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Comparative toxicity of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin to seven freshwater fish species during early life‐stage development
Abstract
The toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to fathead minnow (Pimephales promelas), channel catfish (Ictalurus punctatus), lake herring (Coregonus artedii), medaka (Oryzias latipes), white sucker (Catastomus commersoni), northern pike (Esox lucius), and zebrafish (Danio danio) were observed during early life-stage development after waterborne exposure of fertilized eggs. Species sensitivity based on TCDD-Cegg (TCDD concentration in eggs) was determined by effects observed over a 32-d period for all species except lake herring in which a 100-d period was used. Signs of TCDD toxicity, including edema, hemorrhaging, and craniofacial malformations were essentially identical to those observed in salmonids following TCDD egg exposure and preceded or accompanied mortality most often during the period from hatch through swim-up. The no-observed-effect concentrations and lowest-observed-effect concentrations, based on significant decreases in survival and growth as compared to the controls, ranged from 175 and 270 pg/g for lake herring to 424 and 2,000 pg/g for zebrafish, respectively. Shapes of concentration–response curves, expressed as TCDD-Cegg versus percent mortality, were similar for all species and were consistently steep suggesting that the mechanism of action of TCDD is the same among these species. The LCegg50s (concentrations in eggs causing 50% lethality to fish at test termination) ranged from 539 pg/g for the fathead minnow to 2,610 pg/g for zebrafish. Comparisons of LCegg50s indicate that the tested species were approximately 8 to 38 times less sensitive to TCDD than lake trout, the most sensitive species evaluated to date. When LCegg50s are normalized to the fraction lipid in eggs (LCegg,l50s), the risk to early life stage survival for the species tested ranges from 16- to 180-fold less than for lake trout.
... The AhR is a ligandactivated transcription factor involved in the regulation of thousands of genes across numerous physiological pathways (Brinkmann et al., 2016;Doering et al., 2016). Several isoforms of the AhR are present in fishes, but dysregulation of the AhR2 isoform in fish embryos has been linked to dose-dependent early-life stage mortality and developmental malformations which include pericardial and yolk sac edemas, spinal and cranial deformities, cardiovascular deformities, anemia, and reductions in growth (Clark et al., 2010;Elonen et al., 1998;Van Tiem & Di Giulio, 2011;Vignet et al., 2014). However, PAHs also cause toxicities that are independent of activation of the AhR (Geier et al., 2018;Incardona, 2017). ...
... Exposure of embryos to the parent compound, BAA, resulted in a dose-dependent increase in mortality ( Figure 1A and Table 1), with an LD50 of 26,129 ng/g-egg (Table 1). Zebrafish have previously been shown to be among the least sensitive species of fish to the prototypical agonist of the AhR, TCDD, which has an LD50 of 2.61 ng/g-egg (Doering et al., 2013;Elonen et al., 1998). Consistent with effects of BAA, exposure of embryos to the three alkylated homologs, 4-MBAA, 8-MBAA, and 7,12-DMBAA, resulted in a dosedependent increase in mortality (Figure 1B-D and Table 1), with 8-MBAA being the most potent and 4-MBAA being the least (Table 2). ...
... Representative images of malformations are shown in Supporting Information, Figure S1. The observed malformations are consistent with effects commonly associated with activation of the AhR in fishes and other vertebrates (Elonen et al., 1998;King-Heiden et al., 2012;Tillitt et al., 2017). The prototypical hallmark for activation of the AhR is increased CYP1A (Hahn, 2002). ...
Polycyclic aromatic hydrocarbons (PAHs) are structurally diverse organic chemicals that can have adverse effects on the health of fishes through activation of the aryl hydrocarbon receptor 2 (AhR2). PAHs are ubiquitous in the environment, but alkyl PAHs are more abundant in some environmental matrices. However, relatively little is known regarding the effects of alkylation on the toxicity of PAHs to fishes in vivo and how this relates to potency for activation of the AhR2 in vitro. Therefore, objectives of this study were to determine the toxicity of benz[a]anthracene (BAA) and three alkylated homologues representing various alkylation positions to early life‐stages of zebrafish (Danio rerio), and to assess the potency of each for activation of the zebrafish AhR2 in a standardized in vitro AhR transactivation assay. Exposure of embryos to each of the PAHs caused a dose‐dependent increase in mortality and malformations characteristic of AhR2 activation. Each alkyl homolog had in vivo toxicities and in vitro AhR2 activation potencies different than the parent PAH in a position‐dependant manner. However, there was no statistically significant linear relationship between responses measured in these assays. Results suggest a need for further investigation into the effect of alkylation on the toxicity of PAHs to fishes and greater consideration of the contribution of alkylated homologues in ecological risk assessments. This article is protected by copyright. All rights reserved.
... Sequences and annealing temperatures of oligonucleotide primers used to produce expression constructs…………………………………………………………….……………. 275 Table C7 Relative sensitivity of fishes to the effects of embryo-lethality following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Buckler et al., 2015;Elonen et al., 1998;Guiney et al., 2000;Henry et al., 1997;Park et al., 2014;Tillitt et al., 2016;Toomey et al., 2001;Walker et al., 1991;Yamauchi et al., 2006)……………………………………………...…... 11 Comparison of basal CYP1A transcript abundance among liver, gill, and intestinal tissues in white sturgeon (A). Gill and intestine transcript abundance shown as fold difference from liver transcript abundance. ...
... The dose-response curve for activation of AHR2 of lake sturgeon by TCDD has been published previously with an EC50 of 0.79 nM and is not shown again here ………..……………….……. 196 (Buckler et al., 2015;Elonen et al., 1998;Guiney et al., 2000;Henry et al., 1997;Park et al., 2014;Tillitt et al., 2016;Toomey et al., 2001;Walker et al., 1991;Yamauchi et al., 2006). The seven species used in the study presented here are highlighted in black and list the LD50 (pg TCDD/g-egg). ...
... Of these species, the sensitivity to DLCs of only a few has been well characterized (Walker et al., 1992;, and only a handful of the remaining species have been investigated (Figure 1.3). Due to differences in relative sensitivity to DLCs among fishes that Relative sensitivity of fishes to the effects of embryo-lethality (dose to cause 50 % mortality; LD50) following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Buckler et al., 2015;Elonen et al., 1998;Guiney et al., 2000;Henry et al., 1997;Park et al., 2014;Tillitt et al., 2016;Toomey et al., 2001;Walker et al., 1991;Yamauchi et al., 2006). have been investigated, there is great uncertainty in the risk assessment of DLCs to fishes (Elonen et al., 1998;. ...
Along with overexploitation and habitat loss, pollution is one cause for decreases in populations of fishes. One class of pollutants of particular global environmental concern to fishes are dioxin-like compounds (DLCs). DLCs elicit their toxicity through activation of the aryl hydrocarbon receptor (AHR). Despite this common mechanism of all DLCs, dramatic differences in sensitivity exist among fishes. Sturgeons (Acipenseridae) are an ancient family of fishes in which most species are endangered. It is hypothesized that pollutants, including DLCs, might be contributing to the observed declines in populations because sturgeons have a unique life-style that makes them susceptible to exposure to bioaccumulative chemicals. However, determining sensitivities of sturgeons to DLCs through traditional in vivo toxicity testing is not feasible for practical and ethical reasons. Therefore, the aim of this research was to develop a mechanism-based biological model capable of predicting the relative sensitivity of sturgeons to DLCs. This mechanism-based biological model was developed through investigations into the AHR and AHR-mediated molecular and biochemical responses of white sturgeon (Acipenser transmontanus) relative to teleost fishes and another species of sturgeon. White sturgeon responded to activation of the AHR in a manner that is consistent with responses of teleost fishes (induction of cytochrome P450 1A). Two AHRs with similar levels of expression were identified in white sturgeon, an AHR1 that resembles AHR1s of tetrapods and an AHR2 that resembles AHR2s of other fishes. Both AHR1 and AHR2 of white sturgeon were activated by exposure to five selected DLCs in vitro with effect concentrations less than any other AHR tested to date. These findings were suggestive that white sturgeon might be among the most sensitive species of fish to DLCs. These findings raised the question as to whether other members of the Acipenseridae are similarly sensitive to exposure to DLCs. Therefore, AHR1 and AHR2 were identified in a second species of sturgeon, the lake sturgeon (Acipenser fulvescens). AHR1 of lake sturgeon had the same in vitro sensitivity to activation by the five selected DLCs as AHR1 of white sturgeon, while AHR2 of lake sturgeon was 10-fold less sensitive to activation by the five selected DLCs relative to AHR2 of white sturgeon. AHR2 has been demonstrated to drive adverse effects of DLCs in other fishes, while AHR1 has no known role in mediating toxicities in fishes. Therefore, it was hypothesized that white sturgeon are 10-fold more sensitive to DLCs relative to lake sturgeon in vivo. However, there were uncertainties in whether differences in activation of the AHR are representative of differences at higher levels of biological organization. Therefore, whole transcriptome and whole proteome responses were investigated following exposure to equipotent concentrations of three agonists of the AHR. Equal activation of the AHR of white sturgeon resulted in similar global responses and magnitude of responses across levels of biological organization. This supports the hypothesis that activation of the AHR is predictive of apical level adverse effects of regulatory relevance, such as mortality of embryos. In order to test this hypothesis, AHR1s and AHR2s from seven species of fish of known sensitivity were investigated and the relationship between in vitro and in vivo sensitivities were characterized for the model DLC, 2,3,7,8-TCDD. All AHR1s and AHR2s were activated in vitro by 2,3,7,8-TCDD. There was no significant linear relationship between in vitro sensitivity of AHR1 and in vivo sensitivity among the seven species. However, there was a highly significant linear relationship between in vitro sensitivity of the AHR2 and in vivo sensitivity. The equation of this relationship enables the prediction of the in vivo sensitivity of any species of fish based on in vitro sensitivity of the AHR2. This predictive model could be essential in guiding more objective risk assessments of DLCs to fishes, including endangered species such as sturgeons.
... Early life-stages of fishes are often notoriously sensitive to aromatic hydrocarbon contaminants, although vulnerability among species varies (Elonen et al., 1998). Recent evidence suggests that sturgeons may be particularly susceptible to the toxic impacts of aromatic hydrocarbons such as coplanar PCBs, PCDD/Fs, and some polycyclic aromatic hydrocarbons (PAHs) (Chambers et al., 2012;Doering et al., 2014a,b). ...
... Species of fishes and even some populations differ significantly in their sensitivities to early life-stage toxicities of TCDD and coplanar PCBs. For example, salmonids (bull trout Salvelinus confluentus, lake trout S. namaycush, and brook trout S. fontinalis) are among the most sensitive species, whereas zebrafish Danio rerio and northern pike Esox lucius are approximately three orders of magnitude less vulnerable (Elonen et al., 1998). Similarly, populations of Atlantic killifish Fundulus heteroclitus (Reid et al., 2016) and Atlantic tomcod Microgadus tomcod from contaminated estuaries are orders of magnitude less sensitive to TCDD and coplanar PCBs induced early lifestage toxicities than conspecifics from cleaner locales. ...
... Many studies have demonstrated that young life-stages of fishes are highly sensitive to toxicities from aromatic hydrocarbon contaminants; however, there is considerable variability among species (Elonen et al., 1998), and even populations (Yuan et al., 2006;Wirgin et al., 2011;Reid et al., 2016), in their sensitivities to these contaminants. Where sturgeons lie on the continuum of vulnerabilities is largely unknown although recent reports suggest that their young life-stages may be among the more sensitive of fishes to toxicities from PCBs, TCDD, and PAHs (Chambers et al., 2012;Doering et al., 2014b;Doering et al., 2015). ...
... A variety of toxicities resulting from dysregulation of AhR signaling by DLCs have been characterized in mammals, birds, and fishes, including wasting syndrome, hepatotoxicity, immune suppression, endocrine impairment, and carcinogenesis (Giesy et al., 2002;Spitsbergen et al., 1986;Walter et al., 2000). However, the most severe toxicities typically occur in developing embryos and can include craniofacial and cardiovascular malformations, pericardial and yolk sac edema, and early-life mortality (Cohen-Barnhouse et al., 2011b;Elonen et al., 1998). Although these toxicities are mediated through a single molecular initiating event, activation of the AhR, differences in sensitivity to DLCs can exceed several orders of magnitude both among and within vertebrate taxa (Cohen-Barnhouse et al., 2011b;Doering et al., 2013;Jung & Walker, 1997). ...
... The sensitivity of reptiles to toxicities associated with exposure to DLCs has been unknown to date, which presented a significant challenge for accurate ecological risk assessments for this taxon. Results of the present study demonstrate that in ovo exposure of a model reptile, the common snapping turtle, to four different DLCs caused a dose-dependent increase in early-life mortality as shown previously in numerous species of birds and fish (Cohen- Barnhouse et al., 2011a;Elonen et al., 1998). These studies in birds and fishes have shown that sensitivity to early-life mortality resulting from exposure to the prototypical reference congener, TCDD, can differ by several orders of magnitude both within and among vertebrate taxa (Doering et al., 2013). ...
Reptiles represent the least-studied group of vertebrates with regards to ecotoxicology and no empirical toxicity data existed for dioxin-like chemicals (DLCs). This lack of toxicity data represents a significant uncertainty in ecological risk assessments of this taxon. Therefore, the present study assessed early-life sensitivity to select DLCs and developed relative potencies in the common snapping turtle (Chelydra serpentina) as a model reptile. Specifically, survival to hatch and incidence of pathologies were assessed in common snapping turtle exposed in ovo to serial concentrations of the prototypical reference congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and three other DLCs of environmental relevance, namely, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). In ovo exposure to TCDD, PeCDF, TCDF, and PCB 126 caused a dose-dependent increase in early-life mortality, with median lethal doses (LD50s) of 14.9, 11.8, 29.6, and 185.9 pg/g-egg, respectively. Except for abnormal vasculature development, few pathologies were observed. Based on the measured LD50, common snapping turtle is more sensitive to TCDD in ovo than other species of oviparous vertebrates investigated to date. The potencies of PeCDF, TCDF, and PCB 126 relative to TCDD were 1.3, 0.5, and 0.08, respectively. These relative potencies are within an order of magnitude of World Health Organization (WHO) TCDD-equivalency factors (TEFs) for both mammals and birds supporting these TEFs as relevant for assessing ecological risk to reptiles. The great sensitivity to toxicities of the common snapping turtle, and potentially other species of reptiles, suggests a clear need for further investigation into the ecotoxicology of this taxon. Environ Toxicol Chem 2022;41:175-183. © 2021 SETAC.
... Salmonids are known to be much more sensitive to DLCs embryotoxicity compared to other fish species (Elonen et al., 1998). The rainbow trout (Oncorhynchus mykiss) has been a model fish species in toxicology for several decades, including in studies related to PAHs or DLCs. ...
... These studies were largely used during the development of the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) toxic equivalents concentration (TCDD-TEQs) concept, which allows to predict the toxicity of DLCs alone or in mixture ( Van den Berg et al., 1998). This concept, however, has been challenged several times as significant interspecies differences have been observed in both DLCs relative potencies and relative sensitivities (Eisner et al., 2016;Elonen et al., 1998;Rigaud et al., 2013;Rigaud et al., 2014). First attempts of developing such a toxic equivalent factor (TEF) scheme for PAHs were mostly focused on their carcinogenic effects in mammals (Nisbet and LaGoy, 1992). ...
Salmonids are known to be among the most sensitive fish to dioxin-like compounds (DLCs), but very little is known about the sensitivity of the brown trout (Salmo trutta), which has declined and is endangered in several countries of Europe and Western Asia. We investigated the sensitivity of brown trout larvae to a widespread dioxin-like PAH, retene (3.2 to 320 μg.L−1), compared to the larvae of a salmonid commonly used in toxicology studies, the rainbow trout (Oncorhynchus mykiss). Mortality, growth, cyp1a induction and the occurrence of deformities were measured after 15 days of exposure. Brown trout larvae showed a significantly higher mortality at 320 μg.L−1 compared to rainbow trout larvae. While the occurrence of deformities was only significantly increased at 320 μg.L−1 for the rainbow trout, brown trout larvae displayed pericardial edemas and hemorrhages already at 10 or 100 μg.L−1. cyp1a induction was increased significantly already at ≥3.2 μg.L−1 for the brown trout, versus ≥32 μg.L−1 for the rainbow trout. Least square regression analysis of the concentration-response relationships suggested that S. trutta larvae were at least 2 times more sensitive than O. mykiss larvae for cyp1a induction. The present study suggests that S. trutta larvae are more sensitive than O. mykiss larvae to a potent DLC, retene. As it is possible that S. trutta populations have declined partly because of pollution by DLCs, we recommend generating more data regarding the sensitivity of threatened fish populations, in order to ensure better risk assessment.
... These e ects were not observed in medaka, which might be due to sensitivity di erence between species 20) The toxic outcomes of TCDD exposure are observed a few days before hatching, even if the medaka embryos are exposed at di erent developmental stages. These results are consistent with a previous report 15) . ...
... Elonen et al. 20) has reported that the LC 10 and LC 50 values of TCDD in medaka eggs are 656 and 1110 pg/g-egg, respectively, and these values are almost the same as the calculated LC 10 and LC 50 values at 28-dpf in Test 5 (550 and 753 pg/g-egg, respectively). Therefore, we believe that our xed test method based on the measured concentrations of the exposed compound in both test water and eggs can provide reliable data of toxicities of DLCs. ...
In order to develop an optimal method for the investigation of relative potencies of dioxin-like compounds using Japanese medaka, the present study conducted ve independent early-life stage toxicity tests using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the positive control. First, both short-and long-term e ects of TCDD were examined to determine sensitive and highly reproducible endpoints. Then, the outcome and threshold of TCDD toxicities under di erent experimental conditions, e.g., exposure timing and duration, were compared to develop a cost-e ective method. Finally , we decided to observe yolk sac/pericardial edema, hatching failure, and mortality of medaka embryos/larvae within a 28-d experimental period, after 6-h exposure to TCDD at 0-d post fertilization. We obtained the LC 50 values of TCDD at 28-d post fertilization based on its concentration in water (6.84 ng/L) or in egg (753 pg/g-egg), and the LC 50 values were comparable with those reported in the literature. Thus, in our future studies, relative toxic potencies of dioxin-like compounds will be tested using the method established in this study.
... Thus, any energetically demanding physiological process, such as growth, reproduction, immune function, or behavior, could potentially be impacted when organisms are exposed to toxicants (Adams et al. 1989;Little and Finger 1990;Feist et al. 2005). However, mechanistically linking toxicant exposure across various levels of biological organization (i.e., biochemical to whole organism) is challenging (Clements 2000;Adams et al. 2000), especially since not all species, sexes, or life stages will respond similarly to equivalent contaminant exposures (Elonen et al. 1998;Eisner et al. 2016;Li et al. 2019). Further challenges arise when contaminant effects are explored in nonmodel species, as physiological response to contaminant exposure may vary across taxa with different evolutionary histories. ...
... For example, round stingray embryos from the PCB-exposed population exhibit slower growth and male, compared to female, embryos are relatively lighter than those of their reference counterparts . Given that embryonic development is a vulnerable period in an animal's life (Elonen et al. 1998;Örn et al. 1998), contaminant exposure during pregnancy could impact embryos. Contaminants could have direct metabolic consequences for stingray embryos through maternal offloading (Lyons and Lowe, 2013) or indirect effects by straining female's ability to provide proper nutrition to embryos during development. ...
Organic contaminants are known to affect a suite of physiological processes across vertebrate clades. However, despite their ancient lineage and important roles in maintaining healthy ecosystems, elasmobranchs (sharks, skates, and rays) are understudied with regard to sublethal effects of contaminant exposure on metabolic processes. Perturbations resulting from contaminant exposure can divert energy away from maintaining physiological homeostasis, particularly during energetically challenging life stages, such as pregnancy and embryonic development. Using the round stingray ( Urobatis halleri ) as a model elasmobranch species, we captured adult males and pregnant females (matrotrophic histotrophy) and their embryos from two populations differing in their environmental exposure to organic contaminants (primarily polychlorinated biphenyls (PCBs)). Pregnant females from the PCB-exposed population experienced significant decreases from early- to late-pregnancy in tissue mass and quality not seen in reference females. PCB-exposed pregnant females also failed to maintain plasma urea concentrations as pregnancy progressed, which was accompanied by a loss in muscle protein content. Despite the energetic demands of late-term pregnancy, females had significantly greater liver lipid content than reproductively inactive adult males. PCB-exposed adult males also had high metabolic capacity (i.e., enzyme activity) for most substrate groupings of all sex-site groups, suggesting that males may be even more negatively impacted by contaminant exposure than pregnant females. Evidence that in utero exposure to PCBs via maternal offloading impairs embryo outcomes is accumulating. Embryos from the PCB-contaminated site had lower tissue quality measures and indications that sex-based differences were manifesting in utero as males had higher metabolic capacities than females. This study indicates that accumulated PCB contaminants are not physiologically inert in the stingray.
... Extensive laboratory research suggests that legacy chemicals such as PAHs (Bue et al., 1996;Le Bihanic et al., 2014), PCBs (Tillitt et al., 2016;Di Paolo et al., 2015), PCDDs and PCDFs (Elonen et al., 1998;Peddinghaus et al., 2012), and metals (Barjhoux et al., 2012;Vardy et al., 2014) are toxic to early life stage fish, but these effects are rarely studied in a natural setting. Spawning grounds may be difficult to locate, and there are currently no practical methods for tracking larval fish in the wild (Ellis et al., 2012). ...
... We report contaminant concentrations in sediments collected from areas in proximity to industrial inputs, and from a reference site within the lake that was previously shown to have significantly lower levels of sediment contamination (Painchaud and Laliberté, 2016;Pelletier, 2008). Chemical analyses focus on PAHs, PCBs, PCDDs, PCDFs, and metals/metalloids given that concentrations of these groups of contaminants have been historically high in parts of LSL (ECCC, 2013;Pelletier, 2008), and they are known to be toxic to early life stage fish (Le Bihanic et al., 2014;Tillitt et al., 2016;Elonen et al., 1998;Peddinghaus et al., 2012;Vardy et al., 2014). The collected sediments were also used in a whole sediment contact assay to investigate gene transcription, epigenetic, and organismal effects in developing zebrafish embryos and larvae. ...
Lake Saint-Louis, a shallow fluvial lake near the western tip of the island of Montreal, QC, Canada is an important spawning ground for many species of fish. Sediments in certain areas of the lake are known to be contaminated with high levels of metals and legacy organic chemicals. To improve our understanding of risk to native fish populations, we conducted a study evaluating levels of sediment contamination and potential effects on early life stage fish. Concentrations of PAHs, PCBs, PCDDs and PCDFs were several orders of magnitude higher at two industrial sites (B1 and B2) than at a nearby reference site (IP). Concentrations of 32 metals and metalloids were at least 5-fold higher at B1 and B2 than at IP. Moreover, all available interim sediment quality guidelines (ISQGs) were exceeded at the two contaminated sites,while none were exceeded at the reference site. Biological effectswere evaluated using a sediment contact assay. Zebrafish (Danio rerio) embryoswere exposed to cleanwater (control), or to sediment from IP, B1, and B2 until 120 h post fertilization (hpf).Mortalitywas significantly elevated in fish exposed to the B1, but not the B2 sediment. The frequency of deformities increasedwith increasing contamination, but this trendwas not statistically significant (p > 0.05). Genes that are implicated in the response to PAHs, PCBs, dioxins and furans (cyp1a, cyp1b1, ahr2) were significantly elevated in the 120 hpf larvae exposed to the B1 and B2 sediments. Global DNA methylation, and mRNA expression of genes related to oxidative stress (maft, cat, hmox1, sod2), embryonic development (bmp2b, baf60c), metal exposure (mt2), and DNA repair (gadd45b) were unaffected. Our results suggest that the Beauharnois sector of Lake Saint-Louis is poor quality spawning habitat due to high levels of contamination, and the potential for harmful effects on early life stage fish.
... TCDD toxicity showed characteristic signs in teleost fish from the early life stage, and extensive species differences have been reported. The LC (egg)50 for seven species of fish ranged from 539 pg/g for fathead minnow to 2610 pg/g for zebrafish [23]. After injection and waterborne exposure of Oncorhynchus mykiss, eggs were 421 and 439 pg TCDD/g eggs (LD 50 ), respectively [24]. ...
The climate crisis and growing petroleum demand have put the health of aquatic animals in jeopardy. Fish are sensitive to chemical pollutants in aquatic environments, such as polycyclic aromatic hydrocarbons, dioxins, and dibenzofurans. This study investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone (β-NF) exposure on histopathological and immunohistochemical features and expression patterns of cytochrome P450 1 (CYP1) family genes in black rockfish, Sebastes schlegelii. Histopathological alterations in the liver included congested central vein, sinusoidal dilatation, lymphocyte infiltration, and severe vacuolation within hepatocytes. The most prevalent alterations in TCDD-exposed kidneys were glomerular enlargement, narrowing of tubular lumen, melanomacrophage centers (MMCs), and necrosis. Moreover, CYP1A immunostaining was strong in renal tubules following TCDD exposure. All CYP1 family genes (CYP1A, CYP1B, CYP1C1, and CYP1C2) were significantly increased in the gills, liver, and kidney exposed to TCDD. Similarly, a significant increase of CYP1A mRNA expression in the kidney was observed upon exposure to TCDD (30.9-folds) and β-NF (25.5-folds) compared with that of the control group (p < 0.05). TCDD and β-NF exposure exerted more adverse effects on the kidney than the liver, and TCDD had a greater in vivo toxic effect than β-NF. The combined histopathological, immunohistochemical, and molecular alternations may be helpful for diagnosing chemical contaminant exposure in S. schlegelii.
... The proliferation of the cartilage cells was disrupted, resulting in skeletal defects in Sebastiscus marmoratus exposed to pyrene [68]. TCDD arising as a by-product of different industrial procedures like production of chlorinated insecticides and pesticides, and paper bleaching can lead to deformities of the spine and tail of various fish species [81]. When fishes are exposed to a combination of heavy metals, PAHs, estrogen and other related compounds, they have a higher probability of developing spinal deformities [70]. ...
In the past three decades biological and chemical pollutants have become a serious environmental issue posing major threat to the society, industries and public sectors. Toxic contaminants are produced in most household, agricultural and industrial activities. In the past few years different advanced electrochemical oxidation technologies and low-carbon technologies are being used widely for preventing environmental pollution and remediation of the micropollutants particularly in waterbodies. In these technologies powerful oxidizing agents like hydroxyl radicals are formed electrochemically which degrade organic micropollutants till their mineralization. Fish serves as an effective bioindicator of aquatic health due to their higher trophic position in aquatic food chain and high sensitivity to pollutants. As fishes are consumed by humans globally as a major source of protein, it is also used to indicate the impact of aquatic pollution on human health. In this review we discuss the impact of micropollutants on the fish physiology and the advanced wastewater management techniques used for rapid removal of these pollutants from aquatic ecosystem. Here we discuss both advantages and disadvantages of different commonly used wastewater management techniques which would be beneficial to determine which technology would best suit one’s specific requirements without causing much harm to the environment, a step towards green and sustainable future.
... Finally, the highest BB concentrations of 30 and 50 g/L induced mortality in all unhatched embryos ( Figure 2E). While our search for yolk sac rupture during hatching in zebrafish yielded no result, there were several reports of similar phenomena in other fish species exposed to oil derivatives [61], tetrachlorodibenzo-p-dioxin (TCDD) [62,63], and silver nanoparticles [64]. However, no study suggested a mechanism of yolk sac weakening. ...
The rising concerns about controversial food additives’ potential hazardous properties require extensive yet animal-minimized testing strategies. Zebrafish embryos are the ideal in vivo model representing both human and environmental health. In this study, we exposed zebrafish embryos to eight controversial food additives. Our results indicate that Sodium Benzoate is a Cat.3 aquatic toxicant, while Quinoline Yellow is a strong teratogen. At high concentrations, non-toxic chemicals induced similar phenotypes, suggesting the impact of ionic strength and the applicability of the darkened yolk phenotype as an indicator of nephrotoxicity. Three food additives showed unpredicted bioactivities on the zebrafish embryos: Brilliant Blue could weaken the embryonic yolk, Quinoline Yellow may interfere with nutrient metabolism, and Azorubine induced precocious zebrafish hatching. In conclusion, the zebrafish embryo is ideal for high throughput chemical safety and toxicity screening, allowing systematic detection of biological effects—especially those unexpected by targeted in vitro and in silico models. Additionally, our data suggest the need to reconsider the safety status of food additives Quinoline Yellow, Brilliant Blue, Sodium Benzoate, and other controversial food additives in further studies, as well as pave the way to further applications based on the newly found properties of Brilliant Blue and Azorubine.
... Medaka was less sensitive to brominated dioxins than rainbow trout, as shown by the higher egg-based LD 50 values for medaka (2.42, 99.4, and 15.4 ng/g for TeBDF, PeBDF-1, and PeBDF-2, respectively) than for trout (1.50, 9.56, and 6.19 ng/g, respectively), and similar differences in sensitivity were observed with chlorinated dioxins (Elonen et al., 1998;Zabel et al., 1995). Although non-2,3,7,8-substituted compounds (1,3,6, 8-/1,3,7,9-TeBDD and 2,3,8-TrBDF) are not toxic to medaka, 2,3, 7-TrBDD is known to cause a weak dioxin-like toxic effect in rainbow trout (Hornung et al., 1996). ...
World Health Organization toxic equivalency factors (WHO-TEFs) are recommended for risk management of brominated dioxins in aquatic environments because limited information is available on their toxicity to fish. To validate this approach, we obtained the relative potencies of polybrominated dibenzo-p-dioxins and polybrominated dibenzofurans and mixed-halogenated furans (PXDF, X = Cl/Br) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) based on their toxicity to the early-life stage of Japanese medaka (Oryzias latipes). 2,3,7,8-substituted brominated dibenzofurans caused typical dioxin exposure effects, such as blue-sac disease. The TCDD-relative potency factors (REPs) of test substances were calculated based on the concentrations in water and eggs that caused 20% lethality on day 28 post-fertilization, and were in the order of: 2-chloro-3,7,8-tribromodibenzofuran (REPwater 3.3, REPegg 4.6) > 2,3,7,8-tetrabromodibenzofuran (0.85, 0.92) > 2,3,4,7,8-pentabromodibenzofuran (0.053, 0.55) > 1,2,3,7,8-pentabromodibenzofuran (0.0091, 0.19). The transfer rate from water to eggs was lower for pentabrominated furans than tetrabrominated congeners, and was expected to decrease with the log Kow of the test substance. Although the REPegg value can be used to compare the toxicity potential of brominated dioxins, REPwater may be more suitable for environmental risk assessment because the uptake potential of these compounds from water should be considered. This study is the first to report higher toxicity of a PXDF congener compared with TCDD in vivo, further investigations of the toxicity of mixed-halogenated dioxins and environmental behavior are necessary for environmental risk assessment.
... A similar phenotype was reported byIncardona et al. (2004) following exposure of zebrafish embryos to pyrene, a four-ring PAH.It is unknown why 6-OHCHR caused mortality in zebrafish and not in Japanese medaka.This contradiction suggests the possibility of interspecific differences in oxy-PAH effects, even among teleosts like medaka and zebrafish. While several groups have demonstrated differential toxicity of dioxin-like chemicals to medaka and zebrafish(Elonen et al., 1998;Xu et al., 2018), to our knowledge this is the first study to suggest that there are interspecies, regioselective effects to oxy-PAHs. One possible explanation for the differences in toxicity may be the unique ontogenies of each species. ...
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can enter aquatic environments through runoff, atmospheric deposition, accidental discharge, and oil spills. These compounds can be oxidized photochemically or biologically into oxygenated PAHs (oxy-PAHs) which have been shown to be more toxic compared to parent PAHs. The polar properties of oxy-PAHs increase their mobility within the environment which increases the risk of exposure to fauna and flora compared to parent PAHs. Regioselective toxicity has been observed in several oxy-PAHs and the oxidation state of the oxygen on a specific PAH can have dramatic impacts on the toxicity. Previous studies have found that exposure to hydroxychrysenes at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene (2-OHCHR) being more potent than 6-hydroxychrysene (6-OHCHR). The mechanisms of toxicity of oxy-PAHs are largely unknown and we aimed to characterize the pathways of toxicity of 2- and 6-OHCHR in fish embryos. Our first aim was to characterize the toxic effects in Japanese medaka embryos and to find a sensitive window of development to hydroxychrysenes. We found that 2-OHCHR caused anemia and morality in medaka embryos in contrast to in zebrafish embryos, where 2-OHCHR caused only anemia and 6-OHCHR only caused mortality. A sensitive window to 2-OHCHR toxicity was found between 52-100 hpf which closely coincided with liver development. This led us to our second aim, exploring the metabolism and toxicokinetics of the hydroxychrysenes. We found that although 6-OHCHR was taken up 97.2% ± 0.18 more rapidly than 2-OHCHR, it was also eliminated 57.7% ± 0.36 faster as a glucuronide conjugate. Pretreatment with the general cytochrome P450 inhibitor ketoconazole reduced anemia by 96.8% ± 3.19 and mortality by 95.2% ± 4.76 of 2-OHCHR treatments. In addition, formation of the 1,2-catechol was also reduced by 64.4% ± 2.14. However, while pretreatment with the UGT inhibitor nilotinib reduced glucuronidation of 2-OHCHR by 52.4% ± 2.55 and of 6-OHCHR by 63.7% ± 3.19, it did not alter toxicity for either compound. These results indicated that CYP mediated activation, potentially to the oxidatively active metabolite 1,2-catechol, may be driving the isomeric differences in toxicity. Previous studies have found 2-OHCHR to be a four-fold more potent aryl hydrocarbon receptor (AhR) agonist compared to 6-OHCHR. Therefore, in aim 3, we explored the role of the and oxidative stress in 2-OHCHR toxicity. While treatments with the AhR agonists PCB126 and 2-methoxychrysene (2-MeOCHR) did not cause significant anemia or mortality, pretreatments with AhR antagonist CH-223191 reduced anemia by 97.2% ± 0.84 and mortality by 96.6% ± 0.69. AhR inhibition was confirmed by a significant reduction (91.0% ± 9.94) in EROD activity. Thiobarbituric acid reactive substances (TBARS) concentrations were 32.9% ± 3.56 higher (p<0.05) in 2-OHCHR treatments at 100 hpf compared to controls, indicating oxidative stress. Staining with 2’,7’-Dichlorofluorescin diacetate (DCFDA) revealed 42.6% ± 2.69 of embryos exhibiting high concentrations of ROS in caudal tissues, which is a site for embryonic hematopoiesis. Both muscle and skeletal tissues were affected, as well as some caudal vasculature. Overall, our findings indicate that AhR may mediate 2-OHCHR toxicity, upregulating CYP and potentially forming the 1,2-catechol that generates ROS in the embryos within caudal tissues, potentially disrupting hematopoiesis leading to anemia and subsequent mortality. Further studies should investigate additional key events and construct adverse outcome pathways for oxy-PAHs.
... Early life-stages of fishes are often exquisitely sensitive to toxicities from PCBs and PCDD/Fs. But, finfish species (Elonen et al. 1998), and even populations within species (Nacci et al. 2010;Wirgin et al. 2011), vary by orders of magnitude in their vulnerabilities to these contaminants. For example, lake trout (Salvelinus namaycush) is the most sensitive vertebrate taxon to TCDD known , while other finfishes (e.g., zebrafish Danio rerio) are far less sensitive (Henry et al. 1997). ...
Sturgeon populations worldwide are threatened with extirpation but little is known about their tendency to bioaccumulate contaminants and their sensitivities to environmental burdens of these contaminants. Shortnose sturgeon and Atlantic sturgeon, two species that are federally endangered in the USA, co-occur in the Hudson River (HR) where high sediment levels of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-p-furans (PCDFs) occur. Previous controlled laboratory studies showed that young life-stages of both species are sensitive to toxicities at low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and PCB126 exposure. The objective here was to measure congener-specific hepatic levels of PCBs and PCDD/Fs in HR specimens in order to determine if in situ bioaccumulation of these compounds is sufficiently high to have caused the early life-stage toxicities previously observed. Estimates of hepatic burdens of PCBs and PCDD/Fs were obtained from a small number of specimens of each species collected between 2014 and 2016 and specimens of shortnose sturgeon collected over 30 years earlier and archived in a museum collection. Several significant patterns emerged. Hepatic levels of legacy PCBs and PCDDs were low in specimens of both species but typically higher in shortnose than Atlantic sturgeon, a pattern consistent with their habitat use in the HR. Hepatic burdens in shortnose sturgeon tended to be higher in archived specimens than in more recently collected ones despite expected reduction in archived specimens due to preservation methods. Several inadvertent PCBs congeners were detected at high levels, including PCB11, but their toxicity to natural populations remains unknown. Levels of select PCDFs congeners, 2,3,7,8-TCDF and 2,3,4,7,8 PeCDF, were elevated in some shortnose sturgeon individuals from the HR. Using Relative Potency (ReP) factors derived from white sturgeon, the observed levels of some hepatic PCDFs in HR shortnose sturgeon may have been sufficiently high to impair recruitment of young life-stages in this ecosystem.
... Pyrene exposure increased the deformity rates of Sebastiscus marmoratus embryos, impairing skeleton development by disrupting the proliferation of chondrocytes (cartilage cells) (Shi et al., 2012). The 2,3,7,, a by-product of numerous industrial processes such as chlorinated pesticides production and paper bleaching, can also induce deformities, especially spine and tail ones, on the early life stages of several fish species (Elonen et al., 1998;Giesy et al., 2002). Moreover, Kessabi et al. (2013) found a high incidence of spinal deformities on fish from polluted sites, suggesting a possible correlation between environmental exposure to a mixture of pollutants, such as toxic metals, PAHs, and estrogenic compounds, and spinal deformities. ...
The Iguaçu River basin presents high ecological importance due to its expressive endemic ichthyofauna rate, but chemical pollution may threat this biodiversity. Jordão River is one of the main tributaries of Iguaçu River and contribute to this pollution status, since it drains large agricultural areas receiving domestic and industrial effluents before flowing into the Iguaçu River. The objective of the current study was to evaluate the toxic effects of the Iguaçu, Jordão, and the combination of their waters to the embryo-larval phase of R. quelen, investigating the consequences to the population by means of mathematical modelling. R. quelen fertilized eggs were exposed for 96 h to water samples from Iguaçu River upstream (IR), Jordão River (JR), and downstream of both rivers (MR). The analysis of micropollutants in the water showed that JR presented the most complex mixture of substances and elements, followed by IR, while MR showed the lower number of micropollutants detected. Survival rate was not a sensitive endpoint, while the deformity indices were higher in individuals exposed to water from the three studied sites. Superoxide dismutase activity was increased in MR, while non-protein thiol levels were reduced in MR and JR showing the antioxidant mechanism activation. The mathematical modelling revealed that fish exposed to JR would lead to the greater population reduction (46.19%), followed by IR (40.48%) and MR (33.33%). Although the results showed toxicity in all studied sites, the JR site is the most impacted by micropollutants but decrease its toxicity after dilution with Iguaçu River.
... Pyrene exposure increased the deformity rates of Sebastiscus marmoratus embryos, impairing skeleton development by disrupting the proliferation of chondrocytes (cartilage cells) (Shi et al., 2012). The 2,3,7,, a by-product of numerous industrial processes such as chlorinated pesticides production and paper bleaching, can also induce deformities, especially spine and tail ones, on the early life stages of several fish species (Elonen et al., 1998;Giesy et al., 2002). Moreover, Kessabi et al. (2013) found a high incidence of spinal deformities on fish from polluted sites, suggesting a possible correlation between environmental exposure to a mixture of pollutants, such as toxic metals, PAHs, and estrogenic compounds, and spinal deformities. ...
... We compared the toxicity of TCDD to seven freshwater fish species during early life-stage development. All young fish were found to have edema and head and spinal deformities, but skin discoloration was observed only in some fathead minnows (Pimephales promelas) and medaka (Oryzias latipes) [46]. TCDD sensitivity studies to date have shown that all frogs and toads (order Anura), unlike mammals, fish, and birds, are incredibly insensitive to TCDD [47]. ...
The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates a wide range of biological and toxicological effects by binding to specific ligands. AhR ligands exist in various internal and external ecological systems, such as in a wide variety of hydrophobic environmental contaminants and naturally occurring chemicals. Most of these ligands have shown differential responses among different species. Understanding the differences and their mechanisms helps in designing better experimental animal models, improves our understanding of the environmental toxicants related to AhR, and helps to screen and develop new drugs. This review systematically discusses the species differences in AhR activation effects and their modes of action. We focus on the species differences following AhR activation from two aspects: (1) the molecular configuration and activation of AhR and (2) the contrast of cis-acting elements corresponding to AhR. The variations in the responses seen in humans and other species following the activation of the AhR signaling pathway can be attributed to both factors.
... Apparently, mammalian-based assays are more sensitive to TCDD (rodent > human) and other dioxin-like compounds than fish-based assays (rainbow trout > zebrafish), due to structural differences and affinities of their respectively recruited AhRs Hilscherova et al. 2001;Keiter et al. 2008;Eichbaum et al. 2014). Even within fish, zebrafish is the most insensitive, commonly used test species, as demonstrated in vivo (Elonen et al. 1998;Jönsson et al. 2009;Doering et al. 2012) and in vitro (Creusot et al. 2014). Hence, the regulatory relevance of zebrafishderived reporter assays of the AhR-regulated xenobiotic metabolism TP is in dispute. ...
The “toxicology in the twenty-first century” paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration–response curves. We named such a multi-level inhibitory mechanism that might mask effects as “maisonette squelching . ”
Graphical abstract
A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance.
Synthetic and constitutive promotors interfere with signal transduction (“squelching”) and might increase cellular stress (cytotoxicity).
The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.
... Because the EC 50 value of a contaminant can vary among different endpoints (Yamauchi et al., 2006), the LC 50 is preferred as the toxicity criterion. Data obtained by experiments using recognized model organisms, such as zebrafish (Yamauchi et al., 2006;Elonen et al., 1998), should preferably be adopted. When assessing the ecological risk of a group of compounds (e.g., dioxins or polycyclic aromatic hydrocarbons (PAHs)), the relative potency method can be used to estimate the toxic equivalent quotient (TEQ). ...
Thousands of organic pollutants are intentionally and unintentionally discharged into water bodies, adversely affecting the ecological environment and human health. Screening for organic pollutants that pose a potential risk in aquatic environments is essential for risk management. This review evaluates the processes, methods, and technologies used to screen such pollutants in the aquatic environment and discuss their advantages and disadvantages, in addition to the challenges and knowledge gaps in this field. Combining non-target screening, target screening, and suspect screening is often effective for compiling a list of potential risk compounds and enables the quantitative analysis of these compounds. Sample preparation technologies and pollutant detection technologies considerably affect the results of pollutant screening. The limited amount of chemical and toxicological information contained in databases hinders the screening of organic pollutants with potential risk. Machine learning, high-throughput methods, and other technologies will increase the accuracy and convenience of screening for high-risk pollutants. This review provides an important reference for screening these compounds in aquatic environments and can be used in future pollutant screening and risk management.
... Particularly in the earliest life stage, a high number of embryos in the present study failed to transition from embryo to larvae/alevins; that is, they only partially emerged from the chorion and exhibited ruptured yolk sacs (although this endpoint was not specifically measured). This phenomenon was also noted in a study by Elonen et al. (1998) in lake herring (Coregonus artedi), lake trout, brook trout (Salvelinus fontinalis), northern pike, and rainbow trout when exposed to tetrachlorodibenzo-p-dioxin (TCDD). They hypothesized that it was due to neuromuscular weakness caused by TCDD. ...
Information on the effects of silver nanoparticles (AgNPs) in fish have mostly been generated from standard laboratory species and short‐term toxicity tests. However, there is significant uncertainty regarding AgNP toxicity to native species of concern in North America, particularly in northern freshwater ecosystems. Here, we assessed the chronic toxicity of AgNPs in early life stages of three North American fish species: rainbow trout (Oncorhynchus mykiss), lake trout (Salvelinus namaycush) and northern pike (Esox lucius). Newly fertilized embryos were exposed to nominal aqueous concentrations of 0.1, 0.3, 1.0, 3.0, 10.0, or 30.0 nM AgNPs for 126 (rainbow trout), 210 (lake trout), and 25 (northern pike) days. Endpoints included cumulative developmental time (o C x day or degree‐days to 50% life stage transition), mortality, fork length, embryonic malformations, cumulative survival, and histopathology of gill and liver in larvae/alevins. Results showed life stage‐specific differences in responses, with endpoints during the embryonic stage occurring more often and at lower concentrations, compared to larval/alevin and juvenile stages. Sensitivities among species were highly dependent upon the endpoints measured although developmental time appeared to be the most consistent endpoint across species. At embryonic and larval/alevin stages, northern pike was the most sensitive species (lowest observable effect concentration of 0.1 nM using developmental time). Rainbow trout displayed similar responses to lake trout across multiple endpoints and therefore seems to be an adequate surrogate for trout species in ecotoxicology studies. Moreover, while mortality during individual life stages was not generally affected, the cumulative mortality across life stages was significantly affected, which highlights the importance of chronic, multi‐life stage studies. This article is protected by copyright. All rights reserved.
... A variety of toxicants that have been associated with head-deformities in general include polychlorinated biphenyl (PCB) (Hogan and Brauhn 1975), dioxins (Helder 1981;Elonen et al. 1998), heavy metals (Somasundaram et al. 1984;Sfakianakis et al. 2015), selenium (Hamilton et al. 2005;McDonald & Chapman 2007), polycyclic aromatic hydrocarbons (PAH) (Hannah et al. 1982;Kocan and Landolt 1984), petroleum hydrocarbons (PHC) (Lindén 1978;Tilseth et al. 1984;Vignet et al. 2019), and herbicides (Paganelli et al. 2010). This list is non-exhaustive and mainly used to illustrate that many possibly causative agents exists. ...
This review summarizes the current state of knowledge of pugheadedness in fish. Records in the scientific literature range from detailed descriptions to brief notes and mere remarks. In total, at least 164 species from 60 families were identified to exhibit pugheadedness, with records published over a span of 465 years (1555 − 2020). The main osteological feature behind pugheadedness appears to be shortening or deformation of the parasphenoid bone, which leads to additional deformations of the ethmovomer-and frontal region. Several other deformations and abnormalities of other cranial bones, eyes, and tongue are occasionally observed, depending on the severity of the pugheadedness. Possible cases in elasmobranchs are also encountered, although the developmental causation may differ from actinopterygians, since their crania have a different organization. Natural cases of pugheadedness are found worldwide , covering a wide range of environments and lifestyles (freshwater-, brackish-and marine environments; benthic, neritic and pelagic species). Cases are found in all life-stages, from embryo to mature adults, suggesting that it does not necessarily lead to early-life mortality. There is some evidence for natural selection acting against pugheaded individuals, likely because of e.g. inappropriately functioning mouth parts, sense organs, and possibly brain deformation. High numbers of pugheads are mainly found in aquaculture, but moderate numbers have been found at some localities also in the wild. Abnormally high occurrence in the wild is commonly attributed to pollution, non-normal water chemistry parameters, or temperature. The causation, however, it typically speculated upon. Based on the reviewed literature, there is support for several causative factors, including genetic mutation and embryonic environmental conditions (toxic and non-toxic) affecting development. Pugheadedness, as the term has been used in the literature, is not a single well-defined pathology, but rather a suite of pathological conditions with similar phenotypic expression.
... This contradiction suggests the possibility of interspecific differences in oxy-PAH effects, even among teleosts like medaka and zebrafish. While several groups have demonstrated differential toxicity of dioxin-like chemicals to medaka and zebrafish (Elonen et al., 1998;Xu et al., 2018), to our knowledge this is the first study to suggest that there are inter-species, regioselective effects to oxy-PAHs. One possible explanation for the differences in toxicity may be the unique ontogenies of each species. ...
Exposure to oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene being more potent than 6-hydroxychrysene. To better characterize this phenomenon, embryos of Japanese medaka (Oryzias latipes) were exposed to 2- or 6-hydroxychrysene (0.5, 2, or 5 µM) from 4 h-post-fertilization (hpf) to 7 d-post-fertilization. Following exposure, hemoglobin concentrations were quantified by staining fixed embryos with o-dianisidine (a hemoglobin-specific dye) and stained embryos were imaged using brightfield microscopy. Exposure to 2-hydroxychrysene resulted in a concentration-dependent decrease in hemoglobin relative to vehicle-exposed embryos, while only the highest concentration of 6-hydroxychrysene resulted in a significant decrease in hemoglobin. All tested concentrations of 2-hydroxychrysene also caused significant mortality (12.2% ± 2.94, 38.9% ± 14.4, 85.6% ± 11.3), whereas mortality was not observed following exposure to 6-hydroxychrysene. Therefore, treatment of embryos with 2-hydroxychrysene at various developmental stages and durations was subsequently conducted to identify key developmental landmarks that may be targeted by 2-hydroxychrysene. A sensitive window of developmental toxicity to 2-hydroxychrysene was found between 52-100 hpf, with a 24 h exposure to 10 µM 2-hydroxychrysene resulting in significant anemia and mortality. Since exposure to 2-hydroxychrysene from 52-100 hpf, a window that includes liver morphogenesis in medaka, resulted in the highest magnitude of toxicity, liver development and function may have a role in 2-hydroxychrysene developmental toxicity.
... Sensitivities among fish species vary up to 120-fold. [88][89] Typical findings are excess mortality, oedema, haemorrhages, and craniofacial malformations. So-called blue sac disease of early embryos is associated with high concentrations of TCDD [90] and other dioxin-like compounds. ...
Dioxins and dioxin-like compounds comprise a group of chemicals including polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF), as well as certain dioxin-like polychlorinated biphenyls (dl-PCB), and potentially others. They act via a common mechanism, stimulation of aryl hydrocarbon receptor (AH receptor, AHR), a vital transcription factor in cells. There are very high differences in potency among these compounds, i.e. in the ability to stimulate the receptor. This leads to ten thousand fold or higher differences in doses causing similar toxic effects. Most of these compounds are eliminated very slowly in the environment, animals, or humans, which makes them persistent. They are much more soluble in fat than in water, and therefore they tend to accumulate in lipid or fatty tissues, and concentrate along the food web (bioaccumulation and biomagnification).
PCDD/PCDFs are formed mostly as side products in burning processes, but PCBs were oils manufactured for many purposes. Because of toxicity and persistence, dioxin-like compounds have been regulated strictly since 1980s, and their levels in the environment and animals have decreased by an order of magnitude or more. Therefore the effects on wildlife have clearly decreased, and even populations at the top of the food web such as fish-eating birds or seals have recovered after serious effects on their reproductive capacity and developmental effects in their young especially in 1970s and 1980s. This does not exclude the possibility of some remaining effects.
In humans the intake is mostly from food of animal sources, but because our diet is much more diverse than that of such hallmark animals as white-tailed eagles or seals, the concentrations never increased to similar levels. However, during 1970s and 1980s effects were probably also seen in humans, including developmental effects in teeth, sexual organs, and the development of immune systems.
Both scientists and administrative bodies debate at the moment about the importance of remaining risks. This is very important, because the AH receptors seem to be physiologically important regulators of growth and development of organs, immunological development, food intake and hunger, and in addition regulate enzymes protecting us from many chemicals. Thus a certain level of activation is needed, although inappropriate stimulation of the receptor is harmful. This dualism emphasizes the importance of benefit versus risk analysis. As a whole, regulating the emissions to the environment is still highly important, but one should be very cautious in limiting consumption of important and otherwise healthy food items and e.g. breast feeding.
Distinct toxic effects of high doses of dioxins in humans have been clearly demonstrated by frank poisonings and the highest occupational exposures. Hallmark effects have been skin lesions called chloracne, various developmental effects of children, and a slightly increased risk of total cancer rate. The highest dioxin levels have been ten thousand fold higher than those seen in the general population today.
... In soft water, tricolor shiners were significantly more sensitive to nitrate compared with the other fish, but in moderately hard and hard waters fathead minnows were more sensitive. The fathead minnow is one of the most commonly tested species for regulatory purposes, although they are not necessarily representative of declining species (Dowden and Bennett 1965;Doherty 1983;Thurston et al. 1985;Elonen et al. 1998;Ankley and Villeneuve 2006). Tricolor shiners represent a potentially better surrogate for the protection of imperiled Cyprinella species, such as the endangered blue shiner (Cyprinella caerulea). ...
Growing human populations and increasingly intensive agriculture have resulted in widespread aquatic nitrate pollution. Freshwater mussel populations have been in decline for decades but often are underrepresented in data used for the development of ambient water quality criteria and acute toxicity of nitrate to mussel glochidia has not yet been established. Additionally, toxicity testing with aquatic species often is limited to a few model species; however, relatively little is known about how representative model species are of imperiled species. Therefore, to better define the acute toxicity of nitrate to common and rare aquatic species, we conducted 24-h nitrate acute toxicity tests with glochidia of four species of freshwater mussels, including a federally threatened species (Hamiota altilis) and 7-day tests with larval fish of three species: fathead minnow (Pimephales promelas), tricolor shiner (Cyprinella trichroistia), and tilapia (Oreochromis spp.), across a range of water hardness. Median effective concentrations (EC50s) in freshwater mussel glochidia ranged from 524 to 904 mg/L NO3–N in moderately hard water. In fish, median lethal concentrations (LC50s) ranged from 228 to 1725 mg/L NO3–N and varied with water hardness. Of the species tested, generally sensitivity of the common species was similar to the rare species, although relative sensitivity varied with water hardness. Based on these results, we can conclude that acute lethal effects are unlikely for the fish and mussel species considered here at current environmental levels, but the results of these standardized tests are useful for the development of ambient water quality criteria and other regulatory and management decisions regarding acute nitrate exposures.
... A possible reason could be greater sensitivity of zebrafish larva compared to H4G1.1c2 cells. This assumption is consistent with AhR pathway studies where early life stages of fish have proven to be significantly sensitive to AhRmediated activity (Elonen et al., 1998;Zodrow et al., 2004). Thus, the current in vivo analysis could be a useful tool for detection of mixture AhR-mediated activity. ...
Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the “European Water Framework Directive” (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.
... Lanham et al. 2014) and compromised cardiovascular system function. However, variation among fishes in vulnerability to PCB and PCDD/Fs-induced early life-stage toxicities is great, with LD 50s among species to TCDD spanning at least three orders of magnitude with salmonids being among the most sensitive of taxa and zebrafish among the least (Elonen et al. 1998;King-Heiden et al. 2012). Apparently, sturgeon species are among the more sensitive of fishes on this continuum based on earlier in vitro studies with white sturgeon and lake sturgeon Acipenser fulvescens (Doering et al. 2014ab) and our in vivo studies with Atlantic sturgeon and shortnose sturgeon Chambers et al. 2012). ...
Sturgeon species are imperiled world-wide by a variety of anthropogenic stressors including chemical contaminants. Atlantic sturgeon, Acipenser oxyrinchus, and shortnose sturgeon, Acipenser brevirostrum, are largely sympatric acipenserids whose young life-stages are often exposed to high levels of benthic-borne PCBs and PCDD/Fs in large estuaries along the Atlantic Coast of North America. In previous laboratory studies, we demonstrated that both sturgeon species are sensitive to early life-stage toxicities from exposure to environmentally relevant concentrations of coplanar PCBs and TCDD. The sensitivity of young life-stages of fishes to these contaminants varies among species by three orders of magnitude and often is due to variation in the structure and function of the aryl hydrocarbon receptor (AHR) pathway. Unlike mammals, fishes have two forms of AHR (AHR1 and AHR2) with AHR2 usually being more highly expressed across tissues and functional in mediating toxicities. Based on previous studies in white sturgeon, A. transmontanus, we hypothesized that sturgeon taxa are unusually sensitive to these contaminants because of higher levels of expression and functional activity of AHR1 than in other fish taxa. To address this possibility, we characterized AHR1 in both Atlantic Coast sturgeon species, evaluated its’ in vivo expression in young life-stages and in multiple tissues of shortnose sturgeon, and tested its ability to drive reporter gene expression in AHR-deficient cells treated with graded doses of PCB126 and TCDD. Similar to white sturgeon and lake sturgeon, AHR1 amino acid sequences in Atlantic sturgeon and shortnose sturgeon were more similar to mammalian AHRs and avian AHR1s than to AHR1 in other fishes, suggesting their greater functionality in sturgeon species than in other fishes. Exposure to graded doses of coplanar PCBs and TCDD usually failed to significantly induce AHR1 expression in young life-stages or most tissues of shortnose sturgeon. However, in reporter gene assays, AHR1 drove higher levels of gene expression than AHR2 alone, but their binary combination failed to drive higher levels of expression than either AHR alone. In total, our results suggest that AHR1 may be more functional in sturgeon species than in other fishes, but probably does not explain their heightened sensitivity to these contaminants.
... Calculated EC 50 and LD 50 from the published literature are presented. 3,4,9,11,12 ...
... Some PAHs have impacts on early life stages of fish, including reduced growth, cranio-facial malformations, yolk sac and pericardial oedemas and subcutaneous hemorrhaging (Billiard, et al., 1999;Carls, et al., 1999;Hawkins, et al., 2002). These deformities closely resemble the "blue sac syndrome" that has been described in several fish species, including rainbow trout (Oncorhynchus mykiss), zebrafish (Danio rerio), medaka (Oryzias latipes) and killifish (Fundulus heteroclitus), exposed to certain halogenated aromatic compounds that are agonists for the aryl hydrocarbon receptor (AhR) (Walker and Peterson, 1991;Wannemacher, et al., 1992;Elonen, et al., 1998;Chen and Cooper, 1999;Toomey, et al., 2001). These compounds include coplanar polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), collectively referred to here as planar halogenated aromatic hydrocarbons (pHAHs). ...
Polycyclic aromatic hydrocarbons (PAHs) have been classified as priority pollutants and are the subject of various monitoring programmes in aquatic environments due to their ubiquity and toxicological effects. In this study, the genotoxic and developmental effects of environmental concentrations of PAHs and Lagos Lagoon sediment extracts on different life stages of fish were investigated. The seasonal water physico-chemistry, levels and ecological risk assessment of sixteen (16) priority PAHs were analyzed in water, sediment and dominant fish species sampled from designated zones (Ilaje, Iddo, Atlas cove and Apapa) of the Lagos lagoon. Genotoxic and developmental effects of naphthalene, phenanthrene, pyrene and benzo(a)pyrene (singly and in quaternary mixtures of ratio 12:1:3:1) as well as organic extracts of the Lagos lagoon sediment were tested against Danio rerio (Zebrafish) embryos from 0-72 hours post-fertilization (hpf). Physiological, biochemical and histological studies of sublethal concentrations (1/10th 96hrLC50) of naphthalene, phenanthrene and pyrene were also conducted on Clarias gariepinus (African Catfish) broodstock and embryos for a duration of eight (8) weeks. The water physico-chemistry results revealed significant (P<0.05) increases in conductivity, temperature, salinity and total dissolved solids in the dry season compared to the wet season across the zones. The dominant fish species were Sarotherodon melanotheron (Black-Chinned Tilapia), Gerres melanopterus (Gerres), Liza falcipinnis (Sickle-fin Mullet) and Pseudolithus elongatus (Bobo Croaker) at the Ilaje, Iddo, Atlas cove and Apapa zones respectively. The mean sum PAHs and range were 515.58µg/l (194.94 - 1006.49µg/l), 739.45µg/kg (301.81 - 1290.16µg/kg) and 19.78µg/kg (8.80 - 26.10µg/kg) in water, sediment and fish respectively. Naphthalene was dominant in water and sediment samples while phenanthrene, anthracene, fluoranthene and pyrene were predominant in the fish species across the zones and seasons. PAHs sources were mainly pyrogenic, mixed and pyrogenic in water, sediments and fish respectively. Ecological risk assessment of PAHs in the sediments showed that biological effects will occur frequently at the Apapa zone and occasionally at all other zones in both seasons based on the levels of naphthalene, acenapthylene, acenaphthene, fluorene and benzo(a)anthracene. The sediment pollution classification based on sum PAHs showed that Apapa zone was highly polluted in both seasons while other zones were moderately polluted. Fish species from all the zones were minimally contaminated in both seasons except S. melanotheron. The genotoxicity results revealed that the percentage tail DNA in D. rerio cells in the FPG-modified assay was significantly (P<0.05) increased in the benzo(a)pyrene and quaternary PAHs mixture treatments compared to the solvent control. The results of the developmental toxicity studies showed that percentage hatching was significantly (P<0.05) reduced in the highest phenanthrene (12.33%) and Iddo sediment extract (24.33%) treatments compared to the solvent controls. The number of heartbeats in Danio rerio embryos per minute was significantly (P<0.05) reduced in the phenanthrene and quaternary PAHs mixture treatments. Percentage abnormality was significantly (P<0.05) increased in the highest phenanthrene (100%), quaternary PAHs mixture (68%) and benzo(a)pyrene (43.6%), as well as, sediment organic extract treatments; Iddo (83%) and Ilaje (73.33%). The developmental abnormalities include stunted growth, mild to severe pericardial and yolk-sac oedemas, scoliosis, elongated heart, haemorrhaging, curved tail and tail tip curvature. For the studies on Clarias gariepinus, phenanthrene (1.41 mg/L) was the most toxic, followed by pyrene (1.53 mg/L) and naphthalene (7.21 mg/L) based on the 96 hrsLC50 values. The physiological indices in the C. gariepinus broodstock revealed that there was a significant (P<0.05) increase in the hepato-somatic indices of naphthalene and pyrene-treated male C. gariepinus compared to the solvent control. There was also a reduction in the fecundity of the female C. gariepinus exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene by factors of x2.4, x2.8 and x2.4 respectively compared to the solvent control. The results of the biochemical indices in the gills and liver of PAHs-treated fishes showed no significant (P>0.05) differences except for catalase levels in the liver of phenanthrene-treated female fish that was significantly (P<0.05) lower compared to the solvent control. Histological alterations observed include; oedema, epithelial lifting and hyperplasia, inflammatory cells (gills); vacuolation, haemosederin pigments, sinusoidal congestion (liver) and degenerated zona radiata (ovary). The establishment of environmental standards for PAHs in Nigeria, management of PAHs sources, incorporation of fish early life stages, the FPG-modified comet assay, as well as, the utilization of multi-biomarkers in ecomonitoring programmes are therefore recommended.
... There was no association between contaminant exposure and circulating hormone levels or fecundity in 2 populations of bonnethead sharks (Sphyrna tiburo) occupying areas with different histories of anthropogenic contaminant release (Manire 2002). However, the youngest life stage is often the most sensitive to contaminants (Guillette et al. 1994;Elonen et al. 1998;€ Orn et al. 1998). For instance, juvenile common sole (Solea solea) collected from anthropogenically impacted sites showed reduced growth and lipid stores compared to fish from less impacted sites (Amara et al. 2007). ...
Anthropogenic chemical exposure can result in overall reductions in reproductive success. Using the Round Stingray (Urobatis halleri) as an elasmobranch model with internal gestation, we measured female fecundity and embryo growth from post‐ovulation to near parturition to test the hypothesis that environmental PCB contamination would impair reproductive success. Two sites were sampled from southern California: the mainland site was exposed to legacy PCB contamination (with low exposure to other anthropogenic contaminants), and the offshore reference site at Catalina Island was a separate population with low anthropogenic influence. Contaminant‐exposed embryos weighed less at each stage of development than reference embryos, while accumulating proportionately more liver mass over development. Furthermore, environmental contamination negatively affected male embryos more than female embryos. This is the first study to demonstrate a negative effect of contaminant exposure on elasmobranch embryo growth, with probable fitness costs later in life. This article is protected by copyright. All rights reserved
... Danio rerio is a widely used model for toxicity tests, but its responses could not reflect the reality of the native species of southern Brazil, so we decided to use the Neotropical Astyanax altiparanae, a very common specie in the freshwaters of South America, in order to compare the exposure effects between native and exotic species. In fact, some studies applying other contaminants have showed that responses between native and exotic species can be different (Elonen et al., 1998;Campagna et al., 2008;Murussi et al., 2014;Piancini et al., 2015). ...
Carbon Nanotubes are among the most promising materials for the technology industry. Their unique physical and chemical proprieties may reduce the production costs and improve the efficiency of a large range of products. However, the same characteristics that have made nanomaterials interesting for industry may be responsible for inducing toxic effects on the aquatic organisms. Since the carbon nanotubes toxicity is still a controversial issue, we performed tests of acute and subchronic exposure to a commercial sample of multiwalled carbon nanotubes in two fish species, an exotic model (Danio rerio) and a native one (Astyanax altiparanae). Using the alkaline version of the comet assay on erythrocytes and the piscine micronucleous, also performed on erythrocytes, it was verified that the tested carbon nanotubes sample did not generate apparent genotoxicity by means of single/double DNA strand break or clastogenic/aneugenic effects over any of the species, independently of the exposure period. Although, our findings indicate the possibility of the occurrence of CNTs-DNA crosslinks. Apparently, the sample tested induces oxidative stress after subchronic exposure as shown by activity of superoxide dismutase and catalase. The data obtained by the activity levels of acetylcholinesterase suggests acute neurotoxicity in Astyanax altiparanae and subchronic neurotoxicity in Danio rerio.
... With TCDD exposure, cardiac abnormalities appear shortly before hatching and become progressively more severe until affected fish die at the fry stage from circulatory dysregulation, anemia, hypoxia, and secondary lesions in the brain, retina, liver and other organs . Among fish species, lake trout are exceptionally sensitive to TCDD cardiotoxicity (Elonen et al., 1998), with mortality occurring at tissue concentrations in the low parts-per-trillion range . While less potent, other polychlorinated dibenzo-pdioxins, dibenzofurans, and biphenyls (PCBs) can also contribute to the sac fry syndrome, illustrating the importance of contaminant mixtures in the Great Lakes food webs (Jayasundara et al., 2015;Wright and Tillitt, 1999). ...
As a nonstop pump driven by physiologically complex excitable cells, the heart is especially susceptible to a variety of insults, in particular exogenous chemicals. In humans, for example, heart-related toxicities are the most common adverse drug reaction. Fish living in polluted environments may or may not be able to avoid exposure to cardiotoxic chemicals. Nevertheless, juvenile and adult fish have a variety of robust mechanisms for protecting the heart from toxic chemicals, such as hepatic metabolism. The situation is very different for fish early life history stages, in particular very small embryos and larvae. The developing heart is the first organ to become functional in embryos, at a point very early in its morphogenesis. Owing to the intimate and interacting links between form and function in the developing heart, very subtle cardiotoxicity during embryogenesis can cause serious and long-lasting outcomes. Fish embryos have minimal capacity for metabolic detoxification, and therefore, developmental cardiotoxicity is a real-world environmental health concern for wild populations. This chapter focuses on the exquisite sensitivity of the developing fish heart to chemical contaminants, as demonstrated through aquatic pollution case studies focused on legacy organochlorine compounds and recent oil spills. These studies have revealed a diversity of mechanisms linking cardiomyocyte physiology to heart development, and abnormal development in turn to latent impacts on physiology at later life stages.
... For several other fish species, even higher concentrations were needed to reach LR50. Embryos exposed to water concentration of TCDD of the, comparably, non-sensitive zebrafish (Danio rerio) or shovelnose sturgeon (Scaphirhynchus platorynchus) exhibited a much higher tolerance towards dioxin-like contaminants compared to the previously mentioned salmonid species with LD50s of 2610 and 13,000 pg of TCDD/g ww of egg, respectively (Elonen et al., 1998;Buckler, 2011). Nevertheless, elevated incidences of malformations in embryos in other sturgeon species have been reported at concentrations as low as 50 pg of TCDD/g egg (Chambers et al., 2012). ...
Several eel species of the genus Anguilla are considered endangered due to a severe decline in recruitment. Up to now, the reasons for this threatening development are not fully understood. The eel's highly specialized biology can lead to explicitly high accumulation of globally distributed organic lipophilic contaminants during its continental life. Because of this and due the particular toxicological sensitivity of early life stages of oviparous organisms towards dioxin-like compounds, it is crucial to improve our understanding concerning toxicokinetics and maternal transfer of organic contaminants in eels.
This study presents analytical data on maternal transfer of dioxin-like (dl) compounds in relevant tissue samples taken from artificially matured and non-matured European silver eels (Anguilla anguilla) from German inland waters using gas chromatography coupled with mass spectrometry (GC/MS) and high-resolution mass spectrometry (GC/HRMS). Detected concentrations revealed a lipid-driven transfer of targeted compounds from muscle-fat-reserves to gonads and eggs respectively, with no distinct preferences concerning the chlorination degree of targeted compounds. Dl-PCBs were shown to contribute the major share of toxicity equivalents found in analysed eel tissues. Maternal muscle tissue to egg concentration ratios in wet weight–based samples had a mean of 6.95 ± 1.49 in accordance with the differences in total lipid content in the respective body matrices. Dioxins and furans in analysed samples were (from a toxicological point of view) of less relevance. Furthermore it was shown that muscle concentrations in silver eels could be used in future assessments to make conservative predictions for expected egg concentrations in female eels.
... However, edemas do not always manifest at hatch. Fathead minnow, like Japanese medaka and zebrafish that developed edemas during exposures to dioxin (TCDD) did so rapidly after hatch, while other species did not show such abnormalities for days to weeks post-hatch (Elonen et al., 1998). ...
Naphthenic acid fraction components (NAFCs) are constituents of oil sands process-affected water (OSPW), which is generated as a result of unconventional oil production via surface mining in the Athabasca oil sands region. NAFCs are often considered to be major drivers of OSPW toxicity to various taxa, including fishes. However, the molecular targets of these complex mixtures are not fully elucidated. Here we examined the effects in walleye (Sander vitreus) embryos after exposure to NAFCs extracted from fresh OSPW. Eleutheroembryos (exposed to 0, 4.2 or 8.3 mg/L NAFCs from 1 day post-fertilization to hatch) were subsampled, measured for growth and deformities, and molecular responses were assessed via real-time polymerase chain reaction (PCR). Fourteen genes were evaluated, with a focus on the aryl hydrocarbon receptor (AhR)-cytochrome P450 pathway (arnt, cyp1a1), the oxidative stress axis (cat, gst, sod, gpx1b), apoptosis (e.g. casp3, bax and p53), growth factor signaling (e.g. insulin-like growth factors igf1, igf1b, and igf1bp), and tissue differentiation (vim). NAFC exposure was associated with an increase in the expression of cyp1a1, and a decrease in gpx1b and ribosomal protein rps40. These results indicate that NAFC effects on walleye early-life stages may be mediated through oxidative stress via pathways that include AhR. Crown Copyright
... The embryos (within 6 h post-fertilization) and eleutheroembryos (within 12 h post-hatch; eleuthero-embryo is defined as the phase from hatching to yolk sac absorption, Clay et al., 2008) were kept in a cold container (8-10 • C) and microinjected using a nitrogen-driven pneumatic picopump microinjector (World Precision Instruments LLC, Sarasota, FL, USA). The wet weights of 104 embryos (6 hpf; 0.88 ± 0.025 mg) and 67 eleuthero-embryos (0.304 ± 0.053 mg) were obtained to estimate the exposure dosage in the toxicity tests and agreed with those reported previously (Elonen et al., 1998;Villalobos et al., 2003;González-Doncel et al., 2014). Based on our pilot study, the 96-h LD 50 values for P-CTX-1 exposure estimated for embryos and eleuthero-embryos were 1.71 ng g −1 (or 1.50 pg egg −1 ; 95% confidence interval [CI], 1.39-2.09 ...
... However, edemas do not always manifest at hatch. Fathead minnow, like Japanese medaka and zebrafish that developed edemas during exposures to dioxin (TCDD) did so rapidly after hatch, while other species did not show such abnormalities for days to weeks post-hatch (Elonen et al., 1998). ...
Resistance to chemotherapy and PARPi inhibitors remains a critical challenge in the treatment of epithelial ovarian cancer, mainly due to disabled apoptotic responses in tumor cells. Given mesothelin's pivotal role in ovarian cancer and its restricted expression in healthy tissues, we conducted a drug-screening discovery analysis across a range of genetically modified cancer cells to unveil mesothelin's therapeutic impact. We observed enhanced cell death in low mesothelin expressing cancer cells when exposed to a second mitochondria-derived activator of caspases (SMAC) mimetics, and demonstrated a compelling synergy when combined with chemotherapy in ex vivo patient-derived cultures and zebrafish tumor xenografts. Mechanistically, the addition of the SMAC mimetics drug birinapant to either carboplatin or paclitaxel triggered the activation of the Caspase 8-dependent apoptotic program facilitated by TNF alpha signaling. Multimodal analysis of neoadjuvant-treated patient samples further revealed an association between tumor-associated macrophages and the activation of TNF alpha-related pathways. Our proposed bimodal treatment shows promise in enhancing the clinical management of patients by harnessing the potential of SMAC mimetics alongside conventional chemotherapy.
Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products including building materials, personal care products, paints, and plastics to prevent degradation caused by UV light. Despite widespread occurrence in aquatic environments, little is known regarding effects of BUVSs on aquatic organisms. The aim of this study was to characterize effects of exposure to 2‐(2H‐Benzotriazol‐2‐yl)‐4‐methylphenol (UV‐P) on reproductive success of zebrafish ( Danio rerio ) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV‐P at < 1.5 ng/g‐egg (control), 2.77 ng/g‐egg, and 24.25 ng/g egg, and reared until sexual maturity, where reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV‐P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV‐P exposed males and females, fertility was decreased by 8.75% in the low treatment and by 15.02% in the high treatment, relative to control. In a reproduction assay with UV‐P exposed males and control females, fertility was decreased by 11.47% in the high treatment, relative to the control. Embryonic exposure to UV‐P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of E2 and 11‐KT, and small statistically non‐significant decreases in mRNA abundances of cyp19a1a , cyp11c1 , and hsd17b3. Additionally, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl , was observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV‐P impaired spematogenesis, resulting in decreased sperm quantity. This study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction.
The Aryl Hydrocarbon Receptors (Ahrs) are evolutionarily conserved ligand-dependent transcription factors that are activated by structurally diverse endogenous compounds as well as environmental chemicals such as polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons. Ahr activation leads to several transcriptional changes that can cause developmental toxicity resulting in mortality. Evidence was assembled and evaluated for two novel adverse outcome pathways (AOPs) which describe how Ahr activation (molecular initiating event; MIE) can lead to early life stage mortality (adverse outcome; AO), via either SOX9-mediated craniofacial malformations (AOP 455) or cardiovascular toxicity (AOP 456). Using a key event relationship (KER)-by-KER approach, we collected evidence using both a narrative search, and through systematic review based on detailed search terms. Weight of evidence for each KER was assessed to inform overall confidence of the AOPs. The AOPs link to previous descriptions of Ahr activation, and connect them to two novel key events (KEs), increase in slincR expression, a newly characterized long non-coding RNA with regulatory functions, and suppression of SOX9, a critical transcription factor implicated in chondrogenesis and cardiac development. In general, confidence levels for KERs ranged between medium and strong, with few inconsistencies, as well as several opportunities for future research identified. While majority of the KEs have only been demonstrated in zebrafish with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an Ahr activator, evidence suggests that the two AOPs likely apply to most vertebrates, and many Ahr activating chemicals. Addition of the AOPs into the AOP-Wiki (https://aopwiki.org/) helps expand the growing Ahr-related AOP network to nineteen individual AOPs, of which six are endorsed or in progress, and the remaining 13 relatively underdeveloped.
Polycyclic aromatic hydrocarbons (PAHs) are naturally occurring or anthropogenic organic chemicals that can activate the aryl hydrocarbon receptor 2 (AhR2) and induce toxicity in fishes. Alkyl PAHs have been found to be more abundant than non-alkylated PAHs in certain environmental matrices and there is growing evidence that alkylation can increase potency, dependent on the position of alkylation. However, it is unknown if the effect of alkylation on potency is conserved across species. Additionally, relatively little is known regarding the extent of interspecies variation in sensitivity to PAHs and alkyl PAHs. Therefore, objectives of this study were to characterize potency of benz[a]anthracene (BAA) and three alkylated homologues representing different alkylation positions in nine phylogenetically diverse species of fish using a standardized in vitro AhR2 transactivation assay. BAA and each alkylated homologue activated the AhR2 in a concentration-dependent manner in each species. Position-dependent effects on potency were observed in every species, however these effects were not consistent across species. Interspecies variation in sensitivity to AhR2 activation by each PAH was observed and ranged by up to 561-fold. Alkylation both increased and decreased the range of interspecies variation and sensitivity, but potency of each alkylated homologue relative to BAA ranged by less than an order of magnitude among species. These results represent an early step towards the consideration of alkylated homologues for more objective ecological risk assessments of PAHs to native fishes.
Sediments to be dredged as part of the installation of a harbour crossing in Sydney, Australia, contained measurable concentrations of dioxin-like compounds. To assess the suitability of these sediments for ocean disposal, a defensible sediment quality guideline value (SQGV) for dioxin-like compounds, expressed as pg TEQfish /g dw was required. There were deemed to be too many uncertainties associated with a value derived using effects data from field studies. A similar issue was associated with values based on equilibrium partitioning from sediment to pore water, largely associated with the wide range of reported sediment:water partition coefficients (Koc). Greater certainty was associated with the use of a tissue residue approach based on equilibrium partitioning between sediment and organisms determined using tissue concentrations in fish, the most sensitive aquatic biota, and biota:sediment accumulation factors (BSAFs) as promoted by the USEPA (1993) and fully developed by Steevens et al. (2005). The calculation of an appropriate SQGV used data for dioxin-like compounds in both fish and sediments from Sydney Harbour. A conservative SQGV for dioxin-like compounds of 70 pg TEQ/g dw was deemed to be adequately protective of biota that might be exposed to these contaminants in sediments at the ocean spoil ground. The approach is transferable to similar situations internationally. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2022;00:0-0. © 2022 SETAC.
Sturgeon populations worldwide are threatened with extirpation but little is known about their tendency to bioaccumulate contaminants and their sensitivities to environmental burdens of these contaminants. Shortnose sturgeon and Atlantic sturgeon, two species that are federally endangered in the U.S., co-occur in the Hudson River (HR) where high sediment levels of PCBs and PCDD/Fs occur. Previous controlled laboratory studies showed that young life-stages of both species are sensitive to toxicities at low levels of TCDD and PCB126 exposure. The objective here was to measure congener-specific hepatic levels of PCBs and PCDD/Fs in HR specimens in order to determine if in situ bioaccumulation of these compounds was sufficiently high to cause the early life-stage toxicities previously observed. Estimates of hepatic burdens of PCBs and PCDD/Fs were obtained from a small number of specimens of each species collected between 2014 and 2016 and specimens of shortnose sturgeon collected over 30 yr earlier and archived in a museum collection. Several significant patterns emerged. Hepatic levels of legacy PCBs and PCDDs were low in specimens of both species, but typically higher in shortnose than Atlantic sturgeon, a pattern consistent with their habitat use in the HR. Hepatic burdens from archived specimens of shortnose sturgeon tended to be higher than more recently collected ones despite expected reduction in their burdens due to preservation methods. Several inadvertent PCBs congeners were detected, including PCB11, but their possible toxicity to natural populations remains to be determined in future experiments. Levels of select PCDFs congeners, 2,3,7,8-TCDF and 2,3,4,7,8 PeCDF, were elevated in some shortnose sturgeon individuals from the HR. Using Relative Potency (ReP) factors derived from white sturgeon, the observed levels of some hepatic PCDFs in HR shortnose sturgeon may have been sufficiently high to impair recruitment of young life-stages in this ecosystem.
The aryl hydrocarbon receptor (AHR) has endogenous functions in mammalian vascular development and is necessary for mediating the toxic effects of a number of environmental contaminants. Studies in mice have demonstrated that AHR is necessary for the formation of the renal, retinal, and hepatic vasculature. In fish, exposure to the prototypic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the AHR biomarker cyp1a throughout the developing vasculature and produces vascular malformations in the head and heart. However, it is not known whether the vascular structures that are sensitive to loss of AHR function are also disrupted by aberrant AHR activation. Here, we report that TCDD-exposure in zebrafish disrupts development of 1) the subintestinal venous plexus (SIVP), which vascularizes the developing liver, kidney, gut, and pancreas, and 2) the superficial annular vessel (SAV), an essential component of the retinal vasculature. Furthermore, we determined that TCDD exposure increased the expression of bmp4, a key molecular mediator of SIVP morphogenesis. We hypothesize that the observed SIVP phenotypes contribute to one of the hallmarks of TCDD exposure in fish – the failure of the yolk sac to absorb. Together, our data describe novel TCDD-induced vascular phenotypes and provide molecular insight into critical factors producing the observed vascular malformations.
Dioxin‐like compounds (DLCs) cause early life stage mortality of vertebrates through activation of the aryl hydrocarbon receptor (AHR). A prior study developed a cross‐species quantitative adverse outcome pathway (qAOP) which can predict full dose‐response curves of early life stage mortality for any species of bird or fish exposed to DLCs using the species‐ and chemical‐specific 50% effect concentration (EC50) from an in vitro AHR transactivation assay with COS‐7 cells. However, calculating a reliable EC50 for input into this qAOP requires the maximal response of the concentration‐response curve to be known, which is not always possible for low potency agonists, such as some polychlorinated biphenyls (PCBs). To enable predictions for these low potency agonists, the present study revised this qAOP to use the effect concentration threshold (ECThreshold) from the in vitro AHR transactivation assay as input. Significant, linear relationships were demonstrated between ECThreshold and the dose to cause 0%, 10%, 50%, or 100% mortality among early life stages of three species of birds and seven species of fish for four DLCs, 2,3,7,8‐TCDD, PCB 126, PCB 77, and PCB 105. These four linear relationships were combined to form the revised qAOP. This qAOP using ECThreshold enables prediction of experimental dose‐response curves for lower potency agonists to within an order of magnitude on average, but the prior qAOP using EC50 predicts experimental dose‐response curves for higher potency agonists with greater accuracy. This article is protected by copyright. All rights reserved.
Sturgeons (Acipenseridae) are ancient fishes that have tissue-specific profiles of transcriptional responses to dioxin-like compounds (DLCs) that are unique from those generally measured in teleost fishes. Because DLCs exert their critical toxicities through activation of the aryl hydrocarbon receptor (AHR), this transcription factor has been the subject of intensive study. However, less attention has focused on the aryl hydrocarbon receptor nuclear translocator (ARNT), which is the dimerization partner of the AHR and required for AHR-mediated transcription. The present study sequenced ARNT1, ARNT2, and ARNT3 in a representative species of sturgeon, the white sturgeon (Acipenser transmontanus), and quantified tissue-specific basal transcript abundance for each ARNT and the response following exposure to the model agonist of the AHR, β-naphthoflavone. In common with other proteins in sturgeons, the amino acid sequences of ARNTs are more similar to those of tetrapods than are ARNTs of other fishes. Transcripts of ARNT1, ARNT2, and ARNT3 were detected in all tissues investigated. Expression of ARNTs are tightly regulated in vertebrates, but β-naphthoflavone caused down-regulation in liver and up-regulation in gill, while an upward trend was measured in intestine. ARNTs are dimeric partners for multiple proteins, including the hypoxia inducible factor 1α (HIF1α), which mediates response to hypoxia. A downward trend in abundance of HIF1α transcript was measured in liver of white sturgeon exposed to β-naphthoflavone. Altered expression of ARNTs and HIF1α caused by activation of the AHR might affect the ability of certain tissues in sturgeons to respond to hypoxia when co-exposed to DLCs or other agonists.
Numerous studies of the water-soluble fraction (WSF) from crude oil have concluded that polycyclic aromatic hydrocarbons (PAHs) are the primary causative agents for early life stage (ELS) fish toxicity. Noteworthy is the lack of studies demonstrating that the sum of PAHs are capable of causing toxic effects in ELS fish at the low levels claimed (0.1-5 μg/L) without being part of a complex crude oil mixture. Crude oil and the WSF are composed of thousands of other compounds that co-occur and likely contribute to crude oil toxicity. Based on the available data, it appears that the syndrome of effects (lower heart rate, edemas, and morphological abnormalities) for ELS fish exposed to the aqueous fraction of a crude oil mixture is commonly observed in studies exposing fish embryos to high concentrations of a variety of compounds and may be a nonspecific response. We conclude that the available data support the hypothesis that this syndrome of effects is likely the result of baseline toxicity (not receptor based) due to membrane disruption and resulting alteration in ion (e.g., calcium and potassium) homeostasis. We acknowledge the possibility of some compounds in the WSF capable of causing a specific receptor based toxicity response to ELS fish; however, such compounds have not been identified nor their receptor characterized. Concluding that PAHs are the main toxic compounds for crude oil exposure is misleading and does not result in guideline values that can be useful for environmental protection. Water quality guidelines for any single chemical or suite of chemicals must be based on a complete understanding of exposure concentrations, mechanism of action, potency, and resulting response. This review focuses on the toxic effects reported for fish embryos and the purported toxic concentrations observed in the aqueous phase of an oil/water mixture, the known levels of toxicity for individual PAHs, a toxic unit approach for characterizing mixtures, and the potential molecular initiating event for ELS toxicity in fish. This review also has implications for a large number of studies exposing ELS fish to a variety of compounds at high concentrations that result in a common baseline toxic response.
Polychlorinated biphenyls (PCBs) have left a legacy of environmental contamination, despite being banned from production and active use in the 1970's. PCBs persist in the environment and still have the potential to impact aquatic life. Our objective was to identify data from controlled laboratory studies of PCB‐related adverse effects in fish and conduct a meta‐analysis on mortality, growth, and reproductive (MGR) threshold responses. For each endpoint type, we compiled data on the lowest observed adverse effect concentration (LOAEC) and the degree of effect at the LOAEC as a percentage of control. The LOAECs were expressed as tissue concentrations, so the term lowest observed adverse effect residue‐concentration (LOAER) was used to represent PCB exposures. The lower limit of applicability was set at 0.1 µg/g total PCB tissue concentration, below which adverse MGR effects in fish were not supported by the data. Sensitivity distributions identifying the probability of adverse effects in fish populations or communities, predicted 25% of fish species impacted between 0.1 and 7.5 µg/g. Concentration‐response threshold regressions were developed from the MGR datasets. For example, a 1 µg/g total PCB tissue concentration would predict effects of 17% M, 15% G, and 39% R. The analysis determined the degree of adverse response, with uncertainty estimates, expected across a broad range of PCB tissue exposure concentrations in fish. Data generated from MGR endpoints were combined to determine an approach for overall effect thresholds for PCBs‐related injury in fish. MGR datasets included only laboratory data; however, responses were compared with field‐observed effects. This work provides a comprehensive assessment of PCB‐induced injury in fish utilizing a data‐inclusive approach. This article is protected by copyright. All rights reserved
Dioxin-like compounds (DLCs) elicit adverse effects through activation of the aryl hydrocarbon receptor (AHR). Prior investigations demonstrated that sensitivity to activation of AHR1 in an in vitro AHR transactivation assay is predictive of early life stage mortality among birds. The present study investigated the link between sensitivity to activation of AHR1s and AHR2s and early life stage mortality among fishes. A significant, linear relationship was demonstrated between sensitivity to activation of AHR2 and early life stage mortality among nine fishes, while no relationship was found for AHR1. The slope and y-intercept for the linear relationship between sensitivity to activation of AHR1 and early life stage mortality in birds was not statistically different from the same relationship for AHR2 in fishes. Data for fishes and birds across DLCs were expanded into four significant, linear regression models describing the relationship between sensitivity to activation of AHR and the dose to cause early life stage mortality of 0, 10, 50, or 100%. These four relationships were combined to form a quantitative adverse outcome pathway which can predict dose-response curves of early life stage mortality for DLCs to any bird or fish from species- and chemical-specific responses in an in vitro AHR transactivation assay.
This study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.
The embryotoxicity of extracts of American eels (Anguilla rostrata) was measured to determine whether maternally-derived contaminants contribute to the declining recruitment of eels to Lake Ontario. Sexually maturing, large yellow and silver eels were sampled in 2007 and 2008 from five locations in eastern Canada, including Lake Ontario; positive controls included eels from the Hudson River, United States, and Canal Dessel-Schoten, Belgium (European eel, Anguilla anguilla). Japanese medaka eggs were injected immediately after fertilization with 1 or 10 nL of eel extract, and after 12 days scored for signs of toxicity. Eel extracts did not cause dioxin-like embryotoxicity, reflecting the low concentrations of total dioxin equivalents measured chemically in these same extracts. Embryo mortality and reduced hatching success at high doses of eel extracts may reflect the bioaccumulation of legacy or emerging chemicals of concern. The results were consistent with long-term trends of declining concentrations of persistent organic pollutants (POPs) in tissues of eels and other fish species from Lake Ontario, trends of declining embryotoxicity of eel tissue extracts, and recent increases of recruitment of juvenile eels to Lake Ontario. If dioxin-like compounds contributed in the past to the decline of recruitment and abundance of American eels in Lake Ontario, these data suggest that recruitment should recover, following the same trends as the recovery of lake trout reproduction in Lake Ontario. This article is protected by copyright. All rights reserved
Toxicity and histopathology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish (Danio rerio) early life stages was characterized from 12 to 240 hr postfertilization (hpf) following water-borne exposure of newly fertilized eggs. TCDD did not increase egg mortality (0-48 hpf), nor did it affect time to hatching (48-96 hpf). Egg doses of 1.5 ng [3H]TCDD/g or greater elicited toxic responses in zebrafish larvae. Pericardial edema and craniofacial malformations were first observed at 72 hpf, followed by the onset of yolk sac edema (96 hpf) and mortality (132 hpf). At 240 hpf the ED50s for pericardial edema, yolk sac edema, and craniofacial malformations were 2.2, 2.1, and 1.9 ng [3H]TCDD/g egg, respectively. The LD50, determined at 240 hpf, was 2.5 ng [3H]TCDD/g egg. Severe hemodynamic changes, observed as slowed blood flow in vascular beds of the trunk, head, and gills and slowed heart rate, occurred in TCDD-treated zebrafish prior to or coincident with the onset of gross signs of toxicity. Histological examination of TCDD-treated zebrafish revealed a variety of epithelial tissue lesions including arrested gill development and ballooning degeneration and/or necrosis of the renal tubules, hepatocytes, pancreas, and all major brain regions. Mesenchymal tissue lesions included subcutaneous edema in the head, trunk, and yolk sac, edema of the pericardium and skeletal muscle, and underdevelopment of the swim bladder. This demonstration of zebrafish responsiveness to TCDD early life stage toxicity coupled with the considerable information on developmental biology and genetics of zebrafish provides a foundation for future investigations into the mechanism of TCDD developmental toxicity.
Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) belong to a family of lipophilic halogenated aromatic hydrocarbons that have similar structures, resist chemical and biological degradation, and persist in the environment posing a potential risk to fish, wildlife, and human health. There are more than 400 possible polychlorinated dioxins, dibenzofuran, and biphenyl congeners; however, only 21 are considered highly toxic.1,2 The more potent congeners are planar or coplanar molecules with lateral chlorine substitutions, approximate isostereomers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent PCDD, PCDF, or PCB congener.3 In mammals, TCDD and TCDD-like dioxin, dibenzofuran, and biphenyl congeners evoke similar patterns of toxic responses within a given species, and share a common mechanism of action mediated by binding the cellular aryl hydrocarbon (Ah) receptor and altering gene transcription.3,4 The ability of TCDD and TCDD-like congeners to produce toxicity in mammalian species correlates with their Ah receptor binding affinity and their ability to induce cytochrome P450IA enzyme activity, a gene subfamily that catalyzes monooxygenase reactions and whose expression is regulated by the Ah receptor.4−6
The sensitivity of early life stages of brook trout (Salvelinus fontinalis) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity was investigated. Newly fertilized eggs were exposed for 48 h to water containing either acetone or a range of concentrations of [3H]TCDD dissolved in acetone. Eggs were then transferred to TCDD-free water and observed through development. TCDD concentrations of 101 to 470 pg/g in the eggs caused dose-related increases in sac-fry mortality associated with yolk-sac edema, hemorrhages, and arrested development. These signs of TCDD-induced toxicity resemble blue-sac disease. The NOELs and LOELs for sac-fry mortality were 135 and 185 pg TCDD/g egg, respectively, whereas the LD50 and LD100 (95% fiducial limits) were 200 (179-215) and 324 (283-488) pg/g egg, respectively. The time course and signs of TCDD toxicity to brook trout during early development are essentially identical to those observed in both rainbow trout and lake trout following TCDD exposure of their eggs via water or injection, and in lake trout exposed to maternally derived TCDD. Brook trout sac fry are intermediate in sensitivity to TCDD-induced lethality compared to lake trout and rainbow trout.
The presence of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) in feral lake trout eggs (Salvelinus namaycush) may increase the risk of lake trout early life stage mortality in the Great Lakes. To assess the combined toxicity of PCDDs, PCDFs, and PCBs to lake trout early development, toxic potencies, relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), were determined for individual PCDD, PCDF, and PCB congeners, based on rainbow trout (Oncorhynchus mykiss) early life stage mortality. Newly fertilized rainbow trout eggs were injected with graded doses of 2,3,7,8-TCDD, or a PCDD, PCDF, or PCB congener. LD50 values were determined based on the egg dose that caused mortality from hatching onset to swim-up. Toxic equivalency factors (TEFs) were calculated as 2,3,7,8-TCDD LD50/congener LD50. TEFs were for PCDDs: 2,3,7,8-TCDD = 1.0; 1,2,3,7,8-PeCDD = 0.730;and 1,2,3,4,7,8-HxCDD = 0.319; for PCDFs: 2,3,4,7,8-PeCDF = 0.359; 1,2,3,4,7,8-HxCDF = 0.280; 1,2,3,7,8-PeCDF = 0.034; and 2,3,7,8-TCDF = 0.028; and for PCBs: 3,3',4,4',5-PeCB = 0.005; 3,3',4,4'-TCB = 0.00016; 2,3,3',4,4'-PeCB and 2,3',4,4',5-PeCB < 0.00007. For PCDDs, fish-specific TEFs were 10- to 100-fold higher than TEFs determined in H4IIE rat hepatoma cells, but were similar to TEFs proposed for risk assessment. For PCDFs, fish specific TEFs were similar to TEFs determined in H4IIE cells and TEFs proposed for risk assessment. However, the most significant finding was that for the coplanar PCBs and mono-ortho-chlorinated analogues of the coplanar PCBs fish-specific TEFs were to less than both those determined in H4IIE cells and proposed for risk assessment. Using these fish-specific TEFs, the risk associated with exposure of early life stages of Sake trout to complex mixtures of PCDDs, PCDFs, and PCBs in the Great Lakes can be estimated.
Bottom feeding and game fish, from each of nearly 400 sites throughout the United States were analyzed for 15 PCDD/PCDFs, PCBs, 21 pesticides and herbicides, and 13 other organic chemicals, and mercury. Seven of the PCDD/PCDFs and 15 of the chemicals were detected at over 50 percent of the sites. PCBs were detected at 91% of all sites at a mean concentration of 1.90 μg/g, and exceeded 10 μg/g at 10 sites. 2,3,7,8-TCDD was detected at 70 percent of the sites at a mean concentration of 6.9 ppt and a maximum concentration of 204 ppt. 2,3,7,8-TCDF was detected at 89 percent of the sites at a mean concentration of 13.6 ppt and a maximum concentration of 404 ppt. Fish from 75% of all sites had a total TCDD toxic equivalence concentrations (TEC) due to PCDD/PCDFs, below 10 pg/g. Those with the highest 2,3,7,8-TCDD concentrations also had the highest TEC. p,p1-DDE was the most frequently detected individual analyte (99% of samples), and was found at the highest maximum (14,000 ng/g) and mean (295 ng/g) concentrations. No correlation between specific sources and most of the analytes could be made. However, pulp and paper mills using chlorine appear to be a significant source of 2,3,7,8-TCDD and 2,3,7,8-TCDF in these samples which were collected between 1986 and 1989.
Female zebrafish (Brachydanio rerio) were exposed with their food to various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1, 5, 10 and 20 ng/fish) to investigate effects on oogenesis and reproduction. Doses of ≥5 ng/fish led to dose-related reduction of egg numbers and to lethal anomalies of their offspring (edema and malformations of the notocord). Histology of the ovaries revealed severely impaired development of previtellogenic to vitellogenic oocytes. At doses of 1 ng TCDD/fish no significant toxic effects were observed. The model appears to be useful to study mechanisms of TCDD-induced reproduction toxicity in fish.
Blue-sac disease, an abnormal condition of sac fry, has been recognized for nearly 90 years. At least 23 postulated causes have been advanced, but reports of consistently reproducible results have not been found in literature. Methods whereby blue-sac disease was induced at will are described. Closed-system incubation, whereby metabolic wastes accumulated, resulted in incidences of blue-sac disease roughly proportional to the period of holding and to ammonia concentrations which developed. Blue-sac disease was also induced by adding synthetic nitrogen compounds to water used for incubating eggs and fry.
Fertilized lake trout eggs exposed to vehicle or graded concentrations of [3H]-2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in water for 48 h accumulated 0, < 15, 40, or 400 ppt (parts per trillion) [3H]-TCDD. The exposed eggs were then transferred to flowing [3H]-TCDD free water where they remained throughout early development. Lake trout embryos developed normally in all groups until one week prior to hatch. At this time retrobulbar, meningeal and subcutaneous hemorrhages were evident in many embryos and sac fry that were derived from eggs containing 400 ppt [3H]-TCDD. High mortality prior to or during hatching accompanied these lesions. All sac fry which survived hatching in the 400 ppt TCDD group developed severe subcutaneous edema, with cessation of blood circulation in the yolk sac and body. Necrosis of the retina, brain, and spinal cord occurred in morbid embryos and sac fry. All sac fry in the 400 ppt TCDD group showed arrested development from the time of hatching and all died prior to swim-up. One percent of sac fry in the vehicle control and < 15 ppt TCDD groups, and 2% in the 40 ppt TCDD group, exhibited blue-sac disease, an edematous syndrome identical grossly and nearly identical microscopically to that observed in the 400 ppt TCDD group. Thus, the cardiovascular system appears to be the initial tissue affected in both the TCDD toxicity syndrome and in blue-sac disease of developing lake trout. Lesions in other organs including brain, retina and liver develop as a result of circulatory derangements, anemia and hypoxia.
Chemical methods were developed and evaluated for dissolving the matrix surrounding eggs of channel catfish (Ictalurus punctatus), which permits eggs to be incubated in cylindrical jars. Screening tests identified several dissolving agents, and four solutions appeared most suitable: (1) 1.5% Na2SO3; (2) 1.5% Na2SO3 plus 0.2% papain; (3) 1.5% L-cysteine-HCl plus 0.2% papain; and (4) 1.0% Na2SO3, 0.5% L-cysteine-HCl plus 0.2% papain. Production-level testing on whole egg masses demonstrated that hatching success of channel catfish eggs chemically separated and incubated in jars averaged 20.5% higher than with traditional trough-and-paddle incubation methods. No significant differences in fry deformities or survival were found among the nine treatments and controls. A trend toward higher percent hatch and higher fry viability was detected when eggs were separated from medium-sized egg masses (601–900 g) more than 24 h after the eggs were spawned. Chemical separation of eggs reduced fungal disease problems and labor associated with egg incubation. This process has been used for several years in a variety of cultural situations. Other fish species with adhesive eggs have been successfully incubated with variations of this technique.
The high-resolution gas chromatographic-mass spectrometric analysis of 25 Great Lakes fish extracts confirmed the identities of several 2,3,7,8-tetrasubstituted polychlorinated dibenzofurans (PCDFs) and dibenzo-p-dioxins (PCDDs). The dominant congener in extracts from Lake Michigan, Lake Erie, Lake St. Clair, Lake Huron, and Lake Superior fish was 2,3,7,8-tetrachlorodibenzofuran (TCDF). The fish extracts from Lake Ontario showed significant levels of 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) besides containing considerable amounts of PCDFs. The 2,3,7,8-TCDD equivalents in these extracts were determined by their activities as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. For most of these samples, there was less than a 2-fold difference in the bioassay-estimated 2,3,7,8-TCDD equivalents and the total PCDDs plus PCDFs as determined by GC-MS. The bioassay-derived values were significantly (>2-fold) higher for the Lake Erie and Lake Ontario fish extracts. This difference may be due to several factors including synergistic interactive effects of the congeners in the bioassay induction response or the presence of bioassay-active components that are not detected by GC-MS analysis.
Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were measured in white sucker liver samples from seven pulp and paper mill sites, and three reference sites in Ontario. The mills included five bleached-kraft mills, with and without secondary treatment, and two sulfite-mechanical mills. 2,3,7,8-T4CDD and 2,3,7,8-T4CDF were the dominant congeners detected in both liver and fillet samples at all of the pulp and paper mill sites, with mean 2,3,7,8-T4CDD toxic equivalent concentrations (TEQs) as high as 124 pg g−1 in liver tissue. Concentrations of PCDDs/PCDFs in liver tissues were severalfold higher than in fillet tissue, but this difference can be accounted for by lipid normalization. Biological information, including liver weight, gonad weight, MFO activity (ethoxyresorufin-O-deethylase, EROD), and plasma sex steroids, was measured on individual fish. There was no relationship between TEQs and condition factor, gonadosomatic index, liver somatic index, or circulating plasma 11-ketotestosterone. A weak negative correlation was observed between circulating plasma testosterone and TEQs. Although there was a positive correlation (r = 0.49, p < 0.001) between MFO activity (EROD) and TEQ, one site with very low chlorine use and low TEQs had EROD activity similar to levels observed at more contaminated sites. This finding, along with recent observations that MFO activity is rapidly cleared in pulp-mill-exposed fish, casts doubt on an exclusive causal relationship between MFO activity and PCDDs/PCDFs at these sites.
Embryos of the Japanese medaka were individually exposed to varying concentrations of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a static, nonrenewal system. The EC50 with 95% confidence interval (C.I.) to prevent hatching was 14 (11–17) nanograms (ng) of TCDD equivalents per liter (L) of water (parts per trillion). The LC50 with 95% C.I. for survival to 3 d posthatch was 9 (6–12) ng TCDD equivalents/L. The EC50 with 95% C.I. for embryos with minor lesions and severe, life-threatening lesions were 3.5 (1.3–5.7) ng TCDD equivalents/L and 14 (12.4–15.6) ng TCDD equivalents/L, respectively. In a separate experiment that was terminated prior to the embryos hatching or dying, the EC50 for lesions was calculated to be 2.2 (1.4–3.0) ng TCDD equivalents/L. Based on the amount of TCDD equivalents recovered from dechorionated embryos, the ED50 with 95% C.I. for lesions was calculated to be 0.24 picograms of TCDD equivalents per milligram of dechorionated embryo weight (parts per billion). When the embryos were exposed to [3H]TCDD within 1 to 2 h after fertilization, no concentration dependent increase in visible lesions was observed until after the formation of the liver rudiment (day 4 of development). By exposing Japanese medaka embryos to lethal concentrations of TCDD beginning on different days of embryonic development, it was demonstrated that the sensitive period for toxicity was during liver formation on day 4 or 5 of development. The sensitive period for development was not caused by differences in TCDD absorption across the chorion. When embryos were exposed to [3H]TCDD prior to, during or after liver formation, there was no statistical difference in the dose of TCDD equivalents that crossed the chorion and entered the yolk and embryo.
The sensitivity of early life stages of brook trout (Salvelinus fontinalis) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity was investigated. Newly fertilized eggs were exposed for 48 h to water containing either acetone or a range of concentrations of [3H]TCDD dissolved in acetone. Eggs were then transferred to TCDD-free water and observed through development. TCDD concentrations of 101 to 470 pg/g in the eggs caused dose-related increases in sac-fry mortality associated with yolk-sac edema, hemorrhages, and arrested development. These signs of TCDD-induced toxicity resemble blue-sac disease. The NOELs and LOELs for sac-fry mortality were 135 and 185 pg TCDD/g egg, respectively, whereas the LD50 and LD100 (95% fiducial limits) were 200 (179-215) and 324 (283-488) pg/g egg, respectively. The time course and signs of TCDD toxicity to brook trout during early development are essentially identical to those observed in both rainbow trout and lake trout following TCDD exposure of their eggs via water or injection, and in lake trout exposed to maternally derived TCDD. Brook trout sac fry are intermediate in sensitivity to TCDD-induced lethality compared to lake trout and rainbow trout.
Newly fertilized lake trout (Salvelinus namaycush) eggs were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), or their combination, and sac fry mortality was used to determine toxic potencies. The toxic equivalency factor (TEF) for PCB 126 was 0.0030. The dose-response curve for the PCB 126/TCDD mixture based on TCDD toxic equivalents was not significantly different from that for TCDD alone, suggesting additivity between the two congeners in causing sac fry mortality.
PCB congener 81 (3,4,4′,5-tetrachlorobiphenyl) has been detected in fish tissues from various sites in North America. The embryotoxicity of this compound to medaka (Oryzias latipes) and the induction of hepatic aryl hydrocarbon hydroxylase (AHH) in rainbow trout (Oncorhynchus mykiss) were determined to assess the toxic potency of this compound relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two other non-ortho-substituted PCB compounds, congener 77 (3,3′,4,4′-tetrachlorobiphenyl) and congener 126 (3,3′,4,4′,5-pentachlorobiphenyl). The TCDD toxic equivalency factors (TEFs) estimated for congener 81 from two end points in the medaka embryotoxicity assay were 0.0014 (from mortality data) and 0.006 (from swim bladder inflation data). The TEF estimated for congener 81 from data on AHH induction in rainbow trout was 0.004. All TEFs were greater than those estimated for congener 77 but were less than the TEFs estimated for congener 126. On the basis of these toxicity data, it is suggested that this congener may contribute significantly to the toxic burden of planar halogenated aromatic hydrocarbons in fish.
Toxic equivalency factors (TEFs), which relate the potency of individual polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), are used to assess the risk to fish early life stage development posed by complex mixtures of these congeners in eggs. Newly fertilized rainbow trout (Oncorhynchus mykiss) eggs were injected with graded doses of 5 PCDD, 4 PCDF or 4 nonortho substituted PCB congeners, and sac fry mortality was used to determine TEFs. Potencies of non-ortho substituted PCBs in causing early life stage mortality were much lower than expected based on Ah receptor binding affinities in mammals and 4 mono-ortho and 5 di-ortho substituted PCB congeners did not cause rainbow trout early life stage mortality at doses of 24 300–130 000 ng/g egg. To determine if increased elimination of PCBs, relative to PCDDs and PCDFs, was responsible for the lower toxic potency of PCBs in fish compared to mammals, PCDD, PCDF and PCB congeners were injected into newly fertilized rainbow trout eggs and the percent dose remaining 35 days later at the sac fry stage of development was determined. The percent dose of various PCB congeners remaining in sac fry (62–88%) was similar to that of PCDD and PCDF congeners (78–91%), indicating that increased elimination is not responsible for the extremely low potency of the non-ortho substituted PCB congeners or for the failure of the mono- and di-ortho substituted PCB congeners to cause rainbow trout early life stage mortality.
Vertebrate embryos are extremely sensitive to environmental contaminants known as planar halogenated hydrocarbons (PHHs). The physiological targets that mediate PHH-induced embryotoxicity are not known. We have characterized embryotoxicity in medaka (Orizias latipes) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypic PHH. DNA degradation in cells of the embryonic vasculature and loss of functional integrity of the medial yolk vein were demonstrated in TCDD-exposed embryos. Pharmacological intervention with piperonyl butoxide inhibited TCDD-induced DNA degradation, restored the functional integrity of the medial yolk vein, and protected against the embryotoxicity of TCDD. Treatment of TCDD-exposed embryos with the antioxidantN-acetylcysteine also provided significant protection against the embryotoxicity of TCDD. These results demonstrate that DNA damage and consequent cell death in the embryonic vasculature are key physiological mediators of TCDD-induced embryotoxicity.
This study evaluates the TCDD toxicity in embryos and larvae of the fathead minnow based on body burden. For the fish embryos, the LOAEL with mild lesions was at a tissue dose of 40 pg TCDD/g. The LD50 was 25,710 pg TCDD/g. Embryos exhibited gross lesions such as multifocal hemorrhages, edema, and lower jaw malformation. For the one-month-old larvae, the NOAEL was at 3,590 pg TCDD/g. The LD50 was 70,915 pg TCDD/g. Wasting-type syndrome was observed at a tissue dose of 20,000 pg TCDD/g, and 100% mortality at 163,000 pg TCDD/g.
Freshly fertilized pike eggs were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations of 0.1, 1.0 and 10 ppt (ng/liter) for 96 hours. At all concentrations examined egg development was retarded by 23%, and the growth of fry was also significantly retarded for a long period after exposure. A dose-related mortality was observed. Highest mortality rates occurred during resorption of the yolk and reached almost 100 percent at a concentration of 10 ppt. Death was preceded by development of severe generalized edemas.Histopathologically edemas and hemorrhages were observed, together with alterations of bloodvessel walls. In the liver, two stages of pathological changes were distinguished. The first was characterized by a dilation of sinusoids and a slight swelling of hepatocyte nuclei; in the second stage the nuclei were enlarged up to twice the normal diameter. Hepatocytes were degenerated and varied in size and shape and liver architecture was a almost completely lost.
To characterize the risk that polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) pose to salmonid early life stage survival, we developed a method to expose rainbow trout (Oncorhynchus mykiss) eggs to graded doses of PCDD, PCDF, and PCB congeners, using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a prototype. Rainbow trout eggs were injected 24–50 h post-fertil ization with 0.2 μl of 50 mM phosphatidylcholine (PC) liposomes (control) or 0.2 μl of 5–7 graded doses of TCDD incorporated into 50 mM PC liposomes. Injection volume never exceeded 0.6% egg volume. Immediately following injection, the injection site was sealed with Super glueR, resulting in 92–97% of TCDD dose retained by the egg. Following both egg injection and waterborne egg exposure. TCDD toxicity in rainbow trout was manifested by half-hatching mortality but predominantly by sac fry mortality associated with hemorrhages, pericardial edema, and yolk sac edema. TCDD LD50s, following injection and waterborne exposure of rainbow trout eggs, were 421 (331–489) and 439 (346–519) pg TCDD/g egg (LD50, 95% fiducial limits), respectively. As in rainbow trout, TCDD toxicity in lake trout (Salvelinus namaycush) following the same two routes of exposure was manifested by half-hatching mortality but predominantly by sac fry mortality preceded by hemorrhages and yolk sac edema. LD50s, based on the dose of TCDD in lake trout eggs, were 47 (21–65) and 65 (60–71) pg/g following injection and waterborne exposure, respectively. The egg injection method is ideal for assessing the relationship between early life stage mortality in rainbow trout and graded egg doses of individual PCDD, PCDF, or PCB congeners.
Distribution and elimination of (4-CB) were studied for 1 year after exposing female and male rainbow trout to the compound in water and then transferring the fish to a hatchery raceway. The fish were exposed in December, weights of the maturing eggs and sperm began to increase in June, and the fish began to spawn in October. From January through August elimination of 4-CB was slow with a for whole body elimination of 1.76 years in females and 1.43 years in males. However, during the spawning season whole body elimination was more rapid in both sexes; in females and 0.54 year in males. The increased elimination rate appeared primarily to be due to the voiding of 4-CB containing eggs and sperm as opposed to enhanced elimination of 4-CB from extragonadal tissues. Prior to the enhanced elimination there was a redistribution of 4-CB residues within the fish's body. This was characterized by a depletion of 4-CB from eyes and periorbital fat, followed later in time, by a reduction in 4-CB content of visceral fat. Temporally related to this was an accumulation of 4-CB residues in maturing eggs and sperm. Thus, redistribution of 4-CB occurred during spermatogenesis and vitellogenesis and preceded the enhanced elimination of 4-CB from the whole fish when the gametes were spent from the body. The overall significance of this study is that it underlines the enhancing effect of egg and sperm maturation and spawning on whole body elimination of polychlorinated biphenyls in fish.
The authors conducted chronic toxicity tests to assess the hazard to aquatic orgnaisms which may be exposed to TCDD in water or food after the use of 2,4,5 T in forestry. Some of the toxic characteristics of TCDD in food and water to several major classes of aquatic organisms are reported in the article.
Rainbow trout eggs, yolk sac fry and juveniles were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in different concentrations for 96 h. Exposure of eggs to the lowest concentration, used in this study, 0.1 ppt (10-13 glg), resulted in a significant growth retardation for 72 days. At higher concentrations significant numbers of the forthcoming fry developed generalized edemas and died. Histologically, degeneration and necrosis of liver parenchymal cells were observed. Remaining fry showed teratologic changes as foreshortened maxillas and opercular defects. Administration of TCDD to yolk sac fry had similar effects. Juvenile rainbow trout, exposed to 10 and 100 ppt TCCD for 96 h, showed growth retardation and developed slight edematous changes. At 100 ppt all fry had died within 27 days. Histologically, vacuolization of the liver parenchymal cells and intracellular inclusion bodies in liver, pancreas and stomach were observed.
In this review, we have examined the biochemical and toxic responses produced by halogenated aromatic hydrocarbons and have tried to develop a model for their mechanism of action. These compounds bind to a cellular receptor and evoke a sustained pleiotropic response. In many tissues this response consists of the expression of a battery of enzymes which are, for the most part, involved in drug metabolism, but in other tissues, those which develop toxicity, an additional set of genes is expressed which effects cellular involution, division, and/or differentiation. The toxicity of these compounds appears to be due to the sustained expression of a normal cellular regulatory system, of which we were previously unaware. In future investigations it is hoped that we will learn the nature and physiologic role of this regulatory system. Only then can we hope to understand the mechanism of toxicity of these compounds.
Distribution and elimination of 2,5,2′,5′-tetrachlorobi[14C]phenyl (4-CB) were studied during the egg, sac fry, and fry stages of rainbow trout development. Fertilized eggs were exposed to water-borne [14C]4-CB for 24 hr and transferred to an incubator containing flowing, 4-CB-free water. The whole egg and its component parts (yolk fluid and chorionic membrane) were analyzed separately for 14C immediately after exposure (T0) and 1, 4, 7, 11, 14, and 21 days later. Whole sac fry and its component parts (yolk sac and larva) were analyzed on Days 26, 35, 42, and 49 and whole fry and its component parts (viscera and eviscerated body) on Days 56, 63, 77, 91, and 105. Immediately after exposure (T0), 4-CB content of whole eggs was 0.22 ± 0.01 μg/egg (mean ± SE) and 4-CB concentration on a whole egg wet weight basis was 3.72 ± 0.08 ppm. The majority of 4-CB in the eggs was associated with the yolk fluid where it was apparently associated with water soluble lipoproteins and oil globules composed of triglycerides. During the sac fry stage the majority of 4-CB was associated with the yolk sac. However, as the yolk sac was gradually absorbed the 4-CB was transferred to the larva. The time during sac fry development when the greatest absolute amount of 4-CB was present in the larva was at the very end of the sac fry stage. This was also the time when the oil globules in the yolk sac were first consumed by the sac fry before they began feeding on their own. The highest concentration of 4-CB in the larva occurred at the start of the sac fry stage when the eggs hatched. Thereafter larva 4-CB concentration decreased due to larval growth. The most intriguing finding had to do with a change in the rate of whole body elimination of 4-CB during the transition period from the sac fry to fry stage of development. During the egg and first two-thirds of the sac fry stage, 4-CB elimination was slow (), but during the last one-third of the sac fry stage and throughout the fry stage it was rapid (). This increased rate of elimination resulted in a loss of nearly all of the 4-CB from the fry. Thus, whole body 4-CB concentration dropped precipitously in the fry and this was due primarily to enhanced 4-CB elimination but also dilution by growth. The mechanism for this rapid whole body elimination of 4-CB in rainbow trout fry is not known but may be related to the low fat content of the fry body.
This chapter discusses the extraction of tissue lipids with a solvent of low toxicity. The extraction of lipids from tissues is invariably done with volatile organic solvents, most of which are sold with warnings against excessive inhalation. For each gram of tissue, add 18 ml of extraction solvent and homogenize thoroughly. The mixture should be a well-dispersed suspension. After 30–60 s of mixing, filter the mixture, preferably with a sintered-glass, Buchner funnel under pressure rather than with a vacuum. Materials containing above-average water content, such as plasma, should be extracted with a larger volume of extraction solvent to keep the water in solution. In the case of plasma and whole blood, it is best to add the material to the solvent in small portions with continuous vortexing, if a fine suspension of the nonlipid portion is to be produced. The washing step is a convenient way to reduce the volume of the lipid extract, a factor to consider in working on a large scale. It removes primarily the higher boiling component, and thereby facilitates subsequent evaporative removal of solvent. Removal of solvent from hexane-isopropanol extracts is readily done by vacuum evaporation, but a warmer bath than usual is recommended.
The mammalian aromatic hydrocarbon (Ah) receptor is a soluble protein involved in the regulation of gene expression by halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Little is known, however, about the presence and properties of this receptor in nonmammalian species. In these studies, we sought evidence for an Ah receptor in the liver or liver-equivalent of diverse species of invertebrate and vertebrate animals. Velocity sedimentation analysis of hepatic cytosol labeled with [3H]TCDD gave equivocal results with three species of marine fish. In subsequent studies, photoaffinity labeling with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin was used to identify the Ah receptor. Specific labeling (labeling that could be displaced by an excess of unlabeled ligand) was observed in seven species of teleost and elasmobranch fish, including winter flounder (Pleuronectes americanus), killifish (Fundulus heteroclitus), scup (Stenotomus chrysops), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and dogfish (Mustelus canis and Squalus acanthias). Specific labeling was also found in cytosolic fractions prepared from PLHC-1 fish hepatoma cells and livers of a turtle (Chrysemys picta) and a cetacean, the beluga whale Delphinapterus leucas. The fish Ah receptor was sensitive to conditions of tissue preparation; inclusion of proteinase inhibitors in the homogenization buffer stabilized the receptor in some species. There was heterogeneity in the apparent molecular mass of the largest specifically labeled band in each species; these ranged from 105 to 146 kDa, slightly larger on average than mammalian Ah receptors (95-130 kDa). In contrast to the results obtained with teleost and elasmobranch fish, no specifically labeled polypeptides were detectable in cytosol from two agnathan fish species (hagfish Myxine glutinosa and sea lamprey Petromyzon marinus), the tunicate Ciona intestinalis, or any of nine other invertebrate species representing eight classes in four phyla. Overall these results suggest that the Ah receptor evolved at least 450 million years ago, prior to the divergence of bony and cartilaginous fishes. Although the exact relationship between receptor presence and dioxin responsiveness in these species is uncertain, our data predict that the invertebrate species examined in this study, which appear to lack an Ah receptor protein like that seen in mammals and fish, may be less sensitive than vertebrates to the effects of environmental contaminants that act through this transcriptional regulator.
Edema and cardiovascular dysfunction occur in vertebrates exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development. This study examined cytochrome P4501A (CYP1A) induction in endothelium and its possible association with mortality due to the edema and vascular effects of TCDD in lake trout early life stages. Lake trout (Salvelinus namaycush) eggs were injected at 24-50 hr postfertilization with 0.2 microl of 50 mM phosphatidylcholine liposomes or liposomes containing TCDD to give seven doses ranging from 11 to 176 pg TCDD/g egg. Doses of TCDD greater than 44 pg/g egg elicited hemorrhages; yolk sac, pericardial, and meningial edema; craniofacial malformations; regional ischemia; growth retardation; and mortality at the sac fry stage of development. Expression of CYP1A was assessed at four developmental stages, by immunohistochemical analysis of serial sections of individual fish with monoclonal antibody 1-12-3 to teleost CYP1A. CYP1A staining occurred in endothelial cells of many organs of TCDD-exposed but not vehicle-exposed embryos at 1 week prehatch and sac fry at 2 weeks posthatch. Earlier developmental stages examined were negative for CYP1A expression at any dose of TCDD. The strongest response occurred in sac fry at TCDD doses greater than 88 pg TCDD/g egg but was detected at doses as low as 22 pg TCDD/g egg. CYP1A staining in endothelium appeared at lower doses and was stronger than that in other cell types, in both prehatch embryos and posthatch sac fry. Thus, the vascular system is a major initial site affected by TCDD in lake trout early life stages, and the vascular endothelium is a cell type uniquely sensitive to induction of CYP1A in these developing animals. Based on an index of immunohistochemical staining of CYP1A, endothelial CYP1A induction in sac fry by TCDD occurred with an ED50 of 64-69 pg TCDD/g egg, similar to the dose-response for mortality occurring during the sac fry stage of development (LD50 = 47 pg TCDD/g egg). The correlations seen here suggest that CYP1A or aryl hydrocarbon receptor (AhR) in the endothelium may be linked to early lesions that result in TCDD-induced vascular derangements leading to yolk sac, pericardial, and meningial edema that is associated with lake trout sac fry mortality, but the precise mechanism remains to be determined.
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Toxic equivalency factors for polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in zebrafish liver cells
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Henry TR, Nesbit DJ, Peterson RE. 1996. Toxic equivalency factors for polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in zebrafish liver cells. Abstracts, 17th Annual Meeting, Society of Environmental Toxicology and Chemistry, Washing-ton, DC, USA, November 17–21, p 270.
Lake trout recruitment in the Great Lakes: Relative risks for chemically-induced early life stage mortality
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Embryo toxicity of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD): The embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Oryzias latipes)
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Toxic equivalency factors for polychlorinated dibenzo-p-dioxins
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8-Tetrachlorodibenzo-p-dioxin (TCDD) toxicity at three stages of lake trout egg development
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Lake trout recruitment in the Great Lakes: Relative risks for chemically-induced early life stage mortality
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2 3 7 8-Tetrachlorodibenzo-p-dioxin (TCDD) toxicity at three stages of lake trout egg development
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Toxic equivalency factors for polychlorinated dibenzo-p-dioxins dibenzofurans and biphenyls in zebrafish liver cells
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Lake trout recruitment in the Great Lakes: Relative risks for chemically-induced early life stage mortality
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