Miguel González-Doncel

Toxic effects of an oil spill on fish early life stages may not be exclusively associated to PAHs: studies with Prestige oil and medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs) are assumed to be the primary determinant of oil petroleum toxicity. Since the PAH content in Prestige oil was relatively high, we investigated the effects of different oil fractions (crude or weathered oil -0.05 to 50 g/L, and shaken or sonicated water accommodated fractions, WAFs, 25-100%, v/v) on the embryo-larval development of medaka (Oryzias latipes). Concentrations of summation operator16PAHs analyzed in the incubation medium were highest in the shaken WAF followed by the crude oil, the sonicated WAF and the weathered oil. Both oils (> or =0.25 g/L) induced developmental abnormalities whereas no significant effects were seen in the WAF exposures. In vivo morphometric analysis of the surface of the gallbladder during advanced embryo organogenesis (192 h post-fertilization, hpf) revealed significant dilation in both WAF exposures (>3 x 10(4) microm(2) at > or =25%, v/v, compared to <1.7 x 10(4) microm(2) at 0%, v/v) followed by the crude oil (>2.2 x 10(4) microm(2) at > or =0.05 g/L). Fluorescent aromatic compounds were observed in the gallbladder and the yolk sac of 168-hpf embryos exposed to all oil fractions. Results suggest the presence of components in both oils capable of penetrating the chorion and inducing a toxicity not observed in the WAFs. Hence, the hazard and risk assessment of Prestige oil should not be based solely on the presence of PAHs since proximity or direct contact may induce toxicity not associated exclusively to these compounds. This research offers a new hypothesis for explaining the reported biological observations, which could be correlated to direct oil exposure rather than the traditional mechanism of waterborne PAH exposure. Further research is needed to identify those oil components responsible for toxicity.

Stage sensitivity of medaka (Oryzias latipes) eggs and embryos to permethrin.

The effects of exposure to permethrin on gametes, fertilization and embryonic development were examined in medaka (Oryzias latipes). Following range finding (25, 50, 100, 200 or 300 microg/l) and duration of exposure (0, 120, 144, 168, 192, 216, or 240-h) assays, the relative sensitivity was studied when initiation of exposure to permethrin (100 microg/l, for 192-h) occurred at one of four different stages, i.e., unfertilized egg (0-h), late morula (5-h), early neurula (24-h), and early organogenesis (40-h). The later exposure interval proved the most sensitive. Also, differences were observed in rates of recovery in larvae initially affected following the earliest exposure treatment (0-h, gametes prior to fertilization). Permethrin (100 microg/l) did not affect fertilization success and no lethal effects were observed in embryos. Sublethal effects were primarily observed at hatch. Toxicity endpoints in larvae included: delayed swim bladder inflation; inability of hatchling to respond to stimulus; uncoordinated movements, myoskeletal defects and transient enlargement of gall bladder. These changes were characteristic for all hatchlings exposed to nominal concentrations of 50 microg/l. While certain of the above alterations were reversed within 72-h after hatching, lack of swim bladder inflation and inability to respond to stimuli were two features that persisted with significant incidences. Based on persistence of sublethal effects, results from this work indicate the importance of exposures to gametes and to embryos prior to water hardening. The approach taken herein may better reflect environmental risk conditions than assays limited to exposure of embryonated eggs.

Influence of water hardening of the chorion on cadmium accumulation in medaka (Oryzias latipes) eggs

This report describes a study in which in vitro fertilization methods were used to expose medaka (Oryzias latipes) eggs to cadmium (Cd(2+)). This approach was applied to address the differential sensitivity and cumulative potential of Cd(2+) when exposure was initiated early (before fertilization and water hardening of the chorion) versus later during embryo development (i.e., well after the chorion has undergone water hardening). Following range finding exposures (2.5, 10, 20, 40 or 80 mg/l) under artificially controlled experimental procedures, results from hatching success and embryo malformations showed the earlier exposure interval more sensitive than the assay involving only the embryonated egg. Subsequent accumulation studies have shown that the exposure initiated before fertilization apparently led to more Cd(2+) deposition in the chorion compared to the exposure during embryonated stages of the eggs. Similarly, values for total Cd(2+) indicated higher concentrations in those eggs exposed prior to--and during--water hardening. Results suggest an alteration of the properties of the zona radiata in the early-stage eggs, making it more permeable to the potential exit or entrance of waterborne agents even after water hardening. Ongoing studies must now address the development of more realistic exposure conditions of the gametes by using incubation media with osmolarities similar to surface waters, and by shortening duration for gamete exposure. Also, sensitive methods to localize Cd(2+) and to delineate the transfer from the chorion to the embryo are needed.

Dynamics of BNF-induced in vivo ethoxyresorufin-O-deethylase (EROD) activity during embryonic development of medaka (Oryzias latipes)

This study aimed to characterize quantitatively the temporal basal and induced ethoxyresorufin-O-deethylase (EROD) activity as indicator of cytochrome P4501A (CYP1A) function during embryonic development of medaka (Oryzias latipes). For this purpose, non-invasive methods over fluorescence images of the whole embryo (non-organ-specific [NOS] EROD activity) or specifically of the gallbladder (organ-specific [OS] EROD activity) were used. To induce this EROD activity, embryos were continuously exposed to β-naphthoflavone (BNF; 0.005, 0.05, 0.5, 5 μg/L). Analytical chemistry suggested no signs of BNF dissipation. Mean fluorescence intensity values for EROD induction increased with BNF concentration throughout embryonic development. Significant increments in the NOS activity were seen from exposures to ≥ 0.5 μg BNF/L as early as 2 days post-fertilization (dpf), and in the OS EROD activity as soon as the gallbladder was conspicuous (i.e. 4 dpf). Morphometric in vivo analysis of the gallbladder during embryonic development did not indicate significant dilation after BNF treatment suggesting normal hepatic bile formation. The conditions optimized in this study using intact embryos should allow the quantitation of EROD activity induced by specific chemicals, mixtures and environmental samples in terms of BNF-equivalents, offering a proper estimation of their potency. These results demonstrate the utility of medaka in a fish embryo test for a non-invasive CYP1A analysis expressed as EROD activity, fitting in the three R principles for the minimization of animal use in ecotoxicology evaluations and that are among the objectives of the European Community regulation for the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH).

Stage-dependent ethoxyresorufin-O-deethylase (EROD) in vivo activity in medaka (Oryzias latipes) embryos.

Using medaka (Oryzias latipes) embryos, this study aimed to quantitatively characterize the stage-dependent in vivo ethoxyresorufin-O-deethylase (EROD) as indicator of cytochrome P4501A (CYP1A) activity. Embryos were challenged for 24-h to an agonist (β-naphthoflavone [BNF], 2.5, 5, 10, and 20 μg L(-1)) or to its combination (2.5 μg L(-1)) with an antagonist (α-naphthoflavone [ANF], 25, 50, 100, and 200 μg L(-1)), initiated at four different developmental time points (1, 3, 6, and 9 d post-fertilization [dpf]). Respective induction and competitive inhibition were evaluated over fluorescent images of whole embryo (nonorgan-specific [NOS] EROD activity) and gallbladder (organ-specific [OS] EROD activity). Both flavonoids showed signs of stability in solution. Generally speaking, the mean fluorescence intensity (MFI) values for NOS EROD increased with BNF concentration and exposure challenge. BNF co-exposure with ⩾50 μg ANF L(-1) during the 1-2 and 3-4 dpf challenges lowered NOS EROD to undetectably induced levels. Significant increments in MFIs for OS-EROD were seen from exposures to ⩾2.5 μg BNF L(-1), peaking during the 6-7 dpf challenge regardless of BNF concentration. The simultaneous BNF/ANF incubation showed competitive inhibition for OS EROD activity, although levels were generally detectably induced during all challenges and at all ANF concentrations. The morphometric in vivo gallbladder analysis indicated significant dilation in the 10 dpf-old embryos co-exposed to BNF and 200 μg ANF L(-1). This quantitative approach can be used successfully at 4 dpf at the NOS-EROD or OS-EROD levels, although the NOS-EROD response was sensitive enough to induction or inhibition, even at 2 dpf.

Effects of dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure in growing medaka fish (Oryzias latipes).

In this research work, we addressed the effects of a diet fortified with BDE-47 (0, 10, 100, 1000ng/g) dosed to 4-7 day-old post-hatch medaka fish for 40 days, followed by an 80-day depuration period. BDE-47 accumulation and overall growth were evaluated throughout the dosing period, and its elimination was quantified over the following 60 days. The histological condition of the thyroid gland, liver and gonads from the 1000ng BDE-47-treated fish were assessed 5 and 70days after exposures finished. The phenotypic males to females ratio was also quantified 70days after treatments finished. Sixty days after the BDE-47 exposures, reproductive capacity (i.e. fecundity, fertility and hatchability) was evaluated in mating groups for a 20-day period. BDE-47 exposure via food from larval through juvenile life stages of medaka fish resulted in steady accumulation with time dose-dependently. This accumulation tendency rapidly decreased after dosing ended. The growth rates showed a significant increase only at the highest concentration 70days after exposures finished. The histological survey did not reveal BDE-47-related alterations in the condition of the potential target organs. However, a morphometrical approach suggested BDE-47-related differences in the thickness of the epithelium that lines thyroid follicles. The reproduction studies showed comparable values for the fecundity, fertility and hatching rates. Dietary BDE-47 dosed for 40days to growing medaka fish did not alter the phenotypic sex ratios at maturity. The dietary approach used herein could not provide conclusive evidence of effects on medaka development and thriving despite the fact that BDE-47 underwent rapid accumulation in whole fish during the 40-day treatment.

Stage-dependent effects of chlorpyrifos on medaka (Oryzias latipes) swimming behavior using a miniaturized swim flume

By considering chlorpyrifos (CPF), an organophosphorus pesticide with known mechanisms of action that affect neurobehavioral development, we assessed the validity and sensitivity of a miniaturized swim flume by investigating the effects of the insecticide on swimming behavior in medaka (Oryzias latipes) fish growing stages. Medaka in three developmental periods, namely 0, 20 and 40 day-old post-hatch (i.e. time points 0, 20 and 40, respectively), were exposed to CPF (12.5, 25, 50 and 100 μg/L) for 48 h under semi-static conditions. The CPF half-lives during exposures were evaluated and the swimming patterns in a flume section (arena) were presented on two-dimensional gradient maps of forced movement of fish against water current. A comparative numerical analysis of fish residence times between each time point control and the corresponding CPF groups was performed by dividing arenas into 15 proportional areas. The time point 0 control group gradient map showed a noticeably different swim pattern from those of the ≥12.5 μg CPF/L groups, which was statistically supported by the differences for residence times seen in ≥12 corresponding areas. The control group gradient maps for time points 20 and 40 differed from those of the respective ≥12.5 μg CPF/L groups. The comparative analysis of the residence times in the corresponding 15 areas revealed differences in ≥5 areas for time point 20 and in ≥3 areas for time point 40. The integrative analysis of the gradient maps and the numerical statistics revealed stage-specific effects and a concentration-response relationship between CPF and alterations on forced medaka swimming despite the dissipation of CPF from the water column. These results indicate the validity of the miniaturized swim flume toward a more environmentally realistic scenario for the evaluation of neurodevelopmental and behavioral toxicity in small fish models.

Bioaccumulation, maternal transfer and effects of dietary 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) exposure on medaka fish (Oryzias latipes) offspring

A previous study conducted in our laboratory with growing medaka (Oryzias latipes) showed the capacity of BDE-47 (10-1000ng/g) to bioaccumulate during a 40-day oral exposure. However, the results did not provide evidence for effects during or after the exposure period. In this study, breeding medakas were fed a diet for 40days that contained 1000ng of BDE-47/g. At predefined time points, females (time points 10, 20, 30 and 40), males (time points 30 and 40) and pools of laid eggs (time points 10, 20, 30 and 40) were sampled and collected for: 1) the BDE-47 quantitative analysis in adults in the <24-h-old post-fertilization (hpf) embryos, and in the <24-h-old post-hatch (hph) eleutheroembryos; 2) the evaluation of fecundity, fertility and hatching. Additional pools of embryos collected at time point 40 were evaluated for: 1) the active swimming behavior of the 48hph offspring in the eleutheroembryonic stage; 2) the BDE-47 quantification in the 240hph resultant larvae. BDE-47 accumulated in parents rapidly, and concentrations remained constant at higher levels in males (values within the 50-60ng/g wet weight -w.w.- range) compared with females (70ng/g w.w. range). The BDE-47 concentrations detected in embryos and eleutheroembryos ranged from 200 to 500ng/g w.w. for time points 10-40. Reproductive capacity, hatching and ensuing swim bladder inflation were not affected by parental BDE-47 dietary exposure, nor was the active swimming behavior in eleutheroembryos. The BDE-47 concentration in the 240hph larvae lowered to levels close to those detected in parents. Despite the efficient BDE-47 maternal transfer, these results offered no evidence for BDE-47 effects on fish reproduction or in the early life stages of offspring.