Efficiency of advanced wastewater treatment technologies for the reduction of hormonal activity in effluents and connected surface water bodies by means of vitellogenin analyses in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta f. fario)

Abstract

Endocrine effects in the aquatic environment are in the focus of scientists and media along with debates on the necessity of further steps in wastewater treatment. In the present study VTG responses were compared to evaluate upgrades at wastewater treatment plants (WWTPs). We investigated several advanced sewage treatment technologies at two WWTPs connected to the Schussen, a tributary of Lake Constance, for the reduction of hormonal activity: (1) a powdered activated charcoal filter at the WWTP Langwiese; and (2) a combination of ozonation, sand filter, and granulated activated carbon filter at the WWTP Eriskirch. Rainbow trout and brown trout were either directly exposed to the effluents in aquaria or cages, or in a bypass system flown through by surface water of the Schussen. As a reference, trout were kept in bypass aquaria at the Argen River, which is less influenced by micropollutants. As a biomarker for estrogenicity, we analyzed the yolk precursor protein vitellogenin in immature rainbow trout and brown trout and in trout larvae (100 days post-fertilization) prior to and after the upgrade with the new technologies. Trout of different ages and species were used to detect differences in their sensitivity. At both bypass stations, larvae of brown trout showed significantly higher vitellogenin levels prior to the upgrade compared to negative control levels. Female brown trout exposed at the bypass station downstream of the WWTP showed decreased vitellogenin levels after the upgrade. In 1-year-old immature trout directly exposed to the respective effluents, no significant effects of the upgrades on vitellogenin levels were found. In general, larger effects were observed in brown trout than in rainbow trout, indicating that they are more sensitive test organisms.

Keywords: Endocrine disruption; Fish; Micropollutants; Vitellogenin; Wastewater treatment plant.

Fig. 1

Overview of the study design. PAC powdered activated charcoal

Fig. 2

Vitellogenin concentrations in blood plasma samples of rainbow trout exposed at the WWTP Eriskirch in aquaria connected to the conventional effluent or to the additionally treated effluent in 2013 and 2014; means and standard deviation (SD) are shown. Analyzed by Biosense rainbow trout vitellogenin ELISA kit. N-numbers 2013 females: negative control n = 9, WWTP effluent n = 6, additional treatment n = 8; males: negative control n = 1, WWTP effluent n = 7, additional treatment n = 3. No significant differences with Steel–Dwass test; p > 0.05. N-numbers 2014 females: negative control n = 6, WWTP effluent n = 5, additional treatment n = 4; males: negative control n = 13, WWTP effluent n = 6, additional treatment n = 10. No significant differences with Steel–Dwass test; p > 0.05. No significant differences between years; p > 0.05

Fig. 3

Means of weight (gram) and SD of exposed rainbow trout in 2013 and 2014. For n-numbers see Table 1 and for significant differences see Table 2

Fig. 4

Means of weight (gram) and SD of exposed brown trout in 2013 and 2014. For n-numbers see Table 3. Significant differences with the Tukey–Kramer HSD test: females: neg. control 2013—neg. control 2014 p = 0.0226 and males 2014: neg. control—Argen p = 0.0498 (Asterisks significant differences; *p < 0.05)

Fig. 5

Vitellogenin concentrations in blood plasma samples of rainbow trout exposed in 2013 and 2014 in cages upstream and downstream of the WWTP Langwiese; means and SD are shown. Analyzed with Biosense rainbow trout vitellogenin ELISA kit. N-numbers 2013 females: negative control n = 9, upstream WWTP n = 15, downstream WWTP n = 7; males: negative control n = 1, upstream WWTP n = 2, downstream WWTP n = 4. No significant differences with Steel–Dwass test; p > 0.05. N-numbers 2014 females: negative control n = 6, upstream WWTP n = 9, downstream WWTP n = 13; males: negative control n = 13, upstream WWTP n = 11, downstream WWTP n = 8. No significant differences with Steel–Dwass test; p > 0.05. No significant differences between years; p > 0.05

Fig. 6

a Vitellogenin concentrations in blood plasma samples of rainbow trout exposed in 2013 and 2014 at the bypass stations; means and SD are shown. Analyzed by Biosense rainbow trout vitellogenin ELISA kit. N-numbers 2013 females: negative control n = 9, Argen n = 8, Schussen n = 4; males: negative control n = 1, Argen n = 5, Schussen n = 9. No significant differences with Steel–Dwass test; p > 0.05. N-numbers 2014 females: negative control n = 6, Argen n = 8, Schussen n = 6; males: negative control n = 13, Argen n = 1, Schussen n = 16. No significant differences with Steel–Dwass test; p > 0.05. No significant differences between years; p > 0.05. b Values of a relative to negative control. Neg. control was set to 100 %

Fig. 7

a Absorbance measured in blood plasma samples of 1-year-old brown trout exposed at the bypass stations in 2013 and 2014; means and SD are shown. All samples of a group were analyzed within one semi-quantitative vitellogenin salmonid (Salmoniformes) biomarker ELISA kit (enzyme activity = color intensity is proportional to the concentration of vitellogenin in the sample). N-numbers 2013 females: negative control n = 4, Argen n = 4, Schussen n = 3; males: negative control n = 0, Argen n = 4, Schussen n = 4. No significant differences; p > 0.05. N-numbers 2014 females: negative control n = 6, Argen n = 6, Schussen n = 9; males: negative control n = 10, Argen n = 13, Schussen n = 5. Significant differences with the Tukey–Kramer HSD test: females 2014 neg. control—Schussen p = 0.0231 (Asterisks significant differences; *p < 0.05). b Values of a relative to negative control. Neg. control was set to 100 %. In 2013, no values could be given for males because of absence of males in the neg. control

Fig. 8

a Absorbance measured in homogenates of juvenile brown trout exposed for 99 days post-fertilization at the bypass stations in 2014; means and SD are shown. All samples were analyzed within one semi-quantitative vitellogenin salmonid (Salmoniformes) biomarker ELISA kit (enzyme activity = color intensity is proportional to the concentration of vitellogenin in the sample). Each treatment n = 12. No significant differences with the Steel–Dwass test; p > 0.05. For better comparison, previous results from 2013 prior to the upgrade are also shown. These results were already published in PlosOne by Henneberg et al. 2014 [22]. b Values of a relative to negative control. Neg. control was set to 100 %

Fig. 9

Overview of sampling sites, bypass stations and examined WWTPs at the Schussen River and Argen River, Lake Constance, South Germany