Map of the Mud River watershed.

<p>Sampling sites plus age and extent of surface mining activity. Insets show sequentially larger geographic regions.</p

Toxicity of stream water is driven by osmotic stress.

<p>Stream water, but not sediment pore water, from the Mud River and its tributaries caused greater growth inhibition in osmotic stress-sensitive <i>gpdh-1</i>(<i>ok1558</i>);<i>gpdh-2</i>(kb33) deletion mutants in comparison to wild-type nematodes, demonstrated in samples collected from Sugartree Branch (<b>6A</b> and 6B p=0.0023n=23-55) and Laurel Branch (<b>6C</b> and 6D p=0.0004n=39-51), May 2012. Statistically significant difference from reference site indicated by asterisks. “Size” refers to the optical density (extinction) of each nematode and describes growth since the data shown is for size after three days of exposure. Each exposure was repeated at least two times separately.</p

Growth effects of stream water and sediment pore water.

<p>The growth of wild-type nematodes was affected by stream water (<b>2A</b>, n=27-359) and sediment pore water (<b>2B</b>, n=30-359) from the Mud River and its tributaries, but not by water or sediment pore water from a reference site on the unmined Left Fork. Statistically significant difference from EPA water control indicated by asterisks. “Size” refers to the optical density (extinction) of each nematode and describes growth since the data shown is for size after three days of exposure. Each exposure was repeated at least two times separately. EPA water was used as a laboratory control medium.</p

Mining-impacted tributaries affect the toxicity of the Mud River.

<p>Contributions from mine-affected tributaries have an additive effect on the toxicity of both water (<b>4A</b>, April 2011n=37-251) and sediment (<b>4B</b>, December 2010 n=148-467) from the main branch of the Mud River. Statistically significant difference between upstream and downstream site indicated by asterisks. “Size” refers to the optical density (extinction) of each nematode and describes growth since the data shown is for size after three days of exposure. Each exposure was repeated at least two times separately. Please note the difference in Y-axis scaling.</p

Toxicity of sediment pore water is driven by metals or metalloids.

<p>Stream sediment pore water, but not stream water, caused greater growth inhibition in metals-sensitive <i>pcs-1</i>(tm1748) and <i>smf-2</i> mutants than in wild-type nematodes, demonstrated in samples collected from Mud River sites 5a and 7 in December 2010 (<b>5A</b> p=<0.0001, n=142-533<b>, 5B</b> p=0.0022, n= 42-277), Mud River 5a in April 2011 (<b>5C</b> p=<0.0001 n=55-277) and Berry Branch in April 2011 (5D p=<0.0001, n= 125-482). Statistically significant difference from sediment-treated wild-type nematodes indicated by asterisks. “Size” refers to the optical density (extinction) of each nematode and describes growth since the data shown is for size after three days of exposure. Each exposure was repeated at least two times separately.</p