High concentrations of polycyclic aromatic hydrocarbons (PAHs) in streams can be a significant stressor to aquatic organisms. To understand the likely sources and toxicity of PAHs in Milwaukee-area streams, streambed sediment samples from 40 sites and parking lot dust samples from 6 sites were analyzed for 38 parent PAHs and 25 alkylated PAHs. Diagnostic ratios, profile correlations, principal components analysis, source-receptor modeling, and mass fractions analysis were used to identify potential PAH sources to streambed sediment samples, and land-use analysis was used to relate streambed sediment PAH concentrations to different urban-related land uses. On the basis of this multiple lines-of-evidence approach, coal-tar pavement sealant was indicated as the primary source of PAHs in a majority of streambed sediment samples, contributing an estimated 77% of total PAHs to samples, on average. Comparison with the probable effect concentrations and (or) the equilibrium partitioning sediment benchmark indicates that 78% of stream sediment samples are likely to cause adverse effects to benthic organisms. Laboratory toxicity tests on a 16-sample subset of the streambed sites using the amphipod Hyalella azteca (28-d) and the midge Chironomus dilutus (10-d) measured significant reductions in 1 or more biological endpoints, including survival, in 75% of samples, with H. azteca more responsive than C. dilutus. Environ Toxicol Chem 2017;36:1622-1635. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

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