Non-target trials with Pseudomonas fluorescens strain CL145A, a lethal control agent of dreissenid mussels (Bivalvia: Dreissenidae).

In an effort to develop an efficacious and environmentally safe method for managing zebra mussels (Dreissena polymorpha) and quagga mussels (Dreissena rostriformis bugensis), we initiated a research project investigating the potential use of bacteria and their natural metabolic products as biocontrol agents. This project resulted in the discovery of an environmental isolate lethal to dreissenid mussels, Pseudomonas fluorescens strain CL145A (Pf-CL145A). In previous published reports we have demonstrated that: 1) Pf-CL145A’s mode of action is intoxication (not infection); 2) natural product within ingested bacterial cells lyse digestive tract epithelial cells leading to dreissenid death; and 3) high dreissenid kill rates (>90%) are achievable following treatment with Pf-CL145A cells, irrespective of whether the bacterial cells are dead or alive. Investigating the environmental safety of Pf-CL145A was also a key element in our research efforts, and herein, we report the results of non-target trials demonstrating Pf-CL145A’s high specificity to dreissenids. These acute toxicity trials were typically single-dose, short-term (24-72 h) exposures to Pf-CL145A cells under aerated conditions at concentrations highly lethal to dreissenids (100 or 200 mg/L). These trials produced no evidence of mortality among the ciliate Colpidium colpoda, the cladoceran Daphnia magna, three fish species (Pimephales promelas, Salmo trutta, and Lepomis macrochirus), and seven bivalve species (Mytilus edulis, Pyganodon grandis, Pyganodon cataracta, Lasmigona compressa, Strophitus undulatus, Lampsilis radiata, and Elliptio complanata). Low mortality (3-27%) was recorded in the amphipod Hyalella azteca, but additional trials suggested that most, if not all, of the mortality could be attributed to some other unidentified factor (e.g., possibly particle load or a water quality issue) rather than Pf-CL145A’s dreissenidkilling natural product. In terms of potential environmental safety, the results of these invertebrate and vertebrate non-target trials are encouraging, but it would be unrealistic to think that dreissenids are the only aquatic organisms sensitive to Pf-CL145A’s dreissenid-killing natural product. Additional testing is needed to better define Pf-CL145A’s margin of safety by identifying the sensitivity of other susceptible organisms. The results of these non-target safety trials – in combination with equally promising mussel control efficacy data – have now led to Pf-CL145A’s commercialization under the product name Zequanox ® , with dead cells as the product’s active ingredient. The commercial availability of only dead-cell Zequanox formulations will eliminate the risk of any possible non-target infection by Pf-CL145A, further reducing environmental concerns. During the non-target project reported herein, the limited quantities of Pf-CL145A cells that we were able to culture severely restricted the number and size of our trials. In contrast, the availability of Zequanox will now greatly expand the opportunities for non-target testing. The trials reported herein – exposing non-target organisms under aerated conditions to unformulated, laboratory-cultured cells – clearly point to Pf-CL145A’s potential for high host specificity, but non-target trials with Zequanox – using PfCL145A cells cultured, killed, and formulated using industrial-scale protocols – will be even more important as they will define the nontarget safety limits of the actual commercial products under a wide range of environmental conditions.