Effects of single and combined exposures to benzo(a)pyrene and 3,3'4,4'5-pentachlorobiphenyl on EROD activity and development in Fundulus heteroclitus

The goal of this study was to assess the interactive effects of a polynuclear aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP), and a co-planar halogenated aromatic hydrocarbon (HAH), 3,3'4,4'5-pentachlorobiphenyl (PCB 126) on ethoxyresorufin-O-deethylase (EROD) activity and occurrence of deformities in Fundulus heteroclitus. While each compound administered alone elicited dose-dependent increases in EROD activity, the combined treatment of these two compounds generally did not elicit an additive EROD response. There was a significant correlation between deformity occurrence and EROD induction in embryos dosed with PCB alone but not for embryos dosed with BaP alone, or a combination of BaP and PCB 126.     

Behavioral effects in mummichog larvae (Fundulus heteroclitus) following embryonic exposure to methylmercury

Embryos of the mummichog, Fundulus heteroclitus, were exposed to concentrations of methylmercury (meHg) below those that can cause morphological abnormalities. After hatching, larvae were kept in clean water and tested periodically for ability to capture prey (Artemia salina) or for their swimming performance and predator avoidance. Embryonic exposure to 10 μg/liter meHg resulted in reduced prey capture ability by early larvae, measured as total Artemia caught in 1 and 5 min. However, older larvae recovered from the behavioral deficits, perhaps owing to a compensation for early CNS insult. Fish from a chronically polluted environment (Piles Creek (PC), a tributary of the Arthur Kill), previously shown to have high tolerance to the teratogenic effects of higher concentrations of meHg, proved to be less susceptible to these behavioral effects than fish from a relatively uncontaminated site (EH). Also, the PC control larvae captured prey far more effectively than the EH larvae, although they made more miscues. When tested for swimming performance after embryonic exposure to 10 μg/liter, EH larvae had increased performance when compared to controls. This effect was also transitory. The improved swimming performance did not correspond to improved ability to avoid predation by grass shrimp (Palaemonetes pugio). The increased swimming performance may correspond to greater total activity, making them more likely to attract the predator's attention and resulting in increased capture. Thus, negative, though temporary, effects were observed in both prey capture and predator avoidance after embryonic exposure to meHg.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 micro(micro)M) with or without 0.2 micro(micro)g/l of the CYP1A inducer 3,3',4,4',5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Evaluating prey capture by larval mummichogs (Fundulus heteroclitus) as a potential biomarker for contaminants

We evaluated larval prey capture as a "behavioral biomarker" of contamination by examining feeding behavior of larval mummichogs (Fundulus heteroclitus) from many different sites, including a severely contaminated "Superfund" site, moderately contaminated sites, and reference areas. Prey capture ability was related to sediment contaminant levels. The levels of contaminants at a site were highly correlated with each other, so that the impact of individual contaminants was confounded. The number of captures of brine shrimp by mummichog larvae from all sites was highly variable, but significant negative correlations of prey capture were seen with mercury, lead, zinc, cadmium, and PCBs. As observed previously with adults, polyaromatic hydrocarbons (PAHs) did not appear to impair prey capture ability. The only site in which prey capture rates of 8-day old larvae were severely affected was the most highly contaminated Superfund site, Berry's Creek, NJ. This implies that larval prey capture is not as sensitive a behavioral biomarker for contamination as adult behavior studied previously.     

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.     

Patterns of heritability of decreased EROD activity and resistance to PCB 126-induced teratogenesis in laboratory-reared offspring of killifish (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River,VA, USA

Killifish (Fundulus heteroclitus) from a highly contaminated site on the Elizabeth River are resistant to the acute toxicity and the cytochrome P4501A (CYP1A)-inducing activity of both the sediments from the site and chemically pure polycyclic aromatic hydrocarbons (PAHs). These effects are highly heritable for one generation, but heritable to a lesser degree by subsequent generations, in clean conditions in the laboratory. We show that offspring of this population of Elizabeth River killifish are also resistant to the teratogenicity and P4501A-inducing activity of PCB congener 126, a prototypical coplanar halogenated aromatic hydrocarbon (HAH). Furthermore, the pattern of greater resistance to acute toxicity and P4501A-inducing activity in the first generation and less in subsequent generations is also observed upon exposure to PCB-126.     

温度、光照及仔鱼个体大小对稚海蜇(Rhopilema esculentum)捕食褐牙鲆(Paralichthys olivaceus) 仔鱼的影响

利用实验生态学方法,研究温度(15、19和23℃)、光照(明和暗)、仔鱼大小[全长(3.01±0.08)mm初孵仔鱼和(3.79±0.13)mm孵化后5天仔鱼]对稚海蜇[伞径(21.1±0.4)mm]捕食褐牙鲆仔鱼的影响。结果表明,稚海蜇对初孵仔鱼的捕食率随着水温升高而显著升高。光照条件在各仔鱼密度(10,30,50和80ind/L)下均不显著影响稚海蜇对初孵仔鱼的捕食率,说明光条件并不显著改善稚海蜇捕食仔鱼的能力。在高仔鱼密度(50和80ind/L)下,稚海蜇对孵化后5天仔鱼的捕食率显著低于对初孵仔鱼的捕食率;但在低密度(10和30ind/L)下,稚海蜇对两个不同发育阶段仔鱼的捕食率之间无显著差异。在低仔鱼密度下,稚海蜇对仔鱼的捕食率较低,这会降低仔鱼个体大小对稚海蜇捕食率作用的显著性。这既表明仔鱼逃避被稚海蜇捕食的能力随其发育生长而提高,也反映了稚海蜇对不同个体大小仔鱼捕食作用受仔鱼密度制约。     

Effects of dietary and water-borne oil exposure on larval pacific herring (Clupea harengus pallasi)

Pacific herring (Clupea harengus pallasi) larvae were exposed either directly to the water-soluble fraction (WSF) of crude oil or indirectly via oil-contaminated prey (OCP) (Artemia salina) for <- 28 days, to determine the relative effects of diet and water as routes of contamination. Larvae were affected rapidly by 0·9 ppm WSF, which caused high larval mortality, reduced swimming ability and rapid reductions in feeding rates. Larval length was significantly reduced by 0·7 ppm WSF in 7 days and by 0·3 ppm WSF within 14 days. The WSF exposure also caused similar reductions in larval weights. Highly contaminated prey (6 ppm prey exposure) caused significant mortality, but surviving larvae appeared robust. The OCP did not affect swimming, feeding, or growth. Exposure of larvae was not significantly extended by OCP, which rapidly depurated WSF in clean water (98% in 1 day). Therefore, OCP is probably not an important source of low molecular weight petroleum contamination to larval fish in the marine environment.     

海蜇(Rhopilema esculentum)浮游幼体对褐牙鲆(Paralichthys olivaceus)卵和初孵仔鱼捕食的比较研究

利用实验生态学方法研究不同个体大小的海蜇浮游幼体[平均伞径(2.5±0.1),(4.1±0.2),(11.7±0.5),(21.1±0.6)mm]对不同密度(10,30,50,80ind/L)的褐牙鲆卵[卵径(0.92±0.01)mm]和初孵仔鱼[全长(3.01±0.08)mm]的捕食率,解析海蜇浮游幼体对初孵仔鱼的捕食率随捕食时间(0.5,1,2,3,4,5h)的变化特征。结果表明,各个体组海蜇浮游幼体对卵的捕食率均显著低于对仔鱼的捕食率;它们对卵的捕食率与卵密度和海蜇个体大小的关系不显著,但对仔鱼的捕食率随海蜇个体大小及仔鱼密度的增大而显著升高;伞径21.1mm的个体对仔鱼的捕食率在开始捕食后1h时达到最大值[17.3ind/(predator.h)],此后随捕食时间的延长而逐渐下降。在自然水域中,如果二者发生时空上的匹配关系,海蜇浮游幼体对仔鱼的捕食可能影响褐牙鲆的早期存活及其资源补充量的变动。     

金钱鱼(Scatophagus argus)体色发育及黑斑的形成过程

鱼类的体色图案作为外部形态最显著的特征之一，在动物生存与物种间的交流中起着重要作用。为探究金钱鱼（Scatophagus argus）体色发育及黑斑的形成过程，首先对成年金钱鱼鳍条中色素细胞进行显微观察，采用Olympus体视解剖镜观察了金钱鱼从胚胎色素形成期到仔鱼后期体表色素细胞的种类与分布特征，并跟踪观察了金钱鱼从稚鱼期到成鱼期主要图案黑斑的形成过程，同时对成年金钱鱼的黑斑和非黑斑皮肤进行透射电镜切片观察。结果显示，构成金钱鱼体色共四种色素细胞，分别为：黑色素细胞、黄色素细胞、虹彩色素细胞和红色素细胞，黑色素细胞在胚胎发育阶段最早发生，黄色素细胞和红色素细胞次之，而虹彩色素细胞发生最晚，直到仔鱼后期阶段才观察到。金钱鱼体色仔鱼期（0-24 dph）出现了有趣的变化过程：在0 dph（day post hatching）仔鱼体色较浅到1 dph仔鱼体色明显加深，而2-7 dph仔鱼的躯干部体色出现明显的褪黑现象，在8-24 dph阶段金钱鱼体色又出现了明显的复黑现象，并且在24 dph时通体发黑，在30 dph时虹彩色素细胞在皮肤中的大量出现并形成隐约可见的色素条带，直到幼鱼阶段（50 dph）形成清晰的黑色纵带条纹后不断演变成多个黑斑并在成年金钱鱼中稳定沉积的过程。透射电镜结果发现皮肤中黑色素细胞和虹彩色素细胞数量最多，分布最广。黑斑形成的关键原因是黑素小体和黑色素细胞大量聚集，虹彩色素细胞在其周围大量聚集并形成明显的包围界限。追踪观察了金钱鱼从胚胎色素发育、前期仔鱼、后期仔鱼、稚鱼、幼鱼和成鱼共六个阶段的色素沉积和体色变化过程，为金钱鱼野生群体的年龄鉴定提供了参考，并为进一步的金钱鱼体色改良提供理论指导。     

Induction of phenol-type sulfotransferase and glucuronosyltransferase in channel catfish and mummichog

Conjugation of phenolic xenobiotics and metabolites through sulfation and glucuronidation is an important biotransformation pathway. Sulfotransferases (SULT) are generally considered non-inducible, while some UDP-glucuronosyltransferase (UGT) isoenzymes are co-induced with cytochrome P450-1A by Ah-receptor ligands. To test these assumptions for two fish species, we measured sulfation and glucuronidation of 9-hydroxybenzo[a]pyrene in 3-methylcholanthrene (3-MC) treated channel catfish (Ictalurus punctatus) and in mummichog (Fundulus heteroclitus) from the creosote contaminated Atlantic Wood site in the Elizabeth River, VA. The results show a significant induction of both UGT and SULT activity in 3-MC treated catfish, linked to the expected induction of EROD activity. In mummichog, significant induction of UGT was measured at the contaminated site over the reference site (King's Creek, VA), as well as extremely low SULT activities at both sites. Western blots, using a polyclonal antibody for catfish phenol-type SULT, confirmed the absence of phenol-type SULT in mummichog. Residual, though slightly inducible, SULT activity may be attributed to other SULT isoforms.     

Effects of mercury on the feeding behavior of the mummichog, Fundulus heteroclitus from a polluted habitat

Fish living in contaminated environments may exhibit reduced growth which may be due, in part, to the impact of toxicants on prey capture ability. We studied the effects of methylmercury (meHg) and mercuric chloride (Hg) on feeding of Fundulus heteroclitus from Piles Creek, a polluted habitat in Linden, New Jersey. We found that exposure to 0·01 mg/liter of meHg for one week had less impact on the prey capture rate than exposure to a similar concentration of Hg. Exposure to 0·01 mg/liter for two weeks or to 0·02 mg/liter for one week, of either meHg or Hg, produced significant reductions in feeding rate, compared to controls. In general, the fish were affected to a greater degree by exposure to Hg than to meHg. This is the reverse of the usual order of toxicity, but is in keeping with previous observations on this population, in which we have noted that gametes and embryos had developed tolerance to meHg, but were more susceptible to Hg.     

EROD activities of liver in Mugil so-iuy exposed to benzo (a)pyrene, pyrene and their mixture

Abstract-The effects on hepatic EROD (7-ethoxyresorufin O-deethylase) in Mugil so-iuy exposedto benzo(a)pyrene (BaP), pyrene and their mixtures of equal concentration were investigated, at con-centrations of 0.1, 1.0, 5.0, 10.0, 50.0 μg/dm~3, in experimental condition. Time-effects and dose-response of the biochemical indexs were observed. The results showed that the hepatic EROD activitieswere induced by the exposure of BaP, pyrene and their mixtures at high concentration. Dose-responseconnections were that the hepatic EROD activities were elevated with increasing concentration of the pol-lutants. The combined effect of BaP and pyrene at 1:1 concentration ratio on hepatic EROD activity wasantagonism.     

海水仔稚鱼早期阶段的脂类营养研究概况

简要论述了海水仔稚鱼不同早期阶段的脂类营养 ,包括卵囊的脂类营养 ,胚胎和仔鱼发育期间的脂类代谢 ,仔鱼饥饿期间的脂类 ,轮虫脂类营养的改善 ,卤虫脂类营养的改善 ,以及海水仔稚鱼开口饵料的脂类营养等     

Estrogenic effect of dioxin-like aryl hydrocarbon receptor (AhR) agonist (PCB congener 126) in salmon hepatocytes

Previous studies have shown that aryl hydrocarbon receptor (AhR) agonists possess anti-estrogenic activities and several mechanisms have been proposed to explain the interactions between AhR and estrogen receptor (ER) signalling pathways. In the present study, we show that 3,3'4,4'5-pentachlorobiphenyl (PCB126 - a dioxin-like AhR agonist) produced estrogenic responses in the absence of ER agonist, in fish in vitro system. We exposed salmon primary hepatocytes to PCB126 (1, 10 and 50 pM) and the ER agonist nonylphenol (NP; 5 and 10 microM) singly and also in combination. Vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) levels were analysed by semi-quantitative ELISA. We observed that the protein levels of Vtg and Zr-proteins were significantly induced in a concentration-specific manner in cells treated with PCB126 and NP, singly or in combination. In general, these results show a novel aspect of dioxin-like PCB effect not previously demonstrated in fish system.     

海蜇(Rhopilema esculenta)浮浪幼虫及早期螅状体摄食行为的研究

采用对比实验与显微观察的方法,对海蜇浮浪幼虫及早期螅状体的摄食行为进行了研究。结果表明,海蜇浮浪幼虫期不摄食外界食物;浮浪幼虫附着变态发育成海蜇螅状体1d后,开始摄食其它浮浪幼虫,螅状体发育到第3天摄食能力迅速增强,能够捕食卤虫无节幼体。海蜇早期螅状体的捕食实验结果表明,早期螅状体开始捕食主要受两方面影响,一方面是口的发育,另一方面是刺细胞的发育。     

The effect of long-term depuration on phase I and phase II biotransformation in mullets (Mugil cephalus) chronically exposed to pollutants in River Douro Estuary, Portugal

Pollutants such polycyclic aromatic hydrocarbons (PAHs) are released into the environment by urban communities and industries and the enzymes that catalyse the biotransformation of pollutants play a key role regarding the accumulation of these compounds in fish species inhabiting these areas. In this study the relationship between phase I (EROD activity) and phase II (GST activity) and PAH metabolites was measured in grey mullet (Mugil cephalus) after capture in the Douro estuary, and after long-term depuration in an unpolluted laboratory environment. The results showed a significant decrease in EROD activity after 1 month and in bile metabolites after 4 months in captivity, with both maintaining reduced levels at 4 and 8 months depuration. Liver GST activity did not showed significant changes. This study provides evidence that Douro estuary waters contain bioavailable PAHs that can be associated with the induction of cytochrome P450, and that mullets have the ability to metabolise and eliminate PAHs.     

水母伞径和饵料密度对海月水母(Aurelia sp.1)捕食率的影响

海月水母在全球范围内大量暴发,严重危害海洋生态系统的健康以及鱼类资源的可持续利用。本文以卤虫无节幼体为饵料,初步研究了水母伞径和饵料密度对海月水母个体捕食率的影响以及海月水母的昼夜捕食节律。研究结果表明,海月水母的伞径对其捕食率的影响极显著(P<0.01),在1—8cm范围内,随着伞径的增加个体捕食率线性增加,两者之间的关系式为y=0.044x+0.125(R2=0.9036)。饵料密度对海月水母捕食率的影响显著(P<0.05),在0.25—26.11mgC/L范围内,随饵料密度的增加个体捕食率呈线性增加趋势,关系式为y=0.508x(R2=0.958)。在饵料充足时,海月水母没有昼夜捕食节律。在高饵料密度下海月水母出现过剩捕食,但是捕食行为依然活跃。上述结果既为量化评估海月水母对浮游动物群体的捕食压力提供了基础参数,同时水母过剩捕食行为说明依据生物量和代谢率的推算结果可能低估自然海域水母队浮游动物的捕食压力。     

Bioenhancement of cadmium transfer along a multi-level food chain

Previous studies have shown that metal partitioned to a subcellular compartment containing trophically available metal (TAM) is readily available to predators and may be enhanced by increased binding of metal to heat-stable proteins (HSP - e.g., metallothioneins). The aim of the current investigation was to determine the influence of TAM on the trophic transfer of Cd along an experimental, three-level food chain: Artemia franciscana (brine shrimp)-->Palaemonetes pugio (grass shrimp)-->Fundulus heteroclitus (mummichog). P. pugio were fed for 7 days on A. franciscana exposed to Cd in solution (including (109)Cd as radiotracer) and subjected to subcellular fractionation or fed to F. heteroclitus. An HSP-driven increase in the percentage of Cd associated with TAM (TAM-Cd%) in A. franciscana exposed to 1 muM Cd resulted in a bioenhancement (i.e., a greater than linear increase with respect to A. franciscana exposure) of Cd trophic transfer to P. pugio. Increased dietary Cd exposure did not affect TAM-Cd% in P. pugio nor trophic transfer to F. heteroclitus.