Brook trout Salvelinus fontinalis populations inhabiting groundwater‐fed, headwater streams are sensitive to biotic and abiotic changes caused by anthropogenic actions in their native range but can be very resilient in their introduced range. Brook trout have maintained a self‐sustaining population in the Little Plover River, Wisconsin, despite fish kills during extreme low flows between 2005 and 2009 caused by groundwater pumping and limited precipitation. Increased understanding of brook trout sensitivity and resilience in headwater streams can aid restoration, conservation and eradication efforts. Therefore, we evaluated their movements, survival and spawning behaviours in the Little Plover River using a PIT antenna array (July 2016–July 2019) and spawning redd surveys (2017–2018) paired with estimated groundwater inputs. Tagged individuals often exhibited localised, nocturnal movements for most of the year. However, movements increased and shifted to a diurnal pattern with decreased photoperiod and water temperature beginning during spawning and continuing throughout winter (November–February). Spawning movements primarily (90%) occurred in the upstream direction, with consistent patterns of short (rkm <0.9, 64.8.9–70.7%), moderate (rkm =0.9 – 3.5, 20.0–30.2%) and long (rkm ≥4.5, 4.5–6.2%) distance movements across the three spawning periods. Monthly survival estimates ranged from 72.1 to 98.9% throughout the study (mean =91.5%) and were consistently lowest during winter and spring. Redd surveys indicated spawning peaked in mid‐November and redds were more concentrated in upstream reaches and in areas with higher estimated groundwater inflows in 2017 but not in 2018 when surface flows were higher. Our results increase understanding of brook trout daily and seasonal movements, monthly and seasonal survival, and spawning behaviours including relative to groundwater and surface flows in headwater streams, further informing management, conservation, and eradication efforts.

2,4-Dichlorophenoxyacetic acid (2,4-D) is commonly used to control invasive aquatic macrophytes, including Eurasian watermilfoil (Myriophyllum spicatum) (EWM). Potential influences of 2,4-D on non-target organisms are poorly understood; however, research has suggested the possibility of lethal effects on certain fish species. Lake Ellwood, Wisconsin was treated with 2,4-D to control EWM annually during 2003–2012. Fish surveys following treatment revealed natural recruitment failures of several regionally thriving species, including largemouth bass (Micropterus salmoides) and bluegill (Lepomis macrochirus). We hypothesized that these species had been negatively influenced by the chemical treatments. We monitored the post-chemical treatment fish community and aquatic ecosystem for responses during 2013–2019. Similar data were collected from Cosgrove (EWM absent) and Seidel lakes (EWM present, no chemical treatment) as reference systems. Limnological and submersed aquatic vegetation conditions did not change on Lake Ellwood post-chemical treatment. Total zooplankton density increased immediately post-chemical treatment on Lake Ellwood and then stabilized, whereas total zooplankton density did not change on the reference lakes over time. Analyses indicated immediate increases in recruitment post-chemical treatment for largemouth bass, smallmouth bass (Micropterus dolomieu), bluegill, and yellow perch (Perca flavescens). Back calculation of year class strength confirmed failures occurred during treatment for northern pike (Esox lucius), largemouth bass, bluegill, and black crappie (Pomoxis nigromaculatus). Our results provide evidence to suggest long-term 2,4-D treatments may negatively influence fish through lethal and sublethal mechanisms. Thus, there remains a critical need for directed research on whole-lake herbicide treatment side-effects.