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.

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.

A hepatic metabolomics-based diagnostic approach to assess lethal toxicity of dithiocarbamate fungicide polycarbamate in three marine fish species.

The present study was performed to evaluate the toxic effect of the dithiocarbamate fungicide polycarbamate (PC) on the hepatic metabolic profiles of three marine fish species, red sea bream (Pagrus major), spotted halibut (Verasper variegatus), and marbled flounder (Pleuronectes yokohamae). First, juvenile fish were exposed to graded concentrations of PC for 96h; the 96-h LC50 values obtained were 22-29, 239-553, and 301-364µgL-1 for red sea bream, spotted halibut, and marbled flounder, respectively, indicating that red sea bream possessed higher sensitivity to PC than the two benthic species. Second, the fish were exposed to lethal-equivalent concentration (H group) or sub-lethal (one-tenth of the H group concentrations; L group) for 24 and 96h and gas-chromatography based metabolomics approach was employed to explore the crucial biomarker metabolite associated with lethal toxicity. Of the 53 metabolites identified, only reduced glutathione (GSH) was consistently elevated in the H group for the three fish species at 96h. The calculated cut-off value of GSH (mM) based on receiver operating curve analysis between H group and the other treatment groups (control, solvent control, and L group) was obtained at 0.56mM, which allowed to distinguish between the groups with high confidence for the three fish species. These results are the first to demonstrate the potential of using GSH as a possible biomarker metabolite and its usefulness of threshold cut-off value for diagnosing life-threatening health conditions of fish.

Dietary taurine supplementation ameliorates the lethal effect of phenanthrene but not the bioaccumulation in a marine teleost, red sea bream, Pagrus major.

The present study was performed to evaluate the effect of dietary taurine on the hepatic metabolic profiles of red sea bream (Pagrus major) and on phenanthrene (a polyaromatic hydrocarbon) toxicity and bioaccumulation. The fish were fed a diet supplemented with 0% (TAU0%), 0.5% (TAU0.5%), or 5% (TAU5%) taurine for 40-55d and subjected to phenanthrene acute toxicity and bioaccumulation tests. Taurine deficiency in feed severely affected the hepatic metabolic profiles of fish, which indicated a complementary physiological response to taurine deficiency. For the acute toxicity test, fish were fed the test diets for 55d and were then exposed to 0-893µg/L phenanthrene for 96h. Tolerance to phenanthrene was significantly improved by 0.5% of taurine inclusion in feed relative to TAU0%, but not by 5.0% inclusion. Reduced glutathione in the liver, which acts as an oxygen-free radical scavenger, was associated with a reduction in the toxicity of phenanthrene. For the bioaccumulation test, fish were fed the test diets for 40d and were thereafter chronically exposed to 20µg/L phenanthrene for 13d followed by depuration for 3d. The activity of hepatic biomarker, ethoxyresorufin-O-deethylase, was increased by phenanthrene exposure in the taurine inclusion groups. However, phenanthrene concentrations in the liver and muscle of fish fed TAU5.0% tended to be higher than those of fish fed TAU0% and TAU0.5% during the exposure period. These results indicate that 0.5% of taurine inclusion in feed plays an important role in the alleviation of phenanthrene toxicity but not bioaccumulation. Furthermore, larger amount of taurine inclusion (TAU5%) did not show marked beneficial effects against phenanthrene exposure. This study provides insight about a major concern of environmental contaminants into aquatic environment and can be effectively used for improvement of aquaculture.

Effect of taurine supplementation on hepatic metabolism and alleviation of cadmium toxicity and bioaccumulation in a marine teleost, red sea bream, Pagrus major.

This study was performed to unravel the mechanism of the beneficial action of taurine on marine teleost fish, red sea bream (Pagrus major), by analyzing the hepatic metabolism. Moreover, the ameliorative effects of the nutrient against cadmium toxicity and bioaccumulation were further evaluated. The fish were fed a diet containing 0 % (TAU0 %), 0.5 % (TAU0.5 %), or 5.0 % (TAU5.0 %) taurine for 40-55 days (d) and subjected to cadmium acute toxicity and bioaccumulation tests. Taurine deficiency in feed severely affected growth and the hepatic metabolic profiles of the fish, including a remarkable increase in myo-inositol, aspartate, and ß-alanine in the TAU0 % group, which indicates a complementary physiological response to taurine deficiency. For the acute toxicity test, fish were fed the test diets for 55 d and were then exposed to different dose of cadmium ranging from 0 to 5.6 mg/L for 96 h. Fish fed taurine had a higher tolerance to cadmium than those not fed taurine. For the bioaccumulation test, fish were fed the test diets for 40 d and then were chronically exposed to 0.2 mg/L of cadmium for 28 d followed by depuration for 21 d. Cadmium concentrations in the liver and muscle of fish fed TAU5.0 % were significantly lower than those of fish fed TAU0 % for the first 7 d of exposure and the first 7 d of elimination. Our findings suggest a possible mechanism for the beneficial role played by taurine and that the inclusion of taurine in fish aquaculture feed may reduce cadmium contamination of fish intended for human consumption.

Bioconcentration and Elimination of the Dithiocarbamate Fungicide Polycarbamate in Marine Teleost Fish and Polychaete.

We evaluated the bioconcentration and elimination of polycarbamate, a popular antifoulant classified as a dithiocarbamate fungicide, in a marine teleost fish, mummichog (Fundulus heteroclitus), and a polychaete (Perinereis nuntia). Following polycarbamate exposure and depuration, the calculated bioconcentration factor (BCF) and elimination rate constant (k2) were 3.1 and 0.17 day(-1) in fish and 1.5-7.8 and 0.13-0.18 day(-1) in the polychaete, indicating that the fungicide has low bioconcentration potential in both organisms. Given the BCF of 3.1, the permissible environmental concentration level of polycarbamate in water was calculated to be 6.1 µg L(-1) to satisfy the threshold pesticide residue levels in fish permitted by law, which is far below the permissible levels in Hiroshima Bay, Japan. Therefore, we consider that the current polycarbamate contamination level in terms of bioconcentration is not likely to be an alarming issue in coastal environments.

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.

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.

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.

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.

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.

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.

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.

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.