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.

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.

Cosmopolitan or Cryptic Species? A Case Study of Capitella teleta (Annelida: Capitellidae).

Capitella teleta Blake et al., 2009 is an opportunistic capitellid originally described from Massachusetts (USA), but also reported from the Mediterranean, NW Atlantic, and North Pacific, including Japan. This putatively wide distribution had not been tested with DNA sequence data; intraspecific variation in morphological characters diagnostic for the species had not been assessed with specimens from non-type localities, and the species status of the Japanese population(s) was uncertain. We examined the morphology and mitochondrial COI (cytochrome c oxidase subunit I) gene sequences of Capitella specimens from two localities (Ainan and Gamo) in Japan. Specimens from Ainan and Gamo differed from C. teleta from Massachusetts in methyl-green staining pattern, shape of the genital spines, and shape of the capillary chaetae; we concluded that these characters vary intraspecifically. Species delimitation analyses of COI sequences suggested that worms from Ainan and Massachusetts represent C. teleta; these populations share a COI haplotype. The specimens from Gamo may represent a distinct species and comprise a sister group to C. teleta s. str.; we refer to the Gamo population as Capitella aff. teleta. The average Kimura 2-parameter (K2P) distance between C. teleta s. str. and C. aff. teleta was 3.7%. The COI data indicate that C. teleta actually occurs in both the NW Atlantic and NW Pacific. Given the short planktonic larval duration of C. teleta, this broad distribution may have resulted from anthropogenic dispersal.

Reproductive toxicity of 1-nitronaphthalene and 1-nitropyrene exposure in the mummichog, Fundulus heteroclitus.

After pregnant mummichog were implanted with 1-nitronaphthalene or 1-nitropyrene via cholesterol pellet, we investigated the effects of the chemicals on embryo normality, hatchability and days to hatch of normal embryos, and growth and survival of hatched larvae from normal embryos of the implanted mummichog. Hatchability was the parameter most sensitive to the effects of both 1-nitronaphthalene and 1-nitropyrene. The 4-week lowest-observed-effect concentration (LOEC) of 1-nitronaphthalene, based on the actual concentrations in the eggs in the test, was 447 ng g(-1) wet wt.; and the LOEC and no-observed-effect concentration (NOEC) of 1-nitropyrene were 958 and 344 ng g(-1) wet wt., respectively. The 4-week LOEC of 1-nitronaphthalene, based on the concentration in the water, was estimated at 4.8 µg L(-1) by using the reported bioconcentration factor; and the LOEC and NOEC of 1-nitropyrene, based on the concentration in the water, were estimated at 3.1 and 8.6 µg L(-1), respectively. The reported environmental concentrations of 1-nitropyrene and 1-nitronaphthalene are over three magnitudes lower than the toxicity values we obtained. Therefore, the effects of environmental levels of 1-nitropyrene and 1-nitronaphthalene on fish reproduction, not including genomic effects on embryos, appear to be almost negligible. However, DNA damage has been detected in marine organisms exposed to 1-nitropyrene. Further studies of the genotoxicity of nitrated polycyclic aromatic hydrocarbons at environmental levels are therefore needed to evaluate their ecotoxicological risks.

Examination of digestive enzyme distribution in gut tract and functions of intestinal caecum, in megascolecid earthworms (Oligochaeta: Megascolecidae) in Japan.

Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

Taxonomic identity of a tetrodotoxin-accumulating ribbon-worm Cephalothrix simula (Nemertea: Palaeonemertea): a species artificially introduced from the Pacific to Europe.

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula ( Iwata, 1952 ) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus ( Iwata, 1952 ), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas ( Thunberg, 1793 ) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles ( Jordan and Snyder, 1901 )-also known to contain TTX- consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

Toxicity of degradation products of the antifouling biocide pyridine triphenylborane to marine organisms.

We evaluated the acute toxicities of the main degradation products of pyridine triphenylborane (PTPB), namely, diphenylborane hydroxide (DPB), phenylborane dihydroxide (MPB), phenol, and biphenyl, to the alga Skeletonema costatum, the crustacean Tigriopus japonicus, and two teleosts, the red sea bream Pagrus major and the mummichog Fundulus heteroclitus. DPB was the most toxic of the degradation products to all four organisms. The acute toxicity values of DPB for S. costatum, T. japonicus, red sea bream, and mummichog were 55, 70, 100, and 200-310 µg/L, respectively. The degradation products were less toxic than PTPB to S. costatum and T. japonicus; however, the toxicities of DPB and PTPB to the fish species were similar. We also examined changes in the inhibition of growth rate of S. costatum as well as the percentage of immobilization of T. japonicus as end points of toxicity of PTPB after irradiation of PTPB with 432 ± 45 W/m(2) of 290-700 nm wavelength light. After 7 days of irradiation with this light, the concentration of PTPB in the test solutions decreased markedly. A decrease in toxic effects closely coincided with the decrease in the concentration of PTPB caused by the irradiation. PTPB probably accounted for most of the toxicity in the irradiation test solutions. Because the concentrations of PTPB that were acutely toxic to S. costatum and T. japonicus were <10 % of the corresponding concentrations of its degradation products, PTPB probably accounted for most of the toxicity in the irradiation test solutions.

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.

Tracing and regulating redox homeostasis of model benthic ecosystems for sustainable aquaculture in coastal environments.

Aquaculture in coastal environments has an increasingly important role in the world's food supply; however, the accumulation of organic compounds on seafloors due to overfeeding adversely affects benthic ecosystems. To assess the ecological resilience of aquafarms to nutrient influx, we investigated the redox homeostasis of benthic ecosystems using a marine oligochaete as a model benthic organism in aquaculture fields. Real-time monitoring of the redox potential of a model benthic ecosystem constructed in an electrochemical reactor allowed evaluation of the homeostatic response of the system to nutrient addition. Although the detrimental effects of overfeeding were confirmed by irreversible potential changes in the sediment, redox homeostasis was reinforced through a cooperative relationship between oligochaetes and sediment microorganisms. Specifically, the oligochaetes exhibited reversible changes in metabolism and body position in response to dynamic changes in the sediment potential between -300 and 500 mV, thereby promoting the decomposition of organic compounds. The potential-dependent changes in metabolism and body position were reproduced by artificially manipulating the sediment potential in electrochemical reactors. Given the importance of benthic animals in sustaining coastal ecosystems, the electrochemical monitoring and physiologic regulation of marine oligochaetes could offer an intriguing approach toward sustainable aquaculture.

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.

Anti-viral effects of interferon administration on sevenband grouper, Epinephelus septemfasciatus.

Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the present study, we report cloning and characterization of the IFN gene from the sevenband grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family. The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic Poly(I:C), which is known as an inducer of IFN. It has also been confirmed that injection of recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to sevenband grouper is effective in preventing severe symptom development after NNV infection.

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.

Toxicity of metal pyrithione photodegradation products to marine organisms with indirect evidence for their presence in seawater.

We evaluated the acute toxicities of the metal pyrithiones (MePTs)--copper pyrithione (CuPT) and zinc pyrithione (ZnPT)--to four species of marine algae and a marine crustacean (Tigriopus japonicus). We also performed acute toxicity tests using six of the main MePT photodegradation products: pyridine-N-oxide (PO); 2-mercaptopyridine (HPS); pyridine-2-sulfonic-acid (PSA); 2-mercaptopyridine-N-oxide (HPT); 2,2'-dithio-bis-pyridine ([PS](2)); and 2,2'-dithio-bis-pyridine-N-oxide ([PT](2))-and three marine organisms representing three trophic levels: an alga (Skeletonema costatum), a crustacean (T. japonicus), and a fish (Pagrus major). The acute toxicity values (72-h EC(50)) of CuPT, ZnPT, HPT, (PT)(2), (PS)(2), HPS, PO, and PSA for S. costatum, which was the most sensitive of the test organisms to the chemicals tested, were 1.5, 1.6, 1.1, 3.4, 65, 730, >100,000, and >100,000 microg l(-1), respectively. CuPT was detected in the growth media used for S. costatum tests and in seawater containing HPT or (PT)(2); the concentration of CuPT in seawater containing HPT was highly dependent on the Cu(2+) concentration. These results indicate that in the presence of sufficient Cu(2+), the toxicities of HPT and (PT)(2) should be assessed as CuPT because in Japan MePTs are most frequently used as antifouling booster biocides in conjunction with cuprous oxide.

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.

BMP-2 release and dose-response studies in hydroxyapatite and beta-tricalcium phosphate.

The purpose of this study is to compare in vivo retention of BMP-2 and bone induction in HAp (porosity: 60-80%, pore size: 100-600 mum, sintering temperature: 800 degrees C, surface area: 1 m(2)/g) and beta-TCP (porosity: 75%, pore size: 100-400 mum, sintering temperature: 1050 degrees C, surface area: 4 m(2)/g). We estimated the in vivo release profile of (125)I-labeled BMP-2 and bone induction of hard tissues histologically. The amount of BMP-2 remaining in the beta-TCP at 1 day after implantation was 49.6%, while the amount was 34.0% in the HAp. Furthermore, the HAp and beta-TCP containing 0.0, 0.05, 0.1, 0.3, 0.5, 1.0, 5.0 microg of BMP-2 were implanted into the back subcutis of 4-week old Wistar rats. At 3 weeks after implantation, the ceramics were explanted and evaluated histologically. The HAp/BMP-2 (5.0 microg) system showed 3.0% in the total volume of bone at 3 weeks, while only in the beta-TCP/BMP-2 (5.0 microg) system showed 32.5%. These results indicate that the absorbable beta-TCP block may be an effective bioceramic for bone induction to deliver BMP-2 to the site of action.

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.

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.