Latent effects of early life stage exposure to triclosan on survival in fathead minnows, Pimephales promelas.

The objective of this study was to evaluate the effects of early life stage triclosan (5-chloro-2-(2,4, dichlorophenoxy)phenol, TCS) exposure on hatching, development, and survival in the fathead minnow, Pimephales promelas. Embryonic minnows were exposed to TCS (50 and 100 µg L-1) for 10 days followed by 6 weeks depuration. Mortality and morphological deformities were recorded and quantified during exposure and at the end of depuration. No significant effects on embryonic survival, time to reach the eyed stage, or hatching were found. However, at the conclusion of the depuration period, survival was significantly reduced in TCS exposed fish depending on the concentration. Visual inspection of the exposed fish suggests that mortality is related to spinal deformities, emaciation, and reduced foraging ability. Triclosan exhibits deleterious effects in fish at lower concentrations over longer durations than previously reported. Further, mortality in exposed fish 6 weeks after exposure demonstrates the need for various exposure assays to evaluate effects of TCS.

Antibiotic resistance in triclosan tolerant fecal coliforms isolated from surface waters near wastewater treatment plant outflows (Morris County, NJ, USA).

Triclosan (TCS) is a common antimicrobial agent that has been detected in wastewater treatment plant (WWTP) effluent outflows. A link between TCS exposure and increased antibiotic resistance in microbes has been postulated. The purpose of this study was to evaluate whether fecal coliforms (FC) isolated from surface waters located near (WWTP) outflows display TCS resistance and, if so, whether such organisms exhibit increased resistance to antibiotics. Water samples were collected at two streams in Morris County, NJ that receive WWTP effluent: Loantaka Brook and the Whippany River. Water samples were collected at three sites within each location near the WWTP effluent outflow. Abiotic river parameters were measured and FCs were enumerated for each sample. River parameters were analyzed to determine if TCS or antibiotic resistance was correlated to water quality. Triclosan resistance levels were determined for individual isolates, and isolates were screened against seven classes of antibiotics at clinically relevant levels to assess cross-resistance. At Loantaka Brook, 78.8% of FC isolates were resistant to TCS with an average minimum inhibitory concentration (MIC) of 43.2 µg ml(-1). In addition, 89.6% of isolates were resistant to four classes of antibiotics and all were identified as Citrobacter freundii. There was a significant effect of stream location on mean TCS MIC values in the Loantaka Brook, with effluent isolates maintaining significantly higher MIC values compared to upstream isolates. At Whippany River sites, TCS resistant isolates were detected on 94% of sampling dates with a significant relationship between TCS resistance and multiple antibiotic resistances (≥ three antibiotic classes, p<0.001). TCS resistant isolates were significantly more resistant to chloramphenicol (p=0.007) and to nitrofurantoin (p=0.037) when compared to TCS sensitive isolates. Environmental FC isolates resistant to high level TCS included species of Escherichia, Enterobacter, Serratia and Citrobacter. There was no correlation between river water quality and resistance of isolates to TCS. Presence of isolates not resistant to TCS, but resistant to other antibiotics, were significantly correlated to increased river flow, precipitation, and decreased nutrient levels, suggesting that observed resistance is due to run-off events. This study demonstrates that TCS resistant FC are common in river systems receiving WWTP effluent and display multiple drug resistance.

17alpha-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.

A video-based movement analysis system to quantify behavioral stress responses of fish.

Behavioral alterations can be measured as endpoints for sublethal toxicity, and serve as a tool for environmental risk assessment and analysis of toxicological impact. Numerous technical and biological factors have made sublethal effects on fish behavior difficult to quantify. In order to investigate stress- and contaminant-induced behavioral alterations, a video analysis system was designed by our laboratory. With this system up to 12 fish may be individually housed in 20 L exposure arenas and automatically videotaped at multiple and discrete intervals during an experimental period. Analog video data can then digitized, converted into x,y coordinates, and finally transformed into relevant behavioral endpoints using software designed for tracking fish movement combined with specific algorithms. These endpoints include velocity, total distance traveled, angular change, percent movement, space utilization, and fractal dimension (path complexity). Data from fish exposed to a reference toxicant, MS222, and simulation experiments, are presented to exemplify alterations in fish behavior associated with exposure, and accuracy and precision, respectively. The system provides flexibility to analyze any observed movement behavior, is remotely controlled, and can be transportable. These movement analyses can be used to identify characteristic behavioral responses to a variety of environmentally-relevant stressors, and assist in risk assessment and the development of more sensitive lowest observable effect level and no observable effect level for sentinel species.