Investigating the effects of three trace metals on the viability, embryonic development, and locomotor behavior of the Seminole ramshorn snail at environmentally relevant concentrations.

Trace metal contamination is a widespread issue due to its many natural and anthropogenic sources and known carcinogenic, teratogenic, and reproductive effects. As previous invertebrate trace metal research has primarily focused on model species (Daphnia magna, Chironomidae, etc.), our understanding of effects on non-model invertebrate species remains relatively poor. As such, this study assessed the exposure effects of cadmium, arsenic, and lead on viability, locomotor behavior, and embryonic development of the Seminole ramshorn snail (Planorbella duryi). Exposure treatments of CdCl2 , Na2 HAsO4 • 7H2 O, or Pb (NO3 )2 were prepared at concentrations of 0, 0.01, 0.1, 1, and 10 mg/L and confirmed using inductively coupled plasma optical emission spectroscopy (ICP-OES). Individual adult P. duryi were exposed for 7 days with viability assessed every 24 h, and locomotor behavior was accessed on Days 1 and 7 using ToxTrac v2.97 automated behavior software. Individual embryos from newly laid (<6 h old) embryonic clutches were exposed for 10 days, during which embryonic development stage was documented every 24 h. Based on our results, an additional follow-up study for cadmium was conducted using a lower range of 0-0.1 mg/L to allow for the observation of sublethal endpoints. Adult lead and cadmium exposure resulted in significant mortality in the highest treatments (1 and 10 mg/L), dose-dependent behavioral effects, and delayed embryonic development. Arsenic exposures resulted in little to no impacts for all assessed endpoints. Our results provide new insight into the sublethal impacts of these contaminants and highlight potential for behavior and embryonic development as useful tools for risk assessment. PRACTITIONER POINTS: The exposure effects of lead, cadmium, and arsenic on the viability, embryonic development, and locomotor behavior of a common freshwater snail species was investigated using environmentally relevant concentrations. The severity of impact differed for each trace metal, with cadmium being the most toxic and arsenic the least toxic at concentrations ranging from 0 to 10 mg/L. Embryonic development appeared to be the most sensitive endpoint of those tested in this study, suggesting that exposure may have prolonged effects that extend to population and community levels. The Seminole ramshorn snail serves as a sensitive alternative model species that can be used to assess the impacts of contaminants on freshwater invertebrates in future studies.

Sulfoxaflor causes mortality, decreased locomotion, and altered interactions in pavement ants (Tetramorium caespitum).

This study investigated the exposure effects of sulfoxaflor, a next-generation sulfoximine insecticide, on the viability, locomotor behavior, and nest-mate interactions of the pavement ant (Tetramorium caespitum). Adult worker ants were exposed to 0, 1, 5, 12.5, 25, or 50 mg/L of sulfoxaflor via oral ingestion for 48 h. This short-term exposure to concentrations as low as 1 mg/L had wide ranging effects on multiple locomotive parameters (average speed, mobile average speed, total travel distance), nest-mate interactions (aggression and antennation), and body morphology (abdominal curving). Exposure to sulfoxaflor for 24 h manifested in decreased locomotion, altered intraspecific interactions and the development of abdominal curvature in the 25 and 50 mg/L treatments. Such alterations in mobility and conspecific behavioral parameters would clearly impact the ability of exposed individuals to successfully satisfy resource demands and presents a risk to colony survival.

Assessing the Impacts of Methoxychlor Exposure on the Viability, Reproduction, and Locomotor Behavior of the Seminole Ramshorn Snail (Planorbella duryi).

In the present study, the effects of short-term methoxychlor exposure on the viability, reproduction, and locomotor behavior of adult seminole ramshorn snails (Planorbella duryi) was assessed. To examine impacts on viability and behavior, individuals were exposed to a water control, vehicle control, or 12.5, 50, 100, 250, 500, or 1000 µg/L of methoxychlor for 48 h; and differences in mortality and locomotor behavior assessed using the freely available ToxTrac software. To determine impacts on reproduction, pairs of snails were exposed to a vehicle control and 12.5, 25, 50, 100, and 250 µg/L of methoxychlor for 9 d; and the number of clutches and eggs laid quantified every 24 h. Methoxychlor concentrations in treatments were determined using gas chromatography. Complete mortality was observed in the 500 µg/L and 1000 µg/L treatments after 48 h and in the 250 µg/L treatment after 9 d. Decreases in the number of egg clutches were observed in all treatments, and the number of eggs laid decreased starting in the 25 µg/L treatment. Decreases in average speed, mobile speed, and total distance traveled, as well as a significant increase in frozen events, were also observed. Our results suggest that methoxychlor exposure causes detrimental effects on several nonlethal endpoints in a nonmodel aquatic invertebrate species and that the analysis of locomotor behaviors serves as a reliable, sensitive endpoint for ecotoxicology testing. Environ Toxicol Chem 2019;39:220-228. © 2019 SETAC.

Exposure to levonorgestrel increases nest acquisition success and decreases sperm motility in the male fathead minnow (Pimephales promelas).

Progestins are utilized as a component of human contraceptives, and commonly enter the environment via wastewater treatment plant effluent. Certain progestins activate fish androgen receptors and cause decreases in fecundity and masculinization of females. We used a nest acquisition assay and computer-assisted sperm analysis to examine the effects of levonorgestrel on male fathead minnow (Pimephales promelas) reproductive fitness. Males were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d. Combinations of a control male and a male from one of the treatments were placed into a competitive nesting assay, and the time each male spent holding the nest and time spent exhibiting aggressive behaviors were analyzed at 48 h postexposure. Semen samples were analyzed for total motility, straight-line velocity, curvilinear velocity, average path velocity, linearity, beat cross frequency, and wobble at 0, 30, 60, 90, and 120 s postactivation. Males exposed to either 10 or 100 ng/L of levonorgestrel exhibited increased nest acquisition success and lower levels of aggression compared with control-control pairings, as well as decreases in multiple sperm motion characteristics. Our results suggest that further research is required to ascertain the effects of levonorgestrel on male gamete quality and reproductive behaviors. Environ Toxicol Chem 2018;37:1131-1137. © 2017 SETAC.

Exposure effects of levonorgestrel on oogenesis in the fathead minnow (Pimephales promelas).

The synthetic progestin levonorgestrel is commonly utilized in human oral contraceptives. It enters the environment as a component of wastewater treatment plant effluent, and has been measured at low ng/L concentrations in surface waters. It has been shown to activate fish androgen receptors, causing the physical masculinization of females, changes in reproductive behavior, and decreases in fecundity. In the present study, the effects of levonorgestrel exposure on early-stage oogenesis in the fathead minnow (Pimephales promelas) was examined. Adult females were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d using a flow-through exposure system. The ovaries from each female were then removed via dissection and weighed for gonadosomatic index (GSI) calculations, and oocytes from one lobe preserved in Serra's fixative. Total numbers of late-stage vitellogenic oocytes exhibiting a germinal vesicle were then quantified. In a second exposure, blood plasma samples were collected from adult females and analyzed for vitellogenin concentrations using enzyme-linked immunosorbent assay. Females exposed to both concentrations of levonorgestrel developed male secondary sexual characteristics in a dose-dependent manner, and ovaries contained significantly fewer late stage oocytes. Exposure to 100 ng/L of levonorgestrel resulted in decreased GSI and blood plasma vitellogenin concentrations. The results suggest that female exposure to levonorgestrel alone may have profound effects on reproduction in progestin-contaminated environments. Environ Toxicol Chem 2017;36:3299-3304. © 2017 SETAC.

Exposure to the Contraceptive Progestin, Gestodene, Alters Reproductive Behavior, Arrests Egg Deposition, and Masculinizes Development in the Fathead Minnow (Pimephales promelas).

Endogenous progestogens and pharmaceutical progestins enter the environment through wastewater treatment plant effluent and agricultural field runoff. Lab studies demonstrate strong, negative exposure effects of these chemicals on aquatic vertebrate reproduction. Behavior can be a sensitive, early indicator of exposure to environmental contaminants associated with altered reproduction yet is rarely examined in ecotoxicology studies. Gestodene is a human contraceptive progestin and a potent activator of fish androgen receptors. Our objective was to test the effects of gestodene on reproductive behavior and associated egg deposition in the fathead minnow. After only 1 day, males exposed to ng/L of gestodene were more aggressive and less interested in courtship and mating, and exposed females displayed less female courtship behavior. Interestingly, 25% of the gestodene tanks contained a female that drove the male out of the breeding tile and displayed male-typical courtship behaviors toward the other female. Gestodene decreased or arrested egg deposition with no observed gonadal histopathology. Together, these results suggest that effects on egg deposition are primarily due to altered reproductive behavior. The mechanisms by which gestodene disrupts behavior are unknown. Nonetheless, the rapid and profound alterations of the reproductive biology of gestodene-exposed fish suggest that wild populations could be similarly affected.

Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki).

Endogenous progestogens are important regulators of vertebrate reproduction. Synthetic progestins are components of human contraceptive and hormone replacement pharmaceuticals. Both progestogens and progestins enter the environment through a number of sources, and have been shown to cause profound effects on reproductive health in various aquatic vertebrates. Progestins are designed to bind human progesterone receptors, but they also have been shown to strongly activate androgen receptors in fish. Levonorgestrel (LNG) activates fish androgen receptors and induces development of male secondary sex characteristics in females of other species. Although behavior has been postulated to be a sensitive early indicator of exposure to certain environmental contaminants, no such research on the reproductive behavior of gestagen-exposed fish has been conducted to date. The goal of our study was to examine the exposure effects of a human contraceptive progestin, LNG, on the reproductive development and behavior of the viviparous eastern mosquitofish (Gambusia holbrooki). Internal fertilization is a requisite characteristic of viviparous species, and is enabled by an androgen driven elongation of the anal fin into the male gonopodium (i.e., phallus). In this study, we exposed adult mosquitofish to ethanol (EtOH control), 10ng/L, and 100ng/L LNG for 8d using a static replacement exposure design. After 8d, a subset of males and females from each treatment were examined for differences in the 4:6 anal fin ratio. In addition, paired social interaction trials were performed using individual control males and control females or females treated 10ng/L or 100ng/L LNG. Female mosquitofish exposed to LNG were masculinized as evidenced by the elongation of the anal fin rays, a feature normal to males and abnormal to females. LNG caused significant increases in the 4:6 anal fin ratios of female mosquitofish in both the 10ng/L and 100ng/L treatments, although these differences were not significant between the two treatments. LNG caused significant increases in the 4:6 anal fin ratio of males exposed to 100ng/L, with no effects observed in the 10ng/L treatment. In addition, the reproductive behavior of control males paired with female mosquitofish exposed to 100ng/L LNG was also altered, for these males spent more time exhibiting no reproductive behavior, had decreased attending behavior, and a lower number of gonopodial thrusts compared to control males paired to control female mosquitofish. Given the rapid effects on both anal fin morphology and behavior observed in this study, the mosquitofish is an excellent sentinel species for the detection of exposure to LNG and likely other 19-nortestosterone derived contraceptive progestins in the environment.