Modified Japanese Medaka Embryo-Larval Bioassay for Rapid Determination of Developmental Abnormalities

Abstract

The Japanese medaka (Oryzias latipes) early-life-stage bioassay (18-day test) provides a suitable laboratory model for the evaluation of toxicant impact. The naturally high variability in the time to hatch for medaka embryos is, however, a major limitation in terms of the duration of the test. In this study, the 18-day test was modified to use agitation to synchronize hatch and reduce time to hatch for nonexposed embryos. Then, bioassays were conducted using two different complex mixtures (polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs)) to compare the sensitivity of the measurement endpoints using the 18-day standard protocol (SP) and the modified protocol (MP). Agitation reduced time to hatch by one third or more and improved hatch success (100%). The MP proved to be a more sensitive test method for the sublethal evaluation of a stressor (PAH mixture) that caused reduced hatch length and induced signs of blue sac disease (BSD) including heart deformities, yolk sac-pericardial edema, and cranial-skeletal deformities. In contrast, there was some variability in the sensitivity of the test methods for developmental endpoints for the sublethal evaluation of a stressor (NA mixture) that had no effect on hatch length and was a weak inducer of signs of BSD. Even though the sensitivity of the measurement endpoints were slightly more or less sensitive for the MP vs. SP depending on the mixture tested, the MP using agitation is recommended as a cost-effective and rapid alternative for screening the sublethal impact of toxicants on the early-life stages of fish.

A Perspective on the Toxicity of Petrogenic PAHs to Developing Fish Embryos Related to Environmental Chemistry

“…Shifts in PAH composition and the absence of dose-dependent relationships between particulate oil in water and eggs in oiled-rock column experiments demonstrate the successful adaptation of a procedure originally designed to study the chemistry of slightly soluble hydrocarbons to one that is useful for examining dissolved PAH toxicity without particulate oil as a confounding factor. These observations are consistent with independent contemporary studies of dissolved PAH toxicity (Rhodes et al 2005;Farwell et al 2006) and independent conclusions that dissolved PAHs are much more bioavailable and toxic than those adsorbed from whole oil (Neff et al 2005;Beckles et al 2007;Golding et al 2007). …”

“…Single-compound research (Incardona et al 2004(Incardona et al , 2006 and fractionation studies (Sundberg et al 2005(Sundberg et al . 2006Farwell et al 2006) further demonstrate that certain petrogenic PAHs can be highly toxic. Based on this new research, many of these PAHs should be considered as pharmacologically active compounds with specific cellular targets.…”

“…A suggestion that direct contact with oil is required for PAHs to transfer to fish embryos (Brannon et al 2006) has no support because eggs and other life stages isolated from contact with whole oil or PAH mixtures consistently exhibit severe adverse effects (Carls et al 1998(Carls et al , 1999(Carls et al , 2008Heintz et al 1999;Incardona et al 2005;Rhodes et al 2005;Farwell et al 2006). Paired comparisons of zebrafish embryos exposed to particulate-laden, water-accommodated fractions of crude oil and those isolated from particles by agarose sequestration demonstrate that fish embryos react predominantly to dissolved oil; physical contact did not enhance sensitivity (Carls et al 2008).…”

“…A series of papers have documented that exposure to dissolved polynuclear aromatic hydrocarbons (PAHs) from petrogenic sources such as crude oil are directly toxic to fish embryos at low µg/l aqueous concentrations (Marty et al 1997;Heintz et al 1999Heintz et al , 2000Carls et al 1999Carls et al , 2005Birtwell and McAllister 2002;Rhodes et al 2005;Farwell et al 2006). Early life stages are generally the most sensitive to toxicant insult, hence our studies have focused primarily on embryos.…”

Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish,Carassius auratus

“…In addition, osteoblastic markers such as ALP, osteocalcin, and RANKL mRNA expression were suppressed by the polluted seawater. We believe that one of the toxic causative agents is PAH because bone deformity was reportedly caused in fresh water teleosts (zebrafish and Medaka) (Billiard et al, 2006;Farwell et al, 2006) as well as seawater teleosts (Pacific herring, pink salmon, and sea bass) (Barron et al, 2004;Danion et al, 2011) by PAHs.…”