For the Menidia beryllina test method, the effluent sample was an industrial wastewater spiked with CuSO4, the receiving water sample was a natural seawater spiked with CuSO4, and the reference toxicant sample was bioassay-grade FORTY FATHOMSa synthetic seawater spiked with CuSO4.
Once the solution is spiked with CuSO4, the subsequent tests always showed a spike in CuSO4.