Validation of an enzyme linked immunosorbent assay (elisa) for cyprinus carpio l. vitellogenin,as a biomarker of reproductive disorders

Vitellogenin is a female sex-specific protein, and a validated and suitable method for its assay can be applied as a biomarker of reproductive disorders in male and female aquatic animals. Therefore, the present study was designed to validate an ELISA for measuring plasma vitellogenin in wild carp ( Cyprinus carpio L.) living in the Lake of Trasimeno (Umbria, Italy); plasma samples were taken during pre-spawning, spawning, and post-spawning periods; in addition to vitellogenin, in both male and female carps, plasma changes of estradiol-17 g were monitored together with those of estrogen receptor density in the liver. In females, VTG showed high seasonality, reaching the highest levels in March during the pre-spawning period; the VTG levels correlated with those of estradiol-17 g ( E 2 ), and with the changes of gonadosomatic index (GSI), while a non parallel trend was found in the liver estrogen receptor (ER) density. In forty percent of males, VTG was found to be present in the plasma and changes of ER density in the liver were observed. The data here reported suggest that the common carp can be a useful sentinel species for biomonitoring studies of environmental estrogens, and of their effects on its reproductive biology.     

Estrogenic Effects of Effluents from Sewage Treatment Works

The occurrence of hermaphrodite fish in the lagoons of sewage treatment works led us to hypothesize that sewage effluent might contain a substance, or substances, estrogenic to fish. to test this hypothesis, we placed cages containing rainbow trout in the effluent from sewage-treatment works, and one to three weeks later measured the vitellogenin concentration in the plasma of the fish. Vitellogenin is a protein synthesized by the liver of oviparous fish in response to estradiol stimulation; it is then conveyed by the blood to the ovary, where it is sequestered by oocytes to form the yolk. Thus, the presence of vitellogenin in the plasma is indicative of estrogenic stimulation of the liver. an initial study, at a sewage-treatment works, showed that plasma vitellogenin concentrations rose rapidly and very markedly (over 1000-fold in three weeks) when trout were maintained in the effluent. an extensive nationwide survey was then conducted. Results were obtained from fifteen sewage-treatment works distributed throughout England. in all cases, exposure of trout to effluent resulted in a very pronounced increase (500 to 100,000-fold, depending on site) in the plasma vitellogenin concentration. Induction of vitellogenesis was also observed in carp, but to a much lesser extent than in trout.

The identity of the estrogenic substance is unknown. It is suggested that the two most likely possibilities are ethynylestradiol, originating from pharmaceutical use, or alkylphenol-ethoxylates (APE), originating from the biodegradation of surfactants and detergents during sewage treatment.

Laboratory studies on the potency of ethynylestradiol demonstrated that levels as low as 1 to 10 ng 1^-1 could generate the response shown by the caged fish and that positive responses may arise at 0.1 to 0.5 ng 1^-1. Further work is in progress on the potency of APE.     

Seasonal cycle of gonadal development and serum levels of vitellogenin of the red porgy, Pagrus pagrus (Teleostei: Sparidae)

Histological examination of gonads of female and male red porgy, Pagrus pagrus, reared in captivity, was carried out in order to describe the main gonadal changes related to gametogenesis and the seasonal changes of environmental factors. The gonadosomatic index (GSI) and the concentration of serum vitellogenin (VTG) were also determined. The frequency distribution of gonad development stages and the GSI and vitellogenin concentration during the annual cycle indicated the separation of the female and male reproductive cycles into three main periods. The autumn period when gametogenesis begins (October-November), the period of exogenous vitellogenesis (December-March) or spermiation (December-March), and the spawning season (March-May). The spawning period coincided with an increase in temperature (15-19°C) and daylength. Serum levels of vitellogenin rose significantly in January, reached a peak at the beginning of the spawning period (March, 405.5 µg ml^-1) and remained high until the end of the spawning period (May).     

全氟辛烷磺酸(PFOS)对斑马鱼血浆和组织匀浆中卵黄蛋白原含量的影响

为了研究低剂量的全氟辛烷磺酸(perfluorooctane sulfonate,PFOS)对水生生物的内分泌干扰效应和作用机制,考察了PFOS对斑马鱼(Brachydanio rerio)血浆和组织匀浆中卵黄蛋白原(vitellogenin,VTG)含量的影响。将雄性和雌性斑马鱼分别暴露于0.1、1、10和100μg·L-1的PFOS中进行21d毒性实验,染毒结束后分别检测雄鱼和雌鱼的血浆、头尾组织匀浆液和全鱼匀浆液中的VTG含量。结果显示:(1)PFOS暴露可引起斑马鱼血浆、全鱼和头尾匀浆液中VTG含量的升高,VTG含量的排序为雌鱼(血浆>>全鱼匀浆>头尾匀浆)>>雄鱼(血浆>全鱼匀浆>>头尾匀浆);(2)PFOS暴露所引起的雄鱼血浆和头尾匀浆中VTG含量的升高与剂量呈负相关关系,暴露浓度为0.1μg·L-1时与对照组相比有显著性差异(p<0.05);(3)雌鱼血浆和头尾匀浆中VTG含量的升高与剂量呈倒U型曲线关系,暴露浓度为10μg·L-1时与对照组相比有显著性差异(p<0.05);(4)雄鱼和雌鱼的全鱼匀浆液中的VTG含量与对照组相比均无显著性差异。研究结果表明,PFOS暴露对斑马鱼的内分泌干扰作用明显,其毒性作用机制可能是类雌激素效应,血液和头尾匀浆液中VTG含量能够作为PFOS内分泌干扰效应评价的敏感生物标志物,但响应曲线可能因性别和组织部位的不同有所差异。     

双酚AF对雄性斑马鱼卵黄蛋白原水平与芳香化酶基因表达的影响

双酚AF(4,4'-六氟-2-二酚,BPAF)对生物有机体具有内分泌干扰作用。为研究低剂量BPAF对水生生物的效应,本研究选择成年雄性斑马鱼为研究对象,考察了0.005、0.05和0.5 mg·L~(-1)3种浓度BAPF暴露30 d对血浆中卵黄蛋白原(VTG)含量、2种卵黄蛋白原基因(vtg~(-1)和vtg-3)表达和2种芳香酶基因(cyp 19a与cyp 19b)表达的影响。结果表明:在0.005 mg·L~(-1)浓度暴露30 d后,血浆中VTG含量显著升高,随着暴露浓度的升高,促进作用不显著;BPAF暴露对不同组织中的4种基因存在不同的影响,0.005 mg·L~(-1)BPAF暴露可诱导脑部cyp19b、肝脏中cyp19a和性腺中vtg~(-1)、vtg-3和cyp19b基因表达;0.5 mg·L~(-1)BPAF暴露可导致肝脏中vtg~(-1)、vtg-3、性腺中cyp19a等基因显著上调。实验结果表明,BPAF具有雌性激素样效应,可诱导雄性斑马鱼体内部分组织卵黄蛋白原基因和芳香酶基因的表达。BPAF可引起斑马鱼血浆中的VTG含量的上升,从而干扰由VTG所参与的下丘脑-垂体-性腺轴与免疫系统的正常生理过程。     

Phosphorus in Sediments of Lake Edku, Egypt. The Influence of Chemical and Grain Size Parameters

Lake Edku is one of the Nile Delta shallow brackish water coastal lagoons with communications with the southeastern Mediterranean.

Sediment samples were recovered from the top 10 cm of the lake bottom to study principally their total, inorganic and organic phosphorus. Related sediment parameters such as particle size, organic carbon, carbonate and iron contents were also studied in detail.

It is shown that the lake sediments are generally a sand-silt-clay mud enriched with carbonate (11.4-42%), organic carbon (0.9-11.4%) and iron (1.6-5.6%).

The total phosphorus content in these sediments is 503-1329 μg/g, inorganic phosphorus, 468-954 μg/g and organic phosphorus, 35-526 μg/g.     

烷基酚类化合物的联合雌激素效应

利用鲫鱼卵黄蛋白原诱导作用的雌激素效应研究烷基酚类化合物的联合雌激素效应,通过对鲫鱼血浆卵黄蛋白原含量的相对变化和化合物暴露浓度的非线性回归分析得出17β-雌二醇(E2)、辛基苯酚(PP)、壬基苯酚(p353-NP)及其等毒性同定比例混合物雌激素效应的剂量-反应关系,应用相加作用数学模型根据单个化合物的剂量反应关系可预测混合物效应,各化合物非线性回归分析结果均以Weibull函数拟合效果最好,决定系数R2分别为0.93,0.94,0.96和0.81,半效应浓度值EC50及其95%置信区间分别为0.092(0.080-0.107),63.89(53.90-71.26),152.61(140.11-168.38),11.11(8.54-14.00)μg·l-1;在各个浓度范围内,得出的混合物效应与通过浓度相加或反应相加模型计算得出的混合物效应有很好的一致性,而简单效应相加法得出的结果误差较大;表明了3种雌激素化合物呈现相加作用方式,在低于单个化合物的有效作用浓度下也会产生显著的混合物效应,效应大小取决于化合物的作用性质、暴露量和质量比例.     

Seasonal ovarian cycle in freshwater teleost, Labeo rohita (Ham.) in Tarai region of Uttaranchal

Seasonal ovarian cycle, based on histophysiological changes, and correlative variations in liver were studied in an Indian major carp, Labeo rohita reared in captivity under ambient environmental conditions in Tarai region of Uttaranchal. The oocyte features used for marking ovarian cycle included nuclear-ooplasmic ratio, yolk vesicle incorporation into ooplasm, number and size of nucleolus and differentiation of follicular layer. Based on these features the ovarian cycle in L. rohita was divided into five phases i.e. resting, preparatory, pre-spawning, spawning and post-spawning/regression. The resting and preparatory phases were characterized by the dominating presence of chromatin-nucleolar and yolk-vesicle stage oocytes. The pre-spawning phase ovary was marked by the prevalence of yolk globule stage oocytes having cortical alveoli and differentiating follicular wall. The oocytes containing densely packed yolk-granules and large masses of yolk vesicles and also exhibiting germinal vesicle movement (GVM) signified the ovary of spawning phase. The ovary in regression phase contained pre-ovulatory atretic oocytes and significant number of oogonia. The histophysiological changes in liver showing augmented biosynthetic activity during preparatory and pre-spawning phases seemed well correlated with the gonadal development. The increasing water temperature and day-length apparently was favourable for the gonadal development in L. rohita.     

壬基酚和双酚A对雄性斑马鱼(Danio rerio)卵黄蛋白原mRNA的诱导效应

为了对野外环境中内分泌干扰物更快速有效地进行检测,运用逆转录-聚合酶链反应(RT-PCR)技术检测了17β-雌二醇(E2)、壬基酚(NP)和双酚A(BPA)对雄性成体斑马鱼肝脏合成卵黄蛋白原(VTG)mRNA的诱导作用,并初步探讨了VTGmRNA体内代谢动力学模式.结果表明,随着暴露浓度的升高(E2:20、100、250、500ng·L-1;NP:20、100、250、500μg·L-1;BPA:50、100、250、500、1000μg·L-1),VTGmRNA表达量均显著增加,VTGmRNA表达量与暴露浓度具有明显的剂量-效应关系.在10d的暴露期内,随着暴露时间的延长,VTGmRNA表达量均显著增加,VTGmRNA表达量与暴露时间呈明显的时间-效应关系;停止暴露后,VTGmRNA表达量迅速下降,E2诱导的表达消失得最快(4d),而NP、BPA诱导的表达持续时间相对较长(6d).     

Heavy Metal Concentrations in Sea Urchin Tissues From Egypt, Ireland and United Kingdom

Sea urchins (Paracentrotus lividus) were collected from the Mediterranean coast off Alexandria, Egypt and the Atlantic coast of Ireland to the west of Galway. Samples of another urchin species, Psammechinus miliaris, were collected from the entrance to Southampton Water, U.K. Both the Alexandria coast and Southampton Water receive domestic and industrial waste water inputs whilst the western Irish coast is relatively unpolluted.

Sampled animals were dissected to separate coelomic fluids, Aristotle's lantern, gonads and tissue (digestive tract plus connective tissue). the concentrations of heavy metals (cadmium, copper, lead, nickel and zinc) in the different parts were measured using flame atomic absorption spectroscopy.

Many levels of heavy metals in the different parts were similar in specimens taken across the wide range of sites and the two species. A notable exception was the high level of copper (33 μg g^-1 dry wt.) and zinc 328 μg g^-1- dry wt.) in urchin tissue from Southampton Water.

The metal concentrations in the gonads of Paracentrotus lividus are of particular interest because of human consumption of this species. the highest levels of copper (3.3-5.2 g^-1- dry wt.) and zinc (74-181 μ g^-1- dry wt.) dry wt.) in gonads were found in the samples from Egypt. Data from this study are compared with other results reported from the Mediterranean.

A simple, short term, elevated water column copper uptake experiment was undertaken with Paracentrotus lividus which showed an increase in gonad concentrations of this element.     

Application of High-Volume Fly Ash Concrete to Marine Structures

A new material with high content of fly ash named 'Ashcrete' has been developed. to examine the applicability of Ashcrete to marine structures, strength characteristics, resistance to sea water attack, and safety in the marine environment have been studied. the following results have been obtained:

1) the strength characteristics of high-volume fly ash concrete depend upon the type and proportions of chemical activators as well as curing conditions.

2) Ashcrete using sodium chloride (NaCI) as a chemical activator shows high initial strength and good strength development with age.

3) Ashcrete containing NaCI activator shows good resistance to sea water from the viewpoint of its strength characteristics, volume changes and microstructurcs.

4) Since 1980 many types of large-scale artificial reefs made of Ashcrete have been installed in the sea. As a result of long-term studies and underwater observations, the Japanese Government has demonstrated the durability of the material, safety in the marine environment and attractiveness to fish.

5) It is therefore proposed that Ashcrete might be used in constructing large-scale sea mounts in deep water for the purpose of generating upwelling.     

Total and “Selective” Extraction Methods for Trace Metals in Marine Sediment Reference Samples (Mess-1, NBS 1646)

Trace metals (Cd, Cr, Cu, Mn, Pb and Zn) and Fe were analyzed in two sediment reference samples (NBS 1646, MESS-1) with AAS fitted with graphite furnace, using five different (three total and two selective) extraction procedures.

The comparison of the total digestion methods (hot strong acids) with reference materials gave good results for almost all of them, and the “teflon bomb” was preferred for its rapidity and ease of operation. Some non-referenced data on total and organic carbon, total nitrogen and minerals are given. The two selective leaching extractions (nitric and cold hydrochloric) were also compared with reference values.

The use of certified reference materials (CRM) for environmental chemistry is recommended, together with the determination of organic matter and fundamental mineralogical composition.     

The Brown Trout Population at Loch Fleet Eight Years After Liming

At the start of the Loch Fleet Project in 1984, the Loch and the upper 7 km of its efferent stream were found to be devoid of trout (Salmo trutta) as a result of acidification. Following the liming treatments applied to the catchment, from 1986 the formerly toxic water quality conditions (pH ∼ 4.5, calcium ∼ 1 mgl^-1, elevated aluminium and heavy metal levels) were eliminated, and trout were reintroduced on two occasions, in 1987 and 1988. A total of 520 fish were stocked, at a combined density equivalent to 5.5 kg ha^-1. Surveys of the loch and stream populations were carried out annually until 1993 to monitor their development, using a range of techniques, including electrofishing, gill-netting, seine-netting, spawner trapping and mark-release recapture methods. Length and scale- analysis were used to investigate fish growth.

The trout population in Loch Fleet expanded rapidly as a result of natural spawning in the loch's main feeder stream, augmented by the use of an artificial spawning bed which was constructed at the loch outlet in 1990. in mid-1983 the stock density, estimated by mark-recapture census methods, had increased to 24.9 kg ha^-1. Poor recruitment in the years 1991-93, however, reduced the rate of expansion and resulted in a population comprising mainly older individuals. the poor recruitment in these years was not fully explained but was not caused by water quality and was most likely a result of fry washout by spring spates.

Fish growth rates were high initially and were estimated on the basis of the Elliott trout growth model to be optimal for the prevailing water temperature regime of the loch. By 1991, growth rates had fallen, probably as a result of competition for food, but showed signs of recovery towards the end of the study period in 1993, following the period of lower population densities of young fish.

Trout rapidly repopulated the loch's outlet stream after 1987 but have remained sparse and have shown no signs of spawning within most of the stream. Water analyses have shown that the liming of the Loch Fleet catchment has minimal impact on downstream waters when flows are high, so that potentially toxic acid episodes have not been prevented.     

17β-雌二醇与1-萘酚对雄性罗非鱼（GIFT Oreochromis niloticus）雌激素效应的比较

在实验室条件下,研究了暴露于不同质量浓度1-萘酚（0.064、0.8、2 mg.L-1）与17β-雌二醇（1、10、100μg.L-1）下,对雄性罗非鱼（GIFT Oreochromis niloticus）性腺系数、血清雌二醇质量浓度的影响及血清中卵黄蛋白原的诱导效应,以期对它们的环境雌激素效应有所了解。研究结果显示：暴露于1-萘酚环境下30 d后,雄性罗非鱼的性腺系数有所降低,当质量浓度为2 mg.L-1时,与对照组相比,差异显著（p〈0.05）,雄性罗非鱼血清中雌二醇的量,此时亦达到最大值,为209.3 pg.mL-1,与对照组相比,差异显著（p〈0.05）;但是,血清中卵黄蛋白原并无变化。暴露于17β-雌二醇环境中的雄性罗非鱼,30 d后,其性腺系数有所降低,当质量浓度为10、100μg.L-1时,与对照组相比,差异显著（p〈0.05）;血清中的雌二醇也随着暴露质量浓度的增多,逐渐增多,当质量浓度为100μg.L-1时,差异极显著（p〈0.01）;血清中卵黄蛋白原的质量浓度明显高于对照组,与暴露质量浓度之间呈剂量效应关系。结果显示,17β-雌二醇具有很强的雌激素效应,会使罗非鱼性腺系数下降,诱导血清中雌二醇、卵黄蛋白原的生成;而1-萘酚会使罗非鱼性腺系数下降,诱导血清雌二醇的生成,但是并不诱导卵黄蛋白原的生成,所以推测其雌激素效应可能较弱,需要进一步研究。     

城市污水处理厂进出水中类雌激素暴露影响评价

以稀有鮈鲫（Gobiocypris rarus）为模型动物,利用卵黄蛋白原（VTG）和生长发育指标作为类雌激素污染的生物标志物,评价了沙湖污水处理厂进出水中类雌激素暴露对稀有鮈鲫仔鱼的影响.结果表明：暴露21d后,未稀释进水和一级处理出水对稀有鮈鲫仔鱼VTG有显著诱导,各级处理水对稀有鮈鲫的生长发育也造成了一定的影响,在进水和一级处理出水暴露组中有死亡现象发生.以17β-雌二醇（E2）为阳性对照,对稀有鮈鲫VTG的诱导作标准曲线,计算进水以及各级出水的类雌激素E2的活性当量,发现污水处理厂一级和二级处理工艺对污水的类雌激素活性的去除率分别为47%和95%以上.表明在现有污水处理工艺中,二级处理工艺可去除大部分的类雌激素,而一级处理工艺却不能有效去除类雌激素.     

Endosulfan induced changes in phospholipids in the freshwater female catfish, Heteropneustes fossilis (Bloch)

The catfish, H. fossilis were exposed to endosulfan for 30 days at sub-lethal concentration (0.002 ppm) during different phases of its annual reproductive cycle. Its impact on total (TP) and different phospholipids- phosphatidylcholine (PC), phosphatidylserine (PS), phosphatdylinositol (Pl) and phosphatidylethanolamine (PE) were measured in liver plasma and ovary. On pesticide exposure, during preparatory phase, the hepatic TP PC and PE were declined. The plasma levels of TP, PC and PS were declined with the elevation of PE whereas in ovary only PC was lowered after endosulfan exposure. During pre-spawning phase, the hepatic TP, PC and PE declined in liver plasma and ovary after endosulfan exposure. During spawning phase, only plasma and ovarian phospholipids showed decrease in their levels following endosulfan exposure. In the post-spawning phase, endosulfan elevated the levels of TP, PC and PS in ovary but had no effect on their levels in liver and plasma. During resting phase, the TP, PC and Pl were found to be decreasing its levels. Thus it appears that this pesticide interfere with phospholipids metabolism during annual reproductive cycle of this species.     

Changes in tissue cholesterol and serum cortisol level during four reproductive phases of the male freshwater fish, Notopterus notopterus

The tissue cholesterol and serum cortisol changes during four reproductive phases has been studied in the freshwater fish Notopterus notopterus of male sex during four phases, preparatory (January - March), prespawning (April - May), spawning (August - September) and post spawning (November - December). The cholesterol content of the testis and liver increased during preparatory phase and maximum content of cholesterol was during spawning phase. The serum cortisol estimated by radioimmunoassay (RIA) technique which indicated that the level of the hormone was high during pre-spawning and spawning phases compared to other two phases. The increase in cholesterol during preparatory and spawning phases may be because of increase in cortisol synthesis needed for testicular growth and spermetogenesis during these phases. The gonadosomatic index (GSI) also increased during these phases.     

A Review of Waste Tyre Utilisation in the Marine Environment

Waste tyres pose a major disposal problem on land creating a fire hazard and, in warmer climates, providing breeding pools for mosquitoes. the void space in tyres makes them unsuitable for land burial. Schemes to use shredded tyres for road bases and asphalt filler are being pursued in the USA. Tyre combustion for electricity production is being investigated in the UK.

The widespread availability and durability of tyres has led to their use in the marine environment for breakwaters/coastal defence structures and as artificial reefs for promoting fisheries.

Tyres have a low density and have been used in floating breakwaters. Schemes have been proposed to protect and strengthen shorelines with tyre structures.

The void space in tyres facilitates the construction of artificial reefs to attract fish. the most intensive use is in the south west Pacific and Australia. Tyre surfaces are colonised by algae and a wide range of faunal species, including corals and shellfish. the wide acceptance of tyres as a suitable reef construction material appears to be based largely on these observations. Experience of initial poor deployment practices in the USA led to tyres washing ashore after storms and resulted in the banning or restriction of their use in coastal states of the USA. A review of the scientific literature has yielded limited information on the environmental impact of tyres and in particular the leaching of heavy metals and organic compounds from tyres into sea water.

Preliminary results of tyre dust/sea water leaching studies are presented. These identify zinc as the major leachate (totalling 10mg/tyre after 3 months). Diluted leachates have not shown significant effects of the growth of the phytoplankton Phaeodactylum and Isocrysis.

Further work to characterise the sea water leaching of tyre compounds is recommended.     

Holly (Ilex Aquifolium L.) As Zinc and Cadmium Accumulative Indicator in Biogeochemical Prospecting

The results of previous investigations in mineralized and sterile areas of Tuscany (Italy) appeared to point out the special aptitude of holly (Ilex aquifolium L.) in concentrating cadmium and zinc. in the present paper this behaviour has been verified in a mineralized area of Lombardy (Northern Italy).

Samples of leaves, twigs and barks from holly trees were collected, and metal contents (Zn, Cd, and Pb) have been determined by differential pulse anodic stripping voltammetry (DPASV). for comparison, some samples of wood anemone (Anemone nemorosa L.) and soil were also analyzed.

The data obtained confirm a preferential cadmium and zinc accumulation by holly (up to 16 and 1400 μg g^-1 dry weight, respectively) also depending on different vegetal organs and vegetative stages of plants. Highest contents of lead have been recovered in the outer portion of bark.     

Book Reviews

METHODS FOR ASSESSING THE EFFECTS OF MIXTURES OF CHEMICALS-Report of IPCS Joint Symposium 6, SCOPE 30 and SGOMSEC 3 edited by: V. B. Vouk, G. C. Butler, A. C. Upton, D. V. Parke, S. C. Asher published by: J. Wiley and Sons, Chichester price; £99.00 ISBN 0 471 911232

PHEROMONES OF SOCIAL BEES, by John B. Free, London: Chapman & Hall 1987. £17.50

THE CONSERVATION OF ECOSYSTEMS AND SPECIES, by Gareth E. Jones, Croom Helm (1987), ISBN 0-7099-1463-6, 277 pp. £27.50 hbk; pbk-not available