Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish

Highlights

• Two populations of killifish were behaviorally compared following developmental PAH mixture exposure.

• Early life PAH mixture exposure increased later life mortality.

• Early life PAH mixture exposure caused later life behavioral alterations.

• PAH-adapted killifish were resistant to the mortality and behavioral impacts of embryonic PAH exposure.

• This behavioral testing framework can be used for contaminant assessment.

Abstract

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24 h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3 months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and concentrations increase with human population growth and fossil fuel use (Van Metre et al., 2000, Foan et al., 2010, Yang et al., 2011). Aquatic environments are susceptible to PAH contamination in a variety of ways including oil shipping, oil refining, industrial outfall, wastewater discharges, urban runoff, and atmospheric deposition (Van Metre et al., 2000, Walker et al., 2004, Van Metre and Mahler, 2005, Hylland, 2006). Anthropogenic PAHs are present in the environment as complex mixtures, and the composition is in part dependent on the contribution from pyrolytic and/or petrogenic sources (Van Metre et al., 2000, Van Metre and Mahler, 2005). Pyrolytic PAH mixtures are composed of predominantly high molecular weight (HMW) PAHs such as benzo[a]pyrene (BaP), whereas petrogenic PAHs contain lower concentrations of HMW PAHs (Shen et al., 2013).

PAHs are hydrophobic, and consequently, associated with suspended particulate matter in water or settle into sediments (Benlahcen et al., 1997, Baumard et al., 1998, Cachot et al., 2006, Cachot et al., 2007, Hylland, 2006). Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can be persistent in sediments over time (Cachot et al., 2006, Cachot et al., 2007, Hylland, 2006, Yanagida et al., 2012). For this reason, sediments represent a significant source of exposure for fish embryos (Benlahcen et al., 1997, Baumard et al., 1998, Cachot et al., 2006, Cachot et al., 2007). Fish in both freshwater and marine ecosystems will commonly lay their eggs on sediments or gravel found in river or seabed (Baumard et al., 1998, Cachot et al., 2006, Cachot et al., 2007). Several minnow species are known to bury eggs in sediment (Skinner et al., 2005, Burnett et al., 2007). It is well known that PAHs can transfer through the chorion of the fish embryos (Djomo et al., 1996, Hornung et al., 1996, McElroy et al., 2006). Therefore, it is likely that fish embryos developing on PAH-contaminated sediment and other polluted sediments will be exposed during sensitive ontogenetic time points.

The Elizabeth River (ER) is a highly contaminated subestuary of the Chesapeake Bay watershed, located in the Tidewater region of southeastern Virginia, USA (Walker et al., 2004). Several former wood treatment industries contaminated areas of the river with creosote, a complex mixture of chemicals consisting primarily of unsubstituted polycyclic aromatic hydrocarbons (PAHs) and some heterocyclic and phenolic PAHs (Clark et al., 2013, Fang et al., 2014). Total PAH concentrations at one of the major sites, Atlantic Wood Industries (AW), range from 100 to 500 μg/g in dry sediment (Mulvey et al., 2002, Walker et al., 2004). Sediment pore water from the site has recently been chemically characterized (Fang et al., 2014). The AW site was placed on the Environmental Protection Agency's “National Priorities List” (for Superfund sites) in 1990, and is currently being remediated.

A large number of organic xenobiotics, e.g., dioxins, PCBs, and polybrominated diphenyl ether (PBDE) have been reported to disrupt embryonic development in fish (Cantrell et al., 1996, Henry et al., 1997, Carls et al., 1999, Heintz et al., 1999, Barron et al., 2004, Antkiewicz et al., 2005, Carney et al., 2006, Incardona et al., 2006, Carls et al., 2008). These teratogenic effects have also been reported for individual PAHs and PAH mixtures, and some exposures at environmentally relevant concentrations cause early life stage toxicity (Moles and Rice, 1983, Incardona et al., 2006, Incardona et al., 2009, Incardona et al., 2011). Acute PAH toxicity manifests as cranio-facial and cardiac malformations and pericardial and yolk sac edema that is similar to effects seen with some dioxin-like compounds (DLCs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) (Helder, 1981, Spitsbergen et al., 1991, Walker and Peterson, 1991, Walker et al., 1991).

Fundulus heteroclitus (the Atlantic killifish or mummichog; hereafter referred to as killifish) is a small teleost fish found in Atlantic coastal estuaries from Newfoundland to Florida (Kneib, 1986, Teo and Able, 2003). They are the most abundant intertidal fish species and a major component of food webs in these estuaries. Although killifish are widely distributed, individuals have relatively small home ranges (Lotrich, 1975, Skinner et al., 2005). This high site fidelity and small migration area makes them ideal for studying the effects of anthropogenic contamination and other environmental stressors (Burnett et al., 2007). The killifish inhabiting the AW Superfund site are chronically exposed to PAH-contaminated sediments, but have developed significant resistance to the acute cardiotoxicity and other teratogenic effects of Elizabeth River sediments, PAHs, PCB-126, and several pesticides (Meyer and Di Giulio, 2002, Meyer et al., 2002, Ownby et al., 2002, Clark and Di Giulio, 2012, Clark et al., 2013). Recent studies have also noted other populations of killifish residing throughout the Elizabeth River and their relative resistance and susceptibility to PAH cardiotoxicity and other environmental contaminants (Clark and Di Giulio, 2012, Clark et al., 2013).

In the current study, we focused on the later life behavioral impacts of early developmental exposure to a complex PAH mixture with particular emphasis placed on the comparison between two different populations of killifish. We hypothesized that early embryonic exposure to low dilutions of ERSE would alter larval locomotor activity of the naive King's Creek killifish (KC) (King's Creek is a relatively uncontaminated tributary of the Severn River, VA), but would not alter locomotor activity in the PAH-adapted Atlantic Wood killifish (AW) population. In addition, we postulated that KC killifish exposed to higher dilutions of ERSE would experience increased mortality over time, whereas AW killifish would experience little to no increases in mortality due to their PAH-resistance. To this end, we exposed both the naïve reference KC population of killifish and the PAH-adapted AW killifish to subteratogenic dilutions (dilutions that did not cause overt cardiac abnormalities) of ERSE. After early embryonic exposure to low dilutions of ERSE, larvae were tested for mobility and then raised to three months for additional behavioral testing (startle habituation assay and predator avoidance/novel dive assay). In a separate set of experiments, killifish were treated similarly and raised to 5 months for mortality assessment.

While studies have described the developmental effects of acute individual and mixed PAH exposure, very few studies have examined the later life consequences of early life PAH exposures, particularly alterations to fish behavior (Vignet et al., 2014a, Vignet et al., 2014b). Additionally, there are several tools and assays available for the zebrafish (Danio rerio) model, (Bailey et al., 2013), but few studies have employed these tests to evaluate how early life exposure to PAH mixtures impacts later life behavioral outcomes in a more environmentally relevant model such as the killifish. Little is known about the performance and behavioral costs that may be associated with being a PAH-adapted AW killifish.