Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions

Journal Pre-proof Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions Mary Joy Halog Libatique, Meng-Chou Lee, Han-Yang Yeh, Fu-Jie Jhang PII:

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DOI:

https://doi.org/10.1016/j.chemosphere.2020.126084

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CHEM 126084

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Received Date: 7 October 2019 Revised Date:

21 January 2020

Accepted Date: 31 January 2020

Please cite this article as: Libatique, M.J.H., Lee, M.-C., Yeh, H.-Y., Jhang, F.-J., Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126084. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1

(1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan (2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan (3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan (4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippin es 3320 Corresponding author email: [email protected] Tel: +886–2–2462–2192 ext. 5231 Fax: +886–2–2463–5441 Mobile: +886–978–586–589

Abstract Temperature, light intensity (LI), adsorbent source and concentrations are key external factors affecting algal metabolism and thus metal–accumulation mechanisms. In this study, the alga Sarcodia suiae was exposed individually to a range of temperature (15, 20, and 25 °C), and LI (30, 55, and 80 µmol photons m−2 s−1) at initial arsenate [As(V)] concentration (iconc: 0, 62.5, 125, 250, and 500 µg L−1) conditions, to investigate the variations of total arsenic (TAs) and inorganic arsenic (iAs) accumulation mechanisms in the algal body. Temperature significantly affected TAs and arsenite [As(III)] production and maximum absorption were obtained at 15°C, which was significantly stimulated by increasing iconc. However, the temperature did not affect As(V) production. LI had no significant effect on TAs or iAs production, although maximum absorption was estimated in 80 µmol photons m−2 s−1. The iAs component of TAs was much greater in the temperature experiment particularly under 250–500 µg L−1 iconc than in the LI experiment, is witnessed. Overall, temperature and iconc strongly affected As accumulation. The predominant iAs produced was As(III), regardless of temperature or LI, suggesting that the alga favored As(III) biosorption. Also, visible effects on the morphology of this alga were adverse with increased concentration and environmental factors did affect the difference somewhat. Our results contribute to improving our understanding of the effects of the tested factors on As cycling, which is necessary for maximizing biosorption of algae if utilized for bioremediation studies as well as in the wastewater treatment implementation approach in the environment.

Keywords Total arsenic accumulation . Temperature gradient . Illumination . Inorganic Arsenic . Red macroalga

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Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions

Chemosphere Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1

(1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan (2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan (3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan (4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippines 3320

Corresponding author email: [email protected]

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Graphical Abstract

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Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled

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environmental conditions

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Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1

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(1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan

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(2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan

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(3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan

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(4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippines 3320

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Corresponding author email: [email protected]

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Tel: +886–2–2462–2192 ext. 5231

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Fax: +886–2–2463–5441

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Mobile: +886–978–586–589

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1. Introduction

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A number of incidences of contamination by elevated concentrations of trace elements such as arsenic (As)

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have recently been reported in Southeast Asian countries (Ahmed 2018; Ali 2018). Contamination of food, land, and

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water ecosystem by this element is harmful and has serious effects on human health and the environment (Jia et al.

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2018, Lin et al. 2013, Yokoi and Konomi 2012). In aquatic matrices, inorganic arsenic (iAs) including As(III) and

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As(V) are the major arsenic species with the latter being predominant in oxic environments and the former being

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more available in anoxic environments (Smedley and Kinniburgh 2002). Other than their abundance in the

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environment, the typically occurring inorganic ones, viz. As(V) and As(III) are considered to be more toxic

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compared to their methylated forms, because of their bioavailability and known physiological and toxicological

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effects.. Therefore, understanding the fate of this contaminant is essential.

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Algae are naturally widely distributed in aquatic environments, where As exists as a toxin in various

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concentrations, mainly in the soluble inorganic forms [As(V) and As(III)] (Yin et al. 2012). They have developed

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mechanisms to absorb appreciable amounts of As (Ma et al. 2018) into their cells by using phosphate transporters

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with uptake patterns typical of phosphate through the phosphate-specific transport (PST) system and phosphate-

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inorganic transport (PIT) system and aquaglyceporins (AQP) and hexose permeases (Ybarra and Webb 1998; Thiel

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1988; Cullen et al. 1994; Levy et al. 2005; Zhang et al. 2014). Algae may thus possess metabolic mechanisms for

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taking up As from the environment (Yin et al. 2012) and be of use for improving our understanding of As

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bioaccumulation mechanisms which are believed as an important component involved in the chemical formation of

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iAs speciation .

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The capacity of seaweeds to absorb and tolerate toxic elements is regulated by key external factors that

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affect the efficiency of metabolic processes. Seaweeds grow in a range of environments with different water salinity,

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nutrient content, water movement, temperature, and light conditions (Makkar et al. 2015). In fact, the nutritional

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attributes of seaweeds have been reported to depend on the conditions under which the algae were grown, such as

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water temperature, light intensity (LI), and the nutrient concentration of the water (Misurcová 2012). With regard to

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bioaccumulation, there is a large degree of variability in sensitivity to As, which is thought to be due to biotic factors

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such as species, differing uptake/exclusion pathways, detoxification mechanisms, and prior exposure, as well as

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abiotic factors such as As species, phosphate concentration, pH, and length of exposure (Levy et al. 2005). As such,

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external conditions may have the potential to influence As metabolic processes.

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In general, different species of an element show different degrees of environmental mobility and behavior

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(e.g., Guo et al. 2011). In a previous study, we reported the TAs uptake of Sarcodia suiae was dependent upon

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varying environmental factors (temperature, light intensity, pH, phosphate, exposure duration) when exposed to

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various As(III) concentrations (Lee et al. 2018). As yet, there remains a limited related study of accumulative

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mechanisms in alga especially to the most dominant iAs species [As(V) in the practical environment. Moreover,

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information, whether possible changes of iAs and TAs uptake may happen when differing prior exposure [As(V)]

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may use as exposure source in this algae, have not been elucidated. Therefore it is important to understand the

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effects of As species variability on biological model underlining the influence of temperature, LI and concentrations

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that are crucial in determining the metabolic processes of algae that reportedly affect As bioavailability. The data

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from this study should be added to the literature on the biogeochemical cycling of this element to fill the knowledge

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gaps related to speciation patterns in complex environmental matrices.

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Similar to our previous study, we selected the moderately abundant marine Rhodophyta S. suiae that is

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locally available and present in all seasons in Taiwan (Lin et al. 2018) with the novelty of culturing them artificially

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for years before subjected to exposure treatments to assure uniformity of our algae. This is comparatively different

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to most studies in the toxicology where they collected the model species in the wild and used for exposure studies

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(Abtahi et al. 2013; Tabaraki and Heidarizadi 2018; Geiszinger et al. 2001) which might affect validity of results

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due to the diverse conditions (i.e age, origin) of the algae collected. Besides availability, the established artificial

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culture by this alga in our laboratory, ease of growing them and high sensitivity on high concentrations from the

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limited studies on this alga (Lee et al. 2018; Libatique et al. 2019) that may be useful in bioassessment were the

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criteria set for biomodel selection. The effects of temperature, LI, As(V) concentrations (iconc) on the uptake of iAs

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and TAs in this alga in laboratory conditions have been investigated in 7 days of exposure in a laboratory set-up as

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well as the effects of the factors in morphology to adverse conditions. The obtained results could provide additional

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insights to significant knowledge gap with arsenic species influence and to what extent how these environmental

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factors [temperature, LI, As(V)] modulates As accumulative metabolism, as well as to how these factors impact the

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morphology status of the algae which may be taken for consideration for remediation processes in the practical

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environment.

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2. Materials and methods

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2.1 Arsenic species

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The As species used for exposure treatments and liquid chromatography–inductively coupled plasma–mass

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spectrometry (LC–ICP–MS; X-Series 2, Thermo-Fisher Scientific, USA) preparation and standard analysis was

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sodium arsenate dibasic heptahydrate [As(V); Na2HAsO4 . 7H2O, purity ≥98.0%, Sigma-Aldrich, USA].

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2.2 Macroalga

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The marine macroalga S. suiae was collected in Pingtung County, Southern Taiwan, during winter, in

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February 2017. The alga was brought to the National Pingtung University of Science and Technology and artificially

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cultured in a greenhouse in the University’s Department of Aquaculture. The alga was maintained for two years in

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an Fiberglass Reinforced Plastic (FRP) culture tank containing normal seawater while receiving normal sunlight and

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artificial aeration, and was nutritionally supplemented with F/2 marine enrichment solution (mL−1) (Guillard 1975).

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Biological material was selected for the experiment based on uniformity of size (4.5 ± 0.5 g), shape, color, and

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absence of deformities. After the selection of the alga, it was allowed to acclimate to laboratory conditions for one

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month in a specialized incubator before the experiment commenced, to allow the alga to become accustomed to the

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experimental environment. The incubator was equipped with a temperature–controller, an artificial light with a

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fluorescent bulb, supplemented aeration, and a nutrient medium that mimicked the natural environment.

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2.3 Arsenic exposure and experimental conditions

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The experiment was carried out in a 1 L beaker filled with 800 mL of sterilized normal seawater autoclaved

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at 121 °C for 30 min at 15–20 psi, with salinity adjusted to 32 ± 1‰. Each beaker contained one specimen of alga

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(4.8–5.125 g) and each treatment was replicated three times. The experiment was performed in a laboratory, and the

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alga was cultured in a specialized incubator with temperature controller (SS-980, Tominaga, Taiwan), under the

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specific conditions summarized in Table 1. Briefly, two environmental settings were designed separately in the

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experiment. First, the alga was cultured individually at one of three levels of temperature (15, 20, or 25 °C) provided

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with 55 µmol photons m−2 s−1 intensity of light under 12:12h light/dark cycle. The temperature range used

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corresponds to the regional annual average seawater temperature (13–27℃) in Northeast Taiwan (Taiwan Central

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Weather Bureau) and were based from previous studies in S. suiae (Lee et al. 2018). Second, three LI was also set

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individually for the cultured alga at 30, 55, or 80 µmol photons m−2 s−1 (12:12h light/dark cycle) at 25 °C. LI was

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measured using a Lighting Passport (Asensetek, Taiwan). Each culture was treated with As(V) at five concentrations

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(0, 62.5, 125, 250 and 500 µg L−1) for 7 day, each with three replicates to examine As accumulation within the algal

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body. Cultures without As (0 µg L−1) were used as controls. To provide the necessary nutrition for the alga

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throughout the experiment, F/2 medium (Guillard 1975) was provided once during the start of the experiment. The

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As concentration used in the study were in most cases higher than those measured in natural aquatic systems under

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low pollution levels (Smedley and Kinniburgh 2002; Yan et al. 2016). These concentrations were used to test the

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responses for S. suiae including variations on the mechanisms and the alga’s morphology which mimic the growing

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concern regarding As concentrations progress in the environment.

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2.4 Sample Preparation before As analysis

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After the seven–day experiment, S. suiae samples were washed with deionized freshwater for three times

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and sealed with Ziploc freezer bag, frozen at −80 °C for 24 h, and freeze–dried for 70 h. These samples were

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powdered by crushing manually, labeled and stored in Ziploc bags in refrigerator and stored at 4 °C until analysis.

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2.5 Arsenic speciation Analysis

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As speciation in algal body was extracted as described by Khan et al. 2015 including the operations

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condition for digestion process. It was performed by taking 1.0 g powdered samples in 50 mL self–standing

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polypropylene volumetric tubes and adding 8 mL 50% methanol solvent in 1% HNO3. A microwave accelerated

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reaction system (CEM Microwave Technology Ltd, USA) was used for the sample digestion. Extraction of arsenic

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species in the algal samples was determined by high performance liquid chromatography inductively coupled

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plasma–mass spectrometry (HPLC–ICPMS) (X–Series Thermo–Fisher, Germany) as described by Zhang et al.

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(2009). Arsenic species were separated and quantified by an anion exchange column (Hamilton PRP–X100, 10 µm

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in diameter and 4.6 mm × 250 in diameter mm) with a mobile phase of 2 mM ammonium bicarbonate in 1% MeOH,

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pH 8.0 (Mobile Phase A) and 20 mM ammonium nitrate and 20 mM ammonium phosphate in 1% MeOH, pH 9.2

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(Mobile Phase B). Arsenic species in the samples were identified by comparisons with the retention times of

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standard compounds and quantified by external calibration curves with peak areas.

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2.6 Sample digestion and analysis of total As

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Sample digestion for the TAs analysis was performed based on the method that reported by Zhang et al.

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(2009). Freeze dried algae (0.02 g) were digested in 5 mL of high purity nitric acid (65%, Merck GR grade,

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Germany) overnight and heated in a microwave accelerated reaction system (CEM Microwave Technology Ltd,

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USA). The combustion procedure was as follow: temperature was raised slowly (over a 5–min period), then (1) 55

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°C for 10 min, (2) 75 °C with holding times of 10 min, and (3) the digest was taken up to 95 °C for 30 min. After

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cooling, the digested samples were diluted to 25 mL with Millipore ultrapure water. Arsenic concentration was

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determined by ICPMS) (X–Series Thermo–Fisher, Germany).

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2.7 Morphology analysis

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The algal samples were collected and mounted in a white plastic box filled with seawater to at least 2.54

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cm above the algal surface. The samples were photographed under even lighting, using a camera placed on a tripod,

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to ensure uniformity of distance and image quality. To assess the morpho–response of the alga, we photographed it

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both at the beginning (day 0) and at the end (day 7) of the experiment.

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2.8 Data analysis

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The statistical analyses were carried out using R statistical software. A two–way analysis of variance

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(ANOVA) with Duncan’s multiple range test (DMRT) was performed to compare the main effects of the factors

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(temperature, LI, and iconc and their interaction effects) on the As content of the algal body at the end of the seven–

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day experiment. The significance level was set at p ≤ 0.05, and all graphs were generated using R.

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2.9 Instrumentation

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Algal samples were digested using microwave accelerated reaction system and were analyzed for total

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arsenic using ICP–MS. On the other hand, iAs speciation was analyzed using high performance liquid

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chromatography (HPLC) with ICP–MS. Quality assurance and quality control for the instruments were checked and

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validated. The operating conditions of HPLC and ICP–MS including measurement parameters were followed as

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described by Khan et al. 2015 with which 100 µl was the injection volume of the samples during analysis.

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2.10 Quality Assurance/ Quality Control (QA/QC)

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To validate and assure the quality of results obtained, the analytical methods followed were validated by

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measuring several quality parameters including specificity, linearity, precision, accuracy, and quantitative limits.

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Reagent blanks and Certified Reference Materials (NMIJ CRM 7405–a Hijiki Seaweed) (National Research &

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Development Corporation Industrial Technology Research Institute, Japan) was included to assure the accuracy of

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the test method and it is within 100±20 % of the expected quality control checks. Reagent blank test was carried out

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to test the specificity of the method. Linearity was evaluated from the calibration curves and mathematical equation

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and linear range result was not less than 0.99. Precision was obtained as percent coefficient of variation (CV%) from

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the relative standard deviation divided by the average of recovery of one sample. Accuracy was tested by analyzing

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the CRM. Limit of detection (LOD) and limits of quantification was quantified and reliably differentiated from

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signal–to–noise ratio (S/N) greater than 3 of the former and greater 10 of the latter. Limit of Quantification (LOQ)

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showed at 0.02 mg kg

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respectively thus, confirming the suitability of the method.

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Column recovery for As(III) and As(V) was validated at 106.9% and 115.5 %,

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3. Results and Discussion

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3.1 The effects of temperature on TAs and the iAs produced in the algal body

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To address the gaps in our knowledge about As compounds with respect to the inorganic speciation

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problems of the As metalloid when it is used as an adsorbent source in complex environmental matrices, we

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investigated the metabolic processes of S. suiae with respect to TAs and iAs, together with the effects of varying

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temperature and LI conditions, under various As(V) exposure levels. The details of the production of iAs

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specifically (as opposed to TAs) when algae are exposed to As(V) (which is an important species of As due to its

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toxicological effects) have been documented. The bioaccumulation of As in organisms is dependent not only on the

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total quantity of As but also on its speciation (Duncan et al. 2010; Karadjova et al. 2008), and it is thus necessary to

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understand these aspects due to the potential hazard to both the environment and organisms.

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The effects of temperature on As uptake mechanisms in algae were inconsistent. Previous reports observed

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that metal absorption could be endothermic, exothermic or no significant influence. In the present study, the TAs

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pattern and iAs speciation were observed at three different temperatures (15, 20 and 25 C). The results showed that

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the main effects of temperature in the iAs and TAs were significantly different (p < 0.05) in the 7 days of culture

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(Figs 1, 2, 3). The main effects of temperature on the production of TAs and As(III) in S. suiae under exposure to

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various concentrations of As(V) were similar: both categories of As decreased with increasing temperature (Figs. 1

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and 2). As the temperature increased from 15-25 °C, the uptake decreased ranging from 5.98-257.45 to 5.71-185.75

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mg kg−1 TAs and 0.312-183.74 to 0.185-63.056 mg kg−1 As(III) in 0-500 µg L−1 initial concentration, suggesting

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that chemisorption of these As by the alga might be inhibited at temperature >25 resulting in lower uptake. These

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results also indicate an exothermic process wherein uptake capacity increases with decreasing temperature. This may

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be attributed to several factors, for example: higher temperatures leading to an increase in the capacity of heavy

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metals to transform from the solid phase to the liquid phase; the deactivation of the biosorbent surface; the

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destruction of some of the active sites on the biosorbent surface due to bond ruptures (Meena et al. 2005); or the

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weakness of the biosorption forces between the active sites of the sorbents and the sorbate species and also between

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the adjacent molecules of the sorbed phase (Ahmet and Mustafa 2008).

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The present result, however, is the reverse of the TAs pattern we observed in our previous study (Lee et al.

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2018) with the same alga but using As(III) as the adsorbent source where the uptake amount was significantly

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enhanced with the increased of both fixed factors (temperature and As(III) concentrations). The differences could be

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attributed to the different adsorbent source, which suggests a different pathway and method of regulation of As

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metabolism processes.

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Regarding the main effects of temperature to the quantities of As(V), it can be observed that uptake value

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in the algal body were minimal compared to those of As(III), with a maximum concentration of 13.20 mg kg −1 from

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500 µg L−1 iconc disregard of temperature conditions (Fig. 3). Despite these small quantities of As(V), the As(V)

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content was found to be higher under a temperature of 20–25 °C (Fig. 3) than in 15 °C, suggesting that As(V)

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produced was favored in this temperature, well as this absorption process could be regarded as an endothermic. The

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increasing temperatures that result in increasing metal ion biosorption could be due to the enlarging of the alga’s

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pores during the absorption process at higher temperature, resulting in an increase in the surface area available for

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the sorption, diffusion, and penetration of the metal ions into the algae (Saleem et al. 2007). Also, increasing

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temperature is known to increase the diffusion rate of adsorbate molecules through pores, as a result of decreasing

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solution viscosity, and this would modify the equilibrium capacity of the adsorbent for a particular adsorbate

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(Sulaymon et al. 2013). A similar result was found in a previous study, where the transformation of As was

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increasing the metabolic rates of macroalgae where the transformation of As increased with the metabolic rates of

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macroalgae, which are enhanced under elevated environmental temperature (Zhao et al. 2012).

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Moreover, there was a slight decrease in As(V) uptake under an iconc of 500 µg L−1 and a temperature of

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25 °C compared to that at 20 °C, but this difference was not significant. These findings may be explained as detailed

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above in referenced to Meena et al. (2005) and Ahmet and Mustafa (2008).

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3.2 The effects of LI on TAs and the iAs produced in the algal body

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Light is essential for autotrophic activity, and the availability and intensity of light are major factors

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controlling the productivity of photosynthetic organisms. They play a significant role in determining the metabolism

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and thus the bioaccumulation of substances like metals and metalloids. Indeed, light is one of the strongest factors

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affecting the growth and storage products of algae (Ruangsomboon 2012). This however, was not the case in the

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present study where no significant effects (p ≥ 0.05) of LI or the interaction between iconc and LI (iconc × LI) was

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witnessed, as shown in Figures 4, 5, and 6. Despite no significant main effects of LI being found in the two-way

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ANOVA analysis, it can be seen that concentrations of TAs, As(III), and As(V) absorbed by the alga were increased

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with increasing LI. The results show a direct relationship between LI and iconc, in which an increasing

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concentration of TAs, As(III), and As(V) was evident (Figs. 4, 5, and 6). This suggests that the highest LI used in

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the experiment (80 µmol photons m−2 s−1) could represent the optimal light conditions for As metabolization, and

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that this effect would be further intensified by the possibility and availability of extractable metals (Adams et al.

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2004). The increasing concentrations of TAs and iAs with increasing LI could also be explained by a mechanism

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whereby the additional light that is captured is converted to chemical energy required during metabolic processes

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(Solovchenko et al. 2008), resulting in increasing As volatilization as a positive effect. This result is in agreement

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with the effects of LI on TAs revealed in our previous work (Lee et al. 2018). However, it is interesting to note that

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light that is too intense can put the alga under stress, leading to inefficient metabolic processes, including As

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metabolization and assembly. In fact, it has even been reported that extreme LI can result in reduced growth or death

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of the alga due to photoinhibition (Ruangsomboon 2012). On the other hand, lower LI resulted in lower As

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accumulation in this study, for all categories of As measured. This might be because this is an unfavorable condition

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for the algae, and a low LI might render the plants unable to maintain their metabolic processes properly, and hence

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efficient metabolism is arrested, which results in decreased absorption activity. The differences in TAs and iAs

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production under different LI levels were thus possibly due to the efficiency of irradiance utilization during the

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metabolism/assimilation process.

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3.3 The effects of concentrations

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In general, regardless of temperature or LI, the assembly of TAs and iAs compounds by the alga in our

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study was positively increased with iconc in all respects. With the increase of iconc from 0-500 µg L−1, the uptake

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amount ranged from 5.71-257 mg kg−1 for TAs (Fig. 1), 0.184-183.74 mg kg−1 for As(III) (Fig. 2), and 0.076-10.644

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mg kg−1 for As(V) (Fig. 3), respectively for temperature experiment regardless of operating conditions. While in the

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LI experiment, biosorption was ranged from 5.71-275.44 mg kg−1 for TAs (Fig. 4), 0.184-108.10 mg kg−1 for As(III)

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(Fig. 5), and 0.056-14.250 mg kg−1 of As(V) (Fig. 6), respectively. This may be related to the availability of the

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metalloid: the higher the initial concentration of the As, the more the alga can metabolize, resulting in the

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significantly higher concentrations observed in the higher iconc treatments (Adams et al. 2004). This result was also

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consistent with findings regarding the accumulation of As in algae in other studies, in which steady-state As

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accumulation has been shown to be increased with external As(V) and As(III) concentrations (Bahar et al. 2016;

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Duncan et al. 2013a, b; Lee et al. 2018; Wang et al. 2013, 2017; Wurl et al. 2013).

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While a considerable increase in metal biosorption capacity with an increase in metal concentrations is

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associated with a reduction in removal rate efficiency of the algae. The present results on TAs removal rate

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regardless of temperature or LI as shown in Figures 7 and 8 revealed that the percentage removal as calculated by

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the formula R=[Ci-Ce/Ce] x 100% where R is the removal efficiency (%), Ci is the initial and adsorbed amount of

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arsenic concentrations in equilibrium (µg L−1) (Yan et al. 2010) were decreased with the increased of absorption

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capacity. This phenomenon can be attributed to insufficient available sorption sites where the metal ions could

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interact, allowing for lower removal efficiency. Similar findings had been reported by Sulaymon et al. 2013 where

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an increase of initial concentrations of metal ions (Pb, Cd, Cu, As) to 100 and 200 mg L−1 deplete the percentage

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removal in algae. The report presented by Yan et al. 2010 on Acidithiobacillus ferrooxidans BY-3 is also in

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agreement with these result where an increase of initial concentrations of either iAs(III) or MMA from 500-3000 µg

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L−1 have resulted to 194.26 to 277.22 µg g−1 of iAs(III) and 225.05 to 328.85 µg g−1 of MMA in terms of biosorption

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capacity. Whereas, the removal efficiency has decreased from 77.7 to 18.48% for iAs(III) and from 90.02 to 21.59%

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of MMA(V) in 120 minutes. This clearly shows that increased metal ion concentrations reduced the removal

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efficiency. Hence, it is also possible that excessive concentrations of metal ions may impart greater toxic effects of

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As that prevail and such manifestations may negatively affect the normal metabolic processes, for example, DNA

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damage and destruction of algal cells which result in lower ion optimization as a consequence.

10

276

This, however, was a reverse in terms of iAs in the present findings (Figs. 7 and 8) where an increased

277

removal rate was witnessed even in the same concentrations disregard of temperature or LI conditions. This

278

behavior may be related to the availability of active sites the iAs tend to bind during the chemosorption process. It is

279

reasonable to assume that iAs biosorption is not fully maximized by the alga by the time and that sorption sites

280

could still allow interactions of arsenic species to sorption sites and thus the efficiency of the removal is getting

281

higher. Furthermore, the lower absorption capacity recorded in iAs as compared to TAs most likely to result in

282

higher removal efficiency due to surface area available that will allow the sorbents to bind in the active sites.

283 284

3.4 The effects of the environmental factors in morphology

285

There were noticeable effects of the various cultivation conditions on the external coloration of S. suiae.

286

While there was only a slight change in the color of the alga in the 0 µg L−1 As treatments compared to their initial

287

color, regardless of the other factors, there was however a strong adverse effect with an increase in iconc exposure.

288

The result was adverse in alga exposed to 500 µg L−1 regardless of the environmental factor (temperature or LI),

289

although the factor did affect the difference somewhat (temperature or LI). The change in coloration was observable

290

earlier (for lower iconc treatments) under lower temperature conditions, and stronger for the highest iconc treatment

291

(500 µg L−1; Fig. 9). On the other hand, the effect on the algal color was weaker with lower LI; with the lowest LI it

292

only became noticeable at 250 µg L−1, and even at 500 µg L−1 there was less green and more reddish color visible

293

(Fig. 10). The changes in coloration might be associated with the As metabolic process exacerbated by enhanced

294

uptake amounts of As by the alga, in which the effect of temperature and iconc was shown to be significant by the

295

two-way ANOVA, while that of LI was not. Furthermore, when comparing the plants treated with an iconc of 500

296

µg L−1, it can be seen that the coloration changes in the temperature experiment were more marked than those in the

297

LI experiment. These changes are thought to be a response or adaptation mechanism of the alga to exposure to a

298

stressful environment. As this response of the macroalga to this toxic metalloid is visible to the naked eye and can

299

therefore be easily observed, it could be used as a basis for rapid assessment. Considering morphological changes

300

(i.e coloration) could be the first and most visible response of an organism trying to survive under stress, reflecting

301

its metabolic adjustment and acclimation (Pandey et al. 2012). Moreover, there were reports in literature indicated

302

that arsenic above the threshold limit may decreased photosynthetic pigments (Tahira et al. 2018) for example,

303

chlorophyll and phycoerythrin, and this reduction in pigments might be because of the inhibition of important

11

304

enzymes (Stobart et al. 1985, Aravind and Prasad 2003, Dai et al. 2009) that is necessary for maintaining

305

photosynthetic pigments and good vitality. The reduction of pigments that gives algae and plants their color and

306

allows them to capture energy from light necessary for photosynthesis might also promote a decrease in

307

photosynthetic activity and inhibition of pigment synthesis. There were reports that existence of As in severity

308

interferes with metabolism consequently leading to wilting, curling, necrosis of leaf blades, suppression in the

309

number of leaves and leaf area thereby reducing photosynthesis and biomass accumulation, losses in mineral

310

contents, reduced root elongation, proliferation and nodulation, stunted growth and poor yield (Finnegan and Chen

311

2012; Talukdar 2013). In other studies, localized effects of metal stress include leaf chlorosis, necrosis, turgor loss,

312

reduction in seed germination and a damaged photosynthetic apparatus, finally resulting in the plant death (Dalcorso

313

et al. 2008; Dalcorso et al. 2010). Therefore, coloration changes by the alga during the exposure might also be

314

attributed to toxicity induced by arsenic where photosynthetic machinery was most likely to be affected directly.

315

Therefore it is reasonable to assume that the severity of effects of As in coloration of the algal fronds was promoted

316

remarkably with the rise of As concentration at the end of the experiment regardless of temperature or LI. However,

317

further study is needed to identify this concern.

318 319

3.5 The iAs% uptake of TAs

320

The iAs percentage of TAs in the algal body after the seven–day experiment was ≥ 50% in the samples

321

treated with different temperature in 250-500 µg L−1 iconc where maximum absorption was found in 15 °C (Fig. 11;

322

Fig. S1), but ≤ 50% in the samples treated with different LI among treatments where maximum absorption was

323

found in 80 µmol photons m−2 s−1 (Fig. 12; Fig. S2). Current result is also related to the result of ANOVA analysis

324

showing temperature is most likely to significantly influenced the As accumulation than LI in this alga, well as with

325

iconc

326

these environmental conditions affected the amount of As produced could be explained by their interactions with

327

different environmental factors. It is possible that the differences in the environmental conditions under which these

328

two groups were housed was a confounding factor that affected the plants’ metabolisms. The positive increased of

329

uptake with an increased in iconc may somehow related to the availability of the metalloid, in which the higher the

330

initial concentration of the As, the more alga can metabolize, resulting in the significantly higher concentrations

331

observed in higher initial concentrations (Adams et al. 2004).

which significantly increased in all respects regardless of the temperature/LI conditions. The differences how

12

332

In terms of iAs, As(III) constituted a greater proportion of the iAs than did As(V) in both the temperature

333

(see Fig. S1) and the LI (see Fig. S2) experiments. It is possible that S. suiae could be an As(III) accumulator

334

similarly observed in our previous study when similar alga was exposed to As(III) (Libatique et al. 2019). However,

335

there is little evidence to support this assumption in the currently available data, and thus, further experiment must

336

be assessed to ascertain this hypothesis.

337

4. Conclusion

338

To our knowledge, no studies have yet been reported on TAs and iAs accumulative mechanisms as well as

339

its effects on morphology in marine Rhodophyta under various temperatures (15-25 °C) and LI (55-80 µmol photons

340

m-2 s-1) conditions exposed to As(V). The TAs and As(III) uptake by S. suiae was significantly lower under higher

341

temperature conditions. In contrast, As(V) uptake was enhanced with increased temperature. While the main effect

342

of LI on TAs and iAs production by the alga was not significant. On the other hand, iconc had a significant effect on

343

the TAs and iAs produced regardless of temperature or LI conditions suggesting that uptake was dose-dependently

344

enhanced with increased in iconc which is likely a result of the availability of metal leading to high uptake in high

345

iconc.

346

temperature experiment, but less than 50% in the LI experiment throughout, most likely resulting from significant

347

uptake amounts as affected by both factors (temperature and iconc) even revealed by ANOVA results. Among iAs,

348

As(III) was substantially more abundant in the algal body than As(V), which was only present in minimal quantities

349

indicating that the alga was able to hold As(III) intracellularly. Stronger effects in morphology were observed in

350

higher iconc although the factor (temperature or LI) did affect the difference somewhat. The uptake results depended

351

upon environmental factors (temperature, LI, iconc) that may provide important insights regarding particular culture

352

conditions for efficient wastewater treatment processes for selective algal species. Yet, future studies are needed to

353

further the understanding of As metabolic processes by this alga to ascertain their effectiveness if used for

354

bioremediation.

The iAs percentage of TAs was greater than 50%, in particular, under 250–500 µg L−1 iconc in the

355 356

Acknowledgements

13

357

We are thankful to the entire research team of the Algal Cultivation and Biotechnology Laboratory headed by Dr.

358

Lee, Meng-Chou and to the supporting staff of the Traceability Certification and Inspection Center headed by

359

Director Dr. Nan, Fan-Hua, both of which are housed in the National Taiwan Ocean University.

360

Funding information

361

Compliance with ethical standards

362

Conflicts of interest

363

We declare no conflicts of interest.

364 365

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Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions

16

Figure Captions

Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1 Chemosphere (1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan (2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan (3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan (4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippines 3320 Corresponding author email: [email protected]

17 18 19 20 21

Fig. 1 The effects of temperature (15, 20, and 25 °C), iconc [initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1] and their interaction (iconc × temperature) on the accumulation of TAs in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (*** p ≤ 0.001)

22 23 24 25 26

Fig. 2 The effects of temperature (15, 20, and 25 °C), iconc [initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1] and their interaction (iconc × temperature) on the accumulation of As(III) in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (*** p ≤ 0.001)

27 28 29 30 31 32

Fig. 3 The effect of temperature (15, 20, and 25 °C), iconc [initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1] and their interaction (iconc × temperature) on the accumulation of As(V) in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (ns p ≥ 0.05, ** p ≤ 0.01, *** p ≤ 0.001)

33 34 35 36 37

Fig. 4 The effects of LI (30, 55, and 80 µmol photons m−2 s−1) and iconc (initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1) on the accumulation of TAs in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (ns p ≥ 0.05, *** p ≤ 0.001)

38 39 40 41 42

Fig. 5 The effects of LI (30, 55, and 80 µmol photons m−2 s−1) and iconc (initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1) on the accumulation of As(III) in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (ns p ≥ 0.05, *** p ≤ 0.001)

43 44 45 46 47

Fig. 6 The effects of LI (30, 55, and 80 µmol photons m−2 s−1) and iconc (initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1) on the accumulation of As(V) in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (ns p ≥ 0.05, *** p ≤ 0.001)

1

48 49

Fig. 7 Effect of concentration on percentage removal of Sarcodia suiae at optimum temperature condition (15°C)

50 51 52

Fig. 8 Effect of concentration on percentage removal of Sarcodia suiae at optimum LI condition (80 µmol photons m−2 s−1)

53 54 55

Fig. 9 The coloration of S. suiae prior to the experiment (day 0) and after seven days of culture under the indicated temperature and iconc conditions

56 57 58

Fig. 10 The coloration of S. suiae prior to the experiment (day 0) and after seven days of culture under the indicated LI and iconc conditions

59 60 61 62 63 64

Fig. 11 The effects of temperature (15, 20, and 25 °C), iconc [initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1] and their interaction (iconc × temperature) on the iAs percentage of TAs accumulated in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (** p ≤ 0.01, *** p ≤ 0.001)

65 66 67 68 69

Fig. 12 The effects of LI (30, 55, and 80 µmol photons m−2 s−1) and iconc (initial As(V) concentrations: 0, 62.5, 125, 250, and 500 µg L−1) on the iAs percentage of TAs accumulated in S. suiae after seven days of culture under the indicated conditions. All data are means of three replicates, and error bars indicate one standard deviation. Different letters on bars indicate significant differences between treatments (ns p ≥ 0.05, *** p ≤ 0.001)

70 71 72

2

1

TABLES

2 3 4

5

Table 1 Experimental design Parameters

Treatments

As(V) concentrations (µg L−1)

LI (µmol photons m−2 s−1)

Temperature (°C)

15 20 25

0, 62.5, 125, 250, 500 0, 62.5, 125, 250, 500 0, 62.5, 125, 250, 500

55 55 55

0, 62.5, 125, 250, 500 0, 62.5, 125, 250, 500 0, 62.5, 125, 250, 500

-

30 LI 55 (µmol 80 photons m−2 s−1) ED: exposure duration

6 7

1

Temperature (°C)

Natural seawater (mL)

ED (days)

-

Nutrition (F/2 medium: mL−1) -

800 800 800

7 7 7

25 25 25

-

800 800 800

7 7 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions

16

Figures

Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1 Chemosphere (1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan (2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan (3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan (4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippines 3320 Corresponding author email: [email protected]

17 18 19

*** Temperature *** iconc × Temperature *** TAs accumulation (mg kg ⁻¹)

iconc

300

a

250 b

200 150

0 µg L⁻⁻¹ As(V) ᵢconc 62.5 125

100 50

eef f f

d ff

f

250

fff

500

0 15

20 21

c

c

c

20 25 Temperature (℃ ℃)

Fig. 1

1

iconc

*** *** ***

As(III) accumulation (mg kg ⁻¹)

Temperature iconc × Temperature

22 23 24 25

200 180 160 140 120 100 80 60 40 20 0

a

b

0 µg L⁻⁻¹ As(V) ᵢconc 62.5

c

cd

cd

250

d

de f f

f f 15

ef

500

f f f

20 Temperature (℃ ℃)

125

25

Fig. 2

*** Temperature ns conc × Temperature ** i

As(V) accumulation (mg kg ⁻¹)

iconc

16 14 12 10 8 6 4 2 0

a a 0 µg L⁻⁻¹ As(V) ᵢconc 62.5

b

125

bb bb

b bb

15

20

b bbb

250

b

500

25

Temperature (℃ ℃) 26 27 28 29 30 31 32

Fig. 3

2

iconc

*** ns ns

TAs accumulation (mg kg ⁻¹)

LI iconc × LI 300

a

250 b

200

0 µg L⁻⁻¹ As(V) ᵢconc

b

62.5

150 100 50

c

c

ddd

125

c

250

ddd

ddd

500

0 30 55 80 Light intensity (µmol photons m−2 s−1)

33 34 35 36 37

Fig. 4 iconc

*** ns ns

As(III) accumulation (mg kg ⁻¹)

LI iconc × LI

38 39 40

200 150 a

0 µg L⁻⁻¹ As(V) ᵢconc 62.5

100 b

b 50

c

c ddd

125

ddd

250

c

500

ddd

0 30 55 80 Light intensity (µmol photons m−2 s−1)

Fig. 5

3

iconc

*** ns ns

As(V) accumulation (mg kg ⁻¹)

LI iconc × LI

41 42 43 44 45

18 16 14 12 10 8 6 4 2 0

a a 0 µg L⁻⁻¹ As(V) ᵢconc 62.5

ab

125 bbb

b

bbb

b

250

b bbb

500

30 55 80 Light intensity (µmol photons m−2 s−1)

Fig. 6

1.00 0.90

% Removal

0.80 0.70 0.60

Total Arsenic

0.50

Inorganic Arsenic

0.40 0 46 47 48 49 50

100

200

300 400 Concentration (mg kg-1)

Fig. 7

4

500

600

1.00 Total Arsenic

Inorganic Arsenic

% Removal

0.90 0.80 0.70 0.60 0.50 0.40 0 51 52 53 54 55 56

100

200 300 400 Concentration (mg kg-1)

Fig. 8

57 58 59

Fig. 9

5

500

600

60

61 62 63

Fig. 10

*** Temperature *** ** iconc × Temperature

iAs % of TAs (mg kg ⁻¹)

iconc

100 90 80 70 60 50 40 30 20 10 0

aa

ab ab

b

0 µg L⁻⁻¹ As(V) ᵢconc

c

cd

d

125

e fg g

g 15

gg

20 Temperature (℃ ℃)

250

fg

500 25

64 65

62.5

Fig. 11

6

66 iconc

*** ns ns

iAs % of TAs (mg kg ⁻¹)

LI iconc × LI 100 90 80 70 60 50 40 30 20 10 0

0 µg L⁻⁻¹ As(V) ᵢconc ab

a

a

a

ab c

bc d ee

ee

d

125 250

ee

500

30 55 80 Light intensity (µmol photons m−2 s−1) 67 68

62.5

Fig. 12

69 70 71 72 73

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions

19

Highlights

20 21 22 23 24 25 26 27

Chemosphere Mary Joy Halog Libatique1, 4, Meng-Chou Lee1, 2, 3, Han-Yang Yeh1, Fu-Jie Jhang1

(1) Department of Aquaculture, National Taiwan Ocean University, Keelung City 20224, Taiwan (2) Center of Excellence for Ocean Engineering, National Taiwan Ocean University, Keelung City 20224, Taiwan (3) Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City 20224, Taiwan (4) Provincial Institute of Fisheries, Isabela State University Roxas, Isabela, Philippines 3320

Corresponding author email: [email protected]

• • • •

Temperature impacts the total arsenic (TAs) and As(III) accumulation Light intensity condition did not affect TAs and inorganic arsenic (iAs) accumulation Arsenite[As(III)] produced was comparatively higher than As(V) regardless of temperature or light conditions Increased initial concentration enhanced the biosorption efficiency disregard of temperature or light conditions

1

Author contributions statements Libatique MJH performed the experiment and completed the manuscript text. Lee MC supervised the experiment, helped in experimental design, and reviewed the manuscript. Yeh HY and Jhang FJ helped in the experiment that carried out, helped in data analysis and interpretations.

January 21, 2020

Dear Editors of Chemosphere,

We wish to submit the revised version of our original research article entitled “Total and inorganic arsenic biosorption by Sarcodia suiae (Rhodophyta), as affected by controlled environmental conditions ” for consideration by the CHEMOSPHERE (Environmental Chemistry).

We confirm that this work is original and has not been published elsewhere, nor is it currently under consideration in other publications elsewhere.

Our paper has been approved by all co-authors and we have no conflicts of interest to disclose.

Thank you for considering once again this manuscript. We appreciate your time and look forward to your response. Yours sincerely,

Mary Joy Halog Libatique Contact details: Tel.: +886-2-2462-2192 ext. 5231; Fax: +886-2-2463-5441; Mobile: +886-978-586-589 E-mail: [email protected]