Acute toxicity of polybrominated diphenyl ethers (PBDEs) - Springer Link

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Sep 13, 2011 - Abstract. Background, aim and scope The environmental presence of polybrominated diphenyl ethers (PBDEs), among which. BDE-47 and ...

Environ Sci Pollut Res (2012) 19:708–717 DOI 10.1007/s11356-011-0602-5


Acute toxicity of polybrominated diphenyl ethers (PBDEs) for turbot (Psetta maxima) early life stages (ELS) Lazhar Mhadhbi & José Fumega & Moncef Boumaiza & Ricardo Beiras

Received: 29 March 2011 / Accepted: 29 August 2011 / Published online: 13 September 2011 # Springer-Verlag 2011

Abstract Background, aim and scope The environmental presence of polybrominated diphenyl ethers (PBDEs), among which BDE-47 and BDE-99 are particularly abundant, makes toxicity data necessary to assess the hazard risk posed by PBDE to aquatic organisms. This study examines the effects of BDE-47 and BDE-99 on embryo-larval stages of the marine flatfish turbot. Materials and methods The turbot embryos were exposed at nominal concentrations of BDE-47 and BDE-99 for 6 days. Selected dose levels were relevant for investigating sublethal and lethal effects. Results Both tested compounds caused lethal toxicity as well as non-lethal malformations during embryo development. We found a high toxic potency of BDE-47 compared to BDE-99 (LC50 values for embryos and larvae, respectively, BDE-47: 27.35 and 14.13 μg L−1; BDE-99: 38.28 and 29.64 μg L−1). Discussion The present study shows high sensitivity of fish early life stages (ELS) to PBDE compounds. Based on environmental concentrations of dissolved PBDEs from Responsible editor: Ake Bergman L. Mhadhbi (*) : R. Beiras Toralla Marine Science Station (ECIMAT), University of Vigo, 36331 Vigo, Galicia, Spain e-mail: [email protected] J. Fumega Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Cabo Estai Canido, 36200 Vigo, Galicia, Spain M. Boumaiza Hydrobiology Unit, Environmental Biomonitoring Laboratory, Faculty of Sciences of Bizerte, University of Carthage, Bizerte, Zarzouna 7021, Tunisia

various aquatic ecosystems, waterborne BDE-47 and BDE99 pose little risk of acute toxicity to marine fish at relevant environmental concentrations. Conclusions Turbot fish ELS proved to be an excellent model for the study of ecotoxicity of contaminants in seawater. The results demonstrate harmful effects of PBDE on turbot ELS at concentrations in the range of parts per billion units. Recommendations and perspectives In the perspective of risk assessment, ELS endpoints provide rapid, costeffective and ecologically relevant information, and links should be sought between these short-term tests and effects of long-term exposures in more realistic scenarios. Keywords Brominated flame retardants (BFR) . Polybromodiphenyl ethers (PBDE) . Ecotoxicity . Early life stage (ELS) . Turbot (Psetta maxima)

1 Background, aim and scope Polybrominated diphenyl ethers (PBDEs) are widespread pollutants of increasing environmental concern due to their persistence and high bio-accumulative capacity. They are currently important components in flame retardants and other industrial processes, thus recognized as ubiquitous environmental contaminants (Roberts et al. 2011). They have been present in commercial products used in our daily life, such as electronic equipment, plastics, textiles and building materials (Chou et al. 2010). Concurrent with their increased use, the environmental levels of PBDEs have also risen. Spillage and emissions during production, release from consumer products and disposal of end-of-life consumer products all account for this phenomena. Since PBDEs are polymer additives and

Environ Sci Pollut Res (2012) 19:708–717

are not chemically bound to materials, they are known to leach into the surrounding environment (Hu et al. 2010) and become ubiquitous contaminants. These compounds are not naturally biodegradable in the OECD Test (EU draft RAR 2000), they easily bio-accumulate in fatty tissues and biomagnify throughout food chains (Law et al. 2006; Talsness 2008; Noyes et al. 2010). Comparatively high levels of PBDEs are frequently found in aquatic biotopes from different parts of the world. They have been detected in sediments, sludge, fish and mammals (Talsness 2008; Moss et al. 2009). Likewise, the recent sharp increase in PBDE levels found in human milk in regions of Sweden and North America (Watanabe and Sakai 2003) has raised concern for human health. These compounds are typically produced in three different formulations with varying degrees of bromination, penta-PBDE, octa-PBDE and deca-PBDE (Alaee et al. 2003; La Guardia et al. 2006; Vane et al. 2010). The majority of PBDEs found in environment are 2,2′,4,4′tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99). Generally, in biological samples, the BDE-47 and BDE-99 occur at highest levels (Xu et al. 2009; Hu et al. 2010). The most prevalent PBDE congeners found in maternal and cord blood are BDE-47, BDE-99, BDE-100 and BDE-154 (Guvenius et al. 2003). Estimates based on animal experimental data have been published previously for BDE-47, BDE-99, BDE-100 and BDE-154 (Geyer et al. 2004), indicating long half-lives for all these congeners. Among them, in the large production volumes of brominated diphenyl, tetra- and penta-brominated congeners predominating in biota, BDE-47 was the most abundant (De Boer et al. 1998). The constituents of pentaBDE are the most bio-accumulative and frequently encountered in common organisms, such as fishes (Moss et al. 2009; Van de Merwe et al. 2011). The order of congener dominance in these organisms is typically BDE-47 > BDE99 > BDE-100 (Hu et al. 2010). BDE-47 and BDE-99 were the predominant congeners in market fish suggesting usage of penta-BDE as the main PBDE contamination source (Cheung et al. 2008). The 2,2′,4,4′,5-pentabromodiphenylether (BDE-99) is among the PBDE congeners found more often and at highest levels in sentinel species and human tissues (de Boer et al. 1998; Guvenius et al. 2003; Schecter et al. 2005). Recently, the constituents of penta-BDE technical mixtures have been proposed as persistent organic pollutant (POPs) candidates since they embody all the characteristics of the Stockholm convention definition of POPs: bioaccumulation, toxicity, persistency and long-range transport potential (UNEP 2009). Penta-BDE mixtures, as well as the octa-BDE mixture, have been banned in 2004 in the European Union (EU 2003). However, such regulatory actions can be only partially successful in reducing


environmental concentrations because of continued release from products or materials currently in use (Gewurtz et al. 2011) and PBDEs are still found ubiquitously in marine environments and biota worldwide (Zhang et al. 2010). Our objective was therefore to tests the toxicity of the two environmentally most abundant BDEs (i.e. 47 and 99) on the early life stages (ELS) of turbot. Early life stages are generally considered as more sensitive than juvenile and adults. During early ontogenesis, critical development of tissues and organs takes place, a process which can easily be disrupted by unfavourable environmental conditions including exposure to toxic compounds (Foekema et al. 2008; Kammann et al. 2009). In fish early life stage tests, toxicant effects can be examined through diverse endpoints, such as hatching success, embryo-larval morphology malformations and larval survival (Mhadhbi et al. 2010).

2 Materials and methods 2.1 Chemical analysis 2.1.1 Reagents and solutions All the chemical analyses were performed in the Oceanographic Center of Vigo of the Instituto Español de Oceanografía. Individual standards of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) were obtained as solids from AccuStandard, Inc. (USA). Both PBDE congeners were certified for 100±4% purity and the absence of PBDDs. Primary stock solutions were prepared by dissolving the solids in isooctane. From these solutions, a series of the two chemicals (nominal concentrations shown in Table 1) were prepared in dimethyl sulphoxide (DMSO) and added to the water in the different tanks. Working internal standard octachloronaphthalene solution was prepared in isooctane. All the solutions were stored at 5°C when not in use. All solvents used were designed for organic residue analysis and checked with blanks. Two replicates were prepared for each concentration. 2.1.2 Determination of PBDEs in water The BDEs (47 and 99) were extracted with hexane. The extract were dried over sodium sulphate and concentrated in a vacuum evaporator (Büchi Rotavapor R-200). GC/MS analyses were performed on an Agilent gas chromatograph 6890N equipped with an electronically controlled split/ splitless injection port, coupled to an Agilent 5973N mass selective detector. A concentrated extract (2 μL) was injected onto a CPSil8 CB capillary column (length 30 m,

710 Table 1 Nominal and measured concentrations (percent of nominal concentration) for BDE-47 and BDE-99 in 0-, 24- and 48-h-old solutions for the embryo-larval bioassays

Environ Sci Pollut Res (2012) 19:708–717


Nominal concentration (μg L−1)

Measured concentration (% nominal concentration) t0


Low Medium High


Low Medium High

diameter 0.25 mm, film thickness 0.25 μm). The injection was made in pulsed splitless mode (pulse 58 psi, purge 1.5 min). The injector temperature was 275°C, and the temperature program was 90°C (hold 3 min), increase at 30°C min−1 to 210°C (hold 20 min), increase at 5°C min−1 to 290°C (hold 17 min) and increase at 35°C min−1 to 310°C (hold 5 min). The mass selective detector with quadrupole analyser was operated in the selected ion-monitoring mode under negative chemical ionization using methane as reagent gas. The monitored ions (m/z) were both bromine isotopes (79 and 81) for all PBDE congeners. For the internal standard (octachloronaphthalene), the ion (m/z) was 403.80. The MS transfer line temperature was held at 290°C, and the MS source and quadrupole temperatures were 200°C and 106°C, respectively. 2.2 Biological material Turbot (Psetta maxima) eggs from a single stock of adults were obtained in kind from a fish hatchery (Insuiña S.L., Mougás, Galicia, Spain). Eggs were transported to the laboratory in plastic bags inside portable iceboxes and maintained in aquaria with running natural seawater (salinity 34‰). Eyed eggs were acclimated to laboratory conditions for 24 h at 14±1°C (hatchery rearing temperature) before the experimental exposures to the toxicants. 2.3 Experimental solutions and exposures PBDE congeners were purchased from ChemService, Inc. (Greyhound Chromatography and Allied Chemicals, UK). Stock solutions of PBDEs were dissolved in DMSO (Sigma–Aldrich, Steinheim) and stored in amber glass vials. The following PBDEs were selected based on their abundance in environmental samples: BDE-47 and BDE99. For each toxicant, 12 concentrations in a 2× geometric scale, plus one control with no PBDEs added, were tested, using four replicates for each condition. Nominal concentrations 0.3, 0.75, 1.5, 2.5, 3.125, 6.125, 12.5, 25, 30, 100, 150 and 200 μg L−1 for BDE-47 and 2.5, 5, 8, 16, 20, 25,

2.8 8 24



1.566 (60.18)

1.41 (46.07)

1.351 (49.62)

6.124 (76.66) 13.84 (58.49)

5.51 (67.94) 12.31 (51.61)

4.184 (52.63) 10.56 (43.71)


2.76 (54.62)

2.02 (45.79)

2.24 (57.86)

13.2 39.5

10.57 (81.62) 22.71 (57.39)

8.68 (65.42) 18.92 (50.42)

9.55 (71.24) 18.65 (45.09)

32, 50, 75, 100, 150 and 300 μg L−1 for BDE-99 were used. All experiments were repeated twice consecutively. All treatments, including controls, contained

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