Environ Sci Pollut Res (2011) 18:226–236 DOI 10.1007/s11356-010-0355-6
RESEARCH ARTICLE
Uptake and elimination, and effect of estrogen-like contaminants in estuarine copepods: an experimental study Kevin Cailleaud & Hélène Budzinski & Sophie Lardy & Sylvie Augagneur & Sabria Barka & Sami Souissi & Joëlle Forget-Leray
Received: 15 July 2009 / Accepted: 7 June 2010 / Published online: 4 July 2010 # Springer-Verlag 2010
Abstract Background, aim, and scope In recent years, anthropogenic chemicals which can disrupt the hormonal systems of both humans and wildlife have been raised to a major cause of concern. The aim of the present work was to determine the bioconcentration factors of the two major alkylphenols (AP) of the Seine Estuary [4-nonylphenol (4 NP) and nonylphenol acetic acid (NP1EC)] and of the synthetic estrogen, estrogen ethinylestradiol (EE2), in Eurytemora affinis after exposure in a continuous flow-through system under environmental realistic conditions. Moreover, the elimination of these compounds in copepods from the Seine Estuary has been investigated by measuring concentrations
Responsible editor: Philippe Garrigues K. Cailleaud : H. Budzinski : S. Lardy : S. Augagneur University of Bordeaux 1, CNRS, ISM-LPTC-UMR 5255, 351 cours de la Libération, 33405 Talence, France S. Souissi Université des Sciences et Technologies de Lille, CNRS, UMR 8187 LOG, 32 avenue Foch, 62930 Wimereux, France S. Barka Laboratoire de Toxicologie Marine et Environnementale, UR 09-03, IPEI, Sfax, Tunisia J. Forget-Leray (*) Faculté des Sciences et Techniques du Havre, LEMA-UPRES EA3222 (Laboratoire d’Ecotoxicologie-Milieux Aquatiques), GDR EXECO, 25 rue Philippe Lebon, 76058 Le Havre, France e-mail:
[email protected]
after 1 week in clean water in comparison to background levels. Materials and methods In this study, the dominant copepod species of the Seine Estuary, E. affinis, was exposed at environmental relevant concentrations under laboratorycontrolled conditions to selected waterborn contaminants, a mixture of 4 NP/NP1EC, and a synthetic EE2. The uptake and the elimination of these contaminants by E. affinis have been studied. Results The results show that, at the end of the uptake period, both 4 NP and NP1EC, and also EE2 were accumulated in exposed copepods with respective concentration factors of 324, 3,020, and 5,383. A rapid elimination of these compounds was also observed in copepods placed in clean water since 54% of total NP1EC and 100% of EE2 amounts have been lost after 3 days. Pregnenolone was synthesized after exposure to EE2 and AP mixture. Discussion These results demonstrate that E. affinis has the potency to accumulate but also to eliminate endocrine disrupters which suggests a non-negligible role of this copepod species in the biogeochemical cycles of these contaminants in estuarine ecosystems. Hence, these results also suggest that a transfer of 4 NP, NP1EC, and EE2 to copepod predators and subsequently that secondary poisoning of these organisms might be possible. Estrogen-like contaminants can induce pregnenolone synthesis and affect the reproduction of E. affinis. Conclusions These results suggest the important role of this copepod species in biogeochemical cycles of non-ionic surfactants as well as synthetic steroids in estuarine ecosystems. Recommendations and perspectives E. affinis could be a non-negligible route of exposure for juvenile fish and underline the potential for deleterious effects on copepod predators.
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Keywords 4 NP . NP1EC . EE2 . Crustacean . Estuary . Flow-through system . Pregnenolone
1 Background, aim, and scope Over the last 15 years, concern has grown concerning hormonal system disturbances in aquatic organisms related to anthropogenic contaminant releases. These chemicals, so-called endocrine disrupters, have potential effects on the reproductive system (Metzler and Pfeiffer 2001), especially by mimicking the natural female hormone estrogen through interaction with the estrogen receptor present in mammals and other vertebrates. Thus, the occurrence of reproductive abnormalities has been frequently reported (Jobling et al. 1996; Petrovic et al. 2002). Although the number of published data about endocrine disruption in wild species has substantially increased during the last 10 years for both vertebrate and invertebrate animals (DeFur et al. 1999; Larsson et al. 1999; Oberdörster and Cheek 2000; Porte et al. 2006; Mazurova et al 2008), studies on the specific mechanism by which the endocrine system can be disrupted are scarce. However, some potential hormone-disrupting chemicals have been listed as priority pollutants, depending on their molecular structure, by regulating bodies such as the European Union and the Oslo and Paris Commission. Many chemicals which have endocrine disrupting potential share a common chemical structure with steroid hormones and fall into different categories in relation to their chemical properties (Routledge and Sumpter 1997). At present, the groups of endocrine disrupters which are of major concern are the environmental estrogens and anti-estrogens since experiments have shown their toxicity on vertebrates. Among these contaminants, alkylphenol polyethoxylates (APEs) and natural and synthetic steroid hormones are two well-known groups of environmental endocrine disrupters. APEs are widely used as non-ionic surfactants in various industrial, commercial, and household detergent formulations. Nonylphenol-polyethoxylates (NPEs) are, by far, the most commonly used and synthesized APEs, accounting for about 80% of total APEs (Renner 1997). These molecules are used in numerous applications, principally as detergents and industrial products (coatings, paints, fuels, plastics, paper…). The annual worldwide production of APEs in 1997 exceeded 500,000 metric tons (Renner 1997) with an estimated 60% of this production ending up in water. Thus, the occurrence of high levels of APEs and especially of NPEs in coastal, estuarine, and freshwaters has been extensively documented (Thiele et al. 1997; Ying et al. 2002b). Furthermore, a complex microbial degradation of APEs, which produces metabolites that are more toxic than the parent compounds, has been characterized (Fenner et al. 2002), and these
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metabolites are frequently detected at high levels in aquatic ecosystems. Numerous studies have also suggested the occurrence of steroid estrogens in aquatic environments (Ying et al. 2002a; Williams et al. 2003; Noppe et al. 2006). The most potent steroid, 17α-ethinylestradiol (EE2), a synthetic estrogen widely used as a female contraceptive agent (Purdom et al. 1994) has been detected in different worldwide sewage treatment plant effluents at levels ranging between 0.2–42 ng L−1 (Ying et al. 2002a). In UK, a threshold of 0.1 ng L−1 in water has been therefore proposed for this compound based on chronic toxicity tests (Desbrow et al. 1998). In relation to their high octanol-water partition coefficients (log Kow > 4) both NPEs and EE2 tend to adsorb to organic material and to accumulate in biota. In order to determine the actual impact of these compounds on the aquatic environment and to predict the potential for biomagnification of these compounds through the food chain, it is essential to elucidate their transfers and their metabolic fate in aquatic organisms (Wang et al. 1996). Nevertheless, the majority of the studies have focused on vertebrate species and especially on fish (Staples et al. 1998; Arukwe et al. 2000). Thus, both nonylphenol and EE2 have shown to be experimentally bioconcentrated (Uguz et al. 2003; Labadie and Budzinski 2006) and bioaccumulated by fish species. In addition, hormonal and physiological diseases observed in exposed fish were related to increasing endocrine disrupter levels in fish tissues. However, although planktonic crustaceans constitute an important energetic resource in the aquatic food webs and play a key role in biogeochemical exchanges between sediments and the water column, little attention has been paid concerning their potency to accumulate estrogenic compounds. However, both EE2 and 4 NP have been shown to exhibit endocrine disruptions in copepods (Forget-Leray et al. 2005). In addition, transfer of accumulated estrogenic contaminants from the lower trophic levels to top predators may occur and result in secondary poisoning of these predator species. The Seine Estuary is highly contaminated by organic contaminants, especially by 4 NP and NP1EC (Cailleaud et al. 2007a, b, c). Furthermore, natural sex steroids have been frequently detected in this estuary at levels ranging from 1.8 to 8.3 ng L−1 while synthetic steroids such as EE2 were always at or below the detection limit or the quantification limit (Labadie and Budzinski 2005). Eurytemora affinis is the copepod species that dominates the microzooplankton community of this estuary and, in spite of the high contaminant levels, presents particularly high density in this estuary in comparison to other North-Atlantic estuaries (Devreker et al. 2007). The aim of the present work was to determine the bioconcentration factors of the two major alkylphenols of the Seine Estuary (4 NP and NP1EC) and of the synthetic
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estrogen, EE2 but also to monitor steroid profile in E. affinis after exposure in a continuous flow-through system under environmental realistic conditions. Moreover, the elimination of these compounds in copepods from the Seine Estuary has been investigated by measuring concentrations after 1 week in clean water in comparison to background levels.
2 Materials and methods 2.1 Material design The experimental system described in Fig. 1 was composed of three compartments: the water reservoirs (25 L), the exposure tanks (25 L) containing the copepods, and a recycling tank to remove contaminants from water using activated carbon. All the tanks used during the experiments were glass material disinfected and washed before the use. The three compartments were linked together by a peristaltic pump that draw contaminated water from the water reservoirs to the exposure tanks and eliminated excess water from the exposure tanks into the recycling tank. Contaminant concentrations in water reservoirs and in exposure tanks were identical. Furthermore, in order to avoid copepods to be diffused into the peristaltic pump, a volume of 125 cm3 around rubber tubing extremities was capped with 50-μm mesh. 2.2 Collection of experimental organisms According to Cailleaud et al. (2007a), levels of hydrophobic organic chemicals (HOCs) in E. affinis presents Fig. 1 Schematic view of the laboratory flow-through system. (P peristaltic pump, AP alkylphenol, EE2 17-αethinylestradiol, Ctrl control)
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seasonal variations. Therefore, copepods were sampled during the lowest impacted period, in autumn 2004. Copepods were collected using subsurface tows of WP2 plankton net (200 μm mesh size) at ebb tide, in Tancarville Station in the oligohaline part of the Seine Estuary. Immediately after sampling, the copepods were sorted using 500-μm sieves in order to eliminate predators (especially Mysidacea and Gammaridae), then transferred into isotherm containers using filtered estuarine water and brought back to the laboratory for further precise sorting in order to exclude particulate matter from the sample as previously described (Cailleaud et al. 2007b). After sorting, the copepods were maintained in the laboratory for an acclimatization period of 3 days in a 300-L hydrodynamic canal, under controlled conditions (salinity, 15 PSU; temperature, 10°C; photoperiod, 12/12H). The hydrodynamic canal contained freshly filtered sea water (GF/C Whatman filter, 0.45 μm) sampled in the English Channel, mixed with ultra-pure water in order to reach the selected salinity of 15 PSU. The same water preparation was used for the exposure experiments. During this period, copepods were fed twice a day with algal mixtures (Rhodomonas marina and Isochrysis galbana). Then approximately 250,000 copepods were transferred into the experimental continuous flow-through system (1,000,000 individuals/m3). 2.3 Experimental setups Preliminary tests were performed with the exposure system to ensure that the real exposure concentrations of dissolved APEs and EE2 were within an acceptable range from the selected nominal concentrations of during all the experi-
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ments. Before the exposure started, the tanks and the rubber tubing were saturated with contaminants. Thus, an appropriate flow rate, and a saturation period were characterized for each contaminant class (see Table 1). The contaminants solutions were prepared at high concentrations in acetone in order to introduce low volumes of solvent in the water ( 98%) standard was purchased from Sigma-Aldrich (St Quentin Fallavier, France). Estrone (E1), 17β-estradiol (E2), estriol (E3), testosterone (T), dihydrotestosterone (DHT), androstenedione (A), dihydroan-
drostenedione (DHA), dehydroepiandrosterone (DHEA), progesterone (Pg), 17α-hydroxyprogesterone (OHPg), pregnenolone (Pn), 17 α-hydroxypregnenolone (OHPn), and EE2 were supplied by Sigma-Aldrich (St Quentin Fallavier, France). Deuterated steroids (testosterone-d3, 17β-estradiol-d4, and 17α-ethinylestradiol-d4, purity > 99%), used as internal standard, were obtained from C/D/N Isotopes (Montreal, Canada). Triethylamine (purity > 99%) was used for some SPE procedures (Sigma-Aldrich, St Quentin Fallavier, France). Trifluoroacetic acid (reagent grade, purity > 99%) was obtained from Fisher Scientific Labosi (Elancourt, France). MSTFA (Nmethyl-N(trimethylsilyl)trifluoroacetamide, >97%; Acros Organics, Noisy-Le-Grand, France) was used as silylation agent for gas chromatography/mass spectrometry (GC/MS) analysis, in combination with mercaptoethanol (purity > 99%) and ammonium iodide (purity > 99%), both provided by Acros Organics. Pure water was obtained with a Milli-Q system (Millipore, Molsheim, France). β-glucuronidasearyslsulfatase from Helix pomatia (glucuronidase: 100,000 Sigma units ml−1 and sulphatase, 7,500 Sigma units ml−1) was supplied by Sigma-Aldrich. Sodium acetate tri-hydrate (purity > 99%, Sigma-Aldrich) and acetic acid (purity > 99.5%, VWR International, Strasbourg, France) were used for the preparation of acetate pH buffer. 2.5 Alkylphenols 2.5.1 Extraction Prior to the extraction, freeze-dried copepods were weighed (150 mg dry weight (dw) of a homogenized pool of E. affinis) and water volumes were measured (500 mL). NP1EC and 4 NP were extracted from E. affinis by focused microwaveassisted extraction (10 min at 30 W) with a methanol/ dichloromethane mixture as solvent (3/1, v/v). Organic extracts were concentrated by rotary evaporation and then dissolved into 100 ml of acidified ultra-pure water (pH 2). Before the SPE, the water samples were acidified to approximately pH 2 with 3.5 M HCL. The cartridges Varian BondElut C18 (Interchim, Montluçon, France) were preconditioned with 5 ml of methanol followed by 5 ml of
Table 1 Experimental conditions to saturate the mesocosms with the contaminant solutions and to maintain constant dissolved alkylphenol and EE2 concentrations at the targeted concentrations Sampling time Compounds
NP1EC 4 NP EE2
Flow of water (Lday)
25 50
T0 T 24H Concentrations (ng L-1)
T 42H
T 50H
T 72H
Nominal concentrations
327 360 9.9
318 472 10.1
419 508 10.6
467 520 –
520 480 10
419 416 9.1
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ultra-pure acidified water (pH 2). Then 500 ml of the water sample and 100 ml of the water accommodated copepod extracts were eluted through the cartridge at a flow rate of 10 ml min−1. After the elution, the cartridges were washed with 3 ml of a methanol/acidified ultra-pure water mixture (50/50, v/v). Then the aqueous phase was removed by drying the cartridge for 1 h. At last, the organics were eluted by passing 5 ml of a methanol/dichloromethane mixture (50/50, v/v) and concentrated to 100 μl (for water extracts) and to 500 μl (E. affinis extracts) by warming at 45°C under a gentle nitrogen stream.
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of 250 μl of MSTFA, 15 μl of mercaptoethanol, and 10 mg of NH4I was left for about 10 min at 65°C. Thereafter, this mixture was diluted ten times with MSTFA to obtain the derivatization reagent, 30 μl of which were added to each extract, and samples were kept at 65°C for 30–40 min. Then, 30 μl of dichloromethane were added prior to GC/ MS analysis. 2.6.2 Copepod samples extraction
2.6 Steroid
Copepod samples (100 mg dw) were combined with 2 ml sodium acetate buffer (pH 5.0, 10−2 M) and spiked with EE2-d4. Samples were then homogenized using an UltraTurrax® T25 Basic dispersing tool (IKA® Werke, Staufen, Germany) for 1 min at 6,000 rpm, in 30 ml of methanol/ Milli-Q water (55:45, v/v). Steroids (EE2 and Pn) were then extracted by focussed microwave-assisted extraction (Microdigest 301, Prolabo, Fontenay-sous-Bois, France): 30 W, 5 min, 10 ml of methanol/Milli-Q water (55:45, v/v). Extracts were centrifuged at 4,500 rpm for 5 min, at room temperature. The supernatant was transferred to a vial, and the methanol content of the extracts was evaporated at 60°C under a nitrogen stream. The extract was then fractionated and purified according to the method developed by Labadie and Budzinski (2006). The conjugated fraction was subjected to both solvolysis and enzymatic hydrolysis. Dried Oasis HLB extracts were incubated at 45°C for 30 min in trifluoroacetic acid/methanol/tetrahydrofuran (10 μl/200 μl/800 μl). After neutralization with 0.2 M Na2CO3, the organic solvents were evaporated at 60°C under a gentle nitrogen stream. Sodium acetate buffer (pH 5.0, 10−2 M) and β-glucuronidase-arylsulphatase were added, and samples were further incubated at 55°C for 3 h. These extracts, containing only free steroids, were purified using Oasis HLB and aminopropyl SPE cartridges (Fig. 2). The purpose of this second Oasis HLB step was to switch from aqueous buffer to organic solvent, but also aimed at providing a first clean-up of the extract. Then, samples were derivatized as previously described for the determination of steroids in water before the analysis by GC/MS.
2.6.1 Water samples extraction
2.6.3 Analyses
Water samples from the exposure tanks (500 mL) were spiked with EE2-d4 and extracted on Oasis HLB cartridges (flow rate 15 ml min−1), previously conditioned with 6 ml methanol and 6 ml Milli-Q water (Labadie and Budzinski 2006). The sorbent was rinsed with 5 ml of methanol/water (60/40, v/v), and analytes were eluted with 8 ml of methanol. Extracts were taken to dryness under a stream of nitrogen at 50°C, prior to the silylation step. The derivatization reagent was prepared as follows: a mixture
Separation and detection of the analytes were achieved using an Agilent Technologies gas chromatograph system (6890 series) coupled with an Agilent Technologies mass spectrometer detector (5973 series), used in electron impact mode (70 eV), with the electron multiplier voltage set at 1,500–1,800 V and the source temperature set at 230°C. The separation was performed on an Agilent HP-5MS capillary column (length, 30 m; internal diameter, 250 μm; stationary phase thickness, 0.25 μm), with the following
2.5.2 Analyses Alkylphenols and their metabolites were analyzed using an HPLC separation coupled to electrospray mass spectrometry detection. The HPLC system consisted of an Agilent 1100 series (Agilent Technologies). Chromatographic separation was performed using a reversedphase analytical column (Kromasil C18, Interchim, France) of 150×2.1 mm id×3 μm-film thickness. NP1EC and 4 NP were separated using a mixture of water/ methanol/ammonium acetate (5 mM) (A) and methanol (B) as mobile phase. The solvent program started with an initial B concentration of 60% kept isocratic for 2 min, linearly increased to 80% in 5 min, kept isocratic for 28 min, and linearly decreased to 60% in 3 min, with constant flow rate (0.150 ml/min) and constant column temperature (20°C). An Agilent Technologies mass spectrometer equipped with an electrospray ion (ESI) source was used. The following operation parameters were used: source temperature, 100°C; desolvation temperature, 350°C; desolvation gas flow rate, 10 l/min; ESI+ and ESI− capillary voltage, 4.0 and 3.5 kV. Acquisition was accomplished using the selected ion monitoring mode (SIM). NPE1C (m/z 277) and 4 NP (m/z 219) were identified by their characteristic pattern showing [M-H]ions in the negative ionization mode. The compounds were also identified by co-injection of authentic standards.
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Td3 (m/z 435, M+.) was used for that of all other steroids. EE2-d4 429 (M+.) was used for the quantification of EE2. Furthermore, EE2 molecular ion, m/z, was used to confirm the peak attribution to EE2: for a given peak, the area ratio m/z 425 to m/z 440 was compared with that obtained with an authentic EE2 standard prior to definitive attribution of the peak. 2.7 Quality assurance
Fig. 2 Concentrations of total alkylphenols (a) in E. affinis after exposure via contaminated water during 86 h and in the control groups after 1 week in clean water. The elimination of 4 NP and NP1EC (b) in the control groups are presented from the in situ sampling time to the end of the experiment. 4 NP and NP1EC patterns in exposed copepods after 86 h of exposure (c) are compared with those of the non-exposed copepods and those of water. Means are shown with bars indicating the standard error (n=3). [T0 transfer of the copepods into the flow-through system after 3 days in clean water; T 86H≈1 week in clean water for copepods of the control group]
oven parameters: from 90°C (1 min) at 7.5°C min−1 to 290°C (isothermal 5 min). Helium 6.0 (Linde, Bassens, France) was used as the carrier gas (constant flow, 1 ml min−1). The injection was performed in splitless mode (1 μL injected). The injector temperature was set at 250°C, and the purge flow was set at 60 ml min−1 after 1.5 min. For each series of analyses, blank samples were run, and no steroid was detected (