Abstract:The Joint Danube Survey 3, carried out in 2013 was the world's biggest river research expedition of its kind. The course of the second largest river of Europe passes large cities like Vienna, Budapest and Belgrade and is fed from many tributaries like Inn, Thisza, Drava, Prut, Siret and Argeș. During the 6 weeks of shipping the 2375 km downstream the River Danube from Germany to the Black Sea an enormous number of water samples were analyzed and collected. A wide spectrum of scientific disciplines cooperated in analyzing the River Danube waters. For toxicological analysis, water samples were collected on the left, in the middle, and on the right side of the river at 68 JDS3 sampling points and frozen until the end of the Danube survey. All samples were analyzed with two in vitro bioassays tests (umuC and MTS). water samples came up with toxic signals on two stretches, when activated with S9 enzymes. The override of the limiting value of the umuC investigation with prior S9 activation started downstream Vienna (Austria) and was prolonged until Dunaföldvar (Hungary). This stretch of the River Danube passes a region that is highly industrialized, intensively used for agricultural purposes and also highly populated (Vienna, Bratislava and Budapest). The elevated values may indicate these influences.