Potency matters: Impacts of embryonic exposure to nAChR agonists thiamethoxam and nicotine on hatching success, growth, and neurobehavior in larval zebrafish.

Thiamethoxam (TM) is a neonicotinoid insecticide that acts as a nicotinic acetylcholine receptor (nAChR) agonist. While designed to specifically target invertebrate nAChRs, recent studies have reported adverse effects of neonicotinoid exposure in early life-stage fish. This study examined the health and neurobehavioral impacts of chronic exposure to various concentrations of TM or nicotine (NIC) in early life zebrafish (Danio rerio) in conjunction with in-silico molecular docking to compare their ligand-receptor interactions with vertebrate nAChR. Chronic exposure to both reduced survival by approximately 20% (163 µg TM/l) and 25-100% (≥0.49 µg NIC/l). Hatching and growth were impaired following exposure to ≥0.21 µg TM/l or 4.9 µg NIC/l. Both TM and NIC produced morphological and behavioral indicators of neurotoxicity, with more potent effects following NIC exposure. NIC impaired embryonic motor activity by 40% (49 µg NIC/l), while both TM and NIC significantly altered predator escape response in larvae, specifically the latency and the initial burst movement of the response were impacted. Molecular docking predicted variations in the type and strength of interactions that occur between NIC or TM and vertebrate nAChR. These findings demonstrate that chronic exposure to TM might impact general health and neurobehavior of early-stage zebrafish. Our data support hypotheses that TM presents low affinity for vertebrate nAChR but may still pose an adverse risk to larval fish growth and neurobehavior.

Using morphological, behavioral, and molecular biomarkers in Zebrafish to assess the toxicity of lead-contaminated sediments from a retired trapshooting range within an urban wetland.

The widespread use of lead (Pb) shot in shooting activities, including at former shooting ranges, continues to pose environmental risks. The La Crosse River Marsh (located in Wisconsin, USA) is a biologically diverse urban riparian wetland with a legacy of Pb-contaminated sediment resulting from its use as a trap shooting range from 1929-1963. Within the shot fall zone, shot densities exceed 43,000 pellets/m2 and surface sediments exceed 25,000 mg/kg in some areas. This study used the Zebrafish as a model to determine the acute toxicity of these contaminated sediments. Zebrafish were exposed to sediments containing approximately 13 to 13,450 mg/kg Pb for 5 days (8-120 hr post-fertilization). The toxic responses to sediments were non-monotonic. Only exposure to sediments containing "mid-range" concentrations of Pb (4580 mg/kg) induced mild skeletal malformations and a sluggish C-start response indicating that Pb was marginally bioavailable. Expression of δ-aminolevulinic acid dehydratase (ALA-D) also indicated the potential for uptake of Pb from sediments. Our findings suggest that Pb within the La Crosse River Marsh sediments is not readily bioavailable to Zebrafish, and while this metal poses a minimal acute toxicological risk, toxicity due to chronic exposure of low concentrations of Pb is possible. Further, our data demonstrated that induction of ALA-D gene expression in Zebrafish embryos shows promise as an alternative to ALA-D enzyme activity as a biomarker for acute Pb exposure under lab conditions.

Toxicity of oxidatively degraded quantum dots to developing zebrafish (Danio rerio).

Once released into the environment, engineered nanoparticles (eNPs) are subjected to processes that may alter their physical or chemical properties, potentially altering their toxicity vis-à-vis the as-synthesized materials. We examined the toxicity to zebrafish ( Danio rerio ) embryos of CdSecore/ZnSshell quantum dots (QDs) before and after exposure to an in vitro chemical model designed to simulate oxidative weathering in soil environments based on a reductant-driven Fenton's reaction. Exposure to these oxidative conditions resulted in severe degradation of the QDs: the Zn shell eroded, Cd(2+) and selenium were released, and amorphous Se-containing aggregates were formed. Products of QD weathering exhibited higher potency than did as-synthesized QDs. Morphological endpoints of toxicity included pericardial, ocular and yolk sac edema, nondepleted yolk, spinal curvature, tail malformations, and craniofacial malformations. To better understand the selenium-like toxicity observed in QD exposures, we examined the toxicity of selenite, selenate, and amorphous selenium nanoparticles (SeNPs). Selenite exposures resulted in high mortality to embryos/larvae while selenate and SeNPs were nontoxic. Co-exposures to SeNPs + CdCl2 resulted in dramatic increase in mortality and recapitulated the morphological endpoints of toxicity observed with exposure to products of QD weathering. Cadmium body burden was increased in larvae exposed to weathered QDs or SeNP + CdCl2 suggesting the increased potency of products of QD weathering was due to selenium modulation of cadmium toxicity. Our findings highlight the need to examine the toxicity of eNPs after they have undergone environmental weathering processes.

Binary mixtures of imidacloprid and thiamethoxam do not appear to cause additive toxicity in fathead minnow larvae (Pimephales promelas).

Introduction: Considerable use of neonicotinoid insecticides has resulted in their detection in surface waters globally, with imidacloprid (IM) and thiamethoxam (TM) frequently found together. Neonicotinoids are selective agonists for invertebrate nicotinic acetylcholine receptors (nAChR) leading to paralysis and death. While not overtly toxic to vertebrates, growing evidence suggests that chronic exposure to individual neonicotinoids can cause adverse health effects in fish. This work examined whether chronic exposure to binary mixtures of imidacloprid (IM) and thiamethoxam (TM) would be more toxic to fathead minnow (Pimephales promelas) larvae than either insecticide alone. Materials and Methods: Embryos were exposed to a 1:1 mixture of IM and TM (0.2, 2, 20, 200 or 2,000 µg/L of each pesticide) or a 1:5, 1:10, or 1:20 mixture of IM and TM (0.02 µg/L of IM with 0.1, 0.2, or 0.4 µg/L of TM) for a total of 8 days. Survival, developmental toxicity, embryonic motor activity, and startle escape responses were quantified. Results: Survival and growth were reduced, and hatching induced by exposure to a 1:1 mixture containing > 2 µg/L of each insecticide, but not following exposure to mixtures containing environmentally-relevant concentrations. Acute exposure to a 1:1 mixture did not alter embryonic motor activity; however, chronic exposure to these mixtures resulted in a slight but significant decrease in embryonic movements. Only 1:1 mixtures of high concentrations of IM and TM altered the startle escape response by increasing latency of response; however, a significant proportion of fish exposed to 1:1 mixtures had altered latency and burst speed. Taken together, these behavioral indicators of nAChR activation suggest that in mixtures, neonicotinoids could interfere with nAChR signaling despite their low affinity for the nAChR. Conclusion: Our findings suggest that toxicity of binary mixtures of IM and TM is primarily driven by IM, and that mixtures of IM with TM do not appear to cause significant additive toxicity when compared with our previous studies evaluating each neonicotinoid alone. Given the limited toxicological data available for mixtures of neonicotinoid insecticides in fish, further study is required to better understand the ecological risks these insecticides may pose to aquatic ecosystems.

Chronic Exposure to Environmentally Relevant Concentrations of Imidacloprid Impact Survival and Ecologically Relevant Behaviors of Fathead Minnow Larvae.

Imidacloprid (IM) has emerged as a contaminant of concern in several areas within the United States due to its frequent detection in aquatic ecosystems and its pseudo-persistence, which pose potential risks to nontarget species. We evaluated the sublethal toxicity of IM to fathead minnow larvae following chronic exposure beginning just after fertilization. Our in silico analysis and in vivo bioassays suggest that IM has a low binding affinity for the vertebrate nicotinate acetylcholine receptor (nAChR), as expected. However, chronic exposure to ≥0.16 µg IM/L reduced survival by 10%, and exposure to ≥18 µg IM/L reduced survival by approximately 20%-40%. Surviving fish exposed to ≥0.16 µg IM/L showed reduced growth, altered embryonic motor activity, and premature hatching. Furthermore, a significant proportion of fish exposed to ≥0.16 µg IM/L were slower to respond to vibrational stimuli and slower to swim away, indicating that chronic exposure to IM has the potential to impair the ability of larvae to escape predation. The adverse health effects we observed indicate that chronic exposure to environmentally relevant concentrations of IM may elicit sublethal responses that culminate in a significant increase in mortality during early life stages, ultimately translating to reduced recruitment in wild fish populations. Environ Toxicol Chem 2023;42:2184-2192. © 2023 SETAC.

Geometric Morphometrics as a Tool to Evaluate Teratogenic Effects in Zebrafish (Danio rerio).

Geometric morphometrics allows for the characterization of shape using Cartesian geometric coordinates rather than linear or volumetric measurements, which are dependent upon size and are insufficient to capture geometric shape. By using landmarks on specimens, variations in position, orientation and scale between specimens can be removed to better compare variations in shape. This method has primarily been used in the fields of evolutionary biology and taxonomy. Here we describe how geometric morphometrics can be used to delineate variations in shape caused by teratogenic compounds in zebrafish.

Developmental Toxicity of Triclosan in the Presence of Dissolved Organic Carbon: Moving Beyond Standard Acute Toxicity Assays to Understand Ecotoxicological Risk.

Triclosan (TCS) is an ionizable synthetic antimicrobial that has been found to be a persistent environmental contaminant with potential for bioaccumulation. Standard laboratory assays have shown that TCS is toxic to aquatic organisms; however, varied environmental conditions could impact this risk. For example, we would predict that sorption to dissolved organic carbon (DOC) in natural surface waters would reduce the bioavailability and, therefore, toxicity of TCS. To better understand the potential risk that TCS poses to wild fish, we evaluated the toxicity of TCS to zebrafish in the presence of DOC. Zebrafish were exposed to TCS (0-900 µg TCS/L), DOC (0-25 mg/L), or TCS (0-900 µg TCS/L) together with either 10 or 25 mg DOC/L from 8 to 120 h postfertilization through static waterborne exposure. We compared impacts of TCS alone or in conjunction with DOC on mortality, development, and hatching success. Exposure to TCS in the presence of DOC improves survival and hatching success, and reduces the incidence of developmental toxicity. However, since the presence of DOC did not completely prevent sublethal toxicity, our data suggest that given its bioaccumulation potential, developmental toxicity of TCS under environmental conditions still warrants concern.

Cardiac Toxicity of Triclosan in Developing Zebrafish.

Triclosan (TCS) is an antimicrobial agent found in personal care products that has become prevalent in surface waters. TCS readily bioaccumulates within aquatic organisms, and has been found to be toxic to fish. In larval fishes, exposure to TCS disrupts a variety of developmental processes, impairs hatching success, and causes pericardial edema. In mammals, TCS exposure disrupts excitation-contraction-coupling in cardiac cells, which is associated with reductions in cardiac output. Here, we examine the impacts of TCS on heart function to better understand potential risks that TCS may pose to wild fish. Zebrafish were exposed to 0, 0.4, 40, and 400 µg TCS/L from 8 to 120 h postfertilization via static waterborne exposure with daily renewal. We examined the incidence of pericardial edema, and the impacts on heart structure and heart function. While incidence of pericardial edema increased following exposure to ≥40 µg TCS/L and the structure of the heart was altered, cardiac output was only reduced following exposure to 400 µg TCS/L. A small but significant proportion of embryos showed increased incidence of regurgitation following exposure to ≥0.4 µg TCS/L. Our findings suggest that acute exposure to TCS has the potential to cause subtle cardiac toxicity in developing fish, and further evaluation of the risks TCS pose to wild fish and human health is needed.

Juvenile exposure to vinclozolin shifts sex ratios and impairs reproductive capacity of zebrafish.

Exposure to endocrine disruptors during critical periods of development can impact the sustainability of wild fish populations. Anti-androgenic compounds have received less attention, but are capable of modulating gonad differentiation and maturation, and impairing reproduction in fish. The fungicide vinclozolin (VZ) has been shown to impair reproduction in adult fish, but less is known about its effects following exposure earlier in development. Here we show that waterborne exposure to 400µg VZ/L during critical periods of sex differentiation (21-35 days post fertilization) permanently shifts sex ratios towards females, and alters the maturation of the gonad. Both fecundity and fertility were reduced, even when oogenesis and spermatogenesis recover and sperm motility is not altered. These results demonstrate the need to better understand the impacts of early exposure to anti-androgenic compounds on fish.

Embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prey capture by zebrafish larvae.

As a ubiquitous, persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has the potential to cause lethal deformities in larval fishes. Few studies have examined its impacts on larval growth and craniofacial development in conjunction with feeding capability. The authors used morphological and behavioral assessments to demonstrate that feeding capability of larvae is impaired even when craniofacial structures are not grossly malformed. Zebrafish embryos were exposed to 25 pg TCDD/mL, 50 pg TCDD/mL, or 100 pg TCDD/mL or <0.1% dimethyl sulfoxide for 1 h at 4 h postfertilization and then raised in clean water for 21 d or 90 d to assess craniofacial morphology, feeding capability, and long-term survival. The lower jaw was 5% smaller in 21-d larvae exposed to ≥ 50 pg TCDD/mL, and those larvae caught 10% fewer prey items; survival was reduced by 13% to 23%. The direct cause of TCDD's impacts on feeding capability is not known, but feeding success was correlated with growth, length of lower jaw, and survival. Since low larval mortality rates are key for recruitment, this suggests that exposure to concentrations of TCDD during embryonic development that do not initially cause mortality still has the potential to impact the recruitment success of feral fish. Furthermore, the present work provides additional evidence that behavioral end points are often more sensitive than morphological ones and should be included when assessing the sublethal toxicity of environmental contaminants.

Dioxin induction of transgenerational inheritance of disease in zebrafish.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is an aryl hydrocarbon receptor (AHR) agonist, an endocrine disruptor, and a potent global pollutant. TCDD exposure is associated with diseases of almost every organ system, and its toxicity is highly conserved across vertebrates. While the acute developmental effects of dioxin exposure have been extensively studied, the ability of early sublethal exposure to produce toxicity in adulthood or subsequent generations is poorly understood. This type of question is difficult to study because of the time frame of the effects. With human subjects, such a study could span more than a lifetime. We have chosen zebrafish (Danio rerio) as a model because they are vertebrates with short generation times and consistent genetic backgrounds. Zebrafish have very modest housing needs, facilitating single and multigenerational studies with minimal time and expense. We have used this model to identify transgenerational effects of TCDD on skeletal development, sex ratio, and male-mediated decreases in reproductive capacity. Here we compare these findings with transgenerational effects described in laboratory rodent species. We propose that the zebrafish is a cost-effective model system for evaluating the transgenerational effects of toxic chemicals and their role in the fetal basis of adult disease.

Reproductive and developmental toxicity of dioxin in fish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans.

Quantum dot nanotoxicity assessment using the zebrafish embryo.

Quantum dots (QDs) hold promise for several biomedical, life sciences, and photovoltaic applications. Substantial production volumes and environmental release are anticipated. QD toxicity may be intrinsic to their physicochemical properties, or result from the release of toxic components during breakdown. We hypothesized that developing zebrafish could be used to identify and distinguish these different types of toxicity. Embryos were exposed to aqueous suspensions of CdSe(core)/ZnS(shell) QDs functionalized with either poly-L-lysine or poly(ethylene glycol) terminated with methoxy, carboxylate, or amine groups. Toxicity was influenced by the QD coating, which also contributed to the QD suspension stability. At sublethal concentrations, many QD preparations produced characteristic signs of Cd toxicity that weakly correlated with metallothionein expression, indicating that QDs are only slightly degraded in vivo. QDs also produced distinctly different toxicity that could not be explained by Cd release. Using the zebrafish model, we were able to distinguish toxicity intrinsic to QDs from that caused by released metal ions. We conclude that developing zebrafish provide a rapid, low-cost approach for assessing structure-toxicity relationships of nanoparticles.

Persistent adverse effects on health and reproduction caused by exposure of zebrafish to 2,3,7,8-tetrachlorodibenzo-p-dioxin during early development and gonad differentiation.

Little is understood regarding the impacts of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure during early development on the health, survival, and reproductive capability of adults. Here we use zebrafish to determine whether early life stage exposure to TCDD induces toxicity in adult zebrafish and their offspring. Zebrafish were exposed to graded concentrations of TCDD (0-400 pg/ml) via waterborne exposure for 1 h/week from 0 to 7 weeks of age. The heart and swim bladder were identified as being most sensitive to TCDD exposure during early development. Subtle developmental toxic responses collectively impaired survival, and only zebrafish in the 0, 25, and 50 pg TCDD/ml groups survived to adulthood. Surviving fish exhibited TCDD toxicity in craniofacial structures (i.e., operculum and jaw), heart, swim bladder, and ovary. Exposure to 25 pg TCDD/ml impaired egg production (40% of control), fertility (90% of control), and gamete quality. TCDD-treated males contributed more than females to impaired reproductive capacity. Transgenerational effects were also discovered in that offspring from parents exposed to TCDD during early life stages showed a 25% increase in mortality compared with the F1 of dimethyl sulfoxide fish, reduced egg production (30-50% of control) and fertility (96% of control). Thus, adverse effects resulting from TCDD exposure during early life stages for one generation of zebrafish were sufficient to cause adverse health and reproductive effects on a second generation of zebrafish. In the environment, transgenerational effects such as these may contribute to population declines for the most TCDD sensitive fish species.