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Ecotoxicology and Human Environmental Health

Waterborne and Dietborne Toxicity of Inorganic Arsenic to the Freshwater Zooplankton Daphnia magna Ning-Xin Wang, Yue-Yue Liu, Zhongbo Wei, Liuyan Yang, and Ai-Jun Miao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02600 • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Environmental Science & Technology

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Table of Contents Art

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Waterborne and Dietborne Toxicity of Inorganic Arsenic to

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the Freshwater Zooplankton Daphnia magna

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Ning-Xin Wang†,‡, Yue-Yue Liu‡, Zhong-Bo Wei‡, Liu-Yan Yang‡, Ai-Jun Miao‡*

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†. School of Energy and Environment, Anhui University of Technology, Maanshan,

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Anhui Province 243002, China

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‡. State Key Laboratory of Pollution Control and Resource Reuse, School of the

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Environment, Nanjing University, Nanjing, Jiangsu Province 210023, China

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*Corresponding author:

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A.-J. Miao, School of the Environment, Nanjing University, Mail box 24, Xianlin

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Road 163, Nanjing 210023, Jiangsu Province, China PRC (mailing address),

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86-25-89680255 (phone), 86-25-89680569 (fax), [email protected] (email)

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ABSTRACT: Waterborne and dietborne exposure are both important sources for the

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accumulation of inorganic arsenic (iAs) in aquatic organisms. Although the

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waterborne toxicity of iAs has been extensively investigated, its dietborne toxicity has

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received little attention. The present study examined the acute and chronic toxicity of

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arsenate (iAsV) and arsenite (iAsIII) to the freshwater zooplankton species Daphnia

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magna under both waterborne and dietborne exposure scenarios. The bioaccumulation,

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speciation, tissue and subcellular distributions of arsenic were analyzed to understand

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the mechanisms accounting for differences in toxicity related to different arsenic

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species, exposure scenarios, and exposure duration. The toxicity of iAs increased with

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exposure time, and iAsIII was more toxic than iAsV. Moreover, although dietborne iAs

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had no acute effect on D. magna, it incurred significant toxicity in the

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chronic-exposure experiment. Nevertheless, the toxicity of dietborne iAs was still

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lower than that of waterborne iAs regardless of the exposure duration. This difference

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was found to be caused by the lower bioaccumulation of dietborne iAs, its higher

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distribution in the gut and in the biologically detoxified subcellular fraction, and

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greater transformation to the less toxic dimethylarsinic acid. Overall, the dietborne

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toxicity of iAs should be considered when evaluating the environmental risks posed

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by arsenic.

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■ INTRODUCTION

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Arsenic is ubiquitous in the environment with its concentration in natural waters

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ranging from < 0.5 to 10,000 µg/L.1,2 Among the four oxidation states of arsenic (+5,

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+3, 0, and −3), the pentavalent (iAsV) and trivalent (iAsIII) forms of inorganic arsenic

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(iAs) are the most common in aquatic environments. Organoarsenics, such as

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dimethylarsinic (DMA) and monomethylarsonic acid (MMA), also occur but at much

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lower concentrations. Although the toxicity of trivalent methylarsenics is higher than

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that of iAs,3 the inorganic forms are generally more toxic than organoarsenics and

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their toxicity thus requires comprehensive research and regulatory attention. Most

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studies of arsenic toxicity have been limited to waterborne exposure but in a

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biokinetics study of iAsV performed by our group > 90% of the arsenic

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bioaccumulation in the freshwater zooplankton species Daphnia magna originated

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from its algal foods.4 Therefore, evaluating the dietborne toxicity of arsenic is

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imperative.

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In the limited studies about the dietborne toxicity of arsenic, the organism of

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interest was either the freshwater rainbow trout Oncorhynchus mykiss or the saltwater

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grunt Terapon jarbua.5-7 Dietborne arsenic was shown to reduce the growth of

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rainbow trout, associated with a reduction in the feeding rate, but had no toxic effects

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on T. jarbua. The different results may have been due to the difference in the fish

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species or to the shorter exposure time used by Zhang et al.7 Nevertheless, dietborne

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arsenic toxicity in invertebrates has not been examined. Further, waterborne and

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dietborne toxicity of iAs or other metals/metalloids have rarely been compared 4


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directly.

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The present study investigated the acute and chronic toxicity of waterborne and

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dietborne sources of iAsIII and iAsV to D. magna. Arsenic accumulation and

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speciation in both the daphnids and their algal foods were measured. The distribution

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of arsenic in daphnid tissues was also examined using synchrotron-radiation-based

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micro X-ray fluorescence microscopy (µXRF), conducted at the Shanghai Radiation

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Synchrotron Facility (BL15U1, SSRF, Shanghai, China). The distribution of arsenic

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in five subcellular fractions was further analyzed:8 organelles, heat-sensitive proteins

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(HSPs, mostly enzymes), metallothionein-like proteins (MTLPs), metal-rich granules

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(MRGs), and cellular debris. Organelles and HSPs are the metal-sensitive fractions

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(MSF) while MTLPs and MRGs are sites of biologically detoxified metal (BDM)

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accumulation.8 The main objective of the present study was to testify the dietborne

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toxicity of iAs to the invertebrate D. magna and to reveal the underlying mechanisms

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for the potential toxicity difference between the waterborne and dietborne exposure

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scenarios.

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■ MATERIALS AND METHODS

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Organisms and Culture Conditions. Both the freshwater zooplankton D.

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magna and its foods, the green alga Chlamydomonas reinhardtii, were obtained from

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the Institute of Hydrobiology, Chinese Academy of Science, Wuhan, China. The D.

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magna clone has been maintained in our laboratory for almost 10 years. It is routinely

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reared in aerated tap water that is refreshed every other day. C. reinhardtii is fed daily

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to daphnids < 3 days of age at a concentration of 5×104 cells/mL and at double this

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concentration thereafter. The algal cells used in this study were maintained sterilely in

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WC medium until they reached the exponential growth phase, when they were

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collected by centrifugation (3900 g, 15 min), resuspended in deionized water, and

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stored at 4oC until used as foods.9 Both the daphnids and the alga were cultured in an

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incubator at 23.5±1°C with a light intensity of 50 µmol photons/m2/s and a light-dark

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cycle of 12h:12h. Adult daphnids (7 days old) and neonates (< 1 day old) from the

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same brood were used in the acute and chronic toxicity tests, respectively. The

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simplified M7 medium (SM7, pH = 8.0) described by Miao et al.4 was used as the

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base of all exposure media, to better manipulate the different arsenic species tested.

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Acute Waterborne Exposure. Two 48-h toxicity tests were performed for iAsIII

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and iAsV, at concentrations of 0, 0.5, 1.5, 3, 4.5, and 6 mg/L and 0, 1.5, 3, 4.5, 6, and

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7 mg/L, respectively. A lower concentration range of iAsIII was used due to the higher

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toxicity of this species. Four replicates, each consisting of 15 daphnids in 100 mL of

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experimental medium within a 100 mL beaker, were prepared for each treatment. At

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the beginning of the toxicity tests, daphnids of the appropriate age were collected

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from the stock, depurated in fresh SM7 for 1 h to clear the foods retained in their guts,

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and then transferred to exposure medium lacking any foods. The daphnids were

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depurated for 1 h herein considering the fact that approximately 24 h had passed since

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their last feeding and their guts were relatively clean. During the 48-h experimental

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period, mortality was monitored every 3 h and any dead daphnids were collected and

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washed with fresh SM7 to remove the arsenic weakly adsorbed on their surface. At 6


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the end of the toxicity test, the dead daphnids of each replicate together with those

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that were still alive were dried in an oven at 60°C until their weight remained constant.

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After their dry weight (dw) was measured, the daphnids were digested in 0.5 mL of

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concentrated nitric acid and 2 mL of H2O2, following the same procedure as described

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in Wang et al.10 The arsenic concentration in the digest, and therefore in the daphnids

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([As]daphnid), was then determined by inductively coupled plasma mass spectrometry

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(ICP-MS, NexION300, PerkinElmer, USA), using Ge as the internal standard. The

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tissue and subcellular distributions of arsenic and its speciation were also analyzed

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with details in Supporting Information.

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Acute Dietborne Exposure. Arsenic-containing diets were prepared from a

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mid-exponential phase culture of C. reinhardtii without iAs. The algae were collected

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by centrifugation and resuspended in WC medium containing 0, 1.5, 4.5, or 7.5 mg

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iAsIII or iAsV/L. The algal cell density was initially 1×105 cells/mL and was

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enumerated daily thereafter using a Z2 Coulter counter (Beckman Coulter Inc., CA,

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USA). After 4 days, arsenic content in the algal cells reached steady state. The cells

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were then collected, stored at 4°C, and used within 24 h. The arsenic content in the

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algal foods (including those adsorbed on the cell surface and inside the cells) was

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measured as described in our previous study.9,10

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The other procedures of the dietborne toxicity tests were similar to those in the

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waterborne experiment, except that the daphnids were exposed for 48 h to C.

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reinhardtii at a concentration of 1×105 cells/mL and containing different amounts of

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arsenic. Further, instead of washing with fresh SM7 in the waterborne experiment, the 7



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daphnids were depurated with 1×105 cells/mL of algal foods (without iAs) for 3 h to

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determine [As]daphnid at the end of the dietborne toxicity tests. The depuration time

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was chosen based on our previous finding that [As]daphnid didn’t decrease any more

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after 3 h during a 24-h depuration period.4 Potential arsenic release from the algal diet

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was also measured when the medium was refreshed after 24-h exposure. For this

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purpose, a 10-mL aliquot from each replicate was collected, filtered through a

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0.22-µm polycarbonate membrane, acidified by HNO3, and the arsenic concentration

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determined by ICP-MS as described above.

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Chronic Waterborne Exposure. The exposure time in the chronic toxicity tests

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was 21 days, following the OECD 211 guideline,11 and the same concentration of

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iAsIII and iAsV was used in each treatment: 0, 0.01, 0.03, 0.1, 0.3, and 1 mg/L. To

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ensure waterborne exposure while excluding potential interference from the dietborne

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route, the daphnids were exposed for 12 h to different concentrations of waterborne

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iAs without algal foods (exposure phase) and then transferred to medium without iAs

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but containing non-contaminated algal foods (feeding phase). After the 12-h feeding,

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the daphnids were returned to the toxicity medium without algal foods; this procedure

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was repeated daily. The exposure medium was refreshed every 24 h to minimize

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variations in the concentration and speciation of arsenic. Daphnid mortality was

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monitored daily during the 21-day experiment, together with other parameters such as

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growth and reproduction. Arsenic accumulation was measured at the end of the

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experiment. The other procedures were the same as described above for the

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waterborne acute toxicity test. 8


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Chronic Dietborne Exposure. The experimental procedure was similar to that

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of the above-described dietborne acute toxicity test, except the algal foods were

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pre-exposed to lower concentrations (0, 0.01, 0.03, 0.1, 0.3, and 1 mg/L) of iAs. To

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ensure consistency with the waterborne chronic toxicity experiment, the daphnids

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were cultured in medium without arsenic and algal foods during the first 12 h of each

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day and then in medium containing arsenic-contaminated foods for the remaining 12 h.

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To minimize the release of arsenic from the algal foods, the medium together with the

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foods was refreshed every 24 h.

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Statistical Analysis. Statistical analyses were carried out using SPSS 11.0

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(Chicago, USA). Significant differences were identified based on the results of a

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one-way or two-way analysis of variance with post-hoc multiple comparisons (Tukey

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or Tamhane). The normality (Kolmogorov-Smirnovand Shapiro-Wilk tests) and

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homogeneity of variance (Levene’s test) of the data were examined during the

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analysis of variance. The dose-response curves were fitted by the Logistic equation

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through GraphPad Prism 5 (GraphPad Software Inc., CA, USA) and the LC50 of each

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toxicity test (when available) was thus obtained. The values of LC50 were compared

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by extra sum-of-squares F-test in GraphPad Prism.

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■ RESULTS

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Acute Toxicity. Significant toxicity developed in daphnids exposed for 48 h to

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different concentrations of waterborne iAsV or iAsIII (Figure 1a, c). However, the

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toxicity of iAsIII was higher than that of iAsV and caused the massive, earlier death of

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the daphnids than was the case following iAsV exposure. Specifically, all daphnids 9



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died after an 18-h exposure to 6 mg iAsIII/L but after 42 h for the same concentration

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of iAsV. The toxicity difference between iAsV and iAsIII was also evidenced in the

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waterborne-concentration-based dose-response curves (Figure S1a, Supporting

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Information). The LC50 thus obtained was 3.51 and 1.91 mg/L for iAsV and iAsIII,

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respectively (Table S1, Supporting Information).

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In the dietborne toxicity tests, an exposure-concentration-dependent amount of

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arsenic accumulated in the algal diet, with a significantly (p < 0.05, two-way ANOVA)

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higher amount of iAsV than of iAsIII (Figure S2a, Supporting Information).

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Nevertheless, the toxicity induced by all iAsV or iAsIII concentrations during the

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dietborne exposure was negligible compared to that induced by waterborne exposure

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(Figure 1b, d and Figure S1b, Supporting Information). The arsenic concentration (i.e.,

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0, 1.5, 4.5, and 7.5 mg/L) used in all the toxicity results of the dietborne experiment

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represents the dissolved concentration of iAsV or iAsIII ([As]dis) to which C.

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reinhardtii was exposed during its preparation as algal foods. Despite the

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concentration-dependent release of arsenic from the algal cells into the dissolved

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phase in the dietborne toxicity tests, the highest [As]dis was only 178.7 µg/L (Figure

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S2b, Supporting Information).

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[As]daphnid increased hyperbolically as [As]dis in the toxicity medium

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(waterborne exposure) and in the medium in which the algal foods were cultured

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(dietborne exposure) increased (Figure 1e, f). The correlation between [As]daphnid and

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[As]dis could be well-simulated by the Langmuir isotherm, which showed the

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accumulation of more iAsV than iAsIII especially at high concentrations, regardless of 10


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whether exposure was waterborne or dietborne. Further, significantly (p < 0.05,

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two-way ANOVA) more arsenic bioaccumulated in response to waterborne than to

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dietborne exposure. Based on the arsenic bioaccumulation results, the relative change

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in D. magna mortality at different [As]daphnid was plotted (Figure S1c, d, Supporting

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Information), which still revealed a significant toxicity difference between iAsV and

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iAsIII in the waterborne toxicity tests. The [As]daphnid-based LC50s are also listed in

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Table S1, Supporting Information.

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Chronic Toxicity. Much lower concentrations of both iAs species were used in

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the chronic than in the acute toxicity tests. The second lowest concentration was equal

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to the amount of arsenic allowable in drinking water (10 µg/L) by the WHO.

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Nevertheless, at this concentration significant daphnid death still occurred during the

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21-day exposure period, even in the dietborne experiment (Figure 2a–d). Similar to

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the acute toxicity tests, iAsIII was more toxic than iAsV and waterborne exposure

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resulted in a higher toxicity than did dietborne exposure, based on the mortality rates

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at the end of the chronic toxicity experiment. Accordingly, the [As]dis-based LC50 of

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iAsV and iAsIII was 20.8 and 8.5 µg/L in the waterborne and 153 and 57.4 µg/L in the

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dietborne toxicity tests (Figure S3a, b and Table S1, Supporting Information).

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Nevertheless, the daphnids died earlier in the iAsV than in the iAsIII treatments and in

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the dietborne than in the analogous waterborne treatments, contrasting with the

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observations in the acute toxicity tests. Significant inhibitory effects on daphnid

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growth and reproduction were also observed, with the same extent of inhibition with

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increasing arsenic concentration obtained for the different arsenic species and 11



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exposure scenarios (Tables S2 and S3, Supporting Information).

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Similar to the acute experiments, a hyperbolic correlation between [As]daphnid

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and [As]dis was also obtained in the chronic toxicity tests (Figure 2e, f). However, the

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bioaccumulation of iAsIII and iAsV did not significantly (p > 0.05, two-way ANOVA)

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differ and the bioaccumulation of the same species of arsenic was comparable in the

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different exposure scenarios. Accordingly, [As]daphnid was in the range of 44.9–65.6 µg

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arsenic/g-dw at the highest arsenic concentration used in the four toxicity tests (iAsV

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and iAsIII, waterborne and dietborne). A plot of the mortality data against [As]daphnid

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(Figure S3c, d, Supporting Information) showed that iAsIII was still more toxic than

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iAsV and that toxicity was higher following waterborne than dietborne exposure. The

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[As]dis- and [As]daphnid-based LC50 values are listed in Table S1, Supporting

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Information.

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Tissue Distribution. The daphnids were outlined by their Ca signal and thus

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visible on the µXRF images (Figure 3 and S4). No significant signal of arsenic was

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detected in the daphnids of the control treatment without any exposure of arsenic

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(Figure S4). In the waterborne acute toxicity tests, arsenic was mostly found in the

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daphnid gut, regardless of the arsenic species, but in other parts of the daphnids as

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well. In particular, an obvious arsenic signal was seen in the daphnids’ eggs. By

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contrast, in the dietborne acute toxicity tests arsenic accumulated in the gut but not

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outside of it. In the chronic toxicity experiments, arsenic was also distributed

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throughout the daphnids exposed to waterborne iAsIII or iAsV. In the dietborne

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chronic experiment, the distribution of arsenic was similar to that in the waterborne 12


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experiment; namely, a significant amount of arsenic was detected outside the gut. This

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result contrasted with that obtained in the dietborne acute toxicity experiment.

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Subcellular Distribution. As shown in Table 1, a substantial amount of arsenic

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was detected in all five subcellular fractions. Exposure to iAsIII or iAsV resulted in the

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same subcellular distribution pattern, regardless of the duration of the experiment or

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exposure scenario. Moreover, although in the acute toxicity tests the subcellular

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distribution of arsenic was the same following waterborne vs. dietborne exposure, in

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the chronic experiments significantly (p < 0.05, one-way ANOVA) more arsenic was

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bound to MTLPs (and thus BDM) and less to HSPs (and thus MSF) in daphnids

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exposed to dietborne iAs. In addition, compared to the acute toxicity tests, in the

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chronic experiments more arsenic was found in MRGs and MTLPs (and thus in BDM)

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and less in organelles and HSPs (and thus in MSF), regardless of the arsenic species

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and exposure scenario.

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Arsenic Speciation. As shown in Figure 4, significant oxidation (reduction) of

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iAsIII (iAsV) to iAsV (iAsIII) occurred in all iAsIII (iAsV) toxicity tests. Regardless of

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the exposure duration, exposure scenario, and the valence of the iAs used in the

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toxicity tests, significantly (p < 0.05, one-way ANOVA) more iAsIII than iAsV

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ultimately accumulated in the daphnids. Further, the proportion of both arsenic

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species decreased abruptly when the exposure duration was increased from 2 to 21

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days, as the amount of DMA formed increased over time. In addition to its variation

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with exposure time, the proportion of DMA was higher in daphnids with dietborne

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exposure. Accordingly, DMA accounted for 12.2%–28.1% and 87.4%–88.6% of 13



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[As]daphnid in the dietborne acute and chronic toxicity tests, respectively vs. a

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negligible percentage and 57–66.6% in the corresponding waterborne tests. Arsenic

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speciation in the algal foods used in the dietborne toxicity tests was also analyzed. In

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algae pre-exposed to iAsIII, 44.9% of the cellularly accumulated arsenic consisted of

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iAsIII and 55.1% of iAsV. In the iAsV toxicity test, iAsV was the only arsenic species

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found in the algal cells.

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■ DISCUSSION

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Bioaccumulation. In the acute toxicity tests, more iAsV than iAsIII accumulated

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under both waterborne and dietborne exposures (Figure 1e, f). Similarly,

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Suhendrayatna et al.12 found that arsenic accumulation by D. magna was higher when

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the medium contained iAsV than iAsIII. They proposed that iAsV more easily passes

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through the membrane of the daphnid digestive organ and is more easily metabolized.

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Further, in our study the much higher accumulation of dietborne iAsV than iAsIII may

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have been due to its higher content in the algal foods (Figure S2a, Supporting

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Information).9,10 Nevertheless, the difference in the accumulation of the two iAs

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species in D. magna disappeared in our chronic toxicity experiment, suggesting that

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the same equilibrium is reached after a 21-day exposure.

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Miao et al.4 reported that waterborne and dietborne exposure accounts for < 10%

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and > 90% of the total iAsV accumulation in D. magna, respectively. This conclusion

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was based on the assumption that the bioconcentration factor (BCF) of iAsV in the

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diet was in the range of 10 to 1000 L/g. Since the concentration of dissolved iAs

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(1.5–7.5 mg/L) in the exposure medium of our acute experiment was much higher 14


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than the 1.56 µg/L reported by Miao et al.,4 the BCF of iAs in the algal foods of the

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present study was only 0.25–2.0 L/g. Under this condition, the contribution of

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waterborne exposure to total arsenic bioaccumulation, as the reciprocal of the BCF,

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would increase, which explains the greater accumulation of iAs from the water than

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from the diet in the acute experiment (Figure 1e, f). Therefore, the relative

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contribution of waterborne and dietborne exposure to arsenic accumulation in

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daphnids depends on the concentration of dissolved iAs in the ambient environment.

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Namely, at a low ambient iAs concentration dietborne exposure dominates whereas at

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a high ambient iAs concentration waterborne exposure becomes the dominant factor.

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Transformation. The reduction of iAsV to iAsIII and the subsequent

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methylation of the latter to organoarsenics is an important pathway in the

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detoxification of iAs in aquatic organisms.13,14 Similar to our own observations,

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Suhendrayatna et al.12 found that the arsenic accumulated in D. magna was mainly

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accounted for by iAsIII (55.4–75%) and iAsV (24–44.5%), with trace amounts of DMA

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(0.1–5.4%). Caumette et al.15 also reported 56% iAsV, 10% iAsIII, and 34%

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organoarsenics (e.g., MMA, DMA, and arsenosugars) in the Daphnia pulex collected

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from an arsenic-contaminated lake. Together, our study and those cited above support

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the opinion that iAsIII is an intermediate in the transformation of iAsV to DMA. The

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DMA detected in the daphnids in the dietborne experiment was not from the algal

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foods since only iAs was present in the diet. Given that more DMA was found in

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daphnids from the dietborne than from the waterborne experiment, the significant

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transformation of iAs to organoarsenics seems to take place in the daphnid digestive 15



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tract, where it is mediated by resident bacteria.16

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Distribution of iAs in Different Tissues and Subcellular Compartments. The

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distribution of a metal or metalloid in different tissues of an organism plays critical

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roles in its toxicity. Nevertheless, few studies have examined metal distribution in D.

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magna, as its small size (1–4 mm) precludes its dissection, processing, and

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subsequent analysis by conventional analytical techniques such as atomic absorption

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spectroscopy and ICP-MS. The utility of two microanalytical techniques, laser

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ablation ICP-MS (LA-ICP-MS) and synchrotron radiation-based µXRF, in the

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elemental imaging of D. magna was evaluated by Gholap et al.,17 who demonstrated

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their complementary use in providing an exhaustive chemical profiling of D. magna

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tissue samples. Li et al.18 also used LA-ICP-MS to show that waterborne arsenic was

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distributed throughout D. magna whereas dietborne exposure resulted in larger

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amounts of arsenic in the daphnid gut. In the above-cited study of D. pulex directly

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collected from a lake (and thus with both waterborne and dietborne exposure), µXRF

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mapping showed that the arsenic concentration was ten times higher in the daphnid

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gut than in the surrounding tissues.18 These results are similar to our own. Although

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arsenic accumulation only in the gut was detected following acute dietborne exposure,

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a significant amount of arsenic was detected in other daphnid tissues after the 21-day

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dietborne exposure, suggesting a relocation of arsenic from the gut between days 2

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and 21. Further, skin penetration is generally thought to be the main uptake route of

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waterborne pollutants. Nevertheless, the detection of waterborne arsenic in the

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daphnid’s gut implies that passive drinking may be another non-negligible 16


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accumulation route for this exposure scenario, similar to what we reported before.19

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Intracellular accumulation and the subsequent subcellular distribution of a metal

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or metalloid are directly related to its toxicity.20 Therefore, the potential difference in

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toxicity resulting from the different iAs species, exposure durations, and exposure

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scenarios may reflect their different subcellular distribution patterns. Li et al.18

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exposed D. magna to 1 µM iAsV in the water phase for 3 h and then determined its

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subcellular fractionation by a method similar to that used in our study. Most of the

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arsenic was found in organelles, HSPs, and cellular debris while much less was

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distributed in MRGs and MTLPs. The difference in the subcellular distribution of

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arsenic between that study and ours may be a combined effect of the much lower

338

arsenic concentration and shorter exposure time used by Li et al.,18 given that MTLP

339

synthesis is a function of both pollutant concentration and exposure time.21 The

340

importance of exposure time was further evidenced by our finding of significantly (p

341

< 0.05, one-way ANOVA) more arsenic in BDM and less in MSF in daphnids with

342

chronic than with acute exposure. The subcellular distribution of iAsIII and iAsV from

343

waterborne and dietborne sources was also investigated in the marine fish T. jarbua.7

344

After a 10-day exposure, MTLPs (36.0-46.7%) and cellular debris (31.5-45.4%) were

345

the major arsenic binding sites in the muscle of the fish, with only small amounts

346

(3.5–14.5%) in other fractions. In that study, there was no significant difference in the

347

subcellular distribution of arsenic that had accumulated via the different routes. By

348

contrast, in P. viridis exposed to Cd, the metal was distributed evenly among the

349

different fractions after dietborne exposure, in contrast to the dominant distribution in 17



350

insoluble fractions (organelles, cellular debris, and MRGs) after waterborne

351

exposure.22 Together, these phenomena imply that the subcellular distribution of a

352

metal is a dynamic process, one that depends on the exposure time and exposure

353

scenario but which is also organism- or metal-species-specific.

354

Toxicity of iAsIII vs. iAsV. The two iAs species differ in their toxicity

355

mechanisms, as iAsV mainly interferes in phosphate metabolism while iAsIII binds to

356

intracellular thiols of enzymes and tissue proteins.23 Our study showed that for the

357

zooplankton D. magna, iAsIII was more toxic than iAsV irrespective of the exposure

358

duration and exposure scenario. In a previous study of D. magna, the 24-h lethal

359

concentration of waterborne iAsIII and iAsV was 1.7 and 5 mg As/L, respectively,12 but

360

the literature contains no reports of direct comparisons of the toxicity of waterborne

361

vs. dietborne iAsIII and iAsV. Our finding that iAsIII was more toxic than iAsV cannot

362

be explained by the potential difference in the metals’ bioaccumulation, distribution,

363

or chemical speciation in the daphnids, since there was no significant difference in

364

their tissue or subcellular distribution and chemical speciation. Moreover, in the acute

365

toxicity tests the bioaccumulation of iAsV was even higher than that of iAsIII. Our

366

results suggest that the different arsenic species triggered their different modes of

367

toxic action before biotransformation. It is also possible that arsenic speciation differs

368

in different tissues and subcellular compartments, although the overall speciation was

369

comparable in the whole organism. Differences in arsenic speciation between the gut

370

and surrounding tissues of D. pulex was reported by Caumette et al.15 Moreover,

371

although in our acute toxicity experiment the toxic effects of iAsIII were manifested 18


Page 18 of 35

Page 19 of 35


372

earlier than those of iAsV, the pattern was reversed in the chronic toxicity experiment,

373

which implies the involvement of different modes of action in acute vs. chronic iAs

374

exposure. This hypothesis is supported by the finding of Pane et al.,24 who showed

375

that Mg2+ antagonism is one of the mechanisms underlying the acute toxicity of

376

waterborne Ni to D. magna, while Ni-mediated impairment of respiratory function

377

occurs during chronic exposure.

378

Toxicity of Waterborne vs. Dietborne Exposure. Dietborne exposure is an

379

important route in the bioaccumulation and toxicity of metals in aquatic

380

organisms.25,26 Nevertheless, there are still some challenges in conducting dietborne

381

toxicity tests, such as the release of metals from the algal diets into the dissolved

382

phase, the decrease of food biomass caused by feeding, and the potential nutritional

383

change in the diets due to metal exposure.26 Furthermore, in waterborne toxicity tests

384

potential interference from dietborne sources must be excluded. In the present study,

385

different measures were taken to minimize the potential interference from waterborne

386

(dietborne) arsenic in dietborne (waterborne) toxicity tests. In the dietborne toxicity

387

tests, the daphnids were fed daily and the exposure medium was refreshed

388

simultaneously to minimize the liberation of arsenic from the foods and the decrease

389

in the food biomass during the exposure period. Under this condition, the maximum

390

concentration of arsenic released from the algae was 178.7 µg/L and < 9.0 µg/L in the

391

acute and chronic toxicity tests, respectively. The low concentrations in the

392

experimental media had negligible effects on arsenic accumulation by the daphnids

393

and on the subsequent toxicity. In the waterborne chronic toxicity tests, arsenic 19



394

exposure and feeding were separated to ensure that any ensuing toxicity was not due

395

to the accumulation of arsenic from the algal foods. This is in contrast to the

396

recommendations of OECD 211,11 in which both the algal foods and the pollutant are

397

present in the exposure medium. Therefore, such recommendations result in a

398

combined exposure to both waterborne and dietborne sources, as prior to being

399

ingested by the daphnids the algal foods may adsorb and take up the pollutant from

400

the exposure medium. The intermittent exposure regime implemented in our study

401

was also used in the studies of Hook and Fisher27 and Geffard et al.,28 in which the

402

daily feeding duration was 4 h and 8 h, respectively. Our use of a feeding duration of

403

12 h is more realistic in comparisons of the toxicity of waterborne vs. dietborne

404

exposure. Although we did not examine the potential effects of arsenic on the lipid

405

and protein contents of the food, the arsenic concentration in the exposure medium

406

(especially in the chronic toxicity experiment) was much lower than the EC50 of

407

either iAsIII (132 mg/L) or iAsV (33.5 mg/L) for C. reinhardtii.9,10 Therefore,

408

variations in the nutrient contents of the diet, if any, would be expected to have had

409

limited effects on the toxicity observed herein.

410

During the acute exposure to both iAs species, significant toxicity occurred in

411

the waterborne but not the dietborne experiment. Similarly, in the above-cited study of

412

Suhendrayatna et al.12 the 24-h acute toxicity of waterborne iAsIII and iAsV to D.

413

magna was observed but dietborne arsenic concentrations up to 1130 mg/kg had no

414

toxic effect on daphnid growth. Lima et al.29 reported that foods decreased the toxicity

415

of iAsIII to daphnids, with 96-h LC50 values of 4.34 and 1.5 mg/L with and without 20


Page 20 of 35

Page 21 of 35


416

the addition of foods, respectively. The difference in the toxicity between the

417

waterborne and dietborne exposure scenarios in our study may be partly explained by

418

the lower accumulation of the two iAs species in the daphnids during the dietborne

419

experiment and by the concentration of dietborne iAs only in the daphnid gut. In the

420

latter case, the relatively short duration (2 days) of the study would have not allowed

421

an attack by arsenic of target sites in other tissues. Since the transformation of iAs to

422

organoarsenics serves as an arsenic detoxification pathway, a significant amount of

423

DMA was detected in daphnids from the dietborne but not the waterborne experiment.

424

Thus, DMA also played a non-negligible role in the lower toxicity of dietborne

425

arsenic.

426

In contrast to the acute dietborne toxicity tests, significant adverse effects were

427

observed in the chronic dietborne toxicity experiment although the toxicity was still

428

lower than that in the corresponding waterborne experiment. In the few previous

429

studies of the chronic toxicity of arsenic, dietborne exposure was not separated from

430

waterborne exposure and significant differences in toxicity were reported by the

431

different studies. Spehar et al.30 found no mortality of D. magna after a 14-day

432

exposure to 88–973 µg iAs/L, but death, albeit unexplained, occurred thereafter. Lima

433

et al.29 examined the chronic toxicity of iAsIII to D. magna, with significant mortality

434

occurring between 633 and 1320 µg/L. These concentrations were higher than the

435

LC50s determined in our study (8.5–153 µg/L), perhaps due to the unreported

436

difference in the chemical composition of the experimental medium used in the other

437

studies. For example, the presence of phosphate in the medium may decrease the 21



438

bioaccumulation and toxicity of iAsV.4 In addition, different forms of arsenic were

439

used in those toxicity tests. For instance, Tisler and Zagorc-Koncan31 used As2O3,

440

despite its relatively low solubility, in their chronic toxicity study, which might have

441

led to an underestimation of the toxicity of iAsIII. We found that in contrast to the

442

distribution of arsenic in the dietborne acute toxicity experiment, a substantial amount

443

of arsenic was found outside the gut of D. magna in the dietborne chronic toxicity

444

experiment, which partly explained the significant toxicity under this scenario. The

445

time-dependent variation in the tissue distribution of dietborne arsenic reflects its

446

gradual assimilation, after which it can bind to the respective target sites. Nevertheless,

447

in the chronic experiment the toxicity of dietborne arsenic was still lower than that of

448

waterborne arsenic. This difference may have been due to the lower proportion of

449

more highly toxic arsenic species (i.e., iAs) in the daphnids in the dietborne than in

450

the waterborne experiment. Additionally, in the dietborne experiment more arsenic

451

was subcellularly distributed in BDM and debris, which further lowered its toxicity.

452

Overall, this study directly compared the waterborne and dietborne toxicity of

453

iAs to D. magna in both acute and chronic exposure experiments. Significant toxic

454

effects of dietborne iAs on the survival, reproduction, and growth of D. magna under

455

specific conditions were demonstrated. This exposure scenario should thus be

456

considered in evaluations of the environmental and health risks posed by arsenic to

457

aquatic organisms. Nevertheless, dietborne exposure had a milder impact than

458

waterborne exposure, partly because the former resulted in less bioaccumulation,

459

greater transformation to DMA, reduced distribution to tissues outside the gut, and 22


Page 22 of 35

Page 23 of 35


460

greater distribution in BDM. More detailed studies are needed to further elucidate the

461

molecular mechanisms underlying the differences between the waterborne and

462

dietborne toxicity of iAs.

463

■ ACKNOWLEDGEMENTS

464

We thank three anonymous reviewers for their constructive suggestions on this

465

paper. This work was supported by the National Natural Science Foundation of China

466

(21677068, 21507001, 21237001, and 41271486), the Foundation of the State Key

467

Laboratory for Pollution Control and Resource Reuse (PCRRF16014), and Chinese

468

Public Science and Technology Research funds for ocean projects (201505034).

469

470 471

SUPPORTING INFORMATION This Supporting Information is available free of charge on the ACS Publications

Website.

472

Detailed information about analysis of the tissue and subcellular

473

distributions of arsenic and its speciation. The [As]dis- and [As]daphnid-based

474

median lethal concentration (LC50) of iAsV and iAsIII (Table S1). The toxicity

475

results of growth (Table S2) and reproduction (Table S3) in the chronic

476

experiment. The [As]dis- and [As]daphnid-based dose-response curves of the acute

477

experiment (Figure S1). The concentration of arsenic in the algal food and the

478

concentration of arsenic released into the exposure medium from the algal diets in

479

the dietborne acute experiment (Figure S2). The [As]dis- and [As]daphnid-based

480

dose-response curves of the chronic experiment (Figure S3). Distribution of

481

arsenic in D. magna in the control treatment without any addition of arsenic, as

482

determined by synchrotron-radiation-based µXRF (Figure S4).

483 23



484

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485

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486

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carinata: the influence of microbial transformation in natural waters. Environ.

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Geochem. Health. 2009, 31 (1), 133-141.

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(2) Smedley, P.L.; Kinniburgh, D.G. A review of the source, behaviour and

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distribution of arsenic in natural waters. Appl. Geophys. 2002, 17 (5), 517-568.

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(3) Shaw, J.R.; Glaholt, S.P.; Greenberg, N.S.; Sierra-Alvarez, R.; Folt, C.L. Acute

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toxicity of arsenic to Daphnia pulex: Influence of organic functional groups and

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oxidation state. Environ. Toxicol. Chem. 2010, 26 (7), 1532-1537.

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arsenic in Daphnia magna under different phosphorus and food density regimes.

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Mattson, V.R.; Dawson, T.D.; Lott, K.G. Effects of copper, cadmium, lead, and

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arsenic in a live diet on juvenile fish growth. Can. J. Fish. Aquat. Sci. 2010, 67 (11),

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growth of juvenile rainbow trout. Aquat. Toxicol. 2011, 104 (1-2), 108-115.

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inorganic arsenic in a marine juvenile fish Terapon jarbua after waterborne and

Erickson, R.J.; Mount, D.R.; Highland, T.L.; Russell, H.J.; Jenson, C.T. The

Zhang, W.; Huang, L.M.; Wang, W.X. Biotransformation and detoxification of

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dietborne exposure. J. Hazard. Mater. 2012, 221-222 (4), 162-169.

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biologically detoxified metal (BDM). Mar. Ecol. Prog. Ser. 2003, 249 (1), 183-197.

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of nitrogen and phosphorus on arsenite accumulation, oxidation, and toxicity in

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Chlamydomonas reinhardtii. Aquat. Toxicol. 2014, 157 (7), 167-174.

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(10) Wang, N.X.; Li, Y.; Deng, X.H.; Miao, A.J.; Ji, R.; Yang, L.Y. Toxicity and

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bioaccumulation kinetics of arsenate in two freshwater green algae under different

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phosphate regimes. Water Res. 2013, 47 (7), 2497-2506.

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(11) OECD. OECD Guidelines for the testing of chemicals. Methods Mol. Biol. 2004,

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947, 37-56.

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(12) Suhendrayatna, A.O.; Maeda, S. Arsenic accumulation, transformation and

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tolerance on freshwater Daphnia magna. Toxicol. Environ. Chem. 1999, 72 (1-2),

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1-11.

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(13) Levy, J.L.; Stauber, J.L.; Adams, M.S.; Maher, W.A.; Kirby, J.K.; Jolley, D.F.

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Toxicity, biotransformation, and mode of action of arsenic in two freshwater

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microalgae (Chlorella sp. and Monoraphidium arcuatum). Environ. Toxicol. Chem.

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2005, 24 (10), 2630-2639.

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(14) Zhang, W.; Guo, Z.Q.; Zhou, Y.Y.; Liu, H.X.; Zhang, L. Biotransformation and

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detoxification of inorganic arsenic in Bombay oyster Saccostrea cucullata. Aquat.

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Toxicol. 2015, 158, 33-40.

Wallace, W.G.; Lee, B.G.; Luoma, S.N. Subcellular compartmentalization of Cd

Wang, N.X.; Huang, B.; Xu, S.; Wei, Z.B.; Miao, A.J.; Ji, R.; Yang, L.Y. Effects

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(15) Caumette, G.; Koch, I.; Moriarty, M.; Reimer, K.J. Arsenic distribution and

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speciation in Daphnia pulex. Sci. Total. Environ. 2012, 432, 243-250.

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(16) Freese, H.M.; Schink, B. Composition and stability of the microbial community

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inside the digestive tract of the aquatic crustacean Daphnia magna. Microb. Ecol.

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2011, 62 (4), 882-894.

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(17) Gholap, D.S.; Izmer, A.; De, S.B.; van Elteren, J.T.; Selih, V.S.; Evens, R.; De

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Schamphelaere, K.; Janssen, C.; Balcaen, L.; Lindemann, I.; Vincze, L.; Vanhaecke, F.

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Comparison of laser ablation-inductively coupled plasma-mass spectrometry and

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micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna.

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Anal. Chim. Acta. 2010, 664 (1), 19-26.

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(18) Li, M.T.; Luo, Z.X.; Yan, Y.M.; Wang, Z.H.; Chi, Q.Q.; Yan, C.Z.; Xing, B.S.

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Arsenate accumulation, distribution, and toxicity associated with titanium dioxide

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nanoparticles in Daphnia magna. Environ. Sci. Technol. 2016, 50 (17), 9636-9643.

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(19) Tan, L.Y.; Huang, B.; Xu, S.; Wei, Z.B.; Yang, L.Y.; Miao, A.J. TiO2

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nanoparticle uptake by the water flea Daphnia magna via different routes is

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calcium-dependent. Environ. Sci. Technol. 2016, 50 (14), 7799-7807.

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(20) Wang, W.X.; Rainbow, P.S. Subcellular partitioning and the prediction of

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cadmium toxicity to aquatic organisms. Environ. Chem. 2006, 3 (6), 395-399.

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(21) Tsui, M.T.; Wang, W.X. Biokinetics and tolerance development of toxic metals

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in Daphnia magna. Environ. Toxicol. Chem. 2007, 26 (5), 1023-1032.

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(22) Ng, Y.T.; Wang, W.X. Dynamics of metal subcellular distribution and its

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relationship with metal uptake in marine mussels. Environ. Toxicol. Chem. 2005, 24 26


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(23) Sharma, V.K.; Sohn, M. Aquatic arsenic: toxicity, speciation, transformations,

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and remediation. Environ. Int. 2009, 35 (4), 743-759.

553

(24) Pane, E.F.; Smith, C.; McGeer, J.C.; Wood, C.M. Mechanisms of acute and

554

chronic waterborne nickel toxicity in the freshwater cladoceran, Daphnia magna.

555

Environ. Sci. Technol. 2003, 37 (19), 4382-4389.

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(25) Wang, W.X. Dietary toxicity of metals in aquatic animals: recent studies and

557

perspectives. Chinese Sci. Bull. 2013, 58 (2), 203-213.

558

(26) DeForest, D.K.; Meyer, J.S. Critical review: toxicity of dietborne metals to

559

aquatic organisms. Environ. Sci. Technol. 2015, 45 (11), 1176-1241.

560

(27) Hook, S.E.; Fisher, N.S. Sublethal effects of silver in zooplankton: importance

561

of exposure pathways and implications for toxicity testing. Environ. Toxicol. Chem.

562

2001, 20 (3), 568-574.

563

(28) Geffard,

564

Tusseau-Vuillemin, M.H.; Garric, J. Effects of chronic dietary and waterborne

565

cadmium exposures on the contamination level and reproduction of Daphnia magna.

566

Environ. Toxicol. Chem. 2008, 27 (5), 1128-1134.

567

(29) Lima, A.R.; Curtis, C.; Hammermeister, D.E.; Markee, T.P.; Northcott, C.E.;

568

Brooke, L.T. Acute and chronic toxicities of arsenic(III) to fathead minnows, flagfish,

569

daphnids, and an amphipod. Arch. Environ. Contam. Toxicol. 1984, 13 (5), 595-601.

570

(30) Spehar, R.L.; Fiandt, J.T.; Anderson, R.L.; Defoe, D.L. Comparative toxicity of

571

arsenic compounds and their accumulation in invertebrates and fish. Arch. Environ.

O.;

Geffard,

A.;

Chaumot,

A.;

Vollat,

27


B.;

Alvarez,

C.;


572

Contam. Toxicol. 1980, 9 (1), 53-63.

573

(31) Tisler, T.; Zagorc-Koncan, J. Acute and chronic toxicity of arsenic to some

574

aquatic organisms. B. Environ. Contam. Toxicol. 2002, 69 (3), 421-429.

28


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575

Table 1. The distribution of arsenic in five subcellular fractions (MRGs, MTLPs, Organelles, HSPs, and cellular debris) of Daphnia magna in

576

the acute and chronic toxicity experiments of iAsV and iAsIII under the waterborne and dietborne exposure scenarios. Organelles and HSPs are

577

grouped as metal sensitive fractions (MSF) while MTLPs and MRGs are grouped as biologically detoxified metal (BDM). The data are the mean

578

± standard deviation (n = 4).

579

Acute toxicity experiment Distribution (%)

Waterborne iAs

BDM

MSF

V

iAs

Chronic toxicity experiment

Dietborne III

V

iAs

iAs

Waterborne III

iAs

V

iAs

Dietborne III

V

iAs

iAsIII

MRGs

12.16±0.27

12.76±0.14

13.58±0.20 15.11±0.27

22.14±2.25 19.66±1.01

17.22±2.33 15.77±1.69

MTLPs

31.99±2.03

27.93±0.64

30.32±0.24 25.34±0.49

28.00±1.74 29.89±2.87

38.56±1.95 38.30±0.46

subtotal

44.15±2.30 40.69±0.49

43.90±0.43 40.45±0.76

50.14±0.51 49.55±1.86

55.78±0.38 54.07±1.23

Organelles 22.92±0.51 25.12±1.52

21.41±0.83 21.93±0.98

20.86±0.16 18.58±1.25

16.29±0.25 18.32±0.67

9.61±0.38

4.16±0.98

HSPs

12.21±0.41 11.63±0.26

10.13±0.02

subtotal

35.13±0.92 36.75±1.79

31.54±0.85 31.50±1.67

30.47±0.54 28.43±0.91

20.45±0.73 22.12±0.82

20.73±1.38 22 .55±2.28

24.56±0.41 28.05±2.43

19.39±0.03 22.02±0.95

23.77±0.35 23.81±0.41

Cellular debris

9.57±0.70

580 581

29


9.85±0.34

3.80±0.15


582

■ FIGURE LEGENDS

583

Figure 1. (a-d) Survival of Daphnia magna at different time points during the 48-h

584

acute toxicity experiments assessing the effects of (a, c) waterborne and (b, d)

585

dietborne exposure to (a, b) iAsV and (c, d) iAsIII. The concentrations of iAsV used in

586

the waterborne experiment was 0, 1.5, 3, 4.5, 6, and 7.5 mg/L, and those of iAsIII 0,

587

0.5, 1.5, 3, 4.5, and 6 mg/L. The algae used as foods in the dietborne experiment were

588

pre-exposed to iAs concentrations of 0, 1.5, 4.5, and 7.5 mg/L. (e, f) Increase in the

589

concentration of arsenic that accumulated in D. magna ([As]daphnid) in response to

590

increasing concentrations of iAs ([As]dis) (e) in the exposure medium used in the

591

waterborne acute toxicity experiment and (f) in the medium for culturing the algae

592

used as food in the dietborne acute experiment. The data are the mean ± standard

593

deviation (n = 4).

594

Figure 2. (a–d) Survival of D. magna at different time points in the 21-day chronic

595

toxicity tests assessing the effects of (a, c) waterborne and (b, d) dietborne exposure to

596

(a, b) iAsV and (c, d) iAsIII. The concentrations of iAs used in the exposure medium of

597

the waterborne experiment and in the medium for culturing the algae used as food in

598

the dietborne experiment were 0, 0.01, 0.03, 0.1, 0.3, and 1.0 mg/L. (e, f) Increase in

599

the concentration of arsenic that accumulated in D. magna ([As]daphnid) with increasing

600

concentrations of iAs ([As]dis) (e) in the exposure medium used in the waterborne

601

chronic toxicity experiment and (f) in the medium for culturing the algae used as food

602

in the dietborne chronic toxicity experiment. The data are the mean ± standard

603

deviation (n = 4). 30


Page 30 of 35

Page 31 of 35


604

Figure 3. Distribution of (a, e, i, m, c, g, k, o) Ca and (b, f, j, n, d, h, l, p) arsenic in D.

605

magna (a–h) in the (a, b, e, f) waterborne and (c, d, g, h) dietborne acute toxicity tests

606

of (a–d) iAsV and (e–h) iAsIII, and (i–p) in the (i, j, m, n) waterborne and (k, l, o, p)

607

dietborne chronic toxicity tests of (i–l) iAsV and (m–p) iAsIII, as determined by

608

synchrotron-radiation-based micro X-ray fluorescence spectroscopy (µXRF).

609

Figure 4. Relative distribution of iAsIII, iAsV, and DMA in D. magna, as calculated by

610

the concentration of each arsenic species in the daphnids divided by [As]daphnid, in the

611

waterborne and dietborne (a) acute and (b) chronic toxicity tests of iAsIII and iAsV.

612

The data are the mean ± standard deviation (n = 4).

613

31



614

Page 32 of 35

Figure 1

dietborne

waterborne 150 a

0 1.5

3 4.5

0 1.5

b

6 7.5

4.5 7.5

100 V

iAs Survival (%)

50 0 150 c

1.5 3

0 0.5

4.5 6

0 1.5

d

4.5 7.5

100 III

iAs 50 0

[As]daphnid (µg-As/g-dw)

0

20

30

40 50 0 10 Exposure time (h)

300 e

V

20

30

40

50

f

iAs iAsIII

200 100 0 0

615

10

2

4

6

8 0 2 [As]dis (mg/L)

616

32


4

6

8

Page 33 of 35


617

Figure 2 dietborne

waterborne 150 a

0 0.01

0.03 0.1

b

0.3 1

100 V

iAs Survival (%)

50 0 150 c

d

100 III

iAs 50 0

[As]daphnid (µg/g-dw)

0

5

10

15

80 e

20 0 5 Exposure time (day)

10

15

20

f

iAsV III iAs

40

0 0

0.2

0.4

0.6

0.8

1.0 0 0.2 [As]dis (mg/L)

618 619 620

33


0.4

0.6

0.8

1.0


621

Figure 3

622 623 624 625

34


Page 34 of 35

Page 35 of 35


626 627

Figure 4

150

Relative distribution (%)

100

III

iAs iAsV waterborne

a

DMA dietborne

50 0 150 100

b dietborne waterborne

50 0

iAsIII

iAsV iAsIII Treatments

iAsV

628

35


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