A novel system for embryo-larval toxicity testing of pelagic fish: Applications for impact assessment of Deepwater Horizon crude oil
Graphical abstract
Images and diagrams are from the corresponding author and shapes were created by the corresponding author using Microsoft PowerPoint.
Introduction
Recent research following the BP Deepwater Horizon (DWH) oil spill of 2010 has revealed the potential for specific physiological impacts in fish, notably cardiac impairment and reductions in swim performance, resulting from DWH crude oil exposure (Brette et al., 2014, Esbaugh et al., 2016, Incardona et al., 2014, Mager et al., 2014, Stieglitz et al., 2016). Such findings are in agreement with previous studies examining the effects of crude oil exposure on teleost early life stages (ELSs; embryos/larvae) which reveal a host of common lethal and sub-lethal effects. Many of these effects appear to be the result of cardiotoxicity resulting from acute exposure to polycyclic aromatic hydrocarbons (PAHs) in crude oil during embryonic development (Carls et al., 2008, Carls et al., 1999, Couillard, 2002, Esbaugh et al., 2016, Heintz et al., 1999, Hicken et al., 2011, Incardona et al., 2004). Specifically, acute PAH exposure in teleost ELSs has been shown to induce defects in cardiac function, pericardial and yolk sac edema, neurodevelopmental abnormalities, jaw deformations, and other defects during morphogenesis (Carls et al., 2008, de Soysa et al., 2012, Esbaugh et al., 2016, Incardona et al., 2014, Incardona et al., 2013, Incardona et al., 2011, Incardona et al., 2004, Irie et al., 2011). While teleost ELSs are putatively the life stages of fish most sensitive to crude oil exposure, the scientific literature suggests that effect thresholds vary significantly among species, crude oil compositions, and exposure conditions (Alloy et al., 2016, Esbaugh et al., 2016, Incardona et al., 2014). Due to these differences, it is imperative to conduct toxicity tests replicating the likely exposure scenarios encountered by native, pelagic teleost ELSs in the Gulf of Mexico (GoM) during the DWH spill event. One goal of the injury assessment has been to quantify the effect of the spill on economically and ecologically important teleost fish species in the GoM (McCrea-Strub et al., 2011, Sumaila et al., 2012). However, quantifying toxicity to high-value resident non-model species, such as tuna (Thunnus spp.) and mahi-mahi, or dolphin-fish, (Coryphaena hippurus) following such events using commonly accepted toxicity tests (96-hr bioassays) and endpoints (i.e. LC50) faces many logistical challenges. These challenges result primarily from the difficulty in obtaining and working with such species in a controlled environment.
Marine natural resource impact assessment research in the United States frequently utilizes traditional toxicological testing methods, such as those published by the American Society for Testing and Materials (ASTM) and the United States Environmental Protection Agency (USEPA) (ASTM - E47 Committee, 2005, USEPA, 2002). The use of these prescribed procedures facilitates comparisons among other relevant studies and allows for regulatory applications. This is particularly important for determination of the biological effects of an impact event, whereby acute toxicity tests provide an estimation of the contaminant's toxicity using a commonly accepted metric, such as the median lethal concentration (LC50). Of central importance to such tests is the ability to attain acceptably high levels of survival in control treatments in order to provide a baseline against which treatment effects can be accurately measured. Moreover, control survival of a test is one measure of test quality that provides evidence of organism vigor, physiological quality, suitability of the treatment chambers, and overall test conditions (ASTM - E47 Committee, 2005), particularly in the absence of published guidelines for a specific species and/or life stage (e.g., pelagic fish embryos). In combination with a necessity to maintain high control survival is a need for treatment chambers to contain sufficient numbers of test animals (n) to support high power statistical analyses. An additional benefit to a higher n per replicated treatment chamber is the ability to harvest sufficient numbers of test animals from replicates for use in post-hoc analyses such as morphometric imaging, immunohistochemical analysis, and ecotoxicogenomics (Snape et al., 2004). Accurate determination of the effects of acute and chronic environmental impact events on GoM representative species is aided by robust experimental design incorporating such analyses. In order to obtain high control survival during acute toxicological bioassays, a novel exposure system, the pelagic embryo-larval exposure chamber (PELEC), was designed and assessed for control survival using two different species of GoM-representative pelagic teleosts: mahi-mahi and yellowfin tuna (Thunnus albacares).
Aside from the documented oil contamination of GoM surface waters during the DWH incident (Deepwater Horizon Natural Resource Damage Assessment Trustees, 2016), the pelagic zone of the GoM receives significant ultraviolet (UV) radiation penetration, particularly in summer months (Alloy et al., 2016, Tedetti and Sempéré, 2006, Whitehead et al., 2000). The photo-induced toxicity of crude oil during co-exposure to UV-radiation dramatically decreases the LC50 of PAHs to many aquatic species (Barron et al., 2003, Little et al., 2000, Pelletier et al., 1997), including mahi-mahi embryos 48 h post fertilization (hpf) (Alloy et al., 2016). Given these results, it was hypothesized that existing 96-hr acute lethality estimates for mahi-mahi (Esbaugh et al., 2016) may be underestimated using traditional laboratory bioassays that do not incorporate exposure to levels of UV-radiation present in natural sunlight. Methods utilized for testing photo-induced toxicity of crude oil on 48-hpf mahi-mahi were unsuitable for obtaining 96-hr acute lethality estimates due to the challenges associated with maintaining high control survival over the course of this extended time period which encompasses the hatching and yolk-sac larval period. Consequently, the PELEC system was used to assess the effects of DWH crude oil exposure both with and without the addition of UV-radiation on 96-hr survival of mahi-mahi ELSs to determine potential photo-induced toxicity of DWH crude oil to an economically and ecologically valuable pelagic finfish species of the GoM over an extended time period (>48 hpf).
Section snippets
Control testing of PELEC system
Mahi-mahi and yellowfin tuna (YFT) survival tests (96- and 72-hrs, respectively) were conducted to compare control performance using the two previously utilized methods (i.e., static and agitated beakers) and the newly developed PELEC system. Mahi-mahi embryos were obtained from captive volitionally spawning broodstock maintained in 80,000-L seawater tanks, at the University of Miami Experimental Hatchery (UMEH) on Virginia Key, Florida, USA. Embryos were collected the morning of a spawn and
Control testing of PELEC system
Survival of mahi-mahi embryos after 96-hrs in the PELEC system (89.8% ± 2.12) was significantly greater than in the agitated static exposure (76.8% ± 4.49) and static exposure (67.5% ± 4.79) systems (Fisher's LSD test, P < 0.05)(Fig. 2). One mahi-mahi replicate with full mortality within the agitated static exposure was determined to be an outlier using Grubb's test (P < 0.01), and therefore mean survival data for this exposure system were based on an n of 11. Survival of YFT embryos after
Discussion
The present study demonstrates that for pelagic fish ELSs, the novel PELEC system significantly improves test performance, measured as survival of fish embryos and larvae under test conditions, and the system was employed to test for the impact of natural light on oil toxicity over an extended time period. Results show that co-exposure to UV-radiation increases the acute toxicity of DWH spill oil to a resident, GoM fish species by nearly an order of magnitude. These findings illustrate the
Acknowledgments
The authors thank Abt Associates and the gs2:National Oceanic and Atmospheric Administration for funding this research. Also, the assistance provided by the staff of the Inter-American Tropical Tuna Commission (IATTC) Achotines Laboratory during the YFT testing is much appreciated. This work was supported by funds provided as part of the Natural Resource Damage Assessment (NRDA) for the DWH oil spill. The work of Kimble Chase Custom Glass Shop is also appreciated for constructing the PELEC
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