] Caplan | [END_REF]8], notably in presence of water vapor [9,13]. But also the presence of chromium in nickel alloys may threaten the thermodynamic stability of the tantalum carbides [14]. This may be a problem since these ones play an important role in the reinforcement of cast superalloys [START_REF] Donachie | Superalloys: A Technical Guide[END_REF], notably in the case of the cobalt-based ones [START_REF] Michon | [END_REF]. Indeed, in contrast with some other MC carbides in nickel alloys (e.g. HfC) [17], or with all MC carbides in cobalt-based or iron-based alloys [18,[START_REF] Corona | proceedings of the International Conference on Sustainable Energy and Information Engineering[END_REF], it seems that tantalum carbides exist together with chromium carbides. This is true even for the nickel alloys which are designed to promote the formation of exclusively TaC, i.e. containing as carbon atoms as tantalum atoms. It is maybe possible to favor the development of more TaC in a nickelchromium alloy by decreasing its chromium content in order to lower the efficiency of this carbide-former competitor. But this needs to be experimentally verified as well as the consequences of this Cr decrease for the behavior of the alloy in oxidation at high temperature. One can fear threats on the oxidation resistance as soon as the Cr content becomes lower than a limit. This limit may perhaps depend on the presence of the significant quantity of tantalum required to obtain TaC volume fractions high enough to achieve efficient mechanical strengthening in case of a success of their stabilization by Cr impoverishment.

In this work one examines, for a series of {Ni-0.4C-6Ta, wt.%}-based alloys, the evolution of the carbides population when the Cr content decreases from the classical 30 wt.% value down to 0. One also investigates the consequences of these changes in chromium content on the oxidation behavior at two temperatures which are higher than 1100°C. This study was also extended to alloys with Cr contents beyond 30 wt.%, up to 50 wt.%, since it appeared also interesting to explore also the natures of the carbides and the oxidation behavior when the opposite action is done.

EXPERIMENTAL

Elaboration of the alloys

A series of five Ni-based alloys was synthesized by induction melting and casting under inert atmosphere. All these alloys contain 0.4wt.%C, 6wt.%Ta and a variable Cr content. These alloys are different from one another about their chromium content. The Cr content varies from 10 to 50 wt.%. The studied alloys are named "NTCxCr", in which x is the weight content in Cr. Their targeted chemical compositions are shown in Table 1. For each alloy a mix of metallic elements (Ni, Cr, Ta: Alfa Aesar, C: pure graphite) was prepared by targeting a total mass of about 40 grams. This mix of pure elements was placed in the water-cooled copper crucible of a CELES (France) furnace. This crucible was surrounded by a silica tube to isolate from air the inert internal atmosphere (300 millibars of pure Ar). The mixed elements were heated until reaching 2.5kV. This voltage was maintained during 30 seconds (thermal homogenization). Voltage was then increased until reaching 5 kV. It was maintained 3 minutes for achieving total melting then chemical homogenization. The alloy solidified a first time by decreasing the injected power. It was thereafter melted again according to the same cycle, in order to be sure of the total absence of any not-molten parts in the final ingot.

Preparation of the samples, oxidation tests in furnace and metallographic preparation

The obtained ingots were of a compact conic shape. Each of them was divided into four quarters, by using a Buehler Delta Abrasimet metallographic saw. One of these quarters was prepared as a metallographic sample devoted to the control of the as-cast microstructure. Two others were prepared for the high temperature oxidation tests. These ones were ground all around using 1200-grit SiC papers. A quarter by alloy was exposed at 1127°C in a resistive furnace (Nabertherm) during 24 hours. A second one was exposed at 1237°C, in the same furnace and in the same atmosphere (laboratory air). After total cooling the oxidized samples were all coated by cathodic pulverization of gold. This aimed to give a good electrical conductivity to their surface, despite the presence of the oxide scales. A second coating was then electrolytically applied (Ni 2+ solution heated at 50°C, 2 hours at about 20 mA/cm²). This led to a sufficiently thick shell of electrolytic Ni for protecting the external oxide scales during cutting. The oxidized samples were thereafter cut in two parts, by using the same metallographic saw as described above. The as-cast parts and the parts of oxidized samples were embedded in a cold resin mixture (ESCIL, France). They were ground with SiC papers from 240-grit to 1200-grit, water-washed and ultrasonically cleaned. Final polishing was achieved with a {1µm hard particles}-containing textile disk. The obtained mirror-like samples were thus ready for metallographic characterization.

Bulk, subsurface and surface characterization

A JSM-6010LA Scanning Electron Microscope (SEM) from JEOL (Japan), equipped with an Energy Dispersion Spectrometer (EDS), was used to visualize the as-cast microstructures. Micrographs were taken in Back Scattered Electrons mode (BSE) to carry out surface fraction measurements of the different types of carbides present in the bulk. These measurements were done using the image analysis tool of the Photoshop CS software of Adobe. The subsurface microstructure changes due to oxidation were also observed using the SEM in BSE mode, as well as the oxides formed internally and externally. The depths of the subsurface alloy zones which lost their carbides (from the alloy/external oxide interface), were measured. It was also attempted to value the average thickness of the external oxides. Spot EDS analyses were carried out to assess the chemical compositions in the subsurface (concentration profiles), notably in the extreme surface close to the oxidation front. Spot EDS analyses also allowed identifying the nature of all the present oxides.

RESULTS

Microstructures of the obtained alloys

The five micrographs presented in Figure 1 were taken using the SEM in BSE mode. They illustrate the as-cast microstructures of the alloys. The obtained chemical compositions are displayed in Table 2. Concerning the later one it can be seen that the Cr content is rather well respected for all alloys. This seems to be not the case for tantalum. Indeed, instead to be diminished by possible oxidation during the elaboration as this can be feared by considering the high reactivity of this element, its content appears to be higher than expected. This curious phenomenon is classically met when the global chemical composition of a TaC-rich alloy is assessed by EDS: the TaC carbides emerging on the metallographic surface lead to the enhancement of the concentration in Ta. This phenomenon did not occur for the NTC10Cr alloy (and may be for the NTC20Cr one too). This one obviously contains much less TaC carbides than the other alloys.

About the microstructures one can remark first that the matrix is unsurprisingly dendritic in all cases. Two types of particles are present: chromium carbides (the dark ones) and tantalum carbides (the white ones). The microstructure of the NCT30Cr alloy is rather well-known. Indeed the Ni(bal.)-30Cr-0.4C-6Ta alloy (contents in wt.%) was many times characterized earlier (e.g. in [14]). One can easily recognize the Cr 7 C 3 and TaC carbides, notably. Thus, thanks to the aspect similarities (morphology, tint), the dark particles and the white ones appearing in the NTC10Cr, NTC20Cr and NTC40Cr alloys, can be identified as being Cr 7 C 3 and TaC, respectively. In the first four alloys, one can also see that the peripheries of dendrites are particularly clear. This is due to tantalum segregation during solidification. This phenomenon, classical for such alloys, well helps to distinguish the dendritic structure of the matrix of the NTC10Cr alloy. The NTC50Cr alloy is particular since very dark areas coexist with the dendritic matrix and white areas. These white areas are composed of dendrites peripheries rich in Ta (due to Ta segregation) and of tantalum carbides. The very dark areas are certainly the BCC Cr-based rich part of the matrix which was previously identified in a {Ta, C}-free Ni-50Cr alloy [22] whose matrix structure is very similar to the NCT50Cr one. This can be verified, for the chemical composition point of view, in the Table 3 which gives the results of EDS spot analysis of the two phases (last lines). According to these results, the grey dendrite centers contain more Ni than Cr (45-46wt.%Cr only) while the dark areas contain 60wt.%Cr and more.

In the top part of each micrograph presented in Figure 1 one also gives the values of the surface fractions of the chromium carbides and of the tantalum carbides, except for the NCT50Cr alloy in which it was not possible to distinguish the tantalum carbides from the highly Tasegregated peripheral zones of dendrites. These values are the average values and the standard deviation values calculated from three image analyses. One can see that the surface fraction of chromium carbides increases from the NCT10Cr alloy to the NCT20Cr one. It decreases when arriving to the NCT30Cr. It increases again for the NCT40Cr alloy and decreases again for the NCT50Cr alloy. At the same time, the surface fraction increases steadily from the NCT10Cr alloy to the NCT30Cr alloy and decreases when the Cr content keeps on increasing.

Detailed views of the microstructures of the five alloys after elaboration are given in Figure 2. One can first observe the carbides in better condition. This allows discovering the eutectic origin of the chromium carbides: these ones are acicular and mixed with the peripheral parts of the matrix dendrites (NTC10Cr to NTC40Cr). The tantalum carbides are obviously also of a eutectic nature: script-like shaped and mixed with the outer part of the dendrites too (NTC10Cr to NTC30Cr). The white Ta-rich particles present in the interdendritic areas of the NTC40Cr alloy seem to be morphologically different from the eutectic TaC of the alloys which contain less chromium: they appear to be intermediate to the well-defined script-like shaped TaC (alloys with Cr ≤ 30wt.%) and the more diffuse white interdendritic particles present in the NTC50Cr alloy. The spot analyses performed on the coarsest carbides found in the alloys seemed confirming the stoichiometries of the Cr 7 C 3 carbides (NTC10Cr to NTC40Cr) and of the TaC ones (NTC10Cr to NTC50Cr). In contrast, no concluding result was obtained by this way for the Ta-rich white areas which are present in the NCT50Cr alloy, except the fact that it seems to be very rich in Ta.

Oxidation result at 1127°C

After 24h spent at 1127°C in the laboratory air, more or less complex oxide scales were present around the samples. Except for the two Cr-poorest alloys (NCT10Cr and NCT20Cr) the external scale spalled off during the cooling. It was thus not always possible to extensively specify all the oxidation products. General views of the surface and subsurface deterioration seen in cross-section are provided in Figure 3 while more magnified micrographs can be consulted in Figure 4. The NCT10Cr alloy was obviously covered by a mix of rather thin chromia (constituting the inner layer) and by a thicker complex oxide involving both nickel and chromium (constituting the outer layer). The alloy/external scale is very irregular. This suggests that oxidation was penetrating, and that catastrophic oxidation was imminent. In the subsurface, but very close to the alloy/oxide scale interface, an almost continuous and rather compact complex oxide involving both chromium and tantalum was present. As soon as the chromium content in alloy is equal to 10 wt.%Cr or higher, only chromia is present on the surface of the alloy. Chromia is very present over the NCT20Cr but just small chromia parts remain on surface for the NCT30Cr, NCT40Cr and NCT50Cr alloys. Just below, in the extreme surface part of alloy, the CrTaO 4 oxide is present in significant quantity, but more as juxtaposed oxides islands than an almost continuous subscale as for the NCT10Cr alloy. One can also notice that, when the Cr content in alloy increases along this series, additional CrTaO 4 oxides become to be also present in the whole carbide-free zone (NCT40Cr and NCT50Cr). In contrast they only constitute the outer almost continuous oxide at the extreme surface of alloy for the NCT10Cr, NCT20Cr and NCT30Cr alloys. One must say that, in the case of the NCT50Cr alloy, the outer alloy zone modified by oxidation is more a {BCC Cr-based phase}-free zone than a carbide-free zone as all the other alloys.

Obviously, the running development of a zone which is free of carbides (all alloys except NCT50Cr) or which is free of BCC Cr-based phase (NCT50Cr), demonstrates the diffusion of Cr and Ta toward the oxidation front (Figure 5). SEM/EDS concentration profiles acquired perpendicularly to the oxidation front confirm that Cr diffusion and Ta diffusion occurred. These {Cr and Ta}-depleted depths tend to be deeper than the carbide-free zones but the depths are of the same order of magnitude. The depth of the carbide-free zone depends on the alloy, as is to say on the initial alloy chromium content. It decreases from about 70µm for NCT10Cr to about 40 µm for NCT30Cr. It increases to 60µm for NCT40Cr. In the case of NCT50Cr the depth does not keep on increasing but it decreases, on the contrary. One must notice, for the later alloy, that what is significantly changed in the subsurface is not due to carbide dissolution but to the disappearance of the second phase of matrix (the BCC Cr-based one).

Table 4 also shows few values of thickness of the external oxide scale. Measurement was possible only for the NCT10Cr and NCT20Cr alloys. These ones kept oxide scale parts in quantities high enough to permit thickness evaluation, in contrast with the other alloys which all lost their external oxide scales by spallation at cooling. Since the oxidation process was rather heterogeneous all along each surface, with as results very variable nature and thickness, only a tentative range of thickness is given. Oxidation induced Cr and Ta depletion for all alloys (examples in Figure 5). The chromium content in extreme surface was assessed by three EDS spot analyses. The results (average ± standard deviation) are given in Table 5. For each alloy the decrease in Cr content close to the alloy/external oxides interface was between 5 and 10 wt.%. The local loss in Cr content continuously increases from the NCT20Cr alloy to the NCT50Cr alloy. The NCT10Cr does not obey this evolution because of its almost catastrophic oxidation.

Oxidation result at 1237°C

The metallographic exploitation of the samples oxidized at 1237°C for the same duration of 24 hours was driven similarly to the oxidation tests at 1127°C, by:

• the observation of the cross-sections at ×250 (Figure 6) and ×1000 (Figure 7),

• performing concentration profiles in the subsurface perpendicularly to the oxidation front (Figure 5, graphs of the right side), • the tentative measurement of the external oxide scale thickness (Table 6) when limited spallation at cooling did allow that, • the measurement of the carbide-free depth (Table 6) • and the measurement of the chromium content in extreme surface (Table 7).

The results and observations were globally the same as for the samples oxidized at 1127°C: start of catastrophic oxidation for the Cr-lowest alloy (NCT10Cr), local high thickness of the external scale, very irregular alloy/scale frontier, presence of many types of oxides, minimum of carbide-free depth for the NCT30Cr alloy, existence of a {BCC Cr-phase}-free subsurface for the NCT50Cr alloy, same type of evolution of the Cr-loss in extreme surface from the NCT10Cr alloy to the NCT50Cr one, and same types of Cr and Ta concentration profiles.

Thus the differences between the two temperatures are not qualitative, but they are quantitative: thicker external oxide scales and deeper carbide-free (or BCC Cr phase-free) zones. The chromium contents in extreme surface do not depend on temperature for a given alloy (Figure 8), the better Cr diffusion easiness offsetting the higher Cr consumption.

DISCUSSION

Concerning the effect of the modification of the Cr content on the natures and proportions of the obtained carbides in cast alloys, looking to the SEM/BSE micrographs allowed first feeling that the variation of the chromium content in the chemical composition of the alloys had consequences on the carbides population of the as-cast alloys. Image analysis allowed confirming these trends by quantitative results. The lowest Cr content tested here, 10 wt.%Cr, led to a lowering of the chromium carbides quantity but curiously also of the tantalum carbides one. Adding 10wt.%Cr more induced a logical increase in chromium carbides but also in tantalum carbides. Repeating this addition to reach 30wt.%Cr led to a maximal fraction in TaC carbides and to the rarefaction of the chromium carbides. Choosing a 40wt.%Cr content let the TaC fraction stationary but it favors the formation of more chromium carbides than for 30wt.%Cr. One can suspect a solid solution effect in a first time (sufficient available place for the greatest part of chromium and tantalum in the NCT10Cr alloy) and thereafter new balance between solid solution and carbides when Cr is more present in the chemical composition, with a singular effect in the case of the NCT30Cr alloy in which the chromium tends to stay preferentially in solid solution and to promote the formation of more TaC carbides.

Concerning the oxidation point of view, one can start by reminding that it was earlier observed that the Ni-30Cr-0.4C-6Ta alloy, serving as base for this work, was well oxidation resistant at high temperature. However one must also remind that it presented a high tendency to oxide spallation in thermal cycling. Concerning the strengthening particles for potential high temperature mechanical properties, this alloy presented the problem of co-precipitation of primary tantalum carbides and chromium carbides, despite that the weight contents in C and Ta were chosen to obtain equal atomic contents for these two carbide-forming elements. This molar equivalence of Ta and C was a recipe which was nonetheless efficient for cobalt-based and ironbased alloys. Thanks to its good behavior in isothermal oxidation it was permitted to envisage to decrease the Cr content in order to promote the precipitation of more TaC at the expense of the chromium carbides.

In these alloys, chromium is the most important element for the high temperature oxidation behavior. Its content was here either too low to allow good resistance (the 10wt.%Cr contained in the NCT10Cr alloy), or sufficiently high (30wt.% and beyond). The 20wt.%Crcontaining alloy (NCT20Cr) seems to well behave at the two test temperatures but it is possible that this behavior may degenerate for durations higher than 20 hours. This is suggested by the not perfect compactness and regularity of the chromia scale: after 24 hours at 1127 or 1237°C this chromia scale contains porosities and is a little wavy because of the possible start of inward oxidation. It is possible that this wavy shape of the external chromia scale favored its adherence on the substrate. Indeed the chromia scale had much less spalled off than for the Cr-richer alloys. Concerning spallation, only the alloys with irregular alloy/oxide scale interface were resistant against this phenomenon during cooling. Indeed the NCT30Cr, NCT40Cr and NCT50Cr all lost the main part of their external oxides. It was thought earlier [21] that tantalum oxide which formed just under the chromia scale may threaten the adherence of chromia on substrate. This was found again here for the alloys containing between 30 and 50wt.%Cr which displayed both a very regular alloy/oxide scale interface and an almost continuous sub-oxide of CrTaO 4 .The deleterious effect of the CrTaO 4 sub-layer on the adherence of chromia may be explained by the mismatch of crystalline network between chromia (eskolaite) which possesses a corundum crystal structure (with Cr III replacing Al III at the same locations in the crystal) and the rutile structure of CrTaO 4 [START_REF] Massard | [END_REF]25]. Classically encountered in case of high Ta contents (5 to 9 wt.%) in polycrystalline [26], directionally solidified [27] and single-crystalline [28] nickel-based superalloys, it was reported that tantalum often leads to the formation of more or less continuous sub-layers of CrTaO 4 during high temperature oxidation. This starts rather early and progresses with the growth or coarsening of the subsurface CrTaO 4 islands together with the deepening of the TaC-free zone. The coalescence of these coarsening subsurface oxides progressively results in an almost continuous CrTaO 4 sub-layer. Consequently to the formation of this oxide of both chromium and tantalum, severe spallation of the chromia external scale during post-oxidation cooling was systematically observed for Ta-rich nickel-based, cobalt-based, iron-based and {iron, nickel}-based polycrystalline cast alloys [30][31][32].

In all cases Cr diffused towards the oxidation front, from the solid solution matrix, the dissolving chromium carbides or the disappearing BCC Cr-phase. The two latter acted as important Cr-reservoirs, depending on the alloy. Tantalum diffused too from the solid solution matrix and from the tantalum carbides which are important Ta-reservoirs too. One well knows the consequence for chromium: formation and maintenance of a protective chromia-scale. Contrariwise, for Ta and its oxide, one does not know if its formation, obviously detrimental for the adherence of the external oxide, may act as a supplementary protection against oxidation, added to the one by chromia. It is true that the NCT10Cr alloy, although threatened here and there by a possible local catastrophic oxidation, rather resisted oxidation even at 1237°C, perhaps helped by the subsurface continuous CrTaO 4 oxide. This merits to be deeper studied. Solutions for combatting the oxide spallation problem must be found, why not by an yttrium addition allowing the pegging of the external scale.

CONCLUSION

Decreasing the Cr content of the Ni-30Cr-O.4C-6Ta base (wt.%) induced a visible lowering of the oxidation resistance at the two studied temperatures as soon as the alloy contains only 10wt.%Cr. But the oxidation was not catastrophic of a general manner, at least after 24 hours of exposure to air. It is possible that Ta was responsible of the limitation of the detrimental effect of the Cr lowering down to a so low content. But this Cr decrease did not induced the expected effect, as is to say the stabilization of the TaC carbides known to be very favorable to mechanical strength at elevated temperature. Curiously, but a possible explanation was presented here, this is by increasing the Cr content that one obtains more TaC carbides with few chromium carbides. Since this effect was accompanied by a better resistance against high temperature isothermal oxidation, the way to follow is progressively increasing the Cr content beyond the usual 30wt.%Cr, but not too high to avoid the appearance of the BCC-Cr phase which can be supposed to mechanically weaken the alloy. The optimal Cr content remains to be found, and the oxidation and creep behaviors to be specified.

FIGURE 1 .FIGURE 3 .FIGURE 5 .FIGURE 6 .FIGURE 8 .

 13568 FIGURE 1. As-cast microstructures of the five studied alloys (SEM/BSE micrographs, large view: ×250)

TABLE 1 .

 1 Theoretic compositions for the five studied alloys (weight contents).

	Alloys	Ni	Cr	Ta	C
	"NCT10Cr"	Bal.	10	6	0.4
	"NCT20Cr"	Bal.	20	6	0.4
	"NCT30Cr"	Bal.	30	6	0.4
	"NCT40Cr"	Bal.	40	6	0.4
	"NCT50Cr"	Bal.	50	6	0.4

TABLE 2 .

 2 Obtained chemical compositions for the five alloys (weight contents, 3 EDS full frame analyses).

	Alloys	Ni	Cr	Ta	C*
	"NCT10Cr"	Bal.	10.2 ±0.3	5.9 ±0.2	0.4
	"NCT20Cr"	Bal.	19.8 ±0.2	6.3 ±0.1	0.4
	"NCT30Cr"	Bal.	27.2 ±0.5	10.1 ±0.4	0.4
	"NCT40Cr"	Bal.	40.3 ±0.6	6.9 ±0.5	0.4
	"NCT50Cr"	Bal.	49.6 ±0.6	6.6 ±0.3	0.4

*: cannot be controlled; supposed to be well respected

TABLE 3 .

 3 Obtained chemical compositions for the matrix in the as-cast alloys (weight contents, 3 EDS spot analyses).

	Alloys	Ni	Cr	Ta	C
	"NCT10Cr"	Bal.	10.0 ±0.2	6.8 ±1.2
	"NCT20Cr"	Bal.	19.0 ±0.3	4.6 ±0.2
	"NCT30Cr"	Bal.	26.0 ±0.1	6.4 ±0.3	Not
	"NCT40Cr"	Bal.	38.0 ±0.2	3.8 ±0.3	measurable
	"NCT50Cr"	Grey matrix Black matrix	45.5 ±0.3 60.6 ±0.9	3.8 ±0.1 3.1 ±0.4

TABLE 4 .

 4 Intervals of thickness values for the external oxide scale (first results column) and average ± standard deviation values of the carbide-free depth (second results column) for the five alloys oxidized for 24h at 1127°C

	Alloys	Oxide scale thickness (µm)	Carbide-free zone depth (µm)
	"NCT10Cr"	19-44	66.0 ±9.0
	"NCT20Cr"	7-20	49.3 ±3.1
	"NCT30Cr"	Spalled off	44.3 ±4.3
	"NCT40Cr"	Spalled off	57.9 ±10.6
	"NCT50Cr"	Spalled off	34.2 ±4.9

TABLE 5 .

 5 Average and standard deviation for the chromium content in alloy close to the interface with the external oxide (3 EDS spot analyses)

	Alloys	Chromium content in extreme	Difference to the initial Cr content
	(24h 1127°C)	surface (wt.%)	(wt.%)
	"NCT10Cr"	4.8 ±0.3	-5.4
	"NCT20Cr"	15.5 ±1.3	-4.3
	"NCT30Cr"	21.6 ±0.1	-5.6
	"NCT40Cr"	33.2 ±0.1	-7.1
	"NCT50Cr"	41.1 ±1.1	-8.5

TABLE 6 .

 6 Intervals of thickness values for the external oxide scale (first results column) and average ± standard deviation values of the carbide-free depth (second results column) for the five alloys oxidized for 24h at 1237°C

	Alloys	Oxide scale thickness (µm)	Carbide-free zone depth (µm)
	"NCT10Cr"	17-160	> 300
	"NCT20Cr"	11-15	105 ±5
	"NCT30Cr"	Spalled off	87 ±6
	"NCT40Cr"	Spalled off	170 ±13
	"NCT50Cr"	Spalled off	101 ±6

TABLE 7 .

 7 Average and standard deviation for the chromium content in alloy close to the interface with the external oxide (3 EDS spot analyses)

	Alloys	Chromium content in extreme	Difference to the initial Cr content
	(24h 1237°C)	surface (wt.%)	(wt.%)
	"NCT10Cr"	4.1 ±0.2	-6.1
	"NCT20Cr"	14.5 ±0.3	-5.3
	"NCT30Cr"	21.5 ±0.2	-5.7
	"NCT40Cr"	33.7 ±0.1	-6.6
	"NCT50Cr"	42.7 ±1.1	-6.9