The effects of silver nanoparticles on oyster embryos

Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48 h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48 h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.     

Linkages between cellular biomarker responses and reproductive success in oysters--Crassostrea virginica

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May-June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.     

Linkages between cellular biomarker responses and reproductive success in oysters – Crassostrea virginica

The purpose of these studies was to evaluate if there were relationships between lysosomal destabilization or glutathione concentrations and gamete viability of oysters, Crassostrea virginica. Oysters were collected from field sites during the peak spawning period (May–June) during 2001 and 2002. Lysosomal destabilization rates and glutathione concentrations of hepatopancreas tissues (e.g. digestive gland) were determined. Eggs and sperm from the same adults were also used to conduct embryo development assays with reference seawater collected from a clean site, site water, and also a range of Cd concentrations (the Cd exposures were used to determine if there were differences in susceptibility to pollutants). Baseline embryo development success (e.g. percent normal development when the assays were conducted with reference seawater or site water) was related to lysosomal destabilization, but not glutathione status. However, the susceptibility of embryos to metal toxicity was related to glutathione status, i.e., sensitivity to Cd exposures increased with decreasing glutathione levels. These studies support the hypotheses that there are linkages between embryo development success and susceptibility to pollutant stress and cellular biomarker responses. These kinds of effects on reproductive success could lead to subtle but significant long-term effects on recruitment and viability of oyster populations.     

菲、芘、苯并(a)芘单一暴露及分别与α-萘黄酮(ANF)联合暴露对海水青鳉(Oryzias melastigma)胚胎发育毒性效应的比较研究

为了解和探讨3~5环PAHs对海水鱼类胚胎发育的毒性及作用方式,比较研究了菲(phenanthrene,Phe)、芘(pyrene,Py)、苯并(a)芘(benzo(a)pyrene,BaP)单一暴露和三者各自与α-萘黄酮(α-naphthoflavone,ANF)联合暴露对海水青鳉(marine medaka, Oryzias melastigma)胚胎发育的毒性效应。胚胎体内EROD活性、发育畸形、孵化率和心律等毒性指标被测定,结果显示:Phe,Py和BaP对海水青鳉胚胎体内EROD活性的诱导能力大小为BaP>Py>Phe,各化合物对EROD诱导与发育畸形之间的关系较为复杂,除Phe所引起的EROD诱导与畸形指数之间呈显著相关(r=0.95,p=0.015)外,Py和BaP均无相关性;在100 μg/dm3 ANF影响下,CYP1A活性诱导被抑制,但胚胎发育的畸形指数被显著提高,ANF分别与Phe,Py和BaP的联合暴露对胚胎发育呈潜在的协同作用。本文研究初步表明,3~5环PAHs化合物对海水青鳉胚胎发育的毒性作用方式可能不同;CYP1A活性抑制在PAHs混合物对海水青鳉胚胎发育的毒性作用过程中未起到缓解毒性的作用,CYP1A抑制剂与PAH型CYP1A诱导剂的混合物对鱼类胚胎发育具有潜在的协同毒性作用,现有的PAHs混合物毒性风险评价方法可能低估了实际环境中PAHs的风险;海水青鳉早期生活阶段的心脏发育对PAHs混合物暴露较为敏感,可推荐其作为生物标志物指示PAHs或溢油污染。     

Individual and interactive effects of environmental stress on the embryonic development of the Pacific oyster, Crassostrea gigas. I. The toxicity of copper and silver

Pacific oyster, Crassostrea gigas Thunberg, embryos were exposed to copper and silver salts, alone and in combination, through the completion of embryogenesis and metal effects upon normal embryonic development were monitored. Copper concentrations throughout the test period ranged from 0·0 to 12·0 μg litre^?1 while silver concentrations were between 0·0 and 18·0 μg litre^?1.Administered individually, silver and copper yielded decreasing per cent normal embryonic development with increasing metal concentration. However, both metals exhibited ‘plateaus’ wherein per cent normal embryonic development did not decrease with increasing metal concentration until a particular concentration was attained. A rapid decrease in per cent normal embryonic development occurred with increasing metal concentrations greater than 6·0 μg litre^?1 copper and 11·0 μg litre^?1 silver.The dose-response patterns of copper and silver appear to be similar with copper exhibiting somewhat greater toxicity. At a copper concentration of 10 μg litre^?1 only 50% of the embryos could be considered normally developed, whereas silver concentrations between 16·0 and 18·0 μg litre^?1 yielded 50% normal embryonic development.Response surface methodology indicated that copper and silver interacted additively, with embryonic development affected to a much greater degree by copper than by silver. Optimum development of Crassostrea gigas embryos was greatly influenced by copper concentrations and occurred at all silver concentrations wherein the corresponding copper concentrations were 6·0 μg litre^?1 or less. Above 6·0 μg litre^?1 copper, per cent normal embryonic development was significantly reduced, even at the lowest tested silver concentrations.Abnormal embryos exhibited retarded shell growth, reduced size and extremely erratic swimming behaviour. These sublethal effects probably have a biochemical origin and would limit the organism's capacity to feed properly, escape predation and develop normally, thereby reducing the chances of successful metamorphosis and recruitment into the adult population.     

Global change ecotoxicology: Identification of early life history bottlenecks in marine invertebrates,variable species responses and variable experimental approaches

Climate change is a threat to marine biota because increased atmospheric CO₂ is causing ocean warming, acidification, hypercapnia and decreased carbonate saturation. These stressors have toxic effects on invertebrate development. The persistence and success of populations requires all ontogenetic stages be completed successfully and, due to their sensitivity to environmental stressors, developmental stages may be a population bottleneck in a changing ocean. Global change ecotoxicology is being used to identify the marine invertebrate developmental stages vulnerable to climate change. This overview of research, and the methodologies used, shows that most studies focus on acidification, with few studies on ocean warming, despite a long history of research on developmental thermotolerance. The interactive effects of stressors are poorly studied. Experimental approaches differ among studies. Fertilization in many species exhibits a broad tolerance to warming and/or acidification, although different methodologies confound inter-study comparisons. Early development is susceptible to warming and most calcifying larvae are sensitive to acidification/increased pCO₂. In multistressor studies moderate warming diminishes the negative impact of acidification on calcification in some species. Development of non-calcifying larvae appears resilient to near-future ocean change. Although differences in species sensitivities to ocean change stressors undoubtedly reflect different tolerance levels, inconsistent handling of gametes, embryos and larvae probably influences different research outcomes. Due to the integrative ‘developmental domino effect’, life history responses will be influenced by the ontogenetic stage at which experimental incubations are initiated. Exposure to climate change stressors from early development (fertilization where possible) in multistressor experiments is needed to identify ontogenetic sensitivities and this will be facilitated by more consistent methodologies.     

Toxic chemistry of heavy metals on Isochrysis galbana (parke)in inshore water

Effects of heavy metals on Isochrysis galbana (Parke) were studied in our laboratory. The research focused on toxic effects of metals on algal cells, effects of water-soluble and lipid-soluble organic ligands on metal toxicity and processes of metal uptake by cells. Interactions between two metals and the mechanisms involved were also studied in detail. Finally, some relationships between toxicity and chemical property of metals were revealed.     

Effects of dietary exposure to the pyrethroid pesticide esfenvalerate on medaka (Oryzias latipes)

The pyrethroid insecticide esfenvalerate is widely used on orchard crops throughout California. In the aquatic environment, this compound is likely to accumulate in sediments, food particles and benthic organisms due to its lipophilicity and environmental persistence. This pilot project tested the hypothesis that esfenvalerate is toxic to medaka (Oryzias latipes) when taken up with the diet. For 7 days fish were fed diets, which contained esfenvalerate in three different concentrations (4, 21, 148 mg/kg, measured). Endpoints measured were mortality, fecundity, fertilization and hatching success of embryos, viability of larvae and cellular stress protein (hsp60, hsp70, hsp90) levels. The toxicity of aqueous exposure of medaka to esfenvalerate was also determined. Whereas the 96-h LC50 in the aqueous exposure was <9.4 microg/l, the dietary exposure did not cause mortality. Possible effects of dietary esfenvalerate were seen on fertilization and hatching success and the number of non-viable larvae. Expression of hsp60 and hsp90 showed a dose-dependent response pattern.     

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss)

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid). Male trout were orally exposed for 17 days to 55 microg/kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L. After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.     

The sea-surface microlayer of puget sound: Part I. Toxic effects on fish eggs and larvae

The sea surface is an important habitat for the developmental stages (eggs and larvae) of many fish and invertebrates; it is also a concentration point for anthropogenic contaminants entering the sea. Studies were conducted to determine the extent to which the sea surface of Puget Sound was toxic to the early life history stages of fish. Three urban bays with suspected contamination, a rural reference bay, and a Central Sound site were compared. Surface-dwelling eggs and organisms (zooneuston) were collected with a surface-skimming neuston net and their densities enumerated. Sand sole (Psettichthys melanostictus) embryos were exposed in the field and laboratory to the sea-surface microlayer. To develop a useful year-round approach to monitoring sea-surface toxicity, larval development of anchovies, kelp bass, and sea urchins was also evaluated as an indication of sea-surface microlayer toxicity.During the spawning season (February and March), urban boys in Puget Sound had lower concentrations of sand sole eggs and neustonic organisms on the sea surface than did the rural bayor Central Sound reference sites. Compared to the reference sites, laboratory exposure to surface microlayer samples collected from urban bay sites generally resulted in more chromsomal aberrations in developing sole embryos, reduced hatching success of sole larvae, and reduced growth in trout cell cultures. In situ hatching success of sole eggs was reduced by half or more in urban bays compared to reference sites.Toxicity was associated with visible surface slicks and, in urban bays, increased with increasing surface pressure (dynes cm⁻¹). Results to be reported separately (Part II) indicate that toxicity is strongly correlated with the presence of high concentrations of polycyclic aromatic hydrocarbons and metals in the sea-surface microlayer. The toxicity of SMIC samples was similar when evaluated by sole, anchovy, kelp bass, or sea urchin tests. A sea-surface monitoring program could use sea urchin embryos to evaluate site-specific sea-surface toxicity throughout the year.     

Influence of sample manipulation on contaminant flux and toxicity at the sediment-water interface

Toxicities of sediments from San Diego and San Francisco Bays were compared in laboratory experiments using sea urchin (Strongylocentrotus purpuratus) embryos exposed to pore water and at the sediment-water interface (SWI). Toxicity was consistently greater to embryos exposed at the SWI to intact (unhomogenized) sediment samples relative to homogenized samples. Measurement of selected trace metals indicated considerably greater fluxes of copper, zinc, and cadmium into overlying waters of intact sediment samples. Inhibition of sea urchin embryo development was generally greater in sediment pore waters relative to SWI exposures. Pore water toxicity may have been due to elevated unionized ammonia concentrations in some samples. The results indicate that invertebrate embryos are amenable to SWI exposures, a more ecologically relevant exposure system, and that sediment homogenization may create artifacts in laboratory toxicity experiments.     

Metals in eggs and hatchlings of Dermochelys coriacea (testudines) in Espírito Santo,Brazil and their relation to hatching and emergence success

Certain concentration of metal may influence survival in embryonic stages, reducing the birthrate and hatching emergence. This study aimed to evaluate concentration of metal in eggs and hatchlings of Dermochelys coriacea and correlate metal concentrations in eggs with hatching and emergence success. The samples were collected at Biological Reserve Station, ES, Brazil. At the nests' opening, unhatched eggs and stillborn hatchlings were collected to survey the successful hatching and emergence of D. coriacea. The eggs shell, egg content, and dead hatchlings metal concentrations were compared to hatching and emergence success. A positive correlation was found between the concentration of copper (Cu) in the hatchlings and the success of the emergence (r² = .28, p < .05), and a moderate positive correlation between the concentrations of iron (Fe) and barium (Ba; r² = .44, p < .05) and success hatching. The concentrations of metals in eggs and hatchlings of D. coriacea in the Espírito Santo are below the levels that could be considered harmful to the species. In summary, there is a positive correlation between metals levels and hatching and emergence success.     

Environmental cues and seasonal reproduction in a temperate estuary: a case study of Owenia collaris (Annelida: Polychaeta,Oweniidae)

Coordination of gametogenesis and spawning during restricted breeding seasons increases availability of mates, fertilization rates, and often success of offspring. Orton’s Rule predicts water temperature as the dominant environmental cue for gametogenesis or spawning in temperate invertebrates. Crisp’s Rule predicts that species producing planktotrophic larvae will time their reproduction to ensure optimal nutrition for the larvae. Owenia collaris (Annelida: Oweniidae) is a temperate polychaete that produces planktotrophic larvae that remain in the water column for several weeks. Reproductive phenology and its relationship to the environment were investigated in an estuarine population of O. collaris using field and laboratory studies. Owenia collaris produced mature gametes between March and September each year during the productive season between the spring and fall transitions when day lengths were 12 h or more, alkalinity was 8.1 or higher, and temperature was 11 °C or higher. Gamete production was not related to seasonal changes in salinity or benthic phytoplankton concentrations and gametes were present prior to the spring phytoplankton bloom. In laboratory experiments, production of gametes was influenced somewhat by manipulating day lengths but not adult food. The association with predictable and stable environmental cycles (day length) and a broad spawning season suggest that this species is unlikely to mis-match with larval food availability which would lead to reduced availability of recruits in any given year, supporting Crisp’s Rule. Our data do not support the hypothesis that reproductive timing in this species is driven by access to excess energy required by adults to initiate the production of gametes. Our data also do not support Orton’s premise that temperature is the primary controlling factor of reproduction in this temperate marine invertebrate.     

Effects of ocean warming and acidification on fertilization in the Antarctic echinoid Sterechinus neumayeri across a range of sperm concentrations

The gametes of marine invertebrates are being spawned into an ocean that is simultaneously warming and decreasing in pH. Predicting the potential for interactive effects of these stressors on fertilization is difficult, especially for stenothermal polar invertebrates adapted to fertilization in cold, viscous water and, when decreased sperm availability may be an additional stressor. The impact of increased temperature (2–4 °C above ambient) and decreased pH (0.2–0.4 pH units below ambient) on fertilization in the Antarctic echinoid Sterechinus neumayeri across a range of sperm concentrations was investigated in cross-factorial experiments in context with near future ocean change projections. The high temperature treatment (+4 °C) was also used to assess thermal tolerance. Gametes from multiple males and females in replicate experiments were used to reflect the multiple spawner scenario in nature. For fertilization at low sperm density we tested three hypotheses, 1) increased temperature enhances fertilization success, 2) low pH reduces fertilization and, 3) due to the cold stenothermal physiology of S. neumayeri, temperature would be the more significant stressor. Temperature and sperm levels had a significant effect on fertilization, but decreased pH did not affect fertilization. Warming enhanced fertilization at the lowest sperm concentration tested likely through stimulation of sperm motility and reduced water viscosity. Our results indicate that fertilization in S. neumayeri, even at low sperm levels potentially found in nature, is resilient to near-future ocean warming and acidification.     

In vitro alternatives to whole animal testing. Comparative cytotoxicity studies of divalent metals in established cell lines derived from tropical and temperate water fish species in a neutral red assay

A simple vital dye (neutral red) uptake method for investigation of the acute toxicity of seven divalent metal cations to cell lines derived from the turbot (TF) and bluegill (BF-2) was evaluated. The method was extremely reproducible between replicate experiments on a week to week basis as well as being reproducible between different laboratories. Whilst there were some species-dependent differences in the relative toxicities of the metals there were good correlations between toxicity and the chemical softness (Σp) of the metal ions and also between in vivo and in vitro toxicities. These studies demonstrate that this simple and relatively inexpensive technique carried out in 96-well microplates may be extremely useful for evaluation of structure-toxicity relationships between related groups of compounds.     

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.     

Micronutrient effects on cyanobacterial growth and physiology

Trace metals play crucial roles in the carbon and nitrogen metabolism of cyanobacteria. Physiological responses to metal limitation and toxicity in culture have shown that iron is important for photosynthesis and energy distribution in the cell while both iron and molybdenum are biochemically involved in nitrate reduction and nitrogen fixation. Nitrogen fixation is also relatively sensitive to copper toxicity. Consequently, factors that affect the supply rate, chemical speciation, or the recycling of trace metals can alter patterns of primary productivity and nitrogen metabolism. Overall, three trace metal dependent processes may contribute towards dominance: efficient use of limiting light, nitrogen fixation, and production of extracellular iron binding compounds.     

应用平衡分配模型评价渤海湾和莱州湾沉积物重金属潜在生物毒性

为探讨渤海沉积物中重金属的生物毒性,采集了渤海湾和莱州湾共72个站点的表层沉积物,测定了酸可挥发性硫化物(AVS)、同步萃取金属(SEM)、有机碳(TOC)等参数,应用平衡分配模型(EqP Model)评价了沉积物中重金属的潜在生物毒性。研究显示,有8个站点超过了EqP Model的毒性阈值,显示潜在生物毒性,而61%的站点应无生物毒性。本研究同时测定了沉积物的总含量,应用几种常用的沉积物质量基准进行了评价虽然有部分站点的Cu、Ni、Cr超过了基准值,但与渤海的背景值比较,这些基准值可能并不适用于评价渤海沉积物。研究显示,EqP Model可以更有效的评价沉积物中重金属的活性和生物毒性,尤其针对我国近岸海域的沉积物,TOC和AVS含量偏低而重金属生物毒性偏高的情况可能普遍存在,更应积极考虑应用EqP Model进行重金属生物毒性的评价。     

Seasonal variation of zinc in the barnacle, Balanus balanoides (L.) maintained on a raft in the Menai Strait

A population of the intertidal barnacle, Balanus balanoides (L.) maintained on a raft in the Menai Strait was sampled regularly over a two-year period and pooled fractions analysed for zinc and other trace metals. The seasonal variation of zinc in three fractions (body, other soft tissues and shell) has been followed and related to physiological condition. The body accumulates zinc almost continuously whilst this trace metal accumulates in the ovary only as it develops and is lost in the gametes when these are laid. The newly formed egg-masses therefore may be the more useful sample for indicating zinc pollution.     

Comparison of the effects of water-soluble (EDTA) and particulate (Chelex-100) synthetic ligands on the growth of phytoplankton population in the disphotic zone seawater

To clarify the beneficial roles of naturally occurring organic ligands on the growth of phytoplankton in newly upwelled water, phytoplankton culture experiments using disphotic zone water were conducted to discriminate between the effects of EDTA in the detoxification of certain toxic metal ions and increasing the availability of essential metals. Culture media were prepared by adding EDTA and Chelex-100, separately or in combination, to disphotic zone water samples. Our proposed working hypothesis is that phytoplankton growth can be enhanced by removing toxic metal ions from culture media by Chelex-100 and by detoxification of toxic metal ions or increasing the availability of essential metals by EDTA. A shortening of the lag period and an increase of the specific population growth rate were clearly observed after the addition of Chelex-100; nd EDTA. The effects of EDTA were more considerable than those of Chelex-100; a 17 to 44% in shortening the lag period and a 35 to 56% increase in the growth rate, when comparing the effects of Chelex-100 with those of EDTA. The similar effects of removing toxic metal ion by Chelex-100 as those of detoxification by EDTA suggested that EDTA has a role not only of detoxification but also of increasing the availability of essential metals. The present study suggests that the low productivity in newly upwelled water observed by Barber and Ryther (1969) can be ascribed to both toxic metal ions and a lack of available forms of essential metals because of their low contents of free natural organic ligands.