Lassen, P., Strand, J., Dähllöf, I., Larsen, M.M., Bossi, R., Wiberg-Larsen, P., Long, M., Krüger, T. & Bonefeld-Jørgensen, E.C. 2014. Undersøgelse af hormonforstyrrende kemikalier i vandløb. Aarhus Universitet, DCE – Nationalt Center for Miljø og Energi, 70 s. - Videnskabelig rapport fra DCE - Nationalt Center for Miljø og Energi nr. 85. http://dce2.au.dk/pub/SR85.pdf
In the last decades there has been an increasing focus on how endocrine disrupting compounds affect different animal species’ ability to reproduce. Effluent is an important source for endocrine disrupting compounds in the water environment but there are also contributions from other sources such as run-off from agricultural fields. The occurrence of endocrine disrupting compounds as well as their biological effects has been investigated in different studies in Denmark. But there are missing investigations on the direct correlations between the concentrations of endocrine disrupting compounds in the water environment and their possible biological effect under Danish environmental conditions.
This is a preliminary pilot project which correlates screening of endocrine disrupting compounds in streams with investigations of the effects of the compounds on the reproduction system in large mussels as well as endocrine disrupting effects on mammal cell cultures by in vitro bio analysis for detection of effects on hormone related receptors, comprising oestrogen-, androgen- and Ah-receptor activity.
In the project passive samplers and mussels were exposed simultaneously at different locations in streams, which included sites both with and without sewage effluent admission. The extracts from the samplers were used for analysis of endocrine disrupting compounds as well as for determination of endocrine effects on receptor activity in mammal cell cultures. The mussels were investigated for endocrine effects on male mussels’ sexual development by determining the levels of vitellogenin-like proteins (measured as alkali-labile phosphate, ALP). Vitellogenin-like proteins are egg-yolk proteins which take part in the formation of eggs (oocyter) in normal females. Furthermore, the mussels were investigated for signs of intersex by the formation of egg-like cells in the males’ testis.
Passive samplers were used in this project instead of traditionally water samples in order to obtain optimal information on the presence of the compounds. Two types of passive samplers were used: Polar Organic Chemical Integrative Samplers (POCIS) for sampling polar and semi polar compounds and silicone membranes (SM) for sampling of nonpolar compounds. Based on Performance Reference Compounds (PRC), which were spiked into the membranes before exposure, the amount of water that passed the membranes could be calculated and from these values an average water concentration of the single compounds could be determined.
The results from the chemical analysis showed, not surprisingly, that a range of compounds had higher concentrations downstream for the water treatment plants compared to upstream and other localities. The results also showed that there are other sources as compounds were found at the other localities, especially organotin, PFAS and certain pesticides indicating more diffuse sources. The use of passive samplers as a method for the chemical analysis proved in general to be robust with relative low deviations for duplicate determinations and significant lower detection limits compared to traditionally water analysis. Especially the POCIS-membranes can be regarded suitable for screening of pollutants, which then later on can be included in monitoring programs based to the produced data.
The analyses of the receptor effects in cell cultures were in general highest downstream for the water treatment plants similar to the chemical data. There was observed an increased oestrogen-, androgen- and Ah-receptor activity in the downstream samples. However, the analysis also showed that compounds which inhibit the receptor activities also were present in the same samples. It can therefore be concluded that endocrine disrupting compounds are released from sewage effluents. Furthermore, the results showed that streams which were expected to be relatively unpolluted also contained endocrine disrupting compounds in concentrations that were able to activate all three tested receptor systems. However, it can be difficult to exactly estimate the effects in the environment on the basis of the results. The Results should be considered as biomarkers.
Investigations on the transplanted river mussels (Unio tumidus) showed that alkali-labile phosphate (ALP) in hemolymphe (blood fluid) from males in general was higher downstream for water treatment plants and that the mussels showed signs of endocrine disrupting effects. ALP is used as a biomarker for development of vitellogenin-like (egg-yolk) proteins in males and thereby indicating that the mussels have been subjected to an estrogenic exposure. This is supported by a surprisingly high percentage of males which showed intersex in the form of egg-like cells in the gonad fluid which were found under the determination of the gender. These results were not expected as this type of intersex only has been documented for marine mussels and not for fresh water mussels. More detailed histological investigations are needed in order to finally verify these indications on intersex.
Comparison of results showed a clear statistic correlation between the biologically determined toxic dioxin equivalents (AhR-TEQ) and the frequency of ALP/intersex in male mussels. It is the first time that this correlation ever has been investigated and documented in the environment.
It was more difficult statistically to correlate the measurements of the single chemical compounds with the biological effects. The main reason for this is that the data material is too small (the number of locations) in relation to the number parameters which are measures and that the effects are not related to single compounds but is a net effect from the combined compound mixture, “cocktail effect”. However, there was a clear tendency that the concentrations of certain endocrine disrupting compounds were significant elevated in streams downstream of the water treatment plants and the in vivo investigations of the river mussels as well as the results from the in vitro receptor studies showed that these elevated concentrations will lead to an effect on the organisms present in the environment.
The investigation showed that there also are endocrine disrupting compounds as well as biological effects in water areas which were expected to be relatively unpolluted with these compounds. This confirms that it is difficult to find areas in Denmark which are unaffected by human activities.
The project shows that it has great significance to combine biological effect measurements with measurements of concentrations when the ecological state of the water environment is evaluated. There was found biological effects at all localities even though the concentrations in general were low. The project shows that it is likely that the effects are caused by a combination of many compounds, which are cocktail effects.