Partiton-controlled delivery of toxicants: a novel in vivo approach for embryo toxicity testing

In conventional static or semi-static embryo toxicity assays with fish, the nominal concentrations of hydrophobic chemicals are often used to establish the toxic thresholds, which often far exceed the solubility limits of test compounds. Saturators and continuous-flow diluters have been used to provide stable concentrations below solubility but are complex, use large amounts of test substance, and produce large volumes of waste. We present a partition-controlled delivery (PCD) method that maintains the concentrations of chemicals in test solutions at or below solubility limits for extended exposure times. Concentrations are maintained by equilibrium partitioning of test chemicals from a series of poly(dimethylsiloxane) films loaded with a range of concentrations of each chemical. The efficacy of the PCD assay was tested by comparisons with static (no renewal) and semi-static (24-h renewal) embryo-larval toxicity tests. The test species was Japanese medaka (Oryzias latipes) exposed to retene (7-isopropyl-1-methylphenanthrene), a compound causing blue sac disease (BSD) in fish embryos. In the PCD assay, the median effective concentration (EC50) for BSD was 10 microg/L, below retene's solubility of 17 microg/L. In contrast, the nominal EC50 values for the semi-static 24-h and static assays were about 10 (150 microg/L) and 150 times (2500 microg/L) greater than solubility, respectively. The PCD method is a more sensitive and realistic method for assessing toxicity of nonpolar compounds than (semi)-static assays.     

Cell death, stress-responsive transgene activation, and deficits in the olfactory system of larval zebrafish following cadmium exposure

Cadmium (Cd) is a well-described environmental pollutant known to have adverse effects in fish, including behavioral deficits. We have previously reported the development of an in vivo system that utilizes hsp70 gene activation as a measure of acute 3 h cadmium toxicity in whole living transgenic zebrafish larvae carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. Here, we report that activation of this transgene in olfactory epithelium of zebrafish larvae during 96 h sublethal Cd exposure is predictive of cadmium-induced cell death, altered histological and surface organization of the epithelium, and changes in olfactory dependent behavior. The transgene is first activated in the olfactory epithelium at concentrations below those giving rise to significant defects, but exhibits a more robust response following exposure to Cd at concentrations that begin to cause significant cell death, morphological alterations, and behavioral deficits. Further, the data show that Cd-induced olfactory deficits reported previously in juvenile and adult fish can also occur during larval stages of fish development, and that such behavioral deficits are closely associated with cell death and structural alterations in the olfactory epithelium.     

Comparison of species sensitivity distributions derived from interspecies correlation models to distributions used to derive water quality criteria

Species sensitivity distributions (SSD) require a large number of measured toxicity values to define a hazard level protective of multiple species. This investigation comprehensively evaluated the accuracy of SSDs generated from toxicity values predicted from interspecies correlation estimation (ICE) models. ICE models are log-log correlations of multiple chemical toxicity values for a pair of species that allow the toxicity of multiple species to be predicted from a single measured acute toxicity value for a surrogate species. ICE SSDs were generated using four surrogate species (fathead minnow, Pimephales promelas; rainbow trout, Oncorhynchus mykiss; sheepshead minnow, Cyprinodon varigatus; and water flea, Daphnia magna). ICE-based hazard concentrations (HC5s) from the 5th percentile of the log-logistic distribution of toxicity values were compared to HC5s determined from the acute toxicity of 55 chemicals from the United States Environmental Protection Agency Ambient Water Quality Criteria (AWQC). Measured fish and invertebrate acute toxicity data and HC5s from the AWQC data sets were compared to ICE-based HC5s. Surrogate species choice was found to be an important consideration in developing predictive HC5s. These results illustrated that fish predict fish betterthan invertebrates and D. magna predicted invertebrates better than most fish. For example, a mixed model of predicted fish and invertebrates from fathead minnow and D. magna as surrogate species provided predictive relationships with an average factor of 3.0 (+/- 6.7) over 7 orders of toxic magnitude and several chemical classes (HC5(predicted)/HC5(measured)). The application of ICE models is recommended as a valid approach for generating SSDs and hazard concentrations for chemicals with limited toxicity data.     

An environmental estrogen alters reproductive hierarchies, disrupting sexual selection in group-spawning fish

There is global concern regarding the potential impacts of endocrine-disrupting chemicals (EDCs) on the health of wildlife and humans. Exposure to some estrogens, at concentrations found in the environment impairs reproductive function and behavior. However, nearly all work on endocrine disruption has investigated the effects of exposure on individuals and there is an urgent need to understand impacts on populations. Many fish have mating systems with complex social structures and it is not known whether EDCs will exaggerate or buffer the reproductive skews caused by the dominance hierarchies that normally occur for these species. This study investigated the impact of exposure to the pharmaceutical estrogen ethinylestradiol (EE2) on reproductive hierarchies and sexual selection in group-spawning fish. Breeding zebrafish were exposed to environmentally relevant concentrations of EE2, and effects were determined on reproductive output, plasma androgen concentrations (in males), and reproductive success through microsatellite analyses of the offspring. Reproductive hierarchies in breeding colonies of zebrafish were disrupted by exposure to EE2 at a concentration that did not affect the number of eggs produced. The effect was a reduction in the skew in male paternity and increased skew in female maternity. This disruption in the reproductive hierarchy in group spawning fish, if it occurs in the wild, has potentially major implications for population genetic diversity. Reproductive success in male zebrafish was associated with elevated plasma concentrations of the male sex hormone 11-ketotestosterone and this hormone was suppressed in EE2-exposed males.     

Predicting concentrations of organic chemicals in fish by using toxicokinetic models

Quantification of chemical toxicity continues to be generally based on measured external concentrations. Yet, internal chemical concentrations have been suggested to be a more suitable parameter. To better understand the relationship between the external and internal concentrations of chemicals in fish, and to quantify internal concentrations, we compared three toxicokinetic (TK) models with each other and with literature data of measured concentrations of 39 chemicals. Two one-compartment models, together with the physiologically based toxicokinetic (PBTK) model, in which we improved the treatment of lipids, were used to predict concentrations of organic chemicals in two fish species: rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas). All models predicted the measured internal concentrations in fish within 1 order of magnitude for at least 68% of the chemicals. Furthermore, the PBTK model outperformed the one-compartment models with respect to simulating chemical concentrations in the whole body (at least 88% of internal concentrations were predicted within 1 order of magnitude using the PBTK model). All the models can be used to predict concentrations in different fish species without additional experiments. However, further development of TK models is required for polar, ionizable, and easily biotransformed compounds.     

Effect of sodium hypochlorite on zebrafish swimming behavior estimated by fractal dimension analysis

The behavioral responses of zebrafish (Danio rerio) to sublethal concentrations of sodium hypochlorite (NaClO)-based household bleach were quantified in order to index toxicity of the solution. The swimming behavior of zebrafish was captured using nonplanar 3D stereocameras in combination with 3D coordinate computation with perspective correction (3DCCPC) to compute for actual 3D coordinates. Swimming trajectory and velocity were quantified by fractal dimension analysis. The results showed that, under incremental concentrations of NaClO-based aqueous solutions with the maximum of 0.005% v/v, the fractal dimension of swimming velocity trended to increase with the concentration. The fractal dimension of swimming trajectories trended to increase with pH. Hence, the results have proven that the system is a useful tool to indicate behavioral changes, which may be implemented in biomonitoring systems for acute toxicity bioassay.     

Production,characterization, microbial inhibition,and in vivo toxicity of cold atmospheric plasma activated water

Cold atmospheric plasma activated water (PAW) was produced at various Reactive Nitrogen Oxygen Species (RONS) concentrations. PAW was characterized and in vivo toxicological studies were conducted, using the zebrafish embryo acute toxicity test (OECD 236).Danio rerio (zebrafish), were raised in standard egg PAW of various RONS concentrations and their lethality was monitored. The thresholds for no lethality and simultaneously no obvious morphological phenotypes were determined as approximately 30 mg/L H₂O₂ concentration, while for NO₃⁻ concentration, the values were also correlated to the pH of the solution used. The pH-value of 3.85 was also a measurable threshold for the lethality of the embryos. Toxic and non-toxic PAW were used as medium to assess their inhibitory potential for Escherichia coli and Enterococcus faecium. The results indicated that PAW delayed the recovery time or even resulted in no growth of these microorganisms compared to control samples.     

ABAM, a model for bioaccumulation of POPs in birds: validation for adult herring gulls and their eggs in Lake Ontario

An Avian BioAccumulation Model (ABAM) of persistent organic pollutant (POP) uptake and elimination in adult life-stage of birds was validated by simulation of concentrations of DDE, dieldrin, mirex, and HCB in herring gull eggs in Lake Ontario for the years 1985, 1990, and 1992. These chemicals represented a range of whole-body half-lives of 82-265 days in the gull. Dietary intake of POPs by a female gull was simulated by a dynamic bioenergetics model which included dependence on temperature, photoperiod, egg production, and feeding chicks. Concentrations in the two main prey fish of the gull in Lake Ontario were used for POP exposure. Clearance from the female was based on a two compartment toxicokinetic model. Egg concentrations were estimated from egg/whole body female concentration ratios. Simulated concentrations were compared to measured concentrations in gull eggs from 4 different colonies in the northern part of Lake Ontario. Simulations using a diet of 81% fish and 19% uncontaminated food resulted in the best fit with least variance among predicted and measured data. The mean ratio of predicted to measured concentrations in eggs was 1.0 +/- 0.27 among chemicals, years, and colonies for this exposure scenario. This result was in excellent agreement with field assessments of herring gull diet composition in Lake Ontario of 80-82% fish. The ability to perform accurate a priorisimulations for the range of test conditions employed in the validation constituted a rigorous test of the soundness of the model's structure and parameterization. With species-specific adjustments, ABAM can be regarded as a general model for lipophilic POPs bioaccumulation in birds.     

In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish

The polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are used as fragrance ingredients in perfumes, soaps, and household cleaning products. They are known to be ubiquitously present in the aquatic environment, and because of their lipophilic nature, they tend to bioaccumulate in aquatic biota. In surface waters, concentrations between 1 ng/L and 5 microg/L have been found, depending mainly on the proportion of sewage effluents in the water. In fish, under normal environmental conditions, concentrations in the microgram per kilogram fresh weight (fw) range are found. In a previous study we showed that AHTN and HHCB exert mainly antiestrogenic effects on the human estrogen receptor alpha (ERalpha) and ERbeta in an in vitro reporter gene assay. In the current study, we assessed the in vitro antiestrogenic effects of both musks on zebrafish ERs. Antagonism was observed on zfERbeta, and more pronounced on the newly cloned zfERgamma. Using a transgenic zebrafish assay, we studied antiestrogenicity of the musks in vivo. Dose-dependent antagonistic effects were observed at concentrations of 0.1 and 1 microM AHTN and HHCB. GC-MS analysis showed that the musks bioaccumulated in the fish, with internal concentrations (15-150 mg/kg fw) which were roughly 600 times higher than the nominal test doses. To our knowledge, this is the first time that environmental contaminants are shown to be antiestrogenic in an in vivo fish assay that focuses solely on ER-mediated effects. This makes the transgenic zebrafish assay a promising tool for the rapid detection of both estrogenic and antiestrogenic effects of chemicals in fish.     

Microarray analysis reveals a mechanism of phenolic polybrominated diphenylether toxicity in zebrafish

Polybrominated diphenylethers (PBDEs) are ubiquitous in the environment, with the lower brominated congener 2,2',4,4'-tetrabromodiphenylether (BDE47) among the most prevalent. The phenolic PBDE, 6-hydroxy-BDE47 (6-OH-BDE47) is both an important metabolite formed by in vivo metabolism of BDE47 and a natural product produced by marine organisms such as algae. Although this compound has been detected in humans and wildlife, including fish, virtually nothing is known of its in vivo toxicity. Here we report that 6-OH-BDE47 is acutely toxic in developing and adult zebrafish at concentrations in the nanomolar (nM) range. To identify possible mechanisms of toxicity, we used microarray analysis as a diagnostic tool. Zebrafish embryonic fibroblast (PAC2) cells were exposed to 6-OH-BDE47, BDE47, and the methoxylated metabolite 6-MeO-BDE47. These experiments revealed that 6-OH-BDE47 alters the expression of genes involved in proton transport and carbohydrate metabolism. These findings, combined with the acute toxicity, suggested that 6-OH-BDE47 causes disruption of oxidative phosphorylation (OXPHOS).Therefore, we further investigated the effect of 6-OH-BDE47 on OXPHOS in zebrafish mitochondria. Results show unequivocally that this compound is a potent uncoupler of OXPHOS and is an inhibitor of complex II of the electron transport chain. This study provides the first evidence of the in vivo toxicity and an important potential mechanism of toxicity of an environmentally relevant phenolic PBDE of both anthropogenic and natural origin. The results of this study emphasize the need for further investigation on the presence and toxicity of this class of polybrominated compounds.     

Unifying prolonged copper exposure, accumulation, and toxicity from food and water in a marine fish

The link between metal exposure and toxicity is complicated by numerous factors such as exposure route. Here, we exposed a marine fish (juvenile blackhead seabream Acanthopagrus schlegelii schlegelii) to copper either in a commercial fish diet or in seawater. Copper concentrations in intestine/liver were correlated linearly with influx rate, but appeared to be less influenced by uptake pathway (waterborne or dietary exposure). Influx rate best predicted Cu accumulation in the intestine and liver. However, despite being a good predictor of mortality within each pathway, influx rate was not a good predictor of mortality across both exposure pathways, as waterborne Cu caused considerably higher mortality than dietary Cu at a given influx rate. We show that the use of gill Cu accumulation irrespective of the exposure route as a model for observed fish mortality provided a clear relationship between accumulation and toxicity. Investigation of gill Cu accumulation may shed light on the different accumulation strategies from the two exposure pathways. This correlation offers potential for the use of branchial Cu concentration as an indicator of long-term Cu toxicity, allowing for differences in the relative importance of the uptake pathways in different field situations.     

Mechanisms of acute and chronic waterborne nickel toxicity in the freshwater cladoceran, Daphnia magna

We present evidence that Mg2+ antagonism is one mechanism for acute toxicity of waterborne Ni to Daphnia magna. Acutely, adult D. magna were exposed to either control or 694 microg Ni L(-1) as NiSO4 in moderately soft water (45 mg L(-1) as CaCO3; background Ni approximately 1 microg Ni L(-1)) for 48 h without feeding. Chronically, adults were exposed to either control or 131 microg Ni L(-1) for 14 days (fed exposure). These concentrations were approximately 65% and 12%, respectively, of the measured 48-h LC50 (1068 microg Ni L(-1)) for daphnid neonates in this water quality. The clearest effect of Ni exposure was on Mg2+ homeostasis, as whole-body [Mg2+] was significantly decreased both acutely and chronically by 18%. Additionally, unidirectional Mg2+ uptake rate (measured with the stable isotope 26Mg) was significantly decreased both acutely and chronically by 49 and 47%, respectively, strongly suggesting that Ni is toxic to D. magna due at least in part to Mg2+ antagonism. No impact was observed on the whole-body concentrations or unidirectional uptake rates of Ca2+ during either acute or chronic Ni exposure, while only minor effects were seen on Na+ and Cl- balance. No acute toxic effect was seen on respiratory parameters, as both oxygen consumption rate (MO2) and whole-body hemoglobin concentration ([Hb]) were conserved. Chronically, however, Ni impaired respiratory function, as both MO2 and [Hb] were significantly reduced by 31 and 68%, respectively. Acutely, Ni accumulation was substantial, rising to a plateau between 24 and 48 h of approximately 15 microg g(-1) wet weight--an increase of approximately 25-fold over control concentrations. Mechanisms of acute toxicity of Ni in D. magna differ from those in fish; it is likely that such mechanistic differences also exist for other metals.     

基于斑马鱼胚胎急性毒性测试预测食用油毒性可行性研究

探讨利用斑马鱼胚胎预测食用油毒性的可行性。用乙腈和正己烷联合提取采自我国台湾地区的7个不同品质的猪油样本，并用甲醇溶解，然后用斑马鱼胚胎测试提取物的毒性，最后用非靶标高分辨率复合四极杆质谱仪偶联超高液相色谱/质谱系统进一步分析低毒、中毒、高毒代表性样本中的化学组成，结合ChemSpider、CT-link和Vega软件分析预测有毒物质的结构和功能。结果表明，不同品质的猪油样本毒性差异大，高毒性猪油的特征性质荷比信号相对应的化学成分为脂肪酸的氧化物，是样本的潜在有毒成分。斑马鱼胚胎急性毒性测试可准确地预测猪油品质，该方法为评估食用油安全提供了一个新的思路。     

Environmental risk assessment of fluctuating diazinon concentrations in an urban and agricultural catchment using toxicokinetic-toxicodynamic modeling

Temporally resolved environmental risk assessment of fluctuating concentrations of micropollutants is presented. We separated the prediction of toxicity over time from the extrapolation from one to many species and from acute to sublethal effects. A toxicokinetic-toxicodynamic (TKTD) model predicted toxicity caused by fluctuating concentrations of diazinon, measured by time-resolved sampling over 108 days from three locations in a stream network, representing urban, agricultural and mixed land use. We calculated extrapolation factors to quantify variation in toxicity among species and effect types based on available toxicity data, while correcting for different test durations with the TKTD model. Sampling from the distribution of extrapolation factors and prediction of time-resolved toxicity with the TKTD model facilitated subsequent calculation of the risk of undesired toxic events. Approximately one-fifth of aquatic organisms were at risk and fluctuating concentrations were more toxic than their averages. Contribution of urban and agricultural sources of diazinon to the overall risk varied. Thus using fixed concentrations as water quality criteria appears overly simplistic because it ignores the temporal dimension of toxicity. However, the improved prediction of toxicity for fluctuating concentrations may be small compared to uncertainty due to limited diversity of toxicity data to base the extrapolation factors on.     

Biomarkers of PAH exposure in an intertidal fish species from Prince William Sound, Alaska: 2004-2005

Polycyclic aromatic hydrocarbon (PAH) exposure biomarkers were measured in high cockscomb prickleback (Anoplarchus purpurescens) fish collected from both previously oiled and unoiled shore in Prince William Sound (PWS), Alaska, to test the hypothesis that fish living in the nearshore environment of the sound were no longer being exposed to PAH from the Exxon Valdez oil spill. Pricklebacks spend their entire lives in the intertidal zone of rocky shores with short-term movements during feeding and breeding restricted to an area of about 15 meters in diameter. Fish were assayed for the PAH exposure biomarkers, bile fluorescent aromatic compounds (FAC), and liver ethoxyresorufin O-deethylase (EROD) activity (a measure of cytochrome P450 1A (CYP1A) monooxygenase activity). Bile FAC concentrations and EROD activities were low and not significantly different in fish from previously oiled and unoiled sites. The similar low EROD activity and bile FAC concentrations in fish from oiled and unoiled shores, supports the hypothesis that these low-level biomarker responses were not caused by exposure of the fish to residues of the spilled oil.     

Method for testing the aquatic toxicity of sediment extracts for use in identifying organic toxicants in sediments

Biologically directed fractionation techniques are a fundamental tool for identifying the cause of toxicity in environmental samples, but few are available for studying mixtures of organic chemicals in aquatic sediments. This paper describes a method for extracting organic chemicals from sediments and then re-introducing them into water column toxicity tests in a way that mimics, at least in part, the partitioning processes that govern bioavailability in sediment. This involves transferring solvent extracts of sediment into triolein and then placing the mixture inside low-density polyethylene dialysis tubing in a configuration similar to semipermeable membrane devices (SPMDs) used for environmental monitoring. For four model compounds, SPMDs were shown to effectively maintain water column exposure in static systems for 10-14 d, with partition coefficients similar to K(OW). Toxicity tests indicated that the SPMDs were compatible with four of five freshwater organisms tested and could be used to measure both lethal and sublethal end points. An example application showed good correspondence between organism responses in intactsediment and extracts in SPMDsfor both field-collected and spiked sediments. The SPMD-based method offers a simple, flexible test design, amenable to several different test organisms, and the ability to work with complex mixtures of contaminants while maintaining partitioning behavior similar to that within intact sediments.     

Occurrence and toxicity of 331 organic pollutants in large rivers of north Germany over a decade (1994 to 2004)

We analyzed the detection frequencies and concentrations for 331 organic compounds measured between 1994 and 2004 in the four largest rivers of north Germany, the Elbe, Weser, Aller, and Ems Rivers, and we assessed the potential risk for aquatic fauna using experimental and predicted acute toxicity data for the green alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna, and the fish Pimephales promelas. The detection frequency for most compounds decreased significantly from 1994 to 2004. Polycyclic aromatic hydrocarbons (PAHs) were most frequently detected, while pesticides were the most important chemical group concerning toxicity for the standard test organisms. The predicted toxicity for D. magna was significantly higher than for the other organisms and reached levels envisaging acute toxic effects on the invertebrate fauna, still in 2004. Most of the compounds responsible for potential acute effects on aquatic organisms are currently not considered as priority substances in the European Union, while only 2 of 25 priority substances that have been measured occurred at levels that may be relevant in terms of toxicity for the selected test organisms. We conclude that attenuation of pesticides and other organic toxicants should play an increased role in river basin management.