**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION. The title, authors, and abstract for this completion report are provided below. For a copy of the full completion report, please contact the author via e-mail at mwilkie@wlu.ca. Questions? Contact the GLFC via email at slrp@glfc.org or via telephone at 734-662-3018.**

Effects of Water Alkalinity, pH, and Dosing Regimen on Lake Sturgeon Sensitivity to the Lampricide, 3-trifluoromethyl-4-nitrophenol (TFM)

¹Michael P. Wilkie, ²Lisa O’Connor, ¹Oana Birceanu, and ¹Jonathan M. Wilson.

*Principal Investigator. ¹Department of Biology, Wilfrid Laurier University (WLU), Waterloo, ON, N2L 3C5. ²Great Lakes Laboratory for Fisheries and Aquatic Sciences, Fisheries and Oceans Canada, Sault Ste. Marie, ON.

April 2019

ABSTRACT:

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is used to control invasive sea lampreys in the Laurentian Great Lakes. Applied to rivers and streams, it selectively targets larval sea lampreys (Petromyzon marinus) which have a lower capacity to detoxify and eliminate TFM than non-target fishes. Although relatively uncommon, juvenile lake sturgeon (Acipenser fulvescens) are more vulnerable to TFM-induced non-target mortality than other fishes, particularly when they are less than 10 cm in length and/or living in waters of high alkalinity. The overarching goal of this project was to determine why lake sturgeon are more susceptible to TFM in their early life stages, especially in waters with different alkalinities and to better understand how they take-up and detoxify TFM. The specific objectives were to: (I) Determine how differences in water alkalinity and pH affect TFM uptake, distribution and elimination by juvenile lake sturgeon; (II) Elucidate how gill-mediated acid-base exchange processes and the gill microenvironment influence TFM speciation, uptake and toxicity; (III) Determine if exposure to lower doses of TFM for a longer period of time protects lake sturgeon from TFM toxicity, without reducing sea lamprey mortality.

Rates of TFM uptake by juvenile lake sturgeon, measured using radiolabeled TFM (¹⁴C-TFM), decreased with increasing alkalinity, suggesting that as water alkalinity increased, more TFM is required to cause toxicity in lake sturgeon, as it does in sea lamprey and other fishes. The protective effects of higher alkalinity were confirmed in toxicity tests in which lake sturgeon survival was 100 % when the fish were exposed to TFM in high alkalinity water, compared to low or moderate alkalinity, where partial and complete mortality were observed following exposure to the same TFM dose. Subsequent analysis of TFM toxicity curves revealed that the 12-h LC₅₀ and 12-h LC_99.9 of TFM to lake sturgeon is in fact higher (less toxic) than in sea lamprey at low, moderate and high alkalinity. However, in actual TFM treatments, 1.3-1.5 times the minimum lethal TFM concentration (MLC = LC_99.9) to lamprey is used to maximize mortality. The net effect is that as water alkalinity increases, more and more TFM is required to reach 1.3-1.5 times the MLC, which amplifies its toxic effects on lake sturgeon, increasing the risk of non-target mortality in higher alkalinity waters. Life stage also had a profound effect on TFM sensitivity, with rates of TFM uptake decreasing exponentially as lake sturgeon grew larger. This observation explains why the TFM tolerance of older 1+ sturgeon is greater than in YOY animals.

Neither water alkalinity nor TFM exposure affected gill structure or function in lake sturgeon. Nevertheless, water pH and alkalinity profoundly influenced rates of TFM uptake, which were explained by events taking in place in the gill microenvironment. The gill microenvironment has markedly different water chemistry than the bulk water surrounding the fish, due to the excretion of CO₂ and acid-base equivalents by the gills as water crosses the gills when fish are breathing. Based on work completed on rainbow trout, expired water pH was approximately 1.0 pH unit lower than the inspired, or bulk water pH, due to acidification as it crossed the gills. This effect was most pronounced in water of low alkalinity, which has a low acid buffering capacity. As alkalinity increased there was less acidification due to corresponding increases in the water buffering capacity. Thus, the decreases in TFM uptake we observed as water alkalinity increased were due to decreases in the amount of un-ionized TFM at the gill surface due to less acidification of the water in the gill microenvironment. Ongoing experiments in lake sturgeon should yield similar results.

Measurements of internal TFM and its detoxification products indicated that lake sturgeon used an additional pathway of TFM detoxification, sulfation. It had long been known that TFM was detoxified in the liver by a phase II biotransformation process called glucuronidation, in which a glucuronic acid functional group reacts with TFM to form TFM-glucuronide, which is less toxic, more water soluble and easier to excrete. However, our analysis demonstrated that sulfation, also a Phase II pathway, was an additional mechanism of TFM detoxification. Given these observations, we predicted that exposure to TFM for longer periods to lower doses of TFM would improve their survival during lampricide exposure. When this “long-and-low” approach was tested by exposing lake sturgeon to the 24-h LC_99.9 of larval sea lamprey, all sea lamprey were killed and lake sturgeon mortality was reduced by more than 80 %, but only in high alkalinity waters. There was no reduction in lake sturgeon mortality following “long-and-low” exposure to TFM at moderate alkalinity.

We conclude that the need to apply TFM at concentrations well beyond the MLC of sea lamprey amplifies the risk of non-target mortality in lake sturgeon, particularly in higher compared to low alkalinity waters. Such risks might be reduced, however, by delaying TFM treatments to the fall, when rates of TFM uptake and accumulation by lake sturgeon are much lower due to their larger body sizes. This strategy, combined with the adoption of a “long and low approach” of TFM application, could greatly reduce or eliminate lake sturgeon mortality in high alkalinity waters, without compromising lampricide effectiveness. However, we recommend that further tests in the field be conducted before such measures are adopted on a broader basis.