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Toxicity of lindane, atrazine, and deltamethrin to early life stages of zebrafish (Brachydanio rerio)
Abstract
Fertilized eggs of zebrafish were exposed under flow-through conditions to several concentrations of the following pesticides: lindane 40, 80, 110, 130, and 150 micrograms/liter; atrazine 300, 1300, and 9100 micrograms/liter; deltamethrin 0.5, 0.8, and 1.2 micrograms/liter. Hatching, abnormalities in development (external deformations, edema, etc.), and mortality were recorded over a period of 35 days. At the end of the experiment, the body lengths of the fish were measured. Survival of juvenile fish after 35 days was reduced by increasing concentrations of all xenobiotics tested: lindane enhanced the mortality from 110 micrograms/liter and atrazine from 1300 micrograms/liter, and deltamethrin showed an effect even at the lowest test concentration (0.5 micrograms/liter). Other parameters were affected differently: hatching rate was reduced only by deltamethrin (from 0.8 micrograms/liter): lindane caused a decrease in growth (40 micrograms/liter) but had no effect on the other parameters. Atrazine increased the number of deformations and edema (1300 micrograms/liter) but did not influence hatching rate and growth. The sensitivity of the early life stages to the pesticides was compared with acute toxicity data (LC50) of adult zebrafish. The early life stages were less sensitive to lindane (118 versus 75 micrograms/liter), whereas in the case of atrazine (1300 versus 37,000 micrograms/liter) and deltamethrin (0.5 versus 2 micrograms/liter; 0.5 micrograms/liter was the lowest concentration tested) larvae were more sensitive.
... Ao mesmo tempo, observou-se que, da mesma forma que ocorreu para os ensaios com períodos de exposição mais prolongados, os ensaios com Pimephales promelas em sete dias de exposição mostraram razão igual a quatro. Ainda, merece destaque a razão média igual a 2,8 verificada nos poucos ensaios com Danio rerio (Görge;Nagel, 1990;VanLeeuwen et al., 1990), a qual indica a similaridade de respostas com o Pimephales promelas. ...
... Ao mesmo tempo, observou-se que, da mesma forma que ocorreu para os ensaios com períodos de exposição mais prolongados, os ensaios com Pimephales promelas em sete dias de exposição mostraram razão igual a quatro. Ainda, merece destaque a razão média igual a 2,8 verificada nos poucos ensaios com Danio rerio (Görge;Nagel, 1990;VanLeeuwen et al., 1990), a qual indica a similaridade de respostas com o Pimephales promelas. ...
... Estatisticamente, os limites de previsão são análogos aos de confiança. Enquanto os limites de confiança são restritos ao conjunto de dados utilizados na equação, os limites de previsão (1976, apud Görge, Nagel (1990); d) Görge, Nagel (1990); e) Macek et al. (1976b), apud Görge, Nagel (1990)); f) Jarvinen, Turner (1982); g) Bresch (1991); e **estimativa pela equação: log VCest, Danio rerio = 0,30 + 0,84 log VC, Pimephales promelas. Doherty (1983) permite verificar um limite de previsão de ± 1,6 ordem de magnitude para a equação de regressão, com as espécies de peixe Salmo gairdneri e Lepomis macrochirus, consideradas pelo autor como as únicas de sensibilidade equivalente em termos de ecotoxicidade aguda. ...
... Microinjection is the prevailing and widely accepted method of zebrafish transfection. This method provides high gene expression efficiency (> 80 %) and high survival rate (> 70 %) including applications in genetics [2], virology [3], toxicology [4], and immunology [5]. However, manual injection is labor intensive, time-consuming, prone to error, requires high-resolution microscopy and trained technicians, limiting high throughput transgenesis. ...
Electroporation is regularly used to deliver agents into cells, including transgenic materials, but it is not used for mutating zebrafish embryos due to the lack of suitable systems, information on appropriate operating parameters, and the challenges posed by the protective chorion. Here, a novel method for gene delivery in zebrafish embryos was developed by combining microinjection into the space between the chorion and the embryo followed by electroporation. This method eliminates the need for chorion removal and injecting into the space between the chorion and embryo eliminates the need for finding and identifying key cell locations before performing an injection, making the process much simpler and more automatable. We also developed a microfluidic electroporation system and optimized electric pulse parameters for transgenesis of embryos. The study provided a novel method for gene delivery in zebrafish embryos that can be potentially implemented in a high throughput transgenesis or mutagenesis system.
... Microinjections can be adapted for a variety of target cell types, such as Drosophila embryos, mouse embryos, and zebrafish embryos, which are important for genetic expression evaluations, poison and drug detection, and neurovasculature observations. In particular, zebrafish embryo microinjection (ZEM) technology has been widely used in genetics [114], endocrinology [115], oncology [116], immunology [117], virology [118], toxicology [119] and many other fields. ...
Zebrafish have become a widely accepted model organism for biomedical research due to their strong cortisol stress response, behavioral strain differences, and sensitivity to both drug treatments and predators. However, experimental zebrafish studies generate substantial data that must be analyzed through objective, accurate, and repeatable analysis methods. Recently, advancements in artificial intelligence (AI) have enabled automated tracking, image recognition, and data analysis, leading to more efficient and insightful investigations. In this review, we examine key AI applications in zebrafish research, including behavior analysis, genomics, and neuroscience. With the development of deep learning technology, AI algorithms have been used to precisely analyze and identify images of zebrafish, enabling automated testing and analysis. By applying AI algorithms in genomics research, researchers have elucidated the relationship between genes and biology, providing a better basis for the development of disease treatments and gene therapies. Additionally, the development of more effective neuroscience tools could help researchers better understand the complex neural networks in the zebrafish brain. In the future, further advancements in AI technology are expected to enable more extensive and in-depth medical research applications in zebrafish, improving our understanding of this important animal model. This review highlights the potential of AI technology in achieving the full potential of zebrafish research by enabling researchers to efficiently track, process, and visualize the outcomes of their experiments.Graphical Abstract
... S a typical small multicellular model organism [1], the zebrafish has become increasingly significant in genetics [2], chemical biology [3], and disease modeling [4], [5]. Different from the well-known rodent animal models such as mice, zebrafish is the only vertebrate model organism that can be cultivated in multi-well plates or microfluidic channels, thus enabling high-throughput screening [6]. ...
Zebrafish embryo injection is often required in biomedical research using zebrafish. In the injecting operation, trapping and rotating the zebrafish embryo to achieve a proper posture is essential for the high success rate. We proposed an on-chip platform capable of efficient and automatic trapping and rotating for the injection of zebrafish embryos. A low-cost 3D-printed microchannel is designed to trap zebrafish embryos into the cavity array. The blind-hole design at each cavity can generate microbubbles, and the bubbles exposed to the acoustic wave with a specific frequency can trap and rotate the zebrafish embryos. The progress, including trapping and rotating, can be monitored and executed automatically with computer vision assistance. Experimental results show that we realized on-chip trapping and rotating operations successfully. The success rate of trapping zebrafish embryos was up to 99%, and the time of trapping a single embryo was as low as 0.2 s. Embryo rotation could be achieved by two different modes, including continuous rotation and intermittent rotation. The accuracy and maximal velocity of rotating the embryo reached 5° and 3.5 r/s, respectively. Thus, we believe the proposed efficient automatic on-chip trapping and rotating platform could support the zebrafish embryo injection well.
... According to the U.S. EPA (1996), prometryn is classified as a high-toxic substance, and the 96-h LC 50 values of prometryn to minnow (Phoxinus phoxinus) and silver carp (Hypophthalmichthys molitrix) are 4.5 mg/L and 7.0 mg/L, respectively. In zebrafish (Danio rerio), the 96-h LC 50 of prometryn is 5.3 mg/L, which is much lower than the value of atrazine (37 mg/L), indicating that prometryn might have higher toxicity to fish compared to other triazine herbicides (Abarikwu et al., 2010;Görge et al., 1990;Zhao et al., 2015). However, there are no reports on the effects of low concentrations of prometryn on fish development and growth until now. ...
Triazine herbicides have been widely detected in marine environments because of their extensive usage in
agriculture, but their impact on marine organisms is unclear. In this study, marine medaka (Oryzias melastigma)
embryos were exposed to 0, 1, 10, 100, and 1000 μg/L prometryn, one of the most detected triazine herbicides, to
investigate its potential effects. The results showed that 1, 10, 100, and 1000 μg/L prometryn not only induced
yolk sac shrinkage and heart malformations, but also significantly delayed the hatching time and increased the
heart rate and hatching failure rate of embryos. Moreover, 1, 10, 100, and 1000 μg/L prometryn caused obvious
malformations and decreased the body length of the newly hatched larvae. After 21 d of exposure, increased
larval death rate, decreased body length and width, and higher lipid accumulation were observed in the larvae
from all prometryn groups. Furthermore, prometryn exposure upregulated the expression levels of cardiac
development-related genes GATA, COX, ATPase, SmyD1, EPO, FGF8, NKX2, and BMP4 in the larvae. Transcriptome
analysis revealed that 10 μg/L prometryn upregulated 604 genes, and the topmost pathways of
differentially expressed genes were the complement and coagulation cascades and AMPK signaling pathways.
qPCR results confirmed that prometryn exposure significantly increased the expression levels of the complement
and coagulation cascade genes f2, f5, c3, and c5. This study demonstrated that environmentally relevant concentrations
of prometryn induced significant toxicity in the early life stages of marine medaka. Therefore, the
health risks of herbicides to marine organisms are of great concern.
... Possessing the equivalents of seventy percent of human genes causes the zebrafish to be useful in research into a number of human diseases [13]. Numerous papers on the zebrafish make it apparent that the zebrafish is useful in studies of the environment [14][15][16][17], medicines [18,19], neurotoxicity [20,21], immunotoxicity [22] and cancer [23]. ...
11 active substances used in pesticides were selected. Substances were divided into three groups depending the effect on embryos or fetuses of laboratory animals: 1 – damaging effect on embryos or fetuses (embryotoxic, fetotoxic or teratogenic), 2 – damaging effect on embryos or fetuses, but only at dose toxic for mother (maternal toxicity), 3 – no damaging effect.
Changes for hydra in acute toxicity tests and recovery tests were assessed on an change scale from 0 to 10. The index of the effect on development (TI) for hydras was calculated for every compound. Changes in zebrafish embryos were assessed using a descriptive method. Pearson correlation coefficient showed the correlation between the concentration and the toxic effect in the zebrafish embryos for the substances of the first group.
The study showed that substances having a strong damaging effect on fetuses cause changes that are apparent and easy to evaluate both in hydras and zebrafish embryos. A scoring system was introduced to evaluate the changes of hydras and zebrafish embryos. The point system of evaluation of changes allows quick classification of a substance as potentially embryotoxic, fetotoxic or teratogenic. It allows developing a cheap and fast method alternative to prenatal developmental toxicity studies, a screening method that enables substances of great teratogenic potential to be excluded from studies on laboratory animals.
... The zebrafish (Danio rerio) is a well-established model organism for toxicology, which, since its first application in 1990, has become a popular model for developmental research(49,50) . Zebrafish developmental processes and pathways are well conserved with humans and Zebrafish embryos are easily grown ex utero where their transparency allows for in vivo observation of organ and tissue development(50). ...
Before new chemicals can be put on the market, they must be evaluated for toxicological safety. Evaluating the safety of new chemicals, for either medical, cosmetic or environmental application, is tightly regulated by worldwide legislation. A critical aspect of toxicity evaluation is developmental and reproductive toxicity (DART) testing. Traditionally, DART testing has been conducted in vivo in mammalian model systems. In fact, current EU DART testing guidelines accounts for the majority of animals used and the financial costs of new compound compliance testing. Therefore, because of the need to reduce the financial and animal costs associated with DART testing, there is a growing demand for new alternative model systems for toxicity evaluation. Dictyostelium discoideum is a eukaryotic amoeba which due to its unique developmental cycle has the potential to serve as a non-animal alternative model in DART testing. However, for a new alternative model to be proven effective it must allow for high-throughput screening, whilst maintaining biological complexity; allowing developmental toxicity results to be predictive of mammalian systems. To address these concerns, we developed new high-throughput D. discoideum growth and developmental toxicity assays. We use the assays to characterise toxicity across a broad range of test compounds, thereby revealing a significant relationship between D. discoideum and mammalian toxicity values. Our data demonstrates that D. discoideum has the biological complexity necessary to be predictive of mammalian toxicity. We further assess whether D. discoideum could be used to genetically characterise developmentally toxic compounds. Using next generation functional genomic screens, we show how the developmentally toxicity compounds, lithium and VPA can be globally genetically phenotyped. Using this genetic phenotyping approach, we were also able to identify the biological targets and processes that mediate lithium and VPA toxicity. Together, these studies illustrate the potential of D. discoideum to be developed as a new alternative model in DART testing.
... The hatching rate and surface tension of zebrafish embryos were evaluated at 72 hpf and 24 hpf, respectively. While previous study [33] reported that 0.8 μg/L DM exposure from 6 hpf could reduce hatching rate, our findings suggest that 0.025 mg/L DM exposure from 0.75 hpf could increase the hatching rate, which might due to the higher concentration and longer exposure duration adopted in our study. Previous research and the results of this study indicate that 0.025 mg/L DM could induce cardiotoxicity in larval zebrafish [13]. ...
Increased application of the pyrethroid insecticide deltamethrin has adverse effects on the cardiac system and neurobehavior on the non-target organisms, which has raised the public’s attention. Because of spermidine and melatonin considered to have cardioprotective and neuroprotective characteristics, zebrafish were utilized as the model organism to explore the protective effects of spermidine and melatonin against deltamethrin-induced toxicity. We tested the neurobehavior of zebrafish larvae through a rest/wake behavior assay, and evaluated the levels of the expression of Scn5lab, gata4, nkx2.5, hcrt, hcrtr, and aanat2 by qRT-PCR. Besides that cmlc2 was evaluated by whole-mount in situ hybridization. Results have shown that compared with control group, 0.025 mg/L deltamethrin could significantly disturb the cardiac development, downregulating the expression of Scn5lab and transcriptional factors gata4 and nkx2.5, disturbing cardiac looping, resulting in defects in cardiac morphology and function. Moreover, deltamethrin could alter the expression levels of rest/wake genes and cause hyperactivity in zebrafish larvae. Besides, compared with deltamethrin group, the exogenous 0.01 mg/L spermidine and 0.232 mg/L melatonin could significantly rescue the adverse effects of deltamethrin on the cardiac system and neurobehavior in zebrafish. This indicated that spermidine and melatonin have neuroprotective and cardioprotective effects against deltamethrin-induced adverse effects in zebrafish.
... In Anbetracht der Tatsache, dass die noch über den Dottersack ernährenden Entwicklungsstadien von Fischen gemäß der geltenden europäischen Richtlinie 2010/63/EU zum Schutz der für wissenschaftliche Zwecke verwendeten Tiere (EU, 2010) nicht als geschützt eingestuft werden (Strähle et al., 2012), wurde der Fischembryotest (FET) mit dem Zebrabärbling (Danio rerio) wiederholt als eine vielversprechende Alternative für den akuten Fischtoxizitätstest vorgeschlagen (Braunbeck et al., 2005;Braunbeck et al., 2015;Görge and Nagel, 1990;Lange et al., 1995;Nagel, 2002;Nagel and Isberner, 1998) und schließlich als OECD TG 236 (OECD, 2013) verabschiedet. Insbesondere der Fischembryotest mit dem Zebrabärbling eignet sich zur Vorhersage der akuten Fischtoxizität, da die Fischembryo-Mortalität eine hohe Korrelation mit dem akuten Fischtoxizitätstest aufzeigte (Belanger et al., 2013;Lammer et al., 2009). ...
In der vorliegenden Studie wurde überprüft, inwiefern der Fischembryotest (FET) als Werkzeug zur Bestimmung von spezifischer Toxizität in Embryonen des Zebrabärblings (Danio rerio) dienen kann. Als spezifische Endpunkte wurden dioxinähnliche Wirkung mithilfe des modifizierten Live-Imaging-EROD-Assays und Neurotoxizität mittels des Acetylcholinesterase-Assays untersucht. Diese Assays wurden jeweils mit einem Satz ausgewählter Chemikalien geprüft, die aufgrund ihrer Relevanz für die europäische Wasserrahmenrichtlinie ausgewählt wurden: Methylquecksilberchlorid, Chlorpyrifos, Aroclor 1254, 2,3-Benzofuran, Chinolin, Bisphenol A sowie Paraoxon-methyl (für neurotoxikologische Effekte) und eine Reihe von Sedimenten von Standorten mit bekanntem ökotoxikologischen Hintergrund: Altrip und Ehrenbreitstein am Rhein sowie der Veringkanal im Hamburger Hafen. Um die spezifische Toxizität zu untersuchen und schädliche Wirkungen durch akute Toxizität zu vermeiden, wurde zuerst die Effektkonzentration von 10 % (EC10) bestimmt. Hierbei zeigte sich mit fortschreitender Belastungsdauer, dass mit steigendem log Kow die Diffusionsrate ab- und die Akkumulationsrate zunahm. Eine Akkumulation von Substanzen mit einem hohen log Kow war zum einen mit einer verzögerten Toxizität, zum anderen bei verlängerter Exposition durchaus mit gesteigerter Toxizität verbunden. Neben Teratogenität wurde auch die Bioaktivierung über CYP1A mit einer in dieser Arbeit entwickelten Kombinationsexposition untersucht. Eine gleichzeitige Exposition gegenüber Chlorpyrifos + β-Naphthoflavon führten zu einer 40 %igen Steigerung der Toxizität gegenüber Chlorpyrifos als Monosubstanz. Ein Schwerpunkt der vorliegenden Arbeit lag in der Entwicklung und Optimierung eines modifizierten Live-Imaging-EROD-Assays. Die Auswertung des Assays über verschiedene Entwicklungsstufen hinweg (24, 48, 72, 96 und 120 h) zeigte, dass erst Embryonen mit einer funktionsfähigen Leber (≥ 72 h) und nach β-Naphthoflavon-Stimulation ein deutliches Fluoreszenzsignal in der Leber zeigten, dass nach 96 und 120 h dosisabhängig über die Zunahme von Resorufin im Gastrointestinaltrakt abgebildet werden konnte. Nach diesen Ergebnissen sind 96 und 120 h alte Embryonen die geeigneten Entwicklungsstadien, um die EROD-Aktivität in vivo mithilfe des Live-Imaging-EROD-Assays zu ermitteln. Eine Reduktion der Expositionszeit auf 3 h führte zu einem schnellen, effizienten und einfach handzuhabendem Assay. Die Kombinationsexposition mit Induktoren ermöglichte, erstmals in vivo das gleichzeitige Vorkommen von Agonisten und Inhibitoren von CYP1A in komplexen Umweltproben im Embryo darzustellen und ließ somit die Untersuchung der Rolle der CYP1A-Hemmung in Bezug auf die Toxizität zu. Eine geeignete Bildauswertungssoftware erlaubt schließlich eine Quantifizierung und anschließend eine statistische Auswertung der EROD-Induktion, was seinerseits zu einer Vergleichbarkeit sowie einer erhöhten Akzeptanz des modifizierten Live-Imaging-EROD-Assays beiträgt. Der modifizierte Acetylcholinesterase-Assay mit den Embryonen des Zebrabärblings erwies sich als ein geeignetes Werkzeug zum Nachweis der neurotoxischen Wirkungen bestimmter Substanzklassen sowie auch komplexer Umweltproben. Die vorliegende Studie belegt dabei auch, dass PAKs in komplexen Umweltproben zur AChE-Hemmung beitragen können. Embryonen des Zebrabärblings können ein geeignetes Modell zur Untersuchung neurotoxischer Wirkungen darstellen.
... The chronic exposure of zebrafish females (Danio rerio) for 5 days at two concentrations of DLM (0.5 and 1 μg/L) has determined degeneration of follicles, oogenesis delay and decreased number of primary oocyte (Yön et al., 2009). Eggs' hatching is affected too (Görge et al., 1990;Sharma & Ansari, 2010). Tthe effects of DLM can be observed due disrupting the intern metabolism. ...
The aim of this study was to identify the impact of pyrethroid-type insecticides upon organisms but also the negative influence upon the quality of people lives. Pyrethroids are synthetic chemical mixtures similar to pyrethrin-a compound from a Chrysantemum flowers extract. The degree of aggressiveness of pyrethroid insecticides depends on a number of factors such as dose, route of administration and exposure time. So, the nervous system is the most vulnerable to the toxicity of these compounds, especially to type II pyrethroids. Disturbances due to exposure to compounds of the pyrethroid range lead to disruption of essential functions of the nervous system mainly affecting locomotor coordination and activity of antioxidant enzymes. Deltamethrin (DLM) is a synthetic type II pyrethroid. In insects DLM poisoning may cause paralysis or death. Also, fish and rats have experienced malfunctions of the reproductive system. In humans, exposure to deltamethrin, depending on the amount taken, may lead to headache, nausea, dizziness, tremors, convulsions, fatigue and paralysis.
... Few reports are available on the effects of exposure to deltamethrin on zebrafish. Exposure of zebrafish to deltamethrin has resulted decrease in acetylcholinesterase activity with increase dose of deltamethrin at 0, 1 and 2 toxic unit (TU) exposures on LC 50 5.20 ug/L for 48 hours (Ren et al, 2016), enhancement of lipid peroxidation and reduction of reduced glutathione on exposure for 16 days (Sharma and Ansari, 2012), reduction in hatching rate of embroys at exposure of 0.8 ug/L concentration (Görge and Nagel, 1990) and locomotor deficits in larval zebrafish due to dopaminergic dysfunction on developmental exposure (Kung et al, 2015). However, the acute effect of deltamethrin to develop oxidative stress in zebrafish were not yet evaluated. ...
... They are ideal for DNA or mRNA injection, cell labeling, and transplantation. Therefore, zebrafish embryo microinjection (ZEM) has been widely used in many fields, such as genetics [21], virology [22], toxicology [23], endocrinology [24], immunology [25], and oncology [26]. It is playing an essential role in advancing the field of cell biology, such as in genetics, transgenics, assisted reproduction, and drug discovery. ...
Cell microinjection is a technique of precise delivery of substances into cells and is widely used for studying cell transfection, signaling pathways, and organelle functions. Microinjection of the embryos of zebrafish, the third most important animal model, has become a very useful technique in bioscience. However, factors such as the small cell size, high cell deformation tendency, and transparent zebrafish embryo membrane make the microinjection process difficult. Furthermore, this process has strict, specific requirements, such as chorion softening, avoiding contacting the first polar body, and high-precision detection. Therefore, highly accurate control and detection platforms are critical for achieving the automated microinjection of zebrafish embryos. This article reviews the latest technologies and methods used in the automated microinjection of zebrafish embryos and provides a detailed description of the current developments and applications of robotic microinjection systems. The review covers key areas related to automated embryo injection, including cell searching and location, cell position and posture adjustment, microscopic visual servoing control, sensors, actuators, puncturing mechanisms, and microinjection.
... Atrazine, a widely used herbicide in the US but banned in Europe, has been reported to increase axial deformities in zebrafish (Görge and Nagel, 1990). Whether atrazine interferes with Ar signaling is still unknown, but altered androgen metabolism was observed in fish exposed to atrazine (Moore and Waring, 1998;Spano et al., 2004). ...
Environmental toxicology has been expanding as growing concerns on the impact of produced and released chemical compounds over the environment and human health are being demonstrated. Among the toxic effects observed in organisms exposed to pollutants, those affecting skeletal tissues (osteotoxicity) have been somehow overlooked in comparison to hepato-, immune-, neuro- and/or reproductive toxicities. Nevertheless, sub-lethal effects of toxicants on skeletal development and/or bone maintenance may result in impaired growth, reduced survival rate, increased disease susceptibility and diminished welfare. Osteotoxicity may occur by acute or chronic exposure to different environmental insults. Because of biologically and technically advantagous features - easy to breed and inexpensive to maintain, external and rapid rate of development, translucent larvae and the availability of molecular and genetic tools - the zebrafish (Danio rerio) has emerged in the last decade as a vertebrate model system of choice to evaluate osteotoxicity. Different experimental approaches in fish species and analytical tools have been applied, from in vitro to in vivo systems, from specific to high throughput methodologies. Current knowledge on osteotoxicity and underlying mechanisms gained using fish, with a special emphasis on zebrafish systems, is reviewed here. Osteotoxicants have been classified into four categories according to the pathway involved in the transduction of the osteotoxic effects: activation/inhibition of membrane and/or nuclear receptors, alteration of redox condition, mimicking of bone constituents and unknown pathways. Knowledge on these pathways is also reported here as it may provide critical insights into the development, production and release of future chemical compounds with none or low osteotoxicity, thus promoting the green/environmental friendly chemistry.
... This also creates the opportunity to perform phenotypic comparisons between chemically-induced phenotypes (Hill et al., 2005) and mutant phenotypes. The small size of zebrafish larvae make them particularly wellsuited for chemical (experimental or environmental pollutant) screening because small volumes of chemicals can be used (Görge and Nagel, 1990, Hill et al., 2005, Taylor et al., 2010, Stewart et al., 2014. ...
In recognition of the importance of zebrafish as a model organism for studying human disease, we have created a web-based reference atlas of zebrafish microanatomy for comparing histology and histopathology between model systems and with humans (http://bio-atlas.psu.edu). Fixation, decalcification, embedding, and sectioning were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21°C for 24h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35M produced effective decalcification in 21-day-old juveniles through adults (≥~3Months). To improve section plane consistency in sets of larvae, our lab developed agarose array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue section discontinuity, a common barrier to creating quality sections of zebrafish but not mammalian tissues, was minimized by processing and embedding the formalin-fixed zebrafish tissues in plasticized forms of paraffin wax, and by periodic hydration of the block surface in ice water between sets of sections. Optimal H&E (Hematoxylin and Eosin) staining was achieved through refinement of standard protocols. High quality slide scans produced from glass histology slides were digitally processed to make web-based images similar to what is seen under the highest quality microscopes. Future directions for our bio-atlas include further improvements in tissue processing and the addition of slide collections from other model systems as well as 3D tools for visualizing tissue architecture.
... The zebrafish Danio rerio [Hamilton 1822] is a popular model organism in various biological fields due to its small size, modest maintenance, year-round reproduction, and its advantage as a representative vertebrate system (Grunwald and Eisen 2002;Hill et al. 2005;Lawrence 2007). As the chorion is transparent and thus allows the close observation of developmental progress, D. rerio is also frequently used in Early Life Stage (ELS) tests investigating direct (Görge and Nagel 1990;Roex et al. 2002;Shi et al. 2008) or indirect substance effects (Thellmann et al. 2014) on the embryonic development. The embryo test with D. rerio has been implemented as OECD Technical Guideline 236. ...
Beyond the mere detection of presence or absence of heart beat in zebrafish (Danio rerio) embryos in a fish embryo test conducted referring to the OECD TG 236 at 48 hpf (hours post fertilization) onwards, embryo heart rate may serve as an additional and very sensitive endpoint in ecotoxicological studies. But by including heart rate as a sublethal endpoint, care has to be taken of separating effects exerted by a tested compound from those exerted by temperature. Therefore, profound knowledge on the natural variation of zebrafish heart rates at defined temperatures as a basis for the assessment of gained results is mandatorily needed. As such continuous information in D. rerio is lacking from the literature, we designed a study covering a span of 12°C (from 18 to 30°C in steps of 2°C) to quantify the relationship between heart rate and temperature in D. rerio embryos 48 hpf. Conducting a multiple regression analysis, we found a considerably strong relationship between treatment temperature and the log10 of the heart rate, ranging from 82.8 beats per minute at 18°C to 218.0 beats per minute at 30°C. Our results therefore may serve as a reference for heart rates measured under normal conditions to be able to detect potential effects of contaminants in other studies when working under certain temperatures.
... External fertilization, rapid development, ease of treatment, transparency during embryonic and larval development, all allow monitoring of morphological abnormalities and are some of the attributes making zebrafish embryo testing an adequately sensitive fish bioassay (Cook et al., 2005;Fraysse et al., 2006;De Esch et al., 2012;Pérez et al., 2013). By now, numerous studies of pesticide toxicity have been performed using zebrafish embryos (Görge and Nagel, 1990;Lin et al., 2013;Watson et al., 2014). Many of them have confirmed that exposure of zebrafish to pesticides leads to various nonlethal malformations: yolk sac and pericardial edema, spine and body curvature, heart rate and neural disorders, and craniofacial and swim bladder deformations (Cook et al., 2005;Wiegand et al., 2001;Lin et al., 2013). ...
Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa® EC and GAT Cenit 36 CS, both containing 360 g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC50 values for the technical ingredient, Rampa® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5 mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120 hpf) was 12.1, 10.1 and 24.1 mg/l for technical clomazone, Rampa® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC50/EC50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.
... It causes increased mortality in fishes and benthic macro-invertebrates species, and results in changes in the zooplankton community structure[12,13]. The contamination of seawater can affect early embryonic development in sea organisms such as starfish[14], zebrafish[15]and sea urchins[16]. Lindane is also a carcinogenic endocrine disrupter and is known to exert damaging effects on the reproductive and nervous systems in mammals[17,18]. ...
Lindane is an organochlorine pesticide belonging to persistent organic pollutants (POPs)
that has been widely used to treat agricultural pests. It is of particular concern because of its
toxicity, persistence and tendency to bioaccumulate in terrestrial and aquatic ecosystems. In this
context, we assessed the role of bacteria associated with the sponge Hymeniacidon perlevis in lindane
degradation. Seven bacteria isolates were characterized and identified. These isolates showed
a remarkable capacity to utilize lindane as a sole carbon source leading to a percentage of residual
lindane ranging from 3% to 13% after 12 days of incubation with the pesticide. The lindane metabolite,
1,3–6-pentachloro-cyclohexene, was identified as result of lindane degradation and determined by
gas chromatography–mass spectrometry (GC–MS). The bacteria capable of lindane degradation
were identified on the basis of the phenotypic characterization by morphological, biochemical and
cultural tests, completed with 16S rDNA sequence analysis, and assigned to Mameliella phaeodactyli,
Pseudovibrio ascidiaceicola, Oceanicaulis stylophorae, Ruegeria atlantica and to three new uncharacterized
species. The results obtained are a prelude to the development of future strategies for the in situ
bioremediation of lindane.
... In another study, survival of juvenile zebrafish after 35 days was reduced by lindane (110 mg/l), atrazine (1300 mg/l), and deltamethrin (0.5 mg/l). Hatching rate was also reduced by deltamethrin (from 0.8 mg/l) (Gorge and Nagel, 1990). The male salmon response to female pheromones was shown to be affected at 0.04 mg atrazine/L, with reduced priming effect on milt and reduced plasma sex steroid levels as a result (Waring and Moore, 2004). ...
... The 48 h LC 50 value of deltamethrin in Nile tilapia larvae and fry were found as 1.17 and 1.70 µg/L, respectively, in the present work and here we report deltamethrin to be highly toxic to early life stages of fish. The effect of deltamethrin on the sensitive early life stages of zebra fish (Brachydanio rerio Hamilton, 1822), were examined by Görge and Nagel [11]. The development of larvae was influenced by deltamethrin. ...
Deltamethrin, a synthetic pyrethroid insecticide and contaminating aquatic ecosystems, was investigated in the present study for acute toxicity. The aim of this study was to evaluate LC50 values of deltamethrin on Nile tilapia (Oreochromis niloticus L.1758) larvae and fry after acute exposure to deltamethrin. The 48 h LC50 values for Nile tilapia larvae and fry were estimated as 1.17 and 1.70 μg/L, respectively. The bioassay experiments were repeated 3 times using the static test method. Data were evaluated using the U.S.E.P.A. LC50 computer program based on Finney's Probit Analysis Method. In addition, behavioral changes at each group for deltamethrin concentration were determined. All larvae and fry exposed to deltamethrin showed behavioral effects and the extent of behavioral changes increased with increased deltamethrin concentration. The results are significant for reporting deltamethrin highly toxic to fish early life stages under acute exposure.
... However, in the period of 0-48 hpf, a slightly slower increase of internal concentrations was observed. Görge and Nagel (1990) demonstrated for lindane that eggs showed a lower uptake than later larval stages. A study by Foekema et al. (2012) demonstrated that fish eggs had a lower uptake and excretion of PCBs compared to newly hatched larvae. ...
... Therefore, to evaluate the effect of pesticides on aquatic organisms are important. In this study, fingerlings and larvae were used, because toxins are very influential in the early stages of life, especially during the larval stage, as the effect of deltamethrin on the sensitive early life stages of zebrafish, Brachydanio rerio, were examined by Gӧrge and Nagel (1990). Deltamethrin can be highly toxic to fish. ...
The present research was performed to determine and compare acute toxicity of diazinon and deltamethrin to Spirlin larvae and fingerling. Diazinon and deltamethrin are common usefull agricultural pesticides. LC50 of 24 h, 48 h, 72 h and 96 h was attained by probit analysis software SPSS Version 16. Fish samples (21 fish in each test group) were exposed to different concentrations of diazinon and deltamethrin (diazinon: for fingerling between1-20 ppm and larvae 0.25-2 ppm, deltamethrin:for fingerling between 0.01-0.2 ppm and larvae 0.0025- 0.02) for 96 h and mortality were recorded. The LC5096h of diazinon for fingerlings and larvae were 7.88 and 0.69, respectively. The LC5096h of deltamethrin for fingerlings and larvae were 0.27 and 0.006 ppm, respectively. According to the results, larvae are more sensitive than fingerlings. In the present study, LC50 values indicated that deltamethrin is more toxic than diazinon to this species.
... As another alternative, fish embryos, especially zebrafish embryos, have repeatedly been suggested as potential alternatives to acute fish toxicity testing (Braunbeck et al. 2005b, Görge & Nagel 1990, Lange et al. 1995, Nagel 1986, 1997, 2002, Nagel & Isberner 1998. Upon receipt of the submission to the WNT from UBA in 2006, the OECD rapidly formed an ad hoc expert group in order to address comments received, which held several teleconferences and face-to-face meetings and eventually concluded that additional validation of the FET was required. ...
Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish (Danio rerio) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If-for ethical reasons-not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has-at least for the substances tested so far-compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.
... The turbot embryo-larvae in our study were even less sensitive to high atrazine concentrations than catfish embryo-larvae (at concentration> 65 µg/L) (Birge et al., 1983), Zebrafish (at concentration> 1300 µg/L) (Görge and Nagel, 1990) and Bluegill (at concentration >46 µg/L at 90 days) (Macek et al., 2003). Birge et al. (1983) suggested that exposure of fish embryos to atrazine may induce abnormalities. ...
The present study aimed to evaluate acute toxicity tests for three selected herbicides: Alachlor, Atrazine and Diuron using turbot flatfish. Larvae were more sensitive than turbot embryos to all pesticides. Median lethal concentrations of the selected pesticides during a 48 h and 96 h exposure for turbot embryos and larvae were, respectively: alachlor, 2177 and 2233; diuron, 10076 and 7826 and atrazine, 11873 and 9957 µg/L. On the basis of the obtained acute toxicity data, all compounds were included among substances highly toxic to fish, in the following order: alachlor> atrazine> diuron. At higher concentrations, pesticides caused a significant increase in embryo mortality. Surviving organisms suffered a significant decrease in hatching success, malformations (embryos), pericardial oedema and skeletal deformation (larvae). All herbicides appear to be teratogenic to the turbot early life stage. Furthermore, the three selected pesticides differed in their toxicity to the fish.
... The zebrafish has been extensively used in acute toxicological studies [reviewed by [12,16,5,17]]. Examples include the use of adult zebrafish for the testing of lead and uranium [18], malathion [19], colchicine [20], anilines [21], and metronidazole [22]; and the use of juveniles for testing agricultural biocides [23]. Zebrafish embryos are also being used in toxicity studies (reviewed by [24]). ...
Cannabinoids are natural or synthetic compounds related chemically to Δ -THC, the principle psychotropic constituent of
the hemp plant, Cannabis sativa L. Here, we examine the effects of the cannabinoids Δ -THC, WIN 55,212–2 and CP
55,940, and the cannabinoid antagonist (Am 251). Exposures were either acute (1–12h exposure at 108 hour post
fertilization [hpf]); or chronic (96h exposure starting at 24 hpf. Wild type zebrafish embryos (3,250, including controls),
were cultured individually in 250μl defined buffer in 96-well plates. Geometric range-finding was used to determine the
experimental concentrations At day 5, behavioural analysis (visual motor response test) was carried out in which
movement of individual larvae was analysed using automated video-tracking. With acute exposure, embryos showed a
biphasic response to the dark challenge with Δ -THC, WIN55, 212–2 and CP55,940. This response consisted of stimulation
of locomotor activity at low concentrations, suppression at high doses. With the antagonist AM251 alone, the locomotor
activity was suppressed at high concentrations. With chronic exposure, embryos habituated to the effects of all three
cannabinoids when assayed with the dark challenge phase. Furthermore, the excitation was ameliorated when antagonist
was co-administered with the cannabinoid. We conclude that cannabinoids have similar effects in zebrafish and mammals.
In particular, the acute exposure response resembles behavioural effects reported for adult rodents.
... The zebrafish (Danio rerio) model offers several advantages for screening a large number of compounds in drug discovery and toxicological studies. Because its genetic and physiologic makeup is comparable to that of mammals, this model has continued to gain popularity since its emergence in the 1990s (Gorge and Nagel, 1990). The toxicities of drugs and environmental pollutants are well conserved between humans and zebrafish (Van den Belt et al., 2000;Spitsbergen and Kent, 2003;Teraoka et al., 2003;Hill et al., 2005;Zon and Peterson, 2005;Reimers et al., 2006;Kari et al., 2007;Parng et al., 2007;Brittijn et al., 2009). ...
Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
... [18][19][20][21][22]. Examples include the use of adult zebrafish for the testing of lead and uranium, 23 malathion, 24 colchicine, 25 anilines, 26 and metronidazole, 27 and the use of juveniles and embryos for testing agricultural biocides. 28,29 Not only adult zebrafish, but also zebrafish embryos or larvae are used in toxicity studies (reviewed by Truong et al. 30 ). Examples of this application include the toxicity testing of nanoparticles [31][32][33][34] and chemical com-pounds from different pharmacological classes, 11 and developmental toxicity testing of ethanol [35][36][37] and other compounds. ...
Abstract The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay.
An acute toxicity study of imidacloprid on the larval health and metamorphosis of the giant freshwater prawn Macrobrachium rosenbergii larvae were carried out during the months of January–August 2021 at the aquatic animal raring and experimenting facility in the Cochin University of Science and Technology, Kerala, India. Imidacloprid is a broad-spectrum neonicotinoid insecticide used against sucking pests infecting agricultural crops as both contact poison and stomach poison. The present study investigated the adverse effects of imidacloprid on the survival rate, feeding behaviour, larval quality and incidence of metamorphosis in Macrobrachium rosenbergii larvae. The 48 h median lethal concentrations (LC50 values) and safe concentration of imidacloprid determined for different larval stages (Zoea I–XI) of M. rosenbergii ranged between 0.000635–0.001388 mg l-1 and 0.000055–0.000191 mg l-1, respectively. Larvae exposed to imidacloprid for 48 h showed decrease in survival rate with increase in concentration of imidacloprid. The results of larval condition index (LCI) that indicates the larval quality showed that imidacloprid reduced the larval health significantly (p<0.05) at high concentrations and is potent to delay the incidence of metamorphosis. The feeding rate of larvae exposed to different concentrations of imidacloprid were found to be significantly (p<0.05) reduced due to paralytic effect of imidacloprid. The present work provides relevant data beneficial for the risk assessment of imidacloprid in M. rosenbergii.
In the present investigation, the toxicity of imidacloprid was tested on the embryos (fertilized eggs) and hatchlings of Cyprinus carpio in terms of mortality (%), hatching success (%) and abnormal/malformed hatchling along with their growth performance in toxicant medium. Reported data from acute toxicity tests of imidacloprid was statistically analyzed by Probit Analysis Method and 48 h (hour) LC50 was reported as 78 ppm for embryos of C. carpio. Three sublethal doses of imidacloprid i.e., 7.8 ppm (T1), 15.6 ppm (T2) and 23.4 ppm (T2), were selected for further experimentation along with control. The findings revealed that mortality of exposed eggs (%) was significantly (p<0.05) increased in dose dependent manner as a result hatching rate (%) was decreased as compared to control. Similarly, per cent of abnormal hatchling was also increased significantly (p<0.05) with increase in pesticide concentration. Various types of abnormalities has been reported in different body regions such as whole length curved body, stunted growth, hatchling with single eye, malformed head, flexure in the tail and deformed yolk sac etc. Growth rate was observed as length gain in centimeter (cm), weight gain in grams (g) and growth per cent gain in body weight of hatchling, which was also found to be declined significantly (p<0.05) with increase in pesticide concentration. The findings of the present investigation suggest that imidacloprid has significant effect on the early developmental stages, induces malformations and also decline the growth rate of hatchlings.
Microinjection technique is one of the essential methodologies that used widely in zebrafish research. Microinjection is utilized to perform genetic manipulations within the developing zebrafish model. This technique is used to study a wide range of genetic diseases and gene of interest role in early developmental processes. Thus, quality control for microinjection is an essential factor to ensure experimental reproducibility and consistency. In this technical note, in vitro transcribed synthetic mRNA encoding green fluorescence protein (eGFP), and red fluorescent protein (m-cherry) as well as fluorescein and rhodamine fluorescent dyes were injected into a single-cell zebrafish embryo for volume quality control. Given the importance of having quality control system and methodology to yield similar genetic manipulation within the zebrafish embryo:
•We aimed to establish the unified delivery of injected material into zebrafish one cell stage embryo.
•We aimed to establish consistency of the injected volume into mineral oil droplets that will serve as a quality control parameter to conforms quality control practice to ensure the reproducibility of the microinjection technique.
•The calibration of microinjection droplet size resulted in the visualization of fluorescent protein and dyes in the zebrafish embryo with precise volumes of delivered materials under the control of needle opening, injection pressure and time.
Anthropogenic surface and ground water contamination by chemicals is a global problem, and there is an urgent need to develop tools to identify and elucidate biological effects. Contaminants of emerging concern (CECs) are not typically monitored or regulated and those with known or suspected endocrine disrupting potential have been termed endocrine disrupting chemicals (EDCs). Many CECs are known to be neurotoxic (e.g., insecticides) and many are incompletely characterized. Behavioral responses can identify chemicals with neuroactive properties, which can be relevant to EDC mechanisms (e.g., neuroendocrine disturbances). Two freshwater species, Daphnia pulex and Danio rerio, were evaluated for swimming behavior alterations resulting from 24-hr exposure to 9 CECs: triclosan, triclocarban, chlorpyrifos, dieldrin, 4-nonylphenol, bisphenol-A, atrazine, metformin, and estrone. This is the first step in the development of a bioassay for detecting estrogenic and/or anti-androgenic activity with the goal to evaluate complex mixtures of uncharacterized contaminants in water samples. The second step examines transcriptome alterations following chemical exposure and will be reported in a subsequent report. Significant differences in the swimming behavior response and sensitivity were found across chemicals within a species and across species for a given chemical in this unique optical bioassay system. In the concentration ranges studied, significant behavioral alterations were detected for 6 of 9 CECs for D. pulex and 4 of 9 CECs for D. rerio. These results underscore the utility of this bioassay to identify behavioral effects of sublethal concentrations of CECs as a first step in enabling the exploration of transcriptomic alterations as a second EDC detection step.
In the last decades, the use and misuse of pesticides in the agriculture have increased, having a severe impact on ecosystems and their fauna. Although the various effects of pesticides on biodiversity have been already documented in several studies, to our knowledge no consistent overview of the impact of pesticides in vertebrates, both terrestrial and aquatic, is available. In this review, we try to present a concise compilation of the teratogenic effects of pesticides on the different classes of vertebrates – mammals, birds, reptiles, amphibians and fish.
Liver cancer remains to be a major health concern in the world today. Several major risk factors such as hepatitis viral infection and non-alcoholic steatohepatitis have been well established for causing liver cancer, but the contribution of environmental pollutants to liver inflammation and carcinogenesis remains poorly studied. Here, we aimed at the development of a rapid assay to test selected environmental toxicants for their potential roles in induction of inflammation and stimulation of liver tumorigenesis. By using an established kras oncogene transgenic zebrafish model for liver cancer, we tested a total of eight selected chemicals. First, using LPS (lipopolysaccharides) as a positive control, we confirmed its effects on induction of inflammation and stimulation of liver tumorigenesis as indicated by increases of neutrophils and the size of oncogenic livers respectively. Next, we tested two heavy metals (arsenic and chromium) and five organic toxicants (bisphenol A, lindane, N-nitrosodiethylamine, and 3,3′,4,4′,5-pentachlorobiphenyl [PCB126], and 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]). We observed a good correlation on induction of inflammation and their ability for stimulation of liver tumorigenesis. Most toxicants, namely chromium, bisphenol A, lindane, N-nitrosodiethylamine, and PCB126, resulted in increased inflammation and liver tumorigenesis, while arsenic and TCDD had opposite effects. Thus, our study established a screening system to rapidly assess the effects of candidate chemicals on liver tumorigenesis and inflammation.
Highlighting latest advances in genetics and biochemistry, the completely revised Third Edition reviews the field from basic science, clinical, epidemiological, and regulatory perspectives. Contributions from top opinion leaders in the field bring together developments in molecular embryology and cell biology as they apply to problems in developmental toxicology. It covers testing of pharmaceutical and environmental agents and interpretation of developmental toxicology data, highlighting mathematical and statistical techniques, as well as the effects of toxic exposure on the functional development of various organs. The relationship between maternal and developmental toxicology is examined, in addition to current techniques for studying chemical disposition, metabolism, and placental transfer. Close attention is given to the regulatory aspects of testing and risk assessment. Pre and postconceptional clinical care and genetic factors in clinical developmental toxicology are also discussed.
The effects of 2 currently used commercial pesticide formulations on Pacific sockeye salmon (Oncorhynchus nerka), from fertilization to emergence, were evaluated in a gravel-bed flume incubator that simulated a natural streambed. Embryos were exposed to atrazine at 25 µg/L (low atrazine) or atrazine at 250 µg/L (high atrazine) active ingredient (a.i.), and chlorothalonil at 0.5 µg/L (low chlorothalonil) or chlorothalonil at 5 µg/L a.i. (high chlorothalonil) and examined for effects on developmental success and timing, as well as physical and biochemical growth parameters. Survival to hatch was reduced in the high chlorothalonil group (55% compared with 83% in controls), accompanied by a 24% increase in finfold deformity incidence. Reduced alevin condition factor (2.9–5.4%) at emergence and elevated triglyceride levels were seen in chlorothalonil-exposed fish. Atrazine exposure caused premature hatch (average high atrazine time to 50% hatch [H50] = 100 d postfertilization [dpf]), and chlorothalonil exposure caused delayed hatch (high chlorothalonil H50 = 108 dpf; controls H50 = 102 dpf). All treatments caused premature emergence (average time to 50% emergence [E50]: control E50 = 181 dpf, low chlorothalonil E50 = 175 dpf, high chlorothalonil E50 = 174 dpf, high atrazine E50 = 175 dpf, low atrazine E50 = 174 dpf), highlighting the importance of using a gravel-bed incubator to examine this subtle, but critical endpoint. These alterations indicate that atrazine and chlorothalonil could affect survival of early life stages of sockeye salmon in the wild. Environ Toxicol Chem 2016;9999:1–11.
The triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine) is one of the most used pesticides in North America. Atrazine is principally used for control of certain annual broadleaf and grass weeds, primarily in corn but also in sorghum, sugarcane, and, to a lesser extent, other crops and landscaping. Atrazine is found in many surface and ground waters in North America, and aquatic ecological effects are a possible concern for the regulatory and regulated communities. To address these concerns an expert panel (the Panel) was convened to conduct a comprehensive aquatic ecological risk assessment. This assessment was based on several newly suggested procedures and included exposure and hazard subcomponents as well as the overall risk assessment. The Panel determined that use of probabilistic risk assessment techniques was appropriate. Here, the results of this assessment are presented as a case study for these techniques. The environmental exposure assessment concentrated on monitoring data from Midwestern watersheds, the area of greatest atrazine use in North America. This analysis revealed that atrazine concentrations rarely exceed 20 μg/L in rivers and streams that were the main focus of the aquatic ecological risk assessment. Following storm runoff, biota in lower-order streams may be exposed to pulses of atrazine greater than 20 μg/L, but these exposures are short-lived. The assessment also considered exposures in lakes and reservoirs. The principal data set was developed by the U.S. Geological Survey, which monitored residues in 76 Midwestern reservoirs in 11 states in 1992-1993. Residue concentrations in some reservoirs were similar to those in streams but persisted longer. Atrazine residues were widespread in reservoirs (92% occurrence), and the 90th percentile of this exposure distribution for early June to July was about 5 μg/L. Mathematical simulation models of chemical fate were used to generalize the exposure analysis to other sites and to assess the potential effects of reduction in the application rates. Models were evaluated, modified, and calibrated against available monitoring data to validate that these models could predict atrazine runoff. PRZM-2 overpredicted atrazine concentrations by about an order of magnitude, whereas GLEAMS underpredicted by a factor of 2 to 5. Thus, exposure models were not used to extrapolate to other regions of atrazine use in this assessment. The effects assessment considered both freshwater and saltwater toxicity test results. Phytoplankton were the most sensitive organisms, followed, in decreasing order of sensitivity, by macrophytes, benthic invertebrates, zooplankton, and fish. Atrazine inhibits photophosphorylation but typically does not result in lethality or permanent cell damage in the short term. This characteristic of atrazine required a different model than typically used for understanding the potential impact in aquatic systems, where lethality or nonreversible effects are usually assumed. In addition, recovery of phytoplankton from exposure to 5 to 20 μg/L atrazine was demonstrated. In some mesocosm field experiments, phytoplankton and macrophytes were reduced after atrazine exposures greater than 20 μg/L. However, populations were quickly reestablished, even while atrazine residues persisted in the water. Effects in field studies were judged to be ecologically important only at exposures of 50 μg/L or greater. Mesocosm experiments did not reveal disruption of either ecosystem structure or function at atrazine concentrations typically encountered in the environment (generally 5 μg/L or less). Based on an integration of laboratory bioassay data, field effects studies, and environmental monitoring data from watersheds in high-use areas in the Midwestern United States, the Panel concluded that atrazine does not pose a significant risk to the aquatic environment. Although some inhibitory effects on algae, phytoplankton, or macrophyte production may occur in small streams vulnerable to agricultural runoff, these effects are likely to be transient, and quick recovery of the ecological system is expected. A subset of surface waters, principally small reservoirs in areas with intensive use of atrazine, may be at greater risk of exposure to atrazine. Therefore, it is recommended that site-specific risk assessments be conducted at these sites to assess possible ecological effects in the context of the uses to which these ecosystems are put and the effectiveness and cost-benefit aspect of any risk mitigation measures that may be applied.
Humic acid (HA) is a major component of dissolved organic matter, is ubiquitous in the aquatic environment and influences the biological toxicity of organic pollutants. In this study, we investigated the cytochrome P450 1A (CYP 1A) mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in the gills and liver of zebrafish following exposure to the s-triazine herbicide prometryn with or without HA present. Prometryn induced both CYP 1A mRNA expression and EROD activity. The CYP 1A mRNA expression of zebrafish that were exposed to a combination of prometryn and HA was increased compared to those exposed to prometryn alone. A likely cause for CYP 1A induction is the impact of special components of HA, functioning as aryl hydrocarbon receptor (AHR) agonists. In combination with HA, these increase prometryn levels in tissues. Similar results for EROD activity were evident. In our time course study, CYP 1A mRNA expression reached maximum values during 24 h. This revealed CYP 1A mRNA transcription as a comparatively sensitive toxicity index. In a recovery experiment, we found a faster decrease of CYP 1A mRNA expression to control levels (CK) in gills compared to liver tissue. Following exposure to HA, CYP 1A mRNA expression in liver tissue displayed a faster decrease to CK levels. HA induced enhanced metabolic rates for prometryn. In contrast, recovery regularity of CYP 1A expression in gills was independent of the presence of HA. This result indicates different detoxification mechanisms for HA in liver and gills.
Risk assessments for compounds released to the environment typically rely on single-species toxicity studies to predict concentrations at which effects may be observed. These single-species toxicity studies are usually conducted with a few species, cultured under optimum conditions (diet, temperature, light, etc.) and tested in clean water with constant exposure to the compound of interest. Chronic toxicity data are then extrapolated to the ecosystem during risk assessments to predict concentrations that will not adversely impact the environment. Several approaches have been developed that apply statistical methods to estimate toxicant concentrations adversely affecting a small percentage of single species (e.g., 5%). There are several rarely stated, and infrequently tested, biological and statistical assumptions required to make this extrapolation. One test of the ability to use single-species toxicity data to protect ecosystems is to compare effects on single species with effects on experimental and natural ecosystems (e.g., microcosms, model ecosystems, field). Towards this end, we summarized the chronic single species and experimental ecosystem data on a variety of substances (n = 11), including heavy metals, pesticides, surfactants, and general organic and inorganic compounds. Single-species data were summarized as genus-specific geometric means using the NOEC or EC20 concentration. Genus mean values spanned a range of values with genera being affected at concentrations above and below those causing effects on model ecosystems. Geometric mean model ecosystem no effect concentrations corresponded to concentrations expected to exceed the NOEC of 10 to 52% of genera. This analysis suggests that laboratory-generated single species chronic studies can be used to establish concentrations protective of model ecosystem, and likely whole ecosystem, effects. Further, the use of the 5% of genera affected level is conservative relative to mean model ecosystem data but is a fairly good predictor of the lower 95% confidence interval on the mean model ecosystem NOEC.
Cytopathological alterations in hepatocytes of fish following exposure to xenobiotic compounds represent a powerful tool to reveal sublethal effects of chemicals and to elucidate underlying modes of action. The present communication reviews the available information about ultrastructural changes in fish liver as well as isolated hepatocytes; whereas the discussion of in vivo effects is primarily focused on data from rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), the presentation of in vitro data has been restricted to results from experiments with rainbow trout hepatocytes due to a lack of data from studies with hepatocytes from other species. Both in vivo and in vitro exposure to xenobiotics results in sensitive, selective, and, especially in in vitro experiments, extremely rapid responses of hepatocytes, which, however, may be confounded by internal parameters (species, sex, age, hormonal status) and external parameters (temperature, nutrition, duration of exposure). Thus, transfer of results and conclusions from one experiment to another is usually not possible. Likewise, in vitro results may not necessarily be extrapolated to the situation in intact fish, and effects by acute toxic exposure cannot be translated into sublethal effects.
The aim of the study was to investigate the effect of different concentrations of carbendazim, a commonly used fungicide, on the embryonic development and hatching of Prussian carp, Carassius gibelio (Bloch). The egg samples were incubated with different concentrations of carbendazim - 0.001, 0.006, 0.036, 0.216, and 1.296 mg l⁻¹, for 98 hours. The results of the data showed that carbendazim at concentrations of 0.036 mg l⁻¹ and higher were lethal for Prussian carp embryos. There were no significant differences in the hatching rate, percentage of hatched larvae, or in the percentage of deformed larvae exposed to the two lowest concentrations of carbendazim tested (0.001 and 0.006 mg l⁻¹).The results of the present study showed that carbendazim is harmful to Prussian carp embryos at concentrations of 0.036 mg l⁻¹ and higher.
The present study was conducted to investigate the effect of sub-lethal doses of Cypermethrin (CYP) on
the survival and morphological and biochemical aspects of rohu (Labeo rohita) during early developmental
stages. Newly fertilized eggs were incubated in the presence of sub-lethal concentration of CYP
(20% of LC50 i.e., 8.43 mg L�1) in a well designed flow through system. Increased mortality was found with
increase in exposure time. Deformities like eroded yolk and margins, elongated yolk sac, larvae with
short tail, no eyes or larvae with zigzag movement and paralyzed larvae were observed in CYP treated
group from blastula to fry stage. The activities of antioxidant like Catalase (CAT), Peroxidase (POD) and
Lactoperoxidase (LPO) were decreased from 24 to 96 h in the control group whereas increased in CYP
treated group. The Glutathione reducatse (GR) activity was also increased with time in both the control
and treated groups; however, the activity was significantly higher in CYP treated group. Similarly, the
whole body cortisol level showed an increasing trend with time in both control and treated groups.
However, in CYP exposed group the cortisol level was considerably higher after 24 h exposure but statistically
comparable to the control group after 96 h. It can be concluded from the present results that
CYP even at very low concentration adversely affects the early development of L. rohita and enhances
mortality. The obtained results reveal that CYP may affect the wild population of fish by inducing
oxidative stress and modulating stress response during early ontogenesis.
Suspended sediments can decrease apparent bioconcentration factor of organic pollutants through adsorption. However, it is not clear whether this process can weaken the toxicity of organic pollutants to non-target aquatic organisms. Sediments from Erdaozha, Haihe River were chosen as suspended sediments with atrazine as target pollutant and Brachydaniorerio as target organism to conduct the acute toxic experiment. Results show that without suspended sediments, the 96 h-LC50 of atrazine to Brachydaniorerio is 29.06 mg·L-1 with 95% confidence interval of 24.41~40.70 mg·L-1. When suspended sediments are 7500 mg·L-1 and 15000 mg·L-1, the 96 h-LC50 are 30.74 mg·L-1 and 39.51 mg·L-1, respectively, and their corresponding confidence intervals are 27.17~40.91 mg·L-1 and 30.43~126.93 mg·L-1, respectively. The NOECs of the three contaminate series above are 3, 9 and 15 mg·L-1, respectively. Suspended sediments can weaken the acute toxic of atrazine to Brachydaniorerio.
As a new class of agricultural insecticides, synthetic pyrethroids are widely used to control insect pests. Synthetic pyrethroids have been shown to enter the aquatic environment from agricultural runoff or drift from aerial and ground-based spraying applications posing threat to fishes which are less tolerant to pesticides through direct exposure. These insecticides interfere with the sodium channel of the nervous system resulting in prolonged sodium tail current. Widespread application of these chemicals has warranted the attention of the ecologist to understand the impact of these chemicals on the aquatic environment. In this perspective, an updated account of toxicological evaluation of three type II synthetic pyrethroids, viz. deltamethrin, cypermethrin and fenvalerate in terms of their physico-chemical, metabolic, hematological, histological, behavioral and reproductive aspects with respect to the fishes has been presented which may be useful for policy makers, academics, environmental scientists and agricultural professionals needing ready access to this information. The aim of the present synoptic literature appraisal was to summarize the main effect of current use, type II synthetic pyrethroids (deltamethrin, cypermethrin and fenvalerate) on aquatic environment due to their persistence and accumulation. This article will focus on non-target organisms in inland fresh water environment with special reference to fin fishes and will critically evaluate the toxicity of these pyrethroids in terms of growth inhibition, metabolic disorders, neurotoxicity, reproductive failure, enzymatic dysfunction, haematological alterations, and tissue damages. The rationalized information in this milieu may be useful in ecological risk evaluation and human health management as fish serves as an important bio-indicator for aquatic systems health.
To assess the potential impacts of agricultural pesticides on tropical freshwater ecosystems, the present study developed temperature- specific, freshwater species protection concentrations (i.e. ecotoxicity threshold values) for 8 pesticides commonly detected in Australia's tropical freshwaters. As relevant toxicity data for native tropical freshwater species to assess the ecological risks were mostly absent, scientifically robust toxicity data obtained at ≥20 °C were used for ecologically relevant taxonomic groups representing primary producers and consumers. Species sensitivity distribution (SSD) curves were subsequently generated for predicted chronic exposure using Burrlioz 2.0 software with mixed chronic and converted acute data relevant to exposure conditions at ≥20 °C. Ecotoxicity threshold values for tropical freshwater ecosystem protection were generated for ametryn, atrazine, diuron, metolachlor and imidacloprid (all moderate reliability), and simazine, hexazinone and tebuthiuron (all low reliability). Using these SSD curves, the retrospective risk assessments for recently reported pesticide concentrations highlight that the herbicides ametryn, atrazine and diuron are of major concern for ecological health in Australia's tropical freshwater ecosystems. The insecticide imidacloprid also appears to pose an emerging threat to the most sensitive species in tropical freshwater ecosystems. Exposed temperature-specific approach may be applied for the development of water quality guideline values for other environmental contaminants detected in tropical freshwater ecosystems until reliable and relevant toxicity data are generated using representative native species. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Suspended sediments can decrease the apparent bio-concentration factor of organic pollutants through adsorption. However, whether this process also weakens the toxicity of organic pollutants to non-target aquatic organisms is not clear. Therefore, natural sediments were chosen as suspended sediment examples in this research applying atrazine as the target pollutant and Brachydanio rerio (more recently, Danio rerio (Zebrafish)) as the target organism to conduct acute toxicity experiments. The concentration of atrazine in aqueous solution was measured as a time series. Results show that without suspended sediments, the 96-h LC50 of atrazine to Brachydanio rerio is 29.06 mg/l at 95 % confidence interval (24.41 to 40.70 mg/l). For suspended sediments of 7500 and 15,000 mg/l, the LC50 (i.e., concentration resulting in 50 % mortality) equates to 30.74 and 39.51 mg/l, respectively, and the corresponding confidence intervals are between 27.17 and 40.91 mg/l and between 30.43 and 126.93 mg/l in that order. Probit analysis, which is a type of regression used to analyze binomial response variables, was applied using the Statistical Package for the Social Sciences. For the series of no suspended solids (SS), 7500 and 15,000 mg/l SS, the so-called no-observed-effective concentrations were 3, 9, and 15 mg/l, correspondingly. The uptake quantity and uptake rate of atrazine by B. rerio according to atrazine concentrations in the aqueous solution were computed. The research indicates that suspended sediments can decrease the absorbed rate of atrazine by B. rerio. Thus, suspended sediments weaken the acute toxicity of atrazine to B. rerio.
Abstract Zebrafish embryos are widely used as a model to monitor the effect of chemicals on their survival and hatching at different time epochs. This experimental design generates longitudinal data in which the observations for a given subject are correlated and they are statistically independent across the subjects. This particular nature of the observations suggests the use of generalized estimating equation (GEE) methodology for performing meaningful statistical analysis. However, it has been observed that the researchers (1 , 2) working in this area have been routinely employing statistical methodologies such as analysis of variance (ANOVA) if the data are continuous and logit or probit models if the data are discrete. In our opinion, it is grossly incorrect to use these methods as they do not take into account the correlation structure mentioned above. The sole purpose of this article is to bring out this serious flaw clearly to the attention of the researchers. For illustration, we have studied the effects of two Ayurvedic bhasmas-Tamra bhasma and Suwarnamakshik bhasma-on survival and hatching of zebrafish embryos over certain time duration. The statistical analysis using GEE reveals a weak promotional effect of Suwarnamakshik bhasma and an inhibitory effect of Tamra bhasma on hatching.
The use of chemicals by society has many benefits but contamination of the environment is an unintended consequence. One example is the organochlorine compound hexachlorocyclohexane (HCH). During the 1980s, when HCH was banned in many countries, the brominated flame retardant, hexabromocyclododecane (HBCD), found increasing use. The persistent, bioaccumulative, and toxic characteristics of HBCD are, 30 years later, likely to warrant global action on production and use under the Stockholm Convention on persistent organic pollutants. Historical lessons have taught us that we need to control the use of chemicals and programs are in place worldwide in an attempt to do so.
Small molecules or analytes present in trace level are difficult to be detected directly using conventional surface plasmon resonance (SPR) sensor, due to its small changes in the refractive index induced by the binding of these analytes on the sensor surface. In this paper, a new approach that combines SPR sensor technology with Fe3O4 magnetic nanoparticles (MNPs) assays is developed for directly detecting of deltamethrin in soybean. The Fe3O4 MNPs conjugated with antibodies specific to antigen serves as both labels for enhancing refractive index change due to the capture of target analyte, and "vehicles" for the rapid delivery of analyte from a sample solution to the sensor surface. Meanwhile, SPR direct detection format without Fe3O4 MNPs and gas chromatography (GC) analysis were conducted for detection of deltamethrin in soybean to demonstrate the amplification effect of Fe3O4 MNPs. A good linear relationship was obtained between SPR responses and deltamethrin concentrations over a range of 0.01-1ng/mL with the lowest measurable concentration of 0.01ng/mL. The results reveal that the detection sensitivity for deltamethrin was improved by 4 orders of magnitude compared with SPR direct detection format. The recovery of 95.5-119.8% was obtained in soybean. The excellent selectivity of the present biosensor is also confirmed by two kinds of pesticides (fenvalerate and atrazine) as controls. This magnetic separation and amplification strategy has great potential for detection of other small analytes in trace level concentration, with high selectivity and sensitivity by altering the target-analyte-capture agent labeled to the carboxyl-coated Fe3O4 MNPs.
Two 10-month exposures of malathion and butoxyethanol ester of 2,4-D under continuous-flow conditions were made to determine the effect of these pesticides on reproduction and growth of fathead minnows, Pimephales promelas Rafinesque. Results show that 1/45 and 1/19 of the 96-hour median tolerance limit (TLm) concentrations of malathion and butoxyethanol ester, respectively, will not harm growth and reproduction of fathead minnows during continuous exposure. We suggest that these two fractions applied to TLm concentrations determined for other species and other types of water should estimate accurately safe concentrations for other fishes to these pesticides.
Toxicity data from 25 years of fish life cycle chronic, partial chronic and early life stage tests are examined to determine the utility of the standard fish chronic toxicity endpoints, in particular the growth response, to chemical hazard evaluations. The 173 tests include exposure to metals, pesticides, unclassified organics, inorganic compounds, detergent chemicals and complex effluents. Fry survival was significantly reduced in 57% of all tests at the lowest effect concentration. Fry growth was reduced in 36% and egg hatching in 19% of the tests. Only 60% of the tests include exposure of adult organisms. Reproduction was reduced in 30% of these tests. Adult survival (13%) and growth (5%) were seldom reduced at the lowest effect concentration. Fry survival and growth were very often correlated as equally sensitive. Fry growth was the single most sensitive response in only 14% of the tests reported. In the absence of the growth response data for these 20 chemicals/effluents, the fry survival data would have provided MATC values within a factor of 2 to 7 (). The conclusion is that, as presently derived, the growth response could be deleted from routine applications of the fish early life stage test. The net result would be a significant reduction in the duration and cost of screening tests with no appreciable impact on estimating MATCs for chemical hazard assessments. Possible explanations for the insensitivity of the standard growth response, alternatives, and other relevant observations of this data base are discussed.
Early life stages of fathead minnows (Pimephales promelas) were exposed to permethrin and AC 222, 705 and snails (Helisoma trivolvis) were exposed to permethrin in continuous flow-through exposures for approximately 30 days. Saturated solutions of each pesticide were used to avoid the use of solvents.Survival of newly hatched larvae and early juveniles was found to be the most sensitive measure of effect on fathead minnows of both pesticides. AC 222, 705 was approximately 20 times more toxic to fathead minnows than permethrin by the end of the test period. Based on the chronic limits, as defined in these tests, the predicted chronic no-effect concentrations for fathead minnows were between 0.66 and 1.4 μ/l for permethrin and 0.03 and 0.07 μ/l for AC 222, 705. The no-effect concentration for permethrin and snails was ≥ 0.33 μg/l. The chronic values for these compounds were approximately one-sixteenth and one-fourth of the corresponding 96-h LC50 values, respectively, for fathead minnows in Lake Superior water.The mean bioconcentration factors (BCFs) for permethrin were 2800 for fathead minnows and 800 for snails. The mean BCF for AC 222, 705 and fathead minnows was 4000. Residue concentrations for both pesticides increased with increased water concentrations.
Let $x$ and $y$ be two random variables with continuous cumulative distribution functions $f$ and $g$. A statistic $U$ depending on the relative ranks of the $x$'s and $y$'s is proposed for testing the hypothesis $f = g$. Wilcoxon proposed an equivalent test in the Biometrics Bulletin, December, 1945, but gave only a few points of the distribution of his statistic. Under the hypothesis $f = g$ the probability of obtaining a given $U$ in a sample of $n x's$ and $m y's$ is the solution of a certain recurrence relation involving $n$ and $m$. Using this recurrence relation tables have been computed giving the probability of $U$ for samples up to $n = m = 8$. At this point the distribution is almost normal. From the recurrence relation explicit expressions for the mean, variance, and fourth moment are obtained. The 2rth moment is shown to have a certain form which enabled us to prove that the limit distribution is normal if $m, n$ go to infinity in any arbitrary manner. The test is shown to be consistent with respect to the class of alternatives $f(x) > g(x)$ for every $x$.
The study of developmental patterning has been facilitated by the availability of mutations that produce changes in cell fate, in animals such as Caenorhabditis elegans and Drosophila melanogaster. We now describe a zygotic lethal mutation in the zebrafish, Brachydanio rerio, that also changes how particular embryonic cells develop. Severe pattern deficiencies are observed that are restricted to a single body region, the trunk. The mutation may directly affect mesoderm, as somites do not form in the trunk. Head and tail structures, including tail somites, are relatively undisturbed. The earliest detected expression of the mutation is during gastrulation, when movements of mesodermal cells occur incorrectly. We injected prospective trunk mesodermal cells with lineage tracer dye and observed that in mutants these cells may enter a new body region, the tail, and there may express a new fate appropriate for the changed position.
Environmental toxicant exposure commonly vary in terms of duration and concentration. However, laboratory toxicity tests usually entail continuous exposures to constant concentrations. We compared survival, growth, and toxicant accumulation in early life-stage steelhead trout intermittently or continuously exposed to fenvalerate (FV) for 70 d after fertilization. Acute lethality was assessed in ancillary 96-h LC50 determinations with juvenile fish. Intermittent exposures were daily 4.5-h introductions of toxicant, and continuous exposures were to constant concentrations. All tests were conducted in a flow-through dilution apparatus, and mean concentrations for the entire exposure period were calculated for comparisons between regimens. The respective 96-h LC50 values for intermittently and continuously exposed were 88 and 172 ng/l. In the subchronic study, marked lethality (32%) and reduced terminal weight (50%) were found following exposures to cyclic FV concentrations that yielded an average of 80 ng/l (peak of 461). Continuous exposure to 80 ng FV/l did not affect these parameters. At mean FV concentrations above 20 ng/l, bioaccumulation was greater following intermittent than continuous exposure. Interaction of partitioning and elimination processes may partially explain differences in FV accumulation and subsequent toxicity.
Information on the biological activity of a substance is often obtained from experiments in which the treatments comprise a series of doses of the substance and a zero dose control. The aim of such experiments, particularly in toxicity studies, may be to determine the lowest dose, if any, at which there is activity. A new test procedure is proposed for this situation in the case where the activity of interest is a change in the mean of a single response variate. Tables are given whereby this procedure can be used when all treatments are equally replicated. The advantages of this test over other established tests are discussed.
Effects of Exposure to Heavy Metals on Selected Freshwater Fish
- Sauter
Die Toxizität der Pyrethroide bei Süßwasserorganismen
- Kulzer
Ichtyotoxicite de la decamethrine vis-a-vis de Salmo trutta en fonction de l'age et des conditions experimentales
- Lhoste
Chronic Toxicity of Atrazine to Selected Aquatic Invertebrates and Fishes
- Macek