Chemicals
Potassium dichromate (K2Cr2O7; molecular weight [MW] = 294.18; purity > 99.5%), 3,5-dichlorophenol (3,5-DCP; MW = 163.0; purity > 98.0%), simazine (6-chloro-2-N,4-N-diethyl-1,3,5-triazine-2,4-diamine; MW = 201.66; purity > 99.0%), liquid chromatography mass spectrometry (LC-MS) grade methanol, and ultrapure water were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea; MW = 233.09; purity > 98%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Stock solutions of K2Cr2O7 and 3,5-DCP were prepared by diluting with Milli-Q water from a Milli-Q water purification system (Elix essential UV3 equipped with synergy UV, Merck, Darmstadt, Germany). Stock solutions of simazine and diuron were prepared by diluting with dimethyl sulfoxide (DMSO; product code: D4540, Sigma-Aldrich Japan, Tokyo, Japan). These stock solutions were used in the toxicity tests, as described in the “Growth inhibition tests” and “Sporangia formation inhibition test” sections.
Provasoli’s enriched seawater (PES) medium was prepared by adding 20 mL of PES stock solution (Provasoli 1966) to 1 L of natural seawater filtered through sand, activated carbon, and finally, a glass-fiber cartridge filter (47 mm diameter; 1.2 µm pore size GF/C filter; Whatman, Cytiva, Tokyo, Japan), hereafter referred to as “filtered seawater,” (Practical Salinity Unit [PSU]: 31 ± 1 [mean ± standard error]). The PES medium made from artificial seawater, M-PES medium, was prepared by adding 20 mL of PES stock solution to 1 L of artificial seawater, Marine Art SF-1 (Tomita Pharmaceutical Co. Ltd., Naruto, Tokushima, Japan) filtered through a GF/C filter. Provasoli’s enriched medium (PESI) was prepared by adding 20 mL of PESI stock solution (Provasoli 1966) to 1 L of filtered seawater. Before preparing the media, filtered seawater and Marine Art SF-1 were sterilized using a bottle top filter (0.2 µm pore size; 595–4520; Thermo Fisher Scientific K. K., Tokyo, Japan), and PES and PESI stock solutions were sterilized at 121°C for 20 min in an autoclave (STH307FA, Advantec Toyo Kaisya, Ltd., Tokyo, Japan). The stock solutions and the DMSO (for the solvent control) were diluted 10,000 times with PES medium, M-PES medium, and PESI medium to prepare the test solutions for E. siliculosus, U. aragoënsis, and U. pinnatifida, respectively. M-PES and PES media test solutions for the freshwater tolerance test were prepared by diluting 20 mL of PES stock solution in 1 L of Milli-Q water.
Test Organisms
Ulva aragoënsis strain KU-1532 (= HOK-90, Fig. 1a) and E. siliculosus strain KU-1372 (= Esil 32 male, Fig. 1b) were obtained from the Kobe University Macro-Algal Culture Collection (Kobe, Hyogo, Japan). The KU-1372 strain is a male gametophyte with a fully sequenced genome and is widely used in the fields of physiology and molecular biology. The male and female gametophytes of U. pinnatifida were obtained from Dr. Goro Yoshida at the Fisheries Technology Institute, Japan Fisheries Research and Education Agency.
The E. siliculosus and U. aragoënsis strains were cultured in a Petri dish (diameter = 90 mm; depth = 20 mm, AS ONE corp., Osaka, Japan) with PES medium (Provasoli 1966), under a 12:12 h light:dark cycle (with a light intensity of 58 ± 2 µmol/m2/s) at 12.7 ± 0.1°C and 14.0 ± 0.1°C, respectively, in a temperature-controlled growth chamber (MLR-350; Sanyo, Osaka, Japan, LH-241S; Nippon Medical and Chemical Instruments Co. Ltd., Osaka, Japan). To collect the zoospores, U. aragoënsis was matured at 19.2 ± 1.8°C for a few days under the same light:dark cycle conditions and finely cut with scissors in PES medium and filtered through a nylon mesh filter with a pore size of 10 µm (pluriStrainer; Funakoshi Co., Ltd., Tokyo, Japan). The zoospores in the filtrate were counted using a hemocytometer (Bürker-Türk; AS ONE Corp., Osaka, Japan) under an inverted microscope (IX71; Olympus, Tokyo, Japan) and used for growth inhibition tests as described in the “Growth inhibition tests” section. Cultured E. siliculosus were finely cut with scissors in the PES medium and filtered through nylon mesh filters with pore sizes of 10 µm and 40 µm for toxicity analysis; plumules of the sporophytes on the 10 µm mesh were resuspended in the PES medium and counted with a counting chamber (MPC-200; Matsunami Glass Ind., Ltd., Osaka, Japan) under the inverted microscope and used for the growth inhibition tests as described in the “Growth inhibition tests” section. Approximately 200–400 plumules of sporophytes were transferred to a 24-well plate (305047; Falcon, Corning, NY, USA) that contained 2.5 mL of the PES medium in each well and incubated for 10 to 14 days under the same growth conditions as E. siliculosus (12:12 h light: dark cycle with light intensity of 58 ± 2 µmol/m2/s at 12.7 ± 0.1°C and 14.0 ± 0.1°C). Sporophytes were observed under the inverted microscope, picked before maturation, and used for sporangia formation inhibition tests, as described in the “Sporangia formation inhibition test” section.
The male and female gametophytes of U. pinnatifida were cultured in aerated flasks with the PESI medium (Provasoli 1966) under a 15:9 h light: dark cycle at 21.0 ± 0.5°C in a temperature-controlled growth chamber (LH-241S; Nippon Medical and Chemical Instruments Co., Ltd.). The culture method described by Niwa (2016) was followed to collect the plumules of U. pinnatifida sporophytes (Fig. 1c). The PESI medium was used according to culture conditions as follows: 100 mL of the PESI medium and 0.1 g of male and female gametophytes of U. pinnatifida were homogenized using a blender (7012HBC, Waring; Conair Corporation, Stamford, CT, USA) at 20,000 rpm for 40 s, and the homogenate was diluted 10 times with the PESI medium. The diluted homogenate was cultured in a beaker covered with aluminum foil under a 9:15 h light: dark cycle (with a light intensity of 56 ± 2 µmol/m2/s) at 14.2 ± 0.0°C for over three weeks in a temperature-controlled growth chamber (LH-241S; Nippon Medical and Chemical Instruments Co., Ltd.). The culture solution was filtered through nylon mesh filters with pore sizes of 10 µm and 40 µm. The plumules of the sporophytes on the 10 µm mesh filter were resuspended in the PESI medium and counted using the counting chamber under the inverted microscope and used for growth inhibition tests, as described in the “Growth inhibition tests” section.
Growth Inhibition Tests
Zoospores of U. aragoënsis and plumules of sporophytes of E. siliculosus and U. pinnatifida were used for the tests. The experimental conditions of the test methods are presented in Table 1. Growth inhibition tests were performed as previously described (Hooten and Carr 1998; Wendt et al. 2013), with slight modifications. We conducted the tests in 24-well plates using nominal K2Cr2O7, 3,5-DCP, diuron, and simazine concentrations (Table S1). The growth inhibition tests on E. siliculosus and U. aragoënsis were performed using serial dilutions of the PES medium and M-PES medium (0, 5, 10, 20, 40, and 100% of PES or M-PES medium, for the freshwater tolerance test). The tests were conducted in a temperature-controlled growth chamber. The environmental conditions for the testing period (toxicity tests) are listed in Table S2.
After the tests were performed, a cover glass (φ13 mm, c1100; Matsunami Glass Ind., Ltd.) was placed in the well if the height distribution of the cultured algae was large and over 10 pictures were taken at different focal lengths to obtain fully focused images. The algae were observed and photographed automatically in all the wells using the batch capture and optical modes of the microscope (BZ-X810; Keyence Corporation, Osaka, Japan). Fully focused wide-area images of each well were obtained using the BZ-X analyzer software (Keyence Corporation).
Table 1
Experimental conditions of the test methods using marine macroalgae
Test method | Growth inhibition test | Sporangia formation inhibition test |
Test species | Ulva aragoënsis | Undaria pinnatifida | Ectocarpus siliculosus | Ectocarpus siliculosus |
Test container | 24-well plate | 24-well plate | 24-well plate | 12-well plate |
Volume of test solution | 2.5 mL/well | 2.5 mL/well | 2.5 mL/well | 1.5 mL/well |
Repetition number | 4 | 4 | 4 | 10–17 |
Test chemicals | K2Cr2O7, 3,5-DCP, diuron, simazine | K2Cr2O7, 3,5-DCP | K2Cr2O7, 3,5-DCP, diuron, simazine | K2Cr2O7, 3,5-DCP, diuron, simazine |
Test medium | M-PES medium | PESI medium | PES medium | PES medium |
Set temperature | 20°C | 15°C | 13°C | 13°C |
Light (L): dark (D) cycle | 12:12 h | 9:15 h | 12:12 h | 12:12 h |
Test period | 3 days | 7 days | 7 days or 10 days | 3 days |
Initial biomass (per well) | 1.0×105 of zoospore | About 200–300 of plumules | About 50–150 of shed macroalgae | One grown macroalga from shed macroalgae for 10 days |
Factors | Cell number, length, area, and perimeter | Area | Area | Number of sporangia |
Number of observed individuals (per well) | > 10 | 5 | 10 | 1–2 |
K2Cr2O7, Potassium dichromate; 3,5-DCP, 3,5-dichlorophenol. |
The area, length, circumference, and cell number of over 10 germinating bodies of U. aragoënsis (Fig. 1d), five plumules of sporophytes of U. pinnatifida, and 10 plumules of sporophytes of E. siliculosus per well were measured after the images were corrected for unevenly illuminated background with the subtract background feature and binarized using the ImageJ software 1.53c (Schneider et al. 2012).
Sporangia Formation Inhibition Test
The sporangia formation inhibition tests were performed on the sporophytes of the E. siliculosus before they reached the maturation stage. The tests were performed under the test conditions in Table 1 using a series of nominal K2Cr2O7, 3,5-DCP, diuron, and simazine concentrations as depicted in Table S3. The tests were conducted in a temperature-controlled growth chamber. Environmental conditions during the toxicity testing period are listed in Table S2. After the tests were performed, the number of sporangia in each mature sporophyte (10–17) of the algae was counted using an inverted fluorescence phase contrast microscope (BZ-X810, Keyence Corporation).
Chemical Analysis
The test solutions were sampled at the beginning and end of the growth, and sporangia formation inhibition tests were used to determine the actual toxicant concentrations during the experiment. Simazine, 3,5-DCP, and diuron concentrations in the test solutions were analyzed using solid-phase extraction followed by LC-MS. For 3,5-DCP analysis, an Oasis HLB plus LP cartridge (186000132; Nihon Waters K. K, Tokyo, Japan) was conditioned with 10 mL of methanol followed by 10 mL of Milli-Q water. The water sample was filtered using a PTFE syringe filter unit (pore size 0.45 µm; Membrane Solution, Ltd., Tokyo, Japan), and 8 mL of the sample was used for 3,5-DCP analysis. The cartridge was then washed with 10 mL Milli-Q water and air dried for 1 h. The analytes were eluted in a test tube with 10 mL of methanol, and 100 µL of atrazine-13C3 (1 mg/L in methanol) was spiked as an internal standard and the analytes were further analyzed. Simazine and diuron were extracted from the test solutions using MonoSpin® C18 (GL Sciences Incorporated, Tokyo, Japan), according to the manufacturer’s instructions. The toxicants were extracted from the cartridge in 0.5 mL of methanol, 5 µL of atrazine-13C3 (1 mg/L, in 5 mL methanol solution) was spiked as an internal standard, and the toxicants were subjected to further analyses.
The actual toxicant concentrations were determined using an LC-30AD Prominence system (Shimadzu, Kyoto, Japan). The toxicants were separated on an Inertsil ODS-4 column (2.1 mm i.d. × 50 mm, 2 µm, GL Science Incorporated) equipped with an Inertsil ODS-4 guard cartridge (2.1 mm i.d. × 10 mm, 2 µm, GL Science Incorporated). The mobile phase comprised 5 mM ammonium acetate (solution A) and 5 mM ammonium acetate in methanol (solution B), and the flow rate was 0.2 mL/min with the following gradient: 60–95% B for 3 min, 95% B for 3 min, and 60% B for 4 min to return to the initial condition. The injection volume was 5 µL. Electrospray mass spectrometry analysis was performed for the toxicants (LC-MS-8030; Shimadzu), and analytes were ionized through electrospray ionization in the negative-ion mode. LC-MS was performed in multiple reaction monitoring modes. Analyte-specific detection parameters are presented in Table S4.
Seawater recovery rate and method quantification limit (MQL) were determined for the test chemicals, except K2Cr2O7, in the samples as follows: seawater for the recovery test was spiked with specified amounts of 3,5-DCP, diuron, and simazine and received a pretreatment similar to that of the exposed test samples (3,5-DCP: 10 µg/L; diuron and simazine: 0.5 µg/L). The recovery test was performed seven times, and standard deviations regarding the analyte concentrations and mean recovery rates were determined. The standard deviations multiplied by 10 were taken as MQLs for the analytes. The standard deviations for 3,5-DCP, diuron, and simazine were 0.31, 0.04, and 0.14 µg/L, respectively. Thus, the MQLs of 3,5-DCP, diuron, and simazine were 3, 0.4, and 2 µg/L, and their recovery rates were 105%, 100%, and 90%, respectively. The K2Cr2O7 analysis was performed as follows: the test solution of K2Cr2O7 was filtered using a PTFE syringe filter unit and diluted with ultrapure water. The actual toxicant concentrations of K2Cr2O7 were analyzed using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 8800; Agilent Technologies Japan, Ltd., Tokyo, Japan). The geometric mean values of the toxicant concentrations were used to estimate the 10% effective concentration (EC10), 50% effective concentration (EC50), lowest observed effect concentrations (LOECs), and no observed effect concentrations (NOECs).
Comparison Of The Sensitivity Of The Macroalgal Species
Acute toxicity data, such as EC50 or median lethal concentration (LC50), and chronic toxicity data, such as NOECs, were obtained in this study and from the Ecotox database (https://cfpub.epa.gov/ecotox/). The Ecotox database was also used to retrieve the toxicity effects of test chemicals on the growth, population, reproduction, and mortality of selected plant and algal species, which were compared with the findings of our study. These indicators closely represented growth and reproduction investigated in our study. The data on toxicity values for the test period of less than one day were excluded because this period was not long enough for these indicators to observe the effect of exposure to the pesticide. Where multiple toxicity data based on differences in factors and exposure periods were reported in a previously published study, the minimum toxicity values were selected to compare highly sensitive indicators (Tables S5–9). The geometric mean was used for comparison of the sensitivity of the species for which the toxicity values had been previously reported.
Data analysis
The values of EC10 and EC50 were estimated using R v 4.1.2 (R Core Team 2022) with the drc (logit-log analysis) packages (Ritz et al. 2015), and then multiple comparison tests were performed using R v 4.1.2 (R Core Team 2022) with the multcomp (Dunnett’s test) packages (Hothorn et al. 2008; Ritz et al. 2015). The area, length, circumference, and cell number of U. aragoënsis germinating bodies, and plumules of E. siliculosus and plumules of U. pinnatifida were analyzed using Dunnett’s tests in R v 4.1.2 (R Core Team 2022). Differences between treatment groups were considered significant at p < 0.05.