Effects of chlorpyrifos on the metabolome of the freshwater carp, Cyprinus carpio.

This study investigated the effects of waterborne chlorpyrifos with concentrations of 1 and 100 µg/L for L and H-groups, respectively, on metabolome profiles of carp plasma using (1)H-NMR. Principal component analysis suggests that chlorpyrifos exposure firstly affected in L and H-groups on day 2 or 4, and followed a second effect in both exposure groups on day 14. Levels of metabolites related to the energy production in the body, such as glucose, glycerol, valine, leucine, isoleucine, lactate, alanine, 3-D-hydroxybutyrates and acetoacetate, significantly changed by exposures of chlorpyrifos. Those results suggest that energy production was severely affected in carp. The exposure could also be highly elevated ammonia levels especially in H-group due to severe convulsion in muscle caused by the inhibition of acetylcholinesterase activity.

Accumulation of endocrine disrupting chemicals (EDCs) in the polychaete Paraprionospio sp. from the Yodo River mouth, Osaka Bay, Japan.

This study presents the levels of endocrine disrupting chemicals (EDCs) accumulated by Paraprionospio sp. from the Yodo River mouth, Osaka Bay. Since high concentrations of nonylphenol (NP), bisphenol A (BP), octylphenol (OP), 17ß-estradiol (E2), and estrone (E1) have been measured in sediment from Osaka Bay, some bioaccumulation could be expected particularly in benthic animals. EDCs were analysed in Paraprionospio sp., a dominant benthic species in Osaka Bay. The results showed that Paraprionospio sp. had accumulated varying concentrations (wet weight; w.w.) of NP at 1,460-4,410 ng/g; BP at 22.5-39.6 ng/g; OP at 18.9-45.4 ng/g; E2 at 0.89-4.35 ng/g; and E1 at 0.06-2.50 ng/g. Accumulation of NP and OP were highest among the samples gathered in summer (July 2008), while concentrations of BP, E2, and E1 did not much differs within 3 years. EDC levels in Paraprionospio sp. were apparently greater than those in sediments showing bioaccumulation.

Effects of diphenhydramine exposure on reproduction of mature Japanese medaka (Oryzias latipes).

Diphenhydramine (DPH) is an antihistamine drug. It has been frequently detected in the environment, because it is not completely degraded in wastewater treatment plants. Recent studies have shown the adverse effects of DPH exposure to various aquatic organisms; however, its chronic effects on fish have been poorly elucidated. In this study, several pairs of mature Japanese medaka (Oryzias latipes) were exposed to DPH for a long period to determine the effects of DPH exposure on the subsequent generations, number of spawned and fertilized eggs, expression of sex-related genes, feeding behavior, embryo development, hatching rate, malformations among the hatched larvae, and mortality rate. The number of spawned eggs significantly decreased, when the parent fish were continuously exposed to 31.6 µg/L DPH for over 46 days. DPH exposure also altered the feeding behavior of medaka individuals, and increased the larval mortality rate. The effects of DPH exposure to fish may occur to some extent in the actual aquatic environment, although the risk evaluations in the field are limited.

Exposure of Cattle Breeding Herds to Naturally Co-Contaminated Zearalenone and Deoxynivalenol: The Relevance of a Urinary Mycotoxin Monitoring System for Herd Health and Food Safety.

The widespread presence of Fusarium mycotoxins in animal feed is a global issue, not only for the health of livestock but also for ensure the safety of food as an end product. High concentrations of zearalenone (ZEN) and deoxynivalenol (DON) have been detected in the diets of Japanese Black (JB) and Holstein Friesian (HF) breeding herds. Consequently, we monitored serum biochemical parameters over a long time in both herds, focusing on anti-Müllerian hormone (AMH) levels and acute-phase inflammation. Additionally, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and progesterone levels were measured in the HF herd. The JB herd, a ZEN-dominant model with low DON contamination, demonstrated ZEN levels that exceeded the Japanese limit in the purchased total mixed rations (TMR). Conversely, the HF herd, which primary consumes DON-dominant feed with low ZEN contamination, had high DON levels in the dent corn silage. Specifically, the JB herd's TMR contained 1.79 mg/kg ZEN and 0.58 mg/kg DON, whereas the HF herd's silage had 15.3 mg/kg DON (dried sample) and 0.1 mg/kg ZEN. Enzyme-linked immunoassay were used to measure urinary ZEN-DON levels following confirmation through liquid chromatography-tandem mass spectrometry. Urinary ZEN-DON levels measured were significantly correlated (p < 0.05, r > 0.6) in both herds. In the HF herd, AMH levels increased (p = 0.01) and serum amyloid A (SAA) levels decreased (p = 0.02) when contaminated and at the end of the monitoring period. Additionally, urinary ZEN and DON levels were significantly correlated with SAA levels (ZEN: p = 0.00, r = 0.46; DON: p = 0.03, r = 0.33), with an increase in ZEN and DON levels resulting in higher SAA levels. The JB herd showed no significant differences. Additionally, in the HF herd, 8-OHdG/Cre levels increased significantly during major contamination periods (p < 0.05). Clinical data from the HF herd indicated an increase in mastitis cases and treatment rates during periods of major contamination. Abortion rates in the HF herd decreased from 22.9% (before monitoring) to 8.9% (during the high contamination period) and finally to 1% (at the end of the monitoring period), with corresponding increases in progesterone levels. ZEN-DON contamination adversely affects breeding cattle's productivity, reproductive performance, and health. Therefore, monitoring urinary ZEN-DON is valuable for detecting contaminants and ensuring the safety of food products.

Bioaccumulation of dietary endocrine disrupting chemicals (EDCs) by the polychaete, Perinereis nuntia.

To investigate the biomagnification factor (BMF) of EDCs by the polychaete, Perinereis nuntia, organisms were exposed to EDCs through their diet. BMF values ranged from 0.001 to 0.028 indicating that EDCs were not biomagnified. Elimination rates were (0.20-0.25 day(-1)) and were higher than uptake rate (0.0003-0.003 day(-1)) verifying that EDCs were not biomagnified by P. nuntia due to their fast elimination. The calculated half-life of each EDC in this study varies from 2.76 to 3.45 days. Overall, the findings from this study demonstrated that the studied EDCs are not biomagnified in P. nuntia but exposure does occur from the diet.

Practical Application of a Urinary Zearalenone Monitoring System for Feed Hygiene Management of a Japanese Black Cattle Breeding Herd-Relevance to Anti-Müllerian Hormone and Serum Amyloid A Clarified from a Two-Year Survey.

In this study, a herd of Japanese Black (JB) breeding cattle with sporadic reproductive disorders was continuously monitored for an additional year to assess the effects of the urinary zearalenone (ZEN) concentration and changes in parameters (AMH and SAA) with time-lag variables and herd fertility (reproductive performance). This herd had high (exceeded the Japanese dietary feed regulations) urinary ZEN and rice straw ZEN concentrations (1.34 mg/kg). Long-term data of the herd with positive ZEN exposure revealed a decreasing ZEN concentration in urine and a gradual decrease in the AMH level with age. The AMH level was significantly affected by the ZEN value 2 months earlier and the AMH level in the previous month. The changes in ZEN and SAA values were significantly affected by the ZEN and SAA values in the previous month. Additionally, calving interval data between pre-monitoring and post-monitoring showed a significantly different pattern. Furthermore, the calving interval became significantly shorter between the time of contamination (2019) and the end of the monitoring period (2022). In conclusion, the urinary ZEN monitoring system may be a valuable practical tool for screening and detecting herd contamination in the field, and acute and/or chronic ZEN contamination in dietary feeds may affect herd productivity and the fertility of breeding cows.

Measurement of serum short-chain fatty acid concentrations in cattle after oral administration of difructose anhydride III.

Background and Aim: We previously reported the mitigation effects of difructose anhydride III (DFA III) on mycotoxins, such as zearalenon and sterigmatocystin, based on the urinary concentrations of these molecules in calves. This study was aimed at evaluating the effects of dietary supplementation of DFA III and the fermented status of DFA III in the intestine by comparing serum levels of short-chain fatty acid (SCFAs) in DFA III-supplemented cattle with those in non-supplemented control cattle. Materials and Methods: Serum SCFA concentrations were measured in 30 Japanese Black heifers, aged 9-10 months, from two herds, using gas chromatography on days 0 (before DFA III supplementation), 9, and 14 after DFA III supplementation. Results: A notably different trend was observed for isobutyric acid and enanthic acid, which may reflect the different fermentation status of supplementary DFA III in the intestine. Our results indicate the possibility that this trend observed in the intestinal tract following DFA III administration is associated with changes in the environment of intestinal bacterial flora, which may partially reflect the effects of DFA III supplementation on cattle. Conclusion: Difructose anhydride III supplementation for at least 2 weeks affects the trend of blood SCFA concentrations in cattle. Our results provide evidence supporting the effects of DFA III on the intestinal environment and intestinal barrier function.

¹H NMR-based metabolomics approach to assess toxicity of bunker a heavy oil to freshwater carp, Cyprinus carpio.

Using a ¹H NMR metabolomics approach, the effects of dietary exposure of bunker A heavy oil (0.01, 0.1, 1, and 5% in diet) on freshwater carp, Cyprinus carpio, were examined. Statistical analysis by PCA score plots showed that the amount of metabolites in exposure groups 0.1, 1, and 5% differed from those in the control group. Although no discernible effects on metabolites were noted in the 0.1% exposure group as well as in the lowest concentration (0.01%) group, several metabolites such as amino acid (e.g., leucine, isoleucine, valine, glutamine, histidine, proline, and methionine), 3-D-hydroxybutyrate, and glycerol were elevated, while another metabolite such as formate was reduced in 1 and 5% groups. These changes in the metabolites associated with the tri-carboxylic-acid (TCA) cycle suggest that oil exposure resulted in the disturbance of the TCA cycle in the liver of the carp. Isobutyrate, a marker of anoxia, was also increased in 1 and 5% exposures groups and was directly related to low hemoglobin concentrations leading to reduced oxygen transport by blood. In addition, significant elevation of creatinine in the plasma of carps exposed to 5% heavy oil suggests disturbance in kidney function. Thus, metabolomics approach can detect toxic effects of hazardous pollutants on fish.

Histopathological alterations in gill, liver and kidney of common carp exposed to chlorpyrifos.

Histopathological alterations in gill, liver and kidney of common carp, Cyprinus carpio, intoxicated with sub-lethal concentrations of chlorpyrifos (O,O,-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) pesticide (1 and 100 µg/L) for a period of 14 days were analyzed under light microscope. Gill exhibited hyperplasia and hypertrophy of gill epithelium, blood congestion, dilation of marginal channel, epithelial lifting, lamellar fusion, lamellar disorganization, lamellar aneurysm, rupture of the lamellar epithelium, rupture of pillar cells and necrosis. Alterations in hepatocytes were more pronounced, including nuclear and cellular hypertrophy, cellular atrophy, irregular contour of cells and nucleus, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, pyknotic nucleus, necrosis and melanomacrophages aggregations. Histopathological lesions in kidney were cellular and nuclear hypertrophy, narrowing of tubular lumen, cytoplasmic vacuolation, hyaline droplet degeneration, nuclear degeneration, occlusion of tubular lumen, tubular regeneration, dilation of glomerular capillaries, degeneration of glomerulus and hemorrhage in Bowman's space. The most significant conclusion drawn from this study was that with the increased concentration and duration the toxicosis of chlorpyrifos would be enhanced as shown through the analysis of mean assessment value (MAV) and degree of tissue changes (DTC) also.

Practical Application of Urinary Zearalenone Monitoring System for Feed Hygiene Management of a Japanese Black Cattle Breeding Herd-The Relationship between Monthly Anti-Müllerian Hormone and Serum Amyloid A Concentrations.

This study addresses an advantageous application of a urinary zearalenone (ZEN) monitoring system not only for surveillance of ZEN exposure at the production site of breeding cows but also for follow-up monitoring after improvement of feeds provided to the herd. As biomarkers of effect, serum levels of the anti-Müllerian hormone (AMH) and serum amyloid A (SAA) concentrations were used. Based on the results of urinary ZEN measurement, two cows from one herd had urinary ZEN concentrations which were two orders of magnitude higher (ZEN: 1.34 mg/kg, sterigmatocystin (STC): 0.08 mg/kg in roughages) than the levels of all cows from three other herds (ZEN: not detected, STC: not detected in roughages). For the follow-up monitoring of the herd with positive ZEN and STC exposure, urine, blood, and roughage samples were collected from five cows monthly for one year. A monitoring series in the breeding cattle herd indicated that feed concentrations were not necessarily reflected in urinary concentrations; urinary monitoring assay by ELISA may be a simple and accurate method that reflects the exposure/absorption of ZEN. Additionally, although the ZEN exposure level appeared not to be critical compared with the Japanese ZEN limitation in dietary feeds, a negative regression trend between the ZEN and AMH concentrations was observed, indicating that only at extremely universal mycotoxin exposure levels, ZEN exposure may affect the number of antral follicles in cattle. A negative regression trend between the ZEN and SAA concentrations could also be demonstrated, possibly indicating the innate immune suppression caused by low-level chronic ZEN exposure. Finally, significant differences (p = 0.0487) in calving intervals between pre-ZEN monitoring (mean ± SEM: 439.0 ± 41.2) and post-ZEN monitoring (349.9 ± 6.9) periods were observed in the monitored five cows. These preliminary results indicate that the urinary ZEN monitoring system may be a useful practical tool not only for detecting contaminated herds under field conditions but also provides an initial look at the effects of long-term chronic ZEN/STC (or other co-existing mycotoxins) exposure on herd productivity and fertility.

Detection of zearalenone and its metabolites in naturally contaminated porcine follicular fluid by using liquid chromatography-tandem mass spectrometry.

Zearalenone (ZEN) and its metabolites are important nonsteroidal estrogenic mycotoxins that cause reproductive disorders in domestic animals, especially pigs. We aimed to simultaneously detect ZEN and its metabolites á-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL) in porcine follicular fluid (FF) by liquid chromatography-tandem mass spectrometry. ZEN and α-ZOL, but not ß-ZOL, were detected in all pooled FF samples collected from coexisting follicles (diameter ≥ 6 mm) within 10 ovaries. Furthermore, ZEN and α-ZOL were detected in samples pretreated with ß-glucuronidase/arylsulfatase, but not in those left untreated, suggesting that the FF samples contained glucuronide-conjugated forms of the mycotoxins that may be less harmful to porcine oocytes due to glucuronidation affecting the receptor binding. Nonetheless, the effects of the glucuronide-conjugated forms should be studied, both in vitro and in vivo.

Histopathological effects and EROD induction in common carp exposed to dietary heavy oil.

Common carp, Cyprinus carpio, was exposed to 1% and 5% dietary heavy oil for 2 weeks. Higher polyaromatic hydrocarbons (PAHs) accumulation induced higher hepatic EROD activity in exposure groups compared with the control group. Significant histological alternations were observed in the liver such as nuclear and cellular hypertrophy, accumulation of eosinophilic granules in cytoplasm, nuclear degeneration and fibrillar inclusion in cytoplasm. Severe damage of the kidney was indicated by nuclear and cellular degeneration, and ultimately necrosis in the tubular epithelium cells. In addition, dilated glomerulus with hemorrhage was found in the renal capsule. The mean assessment value (MAV) and degree of tissue changes (DTC), as semi-quantitative analyses, were significantly increased with concentration and duration of exposure. Likewise, frequencies of pathological lesions in both liver and kidney were also increased with concentration and duration of exposure.

Interspecies differences in the accumulation of tributyltin and its metabolites under dietary exposure in sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major.

Interspecies differences in the accumulation of dietary tributyltin (TBT) between sea perch, Lateolabrax japonicus, and red sea bream, Pagrus major, were studied. Although TBTs in both species reached steady-state condition in low-concentration group (L-group, 200 ng/g nominal concentration) by 1 week, it increased up to the end of exposure in high-concentration group (H-group, 3000 ng/g nominal concentration). In H-group, the accumulation rate of TBT in sea perch from 1 to 2 weeks was much higher than in red sea bream, which were 2.4-fold for sea perch and 1.7-fold for red sea bream, although TBT concentrations were similar between sea perch and red sea bream in L-group. Furthermore, in the H-group, the concentrations of TBT at 1 and 2 weeks were about 1.3- and 1.9-fold, respectively, higher in the sea perch than in the red sea bream. On the other hand, DBT residue in red sea bream was about 1.4-fold higher in sea perch for the L-group but concentrations were similar in both fishes for the H-group. These results suggest that red sea bream could metabolize faster the TBT to DBT than sea perch. This study also reveals that fish probably could absorb TBT through the food chain. The uptake of TBT by fish should be regarded in the real environment, because TBT concentration in seawater has been decreasing and now already at significantly low level.

Mitigation of sterigmatocystin exposure in cattle by difructose anhydride III feed supplementation and detection of urinary sterigmatocystin and serum amyloid A concentrations.

We evaluated the effects of supplementing cattle feed with difructose anhydride III (DFA III) by measuring urinary sterigmatocystin (STC) concentrations using 20 Japanese Black cattle aged 9-10 months from one herd. DFA III was supplemented for 2 weeks for 10 animals, and non-treated animals served as controls. The natural STC concentration in the dietary feed was 0.06  mg kg - 1 (mixture of roughage and concentrate) at the beginning of the study (Day 0). The urine STC concentration was measured using liquid chromatography with tandem mass spectrometry 1 d prior to DFA III administration, 9 and 14 d thereafter, and 9 d following supplementation cessation, concomitant with the measurement of serum amyloid A (SAA). The number of heifers in which STC was detected in the urine was low (10 %) in the DFA III group compared to that (60 %) in the control group on Day 9. After 9 d following supplementation cessation (Day 23), STC concentrations were significantly lower ( P = 0.032 ) in the DFA III group than in the control group, although there was no difference in the number of heifers in which urinary STC was detected or in SAA concentrations between the two groups. Our findings demonstrate the effect of DFA III on reducing the urinary concentration of STC in Japanese Black cattle.

Monitoring of PAHs and alkylated PAHs in aquatic organisms after 1 month from the Solar I oil spill off the coast of Guimaras Island, Philippines.

Following the oil spill accident of the Solar I tanker in 2006 off the coast of Guimaras Island in the Philippines, polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs in some aquatic organisms were investigated at Luzaran in Guimaras and Taklong Islands, which were heavily polluted with spilled oil, immediately and 1 month after the accident. The concentrations of total PAHs were 11.9-52.3 ng/g dry weight in fish. Meanwhile, total PAH concentrations in shellfish were 38.0-3,102 ng/g dry weight in Luzaran and 128-236 ng/g dry weight in Taklong. Pyrene, phenanthrene, and fluoranthene were dominant in most fish and chrysene in all shellfish. Significantly higher concentrations of all alkylated homologs were detected in shellfish than in fish. These differences had two possible causes, that is, the differences between fish and shellfish could be attributed to the uptake routes and/or their metabolizing abilities.

Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle.

Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.

Fructo-Oligosaccharide (DFA III) Feed Supplementation for Mitigation of Mycotoxin Exposure in Cattle-Clinical Evaluation by a Urinary Zearalenone Monitoring System.

The potential effect of difructose anhydride III (DFA III) supplementation in cattle feed was evaluated using a previously developed urinary-zearalenone (ZEN) monitoring system. Japanese Black cattle from two beef herds aged 9⁻10 months were used. DFA III was supplemented for two weeks. ZEN concentrations in feed were similar in both herds (0.27 and 0.22 mg/kg in roughage and concentrates, respectively), and below the maximum allowance in Japan. ZEN, α-zearalenol (α-ZOL), and ß-ZOL concentrations in urine were measured using LC/MS/MS the day before DFA III administration, 9 and 14 days thereafter, and 9 days after supplementation ceased. Significant differences in ZEN, α-ZOL, ß-ZOL, and total ZEN were recorded on different sampling dates. The concentration of inorganic phosphate in DFA III-supplemented animals was significantly higher than in controls on day 23 (8.4 vs. 7.7 mg/dL), suggesting a possible role of DFA III in tight junction of intestinal epithelial cells. This is the first evidence that DFA III reduces mycotoxin levels reaching the systemic circulation and excreted in urine. This preventive effect may involve an improved tight-junction-dependent intestinal barrier function. Additionally, our practical approach confirmed that monitoring of urinary mycotoxin is useful for evaluating the effects of dietary supplements to prevent mycotoxin adsorption.

Evaluation of 2,4-dichlorophenol exposure of Japanese medaka, Oryzias latipes, using a metabolomics approach.

In this study, the metabolic effects of waterborne exposure of medaka (Oryzias latipes) to nominal concentrations of 20 (L group) and 2000 µg/L (H group) 2,4-dichlorophenol (DCP) were examined using a gas chromatography/mass spectroscopy (GC/MS) metabolomics approach. A principal component analysis (PCA) separated the L, H, and control groups along PC1 to explain the toxic effects of DCP at 24 h of exposure. Furthermore, the L and H groups were separated along PC1 at 96 h on the PCA score plots. These results suggest that the effects of DCP depended on exposure concentration and time. Changes in tricarboxylic cycle metabolites suggested that fish exposed to 2,4-DCP require more energy to metabolize and eliminate DCP, particularly at 96 h of exposure. A time-dependent response in the fish exposed to DCP was observed in the GC/MS data, suggesting that the higher DCP concentration had greater effects at 24 h than those observed in response to the lower concentration. In addition, several essential amino acids (arginine, histidine, lysine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, and valine) decreased after DCP exposure in the H group, and starvation condition and high concentration exposure of DCP could consume excess energy from amino acids.

Toxic evaluations of sediments in Tokyo Bay, Japan, using Japanese medaka embryos.

Toxic risks of sediments collected from seven sites in Tokyo Bay were evaluated using Japanese medaka embryos. Those sediments with slight pore water were placed in grass petri dishes without overlying water. The most remarkable effect in the field sediment was to cause hatching delay in embryos, and the longest time until hatching took was 12.5 ± 1.6 days post-fertilization (dpf), although that in control group was 10.1 ± 0.7 dpf. A significant delay in hatching was observed at four sites. Because total carbon concentrations were relatively high in sediments at three of these four sites, several chemicals were expected to be residues in these sites and could cause their delay. Although extreme mortality was not observed at all sites, sediments collected from the site close to Kawasaki city induced 10 % mortality. Polycyclic aromatic hydrocarbon (PAH) concentrations were remarkably high at this site compared with other sites, and thus PAH toxicities could be causing the mortality. Concentration of heavy metals such as cadmium, copper, lead, and zinc in sediments were also determined, but no clear relationship was found between toxicities to embryos and the distribution of their concentrations.

Effects of oxygenated polycyclic aromatic hydrocarbons on the early life stages of Japanese medaka.

Oxygenated polycyclic aromatic hydrocarbons (oxyPAHs) are directly discharged into the atmosphere with exhaust gas from diesel engine automobiles and industry and are also generated by photo-oxidation and/or microbial metabolism of parent polycyclic aromatic hydrocarbons (PAHs). They are widely distributed in environment, especially around urban areas. We evaluated the effects of exposure to oxyPAHs as acenaphthenequinone (ANQ), 7,12-benz(a)anthracenequinone (BAQ), 1,4-naphthoquinone (NAQ), and 9,10-phenanethrenequinone (PHQ) in Japanese medaka (Oryzias latipes) embryos. ANQ (>2720 µg/L) caused caving in the yolk sac, and BAQ (>22.4 µg/L) caused tanning of the oil droplet. Additionally, NAQ (>314 µg/L) and PHQ (>734 µg/L) stopped development of medaka embryos beginning 2 days after the start of exposure. The hatched larvae from embryos exposed to ANQ, BAQ, NAQ, or PHQ at 518, 9.86, 36.5, or 80.7 µg/L, respectively, exhibited incomplete development of the cephalic region, incomplete palate, unabsorbed and hypertrophied yolk sac, tubular heart, altered axial curvature, and poor swimming ability. These symptoms were similar to those observed in blue sac disease, which is caused by exposure to PAHs.