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Aquatic Toxicology 148 (2014) 184–194

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

imitations of waterborne exposure of fish early life stages to BDE-47

iguel González-Doncela,∗, Carlos Fernández Torijaa, Eulalia María Beltrána,osé Enrique García-Maurinob, Salvador Sastrec, Gregoria Carbonell a

Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5,-28040 Madrid, SpainDepartment of Cell Biology, School of Medicine, Complutense University, Ciudad Universitaria, E-28040 Madrid, SpainLaboratory of Forest Soils, Department of Forest Ecology, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040adrid, Spain

r t i c l e i n f o

rticle history:eceived 17 October 2013eceived in revised form0 December 2013ccepted 15 January 2014

eywords:olybrominated diphenyl ethersish embryo toxicitynalytical chemistryioaccumulation

a b s t r a c t

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is acknowledged as the most abundant congener of allpolybrominated diphenyl ethers (PBDEs). Despite its limited residence in the water column, most ecotox-icological research using fish early life stages (ELS) has focused on its waterborne bioavailability. Thesestudies have been supported either by chemical analysis in solutions or in tissues after ≤168 h exposuresto relatively high waterborne concentrations with dimethyl sulfoxide (DMSO) as solvent carrier (≤0.5%).Using noninvasive physiological and anatomical features in medaka ELS, we investigated the viability ofwaterborne BDE-47 exposures (100–10,000 �g/L; 1% DMSO) and evaluated the developmental effects inrelation to the actual BDE-47 present in water. Embryos were exposed for 10 days under semi-static (24-hrenewal) conditions and waterborne BDE-47 concentrations (i.e., dissolved) were quantitated daily andtheir accumulation in eleutheroembryonic tissues was analyzed 4 days after exposures finished. BDE-47in solution rapidly decreased after each renewal by >50% in 24 h. This was confirmed by discernible pre-cipitation occurring at ≥5000 �g/L on the bottom of the container and attached to the chorionic filamentsof eggshell. The fast dissipation from water may explain why, besides the subtle, yet significant effectson post-hatching growth (short length at ≥5000 �g/L), no other significant deleterious developmental

effects were observed despite the fact that BDE-47 accumulated in tissues in response to BDE-47 treat-ment. Waterborne BDE-47 exposure was unachievable under traditional semi-static exposure conditions,but was achievable in repeated pulse exposures lasting a few hours whenever the medium was renewed.Hence, this research encourages the use of alternate – more realistic – exposure routes (e.g., particulatematter or sediments) when evaluating early developmental toxicity of BDE-47 or any other PBDE sharingsimilar properties.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Polybrominated diphenyl ethers (PBDEs) are considered to be

mong the most pervasive environmental contaminants. Theyhow high lipophilicity and resistance to degradation, thushey are expected to bioaccumulate effectively in aquatic and

Abbreviations: dpf, days post-fertilization; DMSO, dimethyl sulfoxide; RM,mbryo-rearing medium; ELS, early life stages; LC50, median lethal concen-ration; GC–MS, gas chromatography–mass spectrometry; bpm, heart rate per

inute; hph, hours post-hatch; hpf, hours post-fertilization; MRM, modifiedmbryo rearing medium; PBDEs, polybrominated diphenyl ethers; BDE-47, 2,2′ ,4,4′-etrabromodiphenyl ether; PBB-80, 3,3′ ,5,5′-tetrabromo-1,1′-biphenyl; w.w., weteight.∗ Corresponding author. Tel.: +34 91 347 87 8; fax: +34 91 357 22 93.

E-mail address: miguelgd@inia.es (M. González-Doncel).

166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aquatox.2014.01.015

terrestrial food chains. PBDEs are the organobromine compoundsused to reduce the likelihood and intensity of fire in a variety ofindustrial and consumer products (Darnerud et al., 2001). Theyhave been used as three commercial mixtures (Penta-BDE, Octa-BDE or Deca-BDE) containing different proportions of the 209different congeners. As they do not bind chemically with thematerial being produced, they leach and dissipate continuouslyout of the final product, which results in rising environmen-tal levels in virtually every compartment of the environment,wildlife and humans (McDonald, 2002), and congener 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) is the most prevalent (de Witet al., 2006).

Within the risk assessment framework, the use of exper-imental in vivo and in vitro models has demonstrated theadverse effects of these substances; they affect neurobehavioraldevelopment (Kodavanti and Curras-Collazo, 2010; Williams and

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eSesso, 2010), and the endocrine system by interrupting thehyroid function (Chan and Chan, 2012). BDE-47 is character-zed by low water solubility (11 �g/L) (EPI Suit, 2013) and highipophilicity. These properties explain why, despite its ubiquity,he concentrations reported in the water column were veryow and the compound had a tendency to accumulate in sedi-

ents (de Wit et al., 2006). While municipal sewage is the mostignificant source of PBDE contamination (Kalantzi and Siskos,011), PBDEs end up in suspended particulate matter, sludge andiosolids, with only a low percentage (i.e., <10%) remaining dis-olved in wastewater (Anderson and MacRae, 2006; Peng et al.,009).

For fish, the main BDE-47 exposure routes include uptakehrough respiratory and digestive organs, direct contact throughkin, maternal transfer or, for egg-laying species, direct “translo-ation” from sediments into the embryo. However, toxicologicalDE-47 information for fish early life stages (ELS) has been basedrimarily on waterborne-based studies using zebrafish (Danioerio) as an ontogenetic model. The main observed effects includeevere developmental effects on chorionated (Lema et al., 2007)nd dechorionated (Usenko et al., 2011) embryos. This informationas been backed, whenever available, by BDE-47 analytical deter-inations in solutions when exposures begin. Studies employing

6-h waterborne exposures to zebrafish embryos or eleutheroem-ryos reported induction of genes related to the thyroid function

n embryos and larvae at concentrations of 15,225 �g/L and000 �g/L, respectively (Chan and Chan, 2012). However, theseesults were not backed by an analysis of BDE-47 in the water col-mn. This lack of information on the actual levels and fate of BDE-47

n solution is consistently noted in other studies. Chen et al. (2012)uggested the functional relevance of structural changes in axonalrowth when embryos were exposed statically to 608–9700 �gDE-47/L to therefore assume its ability to enter solution. Zhaot al. (2013) have observed a decrease in locomotor activity aftermbryonic exposures to nominal 500 �g BDE-47/L. One exceptiono these zebrafish studies showing relatively high BDE-47 effectn observed concentrations, is the work by Mhadhbi et al. (2011).hey reported LC50 values of 27.35 and 14.13 �g/L for embryosnd larvae, respectively, when turbot (Psetta maxima) embryosere exposed to BDE-47, which may reveal a high sensitivity

f this species. These authors reported concentrations averag-ng 55% of nominal values, which lowered to 44% after 48 h. Yet,heng et al. (2012) did not observe any molecular or pathologi-al effects in zebrafish ELS with exposure concentrations of up to000 �g/L, suggesting either lack of toxicological effects or thatDE-47 was not available in the water column. Hence, we feelhere is a need to adapt ecotoxicological assays to the informa-ion available on the actual fate of PBDEs in order to developnvironmental protection methods that will eventually allow aonsensus to be reached on the risk of PBDEs exposure in the envi-onment.

To our knowledge, no other significant research works besideshe aforementioned studies into waterborne BDE-47 have beenone with other experimental ELS fish models. Hence in thetandard fish ELS test with medaka (Oryzias latipes) context, weropose a battery of noninvasive tools to examine the physiologicalnd anatomical features in embryos and eleutheroembryos in ordero assess the effects of semi-static waterborne exposure to BDE-7. Toxicological results were correlated with the actual BDE-47oncentrations in the exposure media, measured over time dur-ng different periods, and its accumulation was evaluated in theesulting eleutheroembryos. The information in the present study

hould help in understanding the limitations when performing thenvironmental toxicology and assessment of waterborne persistentrganic pollutants based on the current information from fish ELSoxicity assays.

xicology 148 (2014) 184–194 185

2. Materials and methods

2.1. Chemicals and BDE-47 preparation

BDE-47 (ChemService, West Chester, PA) was dissolved in DMSO(>99.9%; Sigma–Aldrich, St. Louis, MO) to obtain a solution of1 �g/�l, which was stored at −18 ◦C. Using this stock solution, fournominal concentrations (500, 1000, 5000 or 10,000 �g/L, selectedfrom previous studies [Chen et al., 2012; Usenko et al., 2011, 2012]),were prepared in two different media: embryo-rearing medium(RM) (Rugh, 1962); modified RM (MRM) characterized by the pres-ence of only KCl as part of RM’ minor salts, and NaCl, RM’s major saltat a concentration of 1.13 g/L, to maintain the original RM osmolar-ity (37.14 mOsm/kg). Divalent cations Ca2+ and Mg2+ were excludedfrom MRM. We hypothesized the possibility of Ca2+ and Mg2+ form-ing coordination complexes with the aromatic rings of BDE-47 andthereby modifying its solubility (Crabtree, 2009). pH was adjustedto 7.0–7.4 in both media. All the BDE-47 solutions were equili-brated with 1% DMSO to facilitate their solubility in RM and MRM.Although ≤0.05% is a widely accepted maximum recommendedsolvent concentration for aquatic toxicity assays, 1% DMSO has beenproven safe for early development of medaka (González-Doncelet al., 2008; Oxendine et al., 2006).

2.2. Test organism and embryo collection

Golden medaka embryos were obtained from our stock cul-ture, which has been housed in a recirculating system since 2004.The culture conditions were those described elsewhere (González-Doncel et al., 2005). Synchronized-aged embryos, collected 2–4 hafter the daylight cycle started, were immersed in dechlorinated tapwater, separated, rinsed and incubated at 25 ◦C to reach the 10–11blastula stage (i.e., 6–8 h post-fertilization [hpf]) (González-Doncelet al., 2005).

2.3. Embryonic exposures and assessment

Two sets of embryonic exposures to BDE-47, in RM or MRM,were performed in the wells of flat-bottomed 24-well polystyreneplates (Corning Incorporated, Corning, NY. USA). In both sets, plateswere incubated (25 ± 1 ◦C; 16L: 8D) with the respective BDE-47concentrations 24 h before embryo exposures commenced. Indi-vidual embryos (stages 10–11) were distributed (n = 5) by stratifiedrandom assortment into all five wells filled with 600 �l of the cor-responding BDE-47 dilution, which was renewed daily. Controlsconsisted of the embryos in RM or MRM alone, or in combinationwith the vehicle DMSO at 1%. In both sets of experiments, embryoswere exposed during their complete development. For the assess-ment of development, the scoring system by Shi and Faustman(1989) was used, but with some modifications. Briefly, fish wereassessed during five developmental periods (i.e., 76, 124, 196, 244and 316 hpf) under a dissecting microscope (70–80×, OlympusSZX12, Olympus, Tokyo, Japan) using 13 developmental features forscoring development. Each embryo from each replicate was mon-itored in situ by placing the culture plates under the microscopeto obtain a mean value in each replicate per BDE-47 treatment.When fish reached 10 days post-fertilization (dpf), exposures werestopped and individuals were transferred to clean wells with RM orMRM for each corresponding experimental set. Individual hatch-lings were collected daily, placed in borosilicate vials (WheatonScientific, Millville, NJ, USA) with 2 ml RM or MRM, which wasrenewed daily until the experiments finished (i.e., 14 dpf). Mor-

phological and developmental features and sublethal endpoints,such as spinal, cardiovascular, swimbladder and swimming activ-ity (i.e., lethargy, uncoordinated darting or incapacity to respond toprodding), were monitored daily in each individual during the first

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6 hph, and no exogenous food was added. Our experience withedaka has shown that, under semi-static conditions, the yolk sac

ontains sufficient nutrients for the embryo to develop normally forhe first 96 hph. These experiments were performed on four sepa-ate occasions for each experimental set, RM or MRM. On day 9 ofhe experiments, the bottoms of the wells containing the BDE-47olutions and the corresponding embryos from both experimen-al sets were imaged under the microscope using a 90×/10 and a0×/10 objective, respectively.

.4. Measuring the embryonic gallbladder area

In the medaka embryo, the liver and gallbladder are particularlyonspicuous, which allows them to be traced during early devel-pment. This organ is the final recipient of bile fluid excreted byhe liver. At some instances (i.e., presence of toxicants), excessiveepatic bile production or inhibition of bile transport (cholestasis),ay lead to a dilation of the gallbladder (Newman et al., 2001). We

ollowed an in vivo quantitative approach of the gallbladder area as biomarker of exposure to discern the capacity of the embryoniciver to take up BDE-47 and to deposit it intact or as by-products inhe gallbladder. Hence, gallbladder morphometry was monitoredn both the RM and MRM experiments by 192 hpf following meth-ds described elsewhere (González-Doncel et al., 2014). Briefly,ndividual embryos were placed on a dorso-lateral view with theallbladder positioned on the uppermost part of the egg curva-ure. Embryos were photographed digitally through a dissecting

icroscope and the gallbladder was measured by outlining its areaith the Image-Pro Plus software (Media Cybernetics, Silver Spring,D, USA). The values for each area were normalized by calibrating

he corresponding embryo to a fixed averaged horizontal diameterf 1245.9 �m (Iwamatsu, 1994). In each experiment, gallbladderrea measurements were taken from 10 embryos for each BDE-7 treatment (n = 40 embryos per treatment for each experimentalet).

.5. Measuring hatching growth based on body length and weight

Immediately after the development assessment monitoringrocess, body length was evaluated in ≤24 hph-old eleutheroem-ryos, and weight was recorded at 13 dpf.

For body length, individual eleutheroembryos were anes-hetized by immersion in ice-cold water, and by being positionedorsally under the dissecting microscope and photographed. Body

engths were measured from the rostral-most portion of the heado the caudal-most portion of the tail fin using Image-Pro Plus.ish were then recovered in a water bath, returned to their cor-esponding vial and incubated until the end of the experimentaleriod (i.e., 14 dpf) when weight was recorded. Fish measurementsere taken for all surviving ≤24 hph-old eleutheroembryos from

he four independent exposure treatments for each experimentalet (n ≈ 100).

At the end of the experimental period, the individuals from theve replicates, which each BDE-47 treatment consisted of, werenesthetized, sorted into groups of ∼20 individuals (16–25), trans-erred to 1.5-ml eppendorf tubes, blotted dry and weighed. Samplesn = 4 groups of eleutheroembryos per treatment and per experi-

ental set) were frozen immediately at −18 ◦C and stored at −80 ◦Cor the subsequent BDE-47 quantification in fish tissues (see Quan-ification of BDE-47 in fish tissues below).

.6. Cardiac function

Cardiac function, based on heart rate, was evaluated duringhree developmental periods (i.e., 76, 124 and 196 hpf) in bothhe RM and MRM experimental sets. Culture plates were placed

oxicology 148 (2014) 184–194

under the dissecting microscope with a surrounding ambient tem-perature of between 22 and 25 ◦C. Each beat was counted for 15 sand quadruplicated to obtain a heart rate per min (bpm). In eachexperiment, 12 embryos were used per treatment during eachdevelopmental period (n = 48 embryos per treatment).

2.7. Quantification of BDE-47 in exposure solutions

The actual concentrations of the four BDE-47 dilutions in eitherRM or MRM were measured at 0 and 24 h after renewal fol-lowing a 24-h saturation of wells with 600 �l of the solutionwith the respective nominal concentration. Three replicates (wells)were included for each concentration and incubation period. Theactual concentrations of the four BDE-47 dilutions were deter-mined following the procedure described by Polo et al. (2004)using the 3,3′,5,5′-tetrabromo-1,1′-biphenyl (PBB-80) congener(Sigma–Aldrich 717711) as an internal quantification standard.Aliquots of 5–100 �l were sampled in triplicate directly from wellsand were placed in 30-ml headspace vials containing 5 ml of RMor MRM (1% DMSO), 0.1 ml of a 100 ng/ml solution of PBB-80 inthe same medium, and a disposable magnetic stirring bar (Spin-bar, Sigma–Aldrich). Vials were sealed with a headspace aluminumcap furnished with a Teflon-faced septum, and were immersed ina water bath maintained at 95 ◦C. Then, samples were allowedto settle for 5 min before injecting the solid-phase microextrac-tion fiber (100-�m poly-dimethylsiloxane). Afterwards, the fiberwas exposed to the headspace over the sample for 30 min understirring conditions. The fiber was subsequently injected into thegas chromatograph port (a 5-min desorption time at 300 ◦C) andremained inserted until the end of chromatographic analysis. TheGC–MS analysis was performed using gas chromatograph (6890Agilent Technologies, Santa Clara, CA, USA), equipped with a 5973network mass selective detector and an Equity-5 (30 m × 0.25 mmI.D., 0.25 �m) film thickness column. Helium was maintained ata constant flow of 1.2 ml/min and column temperature was pro-grammed from 60 ◦C (initial equilibrium time of 5 min) to 300 ◦Cwith 10 ◦C/min increments and was maintained at 300 ◦C for 5 min.The MS was operated in the selected ion-monitoring mode forthe quantitative analysis (486 and 470 m/z for BDE-47 and PBB-80, respectively). Quantification was performed by the internalstandard procedure (fortified RM or MRM samples 50–0.1 ng/ml ofBDE-47 AccuStandard). The limits of quantification and detectionwere established as 0.1 ng/mL and 0.03 ng/mL, respectively.

2.8. Stability/dissipation of BDE-47

To reduce the likelihood of BDE-47 precipitation, DMSO con-centrations were kept at 1%, as described above. Nonetheless,dissolved BDE-47 quantification revealed an effective dissipationpotential (see Section 3). Thus, a series of studies was performedto discern whether this dissipation was due to chemical instability,precipitation (i.e., by crystal formation) and/or adsorption to thecontainer walls. To firstly ascertain BDE-47 chemical stability,8 ml of a 1000 �g BDE-47/L of RM were prepared and 1 ml wasaliquoted into a series of 2-ml borosilicate vials. Vials were placedin an incubator under the same experimental conditions as thosefor embryo exposures, and were taken at 1-h intervals for 6 h. Ateach interval, 0.5 ml of n-hexane was added to the vial, vortexed,and centrifuged for 2 min at 3000 rpm. A 1-�l volume of the upperhexane layer was injected into the GC–MS system for BDE-47quantification (see Quantification of BDE-47 in exposure solutions)with the help of a calibration curve from 30 to 1000 ng/mL in

n-hexane. Second, to evaluate BDE-47 dissipation from solution,a 1000 �g BDE-47/L of RM was prepared in triplicate and 600 �lwere added to the wells of a flat-bottomed 24-well polystyreneplate, like those used in the ELS assays. The plate was placed in the

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ncubator and 50-�l samples were taken at 1-h intervals for 6 h, sohat the BDE-47 in the water column was quantified accordingly.

.9. Quantification of BDE-47 in fish tissues

A GC–MS analysis was done to determine the BDE-47 concentra-ion in the whole-body tissues of the 14-dpf-old eleutheroembryosrom both treatment sets. These eleutheroembryos were subjectedo a 96-h depuration time and daily renewals with either RM or

RM prior to storing at −80 ◦C. Frozen samples (n = 4 per treatment,er experimental set) were thawed and the body burden of BDE-47as determined following the procedure described by Moliner-artínez et al. (2009) after adapting it to our investigations. The

ools of eleutheroembryos (each pool containing 16–25 fish) wereransferred to 1.5-ml polypropylene microcentrifuge tubes with00 �l of MilliQ water. Each pool was homogenized with a T25igital Ultra-Turrax (IKA, Staufen. Germany), and transferred to a0 ml headspace vial. This procedure was repeated 3 times with00 �l MilliQ water and by combining the extracts in the vial tobtain a volume of 2000 �l. The tube was rinsed twice using 1 ml ofilliQ water (4000 �l) and, once again, by combining the extracts

n the same vial. Then 1 ml of MilliQ water was added to bring thenal volume to 5 ml. After placing a magnetic stirring bar into theube, a 100-�l volume of a 100 ng/ml solution of PBB-80 was useds an internal standard, and the vial was sealed with a headspaceluminum cap furnished with a Teflon-faced septum. BDE-47 wasnalyzed following the procedures described for quantification inxposure solutions. Quantification was performed by building aalibration curve with pools of 25 eleutheroembryos fortified with56–500 ng of the BDE-47/pool, and values were given as �g ofDE-47 per g of eleutheroembryonic w.w. (�g BDE-47/g w.w.).ecoveries ranged from 65% to 105% and were calculated with sixatches of 40 eleuteroembryos fortified at three levels, 25, 250 and500 ng, of BDE-47 by batch.

.10. Data analysis

Data were analyzed for normality with the Kolmogorov–mirnov D test and for homogeneity of variance with Bartlett’sest. Since the data were not distributed normally or their vari-nces were heterogeneous, the Kruskal–Wallis median and theonckheere–Trepsa tests were applied, while the Mann–Whitney Uest with Bonferroni correction was performed to identify any sig-ificant differences between the treatments and the respective 1%MSO control (P < 0.05). The RM and MRM solvent controls were

elected as reference groups after observing no differences withheir respective RM or MRM non-solvent controls for any of thendpoints analyzed. All the statistical analyses were carried outsing the Statistical Package for the Social Sciences software (SPSS

nc., Chicago, IL, USA).

. Results

.1. Embryonic and eleutheroembryonic assessment

Fig. 1 shows how the mean scores increased with developmentver time in the two exposure sets. Both the RM and MRM exper-mental sets revealed similar patterns between the control groupsnd their respective treatments throughout the 316 hpf. Individ-als from the control RM or MRM groups developed normally,ith only minor casual mortalities or morphological abnormal-

ties (≤2%). BDE-47 did not induce significant embryo mortality,

atching failure, or delayed or precocious hatching (Fig. 1).

From the time hatching became imminent, individualleutheroembryos were tracked daily over a 96-h periodFig. 2). As compared to their respective controls, no significant

xicology 148 (2014) 184–194 187

eleutheroembryo mortalities occurred among treatments. Cardio-vascular lesions were negligible. Over 80% of the initially affectedeleutheroembryos in the control and treated groups from bothexperimental sets were able to inflate their swimbladders by96 hph, and it was possible to see this trend even during the first48 h immediately after hatching. Directly after emergence (i.e.,0 hph), 17% and 6% of the control groups in RM and MRM, respec-tively, showed temporary lethargy symptoms, but no anomalousswimming patterns. By 96 hph, values lowered to a prevalence of9% and 5%, respectively. Similar trends, but with different recoveryrates, were seen among the BDE-47-eleutheroembryos in RMor MRM. By 96 hph, weakness was below 15% in all treatments.BDE-47 did not significantly induce myoskeletal lesions afterhatching, and prevalence was below 5%.

3.2. Morphologic assessment: embryonic gallbladder area andpost-hatching growth

The gallbladder control areas in RM and MRM ranged from9970 ± 274 �m to 11,600 ± 279 �m, respectively (Fig. 3). The low-est BDE-47 concentration in RM (500 �g/L) was the only one thatrevealed a slightly significant increase in area (13%) (P ≤ 0.05).In MRM, the gallbladder areas from the BDE-47-treated embryosshowed similar values to those in their respective controls.

Length was measured in all the surviving ≤24 hph-oldeleutheroembryos (Fig. 4) Overall length in the solvent controlsaveraged 5.09 ± 0.03 and 5.07 ± 0.02 mm for RM and MRM, respec-tively. These values were not affected when embryos were exposedto ≤1000 �g/L. BDE-47 at ≥5000 �g/L reduced eleutheroembryoniclength significantly to reach values of ≤5.02 mm in RM and MRM(≤2.2%). Exposure of medaka embryos to BDE-47 did not affecteleutheroembryonic weight (Fig. 4).

3.3. Heart rate

The heart rates in the control and treated groups from bothexperimental sets showed a similar steady increase in trends withdevelopment (Fig. 5), but with no significant differences.

3.4. Quantification of BDE-47 in exposure solutions

The actual BDE-47 concentrations in RM or MRM present in theexposure wells at t = 0 h were comparable to their correspondingnominal concentrations (Fig. 6). The only exception observed wasthe nominal 10,000 �g/L in RM, where concentrations reached only6132 �g/L. After 24 h, the BDE-47 levels dropped to >45% and >65%of the respective initial concentrations in RM and MRM. Irrespec-tive of employing RM or MR, it was not possible to maintain similaractual concentrations in the exposure wells to the correspondingnominal ones, despite renewing the medium every 24 h with DMSOat 1%. Nevertheless for practical, comparative purposes with pub-lished studies, nominal concentrations are used throughout the textto describe individual experiments, whereas actual concentrationsanalyzed at t = 0 h are included with the corresponding nominalconcentrations in the pertinent figures.

3.5. Stability/dissipation of BDE-47

In a first experiment, BDE-47 was assessed for its stability dur-ing incubation in 2-ml borosilicate vials (Fig. 7). The BDE-47 levels,analyzed at 1-h intervals from the upper hexane layer, ranged from

991.4 �g/L (3-h incubation) to 1155 �g/L (6-h), with overall levelsaveraging <15% of the nominal 1000 �g/L value. BDE-47 displayedgood stability after 6-h incubation. A second experiment, carriedout in a polystyrene 24-well plate, which aimed to determine

188 M. González-Doncel et al. / Aquatic Toxicology 148 (2014) 184–194

Fig. 1. The two graphs on the left side represent the embryonic development assessment with the respective 95% confidence intervals for the BDE-47-exposed medakaembryos incubated in RM or MRM. Development scores were summed for each individual embryo or eleutheroembryo. Values were obtained from five replicates (fiveembryos/replicate) and four independent experiments (n = 100), and were averaged to generate the mean and standard deviation for each treatment group. The two graphso hatcht rrespon five r

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n the right side show the median number of days (±25th and 75th percentiles) to

hese two panels show the nominal BDE-47 concentrations and, in brackets, the coon-solvent control or (b) the solvent (1% DMSO). Values represent the response of

he actual presence of BDE-47 in solution, showed an exponen-ial decrease during the 6-h incubation. The concentrations presentn solution lowered to 41% of the initial 1000 �g/L after 3-h incu-ation. By 6 h, only 24% of the initial 1000 �g/L remained in theolution. The regression analysis of the data revealed an expo-ential curve fitting (Fig. 7). Although BDE-47 remained stable

n the exposure containers, it was not possible to maintain itn solution due to its fast dissipation rate from the water col-mn. This dissipation seems to be related to precipitation and/ordsorption to the container surfaces rather than to chemical insta-ility.

Fig. 8 shows the images of the bottoms of the wells contain-ng BDE-47 in RM or MRM, and the corresponding embryos after a-day incubation. Although the BDE-47 stability/dissipation exper-

ments demonstrated the capacity of 1000 �g/L to dissipate fromhe water column, the presence of visually discernible precipitatesas not apparent at this or lower concentrations. BDE-47 precipi-

ation was visually perceptible in the form of crystals or needles at5000 �g/L.

.6. Quantification of BDE-47 in fish tissues

Other than RM-10,000 �g/L, where tissue concentrations

emained comparable to 5000 �g/L, the rest of the treatmentshowed increasing concentrations with BDE-47 treatments in RMnd MRM (Fig. 9). The eleutheroembryos incubated in MRM withigher concentrations (i.e., 5000 and 10,000 �g/L) accumulated

for the BDE-47-exposed medaka embryos incubated in RM or MRM. The x-axes fornding actual concentrations at t = 0 h. The 0 �g/L treatments correspond to (a) the

eplicates (five embryos/replicate) and four independent experiments (n = 20).

more BDE-47 than those that had been incubated in RM with thesame nominal concentrations.

4. Discussion

Different model systems have been used to investigate theaquatic developmental toxicity of PBDEs, and several studies havereported neurobehavioral disorders, reproduction and thyroid asthe main target systems (Chan and Chan, 2012; Chen et al., 2012;Chou et al., 2010; Han et al., 2013). However, currently avail-able information is inconsistent and variable since studies havebeen conducted with different animal models, exposure routes andexperimental procedures. While some authors have evaluated theeffects following dietary exposures of PBDEs (Chen et al., 2010;Chou et al., 2010; Noyes et al., 2011), most developmental stud-ies with fish have relied on the waterborne route by assuming theaqueous bioavailability of this substance.

Despite following the basic procedures previously described inthe literature, we came across some methodological problems inrelation to BDE-47 dilution. When preparing the BDE-47 concen-trations in a step-wise, serial manner, we noticed the presenceof precipitation at concentrations of ≥1000 �g/L in 0.5% DMSO.Chances were that these precipitates would be pipetted from

one concentration to the next, thus dragging aggregates alongserial dilutions and inducing errors in the actual concentrations.In order to avoid or minimize this precipitation, we obtainedeach experimental concentration directly from the stock BDE-47

M. González-Doncel et al. / Aquatic Toxicology 148 (2014) 184–194 189

Fig. 2. Effects of ELS – embryonic – exposure to BDE-47 in the medaka eleutheroembryos incubated in RM or MRM. Evaluations were made from the hatching (0 hph) to thelarval stage (i.e., after four subsequent 24-h intervals). The 0 �g/L treatments correspond to (a) the non-solvent control or (b) the solvent control (1% DMSO). A solid symbolindicates a significant difference from the solvent control (P < 0.05) during the respective developmental period. BDE-47 concentrations are presented as nominal values and,in brackets, the corresponding actual concentrations at t = 0 h. The treatment group values were obtained from four independent experiments, each initially consisting in fivereplicates with five embryos per replicate, which generated a mean (±SE) response.

190 M. González-Doncel et al. / Aquatic Toxicology 148 (2014) 184–194

5000

7500

10000

12500

0a 0b 50 0[479 ]

1,00 0[1,37 5]

5,00 0[5,113 ]

10,00 0[6,132 ]

Ga

llbla

dd

er

are

a (

µm

2)

BDE-47 (µg/L) in RM

5000

7500

10000

12500

0a 0b 500[512 ]

1,00 0[1,051 ]

5,00 0[4,760 ]

10,00 0[10 ,41 6]

BDE-47 (µg/L) in MRM

*

Fig. 3. The gallbladder area in embryos (192 hpf/25 ◦C) as function of exposure to BDE-47 exposure using RM or MRM as exposure media. Both x-axes show the nominalBDE-47 concentrations and, in brackets, the corresponding actual concentrations at t = 0 h. The 0 �g/L treatments correspond to (a) the non-solvent control or (b) the solventc e solvo expe

sc1tenT

Fatefg

ontrol (1% DMSO). An asterisk indicates a significant difference from the respectivf four independent experiments with 10 embryos measured per treatment in each

olution while maintaining 1% DMSO. Yet the occurrence of pre-ipitates was still observed at the higher concentrations (5000 and0,000 �g/L). This precipitation became particularly conspicuous inhe exposure wells after day 5 and progressively increased to the

xtent that it was possible to discern the aggregates of the bromi-ated compound attached to the chorionic filaments of eggshell.he chemical analysis performed at 1-h intervals revealed a fast

4.5

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[479]

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[6,132 ]

Len

gth

(m

m)

*

*

0.50

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0.58

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[5, 113]

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[6,132]

Weig

ht (m

g-w

.w.)

BDE-47 (µg /L) in RM

ig. 4. Eleutheroembryonic (14 dpf) length and weight according to BDE-47 exposure. The

ctual concentrations at t = 0 h. The 0 �g/L treatments correspond to (a) the non-solvent cohe respective solvent control are indicated with an asterisk (P < 0.05). The length values fxperiments, and each one initially contained five replicates per treatment (n = 100, excor RM and MRM represent the mean (±SE) response of four independent experiments,

rouped (n = 4).

ent control (P < 0.05). For each exposure medium, values represent the mean (±SE)riment (n = 40).

BDE-47 dissipation from the water column, which should certainlylimit its bioavailability. This fact can explain, at least partially, thelow deleterious effects we have observed in medaka embryo andeleutheroembryo development.

The methods and results with medaka herein do not coin-cide with some published research works in which zebrafishwas used as an experimental model. Usenko et al. (2011) used

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*

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0a 0b 500

[512]

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[10,416]

BDE-47 (µg/L) in MRM

x-axes show the nominal BDE-47 concentrations and, in brackets, the correspondingntrol or (b) the solvent control (1% DMSO). The values that significantly differ fromor each exposure medium represent the mean (±SE) response of four independentept for those treatments in which casual mortalities occurred). The weight valueswhere all the individuals included in all five replicates from each treatment were

M. González-Doncel et al. / Aquatic Toxicology 148 (2014) 184–194 191

Fig. 5. Heart rate dynamics in medaka fish throughout three embryonic develop-mental periods exposed to BDE-47 in RM or MRM used as exposure media. BDE-47concentrations are presented as nominal values and, in brackets, the correspondingactual concentrations at t = 0 h. The 0 �g/L treatments correspond to (a) the non-smb

dBwAe1ts

Fig. 7. BDE-47 stability within the container and dissipation from the water columnmeasured at 1-h intervals for 6 h of a nominal 1000 �g/L present in RM with 1%DMSO. Stability was measured in the borosilicate vials (represented in the figureby solid circles) after BDE-47 resuspension using hexane extraction and vortexing.Dissipation from the solution was measured in a 24-well polystyrene plate after

Fm

olvent control or (b) the solvent control (1% DMSO). The values in each exposureedium represent the mean (±SE) of four independent experiments in which heart-

eat was measured in 12 embryos selected randomly from each BDE-47 (n = 48).

echorionated zebrafish embryos with DMSO at 0.5% where theDE-47 stock was serially diluted in 0.6% sea salt-reconstitutedater to concentrations ranging from up to 20,000 to 635 �g/L.pparently, there were no signs of BDE-47 aggregates during the

xperiments, with the actual initial concentrations falling within a06 ± 7.8% range of their nominal value, nor was there any men-ion of substance stability, despite exposures being performedtatically. While our research observed only consistent significant

0

2000

4000

6000

8000

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0a 0b 10,0005,000 1,000 500

BD

E-4

in

so

lutio

n (

µg

/L)

Nomin al concen tration of BD E-47 (µg/ L) in RM

0 h 24 h 1

ig. 6. The measured BDE-47 concentrations present in the water column in 0- and 24-h-edia with 1% DMSO. The 0 �g/L treatments correspond to (a) the non-solvent control or (

direct aqueous sample extraction (see Section 2 for Stability/dissipation of BDE-47).Values represent the mean (±SD) of three replicates.

effects for larval length at ≥5000 �g/L, Usenko et al. (2011) reportedmyoskeletal lesions at concentrations of ≥2250 �g/L, as well aseffects on swimming rates at all the concentrations tested. Ques-tions arose such as whether these contrasting results were dueto the role of the chorion acting as a physical barrier, possibledifferences between the exposure media used for diluting BDE-47, or species-differential sensitivity. Using intact – chorionated– zebrafish embryos, Lema et al. (2007) reported delayed hatch-ing and reduced body length at ≥2000 �g/L, and morphologicalabnormalities and cardiac arrhythmias at ≥500 �g/L from post-hatch exposures, although they did not include the actual BDE-47levels during exposures. Zhao et al. (2013) observed hypoactivityafter embryonic and eleutheroembryonic continuous exposure to500 �g BDE-47/L (0.1% DMSO) with 50% daily renewals, but with-out mentioning any BDE-47 stability. In contrast, Zheng et al. (2012)did not report any molecular or pathological effects for exposures(nominal concentrations) up to 200 or 5000 �g BDE-47/L.

The results from the quantification and stability of BDE-47 inthe exposure medium support the hypothesis of the potentialrole of divalent cations on BDE-47 bioavailability. Using MRM,the BDE-47 levels detected at t = 0 h were similar to their respec-

tive nominal concentrations, but this was not achieved with RMwhen dissolving 10,000 �g/L. Therefore, when using MRM, BDE-47 solubility initially increased mainly at high concentrations.

0

2000

4000

6000

8000

0000

0a 0b 10,0005,000 1,000 500

Nominal co nce ntrat ion of B DE-47 (µg/L) in MRM

0 h 24 h

old solutions for the medaka ELS assays where RM or MRM were used as incubationb) the solvent control (1% DMSO). Values represent the mean (±SD) of ten replicates.

192 M. González-Doncel et al. / Aquatic Toxicology 148 (2014) 184–194

Fig. 8. Images show the bottoms of the wells containing BDE-47 in RM or MRM with 1% DMSO after a 9-day incubation at 25 ± 1 ◦C under a 16L:8D photoperiod. The insertedin vivo images show the dorsolateral views of late stage embryos (220 hpf/25 ◦C) from the corresponding wells. BDE-47 concentrations are presented as nominal values and,in brackets, the corresponding actual concentrations at t = 0 h. The 0 �g/L corresponds to images captured for the solvent controls (1% DMSO). Note the presence of crystalsat ≥5000 �g nominal BDE-47/L on the surface of the wells. Similarly, occurrence of precipitation attached to chorionic filaments was visually discernible. This precipitationwas more apparent at the highest BDE-47 concentration. The light blue daylight filter was used for capturing images of the bottoms of wells. For practical purposes, 500 �gnominal BDE-47/L were omitted from the figure.

0

100

200

300

400

500

600

700

800

0a 0b 500[479]

1,000[1,375]

5,000[5, 113]

10,000[6,132]

µg

BD

E-4

7/g

w.w

.

BDE-47 (µg/L) in RM

0

100

200

300

400

500

600

700

800

0a 0b 500[512]

1,000[1,051]

5,000[4, 760]

10,000[10,416]

BDE-47 (µg/L) in MRM

Fig. 9. Uptake of BDE-47 in the whole-body tissues of the 14-dpf-old eleutheroembryos exposed during their complete embryonic development using RM or MRM asincubation media. Both x-axes show the nominal BDE-47 concentrations and, in brackets, the corresponding actual concentrations at t = 0 h. The 0 �g/L treatments correspondto (a) the non-solvent control or (b) the solvent control (1% DMSO). Values are the mean (±SD) of four independent samples.

atic To

HcdiedietwlohtietrrsvaSbf(Luhp

iBuecsicdcuitsDAtilbt2ir2d4w

4ao4an

behavior in juvenile zebrafish (Danio rerio). Aquat. Toxicol. 98, 388–395.

M. González-Doncel et al. / Aqu

owever, by 24 h BDE-47 levels dropped by >50% of their nominaloncentrations. Our studies into BDE-47 stability and dissipationemonstrate that while the compound remained in the container,

t dissipated quickly in the form of aggregates. Hence when consid-ring the nominal concentrations selected in our research, andue to the limited half-life of BDE-47 (i.e., 3.1 h measured in RM),

t was not possible to achieve continuous waterborne embryonicxposures under semi-static (24-h) conditions, but only intermit-ently; that is, in repeated pulse exposures lasting a few hourshenever the medium was renewed. In spite of this experimental

imitation, most of the studies that focus on the aquatic toxicityf waterborne BDE-47 and other structurally similar congenersave based their observations on experimental procedures wherehe medium is renewed every 24 h at the most, and by keep-ng solvent – DMSO – concentrations at ≤0.5%. Using zebrafishmbryos and eleutheroembryos, McClain et al. (2012) investigatedhe developmental toxicity with 48-h static renewals of BDE-49,anging from 1943 to 15,545 �g/L, serially prepared in sea salt-econstituted water with 0.2% DMSO. They reported effects inurvivorship and morphological impairments, such as dorsal cur-atures and cardiac arrhythmias. Apart from the stock solution, thectual exposure solutions levels were not chemically confirmed.imilarly, effects on the thyroid function of zebrafish ELS haveeen described after semi-static conditions (i.e., daily renewal)or waterborne exposures to BDE-47 dissolved in 0.2% DMSOChan and Chan, 2012). This study reported eleutheroembryonicC50 = 5370 �g/L and hatching EC50 = 20,300 �g/L. Nonetheless, val-es were obtained after assuming continuous exposure due toypothetical BDE-47 solubility, so no chemical analysis or signs ofrecipitation were reported.

Our chemical quantification in eleutheroembryos showedncreased BDE-47 accumulation with a concomitant increase inDE-47 solubility. This tendency became even more patent whensing MRM, particularly at higher concentrations where morelevated levels were detected in fish tissues. These results areonsistent with previous BDE-47 quantification in both expo-ure media, which demonstrated that the use of MRM increasesnitial solubility, thus helping to maintain higher BDE-47 con-entrations during the initial hours of incubation. Although theiruration is brief, these intermittent pulses of BDE-47 exposuresould allow embryos to accumulate more substance instead ofsing RM. Lema et al. (2007) quantified BDE-47 concentration

n zebrafish eleutheroembryos after 96-h aqueous post-hatchreatments at 100, 500 or 5000 �g/L in reconstituted water,imilarly to that used by Usenko et al. (2011), but with 0.3%MSO and under semi-static – 48-h renewal – conditions.lthough no chemical analysis was done of BDE-47 in solu-

ion, these authors reported increasing tissue concentrationsn a dose-dependent manner. These values were comparativelyower than those detected in the medaka eleutheroembryos incu-ated in either RM or MRM during embryonic stages. Whilehe 500 and 5000 �g/L exposures resulted in 77.54 ± 7.82 and93.28 ± 21.64 �g/g w.w. in zebrafish, respectively, the same nom-

nal concentrations in the RM- or MRM-medaka eleutheroembryosesulted in 206.09 ± 36.82 or 594.84 ± 74.74 �g/g w.w., and in68.42 ± 28–63 or 694.76 ± 29.63 �g/g w.w., respectively. Theseifferences are probably due, at least in part, to the semi-static –8-h – renewals performed for eleutheroembryo exposures, alongith shorter exposure times.

In more recent studies by Usenko et al. (2013), 10,000 �g of BDE-7/L was chosen with five other PBDE congeners to compare uptakend metabolism in dechorionated zebrafish embryos after a 24-

r a 120-h exposure. The immediate analyses resulted in a BDE-7 uptake of 60.12 ± 13.36 �g/g w.w. and of 553 ± 39.12 �g/g w.w.fter 24 h and 120 h, respectively. However, neither these authors,or Lema et al. (2007) specified whether the detected levels were

xicology 148 (2014) 184–194 193

the result of only the actual accumulation resulting from perme-ation into the organism (i.e., absorption) or of this process togetherwith the adhesion or physical binding of the substance to fish (i.e.,adsorption). This adsorption does not necessarily reflect BDE-47bioavailability. As our methodology describes, the quantification ofBDE-47 present in medaka tissues from embryonic exposures wascarried out with eleutheroembryos that had been previously sub-jected to a 96-h depuration using 24-h renewals in RM or MRM. Wefollowed this approach to avoid the BDE-47 precipitates physicallybinding or adhering to the external eleutheroembryo layer.

Contradictorily, although some authors admit there is not muchlikelihood of high BDE-47 concentrations being reached in surfacewaters (Lema et al., 2007; Usenko et al., 2011), their research worksand others have consistently relied on the waterborne route beingthe main exposure route in fish ELS studies. Our research results,along with low PBDEs solubility, encourage the use of alternateexposure routes when it comes to evaluating early developmentaltoxicity. As these substances are prone to spillage in the environ-ment, impacts on fish are primarily expected in demersal speciesby polluting suspended particulate matter and sediments ratherthan the water column, on the eggs and larvae thriving in thisenvironment, apart from a maternal transfer that must also beconsidered in viviparous species. Consequently, the most feasi-ble exposure routes to be replicated under laboratory conditionsare dietary, or the presence of sediments or particulate mattercontaining the chemical at environmentally realistic concentra-tions. However, ELS do not necessarily involve dietary exposure,especially if the windows of exposure of concern consider theembryonic period. We therefore advocate the use of particulatematter or sediments as principal routes for a potential translocationof the chemical into embryos to assess subsequent effects duringdevelopment.

Acknowledgements

We are grateful to C. del Río for valuable help with fish main-tenance and technical support during the experiments. This workwas jointly supported by Spanish Government Grants CTM201019779-C02-C01 and RTA2010-00004-C02. The medaka fish usedin the experiments were manipulated in accordance with Directive86/609/EEC on the protection of animals for experimental and otherscientific purposes within the European Union. All the experimentswere previously approved by the Animal Care and Use Committeeof our Institute. The contents presented in this work are solely theauthors’ responsibility, and do not necessarily represent the officialviews of the supporting Governmental Institutions.

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