2.1. Study area and sampling
The Shibukuro and Tama Rivers that were targeted in this study are located in the northeastern part of Akita Prefecture, Japan, to the west and south of Hachimantai Akita-Yakeyama volcano. The Shibukuro River has a channel length of 11 km and a basin area of 41 km2 and joins the Tama River. The Tama River has a channel length of 103 km and a basin area of 1219 km2 and joins the Omono River. The Tamagawa hot spring is located in the Tamagawa hot-spring explosion crater at the western foot of Akita-Yakeyama Volcano, which is located about 60 km northeast of Akita City. Most of the Tamagawa hot-spring water is neutralized at a neutralization treatment facility, but some flows directly into the upper section of the Shibukuro River. We selected three sampling sites in the Shibukuro and Tama Rivers. The survey points were selected to include those that are expected to show an effect on aquatic organisms and those that are not expected to show an effect (Fig. 1). Site A is located approximately 3.5 km downstream of the Tamagawa hot-spring facility, Site B is located approximately 3 km farther downstream, at the confluence of the Shibukuro and Tama Rivers, and Site C is located approximately 3 km farther downstream in the Tama River.
All water samples were collected from each site in August 2020. Surface water samples were collected from a depth of 0.3 m below the river surface. Twenty liters of water were taken from each site for chemical analysis for heavy metals and for bioassays using Danio rerio and Daphnia magna. Water samples were transported and kept at 4 ℃ until chemical analysis or bioassay. Water quality parameters were measured by using a pH and EC meter (WQ-310; Horiba, Kyoto, Japan), a dissolved oxygen meter (OM-71; Horiba), a salinity meter (YK-31SA; Mother Tool, Nagano, Japan), and a thermometer (AD-5624; AND, Tokyo, Japan).
2.2. Bioassays using Danio rerio and Daphnia magna
We conducted a bioassay using Danio rerio following OECD TG 212 with minor modification (OECD 1998). First, the sampled river water, which was stored at 4°C, was heated to 25°C using a water bath. Next, a dilution series of six concentrations was prepared using dechlorinated water: control, 6.25%, 12.5%, 25%, 50%, and 100% test water. Fish eggs were obtained by natural mating and unfertilized or abnormally developed eggs were removed under a stereomicroscope. Fertilized eggs within four hours post fertilization were selected and exposed to each concentration for eight days post fertilization (five days post hatching). After exposure, 15 fertilized eggs were transferred to a 100-mL glass vessel (exposure volume, 60 mL) with four replicates for each concentration, for a total of 60 fertilized eggs per treatment. Eggs were observed at the same time each day under a stereomicroscope; abnormal eggs were noted, and dead eggs were removed immediately after observation. The water was changed every two days. The bioassay was conducted at 25 ± 2°C and a photoperiod of 16 h light:8 h dark. After the test was completed, the survival rate was calculated.
The bioassay using Daphnia magna followed OECD TG 202 with minor modification (OECD 2004). First, the sampled river water, which was stored at 4°C, was heated to 25°C using a water bath. Next, a dilution series of six concentrations was prepared using dechlorinated water: control, 6.25%, 12.5%, 25%, 50%, and 100% test water. We selected larvae laid within 24 hours from the bred parent Daphnia and exposed them to each concentration for two days. After exposure, five larvae were distributed to a 100-mL glass vessel (exposure volume, 60 mL) with four replicates for each concentration, for a total of 20 larvae per treatment. Larvae were observed at the same time each day under a stereomicroscope; abnormal larvae were noted, and dead larvae were removed immediately after observation. The water was changed every two days. The bioassay was conducted at 20 ± 2°C and a photoperiod of 16 h light:8 h dark. After the test was completed, the survival rate was calculated.
In all experiments, the Danio rerio and Daphnia magna were handled in a humane manner in accordance with the guidelines of Akita Prefectural University, Japan.
2.3. Chemical analysis for Fe, As, and Al
Sato et al. (2005) reported that the main heavy metals flowing out of Tamagawa hot spring were iron (Fe), arsenic (As), and aluminum (Al). We therefore conducted chemical analyses for these three substances.
Fe concentrations were determined by using an atomic absorption spectrophotometer (AA280FS, Agilent Technologies Japan, Ltd, Tokyo, Japan). Each water sample (200 mL) was placed in a fluororesin beaker to which high-purity nitric acid (5 mL) was added. The beaker was heated for 1 h at 180 ℃ on a hot plate. After the sample cooled to room temperature, nitric acid (2 mL) was added. A portion of the sample was transferred to a test tube and used for flame atomic absorption spectroscopy (FAAS) analysis. Analytical conditions were as follows: flame type, air-acetylene; air mass flow rate, 13.50 L/min; acetylene flow rate, 2.00 L/min; measurement mode, spectral analysis; analytical wavelength, 248.3 nm. The blank value was 0.0350 mg/L.
As concentrations were determined by using an inductively coupled plasma–mass spectrometer (ICP-MS; Agilent 7700x, Agilent Technologies Japan, Ltd). Each water sample (25 mL) was placed in a fluororesin beaker to which nitric acid (0.25 mL) was added. The beaker was heated for 1 h at 150 ℃ on a hot plate. After the sample cooled to room temperature, a portion of the sample was transferred to a test tube and used for analysis. Analytical conditions were as follows: sampling depth, 10 mm; measurement mode, spectral analysis; plasma gas flow rate, 15 L/min; carrier gas flow rate, 0.35 L/min; high matrix introduction (HMI) gas flow rate, 0.60 L/min; collision cell gas (He) flow rate, 10 mL/min; nebulizer pump rotation speed, 0.1 rps; spray chamber temperature, 2 ℃. The internal standard method (with yttrium [Y] as the internal standard) was used in ICP-MS analysis. The blank value was 0.000050 mg/L.
Al concentrations were determined by using an inductivity coupled plasma optical emission spectrometer (ICP-OES; iCAP6300Duo, Thermo Fisher Scientific, Tokyo, Japan). Each water sample (25 mL) was placed in a fluororesin beaker to which nitric acid (1 mL) was added. The beaker was heated for 1 h at 150 ℃ on a hot plate. After the sample cooled to room temperature, a portion of the sample was transferred to a test tube and used for analysis. Analytical conditions were as follows: ICP mode, multi-channel; plasma gas flow rate, 12 L/min; nebulizer gas flow rate, 0.50 L/min; auxiliary gas flow rate, 0.5 L/min; pump speed, 50 rpm. The internal standard method (with yttrium [Y] as the internal standard) was used in ICP-OES analysis. The blank value was 0.0063 mg/L.
2.4. Statistical analysis
Statistical analyses were conducted as reported previously (Horie et al. 2017). We used custom R code and the package Rcmdr (Fox and Bouchet-Valat 2018) to test for homogeneity of variance using Bartlett’s test (significance level, 5%). If the null hypothesis (i.e., the data are homoscedastic) was not rejected, we tested for differences among treatments using Dunnett’s test; otherwise, we used Steel’s test.