Metal Accumulations in Two Extreme-Environment Amphipods, Hadal Eurythenes gryllus and Antarctic Pseudorchomene plebs

: The hadal zone and Antarctic Ocean are two of the least-explored habitats. Knowledge about human impacts on these two extreme environments is limited. Here, we analyzed the metal accumulations of two amphipod species, Eurythenes gryllus , from the Mariana Trench (6040 m, 11.36 ◦ N, 142.41 ◦ E) in the West Paciﬁc Ocean, and Pseudorchomene plebs , from the Ross Sea (600 m, 77.12 ◦ S, 167,67 ◦ E) in the Antarctic. Bioaccumulation of thirteen elements (Na, Mg, K, Ca, Cu, Fe, Al, Cr, Mn, Zn, As, Se, and Cd) in three tissues (exoskeleton, leg muscle, and gut) of the two amphipods was investigated using inductively coupled plasma mass spectrometry (ICP-MS). Comparing the trace element concentrations between the different amphipoda species, we found higher element concentrations in the Antarctic amphipod, and an oligotrophication of the Mariana Trench. The concentrations of Cu, Zn, As, and Se in the three tissues all had a signiﬁcant difference in abundance, and the Cd in the gut of P. plebs was comparably higher than that in E. gryllus, consistent with special environment adaptation. Compared with non-abyssal and shallow water decapoda and amphipoda species, hadal amphipods possessed comparably higher concentrations of Cd and Cr elements and displayed a very high environmental speciﬁcity for amphipods’ metal-element bioaccumulation strategy. This study reveals the amphipods of remote and uncontaminated areas as potential indicator species for metal-element bioaccumulation to measure anthropogenic impacts.


Introduction
Hadal oceans are composed almost exclusively of trenches and are habitats that humans rarely explore and study on Earth [1]. The hadal trench, covering over 800,000 km 2 , is the deepest ocean realm and is commonly referred to as the hadal zone (6000-11,000 m) [2]. These trenches are mainly concentrated around the Pacific Rim [3,4], where oceanic plates move to form trenches along plate boundaries and experience landslides episodically and/or regularly [5]. The funnel effect of trench geomorphology causes the downward transportation of surface sediments. Hence, higher sedimentation rates and concentrations exist in the trench when compared with the neighboring abyssal plain [6,7]. The hadal zone plays an important role in the ocean's ecosystems; although it only occupies less than 2% of marine benthic habitats, it accounts for more than 45% of the total ocean vertical depth [8,9]. whatever the essential or non-essential elements, if their concentrations excee threshold, they might lead to toxicity [36,37]. In the water environment, fish a metals and are enriched by respiration, adsorption, and ingestion [38]. More trace elements are well-established and involved in almost all biological func as cell growth, and play an important role in reproduction [39]. How trace el accumulated in amphipods in the hadal zone or Antarctica is of great interest t and up to now, very little has been known about deep-sea amphipods and w could be used to monitor the impacts of human activities in the future [40].
In this study, two extreme environmental amphipods, hadal E. gryllus an P. plebs, were chosen as the research objects for trace elemental analysis. W evaluate the difference in 13 elements (Na, K, Mg, Al, Ca, Fe, Cr, Mn, Zn, Cu, Cd) of three tissues (exoskeleton, gut, and leg muscle) of two extreme environ phipods. The hadal zone and Antarctic marine environment are largely conside and uncontaminated areas; therefore, we aimed to explore their special metaloaccumulation adaptation strategies to extreme environments and to contribu gional background levels for measuring anthropogenic impacts.

Sample Collection and Preparations
Two extreme environmental amphipods were used in this study. Hadal a E. gryllus, were collected from Mariana Trench (6040 m, 11.36°N, 142.41°E) i Pacific Ocean (Figure 1). Hadal amphipod samples were captured by using mous lander vehicle in deep ocean during the course of sampling campaigns [1 arctic amphipods, P. plebs, were collected in the 34th CHINARE in Antarctic O 2018 January to February. The Antarctic amphipod P. plebs samples were from of the Ross Sea (600 m, 77.12°S, 167,67°E), Antarctica ( Figure 1). The detailed i of amphipod samples, including body weight and length ranges, is shown in T   The amphipods were placed separately in one polyethylene zip-lock bag, then kept stored at −20 • C until transferred to laboratory. We used stainless steel scalpel to cut the amphipod individuals' leg muscle, exoskeleton, and gut tissue. Because of the small size of the two amphipods, several individuals were pooled for metal analyses from each species. Five E. gryllus individuals and thirty P. plebs individuals had metal determinations carried out. The pooled tissue samples were firstly dried 48 h at 110 • C for next evaluation of metal elements.
Transferred 0.2 g of each tissue sample into a digestion flask containing 5 mL nitric acid and digested overnight. The digestion flask was then put into a heating oven at 170°C and remained at this temperature for 4 h. After the samples were cooled, the samples were carried out from the digestion tank. The samples were heated at 100°C for 30 min using a temperature-controlled electric heating plate, and then were mixed with 25 mL or 50 mL water for use. In the meantime, blank test was set. All digested samples were subsequently analyzed for metal content three times. The metal concentration in tissue was recorded as µg/g dry weight.

Quality Assurance
We cleaned labware to avoid any metal contamination using non-ionic soap and rinsed with tap water, labware was soaked in nitric acid (15%) for 24 h and then rinsed in deionized water repeatedly; after that, we dried the labware in laminar flow cabinet for future experiments. All the experiments used super pure-grade reagent. After above-microwave digestion, the determination of metals' chromatographic analysis was conducted with ICP-MS (Agilent 7500CE Series, Agilent Technologies, Palo Alto, CA, USA) according to the People's Republic of China National Standard (GB 5009.268-2016) process.

Statistical Analysis
Each result was reported as the mean value for three analyses. We compared the correlations between the leg muscle, exoskeleton, and gut of P. plebs and E. gryllus. We regarded it as statistically significant when the correlations' p-value ≤ 0.05 (* means significant) and the correlations' p-value ≤0.01 (two * means more significant). The results are expressed as means ± SD. For correlation analysis between the values of different samples, the independent samples t-test and ANOVA analysis were used. The data results were analyzed using SPSS v.26.0 (Chicago, IL, USA) [42].

Regional Variations in the Amphipods' Metal Concentrations
Metal concentrations in the three tissues (leg muscle, exoskeleton, and gut) of the hadal E. gryllus and the Antarctic P. plebs are shown in Table 2. Sorting the 13 metals in the hadal amphipod E. gryllus leg muscle, exoskeleton, and gut tissues, we found that the metal in the leg muscle and exoskeleton showed a similar pattern of abundance. They both had the highest Ca concentrations, and then the Na had the second-highest concentrations; the difference was the level of Cu and Mn concentrations. However, in the gut element, Na had the highest concentration, the second-highest level was Mg, and then the following abundance was Ca, respectively, shown in Table 2. Five elements' (Na, Fe, Al, Mn. and Cd) concentrations in the muscle had a similar accumulation pattern between hadal E. gryllus and Antarctic P. plebs. Additionally, another five elements' (Fe, Al, Mn, Cr, and Cd) concentrations in the exoskeleton and three elements' (Fe, Al, and Cr) concentrations in the gut exhibited no significant differences between the two extreme-environment amphipod species (Table 2). Notes: p-value (a) indicates the muscle concentration comparison between P. plebs and E. gryllus; p-value (b) indicates the exoskeleton concentration comparison between P. plebs and E. gryllus; and p-value (c) indicates the gut concentration comparison between P. plebs and E. gryllus. * means significant (correlation's p-value ≤ 0.05) and ** means more significant (correlation's p-value ≤0.01).
The concentrations of 13 metals in the leg muscle of the hadal E. gryllus and the Antarctic P. plebs are presented in Figure 2. It was obviously shown that Ca, Mg, K, Cr, Cu, Zn, As, and Se were significantly different in the muscle between the two extremeenvironment amphipods (p < 0.01). Among these differential metals, the Ca, Mg, K, Cu, Zn, As, and Se concentrations showed significantly higher concentrations in the Antarctic P. plebs. However, there was no significant difference in the five metals (Na, Fe, Al, Mn, and Cd) between the muscle tissue of the two amphipod species (Figure 2). Figure 3 shows the metal (Ca, Na, Mg, K, Fe, Al, Cr, Cu, Zn, Mn, As, Se, and Cd) levels in the exoskeleton of the hadal E. gryllus and the Antarctic P. plebs. Among the thirteen metals detected, four macro-metals (Na, Mg, K, and Ca) showed significant differences between the two amphipod species, and they were obviously higher in the Antarctic amphipod P. plebs ( Figure 3). The concentrations of the element Ca in the exoskeleton were significantly higher than those in the other two tissues, and the highest concentration of Ca was 17,556.783 µg/g ( Table 2 and Figure 3). Furthermore, it can be seen from Table 2 that two elements (Fe, Al) were not detected significant differences in the leg muscle, exoskeleton, and gut when comparing the two amphipods (p-value >0.05), while the Cr, Mn, and Cd of some tissues showed significant differences between the two amphipods (p-value <0.01) and other elements, such as K, Ca, Cu, and Zn of the three tissues had significant differences between the two amphipod species (p-value <0.01, Figure 3).  Figure 3 shows the metal (Ca, Na, Mg, K, Fe, Al, Cr, Cu, Z levels in the exoskeleton of the hadal E. gryllus and the Antarctic P teen metals detected, four macro-metals (Na, Mg, K, and Ca) sh ences between the two amphipod species, and they were obviously amphipod P. plebs (Figure 3). The concentrations of the element Ca significantly higher than those in the other two tissues, and the h  The mean values of the metals' concentrations in the guts of the two amphipod species are given in Figure 4. The Antarctic amphipod P. plebs showed significantly higher concentrations in the metals Ca, Na, Mg, K, Cu, Zn, Cd, As, and Se than in the hadal amphipod E. gryllus gut tissue. Based on Figure 4 and Table 2, the pattern of the metal concentrations in the gut of the two species can be summarized in descending order as: Na > Mg > Ca > K > Fe > Al > Zn > Cu > As > Mn > Cd > Se > Cr for E. gryllus, and Na > Ca > K > Mg > Zn > Fe > Cu > Al > Cd > Se > As > Mn > Cr for P. plebs. It was obviously shown that element Na was the highest and the concentration of Cr was the lowest for the two amphipod species. Moreover, there existed significant differences in the concentrations of Na, Mg, K, and Ca (p < 0.05), and extremely significant differences in the concentrations of trace elements Cu, Zn, As, and Se (p < 0.01) between the two amphipod species. Such results were consistent with the results of exoskeleton elemental comparisons between the two amphipods. The mean values of the metals' concentrations in the guts of the two amphipod species are given in Figure 4. The Antarctic amphipod P. plebs showed significantly higher concentrations in the metals Ca, Na, Mg, K, Cu, Zn, Cd, As, and Se than in the hadal amphipod E. gryllus gut tissue. Based on Figure 4 and Table 2, the pattern of the metal concentrations in the gut of the two species can be summarized in descending order as: Na > Mg > Ca > K > Fe > Al > Zn > Cu > As > Mn > Cd > Se > Cr for E. gryllus, and Na > Ca > K > Mg > Zn > Fe > Cu > Al > Cd > Se > As > Mn > Cr for P. plebs. It was obviously shown that element Na was the highest and the concentration of Cr was the lowest for the two amphipod species. Moreover, there existed significant differences in the concentrations of Na, Mg, K, and Ca (p < 0.05), and extremely significant differences in the concentrations of trace elements Cu, Zn, As, and Se (p < 0.01) between the two amphipod species. Such results were consistent with the results of exoskeleton elemental comparisons between the two amphipods.   Figures 5 and 6 summarize the metal concentrations in leg muscl gut of the two amphipods. The distribution pattern of the four elemen Ca) in three tissues (leg muscle, exoskeleton, and gut) of the two am sistent (Figures 5 and 6). For example, the distribution order of the elem E. gryllus is listed as gut > leg muscle > exoskeleton, which is similar to shown in Figures 5 and 6, the concentration of Ca in the muscle and two amphipods was significantly higher than the concentrations of N the average concentration of the element Ca was ten times higher than of Mg in Antarctic P. plebs. Moreover, it was interesting that the concen elements Ca and K was listed as exoskeleton > leg muscle > gut for the ronment amphipods.

Metal Analyses in Different Tissues of the Two Amphipods
Figures 5 and 6 summarize the metal concentrations in leg muscle, exoskeleton and gut of the two amphipods. The distribution pattern of the four elements (Na, Mg, K, and Ca) in three tissues (leg muscle, exoskeleton, and gut) of the two amphipods was consistent (Figures 5 and 6). For example, the distribution order of the element Na in the hadal E. gryllus is listed as gut > leg muscle > exoskeleton, which is similar to that for P. plebs. As shown in Figures 5 and 6, the concentration of Ca in the muscle and exoskeleton of the two amphipods was significantly higher than the concentrations of Na, Mg, and K, and the average concentration of the element Ca was ten times higher than the concentration of Mg in Antarctic P. plebs. Moreover, it was interesting that the concentration order of the elements Ca and K was listed as exoskeleton > leg muscle > gut for the two extreme-environment amphipods.  . The distribution patterns of metal elements (Na, Mg, K, Ca, Fe, Al, Cr, Mn, Cu, Zn, As, Se, and Cd) in different tissues of the P. plebs (unit: μg/kg dry weight); each symbol represents a sample and the line represents the mean value. Figures 5 and 6 show, in tissues of the leg muscle, exoskeleton, and gut, the elements' (Fe, Al, Cu, and Zn) average concentrations in two amphipod species were significantly higher than those of another five trace elements, such as Cr, Mn, As, Se, and Cd. Statistical analysis suggested that the accumulation order of the four metal elements (Fe, Al, Cr, and Mn) for both of the two amphipods was exoskeleton > leg muscle > gut (Figures 5 and 6). Additionally, the accumulation of trace element Cr in the exoskeleton was much greater   Figures 5 and 6 show, in tissues of the leg muscle, exoskeleton, and gut, the elements' (Fe, Al, Cu, and Zn) average concentrations in two amphipod species were significantly higher than those of another five trace elements, such as Cr, Mn, As, Se, and Cd. Statistical analysis suggested that the accumulation order of the four metal elements (Fe, Al, Cr, and Mn) for both of the two amphipods was exoskeleton > leg muscle > gut (Figures 5 and 6). Additionally, the accumulation of trace element Cr in the exoskeleton was much greater Figure 6. The distribution patterns of metal elements (Na, Mg, K, Ca, Fe, Al, Cr, Mn, Cu, Zn, As, Se, and Cd) in different tissues of the P. plebs (unit: µg/kg dry weight); each symbol represents a sample and the line represents the mean value. Figures 5 and 6 show, in tissues of the leg muscle, exoskeleton, and gut, the elements' (Fe, Al, Cu, and Zn) average concentrations in two amphipod species were significantly higher than those of another five trace elements, such as Cr, Mn, As, Se, and Cd. Statistical analysis suggested that the accumulation order of the four metal elements (Fe, Al, Cr, and Mn) for both of the two amphipods was exoskeleton > leg muscle > gut (Figures 5 and 6). Additionally, the accumulation of trace element Cr in the exoskeleton was much greater than that in the gut tissue for the two amphipods (Figures 5 and 6). As for the element As, little difference was detected among the three tissues (leg muscle, exoskeleton, and gut) of the two amphipods, and the distribution order of element As in the two species was consistent (Figures 5 and 6).

Discussion
Amphipods are widely distributed on Earth, including both marine and freshwater ecosystems [43]; even in extreme environments, such as the hadal area, scavenging amphipods are also abundant and ubiquitous in deep-sea environments, representing important components of the deep-sea macrofaunal community [25]. Amphipods are usually used as a tool to monitor environmental bioavailable metal concentrations due to their extremely widespread distribution and benthic adaptation. Furthermore, the amphipods accumulate metals in an insoluble form because of their limited excretion [44,45]. At present, with the development of technology more information of amphipods in deep-sea environments are better understood [46][47][48][49]. However, the metal bio-accumulation process and mechanism in deep-sea hadal are still poorly understood.
The results of metal accumulations in the two species amphipods showed that, in the three tissues exoskeleton, leg muscle, and gut, metal Mg, K, Ca, and Na were abundant ( Table 2). The four elements' (Na, Mg, K, and Ca) concentrations in the leg muscle, exoskeleton, and gut of two amphipod species showed that, except for the concentration of the element Na in leg muscle, the enrichment of the four elements in three tissues (leg muscle, exoskeleton, and gut) of the Mariana Trench amphipod was significantly lower than that of the amphipod P. plebs from the Ross Sea, Antarctica (Figures 2-4). Na and K play an important role in maintaining osmotic pressure inside and outside the bone cells and in acid-base balance [50,51]. In the cell, high sodium content results in strengthened biological artery wall contraction and enhanced vascular reactivity, which would contribute to the increase in the arterial blood pressure [52]. Thus, the higher Na levels shown in the Ross Sea amphipods can be inferred from the higher blood pressure and relatively faster circulation of the P. plebs.
The Fe, Cu, Zn, As, and Se accumulated in the exoskeleton, leg muscle, and gut of the Antarctic P. pebs were significantly higher than those in the hadal E. gigyllus (Figures 2-4). This is because of the oligotrophication of the Mariana Trench. The hadal is well-known as a typical food-shortage area covered with nutrient-poor surface water, which corresponds to low-level primary productivity and organic matter vertical deposition [53,54]. Cu intermediate levels worldwide ranged from 30 to 90 µg/g dry weight [55]; See in Table 3 below, our Cu values were below this range, so the amphipods in the Mariana Trench and Ross Sea might be suffering a deficiency of Cu. For Zn, the most values worldwide are in the range 25-110 µg/g dry weight [55]; the Zn value of the Mariana Trench was lower than this range, so it also suffers a deficiency of Zn, but the Zn value of the Ross Sea was in this range, which met the estimated requirements of Zn level for enzymatic activities [56]. In marine ecosystems, contaminants sink in benthic sediments and directly affect benthic communities. Contaminated sediments that are disturbed may cause sediment-bound contaminants to be transported again into the water column and rebuild a wide range of trophic webs [57]. Available data show that other continents' pollution has affected the Antarctic environment by transportation, because pollutants such as pesticides, metals, and some persistent organic pollutants (POPs) were found in the Antarctic [24]. Previous studies showed high Fe, Cu, Zn, As, and Se enrichment factors were affected by human activities in the Antarctic or other parts of the Southern Hemisphere [24,58,59]. Similarly, the research of Planchon et al. showed high concentrations of Fe, Zn, Cu, As, and Se may be the pollutants caused by human activities at mid-latitudes, for instance places like South Africa, South America, and Australia [58], and we also cannot exclude the influences of active volcanoes in Antarctica [60]. In Antarctica, atmospheric transport of POPs is considered to play an important role in pollution transport, and marine transport is also thought to be a physical forcing factor. Global warming would aggravate the problems of POP transportation and deposition [61][62][63].
The E. gryllus amphipod is broadly distributed in every ocean from bathyal, abyssal, to hadal depths. The concentrations of Cr in the three hadal E. gryllus tissues (exoskeleton, leg muscle, and gut) were higher than those in Antarctic P. pebs (Figures 2-4), and the Cr concentration in leg muscle tissue of the two amphipod species had a significant difference (p < 0.01). The high accumulation of Cr has also been observed in Hirondellea gigas of the Mariana Trench [48]. The essential trace element, Cr has a major role in maintaining the body's physiological functions. For example, Bagchi et al. have proven Cr can promote the formation of reactive oxygen species (ROS), reduce the cell-survival rate, and accelerate the apoptosis and death of necrotic cells [64]. Cui et al. showed that in suspended particulate matter (SPM) from the Mariana Trench samples, persistent organic pollutants were not detectable [65]. Therefore, the high concentrations of Cr in the hadal E. gryllus (showed in Table 3) might be related to the adaptation mechanism of the hadal creature.
The concentration order of Al in tissues of E. gryllus was exoskeleton > leg muscle > gut ( Table 2 and Figure 5). The exoskeleton of E. gryllus contained Al as well as a large amount of Ca (Table 2 and Figure 5). Different from the accumulation of other metals, Al was distributed on the exoskeleton surface. In order to know how aluminum accumulated in the hadal creature, Kobayashi et al. performed a metabolome analysis against a hadal amphipod of Hirondellea gigas. Their results showed the aluminum metals extracted from the Mariana Trench could be made into a good material for adapting to high-pressure environments [66], which indicated aluminum trace element plays an important role in adaptation to high-pressure environments.
The hadal zone is a large unknown ecosystem. However, accumulated human activities have influenced the deep-water ecosystems [22]. Additionally, for Antarctica, in the year 1989, the Cd concentrations (µg/g dry weight) in sediments from the Ross Sea (continental shelf) ranged from 0.04 to 0.72 [67]; the range of Cd concentrations in Ross Sea sediment could be considered very close to "background remote ancient" sediments (0.04-0.2 µg/g dry weight) [68]. Now, the Cd concentrations ranged from 0.07 to 267 µg/g dry mass [69]. Different species showed different Cd accumulation; our results for Cd concentrations in the Ross Sea are higher than shallow species (Table 3). Additionally, an abnormally high Cd concentration in the decapods and amphipods in polar areas has been observed in polar crustaceans, ranging from 1.2 (Ceratoserolis trilobitoides) to 6.2 µg/g dry weight (Notocrangon antarcticus) in 1999 and from 1.2 (Waldeckia obesa) to 20.3 µg/g dry weight (Tryphosella murrayi) in 2003; this can be termed Cd-anomaly [55]. That was presumably caused by the Cd-rich deep waters upwelling and the mechanisms of indiscriminate uptake between Cd and Cu, by which Cu-deficient organisms increased uptake of Cd [40,70]. Hydrothermally active areas also supply Mn and Fe elements to influence seawater chemistry in the deep-water over thousands of kilometers [71,72]. Hadal trenches, as a heterotrophic ecosystem, mostly acquired the available food including small phytoplankton, detritus, huge whale falls [73], and terrestrial organic matter which is significantly adjacent to landmasses in the trenches. The problem of ocean pollution has become more serious along with pollutants derived from human activities [74].

Conclusions
The hadal zone is characterized by an extreme environment including low temperature, a shortage of food supply, extremely high hydrostatic pressure, and, often, subduction-zone earthquakes. The Antarctic Ross Sea is another extreme environment featuring extreme low temperatures and a relatively high biological diversity. With the development of technology in the hadal region and in terms of possible pollution, the hadal ecosystem has attracted studies on the toxic mineral impact for marine animals [9]. To evaluate the metal concentrations in amphipods, we analyzed thirteen elements' metal accumulations in two amphipod species, Eurythenes gryllus (Mariana Trench) and Pseudorchomene plebs (Ross Sea, Antarctica) in three tissues (exoskeleton, leg muscle, and gut), and found that most of the elements showed higher concentrations in the Antarctic amphipod, consistent with the oligotrophication of the Mariana Trench. The concentrations of Cu, Zn, As, and Se in the three tissues all had significant differences, and the Cd in the gut of P. plebs was comparably higher than that in E. gryllus, consistent with the special environment. When we compared the leg-muscle trace element accumulations between the hadal amphipods and non-abyssal and the shallow water decapoda and amphipoda species, hadal amphipods possessed comparably higher concentrations in the elements Cd and Cr, which displayed that amphipods' strategy of metal elements shows a very high environmental specificity. The hadal and Antarctic marine environments are largely considered as remote and uncontaminated areas; these results provide us with more data to contribute to measuring anthropogenic impacts.