Aquatic Toxicology 88 (2008) 121–127 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Fish embryos are damaged by dissolved PAHs, not oil particles Mark G. Carls a,∗ , Larry Holland a , Marie Larsen a , Tracy K. Collier b , Nathaniel L. Scholz b , John P. Incardona b a Alaska Fisheries Science Center, Auke Bay Laboratories, 17109 Point Lena Loop Road, Juneau, AK 99801, USA b Ecotoxicology & Environmental Fish Health Program, Environmental Conservation Division, Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, WA 98112, USA article info Article history: Received 18 January 2008 Received in revised form 18 March 2008 Accepted 23 March 2008 Keywords: Fish embryos Dissolved PAH toxicity Mechanism of oil damage Particulate oil abstract To distinguish the toxicity of whole oil droplets from compounds dissolved in water, responses of zebrafish embryos exposed to particulate-laden, mechanically dispersed Alaska North Slope crude oil (mechanically dispersed oil (MDO)) were compared to those of embryos protected from direct oil droplet contact by an agarose matrix. Most polycyclic aromatic hydrocarbons (PAHs) in MDO were contained in oil droplets; about 16% were dissolved. The agarose precluded embryo contact with particulate oil but allowed dif- fusive passage of dissolved PAHs. The incidence of edema, hemorrhaging, and cardiac abnormalities in embryos was dose-dependent in both MDO and agarose and the biological effects in these compartments were identical in character. Although mean total PAH (TPAH) concentrations in MDO were about 5–9 times greater than in agarose, dissolved PAH concentrations were similar in the two compartments. Fur- thermore, mean differences in paired embryo responses between compartments were relatively small (14–23%, grand mean 17%), typically with a larger response in embryos exposed to MDO. Therefore, the embryos reacted only to dissolved PAHs and the response difference between compartments is explained by diffusion. Averaged over 48h, the estimated mean TPAH concentration in agarose was about 16% less than the dissolved TPAH concentration in MDO. Thus, PAHs dissolved from oil are toxic and physical contact with oil droplets is not necessary for embryotoxicity. Published by Elsevier B.V. 1. Introduction Oil occurs in the aquatic environment in both dissolved and par- ticulate phases. While the toxicity of dissolved polycyclic aromatic hydrocarbons (PAHs) has been the focus of considerable research in recent decades, the relative contribution of particulate oil is still poorly understood. Although oil coating is obviously detrimental for organisms reliant on feathers or fur for insulation, oil coat- ing of plankton and other aquatic organisms during various life history stages has received little study. In this paper we focus on the toxic implications to fish embryos of particulate oil generated mechanically without chemical dispersant. Fish embryos (with intact chorions) are useful for toxicological studies involving oil because they are externally simple (essen- tially uniform spheres). They have minimum surface area in contact with surrounding water, do not ingest material and, other than the chorion, they have no external structures such as gills, setae, or feathers that can be fouled. In addition, fish embryos are highly sensitive to PAHs dissolved from oil; the biological effects of pet- ∗ Corresponding author. Tel.: +1 907 789 6019; fax: +1 907 789 6094. E-mail address: Mark.Carls@noaa.gov (M.G. Carls). rogenic PAHs in developing fish have been described in detail and are consistent across species (Marty et al., 1997; Carls et al., 1999, 2005; Heintz et al., 1999, 2000; Kiparissis et al., 2003; Incardona et al., 2004, 2005; Rhodes et al., 2005; Farwell et al., 2006). For example, exposure to low TPAH concentrations (1–5 g/l) is asso- ciated with a syndrome marked by pericardial or yolk sac edema, with other morphological damage, delayed development, or death (Marty et al., 1997; Carls et al., 1999, 2005; Heintz et al., 1999, 2000). Studies in zebrafish embryos showed that the edema that forms with oil exposure is cardiogenic. Exposure to weathered crude oil or individual tricyclic PAHs (fluorene, dibenzothiophene and phenanthrene) caused specific defects in cardiac rate, rhythm, and contractility soon after the heart became functional (Incardona et al., 2004, 2005). Because cardiac function and morphogenesis are inextricably linked (Glickman and Yelon, 2002), these effects of oil exposure result in multiple secondary impacts on cardiac morpho- genesis such as failure to complete looping (Incardona et al., 2005). In each of these previous studies, whole oil was never detected on or in embryos. Nevertheless, a potential causative role for particu- late oil in the observed developmental toxicity has been suggested (Brannon et al., 2006). This highlights the need for an experimental approach to definitively separate the contributions of dissolved and particulate oil to early life stage toxicity in fish. To accomplish this, 0166-445X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquatox.2008.03.014