Occurrence of octylphenol and nonylphenol in the sediment of South China Sea and Malacca Strait, Peninsular Malaysia.

Sediment is the ultimate reservoir of effluent from landmasses. This includes octylphenol (OP) and nonylphenol (NP), two chemical compounds which are known with the ability to disrupt the normal functions of hormones in the organism. To our knowledge, no study of these compounds in the marine sediment of Malaysia has been published to date. Hence, this study presents the level of OP and NP in the sediment of the South China Sea and Malacca Strait, Malaysia. The extraction of compounds was done using the liquid-liquid extraction method and followed by clean-up using solid-phase extraction cartridges. The range of OP in Malacca Strait (1.00-27.16 ng/g dw) was greater than in the South China Sea (5.12-14.16 ng/g dw) whereas a similar range of NP was found in the South China Sea (1.32-23.76 ng/g dw) and Malacca Strait (0.79-27.59 ng/g dw). The concentration of both compounds was consistently high near Redang Island (E2A) and Penang (W32 and W43) suggesting continuous input of these chemicals from this nearby land. Risk quotient (RQ) values of OP showed the potential risk to benthic communities in 4/7 and 21/47 sampling points of the respective South China Sea and Malacca Strait. Both water bodies are located far from the wastewater effluent and yet able to retain these chemicals in their sediment. This suggests that the wastewater treatment system as well as dilution effects do not prevent these chemicals to be ended up in the marine environment.

Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17ß (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ±â€¯28.9 µg/mL (VtgAa), 57.9 ±â€¯30.7 µg/mL (VtgAb) and 12.6 ±â€¯4.8 µg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 µg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ±â€¯733.93 µg/mL) was significantly higher than for VtgAa (150.33 ±â€¯22.35 µg/mL) or VtgC (57.08 ±â€¯6.00 µg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.

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.

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.

Correlation between Polycyclic Aromatic Hydrocarbons in Wharf Roach (Ligia spp.) and Environmental Components of the Intertidal and Supralittoral Zone along the Japanese Coast.

Polycyclic aromatic hydrocarbon (PAH) concentrations in wharf roach (Ligia spp.), as an environmental indicator, and in environmental components of the intertidal and supralittoral zones were determined, and the PAH exposure pathways in wharf roach were estimated. Wharf roaches, mussels, and environmental media (water, soil and sand, and drifting seaweed) were collected from 12 sites in Japan along coastal areas of the Sea of Japan. PAH concentrations in wharf roaches were higher than those in mussels (median total of 15 PAHs: 48.5 and 39.9 ng/g-dry weight (dw), respectively) except for samples from Ishikawa (wharf roach: 47.9 ng/g-dw; mussel: 132 ng/g-dw). The highest total PAH concentration in wharf roach was from Akita (96.0 ng/g-dw), followed by a sample from Niigata (85.2 ng/g-dw). Diagnostic ratio analysis showed that nearly all PAHs in soil and sand were of petrogenic origin. Based on a correlation analysis of PAH concentrations between wharf roach and the environmental components, wharf roach exposure to three- and four-ring PAHs was likely from food (drifting seaweed) and from soil and sand, whereas exposure to four- and five-ring PAHs was from several environmental components. These findings suggest that the wharf roach can be used to monitor PAH pollution in the supralittoral zone and in the intertidal zone.

Pesticide residues in coastal waters affected by rice paddy effluents temporarily stored in a wastewater reservoir in southern Japan.

This paper presents the occurrence, distribution, and environmental impact of pesticide residues in coastal water in southern Japan that receives effluents from a wastewater reservoir temporarily storing surface runoffs of rice paddy fields located near the coastline during the 2005 rice planting season. Concentrations of 14 target pesticides were measured by GC-MS and the hazards posed by the most important pesticides detected were evaluated by acute toxicity tests using a marine diatom, Chaetoceros sp., and a marine amphipod, Hyale barbicornis. Six pesticides (fenobucarb, flutolanil, iprobenfos, mefenacet, phthalide, pyriproxyfen) were detected in the coastal water, with three pesticides (fenobucarb, iprobenfos, and mefenacet) having 100% frequencies of detection. The maximum concentration of mefenacet, at 4.22 microg/L, was at least one magnitude higher than that of fenobucarb and iprobenfos, at 0.27 and 0.19 microg/L, respectively, while the three remaining pesticides had concentrations just around the detection limit of 0.01 microg/L. Consequently, detected concentrations of mefenacet were highly correlated with salinity levels, confirming that the wastewater reservoir is a major source of rice pesticide residues in this particular coastal environment. Hence, the spatial distribution of mefenacet was simulated using their relationship and the results indicate that mefenacet has a tendency to enter and spread to a relatively wide portion of the coastal area during the rice planting season. There is also a possibility that the other pesticides used in rice farming such as fenobucarb and iprobenfos may show similar distribution patterns in coastal waters when they are present in the wastewater reservoir at higher concentrations. These pesticides, however, manifested low acute toxicities to both Chaetoceros sp. and H. barbicornis, suggesting little impact to marine organisms.

Assessment of pesticide residues in freshwater areas affected by rice paddy effluents in Southern Japan.

Pesticide residues in five freshwater areas that are directly affected by rice paddy effluents in southern Japan were measured to determine their maximum concentrations and temporal variations. Water samples were collected every week during the 2005 rice planting season in Kagoshima Prefecture and stations were established in Amori River, Sudo River, Nagaida River (that drains into the bigger Kotsuki River), rice paddy drainage canal, and wastewater reservoir (that collects effluents from rice paddy fields). Of the 14 target pesticides examined, a total of 11 were detected in all stations. Mefenacet, fenobucarb, and flutolanil were the three pesticides with the highest maximum concentrations and were also detected frequently. Analysis of temporal variations of pesticides showed that herbicides had relatively higher concentrations in the earlier stages of the rice planting season, while insecticides and fungicides had relatively higher concentrations at the later stages. There was no significant difference among stations with regards to the temporal patterns of the top three pesticides. The calculated toxic units were less than 1 in all stations, implying low or negligible environmental risk of pesticides detected to freshwater organisms.

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.

A SNP in a Steroidogenic Enzyme Is Associated with Phenotypic Sex in Seriola Fishes.

Vertebrate sex development consists largely of two processes: "sex determination," the initial bifurcation of sexual identity, and "sex differentiation," which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3], in which they are indispensable for proper sex differentiation. By contrast, in many poikilothermic vertebrates, steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17ß-hydroxy steroids relative to the allelic product from the W chromosome, which harbors glycine at that position, by disrupting the hydrogen bond network between the steroid and the enzyme's catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes, whereas W-type mRNA is expressed only in genotypic females. Meanwhile, Cyp19a1 is predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.

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.