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Mixture toxicity of nickel and microplastics with different functional groups on Daphnia magna Dokyung Kim, Yooeun Chae, and Youn-Joo An Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03732 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017
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Environmental Science & Technology 1
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Mixture toxicity of nickel and microplastics with different functional groups on Daphnia magna
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Dokyung Kim, Yooeun Chae, and Youn-Joo An*
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Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea
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*
Corresponding author: Youn-Joo An Tel.: +82-2-2049-6090; Fax: +82-2-2201-6295; Email: anyjoo@konkuk.ac.kr
Abbreviations and nomenclature: HOCs, hydrophobic organic chemicals; MP, microplastic; POPs, persistent organic pollutants; PS, microplastic without functional group; PS-COOH, microplastic with COOH functional group
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ABSTRACT
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In recent years, discarded plastic has become an increasingly prevalent pollutant in aquatic
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ecosystems. These plastic wastes decompose into microplastics, which not only pose a direct
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threat to aquatic organisms but also an indirect threat via adsorption of other aquatic
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pollutants. In this study, we investigated the toxicities of variable and fixed combinations of
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two types of microplastics [one coated with a carboxyl group (PS-COOH) and the other
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lacking this functional group (PS)] with the heavy metal nickel (Ni) on Daphnia magna and
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calculated mixture toxicity using a toxic unit model. We found that toxicity of Ni in
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combination with either of the two microplastics differed from that of Ni alone. Furthermore,
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in general, we observed that immobilization of D. magna exposed to Ni combined with PS-
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COOH was higher than that of D. magna exposed to Ni combined with PS. Collectively, the
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results of our study indicate that the toxic effects of microplastics and pollutants may vary
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depending on the specific properties of the pollutant and microplastic functional groups, and
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further research on the mixture toxicity of various combinations of microplastics and
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pollutants is warranted.
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Keywords: complex toxicity; microplastic; functional group; nickel; Daphnia magna
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1. INTRODUCTION
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The considerable increase in the production and consumption of plastics over the last few
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decades has resulted in a marked increase in the disposal of plastics, and some have been
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discarded into the aquatic environment without being recycled or treated. Discarded plastic
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that enter aquatic ecosystems is eventually degraded into microplastics (MPs), which are
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defined as plastics with particle sizes ≤ 5 mm.1 These plastics can be ingested by aquatic
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organisms, including fish, shellfish, and shrimps, or can adhere to algae and plankton.2,3
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Several previous studies have observed that MPs are taken up by aquatic organisms and
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subsequently have adverse effects on their survival and health.3–8 Although the concentrations
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of microplastics examined in the above-mentioned studies are not comparable with those
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occurring in the field, the findings of these studies do suggest that microplastics have the
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potential to cause adverse effects in organisms. Recently, the presence of MPs in oceans and
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as pollutants in freshwater has become an issue of concern.9–15 Microplastics are flushed
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indirectly into freshwater bodies from various sources, such as industrial plant and washing
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machine effluents,15–18 and plastic products are directly discarded into freshwater sources.
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Numerous studies have confirmed that MPs have spilled into aquatic systems and have
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subsequently combine with or have adsorbed onto other pollutants such as heavy metals,
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persistent organic pollutants (POPs), or hydrophobic organic chemicals (HOCs).19–24 Aquatic
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organisms could ingest MPs that have adsorbed onto other pollutants, which could lead to
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complex toxicities, and a few studies have investigated this phenomenon. For instance,
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Besseling et al.25 observed that the transfer of polychlorinated biphenyls (PCBs) to lugworms
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and the subsequent effects were enhanced with increasing concentrations of plastic. In
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another study, Browne et al.26 demonstrated the transfer of endocrine-disrupting chemicals
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(EDCs) into lugworms via adsorption on MPs. Chua et al.27 evaluated the combined toxicity
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of polybrominated diphenyl ethers (PBDEs) and MPs on marine amphipods, whereas Batel et
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al.28 monitored the effects of benzo[a]pyrene with MPs on nauplii. Similarly, some studies29,30
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have determined the toxicity of fluoranthene adsorbed on MPs using marine mussels, and
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Wardrop et al.31 evaluated the accumulation of PBDEs in rainbow fish. In recent years, four
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mixture toxicity tests have been conducted to evaluate the effects of heavy metals (copper,
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silver, chromium, and gold nanoparticle) combined with MPs32–35 on microalgae and marine
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fish. Thus, several studies have verified that microplastics in aquatic ecosystems combine
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with other pollutants and that there is a potential likelihood that aquatic organisms will
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become exposed to these mixed substances. Therefore, further studies on toxicity using such
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mixed substances are needed.
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In the present study, the toxicity of mixtures of MPs with a heavy metal was assessed in
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the water flea, Daphnia magna. We selected a heavy metal pollutant and designed the study
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based on nickel. The characteristics of MPs vary, including their functional groups, sizes, and
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coating agents. Therefore, the effects of MPs on aquatic organisms may depend on the
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characteristics of the MPs themselves and may differ when combined with different
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chemicals.36,37 To evaluate this phenomenon, we selected two plastics, one containing a
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carboxyl functional group (PS-COOH) and the other lacking this group (PS), and compared
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the mixture toxicities of the MPs combined with nickel. To evaluate mixture toxicities, we
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measured the rate of immobilization of water fleas in the presence and absence of
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microplastics and compared the amount of nickel uptake depending on the type of
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microplastics. Because we assumed that the nickel cation would be more adsorbed onto PS-
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COOH, which has a negatively charged functional group, than onto PS, we hypothesized that
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Daphnia exposed to nickel with PS-COOH would ingest considerably more nickel.
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Accordingly, we hypothesized that nickel with PS-COOH would show much higher toxicity
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than nickel with PS.
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2. MATERIAL AND METHODS 2.1. Test chemicals
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Nickel was purchased from Sigma-Aldrich (St. Louis, MO, USA) as nickel chloride
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(NiCl2, purity 98%), and two types of polystyrene MPs were purchased from Bangs
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Laboratories, Inc. (Fishers, IN, USA). One of the plastics contained a bound carboxyl group
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(PS-COOH), whereas the other plastic lacked this bound functional group. Fotopoulou and
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Karapanagioti38 have demonstrated that virgin microplastics, which do not have a functional
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group, can have a negative charge due to multiple factors in the aquatic environment. We
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therefore selected PS-COOH with a negative charge and PS in a virgin state. We assumed that
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Ni cations would adsorb onto the anionic carboxylate functional group of the MPs (PS-
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COOH) to a greater extent than onto MPs lacking this functional group (PS). The average
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diameter of PS and PS-COOH was 201.5 and 191.3 nm in deionized water (DW) and 194.0
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and 182.7 nm in moderately hard water (MHW)39, respectively. The zeta potentials of the PS
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in each medium were -31.9 and -31.1 mV in DW and MHW, respectively, whereas those of
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PS-COOH were -36.8 and -28.0 mV in DW and MHW, respectively. The surface of the MPs
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was observed using a field emission scanning electron microscope (FE-SEM; S-4300, Hitachi,
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Japan). Scanning electron microscopy (SEM) images of the MPs are shown in Fig. 1.
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2.2. Test species (D. magna)
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Daphnia magna specimens were obtained from the National Institute of Environmental
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Research (NIER, Incheon, Korea). They were cultured in modified MHW at a temperature of
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21 °C under a 16-h:8-h light:dark cycle. The modified MHW contained cyanocobalamin
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(vitamin B12; Daejung, Korea) and sodium selenite (Na2SeO3; Kanto Chemical, Japan). Once
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daily, Daphnia feed was injected with 1 mL of green algae (Chlorella vulgaris) at a
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concentration of 1 × 108 cells/mL. The Daphnia specimens used in all the experiments were
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neonates less than 24 h old.
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2.3. Acute toxicity test on D. magna
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Acute toxicity tests were performed according to the Organization for Economic
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Cooperation and Development (OECD) guidelines for testing chemicals40 for 48 h at 21 °C
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under a 16-h:8-h light:dark cycle and without food. Glass vials (diameter 6 mm, height 75
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mm, and volume 35 mL) were used as test vessels, each containing 10 mL of test solution and
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five daphnids. A control group was used for all experiments. The control group was exposed
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to uncontaminated MHW.
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We initially conducted acute toxicity tests using nickel, PS, and PS-COOH separately.
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For the Ni toxicity test, a 100 mg/L stock solution was diluted with MHW to concentrations
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of 1, 2, 3, 4, and 5 mg/L. Before the toxicity tests, we dispersed the MPs by rolling the stock
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solutions for 30 min using a roller mixer. Thereafter, 100 mg/L stock solutions were prepared
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for each MP and diluted further with MHW to obtain the final MP working concentrations of
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1, 5, 10, 20, and 30 mg/L. The 100 mg/L MP stock solution contained approximately 2.6 ×
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105 particles/mL according to a certificate of analysis of the MPs obtained from Bangs
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Laboratories (Density: 1.05-1.06 g/mL). After a 48-h exposure to the MPs, test organism
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abnormalities, including immobilization and changes in morphology, were recorded. To
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calculate the half-maximal effective concentration (EC50) of the MPs, we conducted
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additional tests with PS using higher concentrations (20, 40, 60, and 80 mg/L). The test
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conditions were the same as described above. The Ni and MP tests were conducted with 12
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and 8 replicates, respectively.
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2.4. Mixture toxicity test on D. magna
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We also carried out three sets of mixture toxicity tests: (i) variable Ni-fixed MP, (ii) fixed
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Ni-variable MP, and (iii) variable Ni-variable MP. Before each test, 10-mL samples of the test
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solutions were prepared in vials with lids, which were continuously shaken at 130 rpm for 24
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h under a 16-h:8-h light:dark cycle in an incubator (21 °C) to mix the Ni and MP in the MHW.
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Five D. magna individuals were placed in each of the four replicate vials, and negative (no Ni,
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no MP) and positive (fixed chemicals only) controls were prepared. The concentrations and
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detailed experimental procedures for each test are described below.
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We also conducted adsorption experiments to confirm differences in the adsorption of Ni
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caused by the presence of the functional group. After 24 h of shaking, samples were filtered
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using filter paper (Whatman 0.45 µm, Whatman, UK) and centrifugal filters (Amicon® Ultra-
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4, Merck Millipore, USA) to remove MPs with adsorbed Ni. The filtrate was analyzed using
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an inductively coupled plasma atomic emission spectrophotometer (ICP–AES) system (JY
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138; Ultrace, Jobin Yvon, France).
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The variable Ni-fixed MP tests (i) were used to confirm the change in Ni toxicity in the
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presence of MP. Ni concentrations were the same as those used in the acute toxicity test,
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whereas the MP concentration was fixed at 5 mg/L, which had no adverse effects on D.
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magna in the MP acute toxicity tests. After a 48-h exposure, observed immobilization was
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recorded, and then living specimens were selected for Ni analysis. The D. magna specimens
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were rinsed several times with distilled water, dried in a 65 °C dry oven, and digested with
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HNO3 (Duksan Pure Chemical, Korea). Ni in each D. magna specimen was measured by
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ICP–MS to determine bioaccumulation.
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Fixed Ni-variable MP tests (ii) were conducted to compare the mixture toxicity of Ni and
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PS or PS-COOH using MP concentrations of 1, 5, 10, 20, and 30 mg/L and Ni at 3 mg/L
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based on the results of the acute toxicity tests in which the 3 mg/L treatment was not a no-
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observed-effect concentration for D. magna in the Ni acute toxicity test (after a 48-h exposure,
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immobilization was recorded).
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Furthermore, variable Ni-variable MP tests (iii) were performed to compare the mixture
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toxicity of Ni and MP at the same concentrations used in each of the respective acute toxicity
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tests, such as 1 mg/L Ni-1 mg/L MP, 2 mg/L Ni-5 mg/L MP, and 5 mg/L Ni-30 mg/L. These
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concentrations were selected for comparison of mixture toxicity with tests (i) and (ii).
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Immobilization was recorded after a 48-h exposure.
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2.5. Statistical analyses
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To assesses whether the differences between the negative controls and exposure groups
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were significant, a Dunnett’s test (version 1.5) was used.41 EC50 values were computed using
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the trimmed Spearman-Karber method.42 A 95% confidence limit was used for all
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comparisons (p < 0.05). To analyze the mixture effect of Ni and MP, the toxic unit (TU)
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approach, which determines the toxicity of a mixture by summing the toxic strength of its
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separate compounds, was used.43 TU values were expressed as concentrations of the mixtures,
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and their sum was expressed as follows:
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ΣTUi = ∑ ,
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where, n is the number of chemicals in the mixture, Ci is the concentration of the individual
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chemicals, and EC50i is the EC50 of the ith mixture components When the 50% adverse effect
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(EC50mix) was observed at TU values < 1, the combined effect was interpreted as a
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synergistic effect. In contrast, when the 50% adverse effect (EC50mix) was observed at TU
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values > 1, the combined effect was interpreted as an antagonistic effect.
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3. RESULTS AND DISCUSSION 3.1. Acute toxicity tests on D. magna
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Among all acute toxicity tests, no immobilization was observed in the negative controls.
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The results of the MP acute toxicity test on D. magna are shown in Fig. 2. The effect of PS
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on D. magna was negligible up to 30 mg/L, which was the highest concentration tested in this
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study. Immobilization of D. magna with increasing PS concentrations of 1, 5, 10, 20, and 30
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mg/L was 0%, 0%, 0%, 12.5%, and 7.5%, respectively, whereas the corresponding
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immobilization induced by PS-COOH was 7.5%, 5%, 10%, 27.5%, and 62.5%. In an
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additional experiment with PS at high concentration (20, 40, 60, and 80 mg/L), the
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immobilization rates of D. magna were 12.5, 45, 62.5, and 90%, respectively. At all the MP
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concentrations tested in the present study, the toxicity of PS-COOH was higher than that of
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PS. This difference in MP toxicity by functional group corresponds with previous studies
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using PS-NH2 and PS-COOH, and the higher toxicity of PS-COOH in this study is possibly
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due to the differences in surface charge.37 Meanwhile, Watt et al.44 confirmed that MPs with
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different functional groups (COOH, NH2) were found at different locations in crab gills. The
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authors suggested that the difference in localization may be because the binding capacity
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within the gill tissue changes depending on the characteristics of the particle surface.
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Similarly, in this study, PS-COOH appeared to exhibit higher toxicity because MPs with
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different functional groups have different binding capacities in Daphnia. In the present study,
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the 48-h EC50 values for PS and PS-COOH were 42.78 and 25.96 mg/mL, respectively (Table.
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1). The results of the Ni acute toxicity tests on D. magna are shown as dark gray bars in Fig.
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3. Immobilization values observed in D. magna at increasing Ni exposure concentrations of 1,
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2, 3, 4, and 5 mg/L were 0%, 3.33%, 25%, 46.67%, and 85%, respectively, whereas the 48-h
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EC50 value of Ni was 3.85 mg/L.
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3.2. Mixture toxicity tests on D. magna
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Analysis of Ni removal from medium in the presence of MPs indicated that the
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difference in adsorption to the two types of MP was not significant. The removal rates of Ni
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were 97.2%, 97.9%, 100.6%, 100.1%, and 99.7% compared to no MP condition for
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increasing PS concentrations (5, 10, 30, 50, and 100 PS mg/L, respectively). The removal
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rates of Ni for PS-COOH were 96.3%, 103.0%, 99.7%, 98.4%, and 97.4%, respectively. As
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MP concentration increased, it was not confirmed that more Ni was adsorbed. There was no
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statistically significant difference in the concentration of nickel adsorbed on the MP based on
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the type of MP. Turner et al.23 measured the amount of Ni adsorbed on a plastic pellet over
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time finding that little was adsorbed as was confirmed in this study. However, it has been
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shown that more Ni adsorbs on the beached pellets than on virgin pellets. On the other hand,
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Holmes21 et al. found that trace metals can be detected in plastic pellets collected from
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England beaches with some comprised of Ni up to 562 µg/g.
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Figure 3 includes the results of the mixture Ni and MP toxicity tests on D. magna
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[variable Ni-fixed PS test (i)]. Immobilization values observed in D. magna in this test were
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2.5%, 12.5%, 12.5%, 30%, and 62.5% for increasing Ni concentrations (1, 2, 3, 4, and 5
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mg/L, respectively), whereas values for the variable Ni-fixed PS-COOH were 7.5%, 12.5%,
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62.5%, 55%, and 75%, respectively. Immobilization values of 7.5% and 2.5% were observed
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in the negative controls (no Ni, no MP) for PS and PS-COOH, respectively. The 48 h-EC50
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values for the variable Ni-fixed PS and variable Ni-fixed PS-COOH tests were 4.67 and 3.14
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mg/L, respectively (Table. 1). These results showed that the immobilization rate observed
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following the variable Ni-fixed PS-COOH treatments was higher than that observed in the Ni
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acute toxicity tests at all Ni concentrations, whereas immobilization in the variable Ni-fixed
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PS tests did not differ significantly from that of the Ni only acute toxicity test. This PS
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mixture toxicity result is consistent with previous studies on population growth inhibition of
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the microalgae Tetraselmis chuii.32 Davarpanah et al.32 evaluated the toxicity of copper on
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these marine microalgae in the presence of MP (polyethylene). They confirmed that the
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toxicity of copper decreased when MP was present; however, this effect was not statistically
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significant. In particular, the percentage immobilization observed in the 3 mg/L Ni with PS-
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COOH treatment was significantly higher than that in the Ni-only exposure group. However,
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because there was no difference in Ni adsorption by the MPs, the difference in mixture
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toxicity was not due to adsorption but due to toxicity of the MPs, which was dependent on the
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respective functional groups, as has been described previously in the literature.37,44
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The results of ICP-MS analysis of Ni bioaccumulation in D. magna indicated that
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individual water fleas ingested more nickel in the presence of an MP (Fig. 4). Although there
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is also the possibility of excretion of Ni by D. magna through digestion in this procedure, we
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attempted to compare Ni accumulation according to presence and type of MPs. Therefore, we
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excluded the effects of depuration. Ni accumulation was generally higher in D. magna in the
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variable Ni-fixed MP tests than it was in the Ni only acute toxicity tests, except at a Ni
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concentration of 5 mg/L. Although not statistically significant, similar trends were observed,
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with that of the D. magna immobilization tests. Similarly, Khan et al.33 confirmed that silver
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uptake increased in the zebrafish intestine when MPs were present. Based on these results, we
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assume that in the variable Ni-fixed MPs tests, the presence of MPs induced higher
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immobilization rates due to the increase in Ni intake and accumulation in D. magna.
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However, at the highest Ni concentration (5 mg/L), the Ni concentrations in D. magna and
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percentage D. magna immobilization in the presence of MPs were lower than those observed
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in the Ni acute toxicity test at that concentration, although these differences were not
252
statistically significant. We were unable to establish the reason for this observation because of
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the lack of surviving D. magna at a Ni concentration of 5 mg/L.
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In the fixed Ni-variable PS tests (ii), the percentage immobilization of D. magna at
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increasing concentrations of PS and PS-COOH (1, 5, 10, 20, and 30 mg/L) was 12.5%, 10%,
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30%, 60%, and 87.5% and 12.5%, 25%, 60%, 75%, and 77.5%, respectively (Fig. 5).
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Immobilization in the negative controls (no Ni, no MP) for PS and PS-COOH was 20% and
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17.5%, respectively. The 48-h EC50 values of the fixed Ni-variable PS and fixed Ni-variable
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PS-COOH treatments were 17.72 and 10.63 mg/L, respectively (Table 1). At low
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concentrations, the percentage immobilization induced by PS and PS-COOH was slightly
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lower than that induced in the negative controls. Comparing the two MP treatments, the
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immobilization rate of D. magna exposed to Ni combined with PS-COOH was higher than
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that of the D. magna exposed to Ni combined with PS at concentrations of 5, 10, and 20
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mg/L. Furthermore, this observed difference was statistically significant at 5 and 10 mg/L.
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Therefore, we inferred that the toxicity of Ni combined with PS-COOH was higher than that
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of Ni combined with PS, which is attributable to the fact that PS-COOH binds more strongly
267
to Ni than does PS. Treatment with MP at 30 mg/L in the mixture toxicity tests showed that
268
percentage immobilization was higher than that in the Ni and MP acute toxicity tests.
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Therefore, these results indicate that the toxicity of Ni increased concentration dependently in
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the presence of MPs.
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In the variable Ni-variable PS tests (iii), the percentage immobilization induced in D.
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magna was 0%, 15%, 30%, 75%, and 100% for the respective increasing treatment
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concentrations, whereas the corresponding values of the variable Ni-variable PS-COOH tests
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were 10%, 22.5%, 80%, 97.5%, and 97.5% (Fig. 6). Immobilization in the negative controls
275
was 0% under both test conditions. The immobilization rates of D. magna exposed to PS-
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COOH were generally higher than those of D. magna exposed to PS. Furthermore, these
277
results were similar to those of the other mixture toxicity tests in this study in that
278
immobilization observed in D. magna exposed to Ni combined with PS-COOH was higher
279
than that of D. magna exposed to Ni combined with PS at intermediate concentrations, which
280
were used in this study.
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3.3. Comparison of the 48-h EC₅₀mix values of mixture toxicity tests
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In the mixture toxicity tests of Ni combined with PS, the EC50mix values were 1.33
284
(variable Ni-fixed PS), 1.16 (fixed Ni-variable PS), and 1.07 (variable Ni-variable PS) mg/L
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(Table 2). For the mixture toxicity tests of Ni combined with PS-COOH, the EC50mix values
286
were 0.96 (variable Ni-fixed PS-COOH), 1.05 (fixed Ni-variable PS-COOH), and 0.78
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(variable Ni-variable PS-COOH) mg/L. All EC50mix values for Ni mixed with PS were higher
288
than 1.00, whereas those of Ni mixed PS-COOH were slightly higher or less than 1.00. In all
289
tests, the 95% confidence intervals of each test did not include 1.00; however, the EC50mix
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values were very close to 1.00. Therefore, we determined that when combined with Ni, PS
291
had a slight antagonistic effect on toxicity, whereas PS-COOH had a slight synergistic effect.
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3.4. Environmental implications
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This study aimed to evaluate the effects of two MPs on Ni toxicity in the water flea D.
295
magna and to compare the mixture toxicity of Ni combined with two types of MPs (PS and
296
PS-COOH), a real phenomenon in the aquatic environment. Overall, we discovered that the
297
toxicity of Ni in combination with either of the two MPs changed compared with that of Ni
298
alone. Compared to the Ni acute toxicity test, the toxicity to D. magna exposed to Ni in
299
combination with PS was mostly lower, whereas toxicity to D. magna exposed to Ni in
300
combination with PS-COOH tended to be higher. Furthermore, the adverse effect on
301
immobilization of D. magna exposed to Ni combined with PS-COOH was generally higher
302
than that of D. magna exposed to Ni combined with PS. Calculation of mixture toxicity using
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a TU model based on these results revealed that PS had a slight antagonistic effect on Ni
304
toxicity, whereas the effect of PS-COOH in combination with Ni was slightly synergistic.
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These results revealed that the effects of MPs and other pollutants may be altered by the
306
specific characteristics of the pollutants and the functional group of the MPs. However, in
307
contrast to our hypothesis that Ni (cation) may be adsorbed onto PS-COOH to a greater
308
extent than onto PS due to the presence of the COOH- (anion) group, the difference in the
309
amount of Ni adsorbed on PS and PS-COOH was not significant. This could be attributed to
310
the fact that nickel is not a substance that adsorbs, much unlike a hydrophobic substance.
311
Therefore, it is important to conduct further detailed investigations on the mechanisms of the
312
binding capacity of MPs according to their functional groups and the higher toxicity of Ni in
313
combination with PS-COOH than with PS. Moreover, we confirmed that the effects of MPs
314
and pollutants may vary depending on the specific properties of the pollutant and MP
315
functional groups. Even though the concentrations of nickel and microplastics tested in this
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study were higher than the concentrations of substances that can exist in the environment, this
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study confirmed that the toxicity of one substance to organism can change when in the
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presence of another substance. This result indicates the possibility that toxicity may change
319
when aquatic organisms are exposed to other substances with microplastics and even that
320
toxicity trends may vary for the same substance depending on the functional group and
321
characteristics of the microplastics. Therefore, further research on mixture toxicity using
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various MPs and pollutants is warranted.
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ACKNOWLEDGMENTS
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This research was supported by the Basic Science Research Program through the
326
National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and
327
Future Planning (2016R1A2B3010445). This study was funded by the Graduate School of
328
Specialization for managing information of chemical risk, and supported by Konkuk
329
University Researcher Fund in 2017. The authors thank the Korean Basic Science Institute
330
for performing FE-SEM, ICP-AES and ICP-MS analyses.
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Table 1. Toxicity values [48-h half-maximal effective concentration (EC50)] of Ni and microplastics (MPs) against Daphnia magna
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Values (mg/L) EC50 (95% confidence limits) Ni only 3.85 (3.83–3.87) PS only 42.78 (42.38–43.17) PS-COOH only 25.96 (25.75-26.17) Variable Ni + 5 mg/L PS 4.67 (4.65–4.69) Variable Ni + 5 mg/L PS-COOH 3.14 (3.12–3.17) 3 mg/L Ni + Variable PS 17.72 (17.72–17.89) 3 mg/L Ni + Variable PS-COOH 10.63 (10.44–10.82) Trimmed Spearman–Karber method; 95% confidence limits, p < 0.05. Test
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Table 2. Forty-eight-hour half-maximal effective concentration-adverse effect (EC50mix) values of Ni and microplastic (MP) mixtures on Daphnia magna
EC50mix Value 95% confidence interval Variable Ni + 5 mg/L PS 1.33 1.32–1.34 3 mg/L Ni + Variable PS 1.16 1.15–1.16 Variable Ni + Variable PS 1.07 1.07–1.08 Variable Ni + 5 mg/L PS-COOH 0.96 0.95–0.97 3 mg/L Ni + Variable PS-COOH 1.05 1.04–1.05 Variable Ni + Variable PS-COOH 0.78 0.78–0.79 Toxic unit model; Pape-Lindstrom and Lydy, 1997, Trimmed Spearman–Karber method; 95% confidence limits, p < 0.05. Test
470 471 472
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Fig. 1. Field emission scanning electron microscopy (FE-SEM) images of microplastics (MPs) without (PS, left) and with (PS-COOH, right) a COOH functional group. Scale bar = 500 nm.
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Immobilization of D.magna (%)
100
Variable MP
478 479 480 481
*
80
60
#
*
40
*
20
0 MP
476 477
#
Variable PS Variable PS-COOH
1
0
2
1
3
5
4
10
5
20
6
30
Concentration (mg/L) Fig. 2. Immobilization of Daphnia magna exposed to microplastics (MPs) for 48-h MP acute toxicity tests; PS (left) and PS-COOH (right). *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.
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Immobilization of D.magna (%)
24
120
Variable Ni-Fixed MP
100
484 485 486 487 488
*
#
* *
80
* *
*
60 40 20 0 Ni MP
482 483
Variable Ni+5 mg/L PS Ni only Variable Ni+5 mg/L PS-COOH
0 5
1 5
2 5
3 5
4 5
5 5
Concentration (mg/L) Fig. 3. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h Ni acute toxicity (middle) and variable Ni-fixed MPs combined toxicity tests; PS (left) and PSCOOH (right). *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Twelve and eight replicates were run for Ni acute toxicity and each combined toxicity test, respectively. Error bars show standard deviations.
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Ni concentration in D. magna (ppb)
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5.5 Variable Ni+5 mg/L PS Ni only Variable Ni+5 mg/L PS-COOH
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0
489 490 491 492 493 494 495 496
1
2
3
4
5
Ni concentration (mg/L) Fig. 4. Accumulation of Ni in Daphnia magna exposed to Ni and microplastics (MPs) for 48h Ni acute toxicity tests (middle) and variable Ni-fixed MPs combined toxicity tests; MPs without (PS, left) and with (PS-COOH, right) a COOH functional group. Measured by inductively coupled plasma mass spectrometry (ICP-MS, n = 4 replicates). Detection limit and limit of quantization concentrations were 0.0071 and 0.0236 µg/L, respectively. Error bars show standard deviations.
497
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Immobilization of D.magna (%)
120
Fixed Ni-Variable MP Ni 3 mg/L+Variable PS Ni 3 mg/L+Variable PS-COOH
100
500 501 502 503 504
* *
*
# 80
* 60
# 40 20 0 Ni MP
498 499
*
1
3 0
2
3 1
3
3 5
4
3 10
5
3 20
6
3 30
Concentration (mg/L) Fig. 5. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h fixed Ni-variable MPs combined toxicity tests; MPs without (PS, left) and with (PS-COOH, right) a COOH functional group. *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.
505
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Immobilization of D.magna (%)
120
Variable Ni-Variable MP
100
508 509 510 511 512
*
*
*
*
*
80 60
* 40
#
*
20 0 Ni MP
506 507
#
#
Variable Ni+Variable PS Variable Ni+Variable PS-COOH
0 0
1 1
2 5
3 10
4 20
5 30
Concentration (mg/L) Fig. 6. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h variable Ni-variable MPs combined toxicity tests; MPs without (PS, left) and with (PSCOOH, right) a COOH functional group. *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.
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171x78mm (96 x 96 DPI)
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