Development of a microplate-based novel toxicity bioassay using Chlorophyta and Phaeophyceae macroalgae.

Macroalgae are one of the main producers in marine environments. However, only a few toxicity test methods have been established that use reference strains of macroalgae to evaluate the effects of chemicals on the growth and reproduction of macroalgae to monitor water quality. We selected reference strains of Chlorophyta, Ulva aragoënsis; Phaeophyceae, Ectocarpus siliculosus; and wakame, Undaria pinnatifida, as test species to establish a microplate-based method to investigate the toxicity of potassium dichromate, 3,5-dichlorophenol, and two common herbicides (diuron and simazine). We determined the growth of the three macroalgae in their early life stages and during the sporangia formation stage in E. siliculosus under laboratory conditions. We observed that the growth and sporangia formation in these algae were impaired in a dose-dependent manner. Additionally, we investigated the sensitivity of these macroalgae by comparing the toxicity values of toxicants used in this study with those obtained from a database. Compared to other microalgae and plant species, macroalgae showed a relatively high sensitivity to organic compounds, including herbicides. Growth tests using U. aragoënsis and E. siliculosus produced reliable results at 0-32 and 25-32 practical salinity units (PSU), respectively. The tests established in this study could test the toxicity of chemical substances in macroalgae and are thus expected to contribute to a better understanding of the environmental risks of chemical substances on aquatic biota. The tests could be applied to all effluent toxicity tests used for the management of seawater and brackish water quality.

Transcriptional responses of the marine diatom Chaetoceros tenuissimus to phosphate deficiency.

The planktonic diatom Chaetoceros tenuissimus sometimes forms blooms in coastal surface waters where dissolved inorganic phosphorus (P) is typically deficient. To understand the molecular mechanisms for survival under P-deficient conditions, we compared whole transcripts and metabolites with P-sufficient conditions using stationary growth cells. Under P-deficient conditions, cell numbers and photosynthetic activities decreased as cells entered the stationary growth phase, with downregulation of transcripts related to the Calvin cycle and glycolysis/gluconeogenesis. Therefore, metabolites varied across nutritional conditions. Alkaline phosphatase, phosphodiesterase, phytase, phosphate transporter, and transcription factor genes were drastically upregulated under dissolved inorganic P deficiency. Genes related to phospholipid degradation and nonphospholipid synthesis were also upregulated. These results indicate that C. tenuissimus rearranges its membrane composition from phospholipids to nonphospholipids to conserve phosphate. To endure in P-deficient conditions, C. tenuissimus modifies its gene responses, suggesting a potential survival strategy in nature.

Single-Cell Level Raman Molecular Profiling Reveals the Classification of Growth Phases of Chaetoceros tenuissimus.

Harmful algal blooms (HABs) are a natural phenomenon caused by outbreaks of algae, resulting in serious problems for aquatic ecosystems and the coastal environment. Chaetoceros tenuissimus (C. tenuissimus) is one of the diatoms responsible for HABs. The growth curve of C. tenuissimus can be observed from beginning to end of HABs: therefore, detailed analysis is necessary to characterize each growth phase of C. tenuissimus. It is important to examine the phenotype of each diatom cell individually, as they display heterogeneity even in the same growth phase. Raman spectroscopy is a label-free technique to elucidate biomolecular profiles and spatial information at the cellular level. Multivariate data analysis (MVA) is an efficient method for the analysis of complicated Raman spectra, to identify molecular features. Here, we utilized Raman microspectroscopy to identify the molecular information of each diatom cell, at the single-cell level. The MVA, together with a support vector machine, which is a machine learning technique, allowed the classification of proliferating and nonproliferating cells. The classification includes polyunsaturated fatty acids such as linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. This study indicated that Raman spectroscopy is an appropriate technique to examine C. tenuissimus at the single-cell level, providing relevant data to assess the correlation between the molecular details obtained from the Raman analysis, at each growth phase.

Relationship closeness of tolerance to two neonicotinoids with their internal body burden in two estuarine resident marine crustaceans.

The estuarine resident crustacean sand shrimp, Crangon uritai, has a higher tolerance to neonicotinoid insecticides than that of the kuruma prawns, Penaeus japonicus. However, the reason for the differential sensitivities between the two marine crustaceans remains to be understood. This study explored the mechanism underlying differential sensitivities based on insecticide body residues after exposing both said crustaceans to two insecticides (acetamiprid and clothianidin) with or without oxygenase inhibitor piperonyl butoxide (PBO) for 96 h. Two graded-concentration groups were formed; group H (1/15-1 times the 96-h LC50 values) and L (one-tenth the concentration of group H). Results showed that the internal concentration in survived specimens tended to be lower in sand shrimp than in kuruma prawns. Co-treatment of PBO with two neonicotinoids not only increased sand shrimp mortality in the H group, but also altered metabolism of acetamiprid into its metabolite, N-desmethyl acetamiprid. Furthermore, molting during the exposure period enhanced bioconcentration of insecticides, but not affects survival. Collectively, the higher tolerance of sand shrimp than that of kuruma prawns to the two neonicotinoids can be explained by lower bioconcentration potential and more involvement of oxygenase in their alleviating lethal toxicity.

Chronological age-related metabolome responses in the dinoflagellate Karenia mikimotoi, can predict future bloom demise.

Karenia mikimotoi is a common harmful algal bloom (HAB)-forming dinoflagellate and has caused severe financial loss in aquaculture. There are limited metabolomic studies on dinoflagellate biology. Here, we examined alterations in metabolic profiles over the growth curve of K. mikimotoi under nitrogen or phosphorus deficiency and further explored a key criterion for the diagnosis of late stationary phase to identify when the dinoflagellate cells will enter bloom demise. The results demonstrate the differential expression of metabolites for coping with chronological aging or nutrient deprivation. Furthermore, an increase in the glucose to glycine ratio in the late stationary phase was indicative of dinoflagellate cells entering bloom demise; this was also detected in the cultured diatom, Chaetoceros tenuissimus, indicating that this may be the general criterion for phytoplankton species. Our findings provide insights regarding chronological aging and the criterion for the prediction of phytoplankton bloom demise.

Desorption of polycyclic aromatic hydrocarbons from polyethylene microplastics in two morphologically different digestive tracts of marine teleosts: Gastric red seabream (Pagrus major) and agastric mummichog (Fundulus heteroclitus).

In this study, we elucidated the desorption potency of polycyclic aromatic hydrocarbons (PAHs) sorbed on microplastics (MP; polyethylene) in the digestive tract of two fish species: gastric red seabream and agastric mummichog. In our in vitro assay system using the real gut sample of unexposed fish, the digestive tract was firstly removed from the fish and divided into three parts. Then, MP that had previously been sorbed with 16 PAHs were incubated with extracts of the gut contents or tissue with buffer or only a buffer. The desorption potency of PAHs was individually assessed for gut contents and tissue, which revealed that PAH desorption from MP was elevated in extracts of the gut contents compared with that in the buffer alone for both fish species. PAH desorption potency was the highest in the midgut for gastric red seabream and in the foregut for agastric mummichog, which indicates that PAH desorption from MP varies among different parts of the digestive tract and among fish with distinct gut morphology. In the midgut contents of red seabream and foregut contents of mummichog, the desorption fraction was 5.6% and 8.1% of the total PAHs sorbed on MP, respectively. The desorption fraction enhancement achieved by adding gut contents extracts tended to be greater with an increase in the n-octanol/water partition ratio, suggesting that enhancement of the desorption fraction in the digestive tract depends on the physicochemical properties of PAHs. Thus, morphological differences in digestive tracts and PAH properties should be considered when evaluating the effect of MP vector on pollutant exposure in fish.

Molting enhances internal concentrations of fipronil and thereby decreases survival of two estuarine resident marine crustaceans.

In aquatic arthropods, molting is a pivotal physiological process for normal development, but it may also expose them to higher risks from xenobiotics, because the organism may take up additional water during that time. This study aimed to assess the effects of molting on bioconcentration and survival after 96-h exposure to insecticide fipronil with or without oxygenase (CYP450s) inhibitor piperonyl butoxide (PBO) of two estuarine resident marine crustacean species: the sand shrimp Crangon uritai and the kuruma prawn Penaeus japonicus, with 96-h LC50 value of fipronil = 2.0 µg/L and 0.2 µg/L, respectively. Two graded concentrations included group high (H) (equivalent to the 96-h LC50 values) and low (L) (one-tenth of the H group concentration). Molting and survival were individually checked, and internal concentrations of fipronil and its metabolites (fipronil desulfinyl, fipronil sulfide, fipronil sulfone) were measured. The results showed that, only fipronil and fipronil sulfone were detected from organism, and that internal concentrations of these insecticides in molted specimens were higher than those of unmolted ones but comparable with those of dead ones. Accordingly, mortality was more frequent in molted specimens than those that were unmolted. Furthermore, involvement of oxygenase and higher lethal body burden threshold may confer higher tolerance to fipronil in sand shrimp than in the kuruma prawn. This study is the first to demonstrate that the body-residue-based approach is useful for deciphering the causal factors underlying fipronil toxicity, but highlights the need to consider physiological factors in arthropods, which influence and lie beyond body burden, molting and drug metabolism.

Integrated transcriptomic and metabolomic analyses reveal mechanism underlying higher resistance of the marine oligochaete Thalassodrilides cf. briani (Clitellata: Naididae) to heavy contamination of sediments with polycyclic aromatic hydrocarbons.

In some coastal areas, sediments are contaminated with various chemical compounds, causing significant threats to marine organisms. Therefore, the development of remediation techniques is important. Here, we focused on bioremediation using marine benthic animals such as aquatic oligochaetes. The oligochaete Thalassodrilides cf. briani is highly resistant to contamination of sediments with toxic chemicals. We examined whether T. cf. briani could decompose high-concentration polycyclic aromatic hydrocarbons (PAHs) in sediments. Furthermore, relevant genes expressed in T. cf. briani exposed to contaminated sediment were comprehensively examined using next-generation sequencing, and its metabolites were identified by metabolomic analysis using gas chromatography-mass spectrometry. T. cf. briani reduced the concentration of 16 PAHs in the sediment from 55,900 to 45,200 ng/g dry weight in 50 days, thereby reducing total PAH concentrations by approximately 20%. The results of transcriptomic analysis suggest that activation of a drug-metabolizing enzyme system may promote the metabolism of harmful chemical substances during excretion of chemicals from the body. According to the results of principal component analysis based on the values of 43 types of metabolomes identified by metabolomic analysis, groups were divided according to the difference in the number of exposure days. In addition, levels of glutamine, which is important for maintaining digestive tract functions, increased. This suggests that the digestive tract function promotes the metabolism and detoxification of foreign substances. Furthermore, transcriptome analysis revealed that glutamate dehydrogenase increased 1.3-fold and glutamine synthetase increased 1.7-fold, confirming the increase in glutamine. Thus, we conclude that T. cf. briani adapted to the polluted sediment by regulating its metabolism.

Microplastic uptake and gut retention time in Japanese anchovy (Engraulis japonicus) under laboratory conditions.

To explore the impact of microplastic (MP) pollution on planktivorous fish, we examined the uptake and retention of MPs by Japanese anchovy (Engraulis japonicus) under laboratory conditions. MP uptake was size selective in adult anchovy-0.3-mm MPs were taken up in significantly larger amounts than 0.85-mm MPs-but not in juveniles. There were no significant differences in the uptake of MPs of three different colors, suggesting that anchovy do not select for MP coloration. More than 90% of the MPs were excreted within 20 h of ingestion, indicating that MP retention time is similar to the processing time of food items. Our findings suggest that Japanese anchovy tend to take up MPs that are equivalent in size to prey items, but that the impacts of MP ingestion are likely to be limited under the current state of oceanic MP contamination.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Immune toxicity of phenanthrene and its combined effects of white spot syndrome virus on the survival of kuruma shrimp (Penaeus Japonicus).

Shrimp inhabiting coasts that are frequented by humans are exposed to various pollutants. Additionally, viral infections that cause serious damage to shrimp populations have been observed in these environments. The present study sought to evaluate the immunotoxic effects of phenanthrene (Phe), a pollutant detected in coastal environments, on kuruma shrimp (Penaeus japonicus). We further examined the survival of shrimp following combined exposure to Phe (30 or 300 µg/L) and white spot syndrome virus (WSSV). Results show that exposure to Phe for seven days decreased immune system-related parameters, including total hemocyte count and phenoloxidase activity in hemolymph (p < 0.05). However, these effects were not detected after three days of exposure. Moreover, a combined exposure assay revealed that shrimp mortality increased following exposure to 300 µg/L Phe and infection with WSSV. The number of WSSV gene copies was also observed to increase in these co-exposed shrimp. Taken together, these results indicate that long-term Phe exposure impairs the immune system of P. japonicus, resulting in fatal proliferation of WSSV. Hence, considering that combined exposure to Phe and WSSV leads to increased mortality of shrimp, it is imperative that the detrimental effects elicited by multiple stresses be considered, and controlled, in areas inhabited by kuruma shrimp.

Alterations of stool metabolome, phenome, and microbiome of the marine fish, red sea bream, Pagrus major, following exposure to phenanthrene: A non-invasive approach for exposure assessment.

The present study aimed to assess the impact of phenanthrene (Phe) on fish health by addressing the alteration of fecal characteristics, in lieu of collecting biomarkers that often involves injurious or even fatal sampling of organisms. The marine fish red sea bream, Pagrus major, was exposed to Phe at a concentration of 18 µg/L for 16 days followed by depuration for 13 days. We collected feces from Phe-exposed or control (Phe-free) fish and then analyzed the fecal metabolite profile (metabolome), carbon utilization of microbiota (phenome), and bacterial 16s rRNA gene sequence (microbiome). Along with the increase in physiological stress markers (SOD and EROD) in serum and liver, we noted the possible role of intestine as a Phe reservoir. Furthermore, abnormal fecal appearance (green coloration) and remarkable changes in fecal characteristics were observed. These changes include alterations of cholesterol and putrescine metabolism and the enhanced utilization of putrescine as a carbon source. Phe also altered the microbial community, with an increase in Phe-degrading bacteria such as Pseudomonas. Interestingly, these enteric impairments were ameliorated by depuration. Taken together, our findings suggest that these alterations in feces were associated with adaptive responses to environmentally relevant Phe exposure scenarios, and that stool samples are potential candidates for exposure assessment in fish.

Estimation of the uptake and gut retention of microplastics in juvenile marine fish: Mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major).

We investigated the impact of microplastics (MPs) on marine fish by estimating the uptake and retention by mummichogs (Fundulus heteroclitus) and red seabreams (Pagrus major) of MPs similar in size (≥0.25 mm) and composition (polyethylene) to MPs detected in fish intestines. Results revealed a correlation between MP concentrations in aquarium water and the content of MPs in the gastrointestinal tracts of exposed fish. More than 95% were excreted from both species within 25 h; this retention time is similar to the processing time of food items. The rate of excretion showed little dependence on MP size, but there was some dependence on fish species and MP shape. These results suggest that MPs similar to those we studied have little direct adverse impact on these two marine fish species.

Ecological risk assessment of an antifouling biocide triphenyl (octadecylamine) boron in the Seto Inland Sea, Japan.

In this study, we derived the predicted no-effect concentrations (PNEC) for triphenyl (octadecylamine) boron (TPB-18) and investigated the occurrence of triphenylboranes (TPBs), including TPB-18, for ecological risk assessment in the Seto Inland Sea, Japan. We tested algal growth inhibition, crustacean immobilization, and reproductive toxicity and performed toxicity tests in fish to assess acute and chronic toxicity and generate the PNEC for TPB-18. The minimum toxicity value was 0.30 µg/L, as determined by the 72-h no-observed-effect concentration (NOEC) for the alga Chaetoceros gracilis. The 5th-percentile of hazardous concentration (HC5), derived from NOECs using the species sensitivity distributions approach, was 0.059 µg/L, which indicated the PNEC of 0.0059 µg/L. In comparison, the highest concentration in seawater sampled from the Seto Inland Sea was 0.00034 µg/L, suggesting that the ecological risks posed by TPB-18 are currently low.

Metabolomics-based approach to explore growth phase-dependent markers in cultured diatom Chaetoceros tenuissimus.

Chaetoceros tenuissimus is a cosmopolitan marine diatom whose metabolism has been little investigated. In this study, we examined the alterations of the metabolite profile between different growth phases (exponential and stationary phase) in cultured C. tenuissimus and identified growth phase-dependent candidate marker metabolite. First, the preparation methodology was optimized, focusing on extraction solvent. Metabolites of the cultured diatom (exponential phase 2.4 × 106 cells/mL, stationary phase 3.1 × 106 cells/mL) were extracted using two solvents with different constituents (solvent 1, methanol:H2O:chloroform = 5:2:2; solvent 2, methanol:ethanol:chloroform = 1:3:1). Gas chromatography mass spectrometry (GC/MS)-based metabolomics successfully detected 43 water-soluble metabolites in both solvents. The metabolic features were dependent on the growth phase: amino acid levels were higher in the exponential phase, whereas sugars and alcohols were more abundant in the stationary phase. Solvent 1 was superior in the recovery of the candidate metabolite that had a retention time of 18.13 min and predominantly contributed to discrimination between the growth phases; the metabolite level was higher in the stationary phase than in the exponential phase. The candidate metabolite was identified as mannonic acid by using GC/MS and liquid chromatography tandem mass spectrometry. Higher levels of mannonic acid during the stationary phase were also observed in other three diatom species. This study provides further insight into the use of metabolomics in the evaluation of physiological conditions of diatoms and suggests that mannonic acid content is a potential biomarker of the growth phase in cultured diatom cells.

Occurrence of neonicotinoids and fipronil in estuaries and their potential risks to aquatic invertebrates.

This study aimed to evaluate and qualify field-based potential risks of seven neonicotinoid and phenylpyrazole (fipronil) insecticides on aquatic invertebrates, including estuary-resident marine crustaceans. One hundred and ninety-three estuarine water samples, with salinity ranging from 0.5 to 32.7, were collected from four estuarine sites in the Seto Inland Sea of Japan, in 2015-2018 and the insecticide levels were measured. Five neonicotinoid and fipronil insecticides were successfully identified, and their occurrence varied temporally. Marine crustaceans were simultaneously harvested every month from one of the estuarine water sampling sites in 2015-2017. Three predominant crustacean species, kuruma prawn (Penaeus japonicus), sand shrimp (Crangon uritai), and mysid (Neomysis awatschensis), were captured and their seasonal presence was species independent. A 96-h laboratory toxicity study with the insecticides using kuruma prawn, sand shrimp, and a surrogate mysid species (Americamysis bahia) indicated that fipronil exerted the highest toxicity to the three crustaceans. Using both toxicity data and insecticide occurrence in estuarine water (salinity ≥10, n = 169), the potential risks on the three marine crustaceans were quantified by calculating the proportion of mixture toxicity effects (Pmix). The Pmix of seven neonicotinoids on the crustaceans was less than 0.8%, which is likely to be too low to indicate adverse effects caused by the insecticides. However, short temporal detection of fipronil (exclusively in June and July) significantly affected the Pmix, which presented the maximal Pmix values of 21%, 3.4%, and 72% for kuruma prawn, sand shrimp, and mysid, respectively, indicating a significant effect on the organisms. As for estuarine water (salinity <10), some water samples contained imidacloprid and fipronil exceeding the freshwater benchmarks for aquatic invertebrates. The present study provides novel insights into the seasonally varying risks of insecticides to estuarine crustaceans and highlights the importance of considering whether ecological risk periods coincide with crustacean presence.

Trans-omics analyses revealed differences in hormonal and nutritional status between wild and cultured female Japanese eel (Anguilla japonica).

Long-term stock decline in the Japanese eel (Anguilla japonica) is a serious issue. To reduce natural resource utilization in Japan, artificial hormonal induction of maturation and fertilization in the Japanese eel has been intensively studied. Recent experiment on feminized (by feeding a commercial diet containing estradiol-17ß for first half year) cultured female eels have shown ovulation problems, which is seldom observed in captured wild female eels. Therefore, the aim of this study is to try to investigate causes of ovulation problem frequently seen in cultured female eels by comparative trans-omics analyses. The omics data showed low growth hormone and luteinizing hormone transcription levels in the brain and low sex hormone-binding globulin transcription levels in the liver of the cultured female eels. In addition, it was found that high accumulation of glucose-6-phosphate and, maltose in the liver of the cultured female eel. It was also found that docosahexaenoic (DHA) acid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA) ratios in cultured female eels were quite different from wild female eels. The data suggested that ovulation problem in cultured female eels was possibly resulted from prolonged intake of a high-carbohydrate diet and/or suboptimal DHA/EPA/ARA ratios in a diet.

Physiological responses of eelgrass (Zostera marina) to ambient stresses such as herbicide, insufficient light, and high water temperature.

This study aimed to elucidate the biological responses of eelgrass (Zostera marina) to artificially induced stresses such as herbicide (Irgarol 1051, Irg) exposure, insufficient light, and high water temperature (27 ± 1.0 °C) by evaluating growth inhibition, photosynthetic activity, and metabolomic profiles. After 14 days, all treatments inhibited growth, but photosynthetic activity was only reduced in the Irg-exposed group. In the Irg-exposed and insufficient light groups, the metabolomic profiles were characterized by decreased levels of sugar (sucrose) and increased levels of amino acids such as glutamine, glycine, and leucine. Biochemical and ultrastructural analyses revealed that the loss of sugar-derived metabolic energy was compensated for by energy generated during autophagic protein degradation. Furthermore, the level of myo-inositol, which has various biological roles and participates in several cellular processes such as cell wall synthesis, stress response, and mineral nutrient storage, was markedly increased in the Irg-exposed and insufficient light groups. A combination of metabolomic analysis with other analyses such as measurement of photosynthetic activity might further elucidate the response of eelgrass to ambient stresses in the natural environment.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Sample treatment optimization for fish stool metabolomics.

Gut microbiota play an essential role in an organism's health. The fecal metabolite profiling content reflects these microbiota-mediated physiological changes in various organisms, including fish. Therefore, metabolomics analysis of fish feces should provide insight into the dynamics linking physiology and gut microbiota. However, metabolites are often unstable in aquatic environments, making fecal metabolites difficult to examine in fish. In this study, a novel method using gas chromatography-mass spectrometry (GC-MS) was developed and optimized for the preparation of metabolomics samples from the feces of the marine fish, red sea bream (Pagrus major). The preparation methodology was optimized, focusing on rinsing frequency and rinsing solvent. Feces (collected within 4 h of excretion) were rinsed three times with sterilized 2.5% NaCl solution or 3.0% artificial seawater (ASW). Among the 86 metabolites identified in the NaCl-rinsed samples, 57 showed superior recovery to that in ASW-rinsed samples, indicating that NaCl is a better rinsing solvent, particularly for amino acids, organic acids, and fatty acids. To evaluate rinsing frequency, fecal samples were rinsed with NaCl solution 0, 1, 3, or 5 times. The results indicate that three or more rinses enabled robust and stable detection of metabolites encapsulated within the solid fecal residue. Furthermore, these data suggest that rinsing is unnecessary when studying sugars, amino acids, and sterols, again highlighting the need for appropriate rinsing solvent and frequency. This study provides further insight into the use of fecal samples to evaluate and promote fish health during farming and supports the application of this and similar analyses to study the effects of environmental fluctuations and/or contamination.