The influence of feeding strategies on trace element bioaccumulation in copepods (Calanoida)

This is the author's final version of the contribution published as:

[inserire: M. Battuello, R. Mussat Sartor, P. Brizio, N. Nurra, D. Pessani, M.C. Abete,

S. Squadrone. The influence of feeding strategies on trace element bioaccumulation

in copepods (Calanoida). Ecological Indicators 74, (2017) 311-320,

10.1016/j.ecolind.2016.11.041

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The influence of feeding strategies on trace element bioaccumulation in copepods (Calanoida).

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M. Battuello1_{, R. Mussat Sartor}1#_{, P. Brizio}2#_{, N. Nurra}1#_{, D. Pessani}1_{, M. C. Abete}2

, S. Squadrone2*

1 _{Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina}

13, 10123 Torino, Italy

2 _{Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, via Bologna 148, 10154 Torino,}

Italy.

*Corresponding author. Tel.: +39 011 2686415; fax: +39 011 2686228; e-mail address:

stefania.squadrone@izsto.it

# _{These authors equally contributed to this work.}

Abstract

Copepods are the most numerous taxonomic group in marine mesozooplankton communities. These planktonic organisms have an essential role in the function of marine trophic webs, as they are the link between phytoplankton and secondary consumers.

The concentrations of 20 essential and non-essential trace elements were investigated by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) in calanoid copepods with different feeding behaviors. The sampling was performed in the Northwestern Mediterranean Sea, at the border between the Northern Tyrrhenian Sea and the Ligurian Sea (Italy). Aluminum, iron, zinc and copper were present in the highest concentrations in herbivorous calanoids (Temora stylifera,

Nannocalanus minor, Neocalanus gracilis). Conversely, the nonessential element - arsenic - and the

essential elements selenium and molybdenum - were present in the highest levels in carnivores (Pontella mediterranea, Candacia ethiopica). In the omnivorous copepod Centropages typicus, metal concentrations were found at an intermediate level between herbivores and carnivores, reflecting the importance of dietary pathways in metal intake and bioaccumulation.

Finally, the bioaccumulation factors (BAFs), expressed as a ratio of the total metal levels in copepods compared to the seawater metal levels, were as follows: herbivores (83699) > omnivores (47855) > carnivores (41648).

Keywords: trace elements, Calanoida copepods, Mediterranean Sea, feeding habits, BAFs.

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1. Introduction

Trace elements are ubiquitous in marine waters and biota, due to their natural origin and to the presence of anthropogenic sources, in particular in coastal areas.

Aquatic invertebrates have a crucial role in the trophic transfer of metals in the marine food chain. These organisms are exposed to inorganic contaminants from both the particulate and the dissolved phases of seawater (Wang, 2002), and mainly accumulate these pollutants by food uptake (Fisher and Reinfelder, 1995). Marine organisms have developed different strategies to deal with the toxicity of metals. The essential trace elements, such as manganese, copper, iron and zinc are under homeostatic control, as they are necessary for many physiological functions, while the nonessential trace elements, such as cadmium, lead, mercury and aluminum are not metabolically regulated (CIESM, 2002). However, marine invertebrates can eliminate inorganic contaminants by excretion (molting, defecation) or by storing them in physiologically-inactive pools inside their body (CIESM, 2002).

In oligotrophic open waters, the contribution of zooplankton excretion to nutrient regeneration is high, providing more than 40% of the nutrients required by phytoplankton (Bode et al., 2015). By contrast, this portion is lower in more productive upwelling regions (<40%), where nutrients become replenished in the euphotic zone from deeper layers (Frangoulis et al., 2005). In general, the majority of nutrients are recycled through the microbial loop (Bode and Varela, 1994).

Copepods are small aquatic crustaceans that constitute the most abundant component of zooplankton, playing a primary role in the energy pathways involved in transfer from primary producers to secondary consumers (Richardson, 2008; Fernández-Severini et al., 2013). The bio-diminution of metals (concentration decreases at a higher trophic level) in the classical marine planktonic food chain (phytoplankton to copepods to fish) is largely due to the efflux of metals by copepods and the very low accumulation of trace elements by marine fish (Wang, 2002).

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Copepods are divided into ten orders; the Calanoida order comprises 2266 species belonging to 42 families of benthic, hyperbenthic and pelagic copepods, which are mainly marine (75%) (Razouls, 2005-2016; Boxshall and Halsey, 2004). Calanoid copepods, thought to have benthic or benthopelagic origins are the most successful colonizers of the pelagic realm (Bradford-Grieve,

2002); calanoids are the major component of plankton in the world's oceans, constituting 55%–95%

of plankton samples. From an ecological point of view, copepods have key roles in the trophic webs of marine ecosystems, oligotrophic ocean gyres, and highly productive upwelling systems (Mauchline, 1998).

This investigation focuses on six of the dominant species of the coastal copepod community in the Northwestern Mediterranean Sea, which belong to five of the most studied calanoid families. These species were used as representatives of the epipelagic copepod community in order to study differences in their feeding behaviors.

The perception of the trophic role of calanoid copepods has changed during recent decades. In the past, they were viewed as purely herbivorous, which acted as a link between primary production and planktivorous fish. Calanoid copepods are thought to use different feeding modes depending on their species-specific mouthparts and the movements that are possible with them (reviewed in Sanders and Wickham 1993, Ohman and Runge 1994, Nejstgaard et al. 1997).

Copepods can potentially obtain food from any known stock of organic matter in dissolved or par-ticulate form; their feeding appendages and behavior allow them to capture particles, phytoplankton or detritus, of just a few microns in size or to attack living zooplankton organisms, such as chaetog-naths, medusae or other copepods (Poulet, 1983; Benedetti et al., 2016). Calanoid copepods can ac-tively search for, capture and choose to ingest or reject potential food particles. If feeding is selec-tive, then the role that copepods play in the ecosystem is partly a result of the strategies they use to obtain their diets (Kleppel, 1993). Trophic relationships are complex, as most calanoid species do

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portunistic omnivorous feeders (Kleppel, 1993; Kiørboe et al., 1996; Kleppel et al., 1996; Vincent and Hartmann, 2001; Calbet, 2008; Escribano and Pérez, 2010; Schukat et al., 2014). Feeding movements of an individual copepod seem to be modified under different food conditions, or rather, according to different sizes and concentrations of food particles (Koehl and Strickler 1981). If cope-pods are herbivorous or carnivorous, and whether they switch between different feeding modes in-fluences phyto- and micro-zooplankton communities, as well as food web interactions (Stibor et al. 2004). However, the natural diet of most copepod species and their trophic positions remain largely unclear. This is especially the case in oligotrophic environments, where the restricted availability of phytoplankton may often be compensated by feeding on heterotrophic protozoans and micro-zoo-plankton (Calbet and Saiz, 2005).

We will now discuss specific calanoid copepods in detail; Centropages typicus is a copepod that has been extensively investigated in the Mediterranean Sea because of its wide distribution (Ji et al., 2013; Razouls and Lepernac, 2006), its role in planktonic communities (Kiørboe and Jiang, 2013), its physiological adaptations and ecological value (Maibam et al., 2015), and its spatial distribution linked to climate forcing (Gaudy and Thibault-Botha, 2007; Molinero et al., 2005). C. typicus is an omnivorous copepod that feeds on a wide spectrum of prey, such as small algae, ciliates, dinoflagellates, and fish larvae (Calbet et al., 2007). The calanoid Temora stylifera is primarily herbivorous (Turner, 1984), and has been thoroughly investigated regarding many biological aspects (Di Capua and Mazzocchi, 2004; Molinero et al., 2005). The calanoids Neocalanus gracilis and Nannocalanus minor, which are also present in subtropical and tropical oceans, are predominantly herbivorous, and graze in the surface layers during the spring and early summer, in order to store lipids in their bodies (Shimode et al., 2009; Doi et al., 2010). The neustonic copepod

Pontella mediterranea is the most common Pontellidae in the Northwestern Mediterranean Sea

(Collard et al., 2015). Neustonic organisms inhabit the surface layer of the water column, a zone particularly susceptible to pollutant accumulation (Garcia-Flor et al., 2008). The Pontellidae are

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omnivorous (Turner, 1978), despite being previously identified as being exclusively carnivorous organisms, although they mainly prey on fish larvae and other copepods (Lillelund and Lasker, 1971). Finally, Candacia ethiopica is a specialized epipelagic, raptorial predator, whose diet includes larvaceans and chaetognaths (Lopez-Irrutia et al., 2004).

In a previous study (Battuello et al., 2016), we investigated the presence of trace elements in marine Mediterranean mesozooplankton, composed of several taxa, copepods, cladocerans, misydaceans/euphausiaceans, chaetognaths and other less abundant groups such as pteropods and siphonophores, collected during all four seasons at three different water depths. Our results demonstrated that metal concentrations in marine zooplankton showed important variations between seasons and water depths, and that zooplankton was a significant metal bioaccumulator, as well as a suitable bioindicator of the presence of trace elements in the studied environment. However, many factors are involved in metal bioaccumulation in marine organisms, e.g. age, diet, trophic level, habitat and environmental availability. Therefore, in the present study, we only analyzed copepods, and in the six aforementioned species belonging to the order Calanoida, focusing on the influence of the different feeding habits (herbivorous, omnivorous and carnivorous) in the bioaccumulation of metals in a zooplanktonic food web.

The aims of the present investigations were:

i) To analyze the concentrations of 20 trace elements in copepod species belonging to the

Calanoida order, including herbivores (Temora stylifera, Nannocalanus minor,

Neocalanus gracilis), omnivores (Centropages typicus), and carnivores (Candacia ethiopica, Pontella mediterranea).

ii) To evaluate the relevance of the different dietary habits of these copepods in the bioaccumulation of trace elements and their transfer through marine food chains.

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2. Materials and methods

2.1 Study area

The sampling site was situated in the Southern Ligurian Sea, in the neritic waters off the Tuscan coast (Fig. 1). The considerable extension of the continental shelf and the limited depth (100 m), even at a great distance from the coast are distinctive features of the investigated region (Chiocci and La Monica 1996). The sampling station (43°28’10” N, 10°01’55” E) was located above the continental shelf at a point in which the bottom depth was 111 m (Fig. 1), corresponding to one of the stations considered in our previous study (Battuello et al., 2016) in order to allow comparison between levels of trace elements in mesozooplankton (previous study) and copepod species (Calanoida order).

2.2 Sampling

Sampling was performed during summer 2015. Surface zooplankton samples were collected at night to allow surface sampling of species involved in nictemeral migrations. Four different samples were collected, as previously described (Battuello et al., 2016) with a WP-2 standard net (mesh size 200 m and diameter 57 cm); (Demina et al., 2009; Rentería-Cano et al., 2011; Bhattacharya et al., 2014). After collection, each plankton sample was washed on board first with filtered seawater from the sampling site, in order to remove terrigenic or inorganic particles, followed by washing with distilled water. After the washing procedure, samples were immediately fixed in 4% neutralized formaldehyde buffered with borax and kept in the dark (Boltovskoy 1981). Each zooplankton sample was divided into two subsamples using a Folsom Plankton Splitter

(Fer-nández de Puelles et al., 2014). One aliquot was used to determine the abundance (ind. m-3_{) of the}

calanoid copepod species investigated. In the laboratory, a qualitative-quantitative analysis of the copepod community was performed on subsamples obtained using the Folsom Plankton Splitter, ranging from 1/10 to 1/25, depending on the total sample abundance (Brugnano et al., 2010).

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over, the entire sample was carefully observed in order to identify rare species (Vives and Shmel-eva, 2007, 2010).

To avoid possible contamination by elements on the surface of the zooplanktonic organisms, each zooplankton sample from the second set of aliquots was washed in the laboratory three times with distilled water. Subsequently, in order to compare the differences in metal content in relation to their different feeding habits (herbivorous, omnivorous, or carnivorous), four subsamples, each of about 500 – 1000 specimens, were selected for each of the six copepod species.

Seawater samples (n=3) were collected during the same sampling at a depth of 1 m, as described in the previous study (Battuello et al., 2016), and subjected to trace element analysis.

2.3 Analysis of trace elements

Calanoid species samples (n=4 for each species, as previously described) were accurately rinsed with Milli-Q water to remove any formaldehyde buffer before detection of trace elements.

Determination of the levels of aluminum (Al), arsenic (As), beryllium (Be), cadmium (Cd), cerium (Ce), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), lanthanum (La), lead (Pb), nickel (Ni), antimony (Sb), selenium (Se), tin (Sn), thallium (Tl), vanadium (V) and zinc (Zn) was performed by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS Xseries II, Thermo Scientific, Bremen, Germany), following the protocol already described (Battuello et al., 2016). In brief, multi-elemental determination was performed by ICP-MS after daily optimization of instrumental parameters and by using an external standard calibration curve; rhodium and germanium were used as internal standards. Analytical performances were verified by processing Certified Reference Materials (Oyster Tissue - SRM 1566b from the National Institute of Standard and Technology), along with blank reagents in each analytical session. The limit of quantification (LOQ), the reference material values and the percentages of recovery obtained are shown in Table S1, while metal concentrations for all samples

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are shown in Table S2. The analytical method was validated according to ISO/IEC 17025 (general requirements for the competence of testing and calibration laboratories).

Seawater samples were subjected to a pre-concentration/matrix elimination process by a chelating

polymer resin, the SPR-IDA Reagent (Suspended Particulate Reagent – Iminodiacetate, by Cetac

Technologies, Omaha, USA) following the manufacturer’s instructions. Resulting extracts were diluted to 3 mL with deionized water. Al, Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn levels were then quantified from seawater samples by ICP-MS.

2.4 Bioaccumulation factors (BAFs)

The ratio of the concentration of metals in copepods compared to the concentration in seawater was evaluated, in order to estimate the bioaccumulation factor (BAF), which takes into account the concentration of metals in an aquatic organism resulting from all possible routes of exposure in a natural environment (McGeer et al., 2003). BAFs were evaluated by groups, considering the feeding strategies of the calanoids, i.e. carnivores, herbivores, omnivores. Metal levels were

expressed as g kg-1 _{in copepods and as g L}-1 _{in seawater.}

2.5 Statistical analysis

The abundance of copepods (reported as ind. m-3_{) from each replicate sample was compared using}

the Kruskal-Wallis test, and statistical analyses were performed using the R software, Version 3.1.2 (R Core Team, 2015); p < 0.05 was considered as statistically significant. No significant differences

were found between the four replicates (Kruskal-Wallis X2 _{= 1.550, dƒ = 3, p = 0.671).}

Before analyzing metal concentrations, we performed the D’Agostino-Pearson normality test to determine the distribution of the values. The one-way analysis of variance (ANOVA) was used to test differences in metal concentrations between herbivores, omnivores and carnivores. Results were considered statistically significant with p values of < 0.05. Statistical calculations were

performed using Graph Pad Statistics Software Version 6.0 (GraphPad Software, Inc., USA).

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3. Results and discussion

3.1 Abundance and diversity of copepods

In total, 98 species of copepods belonging to 24 families and 41 genera were recorded during the survey. Among them, the order Calanoida was represented by 59 species (60.2%) belonging to 17 families (70.8%) and 27 genera (65.8%). The six target species Centropages typicus, Temora

stylifera, Nannocalanus minor, Neocalanus gracilis, Pontella mediterranea and Candacia ethiopica

were observed in all samples. In addition, the herbivorous calanoids Acartia negligens,

Centropages violaceus, Clausocalanus arcuicornis, Clausocalanus furcatus, Clausocalanus pergens, Calocalanus pavo (Mazzocchi and Paffenhöfer, 1998), and the omnivorous (mainly

carnivorous) Pontellina plumata and Pontellopsis regalis (Turner, 1978) were recorded with a 100% frequency. However, only the six target species were analyzed for trace element concentrations, as they were representative of the different feeding habits, and the biomasses of these samples were sufficient to perform the analysis. Conversely, eight species were recorded only once, namely the hyponeuston organisms, Pontellidae Labidocera brunescens and Pontellopsis

villosa (Collignon et al., 2014), along with Euchaeta marina, Paraeuchaeta hebes, Mecynocera clausi, Pleuromamma gracilis, Scolecithricella dentata and Scolecithix bradyi, widely distributed in

the Western Mediterranean Sea, and that inhabit the deepest portions of the water column (Brugnano et al., 2010 and 2012, Fernández de Puelles et al., 2014). In the surface samples, the Shannon-Wiener index diversity (loge) ranged from 2.446 – 2.725, while the abundance of

copepods in the four replicates ranged from 328.12 – 498.02 ind.m-3 _{(167.68 – 368.11 calanoids). Of}

our calanoid copepod targets, the most dominant species in all replicates was N. minor (30.52 ind.m

-3_{; SD 10.01), accounting for 11.26% of the relative abundance of calanoids. Other abundant target}

species were T. stylifera (25.58 ind.m-3_{; SD 16.94) and C. typicus (7.12 ind.m}-3_{; SD 2.68),}

accounting for 9.44% and 2.63%, respectively. Instead, P. mediterranea (1.04 ind.m-3_{; SD 16.94),}

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C. ethiopica (10.52 ind.m-3_{; SD 0.20) and N. gracilis (0.25 ind.m}-3_{; SD 0.13) were present at lower}

abundances.

3.2 Trace elements in seawater and in calanoid copepods

In surface water, the concentrations of dissolved elements were as follows: Zn>Ni>Fe>Al>Pb>Co>Mn>Cu>Cd (Table S3). The trace elements with the highest concentrations were nickel and zinc (10.6 g L-1 _{and 11.4 g L}-1 _{respectively). Metal concentrations were}

comparable to those previously registered in the Mediterranean Sea (Ebling and Landing, 2015, Battuello et al., 2016).

Levels of the analyzed trace elements in all samples species are shown in Table S2, and are

expressed in mg kg-1 _{wet weight (w.w.). Copepods were also grouped together as herbivorous}

(Temora stylifera, Nannocanalus minor, Neocanalus gracilis), carnivorous (Candacia ethiopica,

Pontella mediterraea) or omnivorous (Centropages typicus); results are shown in Table 1 (mean ±

standard deviation) and in Figures 2 and 3. Concentrations of the most represented trace elements (Al, Fe, Cu and Zn) are reported for all the analyzed species in Figure 4, the highest values were registered in the herbivorous calanoida, with important interspecific differences. The highest concentrations of Al and Fe were found in Temora stylifera, while the highest level of Cu was registered in Nannocanalus minor and the highest concentration of Zn in Neocanalus gracilis. The statistical evaluation results (one-way ANOVA) are shown in Table 2.

3.2.1 Nonessential trace elements

The concentrations of nonessential trace elements in copepods are reported in Table 1, Table S2 and Figure 2. Differences in metal levels related to the feeding strategy (herbivorous, omnivorous or carnivorous) were statistically significant (Table 2), apart from antimony and vanadium. The highest Al, Cd, Pb, and Sn levels were recorded in herbivores, while the highest mean level of As was found in carnivorous calanoids.

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Aluminum, antimony and vanadium have been scarcely investigated in marine biota and, to our knowledge, no data are available for these elements in copepod species from the Mediterranean Sea. Al was the most represented nonessential trace element (Figure 4) in all the examined copepod species, reflecting its ubiquity in the aquatic environment and in accordance with a previous report in marine zooplankton (Battuello et al., 2016). The highest levels of Al were found in herbivorous

copepods (68.28 mg kg-1_{), with a high interspecies and inter-group variability (Table S2, Table 1);}

the highest Al concentration was found in the herbivorous copepod Temora stylifera. Antimony is usually present at very low levels in the environment and, in this study, Sb levels were subjected to low variability, with concentrations of 0.02 - 0.03 mg kg-1 _{(Table S2, Figure 2). Vanadium}

concentrations were low in all the studied species, ranging from 0.07 mg kg-1 _{in the herbivorous N.}

gracilisto 0.25 mg kg-1 _{in the carnivorous C. ethiopica (Table S2).}

It has been suggested that cadmium concentrations are usually lower at higher trophic levels of the planktonic food chain (Rainbow, 2002). Accordingly, we found the highest level of Cd in the

herbivorous Neocalanus gracilis (0.35 mg kg-1_{). Considering the calanoid feeding habits, Cd values}

were found to be greatest in herbivores>carnivores>omnivores (Figure 2, Table 1). Compared to other findings in marine mesozooplankton from Mediterranean coastal areas (Miramand et al., 2001; Battuello et al., 2016), we found lower concentrations of this toxic and nonessential element in the analyzed species.

The highest level of lead was found in the carnivorous Candacia ethiopica (1.30 mg kg-1_{) and the}

lowest Pb level was found in the omnivorous Centropages typicus (0.41 mg kg-1_{). Considering the}

mean values, Pb levels were the same (0.97 mg Kg-1_{) in herbivorous and carnivorous calanoids}

(Table 1, Figure 2). Pb concentrations found in copepods were lower than those recorded in a

previous study on mesozooplankton (1.42 mg Kg-1_{) in this coastal environment (Battuello et al.,}

2016). 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292

Arsenic concentrations in seawater usually range from 1 to 5 g L-1_{, and it is mainly present as}

inor-ganic arsenic (Caumette et al., 2011). Shibata and coauthors (1996) demonstrated that phytoplank-ton, herbivorous zooplankphytoplank-ton, and carnivorous zooplankton accumulate arseno-compounds in dif-ferent ways, probably reflecting their difdif-ferent feeding habits. Our findings have shown that As

lev-els in copepods were higher in carnivores (0.20 mg Kg-1_{), followed by herbivores and omnivores}

(Figure 2, Table 1). The differences between the three groups (Table 2) were statistically very

sig-nificant (p<0.0001).In particular, in the carnivorous C. ethiopica, the highest concentrations of As

was found (0.27 mg Kg-1_{) while the lowest (0.09 mg Kg}-1_{) were registered in the herbivorous T.}

stylifera and in the omnivorous C. typicus.

Tin concentrations were found to be low and thus of no concern, according to previous findings (Battuello et al., 2016), and in the following order herbivores> omnivores> carnivores (Table 1, Figure 2).

In marine environments, the presence of the rare earth elements (REE) lanthanum and cerium reflects both their natural occurrence in sediment and in industrial wastewaters (Oral et al., 2010).

REE levels are very low in seawater (pg L-1_{), but zooplankton organisms tend to bioaccumulate}

these elements (Wang and Yamada, 2007; Palmer et al., 2006). In Calanoida copepods, we found that Ce and La concentrations were in the following order herbivores> omnivores> carnivores, with Ce concentrations higher than La levels (Table 1, Table 2, Figure 2).

3.2.2 Essential trace elements

The concentrations of essential elements in the studied copepods are shown in Table 1, Table S2 and Figure 3. The highest levels of cobalt, copper, iron, manganese, nickel and zinc were found in herbivores, and the highest levels of selenium and molybdenum were found in carnivores.

Statistically significant differences were found between the three groups for all the essential elements (Table 2).

Cobalt is a particle-reactive metal and is scavenged by suspended particulate matter in coastal areas

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(Paimpillil et al. 2010). In our study, the lowest Co level (0.03 mg Kg-1_{) was found in the}

omnivo-rous C. typicus, and the highest Co level (0.26 mg Kg-1_{) was found in the herbivorous N. gracilis}

(Table S2, Figure 3).

Some marine organisms are known to absorb significant amounts of copper from the seawater, such as crustaceans, which require this element as an enzyme component and for hemocyanin (Paimpillil

et al. 2010). Copepods do not express hemocyanin, and the requirement of Cu could be restricted to

the activity of enzymes important growth and/or for egg production (Paimpillil et al. 2010).

Copper concentrations in the analyzed copepods ranged from 48.35 mg kg-1 _{in herbivores to 17.96}

mg kg-1 _{in carnivorous calanoids (Table 1, Figure 3), with the highest value in the herbivorous}

Nannocalanus minor (Table S2). These concentrations were an order of magnitude higher than

those found previously in mesozooplankton (Battuello et al., 2016), and the differences between the three groups (Table 2) were statistically very significant (p<0.0001).

Iron is a trace element that is essential for the biological requirements of marine plankton, and in

this investigation, iron was the most represented essential element, second only to the nonessential

but ubiquitous aluminum (Figure 4, Table 1). The highest level of iron was found in Temora

stylifera (78.95 mg kg-1_{), and the lowest level in Candacia ethiopica (25.69 mg kg}-1_{). In fact, we}

found decreasing levels from herbivorous to carnivorous calanoid copepods (Figure 3). These concentrations were an order of magnitude lower than those found previously in mesozooplankton (Battuello et al., 2016).

Manganese is another essential trace element for marine microorganisms, and we found

concentrations ranging from 0.70 mg kg-1 _{in herbivores to 0.14 mg kg}-1 _{in carnivores. These levels}

were lower than those found in Mediterranean marine zooplankton (1.27 mg kg-1_{; Battuello et al.,}

2016).

Chromium levels detected in the three groups of calanoid copepods (herbivores, omnivores, carnivores) were comparable to those recorded in mesozooplankton in the same area and season by

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Battuello and coauthors (2016). The highest levels of chromium (0.19 mg kg-1_{) were recorded in the}

herbivorous N. gracilis (Table 1, Table S2), comparable to the Cr level in zooplankton of our previous study, while we found lower values in herbivores and omnivores (Figure 3).

The trace element molybdenum is detectable in all living organisms as it is an essential micronutri-ent (Eisler 1989). We found a very low Mo contmicronutri-ent in the analyzed calanoids, ranging from 0.03 mg

kg-1 _{in T. stylifera to 0.12 mg kg}-1 _{in C. ethiopica (Table S2). The concentration of the essential}

ele-ment nickel varied greatly between species (Table S2) and groups (Table 1), ranging from 1.47 mg

kg-1 _{in herbivores to 0.24 mg kg}-1 _{in omnivores (Figure 3).}

Previous studies have reported little bioaccumulation of selenium along marine food webs from

al-gae to herbivores and carnivores (Liu et al.,1987), and biological factors such as body size, species

and trophic status could all influence Se concentrations in zooplankton (Goede et al., 1993). The possibility that the feeding habits of zooplankton (herbivorous versus carnivorous) may influence their Se concentrations has been previously suggested (Purkerson et al., 2003; Sakata et al., 2015); in this study, we found the highest Se concentrations in carnivorous calanoids (Table 1, Figure 3). The concentration of Zn was highly variable among calanoids (Table 1, Table S2), even within a particular group of the same feeding behavior (e.g. herbivores), which registered the highest mean

value (48.84 mg kg-1_{). The lowest value was found in the omnivorous Centropages typicus (1.86 mg}

kg-1_).

3.3 Comparison with our previous study on mesozooplankton

In our previous investigation (Battuello et al., 2016), we measured the concentrations of trace elements in marine zooplankton from a Northwestern Mediterranean coastal area without any distinction between taxa and species. We concluded that metal concentrations in marine zooplankton provide important information about bioavailabilities of trace elements in the environment (Battuello et al., 2016). In fact, zooplankton has been shown to be a highly suitable bioindicator in the biomonitoring of metals in marine ecosystems. The present study was performed

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in the same area to allow comparison with our previous findings. In Figure 5, we reported metal levels in Calanoida (considering their feeding behavior) and in marine zooplankton, both collected in summer 2015 in surficial marine waters. Here, we demonstrate that focusing on a particular order or species of marine invertebrate leads to different results in metal concentrations and thus a different significance of the organism as a bioindicator. In fact, lower values of As, Cd, Co, Cr, Fe, Mn, Mo, Pb, Sb and V were detected in calanoid copepods compared to mesozooplankton samples, while Se and Ni levels were similar. The essential elements copper and iron were particularly high in all the Calanoida species examined, compared to marine zooplankton, perhaps reflecting a higher requirement of Cu and Fe in this order of marine invertebrate.

Trace element concentrations in zooplankton have a high heterogeneity, probably due to their complex taxonomic composition. Aquatic invertebrates accumulate both essential and nonessential trace metals, and marine organisms from the same habitat were found to have very different levels of trace metals, even within closely related taxa, e.g. species in the same genus (Moore and Rainbow, 1987; Rainbow et al., 1993; Rainbow, 1998). Several studies have already shown that assimilation efficiencies of metals from food strongly depend on food preferences, feeding strategies, and digestive physiology (CIESM, 2002). In our study, we suggest that the diverse feeding strategies of marine invertebrates may strongly influence their body metal concentrations. In fact, in spite of the high variability in metal concentrations within calanoid species, which could be due to large differences in metal assimilation efficiencies between species, we observed a biodilution of metal levels through this zooplanktonic food web, from herbivores to carnivores. In fact, herbivorous species showed the highest metal concentrations and BAFs for most of the analyzed metals. Biodilution is defined as the decrease in concentration of an element or pollutant as the trophic level increases (Campbell et al. 2005). Primary producers, such as phytoplankton, actively assimilate and passively transport trace elements from the seawater, and accumulate them in their cells (Rentería-Cano et al., 2011). Primary consumers, such as herbivorous zooplankton,

368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392

incorporate these metals - bound by the phytoplankton - into their cells; a higher phytoplankton biomass usually means a lower concentration of metals accumulated by the zooplankton (Pickhardt

et al., 2002).

Biodilution of certain elements has been demonstrated both in freshwater and marine environments, and several studies have documented that metal concentration decreases with increasing trophic levels (Besser et al., 2001; Pickhardt et al., 2002; Quinn et al., 2003; Campbell et al., 2005; Watanabe et al., 2008; Sakata et al., 2015). In particular, Sakata and coauthors (2015) investigated the trophic level-dependent accumulation of elements in biota from Suruga Bay, Japan; their results showed a weak biomagnification for As and Se, and biodilution for Cd, Cu, Mn, Ni, Pb, Sb and Zn. For some trace elements, such as As, Se and Zn, a characteristic trophic transfer related to location was suggested, depending on both the food web structure and environmental factors (Sakata et al., 2015). Some authors have proposed that biodilution of trace elements could be a consequence of the lower surface: mass ratio or to an increasingly efficient excretion of metals at higher trophic levels (Rainbow, 2002; Campbell et al., 2005). In our study, in the calanoid species examined, the body size did not increase with the trophic level and, apart from the differences in physiology that may be unique to each species, the main difference between species was related to the feeding strategy. Some studies have previously demonstrated that the feeding behavior of invertebrates influences the metal burden (Beltman et al., 1999; Besser et al., 2001; Quinn et al., 2003).

3.4 Bioaccumulation factors (BAFs)

Bioaccumulation factors are utilized in determining the level of risk assessment evaluations (Swan-son et al., 1997). The BAF (Bioaccumulation Factor) and BCF (Bioconcentration Factor) represent models for bioaccumulation (Newmann, 1995), and predict partitioning between an exposure medium (such as marine water) and biota (such as copepods). BAFs are usually utilized in natural environments (McGeer et al., 2003), as in the present study.

393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416

BAFs were estimated for Al, Cd, Co, Cu, Fe, Mn, Ni, Pb, and Zn (Figures 6a and 6b). The

average BAFs in copepods were: herbivores: Al>Cu>Fe>Cd>Zn>Mn>Pb>Ni>Co omnivores: Al>Cu>Fe>Cd>Pb>Mn>Zn>Ni>Co carnivores: Fe>Cu>Al>Cd>Zn>Pb>Mn>Ni>Co

While BAF trends were fairly similar between herbivores and omnivores, carnivorous calanoids showed a different pattern of accumulation, with iron (an essential element) rather than aluminum (nonessential but ubiquitous in the environment) being the most bio-accumulated metal.

We have previously demonstrated the high potential of zooplankton as bioaccumulators of metals (Battuello et al., 2016). However, considering the mesozooplankton collected from the same area and in the same season, we found a different trend in the BAFs of metals (Figure 7): Fe>Cd>Cu>Mn>Zn>Pb>Ni>Co>Al.

Moreover, the BAFs for Cd, Co, Mn, Ni and Pb were higher in marine zooplankton than in Calanoida, especially for Cd, Fe and Mn. Therefore, we suggested that these differences in BAFs were probably due to the presence of other zooplankton orders and families in the analyzed samples, having different abilities to accumulate and concentrate metals.

4. Conclusions

This is the first study to analyze the concentrations of 20 trace elements in different species of calanoid copepods, specifically focusing on their feeding habits. Dietary exposure is a significant factor for metal accumulation in marine organisms, such as zooplankton, and feeding strategies were very important for the bioaccumulation and transfer of metals in marine food chains.

In our study, we observed the biodilution of metal levels from herbivorous to carnivorous calanoid copepods. In the perspective of utilizing marine organisms as bio-indicators of metal transfer through the marine trophic web, it is crucial to consider that different organisms have different nutritional requirements and abilities to accumulate or excrete trace elements.

417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

This study has provided new data about the distribution of trace elements in Calanoida copepods, making progress toward the goal of developing a comprehensive understanding of their role as a tool for monitoring marine ecosystems.

Acknowledgements

The Italian Ministry of Health financially supported this study (Project n.14C14).

The authors particularly thank the editor and the reviewers for their constructive suggestions, which have greatly improved the quality of this manuscript.

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Fig.1

: Ligurian Sea (Western Mediterranean): sampling station

654

Fig.2: Box-plot diagrams of nonessential trace elements (mean concentrations ± SD) in herbivorous (n=12), omnivorous (n=4) and carnivorous

(n=8) calanoid copepods. Metal levels are expressed in mg kg-1 _{wet weight (Y axis)}

656

657

658 659 660

Fig.3. Box-plot diagrams of essential trace elements (mean concentrations ± SD) in herbivorous (n=12), omnivorous (n=4) and carnivorous (n=8)

calanoid copepods. Metal levels are expressed in mg kg-1 _{wet weight (Y axis).}

661

662

663 664 665

Fig. 4. Most represented trace elements in the analyzed species (mg kg-1 _{wet weight). The total}

metal values of the represented elements for each species was reported (X axis).

Al As Cd Co Cr Cu Fe Mn Mo Ni Pb Sb Se Sn V Zn 0.01 0.1 1 10 100

herbivorous Calanoida omnivorous Calanoida carnivorous Calanoida zooplankton (previous study)

m g K g-1 lo g sc al e

Fig. 5. Comparison between trace element concentrations in Calanoida copepods and marine

zooplankton (mg kg-1 _{wet weight).}

Temora stylifera Nannocanalus minor Neocanalus gracilis Centropages typicus Candacia ethiopica Pontella mediterranea 0 Zn50 Cu 100Fe 150Al 200 250 300 666 667 668 669 670 671 672 673 674 675 676 677 678 679

Al _Cd Co _Cu Fe _Mn Ni _Pb Zn 0 5000 10000 15000 20000 25000 30000 35000

Fig. 6a. Bioaccumulation factors (BAFs) in calanoid copepods.

0 10000 20000 30000 40000 50000 60000 70000 80000 90000

83699 47855

41648

BAF herbivores BAF omnivores BAF carnivores

Fig. 6b. Total metal BAFs related to feeding groups (ratio between the sum of all metals in calanoid

copepods and in water).

680 681 682 683 684 685 686 687

10 100 1000 10000 100000

herbivorous Calanoida omnivorous Calanoida

carnivorous Calanoida marine zooplankton (previous study)

Al Cd Co Cu Fe Mn Ni Pb Zn

B

A

F

s

Fig. 7. Comparison between BAFs of metals in Calanoida copepods and marine zooplankton.

688 689 690 691 692 693 694 695 696 697 698 699 700 701

Table 1.

Levels of trace elements in calanoid copepods with different feeding strategies (mg Kg -1 _w.w.)

feeding behavior herbivorous (n=12 samples) omnivorous (n=4 samples) carnivorous (n=8 samples)

mean SD mean SD mean SD

Al 68.28 62.01 30.21 1.35 17.62 4.13 As 0.11 0.03 0.09 0.01 0.20 0.10 Cd 0.15 0.17 0.01 0.00 0.02 0.01 Ce 0.09 0.06 0.05 0.01 0.03 0.01 Co 0.10 0.11 0.03 0.01 0.04 0.01 Cr 0.26 0.06 0.19 0.02 0.23 0.01 Cu 48.35 50.43 18.50 0.52 17.96 0.16 Fe 51.59 24.84 30.52 1.13 32.40 9.54 La 0.04 0.03 0.03 0.01 0.01 0.00 Mn 0.70 0.35 0.17 0.01 0.14 0.06 Mo 0.06 0.04 0.10 0.01 0.10 0.02 Ni 1.47 1.70 0.24 0.01 0.71 0.29 Pb 0.97 0.18 0.41 0.02 0.97 0.47 Sb 0.02 0.010 0.02 0.00 0.03 0.01 Se 0.50 0.36 0.43 0.02 0.72 0.05 Sn 0.12 0.05 0.09 0.01 0.08 0.02 V 0.16 0.08 0.12 0.01 0.19 0.10 Zn 48.84 34.93 1.86 0.07 9.02 2.99 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723

Table 2. One way ANOVA, comparison between the three groups (herbivores, omnivores, carnivores)

Trace

element P value Summary of Pvalues

Al P = 0.0237 (P < 0.05) * As P < 0.0001 **** Cd P = 0.0212 (P < 0.05) * Ce P = 0.0026 (P < 0.01) *** Co P = 0.0291 (P < 0.05) * Cr P = 0.0252 (P < 0.05) * Cu P = 0.0357 (P < 0.05) * Fe P = 0.0161 (P < 0.05) * La P = 0.0031 (P < 0.05) * Mn P < 0.0001 **** Mo P < 0.0008 (P < 0.01) *** Ni P = 0.0290 (P < 0.05) * Pb P = 0.0018 (P < 0.01) ** Sb P = 0.4835 (P > 0.05) NS Se P = 0.0283 (P < 0.05) * Sn P = 0.0477 (P < 0.05) * V P = 0.3491(P > 0.05) NS Zn P < 0.0004 (P < 0.01) *** 724 725 726 727 728 729 730 731 732 733 734 735

Table S1. Quantification limit (mg Kg-1_{), reference material values (oyster tissue) and percentages} of recovery. Elemen t LOQ SRM 1566b % Recovery Al 0.010 197.2 ± 6.0 82 As 0.010 7.650.65± 103 Be 0.010 - -Cd 0.010 2.48±0.08 104 Ce 0.010 - -Co 0.010 0.371±0.009 99 Cr 0.010 0.371±0.009 99 Cu 0.010 71.6±1.6 105 Fe 0.010 205.8±6.8 104 La 0.010 - -Mn 0.010 18.5±0.2 95 Mo 0.010 - -Ni 0.010 1.04±0.09 98 Pb 0.010 0.308±0.009 98 Sb 0.010 0.011±0.002 96 Se 0.010 2.06± 0.15 117 Sn 0.010 0.031±0.008 102 Tl 0.010 - -V 0.010 0.0577 ± 0.023 102 Zn 0.010 1424±46 105 736

Table S2.

Calanoida species; concentrations of trace elements (mg Kg-1 _w.w.)

T em or a st yl if er a sample 1 (n= 628 individuals ) sample 2 (n= 614 individuals ) sample 3 (n= 602 individuals ) sample 4 (n= 732 individuals)

mean SD min max

Al 130.18 144.80 135.35 139.21 137.39 6.17 130.1₈ 144.8₀

As 0.08 0.07 0.10 0.09 0.09 0.01 0.07 0.10

Co 0.03 0.03 0.05 0.04 0.04 0.01 0.03 0.05 Cr 0.27 0.30 0.28 0.30 0.29 0.02 0.27 0.30 Cu 11.02 9.58 8.74 8.95 9.57 1.03 8.74 11.02 Fe 83.25 79.85 78.55 74.13 78.95 3.77 74.13 83.25 La 0.07 0.07 0.08 0.09 0.08 0.01 0.07 0.09 M n 0.91 0.85 1.19 1.01 0.99 0.15 0.85 1.19 Mo 0.02 0.03 0.03 0.03 0.03 0.01 0.02 0.03 Ni 0.21 0.22 0.22 0.21 0.22 0.01 0.21 0.22 Pb 0.71 0.74 0.77 0.84 0.77 0.06 0.71 0.84 Sb 0.02 0.01 0.02 0.02 0.02 0.00 0.01 0.02 Se 0.15 0.13 0.13 0.13 0.14 0.01 0.13 0.15 Sn 0.18 0.17 0.15 0.16 0.17 0.01 0.15 0.18 V 0.21 0.24 0.23 0.25 0.23 0.02 0.21 0.25 Zn 41.21 39.23 38.41 39.00 39.46 1.22 38.41 41.21 N an n oc al an u s m in or sample 1 (n=502 individuals ) sample 2 (n= 527 individuals ) sample 3 (n= 594 individuals ) sample 4 (n=663

individuals) mean SD min max

Al 50.25 48.30 51.20 49.98 49.93 1.21 48.30 51.20 As 0.13 0.15 0.15 0.14 0.14 0.01 0.13 0.15 Cd 0.04 0.03 0.04 0.04 0.04 0.01 0.03 0.04 Ce 0.08 0.07 0.07 0.07 0.07 0.01 0.07 0.08 Co 0.03 0.04 0.05 0.04 0.04 0.01 0.03 0.05 Cr 0.28 0.29 0.30 0.28 0.29 0.01 0.28 0.30 Cu 108.25 105.45 103.65 104.08 105.36 2.08 103.6₅ 108.2₅ Fe 44.25 46.54 45.84 44.88 45.38 1.01 44.25 46.54 La 0.02 0.03 0.03 0.03 0.03 0.01 0.02 0.03 M n 0.28 0.35 0.30 0.32 0.31 0.03 0.28 0.35 Mo 0.10 0.09 0.09 0.11 0.10 0.01 0.09 0.11 Ni 0.76 0.86 0.79 0.80 0.80 0.04 0.76 0.86 Pb 1.15 1.02 0.95 0.99 1.03 0.09 0.95 1.15 Sb 0.03 0.03 0.03 0.03 0.03 0.00 0.03 0.03 Se 0.78 0.86 0.85 0.95 0.86 0.07 0.78 0.95 Sn 0.10 0.10 0.09 0.10 0.10 0.01 0.09 0.10 V 0.19 0.18 0.16 0.16 0.17 0.02 0.16 0.19 Zn 21.32 20.00 19.41 17.50 19.56 1.59 17.50 21.32 sample 1 (n=105 individuals ) sample 2 (n= 98 individuals ) sample 3 (n=100 individuals ) sample 4 (n= 105

individuals) mean SD min max

eo ca n a Al 17.02 17.26 17.56 16.75 17.15 0.35 16.75 17.56 As 0.11 0.11 0.12 0.12 0.12 0.01 0.11 0.12 Cd 0.35 0.33 0.33 0.40 0.35 0.03 0.33 0.40

lu s gr ac il is Ce 0.03 0.04 0.04 0.04 0.04 0.01 0.03 0.04 Co 0.22 0.24 0.21 0.24 0.23 0.02 0.21 0.24 Cr 0.19 0.18 0.18 0.19 0.19 0.01 0.18 0.19 Cu 33.08 30.02 29.42 28.00 30.13 2.14 28.00 33.08 Fe 31.55 29.21 32.00 29.01 30.44 1.55 29.01 32.00 La 0.02 0.02 0.01 0.01 0.02 0.01 0.01 0.02 M n 0.79 0.86 0.79 0.75 0.80 0.05 0.75 0.86 Mo 0.03 0.05 0.04 0.04 0.04 0.01 0.03 0.05 Ni 3.02 3.45 3.79 3.33 3.40 0.32 3.02 3.79 Pb 1.18 0.99 0.97 1.30 1.11 0.16 0.97 1.30 Sb 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 Se 0.50 0.48 0.55 0.50 0.51 0.03 0.48 0.55 Sn 0.06 0.05 0.07 0.08 0.07 0.01 0.05 0.08 V 0.07 0.07 0.08 0.07 0.07 0.01 0.07 0.08 Zn 91.75 89.62 82.31 86.30 87.50 4.12 82.31 91.75 C en tr op ag es ty pi cu s sample 1 (n=680 individuals ) sample 2 (n=702 individuals ) sample 3 (n= 726 individuals ) sample 4 (n=598

individuals) mean SD min max

Al 32.14 30.12 29.15 29.44 30.21 1.35 29.15 32.14 As 0.09 0.08 0.09 0.09 0.09 0.01 0.08 0.09 Cd 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 Ce 0.04 0.05 0.05 0.05 0.05 0.01 0.04 0.05 Co 0.02 0.02 0.03 0.03 0.03 0.01 0.02 0.03 Cr 0.18 0.17 0.20 0.20 0.19 0.02 0.17 0.20 Cu 18.00 19.19 18.59 18.22 18.50 0.52 18.00 19.19 Fe 29.29 31.52 29.81 31.44 30.52 1.13 29.29 31.52 La 0.03 0.02 0.03 0.03 0.03 0.01 0.02 0.03 M n 0.18 0.17 0.17 0.16 0.17 0.01 0.16 0.18 Mo 0.09 0.09 0.11 0.10 0.10 0.01 0.09 0.11 Ni 0.22 0.25 0.24 0.24 0.24 0.01 0.22 0.25 Pb 0.39 0.43 0.41 0.39 0.41 0.02 0.39 0.43 Sb 0.02 0.02 0.02 0.02 0.02 0.00 0.02 0.02 Se 0.46 0.41 0.44 0.42 0.43 0.02 0.41 0.46 Sn 0.09 0.10 0.09 0.10 0.10 0.01 0.09 0.10 V 0.13 0.12 0.12 0.12 0.12 0.01 0.12 0.13 Zn 1.95 1.78 1.85 1.86 1.86 0.07 1.78 1.95 C an da ci a h io pi ca sample 1 (n= 513 individuals ) sample 2 (n= 426 individuals ) sample 3 (n= 541 individuals ) sample 4 (n= 510 individuals)

mean SD min max

Al 15.55 15.00 14.11 14.12 14.70 0.71 14.11 15.55

Ce 0.01 0.02 0.02 0.01 0.02 0.01 0.01 0.02 Co 0.05 0.03 0.04 0.04 0.04 0.01 0.03 0.05 Cr 0.22 0.23 0.25 0.22 0.23 0.01 0.22 0.25 Cu 18.21 17.85 17.32 17.98 17.84 0.38 17.32 18.21 Fe 26.51 24.99 25.16 25.94 25.65 0.71 24.99 26.51 La 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 M n 0.19 0.17 0.18 0.18 0.18 0.01 0.17 0.19 Mo 0.11 0.10 0.13 0.13 0.12 0.01 0.10 0.13 Ni 0.54 0.47 0.51 0.49 0.50 0.03 0.47 0.54 Pb 1.29 1.38 1.28 1.25 1.30 0.06 1.25 1.38 Sb 0.03 0.03 0.04 0.02 0.03 0.01 0.02 0.04 Se 0.76 0.77 0.74 0.76 0.76 0.01 0.74 0.77 Sn 0.05 0.06 0.05 0.07 0.06 0.01 0.05 0.07 V 0.23 0.26 0.23 0.26 0.25 0.02 0.23 0.26 Zn 7.12 6.99 6.85 6.68 6.91 0.19 6.68 7.12 sample 1 (n= 392 individuals ) sample 2 (n= 448 individuals ) sample 3 (n= 464 individuals ) sample 4 (n= 512

individuals) mean SD min max

P on te ll a m ed ite rr an ea Al 19.98 21.25 19.58 21.35 20.54 0.89 19.58 21.35 As 0.12 0.14 0.12 0.13 0.13 0.01 0.12 0.14 Cd 0.03 0.02 0.03 0.03 0.03 0.01 0.02 0.03 Ce 0.02 0.02 0.03 0.03 0.03 0.01 0.02 0.03 Co 0.06 0.05 0.05 0.05 0.05 0.01 0.05 0.06 Cr 0.25 0.24 0.20 0.21 0.23 0.02 0.20 0.25 Cu 17.98 17.85 18.52 17.91 18.07 0.31 17.85 18.52 Fe 40.11 38.23 39.14 39.11 39.15 0.77 38.23 40.11 La 0.02 0.02 0.02 0.01 0.02 0.01 0.01 0.02 M n 0.10 0.09 0.10 0.08 0.09 0.01 0.08 0.10 Mo 0.10 0.09 0.10 0.08 0.09 0.01 0.08 0.10 Ni 0.98 0.92 0.91 0.85 0.92 0.05 0.85 0.98 Pb 0.63 0.62 0.65 0.63 0.63 0.01 0.62 0.65 Sb 0.02 0.02 0.02 0.01 0.02 0.01 0.01 0.02 Se 0.65 0.66 0.68 0.71 0.68 0.03 0.65 0.71 Sn 0.10 0.08 0.09 0.10 0.09 0.01 0.08 0.10 V 0.13 0.11 0.12 0.11 0.12 0.01 0.11 0.13 Zn 11.27 11.08 11.17 11.02 11.14 0.11 11.02 11.27

Table S3 Trace elements in seawater (g L-1₎

sample 1 sample 2 sample 3 mean SD

Al 1.46 1.55 1.50 1.50 0.05

738 739

Cd 0.02 0.01 0.02 0.02 0.01 Co 1.41 1.45 1.49 1.45 0.04 Cu 1.30 1.42 1.36 1.36 0.06 Fe 2.38 2.32 2.41 2.37 0.05 Mn 0.52 0.71 0.61 0.61 0.10 Ni 10.77 10.99 10.03 10.60 0.50 Pb 1.50 1.45 1.41 1.45 0.05 Zn 12.02 11.43 10.84 11.43 0.59 740