14The soundscape composition of freshwater habitats is poorly understood. Our goal was 15 to document the occurrence of biological sounds in a large variety of freshwater habitats over a 16 large geographic area. The underwater soundscape was sampled in freshwater habitat categorized 17 as brook/creek, pond/lake, or river, from five major river systems in North America 18 (Connecticut, Kennebec, Merrimack, Presumpscot, and Saco) over a five-week period in the 19 spring of 2008. Over 7,000 sounds were measured from 2,750 minutes of recording in 173 20 locations, and classified into major anthropophony (airplane, boat, traffic, train and other noise) 21 and biophony (fish air movement, also known as air passage, other fish, insect-like, bird, and 22 other biological) sound categories. Anthropogenic noise dominated the soundscape of all 23 habitats averaging 15 % of time per recording compared to less than 2 % for biological sounds.24 Anthropophony occurred in 79 % of recordings and was mainly due to traffic and boat sounds, 25 which exhibited significant differences among habitats and between non-tidal and tidal river 26 regions. Most biophonic sounds were from unidentified insect-like, air movement fish, and other 27 fish sound sources that occurred in 57 % of recordings. Mean frequencies of anthropogenic 28 noises overlapped strongly with the biophony, and comparisons of spectra suggest that insect-29 like and air movement sounds may be more susceptible to masking than other fish sounds. There 30 was a significant decline in biodiversity and biophony with increasing ambient sound levels.31 Our poor understanding of the biophony of freshwater ecosystems, together with an apparent 32 high temporal exposure to anthropogenic noise across all habitats, suggest a critical need for 33 studies aimed at identification of biophonic sound sources and assessment of potential threats 34 from anthropogenic noises. 35 3 36 Introduction 37 Fish sounds were first seriously studied in North America by Abbot [1] who lamented 38 that "the little fishes of our inland brooks and more pretentious denizens of our rivers are looked 39 upon as voiceless creatures…" and concluded that "certain sounds made by these fishes are 40 really vocal efforts….". However, Stober [2] was the first to describe the underwater noise 41 spectra in relation to freshwater fish sounds. He also pointed out the critical lack of data on the 42 ambient noise and fish sounds in freshwater systems. Fortunately, after long neglect there has 43 been a recent surge in interest in the potential impacts of anthropogenic noise on freshwater 44 ecosystems [3-7], but efforts to understand such impacts are hampered by the paucity of data on 45 the natural soundscape composition. The paucity of data on the sound production of freshwater 46 fishes was highlighted in our recent review of the literature which found that sounds have been 47 reported in only 87 species in North America and Europe, but detailed descriptions of sound 48 characteristics are known for only 30 specie...