Michael T. Simonich

Toxicity, uptake kinetics and behavior assessment in zebrafish embryos following exposure to perfluorooctanesulphonicacid (PFOS)

Perfluorooctanesulphonicacid (PFOS), a persistent organic contaminant, has been widely detected in the environment, wildlife and humans, but few studies have assessed its effect on aquatic organisms. The present study evaluated the effect of PFOS on zebrafish embryos. Zebrafish embryos exhibited developmental toxicity of bent spine, uninflated swim bladder, decreased heart rate and affected spontaneous movement after exposure to various PFOS concentrations (0-8mg/L) from 6 to 120h post-fertilization (hpf). The LC(50) at 120hpf was 2.20mg/L and the EC(50) at 120hpf was 1.12mg/L. Continuous exposure to PFOS from 1 to 121hpf resulted in a steady accumulation with no evidence of elimination. PFOS induced cell death at 24hpf was consistently found in the brain, eye, and tail region of embryos. PFOS exposure induced lesions in the muscle fibers with histological examination. Behavior assessment of PFOS in zebrafish embryos elevated the basal rate of swimming after 4 days of exposure, and larvae exposed to PFOS (0.25-4mg/L) for only 1h at 6dpf swam faster with increasing PFOS concentration. Embryos/larvae exposed to 8mg/L PFOS for 24h periods from 1 to 121hpf showed the highest incidence of malformations in the 97-121hpf window. This is the first study to define uptake kinetics and to focus on behavioral consequences following PFOS exposure in zebrafish. Our results further the understanding of the toxicity of PFOS to aquatic organisms and suggest the need for additional research to identify the mode of PFOS toxicity.

Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon

ABSTRACT The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.

Comparative developmental toxicity of environmentally relevant oxygenated PAHs

Oxygenated polycyclic aromatic hydrocarbons (OPAHs) are byproducts of combustion and photo-oxidation of parent PAHs. OPAHs are widely present in the environment and pose an unknown hazard to human health. The developing zebrafish was used to evaluate a structurally diverse set of 38 OPAHs for malformation induction, gene expression changes and mitochondrial function. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to a dilution series of 38 different OPAHs and evaluated for 22 developmental endpoints. AHR activation was determined via CYP1A immunohistochemistry. Phenanthrenequinone (9,10-PHEQ), 1,9-benz-10-anthrone (BEZO), xanthone (XAN), benz(a)anthracene-7,12-dione (7,12-B[a]AQ), and 9,10-anthraquinone (9,10-ANTQ) were evaluated for transcriptional responses at 48hpf, prior to the onset of malformations. qRT-PCR was conducted for a number of oxidative stress genes, including the glutathione transferase(gst), glutathione peroxidase(gpx), and superoxide dismutase(sod) families. Bioenergetics was assayed to measure in vivo oxidative stress and mitochondrial function in 26hpf embryos exposed to OPAHs. Hierarchical clustering of the structure-activity outcomes indicated that the most toxic of the OPAHs contained adjacent diones on 6-carbon moieties or terminal, para-diones on multi-ring structures. 5-carbon moieties with adjacent diones were among the least toxic OPAHs while the toxicity of multi-ring structures with more centralized para-diones varied considerably. 9,10-PHEQ, BEZO, 7,12-B[a]AQ, and XAN exposures increased expression of several oxidative stress related genes and decreased oxygen consumption rate (OCR), a measurement of mitochondrial respiration. Comprehensive in vivo characterization of 38 structurally diverse OPAHs indicated differential AHR dependency and a prominent role for oxidative stress in the toxicity mechanisms.

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.

Automated Zebrafish Chorion Removal and Single Embryo Placement Optimizing Throughput of Zebrafish Developmental Toxicity Screens

The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly characterized permeability enclosing the developing embryo. We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. The robotic embryo placement allocated 6-hpf embryos to 94.7% ± 4.2% of the wells in multiple 96-well trials. The rate of embryo mortality was 2.8% (43 of 1536) from robotic handling, the rate of missed wells was 1.2% (18 of 1536), and the frequency of multipicks was <0.1%. Embryo malformations observed at 24 hpf occurred nearly twice as frequently from robotic handling (16 of 864; 1.9%) as from manual pipetting (9 of 864; 1%). There was no statistical difference between the success of performing the embryo placement robotically or manually.

Microplate Subtractive Hybridization To Enrich for Bacteroidales Genetic Markers for Fecal Source Identification

ABSTRACT The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal Bacteroidales rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related Bacteroidales rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).

Trimethyltin chloride (TMT) neurobehavioral toxicity in embryonic zebrafish

Trimethyltin chloride (TMT) is a neurotoxicant that is widely present in the aquatic environment, primarily from the manufacture of PVC plastic, but few studies have evaluated aquatic neurotoxicity. We have examined TMT dose-dependent malformation and neurobehavioral toxicity in the embryonic zebrafish model. Exposure of embryos to TMT (0-10 μM) from 48 to 72 hours post fertilization (hpf) elicited a concentration-related increase (0-100%) in malformation incidence with an EC(25) of 5.55 μM. TMT also significantly modulated the frequency of tail flexion, the earliest motor behavior observed in developing zebrafish, and the ability to respond to a mechanical tail touch. Exposure to 5 μM TMT from 48 to 72 hpf modulated the photomotor response at 4 and 5 days post fertilization and significantly promoted apoptosis in the tail. Our study demonstrates the morphological and behavioral sensitivity of the developing zebrafish to TMT and establishes a platform for future identification of the affected pathways and chemical modulators of TMT toxicity.

Potential Environmental Impacts and Antimicrobial Efficacy of Silver- and Nanosilver-Containing Textiles

For textiles containing nanosilver, we assessed benefit (antimicrobial efficacy) in parallel with potential to release nanosilver (impact) during multiple life cycle stages. The silver loading and method of silver attachment to the textile highly influenced the silver release during washing. Multiple sequential simulated household washing experiments for fabric swatches in deionized water with or without detergent showed a range of silver release. The toxicity of washing experiment supernatants to zebrafish (Danio rerio) embryos was negligible, with the exception of the very highest Ag releases (∼1 mg/L Ag). In fact, toxicity tests indicated that residual detergent exhibited greater adverse response than the released silver. Although washing the fabrics did release silver, it did not affect their antimicrobial efficacy, as demonstrated by >99.9% inhibition of E. coli growth on the textiles, even for textiles that retained as little as 2 μg/g Ag after washing. This suggests that very little nanosilver is required to control bacterial growth in textiles. Visible light irradiation of the fabrics reduced the extent of Ag release for textiles during subsequent washings. End-of-life experiments using simulated landfill conditions showed that silver remaining on the textile is likely to continue leaching from textiles after disposal in a landfill.

Arsenic (III, V), indium (III), and gallium (III) toxicity to zebrafish embryos using a high-throughput multi-endpoint in vivo developmental and behavioral assay

Gallium arsenide (GaAs), indium gallium arsenide (InGaAs) and other III/V materials are finding increasing application in microelectronic components. The rising demand for III/V-based products is leading to increasing generation of effluents containing ionic species of gallium, indium, and arsenic. The ecotoxicological hazard potential of these streams is unknown. While the toxicology of arsenic is comprehensive, much less is known about the effects of In(III) and Ga(III). The embryonic zebrafish was evaluated for mortality, developmental abnormalities, and photomotor response (PMR) behavior changes associated with exposure to As(III), As(V), Ga(III), and In(III). The As(III) lowest observable effect level (LOEL) for mortality was 500 μM at 24 and 120 h post fertilization (hpf). As(V) exposure was associated with significant mortality at 63 μM. The Ga(III)-citrate LOEL was 113 μM at 24 and 120 hpf. There was no association of significant mortality over the tested range of In(III)-citrate (56-900 μM) or sodium citrate (213-3400 μM) exposures. Only As(V) resulted in significant developmental abnormalities with LOEL of 500 μM. Removal of the chorion prior to As(III) and As(V) exposure was associated with increased incidence of mortality and developmental abnormality suggesting that the chorion may normally attenuate mass uptake of these metals by the embryo. Finally, As(III), As(V), and In(III) caused PMR hypoactivity (49-69% of control PMR) at 900-1000 μM. Overall, our results represent the first characterization of multidimensional toxicity effects of III/V ions in zebrafish embryos helping to fill a significant knowledge gap, particularly in Ga(III) and In(III) toxicology.

Developmental benzo[a]pyrene (B[a]P) exposure impacts larval behavior and impairs adult learning in zebrafish

Polycyclic aromatic hydrocarbons (PAHs) are produced from incomplete combustion of organic materials or fossil fuels, and are present in crude oil and coal; therefore, they are ubiquitous environmental contaminants present in urban air, dust, soil, and water. It is widely recognized that PAHs pose risks to human health, especially for the developing fetus and infant where PAH exposures have been linked to in-utero mortality, cardiovascular effects, and lower intelligence. Using the zebrafish model, we evaluated the developmental toxicity of benzo[a]pyrene (B[a]P). Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 0.4 and 4μM B[a]P. The Viewpoint Zebrabox systems were used to evaluate larval photomotor response (LPR) activity and we identified that exposure to 4μM B[a]P resulted in a hyperactive LPR phenotype. To evaluate the role of aryl hydrocarbon receptor (AHR) in this larval phenotype, we exposed ahr2hu2334 null larvae to 4μM B[a]P. Though ahr2hu2334 larvae did not display hyperactive swimming, these larvae had a decrease in LPR activity, suggesting that AHR2 plays a role in B[a]P induced larval hyperactivity. To determine if developmental B[a]P exposures would produce adult behavioral deficits, a subset of exposed animals was raised to adulthood and tested in a conditioned stimulus test using shuttleboxes. Developmentally exposed B[a]P zebrafish exhibited decreased learning and memory. Together this data demonstrates that developmental B[a]P exposure adversely impacts larval behavior, and learning in adult zebrafish.