Four standard reference materials certified for MeHg and the six total metal concentrations (TORT-2 Lobster Hepatopancreas (NRCC, Ottawa, Canada); DORM-2 Dogfish muscle (NRCC, Ottawa, Canada), NIST 1566b oyster tissue (NIST, Gathersburg, USA), NIST 2976 mussel tissue), as well as a fifth certified only for total metals (Plankton BCR 414 (Geel, Belgium) were used for method validation. Sample weight was varied from 20 to 50 mg for certified reference materials, as well as five replicates of DORM-2 from 5 to 10 mg.
Aquatic invertebrates (39 blue and ribbed mussels, 48 fiddler and green crabs, 29 oysters and clams, 23 sand shrimp, 26 periwinkles, 17 amphipods, 7 polychaete, 19 zooplankton) and small fish (35 killifish, 28 silversides, 5 alewife, 9 stickleback) were collected from six estuaries in New England. Samples were freeze-dried as whole organisms, transferred to 25 mL Teflon screw top vials (machined in-house), with a 15 mm Teflon grinding ball (McMaster Carr, Aurora, OH, USA) added to each sample, and homogenized using a Retsch MM301 ball mill (Haan, Germany). Of the 285 samples included in this study 14 had dry weights of less than 10 mg, and 45 weighed less than 20 mg. Ground samples were stored in glass test tubes (I-Chem, Rockwood, TN, USA) double wrapped in plastic.
A flowchart of the sample preparation protocol is given inFig. 1 . Up to 50 mg of sample was weighed into 7mL PFA Teflon micro-vials (CEM, Matthews, NC, USA). Samples were spiked with an 10 to 50 μL of 50 μg L−1 201Hg and 20 μg L−1 199Hg, depending on sample mass. To each sample was added 2mL 4M HNO3, and the weight recorded. For each batch of 32 samples, three digestion blanks with isotopic Hg spikes, and three standard reference materials were prepared. A duplicate sample was also prepared for every 20 samples, where sufficient sample mass was available. Micro-vials were sealed and heated overnight at 55 °C. A 50 μL aliquot (100 μL for samples less than 10 mg) of leachate was removed for MeHg determination, then vials were re-weighed, and 1.5 mL concentrated HNO3 was added to each vessel before re-capping. Two microvessels were placed inside an HP 500 Plus high-pressure microwave vessel containing a spacer and 10 mL of deionized water. Microwave vessels were loaded into a MARS high pressure microwave (CEM), and the temperature was ramped to 150 °C over 20 min., then held at that temperature for another 10 min. Vessels were allowed to cool to room temperature prior to opening. Digested samples were then transferred to 7 mL PTFE tubes (Savillex, Minnetonka, MN, USA) and weighed. Samples were diluted another 5 times for total method determination.
Between batches, vials for milling were cleaned by scrubbing in Citranox (SPI Supplies, West Chester, PA, USA), followed by soaking overnight in 5% HCl:5% HNO3, then overnight in 10% HNO3, and rinsed with ultrapure water. Microvials for digestion were cleaned by scrubbing with acetone, followed by soaking overnight in 5% HCl:5% HNO3. Vials were then rinsed, and filled with 3 mL concentrated HNO3 and heated to 150°C in the microwave.
Aquatic invertebrates (39 blue and ribbed mussels, 48 fiddler and green crabs, 29 oysters and clams, 23 sand shrimp, 26 periwinkles, 17 amphipods, 7 polychaete, 19 zooplankton) and small fish (35 killifish, 28 silversides, 5 alewife, 9 stickleback) were collected from six estuaries in New England. Samples were freeze-dried as whole organisms, transferred to 25 mL Teflon screw top vials (machined in-house), with a 15 mm Teflon grinding ball (McMaster Carr, Aurora, OH, USA) added to each sample, and homogenized using a Retsch MM301 ball mill (Haan, Germany). Of the 285 samples included in this study 14 had dry weights of less than 10 mg, and 45 weighed less than 20 mg. Ground samples were stored in glass test tubes (I-Chem, Rockwood, TN, USA) double wrapped in plastic.
A flowchart of the sample preparation protocol is given in
Between batches, vials for milling were cleaned by scrubbing in Citranox (SPI Supplies, West Chester, PA, USA), followed by soaking overnight in 5% HCl:5% HNO3, then overnight in 10% HNO3, and rinsed with ultrapure water. Microvials for digestion were cleaned by scrubbing with acetone, followed by soaking overnight in 5% HCl:5% HNO3. Vials were then rinsed, and filled with 3 mL concentrated HNO3 and heated to 150°C in the microwave.