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Bioaccumulation and Metabolic Effects of the Endocrine Disruptor Methoprene in the Lobster, Homarus americanus1
Integrative and Comparative Biology, Feb 2005 by Walker, Anna N, Bush, Parshall, Puritz, Jonathan, Wilson, Thomas, Et al
Bioaccumulation studies
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Adult intermolt lobsters were equilibrated in the laboratory for at least 7 days. Individual lobsters (~570 g) were placed in plastic buckets containing 8 liter filtered seawater. Dilutions of methoprene (50 ppb final concentration) were made from a stock solution (5 mM) prepared in acetone. Control animals were treated with an equal volume of acetone in seawater. Exposures were conducted at 18°C for 4 hr. Thereafter, animals were anesthetized by packing in ice, sacrificed, and tissues were dissected and snap frozen in liquid nitrogen and stored at -80°C. Tissue samples were transported to the Pesticide Analysis Laboratory at the University of Georgia, Athens GA, where they were extracted and analyzed for methoprene content by gas chromatography-mass spectrometry (GC/MS). Sample preparation for GC-MS was essentially as described by Reed et al. (1977). Briefly, tissues (5 g) were homogenized in Na^sub 2^SO^sub 4^ (50 g) and ethyl acetate (300 ml) for 1 min in a motorized blender. After passage through glass fiber filter paper, the filtrate was concentrated using a rotary vacuum evaporator. The residue was re-dissolved in ethyl acetate: toluene (3: 1), vortexed and clarified. The supernatant was defatted by gel permeation chromatography on BioBeads SX-3 (100-200 mesh; Bio-Rad); the eluting solvent was ethyl acetate: toluene (3:1). The sample was further purified by Fluorisil chromatography (serial elution with 6%, 15%, and 50% ether in hexane); material in the 15% fraction was turboevaporated and redissolved in 1 ml methylene chloride. Internal calibration standards were added and the extract was analyzed by gas chromatography-mass spectrometry as described by Noakes et al. (1999); the MS instrument was operated in selected ion monitoring (SIM) mode. Gas chromatographic conditions: column: RTX-5M5 col (Restee, Inc.) 30 m by 0.25 mm ID megabore capillary column; oven temperature program: initial temperature = 70°C, initial hold 2 min; temperature programmed to increase at 20°C per minute to 210°C with a final hold of 10 min. Under these conditions, methoprene had a retention time of 29 min, phenanthrene d-10 retention time = 25.5 min; chrysene d-12 retention time = 33.9 min. Data were expressed as parts per million. The methoprene minimal detection limit varied dependent on sample size but was approximately 0.05 ppm (wet weight). A total of five animals have been exposed, dissected, extracted and analyzed thus far; here, we report representative data from a single animal.
In vivo exposure studies
Post-molt juvenile lobsters (3 cm carapace length) were exposed to 50 ppb methoprene in sterile seawater (1 liter) for 3 hr; controls were held in seawater only. Each animal was then injected through the dorsal carapace with 0.25 ml seawater containing 0.25 mCi Tran-^sup 35^S-Label (ICN Radiochemicals, Irvine, CA). The injection site was sealed with a drop of warm 1% agarose in seawater and the animals were held for 24 hr at 18°C. Animals were anesthetized and tissues dissected and placed on ice. As part of the preliminary studies presented here, a total of two animals have been exposed, dissected, extracted and analyzed thus far. Additional replicate experiments are underway.
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