The copepod T. japonicus was collected at Haeundae Beach (35°9′29.57′′N, 129°9′36.60′′E) from Busan, South Korea, and maintained at the Marine Biotechnology Research Center at the Korea Institute of Ocean Science and Technology (KIOST). The animals were cultured under controlled incubator conditions at 28°C and under a 12:12 h light/dark cycle in 32 practical salinity units (psu) of filtered artificial seawater (ASW; TetraMarine Salt Pro, Tetra™, Cincinnati, OH, USA). The marine microalga Tetraselmis suecica (~6 × 104 cells/mL) was administered as a food source once per day. Species identification was confirmed by morphological characteristics and sequencing of the mitochondrial DNA gene cytochrome oxidase 1 (CO1) which is used as the universal barcoding marker gene (Jung et al. 2006).
Hydrothermal ore samples
Hydrothermal ore particulates were obtained from Dr. Sang-Bae Kim (Korea Institute of Geoscience and Mineral Resources, Daejeon, South Korea). Briefly, hydrothermal ore deposits were collected from hydrothermal vent fields in the Indian Ocean (2°51'34.7"S 61°34'57.8"E) in May 2018 (Fig. 1). Hydrothermal ore deposits were crushed with a Jaw crusher (BB50, Retsch, Germany), cone crusher (HPT, Zenith, Germany) and ground in a rod mill (TI-500ET, CMT Co. Ltd., Japan) at 70 rpm rotational speed for 5 min. Reagents included potassium amyl xanthate (PAX; C5H11OCS2K; collector; ≥ 90% purity; Hong Yuan Industry & Trade Co., Zhejiang, China), sodium oleate (SO; C18H33O2Na; collector; Sigma-Aldrich Corp., St. Louis, MO, USA), and Aerofloat-65 (AF-65; CH3(C3H6O)4OH; frother; American Cyanamid, Wayne, NJ, USA). To investigate hydrothermal ore deposit mineralogy, X-ray diffraction (XRD) analysis (D/Max-2200/PC, Rigaku Corp., Tokyo, Japan) was used. The XRD showed that the samples consisted mainly of sphalerite (ZnS), pyrite (FeS2), chalcopyrite (CuFeS2), galena (PbS), barite (BaSO4), silica (SiO2), and anhydrite (CaSO4) (Fig. S1).
For the flotation tests, 100-g samples were added to 900 mL of deionized (DI) water and mixed at 1,000 rpm impeller speeds with 10 min flotation time. AF-65 (250 mL/ton) was conditioned for 3 min before air was introduced into the suspensions. The process flow for the flotation test is shown in Fig. S2. Rough flotation was conducted at pH 8 and the barite was separated with 300 g/ton SO. It was previously reported that chemisorption occurs between the oleate ions and the barite (Marinakis and Shergold 1985; Deng et al. 2019). Scavenging was conducted with 300 g/ton PAX to separate the sulfides sphalerite, chalcopyrite, galena, and pyrite. Chemisorption might also occur between xanthate ions (X-) and sulfides. Xanthate ion (X-) oxidation to dixanthogen (X2) might occur on the sulfides and act as a redox catalyst (Finkelstein 1997; Peng et al. 2017). The residue from which sulfides and barite were separated was used in the subsequent toxicity experiments. In brief, two leachates were produced by mixing hydrothermal ore and beneficiation with artificial seawater (1 g/L particles in 32-psu artificial seawater) for 12 h at 25 ºC in a glass carboy fitted with a Teflon-coated cap. The leachates were obtained by passing the suspensions through a disposable 0.22 µm syringe filter (BIOFI®). Metal concentrations in the leachates were determined by inductively coupled plasma mass spectromtery (ICP-MS; NexION 2000, Perkim Elmer Inc.) with the commercially-available seaFAST preconcentration system (Elemental Scientific seaFAST SP3). Metal concentrations in the leachates from both ores were analyzed in duplicate.
Effects of the metal leachates on T. japonicus survival, developmental time and fecundity
Acute toxicity tests were conducted on ovigerous female adult T. japonicus subjected to various leachate concentrations (hydrothermal ore type: 0% [control], 10%, 25%, 40%, 55%, 70%, 85%, and 100%; beneficiation type: 0% [control], 20%, 40%, 60%, 80%, and 100%). Ten copepods were transferred to a 12-well culture plate (JET BIOFIL®, Guangzhou, Guangdong, China) containing 4 mL of test solution. They were exposed in triplicate to each of the leachate concentrations for 4 d and 7 d. Dead T. japonicus were observed under a stereomicroscope (SZX2-ILLT; Olympus, Tokyo, Japan) every 24 h to determine the survival rates. Based on the acute toxicity data, low leachate concentration ranges were selected to examine copepod development and fecundity.
Nauplii < 12 h post-hatching were collected to establish the effects of the leachates on the developmental time of the F0 and F1 generations. Ten newly hatched nauplii were transferred to each well of a 12-well culture plate. Each well contained 4 mL of test solution. Based on the acute toxicity data, 0.001%, 0.005%, 0.01%, 0.1%, 0.05%, and 0.1% hydrothermal ore leachate and 0.01%, 0.05%, 0.1%, 0.5%, and 1% beneficiation leachate were selected. There were three replicates per treatment for a total of 120 nauplii. During the leachate exposure period, the copepods were provided with T. suecica (~200,000 cells/mL) and the 50% of the test solution was renewed every 24 h. Developmental stages were examined under a stereomicroscope (SZX-ILLK200; Olympus, Tokyo, Japan) once every 24 h. Development was observed up to the adult stage for 25 d. The average number of days required for development from nauplii to copepodites and ovigerous females were used as endpoints. For the second generation (F1) experiment, ten newly hatched nauplii (F1) from each (F0) female per leachate concentration, were transferred to 12-well tissue culture plates and subjected to the same experimental conditions as those used for the F0 generation experiment.
To examine the effects of the leachates on T. japonicus fecundity, ovigerous female adults were individually exposed to 0.001%, 0.005%, 0.01%, 0.05%, and 0.1% hydrothermal ore leachate and 0.01%, 0.05%, 0.1%, 0.5%, and 1% beneficiation leachate in a 12-well plate. There were ten replicates and 4 mL of test solution per well. The nauplii in each well were counted over 10 d. During the treatment period, the copepods were provided with T. suecica (~200,000 cells/ml) and the 50% test solution was renewed every 24 h.
Transcriptional expression of genes regulating detoxification, antioxidation, and development in response to metal leachates
To examine the expression patterns of the genes regulating detoxification, antioxidation, and development, ~300 copepods were exposed to 0% and 0.1% hydrothermal ore leachate and 0% and 1% beneficiation leachate for 6 h, 12 h, 18 h, and 24 h. Total RNA was extracted with TRIzol® reagent (Invitrogen, Paisley, Scotland, UK) according to the manufacturer’s instructions. Total RNA quantity and quality were validated by spectrophotometry (NanoDrop One; Thermo Fisher Scientific Inc., Madison, WI, USA) at 230 nm, 260 nm, and 280 nm. To synthesize cDNA for RT-qPCR, 2 µg of total RNA and oligo (dT)20 primer were used for reverse transcription with a SuperScript™ III RT kit (Invitrogen, Carlsbad, CA, USA). The RT-qPCR conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 5 s; and 60°C for 10 s in a Rotor-GeneTM SYBR® Green PCR kit (QIAGEN GmbH, Hilden, Germany) with an AriaMx real-time PCR system (Agilent Technologies Inc., Santa Clara, CA, USA). To confirm product amplification, melting curve cycles were run as follows: 95°C for 30 s, 65°C for 30 s, 65°C for 5 s at a 0.5°C increment, and 95°C for 30 s. The RT-qPCR F or R primers used were selected from previous studies and confirmed according to MIQE guidelines (Table S1) (Bustin et al. 2010). The T. japonicus 18S rRNA gene was used to normalize expression level between samples. All RT-qPCR experiments were performed in triplicate. Fold change in relative expression was calculated by the 2−ΔΔCt method (Livak and Schmittgen 2001).
Data were analyzed by one-way ANOVA followed by Tukey’s HSD test (P < 0.05). Levene’s test was used to assess distribution normality and variance homogeneity between samples. All data were expressed as means ± SE. All statistical analyses were performed using SPSS® v. 21 (SPSS Inc., Chicago, IL, USA).