Maturity of spirlin.

<p>Distribution of gonadal maturity (stage 1 = immature; stage 2 = intermediate and, stage 3 = mature) of feral spirlin. 2009–2011. Females: summer Argen n = 35, summer Schussen n = 30, autumn Argen n = 16, autumn Schussen n = 7. Males: summer Argen n = 19, summer Schussen n = 3, autumn Argen n = 19, autumn Schussen n = 8.</p

Location of the sampling sites and bypass systems at the Schussen and Argen rivers in Southwest Germany.

<p>Waste water treatment plant (WWTP) Langwiese and Eriskirch, as well as the storm water over-flow basin (SOB) at the Schussen. Geographic coordinates: S 0 = N47° 45′ 29.40″, E9° 35′ 21.78″, S 1 = N47° 45′ 19.22″, E9° 35′ 25.35″, S 3 = N47° 39′ 16.09″, E9° 31′ 53.35″, S 6 = N47° 37′ 4.73″, E9° 31′ 50.33″S 4 = N47° 44′ 20.46″, E9° 53′ 42.78″, bypass Gunzenhaus = N47° 40′ 44.00″, E9° 32′ 24.77″, and bypass Pflegelberg = N47° 39′ 11.21″, E9° 44′ 30.80″.</p

Model of the study design.

<p>This figure gives an overview of the study design and all performed analyses. Based on their results, we arranged the tests according to their evidence for endocrine disruption.</p

Reproduction test with the mudsnail.

<p>Means and standard deviation of the reproduction test with <i>Potamopyrgus antipodarum</i>. Total embryo number per female in negative (C) and positive controls (PC), in effluent water from the waste water treatment plant Langwiese (STP effluent) and in the two field sediments from sampling sites S 3 at the Schussen river and S 4 at the Argen river (station 3 and station 4) over the seven sampling campaigns. Asterisks indicate significant differences vs. C (one-way ANOVA with Dunnett´s multiple comparison test; p<0.01).</p

Vitellogenin in juvenile brown trout.

<p>Vitellogenin levels in homogenates of juvenile brown trout 99 days post fertilization in 2011/2012, means and standard deviation. Analysed by Biosense rainbow trout vitellogenin ELISA kit. Samples: Neg. control n = 6 (1 out of 6 pos. result, bypass Argen n = 10 (2 out of 10 showed a pos. result), bypass Schussen n = 10 (5 out of 10 showed a pos. result). No significant differences (Steel-Dwass-test: neg. control- bypass Argen p = 1,00, neg. control- bypass Schussen p = 0,5787 and, bypass Schussen- bypass Argen p = 0,4030).</p

Chemical analysis of water and sediment samples.

<p>Chemical analysis of water and sediment samples.</p

E-screen assay (estrogenic activity).

<p>Results of the E-screen assay expressed in 17β-estradiol equivalents (EEQ) in ng/L; means and standard deviation. Only data of samples which showed a low cytotoxicity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098307#pone-0098307-g004" target="_blank">figure 4</a>) were used. WW (Waste water of WWTP Langwiese) n = 4, S 0 n = 5, S 1 n = 4, S 3 n = 7, S 6 n = 6 and S 4 n = 11).</p

E-screen assay (cytotoxicity).

<p>Results of the E-screen assay regarding the cytotoxicity of the analysed samples. Expressed in 1/EC₅₀ Tox (concentration in which 50% of the cells are able to grow) units; means and standard deviation. WW (Waste water of WWTP Langwiese) n = 9, S 0 n = 5, S 1 n = 4, S 3 n = 11, S 6 n = 11 and S 4 n = 11.</p

Dates of the sampling campaigns.

<p>Dates of the sampling campaigns.</p

Semi-quantitative vitellogenin detection in juvenile brown trout.

<p>Absorbance measured in homogenates of juvenile brown trout 111 days post fertilization and 124 days after fertilization exposed in 2012/2013; means and SD. Each sampling analysed with one semi-quantiative vitellogenin salmonid (Salmoniformes) biomarker ELISA kit (enzyme activity = colour intensity is proportional to the concentration of vitellogenin in the sample). Samples March 2013: Neg. control n = 5, bypass Schussen n = 7, bypass Argen n = 6. Significant differences with Steel-Dwass-test: neg. control- bypass Schussen p = 0,0159 and neg. control- bypass Argen p = 0,0221; * = p<0.05. Samples April 2013: Neg. control n = 12, bypass Schussen n = 12, bypass Argen n = 12. No significant differences with Steel-Dwass-test.</p