Fig 1.
Map of sampling area in the Grand River watershed (southern Ontario, Canada).
Fish and surface water were collected from 1 reference (R) site, 2 sites upstream (US), and 4 sites downstream (DS) of municipal wastewater effluent (MWWE) outfalls.
Table 1.
Properties of Waterloo and Kitchener municipal wastewater treatment plants from 2007 to 2011 [51].
Table 2.
Real-time PCR primers used to measure mRNA levels in rainbow darter livers.
R2 and efficiency (E) were calculated from fall 2011 and fall 2012 samples only.
Fig 2.
Concentration of nutrients and pharmaceuticals increases downstream of MWWTPs.
(A) Mean concentration of ammonia (mg/L) and (B) the sum of dominant chemicals of emerging concern (naproxen, ibuprofen, venlafaxine, carbamazepine, and triclosan) in river surface water. Samples were collected in the fall (dark symbols) and in the spring (open symbols) of 2010 (squares), 2011 (diamonds), and 2012 (down-facing triangles). Sites that share a common letter are not significantly different (p > 0.05) as determined by a 2-way ANOVA with a Tukey’s post-hoc test (no interactions were found for season and site).
Fig 3.
Profile of pharmaceuticals and personal care products in surface water.
Mean concentration (± SE) of select pharmaceuticals and personal care products in river surface water through an urban gradient. Samples were collected in the (A, C, E, G) fall and in the (B, D, F, H) spring 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The solid lines indicate the 95% confidence interval calculated from the mean of the data from the rural reference site (R).
Fig 4.
Vitellogenin (vtg) expression is higher in males collected downstream of MWWTP outfalls.
Relative change in vitellogenin (vtg) expression in liver tissues of (A, C) female and (B, D) male rainbow darter collected from sites through an urban gradient, compared with that in R males. Expression was measured in the (A, B) fall of 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles), as well as in the (C, D) spring of 2011. The dashed line indicates the upper 25% critical effect size calculated from the mean of the data from the rural reference site (R) over all 3 years, and the solid line indicates the upper 95% confidence interval from the pooled R. In the spring (panels C, D), * indicates significant (p < 0.05) difference between R and DSK sites as determined by a Student’s t-test.
Fig 5.
Hormone production of ovaries varies through the urban gradient and between seasons.
Normalized (to mean R site) in vitro steroid production of (A, C) estradiol and (B, D) testosterone in stimulated ovarian tissues collected from rainbow darter in the wild through an urban gradient. Measurements were taken in the (A, B) fall of 2007 (red triangles), 2011 (yellow diamonds), and 2012 (blue down-facing triangles), and well as in the (C, D) spring of 2011 (yellow diamonds) and 2012 (blue down-facing triangles). The dashed line indicates the upper 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the upper 95% confidence interval from the pooled R.
Fig 6.
Hormone production of testes is reduced in male exposed to MWWE.
Normalized (to mean R site) in vitro steroid production of (A, C) 11-ketotestosterone and (B, D) testosterone in stimulated testis tissue collected from rainbow darter in the wild through an urban gradient. Measurements were taken in the (A, B) fall of 2007 (red triangles), 2011 (yellow diamonds), and 2012 (blue down-facing triangles), as well as in the (C, D) spring of 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the upper 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the upper 95% confidence interval from the pooled R.
Fig 7.
Impact of MWWE on gonad development is clear in males, but not females.
Proportion of advanced cell types in (A, C) female and (B, D) male gonads of rainbow darter collected through an urban gradient. The proportion of all cell types was assessed through histological analysis of female and male gonads collected in (A, B) fall and (C, D) spring field seasons in 2007 (red triangles), 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the upper 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the upper 95% confidence interval from the pooled R.
Fig 8.
Intersex incidence and severity are consistently higher at sites downstream of MWWTP outfalls.
Intersex (A, C) incidence and (B, D) severity of male rainbow darter collected through an urban gradient in the (A, B) fall of 2007 (red triangles), 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles) and in the (C, D) spring of 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the upper 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the upper 95% confidence interval from the pooled R.
Fig 9.
Changes in gonad somatic index through the urban gradient are variable and difficult to interpret.
Gonad somatic index of (A, C) female and (B, D) male rainbow darter collected through an urban gradient in the (A, B) fall of 2007 (red triangles), 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles) and in the (C, D) spring of 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the 95% confidence interval from the pooled R.
Fig 10.
Liver somatic index is variable through the urban watershed.
Liver somatic index of (A, C) female and (B, D) male rainbow darter collected through an urban gradient in the (A, B) fall of 2007 (red triangles), 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles) and in the (C, D) spring of 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the 25% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the 95% confidence interval from the pooled R.
Fig 11.
Condition factor increases downstream of MWWTP outfalls in fall, but not spring.
Condition factor of (A, C) female and (B, D) male rainbow darter collected through an urban gradient in the (A, B) fall of 2007 (red triangles), 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles) and in the (C, D) spring of 2009 (grey circles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). The dashed line indicates the 10% critical effect size calculated from the mean of the data from the rural reference site (R), and the solid line indicates the 95% confidence interval from the pooled R.
Fig 12.
Principal coordinates (PCO) analysis of biological measures.
(A) Female and (B) male biological measures collected from a reference site (green squares), sites upstream of municipal wastewater treatment plant (MWWTP) outfalls (red downward triangles), or downstream of MWWTP outfalls (blue triangles). Each sampling site is labeled with the season (spring or fall) that data were collected. PCO is based on a Euclidean distance resemblance matrix.
Fig 13.
Canonical analysis of principal coordinates (CAP) ordination with biological variables as vectors.
(A) Female and (B) male biological measures collected from a reference site (green squares), sites upstream of municipal wastewater treatment plant (MWWTP) outfalls (red downward triangles), or downstream of MWWTP outfalls (blue triangles).
Table 3.
Comparison of significant changes observed in female rainbow darter downstream of the Waterloo (DSW) and Kitchener (DSK) MWWTPs in relation to either the rural reference site (R) or the immediate upstream (USW and USK, respectively) reference site.
Comparisons are made for fall and spring field collections.
Table 4.
Comparison of significant changes observed in male rainbow darter downstream of the Waterloo (DSW) and Kitchener (DSK) MWWTPs in relation to either the rural reference site (R) or the immediate upstream (USW, USK respectively) reference site.
Comparisons are made for fall and spring field collections.
Fig 14.
Variability of biological measures decreases with increasing biological complexity.
A comparison of variability in measures of male rainbow darter reproduction across levels of biological organization at a (A) reference site and a (B) MWWE-exposed site from collections in the fall of 2007 (red triangles), 2010 (green squares), 2011 (yellow diamonds), and 2012 (blue down-facing triangles). Measures compared include gene expression of vtg (VTG), in vitro production of testosterone (T), gonad development and relative proportion of spermatozoa (SPZ), and gonad somatic index (GSI).