Water quality samples and liver tissue from one of the deceased fish were sent to the Pennsylvania Animal Diagnostic Laboratory in Kennett Square, Pennsylvania, for analysis. 

To determine whether the IonGen^TM Cu ion generator used in the backyard pond could generate toxic levels of heavy metals, an experiment was carried out to measure the output of Cu, Zn, and silver (Ag) by the IonGen^TM over time. One 1130 L tank was fitted with an IonGen^TM and another 1130 L tank without the IonGen^TM (control). All other parameters between the tanks were identical. Water samples were collected from both tanks before the addition of the IonGen^TM, and then weekly for 12 weeks. The samples were sent to the Pennsylvania Animal Diagnostic Laboratory for analysis.

The water samples were analysed for Cu, Zn, and Ag, using NexION 300D ICP-MS (Perkin Elmer, Shelton, Connecticut, US) by the PADLS New Bolton Centre Toxicology Laboratory, University of Pennsylvania, School of Veterinary Medicine, Kennett Square, PA.  The analytical standards were purchased from SCP (Champlain, New York, US) and trace metal grade nitric acid was purchased from Fisher Scientific (Pittsburgh, PA, US).  All dilutions were done using in-house deionized water (≥18 MO cm) obtained from a Millipore water purification system.

Three millilitre (mL) portions of the water samples were placed in Teflon PFA vials (Savillex, Minnetonka, Minnesota, US) and digested overnight at 70⁰C with 1 mL of concentrated nitric acid.  The digested samples were cooled to room temperature and diluted with deionized water to a final volume of 10.0 mL after addition of 4 internal standards with a final concentration of 20 ppb (⁷⁴Germanium, ¹¹⁵Indium, ⁸⁹Yttrium, ¹⁵⁹Terbium).  Silver was determined by standard mode whereas kinetic energy discrimination (KED) mode using helium as the collision gas was used to analyse Cu and Zn. To ensure the proper instrument operation and accuracy of the results, standard curves were generated for all three elements of interest.  All final concentrations were reported in parts per million (ppm) on a wet weight basis. Method detection limits for Cu, Ag, and Zn were established at 0.005 ppm, 0.001 ppm, and 0.1 ppm, respectively, in the water samples.

Eight of the samples were analysed in duplicate with the values within 20% of each other for all three elements.  Water standard reference material (1643e) with certified mineral values from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, US) was analysed with each batch of samples.