Andrew S. Kane

Noise-induced stress response and hearing loss in goldfish (Carassius auratus).

SUMMARY Fishes are often exposed to environmental sounds such as those associated with shipping, seismic experiments, sonar and/or aquaculture pump systems. While efforts have been made to document the effects of such anthropogenic (human-generated) sounds on marine mammals, the effects of excess noise on fishes are poorly understood. We examined the short- and long-term effects of increased ambient sound on the stress and hearing of goldfish (Carassius auratus; a hearing specialist). We reared fish under either quiet (110-125 dB re 1 μPa) or noisy (white noise, 160-170 dB re 1 μPa) conditions and examined animals after specific durations of noise exposure. We assessed noise-induced alterations in physiological stress by measuring plasma cortisol and glucose levels and in hearing capabilities by using auditory brainstem responses. Noise exposure did not produce long-term physiological stress responses in goldfish, but a transient spike in plasma cortisol did occur within 10 min of the noise onset. Goldfish had significant threshold shifts in hearing after only 10 min of noise exposure, and these shifts increased linearly up to approximately 28 dB after 24 h of noise exposure. Further noise exposure did not increase threshold shifts, suggesting an asymptote of maximal hearing loss within 24 h. After 21 days of noise exposure, it took goldfish 14 days to fully recover to control hearing levels. This study shows that hearing-specialist fishes may be susceptible to noise-induced stress and hearing loss.

Acoustical stress and hearing sensitivity in fishes: does the linear threshold shift hypothesis hold water?

SUMMARY Mammals exposed to loud aerial sounds exhibit temporary threshold shifts (TTS) that are linearly related to increases of sound pressure above baseline hearing levels. It was unknown if this relationship held true for aquatic ectotherms such as fishes. To test this linear threshold shift hypothesis (LINTS) in fishes, we examined the effects of increased ambient sound on hearing of two species differing in hearing capabilities: goldfish (Carassius auratus; a hearing specialist) and tilapia (Oreochromis niloticus; a hearing generalist). Fish were exposed to 1–28 days of either quiet (110 dB re 1 μPa) or continuous white noise. First, we examined the effect of noise sound pressure level (SPL; 130, 140, 160 or 170 dB re 1 μPa) on goldfish hearing thresholds after 24 h of noise exposure. Second, in a long-term experiment using 170 dB re 1 μPa white noise, we continuously exposed goldfish and tilapia for either 7 or 21–28 days. In both experiments, we measured alterations in hearing capabilities (using auditory brainstem responses) of noise-exposed fish. While tilapia exposed to noise for 28 days showed little or no hearing loss, goldfish exhibited considerable threshold shifts that reached an asymptote of up to 25 dB after only 24 h of exposure. There was a positive linear relationship between noise-induced TTS and the sound pressure difference between the noise and the baseline hearing thresholds in goldfish but not in tilapia. A similar relationship was found for published noise-induced threshold shifts in birds and mammals, but the slope of the linear relationship was greater in these groups than for fish. The linear threshold shift relationship provides insights into differential susceptibility of hearing specialist and generalist fishes to noise-induced hearing loss for a given SPL and provides a framework for future research on noise-induced threshold shifts in fishes and other animals.

17α‐Ethinylestradiol alters reproductive behaviors, circulating hormones, and sexual morphology in male fathead minnows (Pimephales promelas)

Ecologically relevant indicators of endocrine disruption in fish must be linked with measures of reproductive success. The ability of male fathead minnows (Pimephales promelas) to compete for, maintain, and defend a spawning substrate is paramount to reproductive success. The present study quantified alterations in male fathead minnow reproductive behaviors after exposure to environmentally relevant concentrations (0, 10, 20, or 40 ng/L) of 17alpha-ethinylestradiol (EE2) for 21 d. A video-based behavioral quantification system examined changes in male-male competitive behaviors (chasing and head-butting) and ability of males to maintain spawning substrates (nibbling and scrubbing). Behaviors analyzed included time under the spawning substrate, frequency of substrate cleaning, and conspecific aggression. Plasma hormone levels (11-ketotestosterone [11-KT], testosterone, and estradiol [E2]), vitellogenin (VTG), secondary male characteristics (tubercle count and dorsal nape pad rank), gonadosomatic index (GSI), and gonad histology also were evaluated. Exposure to 40 ng/L of EE2 decreased the ability of exposed males to compete with control males for spawning substrates (p = 0.09). Furthermore, exposed males displayed reduced frequency of substrate cleaning activities as well as chasing male competitors (p < or = 0.05). 11-Ketotestosterone, testosterone, and E2 were lower, and VTG was notably higher, in EE2-exposed males compared with control males (p < or = 0.03). 17alpha-Ethinylestradiol exposure in males also was associated with reductions in tubercles; lower GSI, gonadal maturity ranks, and number of resorbed tubercles; and presence of an ovipositor (p < or = 0.001). These data reveal alterations in male reproductive behavior that coincide with decreased hormone levels and secondary sex characteristics. Behavioral endpoints to discern potential ecological consequences in fish exposed to low concentrations of endocrine-disrupting chemicals may provide sensitive and functional indices of effect.

The effects of high-intensity, low-frequency active sonar on rainbow trout

This study investigated the effects on rainbow trout (Oncorhynchus mykiss) of exposure to high-intensity, low-frequency sonar using an element of the standard Surveillance Towed Array Sensor System Low Frequency Active (LFA) sonar source array. Effects of the LFA sonar on hearing were tested using auditory brainstem responses. Effects were also examined on inner ear morphology using scanning electron microscopy and on nonauditory tissues using general pathology and histopathology. Animals were exposed to a maximum received rms sound pressure level of 193 dB re 1 microPa(2) for 324 or 648 s, an exposure that is far in excess of any exposure a fish would normally encounter in the wild. The most significant effect was a 20-dB auditory threshold shift at 400 Hz. However, the results varied with different groups of trout, suggesting developmental and/or genetic impacts on how sound exposure affects hearing. There was no fish mortality during or after exposure. Sensory tissue of the inner ears did not show morphological damage even several days post-sound exposure. Similarly, gross- and histopathology observations demonstrated no effects on nonauditory tissues.

Chronic Nanoparticulate Silver Exposure Results in Tissue Accumulation and Transcriptomic Changes in Zebrafish

Increasing utilization of metallic nanomaterials in recent years implies an increasing rate of release to the environment, with potentially serious adverse effects on environmentally important species. Previously, we demonstrated that exposure to nanoparticulate silver for 24-48 h results in dramatic alterations in global gene expression patterns and increased tissue burdens in zebrafish gills. The present study reports outcomes associated with chronic exposure to nanoparticulate silver in zebrafish. Adult female Danio rerio were exposed to 5, 15, 25, or 50 μg/L nanoparticulate silver in a time course up to 28 days. A soluble silver treatment (5 μg/L) was also included. Results indicate that use of flow-through systems for chronic nanometal studies is a viable concept; measured concentrations of approximately 60% of nominal values over the course of the 28-day exposure were observed. Dissolution of nanoparticulate silver was measured twice weekly throughout the exposure ranging between 0.5 and 1.0 μg/L, and was relatively consistent between nanoparticulate silver tanks, with no differences between treatments. Gill samples from the 28-day time point were analyzed for global gene expression patterns and histopathology. Tissue accumulation in both gill and eviscerated carcass was dose-dependent, and remained elevated 4 days after the silver was removed. Microarray analysis also revealed a dose-dependent response pattern, with the largest number of genes affected in the 50 μg/L AgNP exposure. Pathway analysis of affected genes identified a number of GO terms that were significantly over-represented in the high AgNP dataset. These terms are associated with DNA damage repair, cellular restructuring, and developmental processes.

Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus

Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc(2)155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc(2)155, respectively.

Evaluation of Polycyclic Aromatic Hydrocarbons Using Analytical Methods, Toxicology, and Risk Assessment Research: Seafood Safety after a Petroleum Spill as an Example

Background: Polycyclic aromatic hydrocarbons (PAHs) are abundant and widespread environmental chemicals. They are produced naturally and through man-made processes, and they are common in organic media, including petroleum. Several PAHs are toxic, and a subset exhibit carcinogenic activity. PAHs represent a range of chemical structures based on two or more benzene rings and, depending on their source, can exhibit a variety of side modifications resulting from oxygenation, nitrogenation, and alkylation. Objectives: Here we discuss the increasing ability of contemporary analytical methods to distinguish not only different chemical structures among PAHs but also their concentrations in environmental media. Using seafood contamination following the Deepwater Horizon accident as an example, we identify issues that are emerging in the PAH risk assessment process because of increasing analytical sensitivity for individual PAHs, and we describe the paucity of toxicological literature for many of these compounds. Discussion: PAHs, including the large variety of chemically modified or substituted PAHs, are naturally occurring and may constitute health risks if human populations are exposed to hazardous levels. However, toxicity evaluations have not kept pace with modern analytic methods and their increased ability to detect substituted PAHs. Therefore, although it is possible to measure these compounds in seafood and other media, we do not have sufficient information on the potential toxicity of these compounds to incorporate them into human health risk assessments and characterizations. Conclusions: Future research efforts should strategically attempt to fill this toxicological knowledge gap so human health risk assessments of PAHs in environmental media or food can be better determined. This is especially important in the aftermath of petroleum spills. Citation: Wickliffe J, Overton E, Frickel S, Howard J, Wilson M, Simon B, Echsner S, Nguyen D, Gauthe D, Blake D, Miller C, Elferink C, Ansari S, Fernando H, Trapido E, Kane A. 2014. Evaluation of polycyclic aromatic hydrocarbons using analytical methods, toxicology, and risk assessment research: seafood safety after a petroleum spill as an example. Environ Health Perspect 122:6–9; http://dx.doi.org/10.1289/ehp.1306724

Tracking and quantification of single-walled carbon nanotubes in fish using near infrared fluorescence.

Detection of SWCNTs in complex matrices presents a unique challenge as common techniques lack spatial resolution and specificity. Near infrared fluorescence (NIRF) has emerged as a valuable tool for detecting and quantifying SWCNTs in environmental samples by exploiting their innate fluorescent properties. The objective of this study was to optimize NIRF-based imaging and quantitation methods for tracking and quantifying SWCNTs in an aquatic vertebrate model in conjunction with assessing toxicological end points. Fathead minnows (Pimephales promelas) were exposed by single gavage to SWCNTs and their distribution was tracked using a custom NIRF imaging system for 7 days. No overt toxicity was observed in any of the SWCNT treated fish; however, histopathology observations from gastrointestinal (GI) tissue revealed edema within the submucosa and altered mucous cell morphology. NIRF images showed strong SWCNT-derived fluorescence signals in whole fish and excised intestinal tissues. Fluorescence was not detected in other tissues examined, indicating that no appreciable intestinal absorption occurred. SWCNTs were quantified in intestinal tissues using a NIRF spectroscopic method revealing values that were consistent with the pattern of fluorescence observed with NIRF imaging. Results of this work demonstrate the utility of NIRF imaging as a valuable tool for examining uptake and distribution of SWCNTs in aquatic vertebrates.

Viability and induction of tyrosine aminotransferase in rainbow trout hepatocytes cultured on laminin and polylysine in a serum-free medium

The objective of this study was to find an economical substrate for long-term primary culture of rainbow trout hepatocytes in which viability and differentiated liver function could be maintained in a serum-free media. Attachment efficiency of primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes was determined for cells grown on plastic and Falcon Primaria dishes, and dishes coated with ECM (extracellular matrix), Matrigel, fibronectin, collagen type 1, laminin and polylysine. On ECM, Matrigel, laminin and polylysine, attachment efficiency exceeded 90%. On plastic, Falcon Primaria, fibronectin and collagen-coated dishes attachment efficiency was 15–20% and the cells were easily dislodged by pipetting. Cells grown on ECM, laminin and polylysine were maintained up to 22 day with 76%, 60% and 80% viability, respectively. On ECM cells flattened and formed a confluent monolayer on day 2 in culture. Cells grown on matrigel, laminin and polylysine had a more rounded appearance with establishment of cell-to-cell contacts on the second day in culture. Induction of TAT (tyrosine aminotransferase), a marker enzyme for differentiated liver cell functions, was studied with DEX (dexamethasone) in cells grown on laminin and polylysine in serum-free media. Cells grown on laminin and polylysine showed TAT inducibility up to 15 and 19 days, respectively.

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species.

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterussalmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis x M. crysops), and bluegill (Lepomis macrochuris) (n=8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.