Applications, considerations, and sources of uncertainty when using stable isotope analysis in ecotoxicology

Stable isotope analysis (SIA) has become a powerful tool for ecotoxicologists to study dietary exposure and biomagnification of contaminants in wild animal populations. The use of SIA in ecotoxicology continues to expand and, while much more is known about the mechanisms driving patterns of isotopic ratios in consumers, there remain several considerations or sources of uncertainty that can influence interpretation of data from field studies. We outline current uses of SIA in ecotoxicology, including estimating the importance of dietary sources of carbon and their application in biomagnification studies, and we present six main considerations or sources of uncertainty associated with the approach: (1) unequal diet-tissue stable isotope fractionation among species, (2) variable diet-tissue stable isotope fractionation within a given species, (3) different stable isotope ratios in different tissues of the animal, (4) fluctuating baseline stable isotope ratios across systems, (5) the presence of true omnivores, and (6) movement of animals and nutrients between food webs. Since these considerations or sources of uncertainty are difficult to assess in field studies, we advocate that researchers consider the following in designing ecotoxicological research and interpreting results: assess and utilize variation in stable isotope diet-tissue fractionation among animal groups available in the literature; determine stable isotope ratios in multiple tissues to provide a temporal assessment of feeding; adequately characterize baseline isotope ratios; utilize stomach contents when possible; and assess and integrate life history of study animals in a system.     

Screening method for ecotoxicological hazard assessment of 42 pharmaceuticals considering human metabolism and excretory routes

We assessed the ecotoxicological hazard potential of 42 pharmaceuticals from 22 therapeutic groups, including metabolites formed in humans. We treated each parent drug and its metabolites as a mixture of similarly acting compounds. If physicochemical or effect literature data were missing, we estimated these with quantitative structure-activity relationships (QSAR). Additionally, we estimated micropollutant removal efficiency of urine source separation using pharmaceutical information. On average, 50% of a parent drug was metabolized, and 70% was excreted with urine, albeit with large variations among drugs. Metabolism reduced the toxic potential of all but eight drugs. The subsequently modeled risk quotient was mostly below the threshold of one. However, ibuprofen and its metabolites in a mixture could pose an ecotoxicologal risk; and possibly also acetylsalicylic acid, bezafibrate, carbamazepine, diclofenac, fenofibrate, and paracetamol. Lipophilicity and sale quantities of parent drugs alone were insufficient to estimate their ecotoxicological risk. Urine separation could decrease the ecotoxicological risk of some, but not all drugs. The estimated risk quotients were equal in urine and feces, again with large variations among compounds. Because of scientific limitations of the model and inconsistent literature data the results are somewhat uncertain. However, this new approach allows first tier screening of single drugs, thus supporting decision-making.     

天然色素栀子黄的危害评估研究

目的 基于栀子黄色素及其主成分已有的毒理学资料,对栀子黄色素开展危害评估、关键效应和剂量的基准剂量分析.方法 采用系统文献检索方法,制定检索策略,收集栀子黄色素毒理学资料,并根据亚慢性毒性实验结果,应用基准剂量法测算关键效应和剂量.结果 藏红花酸及其酯类是栀子黄色素中的主要成分,主要毒性作用靶器官是肝脏和肾脏,尚未发现...     

Soil ecotoxicity of polycyclic aromatic hydrocarbons in relation to soil sorption,lipophilicity, and water solubility

A data set was generated aiming to predict the toxicity of PAHs to soil organisms. Toxicity data include the effects of 16 PAHs on the survival and reproduction of the soil-dwelling springtail Folsomia fimetaria. The results show that only PAHs with reported log Kow values < or = 5.2 (i.e., naphthalene, acenaphthene, acenaphthylene, anthracene, phenanthrene, fluorene, pyrene, and fluoranthene) significantly affected the survival or reproduction of the test organisms. Threshold values for the toxicity of the individual PAHs could be expressed as pore-water concentrations by the use of reported organic carbon-normalized soil-pore-water partitioning coefficients (Koc values). For the PAHs with a log Kow < or = 5.2, toxicity significantly increased with increasing lipophilicity of the substances (r2 = 0.67; p = 0.012; n = 8), suggesting a narcotic mode of toxic action for most substances. However, the position of anthracene in the regression plot indicated a more specific mode of toxic action than narcosis, and removing this data point yielded the following regression equation: log EC10 (micromol/L) = -0.97 log Kow + 4.0 (r2 = 0.80; p = 0.006; n = 7). Using this quantitative structure-activity relationship (QSAR) to calculate threshold values for the toxicity of the remaining nontoxic substances (benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, dibenz[a,h]anthracene, benzo[a]pyrene, perylene, and indeno[1,2,3-cd]pyrene), the absence of toxicity could, in most cases, be explained by a limited water solubility, indicating that these substances do act by narcosis as the mode of toxic action and that their toxicity is governed by concentrations in the pore-water. The results provide important input to future model predictions of the ecological risk posed by PAH contaminated sites.     

Daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity

Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics.     

Prokaryotic gene profiling assays to detect sediment toxicity: evaluating the ecotoxicological relevance of a cell-based assay

Despite their complexity, ecotoxicological measurements using higher level responses remain a major tool in the assessment of ecosystem integrity. Nevertheless, the past decade saw an increasing number of cell based testing systems have found widespread application in ecotoxicology. One such test is bacterial bioreporters carrying a stress sensitive promoter fused to an easily detectable reporter gene. In the presence of a specific toxic stress,the expression cassette is switched on and the reporter gene is produced. This study evaluated the use of 14 different Escherichia coli bioreporter strains sensitive to different types of toxicity in the assessment of the ecological status of a small river basin in Flanders, Belgium. The river is fed at two geographically separate locations by two distinct and well-described effluents, one from a household sewage treatment facility and one from the discharge of the wastewater treatment facility of a large chemical plant. The results of the bacterial gene profiling assay were related to active biomonitoring results obtained through higher-level responses of caged Dreissena polymorpha, Chironomus riparius, and Cyprinus carpio deployed at the locations sampled for the bacterial assay. The results of the gene induction assay and the active biomonitoring data correlated well and corresponded to the flow dilution data, which is used here as a surrogate forthe chemical pollution gradient present in the river basin.     

Simulating toxicity of carbaryl to Gammarus pulex after sequential pulsed exposure

Aquatic nontarget organisms are typically exposed to sequential pulses of contaminants with fluctuating concentrations. We use the semimechanistic threshold damage model (TDM) to simulate survival of the aquatic invertebrate Gammarus pulex after sequential pulsed exposure to carbaryl and compare itto a simpler model based on time-weighted averages (TWA). The TDM is a process-based model and we demonstrate how to parametrize it with data from an uptake and elimination experimenttogether with data from a survival experiment with sequential pulses. The performance of the two models is compared by the fit to the first survival experiment and the simulation of another, independent survival experiment with different exposure patterns. Measured internal concentrations in the first survival experiment are used to evaluate the toxicokinetic submodel of the TDM. The TDM outperforms the TWA model, facilitates understanding of the underlying ecotoxicological processes, permits calculation of recovery times (3, 15, and 25 days for pentachlorophenol, carbaryl and chlorpyrifos respectively) and enables us to predict the effects of long-term exposure patterns with sequential pulses or fluctuating concentrations. We compare the parameters of the TDM for carbaryl, pentachlorophenol and chlorpyrifos and discuss implications for ecotoxicology and risk assessment.     

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.     

Population Growth Rate Responses of Ceriodaphnia dubia to Ternary Mixtures of Specific Acting Chemicals: Pharmacological versus Ecotoxicological Modes of Action

When considering joint toxic apical effects at higher levels of biological organization, such as the growth of populations, the so-called pharmacological mode of action that relies on toxicological mechanistic effects on molecular target sites may not be relevant. Such effects on population growth rate will depend on the extent to which juvenile and adult survival rates and production rates (juvenile developmental rates and reproduction) are affected by toxic exposure and also by the sensitivity of population growth rates to life-history changes. In such cases, the ecotoxicological mode of action, defined as the crucial life-history trait processes and/or xenobiotic-life-history trait interactions underlying a toxicological effect on population growth rate, should be considered. Life-table response experiments with the crustacean Ceriodaphnia dubia exposed to single and ternary mixtures of nine compounds were conducted to test the hypothesis that joint effects on population growth rates could be predicted from the mixture constituent ecotoxicological mode of action. Joint effects of mixtures containing pharmacologically dissimilar compounds (cadmium, λ-cyhalothrin, and chlorpyrifos) that differentially affected life-history traits contributing to population growth rates were accurately predicted by the independent-action concept. Conversely, the concentration-addition concept accurately predicted joint effects of two different mixtures: one containing pharmacologically similar acting pyrethroids that also affected similarly life-history traits, the other one that included pharmacologically dissimilar compounds (3,4-dichloroaniline, sodium bromide, and fenoxycarb) acting mainly on reproduction rates. These results indicate that when assessing combined effects on population growth rate responses, selection of mixture toxicity conceptual models based on the ecotoxicological mode of action of mixture constituents provided more accurate predictions than those based on the pharmacological mode of action.     

Fractions affected and probabilistic risk assessment of Cu, Zn, Cd, and Pb in soils using the free ion approach

The free ion approach quantifies the toxic effects of cationic metals on soil biota as a function of chemistry. The approach is here extended to calculate the general relationship among toxic effects as the Fraction Affected (FA), soil solution pH, and soil organic matter content (SOM) for Cu, Zn, Cd, and Pb within toxicity data sets from literature. The dependence of FA on SOM is strong, with the FA decreasing as the SOM increases. The dependence of FA upon pH varies; Cd and Zn show strong dependences while for Cu and Pb dependences are weaker. The FA usually decreases as the soil pH increases. When the free ion approach is applied, risks across soils due to different metals can be compared using the FA. The free ion approach may also be applied to probabilistic risk assessment. Risk values, using the joint probability curve approach, were derived for Pb using two field soil data sets. One data set, with higher SOM than that of the Pb toxicity data set, gave a lower risk with the free ion approach than that when the soil chemistry was not considered. The other data set had lower SOM than that of the toxicity data set and gave a higher risk with the free ion approach. Since literature toxicity tests are biased toward low SOM soils of circumneutral pH, using such data to perform classical risk assessment for soils of differing chemical composition can lead to misestimation of risk due to neglecting soil chemistry, especially in soils with extreme pH and/or SOM.     

Recent advances in environmental risk assessment of transformation products

When micropollutants degrade in the environment, they may form persistent and toxic transformation products, which should be accounted for in the environmental risk assessment of the parent compounds. Transformation products have become a topic of interest not only with regard to their formation in the environment, but also during advanced water treatment processes, where disinfection byproducts can form from benign precursors. In addition, environmental risk assessment of human and veterinary pharmaceuticals requires inclusion of human metabolites as most pharmaceuticals are not excreted into wastewater in their original form, but are extensively metabolized. All three areas have developed their independent approaches to assess the risk associated with transformation product formation including hazard identification, exposure assessment, hazard assessment including dose-response characterization, and risk characterization. This review provides an overview and defines a link among those areas, emphasizing commonalities and encouraging a common approach. We distinguish among approaches to assess transformation products of individual pollutants that are undergoing a particular transformation process, e.g., biotransformation or (photo)oxidation, and approaches with the goal of prioritizing transformation products in terms of their contribution to environmental risk. We classify existing approaches for transformation product assessment in degradation studies as exposure- or effect-driven. In the exposure-driven approach, transformation products are identified and quantified by chemical analysis followed by effect assessment. In the effect-driven approach, a reaction mixture undergoes toxicity testing. If the decrease in toxicity parallels the decrease of parent compound concentration, the transformation products are considered to be irrelevant, and only when toxicity increases or the decrease is not proportional to the parent compound concentration are the TPs identified. For prioritization of transformation products in terms of their contribution to overall environmental risk, we integrate existing research into a coherent model-based, risk-driven framework. In the proposed framework, read-across from data of the parent compound to the transformation products is emphasized, but limitations to this approach are also discussed. Most prominently, we demonstrate how effect data for parent compounds can be used in combination with analysis of toxicophore structures and bioconcentration potential to facilitate transformation product effect assessment.     

基于氧化应激对手性三唑类杀菌剂诱导斑马鱼毒性效应的研究进展

三唑类杀菌剂一般具有一个或多个手性中心，因在环境及食品加工过程中具有立体选择性行为，一直是研究的热点与重点。三唑类杀菌剂因大量使用不可避免残留在水体及食品中，随着食物链进入到人体内，因此，基于模式生物斑马鱼的毒理学评价对三唑类杀菌剂评估具有重要意义。本文对近年来手性三唑类杀菌剂诱导斑马鱼毒性效应进行综述，发现其毒性效应大多与氧化应激调控有关，但从手性对映体角度对三唑类杀菌剂对斑马鱼的影响及分子机制阐述尚不清晰。围绕氧化应激通路、线粒体氧化磷酸化及抗氧化酶活性改变等方面，探索三唑类杀菌剂的选择性毒性机制，可为食品中农药类危害物毒性风险评估的控制提供基础数据与研究思路。     

Mixture toxicity of reactive chemicals by using two bacterial growth assays as indicators of protein and DNA damage

The mixture toxicity of reactive chemicals was investigated with a set of bioanalytical tests that quantify not only the toxic effects but also allow the identification of the preferred target of reactive chemicals in bacterial cells. Softer electrophiles such as acrylates react preferentially with thiol groups in proteins and peptides, and harder electrophiles such as epoxides preferentially attack DNA. In addition, some compounds, e.g., benzyl chloride, have no preference for a biological target and damage both DNA and proteins. A thiophenol was used as a model compound representing nucleophiles. We explored if the paradigms of mixture toxicity also hold true for reactive chemicals. Compounds with the same targets and the same modes of action should act concentration additive in mixtures, and compounds with different modes of action should act according to the concept of independent action. In addition, we investigated the potential for interaction of compounds of mixtures of electrophiles or electrophiles plus nucleophiles, which might lead to synergistic or antagonistic effects. The toxicity of mixtures of electrophiles with a single preferred target was consistent with the prediction for concentration addition. Unfortunately, the predictions for independent action did not differ much from those for concentration addition; therefore it was not possible to differentiate between these two models. Mixtures of two groups with different preferred target sites clearly showed concentration addition. In contrast, mixtures of compounds with multiple targets, i.e., compounds that show nonspecific reactivity toward any biological nucleophile, exhibited effects that lay distinctly between the predictions for concentration addition and independent action. We observed neither synergism (higher toxicity than predicted by concentration addition) nor antagonism (lower toxicity than predicted by independent action) for mixtures of electrophiles. Binary combinations of different electrophiles with the nucleophile 4-chlorothiophenol yielded smaller effects than those expected from the prediction for independent action. The degree of antagonism was correlated with the reaction rate constant of the electrophile with the thiol group of glutathione, which indicates that the interaction between the mixture components occur in the toxicokinetic phase and is purely a result of chemical reactivity between the mixture components. Overall, we conclude that the concepts of mixture toxicity apply not only for baseline toxicity and receptor-mediated mechanisms, as has been shown in a large number of studies, but also for reactive mechanisms of toxicity, provided that one has checked beforehand that no chemical reactions occur between the mixture components.     

Comparative ecotoxicological hazard assessment of beta-blockers and their human metabolites using a mode-of-action-based test battery and a QSAR approach

We analyzed nontarget effects of the beta-blockers propranolol, metoprolol, and atenolol with a screening test battery encompassing nonspecific, receptor-mediated, and reactive modes of toxic action. All beta-blockers were baseline toxicants and showed no specific effects on energy transduction nor endocrine activity in the yeast estrogen and androgen screen, and no reactive toxicity toward proteins and DNA. However, in a phytotoxicity assay based on the inhibition of the photosynthesis efficiency in green algae, all beta-blockers were 10 times more toxic than their modeled baseline toxicity. Baseline- and phytotoxicity effects increased with hydrophobicity. The beta-blockers showed concentration addition in mixture experiments, indicating a mutual specific nontarget effect on algae. Using literature data and quantitative structure-activity relationships (QSAR), we modeled the total toxic potential of mixtures of the beta-blockers and their associated human metabolites for the phytotoxicity endpoint with two scenarios. The realistic scenario (I) assumes that the metabolites lose their specific activity and act as baseline toxicants. In the worst-case scenario (II) the metabolites exhibitthe same specific mode of action as their parent drug. For scenario (II), metabolism hardly affected the overall toxicity of atenolol and metoprolol, whereas propranolol's hazard potential decreased significantly. In scenario (I), metabolism reduced the apparent EC50 of the mixture of parent drug and metabolite even further. The proposed method is a simple approach to initial hazard assessment of pharmaceuticals and can guide higher tier testing. It can be applied to other classes of pollutants, e.g., biocides, as well as to environmental transformation products of pollutants.     

Laser ablation ICP-MS Co-localization of mercury and immune response in fish

Mercury (Hg) contamination is a global issue with implications for both ecosystem and human health. In this study, we use a new approach to link Hg exposure to health effects in spotted gar (Lepisosteus oculatus) from Caddo Lake (TX/LA). Previous field studies have reported elevated incidences of macrophage centers in liver, kidney, and spleen of fish with high concentrations of Hg. Macrophage centers are aggregates of specialized white blood cells that form as an immune response to tissue damage, and are considered a general biomarker of contaminant toxicity. We found elevated incidences of macrophage centers in liver of spotted gar and used a new technology for ecotoxicology studies, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), to colocalize aggregates and Hg deposits within the tissue architecture. We conclude that Hg compromises the health of spotted gar in our study and, perhaps, other fish exposed to elevated concentrations of Hg.     

Population consequences of fipronil and degradates to copepods at field concentrations: an integration of life cycle testing with leslie matrix population modeling

The predominant data used in ecological risk assessment today are individual-based rather than population-based; yet environmental policies are usually designed to protect populations of threatened species or communities. Most current methods in ecotoxicology are limited by largely logistic/ technology-driven requirements that yield data for a relatively small number of test species and end points that focus on acute lethality or sublethal nonproduction-based parameters (e.g., biomarkers, mutagenesis, genetic change, physiological condition). A contrasting example is presented here showing the predictive ability of meiobenthos-based full life cycle toxicity testing to extrapolate multi-generational effects of chemicals on variables of import to population growth and maintenance. Less than 24-h-old larvae of a meiobenthic copepod were reared individually in 96-well microplate exposures to parent and degradates of the phenylpyrazole insecticide fipronil. Survival, development rates, sex ratio change, fertility, fecundity, and hatching success were tracked daily for 32 d through mating and production of three broods in spiked seawater. These data were then inserted in a Leslie (Lefkovitch) matrix stage-based population growth model to predict relative rates of population increase (lambda) and changes in net population growth with time and toxicant concentration. Field-reported test concentrations produced strong reproductive (52-88%) and net production (40-80%) depressions for parent (at 0.25 and 0.5 microg/L), desthionyl (0.25 and 0.5 microg/L), and sulfide (0.15 microg/L) moieties as compared to controls. Spiked sediment exposures of 65-300 ng of fipronil/g of dry sediment yielded significantly reduced production rates per female that were 67-50% of control production. The consistent reproductively linked impacts of fipronil and its degradation products at the population maintenance levels suggest risks to sediment-dwelling crustaceans at concentrations well below noneffects for most aquatic test species based on risk assessment data from primarily acute and sub-life cycle toxicity tests.     

我国鸡肉产品中沙门氏菌风险评估的研究进展

本文综述了国内外鸡肉相关产品中沙门氏菌的风险评估研究现状,包括沙门氏菌预测微生物学、评估模型、剂量反应模型及风险评估软件的使用,指出了目前我国鸡肉产品中沙门氏菌风险评估研究中存在的问题,并提出下一步鸡肉中沙门氏菌风险评估工作的研究方向及重点,为政府食品安全监管和理性决策提供科学的理论参考,同时也为消费者日常饮食选择提供风险预警的理论借鉴。     

Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).